Temporal Monitoring and Assessment of Inorganic Nitrogen Content of Soil due to Nitrogen Fertilizers and their Related Cytotoxic Effects

Soil is a natural pool of nutrients and its fertility is reflected by the quantity of different nutrients and their relative ratios. Most of the soils either do not have optimum nutrient contents or get deprived due to numerous reasons like plant uptake, volatilization, leaching, soil erosion etc. Hence, fertilizer supplementation at regular intervals is necessary to provide essential elements and nutrients in a readily available form for the optimum plant growth. Such supplementation works only when the fertilizers are of balanced composition and fertilization is done at right time and in right amount (Bindraban et al., 2015). Among various fertilizers, the global consumption of nitrogen (N) fertilizers is always high and about 70% of soil N for plants is provided by inorganic fertilizers. In the soil, inorganic N exist in two major forms, viz. ammonium nitrogen (NH₄⁺-N) and nitrate nitrogen (NO₃^--N). Therefore, N fertilizers in market are either ammonium based (ammonium sulphate, urea etc.), or nitrate based (calcium nitrate, potassium nitrate, etc.) while some of these are double salt based like ammonium nitrate which provides both the inorganic forms.

Plants show selective uptake of the different N-sources and consequent varied responses which also may be affected by the interaction between soil physico-chemical properties and the environmental factors. Also, this selective uptake of ions by the plants results into different effects on the soil environment especially rhizosphere. Based on the variation in chemical properties of these two inorganic N, their initial fate in soil is different (Li et al., 2012). As plants uptake NH₄⁺, protons (H⁺) are released which make the pH of rhizosphere acidic, whereas nitrate uptake shifts the pH towards alkalinity. In contrast to nitrate, plants cannot safely store elevated levels of ammonium in their cells which also has subsequent toxic effects (Esteban et al., 2016). Earlier reports certify this fact by suggesting that when NH₄+ is used as the chief N source, most of plant species develop toxicity symptoms (Kronzucker et al., 2001; Rogato et al., 2010). On the other hand, nitrate toxicity occurs in plants when higher quantities of nitrates are present in soil (Britto and Kronzucker, 2005; Okushima et al., 2011). There are only handfuls of reports that correlate the effect of external ammonium and nitrate contents to the toxic effects at cellular level (Qin et al., 2011; Arora et al., 2014; Verma et al., 2016).  But there are plenty of reports revealing that plant species grow well if NH₄⁺-N and NO₃^--N are supplied in an appropriate ratio (Kronzucker et al., 1999; Babourina et al., 2007; Bar-Yosef et al., 2009). In view of above cited literature, we have selected two chemically different N-fertilizers, i.e., urea (NH₄⁺-N source) and ammonium nitrate (51% NO₃^--N and 49% NH₄⁺-N) and assessed the temporal changes in inorganic N-contents in soil and their effects at cellular level in plants. Various workers have earlier taken up this aspect, but their analytical approach is different and majorly based on morphological data, growth analysis and yield (Yin et al., 2017; Fashaho et al., 2020; Kumari et al., 2021). To the best of our knowledge, there are very few reports suggesting the genotoxic and cytotoxic effects of fertilizers (Khaldi et al., 2012; Verma and Srivastava, 2017). Hence, considering it an issue of serious concern, the present study is in continuation with our previous works on N- fertilizers (Arora et al., 2014; Verma et al., 2016; Verma and Srivastava, 2018a).

Allium cepa root tip bioassay is used to determine the effects of two N-fertilizers at cellular level and any evident cytotoxic risks associated with it. Earlier, this assay system was used for environmental and toxicological monitoring (Fiskesjo, 1985; Verma and Srivastava, 2018a), but in the present times it has also found application as bioindicator of genotoxicity (Firbas and Amon, 2013). This system bears the merits of providing a rapid screening for the causative pollutants, toxicants, contaminants etc.

The experiment was conducted in the year 2016-2017 at In Vitro Culture and Plant Genetics Unit, Department of Botany, Lucknow University, Lucknow.
 
Soil sampling
 
Collection of soil for the experiment was done from University garden situated at 26°51'53" N latitude and 80°56'15" E longitude at a depth of 30 cm from an area that was not fertilized. One kg sieved soil was air-dried and placed in 6" pots. The physico-chemical properties of the soil used are given in Table 1.


