World Applied Sciences Journal 18 (1): 73-81, 2012ISSN 1818-4952© IDOSI Publications, 2012DOI: 10.5829/idosi.wasj.2012.18.01.47

Corresponding Author: Zafar Iqbal, Department of Chemistry and Biochemistry, University of Agriculture Faisalabad, Pakistan.73

Effect of Nitrification Inhibitors on Nitrogen Leaching and Enzyme Activities

Zafar Iqbal, Khalil-UR-Rahman and Shahzad M.A. Basra1 1 2

Department of Chemistry and Biochemistry, University of Agriculture Faisalabad, Pakistan1

Department of Crop Physiology, University of Agriculture Faisalabad, Pakistan2

Abstract: Nitrification inhibitors (Thiourea and nitrification inhibitor “A”) with urea fertilizer were used toenhance the nitrogen efficiency. Nitrification inhibitors and the amount of nitrogen were applied through ureafertilizer to rice seed in Column Lysimeters. The whole experiment was conducted with following treatmentssuch as; urea application as a control at 2gN/10kg (200 kg N/ha), urea application 2gN/10kg (200 kg N/ha) addedwith 1.0%, 0.5%, 0.1%, 0.05% and 0.01% of thiourea (w/w) and urea application 2gN/10kg (200 kg N/ha) addedwith 1.0%, 0.5%, 0.1%, 0.05% and 0.01% of nitrification inhibitor “A” (w/w) alone and in combination form.Leachate samples were elicited from the Column Lysimeters and observed the NO and NO nitrogen after first,3 2

second, third and fourth weeks. The activity of NO and NO reductase also observed using UV-visible3 2

Spectrophotometer. It was found that all the treatments reduced nitrogen leaching and enzyme activities thatinvolved in nitrification process. It was also observed that the 0.01% of thiourea, nitrification inhibitor “A” andthiourea with nitrification inhibitor “A” for decreased the NO and NO concentration in the leachating samples3 2

and also reduced the activities of NO and NO reductase in the soil after the fourth week.3 2

Key words: Nitrification inhibitor Urea Thiourea Enzyme Reductase

INTRODUCTION A potential method to reduce NO nitrogen leaching

Nitrogen is an essential source for plant growth and of ammonium nitrogen to NO nitrogen [6]. In addition toreproduction. In Pakistan fertilizers are used to increases nitrification, that produces nitrate. The concentrations ofthe crops yield [1]. Nitrogen is converted into available NO - also depend on the level of denitrification, theforms by microorganisms and some enzymes that present process that consumes NO - in the soil. NO reductasein soil [2]. Significant losses from some surface applied (NaR) and NO reductase (NiR) are the major enzymesnitrogen sources can occur through the process of involved in denitrification [7]. It has been reported thatvolatilization. In this process nitrogen is lost as ammonia nitrification inhibitors can reduce nitrogen loss through(NH ) gas. Nitrogen can be lost in this way from manure denitrification [3]. Nitrification inhibitors are chemicals3

and fertilizer products containing urea. Ammonia is an that slow down or delay the nitrification process and alsointermediate form of nitrogen during the process in which used to enhance the nitrogen efficiency in soil [2]. Theurea is transformed to NH . Nitrogen sources are virtually present research experiment was therefore mainly aimed4

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eliminating volatilization losses. Loss of nitrogen from to find out the effects of nitrification inhibitors with ureavolatilization is greater when soil pH is higher than 7.3, air fertilizer on nitrate nitrogen enzyme activities involved intemperature is high, soil surface is moist and there is a lot nitrogen cycling under the soil.of residue on the soil [3]. Water is a good solvent, itdissolves some toxic and hazard substances produced MATERIALS AND METHODSthe water pollution problem. NO and NO concentration3 2

also increased in water with farming fertilizer, pesticide Collection of Samples: The soil samples were collectedor poor sanitary activities. NO nitrogen leaching from from field “Department of Crop Physiology” University of3

agricultural lands also threat against water. NO and NO Agriculture Faisalabad, Pakistan.3 2

contaminated the drinking water, nitrates undergoendogenous reduction and nitrosation of nitrites formed Fertilizer and Inhibitors Applications: Nitrificationthe carcinogens N-nitroso compounds. Nitrites led the inhibitors and the amount of nitrogen were appliedinfant’s disease called methemoglobinemia [4, 5]. through urea fertilizer to rice seed in Column Lysimeters.

