Toxicological and Histological Evaluation of Insecticides and Essential oils in Nano emulsion and formulated Form against Spodoptera littoralis

1.INTRODUCTION

The Egyptian cotton leafworm Spodoptera littoralis (Lepidoptera: Noctüidae) is one of the most destructive pests to about 112 host plants from different families. Infestation often leads to significant crop losses and reduced marketability (Ismail et al. 2020). The most effective control of S. littoralis has been achieved by using chemical pesticides. Extensive use of these pesticides led to the development of resistance and caused many other problems such as environmental pollution and harm to beneficial non-target organisms (Hilliou et al. 2021).

Traditional pesticide formulations suffer from multigate problems such as poorly or insoluble in water, so large amounts of organic solvents are required to solubilize them. Most of the organic solvents contaminated the environment (Sanni and Mutta 2014). Currently researchers the use of Nano formulations of insecticides or essential oils similar traditional formulas, but with enhanced features such as more soluble, slower to release, not prematurely degradable, the effective concentration that is expected to be much lower compared to original materials and they could be formulated in water without organic solvents (Bhattacharyya et al. 2010).

Therefore, this study aims to evaluate the lethal toxicity of the nano - and commercial formulated form of essential oils; peppermint oil and eugenol and insecticides; chlorpyrifos and chlorfenapyr on 4^th instar larvae of S. littoralis. In addition, to analyze the morphological damage caused by tested essential oils and insecticides to the target organs of S. littoralis larvae: the midgut, epithelial cells.

2.MATERIALS AND METHODS

2.1 Insects

Laboratory strain of the S. littoralis was received from Faculty of Agricultural, El-Shatby, Alexandria University, Egypt and was reared on castor bean plant leaves under controlled conditions at 25 ± 1 ºC and 65 ± 5 % R.H for a period 24 hr (16L:8D) without any exposure to insecticides.

2.2. Essential oils (EOs) and insecticides used

peppermint essential oil and eugenol that were obtained from El-Gomhoria Co., for Chemicals supplies in Alexandria, Egypt. The peppermint essential oil main compounds (Eucalyptolss 11.26%, D-menthone 19.7%, Isopropyl-5-methylcyclohexanone 13.26%, Menthol 43.43%, γ-Terpineol 8.07%, Caryophyllene 4.29%) according to Wahba, and Attia (2019).   Two commercial insecticides; chlorfenapyr (challenger 24 % EC) and chlorpyrifos (Indophose 50%EC) were obtained from BASF Chemicals Co. Egypt and Indogolf group Sci., of Pesticides-India respectively.

2.3.Preparation of insecticide nano-emulsions

The oil-in-water nano- emulsions were prepared by the procedure previously reported bySugumar et al. (2013) The chlorfenapyr and chlorpyrifos insecticides were used to prepare 1% nano- emulsion. In the final form the emulsion was prepared by adding one portion of the organic phase containing chlorfenapyr or chlorpyrifos insecticides in 95% technical grade dissolved in one portion of Dimethyl sulfoxide (DMSO) and three portions of olive oil in ratios 1:3:1 (v/v/v) this mixture was added in small portion to a conical flask that containing the aqueous phase which consists of the deionized water containing one portion of surfactant (tween 80), all these steps were carried out   under magnetic stirring at approximately 800 rpm for 15 min. Then the prepared formulation was subjected to ultrasonic emulsification  and subjected to a 75 kHz Sonicator for 30 minutes. Sonicator probe diameter was13 mm dipped into coarse emulsions under cooling conditions. Keeping the emulsion sample beaker in a comparatively bigger beaker containing ice to keep the solution at a constant temperature. Then, the nano-formulated form was characterized to evaluate stability of the new nano-formulation by a dynamic  light  scattering  (DLS)  method  using  Zetasizer Nano ZS (Malvern Instruments, model: ZEN3600, UK).

