Synergistic insecticidal effects of zinc-loaded zeolite nanoparticles combined with essential oils against Callosobruchus maculatus

The chemical constituents of essential oils

Table 1 shows the chemical composition of essential oils extracted from R. officinalis and P. anisum using GC-MS. Based on a fresh plant of R. officinalis weight of extract part, the hydro-distillation yielded about 0.3% w/w. Twenty compounds have been identified, representing 97.23% of the essential oil. These compounds were divided into 43.11% monoterpene hydrocarbons (α-thujene, α-pinene, Camphene, β-pinene, myrcene, γ-terpinene, terpinolene, α-phellandrene, and cymene); 47.81% oxygenated monoterpenes (1,8-cineole, linalool, trans-pinocarveol, Camphor, borneol, terpinen-4-ol, and α-terpineol); 4.59% of sesquiterpene hydrocarbons (caryophyllene, aromadendrene, and humulene); 1.72% of ketone (3-octanone). The major compounds of R. officinalis essential oil were 1,8-cineole (25.36%), α-pinene (23.75%), Camphor (12.66%), and Camphene (8.19%). For P. anisum essential oil, with a yield of 0.35% w/w based on the sample’s fresh weight, twenty-one compounds were recorded, accounting for 96.45%. The essential oil analysis revealed that the oil had a lower quantity of monoterpene hydrocarbons of 3.95% (pinene, carene, limonene, ocimene, and terpinene). The essential oil had rich amounts of oxygenated monoterpenes of 74.71% (linalool, Methyl chavicol, Z-anethole, and E-anethole). The amount of sesquiterpene hydrocarbons was 15.62% (isoledene, longifolene, cedrene, thujopsene, gurjunene, elemene, guaiene, himachalane, and E-isoeugenol), while the oxygenated sesquiterpenes recorded 2.17% (cis-sesquisabinene hydrate, spathulenol, and geranyl isovalerate). P. anisum essential oil contained a high percentage of E-anethole (64.23%), followed by methyl chavicol (8.69%) and longifolene (5.08%). The two oils also differed in their terpene hydrocarbon content. R. officinalis had a higher proportion of monoterpene hydrocarbons (43.11%) than P. anisum (3.95%). In contrast, P. anisum has a higher percentage of oxygenated monoterpenes (74.71%) than R. officinalis (47.81%).

Characterizations of the synthetic zeolites

Figure 1A and B presents the parent zeolite (zeolite-A and zeolite-X). Zeolites mineral profiles were compared to the XRD database and showed perfect matching with PDF card # 73-2340, with Na₁₂Al₁₂Si₁₂O₄₈.27H₂O for zeolite-A (Fig. 1A), and PDF # 39-1380 (1), with Na₂Al₂Si₄O₁₂.8H₂O composition for zeolite-X (Fig. 1B), in respective order. The distinct, sharp, and complete set of both zeolites’ peaks implied good crystallinity. Notably, the synthetic product contains some residue of quartz mineral, which was traced back to the kaolin precursor from which they were formed. Meanwhile, Faujasite-NaX showed a small number of nanoparticle zeolite peaks that seemed to accompany the originally developed zeolite-X material, and this could be seen in light of the similarity in preparation conditions of both zeolites.

Figure 1C and D shows XRD details for Zn-doped zeolites. Both charts compared to the standard references of the PDF-2 database and confirmed the evolution of Zn-doped types of Zn-zeolite-A and Zn-zeolite-X, having respective chemical compositions of Na₅₀Zn₂₃Al₉₆Si₉₆O₃₈₄.216H₂O (Fig. 1C) and (Zn, Na)₂Al₂Si_2.5O₉.72H₂O (Fig. 1D), respectively. As shown in Fig. 1 (C-D) the XRD patterns for Zn-containing phases indicated sharper peaks with higher intensities than those recorded for their un-doped forms (Fig. 1A and B). In addition, zeolite-X implies the presence of minor amounts of zeolite nanoparticles as a secondary accompanying phase that can develop in the zeolite mixtures. This indicates very high crystallinity of many pure phases with no residues of the amorphous metakaolin precursor, which was preserved within the synthetic zeolite powders and appeared in the XRD patterns in the form of a humpy background in Fig. 1 (A and B).

Internal structure testing (SEM and EDS)

The internal textural analysis of zeolite product and its chemical microanalysis give a clear idea about the characteristic morphology and elemental contents of the contained crystals. The internal crystalline texture and the elemental micro-chemical analysis of the dry, unfunctionalized zeolite-A and zeolite-X products were examined using the SEM and EDS tools. The obtained data are given in Fig. 2A and B and Table 2. As can be noticed from Fig. 2A, zeolite-A exhibited its distinctive cubic-shaped crystal form with uniform grain particles in the range of 1–3 μm in size.

