Introduction

Fungicides have been widely used for economic benefit in agriculture and industry for the last few decades, due to their high efficiency and low toxicity (Jin et al. 2017; Zhang et al. 2018). The global fungicide market was worth approximately USD 13.4 billion in 2018 and is forecast to be worth USD 15.7 billion in 2024 (Garside 2019). Imazalil (IMZ; 1-[2-(2,4-dichlorophenyl)-2-(2-propenyloxy) ethyl]-1H-imidazole) is a type of fungicide which is highly beneficial for the prevention and cure of fungal diseases in many plants such as cucumber, tomatoes, citrus fruits, tomato, barley, wheat, and bananas in post-harvest treatments, and it is also used as antimycotic drug in human and veterinary medicine and as a biocide in the formulation of wood and building materials (Bossche et al. 2003; USEPA 2005; Bylemans and Thys 2007; Smilanick et al. 2006; Sepulveda et al. 2015). The derivatives of imidazole and triazole such as IMZ inhibit fungal cell wall synthesis by the inhibition of ergosterol biosynthesis and thus interrupting mechanism of cytochrome P450 enzyme called CYP51 (lanosterol-14-α-demethylase), present in several organisms which acts on cell membrane homeostasis, fluidity, and permeability (USEPA 2005; Correia and de Montellano 2005; Zega et al. 2009; Kuhlmann et al. 2019). They consist of about 25% of fungicides worldwide (Saxena et al. 2015). According to the World Health Organization, extreme allowable residue levels of IMZ in citrus fruits and in bananas should not be more than 5 mg/kg and 2 mg/kg, respectively (Tanaka et al. 2013). In addition to its pest-reducing effects, IMZ has been also detected in soil/sediment, water, and aquatic organisms (Gilbert-López et al. 2012; Belenguer et al. 2014; Masiá et al. 2015; Ruiz-Rodríguez et al. 2015; Xu et al. 2015; Ccanccapa et al. 2016). As IMZ is not specific to fungi, it can interact and inhibit the cytochrome P450 enzymes in non-target organisms like other azoles and may have undesirable effects (Walker 2008; Gottardi and Cedergreen 2019; Kuhlmann et al. 2019). So, cyto-genotoxic assessment of IMZ is inevitable to find its hazards on non-target species.

Allium test is considered as the most frequently used plant cytogenetic assessment method for revealing the cyto-genotoxic effects of fungicides. Different parameters like root growth, mitotic index (MI), and chromosomal aberrations (CAs) can be easily observed. It is a quick, easy, highly accurate, and easily reproducible. It is also accepted as a standard assay by the United Nations Environmental Programme, the United States Environmental Protection Agency, and the World Health Organization (Grant 1994; Rank and Nielsen 1994; Teixeira et al. 2003; Leme and Marine-Morels 2009; Liman et al. 2011; Silveira et al. 2016; Fatma et al. 2018; Bernardes et al. 2019). Root meristems of A. cepa–based comet assay have been widely used to determine DNA damage of pesticides at the level of individual cells, because this technique is quite simple, sensitive, and reliable. A small number of cells are required, and it is also relatively inexpensive compared to other test systems (Türkoğlu 2012; Karaismailoglu 2015; Liman et al. 2015; Silveira et al. 2017; Özkan and Liman 2019).

Despite the mentioned superior properties of the IMZ, its potential adverse effects on non-target plants are not yet known sufficiently. The current study was designed to determine cyto-genotoxic effects of IMZ on A. cepa roots by observing root growth, MI, mitotic phase changes, CAs in ana-telophase cells, and DNA damage.

Materials and methods

Chemicals

The chemicals used in this study were supplied by Sigma Aldrich, Munich, Germany, including IMZ (CAS No 35554-44-0), MMS (CAS No 67-27-3), glacial acetic acid, basic fuchsin hydrochloric acid, potassium disulfite, potassium chloride, sodium chloride, trizma hydrochloride, disodium hydrogen phosphate, potassium phosphate monobasic, normal melting point agarose, low melting point agarose, trizma base, magnesium chloride hexahydrate, triton X-100, sodium hydroxide, di-sodium salt of ethylene diamine tetra acetic acid (EDTA), and ethidium bromide.

