Pediculicidal effect of wood vinegar produced from eucalyptus wood in co-pyrolysis with aromatic herbs against Bovicola caprae (Phthiraptera: Trichodectidae)

Efeito pediculicida do extrato pirolenhoso produzido a partir de madeira de eucalipto em co-pirólise com ervas aromáticas contra Bovicola caprae (Phthiraptera: Trichodectidae)

Abstract

Lice infestation can compromise the productivity of goat farming, and this condition is traditionally controlled with synthetic insecticides that have accelerated the development of parasite resistance and caused contamination of products and the environment. In this context, alternative methods for treating pediculosis in goats have been studied, with a primary focus on plant-derived products, such as wood vinegar (WV). This study evaluated the in vitro pediculicidal activity of two types of wood vinegar produced through co-pyrolysis of Eucalyptus urophylla x Eucalyptus grandis (clone I144) wood and Origanum vulgare (EO) leaves and Thymus vulgaris (ET) leaves on chewing lice of the species Bovicola caprae. Wood samples were obtained from plantations in Macaíba, Rio Grande do Norte State, Brazil. After botanical identification, samples of O. vulgare and T. vulgaris were collected from local farmers. The chemical composition of wood vinegars was characterized using gas chromatography-mass spectrometry (GC/MS). The in vitro lice-killing activity was analyzed using the immersion method at concentrations of 100, 50, 25, 12.5, 6.25, 3.12, and 1.562% (g 100 mL^-1), with all tests performed in quadruplicate. Amitraz was used as the positive control, and distilled water was used as the negative control. A total of 720 lice were distributed among experimental groups, with 10 specimens per plate. Mortality was evaluated at 1, 3, 6, 12, 24, 36, and 48 hours after exposure. At the end of the experiment, the 100% concentration of the wood vinegar showed the highest mortality among the treatments tested. Moreover, mortality rates above 80% indicated a strong insecticidal effect according to the parameters adopted in the study. These findings suggest that both vinegars have promising potential as efficient, ecologically viable pediculicides.

Keywords:
goat production; pediculosis; parasite resistance

Resumo

A infestação por piolhos pode comprometer a produtividade da caprinocultura, e essa condição é tradicionalmente controlada com inseticidas sintéticos, que têm acelerado o desenvolvimento de resistência nos parasitas e causado contaminação dos produtos e do meio ambiente. Dessa forma, métodos alternativos para o tratamento da pediculose em caprinos vêm sendo estudados e, entre eles, destacam-se os produtos de origem vegetal, como o vinagre de madeira (VM). Assim, esse estudo avaliou a atividade pediculicida in vitro de dois tipos de vinagre de madeira produzidos por meio da co-pirólise da madeira de Eucalyptus urophylla × Eucalyptus grandis (clone I144), juntamente com folhas de Origanum vulgare (EO) e de Thymus vulgaris (ET), sobre piolhos mastigadores da espécie Bovicola caprae. As amostras de madeira foram obtidas de plantações localizadas em Macaíba, no estado do Rio Grande do Norte, Brasil. Após a identificação botânica, amostras de O. vulgare e T. vulgaris foram coletadas junto a agricultores locais. A caracterização química dos componentes de ambos os vinagres de madeira foi realizada por cromatografia gasosa acoplada à espectrometria de massas (CG/EM). A atividade pediculicida in vitro foi avaliada pelo método de imersão, nas concentrações de 100, 50, 25, 12,5, 6,25, 3,12 e 1,562% (g 100 mL⁻¹), com todos os testes realizados em quadruplicata. O amitraz foi utilizado como controle positivo e a água destilada como controle negativo. Um total de 720 piolhos foi dividido em grupos experimentais, formados por 10 espécimes por placa. Os intervalos de observação da mortalidade dos insetos foram de 1, 3, 6, 12, 24, 36 e 48 horas após a exposição. Ao final do experimento, a concentração de 100% do vinagre de madeira apresentou a maior mortalidade entre os tratamentos testados. No entanto, em porcentagens superiores a 80%, o produto foi considerado de forte efeito inseticida, de acordo com os parâmetros adotados no estudo. Esses resultados sugerem que ambos os vinagres têm potencial promissor para uso como pediculicidas eficientes e ecologicamente viáveis.

Palavras-chave:
produção de cabras; pediculose; resistência a parasitas

1. Introduction

Goat farming is an essential and diversified sector of the global economy, providing everything from food products to durable consumer goods, and plays an even more crucial socioeconomic role in developing countries, especially in those with semi-arid climates (Naeem et al., 2021; Salgado et al., 2023). In Brazil, according to data from the Brazilian Institute of Geography and Statistics (IBGE, 2023), the goat population is estimated at approximately 12.9 million animals, with 96% of the national herd concentrated in the Northeast region. Ectoparasitism is a common health problem in goats, which directly and indirectly affects their health (Disasa, 2020; Nizamov, 2023). Directly, the hosts suffer from blood loss, allergic, inflammatory, and infectious processes, as well as being indirectly afflicted by anxiety, restlessness, and self- mutilation resulting from the pruritus usually present in infested animals (Nizamov, 2023).

