**2.14. Morphology**

We focused on the investigation of the potential toxic effects caused by nanoclay particles currently used in tons in several industrial applications. Bentonite, montmorillonite, and

**Figure 12.** The structure of smectites. Clays are formed of layered tetrahedral (T) and octahedral (O) sheets.


**Table 3.** Key clay mineral species explored for tissue engineering and regenerative medicine applications with their relevant structural/compositional properties.

kaolin are platy clay particles ranging from nanometers to micrometers, whereas halloysite nanotubes are hollow rod-like particles having a lumen diameter of ∼20 nm and a tube length ranging from 300 nm to 2 μm. Spherical silica nanoparticles were also used as a material mimicking the outer silica layer of the clays. Graphene oxide nanosheets having the shape and size close to smectite clays were selected as a material with a relatively high reported toxicity, 30, which was also confirmed in our study. The typical AFM images demonstrating the geometry and sizes of nanoparticles used in this study are given in **Figure 13**.

The nanoparticles used were suspended in water and added to protozoan media at a range of concentrations. Prior to toxicity investigation, the hydrodynamic diameters and surface potential of nanoparticles were determined using dynamic light scattering (DLS) and electrophoretic mobility measurements in water (**Table 4**) [118].
