Evaluation of the mitodepressive effect, chromosomal aberrations and nuclear abnormalities induced by urea fertilization in the meristematic tissues of Allium cepa L.

ABSTRACT

Urea is one of the most commonly used nitrogen fertilizers in Romania. The cytogenetic objectives of this experience were the evaluation of the effects of cytotoxicity and genotoxicity induced by the action of urea applied in different concentrations (100, 500, 1000, 5000, 10,000 and 20,000 ppm) for 72 h on the meristematic tissues of Allium cepa L., a species very commonly used in cytotoxicity tests. The statistical analysis of the obtained results indicates that with the increase in urea concentration, mitotic activity is inhibited, while the chromosomal aberration rate in the cells in mitosis as well as the frequency of nuclear abnormalities in the interphase cells increases. From this point of view, the main chromosomal aberrations identified were stickiness, laggards and C-mitosis, while nuclear abnormalities were the appearance of a large number of binuclear and multinucleated cells, some with ghost nuclei. In addition, at concentrations of over 1000 ppm and especially at 10,000 and 20,000 ppm urea the presence of cells with one or more micronuclei (even with nine micronuclei) was signalled, indicating a strong clastogenic potential of urea. The results suggest caution in the use of urea, by applying small concentrations, within the environmental limits, for the protection of the environment, but also of humans and animals. The concentrations we recommend to reduce the risk of pollution are between 100 and 500 ppm urea. However, further investigation is needed to confirm these findings.

KEYWORDS:

• Allium cepa L.
• cytotoxicity
• genotoxicity
• urea

Introduction

Considering the importance of global food security, the primary role of modern agriculture is to increase productivity. To enable this, crop plants need different nutrients, of which nitrogen plays a leading role.

Significant amounts of nutrients are extracted by plants from the soil, so the increase in agricultural production relies heavily on the type of fertilizer used to supplement the essential nutrients. The addition of nutrients in any form is essential for maintaining soil quality (Edmeades 2003).

The most important and most used are nitrogen-based fertilizers, but besides the increase in plant productivity, there is a risk of soil and groundwater pollution with nitrates. After its release into the environment, nitrogen may reduce the diversity of terrestrial vegetation and may increase susceptibility to some stress factors such as pests and drought (Gilliam et al. 2016; Van Dobben and De Vries 2017). High urea concentration in the water column may hamper the growth of aquatic plants (Huang et al. 2017). Also, urea fertilization can reduce plant growth in clay soils of subtropical areas (Faustino et al. 2015). The pollutant effect is mainly determined by the excessive quantities used repeatedly over the years. In addition to chemical fertilizers, another group of pollutants is fungicides, and their excessive application can cause some environmental damage (Bonea and Bonciu 2017).

Urea is one of the most popular nitrogen fertilizers in Romania, being the most concentrated solid fertilizer with nitrogen (containing 46% nitrogen active substance) in amide form (NH2). In physico-chemical and microbiological processes, the reserve of nitrogen contained is converted into ammoniacal (NH4 +) and nitric (NO3-) nitrogen.

Nitrogen fertilizers are directly absorbed by plants or transformed into various other forms by the oxidation process, but when the nitrate is not absorbed, it flows into the soil together with water, where the excess nitrogen increases the risk of environmental pollution (Tamme et al. 2009; Sharifi et al. 2011). The human consumption of water or crops contaminated with high nitrate concentration is leading to health injuries (Ikemoto et al. 2002). Through excessive application of fertilizers with nitrogen, high concentrations of nitrates can accumulate in the edible parts of plants (Liu et al. 2014). Generally green leafy vegetables contain the highest levels of nitrates (Prasad and Chetty 2008). The accumulation of nitrates depends on the type of nitrogen fertilizer used and the soil properties (Gunes et al. 1995; Pavlou et al. 2007).

Some plants are used in tests for cytotoxicity and genotoxicity induced by various chemicals in the environment. Chromosomal aberrations induced by chemicals in plants are identical to those induced in animals (Grant 1978).

