Linking the physico-chemical characteristics and ecotoxicology of manufactured. nanomaterials in aquatic and terrestrial environments

Submitted to Environmental Science and Technology Lead, J.R., Smith, E.L., Scott-Fordsmand, J.J., Baun, A., Handy, R.D., Slaveykova, V.L., Tyler, C.R....
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Submitted to Environmental Science and Technology Lead, J.R., Smith, E.L., Scott-Fordsmand, J.J., Baun, A., Handy, R.D., Slaveykova, V.L., Tyler, C.R., Kammer, F., Benedetti, M., Boxall, A., Brust, M., Cumpson, P., Fernandes, T., Hassellov, M., Henry, T.B., Holbrook, R.D., Kookana, R., Masion, A., McClellan-Green, P., Nelson, N., Owen, R., Park, B., Garrod, J., Valsami-Jones, E., Vincent, B. (2008). Linking the physico-chemical characteristics and ecotoxicology of manufactured nanomaterials in aquatic and terrestrial environments. Submitted.

Linking the physico-chemical characteristics and ecotoxicology of manufactured nanomaterials in aquatic and terrestrial environments Jamie R Lead*, Emma L. Smith, Janeck J. Scott-Fordsmand, Anders Baun, Richard D. Handy, Vera I. Slaveykova, Charles R. Tyler, Frank von der Kammer, Marc Benedetti, Alistair Boxall, Mathias Brust, Peter Cumpson, Teresa Fernandes, Martin Hassellov, Theodore B. Henry, R. Dave Holbrook, Rai Kookana, Armand Masion, Patricia McClellan-Green, Noel Nelson, Richard Owen, Barry Park, John Garrod, Eva Valsami-Jones, Brian Vincent. *Lead, J.R., Division of Environmental Health & Risk Management, School of Geography, Earth & Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK; +44 121 414 8147 (phone), +44 121 414 5528 (fax), Email: [email protected] Smith, E.L., Division of Environmental Health & Risk Management, School of Geography, Earth & Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. Scott-Fordsmand, J.J., Department of Terrestrial Ecology, National Environmental Research Institute, Aarhus University, PO Box 314, Vejlsøvej 25, DK-8600 Silkeborg, Denmark. Baun, A., Institute of Environment & Resources, NanoDTU, Technical University of Denmark, Kgs, Lyngby, Denmark. Handy, R.D., Ecotoxicology and Stress Biology Research Group, School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth, UK. Slaveykova, V.I., Environmental Biophysical Chemistry, ISTE_ENAC, Ecole Polytechnique Federal de Lausanne, Station 2, 1015 Lausanne, Switzerland. Tyler, C.R., Environmental and Molecular Fish Biology Group, School of Biosciences, Hatherly Laboratories, University of Exeter, Prince of Wales Road, Exeter, Devon, EX4 4PS, United Kingdom. Benedetti, M. Universite Paris – Diderot – IPGP, Case courrier 7052, 2 place jussieu, 75252 Paris cedex 05, France. 1

Submitted to Environmental Science and Technology

Boxall, A. Environment Department, University of York, Heslington. York, YO10 5DD, UK. Brust, M. Centre for Nanoscale Science, Department of Chemistry, University of Liverpool, L69 7ZD, UK. Cumpson, P. National Physics Laboratory, Teddington, Middlesex, TW11 0LW, UK. Fernandes, T. Environmental Biology Research Group, Faculty of Health, Life and Social Sciences, Canaan Lane Campus, Edinburgh, EH9 2TB, UK. Garrod, J.Chemicals and Nanotechnologies Division, Area 2A Nobel House, 17 Smith Square, London, SW1P 3JR, UK. Hasselov, M. Department of Chemistry, Göteborg University, SE-412 96, Göteborg, Sweden. Henry, T.B., Ecotoxicology and Stress Biology Research Centre, School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth, Devon, PL4 8AA, UK. Holbrook, R.D. Surface and Microanalysis Science Division, National Institute of Standards and Technology, 100 Bureau Drive, Mail Stop 8371, Gaithersburg, MD 20899-8371, USA. Kookana, R. CSIRO Land and Water, Adelaide laboratory, PMB 2. Glen Osmond, 5064, Australia. Masion, A. CEREGE, Europole de l'Arbois, BP 80, 13545 Aix-en-Provence, Cedex 04, France. McClellan-Green, P. Department of Environmental and Molecular Toxicology, North Carolina State University, Box 7633, Raleigh NC 27695 7633, USA. Nelson, N. The Royal Commission on Environmental Pollution, 5-8 The Sanctuary, Westminster, London SW1P 3JS, UK. Owen, Richard. Head, Environment and Human Health, Science Department, Environment Agency, Block 1 Government Buildings, Burghill Rd, Bristol BS10 6BF, UK. Park, B. Oxonica Materials Limited, 7 Begbroke Science Park, Sandy Lane, Yarnton, Kidlington, Oxfordshire OX5 1PF, UK. v.d. Kammer, F., Nanogeosciences Division, Department of Environmental Geosciences Center for Earth Sciences, Vienna University, Althanstrasse 14, A-1090 Wien, Austria.

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Submitted to Environmental Science and Technology Valsami-Jones, E., Department of Mineralogy, Natural History Museum, Cromwell Road, London SW7 5BD, UK. Vincent, B., Polymer and Colloid Group, School of Chemistry, University of Bristol, Cantock’s Close, Bristol, BS8 ITS, UK.

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Submitted to Environmental Science and Technology Abstract As nanotechnology has become a major industry, risks of nanomaterials (NMs) in the environment are a growing concern. In addition to the potential environmental risk of NMs, long term sustainability of the nanotechnology industry, with its immense potential societal benefits, is, in part, dependent on the development of an appropriate risk framework. The current consensus is that an understanding of the physicochemical behaviour of NMs is paramount in the assessment of ecotoxicological impacts and likely mechanisms of biological effects. This understanding will, for instance, allow accurate quantification of exposure, dose and the establishment of a relationship between ecotoxicological testing in the laboratory and effects in natural systems. This critical review is a summation of a Workshop held at the University of Birmingham (November 2007), attended by the authors with a view to address these questions. This paper attempts to understand our current knowledge of the links between physico-chemical characterisation and ecotoxicology in aquatic and terrestrial systems. Recommendations in priority order are the development of analytical methodologies to quantify NM concentration and characteristics in natural systems and the need for an improved understanding of NM fate and behaviour. Finally, there is a need for systematic experiments under realistic conditions which examine and link both suitable ecotoxicological endpoints and physico-chemical characteristics.

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Submitted to Environmental Science and Technology Introduction Nanoscience can be defined as the scientific study of materials on the nanoscale, approximately defined as the length scale between 1-100 nm (1) and nanotechnology as the technological application of this science (2).

Nanomaterials (NMs) can

therefore be defined as materials with at least one dimension in that size range, and importantly with novel properties which are different from the dissolved phase or from larger scale particles (2). Nanoparticles can be defined as an important sub set of NMs, where all dimensions are within 1-100 nm (3). These definitions are not formally agreed as yet and various international bodies (e.g. BSI, ISO, ASTM, OECD) are currently working on precise and formal definitions and nomenclature. The importance of NMs lie in their novel properties and behaviour, compared to the larger scale material.

The economic importance of nanotechnology is in little doubt, with research and development spending globally of many thousands of millions of US dollars every year (4) and estimates of the global market of manufactured goods which will incorporate nanotechnology reaching approximately 2.6 trillion US dollars by 2014 (5). Benefits of nanotechnology and nanoscale materials are still being realised, although they are already used in a range of processes, including remediation of contaminated land and as fuel additives to increase fuel efficiency, whilst appearing in many consumer goods such as cosmetics and sun tan lotions. Increasingly, the benefits, risks and social, economic, human health and environmental impacts of NMs are being discussed (6-8).

