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Jul 21, 2010 - Université de Bordeaux, B.18 av Facultés, F-33405 Talence, France. C. School of Metallurgy and Materials, University of Birmingham, ...
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M. Baalousha et al., Environ. Chem. 2010, 7, 377–385. doi:10.1071/EN10003

Characterisation of structural and surface speciation of representative commercially available cerium oxide nanoparticles M. Baalousha,A,D P. Le Coustumer,B I. JonesC and J. R. LeadA A

School of Geography, Earth and Environmental Sciences, University of Birmingham, Edgbaston B15 2TT, UK. B Universite´ de Bordeaux, B.18 av Faculte´s, F-33405 Talence, France. C School of Metallurgy and Materials, University of Birmingham, Edgbaston B15 2TT, UK. D Corresponding author. Email: [email protected]

Environmental context. Manufactured nanoparticles, increasingly used in a wide range of products, can be released into the natural environment where they might pose a risk to environmental and human health. The nanoparticle characteristics that induce toxic effects, however, are not yet well-known. Understanding the toxicity and the fate and behaviour of nanoparticles in the environment requires precise characterisation of their properties at the nanoscale and the individual particle level. Abstract. The shape, morphology, crystallography, and oxidation state of commercially available cerium oxide nanoparticles as compared with bulk particles were studied by high-resolution transmission electron microscopy coupled to electron energy loss spectroscopy, along with scanning electron microscopy. Nano and bulk particles have the same crystalline structure and morphology as the fluorite-type structure with a mainly octahedral shape enclosed by eight {111} facets, or a truncated octahedral shape enclosed by eight {111} facets and two {002} facets, or eight {111} and two {002} and four {220} facets. Some defects, including twin boundaries and steps and kinks, were observed. Bulk ceria particles contain mainly CeIV, whereas ceria nanoparticles contain a large fraction of CeIII, which decreases after interaction with humic acid and biological media. These properties are likely to play an essential role in the environmental and toxicological behaviour of nanoparticles. Additional keywords: crystallinity, morphology, oxidation state, structure, surface defects.

Introduction

expansion),[6] aggregation and surface adsorption of other species.[9] The chemical and physical properties of NPs, and consequently their toxicity, are determined not only by the large proportion of surface atoms (i.e. as a result of their small size), but also by their crystal structure and the surface-adsorbed species. For instance, the crystal structure determines the shape and morphology of the particles via the surface atomic planes enclosing the particles.[10] Surface properties of nanoparticles play an important role in determining the NP reactivity and potential toxicity. For instance, the toxicity of CdS and CdSe NPs has been shown to be completely controlled by their surface coating, with a reduced toxicity of CdSe NPs coated with organic molecules.[11] Cerium oxide NPs are one of the most widely used nanomaterials in different applications including catalysts, fuel cells, microelectronics, and glass and ceramic applications and as diesel additives to improve fuel oxidation.[12] The use of cerium oxide NPs in a range of applications will certainly result in their release to the environment, where their fate and potential impact are largely unknown. The properties making cerium oxide of interest for industrial application may be the cause of concern for their toxicity. These properties include the high thermodynamic affinity for oxygen and sulfur, potential redox chemistry involving the CeIII/CeIV transformation, and absorption and excitation energy bands associated with its electronic

Manufactured nanoparticles (NPs) can be defined as materials with a particle size range of 1–100 nm, purposely produced for industrial or commercial applications like electronics, cosmetics, sunscreens, paints and coatings, catalysts and lubricants, fuel cells, and medical and health care.[1] As the size of NPs approaches the range ,20–30 nm, their mechanical, electronic, magnetic, optical, chemical reactivity, catalytic properties and potential toxicity may differ significantly from those of their bulk counterparts.[2–4] This different behaviour at the nanoscale can be explained by the high specific surface area and the increased proportion of surface atoms with decrease in particle size,[5] the variation in crystal structure (lattice expansion) with the decrease in particle size, the presence of undercoordinated bonds and the greater disorder at the surface (such as crystal defects and oxidation state),[6] particle shape and other mechanisms.[7] The chemical reactivity of NPs, and potentially their toxicity, may be strongly influenced by the large proportion of surface atoms relative to the ‘bulk’. For instance, the increased fraction of atoms exposed to the surface as the particles approach the nanoscale results in an excess free energy. Thus, NPs are thermodynamically metastable and they tend to minimise their free energy in different ways, including phase transformation,[8] crystal growth, surface structure changes (such as lattice Ó CSIRO 2010

