Introduction

The world’s population is growing rapidly, and it is estimated that the global population will reach 10 billion by 20501. Increasing crop yields and quantities to meet the nutritional needs of this growing population is a primary goal, and one of the most effective approaches to achieving this goal is to minimize crop losses through the application of chemicals known as pesticides2,3. Pesticides are chemicals developed to protect against mice, flies, insects and weeds. Pesticides increase the efficiency of agricultural production and protect crops from weeds, insects, and diseases. In this way, they increase crop yield, quality, and quantity. Each pesticide has a different mechanism of action. Organophosphate and carbamate pesticides act by inhibiting acetylcholinesterase activity, while pyrethroid pesticides bind to voltage-sensitive sodium channels, altering conduction kinetics, and some other pesticides act by inhibiting gamma-aminobutyric acid4. Despite their numerous benefits, widespread, non-selective and improper application of pesticides causes significant damage to ecosystems, leading to toxicity and environmental pollution. It is estimated that approximately 1500 types of pesticides are used worldwide5. A significant amount of these pesticides is lost during application. Only less than 1% of these reach their target6. These lost amounts can have negative effects on living species and ecosystems. Many pesticides are persistent and difficult to degrade, allowing them to persist in the environment for decades. Pesticides can be transported by wind and water, contaminating soils, water, and air, and negatively impacting humans and other non-target organisms (such as beneficial insects, honeybees, amphibians, plants, and animals). Of the approximately 2 million tons of pesticides produced annually, 47.5% are herbicides, 29.5% insecticides, 17.5% fungicides, and the remaining 5.5% are other pesticide types7,8.

Insecticides, a frequently used and important class of pesticides, are compounds primarily intended to eliminate or manage pest populations. Excessive and prolonged use of synthetic pesticides can lead to accumulation in water, soil, air, food, and milk. This accumulation can have toxic effects on humans, plants, animals, and ecosystems. Evidence from previous studies suggests that exposure to some synthetic pesticides can lead to liver and kidney toxicity, primarily through mechanisms involving oxidative stress in experimental animal models. Overuse of synthetic pesticides can also lead to pesticide resistance, the emergence of new pests, and environmental pollution9,10. Methidathion (MTD) is a broad-spectrum non-systemic organophosphate insecticide used since 197211. Organophosphate insecticides are widely used in agricultural applications to increase efficiency against insects and microorganisms. Organophosphate insecticides inhibit acetylcholinesterase activity in target tissues, leading to acetylcholine accumulation and disrupting neuronal transmission. Chronic and subchronic exposure to this group of insecticides can lead to the formation of free radicals and oxidative stress12. MTD is widely used to eradicate sucking and chewing insects and mites that infest agricultural crops such as peaches, pears, apples, mulberries, figs, vegetables, tobacco, alfalfa, sunflowers, greenhouses, and rose gardens13. MTD is a cholinesterase inhibitor, like organophosphorus agents, and in addition to its effects on target organisms, it also has toxicity to non-target organisms such as humans, bees, and aquatic life. Poisoning and even deaths have been reported in humans due to exposure to MTD14,15. It was determined that MTD exposure caused chromosomal abnormalities, decreased mitotic index, and increased micronucleus frequency and sister chromatid exchange frequency in human lymphocytes and induced mutagenic, clastogenic and aneugenic effects16. It has been reported that MTD induces lipid peroxidation and causes biochemical abnormalities and histopathological changes in liver, kidney and heart tissues in subchronic applications17,18. For these reasons, it is considered a class I toxicity agent by the Environmental Protection Agency (EPA). While excessive MTD use causes toxic effects on non-target organisms, it also causes serious environmental pollution by accumulating in soil, air and water ecosystems. MTD has been determined in river waters at concentrations of 0.36–7.5 µg/L but MTD and its derivatives are still used regardless of their toxicity15.

Although MTD is included in the list of banned pesticides in Türkiye, it remains widely used in many countries and poses a potential risk as a persistent environmental pollutant19. Despite its extensive application, there is a marked lack of comprehensive studies addressing its toxicological impacts on non-target plant species. In particular, investigations elucidating the physiological, cytogenetic, biochemical, and anatomical alterations induced by MTD in plant systems are scarce in the literature. To fill this critical gap, the current study used Allium cepa, a globally cultivated and widely consumed crop, as a sensitive bioindicator to provide an integrated assessment of multifaceted toxicity associated with MTD exposure. This study comprehensively demonstrates the biochemical, genetic, and anatomical effects of MTD in plants within a single experimental design. The molecular docking analyses employed in this study reveal potential interactions of MTD with key biomolecules such as DNA, tubulin, topoisomerase, and 5-aminolevulinic acid dehydratase, thus clarifying the toxicity mechanism at the molecular level. Using different concentrations, the toxic effects of MTD were evaluated in detail within the context of a dose-response relationship, yielding important data sets for environmental risk estimation. Furthermore, the study provides compelling evidence that the A. cepa test is an effective model for integrating not only genotoxicity but also physiological, biochemical, and anatomical parameters. The results of this study contribute significantly to the scientific database for the environmental safety of pesticides and the development of sustainable agricultural policies.

