Regular Article
The insecticide spirotetramat may induce cytotoxic and genotoxic effects on meristematic cells of Allium cepa L. and Vicia faba L.
2023 Volume 88 Issue 3 Pages 225-231
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2023 Volume 88 Issue 3 Pages 225-231
Spirotetramat (SPT) is a new insecticide derivative of tetronic acid used extensively in agriculture to enhance the protection of major food crops against scales and aphids. This study aims to determine SPT’s cytotoxic and genotoxic effects using two model plants, Allium cepa and Vicia faba. This evaluation consists of studying the root growth, morphology, and color and the parameters of mitotic index (MI) and chromosome aberrations (CAs) as accurate toxicity markers. Our results showed a significant decrease in mean root length in A. cepa from the 0.0025% concentration. In contrast, a substantial reduction in mean root length in V. faba was recorded only at the 0.02% concentration. Furthermore, the MI was decreased proportionally with increasing concentration and time of exposure to SPT. A significant increase in CAs was observed in A. cepa and V. faba from the 0.0025% concentration after 24 h of treatment. The substantial reduction in MI and abundance of CAs indicated strong genotoxicity of SPT. From the data obtained, it can be concluded that SPT could be absorbed by the exposed plant or other non-target organisms in the proximity, causing damage to agricultural plants, affecting their genomes, and harming the environment.
Spirotetramat (SPT) is an innovative insecticide derivative of tetronic acid developed by Bayer Crop Science (Germany). It possesses a new mode of action that is effective against various phloem-feeding insects, including scales, aphids, and whiteflies (Brück et al. 2009, Ouyang et al. 2011). Therefore, many researchers have explored the effects of SPT on the environment and non-target species. Yin et al. (2014) showed that SPT induces oxidative stress and lipid peroxidation in toad tadpoles at sublethal doses. Furthermore, SPT administration can cause gonadal damage and alter the endocrine system in zebrafish (Zhang et al. 2020). Researchers have long been interested in the genotoxic and cytotoxic effects of environmental contaminants such as pesticides. These molecules are highly bioactive due to their ability to interact with biological macromolecules, including DNA (de Souza et al. 2016).
Many studies use different bioassays to assess the cytotoxic and genotoxic effects of widely used herbicides, insecticides, and fungicides, including the study of Sheikh et al. (2020), which has found that malathion and cypermethrin cause a significant cytotoxic and genotoxic effects on A. cepa roots. Furthermore, the results of Kuchy et al. (2015) on the two insecticides (endosulfan and dichlorvos) and the fungicide carbendazim and the findings of Macar (2021) on the fungicide tetraconazole showed the cytotoxic and genotoxic impact of these pesticides on onion meristematic cells. In addition, significant CAs and mitotic alterations were observed in A. cepa meristematic cells treated with the herbicides diuron (Chauhan et al. 1998) and quizalofop-p-ethyl and cycloxydim (Rosculete et al. 2018).
Modern agriculture’s extensive use of pesticides affects crop genetics, human health, and the environment. Testing the cytotoxic and genotoxic effects of commonly used pesticides is necessary to determine the toxicity to plants and other organisms, requiring their prudent use in agriculture (Sheikh et al. 2020). Therefore, the present study aims to evaluate SPT’s cytotoxic and genotoxic effects on two model plants, A. cepa and V. faba, through the analysis of different morphological and cytological parameters.
The experimental material consisted of healthy onion bulbs (A. cepa, 2n=16), and V. faba seeds were obtained from a local market. Both species were kept in contact with tap water until the roots reached 1.5 to 2 cm in length. Bulbs and seeds were divided into six groups, including the control, and then exposed to different concentrations (0.00125, 0.0025, 0.005, 0.01, and 0.02%) of the insecticide SPT (Movento®, 150 OD, CAS No. 203313-25-1, purity 98.5%). A minimum of three bulbs (five roots to each bulb) and seven seeds were exposed to each concentration tested for five days, with solutions changed daily. Root length was measured every 24 h using a ruler. Other signs of toxicity, such as color changes and root integrity, were also evaluated.
