A sum of 54 EO in the form of Essential Oils (EO) have been incorporated in the present study. These EOs and their respective details of taxa of origin, collection, isolation methodology, yield, composition, and bioactivity have been categorized according to the plant tissues origin and were published in three major clusters.

The first focused on forest plants and includes three species of Juniperus of the Cupressaceae Family [29]; J. phoenicea L. (juniper) from which 17 EOs were retrieved (J 01 – J 17), J. drupacea Labill. (Syrian juniper) with 17 EOs (J 18 – J 34) and J. excelsa M.Bieb. (Greek juniper) with 1 EO (J 35).

The second cluster is based on cultivated plants and contains six Citrus species of Rutaceae Family [30]; C. paradisi Macfad. (grapefruit; C 01 – C 04), C. limon (L.) Osbeck (lemon; C 05 – C 08), C. reticulata Blanco (tangerine; C 09 – C 12), C. sinensis (L.) Osbeck (orange; C 13 – C 16), C. japonica Thunb. (kumquat; C 17), and Citrus x auarantium L. (bitter orange; C 18).

The third cluster is focused on culinary herbs and includes eight taxa from various families [24], each contributing one EO apart from tarhan which contributed two. These herbs are Ruta chalepensis L. (rue; V 01), Echinophora tenuifolia ssp. sibthorpiana (Guss.) Tutin (tarhan; V 05 – V 06), Salvia fruticose Mill. (Greek sage; V 09), Thymbra capitata (L.) Cav. (thyme; V 10), Origanum onites L. (oregano; V12), Foeniculum vulgare Mill. (fennel; V 14), Vitex agnus-castus L. (monk’s pepper; V 15) and Satureja thymbra L. (savory; V 16). All EOs along with their metadata are included in Appendix Table 2.

The essential oils of the study were retrieved mostly by hydro distillation (J 01 – J 35, V 01 – V 16, C 04, C 08, C 12, C 16 – C 18), but also through cold press (C 01, C 05, C 09, C 13), and by combination of them (C 02, C 03, C 06, C 07, C 10, C 11, C 14, C 15). This variability of the essential oil retrieval methodology was included to increase the proposed methodology applicability.

The repellent and larvicidal properties of the EOs were assessed against the Asian Tiger Mosquito in bioassays performed uniformly in all EOs [24, 29, 30].

Starting point of the proposed protocol is the selection of the functional unit, a decision of crucial importance for the indicator’s translation. The calculation of the functional unit is based on the available experimental data and incorporates the amount of the EO used for the bioassay and the total area of the experimental application.

The functional unit is calculated for both repellent and larvicidal experimentation, according to Eqn. 1 and expresses the volume of natural product required for the coverage of 1 m${}^2$.

(1)$$\mathrm{FU}=\left({\mathrm{DD}}_{\mathrm{L}}*{10}^4\right)/{\mathrm{A}}_{\mathrm{L}}$$

FU: Functional Unit in mL per m${}^2$.

DD${}_{\text{L}}$: Laboratory discriminative dose in mL.

A${}_{\text{L}}$: Area of Laboratory application in cm${}^2$.

The EO’s spatial yield constitutes a fundamental figure for structuring the proposed methodology. The calculation of this parameter is based on a methodology recently introduced by authors for the estimation of the essential oil annual crop potentials [7], a methodology that is readily applicable for all classes of natural products. A deviation from this approach is applied for Citrus crops since their prevailing cultivation scheme and the respective plantation density were incorporated instead of the estimated 50% of land coverage.

Next element for the construction of final equation refers to the incorporation of the bioactivity exhibited by the investigated EO. Herein, a binary approach was applied to transform the expression of bioactivity as a clear number ranging from 0 to 1.

In this respect, the repellent coefficient was calculated as a clear number in order to provide a net multiplier of 1 for the optimum result (no landings) and escalate downwards as the number of landings increase. The respective indicator is calculated in accordance with the Eqn. 2.

(2)$$\mathrm{REI}={\left(1+{\mathrm{L}}_{\mathrm{e}}\right)}^{-1}$$

REI: Repellent Efficacy Index,

L${}_{\text{e}}$: Number of Landings.

Respectively the larvicidal coefficient was calculated as a single number in order to provide a net multiplier of 1 for the best result (100% mortality) and escalating downwards as the percent of mortality decreases. This indicator is calculated in the terms of Eqn. 3.

(3)$$\mathrm{LEI}={\mathrm{M}}_{\mathrm{e}}*{10}^{-2}$$

LEI: Larvicidal Efficacy Index.

M${}_{\text{e}}$: Experimental Mortality in %.

