Mg-LDH Nanoclays Intercalated Fennel and Green Tea Active Ingredient: Field and Laboratory Evaluation of Insecticidal Activities against Culex pipiens and Their Non-Target Organisms

(1) Background: Mosquito control with essential oils is a growing demand. This work evaluated the novel larvicidal and adulticidal activity of fennel and green tea oils and their Layered double hydroxides (LDHs) nanohybrid against Culex pipiens (Cx. pipiens) in both laboratory and field conditions and evaluated their effect against non-target organisms; (2) Methods: Two types of nanoclays, MgAl-LDH and NiAl-LDH were synthesized and characterized using PXRD, TEM and SEM, whereas their elemental analysis was accomplished by SEM-EDX; (3) Results: Mg and Ni LDHs were synthesized by the co-precipitation method. The adsorption and desorption of active ingredients were conducted using LC MS/MS, with reference to the SEM-EXD analysis. The desorption process of MgAl-LDH intercalated green tea oil was conducted using ethanol, and reveled significant peaks related to polyphenols and flavonoids like Vanillin, Catechin, Daidzein, Ellagic acid, Naringenin, Myricetin and Syringic acid with concentrations of 0.76, 0.73, 0.67, 0.59, 0.52, 0.44 and 0.42 μg/g, respectively. The larvicidal LC50 values of fennel oil, Mg-LDH-F, and Ni-LDH-F were 843.88, 451.95, 550.12 ppm, respectively, whereas the corresponding values of green tea were 938.93, 530.46, and 769.94 ppm. The larval reduction percentage of fennel oil and Mg-LDH-F reached 90.1 and 96.2%, 24 h PT and their persistence reached five and seven days PT, respectively. The reduction percentage of green tea oil and Mg-LDH-GT reached 88.00 and 92.01%, 24 h PT and their persistence reached five and six days PT, respectively. Against adults, Mg-LDH-GT and Ni-LDH-GT were less effective than green tea oil as their LC95 values were 5.45, 25.90, and 35.39%, respectively. The reduction in adult density PT with fennel oil, Mg-LDH-F, green tea oil, and Mg-LDH-GT reached 83.1, 100, 77.0, and 99.0%, respectively, 24 h PT and were effective for three days. Mg-LDH-GT and Mg-LDH-F increased the predation Cybister tripunctatus (71% and 69%), respectively; (4) Conclusions: For the first time, Mg-LDH-GT and Mg-LDH-F was the best system loaded with relatively good desorption release to its active ingredients and significantly affected Cx. pipiens larvae and adults in both laboratory and field circumstances, and it could be included in mosquito control.