Figure 2B presents the obtained product of Faujasite-NaX (zeolite-X). The micrograph monitors large crystals with pyramidal epics of less than 1 μm in size, accompanied by an ample amount of zeolite nanoparticles (10–15%) and some scattered quartz particles (< 5%). The former SEM result for both zeolites is consistent with the previous XRD data. Table 2 demonstrates the elemental composition of the un-doped zeolites, where the calculated average Si/Al ratios of the crystal composition were 1.11 and 1.75 for zeolite-A and zeolite-X, respectively. Figure 3A and B monitors the SEM morphologies of the obtained zeolite-produced powders after the exchange of their sodium constituents by zinc. The micro-chemical composition is given in Table 2. Obviously, there were no destructive changes in the crystal configuration for both zeolites after doping since the crystal identities were preserved without shape alteration. The only notice was the clear crystal faces and edges. The results of the EDS analysis were collected from an average of three measurement detections for the crystal surfaces of three different crystal generations of the same zeolite species. The respective atomic ratio of Si/Al for zeolite-A was 1.07, and for zeolite-X was 1.6 (Table 2).

Efficiency of zeolite nanoparticles on C. maculatus

The mortality of C. maculatus treated with zeolites at different durations is presented in Table 3. The mortality of the beetles increased with increasing concentration and duration of exposure for all treatments. The results also showed significant differences in mortality between the different types and concentrations of zeolites at each time interval.

The highest mortality rate achieved by zeolite-X was 48.3% after 7 days at 1000 mg/kg, while it was 43.3% for zeolite-A at the same concentration and time interval. The highest mortality was observed for Zn-zeolite-A (51.7%) at 1000 mg/kg after 7 days. Zeolite-X outperformed zeolite-A in insecticidal activity against C. maculatus, while zeolite-A loaded with zinc surpassed Zn-zeolite-X in insecticidal efficacy.

Efficiency of zeolite nanoparticles and R. officinalis combinations on C. maculatus

The mortality of zeolite and R. officinalis combinations after 2, 5, and 7 days against the tested insect is elucidated in Table 4. The concentration- and time-dependent mortality was evident in all treatments compared to the control group that had minimal mortality (3.3%) even after seven days of exposure. Use of R. officinalis essential oil at a single dose produced moderate insecticidal activity with the higher dose (200 mg/kg) causing a higher mortality of 63.3% on the seventh day, compared to 43.3% at the lower concentration (100 mg/kg). When R. officinalis essential oil was used together with zeolites, a high improvement in insecticidal activity was observed. In most cases, the higher the concentration of the essential oil and the zeolites, the higher the mortality. The mixtures of the high concentration of R. officinalis (200 mg/kg) with any of the tested zeolites at 750 or 1000 mg/kg were the most effective. Interestingly, several treatments (R. officinalis (200 mg/kg) combined with Zeolite-X (both 750 and 1000 mg/kg), Zeolite-A (1000 mg/kg), Zn-zeolite-X (1000 mg/kg), and Zn-zeolite-A (both 750 and 1000 mg/kg)) reached 100% mortality by day seven. The fastest and most efficient treatment was the combination of R. officinalis (200 mg/kg) and Zn-zeolite-A (1000 mg/kg) that led to 100% mortality in only five days of exposure.

Efficiency of zeolite nanoparticles and P. anisum combinations on C. maculatus

The mortality of zeolite and P. anisum combinations after 2, 5, and 7 days against the tested insect is presented in Table 5. The findings indicate that P. anisum and R. officinalis essential oils alone or in combination with zeolites were found to significantly increase the mortality of C. maculatus compared to the control. P. anisum was more effective than R. officinalis at the same concentrations. For example, P. anisum (200 mg/kg) produced 63.3% mortality after 2 days, compared to 43.3% in R. officinalis (200 mg/kg). This was consistent throughout the exposure periods, and the P. anisum treatments tended to produce more rapid and more severe lethal effects. The synergistic mixtures of P. anisum or R. officinalis with zeolites also increased the mortality, especially at increased concentrations (1000 mg/kg). It is worth noting that P. anisum-based formulations, such as P. anisum (200) + Zn-zeolite-A (1000) was able to kill 100% of the larvae in 5 days whereas the most effective R. officinalis combination, R. officinalis (200) + Zn-zeolite-A (1000) took 7 days to kill the larvae to the same extent.

Effect of zeolite nanoparticles on progeny production

The mortality of progeny of C. maculatus exposed to zeolites with and without loaded zinc is recorded in Table 6. The results showed that all zeolite treatments caused a significant increase in the mortality of C. maculatus offspring compared to the control. As the concentration of zeolite increases, the mean number of progeny decreases. None of the concentrations applied could suppress the progeny production. All zeolite treatments showed moderate effects on the mortality of progeny of C. maculatus. Zeolite-X and zeolite-A loaded zinc were the most effective treatments with mortality of offspring of 48.43 and 49.64%, respectively at the highest application rate of 1000 mg/kg.

