J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 61 http://jad.tums.ac.ir Published Online: March 31, 2022 Original Article Pediculicidal Activity of Foeniculum vulgare Essential Oil in Treatment of Pediculus capitis as a Public Health Problem Elham Jahanifard1,2, Hoda Ghofleh-Maramazi1, *Mona Sharififard1,2, Mohammad Mahmoodi Sourestani3, Amal Saki-Malehi4, Elham Maraghi4, Sima Rasaei5 1Department of Medical Entomology and Vector Control, School of Public Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 2Social Determinants of Health Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 3Department of Horticultural Science, Faculty of Agriculture, Shahid Chamran University of Ahvaz, Ahvaz, Iran 4Department of Biostatistics and Epidemiology, School of Public Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 5Department of Dermatology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran *Corresponding author: Dr Mona Sharififard, Email: sharififard-m@ajums.ac.ir (Received 2 July 2020; accepted 11 Feb 2022) Abstract Background: Pediculosis, caused by Pediculus spp is an important public health problem in urban and rural areas around the world. Natural compounds such as plant essential oils (EOs) have been suggested as a potential alternative for insect pest control recently. The purpose of this study was to investigate the toxicity of Foeniculum vulgare essential oil against the head louse, Pediculus capitis under laboratory conditions. Methods: Fennel essential oil components were analyzed using GC-mass apparatus. Immersion and contact filter paper bioassays were used to evaluate fennel essential oil toxicity at the two-fold concentrations of 2.5, 5, 10, 20, and 40% against nit and nymph/adult stages of the head louse. Results: Trans-anethole, α-Thujone, and limonene, which consisted of 76.08%, 10.37%, and 5.34% were the most com- ponents of fennel oil respectively. The LC50 values for the adult /nymphs were 11.5, 6.4, 3.9, 3.1 and 2.5% and LC99 values were 29.5, 15.2, 12.8, 10.8, and 7.4% at 10, 20, 30, 45 and 60 minutes after exposure respectively. The lethal times (LT50) for adults/nymphs were 5.2, 8.1, 9.5, 20.5, and 45.8 minutes and LT99 were 138.6, 91.3, 23.8, 21.7, and 13.9 minutes in the concentrations of 2.5, 5, 10, 20 and 40%, respectively. LC50 and LC99 values were 2.32% and 7.36% after 5 days for the eggs. Conclusion: Fennel essential oil at the concentration of 15% after 20min is suggested to develop as an appropriate for- mulation to evaluate in clinical trials. Keywords: Head lice; Pediculus capitis; Pediculusis; Foeniculum vulgare Introduction Pediculus capitis, a blood-sucking insect, belonging to the order Anoplura, the family Pe- diculidae, completes its life cycle on the hu- man head as obligatory ectoparasite (1, 2). The insect feeds several times during the day eve- ry two-three hour. Today, head louse is one of the most important health problems around the world from a village to urban areas (1–3). The main mode of transmission of this infestation is close personal contact and sharing of per- sonal stuff (1–3). Head louse infestation is a global health problem that often infests chil- dren in school ages (4 to 13 years), teachers and family members, and other people who Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 62 http://jad.tums.ac.ir Published Online: March 31, 2022 will be in contact with the infected children (1–5). It is higher in girls and women (2, 3). According to WHO reports approximately 6–12 million people are annually infected by head louse in different areas of the world. This public health problem is prevalent in many de- veloping countries where the primary health- care program of WHO is inefficient and hap- hazard. The prevalence of this infestation world- wide is variable from 0.7–50% and between less than 5% to over 40% among school children, respectively (1–3). The highest prevalence rate of this infesta- tion was seen in Central and South America (33%), followed by Africa (31%), Australia (19%), Asia (18%), North America (8%), and Europe (5%) (6). Epidemiological studies in schools in var- ious countries have shown the different frequen- cies of pediculosis; 13.6% in