Introduction

Stored products are attacked by the remarkable numbers of 1663 insect and 280 mite species (Hagstrum et al. 2013) while grain losses worldwide reach 10–20% due to insect infestations (Islam et al. 2021). The damages of food commodities, in terms of quality and quantity, caused by stored-product pests consist a major issue for food-related industries (Rajendran and Sriranjini 2008). Also, the existence of pests in stored products is linked to the elevated risk of food safety and public health (e.g., spread of pathogens, production of allergens) making their effective management challenging (Hill 2003; Mason and McDonough 2012; Djekic et al. 2019).

Τhe yellow mealworm beetle, Tenebrio molitor L. (Coleoptera: Tenebrionidae) infests a wide spectrum of food materials having plant and animal origin (Hill 2003; Robinson 2005; Hagstrum et al. 2013; Guo et al. 2014). The detoxifying cytochrome P450 enzymes of this species have been reported of markedly high activity, leading to reduced pesticide sensitivity (Pedersen et al. 2020). Previous reports have documented that T. molitor is tolerant to contact insecticides such as alpha-cypermethrin, thiamethoxam, diatomaceous earths (DEs), deltamethrin, and spinosad (Trewin and Reichmuth 1997; Mewis and Ulrichs 2001; Athanassiou et al. 2015; Kavallieratos et al. 2019b). However, the efficacy of insecticides against T. molitor is regulated by abiotic (temperature, relative humidity (RH)) and biotic factors (life stage, type of treated grain commodity) (Kavallieratos et al. 2019b, 2021).

The khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae), is one of the 100 most important invasive species worldwide and it has been recently reported from Greece (Lowe et al. 2000; Athanassiou et al. 2015). This beetle affects a wide range of stored products, survives in extreme abiotic conditions, and is considered one of the most harmful insect pests of stored agricultural products and foodstuffs (Lindgren et al. 1955; Hill 2003; Athanassiou et al. 2016; Kavallieratos et al. 2017b, 2019a; Papanikolaou et al. 2019). The fact that larvae of this species undergo diapause for several years has favored its widespread in several countries of Africa, Asia, and Europe (Aitken 1975; Myers and Hagstrum 2012; Athanassiou et al. 2016, 2019; EPPO 2019). Recent studies have revealed that the management of T. granarium meets certain difficulties due to elevated tolerance to several contact insecticides as surface treatments or grain protectants (e.g., alpha-cypermethrin, beta-cyfluthrin, chlorfenapyr, cypermethrin, deltamethrin, DE, pyriproxyfen, spinosad, thiamethoxam) (Athanassiou et al. 2015, Kavallieratos et al. 2016, 2017a; Ghimire et al. 2017).

The Common Agricultural Policy has highlighted the environmental, food safety, and animal welfare standards and proposed the use of alternatives for the management of stored-product insects (Schillhorn van Veen 1999). The non-synthetic plant protection products are novel effective tools that have the potential to be used alone or in mixtures with synthetic insecticides in the storage environment (Athanassiou et al. 2014). Edible biopesticides of botanical origin may be considered low-risk alternatives according to EC 1107/2009 Articles 22, 47 (EUR Lex 2009), necessitating a smaller experimental dataset for registration purposes thus lower regulatory barrier. Plant secondary metabolites with insecticidal activities (Isman 2006, 2008) are usually selected from libraries of thousands of compounds, originating in biodiversity hotspots like the Mediterranean Basin, to be developed individually into plant protection products. Crude botanicals, representing complex clusters of plant secondary metabolites, may have equally significant or even better plant protection properties, a selective mode of action and help to avoid the emergence of resistant strains of pest species due to the wide variety of secondary metabolites (Isman 2006; Gupta and Birdi 2017; Ntalli and Caboni 2017). Natural botanicals exhibit repellent, antifeedant, sterilizing, ovicidal, or toxic effects on insects (Isman 2006). As members of the botanical insecticides, essential oils (EOs) exhibit a significant range of pesticidal activities (Ntalli et al. 2010a, 2011; Benelli and Pavela 2018a, b; Benelli et al. 2019; Kavallieratos et al. 2020b; Pavela et al. 2019, 2020) and can be prepared in a “green”, easy, and cost-effective way, not employing organic solvents or sophisticated extraction procedures. Among the EO components, the monoterpenes have drawn the greatest attention (Coats et al. 1991). Considering the mode of action of monoterpenes, Houghton et al. (2006) proved that they inhibit acetylcholinesterase (AChE) enzyme activity, although in several cases, there was a lack of direct correlation between insect toxicity and AChE inhibition (Lee et al. 2001; López and Pascual Villalobos 2010, 2014). Monoterpenes have been also reported to affect the cytochrome P4502B1-dependent enzymes in rats that are involved in the activation of genotoxic substances (De Oliveira et al. 1997), as well as to inhibit the α-amylase enzyme activity in the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) (Huang et al. 1999).