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structure.[10] Cerium oxide also shows size-dependent properties, including lattice expansion, oxidation state variation and electron conductivity among others.[6] Cerium oxide NPs have been shown to induce toxicity in bacteria[13] and algae but not Daphnia magna.[14] The toxicity of ceria NPs for bacteria has been shown to be associated with their adsorption and reduction on the bacterium surface. The reduction of ceria NPs can occur after adsorption on bacteria or in solution by molecules released by the bacteria, which are able to reduce NPs.[13] It has been suggested that the reduction of cerium may be responsible for the generation of reactive oxygen species (ROS), and thus their toxicity.[4] Nonetheless, this has not yet been fully proved and other mechanisms may play a role, such as particle dissolution,[15] interaction of NPs with the cell itself, mechanical damage of the cell membrane,[16] NP uptake,[17] adsorption and depletion of nutrients reaching the cells and shading of cells,[18] or a combination of the abovementioned mechanisms. Additionally, physicochemical properties of NPs such as size, shape, surface coating, surface charge, bioavailability, specific surface area and aggregation might have a great influence on their toxicity.[3] The cerium oxide NPs investigated here have been used previously to investigate uptake and toxicity to fresh water algae, and were shown to be more toxic than bulk particles.[18] Although studies of NP toxicity are becoming more established, physicochemical properties of NPs inducing toxicity are not yet well identified, mainly owing to the lack of characterisation of the tested particles. In this paper, we present a detailed understanding of the crystallographic structure of a good example of a commercially available cerium oxide NP that has previously been used in ecotoxicological studies,[18] including the determination of crystal facets, defects and morphology. This paper also investigates the oxidation state of cerium oxide NPs compared with bulk particles and as influenced by their interaction with biological media and natural organic macromolecules (humic substances, HS). The paper provides detailed characterisation data to support the toxicological data presented previously.[18] Materials and methods Materials Cerium oxide nano (25 nm, manufacturer data) and bulk (o5 mm, manufacturer data) particles were purchased from Sigma–Aldrich Ltd (Dorset, UK). Suwannee River humic acid (SRHA) was purchased from the international humic substances society (St Paul, MN, USA). Algal medium was prepared by adding nutrient solutions and 2 mM final piperazine-N,N0 -bis(2ethanesulfonic acid) buffer concentration (Sigma–Aldrich) to stock algal culture (US EPA medium without EDTA).[19] Biological media (M2279) used to grow C3A human hepatocyte cells were purchased from Sigma–Aldrich. According to supplier information, the M2279 medium contains organic molecules including amino acids, sugar and others. The following suspensions were prepared: (i) 20 mg L1 ceria NPs in 10 mM NaNO3; (ii) 20 mg L1 bulk ceria particles in 10 mM NaNO3; (iii) 20 mg L1 ceria NPs in 20 mg L1 SRHA and 10 mM NaNO3; (iv) 20 mg L1 ceria NP algal media þ buffer; and (v) 62.5 mg L1 of ceria NPs in M2279 media. All samples were prepared at pH 7.0 and were kept for 24 h in the dark and at 48C before preparation for transmission electron microscope (TEM) analysis as described in the Accessory publication section. Cerium(III) phosphate and ammonium cerium(IV) nitrate were purchased from Sigma–Aldrich and used as references for