Among the ectoparasites that infest small ruminants, notably ticks, mites that cause mange, fleas, and lice are the most commonly found in goats (Iqbal et al., 2022; Cotticelli et al., 2023; Nizamov, 2023). Different species of lice are etiological agents of pediculosis, the most frequent ectoparasitic disease of veterinary importance and present in practically all areas of the planet, especially during the rainy seasons (Ajith et al., 2019; Nizamov and Iliev, 2023). In caprine pediculosis, species of the suborder Ischnocera, known as chewing or biting lice, are the most prevalent and can inhabit any part of the host's body, especially areas that are difficult to access, such as the less accessible body regions (Benelli et al., 2018; Ballados-González et al., 2023). Lice of the genus Bovicola spp. massively infest goat herds and are the main economic bottleneck in goat production due to the reduced productivity of parasitized animals and the cost of pesticides (Benelli et al., 2018; Prelezov et al, 2022).

Synthetic chemical insecticides remain the most widely used strategy for controlling ectoparasites on goats, including lice of the genus Bovicola. (Benelli et al., 2018; Cotticelli et al., 2023). The main compounds employed belong to the classes of macrocyclic lactones, organophosphates, formamidines, thiazolidines, phenylureas, and pyrethroids (Benelli et al., 2018; Legesse et al., 2022). The effectiveness of synthetic chemical agents in parasite control remains evident when used in accordance with recommended good practices, without underdosing or overdosing (Benelli et al., 2018; Aguiar et al., 2021). However, when used indiscriminately, excessively, and irrationally, these products have shown reduced efficacy and increased mechanisms of parasite resistance, considerably compromising the pharmacological arsenal (Lu, 2023). Furthermore, when misused, they generate residues in animal-derived products and contaminate the environment (Al-Dawood et al., 2023).

Given the increasing resistance to synthetic antiparasitics and the problems associated with chemical pesticides, Candy et al. (2020) and Cotticelli et al. (2023) highlighted the urgent need to develop alternative, efficient, and safe methods of parasite control. Among the possibilities of alternative methods, phytotherapy and pyrolysis products show promising potential against parasites such as ticks (Figueiredo et al., 2019; Salman et al., 2020; Rosário et al., 2023), mites (Akram et al., 2020; Sawitri and Yuningsih, 2020), fleas (Moog et al., 2020; Banuls et al., 2023), and lice (Aguiar et al., 2021; Yingklang et al., 2022). Several methods and technologies have been applied to obtain phytotherapeutics and pyrolysis products from various plants, including wood vinegar (WV), a co-product of wood carbonization (Soares et al., 2021). From this perspective, the wood from Eucalyptus clones, widely used to produce charcoal for metallurgy uses in Brazil, are a potential source of WV, present secondary metabolites with antimicrobial (Soares et al., 2021; Souza et al., 2021; Silva et al., 2023), anthelmintic (Araújo-Filho et al., 2019; Moazeni et al., 2019) and antiparasitic (Soonwera et al., 2018; Madreseh-Ghahfarokhi et al., 2019) biological activity, and can be associated with other plants to obtain a synergistic effect between bioactives. Therefore, this study evaluated the in vitro pediculicidal activity of wood vinegar produced by co-pyrolysis of Eucalyptus wood with aromatic herbs against Bovicola caprae.

2. Materials and Methods

2.1. Type of research and study location

The research was conducted as a quantitative experimental study character using a completely randomized design. This study was conducted in Mossoró (5° 11′ 17″ South and 37° 20′ 39″ West), Rio Grande do Norte State, Brazil, at the Laboratory of Diagnostic and Experimental Parasitology (LPDE) and approved by the Ethics Committee on the Use of Animals (CEUA) of the Universidade Federal Rural do Semi-Árido (UFERSA) under protocol 14/2021. The WV was produced through co-pyrolysis of Eucalyptus wood from plantations in Macaíba (5° 51′ 36″ South and 35° 20′ 59″ West), RN, Brazil.

2.2. Wood vinegar production and refining

WV samples were produced from wood of a hybrid from Eucalyptus urophylla x Eucalyptus grandis (clone I 144) collected from plantations in Macaíba, RN, Brazil, following the methodology described by Santos et al. (2013) and Pimenta et al. (2018). The Eucalyptus trees were 8 years old at the moment of felling. Three trees were selected and cut down. From the trees, 2-cm-thick wood discs were collected at 0, 25, 50, 75, and 100% of the trunk. In this way, 15 wooden discs were obtained. To reduce the sample size, the wood discs were divided into four wedges each and oven-dried for 48 hours at 103 ± 1 °C, according to the methodology described by Araújo et al. (2018). After botanical identification, the herbs were purchased from local organic farmers who do not use pesticides or other agrochemicals in their cultivation. After drying, the wood wedges were carbonized along with dry leaves of Origanum vulgare L. and Thymus vulgaris L., encompassing two experimental treatments. The percentage of herbs was set at 25% of the weight of bone-dry wood, corresponding to roughly 500 g of wood and 125 g of leaves. This percentage was established during preliminary attempts and was considered the best for conducting the experiment.