One of the most commonly used plants for environmental pollution assessment tests is Allium cepa L. (onion). Allium cepa and Allium sativum have long been used as test plants for assessing the cytotoxic effects of various pollutants on the environment (e.g. Levan 1938; Levan and Ostergren 1943; Fiskesjo 1969; Chaurasia 1979). The Allium cepa test is also used for screening and monitoring some environmental chemicals with carcinogenic potential (Ma 1999; Cabaravdic 2010; Sharma and Vig 2012).

The optimal stage of mitosis in which chromosomal aberrations can be observed using specific staining methods is metaphase, but for a very precise identification, the chromosomes banding method is used (Savage 1977). In situ fluorescence hybridization (FISH) is a molecular cytogenetic technique used to significantly improve the identification of chromosomal anomalies (Tucker et al. 1995; Savage and Tucker 1996).

The genotoxic and cytotoxic effects of chemical fertilizers on plants have been demonstrated by many authors (e.g. Arora et al. 2014; Koca 2008; Khaldi et al. 2012). Considering that urea is widely used in Romania, we considered it appropriate to evaluate its cytotoxic and genotoxic potential on the meristematic tissues of Allium cepa L., to assess the eventual damage brought to the environment by the farmers who excessively use this fertilizer.

Materials and methods

Plant material and extract preparation

The onion bulbs (Allium cepa L., 2n = 16, Family Amaryllidaceae) of medium size (3–4 cm in diameter) and medium weight (80–100 g) were purchased from the central square of Craiova, Romania. Urea was purchased from Azomures S.A., from Târgu Mures, Romania. Healthy bulbs, free from signs of disease or pests, were removed by removing the first row of outer leaves and leaving the germ intact. Then they were placed in glass beakers filled with six concentrations of urea solution, each in three repetitions, along with an untreated control which was placed in plain distilled water.

The concentrations of urea were: 100, 500, 1000, 5000, 10,000 and 20,000 ppm (parts per million). These dilutions were made with distilled water in the Agrochemistry Laboratory of the Craiova Faculty of Agronomy.

The onion bulbs were immersed in urea dilutions for 72 h (three days) at 22 ± 2°C. The medium was supplemented from time to time so that the germinal disc of the onion bulbs to be permanently immersed in liquid.

Microscopic preparations

After 72 h of treatment, the meristematic onion roots were harvested at a length of 8–10 mm and transferred to fixative solution (30 ml ethyl alcohol and 10 ml acetic acid) for 24 h. The fixation step is intended to fix the cellular constituents, but at the same time the cells retain their normal structure. The hydrolysis step (carried out with the aim of facilitating the staining process) was performed in 1 N hydrochloric acid solution (hydrochloric acid [82.5 cc] + distilled water [1000 ml]) at 60°C for 6 min. After hydrolysis, the biological material was washed rapidly under tap water to remove traces of HCl.

The staining of the meristematic tissues was carried out at room temperature with a decolourized basic fuchsine solution (Schiff’s reagent), prepared according to the method proposed by Darlington and LaCour (1963). On top of the onion root were added 4 ml Schiff reagent, and after 30 min the meristematic peaks were coloured in intense red-violet colour. The meristematic region where the most mitotic cells are present is about 1 mm in length, as reported by Jensen and Kavaljian (1958). Therefore, for the microscopic study, the coloured tips of onion roots were detached about 1 mm in length.

Five temporary slides were made for each of the variants of the experiment. The slides were coded and the observations were carried out with an optic binocular microscope MBL 2000 (Krüss Optronic, Hamburg, Germany).

The most representative aberrant cells were photographed with a Canon camera model Prima Super 105X (Canon, Melville, NY, USA).

Statistical analyses

The obtained data were statistically analysed with the F-test of analysis of variance (ANOVA) and multivariate analysis and differences between treatment means were compared using the Least Significant Difference [p<0.05 - p<0.001] (LSD)-test at probability level of 0.05% (Botu and Botu 1997). As for cytological analysis, the mitotic index and the percentage of aberrant cells was expressed in terms of the number of cells per 500 scored cells resulting from five roots for each variant.

The mitotic index (MI) was calculated using the following formula:

MI (%) = total number of cells in division/total number of analysed cells × 100.

The percentage of aberrant cells (both chromosomal aberrations and nuclear abnormalities) or relative aberrant cell rate (RAR) was calculated as follows:

RAR (%) = total number of cells with abnormalities/total number of analysed cells × 100.

