Journal of Entomology

Year: 2021 | Volume: 18 | Issue: 1 | Page No.: 8-18
DOI: 10.3923/je.2021.8.18

Toxicity and Growth-Disruptive Effects of Silica, Zinc and Aluminum Oxide Nanoparticles on Spiny Bollworm, Earias insulana

Enas E. Nasr and Shady Selim

Abstract: Background and Objective: Nanoparticles (NPs) are the most efficient technique of pest control without disturbing the potential environmental balance. This study was carried out to compare the toxicity effects of Silica, Zinc and Aluminum oxide NPs on growth, development, malformation and chitin formation parameters of Earias insulana. Materials and Methods: Newly hatched larvae fed on the treated diet for 48 hrs following by the untreated one until pupation. The toxicity impacts of LC₅₀values for each tested NPs were calculated. The fourth instar larvae prepared for scanning electron microscopy and also for defining the chitin formation. Results: The ZnONPs was the highest toxicity, followed by SiO₂ and Al₂O₃ NPs. The treatment with ZnONPs has scored an extreme reduction in the weight compared to other tested NPs. The growth rate inhibition decreased gradually until the fourth instars and then increased slightly in the prepupae and pupae in favor of ZnONPs, while Al₂O₃ NPs was the least effective one. Under-tested NPs, the increase in the larval and pupal periods led to a sharp reduction in the developmental rate and a significant increase in larval deformations. Besides, the least chitin formation ratio was registered in response to ZnONPs. By using electron microscopy, distinct differences were observed in the distribution of NPs on the cuticle surface of the fourth-instars body parts. Conclusion: This study recommends the practical use of ZnO, SiO₂ and Al₂O₃ NPs, respectively, in pest control programs of E. insulana as safe alternative materials.

Keywords: toxicity effects, development, malformation, chitin formation, cotton bollworms, Metal oxide, growth and pest control

INTRODUCTION

Egypt is still one of the leading countries interested in cotton cultivation due to its economic importance. Therefore, the government strived to increase its competitiveness in producing high-quality cotton in various international markets¹. Reports have proven that many pests attack cotton plants during their growth period. Cotton bollworms are prevalent among various insects, causing many economic losses in the rate of cotton production². The Spiny Boll Worm, Earias insulana (Boisd.) Lepidoptera: Noctuidae is one of the most dangerous pests, not only on the cotton plants but also many other crops, in Egypt and other North African countries. Its larvae cause damage to seeds, fibers, flowers and buds, particularly at the final growing stage of the cotton plants. It induces a decrease in the quantity and quality of the lint and the oil extracted from yield³. The extensive spread of pesticides has increased the serious response associated with environmental pollution such as toxic waste, natural enemies and pest resistance. Therefore, it is crucial to look for other alternatives to avoid using conventional pesticides in the control program of the cotton bollworms⁴. In recent years, pest control strategies are designed towards eco-friendly agriculture to maintain environmental balance⁵.

Nanoparticles have become a new generation of pest control techniques and an alternative strategy to the traditional pesticides that have provided effective resolutions to most environmental pollution problems⁶. Pest control with NPs depends on the penetration rate of the active substance through the target living cells, according to the size and properties of the outer surface of the particles compared to the volume rate⁷. The size of the NPs ranges between 10 and 1000 nanometers. A group of materials is useful for making NPs, such as silicates, metal oxides, lipids, emulsions and polymers^8,9. Metal NPs such as ZnO, Al₂O₃, Ag and TiO₂ released the active substance for a relatively long period, causing slow degradation of the living cells as a result of the toxic effects on insects^10,11.

Various studies have tested the toxicity of NPs in controlling many insects, but they have not revealed the most potent compound in protecting cotton plants from E. insulana. El-Ghohary and El-Samahy¹² showed statistical differences toward SiO₂ NPs in reducing cotton bollworms Pectinophora gossypiella (Saunders) and E. insulana (Bois d.) compared to other insecticides. Haroun et al.¹³ proved that ZnO and SiO₂ NPs showed a toxic influence against some stored seed insects at the highest concentration. El-Shennawy and Kandil¹⁴ cleared that treatment with ZnO NPs against E. insulana increased the larval and pupal duration and decreased their weight more than the chitin inhibitor and bio-insecticides that were used. Debnath et al.¹⁵ evidenced that Al₂O₃ and SiO₂ were achieved to be highly toxic pesticides compared to other Nano pesticides against Lipaphis pseudo brassicae. The present study planned to compare the entomotoxic efficacy of tested NPs (SiO₂, ZnO and Al₂O₃) against the larval and pupal stage of E. insulana when treated as newly hatched larvae and its toxicological impacts on growth and developmental parameters. Also, malformation and chitin formation rate induced by tested NPs were studied.