The material was placed inside a stainless-steel container in a laboratory muffle. As Pimenta et al. (2018) described, a water-cooled condenser device was coupled to the metallic container to recover all the liquid products from carbonization. Five carbonization runs were performed for each experimental treatment, totaling 15 samples of total pyrolysis liquids. The carbonization time was 8 hours, and the maximum temperature reached was 450 °C. Once the pyrolysis was complete, the container was removed and left to cool to room temperature. All liquids obtained from each set of five runs were collected into a single container, then stored and refrigerated at 6 °C (Araújo et al., 2018; Soares et al., 2021). This way, three types of WV were obtained. To remove heavy oils and tars, each composed sample was double-distilled under a vacuum of 1.0 mmHg, reaching a maximum temperature of 100 °C. The double-distilled liquid was then stored in a previously autoclaved glass bottle and filtered using a vacuum filtration system with an MF-Millipore membrane filter (0.22 µm porosity; Merck, Darmstadt, Germany). The residue generated during the processes was properly disposed of.

2.3. Gas chromatography and mass spectrometry (GC/MS) analysis

Aliquots of 5 mL were obtained from 100 mL batches of both wood vinegars. Initially, 1.5 mL of concentrated ammonium hydroxide solution (Caledon, UN 2672, Canada) was added to 5 mL aliquots of the samples to increase the pH to 7.0. Then, three 3 mL extractions were carried out using HPLV-grade ethyl acetate (Merck, São Paulo, SP, Brazil). After liquid-liquid extraction, 1 mL of the ethyl acetate extract was transferred to GC vials and promptly analyzed. The GC-MS analyses of the samples were carried out in a Shimadzu QP 2010 gas chromatograph/mass spectrometer (single quadrupole). The components’ separation was performed on a DB-Wax 52 CB column (Agilent, São Paulo, SP, Brazil) with dimensions of 30 m × 0.25 mm × 0.25 μm. The GC injector was kept at 250 °C. The samples (1 μL) were injected with a split ratio of 1:10, and the oven temperature was set at 50 °C for 2 min. From this point, a 2 °C min-1 heating rate was applied from 50 to 240 °C, with the final temperature held for 2 min. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The total running time was 99 minutes. Three injections into the GC/MS were performed for each of the two WV types. It was observed preliminarily that the behavior of the chromatograms and mass spectra was highly reproducible. The mass spectrometry (MS) acquisition range was from m/z 50 to 650 Da. The electron ionization source (EI) and mass spectrometer (MS) interface were held at 250 °C. The solvent cut time was 4 min. Major and minor compounds were detected and identified based on their characteristic electron ionization (EI, 70 eV) mass spectra by comparison with those in the NIST mass spectral library (NIST 23). Most of the chemical compounds reported here exhibited at least 85% mass spectral similarity, while some minor compounds showed greater than 80% similarity.

2.4. Collection of ectoparasites

The search for B. caprae was carried out in goats (Capra hircus Linnaeus, 1758) presented for sale at the Goat Fair in Mossoró, RN, Brazil. The lice were collected manually using a commercial extractor comb on naturally infested and mechanically restrained goats, collecting B. caprae chewing lice directly from the goat hosts (Aguiar et al., 2021). To minimize potential interference with the tests, inspection and collection were performed only on animals that had not received antiparasitic treatment for at least 90 days (Abu et al., 2014; Santos et al., 2019), regardless of sex or age. After collection, the specimens were stored in adequately cleaned, identified, aerated plastic containers and transported to the laboratory in isothermal boxes (Farias et al., 2017). Subsequently, the taxonomic identification of B. caprae was carried out by examining the insects' morphology under a stereoscopic microscope and using taxonomic keys (Guimarães, Tucci, and Battesti-Barros, 2001). Before the experiments began, the viability and morphological integrity of the lice were evaluated under a stereomicroscope, selecting only those that were physiologically active and morphologically preserved, without distinguishing sex or life stage (Heukelbach et al., 2008; Aguiar et al., 2021; França et al., 2021). However, because the nymphs appeared more resistant and numerous, we sought to ensure proportionality between treatments within each collection.