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Submitted to Environmental Science and Technology A number of recent reviews have been published on the subject of nanoparticles in the environment (9-13). The current discussion paper seeks more specifically to understand the relationship between the physico-chemical properties of NMs and their bioavailability in the environment, including biological uptake mechanisms, likely effects and the need for these interactions to be considered in an assessment of their risk. This connection is not in dispute within the ‘environmental nanoscience’ and toxicology community (14), but is poorly understood due in part to the absence of systematic and extensive peer-reviewed literature data. In addition, despite the universal acknowledgement of the relevance of physico-chemical properties to ecotoxicology, supporting data from the literature is haphazard and sporadic. Hansen et al (3) recently reviewed ecotoxicology and toxicology literature assessing which data sets reported the common physico-chemical parameters usually thought to be important in interpreting biological effects. The results are instructive: of 428 studies reviewed, size and some chemical composition of nanomaterials was always measured, but other characterisation was generally lacking. Thus, it is not possible to currently link specific properties of the nanoparticles to observed effects, in a quantitative and mechanistic way.

In addition to the paucity of data in ecotoxicological and environmental fate and behaviour, there is a limit to our conceptual understanding of the exact mechanism(s) of uptake or subsequent toxicity of NMs (at an organism or cellular level) to biota, leaving it difficult to generalise or predict which of the relevant physico-chemical parameters are most important. Logistically, it is essentially impossible to measure all relevant parameters for all relevant conditions, although such a broad brush approach will necessarily be attempted in the early stages of research to try to correlate

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Submitted to Environmental Science and Technology important parameters and define biological mechanisms. It is hoped that some finetuning of this approach may be possible in the short to medium term. Almost certainly this will be performed on a case by case basis for the foreseeable future, until the development of more generally applicable theory.

From the above, it is clear that fully and quantitatively linking physico-chemistry and biology is currently impossible. This paper will not seek to answer these questions definitively, which can only be performed over time by good quality science, but will discuss them within a conceptual framework, to assist in developing future research objectives. Conceptually, the area of interest of this paper is presented in Figure 1, which shows the importance of considering the physico-chemistry of the medium and the characteristics of the NM within the medium, as well as any interactions (external or internal) with the organism of interest. One critical aspect of this diagram is the requirement for characterisation of the NM in environmental compartments under appropriate conditions, which will provide a better understanding of behaviour and effects of NMs for hazard identification and risk assessment. In addition, it will allow effort to focus on assessment of potentially important parameters in order to provide future predictive modelling tools. The protocol for this characterisation is presented in Figure 2, showing the importance of iteratively characterising NMs in the stock solution, in exposure media (before and during exposure to an organism), and once in contact with biological surfaces and taken up by the organism of interest. Not shown explicitly are kinetic behaviour due to physical, chemical and biological processes.

This paper will briefly discuss NM characteristics in the laboratory and their likely transformations and behaviour in the environment, along with their biological uptake

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Submitted to Environmental Science and Technology and effects, leading on to consideration of their physico-chemical properties in ecotoxicological assessment and the need for characterisation. The paper ends with a number of recommendations for future research which the authors believe need to be addressed to provide a sound understanding of the potential ecological risks of NMs.

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Submitted to Environmental Science and Technology Manufactured nanomaterials and their environmental behaviour Manufactured NMs which are purposefully produced by human activity include metals, metal oxides, sulfides and selenides, fullerenes and carbon nanotubes. It is essential to consider these NMs separately from naturally occurring terrestrial and aquatic colloids and nanoparticles (15, 16) and atmospheric natural or adventitious ultrafines and nanoparticles (10). These natural and adventitous NMs, although in the same size range as manufactured NMs, may be produced by erosion, microbial processes or by fossil fuel combustion, for instance. Natural nanoparticles (and colloids) have important environmental functions (15) but have also been shown to have deleterious environmental effects (17). Adventitiously produced nanoparticles (or ultrafines) have been studied in great detail mainly because of their direct human health effects (10, 18), particularly on the cardiovascular system.

Manufactured NMs (subsequently referred to simply as NMs) may be qualitatively different from natural or adventitious nanoscale particles and may have different environmental impacts because of their large and increasing production volume and their structured nature, which may infer upon them unique properties, particularly at the smallest size ranges (19, 20, 21). Representative TEM images of iron oxide nanoparticles (Figure 3a) and carbon nanotubes in association with naturally produced clays (Figure 3b) are shown as examples of both manufactured and natural NMs.

NMs can be composite structures consisting of a core material (usually this is the referred to as the NM) and a shell around the core, either deliberately produced, such as with many quantum dots, or not, as in the oxidation of zerovalent iron NMs to form an iron oxide shell (22). In addition, a surface active agent is almost always used in

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Submitted to Environmental Science and Technology real world applications of NMs. This surface active agent (sometimes referred to as a capping agent) is usually an organic molecule such as a polymer or surfactant. All aspects of the NM will likely affect their relevant behaviour in the environment. The polymer or surfactant layer, for instance, is often used in order to impart colloidal stability and prevent aggregation either by increased charge repulsion or steric effects, and new and targeted NMs with improved stabilizing agents are being produced with increasing pace. Many, for instance, are aimed at crossing biological membrane barriers to aid in drug delivery and other medical applications (23, 24) and these may be of particular concern in the environment because of this characteristic. Stability and ability to cross biological membranes need to be considered when discussing environmental

and

ecotoxicological

effects

and

aggregation-disaggregation

mechanisms are likely to control fate and behaviour of NMs in both aquatic and terrestrial systems. Aggregation may be the default expectation by the majority working in this area, but may not occur as fully, rapidly or irreversibly as expected. For example interactions with a range of organic molecules such as biopolymners, surfactants and other colloids have been found to stabilize CNTs (13). Similarly the interactions with natural organic macromolecules (humic and fulvic acids) may further stabilize NMs (25, 26, 27). In addition, a current concern of nanotechnologists is the (self)assembly of NM building blocks producing more active structures (28) and this aspect of NM structures may need to be considered in future studies.

Nevertheless, on the basis of very little data, but borrowing heavily from the literature on aquatic and terrestrial colloids (15), the general expectation is that NMs, once released into the environment will aggregate relatively quickly. Once this takes place in aquatic systems, they are likely to partition out of the water phase and settle out to

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Submitted to Environmental Science and Technology the sediments. This is likely to be particularly important in the estuarine mixing zone, where salt concentrations increase rapidly, leading to aggregation and precipitation, due to compression of the diffuse double layer and charge neutralization (29). In terrestrial environments and other porous media (soils, sediments and groundwaters), the demobilization of NMs will likely occur through deposition/attachment to immobile soil particles (i.e. heteroaggregation) and also homoaggregation and subsequent straining filtration, resulting in localization of the NMs and a lack of transport (30). This picture of aggregation and reduced transport is complicated by a number of environmental processes. Importantly, our understanding of medium to long term behaviour of NMs is poor: on what timescale do aggregation and disaggregation processes occur? No unifying and predictive theory in complex environmental media yet exists, and the diversity of nanomaterials and environmental systems suggests that a unifying theory will be extremely difficult to develop. In particular, how do conditions or processes increasing stability (soft waters of low ionic strength, presence of humic substances (HS) (26)) and those increasing aggregation (high ionic strengths, presence of organic fibrillar material (31)) interact with diverse and complex structures of NMs? The effect of pH and HS, for instance, although primarily related to NM charge repulsion and attraction, is complex. For instance, at high and low pH values, inorganic NMs are likely to be either negatively or positively charged. Mechanisms of charge generation of fullerenes and CNTs in waters are not fully known (32) and the situation here may require further elucidation. Humic substances have a point of zero charge at about pH 2 (33) and will likely sorb to NMs (26), modifying the surface charge of NMs in natural aquatic media and can therefore stabilize or destabilize NMs in a manner which is pH dependent in a complicated manner. In addition, HS may stabilize material by steric mechanisms

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Submitted to Environmental Science and Technology (34). At low pH values, highly stable NMs will be destabilized and aggregate in the presence of HS (35) due to HS sorption and subsequent reduction of charge. At high pH values and low HS:NM ratios, we might expect patchy surface coverage by HS (36) where both the HS and NM are exposed to the aqueous phase, complicating understanding further. Clearly, fate and behaviour of NMs depends on both the NM characteristics and the relevant environmental conditions (flow, organic matter, pH etc) and are thus difficult to predict currently.

