Effects of Piper nigrum L. Fruit Essential Oil Toxicity against Stable Fly (Diptera: Muscidae)

The efficacy of Piper nigrum L. fruit essential oil (EO) against Stomoxys calcitrans (stable fly), a blood-feeding fly distributed worldwide, was investigated. This study aimed to evaluate the insecticidal activity of EO based on contact and fumigant toxicity tests. Chemical analysis of the EO using gas chromatography–mass spectrometry revealed that sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%) were the major components. The results demonstrated that fly mortality increased with increasing EO concentration and time during the first 24 h of exposure. The median lethal dose was 78.37 µg/fly for contact toxicity, while the 90% lethal dose was 556.28 µg/fly. The median lethal concentration during fumigant toxicity testing was 13.72 mg/L air, and the 90% lethal concentration was 45.63 mg/L air. Our findings suggested that essential oil extracted from P. nigrum fruit could be a potential natural insecticidal agent for control of stable fly. To examine the insecticidal properties of P. nigrum fruit EO, further field trials and investigation into the efficacy of nano-formulations are required.


Introduction
Stable fly [Stomoxys calcitrans (Diptera: Muscidae)], an insect belonging to the order Diptera, is distributed worldwide. Stable flies carry bovine life-threatening viral pathogens including lumpy skin disease virus, bovine herpes virus, and bovine leukosis virus [1]. Furthermore, in the equine industry, stable fly acts as a mechanical vector of equine infectious anemia virus [1,2] and Trypanosoma evansi [3]. Moreover, stable fly plays an important role as the biological vector of the equine stomach nematode Habronema microstoma [4]. This vector has been demonstrated to cause economic impact in equine and livestock husbandry by disease transmission, annoyance, and stress in affected animals [1,5].
Various chemical products have been used to control insect vectors. In an experiment involving stable flies, the toxicity of semiochemicals including aliphatic alcohols, terpenoids, ketones, and carboxylic esters was studied, with the median lethal concentration (LC 50 ) ranging from 16.30-40.41 µg/cm 2 [6]. Recently, cypermethrin (group A) demonstrated an excellent repellent effect with an LC 50 of 1.52 µg/mL [7]. However, the emergence of flies resistant to these chemical substances has been reported worldwide [8,9], and furthermore the chemical residue causes environmental pollution [10].
To control diseases caused by the stable fly vector using green or environmentally friendly products, plant essential oil (EO) has been proposed as an alternative method for insecticidal and repellent insect-vector control. By its volatile properties, EO affects the flight behavior of stable fly and peripheral olfactory networks in the antennae [11]. A repellency study in stable fly using a human hand bioassay demonstrated that various EOs such as patchouli [Pogostemon cablin (Blanco) Bentham], clove (Eugenia caryophyllata

Essential Oil Extraction and Quantification
The yield of P. nigrum L. EO obtained from fresh fruit was 0.96% volume/weight (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C , and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively.

Essential Oil Extraction and Quantification
The yield of P. nigrum L. EO obtained from fresh fruit was 0.96% volume/weight (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C , and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively.

Essential Oil Extraction and Quantification
The yield of P. nigrum L. EO obtained from fresh fruit was 0.96% volume/weight (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C , and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively. The yield of P. nigrum L. EO obtained from fresh fruit was 0.96% volume/weight (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C , and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively. The yield of P. nigrum L. EO obtained from fresh fruit was 0.96% volume/weight (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C , and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively. The yield of P. nigrum L. EO obtained from fresh fruit was 0.96% volume/weight (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C , and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively. The yield of P. nigrum L. EO obtained from fresh fruit was 0.96% volume/weight (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C , and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively. (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C , and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively. (v/w). The oil was clear in color with a pH of 5, a density of 0.87 g/mL at 20 °C , and a refractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively. fractive index of 1.48. The chemical constituents of P. nigrum EO were determined by GC-MS techniques. A total of twenty-one compounds were identified accounting for 99.41% of the total oil including the retention time (min), classes, compounds, formula, chemical structure, peak area (%), and % similarity index (Table 1). A chromatogram of the primary components of P. nigrum EO is shown in Figure 1. The major components were sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%). Limonene and β-caryophyllene were used to quantify the EO, and the concentrations were 0.18% and 0.10% weight/volume (w/v), respectively.

