The natural and eco-friendly role of Cassia angustifolia in reducing carbaryl toxicity at environmentally relevant concentration

Abstract

This study aimed to evaluate the toxicity of carbaryl at environmentally relevant concentrations on the non-target organism Allium cepa L. and to investigate the potential protective effects of Cassia angustifolia Vahl. (Senna) leaf extract (Ca-ex) against this toxicity. Germination parameters, mitotic index (MI), micronucleus (MN), chromosomal aberrations (CAs), comet assays, and spectral shift analyses were employed to assess both toxic and protective effects. Oxidative stress parameters were also investigated. A plant-based bioassay was conducted using root tips and leaves of A. cepa. Exposure to 0.3 µg/L carbaryl resulted in a 41% reduction in germination, a 22% decrease in cell proliferation, and significant increases in DNA fragmentation, MN, CAs, and anatomical damage. Both the decrease in MI rates and the significant increase in MN frequency indicate the cytotoxic effect of carbaryl. In the groups in which Ca-ex was administered together with carbaryl, MI rates increased again and MN frequency decreased, and these findings indicate the protective effect of Ca-ex. Exposure to carbaryl resulted in a significant decrease in chlorophyll levels and a significant increase in malondialdehyde, proline, superoxide dismutase and catalase levels. Administration of Ca-ex in combination with carbaryl improved the oxidative stress parameter levels. The mitigating effect of the extract is likely associated with its phenolic content, and LC–MS/MS analysis identified quercetin, p-coumaric acid, and ferulic acid as major components. While the findings provide promising evidence that C. angustifolia extract can mitigate carbaryl-induced toxicity in A. cepa, it is important to highlight that this study primarily focused on the protective effects in a non-target plant model. Future investigations should aim to explore the compatibility of such extracts with pesticide formulations, their effects on target pests, and their regulatory and economic feasibility in agricultural systems.

Introduction

Pesticides are used to prevent the destruction of crops and to increase yields in agricultural production. Since they are also used against mice, mosquitoes and insects in daily life, they are also distributed in homes, offices, schools, parks and garden^1,2. Pesticides also threaten non-target organisms by contaminating ecosystems and degrading the quality of air, water and soil. Pesticides accumulate in the soil and cause changes in the physicochemical and biological properties of the soil. This phenomenon has been associated with decreased soil health and degradation of flora and fauna. Pesticides reach rivers and groundwater from soil, causing aquatic pollution and jeopardising the quality of water resources. Notably, this contamination has the potential to reach drinking water sources, thereby posing a substantial threat to human health^3,4. El-Nahhal and El-Nahhal⁵ detected 113 pesticide residues in drinking water samples from 31 countries worldwide and reported that 61 of these residues were insecticides, 31 were herbicides and 21 were fungicides. The toxicity classes of these residues were determined to be IA to IV. It is estimated that approximately 385 million acute pesticide poisonings occur annually worldwide, resulting in approximately 11,000 deaths. A multitude of epidemiological studies have demonstrated a correlation between prolonged exposure to pesticides and a plethora of health complications, encompassing respiratory ailments, endocrine dysfunction, digestive disorders, reproductive issues and various forms of cancer^5,6. Many pesticides are also reported to induce oxidative stress-mediated DNA damage, Alzheimer’s disease and neurotoxicity⁷.

Carbaryl is a synthetic carbamate pesticide with the chemical name 1-naphthol-N-methyl carbamate, derived from carbamic acid. Carbaryl is a broad-spectrum insecticide used since 1956 and is widely used for pest control in agricultural fields, poultry, homes and gardens^7,8. It is a highly effective insecticide against beetles, mites, lice and centipedes. Carbaryl is a neurotoxin that kills insects by disrupting the normal function of the nervous system and acting as an inhibitor of choline esterase^8,9. The excessive use of carbaryl has resulted in significant environmental contamination, including of water resources, which are currently facing scarcity. The presence of carbaryl has been detected in water samples from rivers and inland seas at concentrations ranging from 4.320 to 0.2134 μg/L. Furthermore, residues of the carbaryl have been found in sediment at levels of 1.851 μg/g, as well as in organisms such as plankton and fish, with concentrations ranging from 0.4140 to 2.436 μg/g^10,11. Carbaryl has been banned in the European Union and Switzerland since 2007 and is completely prohibited in India. Some applications in Canada were withdrawn in 2019, yet it is still used for agricultural and professional purposes throughout the US. However, in some states, its use is restricted to licensed professionals¹². In response to the environmental and biological impacts of toxic pesticides such as carbaryl, more sustainable, ecological alternatives are gaining attention. These include biological control methods such as predators, parasites or pathogens; plant-based pesticides such as neem oil, garlic extract or pyrethrins; and integrated pest management strategies. These alternatives aim to protect both human health and the balance of ecosystems¹³.

Carbaryl has the potential to enter the food chain via the pollution of aquatic ecosystems and agricultural irrigation, affecting not only seafood but also plant products and, consequently, humans. The US Environmental Protection Agency (EPA) and the French International Agency for Research on Cancer have both classified it as a potential carcinogen for humans¹⁴. It has been reported to cause congenital malformations in the embryos of animal organisms and histopathological changes in various organs, including the heart, liver, kidney, lung and brain¹⁰. Conversely, carbaryl has been observed to disrupt the endocrine system, resulting in fluctuations in hormone levels. Carbaryl intoxication has been demonstrated to reduce testosterone, progesterone and estradiol levels, decrease RNA levels within cells, and inhibit DNA synthesis, thereby limiting protein expression and growth⁷. Pesticides and insecticides cause a wide range of toxicity in plants, including oxidative stress, necrosis, burns, leaf abnormalities, physiological decline, chlorosis, cytogenetic damage and stunting^15,16. Carbaryl has also been demonstrated to induce severe toxic effects in plant life. The occurrence of toxicity symptoms in certain plant life has been documented in the extant literature. Sooksawata et al.¹⁷ reported that exposure to 73.5 and 147.25 mg/kg carbaryl in sunn hemp caused a decrease in chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents. The mechanisms of carbaryl toxicity in plants especially in A. cepa, have not yet been clearly elucidated and further research is required on this subject. This study provides a comprehensive toxicity profile of carbaryl.

