Content uploaded by Pierfrancesco Nardi
Author content
All content in this area was uploaded by Pierfrancesco Nardi on Apr 24, 2018
Content may be subject to copyright.
Pedosphere 28(2): 332–340, 2018
doi:10.1016/S1002-0160(17)60429-6
ISSN 1002-0160/CN 32-1315/P
c
⃝2018 Soil Science Society of China
Published by Elsevier B.V. and Science Press
Nitrogen Release from Slow-Release Fertilizers in Soils with
Different Microbial Activities
Pierfrancesco NARDI1,∗, Ulderico NERI1, Giovanni DI MATTEO1, Alessandra TRINCHERA1, Rosario NAPOLI1,
Roberta FARINA1, Guntur V. SUBBARAO2and Anna BENEDETTI1
1
CREA-Council for Agricultural Research and Economics, Research Center for Agriculture and Environment, Via della Navicella 2,
Rome 00184 (Italy)
2
Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba 305-8686 (Japan)
(Received January 13, 2017; revised March 14, 2018)
ABSTRACT
Soil microbial activity is recognized as an important factor affecting nitrogen (N) release from slow-release fertilizers. However,
studies on the effect of size and activity of soil microflora on fertilizer degradation have provided contrasting results. To date, no clear
relationships exist between soil microbial activity and the release of N from slow-release fertilizers. Hence, the aim of this study was to
better understand such relationships by determining the release of N from three slow-release fertilizers in soils with different microbial
activities. Soils were amended with urea-formaldehyde (UF), isobutylidene diurea (IBDU), and crotonylidene diurea (CDU). Urea, a
soluble fertilizer, was used as the control. Fertilized soil samples were placed in a leaching system, and the release of N was determined
by measuring ammonium-N and nitrate-N concentrations in leachates during 90 d of incubation. Non-linear regression was used to fit
N leaching rate to a first-order model. In all the treated soils, N was released in the order: urea (89%–100%) >IBDU (59%–94%) >
UF (46%–73%) >CDU (44%–56%). At the end of incubation, N released from CDU did not differ (P > 0.05) among soils. On the
contrary, UF and IBDU released significantly lower (P < 0.05) amounts of N in the soil with higher microbial activity and lower pH.
The rate constant (K0) for UF was lower (P < 0.05) in the soil with lower pH. Taken together, our results indicated that soil microbial
size and microbial activity had a marginal effect on fertilizer mineralization.
Key Words: ammonium-N, crotonylidene diurea, isobutylidene diurea, nitrate-N, non-linear regression, urea-formaldehyde
Citation: Nardi P, Neri U, Di Matteo G, Trinchera A, Napoli R, Farina R, Subbarao G V, Benedetti A. 2018. Nitrogen release from
slow-release fertilizers in soils with different microbial activities. Pedosphere.28(2): 332–340.
INTRODUCTION
Slow-release fertilizers (SRFs) are condensation
products obtained by reacting urea, the most common
mineral fertilizer characterized by a high nitrogen (N)
content and relatively low cost, with several aldehy-
des. These SRFs release N at slower rates compared
to conventional N-fertilizers, such as urea releasing N
rapidly by hydrolysis; thus, in theory SRFs facilitate
better N uptake and utilization by crop plants. Poten-
tial benefits from SRFs include improved N use effi-
ciency (NUE), reduced volatilization loss and nitrate
leaching, increased N availability during plant growing
season, and reduced costs of application (i.e., multi-
ple applications of conventional N-fertilizers vs. a sin-
gle application of SRFs) (Allen, 1984). Among SRFs,
urea-formaldehyde (UF), isobutylidene diurea (IBDU),
and crotonylidene diurea (CDU) have gained popu-
larity (Trenkel, 1997). Urea-formaldehyde is obtained
by reacting urea with formaldehyde and consists of
a mixture of chain polymers with different lengths.