The evaluation of the toxicity of various botanical compounds against the stored-product insects has been mainly conducted by applying them as fumigants. For example, according to Regnault Roger and Hamraoui (1995) the oxygenated monoterpenes are more toxic compounds than the non-oxygenated monoterpenes as fumigants against the bean weevil, Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae). In another fumigation assay, Lee et al. (2003) reported that all adults of the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), T. castaneum, and the sawtoothed grain beetle, Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) died when exposed to cineole, l-fenchone and pulegone at 50 mg/ml air 14 h post-exposure. Similarly, Erler (2005) postulated that vapors of γ-terpinene and terpinen-4-ol provided 100% mortality to adults of the confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) at 92.4 and 184.8 mg/l air after 4 days of exposure. The latter concentration had the same effect on the larvae of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). The vapors of carvone at 972 ppm and trans-anethole at 880 ppm killed all exposed adults of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) 1 day post-exposure (López et al. 2008). The use of DL-camphor or estragole as fumigants in combination with low temperature (19 °C) and low pressure (50 mm Hg) successfully controlled the diapausing larvae of the Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) (Mbata et al. 2012). Mbata and Payton (2013) reported that there was variable susceptibility among different life stages of the cowpea weevil, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae) when fumigated with the monoterpenes trans-anethole, estragole, S-carvone, L-fenchone, geraniol, γ-terpinene, and DL-camphor. However, there is little knowledge regarding the use of botanical substances as grain protectants (Islam et al. 2010; Mbata et al. 2012; Osman et al. 2016; Kavallieratos et al. 2020a,b; Pavela et al. 2020) and a paucity of information concerning other than EO originating natural molecules against stored-products insects. Herein, we tested botanical molecules belonging to different chemical groups to delineate for insecticidal activity, while they have already been proved of significant nematicidal activity in the recent years. In specific, we study trans-anethole (phenolic monoterpene) and 2-undecanone (dialkyl ketone) representing EO ingredients (Ntalli et al. 2010a, 2011); (E)-2-decenal (dec-2-enal) and (E, E)-2,4-decadienal (polyunsaturated fatty aldehydes) constituting Ailanthus altissima (Mill.) Swingle (Sapindales: Simaroubaceae) organic extracts (Caboni et al. 2012; Ntalli et al. 2016a); and furfural (aldehyde) and acetic acid (monocarboxylic acid) as ingredients of the fruits of Melia azedarach L. (Sapindales: Meliaceae) (Ntalli et al. 2010b; Caboni et al. 2012; Ntalli et al. 2020). Scanning and transmission electron microscopy studies documented that the acetic acid, (E)-2-decenal, and 2-undecanone provoke irreversible ultrastructural modifications on Meloidogyne incognita (Kofold & White) Chitwood (Rhabditida: Meloidogynidae) J2 (Ntalli et al. 2016b). Interestingly, the insecticidal efficacy of trans-anethole, 2-undecanone, and furfural has been already reported on other insects. In specific, through contact toxicity trials: e.g., trans-anethole vs. T. castaneum (Mondal and Khalequzzaman 2010), 2-undecanone vs. Aedes albopictus (Skuse) (Diptera: Culicidae) (Liu et al. 2014), furfural vs. S. oryzae and the cigarette beetle, Lasioderma serricorne (F.) (Coleoptera: Ptinidae) (Urrutia et al. 2021). To the best of our knowledge, no data exist to date on the activity of the aforementioned molecules for the management of T. granarium and T. molitor. Thus, the objective of the present study was to evaluate the lethal activity of 2-undecanone, acetic acid, trans-anethole, furfural, (E)-2-decenal, and (E, E)-2,4-decadienal as wheat protectants against larvae and adults of T. granarium and T. molitor.

