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Chemosphere 82 (2011) 16291635

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Effect of Azadirachta indica (neem), sodium thiosulphate and calcium chloride

on changes in nitrogen transformations and inhibition of nitrication in soil
incubated under laboratory conditions
M. Kaleem Abbasi , Munazza Hina, Majid Mahmood Tahir
Department of Soil and Environmental Sciences, University of Azad Jammu and Kashmir, Faculty of Agriculture, Rawalakot Azad Jammu and Kashmir, Pakistan

a r t i c l e

i n f o

Article history:
Received 11 June 2010
Received in revised form 8 November 2010
Accepted 16 November 2010
Available online 13 December 2010
Keywords:
Mineralization
Nitrication
Nitrication inhibitor
N-recovery
Nitrogen use efciency
Neem seedcake

a b s t r a c t
A laboratory experiment was conducted to examine the effects of nitrication inhibitors (NIs) neem seedcake (Azadirachta indica) (NSC), sodium thiosulphate (Na2S2O3) and calcium chloride (CaCl2) on changes
in NH
4 N, inhibition of nitrication and recovery of applied nitrogen (N) in soil. Surface soil samples of
015 cm were collected from an arable eld, amended with urea N (UN) at the rate 200 mg N kg1,
UN + NSC, UN + Na2S2O3 and UN + CaCl2 and incubated at 22 C periodically over 50 d. Soil without any
amendment was used as check (control). Results indicated that more than 58% of N applied as NH
4 disappeared over a period of 50 d from the soil mineral-N pool. Some of this N (21%) was accumulated as
NO
3 N while the remaining N was unaccounted for. Addition of nitrication inhibitors NSC, Na2S2O3,
and CaCl2 resulted in a decrease in the extent of NH
4 disappearance by 35%, 44% and 30%, respectively.
1
was accumulated over 50 d (maximum
In the treatment receiving UN alone, 56 mg NO
3 N kg
93 mg kg1) indicated an active nitrication. Application of nitrication inhibitors NSC, Na2S2O3, and
CaCl2 with UN inhibited nitrication by 54%, 64%, and 59%, respectively. Apparent N recovery (ANR) in
the treatment receiving UN alone was 63% that substantially increased to 83%, 89% and 76% in the treatments receiving UN + NSC, UN + Na2S2O3, and UN + CaCl2, respectively indicating 32%, 41% and 20%
increase in N recovery. Among three NIs tested, Na2S2O3 proved superior in inhibiting nitrication and
increasing ANR. The study demonstrated that application of NSC, Na2S2O3, and CaCl2 which are cheap
and easily available NIs inhibited nitrication and improved N recovery efciency of applied N in an
arable soil very effectively. It is suggested that these inhibitors should be tested under eld conditions
for increasing NUE and improving crop productivity.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
Nitrogen is one of the most important nutrients used worldwide
to increase and maintain crop production and is considered a key
element in maintaining the sustainability and economic viability
of farming systems across the world (Fixon and West, 2002). Application of N through chemical fertilizers is the dominant and main
source of N input in the crop production systems world-wide.
Unfortunately, applied N is not utilized efciently in the world agriculture and the recovery of N in soilplant system seldom exceeds
50%. Raun and Johnson (1999) reported that only 33% of the total N
applied for cereal production in the world is actually removed in the
grain, much less than that generally reported. The unaccounted 67%
represented a $15.9 billion annual loss of N fertilizer and with the
increasing costs of N fertilizer due to natural gas shortage, the loss
is now estimated to be worth more than $20 billion dollars annually
Corresponding author. Tel.: +92 (0) 5824 960046; fax: +92 (0) 5824 960004.
E-mail address: kaleemabbasi@yahoo.com (M.K. Abbasi).
0045-6535/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2010.11.044

(Raun and Johnson, 1999). These N losses from agricultural land

pose a major threat to environmental quality worldwide as agriculture activities are reported to contributes up to 90% of NH3 (Boyer
et al., 2002) and 70% of N2O emissions (Janzen et al., 1998). It is generally believed that applied N not taken up by plants escape to the
atmosphere and have adverse effects on the environment including
greenhouse effect, diminishing stratosphere ozone, and surface and
ground water pollution.
Most of the fertilizer N applied to soils for agriculture produc
tion is in the form of NH
4 or NH4 -producing compounds such as
urea or ammonium sulfate (FAO, 1992). Much of this NH4 is usually

oxidized quite rapidly to NO
3 in soil (Schmidt, 1982). The NO3 so
produced is vulnerable to leaching and denitrication, resulting
in 4560% loss of applied N fertilizers (Jarvis, 1996). Losses of applied N through denitrication and leaching resulted in gaseous
emissions to the atmosphere, surface and ground water pollution
leading to low nitrogen use efciency (NUE) and N recovery in
soilplant system (Johnson et al., 1987; Sisworo et al., 1990; Abbasi
and Adams, 2000a). Both eld and lab studies on N losses

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M.K. Abbasi et al. / Chemosphere 82 (2011) 16291635