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into groundwater is the retardation of biological oxidation2

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The whole experiment was conducted with following the UV-Spectrophotometric method using chromotropictreatments such as; urea application as a control acid and NO -N was also estimated by the Afkhami2gN/10kg (200 kg N/ha), urea application 2gN/10kg UV-Spectrophotometer nitrite determination method [9].(200 kg N/ha) added with 1.0%, 0.5%, 0.1%, 0.05% and0.01% of thiourea (w/w) and urea application 2gN/10kg Statistical Analysis: Data was analyzed by applying the(200 kg N/ha) added with 1.0%, 0.5%, 0.1%, 0.05% and Analysis of Variance [10].0.01% of unknown nitrification inhibitor “A” (w/w) aloneand in combination form [8]. RESULTS AND DISCUSSION

Nitrogen Leachating: Leachate samples were elicited from Effect of Nitrification Inhibitors on the Growth: Forthe Column Lysimeters the Nitrate nitrogen and Nitrite observing the effect of nitrification inhibitors (Thiourea,nitrogen were measured by the methods such as. Unknown nitrification inhibitor “A” and Thiourea with

Analysis of Nitrate Nitrogen Leaching: Preparation of grown in the lysimeters in triplicate form with variousReagent: 0.368 g Chromotropic acid was dissolved into nitrification inhibitors treatments such as, 1.0, 0.5, 0.1, 0.05200 mL Conc. H SO and kept the solution in a dark bottle. and 0.01%. All these treatments were applied with the2 4

For analysis of NO nitrogen took 3 mL leachate sample urea. Urea was used as nitrogen source for rice. After first,3

into conical flask, put the flask into cold water for a few second, third and fourth week applying the treatments,minutes, add 1 mL chromotropic acid solution drop by rice growth were observed. The best rice growth wasdrop directly into the leachating sample without mixing observed in the 0.01% of Thiourea with unknownand again put into the cold water for 30 minutes. Mix the nitrification inhibitor “A” treatment.solution and add 6 mL Conc. H SO on the flask wall2 4

without mixing. After adding the Conc. H SO in the Effect of Thiourea on Nitrate Nitrogen (NO -N): The urea2 4

sample swirl the flask and leave to cool at room with the nitrification inhibitor Thiourea was applied withtemperature for 45 minutes until the yellow color the treatments such as 1.0, 0.5, 0.1, 0.05 and 0.01% indeveloped. The absorbance was measured at 430 nm by triplicate form. The effect of nitrification inhibitor Thioureausing UV-Spectrophtometer. on nitrate nitrogen was observed after first, second, third

Analysis of Nitrite Nitrogen Leaching: Preparation of concentration. There was highly significant differenceReagents: The p-nitroaniline (0.2%) solution and between the varying treatments and week. All thediphenylamine (0.5%) solution were prepared in the treatments decreased the NO concentration but in theethanol. For analysis of NO nitrogen took the 5 ml fourth week the treatment 0.01% of Thiourea showed the2

leachate sample add 2 mL of sulfuric acid solution, 1 mL of more efficiency for decreasing the NO -N concentration astriton X-100 solution and 1 mL of p-nitroailine solution shown in the Figure 1. When, urea was applied asinto a 10 mL volumetric flask. The solution was diluted up nitrogen source to the rice. The nitrification inhibitorto 10 mL with distilled water. The solution was allowed to Thiourea inhibited the nitrate loss in the leaching samplesstand for 1 min followed by the addition of 1 mL of and the concentration of NO became low as by thediphenylamine solution. Again the solution was allowed inhibited the nitrification loss [11, 12].to stand for 10 min at the room temperature andabsorbance was measured at 500 nm by using UV- Effect of Nitrification Inhibitor “A” on NO -N: The ureaSpectrophtometer [9]. with the Nitrification inhibitor “A” was applied with the