The degradation and subsequent N release from UF
is driven by the size and activity of soil microflo-
ra (Alexander and Helm, 1990) and factors that in-
fluence microbial activity, such as soil moisture and
temperature. Crotonylidene diurea, a ring-structured
compound, is produced by condensation of urea with
acetic aldehyde. Both microbial activity and hydro-
lysis drive the degradation of CDU. Thus, soil mois-
ture, temperature, and pH influence the CDU-N re-
lease (Trenkel, 1997). Isobutylidene diurea is a single
oligomer compound, formed by the reaction of urea
and isobutyraldehyde. The IDU-N release occurs by
chemical hydrolysis, and its decomposition in soil is
affected by soil temperature, moisture, and pH (Guer-
tal, 2009).
Slow-release fertilizers have been largely studied
under different temperature and soil moisture regimes
∗Corresponding author. E-mail: pierfrancesco.nardi@crea.gov.it.
NITROGEN RELEASE FROM SLOW-RELEASE FERTILIZERS 333
(Engelsjord et al., 1997; Agehara and Warncke, 2005;
Fan and Li, 2010; Fan et al., 2011), whereas the effect
of size and activity of soil microflora on SRF degra-
dation has received less attention with contrasting re-
sults observed (Benedetti, 1983; Koivunen and Hor-
wath, 2004). For example, in the case of UF, a signi-
ficant positive relationship between microbial activity
and N release was found under laboratory conditions
(Trinchera et al., 2010). However, Koivunen and Hor-
wath (2004) observed that both size and activity of soil
microflora have no significant effects on N release. In
addition, due to the role played by the pH in regulating
hydrolysis reactions, the evaluation of N release from
SRFs under different soil pH has implications on their
performance in different soil types. To date, the ques-
tion of whether N release from SRFs proceeds faster in
soils with higher microbial activity is still open. The
aim of this study was therefore to better understand
such relationships by determining N release from UF,
IBDU, and CDU in soils with different microbial acti-
vities. Given that N release from UF should be mainly
driven by microbial activity, we hypothesized a faster
N release from UF in soils with higher microbial acti-
vity.
MATERIALS AND METHODS
Soils and fertilizers
Four soils with different physicochemical and bio-
logical characteristics were selected for this research.
Soils were all classified according to the World Refe-
rence Base for Soil Resources (FAO, 2015), as refe-
rence European classification. The first soil was a Gleyc
Cambisol with sandy loam topsoil (43◦18′31.28′′ N,
11◦49′15.02′′ E), developed on recent alluvial deposits
with seasonal herbaceous crop land use inside the Ce-
sa Research Centre for Agricultural Technologies and
Extension Services (CATES) in the Province of Arez-
zo, Italy. The second soil was a Calcaric Fluvisol with
loam topsoil (43◦40′59.05′′ N, 10◦20′32.06′′ E), deve-
loped on the reclaimed alluvial floodplain of the Arno
River with seasonal herbaceous crop land use inside
the Interdepartmental Centre for Agro-Ecological Re-
search Enrico Avanzi (CIRAA), located near the city
of Pisa, Italy. The third soil was a Cambic Umbrisol
with clay loam topsoil (42◦22′6.83′′ N, 12◦19′44.03′′
E), developed on a volcanic tuff plateau of the Vico
complex with permanent crop (hazelnut orchards) land
use, near Viterbo, Italy. Finally, the fourth soil was a
Relictigleyc Calcaric Cambisol with a loamy sand top-
soil (49◦24′50.27′′ N, 8◦23′56.76′′ E), developed on re-
cent sandy alluvial deposits of the Rhine River with
seasonal herbaceous crop land use, near Limburgerhof,
Germany. Topsoil samples were collected from the 0–
15 cm depth (Ap horizon), dried at room temperature
(approximately 25 ◦C) for 2 weeks, and then passed
through a 2-mm sieve. Main soil physicochemical pa-
rameters are presented in Table I. The N contents in
the SRFs were 380, 320, and 320 g kg−1for UF, IBDU,
and CDU, respectively, and the urea with 460 g kg−1
N was used as a control.
