# Characterization of Nanoparticle Batch-To-Batch Variability
## 3 Results
### Highlights of Variations in Physicochemical Properties between Different Batches per NM
#### 3.2.2 Zinc Oxide NMs  
As shown in Figure 3F all ZnO batches moderately induced DCF oxidation (around 4-fold over control) independent of the synthesis conditions (Figure 3A).  
Zn is known to be a redox-inert metal which does not participate in oxidation-reduction reactions [40; 41; 42]. However, impurities in the sample may have caused ROS-induced DCF fluorescence, although no clear correlation with the impurities indicated in Figure 3D could be identified.  
Figure 2: Flame synthesized silica SilNP012–017. (**A**) Production parameters for the different flame synthesized silica nanoparticles; (**B**) Zeta potential representing the surface charge in –mV at pH \(\sim\)4.3; (**C**) Representative TEM images; (**D**) Selected impurities present in the different flame synthesized SiO\({}_{2}\) NM batches. The mass concentrations of elements above detection limit are shown. The data represent the mean of two assays; (**E**) Particle diameters d\({}_{\rm P}\) in nm: Z-Average of d\({}_{\rm P}\) determined in aqueous suspension using DLS (black bars) and modal value x\({}_{\rm M}\) of the number size distribution in the aerosol phase measured by SMPS (red bars); (**F**) Potential to induce ROS. The test particles were delivered as powder. The graph shows the DCF fluorescence induced by the different batches at 400 \(\upmu\)g/mL relative to a sample without NMs (control). 400 \(\upmu\)g/mL commercial SiO\({}_{2}\) (SilNP01, Aerosil®200, Evonik, Essen, Germany) and 0.3 \(\upmu\)M H\({}_{2}\)O\({}_{2}\) was used as positive controls. The data represent mean values of 2 independent experiments with three replicates \(\pm\) s.e.m.  
All three batches of the third production run of ZnO NMs (ZnONP007--009, 4.95 g zinc acetate) show the lowest and constant amount (<100 \(\mathrm{\SIUnitSymbolMicro g}\)/g) of impurities, while the other runs rise to around 1000 \(\mathrm{\SIUnitSymbolMicro g}\)/g. Depending on the further use of ZnO NMs (e.g., for toxicity testing), the presence of toxic elements such as Cu and Pb and sometimes Cd and Tl should be considered. As found by TEM inspection, at first glance, the primary sizes of the different ZnO NM batches seem to be very similar. Most of the ZnO particles are single-crystalline and have the hexagonal wurtzite structure but show amorphous material in between and around the agglomerated single-crystalline ZnO nanorods (Figure 3C). That would mean that there is no essential influence of the zinc acetate concentration. However, since the particles dominantly exhibit the shape of rods, having a length up to 300 nm and more, presumably the aspect ratio does vary with the zinc acetate concentration. This ratio seems to be higher for NMs produced at increased zinc acetate concentrations. A quantitative assessment of the relative percentage of distinct populations of ZnO NMs appearing as rods with specific aspect ratios in a given sample is quite challenging and requires a high number of individual specimens to be analysed by TEM to reach statistical significance and was therefore not performed in the present work. Nevertheless, in the future such information would be of interest to better understand the impact of geometrical parameters on toxicokinetics and adverse effects.