Materials and methods

Commodity

Hard wheat, Triticum durum Desf. (var. Claudio), free from pests and pesticides, was used in the trials. Wheat was sieved to remove impurities and kept at subzero temperatures for several months. Prior to experimentation, it was warmed under room temperature. Its moisture content was 12.6% as determined by a moisture meter (mini GAC plus, DICKEY-john Europe S.A.S., Colombes, France).

Insects

The insects used in the trials were obtained from colonies that are kept in the Laboratory of Agricultural Zoology and Entomology, Agricultural University of Athens, since 2014. The founding individuals have been collected from Greek storage facilities. We used unsexed adults of T. granarium, < 24 h old; small larvae of T. granarium < 3 mm long (Kavallieratos and Boukouvala 2019); unsexed adults of T. molitor, < 2 weeks old; and small larvae of T. molitor < 10 mm long (Kavallieratos et al. 2019b). Trogoderma granarium was cultured on whole wheat, at 30 °C and 65% RH in continuous darkness (Kavallieratos and Boukouvala 2019). Tenebrio molitor was reared on oat bran with slices of potatoes as a source of moisture (De Vosjoli 2007), at 30 °C and 65% RH in continuous darkness (Kavallieratos et al. 2019b).

Test compounds

The compounds 2-undecanone, acetic acid, trans-anethole, furfural, (E)-2-decenal, and (E, E)-2,4-decadienal (Fig. 1), all over 98% purity, were obtained from Sigma–Aldrich (Buchs, Switzerland).

Fig. 1
figure 1

Test plant secondary metabolites against T. molitor and T. granarium larvae and adults

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Bioassays

Each test substance was tested at 1000 μl/kg and 500 μl/kg wheat. The test concentrations were selected on the basis of preliminary efficacy tests on both insect species. For the test concentration of 1000 μl/kg wheat, a volume of 250 μl of test substance was dissolved in 6 ml pure ethanol and the solution was applied on 0.25 kg wheat. Similarly, 125 μl of test substance were dissolved in 6 ml pure alcohol to treat 0.25 kg wheat, representing the test concentration of 500 μl/kg wheat. Spraying was performed with the airbrush AG-4 (Mecafer S.A., Valence, France) on wheat lots that were laid out on different trays. Additional lots of 0.25 kg wheat treated with same volumes (6 ml) of (a) pure ethanol and (b) water served as controls. After spraying the lots of wheat were transferred separately in 1-l glass containers and were shaken manually for 10 min to achieve equal distribution of test solution on the entire wheat mass. Three samples of 10 g each were obtained per treated lot or control and placed inside small glass vials (7.5 × 12.5 cm diameter and height) with a different scoop. A Precisa XB3200D (Alpha Analytical Instruments, Gerakas, Greece) compact balance was used to weigh the portions of 10 g of wheat on a thin layer. A new layer was used for each weighing. The lid of each vial bore a 1.5-cm diameter hole in the center covered with muslin so as to permit adequate ventilation. The escape of insects was prohibited by coating the upper internal parts of the vials with polytetrafluoroethylene (60 wt % dispersion in water) (Sigma-Aldrich, Chemie GmbH, Taufkirchen, Germany). Coating was conducted with a swap, 24 h before the placement of individuals in the vials. Subsequently, 10 adults of T. granarium or 10 larvae of T. granarium or 10 adults of T. molitor or 10 larvae of T. molitor were separately placed inside each vial. Next, all vials were transferred in incubators set at 30 °C and 65% RH. Mortality of adults or larvae was evaluated under an Olympus stereomicroscope (SZX9, Bacacos S.A., Athens, Greece) at × 57 total magnification by pushing gently each individual with a brush (Cotman 111 No 000, Winsor and Newton, London, UK) to inspect movements after 4, 8, and 16 h and 1, 2, 3, 4, 5, 6, and 7 days of exposure. Different brushes were used for each treatment and controls. All tests were repeated three times for test substances and controls, by preparing new wheat lots and new vials each time.

Statistical analysis

Mortality in the controls, pure alcohol and water, was low (< 5%) for all tested species and life stages. Therefore, no correction was necessary for the mortality values. Before conducting analyses, the mortality data were log (x + 1) transformed to normalize variance (Zar 2014; Scheff and Arthur 2018). Statistical analyses were carried out separately for each tested species and life stage by following the repeated measures model (Sall et al. 2001). The repeated factor was the exposure interval while mortality was the response variable. Concentration and compound were the main effects. The associated interactions of the main effects were considered during analysis. The JMP 14 software (SAS Institute 2018) was used to analyze all data. Means were separated by the Tukey-Kramer honestly significant difference (HSD) test at the 0.05 probability (Sokal and Rohlf 1995).