TABLE I
Selected physicochemical properties of four soils used in this stu-
dy, including a Gleyc Cambisol in the Province of Arezzo, Italy,
a Calcaric Fluvisol near the city of Pisa, Italy, a Cambic Um-
brisol near Viterbo, Italy, and a Relictigleyc Calcaric Cambisol
near Limburgerhof, Germany
Propertya) Arezzo Pisa Viterbo Limburgerhof
Sand (%) 66 40 39 84
Silt (%) 16 38 32 10
Clay (%) 18 22 29 6
Soil textureb) Sandy Loam Clay Loamy
loam loam sand
pH 7.8 7.8 5.2 7.4
EC (dS m−1) 0.19 0.24 0.10 0.22
OM (g kg−1) 10.30 20.20 34.40 15.10
CEC (cmol kg−1) 15.43 17.74 16.47 9.22
Organic C (µg g−1) 6.0 11.7 19.9 8.7
N (µg g−1) 0.6 1.1 1.1 0.9
C/N ratio 9.9 10.5 18.3 9.7
a)EC = electrical conductivity; OM = organic matter; CEC =
cation exchange capacity.
b)According to USDA Soil Taxonomy.
Microbial biomass carbon (MBC) and basal soil respi-
ration
Soil MBC was measured by the chloroform
fumigation-extraction method (Vance et al., 1987) u-
sing air-dried soil samples conditioned by a 21-d in-
cubation in open glass jars at 33 kPa water tension
and 30 ◦C to restore soil microbial activity. Two por-
tions of 20 g (oven-dry basis) moist soil sample were
weighed: the first one (non-fumigated) was extracted
with 80 mL of 0.5 mol L−1K2SO4for 30 min by oscil-
lating shaking at 200 r min−1and then filtered using
Whatman No. 42, and the second one was fumigated
for 24 h at 25 ◦C with ethanol-free CHCl3. Following
fumigant removal, the soil was extracted similarly as
the non-fumigated soil sample. Organic carbon (C) in
the extracts was determined after oxidation with 0.4
mol L−1K2Cr2O7at 100 ◦C for 30 min, and MBC
was calculated as follows:
MBC = EC/kEC (1)
where EC is the difference between organic C extrac-
334 P. NARDI et al.
ted from fumigated and non-fumigated soils and kEC,
the conversion factor, is 0.38 (or 1/2.64) (Vance et al.,
1987).
To measure soil basal respiration, 25 g (oven-dry
basis) moist soil sample was weighed in a vessel and
then placed inside a 1-L stoppered glass jar. The CO2
evolved was trapped, and 2 mL of 1 mol L−1NaOH
was placed in another vessel inside the jar. After 1, 2,
4, 7, 10, 14, 17, and 21 d, CO2was determined by titra-
tion of the excess NaOH with 0.1 mol L−1HCl (Alef,
1995). The average of the 21-d measurements was used
as the basal respiration value for each soil.
It should be noted that soil MBC and basal respira-
tion were only measured once, just prior to the soil in-
cubation. We recognized that this may represent a limit
of our approach as both MBC and microbial activity
can change during the incubation. This excludes the
possibility to determine causal links between microbial
activity and N release from SRFs. On the other hand,
our approach allows for the determination of the rela-
tionships between pre-existing microbial activity and
fertilizer degradation. Therefore, it provided valuable
information on the use of soil microbial activity as a
putative indicator of fertilizer degradation.