In the context of toxicity studies, it is important to identify the effects and mechanisms of action of chemical agents. However, the identification of a solution to the problem under investigation can greatly increase the value of the study. Plant extracts such as Ginkgo biloba, green tea, sage tea, green coffee, grape seed, goat horn and blackberry have recently been used to reduce toxicity caused by chemical agents such as pesticides. Cassia angustifolia Vahl. (Senna) leaf extract is one of these extracts. Its natural habitats are Yemen, Egypt and Saudi Arabia and it is a fast-growing shrub up to 8 m in height. The leaves and bark of C. angustifolia are used as an anti-helmentic for intestinal worms. It is also used as an antipyretic in the treatment of typhoid, splenomegaly, anaemia and cholera caused by Escherichia coli and as a laxative to relieve constipation¹⁸. It exhibits a strong antioxidant activity as it contains many antioxidants such as rhein, rhein-8-glucoside, aloe-emodin, sennosides C and D, anthrone diglucoside, tinnevelin glycoside and 6-hydroxy musizin glycoside, resin, flavonoids, phytosterols and calcium oxalate. Therefore, it has many therapeutic properties, including antidiabetic, anti-inflammatory, anti-cancer and antihyperlipidaemic actions^19,20. The biological, pharmacological and therapeutic effects of C. angustifolia have been reported in many studies. These studies show that C. angustifolia has an important antioxidant power. Given this power, C. angustifolia is expected to reduce the toxic effects induced by chemicals. In this study, the toxicity mitigation capacity of C. angustifolia against carbaryl-induced toxicity in A. cepa was investigated. In this way, both a comprehensive toxicity profile of carbaryl was elucidated and the protective effect of C. angustifolia was determined. Toxic and ameliorative effects were analyzed morpho-physiologically, biochemically, cytogenetically and anatomically. This study confirmed the hypothesis that carbaryl is toxic at the dose detected in the environment and that C. angustifolia can be used as a solution to eliminate/reduce this toxicity problem.

Material and methods

Collection, extraction and quantitative analysis of C. angustifolia

Cassia angustifolia (two-year-old mature plant) leaves were collected from 40.938989, 38.280763 (Bulancak/Turkiye) and later identified by Prof. Dr. Zafer TÜRKMEN at the Department of Botany, Giresun University (Turkiye). A plant specimen bearing the voucher number BIO-Ca-an-1505/2024 was deposited in the herbarium collection. Diseased leaves were excised, and the remaining intact portions were subjected to drying at 35 °C for two days and subsequent pulverisation. 2 g of the sample were incubated in 100 mL of ethanol for 24 h at 35 °C and filtered with Whatman No. 4 filter paper following the incubation period²¹. Following the centrifugation of the filtrate at 5,000 rpm for 10 min., the supernatant was removed at 40 °C using an evaporator. The resulting C. angustifolia extract was then coded as Ca-ex. The quantitative determination of phenolic compounds in C. angustifolia was conducted through LC–MS/MS analysis (Thermo Scientific, ODS Hypersil 4.6*250 mm column). 1 g of Cassia angustifolia was extracted with dichloromethane-methanol solvent (1:4) and the extract was filtered with a 0.45 μm sterile syringe. The elution gradient comprised water with 0.1% formic acid (mobile phase A) and methanol (mobile phase B). The LC–MS/MS analysis was conducted at HUBTUAM (Hitit University) and the detailed informations about analysis profile are given in supplamentary material. The total ion chromatogram is presented in Figure S1, the gradient profile is shown in Table S1, and the ions in MS/MS are listed in Table S2.

Chemical analysis on environmentally relevant concentrations of carbaryl

To determine the toxicity of carbaryl, the amount of carbaryl in the aquatic environment was determined to ensure that the preferred dose was environmentally compatible and realistic. For this purpose, carbaryl was measured at three points in the Pazarsuyu stream (Giresun/Turkiye), which is located near the agricultural areas. Water samples from three different points of Pazarsuyu stream (25 m, 50 m and 100 m distance) were collected and stored in a sterile-refrigerated box at 4 °C until analysed. Samples were analysed within 48 h to avoid changes in sample composition and carbaryl content. The amount of carbaryl in water samples was analysed by HPLC²². The conditions of HPLC analysis and extraction methods are given in supplementary material file.

Experimental groups

Allium cepa bulbs were purchased from Akdeniz Tarım (TR -55-K-009228) and used as a bioindicator in toxicity assays. Carbaryl (CAS Number 63–25-2) was obtained from Merck. Allium bulbs were submerged in distilled water for a period of 24 h to facilitate rooting. As a result of HPLC analysis, the concentration of carbaryl in the samples taken 25 m, 50 mand 100 maway from Pazarsuyu stream was determined in the range of 0.33–0.81 µg/L, respectively. In this study, the lowest dose determined by HPLC analysis was taken as basis and 0.3 µg/L carbaryl was used as environmentally relevant concentration. The Ca-ex doses, were determined based on the IC₅₀ value of the radical scavenging effect indicating antioxidant activity²³ (supplementary material file, Figure S2). In this context, two different doses of 3.5 mg/mL (IC₅₀) and 7 mg/mL (IC₅₀ × 2) were applied and dose-dependent protective effect was also investigated. Allium bulbs were divided into six groups: Group I: Distilled water, Group II: 3.5 mg/mL Ca-ex, Group III: 7 mg/mL Ca-ex, Group IV: 0.3 µg/L carbaryl, Group V: 0.3 µg/L carbaryl + 3.5 mg/mL Ca-ex, Group VI: 0.3 µg/L carbaryl + 7 mg/mL Ca-ex. Group II and Group III were established to ascertain whether varying doses of Ca-ex were toxic, while Group IV was constituted to determine the impact of carbaryl. Group V and Group VI were treated with carbaryl in combination with Ca-ex, with the objective of determining the protective effect of Ca-ex by quantifying the extent to which the effect of carbaryl was reduced. Following a seven-day germination period at room temperature (20 °C) and 60% humidity, root tissues and leaves were collected for further analysis²⁴. Experimental research on plant samples, including the supply of plant material, complies with institutional, national and international guidelines and legislation.

