New Nano-Biopesticide Formulation of Mentha piperita L. (Lamiaceae) Essential Oil Against Stored Product Red Flour Beetle Tribolium castaneum Herbs and its Effect on Storage

In recent years, providing new formulations such as nano-emulsions have been widely used for the target delivery, and enhanced biological functions of pesticides combinations. In this study, contact toxicity of Mentha piperita L. essential oil compared with its nano-emulsion on Tribolium castaneum herbs as well as its effect on wheat grain storage has been investigated. The results indicated that, production of nano-emulsion with this new technique results in considerable decrease of the required EO concentrations. The results concluded that by using nanoemulsion formulation, the effect of essential oil contact toxicity and its durability increases. Hence, the nanoemulsion formulation may represent a new category of biopesticide and this should be considered in the integrated pest management program.


Introduction
Nano-emulsion formulation system is a recent favorable method to improve botanical insecticide characteristics and effectiveness for commercial use [1]. Nano-emulsion formulations have good storage stability under a broad range of temperatures (-10 to 55°C). In nanoemulsion formulation system, the biological performance of the pesticides is improved by using adjuvants and surfactants [2]. Nonionic surfactants are considered safe to be used in chemical, cosmetic, food, and pharmaceutical industries [3]. Formulating essential oils (EOs) into nano-emulsion can be used in food and beverage products, thereby, decreasing the amount of EOs required [4].
Nano-emulsions can be formulated by two kinds of methods such as high-energy and low-energy emulsification methods. High-energy emulsification method comprises high-pressure homogenization and ultra-sonication [5][6][7]. Ultra-sonication is the most widely used method because it is economical and easy to use. Recent studies have shown that the ultrasound technique has developed as a powerful energy-efficient tool for the emulsification process. Ultrasonic emulsification provides reduced droplet diameter and narrow size distribution as compared to other mechanical devices [8,9].
Currently, new trend is started for using the natural plant extracts as well as EOs as natural pesticides to control pests with nanoformulation [1,10] in green pest management [11]. These green and nano-pesticides are safe, low to no mammalian toxicity and have many sites of toxic actions in pests, which lead to high selectivity and low resistance development [12,13]. Essential oils proved to have significant effects against a large number of stored grain insects, acting through ingestion [14] and by contact as well [15,16].
The red flour beetle, Tribolium castaneum (Herbst) is a globally distributed crop pest, infesting a wide variety of stored products worldwide [17]. It is considered a primary pest of flour and other milled products of cereals, and a secondary pest of stored wheat, causing severe damages to these food grains [18,19]. Indeed, heavy losses of grain weight and nutritive contents, reduction of commercial value and frequency of germination were resulted from this insect pest [20].
Many plants may provide potential alternatives to currently used insect-control agents because they constitute a rich source of bioactive chemicals [21], in which active substances extracted from plants may not only act as toxicants, but also as insect growth regulators, repellents, synergists or as phagodeterrents. These substances include a large group of the so-called essential oils (EOS). Essential oils (EOS) are defined as any volatile oils that have strong aromatic components and give a distinctive odor or flavor to a plant [22]. Biopesticides based on essential oils (EOS) appear to be complementary or alternative method in crop production and integrated pest management [23].
The essential oils extracted from M. piperita have also been reported as a source of botanical insecticides [24]. Efficiency of volatile oils derived from M. piperata, Mentha arvensis, M. spicata and Cymbopogon nardus on Adult C. maculatus (F.) were introduced into cowpea seeds.
The toxicity of essential oils to stored-product insects is influenced by the chemical composition of the oil, which in turn depends on the Our study evaluates the efficiency of M. piperita (EO nanoemulsion) in water was formulated using nonionic surfactants such as Tween® 80 by ultrasonic emulsification method, at several concentrations comparing with M. piperita (EO) as possible protecting agents of wheat grains against infestation by the red flour beetle Tribolium castaneum herbs without negative effect on germination of wheat grains. Which may lead to the discovery of new agents for pest control, it may be an effective alternative to conventional synthetic insecticides and encourage the use of these natural oils in nanoemulsion and participate in programs of integrated pest management (IPM).

Compounds
The Peppermint essential oil extraction was used in this study. The non-ionic surfactants Tween 80 (Polysorbate 80) nonionic surfactant was purchased from Al-Nasr Pharmaceutical Chemicals Co. (ADWIC) (Egypt). Acetone was purchased from Al-Nasr Pharmaceutical Chemicals Co. (ADWIC) (Egypt).

Nano-emulsion preparation
The oil-in-water nano-emulsion was formulated using peppermint essential oil, non-ionic surfactant (tween 80) and deionized water. The concentration of M. piperita (EO) (4%, v/v). The preparation method was used as described in a previous study of Massoud et al. [29].