Soil incubation procedure
Modified leaching method of Stanford and Smith
(1972) (Benedetti et al., 1994) was used to measure N
release from N sources. Briefly, 50-g air-dried soils were
mixed with quartz sand at a 1:1 ratio (weight:weight),
followed by the addition of N-fertilizer at the ratio
of 250 mg N kg−1. The mixtures (soil, quartz sand,
and N-fertilizer) were then transferred to the B¨uchner
funnels and incubated at 60% water-holding capaci-
ty (WHC) (pF = 2.5) at 30 ◦C. After 7, 14, 28, 45,
60, 75, and 90 d, released inorganic N (Ning ) was
leached as NO−
3-N and NH+
4-N with 500 mL of 0.01
mol L−1CaSO4solution. To avoid nutrient limiting
effects, after each leaching, a nutrient solution without
N containing 0.002 mol L−1CaSO4, 0.002 mol L−1
MgSO4, 0.005 mol L−1Ca(H2PO4)2, and 0.002 5 mol
L−1K2SO4was added to the mixtures. During the in-
cubation, B¨uchner funnels were covered with punctu-
ated aluminium foil and weighed every 3 d to monitor
soil moisture. Distilled water was added to maintain
soil at 60% WHC. Non-fertilized soil was considered
as the control. Leachates were filtered by Whatman
No. 42 filter paper and analyzed for NH+
4-N and NO−
3-
N with a Technicon II autoanalyser (Bran & Luebbe,
Sydney, Australia). Net NH+
4-N and NO−
3-N on time
from fertilizer release in each soil were calculated after
the subtraction of those of the control soil. Nitrogen
concentrations were reported on a dry matter basis.
Each treatment was replicated three times.
Statistical analysis
Soil MBC and basal respiration were subjected to
one-way analysis of variance (ANOVA). A two-way
ANOVA was performed to test differences in net cu-
mulative Ning (NH+
4-N + NO−
3-N) at the end of in-
cubation (90 d). We performed a 7 ×4×4 three-
way mixed between-within subject ANOVA to test the
effect of soils from Arezzo, Limburgerhof, Pisa, and
Viterbo, fertilizers (CDU, IBDU, Urea, and UF), and
incubation time on soil Ning concentrations during 90-
d incubation. In the mixed ANOVA model, time was
the within-subject factor, whereas soil and fertilizers
were considered as the between-subject factors. The
Mauchly’s test of sphericity indicated a violation of the
sphericity assumption for N concentrations; therefore,
the Greenhouse-Geiser adjusted degrees of freedom of
F-distribution was used (von Ende, 1993). In the two-
way ANOVA, as well as in the three-way ANOVA,
main effects were superseded by significant interacti-
ons. To break down these interactions, we performed
a simple effect analysis with Bonferroni-adjusted sim-
ple effect tests, holding the alpha level at 0.05. We
used non-linear regression to fit N leaching rate to a
first-order model (Agehara and Warncke, 2005) as fol-
lows:
Nrel =N0[(1 −exp(−Kt
0)] (2)
where Nrel (mg N kg−1soil) is the cumulative N re-
leased from each fertilizer at time t(d), N0(mg N
kg−1soil) is the potentially mineralizable N in the ferti-
lizer, and K0(d−1) is the mineralization rate constant.
All data points were used in the regression analysis, al-
though data were reported as mean values. The N0and
K0were considered statistically different at P < 0.05,
if the 95% confidence intervals did not overlap. Data
were analyzed using SPSS version 22.0 software.
RESULTS
Soil MBC and basal respiration
Among the four studied soils, MBC was significan-
tly the lowest (P < 0.05) in Arezzo soil (52.04 µg C
g−1soil) and significantly higher in Pisa (115.57 µg
C g−1soil) and Viterbo (144.31 µgCg−1soil) soils
(Fig. 1). All soils showed significant differences in basal
respiration in the range from 1.78 µg CO2-C g−1soil
in Limburgerhof soil to 12.39 µg CO2-C g−1soil in
Viterbo soil.