Introduction
Hydrotalcite and Hydrotalcite-Like Layered double hydroxides (LDHs) nano sheet have attracted significant attention due to their potential application in different domain of photochemistry, catalysis, polymerization, electrochemistry, environmental science and biomedical applications [1][2][3]. LDHs are synthetic anionic nanoclays or hy drotalcite-like structures, consisting of positively charged metal oxide and sheets of hy droxide with intercalated anions and water molecules. For MgAl-LDH, stacked layers o brucite [Mg(OH)2] have some of its divalent cations as Mg 2+ substituted by trivalent cat ions Al 3+ at the centers of the octahedral sites of the hydroxide sheet, in which the verte contains hydroxide anions that are distributed by three octahedral cations and pointe toward the interlayer region. They are represented by the formula: [M 2+ (1−x) + M 3+ x (OH)2] X (A n − )x/n x − ·mH2O, where M 2+ and M 3+ is a divalent cation such as Mg 2+ , Zn 2+ , Ni 2+ and triva lent cation such as Al 3+ , Cr 3+ and Fe 3+ , respectively. Owing to the partial substitutions o M 3+ for M 2+ , the LDHs sheets are positively charged and need to be neutralized by th intercalation of anions (A n− ), like NO3 − , Cl − , CO3 2− or SO4 2− whereas, the x value is calcu lated by the ratio MIII/MII +MIII and is usually between 0.17 and 0.33, however, higher values have also been reported [4,5]. LDHs possess a well-defined layered structur ( Figure 1) with unique properties. The tunable composition of LDH nanoclays are not th only the result of the divalent or trivalent cations being replaced by other cations, as th interlayer anion is easily replaced by another one and, consequently, their chemical an physical properties will be altered [6]. LDHs exhibit unique adsorption characteristic due to their large surface area, thermal stabilities, high adsorption and desorption [7][8][9] Such materials have been widely used as adsorbents for gas molecules [10][11][12][13] and activ ingredients or ions [14][15][16], and catalysts [17][18][19].  [20].
Mosquitoes are serious pests and the most important vectors of human disease a malaria, yellow, encephalitis, West Nile virus, yellow fever and filarial nematodes therefore, mosquito control is a critical demand worldwide, particularly in the tropic  [20].
Mosquitoes are serious pests and the most important vectors of human disease as malaria, yellow, encephalitis, West Nile virus, yellow fever and filarial nematodes; therefore, mosquito control is a critical demand worldwide, particularly in the tropics and warm areas [21]. Culex pipiens (Diptera: Culicidae) is a nuisance mosquito distributed in Egypt and worldwide, transmitting diseases to humans and animals [22][23][24].
Synthetic insecticides and repellents are widely used to control pests, however their overuse, and misuse, resulted in pest resistance, environmental pollution, and health risks to humans and non-target organisms. Consequently, the discovery of natural alternatives to dangerous synthetic pesticides has drawn medical and economic attention worldwide due to their biodegradability, low toxicity, and ability to overcome insecticide resistance [25][26][27].
Essential oils (EOs) are increasingly popular among organic producers and ecologically concerned consumers due to their acceptability in urban environments, homes, and other sensitive locations. Monoterpenes, biogenetically related phenols, and sesquiterpenes are the main components of essential oils [27]. Effectively EOs were used for insect control [28][29][30].
Green tea leaves contain numerous polyphenols, particularly catechins, which make about 30-40% of the extractable solids in dried green tea leaves [31,32]. Besides polyphenols, it also contains proanthocyanidins (tannins), which are one of the allelochemicals produced by Camellia sinensis against insects [33]. Anti-cancer, antibacterial, antiviral, nematocidal, anti-allergic, cardioprotective, and cholesterol-lowering properties are all found in proanthocyanidins, whereas the ingestion of proanthocyanidins through green tea leaves causes harmful effects on the insects by attacking the midgut after breaking them down to free radicals [34]. Foeniculum vulgare oil contains many bioactive terpenoids, flavonoids, steroids, saponins, and tannins that play a role in the death of many insects including Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi mosquitoes [35,36].
This study aimed to evaluate the larvicidal and adulticidal effects of fennel and green tea oils and their Mg and Ni-LDH Nanoclays against Cx. pipiens in vitro and field evaluations, besides testing their efficacy against non-target predators for the first time. O, Sodium Hydroxide NaOH, hydrolyzed ammonium hydroxide (50%) and decarbonated water purchased from Alfa Aesar, Germany. Two essential oils, Foeniculum vulgare and Camellia sinensis were purchased from the EL CAPTAIN Company for extracting natural oils, plants, and cosmetics "Cap Pharm", El Obor, Cairo, Egypt. All chemicals were used without further purification. (2:1) molar ratio was dissolved in 50 mL decarbonated water and placed in a separatory funnel fitted in one gas inlet-outlet three-neck flask RBF apparatus. Then, 100 mL of NH 4 OH (35%) was prepared separately and placed in the second neck of the gas inlet-outlet RBF apparatus. Separately before the addition, both solutions were degassed by purging purified nitrogen gas for 10 min. The mixed salt solution of Mg(NO 3 ) 2 ·6H 2 O and Al(NO 3 ) 3 ·9H 2 O in the separatory funnel was added dropwise to the solution of NH 4 OH, with vigorous stirring (1500 rpm) at room temperature under blanket of nitrogen by passing nitrogen gas throw the gas inlet-outlet RBF. The Stirring continued for 2 h and the pH was kept constant at pH = 10 by adding 5 mL of NH 4 OH (35%) at an interval of half an hour. The white slurry obtained (in case of MgAl-LDH) centrifuged at (10,000 rpm, RT, for 10 min) and washed several times with decarbonized water and re-centrifuged to remove any impurities. The obtained gelatinous slurries were re-suspended in 50 mL decarbonated water and placed in a Teflon-stainless reactor to be heated in the oven for 14 h at 80 • C then the products were separated by centrifugation (as before). Finally, the slurries were dried at 80 • C for 48 h to obtain MgAl-LDH and NiAl-LDH powder ( Figure 2). In synthesis of Ni LDH, Nickel nitrate hexahydrate Ni (NO 3 ) 2 ·6H 2 O (0.5 M) was used instead of magnesium nitrate hexahydrates and green slurry obtained as final product of Ni-LDH. re-suspended in 50 mL decarbonated water and placed in a Teflon-stainless reactor to b heated in the oven for 14 h at 80 °C then the products were separated by centrifugatio (as before). Finally, the slurries were dried at 80 °C for 48 h to obtain MgAl-LDH and NiAl-LDH powder ( Figure 2). In synthesis of Ni LDH, Nickel nitrate hexahydrate N (NO3)2·6H2O (0.5 M) was used instead of magnesium nitrate hexahydrates and gree slurry obtained as final product of Ni-LDH.

