The complete compositions and the yields of the essential oils obtained from the dried inflorescences of 11 genotypes of Cannabis sativa L., cultivated by CREA-CI (Italy) in an experimental farm located in Rovigo and harvested during the years 2019 and 2020, are reported in Table 2 and Table 3, respectively. In total, 116 compounds were identified, representing 90.6–99.4% of the total composition.

All the EOs, with the exception of Felina 32-2019, were characterized by a predominance of sesquiterpenes, in both their hydrocarbon and oxygenated forms, ranging from 47.1% in Carmagnola-2020 (Table 3) to 78.5% in Fibrante-2019 (Table 2). As reported in Table 4, both the hydrocarbon and oxygenated sesquiterpenes presented significant differences in all the EO compositions as a function of the genotype, the year of cultivation, and their interaction.

In 2019 EOs, sesquiterpene hydrocarbons exhibited a significantly higher relative concentration in Futura 75, followed by Fibrante and Carmagnola. Lower relative abundances were, instead, found in Bernabeo, Eletta Campana, and Felina 32 (Table 2). In 2020 samples, instead, this class of compounds was significantly more abundant in Carmaleonte, Codimono, Eletta Campana, Fibranova, and Futura 75, while their lowest presence was detected in Uso-31 (Table 3). Furthermore, sesquiterpene hydrocarbons were significantly higher in 2020 than in 2019 for each analyzed hemp genotype, except for Carmagnola and Uso-31. Among this chemical class, the main volatile compounds were β-caryophyllene and α-humulene, in accordance with Ascrizzi et al. [9], Vuerich et al. [18], and Menghini et al., 2021 [25], who reported these constituents as being typical of hemp varieties.

Regarding the oxygenated sesquiterpenes, among the 2019 EOs, Carmaleonte and Uso-31 presented higher relative abundances (59.6 and 60.9%, respectively), whilst the lowest was exhibited by Felina 32 (30.9%). Conversely to the hydrocarbon forms, oxygenated sesquiterpenes were significantly more abundant in all the 2019 samples compared to those of 2020. These secondary metabolites are degradation products deriving from the oxidation of the corresponding terpenes due to air exposure (i.e., during prolonged storage), and are considered responsible for the antioxidant activity of many EOs [18,26]. Moreover, higher oxygenated compounds’ relative abundances reflect more favorable growth conditions, as was also confirmed by the higher plant density detected in the 2019 samples. The analyzed samples presented caryophyllene oxide and humulene oxide II as the main components of this chemical class, which were also reported as the main epoxides found in the hemp varieties analyzed by Micalizzi et al. [27]. Furthermore, 14-hydroxy-9-epi-(E)-caryophyllene, reported by Ascrizzi at al. [8], was present in low relative abundances.

The amount of selinene derivative compounds (α-selinene, β-selinene, selina-3,7(11)-diene, and selin-6-en-4-ol) in 2019 and 2020 is notable, as this is a common occurrence in hemp EOs; their presence was more consistent in Futura 75, Eletta Campana, and CS, as already reported in the literature [10,25,27]. Bernabeo EOs in both years were characterized by the presence of higher percentages of neointermedeol, α-bulnesene, and juniper camphor; Fibrante EOs by trans-α-bergamotene and (E)-β-farnesene.

Monoterpenes were poorly represented in all the samples, with no distinction between monoecious and dioecious varieties, but with differences induced by the year of collection and the genotype. They accounted for up to 9.3% in Carmagnola 2020 (Table 3). Overall, 9 hydrocarbons and 14 oxygenated monoterpenes were detected: among the former, α-pinene, β-pinene, and myrcene were the most representative, whilst in the latter, no compounds were revealed in appreciable relative abundances. Among the 2019 EOs, monoterpene hydrocarbons were significantly higher in Carmagnola and Fibrante (2.0 and 2.1%, respectively), and the latter presented a significantly higher content of oxygenated monoterpenes as well (1.3%). Fibrante-2019 also differed from the other genotypes in terms of its higher relative abundance in limonene (0.5%), which reached 0.8% in 2020. In the 2020 samples, hydrocarbon forms were found to be more abundant in Carmagnola (1.8%), while the oxygenated ones in CS (2.3%). However, with the exception of Codimono, 2020 samples were characterized by a significantly higher relative abundance of both hydrocarbon and oxygenated monoterpenes.

