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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Evaluation of the Molluscicidal Effects of Thymus vulgaris and Syzygium aromaticum Oils via Contact and Bait Methods on Monacha cartusiana Snails with Special Emphasize on Neurotoxical, Biochemical, Histopathological and Endocrine Disrupting

H
Heba Abdel Tawab1,*
S
Shawky M. Aboelhadid2
E
Ebtesam A. Yousef3
A
Abdel-Azeem S. Abdel-Baki1
A
Ahmed O. Hassan4
S
Saleh Al-Quraishy5
H
Heba Y. Ahmed6
1Department of Zoology, Faculty of Science, Beni-Suef University, Beni Suef, Egypt.
2Department of Parasitology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt.
3Department of Zoology, Faculty of Science, Sohag University, 82524 Sohag, Egypt.
4Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
5Department Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia.
6Department of Harmful Animals Research, Plant Protectin Research Insititute, Agriculture Research Center.

ABSTRACT

Background: Monacha cartusiana is considered one of the important pests in agriculture and public health sectors. So, its management is critical to global food security. The purpose of this study was to look at the toxicity of Thymus vulgaris (thyme) and Syzygium aromaticum (clove) oil by bait and contact technique. 

Methods: Gas Chromatography-Mass Spectrometry (GC-MS) was used to determine the essential oil’s components. the LC50 of oils via bait and contact testing were determined in the laboratory experiment. Moreover, the molluscicidal activity following the exposure of snails to LC50 were evaluated through using snail soft tissues.

Result: The GC–MS analysis depicted that Thymol (33.89%) and eugenol (32.82%) was the major constituent present in the oils. both oils have ovicidal activity causing complete inhibition of eggs hatchability. Compared to control, the oils showed perturbations in antioxidant/oxidant biomarker, where reduced glutathione significantly decrease and lipid peroxidation increased. Concerning energy reserves biomarker, total lipid level significantly elevated in oils treated snails, While the total protein significantly decreased, Moreover, metabolic enzyme alkaline phosphatase was markedly augmentation. Both oils at the tested doses caused a significant inhibition in acetyl choline esterase activity and downregulation in 17β-estradiol and testosterone hormones. Also, snail digestive glands and ovotestis showed pathological alterations after essential oil exposure. In field application, thyme, clove and binary mixture caused significant reduction in M. cartusiana population by bait assay. Our findings emphasis thyme and clove oil’s potential as an effective biorational molluscicide against pestiferous snails as an alternative to chemocentric management.

INTRODUCTION

Land snails are among the most dangerous pests attacking agricultural crops globally and their economic impact has grown significantly (Barker, 2002). Monacha cartusiana, one of these land snails, is regarded as a snail pest that seriously harms numerous agricultural crops.  It has critical impacts in field crops, fruits, vegetables and ornamental plants nurseries (Abd El-Halim et al., 2021; Murshed et al., 2024). In addition, it acts as a vector for a number of parasites and diseases that affect cultivated plants (Godan, 1983). Pest management in agriculture is crucial for global food security since it ensures crop productivity and quality production (Muhie, 2022).
       
Molluscicides are used extensively to control snail infestations all over the world. However, the indiscriminate application of synthetic molluscicides such as methomyl and other carbamates to control snails poses a serious risk to beneficial and non-target organisms and the emergence of pesticide resistance (Simms et al., 2002; Abdel-Halim et al., 2021; Pathak et al., 2022). To address all of the above issues, there is an urgent need to develop innovative, low-cost, eco-friendly and effective alternative molluscicides.
       
Plant-derived essential oils (EOs)and components are key sources of novel bioactive compounds with broad-spectrum insecticidal activity and they are typically regarded safe for humans and the environment (Dassanayake et al., 2021; Radwan and Gad 2021; Sekar et al., 2025). EOs have been shown to be effective against a variety of pests, including insects, mites, fungi, slugs, snails and nematodes (Isman, 2000; Rana et al., 2011; Yazdani et al., 2014; Sujatha et al., 2015; Pavela and Benelli, 2016; Barua et al., 2017; Da Silva et al., 2018; Klein et al., 2020; Nasiou and Giannakou, 2020).
       
Thymus vulgaris, “thyme,” and Syzygium aromaticum (clove) are well-known aromatic herbs that have long been used as a spice and herbal remedy (Aljabeili et al., 2018; Haro-Gonzalez et al., 2021). The most prevalent fragrant bioactive compounds in thyme and clove essential oils are thymol and eugenol (Bozin et al., 2006; Gavaric et al., 2015; Pandey et al., 2022). The US FDA claims that clove and thyme essential oils are both safe and have a favorable effect on the environment (Food et al., 2007).
       
The pilot study of Ismail and Abdel-Kader (2011) concluded that the flower-bud powder and commercially available eugenol of Syzygium aromaticum seem to have a possible role in the control of land snails. Also, Ali (2017) found  that the aqueous extract of Thymus vulgaris have mollus-cicidal effect against the land snail Eobanica vermiculate.
       
This study expands on the earlier conclusion to examine the molluscicidal effects of thyme and clove against the land snail, M. cartusiana.
 

