Cyto–Genotoxic Effect Causing Potential of Polystyrene Micro-Plastics in Terrestrial Plants

The polystyrene micro-plastics (Ps-MPs) is one of the leading pollutants found in both aquatic and terrestrial ecosystems. While most of the studies on the morphology and cyto-toxicity of MPs have been based on aquatic organisms, their effects on terrestrial plants are still scarcely known. The present study was an attempt to measure the effect of different sizes (80, 100, 200, 500, 1000, 2000, 4000, and 8000 nm) and concentrations (100 and 400 mg/L) of Ps-MPs on the root length and chromosomes of root tip cells of Allium cepa using A. cepa root chromosomal aberration assay. Large size Ps-MPs (4000 and 8000 nm) showed the highest reduction in A. cepa root length; however, the differences were not significant (at p ≤ 0.05), with respect to negative control (Milli-Q water). The mitotic index showed both significant size- and concentration-dependent decreases, being the lowest (12.06%) in 100 nm at 100 mg/L concentration, with respect to the control (25.05%). The chromosomal abnormality index (CAI) and nuclear abnormality index (NAI) showed significant decreases, with respect to negative control. In addition, the induction of micro-nucleated cells was also observed in Allium root tip cells, when treated with MPs of all sizes, which can predict direct DNA damage to the plant cells. Hence, we conclude that most of the MP sizes caused cyto-toxic and nuclear damage by adversely impacting the spindle formation and induction of micro-nucleated cells in Allium cepa root tip cells. To the best of our knowledge, this is the first study that showed the effect of considerable size range of Ps-MP sizes on the root length and cell division in plants.


