Findings of Allium root growth inhibition test is shown in Fig. 1. A significant growth reduction (r =-.991) was observed in clopyralid exposed onion bulbs in a dose dependent manner from 5.69% at 6.25 µg/L (4.31 ± 0.09 cm) to 93.44% at 1000 µg/L (0.3 ± 0.08 cm) compared to control group (4.57 ± 0.11 cm) indicating cytotoxic effects of the clopyralid. EC50 value of clopyralid was calculated as 50 µg/L (2.29 ± 0.1 cm) after 96 h by root growth inhibition test. The root and shoot lengths of Zea mays were reduced by clopyralid (Vettakkorumakankav et al., 2002). Root growth inhibition was also observed by other pyridine-carboxylate auxin herbicides such as triclopyr in Populus trichocarpa (Eslamiamirabadi et al. 2020), fluroxypyr in Oryza sativa (Wu et al., 2010) and other auxin herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) in A. cepa (Fiskesjö et al., 1981; Ateeq et al., 2002). Unlike our result, clopyralid did not show inhibitory effect on Dunaliella primolecta growth (Santin-Montanya et al., 2007).
The percentages of phase and mitotic index in the meristematic root cells exposed to Clopyralid are given in Table 2. Significant reduction of MI between 62.82 ± 0.81 to 55.71 ± 0.52 was observed at clopyralid exposed onion bulbs compared to negative and positive (except 24 and 96 h) control groups. The gradual decrease in the MI in both dose (r=-.957 for 24 h, r=-.919 for 48 h and 96 h, r=-.901 for 72 h) and time (r=-.928 for 25 µg/L, r=-.866 for 50 µg/L, and r=-.878 for 100 µg/L) dependent manner reveals the cytotoxic effects of clopyralid. Clopyralid significantly decreased the percentage of metaphase and anaphase (except 24 h at 25 µg/L) but increased telophase indices compared to negative control group. The other synthetic auxin herbicides like picloram at the highest four concentrations (2.01, 2.68, 3.35, 4.02 mg/L, Özel at al., 2018), triclopyr (El-Khodary et al., 1989), three higher concentrations (2.68, 3.35 and 4.02 mg/L) of 2,4-D at 24 h and 4.02 mg/L of 2,4-D at 48 h (Özkul et al., 2016), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), MCPA and 2,4-D (Fiskesjö et al., 1981) was also reported to inhibit MI in A. cepa root tips. Significant decreases in MI were also detected with 1-Naphthaleneacetamide in human peripheral blood lymphocytes (Kocaman and Güven, 2015), MCPA in Brassica napus L. (Polit, et al., 2014), diclofop in Triticum aestivum (Morrison et al., 1981).
Types and percentages of mitotic abnormalities in clopyralid exposed A. cepa ana-telophase cells are presented in Fig. 2 and Fig. 3, respectively. All the tested concentrations of clopyralid induced CAs with increase in dosage concentration (r = .967 for 24 h, r = .939 for 48 h, r = .937 for 72 h, and r = .913 for 96 h) and time (r = .931 for 25 µg/L, r = .842 for 50 µg/L, and r = .731 for 100 µg/L) duration. No statistical significant difference was observed between the positive control group and the 100 µg/L clopyralid (except at 24 h) concentration. The gradual increase in the CAs reveals the genotoxic effects of clopyralid. Disturbed ana-telophase (Fig. 2a) and chromosome laggards (Fig. 2b) probably occur due to disruptions in the spindle formation and failures in chromosome movement towards poles (Tkalec et al. 2009: Khanna and Sharma, 2013). Stickiness (Fig. 2c) probably occur due to DNA depolymerization, contraction or condensation of chromosomes and partial dissolution of nucleoproteins (Sabeen et al., 2019). The chromosome breakage or fusion, formation of dicentric chromosomes, cross-links between proteins and chromosomes, unequal chromatid exchange or stickiness could be caused anaphase bridge (Fig. 2d, Feretti, et al., 2007: Dutta et al. 2018). Atypical scattering of chromosomes during replication or chromosome segregation may resulted polyploidy (Fig. 2e, Nefic et al. 2013; Palsikowski et al. 2018).Unlike our result, clopyralid did not induce CAs in Chinese hamster lung cells (Wang et al., 2012), in mammalian bone morrow cells (Ilyushina et al., 2019). The other synthetic auxin herbicides like triclopyr (El-Khodary et al., 1989), 2,4-D (Ateeq et al., 2002; Özkul et al., 2016), MCPA, 2,4-D and 2,4,5-T (Fiskesjö et al., 1981) were also reported to induce CAs in A. cepa root tips. The results of mitotic anomalies of the current study are in agreement with the work on plants reported previously such as such as with 2,4-D on Raphanus sativus L. and Phaseolus vulgaris L. (Truta et al., 2011), 2,4-D and dicamba on Arabidopsis thaliana (Filkowski et al., 2003).