MATERIALS AND METHODS

Tested compounds and its consistent
 
Thyme and clove oils were purchased from Purity Factory in the food sector of Kom Abu-Radi Industrial Area, Alwasta, Beni-Suef, Egypt. methyl N- (methylcarbamoyloxy) ethanimidothioate, also known as Methomyl (20% SL), is a carbamate compound purchased from the chemical company of Egypt chem international Agricultural chemical, Egypt, Cairo (purity: 95.5%). The Ministry of Agriculture and Land Reclamation in Egypt has recommended using methomyl to fight land snail infestation on Egyptian fields.
       
Gas Chromatography-Mass Spectrometry (GC-MS) analysis was used to determine the essential oil’s components. Chemical analysis of thyme and clove oils were carried out in the Central Laboratory of the Faculty of Postgraduate Studies for Advanced Sciences, Beni-Suef University, Egypt, using an Agilent Technologies GC system 7890A/5975 Inert MS with Triple Axis Detector. The constituent derivatives were identified by matching their mass spectra to derivative spectra in the Library Search Report [C:\Database\NIST11.L;C:\Database\demo.1].
 
Experimental animals
 
Adult M. cartusiana snails were collected from infested clover fields in Quftan village, Sumasta district, Beni-Suef Governorate and transported to the laboratory in a plastic bag. Snails were acclimatized by keeping them in plastic containers with moist soil and feeding them lettuce leaves for 14 days.
 
Laboratory bioaasay
 
Bait toxicity assay
 
Different concentrations of clove oil (1.25, 2.5, 5, 10, 20, 50 mg/ml) and thyme oil (1.25, 2.5, 5, 10, 20, 50 mg/ml), were tested as poison baits against M. cartusiana and formulated by combining each of these concentrations with 5% molasses + 93% bran. Five grams of bait were placed on the soil surface of each glass box using a plastic sheet. Each tested bait was given to thirty snails (three replicates of ten snails each). Other thirty individuals (three replicates of ten snails each) served as the control group. After 7 days, mortality rates and the LC50 value were calculated according to Finney (1971).
 
Contact toxicity assay
 
The contact toxicity assay was performed using the method described by Asher and Mirian (1981). In brief, two ml of thyme oil (0.32, 0.625 and 1.25 mg/ml) and clove oil (0.625, 1.25, 2.5 and 5 mg/ml) were distributed on the inner surface of a petri-dish and gently rotated in circles. For each concentration, three replicates were used. Equal numbers of untreated snail replicas were given water and were considered the control group. According to Finney (1971), dead animals were counted daily and the mortality % and LC50 were calculated.
 
Ovicidal activities of the Eos
 
After the egg deposition, new clutches were retrieved using a fine hair brush. The eggs were divided into 20-egg batches. Each batch of eggs (up to 24 hours old) was placed in a culture dish containing 5 g of sterile moist soil. One ml of LC50 concentration of each tested compound was applied directly to egg batches. The control group of eggs received distilled water treatment. Each treatment was assessed in three replicates. The hatchability of the eggs was checked daily for seven days.
 
Collection and preparation of tissues
 
The toxicity of EOs against M. cartusiana snails was assessed using the topical application approach (contact action) described by Radwan et al., (2008). For each treatment, triplicates (each of 10 people) were exposed to the LC50 concentrations the thyme and clove oils for 24 hours. The control group received distal water treatment. After removing the snail’s shell, the soft tissue was weighed and homogenized in 10 volumes of ice-cold saline solution (w/v ratio) for 60 seconds using a Polytron Kinemetica homogenizer, then centrifuged at 3000 r.p.m. for 15 minutes. Supernatants were isolated and then stored at -80oC until needed.
 
Biochemical assessment
 
Oxidant/antioxidant defense biomarker
 
The lipid peroxidation (MDA) and glutathione (GSH) levels were evaluated adopting the Ohkawa et al., (1979) and Beutler et al. (1963) methods, respectively, using a reagent kit purchased from Biodiagnostic Company (Egypt).
 
Metabolic enzyme markers
 
Alkaline phosphatase activity (ALP) was measured according to the manufacturer’s instructions for the Spectrum Diagnostic Kit (ALP, Cat. No. AP 10 20). 
 
Energy reserves biomarker
 
Total protein (TP) (Cat. No. TP 20 21) was assessed according to Kinsley and Frankel (1939), meanwhile total lipids (TL)  (Cat. No. TL 20 10) level were estimated by the methods of Zöllner and Kirsch (1962).
 
Neurotoxicity marker
 
Acetylcholinesterase (AChE; EC 3.1.1.7) assay is based on an improved Ellman method, in which thiocholine produced by the action of acetylcholinesterase forms a yellow color with 5,5’-dithiobis (2-nitrobenzoic acid). The intensity of the product color, measured at 412 nm, is proportionate to the enzyme activity in the sample (Ellman et al., 1961).
 
Steroid hormones
 
Hormone concentrations testosterone (T) and 17 β-estradiol (E) were assayed according to the manufacture instructions of T EIA kit (Enzo Life Science, Michigan, USA, ADI-900-065) and E EIA kit (Cayman Chemical Company, Michigan, USA, item no. 582251).
 