Allium root growth inhibition test

Healthy and equal-sized A. cepa bulbs were obtained from local market which were 25–30 mm in diameter. The growth inhibition test was performed according to the modified method proposed by Fiskesjö (1985) as described in Küçük and Liman (2018). Bulbs cleaned from dried roots and brown outer shells were directly exposed to different concentrations of IMZ (0.5, 1, 2.5, 5, 10, 25, 50, and 100 μg/mL) for 96 h which were kept in dark at room temperature as well as distilled water for negative control. To calculate the EC50 value, the average lengths were determined by taking 10 roots from one bulb (50 roots from 5 bulbs) for each application after the exposure time.

Allium ana-telophase test

The Allium ana-telophase test was performed by following the protocol proposed by Rank and Nielsen (1993) with slight modifications. 0.5, 1, and 2 μg/mL of IMZ were exposed to Allium roots whose lengths varied from 2 to 3 cm at the room temperature in the dark for several time intervals (24, 48, 72, and 96 h) as well as distilled water for negative control and 10 μg/mL of MMS for positive control. Three onions were used for each concentration. Five to 8 root tips from each onion were cut about 1 cm in length and fixed for 1 day at 4 °C in Farmer’s solution (1 glacial acetic acid: 3 ethanol, v/v) and then stored in alcohol (70%) at the same temperature. After hydrolysis of roots with 1 N HCl at 60 °C for 8–10 min, Feulgen dye was employed to smear of the root tips at room temperature for 25–30 min. Following rinsing with distilled water thrice for 5 min, squash preparations of the dark stained root tips were prepared with one drop of 45% acetic acid and sealed with finger nail polish. For each application, 5000–5150 cells (1000–1040 cells one slide per bulb) and 500 ana-telophase cells (100 ana-telophase cells one slide per bulb) were counted for MI and CA frequencies using a trinocular light microscope according to Saxena et al. (2005). The following formulas were used in the calculation of MI, phase index, and CA.

$$ \mathrm{MI}\ \left(\%\right)=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{cells}\ \mathrm{in}\ \mathrm{division}}{\mathrm{Number}\ \mathrm{of}\ \mathrm{total}\ \mathrm{cells}}\times 100 $$
$$ \mathrm{Phase}\ \mathrm{in}\mathrm{dex}\ \left(\%\right)=\frac{\mathrm{Particular}\ \mathrm{phase}\ }{\mathrm{Number}\ \mathrm{of}\ \mathrm{cells}\ \mathrm{in}\ \mathrm{division}}\times 100 $$
$$ \mathrm{CA}\ \left(\%\right)=\frac{\mathrm{Total}\ \mathrm{aberrant}\ \mathrm{cells}}{100\ \mathrm{ana}-\mathrm{telophase}\ \mathrm{cells}}\times 100 $$

Comet assay

Genotoxic potential of IMZ on A. cepa roots was assessed with the alkaline comet assay by the instructions of Tice et al. (2000) with slight modifications as stated by Küçük and Recep (2018). The treated root tips as mentioned above were gently sliced to isolate nuclei in 600 μL cold nuclear isolation buffer (0.5% w/v Triton X-100, 2 M Tris 4 mM, MgCl2-6H2O; pH 7.5) and then filtered through 60-μm meshes Nylon filter. At 1200 rpm, the solution was centrifuged, at 4 °C for 7 min. Fifty-microliter 1.5% low melting point agarose was mixed with the pellet of nuclei suspension (50 μL), and then the mixture was smeared onto slides that pre-coated with 1% normal melting agarose and kept on ice for 5 min. After removing cover slips, the slides were placed in a horizontal electrophoresis tank having cold 1 mM EDTA+ 300 mM NaOH buffer (pH ≥ 13) for 20 min at 4 °C and then electrophoresed 300 mA/24 V for 20 min under the same conditions. The slides were neutralized with 0.4 M Tris (pH 7.5) thrice for 5 min. After neutralization, staining was done with ethidium bromide solution (20 μg/mL). A BAB fluorescence microscope (TAM-F, Turkey) was used to analyze on randomly selected 50 comets per slide (150 comets per sample) for evaluation of DNA damage expressed as arbitrary unit at × 400 magnification. The comets were classified in five classes [0 (no damage) to 4 (maximum damage)], according to Kocyigit et al. (2005) as shown in Fig. 1.