Introduction
Plastic is omnipresent and has become one of the main components of our modern-day consumption, including food packaging, cosmetics, personal care, and textiles. Plastic has acquired importance due to its low manufacturing cost, light weight, bioavailability, flexibility, stability, durability, viscosity, and long-lasting ability [1,2]. The World Economic Forum [3] reported that, by 2050, there will be more plastic than fishes in the world's oceans. Human consumption has generated more than 8 billion tons of plastic since 1950s, out of which, only 10% has been recycled, while the rest ended up directly in soil and ocean ecosystems [4].
Plastic is considered non-biodegradable, in general; however, due to various biological, chemical, and physical processes, larger size particles breakdown into smaller ones, which are known as micro-plastics (MPs). MPs with a size range of <5 µm cannot decompose easily or be collected for recycling; hence, they enter directly into the soil, water bodies, food chains, and air [5]. Being ubiquitous in nature and persistent in the environment for longer time, MPs have become a major concern as a pollutant [6,7]. Commercially, high-and low-density polypropylene (PP), polyvinylchloride (PVC), polyethylene (PE), polyethylene tere-phthalate (PET), and polystyrene (Ps) are the most extensively used accumulation of 40 nm and 1 µm polystyrene negatively charged micro-plastic spheres at the root tip and cap cell surfaces in Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum); however, the study did not confirm MP presence inside the cell [44]. MPs have been shown to affect the overall health and development of plants. For example, Qi et al. [45] showed that the application of low-density polyethylene and starch-based biodegradable macro-and micro-sized plastic residues affected wheat growth, while the application of only micro-sized plastics showed more negative effects on both the vegetative and reproductive apparatus of wheat crop, as compared to the macro-plastics. In another study, the application of micro-sized polyethylene, polypropylene, and polystyrene plastic particles had a stronger negative impact on the vegetative growth of juvenile lime trees, as compared to macro-sized in a controlled pot experiment [46].
With plastics becoming a quintessential part of our daily lifestyle, there is an urgent necessity to assess the induced toxicity of MPs of all sizes and shapes on living cells [47]. For the management and risk assessment of polystyrene MPs, eco-toxicity tests employing plant-based models have been recommended by various national and international organizations, including the US Environmental Protection Agency (EPA),United Nations Environmental Program (UNEP), International Program on Plant Bioassay (IPPB), and World Health Organization (WHO) [48]. In order to understand the absorption and toxicity of variable-sized plastic particles on plants, in particular, in vivo models, such as the A. cepa root tip chromosomal aberration assay, would be best suited. The A. cepa assay is the preferred model for studying chromosomal aberrations because it provides better clarity of mitotic phases and the easy detection of chromosomal abnormalities, due to the low chromosome number (2 n = 16) and stability of karyotype. In addition, the inexpensiveness and easy availability of A. cepa bulbs throughout the year also make it an assay of choice [49]. The A. cepa root assay of cyto-genotoxicity of variable-sized Ps-MPs could provide a basic understanding that can be extrapolated in other plants [50][51][52].
Recently some studies have reported on the cyto-genotoxic effects of polystyrene MPs on plants such as Vicia faba and Allium cepa [43,52,53]. The effects of Ps-MPs of sizes 0.5 and 100 nm in V. faba reported an accumulation of 100 nm Ps-MPs into the root tissues of V. faba, which resulted in cyto-genotoxicity, such as micronucleus formation [43]. Another report by Maity et al. [52] measured the cyto-genotoxic nature of 100 nm Ps-MPs employing 25, 50, 100, 200, and 400 mg/L concentrations in A. cepa and confirmed the cyto-genotoxicity, in terms of different chromosomal (clumped chromosomes, laggard chromosomes, ring chromosomes, vagrant chromosomes, multi-polarity, sticky bridge, etc.) and nuclear aberrations. Additionally, Giorgetti et al. [53] reported on the cyto-toxicity of 50 nm size PS-MP nano-beads, which induced chromosomal abnormalities and micro-nucleated cells in A. cepa after MPs accumulation in the cytoplasmic and vacuolar parts of A. cepa.
Considering the toxicity of MPs in terrestrial plants, the present study evaluated the Ps-MPs induced effect on root length and cell division in A. cepa. Our study suggests that MPs, in general, have a negative impact on the root development and cell division of plants, and the severity of the impact can be influenced by the different sizes of MPs present in the plant's vicinity. This work will lay down a foundation for using the A. cepa assay as risk assessment tool for detecting Ps-MP-induced cyto-and nuclear toxicity in higher plants. To the authors' knowledge, this is the first report that included the effects of the considerable size ranges of Ps-MPs on the cell division of A. cepa root tip cells.

Model Plant
The A. cepa bulbs were purchased from the local vegetable market of Kaifeng city, Henan Province, China. Ps-MPs (80,100,200,500,1000,2000, 4000, and 8000 nm; code number: 6-1-0005 to 6-1-0800) and analytical grade chemicals, such as orcein stain, working acids, and glassware, were purchased from Tianjin BaseLine ChromTech Research Centre, located in Tianjin city, China.

Ps-MPs Working Concentrations
Eight different sizes of Ps-MPs were selected for the present study. Two different working concentrations (100 and 400 mg/L) were chosen, based on the earlier studies [10,52]. Concentrations were adopted based on research from study of Maity et al. [52], where 100 and 400 mg/L concentrations of Ps-MPs showed significant cyto-toxic effects on the A. cepa root tip cells. The used concentrations may not represent the Ps-MPs contamination levels, in general, but possibly indicate the realistic concentrations in polluted areas. Ps-MPs working solutions were prepared from stock (procured as 250 mg, 10 mL) using Milli-Q water as a diluent. Prior to use, the solutions were properly homogenized by ultra-sonication for 30 min at 50 MHz.
Fresh, uniform, and equal-sized Allium bulbs with an average weight between 3.5 to 4 g and diameter 2 cm were selected, and dry roots were carefully removed with forceps, leaving behind the initial roots for fresh root growth. Experiment was carried out in triplicates (total of three denuded Allium bulbs); bulbs were incubated in MPs solutions, along with a negative control (Milli-Q water), in incubator for 72 h at 25 • C, with 50-60% humidity level. Time of incubation was selected based on many earlier studies, which proved the efficacy of A. cepa assay as one of the best short-term tests employed in environmental monitoring, providing satisfactory results within 72 h of treatment [49,[54][55][56].