MATERIALS AND METHODS

Study area: This study was carried out in the Entomology Lab, Department of Zoology, Faculty of Science, Zagazig University, Egypt, from August, 2019 to October, 2020.

Tested insects: Newly hatched larvae of E. insulana were obtained from Plant Protection Research Institute, Sharkia Branch, Agricultural Research Center (ARC). The colony was reared on an artificial diet for several consecutive generations without any contamination with insecticides under constant laboratory conditions at 26±2°C, 65±5% RH and 12 hrs (L:D) photoperiod according to Amer¹⁶.

Tested nanoparticles: Silica (SiO₂), Zinc oxide (ZnO) and Aluminum oxide (Al₂O₃) NPs have an average size of 50 nm. These particles have a purity of more than 99%. The SiO₂ NPs and ZnONPs were purchased from Egypt Nanotech Co. limited, 6th October, Giza, Egypt, while Al₂O₃ NPs were purchased from Lab Chemical Trading Co., Cairo, Egypt.

Toxicity values of the tested nanoparticles: To evaluate the lethal concentration LC₅₀ of each tested nanoparticles; five gradient concentrations (1000, 500, 250, 125 and 62.5 ppm) were prepared for each compound by dilution in distilled water. An artificial diet (5 g/ replicate) was made in a petri dish and sprayed with different concentrations for each nanoparticle that was left to dry. Distilled water was used alone to treat the control sample. Newly hatched larvae of E. insulana (10 larvae/replicate) were directly fed on the treated diet for 48 hrs and then replaced with the untreated one until pupation. The diet of the control sample replaced when needed. Each concentration of ZnO, SiO₂ and Al₂O₃ NPs replicated four times. The number of larvae used in the control sample and the number of the replicates equaled with each tested NPs concentration. Mortalities were counted daily until the 18th day to estimate the percentage of the accumulative larval mortality at indicated days after the exposure of newly hatched larvae of E. insulana to different concentrations of the tested NPs consequently; all mortalities were corrected by using Abbott's Eq.¹⁷:

To avoid the harmful effects of NPs overdoses by the toxicity impacts of LC₅₀values for each tested nanoparticle were calculated to determine the growth rate inhibition and the developmental rate of the immature stages of E. insulana when treated as newly hatched larvae. Linear regression variables were also analyzed to predict mortality according to the various potential concentrations. Toxicity index studied according to Sun's Eq.¹⁸:

Growth and developmental parameters: One hundred larvae per nanoparticle were treated using lethal concentration LC₅₀ pre-calculated for SiO₂, ZnO and Al₂O₃ NPs with values of 168.94, 89.41 and 707.85 ppm, respectively. The same numbers of the control sample larvae were studied using distilled water instead of LC₅₀ of the NPs. Each treatment was replicated four times (25 larvae/replicate), where treated larvae were fed on a previously treated diet with LC₅₀ values for each nanoparticle. Additionally, live control larvae were transferred individually to glass tubes 20 mm in diameter, which including 2 g of the untreated diet that was kept under previous laboratory conditions. Larvae and pupae weights were recorded daily using a digital scale from 1st instar larvae until the end of the pupal stage. The pupae were individually kept in Glass jars (250 mL) covered with a white mesh. The growth rate inhibition was calculated as follows:

The developmental rate induced by tested NPs calculated using Dempster's equation¹⁹. Besides, the growth index of the larval and pupal stages of E. insulana was calculated using Pretorius's equations²⁰, as follows:

Larvae preparation for SEM observation: Fourth instar larvae of E. insulana were transferred in primary fixed with 2.5% glutaraldehyde+2% formaldehyde and washed 3×15 min in 0.1 M sodium phosphate buffer pH 7.4+0.1 M Sucrose, post-fixed with 2% sodium phosphate buffer, osmium tetroxide pH 7.4 and washed 3×15 min in 0.1 M sodium phosphate buffer pH 7.4, dehydrated sequentially with ethanol (50, 80, 90, 96, 100%/2×15 min each), contrasted overnight using 70% acetone+0.5% uranyl acetate+1% phosphotungstic acid, after dehydrated with ethanol (in distilled water), at 4°C. Larvae were then coated with gold-palladium membranes and observed in a Jeol JSM-6510 L.V SEM. The microscope was operated at 30 KV at SEM Unit, Mansoura University, Egypt.