Physiological parameters

The criteria proposed by Pehlivan et al.²⁵ were taken into consideration in the measurement of three different physiological parameters.

1. Radicle length (cm) was measured to determine root length,

2. By weighing the bulb weights at the start and conclusion of the 72-h period, weight (g) increase was calculated.

3. Eq. (1) was used to determine the germination percentage.

   1

Cytogenetic parameters

CAs and MN test

CAs and MN were determined using the acetocarmine crushing procedure. After being sliced into 1-cm lengths, the root tips were fixed for two h in a “Clarke” fixator. Washed for 15 min in 96% ethyl alcohol, hydrolyzed for 17 min at 60 °C in 1 N HCl, and then stored for 30 min in 45% glacial acetic acid. Acetocharmine-stained for 24 h, crushed on a slide, viewed seen with a camera-equipped research microscope (Irmeco IM-450 TI model), and captured on photographed at × 400 magnification²⁶. Equation (2) was utilized in order to determine MI. For CAs, MN and MI counts, 10 slides were prepared per group. For CAs and MN, 100 cells per slide and 1,000 cells in total and for MI, 1,000 cells per slide and 10,000 cells in total were analysed. Cells at various phases of mitosis had all damages and counts examined.

2

Spectral analysis of DNA-carbaryl interaction

DNA was initially extracted from A. cepa root tip cells for spectral analysis²⁷. Spectrophotometric analysis was used to evaluate the quantity and purity of DNA. A₂₆₀/A₂₈₀ ratios of roughly 1.8 are recognized as proof of DNA purity. To determine the DNA-carbaryl interaction, the absorbance of the mixture containing different concentrations of carbaryl and DNA was measured in the range of 220–300 nm in a spectrophotometer (Mapada UV-6100PCS)²⁸. All measurements were carried out in triplicate.

Comet analysis

DNA isolation from root cells was carried out using the Sharma et al.²⁷ method. The method suggested by Dikilitaş and Koçyiğit²⁹ was used to analyze Comet. The nuclear slides were kept in ethanol for 24 h and dried in an oven. With the help of a pipette, 100 mL of agarose [1% normal melting point (NMP) dissolved in distilled water at 50 °C] was poured onto the slides and covered with coverslips. To freeze the gel, the slides were refrigerated for 5 min. A mixture of freshly isolated cell solution and 1% low melting point (LMP) agarose was used to create the second layer. 100 µL of the mixture was put on the first layer and covered with a coverslip after the cell suspension and LMP agarose were well combined in distilled water at 40 °C in a ratio of 1/8 on a slide. The coverslips were taken off after the preparations had cooled in the fridge for 5 min. To give the DNA time to relax and dissolve, the preparations were put in the buffer solution in the electrophoresis tank and incubated for 40 min. Subsequently, the preparations were subjected to electrophoresis. Electrophoresis conditions and comet analysis were given in supplementary material file³⁰.

Measurements of biochemical parameters

MDA measurement

For MDA measuremets, 1 mL of 5% trichloroacetic acid was used to homogenize 0.5 g of root tips. After being moved to a fresh tube, the homogenate was centrifuged for 10 min at 10,000 rpm. After being moved to a fresh tube, the supernatant and equal volues of 0.5% thiobarbituric acid were incubated for 30 min at 96 °C in a 20% trichloroacetic acid solution. After cooling, the tubes were centrifuged for 5 min at 10,000 rpm. At 532 nm, the supernatant’s absorbance was read, and the MDA levels were expressed as μM/g FW³¹.

Proline measurement

To determine the levels of free proline, 10 mL of 3% aqueous sulfosalicylic acid were used to homogenize 0.5 g of root tips. Using filter paper, the homogenate was filtered. The filtrate was put in a test tube with 2 mL of glacial acetic acid and 2 mL of acid-ninhydrin, and it was allowed to react for 1 h at 100 °C. An ice bath was used to stop the reaction. 4mL of toluene were used to extract the reaction mixture, and it was agitated for 20 s. After aspirating the toluene-containing chromophore from the aqueous phase and warming it to 21 °C, the absorbance was measured spectrophotometrically at 520 nm using toluene as a blind³². Equation (3) was used to calculate the proline concentration on a fresh weight basis based on a standard curve.

3

Chlorophyll measurement

Chlorophyll extraction and measurement were carried out according to the method proposed by Tümer et al.³³. A leaf sample weighing 0.2 g was collected. The leaves were mashed in a mortar and pestle after being chilled in a refrigerator at 4 °C. In a dark tube, the leaf materials were crushed and extracted using 5 mL of 80% acetone. After filtering, the mixture was moved to a fresh tube, 5 mL of 80% acetone was added, and it was centrifuged at 3,000 rpm. The sediment-free filtrate was moved to a fresh tube. This procedure was carried out again with 80% acetone present. The absorbance of the green chlorophyll solution was measured at 645 and 663 nm using a UV–Vis spectrophotometer. Witham et al.³⁴ equation was used to determine the concentrations of chlorophylls.

Enzyme extraction

The extraction process was done at 4 °C. 5 mL of cold sodium phosphate buffer (50 mM, pH 7.8) was used to crush 0.5 g of root tip in a mortar to homogenize it. After centrifuging the homogenate for 20 min at 10,000 rpm, the supernatant was kept at 4 °C until analysis³⁵. Biochemical analyses were performed in triplicate.