Effect of zeolite nanoparticles and R. officinalis combinations on progeny production

Data presented in Table 7 show the mortality of progeny of C. maculatus treated with zeolite and R. officinalis combinations. All treatments significantly reduced the mean number of progeny and increased the percentage of offspring mortality of C. maculatus compared to the control. The treatment with R. officinalis essential oil (RO) alone at 100 mg/kg resulted in a mean number of progeny of 42, which is significantly lower than the control group (129). Additionally, the mortality of the progeny under this treatment was 35.7%. Increasing the concentration of R. officinalis essential oil to 200 mg/kg further reduced the mean number of progeny to 21 and increased the mortality percentage to 55.2%. The combination of zeolite and R. officinalis essential oil increased the mortality of progeny compared with zeolite alone. Zeolites loaded with zinc and R. officinalis oil mixtures could suppress progeny production at 200 mg/kg of the essential oil and 1000 mg/kg of zeolite.

Effect of zeolite nanoparticles and P. anisum oil combinations on progeny production

The results of Table 8 demonstrate that the progeny of C. maculatus mortality was greatly affected using P. anisum essential oil, either alone or in combination with various zeolites. The progeny mortality rate of the control group was low at 2.1%. On the other hand, the mortality rate was significantly higher when P. anisum essential oil at 100 and 200 mg/kg was used alone (55.0 and 64.6%, respectively). An interesting synergistic effect was also found when P. anisum essential oil was used together with zeolites. The combination of P. anisum at 200 mg/kg with different zeolites was the most effective treatments. A complete mortality was observed with P. anisum (200 mg/kg) and Zeolite-A (750 mg/kg). Moreover, a number of combinations such as P. anisum (200 mg/kg) and either Zeolite-X (750 and 1000 mg/kg), Zeolite-A (1000 mg/kg), or zinc-loaded zeolites (both 750 and 1000 mg/kg) totally inhibited the development of insect progeny.

Toxicity of zeolites, essential oils, and their combinations on C. maculatus

The results of contact toxicity of zeolites, essential oils, and their combinations against C. maculatus are recorded in Table 9. P. anisum oil exhibited higher toxicity than R. officinalis oil against the tested insect, with LC₅₀ values of 126 and 200 mg/kg, respectively. Zeolite-X and zeolite-A had high LC₅₀ values (1407 and 1658 mg/kg, respectively), suggesting lower toxicity than essential oils. Zinc loading in zeolite-X and zeolite-A showed a slight increase in toxicity. The combinations of essential oils (R. officinalis and P. anisum) with zeolites (zeolite-X, zeolite-A, zeolite-X loaded with zinc, and zeolite-A loaded with zinc) significantly lowered LC₅₀ and LC₉₅ values compared to the individual components alone. The LC₅₀ values for the combinations ranged from 161 to 306 mg/kg. The combination of P. anisum oil with zeolite-A loaded with zinc exhibited the lowest LC₅₀ value (161 mg/kg), suggesting the highest toxicity among the tested combinations..

Combined toxic effect of essential oils and zeolites

The results in Table 10 show the effectiveness of two essential oils, R. officinalis and P. anisum, in combination with different zeolite substrates (natural and Zn-loaded Zeolite). All the binary combinations showed positive co-toxicity factors of 20.9 to 30.0, which is a.

sign of synergism. The combination P. anisum + Zn-zeolite-A was the one that produced the highest observed mortality (65%), and the highest co-toxicity factor (30), thus indicating a very strong joint effect. As a rule, the Zn-modified zeolites proved to be more effective in enhancing mortality compared to their non-modified counterparts. Moreover, mixtures of P. anisum oil always had greater co-toxicity factors than the respective combinations of R. officinalis essential oil, indicating greater synergistic effects.

Effect of zeolite nanoparticles on C. maculatus morphology examined by SEM

The SEM images of untreated and treated adults exposed to cowpea seeds treated with zeolite compounds (1000 mg/kg) are shown in Figs. 4 and 5. Zeolite particles revealed a homogeneous distribution of zeolite particles on the cuticle of C. maculatus adults and aggregation between the thorax and abdomen joints compared with untreated adults. Image analysis showed that zeolite particles adhered to all body parts. The results also showed that zeolite treatments induced scratches on the elytra and clear damage in sensilla scatters in some points and absent in others, leaving spaces between these parts in the ventral surface, compared with the normal cuticle shape in untreated beetles of C. maculatus. Zeolite treatments revealed scratches and splits on the cuticle, leading to water loss through dehydration as the water barrier was damaged and died out of desiccation.