Chitin formation of the body wall: The tested NPs with LC₅₀ values were placed in the diet of 10 newly molted of the fourth instar larvae (4 replicates/treatment), none treatment used for the control larvae. Deformed and freshly larvae were weighed after 48 hrs from treatment. The larvae were anesthetized by cooling and dissected longitudinally from the ventral side after removing the head. The gut, various internal tissues and fat bodies were extracted. The body wall was placed in 3 mL of 10% potassium hydroxide (KOH) per each gram of the larval weight to 4 hrs at 100°C and left at room temperature overnight. Chitin extracts were dried at 80°C overnight and then weighed for each larva and compared with chitin larval fresh weight. The chitin formation ratio induced by tested NPs against fourth instar larvae of E. insulana was determined according to equation of Hughes et al.²¹:

Statistical analysis: The toxicity data under LC₅₀ values of the tested NPs were estimated using probit analysis²². Data were used one-way ANOVA, followed by LSD's test to check the significant differences between means by SPSS software.

RESULTS

Toxicity values of the tested NPs against E. insulana: The obtained data in (Table 1) summarized that the LC₅₀ values for SiO₂, ZnO and Al₂O₃ NPs after the treatment of the newly hatched larvae of E. insulana by different concentrations were (168.94, 89.41 and 707.85 ppm, respectively). Moreover, the current results revealed that the highest tested NPs concentrations induced the larval mortality rate (83.78, 86.49 and 54.05%, respectively). Based on these values, E. insulana larvae were more sensitive to ZnO NPs than other tested NPs. The displayed data in (Fig. 1) indicated that the ZnO NPs ratio was the highest toxicity followed by SiO₂ and Al₂O₃ NPs against the newly hatched larvae of E. insulana at (100, 52.92 and 12.63%, respectively), according to the Sun’s toxicity index and based on the LC₅₀ values of the most toxic compound among the various tested NPs.

Accumulative larval mortality induced by tested NPs against E. insulana: The data in Fig. 2a-c showed the cumulative mortality rate of E. insulana larvae (3, 6, 9, 12, 15 and 18 days) after the exposure of the newly hatched larvae to different concentrations of the tested NPs for 48 hrs. The results also showed that the highest ratio of larval mortality, at a concentration (1000 ppm), associated with an increase in the time after treatment (18^th day), where it recorded 85, 87.5 and 57.5% mortalities, while the lowest mortality was 32.5, 35 and 17.5% at 62.5 ppm concentration of SiO₂ (Fig. 2a), ZnO (Fig.2b) and Al₂O₃ NPs (Fig. 2c), respectively. The data in Fig. 2a indicated gradual larval mortality (32.5, 47.5, 65, 75 and 85%, respectively) in ascending order at the various concentrations of SiO₂ NPs. Meanwhile, the mortality was ranged from 35-87.5% under ZnO NPs treatment (Fig. 2b). The results also scored 17.5% at 62.5 ppm and graduated up to 57.5% at 1000 ppm when larvae were treated with Al₂O₃ Nps. The results also showed that the larval mortality rate reached its highest value at a concentration of 1000 ppm on the 18th day after treatment with the tested nanoparticles, where 32.5, 47.5, 65, 75 and 85% mortalities were recorded when treated with various concentrations of SiO₂ NPs, respectively (Fig. 2a). The data in Fig. 2b indicated that the cumulative larval mortality was 35, 62.5, 70, 85 and 87.5%, respectively, according to the ascending order of concentrations in ZnO NP. Also, overall mortality under the various prior concentrations of Al₂O₃ NPs in Fig. 2c was 17.5, 27.5, 40, 47.5 and 57.5%, respectively.