Materials and methods

Plant material and MTD treatment

Allium cepa L. (2n = 16) bulbs purchased from a local market in Giresun province (Türkiye) were used as indicator organisms. The samples were brought to the laboratory, and species identification was confirmed by Prof. Dr. Zafer TÜRKMEN, a faculty member of the Department of Botany at Giresun University (Türkiye). All experimental procedures involving plant material, including its procurement, were conducted in accordance with institutional, national, and international guidelines and regulations. All images used in the article were taken by us or created by us by entering data from the referenced sites.

Methidathion (CAS No: 950-37-8), an organophosphate insecticide procured from Merck, was employed as the toxicant in this study. MTD doses of 15, 30, and 45 mg/L, which were previously used in the biochemical toxicity study conducted by Çavuşoğlu et al.20 on A. cepa and were identified to induce toxic effects, were considered in determining the doses for this study. Four experimental groups of A. cepa bulbs were established: Group I served as the control, Group II was treated with 15 mg/L MTD, Group III with 30 mg/L MTD, and Group IV with 45 mg/L MTD. Sterile glass beakers were used to germinate A. cepa bulbs. The control group (Group I) was treated with tap water, whereas the treatment groups were exposed to MTD at concentrations of 15, 30, and 45 mg/L under room temperature conditions. The germination process lasted for 72 h to obtain root tips and for 144 h to collect leaf samples. Upon completion of the respective exposure periods, root and leaf tissues were harvested and rinsed with distilled water. All samples were subsequently processed for analysis using standard laboratory preparation protocols.

Physiological growth and germination analysis

The assessment of physiological parameters was carried out using the standards set forth by Topatan et al.21.

  1. (a)

    The radicle’s length in centimeters was used to calculate the root’s length;

  2. (b)

    By comparing the bulbs’ initial and final weights, weight increase was calculated;

  3. (c)

    The percentage of germination was determined using Eq. (1).

$${\text{Germination }}\left( \% \right){\text{ }}={\text{ }}\left[ {{\text{Number of germinated bulbs}}} \right]{\text{ }}/{\text{ }}\left[ {{\text{total EquationNumber of bulbs}}} \right]{\text{ x 1}}00$$
(1)

Micronucleus, mitotoic index and chromosomal abnormality test

The aceto-carmine squash technique was utilized to identify micronucleus (MN) and chromosomal abnormalities (CAs). Root tips approximately 1 cm in length were first fixed in Clarke’s fixative for 2 h. The samples were rinsed with 96% ethanol for 15 min and then hydrolyzed in 1 N HCl at 60 °C for 17 min. Next, the roots were treated with 45% glacial acetic acid for 30 min. Staining was carried out by immersing the roots in aceto-carmine solution for 24 h. The root tips were squashed on microscope slides and analyzed under Irmeco IM-450 TI model microscope equipped with a camera, at a magnification of ×40022.

10 slides per group were prepared for CAs, MN counts, and mitotic index (MI) calculations. For CAs and MN frequency, 100 cells were analyzed per slide, totaling 1.000 cells. For MI, 1.000 cells were analyzed per slide, totaling 10.000 cells. Cells at various stages of mitosis were examined to assess all types of damage23.

MI was calculated as a percentage using Eq. (2).

$${\text{MI }}\left( \% \right){\text{ }}={\text{ }}\left[ {{\text{EquationNumber of cells undergoing mitosis}}} \right]{\text{ }}/{\text{ }}\left[ {{\text{total EquationNumber of cells}}} \right]{\text{ x 1}}00$$
(2)

Comet assay

DNA extraction from A. cepa root cells was carried out using the method described by Sharma et al.24. For comet assay analysis, the protocol recommended by Dikilitaş and Koçyiğit25 was followed. Counting and imaging of comets were conducted using the ‘TriTek 2.0.0.38 Automatic Comet Assay’ software. A total of 1.000 cells per group were analyzed, and the DNA content in both the head and tail of the comet was quantified as a percentage. Using the scale suggested by Pereira et al.26, the proportion of tail DNA was used to determine the degree of DNA damage.

MDA measurement

1 mL of 5% trichloroacetic acid was used to homogenize a 0.5 g sample of root tips. After being moved to a fresh tube, the homogenate was centrifuged for 10 min at 12.000 g. Equal amounts of the resultant supernatant, 0.5% thiobarbituric acid, and 20% trichloroacetic acid solution were combined in a new tube and incubated at 96 °C for 30 min. The tubes were centrifuged for 5 min at 10.000 g after cooling. At 532 nm, the supernatant’s absorbance was then determined27.

