Preparation and quantification of crystalline nanosilica for toxicological investigations
Authors: Bellomo Chiara , Pavan Cristina , Rebba Erica , Fiore Gianluca , Mino Lorenzo , Turci Francesco 
Published: March 2022

&lt;p&gt;Crystalline silica (CS) is a well-known human toxicant and inhalation of the airborne particles with size lower than 4 &amp;#181;m is associated to severe occupational diseases, such as silicosis and lung cancer.&lt;sup&gt;1&lt;/sup&gt; The International Agency for the Research on Cancer (IARC) classified CS as carcinogenic to humans and freshly fractured CS is held to be more toxic than aged dust.&lt;sup&gt;1,2&lt;/sup&gt; Fracturing generates on CS a specific family Nearly-Free Silanols (NFS), which are able to destabilize cell membranes,&lt;sup&gt;3&lt;/sup&gt; and some nanometric particles. We aim here to create and assess the possible toxicological impact and the chemical characteristics of the nanometric fraction (nano-CS) formed when CS is fractured.&lt;/p&gt;&lt;p&gt;A highly pure CS of synthetic origin&lt;sup&gt;4&lt;/sup&gt; (&amp;#945;-quartz, micrometric in size) was ball-milled to obtain ultrafine particles. We coupled a dry milling step and a wet milling step, using water as dispersing agent, and we generated particles with specific surface area (SSA) ranging from 37 to 60 m&lt;sup&gt;2&lt;/sup&gt;/g. These SSA values signaled the generation of a relevant nanometric fraction. The increase in SSA paralleled the energy delivered to quartz during the milling, that exceeded by far the energies commonly used in industrial processing. Morphology, crystallinity, size, surface silanols, including quantification of NFS, and membranolytic activity toward red blood cells were assessed. The nano-CS samples exhibited: i) a partial lattice amorphization that increased with the increase of the milling energy; ii) the presence of two distinct domains of scattering that indicated the occurrence of crystallite with nanometric (&lt; 50 nm) and submicrometric (0.8-1 &amp;#181;m) size; iii) a strong tendency to form micrometric agglomerates , which could be partially dispersed with ultrasounds and surfactants in water suspensions; and iv) a moderate membranolytic activity that correlated with the presence of NFS. We selected a nano-CS sample that would be classified as a nanomaterial under EU CLP regulation (&gt;50% of particles in number are &lt; 100 nm).&lt;sup&gt;5&lt;/sup&gt; The nano-CS sample will be used in the next future as a reference material to quantify the nanometric fraction of silica powders and assess the potential exposure to nano-CS in industrial hygiene context.&lt;/p&gt;&lt;p&gt;In conclusion, the preparation and characterization of nano-CS was achieved and the physico-chemical characteristics that could be relevant for silica toxicity were assessed. The nano-CS reference material will be used to quantify nano-CS in industrial scenario, and to clarify the toxic activity of nanometric silica obtained by mechanical fracturing.&lt;/p&gt;&lt;p&gt;&amp;#160;&lt;/p&gt;&lt;p&gt;[1] IARC, &lt;em&gt;Monograph Vol. 100C, &lt;/em&gt;&lt;strong&gt;2012&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;[2] Turci et al, &lt;em&gt;Part Fibre Toxicol, &lt;/em&gt;&lt;strong&gt;2016&lt;/strong&gt;, &lt;em&gt;13&lt;/em&gt;, 32.&lt;/p&gt;&lt;p&gt;[3] Pavan et al, &lt;em&gt;Proc. Natl. Amer. Soc. USA, &lt;/em&gt;&lt;strong&gt;2020&lt;/strong&gt;, &lt;em&gt;117 (45&lt;/em&gt;), 27836&lt;/p&gt;&lt;p&gt;[4] Pastero et al., &lt;strong&gt;2016&lt;/strong&gt;, &lt;em&gt;Cryst. &lt;/em&gt;&lt;em&gt;Growth Des. &lt;/em&gt;16, 4, 2394&amp;#8211;2403.&lt;/p&gt;&lt;p&gt;[5] SCoEaNIHR, E. S., &lt;strong&gt;2010&lt;/strong&gt;, 'Scientific Basis for the Definition of the Term &amp;#8220;nanomaterial&amp;#8221;', &lt;em&gt;European Commission.&lt;/em&gt;&lt;/p&gt;