Experimentation
After incubation of Allium bulbs in Ps-MP solution, root length of each bulb was measured by using a hand ruler. The length of all roots in a single bulb was noted, and average root length was calculated. Cyto-genotoxic study was carried out using A. cepa root tip chromosomal aberration assay. For cyto-genotoxicity assessment, the collected roots were properly washed under tap water, followed by distilled water, and then fixed in Carnoy's fluid for 12 h at 4 • C [56]. Subsequently, the fixed roots were transferred to 70% alcohol and stored in refrigerator at 4 • C temperature until use.

Microscopic Analysis
For the study of chromosomal abnormalities (CAs) and nuclear abnormalities (NAs), microscopic slides were prepared following the protocol of Sharma and Sharma [57]. Briefly, fixed root tips were dipped in 45% acetic acid for 5 min and acid hydrolyzed in watch glass containing 1 N HCl for 10 min using sprit lamp. Further, hydrolyzed roots were stained in 2% aceto-orcein solution for 30 min. The stained root tips were then placed in watch glass containing 2-3 drops of 45% acetic acid for few seconds to remove excess stain. After this, root tips were placed on clean glass slide, and the pointed root tip area was cut using a sharp blade; then, they were carefully covered with coverslip and squashed gently. Nine slides were scored for each MPs size, and a minimum of 1000 cells were scored per slide (10,000 cells/MP size) under a light microscope at 1000× magnification (Olympus CX31).
Mitotic index

Statistical Analysis
Results have been presented as mean ± standard error (S.E.) of nine independent readings. Data were analyzed for the statistical significance between the mean difference of values and control group by one-way ANOVA and Tukey's post-hoc test using SPSS software (ver. 20).

Results
The effect of Ps-MPs on A. cepa showed no significant decrease in root length at p ≤ 0.05, with respect to the negative control ( Figure 1).

Statistical Analysis
Results have been presented as mean ± standard error (S.E.) of nine independent readings. Data were analyzed for the statistical significance between the mean difference of values and control group by one-way ANOVA and Tukey's post-hoc test using SPSS software (ver.20).