2.4. Preparation of essential oils nano-emulsions.

The oil-in-water nanoemulsion was formulated using a modified version of the method described by Sugumar et al., (2014). Eugenol and peppermint oil, surfactant (tween 80) and deionized water. Initially, coarse emulsion was prepared by adding 40ml water to organic phase containing 5% oil and 5% surfactant at ratio 1:1 (v/v) using a magnetic stirrer at 800 rpm for 15m. Then subjected to ultrasonic emulsification using a 75 kHz Sonicator for 30 minutes. Sonicator probe diameter was13 mm dipped into coarse emulsions under cooling condition. Keeping the emulsion sample beaker in a comparatively bigger beaker containing ice to keep solution at constant temperature. Then, the nano-formulated form was characterized to evaluate stability of the new nano-formulation.

2.5. Technique of the tested insect

Insecticides and oils concentrations were applied to the fourth instar larvae of S. littoralis.  Using the method of feeding treatment was applied as Disc dipping technique.The method of Ishaaya and Klein (1990) was conducted and used to evaluate the efficacy of essential oils, pesticides and nano-emulsions of them against fourth instar larvae of the cotton leafworm S. littoralis. Each castor bean leaves disc (2 cm²) was dipped in each essential oil, pesticides and nono-emulsions for 15s. Discs and held vertically to allow the excess solution to drip off at room temperature (25◦C) to dry.  The treated leaves were placed at the bottom of glass jars containing 10 larvae of 4^th instar cotton leaf worm three replicates were made for each concentration and three untreated check with distilled water were used. The percentage of mortality was calculated each concentration for recorded after 24 hours of application to insecticides and after 72 hours to oils, corrected for natural mortality according to Abbott equation (Abbott, 1925). Results were illustrated graphically as probit regression lines, and toxicity LC₅₀ values as well as the slope were obtained according to Finney, (1971).

2.6 Histopathological studies:

The effect of each of the four tested compounds at LC₅₀ values was studied on the cellular structure of 4^th instar larvae surviving treatment. The specimens from S.  littoralis larvae samples were collected and fixed in 10% buffered neutral formalin solution. Paraffin sections of 5 microns thickness were prepared and stained with haematoxylin and eosin according to Bancroft et al. (1990) and examined microscopically. The show in cross section was mid gut  microscopically (200 µm). All sections of S. littoralis larvae were done at faculty of Medicine Alexandria University.

2.7. Statistical analysis

The data were calculated as mean ±SE and analyzed statistically. Statistical significant differences between individual means were determined by one-way analysis of variance (ANOVA) using Costat software. Mean values were analyzed with Tukey’s test at the 0.05 level or less.

3.RESULTS

3.1.Toxicity of nano and traditional formulation of chlorpyrifos and chlorfenapyr on fourth instar larvae of Spodoptera littoralis.

The toxicity of chlorpyrifos and chlorfenapyr, formulations for nano and traditional formulate tested against the fourth instar larvae of S. littoralis are given in Table (1) and Figure (1). It was shown that nano chlorpyrifos was 10.33 times more toxic than traditional formula while nano chlorfenapyr was 2.77 times more toxic than traditional formula after 24hr post-treatment. While LC₅₀ values were 0.0031% and 0.0061 % for  both traditional formulations of chlorpyrifos and chlorfenapyr, respectively.

The data show a very narrow effect on confidence limits in statical analysis Also, the sharpener of slope. Comparing the slope values of the toxicity lines of these insecticides data in Table (1) show that commercial chlorpyrifos had a high slope value of toxicity line with a 1.92 slope value.

Table 1. Toxicity of nano emulsion and traditional formulations of chlorpyrifos and chlorfenapyr on fourth instar larvae of Spodoptera littoralis.

Fig. (1): Toxicity of nano emulsion and traditional formulations of chlorpyrifos and chlorfenapyr on fourth instar larvae of Spodoptera littoralis treated with feeding methods.