All the results were expressed as the mean of three replicates per treatment ± standard error (SE).

Results

Based on ANOVA results (Table 1), there are statistically significant differences between mitotic index and total aberrant cell count (p < 0.05) between the different urea treatment variants.

Table 1. Variance analysis (ANOVA) of studied traits of Allium cepa L.

Traits	SS	df	MS	F
Mitotic index (MI%)	4336.56	6	722.76	82.08*
Total aberrant cells	467,746.3	6	77,957.71	809.65*

Abbreviations: SS, sum of squares; df, degrees of freedom; MS, mean sum of squares; F, F statistic test.

*Significant at p < 0.05.

From the point of view of the mitotic index and the frequency of the mitotic phases, the cytotoxic effects of urea on the meristematic tissues of Allium cepa L. are shown in Table 2. Thus, it was observed that all treatment variants (V1, V2, V3, V4, V5 and V6) resulted in a significant reduction (p < 0.05) of the mitotic index relative to the control. The highest MI was recorded in the control (44.2 ± 2.43%), after which the mitodepressive effect of urea was quantified by decreasing values from 31.2 ± 1.27% (V1/100 ppm urea) to only 3.4 ± 1.16%, for V6 variant, treated with 20,000 ppm urea.

Table 2. Effect of different concentrations of urea on cytological parameters in the meristematic tissues of Allium cepa L.

Variant/concentration	Analysed cells	MI ± SE %	I	Mit	P	M	A	T
Control	500	44.2 ± 2.43	279	221	48	39	53	81
V1/100 ppm	500	31.2 ± 1.27*	344	156	28	24	30	74
V2/500 ppm	500	26.2 ± 1.73*	369	131	36	12	25	61
V3/1000 ppm	500	12.4 ± 2.54*	438	62	20	6	8	28
V4/5000 ppm	500	8.2 ± 0.11*	459	41	13	4	6	18
V5/10,000 ppm	500	4.6 ± 1.5*	477	23	7	3	2	11
V6/20,000 ppm	500	3.4 ± 1.16*	483	17	5	1	3	8
LSD 5%		2,42

Abbreviations: MI%, mitotic index; I, number of cells in interphase; Mit, number of cells in mitosis; P, number of cells in prophase; M, number of cells in metaphase; A, number of cells in anaphase; T, number of cells in telophase; SE, standard error.

*Significant at p < 0.05.

The largest number of cells in mitosis belonged to the control (221), the difference of 279 cells being interphase. As the concentration of urea increased, in all treatment variants the number of cells in mitosis decreased from 156 cells (V1) to 17 cells (V6), while the number of cells in the interphase increased from 344 cells (V1) to 483 cells (V6), thus confirming the strong mitodepressive effect of urea in the meristematic tissues of Allium cepa L.

The most frequent stage of mitosis was telophase, followed by prophase, anaphase, and metaphase. However, as the urea concentration increased, the number of cells in telophase decreased from 81 (control variant) to eight cells (V6/20,000 ppm), while in metaphase, the number of cells ranged from 39 (control) to just one metaphase cell identified in V6.

Regarding the genotoxicity effect, as evidenced by the total number of aberrant cells, it was observed that it increased significantly (p < 0.05) with increasing urea concentration (Table 3). Thus, if in the control variant only 4 ± 0.66% of abnormal cells were recorded, in the other variants (V1–V6) the genotoxic effect had significantly higher values compared to the control, from 99 ± 5.78% aberrant cells (V1/100 ppm) to 451 ± 7.51% (V6/20,000 ppm). Percentage, relative aberrant cell rate (RAR) was 0.8% for the control variant, while directly proportional to urea concentration, RAR values increased progressively from 19.8% (V1) to 90.2% (V6), as shown in Figure 1.

Table 3. Total number of aberrant cells (cells with nuclear anomalies and cells with chromosomal aberrations) identified in meristematic tissues of Allium cepa L. treated with different urea concentrations.