2.5. In vitro analysis of pediculicidal activity

The analysis of in vitro pediculicidal activity was carried out according to the methodology of Heukelbach et al. (2006), adapted for goat lice, where these, attached to fur, were completely immersed in 1 mL of WVs at different concentrations (100%, 50%, 25%, 12.5%, 6.25%, 3.12% and 1.56%) and in controls for 1 min. For the positive control, 12.5% amitraz (Ibatox – Ibasa, Porto Alegre, RS, Brazil) was diluted as indicated by the manufacturer, and distilled water was used as the negative control. The experiment was carried out in quadruplicate, with 720 chewing lice randomly distributed at a rate of 10 specimens per plate across the seven treatment groups and two control groups for each WV tested (Legesse et al., 2022). Immediately after immersion, the insects were placed in Petri dishes containing goat hair and lined with filter paper moistened with 200 µL of water to prevent the parasites from drying out (Burkhart and Burkhart, 2001; Heukelbach et al., 2006). The Petri dishes containing the lice were kept at room temperature throughout the tests, and observations to assess mortality were conducted at 1, 3, 6, 12, 24, 36, and 48 hours post-test initiation. The death of chewing lice was defined by the complete absence of movements in the abdominal segments of the insects and by the lack of motility of the antennae or legs, with or without stimulus from pincers (Burkhart and Burkhart, 2001; Heukelbach et al., 2008).

2.6. Data analysis

The pediculicidal efficacy of the wood vinegar from the co-pyrolysis of wood and wood combined with aromatic herbs, using the products at concentrations of 100, 50, 25, 12.5, 6.25, 3.12, and 1.56% (g 100 mL^-1). The lethal effect was evaluated at 1, 3, 6, 12, 24, 36, and 48 hours. After each interval, the percentage of lice mortality was calculated according to Equation 1 described by Islam et al. (2018), Aguiar et al. (2021), and Legesse et al. (2022) (Equation 1):

$$\text{\% Mortality}=\frac{\text{Number of dead lice}}{\text{Total number of lice}}\times 100$$ (1)

The means and standard deviations were calculated from the mortality percentages obtained in each experimental run. Additionally, the insecticidal effect of the WVs was classified based on the mean mortality values as follows: strong, mortality > 80%; moderate, mortality from 80 to 60%; weak, mortality from 60 to 40%; little or none, mortality < 40% (Gemeda et al., 2014). Before analysis, the percentage data were arcsine-transformed and tested for normality using the Shapiro-Wilk test. The Kruskal-Wallis test, the Dunn post-test, and the Friedman test were used to assess statistical differences among groups, group comparisons, and time intervals. Microsoft Excel was used to determine measures of central tendency and standard deviation and to perform data transformations. The other statistical tests were also performed using the BioEstat software (version 5.3). The lethal concentration 50 (LC50) was calculated using Probit regression in Microsoft Excel. The significance level adopted was 5%.

3. Results

Table 1 displays the carbonization run results for eucalyptus wood in combination with Origanum vulgare and Thymus vulgaris. The results obtained for WV from co-pyrolysis of wood and aromatic herbs are close to the gravimetric yields observed for the carbonization of eucalyptus wood only. When marjoram was combined with eucalyptus wood, the yield of pyrolysis liquid did not increase significantly, despite the same gas pattern, compared with the control treatment with wood only. This pattern is consistent with results cited in the literature for eucalyptus clones (Araújo et al., 2018; Pimenta et al., 2018) and industrial carbonization (Pimenta et al., 2023). Both experimental treatments produced raw pyrolysis liquids with yields exceeding 40%. This is an interesting characteristic from an industrial standpoint, as when charcoal and liquid yields are maximized, the economic balance is favorable since these are saleable products. Purified WV yields from the following vacuum distillation showed no significant difference between the two treatments. For both, the refining results were consistently above 95%, which is consistent with the results previously reported by Pimenta et al. (2023).

Table 1
Gravimetric yields determined for the carbonization runs.

Material	Gravimetric Yields (%)
	Charcoal	Liquids	Gases
Eucalyptus wood + EO	34.6	43.3	22.1
Eucalyptus wood + ET	34.6	41.2	24.2

• Gravimetric yields in charcoal, pyrolysis liquids, and gases calculated based on the initial weight of bone-dry wood plus Origanum vulgare (EO) or Thymus vulgaris (ET).

Refining is essential to remove heavy oils and tar pitch, usually present in WV. Distillation is the most effective and reliable method for producing high-quality products (Higashino et al., 2005; Pimenta et al., 2023). During distillation, polycyclic aromatic hydrocarbons, volatile organic compounds, and other contaminants are completely removed from the WV (Pimenta et al., 2000; Pimenta et al., 2023). This way, we guarantee that the effect of the refined WV is exclusively due to the biologically active components in its composition.Table 2 lists the annotated compounds in both types of WV (EO and ET). The GC/MS analysis showed that the tested products exhibited similar compositions. A total of 89 compounds could be identified. However, the two WVs differ. Among the compounds, 12 were present exclusively in the EO, and another 13 were only found in the ET. Phenolic compounds and furfural stood out as the main components in both WVs. Furfural has the highest percentage in the ET, at 23.12%. Meanwhile, eucalyptus WV contained phenol as one of its components at a concentration of 7.46%.