Mexico, 26.6% in Jordan, 15.3% in South Africa, 23.32% in Thai- land, 26.4% in Nigeria, and 28.3% in England (1–3). Head louse infestation in different regions of Iran has been reported as less than 6% to 30% (4). Varied prevalence rates have been observed in different provinces of Iran such as 4% in Urmia, 13.5% in Hamedan, 1.8% in Ker- man, 4.7% in Sanandaj, 7.6% in Qom Prov- ince, 27% in Sistan-Balochistan Province, and 0.47% in Isfahan (1–4). Pediculus capitis infestation is increasing in some areas in Iran along with other com- municable diseases (5). Growth of population, people's immigration from villages to cities, marginalization, and the establishment of sat- ellite settlements with minimal health facili- ties and welfare services can be the reasons for this increase (5). Although head louse does not transmit any disease to humans, it causes prob- lems such as itching, skin lesions, lymph nodes, and secondary fungal and bacterial infections, including yellow ulcers in severe infestation (6–8). It can be caused anemia, particularly in children, headaches, insomnia, bad morals, rest- lessness, and decentralization, especially in chil- dren, social embarrassment, isolation, and men- tal stress (1–8). Physical methods such as combing, sepa- rating hair lice from the hair, and scrubbing hair have been used to remove head louse infesta- tion in the past (9, 10). Accurate differentia- tion of nits and hair casts plays important role in treating P. capitis (11). Nowadays, anti-pe- diculosis compounds are recommended to treat the infestation (9, 10). Chemical control, as the main treatment for head lice, involves the use of a wide range of synthetic neurotoxic insec- ticides such as Permethrine %1, Malathion 0.5%, Lindan 1%, Permethrin 5%, Crotamiton 10%, Ivermectin 0.5% , Spinodad 0.9%, Pyre- thrins plus piperonal butoxide and Benzyl al- cohol 5% (9, 10). Most of these compounds may have harmful effects on the patients (10). The high prices of anti-lice products and lice resistance concerns in several countries, includ- ing Iran (5, 7). Therefore, today, the development of new anti-lice combinations with higher safety and performance is considered a serious ne- cessity. Alternative control compounds with novel mode of action, low mammalian toxicity and harmless environmental impact are needed to be developed in order to prevent and control vector- borne diseases. It seems the plant based on products and their main components, such as meutropoinids are good alternatives to chem- ical insecticides because of easily their extract- ed, biodegradability and little toxicity against mammals (12). Also, they are effective against a wide range of insect pests, including head lice, and unlike chemical pesticides, the problem of resistance development is occurring slowly (12, 13). Many of these compounds are found in the markets in unusual products offered as pedic- ulicides without proper evaluation (14). The anti-lice properties of the plant compounds may be enhanced by their lipophilic performance and cause better penetration and greater bioavail- ability in the insect body (15, 16). Several fac- tors which include the lipophilicity of the prod- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 63 http://jad.tums.ac.ir Published Online: March 31, 2022 ucts, the rate of diffusion through the cuticle and some physicochemical variables such as the density and the molecular structure of the EO components may affect the penetration rate and finally, contact toxicity of the EOs (18-19). So far, numerous plant essential oils have been studied to determine their pediculicidal prop- erties against head lice around the world (8, 13–26). Fennel (Foeniculum vulgare) is an aromatic herb of the family Apiaceae. This plant is known as a native plant in the Mediterranean and South- ern Europe, and warm weather is favorable for its growth. It is distributed in different parts of Iran, including Gorgan, northern Manjil, Balu- chistan and Azerbaijan (27). Fennel is one of the medicinal plants that have been introduced in Iran's herbal pharmacopoeia (27), which is known for its medicinal properties, including anti-nausea, digestibility, and diuretic