electron energy loss spectroscopy (EELS) analysis. A small amount of each salt was suspended in ultra-high-purity water and a droplet of each solution was dried slowly on a TEM grid. HR-TEM-EELS High-resolution transmission electron microscopy (HR-TEM) is a powerful and unique tool for structural characterisation as well as for identifying and quantifying the chemical composition, electronic structure and valence state at high spatial resolution (,1 nm), when coupled to EELS, allowing direct analysis of single NPs. One of the vital features of HR-TEM is the atomic-resolution real-space imaging of thin samples (,10–20 nm, making it valuable for NPs), revealing the atomic distribution on nanocrystal surfaces, allowing determination of interatomic distances and crystal facets, and therefore, determining NP morphology.[20] Here, 125 single particles were counted to measure particle size and 35 individual NPs to gain deeper information on particle morphology. EELS has been used to determine the oxidation state of cerium oxide nanoparticles suspended in different media as described above, and 11–21 spectra were collected on each sample. Data are expressed as mean values  standard deviation and statistical analyses to determine if differences are significant (P o 0.05) were performed using Excel applying the ANOVA test (singlefactor) followed by the Tukey test. The HR-TEM used here is an FEI Tecnai F20 field emission gun (FEG) coupled with an X-ray energy dispersive spectrometer (X-EDS) from Oxford Instruments (Oxfordshire, UK) and EELS from Gatan, Inc. (Pleasanton, CA, USA). Conditions were chosen so as to minimise possible degradation of the sample under the electron beam, including an accelerating voltage of 200 keV, a field emission gun at emission 3, gun lens 2–3 (apparent size) and extraction voltage of 3800–4400 eV and spot size 2–3. The aperture of the second condenser lens was nominally 50 mm and the objective aperture was nominally 40 mm. The spatial resolution was better than 0.24 nm point to point with 0.12-nm line resolution. The spatial resolution allows the imaging of macromolecules as well as crystal atoms. Contrasted brightfield (CBF) was used for morphological and structural analysis.[21] TEM micrographs were collected on a Gatan TV camera. DigitalMicrograph software was used to calculate the Fourier Transform (FT) of the lattice fringes and generate picture of the structure corresponding to the reciprocal space of the lattice fringes, allowing precise calculations of the atomic distances, similarly to selective area electron diffraction (SAED). TEM samples were prepared by ultracentrifugation at 30 000 rpm (150 000g at 108C) using a Beckman ultracentrifuge (L7–65 Ultracentrifuge, Beckman Coulter, High Wycombe, UK) with a swing-out rotor SW40Ti as described in Wilkinson et al.[22] The EELS spectra were collected with a Gatan PEELS 666 parallel electron energy spectrometer and EL/P data acquisition software. The energy spread of the incident electron beam (as measured by the full width at half maximum (FWHM) of the zero-loss peak) is ,1.0 eV. All spectra were collected in diffraction mode (i.e. image coupling to the spectrometer). Scattering within the field emission gun of the Tecnai F20 produces a small number of non-collimated electrons that can degrade energy resolution and cause perturbation of the energyloss background. These non-collimated electrons were effectively blocked by using a 10-mm selected area aperture. The EELS spectrum Ce M-edge was collected with an illumination angle 2a ¼ 8.2 mrad, a collection angle 2b ¼ 8.9 mrad, a 1.0-mm

Structure and surface speciation of ceria nanoparticles

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Fig. 1. Typical scanning electron microscopy (SEM) micrographs of: (a) bulk ceria particles showing different shapes of particles including octahedron (1), truncated octahedron (2) and truncated hexagonal pyramid (3); (b) ceria nanoparticles (NPs); and typical transmission electron microscopy (TEM) micrograph of (c) bulk ceria particles showing different shapes of particles including mainly rhombus (1) and hexagonal (2) shapes, and (d) ceria NPs showing mainly rhombus shape.