SOD measurement

For the SOD activity assay, the reaction mixture was prepared as follows: 1.5 mL 0.05 M sodium phosphate buffer, 0.3 mL 130 mM methionine, 0.3 mL 750 μM nitroblue tetrazolium chloride, 0. 3 mL 0.1 mM EDTA-Na₂, 0.3 mL 20 μM riboflavin, 0.01 mL enzyme extract, 0.01 mL 4% insoluble polyvinylpyrrolidone and 0.28 mL de-ionised water. The prepared solution was placed in sterile tubes. The tubes were exposed to a fluorescent lamp (15 W) for 10 min to start the reaction, and they were left in the dark for 10 min to finish it. The absorbance at 560 nm was measured³⁶. The quantity of SOD needed to cause 50% inhibition of nitroblue tetrazolium chloride reduction under experimental circumstances was defined as one unit of SOD activity, and it was measured as U/mg FW.

CAT measurement

UV–Vis spectrophotometry was used to measure the CAT activity in 2.8 mL of total volume at 25 °C (1.5 mL 200 mM monosodium phosphate buffer, 1.0 mL distilled water and 0.3 mL 0.1 M H₂O₂) reaction mixture. 0.2 mL of enzyme extract was added to start the reaction. Monitoring the drop in absorbance at 240 nm brought on by H₂O₂ consumption allowed for the measurement of CAT activity. CAT activity was measured as OD_240nm min/g and reported as units per minute per fresh weight of root tip³⁷.

Anatomical observations

The tips of the roots were cut into 1-cm lengths, sandwiched between pieces of foam, and then sectioned transversely with a razor blade in a single motion. Following 2 min of staining with 5% methylene blue on a slide, the sections were covered with a coverslip, viewed under research microscope (Irmeco IM-450 TI model) equipped with a camera attachment, and photographed at × 200 magnification³⁸.

Statistical analysis

The statistical analysis was conducted utilising the SPSS (IBM SPSS Statistics 22) software programme. The mean and standard deviation values were calculated for the entire dataset. The analysis employed the one-way ANOVA test to ascertain within-group differences for the measurements obtained. The 'one-way ANOVA’ method was implemented to identify the differences between groups for each analysis. The ‘Duncan’ test, a post-hoc test, was selected to determine the differences. In the statistical analysis, p-values less than 0.05 were considered to be statistically significant.

Results and discussion

Physiological findings

As demonstrated in Table 1, the alterations in the physiology of A. cepa bulbs induced by carbaryl are evident. Groups I (control), II and III exhibited the highest levels of germination, root elongation and weight gain. The physiological parameter values measured in these groups did not differ significantly (p > 0.05). In Group IV, where carbaryl was applied, significant (p < 0.05) decreases were detected in all parameters. Compared to the control group, there was a 41% decrease in germination, around 3.21 times less root growth and 4.27 times less weight gain. Groups V and VI, which received Ca-ex in addition to carbaryl, showed significant (p < 0.05) increases in the values of chosen physiological parameters. These increases were more pronounced in Group VI, where 7 mg/mL dose of Ca-ex was applied. Compared to Group IV, which was exposed to carbaryl, Group VI had 17% more germination, 1.89 times more root elongation and 2.96 times more weight gain. These findings are consistent with the limited number of studies examining the effect of carbaryl on various plant species. Sengupta et al.³⁹ reported that 75, 100 and 150 ppm doses of carbaryl decreased root elongation and body weight of Vigna sinensis (L.) Endl. (cowpea) seeds in a dose-dependent manner. Goswami et al.⁴⁰ found that three different carbaryl concentrations significantly reduced the germination percentage and early seedling growth of Medicago sativa L. (alfalfa) seeds. The inducing of physiological delay in growth by carbaryl exposure can be attributed to many mechanisms. Some of these mechanisms include decreased mitotic activity that provides growth and development, disruptions in nutrient and mineral intake, and DNA damage that prevents cell proliferation^41,42. In addition, carbaryl indirectly inhibits germination by triggering oxidative stress in plants. It has been observed that free radicals generated by carbaryl exposure inhibit the giberillic acid and abscisic acid pathways during germination^43,44.

Table 1.

Protective effects of Ca-ex against the physiological toxicity of carbaryl.

Groups	Germination (%)	Root length (cm)	Initial weight (g)	Final weight (g)	Weight gain (g)
Control	100a	10.1 ± 0.46a	15.6 ± 0.65	26.7 ± 0.53	+ 11.1a
3.5 mg/mL Ca-ex	100a	10.2 ± 0.48a	15.4 ± 0.61	27.0 ± 0.43	+ 11.6a
7 mg/mL Ca-ex	100a	10.4 ± 0.37a	15.7 ± 0.76	27.2 ± 0.39	+ 11.5a
0.3 µg/L carbaryl	59d	3.14 ± 0.22d	15.8 ± 0.58	18.4 ± 0.63	+ 2.60d
Carbaryl + 3.5 mg/mL Ca-ex	65c	4.66 ± 0.35c	15.3 ± 0.71	21.2 ± 0.50	+ 5.90c
Carbaryl + 7 mg/mL Ca-ex	76b	5.94 ± 0.20b	15.3 ± 0.63	23.0 ± 0.70	+ 7.70b

The data is displayed as mean ± SD. 10 bulbs were used to measure weight and root length, while 50 bulbs were used to measure germination. In the same column, means denoted by various letters^(a-d) are statistically significant (p < 0.05).