suggested that if nitrication is reduced in agricultural systems,

plants have more time to take up available N, thereby improving
N recovery and uptake and reducing NO
3 leaching and associated
off farm environmental impacts (Subbarao et al., 2006).
A potential method to reduce N losses occurred due to nitrica
tion is the retardation of biological oxidation of NH
4 N to NO3 N
i.e. inhibition of nitrication (McTaggart et al., 1997; Serna et al.,
2000). The most common practice currently being considered for
inhibiting nitrication is the use of nitrication inhibitors (NIs)
(Abbasi et al., 2003; Di and Cameron, 2005; Zaman et al., 2009).
Nitrication inhibitors have been used in agriculture to improve
fertilizer efciency and minimize denitrication and/or leaching
losses of NO
3 by maintaining applied fertilizer N in the soil as
NH
4 N (McTaggart et al., 1997).
During last decade different synthetic NIs have been developed
which block enzymatic pathway, mainly ammonium mono-oxygenase (AMO) of bacteria responsible for NH
4 oxidation. AMO has wide
range of substrate for catalytic oxidation, and the inhibitory effects
of many NIs compounds are due to competition for the active site
of enzyme (McCarty, 1999). Many of these compounds used as NIs,
specially those having thiono-S can bind to Cu in the active site of
AMO enzyme and inactivate it, while others such as heterocyclic N
compounds inhibit AMO activity by their N ring (McCarty, 1999). Beside synthetic NIs, many chemicals and plant materials/extract can
inhibit nitrication. These chemicals and plant products are less
expensive and easily available than synthetic NIs. Different chloride
salts, for instance KCl, CaCl2, MgCl2 and NaCl have been found to reduce nitrication rate in laboratory and eld conditions (Agrawal
et al., 1985; Sloan and Anderson, 1998). Similarly, sodium thiosulphate (Na2S2O3) found to be an active nitrication inhibitor. It was
reported that the addition of 32 mg S kg1, as sodium thiosulfate,
inhibited the nitrication process from 5580% (Goos, 1985). Sallade
and Sims (1992) reported that thiosulfate when incorporated with
(NH4)2S04, NH4NO3, and urea, providing 85%, 100%, and 81%
inhibition of nitrication, respectively after 8 weeks of incubation.
Nitrication inhibitory properties of plant materials such as Karanj
(Pongamia glabra), neem (Azadirachta indica) and tea (Camellia
sinensis) waste have been identied (Majumdar, 2002; Kiran and
Patra, 2003a). Extracts from neem leaves, seeds, and bark possess a
wide spectrum of antibacterial action against Gram-negative and
Gram-positive microorganisms, and also act as an active nitrication inhibitor (Mishra et al., 1975; Santhi et al., 1986; Biswas et al.,
2002; Kumar et al., 2007).
Our objective was to evaluate the effect of neem seedcake
(plant material) and two chemicals sodium thiosulphate and calcium chloride on the changes in NH
4 N, inhibition of nitrication
and recovery of added N in soil incubated under controlled laboratory conditions.

2. Materials and methods

2.1. Soil sampling/collection
The soil used in this study was collected from an arable eld located at the research farm, Faculty of Agriculture, Rawalakot, Azad
Jammu and Kashmir. Detailed soil survey/classication and soil
prole studies of the area have not been done so far. The eld
was barren at the time of sampling but previously maize and
wheat were cultivated during the growing season. The selected
area was uniform in topography and well leveled. Soil samples
were collected from 0 to 15 cm depth at random from ve locations. The eld moist soil was sieved through a 4 mm sieve to eliminate coarse rock and plant material, thoroughly mixed to ensure
uniformity and stored at 4 C prior to use until the incubation
was initiated. A sub-sample was air dried and sieved through

2 mm mesh to determine selected soil physical and chemical characteristics (Table 1). Particle-size distribution was determined
after the organic matter was removed with 30% H2O2, by the Bouyoucos hydrometer method. Bulk density was determined by the
core method. Soil organic matter was determined using a modied
Mebius method (Nelson and Sommers, 1982). Total N was determined by the Kjeldahls digestion, distillation and titration method
(Bremner and Mulvaney, 1982), available P by the Olsen extraction
method (Olsen and Dean, 1982) and available K was extracted with
1 N ammonium acetate, adjusted to pH 7 and was determined
amephotometrically (Simard, 1993). CEC of the soil was determined by leaching the soil with KCl followed by extraction of
exchangeable K+ by ammonium acetate (Rhoades, 1990). K+ in
solution was determined amephotometrically. Soil pH was measured with a glass electrode, samples having been diluted with
water (the ratio of soil to water was 1:2.5).
2.2. Laboratory incubation
Field-moisture soil samples were placed into polyethylene bags,
adjusted to a water content of 60% of water-holding capacity
(equivalent to eld capacity), and pre-incubated at 22 C in the
dark to stabilize the microbial activity. The treatments include:
(a) ve N treatments including control with and without nitrication inhibitors (NIs) i.e. control, UN, UN + NSC, UN + Na2S2O3, and
UN + CaCl2; (b) nine incubation periods i.e. 0, 5, 10, 15, 20, 25,
30, 40 and 50 d. The treatments were replicated three times. Altogether, a total of 135 experimental units were used at the start of
the experiment. Ten days after the pre-incubation, 100 g of soil was
weighed and transferred into 200 mL glass jars. Phosphorus was
added to all jars at the rate of 90 mg P2O5 kg1 soil in the form of
single super phosphate (SSP) while potassium sulphate was added
as K source at the rate of 60 mg K2O kg1 soil. Similarly, urea N
(UN) was added as N source to all jars except control at the rate
of 200 mg N kg1. For the treatments subject to inhibitors application, neem seed cake (NSC) was added at 20% of N applied i.e.
40 mg kg1, sodium thiosulphate (Na2S2O3) was added at a rate
of 64 mg S kg1 (Sallade and Sims, 1992) while calcium chloride
CaCl2 was applied at the rate of 50 mg Ca kg1 soil i.e. 0.25, Ca/
N eq. wt. ratio (Sloan and Anderson, 1998). All the amendments
were mixed well into the soil. Soil without adding any amendment
was used as control. After adding different amendments, all the
jars were weighed and their weight was recorded. The jars were
covered with paralms with 34 small holes on the top to allow
Table 1
Some selected physicochemical properties of the soil
used in incubation experiment.
Parameter

Value

Soil bulk density (Mg m3)

Particle density (Mg m3)
Porosity (%)
Particle-size distribution
Sand (%)
Silt (%)
Clay (%)
Textural class
Soil pH
CEC (cmol kg1)
Organic matter (g kg1)
Organic C (g kg1)
Total N (g kg1)
C:N ratio
Total mineral N (mg kg1)
Available P (mg kg1)
Available K (mg kg1)

1.21 (0.03)
2.68 (0.05)
52 (3.18)
55 (2.33)
31 (1.86)
14 (1.15)
Sandy loam
7.1 (0.18)
6.4 (0.29)
112 (5.78)
6.5 (0.24)
0.39 (0.02)
9.6 (0.97)
6.6 (0.39)
87.0 (8.39)

Values in the parenthesis are the standard error of

means (SEM) i.e. n = 3.