Assay of Nitrie Reductase (Nir) and Nitrate Reductase form. The effect of Nitrification inhibitor “A” on nitrate(NaR) Activities: For the assay of NaR and NiR nitrogen was observed after first, second, third and fourthactivities, 1 ml of 1% KNO solution and 0.5% NaNO week and it decreased the NO -N concentration. There3 2

solution were added to 1.0 g soil, respectively. The was highly significant difference between the varyingmixture were incubated at 30°C for 24 hours and the treatments and week. All the treatments decreased theamount of reduced NO -N and NO -N were estimated to NO concentration but in the fourth week the treatment3 2

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represent the activities of NaR and NiR respectively 0.01% of nitrification inhibitor “A” showed the more(Guan, 1986). And the reduced NO -N was measured by efficiency for decreasing the NO -N concentration as3

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Unknown nitrification inhibitor “A”) Rice seeds were

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and fourth week and it decreased the NO -N3

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treatments such as 1.0, 0.5, 0.1, 0.05 and 0.01% in triplicate

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Fig. 1: Effect of Thiourea on Nitrate nitrogen

Fig. 2: Effect of Nitrification inhibitor "A"on Nitrate nitrogen

shown in the Figure 2. When, urea was applied as inhibited the NO loss in the leaching samples and thenitrogen source to the rice. The Nitrification inhibitor concentration of NO became low as by the inhibited the“A” inhibited the NO loss in the leaching samples and nitrification loss [12].3

the concentration of NO became low as by the inhibited3

the nitrification loss [11, 13] Effect of Thiourea on Nitrite Nitrogen (NO -N): The urea.

Effect of Thiourea with Nitrification Inhibitor “A” on the treatments such as 1.0, 0.5, 0.1, 0.05 and 0.01% inNO -N: The urea, Thiourea with Nitrification inhibitor triplicate form. The effect of nitrification inhibitor Thiourea3

“A” was applied with the treatments such as 1.0, 0.5, 0.1, on NO -N nitrite nitrogen was observed after first, second,0.05 and 0.01% in triplicate form. The effect of Thiourea third and fourth week and it decreased the nitrite nitrogenwith Nitrification inhibitor “A” on NO -N was observed concentration. There was highly significant difference3

after first, second, third and fourth week and it decreased between the varying treatments and week. All thethe NO -N concentration. There was highly significant treatments decreased the nitrate concentration but in the3

difference between the varying treatments and week. All fourth week the treatment 0.01% of Thiourea showed thethe treatments decreased the NO concentration but in the more efficiency for decreasing the NO -N concentration as3

fourth week the treatment 0.01% of Thiourea with shown in the Figure 4. When, urea was applied asNitrification inhibitor “A” showed the more efficiency for nitrogen source to the rice. The nitrification inhibitordecreasing the NO -N concentration as shown in the Thiourea inhibited the NO loss in the leaching samples3

Figure 3. When, urea was applied as nitrogen source to and the concentration of NO became low as by thethe rice. The Thiourea with Nitrification inhibitor “A” inhibited the nitrification loss [11, 12].