EELS aperture and 0.2 eV per channel energy dispersion. Ceria particles are very beam-sensitive, with reduction of Ce4þ to Ce3þ with time through irradiation-induced reduction.[23] A time series of EELS spectra was collected every 3 s for 3 min. No irradiation damage was observed within the first 9 s, and therefore the collection time for these spectra was confined to 3 s to prevent any irradiation damage. The Ce M4–5 (see Accessory publication) core-loss ionisation edges and the corresponding low-loss EELS spectrum, including the zero loss peak, were acquired consecutively from the same specimen region. A detailed description of HR-TEM-EELS data analysis is given in the Accessory publication.

octahedron (2), and truncated hexagonal pyramid (3). Therefore, SEM can provide information about the shape and morphology of the bulk particles, but no crystallographic information (i.e. crystal structure and facet identification). Fig. 1c is a typical TEM image of bulk ceria particles, which reveals the shape of the bulk particles, but does not allow resolution of the crystal structure of the particles owing to their large thickness. Particle shapes are mainly rhombus- (1) and hexagonal- (2) shaped, though these shapes represent a projection of the particle in two dimensions. Fig. 1d is a typical low-magnification TEM micrograph of ceria NPs showing that the dominant particle shape is rhombus. Fig. 1c is a twodimensional projection of the particles, which does not allow reconstruction of the three-dimensional shape of the particles, and it does not illustrate the crystallographic structure, which is investigated in more detail below using the high-resolution images. Nonetheless, the aspect ratio can be calculated and this suggests that the majority of the particles have an aspect ratio of 1 (rhombus shape, 44%, or 1.1–1.5 rhombus shape with unequal side lengths, 42%) based on 125 single-particle analyses. HR-TEM allows direct observation of the lattice images of thin samples (,10–20 nm), making it a very useful technique for the characterisation of NPs (35 individual NPs were observed and investigated in detail). Representative HR-TEM

Results and discussion Morphology and crystal structure Figs 1a and b show typical scanning electron microscopy (SEM) micrographs of bulk and nano ceria particles. Clearly, SEM provides semi-3-D images that resolve the shape and morphology of the bulk (Fig. 1a), but not the nano (Fig. 1b) particles, owing to the limited resolution (,5 nm) of the SEM. For bulk CeO2 particles, the particles are relatively well defined and the facets are apparent, but cannot be indexed (Fig. 1a). The observed shapes include octahedron (1), truncated 379

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Fig. 2. High-resolution transmission electron microscopy (HR-TEM) micrographs showing a projection along the o1104 direction of (a) ceria nanoparticle (NP) with four {111} facets (white solid lines), two sets of {111} planes (black solid lines, distance between atomic planes (d) ¼ 0.32 nm), one set of {002} planes (black dashed lines, d ¼ 0.27 nm) and one set of {220} planes (white dashed lines, 0.19 nm), particle size ¼ 11.1  16.7 nm, and illustrating the formation of twin boundary indicated by the arrow; (b) fast Fourier transform of (a); (c) ceria NP with a hexagonal projection along the o1104 direction with four {111} facets (solid lines, d ¼ 0.32 nm), two {002} facets (dashed lines), particle size ¼ 8.1  12.1 nm; and (d) fast Fourier transform of (c).

micrographs of the different shapes of cerium oxide NPs observed at lower magnification (Fig. 1d) are shown in Figs 2 and 3, which show the lattice images of ceria NPs along a o1104 direction. These micrographs allow determination of the crystal structure of the NPs, as well as the terminating planes, based on the interplanar distances and angles as well as the diffraction pattern along the o1104 direction calculated by fast Fourier transform (FFT). A discussion and representations of the different atomic planes and possible facets that can limit the ceria particles are given in Figs A3 and A4 in the Accessory publication. Fig. 2a shows a 2-D projection along a o1104 direction of the lattice image of ceria NP with a rhombus shape. Two sets of {111} planes (black solid lines, 0.32 nm), one set of {002} planes (black dashed lines, distance between atomic planes ¼ 0.27 nm) and one set of {220} planes (white dashed lines, 0.19 nm) are observed as indicated in Fig. 2a. These distances are in good agreement with the fluorite (CaF2) structure of ceria (lattice parameter, a ¼ 0.54 nm) as determined by TEM and X-ray diffraction (XRD).[24] Complementary XRD data, not shown here, show patterns typical of cerium oxide (CeO2), in agreement with the TEM data. This particle is enclosed by four {111} planes (white solid lines) as indicated in Fig. 2a and