Antioxidant enzyme activity

The extraction procedure was carried out at + 4 °C. 0.5 g sample of root tips was ground and homogenized in a mortar with 5 mL of cold sodium phosphate buffer. The resulting homogenate was then centrifuged at 10.500 g for 20 min, and the supernatant was stored at + 4 °C until further analysis28. Beauchamp and Fridovich29 approach was used to measure the activity of the SOD enzyme. 1.5 mL of 0.05 M sodium phosphate buffer, 0.3 mL of 130 mM methionine, 0.3 mL of 750 µM nitroblue tetrazolium chloride, 0.3 mL of 0.1 mM EDTA-Na2, 0.3 mL of 20 µM riboflavin, 0.01 mL of enzyme extract, 0.01 mL of 4% insoluble polyvinylpyrrolidone, and 0.28 mL of de-ionized water were added to a total reaction volume of 3 mL. After transferring the mixture to sterile tubes, the tubes were exposed to a 15 W fluorescent lamp for 10 min to start the reaction. After that, the tubes were left in the dark for 10 min to stop the reaction. At 560 nm, absorbance was measured. The quantity of enzyme needed to provide a 50% inhibition of the nitroblue tetrazolium chloride reduction under the specified experimental conditions was referred to as one unit of SOD activity30. CAT activity was measured according to Beers and Sizer31 method. 1.5 mL of 200 mM monosodium phosphate buffer, 1.0 mL of distilled water, and 0.3 mL of 0.1 M H₂O₂ made up the reaction mixture (total volume: 2.8 mL), which was then incubated at 25 °C. 0.2 mL of enzyme extract was added to start the reaction. By monitoring the drop in absorbance at 240 nm, which is equivalent to the consumption of H2O₂, CAT activity was tracked. The rate of change in absorbance (OD240 nm) per minute per gram of fresh weight of the root tip (min/g) was used to express enzyme activity32.

Chlorophyll measurement

The procedure outlined by Kaydan et al.33 was followed for the measurement and extraction of chlorophyll. A 0.2 g leaf sample, previously cooled to + 4 °C, was crushed and ground using a mortar. The extraction was performed by adding 5 mL of 80% acetone to the crushed leaf sample in a dark environment. The resulting mixture was then filtered and transferred to a new tube, with an additional 5 mL of 80% acetone added. The sample was centrifuged at 3.000 rpm, and the filtrate was carefully transferred to a new tube, ensuring no sediment remained. This process was repeated with 80% acetone. Finally, UV/VIS spectrophotometer was used to test the green chlorophyll solution’s absorbance at 645 and 663 nm. The formulae supplied by Witham et al.34 were used to determine the concentrations of chlorophyll a and chlorophyll b.

Molecular Docking to assess MTD toxicity

The molecular docking method was employed to explore the potential toxicity of the MTD in a computational environment. This approach utilized the B-DNA form, as elucidated by Watson and Crick, which is the most abundant DNA form in nature35, to examine the direct interaction between MTD and DNA. Three different DNA sequences were used for this purpose: B-DNA dodecamer D (5’-D(CGCGTTAACGCG)-3’) (PDB: 195D)36, DNA (5’-D(TTGGCCAA)-3’) (PDB: 1CP8)37, and B-DNA dodecamer (5’-D(CGCGAATTCGCG)-3’) (PDB: 1BNA)38. Additionally, topoisomerase I (PDB: 1K4T)38 and topoisomerase II (PDB: 5GWK)35,40 were included, as these enzymes relieve the tension in the supercoil formed during DNA replication and protect DNA from temperature-related damage. Alpha and beta tubulin proteins (PDB: 6RZB)41,42 were also considered, as these proteins form spindle fibers (microtubules) that pull chromosomes toward the poles during anaphase. During chlorophyll synthesis, the following enzyme forms were used: protochlorophyllide oxidoreductase (PDB: 2XDQ)43,44, which reduces protochlorophyllide to chlorophyll in a light-dependent manner; glutamate-1-semialdehyde 2,1-amino mutase (PDB: 5HDM)45, which catalyzes the isomerization of glutamate-1-semialdehyde to 5-aminolevulinate; and 5-aminolevulinic acid dehydratase (PDB: 1W1Z)46,47, which facilitates the formation of porphobilinogen, the basic unit of tetrapyrroles, by asymmetrically combining two molecules of 5-aminolevulinic acid. The ligand MTD (PubChem ID: 13709)48 was retrieved from PubChem. The macromolecules were corrected for hydrogen atom deficiencies using the Maestro BioLuminate 5.0 software49. Interactions between the macromolecules and the ligand were carried out with AutoDock Vina version 4.2.650, and the Biovia Discovery Studio 2020 Client51 was used to visualize the outcomes.