A first step towards further understanding of the environment fate and behaviour is to have the analytical capability to detect low concentrations of different NMs in different physico-chemical forms, usually against a high background of natural nanoparticles, with a similar structure and chemistry. Essential advances are being made with advanced separation, spectroscopic and microscopic methods such as flow field flow fractionation – inductively coupled plasma – mass spectrometry (FlFFFICP-MS) (37) and two-dimensional Field-Flow Fractionation-Liquid Chromatography (38) but much work remains to be performed.

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Submitted to Environmental Science and Technology Biological uptake and effects Biological uptake

As shown in Figure 1, an initial step in the biological uptake

mechanisms, which will be strongly linked to fate and behaviour and to NM properties, is movement from the bulk solution (fresh or marine water, soil pore water or gut luminal fluid, for instance), where transport through a diffusive or unstirred layer may contain high concentrations of mucus type secretions close to the organism. Both the properties of this medium (flow, viscosity, chemistry) and the behaviour and properties of the NM in this medium will affect movement and uptake.

Uptake of chemicals including NMs may be either by passive diffusion processes or by active uptake, both of which are likely to be dependent on species (39) and NM surface properties. For instance, diffusive transport both through the unstirred layer, mucus and through and between cells is likely to be size dependent, with only the smallest material (less than a few nm) able to be taken up. For example, non-specific diffusion can occur for NPs smaller than e.g. 2nm (corresponding to the largest globular protein that can pass through the intact bacterium cell walls) (40). Uptake kinetics can be size and shape dependent (41). Uptake and attachment is also likely to be partly charge dependent with electrostatic interactions playing a role (42), although other non-electrostatic forces will also be crucial. Such differences in uptake kinetics, may be reflected in the dose-response kinetics and it can be speculated as to whether or not the standard dose-response relationship as observed for conventional chemicals applies to NM (43). The precise uptake rate and specific routes onto and through the cells for each individual manufactured NM in a particular species will depend on the details of the organism’s surface chemistry (e.g. presence of mucus) and external anatomy (e.g. soft bodied organisms versus those with a hard carapace), as well as the

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Submitted to Environmental Science and Technology behaviour on the NP on/in the cells, and the organism’s lifestyle (e.g. benthic vs pelagic). Furthermore, it has been shown that gram negative and gram positive bacteria have widely different penetration potential for NM (44).

In addition to uptake through intact cell structures, damage to cell membranes and walls may result from processes such as reactive oxygen species (ROS) production (44) and inflammation (45), alteration of membrane permeability (44) or direct physical interactions or other processes. Such damage may result in entry to cells of NMs (46, 47). Highly saline cell media may limit uptake and effects due to enhanced aggregation potentially reducing bioavailability (48), although aggregates of NMs may be more toxic than bulk scale material of the same size and biological effects (49) may result despite aggregation.

Biological effects

In mammalian and human toxicology, a major thrust of work

has been on the production of ROS, oxidative stress, inflammation and related effects. In aquatic and terrestrial environments, the literature is both sparser and more equivocal. While oxidative stress has been shown in fish (45) and in bacteria (42), other studies have indicated that this may be not be the most important mechanism (50, 51) or may not be operative. Effects on respiration (52) have been observed from single walled CNTs in fish, while algal toxicity due to zinc oxide particles has been shown to be related to dissolved zinc ion toxicity released from the nanoparticle (53). Nanoparticles may potentially also modify toxic outcomes of other xenobiotics in the environment such as dissolved metals or organic pollutants by modifying their speciation, transport, or bioavailability (15). An example of this in the aquatic

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Submitted to Environmental Science and Technology environment is the uptake of arsenic (54) and cadmium (55) into carp which has been shown to be enhanced in the presence of titanium dioxide nanoparticles.

Laboratory studies have shown that toxicity and other biological effects may be a function of experimental preparation such as nature of the test medium and light conditions (44, 51) as well as the dissolution of the NM (39, 53) and nature of the test organism. The aggregation of NMs is thought to be one of the key determining factors for bioaccumulation and ecotoxicity of nanoparticles, as actual dose and exposure will likely change with degree of aggregation. Aggregation is difficult to control in conventional ecotoxicological experiments where the exposure concentrations are produced by a dilution series from a stock solution in order to prepare the concentrations needed for the concentration-response experiment. Clearly, producing concentrations in this way may change aggregation status where kinetics of aggregation will be concentration dependent. Further problems encountered include media at artificially high ionic strength to ensure healthy organisms, thus inducing aggregation of NMs, and thereby reducing the effective exposure concentrations. Additionally, NM concentrations in laboratory investigations are likely to be extremely high compared with likely environmental concentrations (although it is possible that environmental hotspots may occur), again with concomitant changes in exposure and dose. Hence, NMs may be aggregated to a different extent and the doseresponse curve will not only reflect the inherent toxicity of the particles but also be heavily influenced by the degree of aggregation; toxicity may therefore be different in the laboratory when compared to the natural environment, and the traditionally used procedures to estimate predicted no-effect concentration (PNEC) for risk assessment purposes may not be valid. Various experimental approaches have attempted to

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Submitted to Environmental Science and Technology address this concern, for example, the use of stirring/sonication techniques or solvents to disperse the NMs in solution (49, 56). However, these techniques are also fraught with problems: surface chemistry may be altered and added solvents have potential effects. Consequently, such methods require extensive testing on a case by case basis before routine data gathering can be performed.

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Submitted to Environmental Science and Technology Physico-chemical characterisation and nature of NMs The suitable characterization of NMs and their physico-chemical nature is clearly essential in understanding their biological uptake (12) but the question remains as to which characteristics displayed by NMs are significant and upon which environmental factors these depend. For NMs, there are 5 essential components in this regard and they are considered below.

Firstly, we need to consider the NM itself e.g. nature of core and shell including chemistry and crystallography, and capping agent or other surface active material such as co-solvents and their contaminants. One challenge for ecotoxicologists is to test the toxicity of the nanomaterial itself and eliminate confounding effects of vehicle solvents or unrealistic solvent-induced effects in the nanomaterial. An example of this difficulty is research that investigated the toxicity of C 60 and reported oxidative injury in brains of fish (43), but failed to adequately account for the effects of the tetrahydrofuran (THF) vehicle used to generate the aqueous fullerene aggregates. Subsequent work demonstrated that C60 aggregates formed using THF may retain THF (32), and further tests demonstrated that toxicity may be associated with THF decomposition products rather than C60 (57), although, in general, physico-chemical characterisation was limited.

Secondly, we need to consider the dissolution of the NM, especially in the case of inorganic NMs. For the highly soluble zinc oxide, toxicity has been shown to correlate with the dissolved Zn2+ (aq) ion, rather than the zinc NM in both human cells (58) and algae (53). In less soluble materials such as silver or titanium dioxide there

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Submitted to Environmental Science and Technology may be a mixtures effect between dissolved ions and the NM itself, and this will need to be investigated for relevant NMs.