 
Application of fertilizers
 
Soil in pots was treated with selected N-fertilizers, urea and ammonium nitrate. Three treatments as Control (T₁) which was without any additive, soil supplemented with urea at the rate of 200 mg N kg^-1 soil (T₂) and soil supplemented with ammonium nitrate at the rate of 200 mg N kg^-1 soil (T₃) were made. Three replicates, consisting of 3 pots each were taken per treatment i.e. 9 pots per treatment.
 
Estimation of inorganic N nutrients
 
Temporal monitoring of inorganic N (Ammonium-N (NH₄⁺-N) and nitrate-N (NO₃^--N) contents) was done on 1, 5, 10, 15, 20, 25 and 30 days after treatment (DAT). The ratio between NH₄+-N and NO₃^--N content was calculated as NH₄⁺-N per unit NO₃^--N.
 
Estimation of NH4+-N
 
Nessler’s method was used to analyse NH₄+-N in soil samples (Peech et al., 1947). Soil suspension was prepared by using 100 g of soil sample, taken in a conical flask and then 200 ml of acidified NaCl solution was added to it. The suspension was vigorously shaken for about 30 min. then filtered. The leachate obtained was taken and volume was made up to 100 ml using acidified NaCl. Further NH₄+-N was determined using Nessler’s method. The transmission (%) of the solution was read in a spectrophotometer at 410 nm.
 
Estimation of NO3--N
 
Phenoldisulphonic method (Bremner, 1965) was adopted to analyse the NO₃--N in soil samples. For soil suspension, 50 g of soil was taken and 250 ml of extraction solution (1 part of CuSO₄ and 5 parts of Ag₂SO₄) was added to it. The soil suspension was shaken for 10 min and then 0.4 g Ca (OH)2 was added. Further shaking of 5 min was done and then 1 g of MgCO₃ was added. Suspension was then filtered. Ten ml of clear filterate was taken and evaporated to dryness in evaporating dish. Three ml of phenol disulfonic acid was added and it was left for 10 min. Then 15 ml of cold water was added to it. Sodium hydroxide (12%) was added slowly to the solution until it became distinctly alkaline which was indicated by the development of yellow color. Finally, 3 ml more of sodium hydroxide solution was added to it. The transmission (%) of the solution was read in a spectrophotometer at 420 nm.
 
Cytotoxicity analysis
 
Healthy onion bulbs of medium size were properly cleaned. Their outer scales and brownish bottom plates were removed with a precaution of not damaging the ring of root primordia. The experiment was designed to run in three replicates with each replicate consisting of three pots and each pot had one onion bulb placed in the centre. The same soil was used for sowing fresh onion bulbs at 1, 5, 10, 15, 20, 25 and 30 DAT. Roots were harvested on day 3 of each sowing.

For cytological analysis, roots were fixed in Carnoy’s fluid (1 part glacial acetic acid + 3 part ethanol) for 24 hrs and then preserved in 70% ethanol. Fixation and slides preparations were done by following modified protocol of Sharma and Sharma (1980). The mitotic analysis was done on temporary slides. The dividing cells were observed under microscope. Approximately 500 cells from at least 5 random root meristems per replicate were screened to determine the mitotic index percentage (MI%).
   

              

 
Statistical analysis
 
The data obtained was evaluated statistically with the statistical software SPSS for Windows ver. 15.0, (SPSS Inc., Chicago, Ill., USA). The data was analysed by One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) and significance was measured at p≤0.05. The correlation amongst the parameters was assessed by Pearson Correlation Coefficient analysis (Zou et al., 2003).

The data was collected every 5 DAT till one month. Control (T₁) had very low amount of NH₄⁺-N and NO₃^--N as compared to other treatments (Fig 1a). Evaluation of the two treatments, T₂ and T₃, revealed that the overall inorganic N (NH₄⁺-N and NO₃^--N) content range was higher in T₃ treatment. Temporal variations in NH₄⁺-N and NO₃^--N contents in individual treatments, were significantly different.