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with the nitrification inhibitor Thiourea was applied with

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Fig. 3: Effect of Thiourea with Nitrfication inhibitor "A" on Nitrate nitrogen

Fig. 4: Effect of Thiourea on Nitrite nitrogen

Effect of Nitrification Inhibitor “A” on Nitrite Nitrogen Effect of Thiourea with Nitrification Inhibitor “A” on(NO -N): The urea with the Nitrification inhibitor “A” Nitrite Nitrogen (NO -N): The urea, Thiourea with2

was applied with the treatments such as 1.0, 0.5, 0.1, 0.05 Nitrification inhibitor “A” was applied with the treatmentsand 0.01% in triplicate form. The effect of Nitrification such as 1.0, 0.5, 0.1, 0.05 and 0.01% in triplicate form. Theinhibitor “A” on NO -N was observed after first, second, effect of Thiourea with Nitrification inhibitor “A” on2

third and fourth week and it decreased the nitrite NO -N was observed after first, second, third and fourthnitrogen concentration. There was highly significant week and it decreased the NO -N concentration. Theredifference between the varying treatments and week. was highly significant difference between the varyingAll the treatments decreased the NO concentration but in treatments and week. All the treatments decreased the2

the fourth week the treatment 0.01% of Nitrification nitrate concentration but in the fourth week the treatmentinhibitor “A” showed the more efficiency for decreasing 0.01% of Thiourea with Nitrification inhibitor “A” showedthe nitrite nitrogen concentration as shown in the the more efficiency for decreasing the NO -NFigure 5. When, urea was applied as nitrogen source to concentration as shown in the Figure 6. When, urea wasthe rice. The Nitrification inhibitor “A” inhibited the NO applied as nitrogen source to the rice. The Thiourea with2

loss in the leaching samples and the concentration of Nitrification inhibitor “A” inhibited the NO loss in theNO became low as by the inhibited the nitrification loss leaching samples and the concentration of nitrite became2

[12, 13]. low as by the inhibited the nitrification loss [11, 12].

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Fig. 5: Effect of Nitrification inhibitor "A" on Nitrite nitrogen

Fig. 6: Effect of Thiourea with Nitrification inhibitor "A" on Nitrite nitrogen

Effect of Thiourea on Nitrate Reductase (NaR): The Effect of Nitrification Inhibitor “A” on Nitrate Reductaseurea with the nitrification inhibitor Thiourea was (NaR): The urea with the Nitrification inhibitor “A”applied with the treatments such as 1.0, 0.5, 0.1, 0.05 and was applied with the treatments such as 1.0, 0.5, 0.1, 0.050.01% in triplicate form. The effect of nitrification and 0.01% in triplicate form. The effect of Nitrificationinhibitor Thiourea on NO reductase was observed inhibitor “A” on NO reductase was observed after first,3

after first, second, third and fourth week and it second, third and fourth week and it decreased thedecreased the NO reductase activity. There was NO reductase activity. There was highly significant3

highly significant difference between the varying difference between the varying treatments and week.treatments and week. All the treatments decreased the All the treatments decreased the NO reductaseNO reductase activity but in the fourth week the activity but in the fourth week the treatment 0.01% of3

treatment 0.01% of Thiourea showed the more Nitrification inhibitor “A” showed the more efficiencyefficiency for decreasing the NO reductase activity as for decreasing the NO reductase activity as shown in3

shown in the Figure 7. When, urea was applied as the Figure 8. When, urea was applied as nitrogen sourcenitrogen source to the rice. The nitrification inhibitor to the rice. The Nitrification inhibitor “A” inhibited theThiourea inhibited the NO reductase that involved in the NO reductase that involved in the nitrification process3

nitrification process [14, 15]. [12, 13, 15].