Thirdly, we need to consider the primary unitary size of the NM, since this is the most important identifier of the NM. The nanoscale region may be regarded as a transition zone between bulk materials and the dissolved phase, in which changes in material properties with size occur based on changes in specific surface area, surface free energy, structural variations (bond lengths, crystal defects etc) and size-based qauntization effects (59). However, in laboratory test solutions, primary particles alone may not be present to any great extent due to aggregation unless specifically stabilized by additives (12, 48). In natural waters, there is likely to be complex behaviour between aggregation and disaggregation, as discussed.

Therefore, fourthly, the aggregation behaviour of NMs is extremely important. Equally important to NM behaviour is that aggregate size, morphology and kinetics may change with NM type and other environmental factors. Aggregation processes clearly influence fate, behaviour and dose as discussed. Nevertheless, the potential reduction in biological effects with aggregation should not be over-emphasised and requires testing. For instance, aggregation is not necessarily a permanent loss mechanism to natural waters as re-suspension, disaggregation and other processes may occur before burial within deep sediments and permanent loss (15). In addition, some evidence indicates that large, discrete particles are considerably less toxic than similarly sized aggregates of NMs with the same chemistry. This may perhaps be explained because novel properties of the NM may persist in the aggregated form. For instance, for some aggregate morphologies, specific surface area (SSA) is

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Submitted to Environmental Science and Technology independent of aggregate size (60) and SSA is often cited as a key parameter in NM toxicity but there are also indications that reactivity e.g. sorption of cations, does not always scale with SSA measured in the dry state (61).

Surface properties including area, chemistry, contamination and charge are the fifth aspect of NM characterisation which need consideration and are dependent on both the first point about the nature of the NM itself and the media conditions. In fact, there are likely to be specific feedback links and relationships between these different aspects which will influence interactions between these different components and strongly impact on NM behaviour.

Once these potentially important characteristics of NMs have been identified, how are they measured in practice? This is a far from trivial task, but achievable, and an extensive but not definitive list of techniques is given in Tables 1-3. A number of methods give overlapping information, so they do not directly map onto the 5 important aspects described above. For instance, the essential technique of transmission electron microscopy (TEM) will give information on particle size (potentially with a sub-nm resolution), morphology, shape and crystallography. With suitable additional detectors and data analysis, chemical information (elemental and bonding) may be obtained, along with particle size distributions (PSDs), SSA and fractal dimensions. The latter can be interpreted in terms of aggregation mechanisms. However, an important difficulty in sample preparation and data interpretation is introduced as many of the nanometrology methods such as EM and the BET method for measurement of SSA are performed under ultra high vacuum (UHV). Under these conditions, water is completely removed from the system. However, relevant NM

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Submitted to Environmental Science and Technology ecotoxicology and chemistry experiments are performed in aqueous systems and there is uncertainty in data interpretation due to the drying process; how are aggregation and surface chemistry changed by dehydration, for instance. In the natural environment in both aquatic and terrestrial systems or in laboratory experiments using natural organic macromolecules, this problem is exacerbated (15). The uncertainty can be reduced by use of suitable preparation techniques (62), which need validating for systems containing NMs, and by not over-interpreting data. For instance, BET will not give absolute SSA values, but trends and comparisons between samples will be instructive.

Assuming logistics are not a difficulty and in particular that there are no restraints in performing extensive measurements on a limited sample mass, a key problem is in relating the measured parameter to its property in suspension or on/within the organism. Clearly, characterization within a number of compartments (stock, exposure solution before and after exposure to the organism, at the biotic-abiotic interface and within the organism) is required as shown in Figure 2. Full and correct interpretation of these data ideally relies on an iterative procedure between data collected from these different compartments. Characterisation of the raw NM is essential to understand the fundamental NM characteristics (core, shell and capping agent). However, once in the exposure solution, NM aggregation, surface properties and dissolution state may alter substantially due to changes in pH, ionic strength and other parameters. Low concentrations of NMs are likely in the natural environment (63) but in postulated primary sinks such as river and estuarine sediments, concentrations of potentially persistent NMs will rise over time. Lastly, the high concentrations currently used in organism exposures at the laboratory scale (45) will

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Submitted to Environmental Science and Technology likely lead to more rapid aggregation of NMs, leading to reduced exposure concentrations compared to the added dose concentration. The order of addition and ageing of nanomaterials in experiments also requires substantial thought. For all these reasons, actual exposure cannot be determined on the ‘raw’ NM or calculated simply as the mass concentration added in exposure experiments.

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Submitted to Environmental Science and Technology Linking ecotoxicology and physico-chemistry We have now critically reviewed relevant aspects of both NM biology and physicochemistry and it is quite clear that there is a strong link between the two and understanding the precise nature of this link is essential but not fully known at present. Nevertheless, there is reason to be optimistic about progress in the next decade, given the early recognition of the importance of the physico-chemistry by the ecotoxicological community. The situation is perhaps analogous to the development of our understanding of trace metal speciation, bioavailability and toxicity. In the 1960-1970s, the importance of biological aspects such as sex, age etc of the organism was thought to be of primary importance. Solution speciation was later shown to be critical leading to the development of the free ion activity model (FIAM) (64), which state that biological effects are proportional to the free (hydrated) ion in solution. Later, the biotic ligand model (BLM) (65) was developed which switched the focus from the solution speciation to the abiotic-biotic interface, but still with considerable solution phase chemistry involved. The BLM is now used as a regulatory tool in sitespecific studies by the USEPA and the EU. Other more sophisticated and potentially predictive dynamic models based on mass transport, chemical kinetics and equilibrium chemistry are becoming available (66), although bioavailability and toxicity are organism dependent. Similar progress must be achieved with NMs.

Referring to Figure 1 and the earlier discussion, specific links between physicochemistry and bioavailability and subsequent effects are related to three specific areas: 1) the fate and behaviour (transport) of the NMs in different environmental compartments will indicate whether exposure at significant levels is likely. A simplified example is again related to aggregation. Ecotoxicological studies on

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Submitted to Environmental Science and Technology organisms within the water column may be less relevant if the NM of interest aggregates rapidly and completely. If this is the case benthic organisms (e.g. biofilms, macroalgae, worms and shellfish) are the environmentally relevant target organisms. However, for screening studies and as NMs may exist as reasonably stable dispersions over relatively long time periods, pelagic receptor organisms will still serve important purposes. 2) Local scale transport from the bulk water to the organism (Figure 1) is directly tied to bioavailability and biological effects and here the important processes will be organism dependent. For instance, transport through the diffusion layer around algal, bacterial and fish gill cells will be a controlling influence, and this will, at least in part, depend upon diffusion coefficient (size) of the NM and the thickness of the boundary layer. In addition, electrostatic and steric repulsion and attraction mechanisms will also play a role in attachment of a NM to an organism in many circumstances. 3) The exposure dose (to which the organism is exposed in toxicity studies) is not necessarily identical to the added dose, due to processes of aggregation, dissolution and changes in surface chemistry. The dose-response curve is thus poorly defined for NMs in ecotoxicology experiments, not only in terms of the biological response on the secondary (response) axis but also in terms of defining the relevant dose metric on the primary axis. All of this emphasises the need for measurement and characterisation, but also highlights the problem of determining and expressing concentrations which do not necessarily exist in a wholly dissolved or homogenous form.

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Submitted to Environmental Science and Technology Recommendations The following list is a series of recommendations in priority order which it is hoped will feed into further basic and fundamental research and inform the academic, regulatory, policy making and industrial communities involved with NMs.