Histological studies
 
The digestive gland and ovotestis of the surviving M. cartusiana snails were removed and preserved in Bouin’s solution for histological studies. According to Mohamed and Saad (1990), these samples were first dehydrated in gradient ethanol, then embedded in paraffin wax and then cut into sections and stained with hematoxylin and eosin. Slides of these organs were examined for any histological changes in comparison to control snails.
 
Field application
 
The field efficacy of clove and thyme oil as bait against Monacha snails was compared to the prescribed methomyl compound (2%). At Quftan Village, Sumsta District, Beni- Suef Governorate, twelve clover-growing plots (each 30 m2) were selected and infested with M. cartusiana. For each evaluated compound and the control, three replicates were used. The space between the plots is maintained at least at four metres. The most potent lab-tested concentrations of thyme (50 mg/ml) and clove oils (20 mg/ml) were selected for evaluation against M. cartusiana in the field after treatment, live snails were counted in each plot both before and after the application. Henderson and Tilton (Henderson and TILTON, 1955) determined the decline in snail population after 21 days of treatment.
 
Statistical analysis
 
Our data was analyzed by one-way analysis of variance (ANOVA) using SPSS (version 20) statistical program (SPSS Inc., Chicago, IL, USA). Analysis of differences between the means of different groups was performed using Duncan’s multiple range and two tailed paired t-test. The lethal concentration values and respective 95% confidence limit (CL) of LC50 was calculated by Probit analysis (Finney, 1971).

RESULTS AND DISCUSSION

GC Mas analysis of thyme and clove oil
 
The GC-MS analysis identified the primary components of thyme oil as thymol (33.89%), p-cymene (20.48%), carvacrol (4.65%), terpinen-4-ol (3.71%) and linalool (7.74%). For clove oil, the main constituents were eugenol (32.82%), heneicosane (11.32%), acetophenone (11.16%) and benzothiophene-3-carbohydrazide (9.91%) as shown in (Table 1).

Table 1: Chemical composition of thyme oil and clove oil as detected by GC-MS analysis.


 
Molluscicidial activity of thyme and clove oil against M. cartusiana snails
 
The molluscicidal activity of thyme and clove oils was confirmed through bait and contact toxicity assays (Table 2) with both methods showing dose-dependent effects. In bait toxicity, thyme oil exhibited higher mortality (93.33% at 50 mg, P<0.001 ) compared to clove oil (80.00%, P<0.05), with LC50 values of 15.45 mg and 16.02 mg, respectively. Contact toxicity assays demonstrated greater efficacy, with thyme oil causing 100% mortality at 0.625 mg and achieving an LC50 of 0.21 mg, compared to clove oil’s LC50 of 0.548 mg.

Table 2: Molluscicidal activity, LC50, LC90 and 95% confidence limit (CL) of clove and thyme against M. cartusiana snail after 24 h of exposure by Bait and contact toxicity assay.


 
Ovicidal activities of the Eos
 
Both oils showed significant ovicidal activity at LC50 concentrations (Fig 1), causing 100% un-hatchability.

Fig 1: Ovicidal activity of thyme (B) and clove (C) oil against M. cartusiana snail after 7 days of LC50 exposure in compared to control (A), EM: embryo.


 
Biochemical evaluation
 
Sub-lethal doses of thyme and clove oils induced oxidative stress, evident from elevated MDA levels and decreased non-enzymatic antioxidant GSH levels in treated snails (Fig 2). Thyme oil caused a greater increase in lipid peroxidation than clove oil (P<0.001).

Fig 2: Oxidant/antioxidant stress biomarkers in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls.


       
Fig 3 shows that the LC50 doses of thyme and clove oil significantly increased ALP activity in snail tissues (P<0.001; P<0.05, respectively) when compared to control snails. Comparing thyme oil to clove oil, thyme oil significantly increased ALP (P<0.001).

Fig 3: Metabolic enzyme markers in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls.


       
Thyme and clove oils significantly increased lipid levels (P<0.001) and decreased total protein levels in treated snails (Fig 4). Following exposure to thyme and clove Eos at LC50 concentration, levels of T significantly decreased (P<0.01, P<0.001, respectively). Additionally, when compared to control, both oils had the same effect on the level of E (P<0.001, P<0.001) (Fig 5). Clove oil caused more severely endocrine disruption (P<0.001) than thyme oil did.

Fig 4: Energy reserves biomarker in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls.



Fig 5: Steroid hormones in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls.


       
Both oils significantly reduced acetylcholine esterase (AChE) activity (Fig 6), with thyme oil exhibiting stronger inhibition P<0.001.

Fig 6: Acetylcholinestrase activity in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls and treated group.



Histopathological evaluation
 
Histological analysis
 
The results of the current investigation demonstrated that LC50 dose of thyme oil caused rupturing of the germinal epithelium in the digestive gland (Fig 7B), as well as deformed sperm and ova in the hermaphrodite gland of M. cartusiana snails (Fig 8B). On the other hand, clove oil significantly reduced the amount of sperm and ova along with severe damage (Fig 8C). Additionally, the digestive gland displayed abnormalities such as inflammation, DC degeneration and tubular disturbances with loss of their characteristic form (Fig 7C).