Anatomical observations

Root tips were excised into 1 cm segments and placed between styrofoam for stabilization. Cross sections were obtained in a single motion using a razor blade. After that, the sections were put on microscope slides and stained for two minutes using a 5% methylene blue solution. After staining, the sections were placed under a coverslip and seen at 200x magnification using Irmeco IM-450 TI type microscope with a camera. Images were captured for further analysis52.

Statistical analysis

SPSS Statistics 22 (IBM SPSS, Turkey) was used to perform statistical analyses on the data. The differences between groups were assessed using Duncan’s multiple range test after one-way analysis of variance (ANOVA). A p-value of less than 0.05 was considered statistically significant, and the findings are shown as mean ± standard deviation (SD).

Results and discussion

Physiological growth and germination analysis

The physiological toxicity induced by MTD treatment is summarized in Table 1. The control group showed the highest values for germination, root elongation, and weight gain, with 100% germination, 8.81 cm root elongation, and a weight gain of 10.1 g. MTD treatment resulted in a statistically significant (p < 0.05) reduction in all three physiological parameters, with the magnitude of the decrease being dose-dependent. Compared to the control group, germination, root elongation, and weight were reduced by 44%, 3.0-fold, and 5.3-fold, respectively, in group, which received the highest MTD dose (45 mg/L). It is believed that the physiological changes observed in A. cepa bulbs following MTD treatment may be due to the damage caused to the roots, which interferes with the uptake of water and nutrients by root cells and reduces mitotic activity in those cells. Some supporting literature reinforces this idea. Roots play a crucial role in the supply of water and minerals. Damage or loss of root tissue significantly impacts plant development and nutrient absorption53. A comprehensive study investigating the physiological changes induced by MTD in plant cells is lacking in the literature. Therefore, our results were compared with those from studies on the physiological toxicity of other insecticides in A. cepa, and a consistent pattern was observed. Kutluer et al.54 reported that azadirachtin insecticide treatment at doses of 5, 10, and 20 mg/L inhibited germination, root elongation, and weight gain in A. cepa, with these effects being dose-dependent. Ayhan et al.55 found that cypermethrin applied at a concentration of 6 mg/L resulted in a decrease in germination, root elongation, and weight gain in A. cepa bulbs. Himtaş et al.56 observed a reduction in germination, root elongation, and bulb weight in A. cepa treated with permethrin at a concentration of 100 µg/L. Kutluer et al.54 reported that insecticides caused damage to the epidermis and cortex cells, as well as thickening of the conduction tissues in A. cepa roots, reducing water and mineral uptake and leading to a decline in physiological processes such as germination, root elongation, and weight gain. Mitotic activity is a critical factor for growth and development. A reduction in mitotic activity prevents root elongation and inhibits plant growth. Haq et al.57 found that insecticide applications reduced mitotic activity by causing plant cells to remain in the interphase and prophase stages for extended periods. The structural damage and decrease in mitotic activity observed in stem cells following MTD treatment in this study are consistent with the findings in the literature, which explain the observed decrease in physiological parameter values.

Table 1 Dose-dependent effects of MTD on physiological parameters.
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Genotoxicity findings

The genotoxic effects induced by MTD treatment are illustrated in Figs. 1, 2, 3 and 4; Tables 2 and 3. The control group (Group I) exhibited the highest MI and DNA content, along with the lowest frequencies of MN and CAs. MTD treatment led to statistically significant (p < 0.05) reductions in MI values, as well as significant (p < 0.05) increases in MN and CAs frequencies. In comparison to the control group, MI decreased by approximately 19.1%, while MN frequency increased by about 186-fold and vagrant chromosomes—the most frequently observed CA—showed a 312.5-fold increase in Group IV, which received the highest dose of MTD. The comet assay results revealed that MTD exposure caused a significant (p < 0.05) decrease in head DNA percentage and a corresponding increase in tail DNA percentage (Fig.S1). In Group IV, the head DNA content decreased by approximately 68.3%, while tail DNA content increased by the same proportion compared to the control. MTD treatment also induced various CAs in root tip meristem cells, such as vagrant chromosome, sticky chromosome, unequal chromatin distribution, fragment, nuclear bud, bridge and binuclear cells. There are very few studies in the literature that investigate the genotoxic effects of MTD in A. cepa root tip cells. In one such study, Ünal et al.16 reported a decrease in the MI in A. cepa root cells exposed to four different concentrations of MTD (7.5–45 mg/L), with reductions observed at all doses. They also noted the induction of CAs, including chromosome adhesion, C-mitosis, fragments, bridges, lagging chromosomes, and multipolarity. Although studies specifically focusing on MTD-induced genotoxicity in A. cepa are limited, there is a broader body of research addressing the genotoxic effects of other insecticides, and our findings are consistent with those reports. For instance, Çavuşoğlu et al.58 observed a dose-dependent reduction in MI and an increase in MN frequency and CAs—such as sticky chromosomes, vagrant chromosomes, bridges, and unequal chromatin distribution—in A. cepa bulbs treated with lambda-cyhalothrin at concentrations of 1.75, 3.50, and 7.00 g/L. Tütüncü et al.59 found that methiocarb application at doses of 2.5, 5.0, and 7.5 mg/L led to a decrease in MI and an increase in MN frequency and CAs, including fragments, adhesive chromosomes, bridges, unequal chromatin distribution, and vagrant chromosomes. In another study, Kutluer et al.54 employed the Comet assay and demonstrated that azadirachtin exposure at 5, 10, and 20 mg/L induced DNA damage in A. cepa root cells, evidenced by a dose-dependent decrease in the percentage of head DNA and an increase in tail DNA percentage.