3.2Toxicity of nano and traditional formulate emulsions of peppermint oil and eugenol on fourth instar larvae of Spodoptera littoralis.
The toxicity of nano and traditional peppermint oil and eugenol formulations of nano and traditional formulate tested against the fourth instar larvae of S. littoralis are given in Table (2) and Figure (2). Traditional peppermint oil and eugenol formulations shows insecticidal toxicity. LC₅₀ values were 3.76% and 3.82% for traditional peppermint oil and traditional eugenol, respectively. The LC₅₀ values were 1.48%, 2.46% for nano peppermint oil and nano eugenol respectively. While, after 72hr post-treatment. In general, two nano forms have highly significant effect than the traditional forms. Also, our data indicated that the nano- peppermint oil showed more effective 2.54 folds than raditional peppermint oil, while nano- eugenol was most effective 1.55 folds than traditional eugenol.  The data show a very narrow effect on confidence limits in statical analysis which mean the very sharp data calculate the effect of eugenol or peppermint oil nano or traditional formulation which give high confidence limit for the results obtained. Data in Tables (2) show that peppermint oil and eugenol had very narrow slop values ranging between 1.68 to 1.67.

Table 2. Toxicity of nano and traditional formulation emulsions of on peppermint oil and eugenol fourth instar larvae of Spodoptera littoralis.

Fig. (2): Toxicity of nano and traditional formulation emulsions of on peppermint oil and eugenol fourth instar larvae of Spodoptera littoralis treated with feeding methods.

1.1.      Effect of physical charactrisation of nano-emulsion

The data show in Table (3) that formulations droplet size decrease from 401.2 to 177.6 nm after sonication for chlorpyrifos traditional and nano formulation Fig (3). However, the droplet size was 1453 to 90.23 nm after sonication for chlorfenapyr traditional and nano formulation Fig (4) . However, the droplet size was 543.7 to 188.6 nm after sonication for eugenol conventional and nano formulation Fig (5).  Also, formulations droplet size decrease from 599.8 to 66.16 nm after sonication for peppermint oil traditional and nano formulation Fig (6).The droplet size was 177.6 to 90.23 nm for chlorpyrifos and chlorfenapyr nano formulation respectively.  Also, Fig (7) show the appearance of emulsions prepared of  nano-emulsion (O/W) of eugenol, peppermint oil essential oil, chlorpyrifos, chlorfenapyr before Sonication and After Sonication. This indicates that when the droplet size decreased in comparison between nano and Traditional formulation, the toxicity increased 2.27 folds of chlorpyrifos and 16.10 fold of chlorfenapyr.

the droplet size varied from 188.6 to 66.16 nm (Table 3) for eugenol and peppermint oil, respectively. The droplet size decreased in comparison between nano and traditional coarse, the toxicity increased 9.07 folds of peppermint oil and 2.88 fold of eugenol Which indicates that the toxicity of both eugenol and peppermint oil increase with decreeing droplet diameter.

Table (3) show that PDI (poly dispersity index) value decrease from 0.885 to 0.460 nm and 0.603 to 0.398 nm after sonication for chlorpyrifos and chlorfenapyr respectively, and also, it was decrease from 0.616 to 0.366 nm and 0.976 to 0.206 nm after sonication for eugenol and peppermint oil respectively.

Table (3). Physicochemical properties for different course and nano-emulsions.

Histological Studies:

Histopathological effect for the tested insecticides on midgut tissues of the cotton leafworm, Spodoptera littoralis 4^th instar larvae are shown in Fig (8–12).

1.1.     Histological studies on midgut of Spodoptera larvae

        The foregut of lepidopteran larvae is composed of an esophagus and an enlarged tisenes, which holds food prior to its passage into the midgut then the hindgut. The transverse sections of treated and check untreated larvae were determinate at a thick of 5μm which were cut in medial larval body where found the midgut.  The slides were examined by microscope (100X, 400X). The aim of this study to evaluate the effects of two insecticides (Chlorpyrifos and Chlorfenapyr) and two essential oils (eugenol and peppermint oil) on histological structure and their function on the cell level, comparing with untreated 4^th instar larvae of cotton leaf worm midgut section.

      Cross sections of untreated 4^th instar larvae of Spodoptera littoralis which showed that midgut was performed after 24 hrs of feeding on fresh discs of castor bean leaves (Figure 8).

Figure (8): Transverse section of untreated 4^th instar larvae cotton leaf worm midgut.