Table 2
Annotated compounds in WVs from Eucalyptus wood combined with Origanum vulgare (EO) or Thymus vulgaris (ET).

Compounds (in order of elution)	Eucalyptus wood + EO	Eucalyptus wood + ET
1. Furfural	11.32	23.12
2. 2-methoxy-phenol	10.83	11.03
3. Phenol	7.46	*
4. Borazine, 2,4-dimethyl-	*	3.8
5. 2-Furancarboxaldehyde, 5-methyl-	3.88	3.78
6. Phenol, 2-methoxy-4-methyl-	3.89	3.08
7. 2-Cyclopenten-1-one	2.81	3.91
8. 2-Cyclopenten-1-one, 2-methyl-	2.80	3.03
9. Acetic acid	6.80	6.71
10. 1-Hydroxy-2-butanone	1.42	2.24
11. Butanoic acid	1.34	1.92
12. 2-Cyclopenten-1-one, 3-methyl-	1.88	1.6
13. 2-Cyclopenten-1-one, 2,3-dimethyl-	1.60	1.47
14. Cyclopentanone	1.90	1.81
15. Methyl 2-furoate	1.38	1.24
16. Propanoic acid	2.41	2.29
17. Phenol, 3-methyl-	1.26	0.92
18. 2-Cyclopenten-1-one, 2,3-dimethyl-	1.03	1.07
19. Phenol, 4-methyl-	2.57	0.56
20. 2-Butanone, 1-(acetyloxy)-	0.94	*
21. 3-Hexen-2-one, 3-methyl-	0.91	*
22. 1,2-Cyclopentanedione, 3-methyl-	0.64	0.46
23. Phenol, 2-methoxy-3-methyl-	0.71	0.53
24. 2-Cyclopenten-1-one, 3,4-dimethyl-	0.77	0.77
25. 2,3-Pentanedione	0.98	0.32
26. 3-(Diethylamino)-1,2-propanediol	0.46	*
27. Phenol, 2,6-dimethoxy-	0.6	0.2
28. 2-Furanmethanol	0.50	*
29. 2-Cyclopenten-1-one, 3-ethyl-	0.49	0.44
30. 3-Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-, (R)	0.87	*
31. 1-Buten-3-yne, 1-(1,1-dimethylethoxy)-, (E)-	*	0.54
32. 2-Cyclopenten-1-one, 2,3,4-trimethyl-	0.44	0.47
33. 2-Methoxy-5-methylphenol	0.48	0.38
34. Furan, 2-(methoxymethyl)-	0.40	0.25
35. Phenol, 2,4-dimethyl-	*	0.51
36. Bicyclo[2.2.2]octane, 2-methyl-	*	0.36
37. Phenol, 2,6-dimethyl-	0.38	0.32
38. 3-Ethenyl-3-methylcyclopentanone	0.27	0.38
39. Butanoic acid, 2-hydroxy-, methyl ester	0.23	0.18
40. Pentanoic acid, 3-methyl-	0.86	0.59
41. 2-Cyclopenten-1-one, 2,3-dimethyl-	0.34	0.35
42. 2-Cyclopenten-1-one, 3-ethyl-2-hydroxy-	0.33	0.33
43. (R)-(+)-3-Methylcyclopentanone	0.23	*
44. 2-Cyclopenten-1-one, 3-ethyl-2-hydroxy-	0.33	0.18
45. Pentanoic acid, 4-methyl-	0.32	0.17
46. 2,3-Dimethoxytoluene	0.27	0.28
47. Bicyclo[3.3.1]nonane	0.35	*
48. 1,3-Hexadiene, 3-ethyl-2-methyl-, (Z)-	0.28	*
49. Cyclopentanone, 2-methyl-	0.26	0.32
50. Ethylbenzene	*	0.23
51. Phenol, 3,5-dimethyl-	0.44	0.42
52. Cycloheptanone, 2-ethyl-	*	0.05
53. Pentanoic acid	0.35	0.28
54. Adrenalone	0.30	0.2
55. 2-Acetyl-5-methylfuran	0.30	0.22
56. 3-Cyclohexene-1-carboxaldehyde, 1-methyl-	*	0.28
57. Vinyl butyrate	0.21	0.28
58. 2-Butanol, 1-methoxy-	0.28	*
59. Cyclohexaneacetic acid, .alpha.-ethyl-	0.25	0.26
60. Phenol, 2-methoxy-4-propyl-	0.27	0.22
61. 1,4-Dioxin, 2,3-dihydro-	0.19	0.24
62. Cyclopentanone, 3-methyl-	*	0.26
63. 2-Hydroxy-3-pentanone	0.25	0.24
64. Phenol, 2-methyl-5-(1-methylethyl)-	0.2	0.1
65. 2-Cyclohexen-1-one	0.24	0.22
66. 3-Pentanol	*	0.27
67. 2-Cyclopenten-1-one, 3-methyl-	0.24	0.28
68. Pyrazole-4-carboxaldehyde, 1-methyl-	*	0.23
69. 3-Furaldehyde	0.23	0.34
70. 2-Propen-1-ol	0.23	0.2
71. 4-Hepten-3-one, 4-methyl-	*	0.19
72. Furan, 2,5-dihydro-3,4-dimethyl-	0.22	0.15
73. 1H-Imidazole-1-carboxamide, N, N-diethyl-	0.21	0.11
74. Eucalyptol	0.19	*
75. Phenol, 2-ethyl-	0.29	0.17
76. 2-Cyclopenten-1-one, 3-ethyl-2-hydroxy-	0.20	0.21
77. 2,3-Pentanedione	0.24	0.32
78. 4-Hexen-3-one, 4,5-dimethyl-	0.19	*
79. Butyrolactone	0.12	0.12
80. 2-Cyclohexen-1-one, 2-methyl-	0.17	0.15
81. 3-Heptyne	0.14	0.16
82. Cyclooctene	0.16	0.13
83. 2-Hexanol, 2-methyl-	0.16	0.18
84. 3-Penten-2-one, (E)-	0.18	*
85. Thymol	0.30	0.7
86. Ethanone, 1-(2-furanyl)-	3.53	2.94
87. 2H-Pyran-3(4H)-one, dihydro-	0.10	*
88. Pentanoic acid, 3-methyl-	0.15	0.09
89. Crotonic acid	0.19	0.22