Growth, developmental and malformation parameters induced by the tested NPs against E. insulana: The current results in (Table 2) showed significant differences between the treated larvae with the tested NPs and untreated larvae in all treatments. The comparison data between tested NPs indicated statistical differences in larval weights in the direction of ZnO NPs at 4.55, 17.85 and 11.08 mg for the 2nd, 4th instars and prepupae, respectively. Moreover, SiO₂ topped Al₂O₃ NPs with values 8.93, 31.50 and 19.33 mg at the same previously mentioned ages, respectively. Otherwise, there were no significant differences between SiO₂ and ZnO NPs in the weights of 2nd, 3rd instars and pupae at 3.67, 15.35 and 14.45 mg compared to 3.29, 12.97 and 10.75 mg, respectively.

The growth rate inhibition values in (Table 3) decreased gradually until the fourth instar larvae at 59.85, 77.25 and 29.96% for SiO₂, ZnO and Al₂O₃ NPs, respectively, then these values increased slightly in the prepupae and pupae. The results also indicated that ZnO NPs induced the growth rate inhibition during the various instar larvae and pupae at 75.09, 84.65, 72.87, 77.25, 83.14 and 78.88%, respectively, while Al₂O₃ NPs was the least effective one at 55.19, 49.61, 38.77, 29.96, 45.45 and 56.13%, respectively.

Data in (Fig. 3) revealed that tested NPs affected the developmental rate of the larval and pupal stages of E. insulana when treated as newly hatched larvae, where it severely reduced in the larval stage at 4.48, 3.67 and 5.41%, for the tested NPs, respectively, vs. 7.19% of the untreated larvae. Furthermore, the pupal developmental rate prolonged to 8.10, 6.94 and 8.45% for the tested NPs, respectively, vs. 11.30% of the untreated pupae. Moreover, data in (Fig. 4) showed that the growth index in the larval stage was 1.47, 2.05 and 2.57% on ZnO, SiO₂ and Al₂O₃ NPs, respectively, vs. 6.65% of the untreated larvae. The highest pupal growth index was 2.96% on Al₂O₃ NPs, while the lowest was 2.08% on ZnONPs, vs. 10.17% of the untreated pupae. The larval and pupal malformation rates are displayed in (Fig. 5), where the larval deformation significantly was increased at (12.5, 17.5 and 7.5% for SiO₂, ZnO and Al₂O₃ NPs, respectively) compared to 2.5% of control larvae. These ratios were not associated with the larval mortality rate, but actually, they were related to the morphological changes in the lived larvae. The current results exhibited an evident decrease in the pupal malformation percentages with 7.5, 2.5 and 5% for the tested NPs, respectively, without any malformed pupae in the control sample.

Under the electron microscopy (SEM), distinct differences were showed in the distribution of NPs on the epicuticle surface of different body parts (head, thorax and abdomen) of the spiny bollworm fourth-instars, (Fig. 6a-h). Larval dwarfism, severe malformed thorax cuticle and head capsule were displayed in (Fig. 6a) creating due to the treatment with SiO₂ NPs also, abnormal abdominal cuticle induced by a high density of SiO₂ NPs (Fig. 6b). The highest deformations were represented in (Fig. 6c-d) by the stiffness of the epidermal wax layer resulting from ZnO NPs, in the thorax and abdomen, which has caused the death of the larvae.

However, a low density of Al₂O₃ NPs showed on the thorax and abdomen cuticles (Fig. 6e-f), causing less larval dwarfism than other tested NPs. Moreover, the untreated 4th instar larvae appeared without any abnormalities with typical head, thorax (Fig. 6g) and abdomen cuticles (Fig. 6h).

Chitin of the body wall induced by the tested NPs against the fourth instars: The fresh weight of the 4th instar larvae was measured before the viscera have removed, as shown in (Table 4). The results recorded significant differences in the dry chitin weight between the tested NPs at 1.05, 0.65 and 1.62 mg, respectively, compared to 3.06 mg of the control larvae. The ratio of chitin formation was calculated according to the fresh weight of the fourth instar larvae.

The obtained results in (Fig. 7), indicating that the tested ZnO NPs could be considered the least chitin formation ratio in the fourth instar followed by SiO₂ NPs and finally Al₂O₃ NPs, were recorded 16.91, 21.94 and 29.09%, respectively, compared to 39.01% of control larvae.