Fig 7: Light micrographs showing effect of thyme and clove oil at LC50 on the digestive gland of M. cartusiana snails.



Fig 8: Light micrographs showing effect of thyme and clove oil at LC50 on ovotests of M. cartusiana snails.


 
Field efficacy
 
Field studies demonstrated significant reductions in the M. cartusiana population after treatment with thyme and clove oils, achieving reductions of 90.94% and 93.56%, respectively Table 3.

Table 3: Field performance of thyme, clove and binary mixture against land snails, M. cartusiana, comparing with control after 21 days of treatment.


       
The chemical compositions of thyme and clove oils in this study are consistent with those reported by Haro-Gonzalez et al. (2021) and Pandey et al. (2022), though slight variations are expected due to factors like geographic origin, cultivation conditions and extraction techniques (Pavela and Benelli, 2016). The phenolic compounds thymol and eugenol are recognized for their potent bioactivity and are likely responsible for the strong molluscicidal effects observed (Radwan and Gad, 2021).
       
The high mortality rates in both bait and contact assays confirm the potent molluscicidal properties of thyme and clove oils, with thyme oil showing slightly greater efficacy. These findings align with earlier work by El-Zemity and Radwan (2001) and Abdel-Rahman and Amal (2020), reinforcing the topical effectiveness of essential oils against snails.
       
The ovicidal activity observed supports the hypothesis that essential oils interfere with embryonic development, as previously reported by Ali (2017). This may be attributed to disruption of hormonal regulation or direct damage to egg membranes.
       
Biochemical assays indicated that sub-lethal doses of both oils induce oxidative stress, evidenced by increased MDA and decreased GSH levels. These results echo findings by Lazarevic et al. (2020) and Gad et al. (2023), who observed similar oxidative responses in invertebrates exposed to essential oils.
       
Increased ALP activity, especially with thyme oil, suggests lysosomal membrane destabilization and cellular damage, consistent with prior observations (Shekari et al., 2008; Mobarak et al., 2015). The accompanying increase in lipid levels and reduction in protein content may be due to stress-induced catabolism and impaired metabolic functions, in line with Capowiez et al., (2015).
       
The endocrine-disrupting effects, particularly the reduction in testosterone and estradiol levels, indicate interference with steroidogenesis, supporting conclusions by Desouky et al., (2022). The more severe hormonal disruption caused by clove oil implies differential activity of its constituents on endocrine pathways.
       
AChE inhibition by both oils, especially thyme oil, suggests neurotoxicity. This is likely due to thymol and eugenol, known AChE inhibitors (Liao et al., 2016; El Gohary et al., 2021). Such inhibition can lead to overstimulation of the nervous system and ultimately, death.
       
Histopathological changes observed in the digestive and hermaphrodite glands reinforce the biochemical and hormonal findings. The degeneration of reproductive tissues indicates a significant impact on fertility, as similarly reported by Desouky et al., (2022) and Hategekimana and Erler (2020).
       
Finally, the field efficacy results highlight the practical potential of these essential oils as eco-friendly molluscicidal agents. The comparable performance to methomyl suggests their possible integration into integrated pest management strategies, as supported by previous field studies (Ismail and Abdel-Kader, 2011; Powell and Bowen, 1996).

CONCLUSION

Our research highlights the potential of thyme and clove oil as a biorational molluscicide against land snails as an alternative for the chemical managements.

ACKNOWLEDGEMENT

This work was supported by Ongoing Research Funding Program  (ORF-2025-03), King Saud University, Riyadh, Saudi Arabia.
 
Authors contributions
 
Conceptualization; Heba Abdel Tawab, Ebtesam A. Yousef, Heba Y. Ahmed; Data curation; Heba Abdel Tawab, Heba Y. Ahmed, Abdel-Baki, A.A.S., Ahmed O Hassan; Formal analysis; Heba Abdel Tawab, Heba Y. Ahmed; Funding acquisition; Ahmed, Abdel-Baki, A.A.S., Saleh Al-Quraishy; Investigation; Heba Abdel Tawab, Ebtesam A. Yousef; Methodology Heba Abdel Tawab, Ebtesam A. Yousef, Heba Y. Ahmed Supervision; Shawky M Aboelhadid, Ahmed, Abdel-Baki, A.A.S; Validation; Saleh Al-Quraishy, Shawky M. Aboelhadid, Ahmed O Hassan; Visualization; Heba Abdel Tawab; Heba Y. Ahmed Roles/Writing - original draft; Heba Abdel Tawab, Writing - review and editing; Ahmed, Abdel-Baki, A.A.S., Shawky M. Aboelhadid.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Ethics approval
 
Not applicable.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships.