• In the table, asterisks (*) mean the compound was not annotated in the respective WV.

From the in vitro assays to evaluate the pediculicidal activity of the wood vinegars from wood and EO (O. vulgare) or ET (T. vulgaris) at the different concentrations tested on B. caprae, the highest mortality rate for insects was observed in the experimental groups treated with the 100% EO vinegar, causing the death of more than 80% of the parasites and thus classified as having a strong insecticidal effect (Table 3).

Table 3
Statistical comparison of mortality means and standard deviation of the pediculicidal effect on Bovicola caprae as a function of concentration of Eucalyptus wood with Origanum vulgare WV and positive and negative controls.

Experimental groups
	WV concentration (% or g 100 mL-1)
C+	100	50	25	12.5	6.25	3.125	1.562	C-
49.0 ± 36.5 B	82.1 ± 12.6 A	51.8 ± 22.6 ABCG	35.4 ± 25.5 CBDEFGH	23.6 ± 28.8 DBEFGH	21.1 ± 24.2 EFGH	20.0 ±25.2 FGH	25.4 ± 24.6 GBH	15.7 ± 20.1 H

• Means followed by different letters are statistically dissimilar by the Kruskal-Wallis test, post-hoc Dunn at 95% probability C+ = positive control (amitraz at 12.5%); C- = negative control (distilled water).

Considering, in turn, the analysis of the ET's pediculicidal impact, the 100% WV demonstrated the best performance on the groups that received the treatments. This concentration caused more than 50% mortality among ectoparasites and was classified as having weak insecticidal activity. However, it was statistically similar to the amitraz result at 12.5%, which was used as a positive control (Table 4). Throughout the experimental groups' observation period, at intervals of one, three, six, twelve, twenty-four, thirty-six, and forty-eight hours after the assessed products and controls, the most significant percentage of lethality value was identified at 48 hours for the EO vinegar (Table 5) and ET (Table 6).

Table 4
Statistical comparison of mortality means and standard deviation of the pediculicidal effect on Bovicola caprae as a function of concentration of Eucalyptus wood with Thymus vulgaris WV and positive and negative controls.

Experimental groups
	WV concentration (% or g 100 mL-1)
C+	100	50	25	12.5	6.25	3.125	1.562	C-
43.2 ± 34.4 BA	57.5 ± 20.4 A	48.2 ± 26.2 CAD	25.7 ± 24.0 DB	19.0 ± 22.4 EBD	21.0 ± 27.0 FBDE	13.0 ± 27.0 GDEF	14.3 ± 15.4 HDEFGI	11.4 ± 17.7 IDEFG

• Means followed by different letters are statistically dissimilar by the Kruskal-Wallis test, post-hoc Dunn at 95% probability C+ = positive control (amitraz at 12.5%); C- = negative control (distilled water).

Table 5
Statistical comparison of mortality means and standard deviation of the pediculicidal effect on Bovicola caprae as a function of concentration of Eucalyptus wood with Origanum vulgare WV and positive and negative controls, according to the exposure time.