Results
The effect of Ps-MPs on A. cepa showed no significant decrease in root length at p ≤ 0.05, with respect to the negative control ( Figure 1). No significant differences were observed in root length of A. cepa bulbs, but larger sized MPs showed the highest reduction in root length, as compared to the negative control, which could depict the toxic nature of large-sized MPs. At 100 mg/L concentration, as compared to the control (1.101 cm), a reduction in root length was observed in 4000 nm size Ps-MP (0.49 cm), while at 400 mg/L, the concentration reduction in the root length was found in 8000 nm-size Ps-MP (0.541 cm). No significant differences were observed in root length of A. cepa bulbs, but larger sized MPs showed the highest reduction in root length, as compared to the negative control, which could depict the toxic nature of large-sized MPs. At 100 mg/L concentration, as compared to the control (1.101 cm), a reduction in root length was observed in 4000 nm size Ps-MP (0.49 cm), while at 400 mg/L, the concentration reduction in the root length was found in 8000 nm-size Ps-MP (0.541 cm).
The cyto-genotoxic effect of Ps-MPs in the root tip cells of A. cepa was assessed by studying indices such as the mitotic, chromosomal, and nuclear abnormality indices. We observed a statistically significant decline in the mitotic index, in a size-dependent manner, for the tested Ps-MPs. The 100 nm-sized Ps-MPs showed a significant decrease in mitotic index (12.06 ± 0.284%) in the root tip cells at 100 mg/L concentration, as compared to the negative control (25.05 ± 0.917%) at p ≤ 0.05 after 72 h incubation ( Figure 2 and Table 1).
In the present study, phase indices were also calculated, as compared to the control, with decreases in the anaphase index (ARI) and telophase index (TLI); increases in the prophase index (PRI) and metaphase index (MTI) were observed in the root tip cells when treated with different sized Ps-MPs (Table 1).
The cyto-genotoxic effect of Ps-MPs in the root tip cells of A. cepa was assessed by studying indices such as the mitotic, chromosomal, and nuclear abnormality indices. We observed a statistically significant decline in the mitotic index, in a size-dependent manner, for the tested Ps-MPs. The 100 nm-sized Ps-MPs showed a significant decrease in mitotic index (12.06 ± 0.284%) in the root tip cells at 100 mg/L concentration, as compared to the negative control (25.05 ± 0.917%) at p ≤ 0.05 after 72 h incubation ( Figure  2 and Table 1).  In the present study, phase indices were also calculated, as compared to the control, with decreases in the anaphase index (ARI) and telophase index (TLI); increases in the  To record the percentage of cyto-toxicity of the Ps-MPs in plants, the A. cepa root chromosomal aberration assay was used. The induction of diverse types of chromosomal aberrations (CAs) was reported in all the Ps-MP sizes, while no aberrations were detected in the negative control (Table 2).
It was observed that the roots, when treated with a 100 mg/L concentration of 80 nm  (Table 2). Some representative pictures of normal mitotic phases and chromosomal aberrations induced in A. cepa root tip cells, following the treatment of different sized Ps-MPs were shown in Figure 3.    The Ps-MPs induced cyto-genotoxicity was observed by calculating the chromosomal abnormality index (CAI). In our study, CAI showed a statistically significant (at p ≤ 0.05) size-dependent decrease, where 200 nm-sized Ps-MP showed the highest CAI (31.65%), and the least was observed in 4000 nm MP (6.759%)-treated Allium root tip cells after 72 h of incubation (Figure 4).

Conc. (mg/L) DO (%) BG (%) BK (%) RC (%) CAI (%) * MN (%) NB (%) NAI (%)
We also detected two types of nuclear abnormalities (NAs), namely micronuclei (MN) and nuclear bud (NB), in our test samples, whereas no NAs were detected in the negative control. In the present study, variations in the occurrence of MN and NB in the tested sizes and concentrations of MPs were found to be statistically significant, as compared to the negative control ( Figure 5 and Table 2).
The maximum percentage of MN (2.43%) was observed in root tip cells when treated with Ps-MP of size 2000 nm at 400 mg/L, while NB (4.93%) recorded the highest percentage for 500 nm at 100 mg/L concentration. NBs were observed in all sizes of MPs, except for 8000 nm at 100 mg/L and 4000 nm at 400 mg/L after 72 h incubation, indicating the prominent cellular toxicity of Ps-MPs on the root tip cells of A. cepa. Overall, 100 mg/L concentration of the studied Ps-MPs showed high cyto-toxic effect, while 400 mg/L concentration induced nuclear abnormalities in the Allium root tip cells.   The Ps-MPs induced cyto-genotoxicity was observed by calculating the chromosomal abnormality index (CAI). In our study, CAI showed a statistically significant (at p ≤ 0.05) size-dependent decrease, where 200 nm-sized Ps-MP showed the highest CAI (31.65%), and the least was observed in 4000 nm MP (6.759%)-treated Allium root tip cells after 72 h of incubation (Figure 4). We also detected two types of nuclear abnormalities (NAs), namely micronuclei (MN) and nuclear bud (NB), in our test samples, whereas no NAs were detected in the negative control. In the present study, variations in the occurrence of MN and NB in the tested sizes and concentrations of MPs were found to be statistically significant, as compared to the negative control ( Figure 5 and Table 2).

