# Application of the _Aliium_ Test in Toxicity Studies of Lead and Copper: A Cytological Perspective
## 4 Discussion  
Heavy metals are more persistently present in our lives and activities due to the high levels of industrialization [68; 69], making it essential to better understand the maximum permissible limits [70]. Over time, their accumulation can ultimately trigger significant inhibition of the physiological activity of both plants and humans [71]. One of the major issues regarding heavy metals is the significant variation in regulations and the wide margin of error concerning the maximum allowable limits for heavy metals. It remains unclear at what precise concentration a heavy metal becomes toxic [33; 34; 35; 72; 73]. Cellular toxicity effects can be demonstrated by phenomena such as irreversible vacuolization, where the mitotic spindle becomes blocked and displaced to the outer edge of the vacuole, ultimately leading to the cessation of the cell division process. Irreversible vacuolization not only halts the process of cell division but is also associated with the phenomenon of programmed cell death (PCD) [74]. Irreversible vacuolization and dynamic vacuole formation in the process of toxicity are observed in several species, including _Allium cepa_[34; 75]. The _Allium_ test enabled the identification of genotoxic effects and the comparison of the toxicity levels of \(Cu^{+2}\) and \(Pb^{+2}\). According to the experiment, which involved a direct proportional relationship between the \(Cu^{+2}\) and \(Pb^{+2}\) treatments, the genotoxic impact of concentration and exposure time were revealed. The findings revealed that exposure to increased doses of lead and copper significantly reduces the mitotic index (MI), while increasing the abnormalities index (AI). On the other hand, the ANOVA test evaluating dose and exposure time was significant only in the case of copper, reflecting that as the dose increases, the acute effect of copper becomes more pronounced. This was also observed in the correlation analyses. The impact of copper is reflected in the observation that the MI was inhibited by 50% (IC\({}_{50}\)) at lower concentrations, starting from a dose of 0.50 mM. Copper exhibited a stronger negative correlation between the mitotic index (MI) and the abnormalities index (AI) (\(-0.69\)), reflecting the genotoxicity that causes disruptions at the cellular level. In contrast, for lead, the IC\({}_{50}\) was observed at concentrations starting from 0.75 mM, exhibiting a negative correlation between the mitotic Index (MI) and the abnormalities index, which, at \(-0.29\), was lower than that of copper. The cumulative effects of heavy metals, particularly lead, become evident, with a significant inhibition of cell division observed at high concentrations (1 mM) after 72 h.  
In plants, PCD should be termed as apoptotic-like PCD (AL-PCD), as suggested by [PERSON] et al., 2008 [34; 76]. Studies demonstrate the relevance of genotoxicity and AL-PCD induction in essential biomarkers for stress in _Allium_ species [76], suggesting that there are some similarities between AL-PCD and apoptosis, although they are clearly different in each process. Specific AL-PCD processes, i.e., chromatin condensation, vacuolization, and DNA fragmentation, were detected [34,76]. However, the _Allium_ test proved effective in detecting genotoxic effects and comparing the toxicity of copper and lead. Our correlation analyses results indicate a higher impact of copper on mitotic activity compared to lead. On the other hand, the limitations of the study did not clearly reflect the acute and chronic effect differences between lead and copper. Furthermore, according to studies in literature, hormesis can have beneficial stimulatory effects at low doses, promoting cellular repair and an increase in the mitotic index. Studies demonstrate the capacity of the _Allium_ test to detect the hormesis effect in cellular division [77]. However, the concentrations used in this study (0.25-1.00 mM) did not reveal a cellular hormesis response to copper and lead.