proper- ties. Different parts of the fennel plant are con- sidered for essential oil extraction. There are more than 30 components in fennel essential oils, the most important of which are trans-an- ethole, funchon, limonene, α-pinene and estrag- ole (28–30). The main fennel oils have acari- cidal, anti-fungal, antibacterial properties, and have recently been shown to have a repellent effect against insects (28–30). Its efficiency has been proven against insect-borne diseases such as the mosquitoes Culex pipiens and Aedes ae- gypti (29, 30). The aim of this study was to determine the toxicity of F. vulgare essential oil against head lice under laboratory condi- tions. Materials and Methods Essential oil extraction The fennel plant was harvested from Razan area of Hamedan Province during the harvest- ing season in late summer and early autumn. The dried seed was used for essential oil ex- traction (28–30). The seed was crushed and pow- dered with an electric mill and was extracted by the Clevenger apparatus by water distilla- tion. For this purpose, every 100 to 150g of crushed fennel seed was extracted for 4 hours using a Clevenger apparatus. Extracted essen- tial oils were stored in the refrigerator at 4 °C and in the dark glass until the test was per- formed. Determination of Essential Oil Components Gas-chromatography-mass spectrometer (GC-MS) was used for the analysis and iden- tification of fennel essential oil components (GC Agilent 7890, MS Agilent 5975). It was equipped with HP–5MS column (30m× 0.25 mm× 0.25μm). For this purpose, the essential oil samples were first rehydrated with sodium sulfate (Na So4) and diluted with dichloro- methane, which was especially spectrophoto- metric. Then 0.2μl of diluted oil was taken by the micro sampler and injected into the GC- MS apparatus. The essential oil constituents were identified by comparing their retention indices, and mass spectra fragmentation with those in a stored Wiley 7n.1 mass computer library and those of the National Institute of Standards and Technology (NIST). Bioassay tests Adults, nymphs, and eggs of P. capitis were collected from the head of children 6–13 years old, who attended primary schools in Karoon County, Khuzestan Province from three schools over a 2-month period. The children were not previously treated with anti-lice products for at least 1 month and the head lice were col- lected using a fine- toothed anti-louse metal comb and transported to the Medical Entomol- ogy Laboratory of Ahwaz Jundishapur Uni- versity of Medical Sciences in glass jars with screw caps. Adults and nymphs test Fennel oil was dissolved in ethanol as sol- vent to obtain the following two-fold doses: 2.5, 5, 10, 20 and 40% (= 0.39, 0.77, 1.5, 3.1, 6.2mg/cm2). For evaluating pediculicidal activ- ity, the contact bioassay method was used. Pe- tri dishes lined with Wathman no.1 filter pa- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 64 http://jad.tums.ac.ir Published Online: March 31, 2022 pers (9cm in diameter) were treated with 1ml of different concentrations of EOs and control filter paper received 1ml of ethanol. After dry- ing of filter papers, batches of 10 adults and 4–5th instar nymphs of head lice were placed on each petri dish, containing a few strands of human hair, and the dishes were covered with lids. Treated and control groups were left in Petri dishes for 15min at 65±5% humidity in the dark chamber and incubated at 35±2 °C and then placed in Petri dishes with untreated filter papers and incubated under mentioned conditions (18, 31). Lice, exposed to the es- sential oil within 2h after collection. A control test was performed with lice placed on solvent ethanol-impregnated filter paper dried for 5 min. The plates with adults and nymphs of lice were observed by stereomicroscope at 10, 20, 30, 40, and 60min after exposure. Head louse death was defined as the ab- sence of movement of limbs and gut, with or without stimulation using forceps. Experiments were repeated at least three times (18). Ovicidal test Hair-containing eggs were cut from the stu- dent's heads with scissors and placed in a con- tainer and transported to the laboratory of Med- ical Entomology for bioassay