Fig. 1
figure 1

Structural chromosomal abnormalities induced by MTD in Allium cepa root meristem cells. Bar = 10 µM. micronucleus (a), vagrant chromosome (b), sticky chromosome (c), unequal distribution of chromatin (d), fragment (e), nucleus bud (f), bridge (g), binuclear cell (h).

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Table 2 Alterations in mitotic index and frequencies of chromosomal abnormalities induced by MTD.
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Table 3 Quantitative evaluation of MTD-induced genotoxicity through comet assay.
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The decrease in MI observed following MTD application, an indicator of cytotoxicity, may be attributed to damage to tubulin proteins, which are important in the formation of microtubules that play a role in chromosome segregation during mitosis. Similar results reported in the literature confirm this relationship. Oliver et al. 60 demonstrated that pesticides inhibit mitosis in plant species by specifically disrupting microtubules or microtubule organizing centers. Similarly, Srivastava et al.61 observed that insecticides depolymerize microtubules, inhibiting cell division. Fernandes et al.62 demonstrated that pesticides bind to tubulin proteins, preventing polymerization and consequently inhibiting microtubule formation. In this study, the interaction of MTD with alpha and beta tubulin proteins was investigated using molecular docking in a computational environment to mechanistically support the observed decrease in MI. The binding energy between MTD and alpha and beta tubulin was found to be -4.50 kcal/mol and − 4.91 kcal/mol, respectively. Hydrogen bonds were identified within the amino acid chains of both proteins, which could induce distortions in the three-dimensional structure of the tubulin protein. Such disruptions may impair the protein’s function, potentially affecting microtubule organization and impairing chromosome movement during mitosis, leading to reduced mitotic activity. The interaction of MTD with tubulin proteins is considered moderate compared to that of colchicine, a standard tubulin inhibitor. Klejborowska et al.63 reported that various colchicine derivatives they synthesized exhibited binding energies lower than − 8.70 kcal/mol, while the calculated binding energy for colchicine was − 8.09 kcal/mol, and they reported strong tubulin inhibition. While no study in the literature has examined the interaction between MTD and tubulin via molecular docking, some studies have investigated the interaction of other insecticides with tubulin. Himtaş et al.56 reported that the permethrin insecticide interacted with tubulin alpha and beta proteins, with binding energies of -8.62 kcal/mol and − 7.71 kcal/mol, respectively.

Fig. 2
figure 2

Molecular interactions between MTD and tubulin proteins. MTD-tubulin alpha (a), MTD-tubulin beta (b). Green line : hydrogen bonding, red line: unfavorable positive-positive, pink line : Pi-Pi stacked.