Characterization of LDH
Powder X-ray diffraction (PXRD) patterns of LDH were investigated using X, Per PRO Panalytical with Cu Kα radiation (λ = 1.5406 Å). Diffraction patterns were at the 2 θ range (4-80) with a scanning rate of 2.4°/min. Particle morphologies of LDH were ex amined by field transmission electron microscopy (HR-TEM, JSM-7100F); images wer recorded with JEOL JEM-2100-115 high-resolution transmission electron microscopes. I each case, the accelerating voltage was 200 kV. The surface images of nanoparticles wer then recorded using a Quanta FEG 250 scanning electron microscope (FEI Company Hillsboro, OR, USA) at EDRC, DRC, Cairo. Samples were mounted onto SEM stubs. Th applied SEM conditions were: a 10.1 mm working distance, with an in-lens detecto combined with energy-dispersive X-ray spectroscopy (EDX) for the determination of th metal composition.

Adsorption of Active Ingredient Study
The adsorption experiments of the active ingredient of both Foeniculum vulgar (fennel oil) and Camellia sinensis (green tea oil) onto MgAl LDH and NiAl LDH wer employed at room temperature according to oil miscibility (solubility) using the follow ing protocol, About 2 g of M (II) Al-LDH was dispersed in 50 mL proper solvent, water i case of the adsorption of green tea and chloroform in case of the adsorption of fennel oi and placed in a three-neck (RBF) followed by degassing through purging nitrogen gas fo 10 min. Then, 15 mL of well-mixed (5 mL green tea oil + 10 mL deionized water) in case o green tea oil adsorption which is miscible in water while, well-mixed (5 mL of fennel oil 10 mL chloroform) in case of fennel oil adsorption which is immiscible in water and miscible in chloroform was added and stirred gently for 8 h. The resulting slurry wa centrifuged, washed with a small amount of the same solvent, then well dried at 60 °C fo

Characterization of LDH
Powder X-ray diffraction (PXRD) patterns of LDH were investigated using X, Pert PRO Panalytical with Cu Kα radiation (λ = 1.5406 Å). Diffraction patterns were at the 2 θ range (4-80) with a scanning rate of 2.4 • /min. Particle morphologies of LDH were examined by field transmission electron microscopy (HR-TEM, JSM-7100F); images were recorded with JEOL JEM-2100-115 high-resolution transmission electron microscopes. In each case, the accelerating voltage was 200 kV. The surface images of nanoparticles were then recorded using a Quanta FEG 250 scanning electron microscope (FEI Company, Hillsboro, OR, USA) at EDRC, DRC, Cairo. Samples were mounted onto SEM stubs. The applied SEM conditions were: a 10.1 mm working distance, with an in-lens detector combined with energy-dispersive X-ray spectroscopy (EDX) for the determination of the metal composition.

Adsorption of Active Ingredient Study
The adsorption experiments of the active ingredient of both Foeniculum vulgare (fennel oil) and Camellia sinensis (green tea oil) onto MgAl LDH and NiAl LDH were employed at room temperature according to oil miscibility (solubility) using the following protocol, About 2 g of M (II) Al-LDH was dispersed in 50 mL proper solvent, water in case of the adsorption of green tea and chloroform in case of the adsorption of fennel oil, and placed in a three-neck (RBF) followed by degassing through purging nitrogen gas for 10 min. Then, 15 mL of well-mixed (5 mL green tea oil + 10 mL deionized water) in case of green tea oil adsorption which is miscible in water while, well-mixed (5 mL of fennel oil + 10 mL chloroform) in case of fennel oil adsorption which is immiscible in water and miscible in chloroform was added and stirred gently for 8 h. The resulting slurry was centrifuged, washed with a small amount of the same solvent, then well dried at 60 • C for 10 h, grinding with a porcelain mortar was then performed so as to be ready for the fieldwork.