⃰ MgT; Malpighian Tubules, CM; Circular Muscles, PM; Peritrophic Membrane, MV; Microvilli, LM; Longitudinal Muscles, Ep; Epithelium, F; Food.

1.2.    Effects of Nano and traditional treatments of Chlorpyrifos on 4th instar larvae of Spodoptera littoralis midgut

      Traditional treatment of Chlorpyrifos in comparison with the untreated 4^th instar larvae of Spodoptera littoralis midgut which is illustrated in Fag (9, A), the data showed that undifferentiated epithelial cell, appears on lumen and large vacuoles after treatment by traditional formulation of chlorpyrifos. While after treatment the 4^th instar larvae of cotton leaf worm by nano-formulation of chlorpyrifos, the data revealed separated peritrophic membrane, microvilli and many small vacuoles (Fig 9, B). This data are in agreements a with which found before that nano- chlorpyrifos is 10.33 times toxic than traditional chlorpyrifos due to nano more damage inside the cell. Also, data agree with which found before by El-Sabrout et al (2013) and Abd-El-Aziz et al (2019) that nano treatment were observed large numbers of vacuoles which meaning decrease in epithelial cell which lead to decreasing of insect digestion activities.

1.3.Effect of Nano and traditional treatments of Chlorfenapyr on 4th instar larva of Spodoptera littoralis midgut

Traditional treatment of chlorfenapyr in comparison with the untreated 4^th instar larvae of Spodoptera littoralis midgut which is illustrated in Fag (10, A), the data showed that undifferentiated epithelial cell after treatment by traditional formulation of chlorfenapyr. While after treatment the 4^th instar larvae of cotton leaf worm by nano-formulation of chlorfenapyr, the data revealed incomplete peritrophic membrane, clear separated malpighian tubules and many separated vacuoles (Fig 10, B).  the data revealed that nano-treatment of chlorfenapyr showing incomplete peritrophic membrane and smaller size of epithelial cell and undifferentiated epithelial cell which in agree with found before by (Saleh et al. (2021).

1.5.Effect of Nano and traditional treatments for eugenol on 4th instar larva of Spodoptera littoralis midgut.

Traditional treatment of eugenol in comparison with the untreated 4^th instar larvae of Spodoptera littoralis midgut which is illustrated in Fag (11, A), the data showed that slight effect on undifferentiated epithelial cell and incomplete peritrophic membrane after treatment by traditional formulation of eugenol. While, after treatment the 4^th instar larvae of cotton leaf worm by nano-formulation of eugenol, the data revealed high effect of separated peritrophic membrane, cutting on theLongitudinal Muscles, cutting in Circular Muscles, Microvilli and many vacuoles (Fig 11, B). The data shows that nano- eugenol effect on midgut section by separated peritrophic membrane which agree with which found before by Hanan et al., (2020) and clear that junk food which contain dangerous tupes of oils and lipids can does a serious bad effect on cell performance.

1.6.Effect of Nano and traditional treatments of peppermint oil on4th instar larva of Spodoptera littoralis midgut

     Traditional treatment of peppermint oil in comparison with the untreated 4^th instar larvae of Spodoptera littoralis midgut which is illustrated in Fag (12, A), the data showed that cutting in circular muscle, appears of vacuoles and absent peritrophic membrane after treatment by traditional formulation of peppermint oil. While after treatment the 4^th instar larvae of cotton leaf worm by nano-formulation of peppermint oil, the data revealed effect on cutting the peritrophic membrane, effect the circular muscles, appears of microvilli, effet on basement membrane and large vacuoles (Fig 12, B).   The data shows that the layers of both longitudinal and circular muscles were separated from epithelial layer with nano- peppermint oil treatment which in agree with which found by Abd-El-Aziz et al (2019). Other traditional pesticide formulations are relatively poor, which enables pesticide absorption and penetration difficult, severely limiting their biological activity. The size of the droplets can considerably increase their adherence and permeability on the target insects by increasing the biological activity of insecticide and/or decreasing their applied amounts. A nanoemulsion with smaller droplets, high wetting performance, rapid diffusion, and penetration at the target surface is advantageous for increasing pesticide biological activity.