testing. Ten via- ble louse eggs were dipped in 1ml of EOs so- lutions of 2.5, 5, 10, 20 and 40 % (= 6.2, 3.1, 1.5, 0.77 and 0.39mg/cm2) for 1 minute and then distributed in Petri dishes lined with damped Wathman no.1 filter paper. The control group was treated with ethanol as a solvent. Treated and control groups were incubated at 35±2 °C and 65±5% humidity chamber in darkness. The louse egg hatching was monitored daily under microscopic inspection until 7 days after hatch- ing of the control group. Louse eggs with closed operculum and nymphs inside were the crite- rion for embryo mortality. Experiments were repeated, at least, three times (14–16, 18). Data Analysis The probit regression model was used for determining lethal doses (LD50 and LD99) and lethal times (LT50 and LT99). The P-value and χ 2- tests were used to assess the significance and goodness of fit to the probit regression mod- els, respectively. The mortality means with fen- nel oil were corrected with Abbott`s formula using natural mortality data. Abbot`s formula (Abbot): Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) software (version 16). A p-value of less than 0.05 was considered statistically sig- nificant. Results Essential oil extracted An average of 1.8ml of essential oil was extracted from 150 grams of fennel seed. The density of 1ml of fennel essential oil was cal- culated to be 0.97g/ml. Approximately 19 main compounds were identified in the fennel es- sential oil, the highest amount was related to trans-anethole (76.08%), α-thujoneα (10.37%), d1-limonene (5.34%) respectively and then the combination of methyl chavicol (3.55%) (Es- tragol) (Table 1). Bioassay test Results The contact toxicity of fennel essential oil against head lice resulted in significant differ- ences in mortality means over the times of 10, 20, 30, 45 and 60 minutes in a constant dose (F= 154.528, df= 4, P< 0.001). In other words, the effect of time in the means’ mortality was significant, so the mortality increases with time increasing. Significant differences were ob- served between fennel essential oil doses, i.e., the effect of dose was significantly on the head lice mortality means (F= 265.658, df= 5, P< 0.001) and the mortality increased with essential oil increasing dose (Table 2). http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 65 http://jad.tums.ac.ir Published Online: March 31, 2022 Probit analysis of head lice mortality rates (adult/nymph) at different times is presented in Table 3. As shown, the P values of the mod- el were < 0.05 at different times which indi- cates that the data fit the Probit model. Also, heterogeneity factors are < 1 which confirms the data accuracy (Table 3). When the het- erogeneity factor (which equals the chi-square divided by degrees of freedom) is > 1, a plot of the data should be examined because the data do not fit the model (32). The concentrations need to eliminate 50% and 99% of adult/nymph stages at different times are presented in Table 3. With increas- ing time from 10min to 60min, LC50 values de- creased from 16.05 to 7.38% and LC99 changed from 39.5 to 2.46%. A comparison of the le- thal doses of 50% and 99% of head lice mor- tality at different times showed no overlap be- tween the confidence limits (CL) of LC50 val- ues at 10min with the other times (20, 30, 45 and 60min). So, it can be concluded that the effect of lethal doses on head lice mortality was significantly different. However, there was an overlap between the confidence limits of LC50 at 20, 30, 45 and 60 minutes, indicating no sig- nificant differences between the 50% lethal dos- es at these times. Comparison of LC99 confi- dence limits after different time periods showed CL overlaps at 20, 30 and 45 minutes and so there were no significant differences in LC99 values at these time periods, but the highest overlap can be seen in the confidence limits of LC99 between 20 and 30min and also between 30 and 45min. LC99 values at 10 minutes were significantly different from these values at other times due to a lack of overlap in confidence limits. The lethal doses of 50% and 99% of fennel essential oil against head lice nit were 2.3% and 7.4% after 5 