Adsorption Data Analysis and Drug Loading
To investigate the active ingredients, 2 g of LDH loaded essential oil was stirred vigorously for 3 h on absolute ethanol. The slurry collected by centrifugation and supernatant was divided into two portions; one of them concentrated until all the ethanol was vaporized, then a nonpolar solvent was used to dissolve the residue to be analyzed using GC/MS analysis to determine whether any volatile or nonpolar active ingredients were present or not. The other portion was injected directly into the LC MS/MS, to determine the non-volatile or polar active ingredients like polyphenols of flavonoids, using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with an Exion LC AC system for separation and SCIEX Triple Quad 5500 + MS/MS system equipped with an electrospray ionization (ESI) for detection. The separation was performed using ZORBAX SB-C18 Column (4.6 mm × 100 mm, 1.8 µm). The mobile phases consisted of two eluents: A) 0.1% formic acid in water; B) acetonitrile (LC grade). The mobile phase was programmed as following, 2% B from 0-1 min, 2-60% B from 1-21 min, 60% B from 21-25 min, 2% B from 25.01-28 min. The flow rate was 0.8 mL/min and the injection volume was 3 µL. For the MRM analysis of the selected polyphenols, positive and negative ionization modes were applied in the same run with the following parameters: curtain gas: 25 psi; Ion Spray voltage: 4500 and−4500 for positive and negative modes, respectively; source temperature: 400 • C; ion source gas one and two were 55 psi with a declustering potential: 50; collision energy: 25; collision energy spread: 10.
Whereas, the GC/MS analysis was performed by Thermo Scientific, Trace GC Ultra/ISQ Single Quadrupole MS, and TG-5MS fused a silica capillary column (30 m, 0.251 mm, 0.1 mm film thickness). An electron ionization system (ionization energy of 70 eV) was used for the GC/MS detection. Helium, a carrier gas, was used with a constant flow rate of 1 mL/min. The injector and MS transfer line temperature was set at 280 • C. The oven temperature was programmed initially as 50 • C (hold 2 min) to 150 • C at an increasing rate of 7 • C/min, then to 270 at an increasing rate 5 • C/min (hold 2 min), then to 310 • C as a final temperature at an increasing rate of 3.5 • C/min (hold 10 min). The quantification of the identified components was investigated using a percent relative peak area. Tentative identification of the compounds was analyzed by comparing their relative retention time and mass spectra with those of the NIST, WILLY library data of the GC/MS system. Moreover, the identification was accomplished using computer search user-generated reference libraries, incorporating mass spectra. Peaks were examined by single-ion chromatographic reconstruction to confirm their homogeneity. In some cases, when identical spectra have not been found, only the corresponding component's structural type was proposed based on its mass spectral fragmentation. Reference compounds were co-chromatographed when possible, to confirm GC retention times.

In Vitro Larvicidal Efficacy
Crude oils and their nanocomposite were carried out for their larvicidal efficacy [37] against the early fourth instar larvae, Cx. pipiens. Oils were added to a solvent consists of dechlorinated water plus 5 mL Tween 20. Twenty larvae were placed in a 500 mL glass beaker containing 250 mL of crude oils or their nanocomposite. Different concentrations of essential oils (125, 250, 500, 1000, and 2000 ppm) were tested [38]. Each experiment and the control group (treated with the solvent only) were replicated three times. Larval mortalities were recorded 0.5, 2, 8, 24, and 48 h post-treatment (PT).

Larvicidal Field Evaluation
Field evaluation of fennel and green tea oils and their Mg-LDH were evaluated against the larval and pupal mosquito population in stagnant water ditches (avg. 350 m × 4.5 m by 0.65 m deep) at Shablanga village, Qalyubiya Governorate, Egypt, where water was relatively stable with a high mosquito density. The LC 95 X2 of each oil and its Mg-LDH were applied. The control site was treated with chlorinated water. Three replicates were used for each treatment. Mosquito samples per site were taken prior to treatment and daily PT for a week. The fourth instar larvae in the field water were collected from each site using an enamel plate (450 mL) at each of the larvicides to transport the sample to the laboratory to evaluate the efficacy of the selected larvicides on the mosquito population [39].

In Vitro Adulticidal Efficacy
Adult mosquito susceptibility testing was conducted using modified CDC bottle bioassays [40]. Pure ethanol was used to make different concentrations of each oil (2, 5, 10, 15, and 25%). Oils loaded in Mg-LDH were prepared with different concentrations (0.5, 1, 2, 3, and 4%). The bottles were coated with the desired concentrations and left open to evaporate the solvent overnight at 28 ± 2 • C. Three bottles were used for each concentration. A hand aspirator was used to release adult mosquitoes (10-15, aged 3-4 days) fed on a 10% sucrose solution into each bottle. 10, 20, 30, 40 and 60 min were used as exposure times. The mosquitoes in the bottles were removed. Mosquito groups were placed in separate paper cups containing a 10% sucrose solution, and mortality was measured after 24 h. For each concentration, three replicates were made.

Adulticidal Field Evaluation
The efficacy and stability of fennel and green tea oils and their Mg-LDH were performed on the adult mosquitoes [41] at Shablanga village, as mentioned before, where animal barns were located inside houses near some irrigation ditches. Each oil and its Mg-LDH (LC 95 X2) were sprayed in three rooms/home. Three rooms were sprayed with dechlorinated water as a control group. The reduction in adult mosquitoes was calculated [42]. To determine the persistence or stability of selected adulticidal, ten adult mosquitoes from the same house were captured through the aspirator and put into a device on the wall in the room for 30 min. Then, the last adult mosquito was killed (WHO 2016).