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M.K. Abbasi et al. / Chemosphere 82 (2011) 16291635

O2 exchange within glass jars and incubated in an incubator at

22 C. The jars were arranged in the incubator according to completely randomized design. Soil moisture was checked/adjusted
after every 2 d by weighing the glass jars and adding the required
amount of distilled water when the loss was greater than 0.05 g.
During this process care was taken not to disturb the soil either
through stirring or shaking.
2.3. Soil extraction and analysis
Samples of all treatments incubated for different timings were
analyzed for total mineral nitrogen (TMN) and ammonium-N

(NH
4 N). Initial concentration of TMN and NH4 N at day 0 was
determined by extracting soil samples with 200 mL of 1 M KCl
added directly to the ask immediately after incorporation of each
amendment. Thereafter, triplicate samples from each treatment
were removed randomly from the incubator at different incubation
periods and extracted by shaking for 1 h with 200 mL of 1 M KCl
followed by ltration through Whatmans No. 40 lter paper. The
mineral N-contents of the extract were determined by using the
steam distillation and titration method of Keeney and Nelson
(1982). Aliquots (40 mL) of the extracts were pipetted into a distillation ask and steam distillation was carried out after adding MgO
for trapping NH3 in case of NH
4 N while for the determination of
TMN, magnesium oxide (MgO) + Deverdas alloy were added for

trapping NH3 evolved from NO
3 NO2 . The distillate was then
collected in 5 mL of boric acid containing bromocresol green/
methyl red mixed indicator and titrated against 0.05 M HCl. NitrateN was calculated by subtracting NH
4 N from total mineral
N. Any NO2 present would have been included in NO
3 fraction.
The percent nitrication inhibition was calculated as per Sahrawat (1980) as follows:

% Inhibition of nitrification C  S=C  100

where C NO
3 N concentration in urea treated soil
350

S NO
3 N concentration in urea treated soil with test compounds i.e. NIs
2.4. Statistical analysis
The data were statistically analyzed by multifactorial analysis of
variance (ANOVA) using the software package Statgraphics, 1992.
Least signicant differences (LSD) are given to indicate signicant
variations between the values of either treatments or time intervals. Condence values (P) are given in the text for the signicance
between treatments, time intervals, and their interactions.
3. Results
3.1. Changes in NH
4 N
In the control soil without added N, initial NH
4 concentrations
(17 mg kg1) increased slowly but progressively with time. The
maximum accumulation of 37 mg kg1 was recorded at day 20
indicating 2-fold increase in NH
4 N relative to the concentration
at the start of the experiment (Fig. 1). The concentration declined
slowly in the later stages of incubation and reached to background
level at the end (50 d). Addition of UN in soil signicantly
(P 6 0.05) increased the initial NH
4 N concentration at day 5
(Fig. 1). At this time the concentration of NH
4 N in the UN treatment without NIs was close to 200 mg N kg1. After days 5, NH
4 N
signicantly (P 6 0.05) decreased over time and at the end concentration reached to a background level recorded at day 0. Averaged
across different timings, NH
4 N in the treatment receiving UN
was 109 mg kg1 (Fig. 2). Taking into account the NH
4 N in the
control (25 mg kg1), concentration of NH
4 N in the UN treatment
was 84 mg kg1 indicating that 58% of the NH
4 had disappeared
from the mineral-N pool. In the soil to which NIs were added with
UN, the pattern of changes in NH
4 N was quite different (Fig. 1).
Following initial hydrolysis, NH
4 N concentrations in the urea
120

T1
T2
T3
T4
T5

300

T1
T2
T3
T4
T5

100

250

-1

NO3 -N (mg kg )

200

60

NH4- -N (mg kg-1)

80

150

40
100

20

50

0
0

10

20

30

Days after N application

40

50

10

20

30

40

50

Days after N application


Fig. 1. Changes in the concentration of NH
4 N and accumulation of NO3 N in a soil collected from an arable led, amended with urea N with and without nitrication
inhibitors and incubated at 22 C under controlled laboratory conditions. The legends indicate: T1 = Control; T2 = urea N (UN) without nitrication inhibitors (NIs);
T3 = UN + Neem seedcake; T4 = UN + Na2S2O3; T5 = UN + CaCl2.

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M.K. Abbasi et al. / Chemosphere 82 (2011) 16291635

250

70

60
200

-1

NO3 -N (mg kg )

150

NH4- -N (mg kg-1)

50

100

40

30

20
50
10

0
T1

T2

T3

T4

T5

Differernt N treatments

T1

T2

T3

T4

T5

Different N treatments


Fig. 2. Over all changes (average over 50 d incubation) in NH
4 N and accumulation/inhibition of NO3 N in a soil collected from an arable led, amended with urea N with
and without nitrication inhibitors and incubated at 22 C under controlled laboratory conditions. The symbols on X-axis indicate: T1 = Control; T2 = urea N (UN) without
nitrication inhibitors (NIs); T3 = UN + Neem seedcake; T4 = UN + Na2S2O3; T5 = UN + CaCl2.