 
Temporal monitoring of inorganic N content
 
In case of T₁, soil NH4+-N content followed a continuous decrease since 1 DAT (Fig 1a). Treatment T2 showed an increase in soil NH4+-N till 5 DAT and reached up to 80 mg kg^-1, then between 10 DAT to 30 DAT a decline from 74 mg kg^-1 to 18 mg kg^-1 was observed in the content (Fig 1b). In T₃, periodic fluctuations were observed in the amount of NH₄⁺-N i.e., no continuum in the trend was observed. There was a decrease from 162.2 mg kg^-1 on 1 DAT to 99.5 mg kg^-1 as on 10 DAT then again, increases till 30 DAT (Fig 1c). Regarding NO₃--N content, T₁ showed a continuous decrease till 30 DAT. Urea (T₂) showed a contrary result to NH₄⁺-N, from 1 DAT to 15 DAT, there was a drop and then elevation in NO₃^--N content. This increase in NO₃^--N could be due to nitrification process. In case of T₃, NO₃^--N content of the soil also followed similar trend as that of NH₄⁺-N, that is, it was observed to be 88 mg kg^-1 initially on 1 DAT and then a decline was obtained till 10 DAT. There was a continuous rise of nitrate amount from 15 DAT to 30 DAT. A correlation analysis showed that, NH₄⁺-N and NO₃^--N contents in various treatments were almost ideally correlated with each other, that is, in T₁ and T₃ an ideal positive correlation with r = 0.95 and 0.98 respectively, at p≤0.05, 0.01 and 0.001 was obtained where as in T₂ a negative correlation with r = -0.92 at p≤0.05 and 0.01 was obtained (Fig 2a-c).

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Relation of MI with inorganic N content
 
Cytological analysis of root meristems revealed that in T₁ and T₂, there was an increase in MI from 1 DAT to 5 DAT which then decreased thereafter till 30 DAT (Fig 1a and b). In case of T₃, from 1 DAT to 10 DAT, MI increased and then decreased till 30 DAT (Fig 1c). Maximum MI of 35% on 10 DAT was observed in T₃, whereas in T₂ treated roots maximum MI was 5.8% on 5 DAT which was even lower than T₁. In case of T₂, changes in NH4+-N content in soil was significantly correlated with MI, r = 0.76 at p≤0.05, where as in T3, NO₃^--N showed a significant negative correlation with MI, r = -0.74 at p≤0.05 and NH4+-N did not show any significant relation with MI.
 
Relation of MI with NH4+-N and NO3--N ratio
 
Data for ratio of NH₄+-N to one unit of NO₃--N was analysed in each treatment and varied trends were obtained, viz. in T₁ ratio increased till 10 DAT then decreased till 20 DAT and again increased till 30 DAT. In T₃, it initially increased till 10 DAT then kept on decreasing till 30 DAT. In urea (T₂), initially the ratio increased till 15 DAT and then reduced up till 30 DAT. In the treatments, ratio at which MI% was at its peak, considered to be the optimum ratio for that particular treatment. In T₁ on 5 DAT, 14.2% MI at ratio 12.52, in T₂ on 5 DAT, 5.8% MI at ratio 64.5 whereas in T₃, highest of all MI values that is 35% at ratio 6.22 was obtained on 10 DAT (Fig 3). These can be considered as optimum ratios because beyond these ratios lower values of MI were observed in all the treatments. No significant correlation between ratio and MI was observed in any of the treatments.