NITROGEN RELEASE FROM SLOW-RELEASE FERTILIZERS 335
Soil effects
At the end of the incubation period, CDU released
approximately 51% of the applied N with Ning concen-
trations being 110.3 and 138.9 mg N kg−1soil for Arez-
zo and Viterbo soils, respectively (Table II). However,
the difference was not statistically significant (P >
0.05). Isobutylidene diurea released more than 80% of
the applied N with a significantly lower amount of N
detected from the Viterbo soil (147.9 mg N kg−1soil;
P < 0.05). The recovered urea-N ranged from 89% to
100% of the applied N, with non-significant differences
among soils. Nitrogen released from UF was signifi-
cantly lower in Viterbo soil (116.4 mg N kg−1soil;
P < 0.05) than the other soils.
Fertilizer effects
In all soils, fertilizers released a decreasing amount
of N in the order: urea >IBDU >UF >CDU (Table
II). Net cumulative concentration of Ning was signifi-
cantly different (P < 0.05) among fertilizer treatments
for the Arezzo soil. In Limburgerhof and Pisa soils, net
cumulative Ning concentration was significantly higher
(P < 0.05) for urea and IBDU treatments than UF
and CDU treatments, with no differences between the
former two fertilizer treatments. In Viterbo soil, the
net cumulative Ning released from CDU and UF was
not statistically different (P > 0.05).
Dynamics of N concentration as affected by soil type,
fertilizer, and incubation time
A two-way ANOVA was performed to evaluate the
effects of soil and fertilizer on net Ning at the end
of incubation (90 d) (Table III). However, the main
effects of soil (F= 29.40, P < 0.001) and fertilizer
(F= 177.05, P < 0.001) were both significant and su-
perseded by significant soil and fertilizer interactions
(F= 6.54, P < 0.001). The three-way mixed between-
within subject ANOVA showed that incubation time
was the within-subject factor of Ning concentration,
whereas fertilizer and soil type, factorially combined
with incubation time, were the between-subject fac-
tors. For NH+
4-N and NO−
3-N, as well as for Ning, there
were significant main effects for both within- and bet-
Fig. 1 Microbial biomass C and basal respiration in four soils prior to the incubation. Values are means with standard deviations
shown by vertical bars (n= 3). Different letters indicate statistically different means between soils at P < 0.05.
TABLE II
Net cumulative concentrations of inorganic N (NH+
4-N + NO−
3-N) in four soils amended with slow-releas fertilizers (crotonylidene
diurea (CDU), isobutylidene diurea (IBDU), urea, and urea-formaldehyde (UF)) at the end of the incubation period (90 d)
Soil Net cumulative concentration of inorganic N
CDU IBDU Urea UF
mg N kg−1soil
Arezzo 127.8 ±3.4a)aAb) 217.7 ±5.8aB 249.8 ±17.7aC 168.1 ±5.8aD
Limburgerhof 132.6 ±10.4aA 220.8 ±8.9aB 222.6 ±8.6aB 181.8 ±8.7aD
Pisa 138.9 ±5.5aA 235.8 ±3.2aB 249.5 ±5.6aB 175.8 ±4.8aD
Viterbo 110.3 ±3.8aA 147.9 ±3.2bB 241.2 ±2.8aC 116.4 ±6.3bA
a)Means ±standard errors (n= 3).
b)Means followed by different lowercase letters within a column and uppercase letters within a row are statistically different for a given
soil and fertilizer, respectively (P < 0.05).