Fig. 1
figure 1

Comet scores of DNA damage in A. cepa roots at × 400 magnification. 0 no visible damage, 1 low level damage, 2 moderate damage, 3 high level damage, 4 maximal damage

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Data analysis

Duncan’s multiple range tests were used for statistical evaluation of the results (mean ± standard deviation) using SPSS 23.0 version (SPSS Inc., Chicago, USA) at p ≤ 0.05. Pearson correlation analysis was also used to determine dose-response and dose-time relationships at p = 0.01 significance level.

Results and discussion

The EC50 of IMZ was found to be 1 μg/mL (50.72%) by Allium root growth inhibition test (Fig. 2). IMZ decreased root growth within the range of 35.4 to 96.07% compared to the control group in a dose-dependent manner (r = −0.969). The factors affecting root growth are generally related to suppression of apical meristematic activity (Webster and Macleod 1996), cell cycle during differentiation (Fusconi et al. 2006), or enzymes that are related to cell division (Silveira et al. 2017). IC50 of IMZ for Chironomus riparius was found to be 0.11 ± 0.01 and 0.09 ± 0.01 μmol/L for R- and S-imazalil, respectively (Kuhlmann et al. 2019). EC50 of IMZ for Pseudokirchneriellasubcapitata was found to be 0.623 μg/mL for 72 h. LC50 of IMZ was found to be 0.882 μg/mL for Daphnia magna and 2.324 μg/mL for Danio rerio (Li et al. 2019), 541 μg/g in the artificial soil test and 12.8 μg/cm2 for Eisenia foetida (Van Leemput et al. 1989), and 173.7 μM for Phallusia mammillata (Pennati et al. 2006).

Fig. 2
figure 2

Allium root growth inhibition test at different IMZ concentrations after 96 h. *Different letters are significantly different at p ≤ 0.05.

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Table 1 shows MI and mitotic phase indices of IMZ obtained using Allium test. All concentrations of IMZ statistically decreased MI compared to the control group not only dose-dependently (for 24 h r = − 0.885, for 48 h r = − 0.924, for 72 h r = − 0.855, and for 96 r = −0.831) but also time-dependently (for 0.5 μg/mL r = − 0.943, for 1 μg/mL r = − 0.94, and for 2 μg/mL r = − 0.905). The reduction in MI for IMZ was not lower than MMS. IMZ statistically decreased the prophase index but increased the telophase index compared to the control group. It also increased metaphase and anaphase indices but was not found statistically significant except for the anaphase index at 96 h. A significant decrease in MI by IMZ was indicative of cytotoxic damage and may occur due to inhibition of DNA polymerase by the inhibition of specific proteins (Hidalgo et al. 1989), the changes of cell durations (El-Ghamery et al. 2000; Sudhakar et al. 2001; Rajeshwari et al. 2016), or mitotic stage duration changes (Tkalec et al. 2009) or mitodepressive regulation of chemicals (Sharma and Vig 2012), and/or ROS disturbance homeostasis (Livanos et al. 2012). IMZ showed also toxicity on isolated rat hepatocytes at 0.75 mM (Nakagawa and Moore 1995), on the mouse fibroblast L929 cells at 50 μM (Maruyama et al. 2007), and on Danio rerio embryos at 10 mM and above (Şişman and Türkez 2010). Hatch ability in chickens was inhibited by in ovo exposure of IMZ at 2 mg/egg (Matsushita et al. 2006). IMZ induced acute cell death higher than 7 μg/mL on Scaphechinus mirabilis and Strongylocentrotus nudus (Hosoya and Mıkamı 2008). IMZ was found as toxic in vivo but nontoxic or moderate toxic in vitro by Ames and micronucleus test (Ilyushina et al. 2019). Unlike our result, 2.5 μg/mL of IMZ did not show toxicity in the human intestinal Caco-2 cells after 48-h incubation (Sergent et al. 2009). Leaf chlorophyll content and growth in Phragmites australi were also found to be similar between the control group and 10 μg/mL of the IMZ exposed groups after 24 days (Lv et al. 2017).