The Efficacy against Non-Target Predators
An evaluation of the larvicidal and adulticidal activity of fennel and green tea oil was conducted, before and after being loaded by layered double hydroxide (LDH) in both laboratory and field conditions. The efficiency of fennel and green tea oils were examined against some selected common predators captured using a standing diver in different mosquito larval habitats as Gambosia affinis, Cybister tripunctatus, and Sphaerodema urinator. The predators were collected alive in plastic bags half-filled with water from the field to the laboratory and identified in the Entomology Department at Benha University, Egypt [43].

Data Analysis
For analyzing the data, we applied the one-way analysis of variance (ANOVA), and multiple comparisons were carried out applying Tukey's test and Probit analysis for calculating the lethal values using the computer program PASW Statistics 2009 (SPSS version 22). The significance level was set at p < 0.05.
The percent reduction in larval and pupal density was calculated [42] according to the following formula: where C1 = Pre-treatment immature density in the control site. C2 = post-treatment immature density in the control site. T1 = pre-treatment immature density in the treated site. T2 = post-treatment immature density in the treated site.

Characterization of M(II)LDH
The phase determination of synthesized LDH hydrotalcite-like materials was conducted by using XRD. Figure 3 shows the powdered X-ray Diffraction Patterns of unloaded MgAl-LDH and loaded MgAl-LDH with green tea oil. The diffraction pattern confirmed that, LDH synthesized perfectly with high crystallinity due to the appearance of strong and sharp peaks in both Mg LDH and MgLDH-GT. The values of 2θ = 11, 23, 34, 38, 45 and 60 were attributable to (003), (006), (009), (015), (012), (110) and (113) planes of reflection specific to MgAl-LDH. P-XRD diffractogram of the synthesized MgAl LDH revealed a hydrotalcite structure with characteristic reflections, starting from intense and sharp basal 00l reflections of 003, 006 and 009 planes that showed a low reflection angle, less than 35 (35 • > 2θ). Whereas, little broad 0kl planes of reflection of 012 and 015 clearly showed moderate reflection angle in range of (2θ = 38 • and 45 • ), and finally, the sharp hk0 and hkl planes of reflections of 110 and 113, in the highest region angle (2θ = 45-60 • ). Referring to the XRD library, the data obtained fund to be in consistent with the structure of Mg Al LDH.
where C1 = Pre-treatment immature density in the control site. C2 = post-treatment immature density in the control site. T1 = pre-treatment immature density in the treated site. T2 = post-treatment immature density in the treated site.