Results

Effectiveness against T. molitor

Between and within exposure intervals, all main effects and related interactions were significant (P < 0.05) for both T. molitor adults and larvae, except for the interaction exposure × compound × concentration for T. molitor larvae which was not significant (Table 1). Concerning T. molitor adults, furfural was the most effective compound at 500 μl/kg wheat, followed by trans-anethole causing 41.1% and 21.1% mortalities, respectively, after 7 days of exposure (Table 2). The overall mortality provided by the other compounds did not exceed 13.3%. (E, E)-2,4-decadienal was the least effective compound for both concentrations since it killed 5.6% and 41.1% of the exposed adults at 500 μl/kg and 1000 μl/kg wheat respectively. After 3 days of exposure, 2-undecanone caused 60.0% mortality at 1000 μl/kg wheat, while it reached 98.9% 7 days post-exposure. The compound acetic acid and furfural provided 94.4% and 92.2% mortalities after 7 days of exposure respectively. Mortality caused by trans-anethole and (E)-2-decenal reached the moderate levels of 64.4% and 56.7%, respectively, after 7 days of exposure.

Table 1 MANOVA parameters about the main effects and associated interactions for mortality levels of T. molitor and T. granarium adults and larvae between and within exposure intervals (in all cases error df = 96)
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Table 2 Mean mortality (% ± SE) of T. molitor adults over selected exposure intervals on wheat, treated with 2-undecanone, acetic acid, trans-anethole, furfural, (E)-2-decenal, and (E, E)-2,4-decadienal
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Regarding T. molitor larvae, the overall mortality at 500 μl/kg wheat did not exceed 47.8% after 7 days of exposure for 2-undecanone and (E)-2-decenal (Table 3). The compound acetic acid killed 7.8% while both trans-anethole and furfural killed 11.1% of the exposed larvae at 500 μl/kg wheat 7 days post-exposure. At 1000 μl/kg wheat, furfural provided the highest larval mortality (i.e., 66.7%) after 5 days of exposure, while 2-undecanone and (E)-2-decenal caused the highest mortalities to larvae (i.e., 87.8% and 80.0% respectively) 7 days post-exposure. In contrast, the lowest mortality (i.e., 16.7%) was caused by trans-anethole after 7 days of exposure at 1000 μl/kg wheat.

Table 3 Mean mortality (% ± SE) of T. molitor larvae over selected exposure intervals on wheat, treated with 2-undecanone, acetic acid, trans-anethole, furfural, (E)-2-decenal, and (E, E)-2,4-decadienal
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Effectiveness against T. granarium

Between and within exposure intervals, all main effects and related interactions were significant (P < 0.05), for both T. granarium adults and larvae, except the interaction compound × concentration for T. granarium adults which was not significant (Table 1). Concerning T. granarium adults, > 95% of the exposed individuals were dead on wheat treated with (E)-2-decenal 5 days post-exposure at 500 μl/kg wheat. Complete (100%) mortality of T. granarium adults was reached by (E)-2-decenal after 7 days of exposure at 500 μl/kg wheat or after 5 days at 1000 μl/kg wheat. Similarly, acetic acid killed all adults 7 days post-exposure at 1000 μl/kg wheat (Table 4). (E, E)-2,4-decadienal killed the same percentage of adults (i.e., 94.4%) after 7 days of exposure at both concentrations. 2-undecanone provided 94.4% mortality to T. granarium adults 6 days post-exposure at 1000 μl/kg wheat. The mortalities caused by all other compounds ranged between 64.4 and 81.1% at 500 μl/kg wheat, or between 82.2 and 92.2% at 1000 μl/kg wheat after 7 days of exposure.