Genotoxicity evaluationDifferent SPT concentrations (0.00125, 0.0025, 0.005, 0.01, and 0.02%) were tested on A. cepa bulbs and V. faba seeds to find possible signs of genotoxicity with root lengths between 1.5–2 cm, for 12 h and 24 h. The treated bulbs and seeds were carefully washed and then transferred to a solution of ethanol/acetic acid: 3v/1v for 24 h and stored in 70% ethanol at 4°C until use. The rootlets were rinsed with distilled water and hydrolyzed in 1 M HCl solution for 5 min at 60°C and, in the end, stained with Schiff’s reagent for 20 min (Sharma and Sharma 2014). Apical meristems were crushed in 45% acetic acid drop and examined under a light microscope at ×40 magnification. At least 1,000 cells on each slide and five slides for each concentration were considered to determine the MI and CAs (Fiskesjo 1985, Bakare et al. 2000).
The percentage of CAs in cells, as well as the MI and chromosomal aberration frequency (AF), were measured using the formulas (Akwu et al. 2019):
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Data for root length, MI, and CAs were statistically analyzed using Graph pad prism version 9.2.0 (Graph Pad Software, LLC, CA, USA). Statistical results were compared using one-way ANOVA and Dunnett’s multiple comparison test. All values were expressed as mean±SD and were considered statistically significant when p＜0.05.
The results in Table 1 demonstrate changes in root length and morphology of A. cepa and the Table 2 for V. faba compared to the control after exposure to different SPT concentrations. The inhibitory effect of SPT increased with increasing concentration and exposure time. Thus, maximum root growth was observed in control, with mean lengths of 10.77±0.06 cm and 10.48±0.05 cm after 120 h in A. cepa and V. faba, respectively. Therefore, no significant effect of the 0.00125% concentration on the mean root length of A. cepa and the 0.00125%, 0.0025%, 0.005%, and 0.01% concentrations on the mean root lengths of V. faba was observed during the five days of treatment. However, exposure of A. cepa roots to the 0.0025%, 0.005%, 0.01%, and 0.02% concentration range caused a significant decrease in root growth with values of 4.26, 3.87, 3.61, and 3.58 cm, respectively. Similarly, a considerable reduction (p＜0.05) in root length in V. faba was noted at the 0.02% concentration (4.15 cm) compared to the control. Simultaneously, the inhibitory effect of SPT increased positively with concentration and time of exposure, whose value increased from 35.85% (0.00125%) to 61.1% (0.02%) after 120 h of exposure. Furthermore, it was remarked that A. cepa roots were more sensitive to SPT than V. faba roots (61.1% and 53.54% inhibition, respectively).
| Control and SPT treatments | Nr | Mean root length (cm) and inhibition (%) affected by SPT treatment | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 00 h | 24 h | 48 h | 72 h | 96 h | 120 h | Mean of longth±SD | Form and color | ||||||||
| Mean±SD | In% | Mean±SD | In% | Mean±SD | In% | Mean±SD | In% | Mean±SD | In% | Mean±SD | In% | ||||
| Control | 15 | 1.80±0.04 | — | 4.18±0.03 | — | 5.65±0.03 | — | 7.32±0.06 | — | 9.57±0.04 | — | 10.77±0.06 | — | 7.49±2.17 | Straight, white |
| 0.00125% | 15 | 1.78±0.04 | — | 3.61±0.08 | 13.54 | 4.57±0.02 | 19.12 | 5.18±0.08 | 29.24 | 6.43±0.04 | 32.62 | 6.91±0.03 | 35.85 | 5.34±1.34 | Straight, white |
| 0.0025% | 15 | 1.80±0.01 | — | 3.31±0.05 | 20.05 | 3.99±0.01 | 29.39 | 4.37±0.06 | 40.31 | 4.56±0.06 | 52.36 | 5.09±0.03 | 52.74 | 4.26±0.66** | Straight, white |
| 0.005% | 15 | 1.83±0.02 | — | 2.96±0.07 | 29.19 | 3.44±0.02 | 29.19 | 3.92±0.07 | 46.45 | 4.33±0.04 | 54.76 | 4.71±0.03 | 56.27 | 3.87±0.69** | Straight, white |
| 0.01% | 15 | 1.75±0.06 | — | 2.64±0.05 | 37 | 3.32±0.02 | 39.12 | 3.55±0.05 | 51.51 | 4.27±0.04 | 55.39 | 4.30±0.04 | 60.08 | 3.61±.69*** | Slimy, white |
| 0.02% | 15 | 1.82±0.01 | — | 2.61±0.1 | 37.8 | 3.29±0.01 | 41.76 | 3.64±0.05 | 50.43 | 4.17±0.02 | 56.43 | 4.19±0.04 | 61.1 | 3.58±.66*** | Slimy, white |
Data are shown as mean±SD, Nr: Number of roots, In (%): The inhibition percentage, ** p＜0.01 and *** p＜0.001 versus control, used one-way ANOVA; Dunnett test.