Characterization of M(II)LDH
The phase determination of synthesized LDH hydrotalcite-like materials was con ducted by using XRD. Figure 3 shows the powdered X-ray Diffraction Patterns of un loaded MgAl-LDH and loaded MgAl-LDH with green tea oil. The diffraction pattern confirmed that, LDH synthesized perfectly with high crystallinity due to the appearanc of strong and sharp peaks in both Mg LDH and MgLDH-GT. The values of 2θ = 11, 23 34, 38, 45 and 60 were attributable to (003), (006), (009), (015), (012), (110) and (113 planes of reflection specific to MgAl-LDH. P-XRD diffractogram of the synthesized MgAl LDH revealed a hydrotalcite structure with characteristic reflections, starting from intense and sharp basal 00l reflections of 003, 006 and 009 planes that showed a low re flection angle, less than 35 (35° > 2θ). Whereas, little broad 0kl planes of reflection of 01 and 015 clearly showed moderate reflection angle in range of (2θ = 38°and 45°), and fi nally, the sharp hk0 and hkl planes of reflections of 110 and 113, in the highest region angle (2θ = 45-60°). Referring to the XRD library, the data obtained fund to be in con sistent with the structure of Mg Al LDH.  The morphological and microstructural of LDHs investigated Using Transmission Electron Microscopy (TEM) showed, as in Figure 4, a well-designed hexagonal shape with a particle size dimension of 100 nm for MgAl-LDH (Figure 4a), and a thin plate structure for NiAl-LDH with a particle size 200 nm (Figure 4b), respectively. Mg and Ni LDH intercalated green tea and fennel oil showed the bulky aggregations on a TEM micrograph, as showed in Figure 4c-f, due to the high absorbability made by LDH with little increase in particle size, especially in the case of MgAl-LDH intercalated green tea as, 0.2 µm, 200 nm, 0.2 µm and 200 nm for MgLDH-GT, MgLDH-F, NiLDH-GT and NiLDH-F, respectively. To investigate the surface morphology, field-emission scanning electron microscopy (FESEM) was used. Figure 5 revealed that the unloaded MgLDH and NiLDH appeared in two types: sphere-like and plate-like in the order of 50 µm to 100 µm, respectively. In addition, SEM-EDX was used to determine the elemental analysis of the metal surface constituents or atomic composition for the MgLDH and NiLDH intercalated green tea and fennel oils as shown in (Figure 6). Based on, the SEM-EDX results, the best loading occurred between MgAl-LDH and green tea oil, which may explain the potential larvicidal and adulticidal actions of MgLDH-GT against Cx. pipiens and the related non-target species. That interpretation comes from the weight percentage of the carbon atom present on the surface of the sample at a point as the higher the carbon content, the higher the active ingredients loaded. The loaded MgLDH-GT presented a carbon percentage of 14.52% while unloaded MgLDH showed 2.9% (Figure 6a,e), whereas 4.45, 6.28 and 6.3% were recorded for the carbon content of MgLDH-F, NiLDH-GT and NiLDH-F, respectively, as shown in (Figure 6b-d). The LCMS/MS results, shown in Figure 7, are consistent with the SEM-EDX results shown in Figure 6, that means when the number of active ingredients detected by LCMS/MS increased the carbon weight percentage detected by SEM-EDX also increased. That will be observed if we compare SEM-EDX of unloaded Mg LDH, which contains a low carbon content of 2.9%, and Mg LDH loaded green tea oil that contains a high carbon content of 14.52%. The higher carbon content explains the more active ingredient loading. electron microscopy (FESEM) was used. Figure 5 revealed that the unloaded and NiLDH appeared in two types: sphere-like and plate-like in the order of 50 µ µm, respectively. In addition, SEM-EDX was used to determine the elemental an the metal surface constituents or atomic composition for the MgLDH and NiLD calated green tea and fennel oils as shown in (Figure 6). Based on, the SEM-EDX the best loading occurred between MgAl-LDH and green tea oil, which may exp potential larvicidal and adulticidal actions of MgLDH-GT against Cx. pipiens an lated non-target species. That interpretation comes from the weight percentag carbon atom present on the surface of the sample at a point as the higher the content, the higher the active ingredients loaded. The loaded MgLDH-GT pre carbon percentage of 14.52% while unloaded MgLDH showed 2.9% (Figu whereas 4.45, 6.28 and 6.3% were recorded for the carbon content of Mg NiLDH-GT and NiLDH-F, respectively, as shown in (Figure 6b-d). The LCMS sults, shown in Figure 7, are consistent with the SEM-EDX results shown in Figu means when the number of active ingredients detected by LCMS/MS increased bon weight percentage detected by SEM-EDX also increased. That will be observ compare SEM-EDX of unloaded Mg LDH, which contains a low carbon content and Mg LDH loaded green tea oil that contains a high carbon content of 14.5 higher carbon content explains the more active ingredient loading.

Desorption of Active Ingredient and Drug Loading
To ensure the existence of volatile and nonvolatile active ingredients released from nanoclay systems and to rationalize the insecticidal activity reasons, the desorption was conducted as explained in the experimental section using ethanol as the solvent and Mg LDH loaded green tea as template of nanoclays loaded with natural oil followed by an analysis of the supernatant produced from the desorption process using a chromatography technique. Results of volatile active ingredients determined by GC/MS showed neglected very small uproarious signals (background) in a chromatogram, confirming that there are no significant peaks related to volatile active ingredients. This may be due to the low affinity of LDHs nanoclays towards volatile active ingredients or the reason for the absence of this volatile compound may be they not having the opportunity to be loaded as a result of their high volatility.
Polyphenol and flavonoid nonvolatile active ingredients were detected using LC MS/MS. According to Figure 7 and Table 1, significant peaks related to polyphenols and flavonoids of Vanillin, Catechin, Daidzein, Ellagic acid, Naringenin, Myricetin and Syringic acid with concentrations of 0.76, 0.73, 0.67, 0.59, 0.52, 0.44 and 0.42 ug/g, respectively, were shown compared to their standard concentration of 0.05 g/mL (Figure 7a,b). The detected flavonoids and polyphenols, as active ingredients, were proven to have insecticidal effect in many oil and plant extracts [44,45]. The last findings explain the potential insecticidal activities of MgLDH loaded green tea oil. These findings are consistent with those announced in our previous study [46], which discussed the results of the HPLC analysis of raw green tea oil (Camellia sinensis) and confirmed that green tea oil contains some polyphenolic and flavonoid active ingredients, which transferred to the MgLDH nanoclays and were effectively released to be detected using LCMS/MS in our current study.