Experimental groups	Exposure time (hours)
	1	3	6	12	24	36	48
C+	37.5 ± 32.0 a	15.0 ± 13.0 a	25.0 ± 29.0 a	25.0 ± 30.0 a	70.0 ± 29.4a	82.5 ± 23.6 a	87.5 ± 19.0 a
100	80.0 ± 18.3 a	82.5 ± 17.1 a	72.5 ± 15.0 a	82.5 ± 12.6 a	87.5 ± 5.0 a	85.0 ± 10.0 a	85.0 ± 10.0 a
50	42.5 ± 9.6 ab	35.0 ± 12.9 ab	32.5 ± 15.0 b	37.5 ± 22.2 ab	60.0 ± 14.1 ab	72.5 ± 17.1 ab	82.5 ± 9.6 a
25	15.0 ± 5.8 b	17.5 ± 9.6 ab	15.0 ± 5.8 b	17.5 ± 5.0 ab	45.0 ± 10.0 ab	57.5 ± 5.0 ab	80.0 ± 11.5 a
12.5	2.5 ± 5.0 b	7.5 ± 15.0 ab	0.0 ± 0.0 b	5.0 ± 10.0 ab	25.0 ± 17.3 ab	55.0 ± 19.1 ab	70.0 ± 11.5 a
6.25	2.5 ± 5.0 b	0.0 ± 0.0 b	2.5 ± 5.0 ab	7.5 ± 9.6 ab	25.0 ± 10.0 ab	47.5 ± 5.0 ab	62.5 ± 5.0a
3.125	5.0 ± 10.0 b	7.5 ± 15.0 ab	7.5 ± 15.0 ab	10.0 ± 14.1 ab	15.0 ± 17.3 ab	42.5 ± 17.1 ab	52.5 ± 15.0a
1.562	5.0 ± 5.8 b	5.0 ± 5.8 b	5.0 ± 5.8 b	12.5 ± 5.0 ab	32.5 ± 9.6 ab	52.5 ± 9.6 ab	65.0 ± 10.0a
C-	5.0 ± 10.0 ba	0.0 ± 0.0 b	0.0 ± 0.0 b	5.0 ± 5.7 ab	20.0 ± 18.2 ab	32.5 ± 9.6 ab	47.5 ± 19.0a

• Means followed by different letters are statistically dissimilar by the Friedman test at 95% probability. C+ = positive control (amitraz at 12.5%); C- = negative control (distilled water).

Table 6
Statistical comparison of mortality means and standard deviation of the pediculicidal effect on Bovicola caprae as a function of concentration of Eucalyptus wood Thymus vulgaris WV and positive and negative controls, according to the exposure time.

Experimental groups	Exposure time (hours)
	1	3	6	12	24	36	48
C+	35.0 ± 24.0 ab	10.0 ± 8.1 b	10.0 ± 11.5 b	22.5 ± 20.6 ab	65.0 ± 23.8 ab	72.5 ± 28.7 a	87.5 ± 9.5a
ET100%	50.0 ± 18.2 ab	45.0 ± 13.0 ab	47.5 ± 12.5 ab	42.5 ± 17.1 b	55.0 ± 19.1 ab	77.5 ± 9.5 ab	85.0 ± 10.0a
ET50%	47.5 ± 21.0 ab	35.0 ± 35.1 ab	35.0 ± 30.0 b	37.5 ± 27.5 ab	42.5 ± 26.2 ab	65.0 ± 13.0 ab	75.0 ± 6.0a
ET25%	10.0 ± 8.1 b	15.50 ± 13.0 ab	12.5 ± 12.5 ab	15.0 ± 17.3 ab	20.0 ± 22.0 ab	45.0 ± 19.1 ab	62.5 ± 17.0a
ET12.5%	10.0 ± 5.0 ab	5.0 ± 10.0 ab	2.5 ± 5.0 ab	5.0 ± 10.0 b	17.5 ± 22.1 ab	42.5 ± 19.0 ab	50.0 ± 20.0a
ET6.25%	2.5 ± 5.0 ab	0.0 ± 0.0 b	2.5 ± 5.0 ab	5.0 ± 6.0 ab	17.5 ± 17.0 ab	52.5 ± 17.0 ab	65.0 ± 10.0a
ET3.125%	2.5 ± 5.0 ab	2.5 ± 5.0 ab	2.5 ± 5.0 ab	0.0 ± 0.0 b	5.0 ± 6.0 ab	45.0 ± 24.0 ab	62.5 ± 22.1a
ET1.562%	7.5 ± 9.5 ab	5.0 ± 6.0 ab	2.5 ± 5.0 b	5.0 ± 6.0 ab	12.5 ± 5.0 ab	22.5 ± 9.5 ab	45.0 ± 6.0a
C-	2.5 ± 5.0 a	0.0 ± 0.0 a	0.0 ± 0.0 a	0.0 ± 0.0 a	15.0 ± 19.1 a	27.5 ± 21.0 a	35.0 ± 17.3a

• Means followed by different letters are statistically dissimilar by the Friedman test at 95% probability. C+ = positive control (amitraz at 12.5%); C- = negative control (distilled water).

