# Size Effect of Silver Nanoparticles Derived from Olive Mill Wastewater in THP-1 Cell Lines
## 3 Results and Discussion  
OMWW is produced in large quantities in the Mediterranean area because most of the agricultural companies produce olive oil in winter [13]. However, the OMWW is characterized by a great concentration of polyphenols (up to 10 g/L) that represents a big problem for disposal. Since in the green processes polyphenols are involved to produce metallic NPs [14], this agricultural waste can be useful to obtain nanomaterials with specific physicochemical properties [15]. In general, metal oxide and metallic NPs were used to purify OMWW, as described in different works [16; 17; 18]. In our case, we synthesized AgNPs with two different sizes starting from solutions of AgNO\({}_{3}\) (1 mM and 1.5 mM) to which we added OMWW after its purification from solid residues. The OMWW was used as a pristine medium without the extraction of polyphenols that were directly involved in the synthetic route as reducing and capping agents. The entire process occurred at room temperature and for this, the energy consumption was minimal; this is fundamental to meet the scope of the green chemistry principles [19]. In order to understand the quantity of polyphenols in the OMWW and in the solution containing AgNPs after synthesis, a Folin-Ciocalteu analysis was carried out. We quantified 24 \(\pm\) 1.7 mg/L as reported in Table 1.  
The TEM images shown multifaceted spherical nanostructures, both for 30 nm and 50 nm (Figure 2a,b). The statistical analysis was carried out on the TEM images measuring 100 of each NP type with a Gaussian fitting using ImageJ software. The NPs size were 30 \(\pm\) 5 nm and 50 \(\pm\) 6 nm. (Figure 2c,d). The UV-Vis spectra of the two sizes of AgNPs are reported in Figure 2e,f, showing the Localized Surface Plasmon Resonance (LSPR) absorption peaks at c.a. 410 nm and 430 nm for the 30 nm AgNPs and 50 nm AgNPs, respectively. The FTIR analysis of the two sizes of NPs (Figure 2g,h) exhibited peaks for the -OH, -CH\({}_{2}\), -CH\({}_{3}\) stretching modes corresponding to the phenolic groups. In the 1742 cm\({}^{-1}\) and 1642 cm\({}^{-1}\) wavelengths, the bands corresponded to the -C=O stretching vibration and the -C=C- of the aliphatic vinyl ethers. At 1417 cm\({}^{-1}\), we found vibrations ascribed to the -CH and the 1455 cm\({}^{-1}\) peak corresponding to the -C=C- bonds. The peaks at 850 cm\({}^{-1}\), 1038 cm\({}^{-1}\), 1108 cm\({}^{-1}\) \begin{table}
\begin{tabular}{c c} \hline \hline
**Sample** & **Total Polyphenols (mg/L)** \\ \hline Olive Mill Wastewater (OMWW) & 24 \(\pm\) 1.7 mg/L \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Quantification of total polyphenols in OMWW. 1163 cm\({}^{-1}\), 1209 cm\({}^{-1}\) and 1233 cm\({}^{-1}\) were related to the -C-H, C-O, -C-O-C-, -O-C-, -O-H, -C(=O)-O- stretching vibrations. These results meant that the polyphenols from the OMMW were also capping agents on the NPs surface. We also analyzed the UV-Vis absorption of pristine OMMW. The UV-Vis absorption showed an intense absorption band in 230 nm that was associated with carbon bonds of the aromatic phenolic compounds (Figure 3a). In the infrared spectrum of the OMMW, -OH groups were represented by the band between 3650 and 3000 cm\({}^{-1}\). The 2924 cm\({}^{-1}\), and 2884 cm\({}^{-1}\) bands corresponded to the CH\({}_{2}\) and CH\({}_{3}\) stretching of polyphenic molecules. The 1708 cm\({}^{-1}\) and 1666 cm\({}^{-1}\) bands corresponded to the -C=O whereas the peak at 1587 cm\({}^{-1}\) was related to the -C=C- vibration of aromatic rings. Lastly, 1396 cm\({}^{-1}\), 1260 cm\({}^{-1}\) and 1068 cm\({}^{-1}\) peaks were related to -OH, -C-O and C-O-C- bonds, respectively. Figure 2: (**a**,**b**) Representative TEM images of 30 nm AgNPs and 50 nm AgNPs derived from the OMMW synthetic route; statistical analysis with Gaussian fit for 30 nm AgNPs (**c**) and 50 nm AgNPs (**d**); UV–vis of 30 nm AgNPs (**e**) and 50 nm AgNPs (**f**). FTIR spectra of 30 nm AgNPs (**g**) and 50 nm AgNPs (**h**).