Contact Toxicity Test
The contact toxicity of P. nigrum EO against S. calcitrans was studied at various EO concentrations. The test was validated using acetone, with no insecticidal activity, as a negative control. When EO treatments at 43.25 µg/µL, 86.50 µg/µL, 216.25 µg/µL, and 432.50 µg/µL were compared with cypermethrin, EO was found to have little to no insecticidal efficacy (

Fumigant Toxicity Test
The fumigant toxicity of P. nigrum EO against S. calcitrans was assessed at various EO concentrations. The test was validated using acetone, which has no insecticidal activity, as a negative control. At EO concentrations < 25.95 mg/L, the EO had little or no insecticidal action for S. calcitrans (Table 4). From 6-24 h after treatment, the EO at a concentration of 25.95 mg/L demonstrated insecticidal activity comparable to cypermethrin. The results showed that S. calcitrans mortality increased with the increased EO concentration and time. The interaction between concentration and time was statistically significant for S. calcitrans mortality (time, F (2.28, 31.97) = 75.47, p < 0.001; treatment, F (6, 31.97) = 158.11, p < 0.001; treatment × time, F (13.70, 31.97) = 24.20, p < 0.001). The LC 50 and LC 90 values were 13.72 mg/L air and 45.63 mg/L air, respectively (Table 5).