days, respectively. Con- sidering the model's P values and the hetero- geneity factor calculation which is < 1 also show that the observed data fit the Probit model ap- propriately. Head Lice Lethal Time The times required killing 50% and 99% of head lice in adult and nymph stages exposed to different concentrations of fennel essential oil are shown in Table 4. It would take 13.9, 21.7, 23.8, 91.3 and 138 minutes to kill 99% of head lice at concentrations of 40, 20, 10, 5 and 2.5%, respectively, which indicate an in- verse relationship between lethal times and es- sential oil concentrations. Also, the negative slope values in Table 4 indicate a decrease in lethal times with the concentration increasing of fennel essential oil. A comparison of the con- fidence limits of LT99 at different concentra- tions also indicates a significantly different be- tween the time of lethality at concentrations of 2.5% and 5% with other lethal times. The 99 % lethal time at a concentration of 10%, 20% and 40% had no significant difference due to their overlap of confidence limit. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 66 http://jad.tums.ac.ir Published Online: March 31, 2022 Table 1. Storage time and percentage of essential oil components of fennel using gas chromatography )%( Retention time Fennel essential oil components 0.97 0.13 0.28 0.06 0.32 0.11 0.16 5.34 0.35 0.11 10.37 0.21 3.55 0.17 0.05 0.68 76.08 0.01 7.896 8.314 9.041 9.121 9.562 9.939 10.546 10.672 10.735 11.564 12.463 14.088 15.655 16.657 16.937 17.218 18.247 24.187 α-Pinene Camphene Sabinene β-pinene β-Myrcene 1-Phellandrene Cymene d1-Limonene)Limonene( 1,8-Cineole Terpinene α-Thujone Camphor Methyl chavicol ) Estragol( α-Fenchyl acetate Carvone Anisaldehyde Trans-Anethole delta-Cadinene Table 2. Mean mortality percentages of head louse at adult/nymph stages exposure to different concentrations of fen- nel essential oil by contact bioassay Essential oil concentration (%) Time after exposure (min) Mortality mean (%) ± SE 40 10 20 30 45 85±4.0 90±0.0 90±0.0 90±0.0 60 90±0.0 10 67.5±4.8 20 77.5±4.8 20 30 90±0.0 45 90±0.0 60 90±0.0 10 42.5±4.8 20 75±2.9 10 30 80±5.8 45 85±2.9 60 87.5±4.8 10 25±4.1 20 45±2.9 5 30 57.5±6.3 45 65±2.9 60 80±4.1 10 6±2.4 20 20±3.2 2.5 30 34±4 45 42±3.7 60 48±3.7 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 67 http://jad.tums.ac.ir Published Online: March 31, 2022 Table 3. Lethal concentrations (%) of fennel essential oil against adult and nymphs and egg of head lice using filter paper contact P Slope (±SE) LC99 (95%CL) LC50 (95%CL) Time (min) Head lice <0.001 21.4 (22) 0.07 (±0.01) 29.5 (23.2-36.9) 11.1 (9.5-21.6) 10 Adult/ nymph <0.001 7.9 (22) 0.22 (±0.04) 15.2 (13.6-20.3) 6.4 (2.8-7.2) 20 <0.001 18.9 (22) 0.26 (±0.02) 12.6 (10.6-16.4) 3.9 (1.8-5.6) 30 <0.001 14.4 (22) 0.30 (±0.05) 10.8 (8.9-13.9) 3.1 (1.2-4.6) 45 <0.001 3.9 (22) 0.90 (±0.18) 7.4 (5.9-11.7) 2.5 (2.1-3.7) 60 <0.001 14 (21) 0.50 (±0.09) 7.4 (6.1-11.9) 2.3 (1.5-4.3) 5 days Egg 95% CL: 95% confidence Limits of lethal concentrations of 50% and 99% head lice, : chi- square (degree of freedom), P: significance level of Probit model Table 4. Lethal times (LT50 and LT99) of head louse (adult / nymph) at different concentrations of fennel essential oil in vitro (min) 95% CL: 95% confidence Limits of lethal times of 50% and 99% head lice, a: chi-square (degree of freedom), P: significance level of Probit model Discussion In this study, the highest percentages of fennel EO components were belonged to trans- anethole, α -thujone, and limonene which con- stituted of 76.08%, 10.37%, and 5.34%, respec- tively. This result is consistent with the other findings. Trans-anethole (32% and 30%, respec- tively), limonene (28% and 18%, respectively) and fenchone (10% in both cases) were the main compounds identified in the fennel EOs from Cape Verde and Portugal, respectively (30). Trans-anethole constituted 72% of the fen- nel oil composition in the study by Zoubiri et al. 2014 (29). The results of Lee (2004) on the properties of fennel essential oil against two Dermatophagoides dust mites and the essential oil constituents showed that the highest percent- age of essential oil components were belonged to