amended soil containing NSC, Na2S2O3 and CaCl2 remained at

approximately 200 mg kg1 from day 10 to day 25 of the incubation. After days 25, NH
4 N in the treatment receiving NSC and
CaCl2 declined without the corresponding increase in NO
3 N
(Fig. 1). However, the rate and amount of NH
4 disappearance
was much smaller than in soil amended with UN without NIs. At
the end of incubation (at day 50), 38 mg kg1 of NH
4 N was left
in the mineral-N pool in the treatment receiving UN without NIs
1
while a substantial amount of NH
4 N i.e. 101, 142, 107 mg kg
was still persisted in the treatments receiving UN + NSC, UN +
Na2S2O3 and UN+ CaCl2, respectively. Overall treatments effect
(050 d) indicated that NH
4 N in the treatment receiving UN +
NSC, UN + Na2S2O3 and UN + CaCl2 was 180, 197, 168 mg kg1,
respectively (Fig. 2). Taking into account the NH
4 N in the control
(25 mg kg1), NH
4 N in UN + NSC, UN + Na2S2O3 and CaCl2 was
154, 172, 143 mg kg1, respectively. On the basis of N applied
(200 mg kg1), NH
4 disappeared from NIs treatments was 23%,
14%, 29% compared with 58% that had been disappeared from urea
amended soil without NIs.
3.2. Inhibition of nitrication
The concentration of NO
3 N at the start of the experiment was
generally low (35 mg kg1) (Fig. 1). In the control soil without
added N, initial NO
3 N increased slowly and a maximum of
1
21 mg NO
was recorded at day 30. The concentration of
3 N kg
NO
3 N in soil amended with UN (without NIs) was signicantly
(P 6 0.05) increased with time and a maximum of 93 mg of
1
NO
soil was accumulated at day 30, thereafter the con3 N kg
centration decreased (Fig. 1). In the soil to which NIs were added,
the extent and rate of NO
3 accumulation was generally lower. The
concentration of NO
3 N in the treatment receiving UN + NSC,
UN + Na2S2O3 and UN + CaCl2 ranging between 1337, 1228 and
1432 mg kg1, respectively. Average across different timings,
accumulation of NO
3 N in the treatments receiving UN, UN + NSC,
UN + Na2S2O3 and UN + CaCl2 was 56, 26, 20, 23 mg kg1, respectively (Fig. 2). These values indicated that NIs NSC, Na2S2O3 and
CaCl2 were able to inhibit nitrication by 54%, 64%, and 59%,

respectively. Among different NIs used, Na2S2O3 proved superior

in inhibiting nitrication compared to NSC and CaCl2. The accumu1
lation of NO
3 N at the end of incubation (day 50) was 65 mg kg
in the treatment receiving UN alone while the corresponding value
for inhibitor treatments (average) was 31 mg kg1.
The net rates of nitrication showed similar trend that recorded
for accumulation of NO
3 N (Table 2). Net rates in the control soil
almost unchanged between day 10 to day 30 ranging between 0.6
and 0.9 mg kg1 d1 and thereafter rates declined to 0.26 mg kg
1 1
d at day 50. Nitrication rates signicantly (P 6 0.05) increased
in the urea amended soil ranging from 2.97 to 3.67 mg kg1 d1 between day 10 to day 30 and thereafter rates declined. Comparative
analysis showed that accumulation of NO
3 N in UN amended soil
was 46 times higher than the control soil throughout the incubation. In comparison with UN amended soil, nitrication rates in soil
amended with UN + NIs were low, ranging from 0.50 to
1.93 mg kg1 d1. Average across different timings, net rates of
nitrication in the soil treated with UN, UN + NSC, UN + Na2S2O3
and UN + CaCl2 was 2.66, 1.29, 1.01 and 1.08 mg kg1 d1, respectively showing that nitrication inhibitors NSC, Na2S2 O3, and CaCl2
decreased nitrication rates by 51%, 62% and 60%, respectively over
the treatment receiving UN without NIs.
3.3. Apparent recovery of applied N
Data on the changes in total mineral N (TMN) in response to
different treatments is presented in Table 3. In the soil amended
with UN without NIs, N had been disappeared shortly after fertilizer application and 65% of the applied N had been depleted
from the mineral-N pool by the end of incubation. The applied
N also disappeared from soils amended with NIs but the extent
and rate of depletion was lower. Average across different timings
(050 d), TMN recovered from applied N was 127, 167, 178, and
152 mg kg1 in the soil amended with UN, UN + NSC, UN + Na2S2O3 and UN + CaCl2, respectively (Table 4). The apparent recovery of applied N in these treatments was 63%, 83%, 89%, and 76%,
respectively. These values indicated that application of NIs NSC,
Na2S2O3 and CaCl2 leading to a 32%, 41%, and 20% increase in

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M.K. Abbasi et al. / Chemosphere 82 (2011) 16291635

Table 2
1
day1) in soil amended with urea N (200 mg N kg1 soil) with and without nitrication inhibitors over 50 d of laboratory incubation.
Net rates of nitrication (NO
3 N mg kg
Treatments

1
NO
day1 soil)
3 N (mg kg
Days after N application
5
10

15

20

25

30

40

50

T1
T2
T3
T4
T5
LSD (P 6 0.05)

1.20
1.80
1.80
1.80
1.80
0.22

0.60
3.67
1.93
1.27
1.13
0.57

0.70
3.65
1.35
0.95
1.10
0.43

0.60
3.12
1.00
0.80
0.84
0.51

0.60
2.97
1.10
0.83
0.77
0.41

0.30
1.85
0.77
0.55
0.63
0.20

0.26
1.22
0.56
0.50
0.54
0.21

0.9
3.0
1.9
1.4
1.8
0.61

LSD (P 6 0.05)

Treatments effect averaged over 550 d (mg kg1 day1 soil)

T1
T2
T3
0.65
2.66
1.29
Timings effect averaged over 550 d (mg kg1 d1 soil)
5
10
15
20
1.68
1.80
1.72
1.55

T4
1.01
25
1.27

T5
1.08

30
1.29

40
0.81

0.11
0.44
0.18
0.26
0.21

LSD (P 6 0.05)
0.17
50
0.62

LSD (P 6 0.05)
0.21

T1 = control; T2 = urea N (UN) without nitrication inhibitors (NIs); T3 = UN + neem seedcake; T4 = UN + Na2S2O3; T5 = UN + CaCl2.