DISCUSSION

Results of the toxicity index of NPs against the newly hatched larvae of E. insulana showed that ZnO NPs were the most potent compound, followed by SiO₂ and Al₂O₃ NPs. These results agreed with the findings of Derballah et al.⁴ confirmed that ZnO NPs were the highest toxicity against the first instar larvae of P. gossypiella, followed by silica, with LC₅₀ values at 11.29 and 37.78 ppm, respectively. Also, Haroun et al.¹³ proved that ZnO and SiO₂ NPs were the most efficient treatments on the C. maculatus and S. oryzae at the highest concentration while T. castaneum displayed high defense against the tested components; they have recorded mortality at 98, 98,3 and 57% of SiO₂ NPs for the previously mentioned insects, respectively, moreover 88.3, 100 and 36% of ZnO NPs, respectively. In the same direction, El-Shennawy and Kandil¹⁴ indicated that ZnO NPs was the most toxic compound against E. insulana larvae when treated by it in the first instar larvae, compared to other traditional insecticides. The current results disagreed with those of Debnath et al.¹⁵ who reported that Al₂O₃ NPs were more toxic than ZnO NPs against L. pseudo brassicae.

The ratio of the cumulative larval mortality of E. insulana scored the highest mortalities when larvae were treated with various concentrations of ZnO then SiO₂ NPs. The current results are due to the absorption of ZnO and SiO₂ NPs through the larval cuticle lipid, which is considered lethal due to desiccation. As NPs have a massive surface area, they caused deformation and abrasion in the water barrier of the insects' cuticle. The bodies of insects began to lose water and finally died. The dead larvae became excessively dried and shrunken compared to the live ones. These results concurred with Rahman et al.²³ indicated the absorption of ZnO and SiO₂ NPs into the cuticular lipids and the penetration of the water barrier of E. insulana. Causing an imbalance of the protective wax layer resulted in the desiccation and death of these insects. Goswami et al.¹⁰ investigated the insecticidal features of ZnO and Al₂O₃ NPs to control many insect pests. These results also agreed with Osman et al.²⁴ showed that the highest larval mortalities scored 86 and 83% at the maximum concentration of ZnO and SiO₂ NPs, respectively. These results confirmed with El-Ghoharyand El-Samahy¹² illustrated that SiO₂ NPs were the most operative treatment against E. insulana compared to other bio-insecticides after 15 days post-treatment. Additionally, Benelli²⁵ hypothesized that the mortality rate induced by the treatment of ZnO NPs could be caused by the passage of these ultra-small particles through the cuticle of mosquito larvae and intervene in molting processes.

There are no significant differences between the weights of the first, third instar larvae and pupae of E. insulana when treated with ZnO and SiO₂ NPs. Although most of the results showed significant differences in the weight of the larvae and pupae when treated with ZnO NPs compared to SiO₂ and Al₂O₃ NPs, at the same time, when compared to the untreated larvae. This result was due to the small size, surface area of zinc particles and their high density compared to other NPs. Several studies managed to clarify the structural properties of ZnO NPs, as it characterized by high density and sharp peaks that reflected the greater crystal size and high crystallinity^{26, 27}. Furthermore, SiO₂ NPs were less crystalline and intensive than ZnO NPs. The current results agreed with Farnaz et al.²⁸ indicated that Al₂O₃ NPs were the low density and less diversified in crystallization and it has spherical shapes compared to the hexagonal forms of ZnO NPs. According to the previously listed data, El-Samahy et al.²⁹ showed that one of the most significant characteristics of SiO₂ NPs is their capacity to prevent insects from feeding directly after food treatment with these particles. In the same context, El-Shennawy and Kandil¹⁴ emphasized that when using ZnO NPs against newly hatched larvae of E. insulana, it caused highly decrease in larval and pupal weight by approximately 10 and 13.6 times, respectively, compared to the control. The reduction in larval and pupal weight recorded 91 and 93.7%, respectively.