Cytogenetic findings

The effects of carbaryl on cell proliferation and MN frequency in A. cepa root meristem cells are given in Table 2. MI is an indicator of cell proliferation and carbaryl caused a significant decrease in MI rate (p < 0.05). 0.3 µg/L carbaryl treatment caused a 22% decrease in the number of dividing cells compared to the control. Low level of MN was detected in control and Ca-ex only groups, which indicates that Ca-ex alone does not induce MN formation. In the 0.3 µg/L carbaryl treated group, a high level (86.6 ± 4.72) was determined. Both the decrease in MI rates and the significant increase in MN frequency indicate the cytotoxic effect of carbaryl. In the groups in which Ca-ex was administered together with carbaryl, MI rates increased again and MN frequency decreased, and these findings indicate the protective effect of Ca-ex. The types and frequencies of CAs induced by carbaryl and Ca-ex treatment are given in (Table 2 and Fig. 1). Low and non-statistical CAs presence was detected in control and Ca-ex only groups (p > 0.05). Carbaryl exposure caused CAs such as vagrant chromosome, sticky chromosome, and unequal distribution of chromatin, fragment, bridge, nucleus bud, disorientation and multipolar anaphase in root meristem cells. Among all CAs, vagrant chromosome formations were detected at the highest frequency and multipolar anaphase at the lowest level. In Group V and Group VI treated with Ca-ex at doses of 3.5 and 7 mg/mL together with carbaryl, significant (p < 0.05) decreases in CAs frequencies were recorded. These improvements in genotoxicity values were found to be more pronounced in Group VI, which received 7 mg/mL Ca-ex. Compared to Group IV exposed to carbaryl, the vagrant chromosome, the most commonly observed CAs, was reduced approximately 1.74-fold in Group VI. Consequently, CAs induced by carbaryl application may have caused cell cycle delays. This delay also explains the decreases in MI and and is also a possible cause of the decreases in root elongation and germination percentage observed in the previous analysis. The regression of genotoxic effects of Ca-ex also positively affected cell proliferation and physiological development.

Table 2.

Effects of carbaryl and Ca-ex applications on MI, MN and CAs frequency.

CAs types	Control	3.5 mg/mL Ca-ex	7 mg/mL Ca-ex	0.3 µg/L carbaryl	Carbaryl + 3.5 mg/mL Ca-ex	Carbaryl + 7 mg/mL Ca-ex
MI(%)	906 ± 13.7a (9.06)	908 ± 13.2a (9.08)	902 ± 10.7a (8.02)	703 ± 9.68d (7.03)	766 ± 10.1c (7.66)	804 ± 9.57b (8.04)
MN	0.40 ± 0.52d	0.50 ± 0.53d	0.20 ± 0.42d	86.6 ± 4.72a	72.5 ± 3.47b	55.1 ± 3.28c
VC	0.40 ± 0.52d	0.20 ± 0.42d	0.30 ± 0.48d	69.6 ± 3.50a	50.2 ± 3.16b	39.9 ± 3.90c
SC	0.30 ± 0.48d	0.52 ± 0.53d	0.20 ± 0.42d	60.6 ± 3.27a	46.8 ± 3.16b	32.4 ± 2.88c
UDC	0.50 ± 0.53d	0.30 ± 0.48d	0.20 ± 0.42d	51.1 ± 2.96a	41.1 ± 2.85b	29.2 ± 3.32c
F	NDO	NDO	NDO	41.5 ± 2.99a	29.3 ± 2.87b	20.6 ± 2.01c
B	NDO	NDO	NDO	29.8 ± 2.66a	20.0 ± 2.54b	13.2 ± 2.35c
NB	NDO	NDO	NDO	23.5 ± 2.80a	15.8 ± 1.87b	10.0 ± 1.41c
D	NDO	NDO	NDO	15.9 ± 1.66a	10.4 ± 1.51b	5.20 ± 1.14c
MA	NDO	NDO	NDO	9.90 ± 1.45a	5.30 ± 1.16b	3.00 ± 1.05c

The data is displayed as mean ± SD (n = 10). 1,000 cells were analyzed to determine MN and CAs, and 10,000 cells were analyzed to determine MI. In the same line, means denoted by various letters^(a-d) are statistically significant (p < 0.05). MI mitotic index, MN micronucleus, VC vagrant chromosome, SC sticky chromosome, UDC unequal distribution of chromatin, F fragment, B bridge, NB nucleus bud, D disorientation, MA multipolar anaphase. NDO no data observed.

Fig. 1.

CAs caused by carbaryl insecticide. Bar = 10 µm.

Comet analysis is used to detect DNA fragmentation and the results of comet assay are given in Table 3. Group IV that received carbaryl treatment showed significant (p < 0.05) increases in tail DNA percentage and significant (p < 0.05) decreases in head DNA. Compared to the control group, the amount of head DNA decreased by approximately 43% in Group IV and the amount of tail DNA significantly increased. Improvement in comet findings was observed in the groups administered Ca-ex together with carbaryl. Compared to Group IV exposed to carbaryl, head DNA increased by approximately 29% in Group VI exposed to Carbaryl + 7 mg/mL Ca-ex.

Table 3.

Comet data for determining DNA damage.

Comet parameters	Control	3.5 mg/mL Ca-ex	7 mg/mL Ca-ex	0.3 µg/L carbaryl	Carbaryl + 3.5 mg/mL Ca-ex	Carbaryl + 7 mg/mL Ca-ex
Head diameter (px)	28.000	30.000	32.000	48.000	34.000	26.000
Head density	170.201	165.055	145.467	249.407	187.504	207.758
Head DNA (%)	97.5 ± 1.27a	98.0 ± 1.15a	98.5 ± 1.43a	53.6 ± 1.51d	62.5 ± 1.27c	82.4 ± 1.71b
Tail length (px)	4.000	3.000	1.000	71.000	59.000	22.000
Tail density	4.325	3.441	2.168	216.337	112.273	44.343
Tail DNA (%)	2.50 ± 1.27d	2.00 ± 1.15d	1.50 ± 1.43d	46.4 ± 1.51a	37.5 ± 1.27b	17.6 ± 1.71c
Tail Moment	0.280823	0.212094	0.184949	21.187	13.171	2.880

The data is displayed as mean ± SD (n = 10). 1,000 cells from each group were examined for DNA damage. In the same line, means denoted by various letters^(a-d) are statistically significant (p < 0.05). The percentage of tail DNA served as the basis for the Comet scale, which indicated DNA damage. Damage can be classified as minimal or nonexistent (≤ 5%), low (5–20%), moderate (20–40%), high (40–75%), or severe (≥ 75%).