Table 3

1
Changes in the total mineral N (NH
soil) with and without nitrication inhibitors over 50 d of laboratory incubation.
4 N NO3 N) in soil amended with urea N (200 mg N kg
Treatments


1
soil
Total mineral N (NH
4 N NO3 N) mg kg
Days after N application
0
5
10
15

20

25

30

40

50

LSD (P 6 0.05)

T1
T2
T3
T4
T5
LSD (P 6 0.05)

20
28
27
25
25
NS

54
179
243
251
234
24.0

39
149
223
224
219
20.77

48
144
199
214
156
19.20

39
123
151
192
142
18.14

37
103
133
170
139
16.93

6.34
18.13
17.41
17.97
7.14

30
291
309
310
293
18.18

39
247
294
295
268
19.68

44
226
271
274
243
21.46

T1 = control; T2 = urea N (UN) without nitrication inhibitors (NIs); T3 = UN + neem seedcake; T4 = UN + Na2S2O3; T5 = UN + CaCl2.

Table 4
Fate and apparent recovery of fertilizer N (UN at the rate of 200 mg N kg1 soil) applied to soil with and without nitrication inhibitors over 50 d of laboratory
incubation.
Treatments

TMN (mg kg1 soil,

mean of 50 d)

TMN recovered from

applied N (mg kg1 soil)

N-unaccounted for
(mg kg1 soil)

ANR (%)

Increase in N recovery
due to NI (%)

T1
T2
T3
T4
T5

39
166
206
217
191

127
167
178
152

73
33
22
48

63
83
89
76

32
41
20

T1 = control; T2 = urea N (UN) without nitrication inhibitors (NIs); T3 = UN + Neem seedcake; T4 = UN + Na2S2O3; T5 = UN + CaCl2, ANR = apparent nitrogen
recovery.

the apparent recovery of added N. If we considered the TMN left

at the end of incubation (at day 50), the N recovered in soil
amended with UN alone, UN + NSC, UN + Na2S2O3 and UN + CaCl2
was 66, 96, 133, 102 mg kg1 and the apparent recovery of applied N for UN alone, UN + NSC, UN + Na2S2O3 and UN + CaCl2
was 33%, 48%, 67% and 51%, respectively leading to a 45%,
100%, and 55% increase in the apparent recovery of added N
due to the application of NIs.

4. Discussion
In the present investigation, a signicant increase in NH
4 N
concentration was detected during incubation in the control soil
reecting mineralization of organic N present in the soil. The high1
est concentration of 37 mg NH
was found by day 20, demon4 kg
strating signicant N cycling and turn over in the control soil
without N. Expressed as percentage of total organic N initially
present in the soil (390 mg kg1), the N mineralized during the

incubation was 9.5% similar to those reported by Haer and Benbi

(2003) i.e. 2.7% to 8.8% in the arable soils.
Following the addition of UN with and without NIs, the highest
1
concentration of NH
4 N (278298 mg ) was found at day 5 indicated that virtually all the added UN was hydrolyzed within the
rst 5 d period. In the soil amended with UN, a substantial proportion of the added N disappeared from the mineral-N pool after day
5 till the end if incubation. During NH
4 disappearance, concentra
1
tions of NO
3 N increased to a maximum of 93 mg of NO3 N kg
at day 30 indicating that nitrication was also occurring. However,
it is worthnoting that the depletion of NH
4 N during same period
was greater than 200 mg kg1 indicating that accumulation of

NO
3 N was much lower than the rate of NH4 depletion. The pat
tern of changes in NH4 N and accumulation of NO
3 N observed
here was almost similar to that found in our previous study in
grassland soil where more than 50% of N applied as NH
4 disappeared over a period of 42 d and only 35 mg kg1 was accumulated

as NO
3 N (Abbasi and Adams, 2000b). The NH4 N disappeared
may not be leached down because of experimental procedure

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M.K. Abbasi et al. / Chemosphere 82 (2011) 16291635

adopted in the experiment. Chances of immobilization could not be

neglected but our earlier study suggested that immobilization normally occurred in the earlier stages of incubation and later on
immobilized N released into mineral N (Abbasi et al., 2001). We
have concluded in our earlier studies that under the experimental
conditions reported here denitrication is the main mechanism of
N loss occurred after nitrication of NH
4 (Abbasi and Adams,
2000a,b). Application of NIs to urea amended soils delayed the extent and rate of NH
4 reduction and the disappearance was much
smaller relative to the soil without NIs. The reduction in NH
4 disappearance was due to inhibition of nitrication by NIs thereby
maintaining NH
4 N for longer period in the mineral-N pool. It appears that the additions of NIs inhibited nitrication and consequently denitrication by restricting the supply of NO
3 to the
denitrifying organisms. Our ndings are similar to those reported
by others (Freney et al., 1993; Chen et al., 1994; Rochester et al.,
1996; Abbasi and Adams, 2000b). However, the process of denitrication and production of N2O be smaller in arable soil decient in
organic matter compared to grassland soil due to less availability
of organic C. Therefore, only N2O production may not be indicative
of the extent of the N unaccounted for in the present study.
Extents of nitried N (NO
3 N) as percent of total mineral