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The decrease in DNA content, the induction of DNA tail formation, and the increase in the frequency of MN and CA following MTD treatment can be explained by different mechanisms. One of these mechanisms is the interaction of free radicals generated by MTD with DNA, while another is a direct interaction with DNA. Similarly, Kaur and Kaur64 reported that reactive oxygen species produced by pesticides cause oxidative stress and disrupt DNA and repair mechanisms, leading to CAs. Furthermore, Aldana-Salazar et al.65 found that pesticides directly interact with DNA, causing chromosome damage. In this study, to elucidate the genotoxicity mechanisms of MTD, the interaction of MTD with topoisomerase I and topoisomerase II enzymes, which are involved in the unwinding of the supercoiled structure during DNA replication, as well as the interaction of MTD with DNA, was investigated using molecular docking in a computational environment. MTD exhibited an inhibition constant of 2.84 mM with topoisomerase I and 4.10 mM with topoisomerase II. Topoisomerase I formed hydrogen bonds with MTD at the glutamic acid residue within its structure, while topoisomerase II formed hydrogen bonds between the glutamine and tyrosine amino acids and MTD. These interactions could result in disruptions to the three-dimensional structure of the topoisomerase enzymes, leading to a loss of function. Such a loss of function could impair the ability to relieve supercoiling tension during DNA replication, ultimately causing replication errors. No prior molecular docking studies in the literature have investigated the interaction between pesticides and topoisomerase enzymes. The interaction of MTD with DNA was investigated using three different DNA sequences, revealing binding energies ranging from − 5.09 kcal/mol to -6.21 kcal/mol. In the case of the 1BNA sequence, MTD interacted with guanine and cytosine nucleotides through hydrogen, pi-sulfur, and carbon-hydrogen bonding. For the 1CP8 sequence, MTD interacted with cytosine, guanine, and adenine bases, while in the 195D sequence, it interacted with all bases via pi-sulfur, hydrogen, and carbon-hydrogen bonds. The molecular docking results demonstrate that MTD can directly interact with DNA, which provides a crucial insight into the DNA and CAs induced by MTD. Although there is no study investigating the interaction between MTD and DNA, there are studies on the interaction of standard DNA and topoizomerase inhibitors. Doxorubucin, which interacts with DNA through intercalation, causes genotoxic effect and shows the property of an anticancer, has a binding energy of -4.99 kcal/mol66. In this study, MTD, which exhibits binding energy between − 5.09 and − 6.21 kcal/mol with different DNA sequences, showed a similar level of interaction to almost doxorubicin. For etoposide, a well -known topoisomerase inhibitor, the binding energy is reported in the literature as -22.4 kcal/mol67. In this study, binding energies in the − 5.09 and − 6.21 kcal/mols calculated for MTD-topoisomerase interactions are low compared to etoposide. Molecular docking results indicate that MTD shows the genotoxic effect mostly by interacting with DNA and tubulins.

Fig. 3
figure 3

Molecular interactions between MTD and topoisomerase enzymes. MTD-topoisomerase I (a), MTD-topoisomerase II (b). Dark green line: hydrogen bonding, red line: unfavorable positive-positive, pink line: Pi-alkyl, orange line: Pi-cation, yellow line: Pi-sulfide, light green line: Carbon-hydrogen bond.

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Fig. 4
figure 4

Molecular interactions between MTD and DNA sequences. 1BNA (a), 1cp8 (b), 195d (c) Orange line : attractive charge, dark green line: hydrogen bonding, yellow line : Pi-sulfide, light green line: carbon-hydrogen bond.

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Biochemical findings

The biochemical toxicity induced by MTD treatment is shown in Table 4; Fig. 5. MTD treatment led to a statistically significant (p < 0.05) increase in MDA levels and a significant (p < 0.05) decrease in chlorophyll content. In Group IV, which received the highest MTD dose, the MDA level increased approximately 3.0-fold, while chlorophyll a and chlorophyll b levels decreased by approximately 2.88-fold and 3.32-fold, respectively, compared to the control group. SOD and CAT activities initially increased at the 15 and 30 mg/L doses of MTD, but decreased again at the 45 mg/L dose. Despite this decline, both SOD and CAT activities remained higher than those observed in the control group. In Group III, SOD activity increased 1.87-fold, and CAT activity increased 4.52-fold compared to the control group. The number of studies on the biochemical toxicity induced by MTD in A. cepa root cells is limited. One such study by Çavuşoğlu et al.68 reported dose-dependent increases in root MDA and proline levels, as well as SOD and CAT enzyme activities following MTD treatment at doses of 15, 30, and 45 mg/L. While studies on MTD’s biochemical toxicity in A. cepa are scarce, there are several studies examining its effects in other organisms. Aslanturk et al.69 observed increased MDA levels and elevated SOD, CAT, and glutathione peroxidase (GPX) enzyme activities in Lymantria dispar larvae exposed to MTD. Although research on MTD-induced biochemical toxicity is limited, there are numerous studies investigating the effects of other pesticides on biochemistry. Yildiztekin et al.70 found increased MDA, proline, and H2O2 levels, along with elevated SOD, peroxidase (POD), and CAT enzyme activities, in Lycopersicum esculentum (tomato) plants exposed to acetamiprid, imidacloprid, abamectin, and thiomethoxam insecticides. The study also reported a decrease in chlorophyll and carotenoid contents. There are also many literature studies reporting that pesticides and many other chemicals cause biochemical changes by inducing oxidative stress71,72,73.