| Control and SPT treatments | Nr | Mean root length (cm) and inhibition (%) affected by SPT treatment | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 00 h | 24 h | 48 h | 72 h | 96 h | 120 h | Mean of longth±SD | Form and color | ||||||||
| Mean±SD | In% | Mean±SD | In% | Mean±SD | In% | Mean±SD | In% | Mean±SD | In% | Mean±SD | In% | ||||
| Control | 7 | 1.80±0.02 | — | 3.38±0.04 | — | 4.78±0.02 | — | 6.24±0.05 | — | 8.55±0.03 | — | 10.48±0.05 | — | 6.68±2.85 | Straight, white |
| 0.00125% | 7 | 1.82±0.01 | — | 3.1±0.04 | 8.83 | 4.41±0.03 | 7.75 | 5.08±0.04 | 18.86 | 5.48±0.05 | 35.91 | 6.00±0.01 | 42.75 | 4.81±1.12 | Straight, white |
| 0.0025% | 7 | 1.81±0.01 | — | 3.17±0.06 | 6.22 | 4.32±0.04 | 9.63 | 5.00±0.02 | 19.88 | 5.41±0.04 | 36.73 | 5.62±0.04 | 46.38 | 4.70±0.99 | Straight, white |
| 0.005% | 7 | 1.86±0.06 | — | 3.12±0.02 | 7.7 | 4.25±0.03 | 11.09 | 4.97±0.02 | 20.36 | 5.19±0.02 | 39.77 | 5.2 2±0.04 | 50.2 | 4.54±0.88 | Slimy, brown |
| 0.01% | 7 | 1.83±0.07 | — | 3.35±0.05 | 7.99 | 4.28±0.03 | 10.47 | 4.90±0.03 | 21.48 | 5.10±0.03 | 40.36 | 5.15±0.03 | 50.86 | 4.55±0.75 | Slimy, dark brown |
| 0.02% | 7 | 1.84±0.02 | — | 3.08±0.02 | 8.88 | 3.85±0.04 | 19.46 | 4.37±0.03 | 29.97 | 4.58±0.04 | 43.05 | 4.87±0.03 | 53.54 | 4.15±0.70* | Slimy, dark brown |
Data are shown as mean±SD, Nr: Number of roots, In (%): The inhibition percentage, * p＜0.05 versus control, used one-way ANOVA; Dunnett test.
Regarding the morphological aspect of the roots, changes in structure and color were noted especially in V. faba roots treated from the 0.005% concentration appeared slimy brown to slimy dark brown compared to the control. However, roots exposed to the 0.01% and 0.02% concentrations showed necrosis after 120 h of treatment.
The effect of different concentrations of SPT on MI of A. cepa is shown in Fig. 1A (12 h) and 1B (24 h). Significant decrease in MI was detected in meristematic cells of A. cepa et V. faba exposed to SPT compared to the control. Analysis of the results in Fig. 1A and 1B revealed that the control has the highest MI in both A. cepa and V. faba (59.26±0.88% and 59.90±0.40%, respectively) (12 h) and (60.74±0.45% and 60.14±0.48%, respectively) (24 h). It is reported that treatment of cells with the concentrations 0.00125% and 0.0025% did not impair the rate of cell division. In contrast, the values were as high as the control (12 h and 24 h) in A. cepa (60.00±048% and 60.16±0.47%, respectively) and V. faba (59.30±0.32% and 59.92±0.75%, respectively). However, cytotoxic effects were observed from the 0.005% concentration, accompanied by a significant decrease in MI (p＜0.001) after 12 h and 24 h of exposure at the 0.005%, 0.01%, and 0.02% concentrations.