Finally, all above mentioned figures were incorporated in the calculation of the Land Equivalent Unit indicator. This indicator refers to the annual volume of essential oil production from a land area of 1 hectare and simultaneously incorporates the land use efficacy by each natural product that is evaluated. In specific, this indicator is calculated in the terms of Eqn. 4, reflecting both larvicidal and repellent activities.

(4)$$\mathrm{LEU}=\left({\mathrm{P}}_{\mathrm{NP}}/\mathrm{FU}\right)*{\mathrm{B}}_{\mathrm{CO}}$$

LEU: Land Equivalent Units in m${}^2$ per hectare & year,

P${}_{\text{NP}}$: Spatial yield of natural product in mL per hectare and year.

FU: Functional Unit of Repellent or Larvicidal application in mL per m${}^2$,

B${}_{\text{CO}}$: Bioactivity coefficient, REI or LEI respectively.

The distribution of the 54 EOs in the 5 bioactivity clusters is depicted in Fig. 2. In the high activity cluster are included twenty-two EOs, with fifteen of them exhibiting zero landings.

These last EOs belong to various taxa and include samples originating from tarhan (V 06), oregano (V 12), thyme (V 10), Greek sage (V 09), juniper leaf (J 02, J 05) and berry (J 16, J 17), orange fruit (C 16), Syrian juniper leaves (J 18, J 19, J 24, J 30) and bitter orange (C 18). In this high activity cluster were also included two more EOs presenting REI higher than 0.80. These were the EOs from tarhan (V 06) and juniper leaves (J 10). In the cluster of potent were assigned 5 EOs, with REI 0.80 and originating from Syrian juniper berry (J 21) and leaves (J 25), juniper leaves (J 14), grapefruit (C 04), kumquat (C 17). In the group of moderate activity were included four EOs, with two of them exhibiting REI of 0.53 (tarhan, V 05; Syrian juniper berries, J 23), while the REI of juniper berries (J 11) EO was 0.47 and that from monk’s pepper (V 15) 0.43. Two EOs from the leaves of juniper (J 01, J 15) were found of mild activity exhibiting REI values 0.38 and 0.35 respectively. In the low activity cluster were assigned 26 EOs from which only three presented REI above 0.20 and these were originated from tangerine (C12), fennel (V 14) and Greek juniper (J 35), while all other EOs were assigned REI values close to or lesser to 0.10.

In the present case not all the investigated EOs were evaluated. This happens because limitations in the laboratory scale production of the EOs did not produce adequate quantities for duplicate testing. In specific 10 EOs were omitted and therefore concluding to 44 samples the distribution of which in the 5 bioactivity clusters is depicted in Fig. 3.

In the high activity cluster are included 11 EOs from which the four that exhibited 100% larval toxicity originate from oregano (V 12), thyme (V 10), juniper leaves (J 14) and Syrian juniper leaves (J 24). In the same group were also assigned seven more EOs presenting LEI values above 0.81. These were in LEI descending order: 0.96 savory (V 16) and Syrian juniper leaves (J 25); 0.94 Syrian juniper leaves (J 19, J 26) and orange (C 14); 0.90 juniper leaves (J 15); 0.84 orange (C 15). In the cluster of potent bioactive EOs were included the EOs from fennel (V 14), rue (V 01), kumquat (C 17), lemon (C 05), juniper leaves (J 02), Syrian juniper leaves (J 18), and tarhan (V 06). The EOs that were found to exhibit moderate activity were these from Syrian juniper berries (J 33), orange (C 16, C 13) and tangerine (C 11). In the mild activity cluster were assigned seven EOs and more specifically these from tarhan (V 05), Syrian juniper berries (J 23), lemon (C 06, C 07), grapefruit (C 02), juniper berries (J 11) ans Syrian juniper berries (J 21). Finally, in the inactive cluster were catalogued fifteen EOs of various origin.

Based on the fact the final application of any repellent scopes to protect the exposed human skin, as operational unit for the repellent land footprint was chosen the coverage of the 50% of the average human skin’s surface. Considering that the area of the human skin varies between individuals from 1.5 to 2.0 m${}^2$ an average of 1.75 m${}^2$ was considered for the calculation of the repellent operational unit to 0.875 m${}^2$ [35]. This operational unit was utilised for the transformation of repellent LEU from m${}^2$/ha to number of applications per hectare. Then the number of applications per hectare was transformed to land area per application concluding thus to the land footprint per application for each EO. These results are presented in Fig. 4 and shortly discussed in follow.