Bioassay technique
Contact toxicity bioassay using thin film residue: The insecticidal efficacy of the M. piperita free EO and its formulation (nano-emulsion) was carried out by residual film technique against the adults of T. castaneum. The residual film was done directly on petri dish (9 cm) without any grains according to Qi and Burkholder [30] Broussalis et al. [31].
M. piperita free EO and its formulation (nano-emulsion) concentrations were (10, 15 and 30 µl) to T. castaneum every concentration were mixed with 1 ml acetone by using micropipette, for each concentration in petri-dish. The dilutions were spread uniformly along the whole surface of the petri dishes. The solvent was allowed to evaporate leaving thin film on the floor of the dishes. After evaporation of acetone, 20 adults were placed in each petri-dish.
Three replicates were carried out of each concentration and control. Mortality percentages were recorded after 24, 48, and 72 hours of treatment and LC 50 values were calculated according to Finney [32]. The toxicity index (TI) of nano-emulsion or free EO calculated by the equation according to Sun, [33] and Yamamoto et al., [34]: • Toxicity index (TI)=(LC 50 of nano-emulsion or EO)/(LC 50 of EO). • Based on LC 50 calculated after 72 hrs.
• The compound has TI less than 1 (TI<1) have high toxicity.
Sixty gram of grains were used in each concentration and then divided into three equal replicates in 0.4 Litter glass jars. After evaporation of acetone, the treated grains were infested by newly emerged adults (10 pairs). Mortality was recorded every week for two weeks. The number of progeny was recorded after six weeks of infestation. Notably, it has been previously elucidated that when the solvent evaporates, the nano-emulsion retains its properties [35].
Germination tests: Germination test were done for the treated wheat grains with M. piperita free EO and its formulation (EO nanoemulsion), after one month of storage, accomplished for wheat according to Qi and Burkholder [30] with slight modification. Sixty seeds from each treatment with essential oils were divided into three replicates, placed on petri dishes containing cotton layer instead of (filter paper) soaked with tap water and covered with tissue paper. Germinant seed were recorded after 4 days for wheat. The obtained results of germination test were recorded for all treatment and control.

Statistical analysis
The toxicity data was analyzed using probit analysis to estimate the LC 50 (Ldp line). All data were analyzed using one-way ANOVA. Significant differences between treatments were determined using Duncan's test (p<0.05).

Contact toxicity bioassay of the M. piperita free EO and (EO nano-emulsion) against adults of T. castaneum using thin film residue
The insecticidal activity of the nano-emulsion M. piperita and free M. piperita EO was evaluated by direct contact toxicity (Film residue) against T. castaneum. The results presented in Table 1 and Figure 1 indicated that after 24 hr, 48 hr, 72 hr from exposure the lethal concentration LC 50    The toxicity index (TI) for T. castaneum accounted 0.455 based on LC 50 after 72 hr of exposure as show in Table 2 while for free was EO 1.00 as show in Table 1.
However, the mortality in T. castaneum was increased with increasing exposure time and concentration of nano-emulsion or free EO in a concentration-dependent manner. A significant difference (p<0.05) between M. piperita nano-emulsion and free EO at the concentration 10 µl was observed (Table 3). It is observed that M. piperita nano-emulsion was more effective on the adult of T. castaneum as shown in Table 3 after 24 hr of application which caused 45.00% death compared to free EO 11.67% at the same concentration 10 µl. While the concentration of 15 µl caused 50.00% mortality compared to the free EO 31.67% after 24 hr of application. And also conc. 30   The residual effect of M. piperita oil (nano-emulsion and free EO) against the adult of T. castaneum was tested at intervals after 48 h and 72 hr (Table 3). After 48 hr, the calculated data indicated that the nano-emulsion at 10 µl conc. was more efficient showing 51.67% compared with 11.67% mortality for the free oil.
Moreover, the concentrations 15 µl and 30 µl of the nano-emulsion gave 63.33 and 98.33% mortality versus 35.00 and 76.67% mortality for the free oil, respectively. Furthermore, the nano-emulsion showed more residual effect than among the three used concentrations 10, 15 and 30 µl resulting adult mortality of 65.00, 80.00 and 100% compared with 11.67, 38.33 and 80.00% in the free oil, successively.

Efficacy effect M. piperita free EO and EO nano-emulsion upon adults of T. castaneum and wheat grain storage
In this study the mortality of T. castaneum was evaluated throughout 1,2 weeks and subsequently the adult's emergence post treating wheat grain with M. piperita nano-emulsion formulation and the free (EO) of the tested oil (Table 4).

Essential Oil
Conc. µl   Table 4: Effect of M. piperita (EO nano-emulsion) and M. piperita free EO on mortality and emergence of T. castaneum adults.

Mean of Emerged Adults
The results indicated that the highest percentage of T. castaneum adult mortality (100%) was obtained post the exposure to the treated wheat grains with nano-emulsion concentration 800 and 500 µl after one week and two weeks, respectively. Meanwhile, this highest mortalities, 90 and 100% needed high concentrations amounted to 1000 µl and 2000 µl with M. piperita free (EO) after one and two weeks of exposure, consequently. The results in Table 4 showed that significant differences (p<0.05) in the emergence of T. castaneum adult between concentrations of M. piperita free EO, M. piperita nanoemulsion and control after 6 weeks and 3 months.
The number of emerged T. castaneum adult decreased with increasing concentration gradient of the M. piperita nano-emulsion and the free EO. After 6 weeks, concentration 200, 500 and 800 µl of nano-emulsion, gave no adult emergence, while after 3 months concentration 500 and 800 µl gave no adult emergence was observed (Table 4). Meanwhile, free EO concentration 800, 1000 and 2000 µl gave no adult emergence, for 6 weeks and 3 months.