Variant/concentration	Total aberrant cells (X ± SE)	CB	CM	MN	S	L	C-M
Control	4 ± 0.66	3	-	-	-	-	1
V1/100 ppm	99 ± 5.78*	24	15	-	23	28	9
V2/500 ppm	118 ± 5.78*	31	18	-	26	29	14
V3/1000 ppm	139 ± 6.35*	38	21	-	28	36	16
V4/5000 ppm	200 ± 6.93*	46	32	10	37	47	28
V5/10,000 ppm	384 ± 3.47*	81	68	51	56	69	59
V6/20,000 ppm	451 ± 7.51*	109	87	60	64	70	61
LSD 5%	17.22

Abbreviations: CB, binucleated cells; CM, multinucleated cells; MN, cells with micronuclei; S, sticky chromosomes; L, laggards chromosomes; C-M, C-mitosis; SE, standard error.

*Significant at p < 0.05.

Figure 1. The relative aberrant cell rate (RAR%) in meristematic tissues of Allium cepa L. treated with different urea concentrations (ppm).

In terms of chromosomal aberrations, chromosomes of the laggard type (Figure 2(a)), sticky (Figure 2(b, c)) and C-mitosis (Figure 2(d)) were the most frequent. On the other hand, the most common nuclear abnormalities were binucleated cells (Figure 3(a)) and multinucleated cells, some of which identified with ghost nuclei (Figure 3(b)), especially in the 10,000 ppm treated urea (V5) and 20,000 ppm (V6), respectively. Other nuclear abnormalities recorded were cells with one or even nine micronuclei (Figure 3(c, d)).

Figure 2. Some chromosomal aberrations induced by exposure to different concentrations of urea in the meristematic tissues of Allium cepa L.: (a) laggard chromosome; (b, c) sticky chromosomes; (d) C-mitosis.

Figure 3. Some interphase nuclear anomalies induced by exposure to different concentration of urea in meristematic tissues of Allium cepa: (a) binucleated cells; (b) multinucleated cells with ghost nuclei; (c) cell with three micronuclei; (d) cell with nine micronuclei.

Discussion

Urea can be applied in the field both in granulated form but especially in solution, along with other treatments. The amount of applied urea varies with crop type, climate and soil, between 100 and 400 kg ha^–1. When applying urea in a solubilized form, concentration in the treatment solution varies depending on the crop plant, vegetation period and temperature (Table 4).

Table 4. Usual concentrations of urea in the solubilized form to the main crop plants.

Culture plant	Application conditions	Concentration (%)
Wheat and barley	Stage of the stalks formation, temperature 15°C	8
	Stage of the stalks formation, temperature 20°C	6
Maize	During the vegetation period, at the stage of 6–8 leaves	2,5
Sunflower	Up to the stage of developing floral button	2,5
Fruit trees	Before the first winter treatment	5
Vegetables	During the vegetation period	2,5

Urea contains amide nitrogen, a form of organic nitrogen which, in a first phase, decomposes into ammoniacal nitrogen and then into nitric nitrogen. In this situation, urea nitrogen will be more quickly taken up by plants, especially if the brightness is lower and especially in the younger stages of culture. So a higher nitrogen concentration and a stronger absorption of plants (associated with nebulous conditions) increase the risk of plant poisoning, especially if excessive doses of urea are used. Considering all these aspects, we can assume that urea can influence the cell cycle mechanism due to its results because it has a very high cytotoxic and genotoxic potential even from concentrations above 500 ppm.

Mitotic index value can show the cytotoxicity of a chemical agent. Variability of this index in our experience indicates a high level of cytotoxicity, manifested by a significant mitodepressive effect. Similar findings came from several researchers who showed that the variation in the mitotic index of Allium cepa L. treated with various chemicals suggests cytotoxicity (Marcano et al. 2004; Celik et al. 2005; Rao et al. 2005; Thaís et al. 2007).

The mitogenic mutagenic effect as well as the clastogenic effect of urea on the meristematic tissues of Allium cepa L. was also demonstrated by Verma et al. (2016) by cell division anomalies and interfacial nuclear abnormalities occurring in onset meristematic cells after the urea was added to the soil at a rate of 200 mg N kg^–1 soil. The authors also noted that the percentage of nuclear abnormalities was higher for urea compared to ammonium nitrate, the second fertilizer studied by the respective authors. So urea can affect the cell cycle even more than other chemical fertilizers with nitrogen. In fact, as other authors suggest (Chaurasia 1979; Chaurasia and Sinha 1987), it is highly likely that residues or metabolites of urea cause some damage to chromosomes due to the fact that nitrogen is a major component of them (protein and DNA).