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Urea undergoes hydrolysis by the action of urease and releases NH₄⁺ and carbon dioxide, thereby providing NH₄+ as sole N source for plant, whereas ammonium nitrate being the salt of strong acid undergoes ionization and releases NO₃- and NH₄+ ions. Final fate of NH₄+ (from either source) is to undergo nitrification and get converted into nitrates. This fact is in consonance with our results wherein T₂, temporal changes in NH₄⁺-N and NO₃^--N contents are negatively correlated but in T₃ a positive correlation was obtained. Both the ions after immediate ionization are available for plant in case of double salt fertilizer ammonium nitrate. This is a known fact that plants can absorb and utilize N as NH₄⁺ and NO₃- present in soil solution (Oh et al., 2008), which then within plant undergoes various processes of assimilation, transformation and mobilization (Oh et al., 2008). In present analysis, in case of T₂ NH₄⁺-N levels are significantly correlated with MI showing NH₄⁺-N is the sole N-source utilized by plants initially. Among the treatments, T₂ seems to cytotoxic. Initially till 5 DAT, MI is increasing but 10 DAT MI showed a decline due to higher NH₄⁺-N accumulation. Also, previous studies by the same group, on the fertilizer has revealed that it is not only mitodepressive but also promotes mitotic anomalies especially interphase anomalies (Verma et al., 2016). The reasons may be toxicity of NH₄+-N and its accompanied effects. Mitotic index is the measure of cell division and growth and is a potential parameter for cytotoxicity (Fiskesjo, 1985; Bianchi et al., 2016), further it has also been regarded as a cytotoxicity biomarker (Verma and Srivastava, 2018b). In view of cited literature low MI in present case reflect the cytotoxic effects in plant. As NH₄⁺-N is taken up by plants it releases protons in soil reducing the pH of soil which causes NH₃⁺ to bind with protons and form NH₄⁺-N ions, this build-up of NH₄⁺-N concentration in soil hampers the uptake of other ions and induces salinity stress (Cabrera, 2001). In a recent report, it has been shown that higher levels of ammonium in rice plants, induces reactive oxygen species (ROS) mediated reactions (Yang et al., 2020). The excessive generation of ROS in plants is due to environmental stress like drought, salinity (Mittler, 2002) in response to ammonium toxicity. Further the results can be supported by the previous reports of Babourina et al., (2007), Bittsanszky et al., (2015), suggesting the NH₄⁺-N toxicity in plants when NH₄⁺-N is the sole N source. Their accumulation in soil causes acidification of soil and when they accumulate in plant cells acidification of cytosol occurs. Various morphotoxic and cytotoxic effects of NH₄⁺-N in plants is well known in literature as shown by Liu et al. (2013) and Arora et al. (2014). Toxicity of NH₄⁺-N caused inhibition of primary root growth by inhibiting cell elongation and division and even led to root cell death as reported by Qin et al., (2011). It also causes disturbed phytohormone and polyamine levels (Britto and Kronzucker, 2013).

Mitotic index seems to be higher in case of T₃ as both the forms of nitrogen, can be utilized by the plants. Though in case of T₃, plants get both forms of N but the peak of MI was obtained when the ratio of the two ions was optimum i.e., at 10 DAT. It is possible that a lower or higher ratio between the two ions may not be effective for the root growth; therefore low values of MI were obtained. When mixed N nutrition is supplied to the plants, protons are generated during NH₄⁺-N assimilation which can be utilized for NO₃^--N reduction hence regulate the intercellular pH (Li et al., 2012). Rhizospheric pH got neutralized when both the forms of N were supplied as reported by Hinsinger et al., (2003). Nitrogen as NH₄⁺ can lead to release of protons which can decrease the rhizospheric pH. At low pH there is more ammonium formation as reaction proceeds in forward direction. This may lead to accumulation of ammonium in soil because of limited uptake by plant. Whereas N as NO₃^- can take up protons from the rhizosphere, which causes an increase in the rhizospheric pH. Being opposite charge ion, nitrate protects the root cells from NH₄⁺ induced depolarization of plasma membrane by charge balancing (Wang et al., 1993). It causes decrease in internal ratio of cations and anions in plants (Britto and Kronzucker, 2002). Kronzucker et al., (1999) concluded in their study on NO₃^- - NH₄⁺ synergism in rice that presence of NO₃^- enhanced the NH₄+ fluxes, NH₄⁺ metabolism and cytosolic NH₄⁺ accumulation. Whereas presence of NH₄⁺ repressed the NO₃^- fluxes, accumulation and metabolism to the great extent. They also showed that net N-acquisition and N-translocation was enhanced when both the inorganic form of N was provided. However, it has been observed that plants show preference for the form of N available (Britto and Kronzucker, 2013) though depends on certain factors like, soil pH, aeration of soil etc. (Masclaux-Daubresse et al., 2010). Many reports have suggested the role of synergism between NH₄⁺ and NO₃^- nutrition (Marschner, 2012). Lastly if we compare all the treatments, drawback/constraint with urea (T₂) is that NH₄⁺-N levels are very high initially till one week contributing to growth to certain limit beyond which it causes toxic effects as seen in present and previous studies. The reason is that here nitrate is not contributing to alleviate the toxic effects of its counter ion. In ammonium nitrate, the advantage is availability of both the ions for selective uptake by plants as well as counter balance of each other’s ill effects and synergistic enhancement of growth.

Present study gives an idea about the cytotoxic effects of two fertilizers, urea and ammonium nitrate. Urea fertilizer provides single source of nitrogen i.e., NH₄⁺-N which shows cytotoxic effects, whereas double salt fertilizer ammonium nitrate yields both ions on ionization, which nullify each other’s negative effects. Such studies are warranted in the field of agriculture management, where input of fertilizers is meant to increase the yield.