The amount of the compounds belonging to this class of secondary metabolites is very different from those reported by Bertoli et al. [21], Benelli et al. [10] and Nissen et al. [28], in which the main detected chemical class was represented by monoterpenes, with significant differences between monoecious and dioecious genotypes; in these papers, however, the starting material consisted of the fresh inflorescences, rather than the dried ones. Assuming that the monoterpene content decreased in the drying process and storage [26,27,29,30], the predominance of sesquiterpenes in the examined EOs might be due, at least in part, to the drying process, which might have induced some changes in the chemical composition of the starting material, such as (i) evaporation of the low boiling-point compounds [27], and (ii) the induction of oxidative reactions, as in the conversion of β-caryophyllene in caryophyllene oxide, and α-humulene in humulene oxide II [30]. Nevertheless, few of the literature studies used the dried hemp inflorescences for the collection and characterization of the EOs.

Finally, cannabinoids were the second relevant class of compounds in all the EOs, excluding that of Felina 32-2019, in which they were the main chemical group, accounting for the 53.4% of the total (Table 2). The detected cannabinoids were cannabidiol, cannabichromene, and Δ⁹-tetrahydrocannabinol, but the first was the most abundant, with relative percentages ranging from 11.8 to 51.5% in Uso-31-2019 and Felina 32-2019, respectively (Table 2). In 2020 samples, these metabolites were detected in a greater relative amount in Carmagnola (37.7%) than in Bernabeo (15.0%, the lowest). In general, cannabinoids were meaningfully higher in the 2020 samples, except for Bernabeo and Felina 32, but according to the two-way ANOVA (Table 4), no significant differences were present between the two years of cultivation for this chemical class, which, on the contrary, showed significant differences among the genotypes. As for the sesquiterpenes, the predominance of cannabinoids in the EOs might be imputable to the drying process, during which the decarboxylation of cannabinoid acids into their relative volatile forms takes place [30].

Nevertheless, all the detected classes of compound, excluding the already mentioned cannabinoids, presented significant differences as a function of genotype (G), year of cultivation (Y), and genotype–year interaction (G × Y), as evidenced by the two-way ANOVA (Table 4), which was also conducted on the chemical compounds, selected by the SIMPER test, which contributed to at least 1.00% of the dissimilarities in the composition between 2019 and 2020 EO samples. The statistical analysis evidenced that 16 compounds were responsible for an overall dissimilarity contribution of 32.94%, and only seven accounted for a total dissimilarity contribution above 55%. They comprised (i) the two main sesquiterpene hydrocarbons, β-caryophyllene and α-humulene, (ii) four oxygenated sesquiterpenes, caryophyllene oxide, 14-hydroxy-9-epi-(E)-caryophyllene, and the two isomers of caryophylla-4(14),8(15)-dien-5-ol, and (iii) one cannabinoid, cannabidiol: all the selected chemicals had significant differences based on the genotypes, year of cultivation, and their interaction.

The essential oil extraction yield ranged from 0.03 to 0.12% w/w for the samples collected in 2019, and from 0.03 to 0.23% w/w for the 2020 samples, where the highest yield was comparable to those already published in the literature [20]. A statistically significant difference in percentage was found among genotypes, year of cultivation, and the interaction between genotype and year (Table 4). Regarding the dependence of the EO extraction yield on the hemp genotype, several studies are present in the literature [18,19,22,28,31], while no references are available for its dependence on the year of cultivation.

For the 2019 samples, the significantly highest EO extraction yield was obtained by Futura 75 (0.12%), followed by Carmagnola (0.09%) and Eletta Campana (0.09%), while the lowest (0.03%) from Bernabeo, Felina 32 and Uso-31. Regarding the 2020 samples, instead, Codimono, followed by Eletta Campana > CS > Fibrante (0.23% > 0.20% > 0.20% > 0.16%, respectively), were the most productive cultivars, whilst Uso-31 and Bernabeo were the least productive ones (0.03% and 0.05%, respectively). For Uso-31 (chemotype V), whose inflorescences are characterized by a negligible level of cannabinoids, the lowest EO extraction yield was found irrespective of the year. A positive correlation between the accumulation of total cannabinoids and total terpenes, both synthesized in glandular trichomes [22], was already reported [32,33,34], and explains the low EO yield obtained for Uso-31. Considering both the years of cultivation, all the 2020 samples were characterized by a significantly higher EO extraction yield than the 2019 ones (Table 2 and Table 3), and this could be at least partly due to the different meteorological conditions occurring in the two subsequent growing seasons, particularly regarding rainfalls and average temperature (Table 5). Higher temperatures might, indeed, cause a higher spontaneous evaporation degree of the EO from the plant trichomes.