Table 4 Dose-dependent biochemical alterations induced by MTD.
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The observed increase in root MDA levels following MTD treatment suggests that damage to the cell membranes may have occurred. Specifically, MTD likely caused damage to the root cell membranes, leading to lipid peroxidation, which consequently resulted in the formation of MDA. MDA, being a highly reactive metabolite, is commonly used as a marker for oxidative stress. This hypothesis is supported by several studies in the literature. Ayala et al.74 described lipid peroxidation as a process in which oxidants, such as free radicals, target lipids containing carbon-carbon double bonds, especially polyunsaturated fatty acids. These free radicals or ROS can directly damage lipids, resulting in damage to cells, tissues, and organs. Ayala et al.74 also pointed out that exposure to environmental factors, such as insecticides and herbicides, is one of the primary sources of ROS production. The increase in root SOD and CAT activities following MTD treatment suggests that this response may represent a self-protection mechanism by the plant cells. Specifically, MTD may have elevated free radical production within root cells, prompting an increase in SOD and CAT synthesis to protect the cells from oxidative damage. This hypothesis is supported by several studies in the literature. Sule et al.75 noted that pesticides can enhance the levels of NADPH oxidases and superoxide radicals, leading to increased ROS production within cells. Excessive ROS can cause toxicity by promoting lipid, protein, and DNA oxidation. In response, cells increase the synthesis of enzymatic antioxidants such as SOD and CAT. On the other hand, the reduced activities of SOD and CAT observed at the 45 mg/L MTD dose may be associated with the disruption of the protein structures of these enzymes. SOD and CAT are both protein-based enzymes, and an overdose of MTD could potentially cause a decline in enzyme activity by denaturing or otherwise damaging their structures. This idea is supported by Sipahi Kuloğlu et al.76, who reported that high doses of pesticide exposure can lead to decreased SOD and CAT activities due to the denaturation or disruption of their protein structures. Together, these findings suggest that while low to moderate doses of MTD may stimulate antioxidant enzyme activity as a protective response, higher doses may impair this defense system by damaging the enzymes themselves.

The decrease in chlorophyll content observed following MTD application may be attributed to several factors, including damage to leaf tissue, inactivation of Mg and Mn (essential for chlorophyll synthesis), increased ROS production in leaves, and disruption in the structure of enzymes responsible for chlorophyll synthesis. This hypothesis is supported by the literature. Zobiole et al.77 reported that chlorotic symptoms in leaves and the inhibition of Mg and Mn, which are crucial for chlorophyll synthesis and photosynthesis, led to a decrease in chlorophyll content and a subsequent reduction in photosynthetic rates. Sharma et al.78 further supported this idea, stating that pesticide application induces oxidative stress by increasing ROS formation in leaves. This oxidative stress leads to the degradation of chlorophyll pigments and proteins, thereby diminishing the plant’s photosynthetic efficiency. Additionally, Topatan et al.21 found that etoxazole exposure inhibited the activities of key enzymes involved in chlorophyll synthesis, such as glutamate-1-semialdehyde aminotransferase and protochlorophyllide reductase, which contributed to a reduction in chlorophyll levels. In order to support the effect of MTD at the pigment level, interactions with three different enzymes in chlorophyll synthesis were investigated. MTD interacted with 5-aminolevulinic acid dehydratase via hydrogen bonds, pi-sulfur interactions, and pi-sigma bonds at various amino acid residues, releasing a binding energy of -5.34 kcal/mol. MTD also interacted with glutamate-1-semialdehyde 2,1-amino mutase through pi-sigma, pi-sulfur, and hydrogen bonds. Additionally, it interacted with the enzyme protochlorophyllide oxidoreductase, forming hydrogen, carbon-hydrogen, and pi-pi stacked bonds, with an inhibition constant of 713.92 mM. These interactions suggest that MTD may disrupt or completely halt chlorophyll synthesis, leading to a reduction in chlorophyll content and causing developmental disorders, particularly in photosynthesis. The decreases in the physiological parameters observed in this study support this hypothesis. While there are no findings reporting the interaction of MTD with enzymes involved in pigment synthesis, there are studies reporting the interactions of various inhibitor substances. Nogara et al.79 investigated the interactions of phenylselenic acid, which exhibits inhibitory properties for the 5-aminolvaleric acid dehydratase enzyme. They reported that the inhibitor accessed the enzyme’s active site, forming electrostatic interactions with ASP217, π-π stacking with phe330, and hydrogen bonds with Arg301 and Lys291, resulting in a binding energy of -5.9 kcal/mol.

Fig. 5
figure 5

Molecular interactions between MTD and key enzymes involved in chlorophyll biosynthesis. MTD-5-aminolvulinic acid dehydratase (1W1Z) (a), MTD-glutamate-1-semialdehyde 2,1-amino mutase (5HDM) (b), MTD-protochlorophyllide oxidoreductase (2XDQ) (c). Pink line: Pi-Pi stacked, yellow line: Pi-sulfide, green line: hydrogen bonding, purple line: Pi-sigma.