Discussion
This investigation utilized P. nigrum fruit EO for in vitro contact and fumigant toxicity testing against wild-caught stable flies from a rural horse farm. Since the wild-caught flies varied in age, sex, and nutritional and physiological status, these factors should be considered when comparing the results from this study with those of laboratory studies [26]. While the laboratory-reared stable fly is suitable for insecticide-sensitivity and vectorcompetency experiments under controlled conditions [1,27], the field-caught stable fly might better represent the actual response to the plant's insecticidal toxicity under real field conditions [16,28].
Previous reports have described the repellent and insecticidal effects of P. nigrum EO. The fumigation activity of P. nigrum EO has been reported against Tribolium castaneum (Coleoptera: Tenebrionidae) [25,29]. EOs obtained from black pepper had repellent, insecticidal, oviposition inhibitory, and acetylcholinesterase (AChE) enzyme inhibitory activities against maize weevil, Sitophilus zeamais [30]. A fumigation toxicity assay measured an LC 50 of 0.287 µL/cm 3 and 0.152 µL/cm 3 after 24 and 48 h exposure periods, and a contact toxicity assay reported an LC 50 of 0.208 µL/cm 2 and 0.126 µL/cm 2 after 24 and 48 h exposure periods, respectively [30]. In addition, chitosan-encapsulated nanoparticles of P. nigrum EO demonstrated insecticidal activity via the AChE function against insect pest species T. castaneum and Sitophilus oryzae [31].
This study noted a contact toxicity LD 50 of 78.37 µg/fly and an LD 90 of 566.28 µg/fly 24 h after exposure, which differs slightly from the toxicity of citrus EO for the stable fly (LD 50 of 105.88 µg/fly and LD 90 of 499.25 µg/fly). However, the fumigant toxicity values of an LC 50 of 13.72 mg/L air and an LD 90 of 45.63 mg/L air were consistent with a previous report (LC 50 of 13.06 mg/L air and LC 90 of 43.13 mg/L air) [16]. Furthermore, the percentage of mortality from citrus EO at 25.20 mg/L air was similar to that of 1% cypermethrin, from 2-24 h after treatment. This was somewhat better than the results of the current study (25.95 mg/L air from 6-24 h after treatment). Limonene (93.79%) is the major compound found in citrus EO [16], while sabinene (24.41%), limonene (23.80%), β-caryophyllene (18.52%), and α-pinene (10.59%) are the major compounds found in P. nigrum EO. Prior studies have reported the insecticidal effects of compounds found in Plants 2023, 12, 1043 8 of 13 essential oil. Sabinene is reportedly insecticidal against Aphis pomi [38], has a repellent effect against Tribolium castaneum [39], and exhibits strong fumigant toxicity against maize weevils [40]. Limonene has insecticidal activity against mealybugs and scale insects [41], horn flies [42], and the tomato leaf miner [43]. Combinations of these compounds can exert insecticidal activity. Sabinene has additive effects with α-pinene, 1,8-cineole, 1-octen-3-ol, and linalool, and limonene has a synergistic effect with α-pinene and sabinene [44]. βcaryophyllene and α-pinene are toxic to Aphis gossypii Glover (Homoptera: Aphididae) by inhibiting AChE activity, polyphenol oxidase, carboxylesterase, and glutathione Stransferases in A. gossypii [45].
The cytotoxicity of P. nigrum EO has been investigated on a normal cell line, human fetal lung fibroblast (MRC-5), and five different tumor cell lines including human cervical carcinoma (HeLa), human myelogenous leukemia (K562), lung adenocarcinoma (A549), colon cancer (LS-174), and melanoma (FemX). The results indicate that P. nigrum EO exhibited lower cytotoxic activity, with an IC 50 of 25.57-56.74 µg/mL after 48 h of treatment compared with cisplatin (IC 50 , 2.34-9.08 µg/mL) as a positive control [46]. The toxicity of P. nigrum EO at concentrations of 5, 10, or 50 mg/kg was evaluated for 21 days in adult male albino Swiss mice. None of the P. nigrum EO doses induced signs of toxicity, such as irritability, reaction to contact, gripping the tail, twisting, writhing, grip strength, ataxia, tremor, convulsion, lacrimation, piloerection, changes in respiratory frequency, changes in body weight, or abnormalities in biochemical parameters [47]. Based on the LD 90 of 556.28 (325.89-1475.26) µg/fly and the LC 90 of 45.63 mg/L air obtained in the current study, we expected that the P. nigrum EO used in this study should not affect horses and livestock, the primary hosts of the stable fly.
Although this study did not investigate the molecular mechanism of EO effect, the insecticidal activities of EO have been reported. Plant-derived EOs, primarily monoterpenes, exhibit their neurotoxic effects by the inhibition of AChE in the octopamine pathway in insects, alongside the essential detoxification enzymes glutathione S-transferase and catalase. They are also capable of inhibiting gamma-aminobutyric acid receptors [25,48,49]. However, P. nigrum EO and the compounds sabinene, limonene, β-caryophyllene, and α-pinene should be the subjects of future studies of the EO insecticidal mechanism of action.

Conclusions
This study demonstrated the toxicity of unripe P. nigrum fruit EO, primarily containing sabinene, limonene, β-caryophyllene, and α-pinene, as an environmentally friendly insecticidal agent against stable fly (S. calcitrans), using contact and fumigant toxicity tests. The synergistic effects of the components of this EO on the insect's neuromuscular system should be investigated further. As there are limited reports available discssing the insecticidal activities of P. nigrum fruit EO on horse and livestock vectors such as horse fly, horn fly, and midges, the study of P. nigrum fruit EO should be expanded and its nano-formulation for open field trials should be implemented.

Ethical Statement
This study protocol was approved by the Faculty of Veterinary Science, Mahidol University Animal Care and Use Committee (Ref. MUVS-2020-12-63).

Insects
Populations of S. calcitrans used in this study were collected using Nzi Traps [50], from a horse farm in Nakhon Pathom Province (13 • 45 43.4 N, 100 • 08 15.7 E), between March and May 2021. To the best of our knowledge, insecticides had never been used on that farm. The traps were set up at the collection site from 16:00 to 19:00. All flies were stored in plastic cups and transported in Styrofoam boxes containing ice packs to the Pharmacology Laboratory, Faculty of Veterinary Science, Mahidol University. Male and female S. calcitrans were tested and were selected from groups of undamaged flies under a stereomicroscope (SMZ745, Nikon, Tokyo, Japan). Blood-fed flies were separated out of this study. The samples were preserved between 27-29 • C and 70-80% relative humidity upon arrival at the laboratory until testing.