Genotoxicity findings of this study are in agreement with similar literature studies. Amer and Farah⁴⁵ reported that carbaryl treatment caused meiotic CAs such as chromosome delay, polyploidy, fragmentation, stickiness and bridging in Vicia faba pollen mother cells. Asita and Hatane⁴⁶ found that 20 g/kg carbaryl decreased MI and promoted sticky chromosome development. Sarangi⁴⁷ found that three different doses of carbaryl inhibited MI, increased MN frequency, caused CAs and sister chromatid exchanges in peripheral erythrocytes of Channa punctatus. Meeker et al.⁴⁸ observed that environmental exposure to carbaryl caused increased DNA damage in human sperm cells. The decreases in MI and head DNA and the increases in the percentage of MN, CA and tail DNA observed in this study indicate the genotoxic effect of carbaryl. The genotoxicity caused by carbaryl exposure can be explained by different mechanisms. The formation of sticky chromosomes may be attributed to carbaryl forming noncovalent bonds with DNA, condensing chromatin fibers and preventing their separation. Fragments or bridges may arise from carbaryl causing DNA double-strand breaks or triggering faulty repair mechanisms. Vagrant chromosomes and unequal chromatin distribution may be related to microtubule disorganization and impaired kinetochore function. Multipolar anaphase and disorientation may be due to carbaryl-induced disruptions in the cytoskeleton and centrosome division. Nuclear buds may be interpreted as an attempt to expel genomic material from the nucleus as a result of DNA damage or excessive replication. Carbaryl-induced oxidative stress also plays an important role in the emergence of chromosomal abnormalities^8,48,49. Literature studies also confirm DNA and spindle interactions with carbaryl. Meeker et al.⁴⁸ found that carbaryl acts as a spindle disrupting agent in Chinese hamster cells and that carbaryl or its metabolic intermediates act directly or indirectly as alkylating agents on DNA bases. Önfelt⁴⁹ reported that carbaryl is a spindle disrupting agent. The other mechanism is that carbaryl triggers oxidative stress formation by increasing the free radical level in the cell. Fattahi et al.⁸ reported that carbamate insecticides accelerate free radical formation and induce peroxidation and degradation of intracellular macromolecules. They also found that carbaryl inhibited DNA synthesis and decreased RNA levels, leading to a decrease in protein synthesis.

In this study, carbaryl-DNA interaction was investigated and tried to be elucidated by spectral analysis. The spectroscopic absorption plot of the DNA-carbaryl interaction is given in (Fig. 2). DNA at 0.15 mg/mL has an absorbance value of 1.95 at 260 nm. Addition of different concentrations of carbaryl to the DNA solution caused significant increases in absorption. DNA mixtures containing 0.15, 0.3 and 0.6 µg/L carbaryl had absorbance values of 2.11 ABS, 2.49 ABS and 2.66 ABS at 260 nm, respectively. Increases in absorbance at 260 nm between 8.2% and 36.4% were seen when carbaryl was added to the DNA solution. The gradual increase in absorbance after the addition of carbaryl to the DNA solution indicates a hyperchromic shift. UV–Vis absorption spectroscopy is a popular and efficient technique for studying DNA binding. Following binding of a compound to DNA, it causes changes in the helical structure and steric configuration of DNA. These changes trigger changes in the absorption spectrum. A hyperchromic change, which results a change in absorbance, indicates that carbaryl binds to DNA in an intercalation mode, the formation of a DNA-carbaryl binary complex and damage to the DNA double helix structure^50,51. Chemical agents and DNA typically interact non-covalently through a variety of binding mechanisms, including intercalation. Intercalation involves the stacking of agents between DNA double strands in the absence of covalent bonding. This stacking has a promutagenic effect by breaking DNA, unwinding supercoiled DNA and interfering with the ability of DNA to interact with binding and regulatory proteins⁵². All of these potential modifications to the DNA structure after contact further support the cytotoxic and genotoxic properties of carbaryl. There are similar studies reporting DNA-carbaryl interactions in the literature. Zhang et al.⁵¹ observed a 21% increase in absorbance after carbaryl interaction with calf thymus DNA, indicating a hyperchromic and stated that this increase was indicative of DNA damage.

Fig. 2.

Spectral shift observed in DNA-carbaryl interaction.

Biochemical findings

Biochemical changes induced by carbaryl in A. cepa are given in (Fig. 3). Groups I (control), II and III treated only with two different doses of Ca-ex had the highest chlorophyll levels and the lowest MDA, proline, SOD and CAT enzyme activities. The values of the biochemical parameters tested in these groups were not significantly different (p > 0.05). Exposure to carbaryl resulted in a significant decrease in chlorophyll levels and a significant increase in MDA, proline, SOD and CAT levels (p < 0.05). In comparison with the control group, the levels of MDA increased by 3.06-fold, proline levels increased by 2.65-fold, SOD activity increased by 2.27-fold and CAT activity increased by 2.31-fold in Group IV. Conversely, chlorophyll a and chlorophyll b levels exhibited a decline of 59% and 54.8%, respectively. Administration of Ca-ex in combination with carbaryl improved these abnormalities. Similar biochemical changes caused by carbaryl in various organisms have been reported in the literature. Habib et al.⁵³ found that carbaryl exposure caused a decrease in chlorophyll, carotenoid, phycobilin and protein levels and an increase in MDA, SOD, CAT and ascorbate peroxidase activities in Calothrix brevissima, a bacterial species. Tütüncü et al.⁵⁴ reported that methiocarb, a carbamate pesticide like carbaryl, increased MDA levels and SOD and CAT enzyme activities in A. cepa root cells.

Fig. 3.

C. angustifolia leaf extract’s ability to protect against the biochemical toxicity induced by carbaryl. Group I: Control, Group II: 3.5 mg/mL Ca-ex, Group III: 7 mg/mL Ca-ex, Group IV: 0.3 µg/L carbaryl, Group V: Carbaryl + 3.5 mg/mL Ca-ex, Group VI: Carbaryl + 7 mg/mL Ca-ex. The data is displayed as mean ± SD (n = 10). The means denoted by various letters (a–d) are statistically significant (p < 0.05). MDA: µM/g, SOD: U/mg, CAT: OD_240nm min/g, Chl a and b: mg/g, proline: (µmole/g).

