## 2 Results and Discussion
### Shelf-Life Study
#### 2.3.1 Evolution of Chemical Characteristics over Time
Table 8 shows that TTC varied significantly with storage time, EVOO sample, and their interaction, as well as the interaction between sample and bottle size and the three-way interaction. TTC ranged from an initial value of 387.7 mg/kg to 345.5 mg/kg for the 250 mL bottle and to 322.0 mg/kg for the 500 mL bottle in the case of EVOO 1, and from an initial 407.8 mg/kg to 328.5 mg/kg for the 250 mL bottle and to 337.8 mg/kg for the 500 mL bottle for EVOO 2. These values are considered good for high-quality EVOOs after one year of storage. As shown in Figure 3, initial locopherol content was higher in  
Figure 3: Evolution of tocopherols (**A**) \(\alpha\)-, (**B**) \(\beta\) + \(\gamma\), (**C**) \(\delta\), (**D**) Total content in the two oils stored in the two bottles over storage. 0, time 0; 1, 2 months; 2, 5 months; 63, 12 months; 250 mL and 500 mL are the bottles’ sizes. In each graph, different letters over the bars indicate significant differences at \(p\) < 0.05. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Factors**} & \multicolumn{2}{c}{\(\alpha\)**-Tocopherol**} & \multicolumn{2}{c}{\(\beta\) + \(\gamma\)**-Tocopherol**} & \multicolumn{2}{c}{\(\delta\)**-Tocopherol**} & \multicolumn{2}{c}{**Total Tocopherol} \\  & **Pr(\(\circ\)F)** & **Signif.** & **Pr(\(\circ\)F)** & **Signif.** & **Pr(\(\circ\)F)** & **Signif.** & **Pr(\(\circ\)F)** & **Signif.** \\ \hline EVOO & \(2.47\times 10^{-7}\) & *** & \(<\)\(2\times 10^{-16}\) & *** & \(0.00768\) & ** & \(0.0491\) & * \\ Time & \(<\)\(2\times 10^{-16}\) & *** & \(9.41\times 10^{-16}\) & *** & \(1.39\times 10^{-11}\) & *** & \(<\)\(2\times 10^{-16}\) & *** \\ Bottle Size & \(0.40076\) & & \(0.0437\) & * & \(0.27455\) & & \(0.7204\) & \\ EVOO:Time & \(4.72\times 10^{-9}\) & *** & \(2.05\times 10^{-6}\) & *** & \(2.74\times 10^{-6}\) & *** & \(8.16\times 10^{-8}\) & *** \\ EVOO:Bottle Size & \(0.00065\) & *** & \(0.1288\) & & \(0.21125\) & & \(0.0052\) & ** \\ Time:Bottle Size & \(0.36106\) & & \(0.0767\) & - & \(0.5705\) & & \(0.2958\) & \\ EVOO:Time:Bottle & \(0.0188\) & * & \(0.0279\) & * & \(0.29124\) & & \(0.0251\) & * \\ Size & \multicolumn{2}{c}{} & \multicolumn{2}{c}{} & \multicolumn{2}{c}{} & \multicolumn{2}{c}{} & \multicolumn{2}{c}{} & \multicolumn{2}{c}{} \\ \hline \hline \end{tabular}  
* Pr(\(\circ\)F) indicates the level of probability that variable has no effect on that parameter, on a scale from 0 (it has an effect at 100%) to 1 (it has an effect at 0%). Significance of the effects: ***, from 99.9 to 100%; ***, from 99.9%; **, from 99.9 to 99.9%; **, from 99.9%; **, from 99.9%; **, from 99.9%; **, from 99.9%; **, from 99.9%; no symbols, from 0 to 99%.  
\end{table} 
Table 8: Significance of the effects of individual factors and their interactions on the total tocopherol content (TTC), and on each tocopherol over time, as evaluated by 3-way ANOVA. EVOO 2 but decreased markedly after t1, falling below the levels observed in EVOO 1. Both oils showed a general decreasing trend, with opposite effects from bottle size. **Volatile compounds**: Figure 4 shows the evolution of selected VOCs such as hexenal, (_E_)-2-hexenal, total LOX VOCs, rancid VOCs, microbial VOCs, and 2 + 3-methylbutanal over time. Table 9 summarizes the significance of each factor and their combination as evaluated using 3-way ANOVA. Detailed data are provided in Table S7 of the Supplementary Material. VOCs are key markers in differentiating oils from a sensory perspective [5; 31; 36]. Major VOCs groups in EVOO include the following: (i) LOX-pathway-derived molecules, linked to green and fruity notes; (ii) oxidation-related molecules (e.g., octane, saturated C5-C10 aldehydes, mono- and di-unsaturated C7-C10 aldehydes); (iii) molecules linked to microbiological defects (with a wide spectrum resulting from numerous microbial processes) [27; 31; 54; 55]. Hexanal and (_E_)-2-hexenal: According to Table 9, hexanal, which can be formed via both the LOX-pathway and oxidation, showed significant variation with all factors except the time-bottle size interaction.