Table 1 Effect of IMZ on mitotic and phase indices in A. cepa roots
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The types and rates of CAs caused by the IMZ in A. cepa root meristem cells are given in Figs. 3 and 4. IMZ prompted total CAs by showing the disturbed ana-telophase, stickiness, anaphase bridges, chromosome laggards, and polyploidy in ana-telophase cells of A. cepa in a dose-dependent manner compared to the control group, suggesting genotoxic effect of IMZ. However, the increases between IMZ groups were not statistically significant. Stickiness (at 0.5 μg/mL for 24 h, 2.6%) was the most common CAs, and the least seen CAs was polyploidy (at 0.5 μg/mL for 24 h, 1.4%). Disturbed ana-telophase (Fig. 4a) and chromosome laggards (Fig. 4c) may result from degraded microtubules or inhibition of movement of chromosomes to opposite poles (Evseeva et al. 2005; Kumari et al. 2009; Rajeshwari et al. 2016; Singh and Roy 2017). Stickiness (Fig. 4b), an indicator of toxicity, may be caused by formation cross-linking of DNA-DNA or DNA-protein (Amin 2002; Barbério et al. 2011). Anaphase bridges (Fig. 4d) may cause chromosome laggards by showing clastogenic effect due to the formation of dicentric chromosomes, stickiness, changes in replication enzyme activity, breakage or fusion of chromosomes, and unequal chromatid exchange (El-Ghamery et al. 2000; Luo et al. 2004; Dutta et al. 2018). Polyploidy (Fig. 4e) may result from abnormal segregation of chromosomes during the cell division (Nefic et al. 2013; Palsikowski et al. 2018). In addition to ana-telophase anomalies, c-metaphase and binuclear cells in other cells were also observed. Cytokinesis inhibition at any cell cycle control point (Ateeq et al. 2002) may induce binuclear cells (Fig. 4f). C-metaphase (Fig. 4g) may occur due to spindle failure or an imbalance in amount of proteins responsible for the formation of nuclear chromatin (Odeigah et al. 1997; Mesi and Kopliku 2013). IMZ also induced micronucleus frequency and CAs in human peripheral lymphocytes (Şişman and Türkez 2010).

Fig. 3
figure 3

IMZ induced CAs (a) in ana-telophase cells and other anomalies (b) in A. cepa root cells. *Different letters for each treatment time are significantly different at p ≤ 0.05.

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Fig. 4
figure 4

Anomalies induced by IMZ in A. cepa roots. a Disturbed anaphase-telophase. b Stickiness. c Chromosome laggards. d Anaphase bridge. e Polyploidy. f Binuclear cell. g c-Metaphase. Scale bars 10 μm

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The genotoxic effects of IMZ in the A. cepa root meristematic cells were evaluated by comet assay (Fig. 5). There was more DNA damage by the IMZ groups compared to the control group. A dose-dependent (for 24 h r = 0.94, for 48 h r = 0.971, for 72 h r = 0.954, and for 96 r = 0.946) and time-dependent (for 0.5 μg/mL r = 0.961, for 1 μg/mL r = 0.943, and 2 μg/mL r = 0.92) increases of DNA damage between 95.67 ± 6.03 and 153 ± 2.65 were observed after IMZ applications. Similarly, IMZ induced DNA damage observed by the alkaline comet assay in mouse hepatocytes (Đikić et al. 2012) and in human lymphocytes (Ramirez and Cuenca 2002; Vindas et al. 2004). After chronic IMZ exposure in mice for 15 weeks, reactive oxygen species (ROS) were also increased in mouse hepatocytes, resulting to oxidative stress (Jin et al. 2018). Oxidative stress produced by IMZ at high concentrations in different organisms was linked to cell death by damaging cell membranes according to previous studies (Heusinkveld et al. 2013; Prado et al. 2015; Pereira et al. 2019).

Fig. 5
figure 5

IMZ induced DNA damage in A. cepa roots. *Different letters in the same columns for each treatment time are significantly different at p ≤ 0.05.

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Conclusions

IMZ showed not only cytotoxic effect by decreasing inhibition of root growth and MI but also genotoxic effect by increasing CAs and DNA damage in A. cepa roots. Further studies are therefore needed to clarify IMZ’s cyto-genotoxic mechanisms on plants.