The biochemical changes exhibited by carbaryl exposure may be explained by the induction of oxidative stress. Önfelt⁴⁹ showed that carbaryl caused increased lipid peroxidation in V79 Chinese hamster cells. There is also information in the literature that pesticides increase membrane damage by generating ROS and increase MDA levels by causing lipid peroxidation⁵⁵. The increase in proline levels caused by carbaryl exposure suggests that it is a mechanism developed by the plant for defence against stress. This theory is supported by data in the literature showing that proline accumulation in plants is positively correlated with tolerance to abiotic stress; increases resistance in plants exposed to various stress factors such as salinity, heavy metals and xenobiotics, and is considered a sign of stress sensitivity in plants⁵⁶. Oxidative stress induced by carbaryl also increases antioxidant enzyme levels. Habib et al.⁵³ reported that many pesticides directly increase radical production and the increases observed in SOD and CAT enzyme activity in the presence of carbaryl are due to the role of these enzymes in free radical scavenging. Carbaryl caused a decrease in photosynthetic pigment levels as well as disturbances in the antioxidant balance of the cell. The decrease in chlorophyll pigment levels may be associated with the change in ion concentration in leaves and inhibition of the synthesis of enzymes involved in chlorophyll synthesis⁵⁷. Lei et al.⁵⁸ found a decrease in Na, Ca and K ion concentrations as a result of pesticide exposure and a corresponding decrease in the synthesis of photosynthetic pigments. The results obtained in biochemical analyses are consistent with the physiological and cytogenetic parameter data of this study. Especially the decrease in chlorophyll levels is an important reason for the regression in physiological development. Oxidative stress indicated by increased MDA and proline levels is the main cause of chromosomal abnormalities and decreased cell cycle.

Anatomical findings

The root cells of Group I (control) and Groups II and III, which received two varying dosages of Ca-ex, showed no signs of injury under a microscope. On the other hand, carbaryl treatment caused severe epidermis cell damage, thickening of the cortex cell wall and flattened cell nucleus damage in the root cells of Group IV (Fig. 4). In the root cells of Group V and Group VI, which were treated with Ca-ex at doses of 3.5 and 7 mg/mL together with carbaryl, the severity of the observed damages decreased. This improvement was more pronounced in the root cells of Group VI treated with 7 mg/mL dose of Ca-ex. There is no comprehensive research in the literature on the anatomical damage caused by carbaryl in plant root tip cells. However, there are studies reporting that carbamate pesticides with similar structure induce anatomical damages. Tütüncü et al.⁵⁴ reported that three different doses of methiocarb, a carbamate insecticide, caused anatomical damages such as necrosis, epidermis cell damage and thickening of the cortex cell wall in A. cepa root meristem cells. Himtaş et al.²⁶ found that permethrin exposure at a dose of 100 µg/mL promoted anatomical damages such as epidermis cell damage, flattened cell nucleus, cortex cell damage, thickening of the cortex cell wall and indistinct conduction tissue in A. cepa root tip meristem cells. Cell wall thickening in root cells may be a morphological defence mechanism developed by the plant to prevent the penetration of carbaryl^59,60. Similarly, Çakır et al.⁶¹ and Yılmaz et al.⁶² reported that the passage of chemical agents into the cell was limited by thickening of the cell wall in roots. Microscopic analysis showed a marked increase in the number of cortex and epidermis cells in roots exposed to carbaryl. This alteration increases the contact between the cells, generating mechanical pressure. The epidermis cells and their nuclei can be deformed as a result of this pressure. This claim is supported by the literature indicating that plants develop alkaloids, terpenoids, phytochelatin accumulation⁶³, physical change (increase in cell number and cell layer)²⁵ and morphological adaptation (thickening of the cell wall and indistinction of the conduction tissue)²⁶ as defence against toxic agents. Genomic instabilities and chromosomal alterations detected in cytogenetic analyses and the comet test may be the causes of the observed changes in cell nucleus shape^64,65.

Fig. 4.

Appearance and frequencies of meristematic cell damages in 0.3 µg/L carbaryl group. Bar = 10 µm. No damage is denoted by ( −), light damage by ( +), moderate damage by (+ +), and severe damage by (+ + +).

Protective role of Cassia angustifolia

Carbaryl exposure caused negative changes in all selected physiological, cytogenetic, biochemical and anatomical parameters of A. cepa. In this context, germination root elongation, weight gain, MI and chlorophyll levels decreased, while DNA damage, MN and CAs numbers, MDA, proline, SOD and CAT levels increased. 3.5 and 7 mg/mL Ca-ex treatments again caused an improvement in all selected parameters, although not at a level to reach the values of the control group and this effect was more noticeable at 7 mg/mL Ca-ex treatment. Ca-ex showed an increasing ameliorative effect with increasing dose in all parameters. Administration of 3.5 and 7 mg/mL Ca-ex resulted in a 16 and 35% reduction in the frequency of MN induced by carbaryl, respectively. Ca-ex provided 27–42% protection against vagrant chromosome levels, the highest frequency among CAs. Abnormalities identified in cytogenetic analyses were associated with the induction of oxidative stress. The 25–43% decrease in MDA level detected in the carbaryl-treated group indicates that oxidative stress also decreased with Ca-ex application. Briefly, an improvement was observed in all parameters at different rates. The regressive effect of C. angustifolia on carbaryl toxicity is in parallel with the literature studies. Bellassoued et al.⁶⁶ reported that C. angustifolia leaf extract caused significant reductions in carbon tetrachloride-induced lipid peroxidation in the liver tissues of adult rats, prevented the formation of pathological liver lesions, and had the potential to remove free radicals and alleviate hepatotoxicity. Osman et al.⁶⁷ showed that C. angustifolia extract reduced oxidative stress in kidney tissue and provided a protective effect against streptozotocin-induced diabetes by reducing blood glucose levels, increasing plasma insulin levels, reducing weight loss and improving renal enzyme levels such as CAT and glutathione (GSH) in diabetic rats. Karajibani et al.¹⁸ determined that C. angustifolia exposure in hyperlipidemic male Wistar rats for a brief period of time resulted in a significant drop in blood cholesterol, triglyceride, and LDL-C levels while increasing HDL-C levels and body weight.