NNH
4 N NO3  N in soil was much higher in the treatment
receiving UN without NIs (34%) as compared to UN + NSC (13%),
UN + Na2S2O3 (9%) and UN + CaCl2 (12%), indicating that nitrication was inhibited much more strongly by Na2S2O3 than NSC and
CaCl2. Percent nitrication inhibition was estimated to nd out
the effectiveness of the three inhibitors in inhibiting nitrication.
Percent nitrication inhibition by NSC, Na2S2O3 and CaCl2 was
ranging between 3265%, 5072%, and 3270%, respectively indicating Na2S2O3 superior over NSC and CaCl2. Inhibition was highest
between 20 and 30 d incubation and thereafter, inhibition decreased. But still the accumulation of NO
3 N in soil amended with
UN was 23-fold compared to the NO
3 N level in NIs treatment at
day 50. This demonstrated the impact of inhibitors to effectively
limit nitrication till the end of incubation. Khalil et al. (2009) reported that nitrication inhibition effect of NIs used was up to
5 weeks under both laboratory and greenhouse conditions. Our results were in accordance with those reported by Majumdar (2002)
where application of Karajan inhibited nitrication by 57%, 62%,
47%, 31% and 12% at 15, 30, 45, 60 and 75 d after incubation,
respectively. In another study Majumdar et al. (2002) reported that
Nimin coated urea, urea + DCD, urea + thiosulphate and neem
coated urea reduced total N2ON emission by 63%, 49%, 35% and
9%, respectively, as compared to urea treatment. Similarly, the percent nitrication inhibition (NI) by neem oil coated urea (NOCU)
was calculated and found that all of the neem oils caused nitrication inhibition ranging from 4.0% to 30.9% (Kumar et al., 2007).
Santhi et al. (1986) reported that addition of neem cake to soil signicantly reduced the population of nitrifying bacteria from
320  102 g1 at the start of experiment to 26  102 g1 after 30 d
incubation. With regard to Na2S2O3, Sallade and Sims (1992) reported that addition of thiosulphate to soil inhibited nitrication
by 81% in week eight to 26% at week 12. Reducing conditions created by the oxidation of thiosulfate to tetrathionate (S2 O2
6 ) in soils
were hypothesized by Goos (1987) to increase the concentrations
of Mn2+ and Fe2+ to inhibitory levels. Thiosulphate also inhibits
NH
4 oxidation by heterotrophic nitriers and is interconverted
with tetrathionate by many soil microorganisms which is a soil
urease inhibitor (LeFaou et al., 1990; Malla et al., 2005).
Application of NSC, Na2S2O3 and CaCl2 in urea amended soils

exhibited greater total mineral NNH
4 N NO3 N concentrations at the end of the incubation (day 50) (133, 170, and
139 mg kg1) than soil amended with only UN (103 mg kg1). Similarly, soil with urea N (without NIs) exhibited substantially higher
N loss i.e. 73 mg kg1 compared with the loss recorded for NIs

treatments i.e. 33, 22, 48 mg kg1 respectively. One explanation

for higher TMN and less N loss by NIs amended soil could be the
ability of NIs to maintain applied N in NH
4 form and to inhibit
the accumulation of NO
3 in the mineral-N pool. However, the exact
mechanisms involved in the process are not fully understood. The
apparent N recovery (ANR) of applied N calculated on the basis of
TMN recovered during 50 d incubation (average of 50 d). The ANR
in the soil treated with UN without NIs was 63%. Addition of nitrication inhibitors NSC, Na2S2O3 and CaCl2 substantially increased
recovery efciency of applied N to 83%, 89%, and 76%, respectively
indicating 32%, 41% and 20% increase in ANR. These results indicating that the applied NIs NSC, Na2S2O3 and CaCl2 which are cheap
and easily available decreased N loss and increased recovery of
added N as efciently as nitrapyrin, dicyandiamide, and other
expensive synthetic NIS (Agrawal et al. 1985; Malla et al., 2005;
Mohanty et al. 2008). A 26% increase in the recovery of added N
was observed in our previous study following the application of
nitrapyrin in grassland soil incubated for 42 d period (Abbasi
et al., 2003). Kiran and Patra (2003b) used essential oil bearing
plants M. spicata, Artemisia and DCD as nitrication inhibitor and
reported that the apparent N recovery by plants was the highest
(75.1%) with M. spicata oil followed by Artemisia oil (62.8%), DCD
(46.2%) and urea without NIs (35.5%). The increase in ANR was
due to the inhibition of nitrication by NIs and decreased and delayed in disappearance of NH
4 N. Nitrication inhibitors inhibit
the NH
4 mono-oxygenase enzyme which is responsible for converting soil NH
4 to hydroxyl amine which is further oxidized to

NO
2 nitrite and then to NO2 (Prasad and Power, 1995; Malla
et al., 2005). The lesser availability of NO
2 on application of nitrication inhibitors reduced N losses leading to increase the apparent
recovery of applied N. Delayed nitrication and the subsequent decline of N2O emission with the inhibitors under cropped and laboratory conditions were also recently reported by Khalil et al.
(2009). Among the three NIs used in the study, Na2S2O3 proved
superior over NSC and CaCl2 in increasing recovery efciency of applied N in soil. Reduction in N2O emission on the application of
nitrication/urease inhibitors along with urea ranged from 5% with
hydroquinone to 31% with thiosulphate in rice and 7% with hydroquinone to 29% with DCD in wheat crop (Malla et al., 2005). The
authors reported thiosulphtae superior over neem (Azadirachta melia) cake, coated calcium carbide, neem oil coated urea and dicyandiamide (DCD) in reducing denitrication and N2O emissions
because of the dual effect on nitrication inhibition and urea
hydrolysis.

5. Conclusions
Use of nitrication inhibitors is one of the strategies to improve
NUE of nitrogenous fertilizers in agriculture. Similarly, the environmental threats caused by gaseous emissions and ground water pollution because of denitrication and nitrate leaching can be
reduced or minimized by the use of nitrication inhibitors. The
work presented here demonstrated positive inuence of NIs on N
transformations in soil. In fact, by delaying and decreasing the extent of NH
4 oxidation, NIs were able to contribute maintaining
NH
4 N in mineral-N pool for longer period of time. As a result,
the recovery efciency of applied N increased by 32%, 41%, 20%
due to the use of NSC, Na2S2O3 and CaCl2, respectively. Nitrication
inhibitors NSC, Na2S2O3 and CaCl2 also inhibited nitrication by
54%, 64% and 59%, thereby effectively inhibited NO
3 accumulation
i.e. substrate for N2O emission and nitrate leaching. Therefore,
inhibition of nitrication would be benecial for environmental
protection. Some cheap and easily available inhibitors like neem
seed cake, calcium chloride, and sodium thiosulphate decreased
accumulation of NO
3 N and increased recovery of added N as