1) A first and most immediate priority is the investigation of analytical techniques which are capable of measuring the concentration and fractionation/properties of NMs in both environmental compartments (waters, soils and sediments) and biological tissues. In particular, in the environment and in biological tissues, techniques must be able to discriminate between high concentration natural materials and lower concentrations manufactured NMs. Likely these techniques will be based on both size discrimination and elemental/isotopic analysis, supplemented with single particle analysis characterisation such as EM and force microscopy. A good example is the use of flow field-flow fractionation with inductively coupled plasma mass spectrometry (FlFFF-ICP-MS, 37). Application to NMs which are likely to be rare in the natural environment (Ag and Ce rather than Fe and Zn) will also be most productive, followed by the use of suitable isotopically labelled NMs as tracers. 2) In parallel, it is essential to investigate the effects of NM properties (core, shell and capping agents, size and other parameters) and solution conditions (pH, ionic strength, organic matter) on NM fate and behaviour. Advances will be made when further information on the following are produced: a. Data from systematic laboratory studies, including mesocosm-type work and within-field studies for elucidation of mechanisms of NM

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Submitted to Environmental Science and Technology interaction with natural substances. Data will particularly focus on chemistry, transport and distribution in both aquatic and terrestrial systems. Modifications to existing theories in colloid and solute transport in natural waters and soils are likely. b. Application of state of the art separation, spectroscopy and microscopy methodologies to detect NMs in natural systems at realistic levels. The information gathered here will allow an improved understanding of whether exposures at concentrations potentially capable of causing harmful effects actually occur and, if so, to what extent and in which environmental compartments. 3) Systematic exposure studies of important NMs to a wide variety of invertebrate and vertebrate organisms. Impact on bacteria, algae, crustaceans and selected fish species are excellent starting organisms. An understanding of potential target tissues and biological effects needs to be gained and correlated with specific properties of the NMs. This will allow improvements to be made in our rudimentary understanding of the link between physico-chemistry and biological impacts. The criteria listed below need to be borne in mind and acted upon in ecotoxicology studies: a. Due to various reasons, exposures of NMs to organisms both in laboratory tests and in the environment are not equilibrium processes making the use of partition coefficients of limited relevance. In terms of solution behaviour, time-dependent processes such as aggregation and mass transport from the bulk suspension to the biological surfaces need to be considered. In bioaccumulation studies, focus should be on uptake and elimination rates and fluxes rather than at steady-state concentrations.

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Submitted to Environmental Science and Technology b. There is not necessarily a direct and linear relationship between added dose and exposure dose of NMs. The main reason for this is aggregation (higher concentrations may lead to loss and localisation of added material), but other processes may be important such as dissolution, leading to loss of NMs and production of potentially toxic dissolved ions. For inorganic NMs, separating the effects of truly dissolved and nanoparticulate metal is essential. The dose in doseresponse experiments is still poorly defined and needs particular care in defining over the course of exposure and not simply at the point of NM addition. c. Related to point b, experiments with long exposure times and low doses will mimic realistic environmental conditions more appropriately than high dose-short exposure time studies, as are performed currently. Thus low dose, long-term exposures need to be performed as a matter of priority which might better account for persistence and bioaccumulation. d. Accumulation over time in the organism and in the environment, especially in sediments and soils is likely, so emphasis needs to be put on both persistence and bioaccumulation/bioavailability and other long term effects. Traditional regulatory measures of such properties such as octanol-water partition coefficients are unlikely to be relevant because of the ampiphilic nature of many NMs and the non-equilibrium conditions. e. A further difficulty in relating ecotoxicology studies to environmental behaviour is that the media composition is likely to be significantly

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Submitted to Environmental Science and Technology different in the laboratory compared to the environment and this will profoundly affect NM physico-chemistry. Exposure studies are often performed at high ionic strengths, high Ca concentration and neutral pH in the absence of organic matter. These conditions are chosen for simplicity, and to enable to test organisms to grow satisfactorily, in order to standardise test methods. They also allow rapid data collection on possible toxicity. However, to improve the extrapolation of results from laboratory toxicity tests to environmental conditions some improvements are needed from the regulatory tests used today. As an initial step, the addition of humic substances to test media is being considered in ecotoxicology studies. Experiments to optimise condition for both NMs and organisms need to be performed. In addition, the selection of suitable end-points may be required, which can detect stress induced by the NMs in organisms grown in sub-optimal conditions. 4) At this stage, a small number of NMs (based on previous steps and on knowledge of production volumes and projected future use) needs to be selected and all aspects of physico-chemistry and bioavailability over the full life cycle of those NMs need to be assessed, as has been recently performed by the OECD. This is particularly useful for regulatory purposes, although fundamental scientific studies will require a more complete flexibility in experimental design and a panel of standard NMs will be less useful. 5) Based on the data produced by above research, practical risk assessment models need to be developed, parameterised and tested.

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Submitted to Environmental Science and Technology 6) Finally, there is a need for high-throughput screening using in vitro methods for examining NMs, again based on earlier steps.

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Submitted to Environmental Science and Technology 7) Acknowledgements or Disclaimer

Certain commercial equipment, instruments or materials are identified in this paper to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

The authors would like to acknowledge the Natural Environment Research Council for a Knowledge Exchange grant (NANONET, NE/E002889/1) which provided financial support or this review.

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Submitted to Environmental Science and Technology References (1) Borm, P. J. A.; Robbins, D.; Haubold, S.; Kuhlbusch, T.; Fissan, H.; Donaldson, K.; Schins, R.; Stone, V.; Kreyling, W.; Lademann, J.; Krutmann, J.; Warheit, D. B.; Oberdorster, E., The potential risks of nanomaterials: a review carried out for ECETOC. Particle and Fibre Toxicology 2006, 3, 11. (2) The National Nanotechnology Initiative. The National Nanotechnology Initiative Strategy Plan. 2004. (3) Hansen, S. F.; Larsen, B. H.; Olsen, S. I.; Baun, A., Categorization framework to aid hazard identification of nanomaterials. Nanotoxicology 2007, 1 243-250. (4) Roco, M. C., International perspective on government nanotechnology funding in 2005. Journal of Nanoparticle Research 2005, 7, 707-712. (5) Lux Research. The Nanotech Report. 2006. (6) The Royal Society. Nanoscience and nanotechnologies: opportunities and uncertainties. 2004 (7) Institute of Occupational Medicine. Nanoparticles: An occupational hygiene review. 2004. (8) DEFRA. Characterising the potential risks posed by engineered nanoparticles. 2005. (9) Moore, M. N., Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environment International 2006, 32, 967-976. (10) Biswas, P.; Wu, C., Nanoparticles in the Environment. Journal of the Air and Waste Management Association 2005, 55, 708-746. (11) Hester, R. E.; Harrison, R. M., Nanotechnology: Consequences for human health and the environment. RSC Publishing: 2007; Vol. 24, p 134. (12) Handy, R. D.; Kammer, F. v. d.; Lead, J. R.; Hassellov, M.; Owen, R., The ecotoxicology and chemistry of manufactured nanoparticles. Ecotoxicology in press. (13) Nowack, B.; Bucheli, T. D., Occurence, behaviour and effects of nanoparticles in the environment. Environmental Pollution 2007, 1-18. (14) Warheit, D. B., How meaningful are the results of nanotoxicology studies in the absence of adequate material characterisation? Toxicological Sciences 2008, 101, 183-185. (15) Lead, J. R.; Wilkenson, K. J., Aquatic Colloids and Nanoparticles: Current Knowledge and Future Trends. Environmental Chemistry 2006, 3, 159-171. (16) Wilkenson, K. J.; Lead, J. R., Environmental Colloids and Particles: Behaviour, Separation and Characterisation. John Wiley and Sons Ltd: Chichester, 2007. (17) Fabian, N. J.; Albright, L. B.; Gerlach, G.; Fisher, H. S.; Rosenthal, G. G., Humic acid interferes with species recognition in zebrafish. Journal of Chemical Ecology 2007, 33, 2090-2096. (18) Harrison, R. M.; van Grieken, R. E., Atmospheric Particles. Wiley and Sons: Chichester, 1998. (19) Madden, A. S.; Hochella, M. F. J.; Luxton, T. P., Insights for size-dependant reactivity of hematite nanomineral surfaces through Cu2+ sorption. Geochimica Cosmochmica Acta 2006, 70, 4095-4104. (20) Madden, A. S.; Hochella, M. F. J., A test of geochemical reactivity as a function of mineral size: manganese oxidation promoted by hematite nanoparticles. Geochimica Cosmochmica Acta 2005, 69, (2), 389-398. (21) Waychunas, G. A.; Kim, C. S.; Banfield, J. F., Nanoparticulate iron oxide minerals in soils and sediments: unique properties and contaminant scavenging mechanisms. Journal of Nanoparticle Research 2005, 7, 409-433.