Table 4 Mean mortality (% ± SE) of T. granarium adults over selected exposure intervals on wheat, treated with 2-undecanone, acetic acid, trans-anethole, furfural, (E)-2-decenal, and (E, E)-2,4-decadienal
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As far as T. granarium larvae are concerned, no compound was lethal to all individuals even after 7 days of exposure at 500 μl/kg wheat (Table 5). (E)-2-decenal was the most effective tested compound at 500 μl/kg wheat since it provided 87.8% mortality at the end of the experiment followed by (E, E)-2,4-decadienal which caused 80.0% mortality. At 1000 μl/kg wheat, (E, E)-2,4-decadienal killed 90.0% of the larvae on wheat after only 2 days of exposure, while all individuals were dead 4 days post-exposure. (E)-2-decenal was also highly effective because it killed 94.4% of larvae after 4 days of exposure. Two days later, all exposed larvae were dead on wheat treated with (E)-2-decenal at 1000 μl/kg wheat. 2-undecanone, acetic acid, and furfural provided moderate mortalities, ranging between 11.1 and 46.7% at 500 μl/kg wheat or between 54.4 and 76.7% at 1000 μl/kg wheat 7 days post-exposure. The compound trans-anethole caused the lowest mortality scoring 8.9% at 500 μl/kg wheat and 28.9% at 1000 μl/kg wheat after 7 days of exposure.

Table 5 Mean mortality (% ± SE) of T. granarium larvae over selected exposure intervals on wheat, treated with 2-undecanone, acetic acid, trans-anethole, furfural, (E)-2-decenal, and (E, E)-2,4-decadienal
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Discussion

The published information on the potential of substances of botanic origin as effective grain protectants is sporadic. For instance, the binary combinations of the DE SilicoSec with cinnamaldehyde or eugenol on wheat exhibited synergistic activity by causing higher mortalities to adults of S. oryzae than the DE or the monoterpene alone (Islam et al. 2010). Mbata and Payton (2013) showed that mated female individuals of C. maculatus did not lay eggs οn beans that had been treated with E-anethole, estragole, S-carvone, L-fenchome, geraniol, γ-terpinene, and DL-camphor. The exposure of the 4th instar larvae of T. granarium on wheat treated with 0.5 ml diluted caraway oil οr 2 ml diluted carvone for 2 days led to serious histological decays of their midgut. Also, the midgut and the ovarioles of the emerged adult individuals suffered several histological abnormalities (Osman et al. 2016). Recently, Kavallieratos et al. (2020a) found that the sesquiterpene isofuranodiene is highly effective against adults of T. granarium after 3 days of exposure at 1000 μl/kg wheat.

According to our results, the lethality of T. molitor adults, after exposure to 1000 μl/kg wheat of test substances for 7 days, ranged between 56.7 and 98.9%. Regarding T. molitor larvae, the assessed lethality was between 16.7 and 87.8%. This fact demonstrates that adults are more susceptible to the test compounds than larvae. Previous studies have documented that the adult stage of T. molitor is more susceptible than larval stage to several contact insecticides as grain protectants. For instance, Kavallieratos et al. (2019b) reported that deltamethrin, pirimiphos-methyl, SilicoSec, and spinosad killed more adults than small or large larvae on wheat, barley, and maize. Recently, Kavallieratos et al. (2021) documented that pirimiphos-methyl provided significantly higher mortality to T. molitor adults than small and large larvae under different combinations of temperature (20, 25, 30 and 35 °C) and RH levels (55 and 75%) on stored barley. In contrast, the monoterpenes eugenol, caryophyllene oxide, α-humulene, α-phellandrene, and α-pinene were more lethal to larvae than adults of T. molitor in toxicity tests 2 days post-exposure (Martínez et al. 2018). The authors hypothesized that differences in penetration and metabolization of the compounds into the bodies of adults and larvae could explain the different levels of the observed toxicity. Whether the monoterpenes tested by Martínez et al. (2018) remain more lethal to larvae than adults if applied as grain protectants merits further investigation. The method of application, i.e., application of the molecules directly as liquids on the thorax, application via fumigation, and application on grains, may affect insects differently. In fact, according to our results, the monoterpene trans-anethole was more toxic to adults than larvae as wheat protectant. In the same frame, the pyrethroid insecticides exhibit different performance towards adults and larvae according to the method of application, i.e., surface treatment vs. grain protectants (Athanassiou et al. 2015, Kavallieratos et al. 2019b, 2021). The problem of performance of insecticides and substances that exhibit insecticidal properties within the developmental stage of T. molitor could be faced up with the combination of compounds that exhibit elevated mortality levels to movable stages of this species which are responsible for the infestations of stored products. Further experimentation is needed to clarify this issue.