Germination
The effect of nano-emulsion on germination of wheat grains was evaluated at different concentrations (100, 200, 500 and 800 µl) to study the adverse effect response.
The results in Table 5 showed non-signi icant differences (p>0.05) in seed germination between M. piperita nano-emulsion, free EO treatments and control. Results showed that M. piperita nanoemulsion concentrations (100, 200, 500 and 800 µl) gave germination percentages respectively (98.33, 96.67, 95.00 and 93.33%).  Table 5: Germination of wheat grains with M. piperita nano-emulsion and M. piperita free EO after storage one month of treatment.
The germination percentages slightly decreased with increasing the concentration of M. piperita nano-emulsion. These results indicated that the M. piperita nano-emulsion had a slight effect on the germination as compared to the control. While that free EO reduced the germination percentage of wheat grains, especially with high concentrations, compared to the control and/or M. piperita nanoemulsion formulation.

Discussion
In the present study laboratory experiments were carried out to evaluate the effect of M. piperita essential oil and its nano-emulsion formulation against the most common stored grain pest's adult of T. castaneum. Two methods of application (thin film residue and mixing with the grains) were used in this evaluation, and their effect on germination and storage. It is an attempt to control these stored grain pests and avoid the environmental risks and mammalian toxicity arose from chemical insecticides. The results of contact toxicity in thin film residue method using glass Petri dish and contact toxicity using treatment with wheat grains method, showed that with increasing concentration and the exposure time for both insects, increased the mortality agreed with results are similar to those obtained by researches using other essential oils studies [37][38][39][40]. And the effect in a long-term agreed with Sabbour and Abd El-Aziz [41].
They found that oils (Bulk and Nano) had bio-residual efficacy against E. kuehniella moths. The percentage of emerged moths was extremely significantly decreased with Purslane oil in Bulk phase (10%) in comparison with untreated control (57%) during tested storage interval (120 days). While, Purslane oil in Nano phase completely suppressed the percentage of emerged moths (zero%) comparing with untreated control (66%).
Certainly, when the essential oil formulated as a nano-emulsion leads to smaller particle size and increase biological activity due to increased surface area therefore more opportunity of the formulation to come in contact with the target insect. Whereas, the lower mortality caused by M. piperita EO with the biggest particle size indicates that the smaller the particle size, the greater the probability of higher efficacy.
This finding is consistent with the results are similar to those obtained by researches using other essential oils studies of Yang et al. [42]. The nanoparticles and bulk of garlic essential oil were tested against Tribolium castaneum adults. The control efficacy of both nanoparticles and free garlic essential oil were recorded 80% and 11%, respectively. And Anjali et al. [1]; Nenaah [43]; Pant et al. [44]; Sugumar et al. [45]; Abouelkassem et al. [46]; Nenaah et al. [35]; Oliveira et al. [47,48]; Choupanian et al. [49]; Mossa et al. [50]; Choupanian et al. [51] and Louni et al. [52]. The study showed that M. piperita essential oil based nano-emulsion formulations were able to increase the mean mortality rate of T. castaneum compared to the M. piperita EO, in addition to this nano-emulsion method can improve performance of M. piperita oil and maintains its properties in a longterm.
The study has also shown that significantly more effective against adult of T. castaneum with respect to adult mortality and emergence between concentrations of M. piperita free EO, M. piperita nanoemulsion and control after 6 weeks and 3 months. It has been reported that one of the main mechanisms of plant oil action is their ability to penetrate the chorion of eggs via the micropyle and cause the death of developing embryos through sphyxiation [53].
The possible explanation for these results caused considerably higher reductions of adult emergence to smaller particle size and increase biological activity due to increased surface area when the essential oil formulated as a nano-emulsion therefore more opportunity of the formulation to come in contact with the eggs and the higher ability to penetrate into the insect's body. Reduction of adult's emergence may have been achieved through a combination of high mortality of eggs and larvae immediately a ter eclosion and contact with essential oil [54][55][56][57][58].

Conclusion
In our results showed that M. piperita EO had effect on germination of wheat grains, so M. piperita EO was the highest treatment that reduced germination percentages of wheat grains. The international germination threshold required by seed exportation is 90%, so the M. piperita nano-emulsion could be used to protect wheat used as seed.
The obtained results are in line with Zayed, reported that M. piperita nano-emulsion had a slight effect on the germination as compared with M. piperita EO.
However, these results are similar to those obtained by researches using other essential oils found that both nano-emulsion and camphor EO did not show any effect on germination especially at the concentration of LC 50 and LC 90 . Derbalah and Ahmed, found that the spearmint oil was the highest treatment that reduced the germination percentage of wheat grains. Arya and Tiwari, found that mustard oil at 2% concentration clearly reduced the wheat grains germination.