corresponds to the projection shown in Fig. A4a. The Fourier transform (Fig. 2b) shows only four spots corresponding to the {111} planes. The 3-D shape of the particle is probably an octahedron surrounded by eight {111} facets with sharp edges as shown in Fig. A4e, in good agreement with the octahedron shape observed by SEM for the bulk particles. Fig. 2c shows a lattice image of another particle in a 2-D projection along its o1104 direction with a hexagonal shape. Fig. 2d shows the Fourier transform performed on the selected particle to avoid contributions from other particles. The terminating planes were identified to be four {111} facets (solid line) and two {002} facets (dashed lines) as indicated in Fig. 2d and correspond to the projection shown in Fig. A4b. The 3-D shape of this particle is probably a truncated octahedron surrounded by eight {111} and two {002} facets as shown in Fig. A4f, again in good agreement with the shape observed by SEM for the bulk particles. Fig. 3a shows a further example of an NP lattice image along the o1104 direction with an octagonal projection, together with the Fourier transform in Fig. 3b. Two sets of {111} planes (solid lines), one set of {002} planes (black dashed lines) and one set of {220/110} planes (white dashed lines) are observed, which correspond to the projection shown in Fig. A4d. For this 380

Structure and surface speciation of ceria nanoparticles

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Fig. 3. High-resolution transmission electron microscopy (HR-TEM) micrograph of ceria nanoparticle (NP) showing a projection along o1104 (a) showing a distorted octagonal projection along the o1104 direction with four {111} facets, two {002} facets and two {220} facets, two sets of {111} planes (solid lines, distance between atomic planes (d) ¼ 0.32 nm), one set of {002} planes (black dashed lines, d ¼ 0.27 nm) and one set of {220} planes (white dashed lines, 0.19 nm), particle size ¼ 7  11 nm; (b) fast Fourier transform of (1); (c) a particle with a rhombus projection along the o1104 direction with four {111} facets, two sets of {111} planes (solid lines, d ¼ 0.32 nm), one set of {002} planes (black dashed lines, d ¼ 0.27 nm) and one set of {220} planes (white dashed lines, 0.19 nm), particle size ¼ 11  11 nm. Fig. 3d also shows imperfections in the crystal growth including steps and kinks. (d) fast Fourier transform of (c).

particle, the Fourier transform shows two sets of {111} atomic planes, one set of {002} and one set of {220} atomic planes. The terminating planes were identified to be four {111} (solid lines), two {002} and two {220} planes as indicated in Figs 3a and b. The 3-D shape of that particle is probably a truncated octahedron surrounded by eight {111}, four {220} and two {002} facets as shown in Fig. A4h, again in good agreement with the shape observed by SEM for the bulk particles. Another particle with a rhomboid shape is shown in Fig. 3c with the corresponding Fourier transform in Fig. 3d. This particle is again an octahedron terminated by eight {111} atomic planes, though it has a defect known as a step and kink (discussed later). Together, low- and high-resolution TEM micrographs suggest that the majority of the observed particles in this study have an octahedral shapes with eight {111} facets, with a lower proportion of truncated octahedral shape with eight {111} facets and two {002} facets or a truncated octahedral shape with eight {111}, four {220} and two {002} facets. The shape of the NPs is principally determined by their surface free energy, which is directly related to the surface area of the crystallite and ensuring that only surfaces of low energy are exposed, and this

results in a truncated octahedral shape here. The {111} surfaces are the most dominant surfaces, in good agreement with the principles of thermodynamics (it has the lowest surface energy) and also in good agreement with previous studies.[10,24] Additionally, the majority of the observed particles are single crystals, with some particles having twin boundaries (indicated by the arrow, Fig. 2a) along the {111} contact plane and appearing as symmetric bicrystals, and some other particles having edge-type dislocation (steps and kinks) (Fig. 3c). These features are induced by the mechanisms of crystal growth to minimise the free energy of the crystal. Twin crystals are known to be more stable (lower surface energy) compared with the simple ones. Twinning is frequently observed, for example in rutile-structured metallic nanocrystals, usually on the {111} atomic planes.[20] Edge dislocation defects, which are frequent in the step-kink mechanism, producing rough structures, are known to enhance the catalytic properties of nanoparticles, and are likely to play an important role in nanoparticle toxicity. Defects such as step edges and oxygen vacancies (discussed below) are the most reactive sites on the surface of metal oxides.[25] 381