Our study shows that the overall mortality of T. granamum adults ranged between 82.2 and 100% at 1000 μl/kg wheat after 7 days of exposure. In fact, (E)-2-decenal killed all exposed adults and larvae 6 days post-exposure. This is one of the most important findings since it is well documented that the larva is the most harmful stage of T. granarium due to the rapid population growth on numerous grains (Athanassiou et al. 2016; Kavallieratos et al. 2017b) or non-grain commodities (Kavallieratos et al. 2019a). Furthermore, larvae, contrary to adults, are tolerant to several insecticides of chemical or natural origin either as grain protectants (i.e., cypermethrin, deltamethrin, pirimiphos-methyl, SilicoSec, s-methoprene, spinosad) (Kavallieratos et al. 2017a) or as surface treatments (i.e., alpha-cypermethrin, deltamethrin, thiamethoxam) (Athanassiou et al. 2015; Kavallieratos and Boukouvala 2018). So far, only few chemical insecticides have provided elevated management of T. granarium larvae and adults such as chlorfenapyr, pirimiphos-methyl and a mixture of acetamiprid plus d-tetramethrin plus piperonyl butoxide (Kavallieratos et al. 2017a, Kavallieratos and Boukouvala 2018, 2019). Surprisingly, there is limited knowledge regarding the efficacy of botanicals as effective grain protectants against T. granarium. For example, a recent study Kavallieratos et al. (2020a) showed that although isofuranodiene provided > 96% mortality to T. granarium adults, it killed < 38% of the exposed larvae 7 days post-exposure. However, similar findings to the current study have been reported by Kavallieratos et al. (2020b) who found that the EO of Mentha longifolia (L.) Huds. (Lamiales: Lamiaceae) completely (100%) suppressed larvae and adults of T. granarium after 2 days and 16 h of exposure, respectively, at 1000 μl/kg wheat. In the same study, the EO of Dysphania ambrosioides (L.) Mosyakin and Clemants (Caryophyllales: Amaranthaceae) was also highly effective by killing all adults of T. granarium and > 95% of larvae at the same concentration 2 and 4 days post-treatment. Therefore, it becomes evident that botanicals, either as single compounds or as constituents of EOs, are effective agents against this species and compatible to synthetic insecticides.

The tested substances exhibit a safe profile within mammalian systems, an issue that may enable them to be endorsed as components of a biorational strategy in the frame of integrated management of infestations and losses of stored food commodities. In specific, furfural, trans-anethole, 2-undecanone, and (E)-2-decenal are EU Food Improvement Agents (EUR Lex 2012) as well as food additives Generally Recognized as Safe (GRAS) in the USΑ (USDHHS 2021). 2-undecanone is a natural flavor ingredient that is not genotoxic while it does not meet safety issues regarding sensitization of the skin (Api et al. 2019). The compound acetic acid is a normal body metabolite in mammals and can be found naturally in a wide variety of foods. It is frequently used as a preservative and its commonest use is as vinegar (Pravasi 2014). (E, E)-2,4-decadienal is used as a synthetic flavoring and fragrance material. Toxicity studies on certain strains of male and female rats and mice determined the no-observed-adverse-effect level at 100 mg/kg body weight. Moreover, (E, E)-2,4-decadienal was not mutagenic in vitro or in vivo (Chan 2011). (E, E)-2,4-decadienal and (E)-2-decenal are safe at maximum use level of 5 mg/kg feed for all animal species. No safety concern would arise for the consumer from the use of these compounds up to the highest safe levels in feed (Bampidis et al. 2019).

Our study gives the first insight into the use of 2-undecanone, acetic acid, trans-anethole, furfural, (E)-2-decenal, and (E, E)-2,4-decadienal as possible natural insecticides against different developmental stages of T. molitor and T. granarium infesting stored wheat. Among these substances, 2-undecanone was the most effective against T. molitor adults and larvae while (E)-2-decenal was 100% lethal for both life stages of T. granarium followed by (E, E)-2,4-decadienal. Additional studies are necessary to clarify the spectrum of efficacy of the tested substances by including more stored-product insect species and food commodities under different levels of temperature and RH levels. Currently, we are in the process of testing the toxicity of mixtures of the compounds to demonstrate any synergic, antagonistic, or additive effects, along with the ultrastructural deformations on stored-product insects.