The results of DNA damage in A. cepa root tips treated with clopyralid is summarized in Fig. 4. Clopyralid exposed root tips showed significantly higher DNA damage (between 124 ± 4.58 to 166.67 ± 2.52 Arbitrary Unit) than the negative control group (between 11.67 ± 1.53 to 13.67 ± 0.58 Arbitrary Unit). Induction of DNA damage was found to be dose (r = .972 for 24 h and 48 h, and r = .981 for 72 and 96 h) and time (r = .954 for 25 µg/L, r = .933 for 50 µg/L, and r = .915 for 100 µg/L) dependent. Clopyralid did not induce genotoxicity on Salmonella typhimurium and mice CD-1 strains (Ilyushina et al., 2019). DNA damage by using comet assay was also observed by other synthetic auxin herbicides like 2,4-D and dicamba on Phaseolus vulgaris root tips (Cenkci et al., 2010), triclopyr on Anguilla anguilla L. (Guilherme et al., 2015), 2,4-D on human Caco-2 cells (Syberg et al., 2015), Chinese hamster ovary cells (González et al., 2005, Laborde et al., 2020), on Clarias batrachus (Ateeq et al., 2005),on Cnesterodon decemmaculatus (de Arcaute et al., 2016), and on Syrian hamster embryo cells at 11.5 µM and 23 µM of 2,4-D (Maire et al., 2007), MCPA and 2,4-D on epithelioma papillosum cyprini cell line (Bokán et al., 2013), MCPA on mussel gill (Emmanouil et al., 2008). But negative results by using comet assay was reported with other synthetic auxin herbicides such as 2,4-D on A. cepa (Özkul et al., 2016), dichlorpop and mecoprop on epithelioma papillosum cyprini cell line (Bokán et al., 2013).
The cytotoxicity and genotoxicity of mechanism of clopyralid has not yet been fully elucidated. The suppression of root growth and reduction of MI associated with enhanced genotoxicity by clopyralid could be due to inhibition of cell division in root tips by inducing ethylene biosynthesis through the synthesis of ACC and ABA leading to ROS overproduction (Cremlyn 1991; Tu et al., 2001; Grossmann, 2003, 2010; Sunohara and Matsumoto, 2008; Christoffoleti et al., 2015; Hura 2019, Abou-Zeid et al., 2020). But high levels of ROS can also stimulate the ethylene and ABA biosynthesis (Xing et al., 2004; Kim et al., 2008, McCarthy-Suárez, 2017). Overproduction of ROS by auxin herbicides can damage the membranes, nucleic acids, proteins and enzyme activities (Bradberry et al., 2004; González et al., 2005; Rodríguez-Serrano et al., 2014).
Clopyralid induced cytotoxicity and genotoxicity on the A. cepa roots by a dose dependent decline in root growth and MI, and by a dose and time dependent increase in CAs and DNA damage. Clopyralid should be used carefully at appropriate doses. The cytotoxic and genotoxic mechanisms of clopyralid should be investigated with using both plant and animal models.