Three bulbs and seven seeds were treated in each concentration, including the control after 12 h and 24 h. The data are reported as mean±SD (n=5,000 cells).
The aberration percentage and the different CAs induced by SPT are presented in Figs. 2A, B, 3A–D, 4, and 5. The increase in the CAs depends on the rise in the concentration of SPT and the period of treatment. Compared to the control, no significant effect on the percentage of CAs was recorded after treatment with the concentrations 0.00125% and 0.005% (12 h), and 0.00125% (24 h) in A. cepa and V. faba. While a significant increase (p＜0.001) in CAs (%) was revealed in cells treated with the concentrations 0.005%, 0.01%, and 0.02% (12 h) and at 0.0025%, 0.005%, 0.01%, and 0.02% (24 h) compared to the control. The highest percentages of CAs in A. cepa and V. faba were 7.72±0.74% and 8.04±0.65%, respectively, after 24 h treatment with SPT at the 0.02% concentration, and the lowest was 0.94±0.15% and 0.78±0.14%, respectively, at the 0.00125% concentration (12 h). The most common types of CAs were multipolar in A. cepa (38.95±3.59%) (12 h) and (37.73±3.27%) (24 h) and in V. faba (31,17±1.59%) (12 h) and (31.44±3.64%) (24 h), followed by break in A. cepa (21.84% ± 3.53%) (12 h) and (31.29±5.37%) (24 h) and in V. faba (25.63±2.03%) (12h) and (25.63±2.03%) (24 h), stickiness in A. cepa (19.55±0.96%) (12 h) and (20.30±3.86%) (24 h) and in V. faba (20.48±3.63%) (12 h) and (20.41±1.54%) (24 h), vagrant in A. cepa (19.21±1.58%) (12 h) and (17.71±3.42%) (24 h) and in V. faba (14.33±1.81%) (12 h) and (14.33±1.81%) (24h), bridge in A. cepa (15.20±5.07%) (12 h) and (12.78±5.33%) (24 h) and in V. faba (14.90±2.71%) (12 h) and (18.22±1.18%) (24 h), C-mitosis in A. cepa (3.62±1.50%) (12 h) and (3.72±1.36%) (24 h) and in V. faba (1.94±1.34%) (12 h) and (1.98±1.43%) (24 h), and M-nucleus in A. cepa (0.15±0.15%) (12 h) and (0.25±0.57%) (24 h) and in V. faba (0.49±1.10%) (12 h) and (0.49±1.10%) (24 h) (Fig. 3A–D).
Three bulbs and seven seeds were treated in each concentration, including the control after 12 h and 24 h. The data are reported as mean±SD (n=5,000 cells).
Three bulbs and seven seeds were treated in each concentration, including the control after 12 h and 24 h. The data are reported as mean±SD (n=5,000 cells).
vagrant (A1) with stickiness (A2, 3), multipolar (B1) and vagrant (B2), break (C1) and c-mitosis (C2) with multipolar (C3) and bridge (C4), stickiness (D1) and break (D2, 3), multipolar (E1, 2) with a bridge (E3), break (F1), multipolar (G1) with bridge (G2) and vagrant (G3), bridge (H1) with micro-nucleus (H2). After 12 h and 24 h. Scale bar=10 µm.
vagrant (A1) and stickiness (A2, 3), c-mitosis (B1, 3) and micro-nucleus (B2), stickiness (C1) and multipolar with bridge (C2, 3), micro-nucleus (D1), break (E1–3) with bridge (E4) and stickiness (E5), vagrant (F1) and stickiness (F2–4), break (G1, 2) and bridge (G3). After 12 h and 24 h. Scale bar=10 µm.
According to the data obtained, it was found that the application of SPT caused cytotoxic and genotoxic effects in the root tip cells of A. cepa and V. faba. These effects are manifested by inhibition of root growth, changes in root color and structure, a decrease in the MI, and increased CAs with the appearance of different types of chromosomal abnormalities.
Similar results were obtained by the study of Sheikh et al. (2020) on the two pesticides malathion and cypermethrin and the research of Macar (2021) on the fungicide Tetraconazole, which revealed that these pesticides can cause severe growth retardation, genotoxic damage and meristematic cell disorders on A. cepa. According to Wierzbicka (1988), root growth during cell differentiation is directly related to meristematic activity, and cell elongation. This elongation is also based on other factors, such as an enzymatic activity that promotes cell elongation and cell membrane release during cell differentiation (Silveira et al. 2017). Thus, the slowing of root growth may be due to the inhibitory effect of SPT on the enzymatic activity that promotes the elongation of the meristematic zone.