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Fig. 4. (a) Representative electron energy loss spectra for cerium phosphate, cerium ammonium nitrate (representing respective CeIII and CeIV standards), cerium oxide nanoparticles (NPs), cerium oxide NPs with humic acid and biological media and bulk cerium oxide particles; (b) percentage fraction of CeIII in the same samples as calculated from electron energy loss spectroscopy (EELS) analysis according to the peak area evaluation method; and (c) percentage fraction of CeIII in the same samples as calculated from EELS analysis according to the second derivative method. SRHA, Suwannee River humic acid; M1, algal biological media þ buffer; and M2, biological media (M2279) used to grow C3A human hepatocyte cells.

Determining the crystal structure, morphology and terminating facets of NPs is important in determining their properties and potential (eco)toxicity. For instance, the surface free energy, stability and reduction of ceria particles depend strongly on the type of the terminating planes.[26] The reactivity of the surface atoms is a function of the atomic plane they belong to, and can be affected by defects in the crystallographic structure. Different atomic planes have different atom densities, electronic structure and bonding and chemical reactivities,[20] and are likely to affect NP toxicity, which needs further investigation. The oxidation and reduction sites can be located at different crystal facets, suggesting that crystal facets can help in separating electrons and holes.[27] A similar behaviour can control the toxicity of NPs, that is, specific atomic planes might have different toxicities, an interesting topic that also requires further investigation. Pal et al. suggested that nanoscale size and the presence of {111} planes combine to promote the biocidal effect of silver nanoparticles.[28] However, such detailed studies are usually complicated by the presence of a mixture of shapes, morphologies and terminating facets, especially in commercially available NPs. Therefore, synthesis of NPs with specific facets is an ultimate necessity in order to address this research area. Additionally, facet-specific redox and other reactions may have important consequences for NP–contaminant interaction. Controlled synthesis of gold, silver and platinum NPs of different

shapes and therefore with different atomic facets has been performed by varying the molar ratio of the capping agent and the metal salt.[29,30] Surface speciation For metallic and metal oxide NPs, the oxidation state of the surface atoms might change as a function of their size or adsorbed species.[6,31,32] For instance, cerium forms two wellcharacterised oxides: CeO2 and Ce2O3. Fig. 4a shows EELS spectra for cerium phosphate and cerium ammonium nitrate (respective CeIII and CeIV standards), ceria NPs in NaNO3, ceria NPs in SRHA, ceria NPs in algal cell media and buffer, ceria NPs in M2279 media and ceria bulk particles. Fig. 4a shows two peaks at ,880–883 and ,897–901 eV, indicating the cerium M5 and M4 edges (double white line; see discussion about their origin in the Accessory publication). From the CeIII and CeIV reference spectra, it can be seen that the M5,4 peaks have different positions on the energy scale and different shapes and heights. The CeIV M5 and M4 edges consist of two main symmetrical maxima at ,883 and 901 eV. They have equal heights and are followed by two peaks of lower intensity (shoulders) at ,889 and 906.4 eV. The CeIII M5 and M4 edges consist of a single peak each at ,880 and 897 eV, with a higher M5 peak. The bulk ceria particles have a spectrum similar to that of CeIV, indicating the dominance of the CeIV 382