336 P. NARDI et al.
TABLE III
Three-way mixed analysis of variance results for the effects of slow-release fertilizers (crotonylidene diurea, isobutylidene diurea, urea,
and urea-formaldehyde), soil type (Arezzo, Limburgerhof, Pisa, and Viterbo), and incubation time (7, 14, 28, 45, 60, and 90 d) on soil
net N concentrations
Factor(s) Inorganic N NH+
4-N NO−
3-N
dfa) Fvalue Pvalue df Fvalue Pvalue df Fvalue Pvalue
Between subjects
Soil type (ST) 3 29.40 <0.000 1 3 592.22 <0.000 1 3 488.74 <0.000 1
Fertilizer 3 177.05 <0.000 1 3 43.51 <0.000 1 3 174.45 <0.000 1
ST ×fertilizer 9 6.54 <0.000 1 9 134.76 <0.000 1 9 39.33 <0.000 1
Error 32 32 32
Within subjects
Incubation time (IT) 3.21 1 172.3 <0.000 1 3.85 651.22 <0.000 1 2.78 633.12 <0.000 1
IT ×ST 9.64 22.7 <0.000 1 11.55 203.19 <0.000 1 8.35 100.08 <0.000 1
IT ×fertilizer 9.64 951.5 <0.000 1 11.55 369.75 <0.000 1 8.35 618.48 <0.000 1
IT ×ST ×fertilizer 28.90 16.3 <0.000 1 34.65 197.47 <0.000 1 25.04 77.67 <0.000 1
Error 103 32 32
a)Degree of freedom.
ween-subject factors. However, these effects were obta-
ined in the context of significant three-way interacti-
ons: F= 197.47, P < 0.001 for NH+
4-N, F= 76.67,
P < 0.001 for NO3N, and F= 16.27, P < 0.001 for
Ning.
Crotonylidene diurea-amended soils
Net Ning concentrations were quite low during the
first 14 d in all soils amended with CDU with the
exception of Pisa soil, and then increased significan-
tly from days 15 to 28 in all the soils, but general-
ly decreased afterwards (Fig. 2). In particular, on day
7, net Ning concentration was in the range of 14%,
6%, 5%, and 3% of the applied N in Pisa, Arezzo,
Limburgerhof, and Viterbo soils, respectively, with si-
gnificant differences between Pisa and Limburgerhof
soils (P < 0.05) and between Pisa and Viterbo soils
(P < 0.01). The net Ning concentration significantly
decreased (P < 0.01) in Pisa soil by day 14. From days
15 to 28, net Ning concentrations increased 3.5-fold to
27.5 mg N kg−1soil, 11-fold to 43.3 mg N kg−1soil,
15-fold to 53 mg kg−1soil than the former period (from
days 8 to 14) in Pisa, Limburgerhof, and Viterbo soils,
respectively (P < 0.001). On day 28, Arezzo soil ex-
hibited the significantly lowest net Ning concentration
(P < 0.001) compared to the other three soils. From
days 29 to 45, net Ning concentration significantly de-
creased in Viterbo soil (P < 0.05) and remained quite
constant in Limburgerhof and Pisa soils. Nitrogen dy-
namics differed among soils. Pisa soil, with the excep-
tion of the first week, was clearly dominated by the
prevalence of NO−
3-N during the incubation period.
Yet, NO−
3-N was prevalent in Arezzo soil for at least
the first three-incubation periods and in Limburgerhof
soil from days 29 to 45 and 46 to 60. In Viterbo soil,
NO−
3-N was almost absent during the first two weeks,
but increased afterwards.
Isobutylidene diurea-amended soils
Isobutylidene diurea released N at very low rate
during the first week of incubation, with net Ning con-
centration consistently less than 17 mg N kg−1soil,
with non-significant (P > 0.05) differences among soils
(Fig. 2). Conversely, approximately 68%, 76%, and
79% of the applied N was recovered in Arezzo, Pisa,
and Limburgerhof soils, respectively, during the next
two incubation periods, i.e., days 7–14 and 15–28. Dis-
tinct peaks in net Ning concentration were also found
for the Viterbo soil during the same incubation periods,
but the amount of N released was significantly lower
(P < 0.05) compared to the other three soils. During
days 8–14, net Ning concentration increased 10-fold to
83 mg N kg−1soil in Arezzo soil and 64 mg N kg−1
soil in Limburgerhof soil, 7-fold to 16 mg N kg−1soil
in Pisa soil, and approximately 4-fold to 32 mg kg−1
soil in Viterbo soil (P < 0.001) compared to those du-
ring days 0–7. From days 15 to 28, net Ning concentra-
tion increased 2-fold in Limburgerhof and Viterbo soils
(P < 0.001), but it remained almost constant in Arez-
zo soil (P= 1.000), and significantly decreased in Pisa
soil (P < 0.001) compared to the former duration. Af-
ter 28 d, net Ning concentration significantly decreased
in all soils and became negligible for the remaining pe-
riod. Nitrogen was nitrified faster in Arezzo, Pisa, and
Limburgerhof soils, but persisted in NH+
4-N in Viterbo
soil.