Discussion
The ubiquitous nature of MPs in the environment endangers the terrestrial ecosystem to a great extent. MPs occur in soil in different forms; their bioavailability to plants and soil organisms increases with size and depends on soil characteristics such as particle size and density, abundance/co-occurrence, chemical characteristics, etc. [60,61]. The present study planned to measure the effects of different sizes and concentrations of Ps-MPs on root length and plant root tip cell chromosomes. The insignificant reduction in A. cepa root growth was observed when treated with large-sized MPs; this might be due to the direct contact of larger-sized MPs with the root pores, which blocked and hindered the root apical meristem activity. It is known that the larger-sized Ps-MP particles cannot easily enter into the plant cell and, therefore, are adsorbed to the root surfaces, thereby inhibiting the uptake and absorption of water, as well as the essential nutrients required for growth and development in plants [62]. Bosker et al. [63] found that MPs of 4.8 µm size can accumulate in the pores of seed capsule of Lepidium sativum (cress) and significantly reduce the germination rate and root growth after 8 and 24 h exposure, respectively. Researchers have reported on the accumulation of different size MPs on the root surfaces of alfalfa and rice [64], Vicia faba [43], A. cepa [52], and wheat [44]. The particle size of MP acts as an important factor in determining their interaction with living tissues and cells; hence, their accumulation and distribution [65].
Increase in the cyto-genotoxicity of any toxicant can be recognized by the decline in the mitotic index of the cells [66]. In the present study, the significant size-and concentration-dependent decrease in the mitotic index was observed, with respect to the control. Gopinath et al. [1], in their study, attributed the decrease in the MI of A. cepa root cells to the potential of MPs in DNA synthesis inhibition, abduction of mitotic phases, and slow cell progression. A similar cytotoxic effect of 100 nm-sized Ps-MPs at 100 mg/L concentration was reported earlier by Maity et al. [53] and Jiang et al. [43]. Many earlier studies have reported on the significant toxicity of different sized MPs on the normal mitotic cell cycle of plants [67,68].
MPs were found to result in mito-depressive effects in plants by blocking the G2 phase of the normal mitotic cell cycle. This can further lead to the inhibition of regulators and DNA replication impairment, which can cause disturbances in the prophase index [52,69,70]. In the present study, phase indices were calculated to investigate the inhibition of mitotic cell division and increases in the prophase index (PRI) and metaphase index (MTI), while decreases in the anaphase (ARI) and telophase index (TLI) of A. cepa were observed when treated with different sizes and concentrations of Ps-MPs. The G2 phase inhibition of the cell cycles of the root tip cells can disturb the normal prophase index [52], but this was not significant in our study.
CAI results indicate the high cyto-genotoxic potential of small-and large-sized Ps-MPs. The cyto-genotoxicity can be attributed to a hindrance in DNA and protein synthesis. Gopinath et al. [1] observed that MPs can interrupt the nucleic acid metabolism, thus affecting protein and DNA synthesis, resulting in a range of chromosomal and nuclear abnormalities. Hindrances in nucleic acid and protein synthesis can alter the cell/nucleus volume, producing giant nucleus cells [71]. Many reports have indicated significant genotoxic effects of MPs on wheat [43], ryegrass [72], V. faba [44], spring onion [73], and A. cepa [52]. Maity et al. [52] reported significant fluctuations in MI and the induction of various chromosomal aberrations in A. cepa root tip cells when treated with different concentrations (25,50,100,200, and 400 mg/L) of 100 nm-sized Ps-MPs. The underlying mechanism behind the genotoxic effects of MPs is largely unknown, but it might be due to the accumulation of MPs in the root tissues, thereby blocking cell wall pores and disrupting the transport of essential nutrients [44].
Irrespective of the size and concentration used in the present study, we observed the highest percentages of CC, CM, DLA, VG, BG, BK, etc. Chromosomal aberrations lead to destabilization of the genome, resulting in various genotoxic and mutagenic effects, including disturbances or malformation in spindle fibers, chromosome movement failure, breakage and fusion of chromosomes/chromatids, chromosomal pole shifting due to microtubule depolymerization, and alteration in the activation of the enzymes necessary for replication [49,52,74,75].The induction of c-mitosis can be due to hindrance in disulfide bonds formation, which effects the tubulin organization required for spindle microtubules [51,52]. Subsequently, laggard chromosomes can arise due to spindle disturbances or failure in anaphasic and pro-metaphasic movement [76,77], whereas clumped or sticky chromosomes can be formed due to the faulty functioning of specific non-histonic proteins, which are important for chromatid segregation, as well as chromosomal organization and separation [78,79]. Vagrants may arise due to the action of external agent's c-mitotic force, resulting in the movement of chromosome away from the anaphasic pole or equatorial plate [80]. Chromosomal breaks are formed due to the unfinished or mis-repaired DNA molecules required for linear continuity of the chromosome's structure [81], while the anaphasic bridge results from chromosomal breakage, stickiness, fusion, para-centric inversion, and altered functioning of the replication enzymes [82]. The ring chromosome can arise due to breaks in the chromosome arms and fusion of the proximal broken ends, which leads t oa loss of the distal material or rings; they can be formed by telomere dysfunction, triggering the fusion of reactive chromosome ends [52,83,84].
Ps-MPs also induced the formation of NB and MN in the A. cepa root tip cells, which mainly results from acentric or lagging chromatid fragments or a whole chromosome that is not present in the daughter nuclei after mitosis. These lagging fragments are not able to correctly attach to the spindle during chromosomes segregation in anaphase stage of mitosis; thus, they are unable to pass to the daughter nuclei [85]. These microstructures are enclosed by a nuclear membrane, and they are structurally similar but smaller in size, as compared to the main nuclei [86]. MN formation indicates the genotoxicity of MPs, due to DNA damage, hence the chromosomal instability. The formation of NB in A. cepa root tip cells might be due to the direct breaking action of the Ps-MPs on the genetic material or inhibition of spindle fibers formation [50,87]. The action of MPs on root tip cells may lead to cytokinesis inhibition, resulting in the formation of MN cells [88]. From the present study, it comes into sight that the impacts of different sizes of micro-plastics are varied and can be dependent on the type of plant, type and size of the plastics, concentration, and different experimental conditions.