Within the same observation interval, the mortality rates for positive and negative controls also reached their highest levels. Furthermore, in the first hours (1 and 3 hours) for the EO vinegar, except for the positive control and the 6.25% concentration, high mortality means were presented for the EO, followed by a reduction in these values. A similar pattern was observed for the ET vinegar at the first evaluation time (1 hour), except for the negative control and the 25% concentration, with high mortality and decreased lethality in the subsequent observation. Based on the experimental data and appropriate statistical tests, the concentrations of the two types of wood vinegar required to kill half of the individuals in the experimental groups were estimated. This measurement, known as the lethal concentration 50 (LC50), was 49.90% and 90.49% for the EO and ET wood vinegars, respectively.

4. Discussion

The result that most demonstrated pediculicidal activity in the experiment was the EO vinegar, which had a strong insecticidal effect. A key characteristic of WVs is the synergistic interaction among their constituents, in which one compound can enhance the biological efficacy of another. This results in a synergistic effect that yields a cumulative outcome greater than the sum of the individual actions (Gama et al., 2023; Mota et al., 2025). Based on the chemical profiles of eucalyptus wood vinegar reported by Pimenta et al. (2018) and Oliveira et al. (2024), this pyrolysis product has a complex composition, containing a wide range of substances, with furfural as its main component. In this sense, the insecticidal activity presented by the wood vinegar can be essentially associated with the high furfural content, which, according to Chai et al. (2013), can inhibit the activity of the enzyme tyrosinase (polyphenol oxidase), which in insects is responsible for the sclerotization process of the exoskeleton and also plays a role in the immunity of these animals against pathogens through the synthesis of opsonins, encapsulation processes, and nodule structuring. However, in the present work, despite ET having a higher furfural content, EO exhibited a comparable pediculicidal effect after 48 hours of exposure.

The exoskeleton in insects serves as external protection. With sclerotization inhibition, the external skeleton loses rigidity, resulting in softening of the body. In addition, the interruption of molecular synthesis and immunological processes in these animals can contribute to their mortality (Chase et al., 2000; Chai et al., 2013; Neves, 2022). Another factor that may explain the louse-killing activity of EO vinegar is the high concentration of monoterpenes in oregano, especially eucalyptol and thymol (Maithani et al., 2023). According to Mohammedi (2017), these two components can exert insecticidal effects through different mechanisms, including neurotoxicity that acts as a positive modulator of gamma-aminobutyric acid (GABA) receptors and inhibits the insect nervous system.

According to Silva et al. (2017) and López and Pascual-Villalobos (2010), monoterpenes act together in the cholinergic system of insects. These compounds act by inhibiting the enzyme acetylcholinesterase (AChE), which regulates nerve impulse transmission, thereby causing paralysis and death of the parasites (López and Pascual-Villalobos, 2010; Silva et al., 2017). In addition, it is essential to highlight that, in agreement with Urrutia et al. (2021), the insecticidal activity of plant products, including WV, can be mediated by the synergistic action of minor components, which may act in concert with the main compounds to contribute to biological activity. Further corroborating the insecticidal effect of EO vinegar, Mattos et al. (2019) describe the pesticide capacity of WVs produced from different biomass sources on moths, beetles, termites, aphids, and mosquito species.

On the other hand, regarding the biocidal effect presented by the ET extract on lice, classified as weak according to parameters established by Gemeda et al. (2014), it is likely that compounds present exclusively in thyme act antagonistically to the other bioactives responsible for the insecticidal activity, reducing the overall efficiency of the extract. Regarding the performance of the extracts in the mortality of B. caprae specimens throughout the observation period of this research, a study by Burkhart and Burkhart (2001) describes similar behavior. The authors report that monitoring to assess lice mortality should extend for 24 hours, even if vital signs are already absent, to avoid overestimated lethality. After 24 to 36 hours, the physiological death of the parasites is considered normal (Burkhart and Burkhart, 2001), which may explain the high mortality rates at 48 hours.

Also linked to the period of observation of lice mortality is the supposed inconsistency in lethality rates, which are high in the first few hours and then drop. A likely explanation for this behavior is the phenomenon known as " lice resurrection” (Heukelbach et al., 2006). In this mechanism, ectoparasites can remain for long periods without breathing, closing their respiratory spiracles to avoid death by asphyxiation and, in addition, assuming a state of apparent death (Burkhart and Burkhart, 2006). From this perspective, wood vinegar, especially EO extract, emerges as a promising alternative to the synthetic compounds used to control lice in goats. This alternative approach is relevant because treating caprine pediculosis with synthetic substances poses a risk of contaminating goat farming products and the environment. Furthermore, bioproducts can serve as promising alternatives, helping reduce the progression of chemical drug resistance (Silva et al., 2026).

Acknowledgements

This work was carried out with the support of CNPq (National Council for Scientific and Technological Development – Brazil), Process No. 131375/2023-7. No potential conflict of interest was reported by the author(s). The authors also acknowledge the institutional support of Universidade Federal Rural do Semi-Árido (UFERSA).

Data Availability Statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  24 July 2026
• Date of issue
  2026

History

• Received
  25 Feb 2026
• Accepted
  17 May 2026