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    Evaluation of the Molluscicidal Effects of Thymus vulgaris and Syzygium aromaticum Oils via Contact and Bait Methods on Monacha cartusiana Snails with Special Emphasize on Neurotoxical, Biochemical, Histopathological and Endocrine Disrupting

    H
    Heba Abdel Tawab1,*
    S
    Shawky M. Aboelhadid2
    E
    Ebtesam A. Yousef3
    A
    Abdel-Azeem S. Abdel-Baki1
    A
    Ahmed O. Hassan4
    S
    Saleh Al-Quraishy5
    H
    Heba Y. Ahmed6
    1Department of Zoology, Faculty of Science, Beni-Suef University, Beni Suef, Egypt.
    2Department of Parasitology, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62511, Egypt.
    3Department of Zoology, Faculty of Science, Sohag University, 82524 Sohag, Egypt.
    4Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
    5Department Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia.
    6Department of Harmful Animals Research, Plant Protectin Research Insititute, Agriculture Research Center.

    ABSTRACT

    Background: Monacha cartusiana is considered one of the important pests in agriculture and public health sectors. So, its management is critical to global food security. The purpose of this study was to look at the toxicity of Thymus vulgaris (thyme) and Syzygium aromaticum (clove) oil by bait and contact technique. 

    Methods: Gas Chromatography-Mass Spectrometry (GC-MS) was used to determine the essential oil’s components. the LC50 of oils via bait and contact testing were determined in the laboratory experiment. Moreover, the molluscicidal activity following the exposure of snails to LC50 were evaluated through using snail soft tissues.

    Result: The GC–MS analysis depicted that Thymol (33.89%) and eugenol (32.82%) was the major constituent present in the oils. both oils have ovicidal activity causing complete inhibition of eggs hatchability. Compared to control, the oils showed perturbations in antioxidant/oxidant biomarker, where reduced glutathione significantly decrease and lipid peroxidation increased. Concerning energy reserves biomarker, total lipid level significantly elevated in oils treated snails, While the total protein significantly decreased, Moreover, metabolic enzyme alkaline phosphatase was markedly augmentation. Both oils at the tested doses caused a significant inhibition in acetyl choline esterase activity and downregulation in 17β-estradiol and testosterone hormones. Also, snail digestive glands and ovotestis showed pathological alterations after essential oil exposure. In field application, thyme, clove and binary mixture caused significant reduction in M. cartusiana population by bait assay. Our findings emphasis thyme and clove oil’s potential as an effective biorational molluscicide against pestiferous snails as an alternative to chemocentric management.

    INTRODUCTION

    Land snails are among the most dangerous pests attacking agricultural crops globally and their economic impact has grown significantly (Barker, 2002). Monacha cartusiana, one of these land snails, is regarded as a snail pest that seriously harms numerous agricultural crops.  It has critical impacts in field crops, fruits, vegetables and ornamental plants nurseries (Abd El-Halim et al., 2021; Murshed et al., 2024). In addition, it acts as a vector for a number of parasites and diseases that affect cultivated plants (Godan, 1983). Pest management in agriculture is crucial for global food security since it ensures crop productivity and quality production (Muhie, 2022).
           
    Molluscicides are used extensively to control snail infestations all over the world. However, the indiscriminate application of synthetic molluscicides such as methomyl and other carbamates to control snails poses a serious risk to beneficial and non-target organisms and the emergence of pesticide resistance (Simms et al., 2002; Abdel-Halim et al., 2021; Pathak et al., 2022). To address all of the above issues, there is an urgent need to develop innovative, low-cost, eco-friendly and effective alternative molluscicides.
           
    Plant-derived essential oils (EOs)and components are key sources of novel bioactive compounds with broad-spectrum insecticidal activity and they are typically regarded safe for humans and the environment (Dassanayake et al., 2021; Radwan and Gad 2021; Sekar et al., 2025). EOs have been shown to be effective against a variety of pests, including insects, mites, fungi, slugs, snails and nematodes (Isman, 2000; Rana et al., 2011; Yazdani et al., 2014; Sujatha et al., 2015; Pavela and Benelli, 2016; Barua et al., 2017; Da Silva et al., 2018; Klein et al., 2020; Nasiou and Giannakou, 2020).
           
    Thymus vulgaris    , “thyme,” and Syzygium aromaticum (clove) are well-known aromatic herbs that have long been used as a spice and herbal remedy (Aljabeili et al., 2018; Haro-Gonzalez et al., 2021). The most prevalent fragrant bioactive compounds in thyme and clove essential oils are thymol and eugenol (Bozin et al., 2006; Gavaric et al., 2015; Pandey et al., 2022). The US FDA claims that clove and thyme essential oils are both safe and have a favorable effect on the environment (Food et al., 2007).
           
    The pilot study of Ismail and Abdel-Kader (2011) concluded that the flower-bud powder and commercially available eugenol of Syzygium aromaticum seem to have a possible role in the control of land snails. Also, Ali (2017) found  that the aqueous extract of Thymus vulgaris have mollus-cicidal effect against the land snail Eobanica vermiculate.
           
    This study expands on the earlier conclusion to examine the molluscicidal effects of thyme and clove against the land snail, M. cartusiana.
     