Conclusions
The present study indicated cyto-genotoxic potential of Ps-MP particles in A. cepa root tip cells by induction of high percentage of chromosomal and nuclear aberrations. The study also suggests the suitability of A. cepa root chromosomal aberration as a reliable cyto-genotoxicity test, as A. cepa showed common basic genetic constitution throughout the eukaryotic organisms; the results can be helpful in the extrapolation of data in other higher animals and mammals used as test models. Our study is the first report based on the toxic effects of considerable size ranges of Ps-MPs on A. cepa root tip cells. Both large-and small-sized MPs showed the morpho-toxicity, cyto-genotoxicity, and induction of nuclear abnormalities in A. cepa root tip cells by reduction in the root growth, mitotic index, and induction of high percentage of chromosomal and nuclear abnormalities. The occurrence of clastogenic aberrations in the present study suggests the chromosomal breakage and spindle disrupting potential of Ps-MPs in terrestrial plants, irrespective of their size and concentration. Based on the results of the present study, environmental risk assessment and proper management of plastic debris of any type, size, and concentration are required before disposal, in order to avoid detrimental effects on the target, as well as non-target, living organisms. In the future, more research needs to be carried out, in order to address the knowledge gaps regarding the co-toxicity potential of MPs and other pollutants, as well as the mode of toxicity caused by MPs on plants, which will be of great importance.  Acknowledgments: Authors would like to thank the College of Geography and Environment Science (Henan University, China) for providing laboratory facilities for performing the experiments. Mandeep Kaur would also like to thank Tengfei Lee for helping tosetup the experiments. All authors would also like to thank Parvinder Pal Singh for insights into data analysis and Sapinder Bali for reviewing and editing the manuscript.