trans-anethole, fenchone and estragole with 53.2, 14.2, and 12.7%, and fenchone had high lethal activity against dust mites (28). Fennel essential oil showed appropriate po- tential for treating the head louse infestation in nit and nymph/adult stages in our study. At the concentration of 10%, it kills 50% and 99% of the adult/ nymph after 9.5 and 24 minutes, re- spectively. The lethal times of 50% and 99% mortality decreased to 8 and 21.7 minutes at the concentration of 20%. The LC50 and LC99 values decreased with an increase in exposure times. After 20 minutes, these values were calculated as 6.4 and 15.4%. Fennel EOs from Cape Verde and Portu- gal resulted in 99% mortality of Ae. aegypti larva at 37.1 and 52.4µl l-l, respectively (30). P a Slope ( SE) LT99 (95%CL) LT50 (95%CL) Concentration (%) 0.001 10.1 (18) -1.5±0.21 138 (107.3-214.4) 45.8 (38.9-58.1) 2.5 0.001 8.2 (18) -0.68±0.2 91.3 (74.6-110.3) 20.5 (12.1-26.2) 5 0.001 10.9 (18) -0.52±0.25 23.8 (18.8-30.2) 9.5 (8.8-14.6) 10 0.001 6.2 (18) -0.3±0.19 21.7 (15.5-35) 8.1 (6.5-17.8) 20 <0.001 12.2 (18) -0.37±0.27 13.9 (9.8-32.5) 5.2 (3.6-8.4) 40 http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 68 http://jad.tums.ac.ir Published Online: March 31, 2022 Application of 40 and 60mg.l-1 fennel es- sential oil eliminated 50% and 90% of the sec- ond instar larval population of Culex pipiens after 2 hours and 4 hours, respectively (29). Repellency of antitol and estragole compo- nents of fennel essential oil has been reported against stored grain pests, Rhyzopertha domi- nica, Sitophilus zeamais and Tribolium con- fusum. The estragole type of fennel essential oil showed more repellency than the anethole type against the three studied pests (26). The mentioned studies indicate that the fennel es- sential oil has toxicity or repel activity against insect pests. The results of these studies are consistent with our study in terms of the major constitu- ent of fennel essential oil which is trans-ane- thole and the potential of this essential oil for repelling or killing of insect pests. Differences in the obtained results can be attributed to the pest species, plant phenology affected by the geographical condition and climate as well as the bioassay test. Adulticidal and ovicidal activity against P. capitis has been reported for some essential oils. Many essential oils that are recommend- ed for the treatment of head lice including eu- calyptus, rosemary, geranium, tea tree, lemon, and their components were studied for possi- ble adulticide and repellent effects on head and body lice (13–27). Tea tree (Melaleuca alternifolia) and nerolidol essential oils alone and in combina- tion showed that tea oil with 1% concentra- tion caused mortality of 100% of adult/nymph in 30min and had a better effect than nerolidol oil. The toxicity of nerolidol against head louse egg was better than tea essential oil (50% egg lethality at 1% concentration for 5 days). Com- bining these two substances together killed the entire lice population within 30 minutes (18). The lethal toxicity of wild bergamot, clove, lav- ender, tea tree, and verbena essential oils was evaluated against adult head lice using im- pregnated filter paper bioassay method. Clove oil, diluted either in coconut oil or sunflower oil, demonstrated the best adulticidal activity of > 90% mortality within 2h in lice exposure to 30min contact toxicity (14). The LT50 val- ues were calculated as 2.5, 8.1, 9.5, 20.5, and 45.8 minutes in the concentration of 40, 20, 10, 5 and 2.5% (equal to 6.2, 3.1, 1.5, 0.77 and 0.39mg/cm2) respectively in our study. Thymus vulgaris, Aloysia polystachya and Aoysia citriodora EOs showed that Thyme es- sential oil has significant toxicity for adult and egg stages as well as knock down against the eggs and adults of P. capitis by fumigant and contact toxicity bioassays. The calculated KT50 values for adults at doses of 0.84, 0.63, 0.42 and 0.21mg/cm2 were 3.93, 6.30, 6.49 and 9.90 minutes in contact bioassay, respectively (22). Three Origanum species essential oils have been shown to decrease the rate of limb, bowel, and abdomen movements of head louse sig- nificantly at the concentration of 1%, produc- ing more