The sustainability assessment of repellent usage of EOs, highlighted as best of the best these from savory (V 16), oregano (V 12), tarhan (V 06), Greek sage (V 10), thyme (V 09) and juniper (J 17) exhibiting a land footprint per application less than 10 m${}^2$. All these EOs exhibited REI value of 1, while were also include in the top ten of spatial yield. This group of EOs constitutes the primary target for further research elaborating on the minimum landing inhibition EO concentration which will greatly improve their land footprint. Under this perspective the utility of the proposed methodology maybe expanded beyond the ex-ante assessment and include also more advanced research results. This expansion precludes the replacement of the discriminative dose with the indicated minimum EO concentration, which requires extensive research resources. Therefore, the present assessment is most valuable for the decision of further experimentation and the selection of its subjects. In this respect as most viable targets, maybe considered these from juniper leaf (J 02, J 05) and berry (J 16), orange fruit (C 16), Syrian juniper leaves (J 18, J 19, J 24, J 30) and bitter orange (C 18). In addition, the slope of Fig. 4 indicates that the repellent discriminative dose of the initial screening maybe significantly lowered to highlight bioactivity differences among the various EOs.

On the other hand, another group of EOs that presented land footprint less than 40 m${}^2$ without being among the most efficient and productive in terms of spatial yield were these from Syrian juniper (J 23, J 21), juniper (J 11, J 10, J 19, J 14) and grapefruit (C 04), while in the present group was also included the most productive EO (J 06) exhibiting a land footprint of 34.57 m${}^2$. This former group includes EOs that may be considered as viable targets for complementary research in terms of their production potentials. This group though less sustainable is also of significant importance because of the market characteristics of the end-products in which a series of other values like odour, persistence, allergies, irritation etc. must be considered. It is thus profound that although valuable the proposed methodology it must be complemented by expansion of the research subjects to ameliorate market up-take of preliminary bioprospecting results.

The fundamental assumption for the larvicidal application unit was defined by the large-scale interventions, which in municipal level are mostly focussed on catch-basins treatment. The size of them varies between countries but also in relation to the drainage system grade; primary catch-basins dimensions escalate from 0.25 by 0.25 m to 0.5 by 0.7 m. For the purposes of the study as application unit was selected an intermediate size rectangle catch-basin 0.5 by 0.5 m the surface of which was calculated to 0.25 m${}^2$. Same as for the repellent footprint this application unit was translated in quantity of EO suggested and correspondingly to the respective land footprint. These results are presented in Fig. 5 and shortly discussed in follow.

A first remark on the larvicidal land footprint of the EOs reflects the comparison in between the two uses considered in the present study, which indicates larvicidal use as more sustainable than repellent, since in all the EOs from oregano (V 12), Greek sage (V 10), juniper (J 14) and Syrian juniper (J 24) that presented LEI and REI value of 1, as more sustainable was proven the larvicidal use which in oregano presented a larvicidal land footprint of 1.79 m${}^2$ less than half of the repellent (4.74 m${}^2$). The same land footprint gap in between larvicidal and repellent use was found for the EOs from Greek sage and Syrian juniper in which the repellent use required 2.65 more land than the larvicidal, while the EO from juniper with larvicidal and repellent land footprints of 11.77 vs. 39.03 m${}^2$, required 3.35 more land in the case of the repellent use.

Another significant difference is observed in the bioactivity coefficient of the six more sustainable EOs that presented land footprint less than 10 m${}^2$. In specific, savory (V 16) the more sustainable of the EOs presented LEI value of 0.96, while among the rest fennel’s (V 14) LEI was 0.8, tarhan’s (V 06) 0.66 and rue’s (V 01) 0.75, and only oregano (V 12) and Greek sage (V 10) presented LEI value of 1. These results advocated by the distribution of the EOs larvicidal land footprint distribution presented in Fig. 5 suggest that the larvicidal discriminative dose was more efficient in the bioactivity evaluation of the EOs than the repellent.

The performed ex-ante sustainability assessment results indicated in this case also potential directions and targets for further research. Beside the already mentioned EOs as potent active ingredients deserving more focus were indicated 15 more EOs that presented land footprint less than 20 m${}^2$. Prominent among them were the orange (C 13, C 14, C 15) EOs, which being crude and processed industrial by-products present intriguing aspects encompassing the circular economy and fostering the bio-based industrial solutions. Then the numerous juniper (J 15, J 14, J 06, J 02, J 11) and Syrian juniper (J 23, J 26, J 25, J 18) EOs along with these from tarhan (V 05) and orange fruit (C 16) maybe further evaluated in terms of ecotoxicity along with their land footprint to delineate this crucial characteristic for field application that will be also environmentally neutral.