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Submitted to Environmental Science and Technology (22) Li, L.; Fan, M.; Brown, R.; van Leeuwen, J.; Wang, J.; Wang, W.; Song, Y.; Zhang, P., Synthesis, Properties, and Environmental Applications of Nanoscale IronBased Materials: A review. Critical Reviews in Environmental Science and Technology 2006, 36, (5), 405-431. (23) Doty, R. C.; Tshikhudo, T. R.; Brust, M.; Fernig, D. G., Extremely stable watersoluble Ag nanoparticles. Chemistry of Materials 2006, 17, 4630-4635. (24) Hoppe, C. E.; Lazzari, M.; Pardinas-Blanco, I.; Lopex-Quintela, M. A., One-step synthesis of gold and silver hydrosols using poly(N-vinyl-2-pyrrolidone) as a reducing agent. Langmuir 2006, 22, 7027-7034. (25) Chen, K. L.; Elimelech, M., Influence of humic acid on the aggregation kinetics of fullerene (C60) nanoparticles in monovalent and divalent electrolyte solutions. Journal of Colloidal and Interface Science 2007, 309, 126-134. (26) Hyung, H.; Fortner, J. D.; Hughes, J. B.; Kim, J. H., Natural Organic Matter Stabilizes Carbon Nanotubes in the Aqueous Phase. Enviromental Science Technology. 2007, 41, (1), 179-184. (27) Giasuddin, A. B. M.; Kanel, S. R.; Choi, H., Adsorption of Humic Acid onto Nanoscale Zerovalent Iron and Its Effect on Arsenic Removal. Environmental Science and Technology 2007, 41, 2022-2027. (28) Renn, O.; Roco, M. C., Nanotechnology and the need for risk governance. Journal of Nanoparticle Research 2006, 8, 153-191. (29) Lead, J. R., In Nanotechnology: Consequences for human health and the environment, Hester, R. E.; Harrison, R. M., Eds. RSC Publishing: 2007; Vol. 24, p 134. (30) Espinasse, B., Hotze, E.M., Wiesner, M.R. Transport and retention of colloidal aggregates of C-60 in porous media: Effects of organic macromolecules, ionic composition, and preparation method. Environmental Science & Technology 2007, 41, 7396-7402. (31) Buffle, J.; Wilkinson, K.J.; Stoll, S.; Filella, M., Zhang, J. A Generalized Description of Aquatic Colloidal Interactions: The Three-Colloidal Component Approach. Environmental Science and Technology 1998, 32 (19), 2287-2899. (32) Brandt, J.; Leocanet, H.; Wiesner, M. R., Aggregation and deposition characteristics of fullerene nanoparticles in aqueous systems. Journal of Nanoparticle Research 2005, 7, 545-553. (33) Lead, J. R.; Hamilton-Taylor, J.; Hesketh, N.; Jones, M. N.; Wilkenson, A. E.; Tipping, E., A comparative study of proton and alkaline earth metal binding by humic substances. Analytica Chimica Acta 1994, 294, 319-327. (34) Tipping, E. Cation Binding by Humic Substances, Cambridge University Press, UK. 2005 pp 444 (35) Baalousha, M.; Manciulea, A.; Cumberland, S.; Kendall, K.; Lead, J. R., Aggregation and surface properties of iron oxide nanoparticles; influence of pH and natural organic matter. Environmental Toxicology and Chemistry in press. (36) Gibson, C. T.; Turner, I. J.; Roberts, C. J.; Lead, J. R., Quantifying the Dimensions of Nanoscale Organic Surface Layers in Natural Waters. Environmental Science and Technology 2007, 41, 1339-1344. (37) Stolpe, B.; Hassellov, M.; Andersson, K.; Turner, D. R., High resolution ICPMS as an on-line detector for flow field-flow fractionation; multi-element determination of colloidal size distributions in a natural water sample. Analytica Chimica Acta 2005, 535, 109-121. (38) Yohannes, G.; Wiedmer, S. K.; Hiidenhovi, J.; Hietman, A.; Hyotylainen, T., Comprehensive two-dimensional field-flow fractionation-liquid chromatography in

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Submitted to Environmental Science and Technology the analysis of large molecules. Analytical Chemistry 2007, 70, 3091-3098. (39) Morones, J. R.; Elechiguerra, J. L.; Camacho, A.; Holt, K.; Kouri, J. B.; Ramirez, J. T.; Yacaman, M. J., The bactericidal effect of silver nanoparticles. Journal of Nanotechnology 2005, 16, (10), 2346-2353. (40) Kloepfer, J. A.; Mielke, R. E.; Nadeau, J. L., Uptake of CdSe and CdSe/ZnS Quantum Dots into Bacteria via Purine-Dependant Mechanisms. Applied and Environmental Microbiology 2005, 71, 2548-2557. (41) Chithrani, B. D.; Chan, W. C. W., Eluciating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes. Nano Letters 2007, 7, 1542-1550. (42) Thill, A.; Zeyons, O.; Spalla, O.; Chauvat, F.; Rose, J.; Auffan, M.; Flank, A. M., Cytotoxicity of CeO2 nanoparticles for Escherichia coli. Physico-chemical insight of the cytotoxicity mechanism. Environmental Science and Technology 2006, 40, 61516156. (43) Oberdorster, E., Manufactured Nanomaterials (Fullerenes, C60) Induce Oxidative Stress in the Brain of Juvenile Largemouth Bass. Environmental Health Perspectives 2004, 112, (10), 1058-1062. (44) Adams, L. K.; Lyon, D. Y.; Alvarez, P. J. J., Comparative eco-toxicity of nanoscale TiO2, SiO2 and ZnO water suspensions. Water Research 2006, 40, (19), 3527-3532 (45) Federici, G.; Shaw, B. J.; Handy, R., Toxicity of titanium dioxide nanoparticles to rainbow trout (Oncoryhnchus mykiss): Gill injury, oxidative stress, and other physiological effects. Aquatic Toxicology 2007, 84, 415-430. (46) Brayner, R.; Ferrari-Iliou, R.; Brivois, N.; Djediat, S.; Benedetti, M. F.; Fievet, F., Toxicological Impact Studies Based on Esherichia coli Bacteria in Ultrafine ZnO Nanoparticles Colloidal Medium. Nano Letters 2006, 6, (4), 866-870 (47) Sondi, I.; Salopek-Sondi, B., Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. Journal of Colloidal and Interface Science 2004, 275, 175-182. (48) Handy, R. D.; Henry, T. B.; Scown, T. M.; Johnstone, B. D.; Tyler, C. R., Manufactured nanoparticles:their uptake and effects on fish - A mechanistic analysis. Ecotoxicology in press (49) Handy, R. D.; Shaw, B. J., Toxic effects of nanoparticles and nanomaterials: Implications for public health, risk assessment and the public perception of nanotechnology. Health, Risk and Society 2007, 9, 125-144. (50) Lyon, D. Y.; Fortner, J. D.; Sayes, C. M.; Colvin, V. L.; Hughes, J. B., Bacterial Cell Association and Antimicrobial Activity of a C 60 Water Suspension. Environmental Toxicology and Chemistry 2005, 24, (11), 2757-2762. (51) Lyon, D.; Adams, L. K.; Falkner, J.; Alvarez, P., Antibacterial activity of fullerene water suspensions:Effects of preparation method and particle size. Environmental Science and Technology 2006, 40, 4360-4366. (52) Smith, C. J.; Shaw, B. J.; Handy, R. D., Toxicity of single walled carbon nanotubes to rainbow trout, (Oncorhynchus mykiss): Respiratory toxicity, organ pathologies and other physiological effects. Aquatic Toxicology 2007, 82, 94-109. (53) Franklin, N. M.; Rogers, N. J.; Apte, S. C.; Batley, G. E.; Gadd, G. E.; Casey, P. S., Comparative Toxicity of Nanoparticulate ZnO, Bulk ZnO and ZnCl2 to a Freshwater Microalga (Pseudokirchneriella subcapitata): The importance of particle solubility. Environmental Science and Technology 2007, 41, 8484-8490. (54) Sun, H.; Zhang, X.; Niu, Q.; Chen, Y.; Crittenden, J. C., Enhanced Accumulation of Arsenate in Carp in the Presence of Titanium Dioxide Nanoparticles. Water Air