than 90% mortality after 12h using the adult immersion test for 5min (27). Nonconformity in the results can be relat- ed to differences in the components of the plant essential oil, the head lice susceptibility, and the bioassay method. Variation in insect responses to different essential oils has been studied previously. The qualitative and/or quan- titative chemical composition among plant spe- cies may be different and it definitely affects the obtained results (25). Conclusion Fennel oil is a potent and useful compound for human head louse treatment. Concentra- tions of 12.6% to 15.2% of this essential oil killed 99% of adult/nymph at 30 and 20 minutes. It also kills 99% head louse nit in 5 days. But further research is necessary to evaluate the safety of this plant EO on human health and to develop appropriate formulation for improving the pediculicidal activity in clinical trials. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 69 http://jad.tums.ac.ir Published Online: March 31, 2022 Acknowledgements This article is part of Hoda Ghofleh Marema- zi’s MSPH thesis. It was financially supported by Ahvaz Jundishapur University of Medical Sciences (AJUMS). (Project No. U-96076). Ethical considerations This article was approved by AJUMS (Eth- ical Code IR.AJUMS.REC.1396.418). Conflict of interest statement Authors declare that there is no conflict of interest. References 1. Nejati J, Keyhani A, Kareshk AT, Mahmoudvand H, Saghafipour A, Kho- raminasab M, Oliaee RT, Mousavi SM (2018) Prevalence and risk factors of pe- diculosis in primary school children in South West of Iran. Iran J Public Health. 47(12): 1923–1929. 2. Hatam-Nahavandi K, Ahmadpour E, Pashaza- deh F, Dezhkam A, Zarean M, Rafiei- Sefiddashti R, Salimi-Khorashad A, Hos- seini-Teshnizi S, Hazratian T, Otranto D (2020) Pediculosis capitis among school- age students worldwide as an emerging public health concern: a systematic review and meta-analysis of past five decades. Parasitol Res. 119(10): 3125–3143. 3. Moosazadeh M, Afshari M, Keianian H, Nezammahalleh A, Enayati AA (2015) Prevalence of head lice infestation and its associated factors among primary school students in Iran: a systematic review and meta-analysis. Osong Public Health Res Perspect. 6(6): 346–356. 4. Farzinnia B, Hanafibejad AA, Raeiskarami SR, Jafari T (2004) Epidemiology of head lice in primary school girls in Qom, 2002. Hormozgan Med J. 2(8): 103–108. 5. Zahirnia AT, Bhuthehia J (2005) Evalua- tion of three types of daphnostam sham- poo 0.2%, permethrin 1% and lindin 1% in the treatment of head lice infection in elementary school female students. J Ma- zandaran Univ Med Sci. 15(49): 16–24. 6. Toloza AC, Zygadlo J, Biurrun F, Rotman A, Picollo MI (2010) Bioactivity of Ar- gentinean essential oils against perme- thrin-resistant head lice, Pediculus hu- manus capitis. J Insect Sci. 10(1): 1–8. 7. Rafinejad J, Nourollahi A, Biglarian A, Ja- vadian E, Kazemnejad A, Doosti S (2011) The comparison of the effect of perme- thrin shampoo and lindane lotion on the treatment of head lice (Pediculus Hu- manus Capitis) in the primary school pu- pils. J Mazandaran Univ Med Sci. 21(83): 35–41. 8. Toloza AC, Vassena C, Picollo MI (2008) Ovicidal and adulticidal effects of mon- oterpenoids against permethrin resistant human head lice, Pediculus humanus capi- tis. Med Vet Entomol. 22(4): 335–339. 9. Verma P, Namdeo C (2015) Treatment of Pediculosis capitis. Indian J Dermatol. 60(3): 238–247. 10. Feldmeier H (2012) Pediculosis capitis: new insights into epidemiology, diagno- sis and treatment. Eur J Clin Microbiol Infect Dis. 31(9): 2105–2110. 11. Boukan A, Mohebi L, Rashti R, Boukan A, Oshaghi MA (2022) Pediculosis Capi- tis; the Importance of Accurate Differen- tiation of Nits and Hair Casts. Int J Trop Insect Sci. 42(1): 647–650. 12. Koul O, Walia S Dhaliwal GS (2008) Es- sential oils as green pesticides: potential and constraints. Biopestic. 4(1): 63–84. 13. Rassami W, Soonwera M (2013) In vitro Pediculicidal activity of herbal shampoo base on Thai local plants against head louse (Pediculus humanus capitis De Geer). Parasitol Res. 112(4): 1411–1416. http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 70 http://jad.tums.ac.ir Published Online: March 31, 2022 14. Candy K, Nicolas P, Andriantsoanirina V, Izri A, Durand R (2018) In vitro effica- cy of five essential oils against Pedicu- lus humanus capitis. Parasitol Res. 117 (2): 603–609. 