Desorption of Active Ingredient and Drug Loading
To ensure the existence of volatile and nonvolatile active ingredients released from nanoclay systems and to rationalize the insecticidal activity reasons, the desorption was conducted as explained in the experimental section using ethanol as the solvent and Mg LDH loaded green tea as template of nanoclays loaded with natural oil followed by an analysis of the supernatant produced from the desorption process using a chromatography technique. Results of volatile active ingredients determined by GC/MS showed neglected very small uproarious signals (background) in a chromatogram, confirming that there are no significant peaks related to volatile active ingredients. This may be due to the low affinity of LDHs nanoclays towards volatile active ingredients or the reason for the absence of this volatile compound may be they not having the opportunity to be loaded as a result of their high volatility.
Polyphenol and flavonoid nonvolatile active ingredients were detected using LC MS/MS. According to Figure 7 and Table 1, significant peaks related to polyphenols and flavonoids of Vanillin, Catechin, Daidzein, Ellagic acid, Naringenin, Myricetin and Syringic acid with concentrations of 0.76, 0.73, 0.67, 0.59, 0.52, 0.44 and 0.42 µg/g, respectively, were shown compared to their standard concentration of 0.05 g/mL (Figure 7a,b). The detected flavonoids and polyphenols, as active ingredients, were proven to have insecticidal effect in many oil and plant extracts [44,45]. The last findings explain the potential insecticidal activities of MgLDH loaded green tea oil. These findings are consistent with those announced in our previous study [46], which discussed the results of the HPLC analysis of raw green tea oil (Camellia sinensis) and confirmed that green tea oil contains some polyphenolic and flavonoid active ingredients, which transferred to the MgLDH nanoclays and were effectively released to be detected using LCMS/MS in our current study.

Larval Laboratory Experiments
The larvicidal effects of fennel, F. vulgare and green tea oils, C. sinensis and their LDH were evaluated against the early 4th larvae, Cx. pipiens. The mortality (MO) % PT with 2000 ppm for 48 h with fennel oil, and its LDH reached 100% MO (Table 2), whereas MO% for green tea oil, Mg-LDH-GT, and Ni-LDH-GT were 95.00, 100.00, and 96.67%, respectively (Table 3).  The LC 50 Table 4). The data LC 50 values of fennel and green tea nanoparticles (Mg and Ni LDH) and fennel and green tea oils showed there are significant differences (F = 13.4; df = 2,27; p = 0.001) between all treatments except the control.

In Vitro Adulticidal Effect
The adulticidal effects of the applied materials was evaluated against the Cx. pipiens (3-4 days old) and indicated that Mg-LDH-F and Ni-LDH-F were more highly effective than fennel oil, where the mortality reached 100%, 93.3% and 86.7%, 24 h PT, respectively, and their LC95 values were 14.16, 33.16, 72.40%, respectively (Table 5).

In Vitro Adulticidal Effect
The adulticidal effects of the applied materials was evaluated against the Cx. pipiens (3-4 days old) and indicated that Mg-LDH-F and Ni-LDH-F were more highly effective than fennel oil, where the mortality reached 100%, 93.3% and 86.7%, 24 h PT, respectively, and their LC 95 values were 14.16, 33.16, 72.40%, respectively (Table 5). The different superscript letters indicate that the differences between the groups are significant by (one-way ANOVA, Tukey's range test, p < 0.05).
Moreover, Mg-LDH-GT and Ni-LDH-GT were more effective than green tea oil, where the mortality reached 93.3%, 83.33%, and 76.67%, 24 h PT, respectively, and their LC 95 values were 8.84, 29.14, and 35.39%, respectively (Table 6). The different superscript letters indicate that the differences between the groups are significant by (one-way. ANOVA, Tukey's range test, p < 0.05).

The Efficacy against Non-Target Predators
The efficiency of oils and their Mg-LDH were examined against some predators, such as G. affinis, C. tripunctatus, and S. urinator. There was no significant difference between the mean of predation of both fennel and green tea oils. In the case of Mg-LDH-GT and Mg-LDH-F, the predation rate of the beetle, C. tripunctatus increased

The Efficacy against Non-Target Predators
The efficiency of oils and their Mg-LDH were examined against some predators, such as G. affinis, C. tripunctatus, and S. urinator. There was no significant difference between the mean of predation of both fennel and green tea oils. In the case of Mg-LDH-GT and Mg-LDH-F, the predation rate of the beetle, C. tripunctatus increased (71% and 69%), respectively, compared to green tea and fennel oils (55.67, and 52.33, respectively). On the other hand, the predation rate without treatments reached >97% (Table 8). a, b and c: There is no significant difference (p > 0.05) between any two means, within the same column that have the same superscript letter. A, B and C: There is no significant difference (p > 0.05) between any two means for the same attribute, within the same row that have the same superscript letter. * Control with free Mg-LDH-F, ** control with free Mg-LDH-GT, *** control without treatment.