The strong clastogenic effect of urea was also reported in animal cells. Thus, the maximum rate of chromosome aberrations and changes in sister chromatid in Chinese hamster cells can cover more than two cell cycles (Thust et al. 1980). In addition, soil fertilization with different doses of urea for 60 days had a negative effect on the activity of the earthworm population (Eisenia fetida), from the point of view of populations, biomass, number of cocoons, juveniles, etc. (Rai et al. 2014).

Many researchers consider the appearance of the micronucleus (MN) as a clear sign of cytotoxicity in both animal (Faccioni et al. 2003; İz et al. 2010) and plant cells (Kontek et al. 2007; Leme and Marin-Morales 2009). At concentrations of 5000, 10,000 and 20,000 ppm, urea showed both an aneugenic effect and a clastogenic effect in the meristematic tissues of Allium cepa L., the occurrence of large and small micronuclei being obvious. As some authors mention (Leme and Marin-Morales 2009), the appearance of large micronuclei indicates the aneugenic effect due to chromosome loss, while the appearance of small micro-nuclei indicates a clastogenic effect due to chromosomal ruptures.

However, it is clear that urea can act as a true mutagen in Allium cepa L. The formation of meristematic cells can be considered as a biochemical process rather than as a biophysical process, as Haldane has introduced it since 1954; from the point of view of the mitosis phase biochemistry, it has been studied extensively, and this much orchestrated process has been found to involve hundreds, if not thousands, of cellular proteins (Mitchison and Salmon 2001).

The occurrence of chromosomal aberrations indicates the harmful effect of a chemical agent on plant cells (Nag et al. 2013). In our experience, sticky chromosomes appeared particularly in anaphase but also in metaphase, and this phenomenon is probably based on the partial dissociation of nucleoproteins. Similarly, Asita and Mokhobo (2013) suggested that the induction of sticky chromosomes in Allium cepa under the influence of pesticides indicates abnormal DNA condensation, abnormal chromosomal wrapping, and inactivation of the axes, and all these anomalies in cell division can have adverse effects on both the environment and human health.

It has been shown that, in general, chemicals that induce genotoxicity in meristematic Allium cepa L. cells may produce similar effects in human lymphocytes (Grover et al. 1990). Also, some studies performed on mice suggest that the cardiovascular system may be a target for urea (Abaurre et al. 1992; Massy et al. 2005). On the other hand, urea can seriously affect fish populations living in waters polluted with the residues of this fertilizer, producing histopathological changes (Srivastava and Srivastava 1979). To reduce environmental pollution, efficient use of agricultural inputs, including urea fertilizer, is mandatory. Imbalanced use of nitrogen fertilizer can reduce crop yields and can even adversely affect soil biological properties. As Bhatt et al. (2016) suggests, application of integrated fertilizer with NPK and farmyard manure sustained higher yields and soil biological properties compared to 100% N application under a rice–wheat cropping system in Mollisols. For assessing the sustainability of agricultural management there are useful tools, e.g. agro-ecosystem models (DNDC – DeNitrification and DeComposition) (Dutta et al. 2017).

In conclusion, at concentrations over 500 ppm, urea induced a strong cytotoxic effect in Allium cepa, inhibited mitotic activity and caused the appearance of several types of chromosomal aberrations and nuclear abnormalities. The occurrence of large numbers of cells with micronuclei and multinucleated cells with ghost nuclei can be considered as clear cytotoxicity signs. Therefore, we recommend applying urea at low concentrations (100–500 ppm) to reduce the risk of environmental pollution and damage to plant and animal health.

However, further investigation is needed to confirm these findings. Due to the global demographic explosion and consequently the increase in the need for food, modern agriculture will face a major challenge in the coming years: to provide food for the entire population, but at the same time not to cause serious damage to the environment, humans and animals.

Disclosure statement

No potential conflict of interest was reported by the authors.