Sedimentation kinetics of Titanium dioxide nanoparticles (TiO2) and Multi-walled carbon nanotubes in artificial freshwater by Centrifugal Separation Analysis (CSA) LUMiSizer 651

Environmental and human exposure to engineered nanomaterials (ENMs) is constantly increasing due to the exponential growth of marketed nano-based products [1]. Despite manifold benefits, some adverse effects of ENMs to biota have been shown [2, 3]. In order to correctly interpret toxicity data, the behavior of ENMs in dispersions should be investigated to link the characterization data with toxicological results. To achieve this goal, the behavior of pristine ENMs in an artificial freshwater used in ecotoxicological testing was studied by Centrifugal Separation Analysis (CSA) LUMiSizer 651, allowing to obtain sedimentation rates of ENMs in dispersion at force gravity over long-time scale to support nanotoxicology. Sedimentation kinetics of two different ENMs, with regards to chemical composition, shape and size, i.e. the inorganic Aeroxide® P25 TiO2 and the organic NanocylTM NC7000 Multi-Walled Carbon Nanotubes (MWCNTs), the latter provided within the EU-FP7 SUN Project, was investigated by the Multi-wavelength Dispersion Analyzer LUMiSizer 651 (L.U.M. GmbH, Berlin), over a maximum of 30 days. According to chronic ecotoxicological studies, each ENM was dispersed in the reconstituted artificial freshwater AFW OECD 203 [4] at the following concentrations: 5, 10, 15, 20, 50, 100 mg/l for P25 and 6, 10, 14, 20, 40 and 100 mg/l for NC 7000. Humic acid (0.2% w/w) was added to NC7000 dispersions in order to avoid the very fast sedimentation that would not allow settling measurements. Specifically, LUMiSizer 651 measured the intensity of the transmitted light as function of time and position, at 470 nm and 25°C, over the entire sample length (Fig. 1). Exploiting Lambert-Beer’s law, transmittance values at the midpoint of the length of the cuvette (i.e.11 mm), for different relative centrifugal force (RCF) and ENM concentrations tested, were converted to absorbance and then to ENMs concentration. The settling rate constant, k, was calculated at different RCF and tested concentrations by the equation C=(C0-plateau)·et+plateau, in order to obtain k values at force gravity for each ENM concentration. The overall results showed that ENMs sedimentation was affected by the initial concentration: k at force gravity increased with the concentration for both ENMs but with different rates. As far P25, k values ranged from 1.02·10-4 to 1.85·10-4, from the lowest to the highest concentration tested, fitting with linear regression (slope=9.2·10-3 ; R2 =0.969); k values for NC7000 ranged from 3.28·10-5 to 4.13·10-4, fitting again with linear regression (slope=6.3·10-6; R2 =0.945). According to Quik et al., 2014 [5], sedimentation rates were calculated from k values, using the sedimentation length, measured from the top to the midpoint of the cuvette (h=11mm). The obtained results are in line with previously published studies corresponding to an average centrifugal acceleration of 189 xg over the whole sample. To our knowledge, this is one of the first studies that calculates the sedimentation rates of TiO2 P25 and MWCNT NC7000 in an ecotoxicological medium with the CSA LUMiSizer up to 38 days. One of the main advantage to use this technique is the possibility to obtain sedimentation rates of different ENMs in a liquid dispersion over a long-time scale, supporting the development of fate and exposure models for ENMs, especially from the chronic exposure point of view. We therefore propose the LUMiSizer 651 as an alternative to other classic techniques, e.g. UVspectrophotometer, to assess the stability of ENM dispersions for (eco)toxicological testing over long-time scale.