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Submitted to Environmental Science and Technology Soil Pollution 2007, 178, 245-254. (55) Zhang, X.; Sun, H.; Zhang, Z.; Niu, Q.; Chen, Y.; Crittenden, J. C., Enhanced bioaccumulation of cadmium in carp in the presence of titanium dioxide nanoparticles. Chemosphere 2007, 67, 160-166. (56) Scott-Fordsmand, J. J.; Krogh, P. H.; Lead, J. R., Nanomaterials in Ecotoxicology. Intergrated Environmental Assessment Management 2008, 4, 126-8 (57) Henry, T. B.; Menn, F.; Fleming, J. T.; Wilgus, J.; Compton, R. N.; Sayler, G. S., Atrributing Effects of Aqueous C60 Nano-Aggregates to Tetrahydrofuran Decomposition Products in Larval Zebrafish by Assessment of Gene Expression. Environmental Health Perspectives 2007, 115, (7), 1059-1065. (58) Brunner, T. J.; Wick, P.; Manser, P.; Spohn, P.; Grass, R. N.; Limbach, L. K.; Bruinink, A.; Stark, W. J., In vitro cytotoxicity of oxide nanoparticles: comparison to asbestos, silica and the effect of particle solubility. Environmental Science and Technology 2006, 40, 4374-4381. (59) Wiggington, N. S.; Haus, K. L.; Hochella, M. F., Aquatic environmental nanoparticles. Journal of Environmental Monitoring 2007, 9, 1285-1432. (60) Cwiertny, D. M.; Handler, R. M.; Schaefer, M. V.; Grassian, V. H.; Scherer, M. M., Interpreting nanoscale size-effects in aggregated Fe-oxide suspensions: Reaction of Fe(II) with Goethite. Geochemica Cosmochemica Acta 2008, 72 (5) 1365-1380. (61) Zhang, Z.; Buffle, J.; Alemani, D., Metal flux and dynamic speciation at (bio)interfaces. Part II: Evaluation and compilation of physicochemical parameters for complexes with particles and aggregates. Environmental Science and Technology 2007, 41, 7621-7631. (62) Balnois, E.; Wilkenson, K. J.; Lead, J. R.; Buffle, J., Atomic force microscopy of humic substances: effects of pH and ionic strength. Environmental Science and Technology 1999, 33, 1311-1317. (63) Boxall, A. B. A.; Tiede, K.; Chaudhry, Q., Engineered nanomaterials in soils and water:how do they behave and could they pose a risk to human health? Nanomedicine 2007, 2, 919-927. (64) Campbell, P.G.C. Interactions between trace metals and aquatic organisms: a critique of the free-ion activity model In: Tessier, A. Turner, R. (Eds) Metal Speciation and Bioavailability in Aquatic Systems. 1995 John Wily, New York pp45102. (65) Paquin, P. R.; Gorsuch, J. W.; Apte, S. C.; Batley, G. E.; Bowles, K. C.; Campbell, P. G. C.; Delos, C. G.; Di Toro, D. M.; Dwyer, R. L.; Galvez, F.; Gensemer, R. W.; Goss, G. G.; Hogstrand, C.; Janssen, C. R.; Mcgeer, J. C.; Naddy, R. B.; Playle, R. C.; Santore, R. C.; Schneider, U.; Stubblefield, W. A.; Wood, C. M.; Wu, K. B., The biotic ligand model: a historical overview. Comparative Biochemical Physiology C 2002, 133, 3-35 (66) van Leeuwen, H. P.; Town, R. M.; Buffle, J.; Cleven, R. F.; Davison, W.; Puy, J.; van Riemsdijk, W. H.; Sigg, L., Dynamic speciation analysis and bioavailability of metals in aquatic systems. Environmental Science and Technology 2005, 39, 85458556.

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Submitted to Environmental Science and Technology Figure 1. Important factors for consideration in linking the physico-chemical characteristics and ecotoxicology of manufactured nanomaterials in the environment, highlighting the importance of characterisation of the nanomaterials at/on/within the receiving environment. The state of the nanomaterials (NM) as either particle, aggregated or dissolved will be in part determined by the material itself and part determined by the receiving environment, this will in turn affect reactivity and transport and is of major importance. The surface modification of the NM by its environment must be considered as well as the potential for it to behave as a pollutant vector transporting chemicals and either enhancing or decreasing their mobility. Once in contact with the organism/cell effects may be physical or uptake may occur by passive or active transport and these will all be dependent on the form of the NM and the potential for biomodification.

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Submitted to Environmental Science and Technology Figure 2. Characterisation protocol for NMs in assessing ecotoxicology. It is important to know the form and function of the nanomaterials of interest at each of these four stages to fully understand their potential effects. Firstly, characterisation of the raw NM (on a batch to batch basis as physico-chemical properties may change between batches). Secondly their form in the environment may be different in terms of aggregation or surface modification, which needs to be considered in their fate and transport. Thirdly, the manner in which NMs interact at the biological interface and what characteristics are important for uptake need to be assessed. Once within an organism, the extent of uptake accumulation, concentration within the organism and the form in which the NM is accumulated, need to be quantified.

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Submitted to Environmental Science and Technology Figure 3 Representative TEM images nanoparticles from environmental type samples a)TEM micrograph of iron-rich nanoparticles extracted from a humus rich soil horizon (Ah horizon of a gleyic podzol). b) This image shows that NOM and clay (kaolin) can quickly attach to multi-wall carbon nanotubes in the aqueous environment.

