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Dispersion Stability of Nanoparticles in Ecotoxicological Investigations: the Need for Adequate Measurement Tools

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Dispersion Stability of Nanoparticles in Ecotoxicological Investigations: the Need for Adequate Measurement Tools J Nanopart Res DOI 10.1007/s11051-011-0298-y RESEARCH PAPER Dispersion stability of nanoparticles in ecotoxicological investigations: the need for adequate measurement tools Ratna Tantra • Shingheng Jing • Sivaraman K. Pichaimuthu • Nicholas Walker • James Noble • Vincent A. Hackley Received: 20 October 2010 / Accepted: 14 February 2011 Ó Crown Copyright 2011 Abstract One of the main challenges in nanoeco- that nanoparticle dispersion made in the fish medium toxicological investigations is in the selection of the was less stable compared to corresponding dispersion most suitable measurement methods and protocols in de-ionised water. Stability of these dispersions was for nanoparticle characterisation. Several parameters monitored using various techniques, for a period of have been identified as being important as they 3 days. Our findings have shown that dispersion govern nanotoxicological activity, with some param- stability can be suitably assessed by monitoring: eters being better defined than others. For example, as (a) surface charge, (b) sedimentation events and a parameter, there is some ambiguity as to how to (c) presence of agglomerates, through time. The measure dispersion stability in the context of ecotox- majority of techniques employed here (zeta potential, icological investigations; indeed, there is disagree- particle size via DLS, fluorescence and UV–Vis ment over which are the best methods to measure spectroscopy and SEM) were shown to provide nanoparticle dispersion stability. The purpose of this useful, complementary information on dispersion article is to use various commercially available tools stability. Nanoparticle Tracking Analysis (NTA) to measure dispersion stability and to understand the provides useful, quantitative information on the information given by each tool. In this study, CeO2 concentration of nanoparticles in suspension, but is was dispersed in two different types of media: limited by its inability to accurately track the motion de-ionised water and electrolyte-containing fish of large agglomerates found in the fish medium. medium. The DLS mean particle size of freshly dispersed sample in DI water was *200 nm in Keywords Nanoparticles Á Dispersions Á diameter. A visual sedimentation experiment showed Nanomaterial characterisation Á Nanometrology Á Environmental and health effects R. Tantra (&) Á S. Jing Á S. K. Pichaimuthu Á J. Noble National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, UK e-mail: [email protected] Introduction N. Walker School of Biosciences, University of Exeter, The potential toxicity of nanoparticles has attracted Geoffrey Pope Building, Exeter EX4 4QD, UK attention in recent years and consequently has presented a dilemma to toxicologists and risk asses- V. A. Hackley National Institute of Standards and Technology, 100 sors in general (Boverhof and David 2010). The main Bureau Drive, Gaithersburg, MD 20899-8520, USA research bottleneck is in the ability to reliably link 123 J Nanopart Res bioassay data with the relevant physicochemical 2008). Others have also equated this to indicate the properties of the nanoparticle preparations. This in level of ‘resistance to sedimentation’ and coagulation turn is governed by the lack of agreed, validated (or flocculation), which can be expressed as the protocols for the evaluation of nanoparticle toxicity ‘constancy of the number of particles per unit volume’ (Tiede et al. 2008). A significant hurdle, in the case of in the dispersion (Kissa 1999). If the dispersion is physicochemical characterisation tests, is the lack unstable, then we expect: (a) lower zeta-potential of suitable tools to measure, low concentrations of values (irrespective of polarity), (b) increase in par- nanoparticles (ng/L or less) reliably in complex media ticle size with time as particles agglomerate and (Simonet and Valcarcel 2009). This requires tech- (c) decrease in particle concentration with time in the niques that can offer: sensitivity, high selectivity, and upper dispersion layer as larger agglomerates sedi- ‘representativeness’ of the entire sample population. ment out. Due to the lack of suitable tools, past researchers such The main question put forward in this study is: as Powers et al. (Powers et al. 2006) have put forward ‘Do different methods lead to different results and, if a strategy ‘to characterise with as many methods as so, to what extent do they impact the final interpre- possible’; in doing so, researchers are allowed to ‘pick tation of the state of dispersion?’ To address this and choose’ on what or how to perform the charac- question, CeO2 (an industrially relevant nanoparticle) terisation step. In reality, different tools yield different is dispersed in both deionized (DI) water and fish information and their accuracy will be very much medium; in the past, the dispersion of CeO2 nanopar- dependent on the strengths and limitations of the ticles in DI water at concentrations of 50 mg/L or less, individual techniques. For example, it is known that was shown to be stable after a period of 3 days. We for the measurement of particle size, dynamic light will assess the state of dispersion through the use of scattering (DLS) will give a different answer relative various measurement techniques, viz.: visual inspec- to imaging-based techniques such as atomic force tion of sedimentation, UV–Vis and fluorescence microscopy (AFM); this difference is linked with spectroscopy, DLS, scanning electron microscopy sample preparation issues and with dry versus wet (SEM), zeta-potential measurements (microelectro- measurements. An excellent example of these appar- phoresis) and nanoparticle tracking analysis (NTA). ent size discrepancies can be found in the report of The media analysed was selected based on previ- analysis for NIST reference material 8012 (gold ous studies highlighting the differences in stability nanoparticles, nominally 30 nm) (Reference Material between DI water and fish medium, thereby provid- 8012—Gold Nanoparticles), which was characterised ing a good model for comparison. by six independent sizing methods; the reported Tools were chosen on the basis that they are: reference values for DLS and AFM are consistently (a) commercially available, and (b) the measurand is different, with DLS typically larger compared with different. Out of all the techniques employed here, AFM and other microscopy methods. NTA is considered a relative newcomer to the The objective of this study is to further explore the nanoparticle analysis toolkit. NTA technology has impact of using different tools to measure other the capability to measure particle size and number parameters. In particular, we are interested to study concentration in situ; particle size measurement those parameters, whose importance may be widely involves tracking the Brownian motion of individual recognised, but which have no defined guidelines for particles using a digital camera and tracking software the best methods/tools to use for their measurement. (Malloy and Carr 2006). As with DLS, the hydrody- In the nanoecotoxicological context, ‘dispersion sta- namic size of the particle is reported, calculated bility’ is an important parameter, as this relates to the through the Stokes–Einstein equation. Although NTA assessment of fate, exposure and subsequent bio- is a relatively new technique with limited validation availability (thus biological effect); in general, the data (Filipe et al. 2010), we have selected this method more stable the dispersion, the higher the bioavail- as it is less sensitive than DLS to signal domination ability. In the past, researchers have equated this by larger particles and offers some complementary parameter to the monitoring of particle size change information, such as particle number concentration, through time (agglomerate formation) and corre- unattainable from the other methods used in this sponding zeta-potential measurements (Handy et al. study. 123 J Nanopart Res Experimental details Table 1 Nanoparticle dispersion and analysis Chemical formula Concentration Materials mmol/L Calcium chloride CaCl2 4.87 Nanocrystalline cubic CeO2 powder (NanoGrain 1 Magnesium sulphate MgSO4 1.92 CeO2 CMP, 99.95 %) was obtained from Umicore (Olen, Belgium). The manufacturer specifies a median Sodium bicarbonate NaHCO3 1.54 particle size of (70 ± 11) nm for particles dispersed Potassium chloride KCl 0.15 in water after 24 h and based on an X-ray disc centrifuge (XDC) method. The Brunauer–Emmett– Teller—commonly called BET—specific surface area is specified as (30 ± 3) m2/g. The supplier also volume with a final nanoparticle concentration of specifies an isoelectric point (IEP) of 8.5 ± 0.5. The 50 mg/L. In order to ensure homogeneity, a glass rod primary particles in suspension appear to exist as was used to gently mix the final dispersion. Disper- small compact aggregates of primary crystallites. sions were stored in a separate pre-cleaned 1 L media The CeO2 particles were dispersed in one of two glass bottle. Optical images showing the state of the aqueous media: de-ionised (DI) water and fish dispersion in the bottles were recorded using a digital medium at pH 7.0, as measured according to camera at set intervals over the analysis period of guidelines in ISO-7346/3 (1996). The composition 3 days. Sample bottles were stored in the dark, when of the fish medium is presented in Table 1, with salts images were obtained care was taken not to disturb obtained from Sigma-Aldrich, UK. the suspension. Nanoparticle powders (50 mg) were weighed, At various
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