    MATERIALS AND METHODS

    Tested compounds and its consistent
     
    Thyme and clove oils were purchased from Purity Factory in the food sector of Kom Abu-Radi Industrial Area, Alwasta, Beni-Suef, Egypt. methyl N- (methylcarbamoyloxy) ethanimidothioate, also known as Methomyl (20% SL), is a carbamate compound purchased from the chemical company of Egypt chem international Agricultural chemical, Egypt, Cairo (purity: 95.5%). The Ministry of Agriculture and Land Reclamation in Egypt has recommended using methomyl to fight land snail infestation on Egyptian fields.
           
    Gas Chromatography-Mass Spectrometry (GC-MS) analysis was used to determine the essential oil’s components. Chemical analysis of thyme and clove oils were carried out in the Central Laboratory of the Faculty of Postgraduate Studies for Advanced Sciences, Beni-Suef University, Egypt, using an Agilent Technologies GC system 7890A/5975 Inert MS with Triple Axis Detector. The constituent derivatives were identified by matching their mass spectra to derivative spectra in the Library Search Report [C:\Database\NIST11.L;C:\Database\demo.1].
     
    Experimental animals
     
    Adult M. cartusiana snails were collected from infested clover fields in Quftan village, Sumasta district, Beni-Suef Governorate and transported to the laboratory in a plastic bag. Snails were acclimatized by keeping them in plastic containers with moist soil and feeding them lettuce leaves for 14 days.
     
    Laboratory bioaasay
     
    Bait toxicity assay
     
    Different concentrations of clove oil (1.25, 2.5, 5, 10, 20, 50 mg/ml) and thyme oil (1.25, 2.5, 5, 10, 20, 50 mg/ml), were tested as poison baits against M. cartusiana and formulated by combining each of these concentrations with 5% molasses + 93% bran. Five grams of bait were placed on the soil surface of each glass box using a plastic sheet. Each tested bait was given to thirty snails (three replicates of ten snails each). Other thirty individuals (three replicates of ten snails each) served as the control group. After 7 days, mortality rates and the LC50 value were calculated according to Finney (1971).
     
    Contact toxicity assay
     
    The contact toxicity assay was performed using the method described by Asher and Mirian (1981). In brief, two ml of thyme oil (0.32, 0.625 and 1.25 mg/ml) and clove oil (0.625, 1.25, 2.5 and 5 mg/ml) were distributed on the inner surface of a petri-dish and gently rotated in circles. For each concentration, three replicates were used. Equal numbers of untreated snail replicas were given water and were considered the control group. According to Finney (1971), dead animals were counted daily and the mortality % and LC50 were calculated.
     
    Ovicidal activities of the Eos
     
    After the egg deposition, new clutches were retrieved using a fine hair brush. The eggs were divided into 20-egg batches. Each batch of eggs (up to 24 hours old) was placed in a culture dish containing 5 g of sterile moist soil. One ml of LC50 concentration of each tested compound was applied directly to egg batches. The control group of eggs received distilled water treatment. Each treatment was assessed in three replicates. The hatchability of the eggs was checked daily for seven days.
     
    Collection and preparation of tissues
     
    The toxicity of EOs against M. cartusiana snails was assessed using the topical application approach (contact action) described by Radwan et al., (2008). For each treatment, triplicates (each of 10 people) were exposed to the LC50 concentrations the thyme and clove oils for 24 hours. The control group received distal water treatment. After removing the snail’s shell, the soft tissue was weighed and homogenized in 10 volumes of ice-cold saline solution (w/v ratio) for 60 seconds using a Polytron Kinemetica homogenizer, then centrifuged at 3000 r.p.m. for 15 minutes. Supernatants were isolated and then stored at -80oC until needed.
     
    Biochemical assessment
     
    Oxidant/antioxidant defense biomarker
     
    The lipid peroxidation (MDA) and glutathione (GSH) levels were evaluated adopting the Ohkawa et al., (1979) and Beutler et al. (1963) methods, respectively, using a reagent kit purchased from Biodiagnostic Company (Egypt).
     
    Metabolic enzyme markers
     
    Alkaline phosphatase activity (ALP) was measured according to the manufacturer’s instructions for the Spectrum Diagnostic Kit (ALP, Cat. No. AP 10 20). 
     
    Energy reserves biomarker
     
    Total protein (TP) (Cat. No. TP 20 21) was assessed according to Kinsley and Frankel (1939), meanwhile total lipids (TL)  (Cat. No. TL 20 10) level were estimated by the methods of Zöllner and Kirsch (1962).
     
    Neurotoxicity marker
     
    Acetylcholinesterase (AChE; EC 3.1.1.7) assay is based on an improved Ellman method, in which thiocholine produced by the action of acetylcholinesterase forms a yellow color with 5,5’-dithiobis (2-nitrobenzoic acid). The intensity of the product color, measured at 412 nm, is proportionate to the enzyme activity in the sample (Ellman et al., 1961).
     
    Steroid hormones
     
    Hormone concentrations testosterone (T) and 17 β-estradiol (E) were assayed according to the manufacture instructions of T EIA kit (Enzo Life Science, Michigan, USA, ADI-900-065) and E EIA kit (Cayman Chemical Company, Michigan, USA, item no. 582251).
     