The current results also indicated that the growth rate inhibition values decreased gradually until the fourth instar larvae. In addition, ZnO NPs caused the highest growth rate inhibition during the various instar larvae and pupae of E. insulana, while Al₂O₃ NPs was the least effective one. These outcomes are similar to those recorded by Massey³⁰ who observed a reduction of feeding efficacy and growth rates of the Spodoptera by treating them with SiO₂ NPs. The results of many previous studies are consistent with the current data in the growth rate inhibition when treated with different types of NPs against some insects, such as ZnO and Al₂O₃ NPs by Goswami et al.¹⁰ and Salem et al.³¹; SiO₂ NPs by Debnath et al.³²; ZnO by Kadarkarai et al.³³.

The present results showed significant differences between the larval and pupal periods of E. insulana for different treatments; meanwhile, these differences did not appear between SiO₂ and Al₂O₃ NPs treatments in the pupal period. These results are attributed to the increase in the developmental periods when dealing with NPs more than the control samples. At the same time, they recorded a remarkable decrease in the developmental rate of larval and pupal stages. These results agreed with previous reports of development duration by El-Shennawy and Kandil¹⁴ recorded that the larval and pupal periods were exceptionally prolonged post-treatment with ZnO NPs against newly hatched larvae of E. insulana. Whereas, El-Helaly et al.³⁴, Shaker et al.³⁵ revealed that the newly hatched larvae of S. littoralis were treated with SiO₂ NPs at different concentrations gave prolonged larval and pupal durations. Furthermore, the treatment of the second instar larvae of S. littoralis with tested NPs at LC₅₀ values significantly increased the larval duration, as compared with the control. In this regard, the results of Al-Bandari and EL-Healy³⁶ also indicated that a highly toxic activity of SiO₂NPs after 15 days post-treatment against S. littoralis negatively affected the developmental rate of larvae and pupae.

Results of the larval and pupal malformations rate agreed with El-Shennawy and Kandil¹⁴ showed that the highest ratio of malformations in E. insulana larvae, when treated with ZnO NPs was 12% compared to untreated larvae at 1%. The deformations of the treated larvae appeared as the abnormal color of larval bodies, resulting from cuticle pigment and disability to shedding out the previous cuticle, which causes the failure of molting or metamorphosis and then death. Likewise, the pupa couldn’t fully form its cocoon and thus the pupation process has not been completed. The toxic effects of NPs were attributed to their small size and large surface area, which helped to penetrate the cuticle cells and increase the interaction with them, according to Medina et al.⁷.

Treatment with ZnO NPs scored the least ratio of chitin formation in the fourth instar larvae, followed by SiO₂ NPs and finally Al₂O₃ NPs, compared to control larvae. These interpretations were in agreement with the previous findings of many authors, such as Martinez and Van Emden³⁷, El-Sabrout et al.³⁸, Abdelgaleil and El-Sabrout³⁹. They studied the insecticidal effects on cuticle stretching and inhibiting the larval growth rate of the cotton leaf worm, consequently on chitin formation. Furthermore, nanoparticle action is based on drying the insect's cuticle by absorbing the lipids, which produces damage in the cell membrane, causing cell breakdown and insect dead⁴⁰. Previous studies have confirmed that NPs of metal oxides are efficient against insects. Hence, NPs have been applied in the novel formulations of insecticides^31,41,42.

CONCLUSION

To sum up, the effectiveness of zinc, silica and aluminum nanoparticles was tested as insecticides against the Spiny Bollworm, causing growth disturbances and abnormalities in the larvae and pupae. The possibility of using these unconventional agents to control the Spiny Bollworm is possible if commercial production at a low cost is taken into consideration. Hence, the tested nanoparticles find a promised place in pest control strategies due to their low environmental risks.

SIGNIFICANCE STATEMENT

This study discovers three nanoparticle oxides that can be used in the control of agricultural pests, particularly those cause losses in cotton production in Egypt. Also, Low doses of nanoparticles, as environmentally friendly tools, give positive results. This study will help the researchers to discover other nanoparticles that many researchers have not been able to explore, which could have an actual effect on integrated pest management programs. This research can also be considered a new point for integrating nanoparticles with other chemical pesticides to reduce their toxicity to humans and other non-target organisms.

ACKNOWLEDGMENT

The authors are grateful to the Department of Zoology, Faculty of Science, Zagazig University, Egypt, for providing all the necessary laboratory facilities.

REFERENCES