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be induced by the vacuum in the TEM as reported by Zhang et al.[35] Clearly, more investigation is required to assess the variability of the reported proportion of CeIII in the different studies, in particular, the effect of the technique used and particle synthesis method, shape and morphology. Reduction of ceria NPs occurs by the removal of surface oxygen atoms; surface energy calculations suggest that an oxygen termination at the top of the O–Ce–O layer is more stable than a Ce-layer termination.[26] Additionally atomic force microscopy[37] and scanning tunnelling microscopy[38] confirm the same observation and suggest that cerium reduction is accompanied by oxygen deficiency (oxygen vacancies). Removal of one oxygen atom can result in the reduction of two CeIV to CeIII by the excess electrons from the removal of the oxygen.[37] Such a removal of surface oxygen atoms results in a patchy surface with islands of oxygen. Further, Figs 4b and c show statistically different averages (ANOVA test (single-factor) followed by Tukey test, P o 0.05) of the fraction of CeIII in the NP suspended in the different media compared with the NPs suspended in NaNO3 solution. The percentage fraction of CeIII in the NPs (62–63%) decreases after interaction with algal biological medium (48–53%), M2279 medium (44–52%) and SRHA (16–18%), depending on the calculation method, suggesting the oxidation of the cerium oxide NPs, in good agreement with the data presented in Wu et al.[32] The organic molecules (electron donors) present in these solutions supply the cerium atoms with the oxygen required to compensate for the oxygen vacancies originating from the size, surface and defect effects. Nonetheless, even after the interaction of NPs with SRHA or biological media, there is still a certain fraction of CeIII ions at the surface of the NPs, i.e. the surface coating causes partial oxidation only of the surface atoms. Such a reaction may explain the reduced toxicity of NPs in the presence of humic substances,[39,40] although other studies suggest that other organic molecules (produced by bacteria) may further reduce the NPs, resulting in an increased fraction of CeIII.[13] Although the redox potential of Ce4þ/Ce3þ is higher than that of the HA, the presence other ligands in the water may shift the redox potential of Ce4þ/Ce3þ. Oxidation of Ce3þ to Ce4þ is a common process in the natural environment.[41,42] It has been shown that Ce3þ can be oxidised to Ce4þ in the presence of carbonate,[43] and Mn (hydro)oxides and Fe oxyhydroxide surfaces.[44,45] Much of the unusual catalytic chemistry involved with nanoceria is believed to be due to oxygen vacancies at the surface of the particles. These oxygen vacancies are characterised by cerium(III) atoms in the centre of the vacancy surrounded by adjacent cerium(IV) atoms.[46] The presence of cerium(III) at the surface of nanoceria is unique to the centre of the oxygen vacancy and the relatively high abundance of these vacancies in nanoceria may be responsible for the altered redox chemistry of nanoceria v. bulk cerium.[47] There is contradictory evidence concerning the role of cerium oxide nanoparticles as antioxidants[48] or pro-oxidants.[4] For instance, cerium oxide NPs have been shown to protect cells against oxidative stress through superoxide dismutase (SOD) mimetic activity, and that was related to surface CeIII sites.[48] However, cerium oxide nanoparticles have been shown to produce oxidative stress in human lung cancer cells[49] and bacterial cells.[13] It has been suggested that NPs able to be oxidised and reduced are cytotoxic and even genotoxic for cellular organisms.[4] Thus, knowledge of the oxidation state of atoms that constitute the NPs, or more specifically the surface atoms, is of considerable importance in