    Histological studies
     
    The digestive gland and ovotestis of the surviving M. cartusiana snails were removed and preserved in Bouin’s solution for histological studies. According to Mohamed and Saad (1990), these samples were first dehydrated in gradient ethanol, then embedded in paraffin wax and then cut into sections and stained with hematoxylin and eosin. Slides of these organs were examined for any histological changes in comparison to control snails.
     
    Field application
     
    The field efficacy of clove and thyme oil as bait against Monacha snails was compared to the prescribed methomyl compound (2%). At Quftan Village, Sumsta District, Beni- Suef Governorate, twelve clover-growing plots (each 30 m2) were selected and infested with M. cartusiana. For each evaluated compound and the control, three replicates were used. The space between the plots is maintained at least at four metres. The most potent lab-tested concentrations of thyme (50 mg/ml) and clove oils (20 mg/ml) were selected for evaluation against M. cartusiana in the field after treatment, live snails were counted in each plot both before and after the application. Henderson and Tilton (Henderson and TILTON, 1955) determined the decline in snail population after 21 days of treatment.
     
    Statistical analysis
     
    Our data was analyzed by one-way analysis of variance (ANOVA) using SPSS (version 20) statistical program (SPSS Inc., Chicago, IL, USA). Analysis of differences between the means of different groups was performed using Duncan’s multiple range and two tailed paired t-test. The lethal concentration values and respective 95% confidence limit (CL) of LC50 was calculated by Probit analysis (Finney, 1971).

    RESULTS AND DISCUSSION

    GC Mas analysis of thyme and clove oil
     
    The GC-MS analysis identified the primary components of thyme oil as thymol (33.89%), p-cymene (20.48%), carvacrol (4.65%), terpinen-4-ol (3.71%) and linalool (7.74%). For clove oil, the main constituents were eugenol (32.82%), heneicosane (11.32%), acetophenone (11.16%) and benzothiophene-3-carbohydrazide (9.91%) as shown in (Table 1).

    Table 1: Chemical composition of thyme oil and clove oil as detected by GC-MS analysis.


     
    Molluscicidial activity of thyme and clove oil against M. cartusiana snails
     
    The molluscicidal activity of thyme and clove oils was confirmed through bait and contact toxicity assays (Table 2) with both methods showing dose-dependent effects. In bait toxicity, thyme oil exhibited higher mortality (93.33% at 50 mg, P<0.001 ) compared to clove oil (80.00%, P<0.05), with LC50 values of 15.45 mg and 16.02 mg, respectively. Contact toxicity assays demonstrated greater efficacy, with thyme oil causing 100% mortality at 0.625 mg and achieving an LC50 of 0.21 mg, compared to clove oil’s LC50 of 0.548 mg.

    Table 2: Molluscicidal activity, LC50, LC90 and 95% confidence limit (CL) of clove and thyme against M. cartusiana snail after 24 h of exposure by Bait and contact toxicity assay.


     
    Ovicidal activities of the Eos
     
    Both oils showed significant ovicidal activity at LC50 concentrations (Fig 1), causing 100% un-hatchability.

    Fig 1: Ovicidal activity of thyme (B) and clove (C) oil against M. cartusiana snail after 7 days of LC50 exposure in compared to control (A), EM: embryo.


     
    Biochemical evaluation
     
    Sub-lethal doses of thyme and clove oils induced oxidative stress, evident from elevated MDA levels and decreased non-enzymatic antioxidant GSH levels in treated snails (Fig 2). Thyme oil caused a greater increase in lipid peroxidation than clove oil (P<0.001).

    Fig 2: Oxidant/antioxidant stress biomarkers in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls.


           
    Fig 3 shows that the LC50 doses of thyme and clove oil significantly increased ALP activity in snail tissues (P<0.001; P<0.05, respectively) when compared to control snails. Comparing thyme oil to clove oil, thyme oil significantly increased ALP (P<0.001).

    Fig 3: Metabolic enzyme markers in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls.


           
    Thyme and clove oils significantly increased lipid levels (P<0.001) and decreased total protein levels in treated snails (Fig 4). Following exposure to thyme and clove Eos at LC50 concentration, levels of T significantly decreased (P<0.01, P<0.001, respectively). Additionally, when compared to control, both oils had the same effect on the level of E (P<0.001, P<0.001) (Fig 5). Clove oil caused more severely endocrine disruption (P<0.001) than thyme oil did.

    Fig 4: Energy reserves biomarker in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls.



    Fig 5: Steroid hormones in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls.


           
    Both oils significantly reduced acetylcholine esterase (AChE) activity (Fig 6), with thyme oil exhibiting stronger inhibition P<0.001.

    Fig 6: Acetylcholinestrase activity in M. cartusiana exposed to sublethal LC50 of thyme and clove oil along with their respective controls and treated group.