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Anatomical observations

The anatomical changes induced by MTD treatment in root tip meristem cells are summarized in Table 5 and depicted in Fig. 6. No significant damage was observed in the root tip cells of the control group. In contrast, MTD treatment led to damage in the epidermal and cortical cells, with observable changes in cell morphology, such as flattened nuclei and bi-nucleolus cells. Additionally, the severity of these cellular damages increased in a dose-dependent manner as the concentration of MTD was raised. Although there is no comprehensive study in the literature specifically investigating the anatomical changes induced by MTD in A. cepa root meristem cells, several studies have explored anatomical alterations caused by other insecticides. Tütüncü et al.59 reported that methiocarb, applied at doses of 2.5, 5.0, and 7.5 mg/L, resulted in necrosis, damage to epidermal cells, and thickening of the cortex cell wall in A. cepa root cells. Kurt et al.80 found that profenofos, applied at doses of 25, 50, and 100 µg/L, led to epidermal and cortical cell damage, thickening of the cortex cell wall, and flattening of cell nuclei in A. cepa root tip meristem cells. The extent of these damages increased with higher doses. In another study, Kutluer et al.54 observed that azadirachtin treatment at doses of 5, 10, and 20 mg/L caused damage to epidermal and cortical cells, accumulation of substances in cortex cells, thickening of the cortex cell wall, flattening of the nucleus, and thickening of conduction tissues in A. cepa root cells.

It is believed that the damage observed in the root anatomy following MTD application results from the defense mechanisms developed by the plant to limit the insecticide’s entry into the cell. Microscopic examinations revealed a significant increase in the number of epidermal and cortex cells in the MTD-treated groups compared to the control group. While this increase acts as a barrier to restrict MTD’s entry, the elevated cell number creates mechanical pressure. This pressure may lead to deformations in the epidermal and cortex cells, as well as their nuclei. This hypothesis is supported by several studies. Kesti et al.81, Tümer et al.82, Kutluer et al.83 have reported that A. cepa roots develop defense mechanisms against pesticide/chemical exposure, such as an increase in epidermal and cortex cell numbers and thickening of the conduction tissue, which can result in deformities in the epidermal and cortex cells, as well as their nuclei. Another possible cause of the observed deformation in epidermal cells could be the disruption of the cell membrane structure. Upon MTD uptake, epidermal cells are particularly affected. The insecticide may cause lipid destruction in the cell membranes, which disrupts their integrity and leads to deformities in the epidermal cells. Furthermore, the entry of MTD into stem cells could alter intracellular pressure, potentially causing changes in the shape of the cell nucleus. In support of these mechanisms, Üstündağ et al.22 and Özkan et al.84 also found similar responses in A. cepa roots exposed to pesticides. An additional defense strategy employed by the plant is the increased synthesis of chemicals such as alkaloids, phenolic compounds, and terpenoids, as well as proteins like metallothionein and phytochelatins85. In our study, the increased number of nucleoli observed in the MTD-treated groups—critical for ribosomal RNA synthesis, ribosome formation, and protein synthesis—serves as a clear indicator of this adaptive response. It is likely that root cells increased nucleolar production to synthesize or boost the levels of protective chemicals and proteins to mitigate the toxic effects of MTD.

Table 5 Severity of meristematic cell damage induced by MTD.
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Fig. 6
figure 6

Structural impairments in meristematic cells following MTD exposure. Bar = 10 µM. epidermis cells in control (a), epidermis cell damage (b), cortex cells in control (c), cortex cell damage (d), nucleus in control (e), flattened nucleus (f), mono- nucleolus cell (g), bi-nucleolus cell (h).

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Conclusion

MTD, which has been widely used to control insects and mites on fruits and vegetables for more than 50 years, has been shown in A. cepa test material to cause toxic effects to non-target organisms when a certain dose level is reached. MTD promoted physiological, cytogenetic, biochemical and anatomical toxicity in A. cepa, a eukaryotic organism. This toxicity has been demonstrated experimentally using currently accepted methods of analysis and in detail in a computerized environment. Therefore, we believe that this study is the most comprehensive study on MTD toxicity with the help of many different parameters and will make a great contribution to the literature. Molecular docking analyses have provided important insights into the potential interactions of MTD with key macromolecules such as DNA, tubulin, topoisomerase, and 5-aminolevulinic acid dehydrate. However, these interactions can vary under the influence of multiple biological factors, such as intracellular metabolic processes, membrane permeability, enzyme kinetics, and cellular defense mechanisms. In this context, prospective in vivo and in planta studies are essential to confirm the molecular docking findings and assess the toxic effects of MTD from a more holistic and mechanistic perspective. However, while the Allium cepa model used in this study is a reliable tool for environmental toxicity and genotoxicity studies, it should be noted that the effects of MTD may vary in other plant species or different non-target organisms. This clearly highlights the need for comprehensive toxicity studies in different model organisms.

In agricultural production, determining effective doses for pesticide applications against non-target organisms while not being toxic to target organisms is crucial. In this context, farmers’ meticulous adherence to recommended doses and application times is a critical measure to prevent excessive and unnecessary pesticide use. Furthermore, appropriate preventative measures must be taken to prevent environmental pollution and adverse effects on non-target organisms during pesticide applications. Otherwise, both ecosystem health and environmental contamination may be jeopardized, and environmental contamination may become inevitable.