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Submitted to Environmental Science and Technology

Table 1a. Methods to determine the bulk particle concentration method

obtained Information

sample preparation

limitations

gravimetry (balance)

particle concentration of inorganic and organic particles / macromolecules with particle size above the nominal cut-off of the membranes used for filtration. Further analysis possible estimation of particle concentration non-invasive

membrane filtration, clean-up, drying. Typical membranes used are: 1000 MWCO Amicon, 15 nm PC-track-etched or 20 nm Whatman Anopore suspension in flow-trough cuvettes or single samples in cuvettes

nephelometry (nephelometric turbidity detection)

estimation of particle concentration non-invasive

suspension in flow-trough cuvettes or single samples in cuvettes

TOC total organic carbon analyzer

concentration of dissolved, colloidal or particulate organic carbon, depending on sample preparation/ fractionation prior analysis concentration of colloidal particles, average equivalent particle size from breakdown plasma geometry

solution, suspension

large sample volumes required at low particle concentrations, perturbations by disruption of bacteria, particles < cut-off are lost low sensitivity, interfered by light absorbing substances as natural organic matter, signal dependent on particle concentration, size, refractive index and shape sensitive, close to no interference from natural organic matter, signal dependent on particle concentration, size, refractive index and shape organic substance sorbed to surfaces of larger entities will only be partly determined

UV-VIS spectrometry

Estimation of soluble or colloidal organic substance

suspension in flow-trough cuvettes or single samples in cuvettes

Fluorescence spectrometry

Estimation of soluble or colloidal organic substance

(dilute) suspension in flowtrough cuvettes or single samples in cuvettes

FCS fluorescence correlation spectroscopy

Number of particles, equivalent hydrodynamic size and distribution

very diluted suspensions

turbidimetry (photometer)

LIBD laser induced breakdown detection

37

dilute suspension

most sensitive method available, concentration from breakdown rate and particle size, hence critical regarding suitable calibration Signal depends on concentration and extinction coefficient, interference from turbidity generated by particles (see UV/VIS turbidity measurements) Only for fluorescent NPs or labelled NPs

One of the most sensitive techniques, but applicable only in the case of the fluorescent or labelled NPs

Submitted to Environmental Science and Technology Table 1b. Methods for particle composition. method

obtained Information

sample preparation

limitations

ICP-OES

elemental concentration for most elements

direct analysis of suspensions containing particles below 1 µm

ICP-MS

elemental concentration for most elements, higher sensitivity than ICP-OES for most elements

direct analysis of suspensions containing particles below 1 µm

element detection / concentration with outstanding spatial resolution > ~10 nm

single particle analysis on filter membrane (SEM) or sample support grid (TEM)

sensitivity differs for each element, batch method, destructive, particle size selective separation in nebulizer possible sensitivity differs for each element, batch method, destructive, particle size selective separation in nebulizer possible lacks sensitivity, qualitative / semi-quantitative method predominantly for main elements or trace elements in particulate form or abnormal high concentrations

only in TEM, crystal structure of single particles (high spatial resolution)

Single particles support grid

mineralogical composition

dry powder, thin ceramic support

EDX or ADX in

electron

microscopy (SEM/TEM) TEMdiffractometry x-ray diffractometry

on

(XRD) Fourier Transform

Bulk chemical bonding organic and inorganic

environment,

sample film

on

low sensitivity, comparably large amount of sample required

dry powder, thin film

semi-quantitative

Solution

Semi-quantitative

dry powder, thin film

semi-quantitative

solid phase

semi-quantitative

Infrared Spectroscopy FTIR ATR-FTIR Raman spectroscopy X-ray photoelectron

Surface FTIR in solution Bulk chemical bonding organic and inorganic

environment,

high resolution surface chemical bonding environment

Spectroscopy, XPS

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Submitted to Environmental Science and Technology Table 1c. Methods for determination of particle size, size distributions, shape and morphology method

obtained Information

XAF x-ray

absorption

fine

structure light microscopy

scanning

Particle number, size, distribution, morphology

size-

electron

particle number, size, distribution, morphology

size-

microscopy (SEM) transmission

x-ray

microscopy (TXM)

scanning

electron microscopy force

bonding

electron

microscopy (TEM)

atomic

molecular

particle number, size, morphology

transmission

environmental

near-field environment

microscopy

(AFM) dynamic light scattering (PCS or DLS)

sedimentation analysis

Particle number, size, sizedistribution, morphology; observation in suspension sample, no drying or dehydration of the sample necessary, spectroscopy possible on few elements particle number, size, sizedistribution, morphology; observation in wet sample particle number, size, sizedistribution, morphology, surface chemistry (charge heterogeneity), force measurements mean hydrodynamic radius of particles, diffusion coefficient, particle size distributions only in systems with low complexity

sample preparation

limitations

powders, pastes or even liquid samples

synchrotron source necessary, insufficient detection limits for trace elements

droplet on glass support Single particles on sample support grid/ film particles on membrane or sample support stable suspension

low resolution, only for particles > 1 µm time consuming for quantitative information, preparation artifacts from drying time consuming for quantitative information, preparation artifacts from drying time consuming for quantitative information; resolution between light microscopy and SEM

particles on membrane or sample support particles on flat surface (mica), dry or under a thin film of water dilute particle suspensions

time consuming for quantitative information, resolution below SEM

particle size distributions from determined settling velocities of particles between ~ 1 and 100 µm particle size distribution, further analysis of obtained fractions possible, enrichment of the particle fraction particle size distribution from sedimentation velocities and diffusion gradients generated by high centrifugal forces; analytical and preparative techniques for particles from 0.001 – 1 µm chromatographic fractionation according to molecular weight, highresolution MW distributions high-resolution particle size distribution of particles between 0,001 and ~ 1µm; further analysis on subfractions with defined particle size possible estimation of surface charge (by determination of the zeta-potential)

particle suspension

N2-absorption (BET)

specific surface area

powder

adsorption experiments

sorption capacity, sorption/desorption kinetics, isotherms

stable, high concentrated samples

sequential filtration

centrifugal

sedimentation

techniques

size-exclusion chromatography (SEC) field-flow fractionation (FFF)

electrophoretic mobility

39

particle suspension

numerous artifacts, no information about elemental composition

only intensity weighted distributions for nonspherical, nonhomogeneous particles, signal dominated by larger particles, difficulties in heterogeneous or polydisperse samples limited to particles > 1µm, errors from particles of different density and shape errors by particle surface charge differences and particle shape, low resolution, filtration artifacts

particle suspension

errors by particles with different shape and density

particle suspension < 0.2 µm

risk of sample loss, MW calibration is problematic

particle suspension

errors by particles with different shape and density, requires a well suited combination of carrier composition and membrane surface chemistry to minimize system losses average values, limited information about charge-heterogeneity, problems in interpretation with heterogenic samples difficulties with aggregated or microporous structures; the value determined for the dry powder must not be valid for particles in aqueous dispersion large sample amounts and concentrations, numerous artifacts

particle suspension

Submitted to Environmental Science and Technology differential gradient in thin films DGT Time-of-Flight

Secondary

Ion Mass Spectrometry

concentration of elements in real solution or only labile complexed, provides also a pre-concentration of metals in the ion-exchange gel Ionized inorganic and organic surface material

particle suspension

limited to cationic metals

Ultrahigh vacuum compatible particles, films.

Similar to SEM but with crosssectional abilities (3-D imaging) and TEM sample preparation Surface specific elemental composition

particles on membrane or sample support Ultrahigh vacuum compatible particles, films,

Destructive, surface specific, improved detection of specific ionized material, spatial resolution approximately 1 μm time consuming for quantitative information, partly destructive

Number of particles, diffusion coefficients and equivalent hydrodynamic radius and distributions are determined from the correlation function

very dilute suspensions

(ToF-SIMS) Focused Ion Beam (FIB)

Scanning Auger Microscopy (SAM) and x-ray photon spectroscopy (XPS) FCS

fluorescence

correlation spectroscopy

40

Challenging sample preparation, strict surface cleanliness requirements One of the most sensitive techniques, but limited to fluorescent or labelled NPs

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