15. Yang YC, Lee HS, Clark JM, Ahn YJ (2004) Insecticidal activity of plant es- sential oils against Pediculus humanus capitis (Anoplura: Pediculidae). J Med En- tomol. 41(4): 699–704. 16. Yang YC, Lee SH, Lee WJ, Choi DH, Ahn YJ (2003) Ovicidal and adulticidal effects of Eugenia caryophyllata bud and leaf oil compounds on Pediculus capitis. J Agric Food Chem. 51(17): 4884–4888. 17. Soonwera M, Wongnet O, Sittichok S (2018) Ovicidal effect of essential oils from Zin- giberaceae plants and Eucalytus globulus on eggs of head lice, Pediculus humanus capitis. De Geer. Phytomedicine. 47: 93– 104. 18. Di Campli E, Di Bartolomeo S, Pizzi PD, Di Giulio M, Grande R, Nostro A, Cel- lini L (2012) Activity of tea tree oil and nerolidol alone or in combination against Pediculus capitis (head lice) and its eggs. Parasitol Res. 111(5): 1985–1992. 19. Abdel-Ghaffar F, Semmler M, Al-Rasheid K, Klimpel S, Mehlhorn H (2010) Com- parative in vitro tests on the efficacy and safety of 13 anti-head-lice products. Par- asitol Res. 106(2): 423–429. 20. Soonwera M (2015) Pediculicidal Activi- ties of herbal shampoos from Zingiber officinale Roscoe and Camellia sinensis (L.) Kuntze against head louse (Pedicu- lus humanus capitis De Geer: Phthirap- tera). J Agri Tech. 11(7): 1493–1502. 21. Ghavami MB, Ahmadi S (2017) Effective- ness of eucalyptus and cinnamon essen- tial oils compared to permethrin in treat- ment of head lice infestation. J Adv Med Biomed Res. 25(112): 86–98. 22. Gutiérrez MM, Werdin-González JO, Stef- anazzi N, Bras C, Ferrero A (2016) The potential application of plant essential oils to control Pediculus humanus capitis (Anoplura: Pediculidae). Parasitol Res. 115(2): 633–641. 23. Bagavan A, Rahuman AA, Kamaraj C, Elan- go G, Zahir AA, Jayaseelan C Santhosh- kumar T, Marimuthu S (2011) Contact and fumigant toxicity of hexane flower bud extract of Syzygium aromaticum and its compounds against Pediculus humanus capitis (Phthiraptera: Pediculidae). Par- asitol Res. 109(5): 1329 –1340. 24. Sittichok S, Wongnet O, Soonwera M (2018) New Thai herbal shampoos as pedicu- licides for killing head louse, Pediculus humanus capitis De Geer (Phthiraptera). Asian Pac J Trop Biomed. 8(2): 106–112. 25. Bedini S, Bougherra HH, Flamini G, Cosci F, Belhamel K, Ascrizzi R, Conti B (2016) Repellency of anethole-and estragole-type fennel essential oils against stored grain pests: the different twins. Bull Insectol. 69(1): 149–157. 26. Arserim SK, Cetin H, Yildirim A, Limoncu ME, Cinbilgel I, Kaya T, Ozbel Y, Balcioglu IC (2021) The Toxicity of Es- sential Oils from Three Origanum Spe- cies against Head Louse, Pediculus hu- manus capitis. Acta Parasitol. 66(3): 1003– 1011. 27. Shams Ardekani M HA, Jamshidi A, Abdi K (2005) The study of volatile oil of Foeniculum vulgare Miller. In their tis- sue culture and comparison with the whole plant. J Med Plants. 3(15): 73–80. 28. Lee HS (2004) Acaricidal activity of con- stituents identified in Foeniculum vulgare fruit oil against Dermatophagoides spp. (Acari: Pyroglyphidae). J Agric Food Chem. 52(10): 2887–2889. 29. Zoubiri S, Baaliouamer A, Seba N, Chamouni N (2014) Chemical composi- tion and larvicidal activity of Algerian Foeniculum vulgare seed essential oil. Arab J Chem. 7(4): 480–485. 30. Rocha DK, Matos O, Novo MT, Figueire- do AC, Delgado M, Moiteiro C (2015) http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 61–71 E Jahanifard et al.: Pediculicidal Activity of … 71 http://jad.tums.ac.ir Published Online: March 31, 2022 Larvicidal activity against Aedes aegypti of Foeniculum vulgare essential oils from Portugal and Cape Verde. Nat Prod Com- mun. 10(4): 677–682. 31. WHO (1981) Instructions for Determining the Susceptibility or Resistance of Body Lice and Head Lice to Insecticides. WHO/ VBC/81.808, Geneva. 32. Robertson JL, Russell RM, Presler HK, Savin NE (2017) Bioassays with Arthro- pods. CRC Press, Taylor and Francis Group. Boca Raton, London and New York. http://jad.tums.ac.ir/