    Histopathological evaluation
     
    Histological analysis
     
    The results of the current investigation demonstrated that LC50 dose of thyme oil caused rupturing of the germinal epithelium in the digestive gland (Fig 7B), as well as deformed sperm and ova in the hermaphrodite gland of M. cartusiana snails (Fig 8B). On the other hand, clove oil significantly reduced the amount of sperm and ova along with severe damage (Fig 8C). Additionally, the digestive gland displayed abnormalities such as inflammation, DC degeneration and tubular disturbances with loss of their characteristic form (Fig 7C).

    Fig 7: Light micrographs showing effect of thyme and clove oil at LC50 on the digestive gland of M. cartusiana snails.



    Fig 8: Light micrographs showing effect of thyme and clove oil at LC50 on ovotests of M. cartusiana snails.


     
    Field efficacy
     
    Field studies demonstrated significant reductions in the M. cartusiana population after treatment with thyme and clove oils, achieving reductions of 90.94% and 93.56%, respectively Table 3.

    Table 3: Field performance of thyme, clove and binary mixture against land snails, M. cartusiana, comparing with control after 21 days of treatment.


           
    The chemical compositions of thyme and clove oils in this study are consistent with those reported by Haro-Gonzalez et al. (2021) and Pandey et al. (2022), though slight variations are expected due to factors like geographic origin, cultivation conditions and extraction techniques (Pavela and Benelli, 2016). The phenolic compounds thymol and eugenol are recognized for their potent bioactivity and are likely responsible for the strong molluscicidal effects observed (Radwan and Gad, 2021).
           
    The high mortality rates in both bait and contact assays confirm the potent molluscicidal properties of thyme and clove oils, with thyme oil showing slightly greater efficacy. These findings align with earlier work by El-Zemity and Radwan (2001) and Abdel-Rahman and Amal (2020), reinforcing the topical effectiveness of essential oils against snails.
           
    The ovicidal activity observed supports the hypothesis that essential oils interfere with embryonic development, as previously reported by Ali (2017). This may be attributed to disruption of hormonal regulation or direct damage to egg membranes.
           
    Biochemical assays indicated that sub-lethal doses of both oils induce oxidative stress, evidenced by increased MDA and decreased GSH levels. These results echo findings by Lazarevic et al. (2020) and Gad et al. (2023), who observed similar oxidative responses in invertebrates exposed to essential oils.
           
    Increased ALP activity, especially with thyme oil, suggests lysosomal membrane destabilization and cellular damage, consistent with prior observations (Shekari et al., 2008; Mobarak et al., 2015). The accompanying increase in lipid levels and reduction in protein content may be due to stress-induced catabolism and impaired metabolic functions, in line with Capowiez et al., (2015).
           
    The endocrine-disrupting effects, particularly the reduction in testosterone and estradiol levels, indicate interference with steroidogenesis, supporting conclusions by Desouky et al., (2022). The more severe hormonal disruption caused by clove oil implies differential activity of its constituents on endocrine pathways.
           
    AChE inhibition by both oils, especially thyme oil, suggests neurotoxicity. This is likely due to thymol and eugenol, known AChE inhibitors (Liao et al., 2016; El Gohary et al., 2021). Such inhibition can lead to overstimulation of the nervous system and ultimately, death.
           
    Histopathological changes observed in the digestive and hermaphrodite glands reinforce the biochemical and hormonal findings. The degeneration of reproductive tissues indicates a significant impact on fertility, as similarly reported by Desouky et al., (2022) and Hategekimana and Erler (2020).
           
    Finally, the field efficacy results highlight the practical potential of these essential oils as eco-friendly molluscicidal agents. The comparable performance to methomyl suggests their possible integration into integrated pest management strategies, as supported by previous field studies (Ismail and Abdel-Kader, 2011; Powell and Bowen, 1996).

    CONCLUSION

    Our research highlights the potential of thyme and clove oil as a biorational molluscicide against land snails as an alternative for the chemical managements.

    ACKNOWLEDGEMENT

    This work was supported by Ongoing Research Funding Program  (ORF-2025-03), King Saud University, Riyadh, Saudi Arabia.
     
    Authors contributions
     
    Conceptualization; Heba Abdel Tawab, Ebtesam A. Yousef, Heba Y. Ahmed; Data curation; Heba Abdel Tawab, Heba Y. Ahmed, Abdel-Baki, A.A.S., Ahmed O Hassan; Formal analysis; Heba Abdel Tawab, Heba Y. Ahmed; Funding acquisition; Ahmed, Abdel-Baki, A.A.S., Saleh Al-Quraishy; Investigation; Heba Abdel Tawab, Ebtesam A. Yousef; Methodology Heba Abdel Tawab, Ebtesam A. Yousef, Heba Y. Ahmed Supervision; Shawky M Aboelhadid, Ahmed, Abdel-Baki, A.A.S; Validation; Saleh Al-Quraishy, Shawky M. Aboelhadid, Ahmed O Hassan; Visualization; Heba Abdel Tawab; Heba Y. Ahmed Roles/Writing - original draft; Heba Abdel Tawab, Writing - review and editing; Ahmed, Abdel-Baki, A.A.S., Shawky M. Aboelhadid.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Ethics approval
     
    Not applicable.

    CONFLICT OF INTEREST

    The authors declare that the research was conducted in the absence of any commercial or financial relationships.

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