The improvement provided by C. angustifolia leaf extract in selected physiological, cytogenetic, biochemical and anatomical parameter values can be explained by the antioxidant activity and pharmaceutical properties of C. angustifolia. LC–MS/MS chromatogram of phenolic compounds in the structure of C. angustifolia leaf extract is shown in (Fig. 5). As a result of LC–MS/MS analysis, the highest amount of 476.229 µg/g kaempferol and 125.540 µg/g rutin were determined in C. angustifolia leaves. These substances were followed by quercetin > p-coumaric acid > ferulic acid, which were above the level of 10 µg/g. Below 10 µg/g level, benzoic acid > salicylic acid > epicatechin > caffeic acid > vannillin > catechin > naringenin > syringic aldehyde > oleuropein were detected. Kaempferol, most abundant in C. angustifolia leaf extract, has anti-inflammatory, antidiabetic, cardioprotective, neuroprotective, antioxidant, antibacterial, and anti-cancer properties⁶⁸. A flavonoid present in plants including buckwheat, apples, and tea, rutin is the second most prevalent substance in C. angustifolia leaf extract. It has been reported that rutin protects the liver, kidneys, and heart. The primary pharmacological effect of rutin is to increase antioxidant capacity. It also has anti-inflammatory and antiapoptotic effects. In addition, rutin also shows strong metal chelating properties. Thus, it can prevent peroxidation caused by metal ions⁶⁹. Quercetin is a bioflavonoid, one of the three substances with levels above 10 µg/g. It possesses potent anti-inflammatory and antioxidant qualities. It is known to act as an antioxidant against various radicals, especially superoxide radical, lipid peroxyl radical and hydroxyl radical. It has also been reported that quercetin helps in cancer treatment by blocking the flow of nutrients and oxygen to cancerous cells⁷⁰. The second substance with a level above 10 µg/g is p-coumaric acid, a polyphenol found mainly in plants. Recent studies have reported that p-coumaric acid has antioxidant, antimutagenic, anti-inflammatory, antimelanogenic, antiplatelet, cardioprotective and immunomodulatory properties. It is also known to reduce the growth of cancer cells⁷¹. Ferulic acid, the last substance above 10 µg/g, is a water-soluble antioxidant. It participates in the process by which cells scavenge free radicals, or ROS. It also has antiallergic, anti-inflammatory, antihyperlipidemic and hepatoprotective activity⁷². C. angustifolia leaf extract contains nine other substances whose protective roles are not discussed in detail since their amounts are below 10 µg/g. Among these substances, especially catechins and epicatechins are known to have higher antioxidant power than vitamins, to take an active role in the elimination of superoxide and hydroxyl radicals from cells, and to inhibit the formation and growth of cancer cells⁷³. The protective effects of plant extracts are primarily attributed to their content of various biologically active compounds, especially phenolic substances. The protection exhibited by C. angustifolia against carbaryl-induced toxicity may be associated with the cumulative effects of phenolic compounds identified through LC–MS/MS analysis. Similarly, in the literature, the protective effects of various plant extracts against chemically induced toxicity have also been reported in our previous studies^74–78.

Fig. 5.

LC–MS/MS chromatogram and phenolic substance profile of C. angustifolia.

C. angustifolia, which can grow easily without the need for specific cultivation conditions, offers a significant advantage in terms of cost-effectiveness and practical applicability due to its notable toxicity-reducing effects even at low doses. Furthermore, its natural composition prevents secondary toxicity or environmental pollution, making it particularly valuable from an ecological sustainability perspective. In this study, the Allium assay was employed to assess carbaryl-induced toxicity and to evaluate the protective potential of C. angustifolia against this toxicity. As a bioindicator plant containing eukaryotic DNA, Allium cepa provides reliable and consistent results that can serve as early warnings for other biological systems. However, considering its relatively low oxidase enzyme concentrations and limited substrate specificity compared to other organisms, it is crucial to investigate these effects across a broader range of biological models. The findings of this study should be communicated to relevant regulatory authorities to raise awareness and encourage the implementation of necessary measures, particularly concerning pesticide use, within the framework of legal and environmental regulations.

Conclusion

The current study demonstrates that carbaryl exposure causes significant morphotoxic and cytogenotoxic effects in Allium cepa, as evidenced by decreased germination rates, decreased photosynthetic pigment content and mitotic index, and increased levels of oxidative stress, chromosomal aberrations and micronuclei. The results demonstrate that carbaryl exhibits toxicity even at low concentrations, highlighting the environmental risks associated with pesticide overuse and the need for safer and more sustainable practices. Furthermore, Cassia angustifolia extract was found to mitigate the toxic effects of carbaryl, likely due to its antioxidant properties. The use of the Allium test system allows for an integrated assessment of genotoxic, physiological, and anatomical changes, strengthening its applicability in environmental toxicity testing.

However, it is important to note that this study focused solely on plant systems and did not assess the interaction of Cassia angustifolia extract with carbaryl in terms of insecticidal efficacy. Additionally, practical challenges such as formulation complexity, regulatory considerations, and cost implications of incorporating plant extracts into commercial pesticide products must be considered. Future studies should investigate these aspects, particularly the potential antagonistic or synergistic effects of Cassia angustifolia extract on target pests. Despite these limitations, the study provides a strong foundation for further research on natural, plant-based strategies to reduce pesticide-induced toxicity in agroecosystems.

Author contributions

A.B: investigation; methodology; visualization; writing-review and editing. E.Y: conceptualization; methodology; data curation; software; visualization; writing—review and editing. K.C: conceptualization; data curation; investigation; methodology; visualization; writing—review.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-16685-z.