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M.K. Abbasi et al. / Chemosphere 82 (2011) 16291635

efciently as nitrapyrin, dicyandiamide, and other expensive synthetic NIS. Therefore, the use of such materials, which are produced
by less energy intensive processes, should be encouraged to farmers to increase nitrogen use efciency and improve crop productivity. However, these benets need to be further investigated in eld
scale experiments to assess the N use efciency and N balances in
soilcrop systems.
References
Abbasi, M.K., Shah, Z., Adams, W.A., 2003. Effect of the nitrication inhibitor
nitrapyrin on the fate of nitrogen applied to a soil incubated under laboratory
condition. J. Plant Nutr. Soil Sci 166, 513518.
Abbasi, M.K., Shah, Z., Adams, W.A., 2001. Mineralization and nitrication potentials
of grassland soils at shallow depth during laboratory incubation. J. Plant Nutr.
Soil Sci. 164, 407502.
Abbasi, M.K., Adams, W.A., 2000a. Gaseous N emission during simultaneous
nitricationdenitrication associated with mineral N fertilization to a
grassland soil under eld conditions. Soil Biol. Biochem. 32, 12511259.
Abbasi, M.K., Adams, W.A., 2000b. Estimation of simultaneous nitrication and
denitrication in grassland soil associated with urea-N using15 N and
nitrication inhibitor. Biol. Fertil. Soils 31, 3844.
Agrawal, M.P., Shukla, A., Singh, M., 1985. Nitrication inhibition of added
nitrogenous fertilizers by potassium chloride in soil. Plant Soil 86, 135139.
Biswas, K., Chattopadhyay, I., Banerjee, R.K., Bandyopadhyay, U., 2002. Biological
activities and medicinal properties of neem (Azadirachta indica). Curr. Sci. 82,
13361345.
Boyer, E.W., Goodale, C.L., Jaworsk, N.A., Howarth, R.W., 2002. Anthropogenic
nitrogen sources and relationships to riverine nitrogen export in the
northeastern USA. Biogeochemistry 57, 137169.
Bremner, J.M., Mulvaney, C.S., 1982. NitrogenTotal. In: Page, A.L., Miller, R.H.,
Keeney, D.R. (Eds.), Methods of Soil Analysis, Part 2. Chemical and
Microbiological Properties; SSSA Madison WI, pp. 595624.
Chen, D.L., Freney, J.R., Mosier, A.R., Chalk, P.M., 1994. Reducing denitrication loss
with nitrication inhibitors following presowing applications of urea to a cotton
eld. Aust. J. Exp. Agric. 34, 7583.
Di, H.J., Cameron, K.C., 2005. Reducing environmental impacts of agriculture by
using a ne particle suspension nitrication inhibitor to decrease nitrate
leaching from grazed pastures. Agric. Ecosyst. Environ. 109, 202212.
FAO, 1992. Fertilizer use by crop. International Fertilizer Industry Association,
International Fertilizer Development Center and Food and Agriculture
Organization of the United Nations, Rome.
Fixon, P.E., West, F.B., 2002. Nitrogen fertilizers: meeting contemporary Challenges.
Ambio 31, 169176.
Freney, J.R., Chen, D.L., Mosier, A.R., Rochester, I.J., Constable, G.A., Chalk, P.M., 1993.
Use of nitrication inhibitors to increase fertilizer nitrogen recovery and lint
yield in irrigated cotton. Fertil. Res. 34, 3744.
Goos, R.J., 1987. Ammonium thiosulfate as a urease inhibitor: suggested
mechanism. In: Proc. Nineteenth North Central Extension Industry-Soil
Fertility Conf. St. Louis, MO, 2829 October 1987. Potash Phosphate Inst., W.
Lafayette, IN.
Goos, R.J., 1985. Identication of ammonium thiosulfate as a nitrication and urease
inhibitor. Soil Sci. Soc. Am. J. 49, 232235.
Haer, H.S., Benbi, D.K., 2003. Modelling nitrogen mineralization kinetics in arable
soils of semiarid India. Arid Land Res. Manage. 17, 153168.
Janzen, H.H., Desjardins, R.L., Asselin, J.M.R., Grace, B., 1998. The health of our air
toward sustainable agriculture in Canada. Publ.1981/E. Research Branch,
Agriculture and Agricultural Food Canada, Ottawa, ON.
Jarvis, S.C., 1996. Future trends in nitrogen research. Plant Soil 181, 4756.
Johnson, C.J., Bonrud, P.A., Dosch, T.A., Kilness, A.W., Serger, K.A., Busch, D.C., Meyer,
M.R., 1987. Fatal outcome of methaemoglobinaemia in an infant. J. Am. Med.
Assoc. 257, 27962797.
Keeney, D.R., Nelson, D.W., 1982. Nitrogen-Inorganic forms. In: Page, A.L., Miller,
R.H., Keeney, D.R., (Eds.). Methods of Soil Analysis, Part 2. Chemical and
Microbiological Properties; SSSA Madison WI, pp. 643693.
Khalil, M.I., Gutser, R., Schmidhalter, U., 2009. Effects of urease and nitrication
inhibitors added to urea on nitrous oxide emissions from a loess soil. J. Plant
Nutr. Soil Sci. 172, 651660.
Kiran, U., Patra, D.D., 2003a. Medicinal and aromatic plant materials as nitrication
inhibitors for augmenting yield and nitrogen uptake of Japanese mint (Mentha
arvensis L. Var. Piperascens). Bioresour. Technol. 86, 267276.
Kiran, U., Patra, D.D., 2003b. Inuence of natural essential oils and their by-products
as nitrication retarders in regulating nitrogen utilization for Japanese mint in