The MI is a marker of cytotoxicity caused by pollutants agents (Leme and Marin-Morales 2009). It’s also used to determine the proportion of dividing cells and those in cell arrest during the cell cycle (Rojas et al. 1993). In this work, the decrease in MI after 12 h and 24 h of SPT exposition demonstrated its significant potential cytotoxic effect. Our result indicates that SPT inhibits the proliferation of A. cepa and V. faba cells which resulted in a substantial decrease in MI at high concentrations. However, research on SPT-induced mitotic degradation is scarce.
Different studies tested different pesticides, such as the herbicide atrazine (El-Ghamery et al. 2000), abamectin (Kalefetoğlu Macar 2020), and malathion and cypermethrin (Sheikh et al. 2020). Furthermore, de Souza et al. (2016) suggested that the decrease in MI is associated with the increase in the frequency of CAs due to the potent antiproliferative effect of the different herbicides tested. Furthermore, a significant reduction in MI could be attributed to the inhibition of the G2 phase (El-Ghamery et al. 2000). Our results suggest that potential cytotoxicity is caused by SPT, and this cytotoxicity, according to Bonciu et al. (2018) is due to a perturbation of regular cell cycle progression.
CAs induced by SPT were observed at all concentrations. SPT insecticide showed a significant genotoxic effect from 0.005% (12 h) and 0.0025% (24 h) concentrations inducing cell cycle disruption. Also, it caused abnormal segregation of chromosomes so that different chromosomal abnormalities were formed, the most frequent being multipolar, break, stickiness, vagrant, bridge, c-mitosis, and micro-nucleus. Similar genotoxic results are obtained in studies conducted on pesticides acting as genotoxic and mutagenic agents (Adesuyi et al. 2018, Kumari et al. 2020, Macar 2021). Analysis of the data showed that the most frequent CAs were multipolar anaphase. According to Khallef et al. (2019), multipolarity at the anaphase stage may correspond to instability at the mitotic spindles. Chromosomal abnormalities, such as chromosomal breaks and bridges, indicate clastogenic activities (Sabeen et al. 2020).
Chromosome break and sticky are the abnormalities often observed in this study. According to Kuchy et al. (2015), excessive chromosome condensation or improper nucleoprotein synthesis may contribute to the frequency of stickiness. Uneven translocation, chromosome segment inversions and the generation of chromatin bridges as a result of stickiness leading to failure to achieve chromosome separation at anaphase (Evseeva et al. 2005). The chromosome bridge is formed at the anaphase stage, which can be attributed to unequal exchanges leading to the origin of dicentric chromosomes that are equally attracted to both poles of the cell (Sax and Sax 1968). Bridge formation may also be due to fusion, breakage of chromosomes and chromatids or by changes in the activation of replication enzymes (Luo et al. 2004). Another significant aberration was chromosome vagrant. This aberration might be produced by the maldistribution of chromosomes during anaphase (Fiskesjo 1985) or by the disruptions in the spindle’s fiber formation due to the effects of chemicals (Haliem 1990). Micro-nucleus and c-mitosis occurred at a low frequency. Their existence might be explained by the spindle apparatus’ inability to organize and function normally (Rosculete et al. 2018). In addition, Fenech et al. (2011) indicate that the micronucleus may be the result of acentric chromatid fragments and acentric fragments that indicate clastogenic potency, or it may be a whole chromosome that indicates aneugenic activity and is not integrated into the main nucleus through nuclear division. The concept of “c-mitosis” was established by Levan (1938) to indicate the effect of a chemical that inhibits the assembly of spindle microtubules by dissociating disulfide bonds, such as the effect of colchicine.
This research is the first one showing the cytotoxic and genotoxic effects of SPT on the meristematic cells of A. cepa and V. faba. The decrease in root growth accompanied by a decrease in the MI and an increase in CAs are signs of cytotoxicity and genotoxicity. Therefore, the exposition and the continuous accumulation of SPT may endanger other living organisms other than the targeted species.