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Fig. 7: Effect of Thiourea on Nitrate reductase (NaR)

Fig. 8: Effect of Nitrification inhibitor "A" on Nitrate reductase (NaR)

Effect of Thiourea with Nitrification Inhibitor “A” on The Thiourea with Nitrification inhibitor “A” inhibited theNitrate Reductase (NaR): The urea, Thiourea with NO reductase that involved in the nitrification processNitrification inhibitor “A” was applied with the treatments [11, 13, 15].such as 1.0, 0.5, 0.1, 0.05 and 0.01% in triplicate form. Theeffect of Thiourea with Nitrification inhibitor “A” on NO Effect of Thiourea on Nitrite Reductase (NiR): The urea3

reductase was observed after first, second, third and with the nitrification inhibitor Thiourea was applied withfourth week and it decreased the NO reductase activity. the treatments such as 1.0, 0.5, 0.1, 0.05 and 0.01% in3

There was highly significant difference between the triplicate form. The effect of nitrification inhibitor Thioureavarying treatments and week. All the treatments on NO reductase was observed after first, second, thirddecreased the NO reductase activity but in the fourth and fourth week and it decreased the nitrite reductase3

week the treatment 0.01% of Thiourea with Nitrification activity. There was highly significant difference betweeninhibitor “A” showed the more efficiency for decreasing the varying treatments and week. All the treatmentsthe NO reductase activity as shown in the Figure 9. decreased the NO reductase activity but in the fourth3

When, urea was applied as nitrogen source to the rice. week the treatment 0.01% of Thiourea showed the more

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Fig. 9: Effect of Thiourea with Nitrification inhibitor "A" on Nitrate reductase (NaR)

Fig. 10: Effect of Thiourea on Nitrite reductase (NiR)

efficiency for decreasing the NO reductase activity as week the treatment 0.01% of Nitrification inhibitor “A”2

shown in the Figure 10. When, urea was applied as showed the more efficiency for decreasing the NOreductase activity as shown in the Figure 11. When, urea

Thiourea inhibited the NO reductase that involved in the was applied as nitrogen source to the rice. The2

nitrification process [11, 14, 15]. Nitrification inhibitor “A” inhibited the NO reductase that

Effect of Nitrification Inhibitor “A” on Nitrite Reductase(NiR): The urea with the Nitrification inhibitor “A” was Effect of Thiourea with Nitrification Inhibitor “A” onapplied with the treatments such as 1.0, 0.5, 0.1, 0.05 and Nitrite Reductase (NiR): The urea, Thiourea with0.01% in triplicate form. The effect of Nitrification inhibitor Nitrification inhibitor “A” was applied with the treatments“A” on NO reductase was observed after first, second, such as 1.0, 0.5, 0.1, 0.05 and 0.01% in triplicate form. The2

third and fourth week and it decreased the NO reductase effect of Thiourea with Nitrification inhibitor “A” on2

activity. There was highly significant difference between nitrite reductase was observed after first, second, thirdthe varying treatments and week. All the treatments and fourth week and it decreased the NO reductasedecreased the nitrite reductase activity but in the fourth activity. There was highly significant difference between

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nitrogen source to the rice. The nitrification inhibitor

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involved in the nitrification process [11-13, 15].

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Fig. 11: Effect of Nitrification inhibitor "A" on Nitrite reductase (NiR)

Fig. 12: Effect of Thiourea with Nitrification inhibitor "A" on Nitrite reductase (NiR)

the varying treatments and week. All the treatments 2. Riley, W.J., I.O. Monasterio and P.A. Matson, 2001.decreased the NO reductase activity but in the fourth Nitrogen leaching and soil nitrate, nitrite and2

week the treatment 0.01% of Thiourea with Nitrification ammonium levels under irrigated wheat in Northerninhibitor “A” showed the more efficiency for decreasing Mexico. Nutrient Cycling in Agroecosystems,the NO reductase activity as shown in the Figure 12. 61: 223-236.2

When, urea was applied as nitrogen source to the rice. 3. Zhu, J.G., G. Liu, Y. Han, Y.L. Zhang and G.X. Xing,The Thiourea with Nitrification inhibitor “A” inhibited the 2003. Nitrate distribution and denitrification in theNO reductase that involved in the nitrification process saturated zone of paddy field under rice/wheat2

[11, 12]. rotation. Chemosphere, 50: 725-732.

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