1635

sandy loam soils of subtropical central India. Agric. Ecosyst. Environ. 94, 237
245.
Kumar, R., Devakumar, C., Sharma, V., Kakkar, G., Kumar, D., Panneerselvam, P.,
2007. Inuence of physicochemical parameters of Neem (Azadirachta indica A
Juss) oils on nitrication inhibition in soil. J. Agric. Food Chem. 55, 13891393.
LeFaou, A., Rajagopal, B.S., Daniels, L., Fauque, G., 1990. Thiosulfate, polythionates
and elemental sulfur assimilation and reduction in the bacterial world. FEMS
Microbiol. Rev. 75, 351382.
Majumdar, D., 2002. Suppression of nitrication and N2O emission by karanjin a
nitrication inhibitor prepared from karanja (Pongamia glabra Vent).
Chemosphere 47, 845850.
Majumdar, D., Pathak, H., Kumar, S., Jain, M.C., 2002. Nitrous oxide emission from a
sandy loam inceptisol under irrigated wheat in India as inuenced by different
nitrication inhibitors. Agric. Ecosyst. Environ. 91, 283293.
Malla, G., Bhatia, A.H., Pathak, H., Prasad, S., Jain, N., Singh, J., 2005. Mitigating
nitrous oxide and methane emissions from soil in ricewheat system of the
Indo-Gangetic plain with nitrication and urease inhibitors. Chemosphere 58,
141147.
McCarty, G.W., 1999. Mode of action of nitrication inhibitors. Biol. Fertil. Soils 29,
19.
McTaggart, I.P., Clayton, H., Parker, J., Swan, L., Smith, K.A., 1997. Nitrous oxide
emissions from grassland and spring barley, following N fertilizer application
with and without nitrication inhibitors. Biol. Fertil. Soils 25, 261268.
Mishra, M.M., Nelakantan, S., Khandelwal, K.C., Bhardwaj, S.K., Vuxs, S.R., 1975.
Margosa (neem) seed cake as an inhibitor of nitrication. Soil Biol. Biochem. 7,
183184.
Mohanty, S., Patra, A.K., Chhonkar, P.K., 2008. Neem (Azadirachta indica) seed kernel
powder retards urease and nitrication activities in different soils at contrasting
moisture and temperature regimes. Bioresour. Technol. 99, 894899.
Nelson, D.W., Sommers, L.E., 1982. Total carbon, organic carbon and organic matter.
In: Page, A.L., Miller, R.H., Keeney, D.R., (Eds.). Methods of Soil Analysis, Part 2.
Chemical and Microbiological Properties;SSSA Madison WI, pp. 539577.
Olsen, S.R., Dean, L.A., 1982. Phosphorus. In: Page, A.L., Miller, R.H., Keeney, D.R.,
(Eds.). Methods of Soil Analysis, Part 2. Chemical and Microbiological
Properties;SSSA Madison WI, pp. 10351049.
Prasad, R., Power, J.F., 1995. Nitrication inhibitors for agriculture, health and the
environment. Adv. Agron. 54, 233281.
Raun, W.R., Johnson, G.V., 1999. Improving nitrogen use efciency for cereal
production. Agron. J. 91, 357363.
Rhoades, J.D., 1990. Cation exchange capacity. In: Page, A.L., Miller, R.H., Keeney,
D.R., (Eds.). Methods of Soil Analysis, Part 2. Chemical and Microbiological
Properties; SSSA Madison WI, pp. 149158.
Rochester, I., Constable, G., Safgna, P., 1996. Effective nitrication inhibitors may
improve fertilizer recovery in irrigated cotton. Biol. Fertil. Soils 23, 16.
Sahrawat, K.L., 1980. On the criteria for comparing the ability of compounds for
retardation of nitrication in soil. Plant Soil 55, 487490.
Sallade, Y.E., Sims, J.T., 1992. Evaluation of thiosulfate as a nitrication inhibitor for
manures and fertilizers. Plant Soil 147, 283291.
Santhi, S.R., Palaniappan, S.P., Purushothaman, D., 1986. Inuence of neem leaf on
nitrication in a lowland rice soil. Plant Soil 93, 133135.
Schmidt E.L., 1982. Nitrication in Soil. In: Stevenson, F.J. (Ed.). Nitrogen in
Agricultural Soils; American Society of Agronomy-SSSA: Madison, WI, pp. 253
288.
Serna, M.D., Nanuls, J., Quinones, A., Primo-Millo, E., Legaz, F., 2000. Evaluation of
3,4-dimethylpyrazole phosphate as a nitrication inhibitor in a citruscultivated soil. Biol. Fertil. Soils 32, 4247.
Simard, R.R., 1993. Ammonium acetate-extractable elements. In: Carter, M.R. (Ed.),
Soil Sampling and Methods of Analysis. Lewis Publishers, Boca Raton, FL, pp.
3942.
Sisworo, W.H., Mitrosuhardjo, M.M., Rasjid, H., Myers, R.J.K., 1990. The relative roles
of N xation, fertilizer, crop residues and soil in supplying N in multiple
cropping systems in a humid, tropical upland cropping system. Plant Soil 121,
7382.
Sloan, J.J., Anderson, W.B., 1998. Inuence of calcium chloride and ammonium
thiosulphate on bermudagrass uptake of urea nitrogen. Commun. Soil Sci. Plant
Anal. 29, 435446.
StatGraphics Manugistics, Inc., 1992. Statistical graphics system by statistical
Graphics Corporation, Reference Manual. Version 6. Manugistics, Inc., Rockville,
Muryland, USA.
Subbarao, G.V., Ishikawa, T., Ito, O., Nakahara, K., Wang, H.Y., Berry, W.L., 2006. A
bioluminescence assay to detect nitrication inhibitors released from plant
roots: a case study with Brachiaria humidicola. Plant Soil 288, 101112.
Zaman, M., Saggar, S., Blennerhassett, J.D., Singh, J., 2009. Effect of urease and
nitrication inhibitors on N transformation, gaseous emissions of ammonia and
nitrous oxide, pasture yield and N uptake in grazed pasture system. Soil Biol.
Biochem. 41, 12701280.