oxidation state, whereas the ceria NPs have a spectrum intermediate between CeIII and CeIV, and have features of both, indicating the presence of a mixture of both oxidation states. Additionally, ceria NPs interacting with SRHA and with biological media show a shift to higher energies compared with CeIII, and a shape closer to that of the CeIV standard, indicating the presence of a mixture of oxidation states and possibly the oxidation of the ceria NPs in the presence of humic substances and biological media. The ratio between the M4 and M5 peaks is linearly proportional to the oxidation state of cerium.[23,33] Thus, quantification of the oxidation state of cerium in ceria particles can be accomplished if this ratio is known for the CeIII and CeIV standards. There are different methods to quantify the M5/M4 intensity ratio including: (i) peak area evaluation after exponential pre-peak background subtraction and post-peak continuum subtraction; (ii) second derivative conversion with subsequent peak area evaluation between zero crossings[33,34]; (iii) a linear combination of CeIII and CeIV spectra[23]; and (iv) peak modelling and fitting (highly elaborate). The results depend somewhat on the evaluation method.[34] The ratio of M5/M4 peak intensities was quantified by methods (i) and (ii) (described in detail in the Accessory publication) for the set of data presented in Fig. 4a. The ratio M4/M5 for CeIII and CeIV was found to be 0.94 and 1.16 by the area evaluation method and 0.82 and 1.13 by the second derivative method respectively, in good agreement with previously reported values.[31,34] The concentration of CeIII in each sample was calculated by linear interpolation according to Eqn A2, and the results are presented in Figs 4b and c. The relatively high variability in the fraction of CeIII in the NPs (large standard deviation) can be explained by the range of particle sizes of the nanoparticles (14.9  6.3 nm or in the range 7–20 nm as determined by TEM based on 125 particles; data not presented here). In addition, the reduction of ceria particles depends on the crystal terminating facets (atomic planes), which are different for the different particles, as discussed above. Such variability is not observed for the bulk particles owing to the limited effect of particle size. It is worth mentioning that this variability (standard deviation) is smaller for the second derivative method compared with the peak area evaluation method, suggesting that it could be a better method for EELS data analysis. The results show that, whereas bulk particles are dominated by CeIV, the ceria NPs contain a large fraction (62–63%) of CeIII ions, suggesting the reduction of the cerium oxide NPs compared with larger particles. This is in good agreement with previous studies suggesting the reduction of cerium oxide with decrease in particle size, to complete reduction to Ce2O3 at ,3-nm diameter.[31] Although these results suggest that bulk particles contain mainly CeIV ions, they may contain a certain fraction of CeIII ions that do not influence the EELS spectra, owing to their low contribution to the total. We estimate that a proportion of 410% CeIII would be discernable by our EELS method. Nonetheless, our results show a higher proportion of CeIII compared with the values (,45%) reported by Wu et al. using the same technique (EELS) and covering the same size range.[31] The amount of Ce2O3 in ceria NPs is a strong function of the particular synthesis method used to synthesise the particles[31] and the characterisation technique used,[35] which explains the small differences between our results and previously published work using X-ray photoelectron spectroscopy[35] and calculation from lattice relaxation measured by X-ray diffraction using Vegard’s law.[36] Additionally, the high proportion of CeIII might 383

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toxicity studies, as it might dictate their toxicity[4] or protective effect.[48]

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Conclusions The majority of observed nano and bulk particles are single crystals with the same crystalline structure as the fluorite-type structure and the same morphology with mainly an octahedral shape enclosed by eight {111} facets, or a truncated octahedral shape enclosed by eight {111} facets and two {002} facets, or eight {111} and two {002} and four {220} facets. Additionally, a few particles contain structural defects, such as twin boundaries and steps and kinks. However, the surface speciation of nanoparticles and bulk particles are significantly different and are altered in the presence of natural organic matter and biological media. Bulk ceria particles contain mainly CeIV, whereas ceria NPs contain a large fraction of CeIII, which decreases after interaction with HA and biological media. Electron microscopy coupled to spectroscopic detectors such as EELS are very important tools for characterising nanoparticles at high resolution, i.e. at the nanometre scale and individual particle level. Such detailed characterisation is very important in the quest for nanoparticle characteristics responsible for toxic effects. Accessory publication The Accessory publication provides a detailed description of the TEM-EELS methodology, the different methods for EELS data analysis (Figs A1, A2), a description of the crystallographic structure of cerium oxide and its projection along the o1104 direction (Fig. A3) and the different possibilities of particle shapes, their projection along the o1104 direction and the observed atomic planes, together with the distances and angles between them (Fig. A4). Acknowledgement This work was funded by the Natural Environment Research Council (NE/ D004942/1) and supported by the Facility for Environmental Nanoscience Analysis and Characterisation (FENAC).

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