Discussion
The most efficient strategy to prevent mosquito bites and disease transmission is synthetic insecticides to kill larvae in water bodies and adults in the air. To avoid pest resistance and environmental contamination, natural alternatives are in high demand. EOs and plant extracts are relatively safe, readily available, and biodegradable [26] and could be used as natural alternatives for mosquito control [47][48][49].
Our results indicated that the fennel oil, F. vulgare and green tea oil, and C. sinensis oils have larvicidal and adulticidal activity, and their LDH Nanoclays increased their efficacy and persistence.
Similar to our findings, fennel oil was effective against larvae and adults of Culex quinquefasciatus [36,50], Anopheles atroparvus [51], and Aedes aegypti [35]. In another study, Zoubiri, et al. [52] showed that 40 mg/L of fennel oil, F. vulgare oil, was sufficient to cause 50% mortality in the second larval instar of Cx. pipiens after 2 h of exposure. Furthermore, a concentration of 60 mg/L resulted in 90% mortality for the fourth instar larvae after 4 h of exposure.
With almost half of the doses applied in the field, the larval reduction percentage of fennel oil and Mg-LDH-F reduced larval density (92% and 96.2%, 24 h PT) and their effect lasted (reduction% > 50%) for 5 and 7 days PT, respectively. Those of green tea oil and Mg-LDH-GT were 88.00 and 90.01%, respectively, 24 h PT and persisted for six days PT, respectively. On the other hand, the reduction in adult density PT with fennel oil, Mg-LDH-F, green tea oil, and Mg-LDH-GT reached 83.00, 100, 76.67, and 97.00%, respectively, and was effective for three days. To the best of our knowledge, there was no previously filed application for the use of nanoparticles against mosquitoes.
The tea leaves C. sinensis are used to produce a non-alcoholic drink that really is drunk all over the world for its psycho-activity and health benefits [33]. Graham (1992) found that the immature tea leaves are high in methylxanthines (caffeine, theophylline), catechins (catechin, gallocatechin, catechin gallate), flavonoids, vitamins, proteins, glycosides (kaempferol, myricetin), and that the catechins have antimalarial, antiviral, antibacterial, anticarcinogenic, antioxidant, anti-inflammatory, anti-arthritis, anti-aging properties (Sannella et al., Afzal et al., 2015). Proanthocyanidins were found to be the most abundant bioactive chemicals in the C. sinensis leaf extract [33].
Mosquito predators, such as Gambusia sp., are released worldwide for biological mosquito control due to their excellent predation effectiveness [59]. This work indicated the safety of the applied materials against three non-target organisms. Similarly, several studies have investigated the impact of the acute toxicity of nanoparticles on the aquatic non-target species, and did not detect toxicity within the silver nanoparticles produced using plant extracts toxic to mosquito larvae [69].
The present work indicated that the predation rates of C. tripunctatus and S. urinator were slightly decreased and increased after being subjected to crude oils and LDH, respectively, when compared to the free Mg-LDH control group. Correspondingly, a noticed increase in the predation rate of the tadpoles, Hoplobatrachus tigerinus, against the larvae of Ae. aegypti, in the laboratory and in an aquatic environment, treated with ultra-low doses of AgNP, was recorded [60]. AgNP of Nicandra physalodes was safe for the non-target aquatic organism Diplonychus indicus (LC 50 and LC 90 values were 1032.81 and 19,076.59 µg/mL, respectively) [61]. Zornia diphylla AgNP is safe for some non-target organisms as Chironomus circumdatus, Anisops bouvieri and Gambusia affinis (LC 50 = 613.11 − 6903.93 µg/mL), if compared to target mosquitoes [62].
Some oils have a neurotoxic effect and interfere with the neuromodulator octopamine and gamma-aminobutyric acid, GABA, and gated chloride channels [27]. Few studies reveal the mode of action of green-synthesized nanoparticles, which may be related to exoskeleton penetration, or nanoparticles that could bind to sulfur from proteins or to phosphorus from DNA, inducing the rapid denaturation of enzymes and organelles, followed by the decrease in membrane permeability and disturbance in proton motive force affecting cellular function and cell death [59]. In addition, nanoparticles enter the cuticle membrane of mosquito larvae, then move to their intestine and damage their DNA banding pattern [63].