Essential Oil Extraction and Quantification
Piper nigrum L. fruit (Figure 2) was obtained from Nakhon Pathom Province in central Thailand (13 • 45 12.3 N, 100 • 16 13.2 E) and was pesticide-free. The plant was identified and delivered to the Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University (PBM No.005504-6). The EO was extracted from 50 kg of fruit, within 6 h of arrival, by the steam distillation process, where the fruits were immersed in water and the system was heated to 100 • C. The EO produced was stored in amber bottles at 4 • C until use. The EO yield is expressed in % (v/w) based on the fresh plant material weight.
Populations of S. calcitrans used in this study were collected using Nzi Traps [50], from a horse farm in Nakhon Pathom Province (13°45′43.4″ N, 100°08′15.7″ E), between March and May 2021. To the best of our knowledge, insecticides had never been used on that farm. The traps were set up at the collection site from 16:00 to 19:00. All flies were stored in plastic cups and transported in Styrofoam boxes containing ice packs to the Pharmacology Laboratory, Faculty of Veterinary Science, Mahidol University. Male and female S. calcitrans were tested and were selected from groups of undamaged flies under a stereomicroscope (SMZ745, Nikon, Tokyo, Japan). Blood-fed flies were separated out of this study. The samples were preserved between 27-29 °C and 70-80% relative humidity upon arrival at the laboratory until testing.

Essential Oil Extraction and Quantification
Piper nigrum L. fruit (Figure 2) was obtained from Nakhon Pathom Province in central Thailand (13°45'12.3″ N, 100°16'13.2″ E) and was pesticide-free. The plant was identified and delivered to the Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University (PBM No.005504-6). The EO was extracted from 50 kg of fruit, within 6 h of arrival, by the steam distillation process, where the fruits were immersed in water and the system was heated to 100 °C. The EO produced was stored in amber bottles at 4 °C until use. The EO yield is expressed in % (v/w) based on the fresh plant material weight. The EO's physical properties were analyzed as follows: color was visually verified by three different individuals, pH was measured with pH-indicator strips (Merck, Darmstadt, Germany), density was measured with a density meter (DA-100M, Tokyo, Japan), and the refractive index was calculated with a refractometer (RX-5000CX, Atago, Tokyo, Japan). The EO's physical properties were analyzed as follows: color was visually verified by three different individuals, pH was measured with pH-indicator strips (Merck, Darmstadt, Germany), density was measured with a density meter (DA-100M, Tokyo, Japan), and the refractive index was calculated with a refractometer (RX-5000CX, Atago, Tokyo, Japan).
The EO's chemical composition was determined using a GC-MS model 7890A-MS5975C (Agilent Technologies, Santa Clara, CA, USA) equipped with a DB-5HT capillary column (length, 30 m; inner diameter, 0.25 mm; film thickness, 0.1 µm). The sample was injected in split mode with a 1:10 split ratio. Helium was used as the carrier gas, with a flow rate of 1 mL·min −1 . The injection port temperature was 250 • C, and the column temperature schedule was as follows: 50 • C for 2 min followed by a rise to 250 • C at a rate of 10 • C per minute, then maintained at 250 • C for 5 min. The MS included an ion source temperature of 230 • C, an ionization energy of 70 eV, and a mass scan range of 350-550 amu. Components were identified by comparing their mass spectra with data from the Wiley 7th edition mass spectrometry library. Concentrations of the principal components were calculated by comparing the peak area of the sample with the peak area of the standard.

Contact Toxicity Test
Contact toxicity of the EO from P. nigrum L. to S. calcitrans was achieved by topical application as described by Leesombun et al., with some modifications [22]. Preliminary studies were performed to determine appropriate test range concentrations. The various concentrations of P. nigrum EO were prepared in a 1.5 mL microcentrifuge tube using ace-tone as a solvent. Five EO concentrations were used in this trial: 43.25 µg/µL, 86.50 µg/µL, 216.25 µg/µL, 432.50 µg/µL, and 865.00 µg/µL, representing 5%, 10%, 25%, 50%, and 100% (v/v) of S. calcitrans (mixed sexes). Flies were anesthetized at −20 • C for 30-45 s, and 0.5 µL of EO at each concentration was applied directly to the fly's chest using a micropipette. Acetone and cypermethrin (1% w/v) were applied at the same volume for negative and positive controls, respectively. Each treatment was performed in three iterations on 10 flies each time. After application, the treated flies were placed in a clear plastic cup (10 flies/cup), and the cup was covered with a fine mesh fabric and fastened with rubber bands. A honey solution (10%) on cotton was layered over the mesh fabric, for the flies to consume. The flies were brought to recovery at 27-29 • C and 70-80% relative humidity. Mortality was recorded at 1, 2, 4, 6, 12, and 24 h after treatment. Flies were determined to be dead when they did not move after being mechanically stimulated with a brush.

Fumigant Toxicity Test
Fumigant toxicity of the EO from P. nigrum active against S. calcitrans was examined according to the procedures reported by Leesombun et al., with some modifications [22]. This test was performed in a 1 L clear plastic container with a lid. Preliminary studies were performed to determine the appropriate range of test concentrations. Different amounts of EO (0.47, 0.93, 1.87, 2.8, and 4.67 mg) dissolved in 100 µL of acetone were pipetted separately onto a 55-mm diameter Whatman No. 1 filter paper (GE Healthcare, Buckinghamshire, UK) and placed on the bottom of a glass petri dish (55 mm diameter). The solvent on each filter paper in the petri dish was allowed to evaporate for 2-3 min, and then the petri dish was covered with a fine mesh fabric which was fastened with rubber bands to prevent contact between the filter paper and the flies. Acetone and cypermethrin (1% w/v) were used as negative and positive controls, respectively. The petri dishes were placed on the bottom of the plastic container. Honey solution (10%) on cotton wool was placed on the bottom of the plastic container. Mixed sexes of S. calcitrans were used for testing. Flies were anesthetized at −20 • C for 30-45 s, placed in the plastic containers, and each container was closed. Each treatment was performed with ten flies in 3 iterations. Flies were allowed to recover and were maintained at 27-29 • C with 70-80% relative humidity. Mortality was recorded at 1, 2, 4, 6, 12, and 24 h after treatment. Flies were determined to be dead when they did not move.

Data Analysis
Insect mortality was calculated and the mortality data were corrected using Abbott s formula [51], when the control mortality ranged between 5-20%. Normality and homogeneity of the variables were verified using the Shapiro-Wilk and Levene tests, respectively. Statistical comparisons of mortality between treatments were analyzed by one-way analysis of variance (ANOVA) followed by post hoc analysis (Tukey's honest significant difference test), in SPSS software version 21.0 (SPSS, Chicago, IL, USA). Repeated ANOVA and Greenhouse-Geisser corrections were applied to compare the effects of the treatment concentrations and exposure times on mortality, using SPSS version 21.0 software. Exposure interval was the repeated factor and insect mortality was the response variable, with treatment as the main effect. A p value < 0.05 was considered significant. Probit analysis to calculate toxicity values, mean lethal dose (LD 50 ) and 90% lethal dose (LD 90 ) at 24 h after treatment, and mean lethal concentration (LC 50 ) and 90% lethal concentration (LC 90 ) at 24 h after processing was performed using LdP series software (Ehab Mostafa Bakr, Dokki, Cairo, Egypt). The software can be downloaded free of charge from http://www.ehabsoft.com/ldpline/ and was accessed on 6 November 2022.