Urea-amended soils
As expected, urea released N very fast, and almost
all N added was recovered during the first 2 weeks of
NITROGEN RELEASE FROM SLOW-RELEASE FERTILIZERS 337
Fig. 2 Net concentrations of inorganic N (Ning ) containing NH+
4-N and NO−
3-N in four soils (Arezzo, Limburgerhof, Pisa, and Viterbo)
amended with slow-release fertilizers (crotonylidene diurea (CDU), isobutylidene diurea (IBDU), urea, and urea-formaldehyde (UF))
during the 90-d incubation.
incubation (Fig. 2). The remaining N was recovered on
day 14, with no difference (P > 0.05) among soils.
Urea-formaldehyde-amended soils
In soils amended with UF, net Ning concentration
decreased with incubation time in Pisa, Arezzo, and
Limburgerhof soils (Fig. 2). The UF-N recovered on
day 7 was 26%, 24%, and 19% of the applied N in Pisa,
Arezzo, and Limburgerhof soils, respectively, with no
significant difference. During the same incubation peri-
od, approximately 10% of the applied N was recovered
in Viterbo soil with significant differences (P < 0.01)
compared to Pisa and Arezzo soils. During days 15–
28, a significantly higher net Ning concentration (52.5
mg kg−1soil) was released in Viterbo soil than Arez-
zo and Limburgerhof (P < 0.001) and Pisa (P < 0.05)
soils. The released N was nitrified very fast in Pisa soil,
as NO−
3-N was the prevalent N form during the incu-
bation period. Arezzo soil followed similar dynamics,
with NO−
3-N concentration always higher than that of
NH+
4-N. The balance between the two N forms was dif-
ferent in Limburgerhof soil because of the prevalence
of NH+
4-N during days 0–7 and the presence of both
NH+
4-N and NO−
3-N during days 8–14, whereas NO−
3-
338 P. NARDI et al.
N dominated the remaining incubation period. As ob-
served for the other treatments, Viterbo soil showed
different N dynamics, with NH+
4-N as the dominant
form during the first 28 d of incubation but higher
NO−
3-N concentration during days 29–45 and 46–60.
Nonlinear regression
The first-order model generally fit the obtained da-
ta (Fig. 3, Table IV), with the only exception of Arez-
zo soil fertilized with CDU, where the model predic-
ted an unrealistic N0. The analysis of residuals (data
not shown) revealed systematic patterns in their dis-
tribution; thus indicating that the observed data for
CDU-amended Arezzo soil were not well described by
the model. Therefore, both N0and K0predicted for
Arezzo soil amended with CDU were excluded and not
compared with the other regression coefficients. Non-
significant differences (P > 0.05) in both N0and K0
were observed in the other soils treated with CDU (Ta-
ble IV). For IBDU-amended soils, N0was significantly
lower (P < 0.05) in Viterbo soil than the other three
soils, whereas no statistical differences were detected
for K0. In soils amended with urea, Limburgerhof soil
showed the lowest N0, and K0values were not sta-
tistically different among the four soils. Interestingly,
for UF-amended soils, both N0and K0were signifi-
cantly lower in Viterbo soil (P < 0.05) than the other
soils, and the highest K0was observed in Arezzo soil,
followed by Pisa and Limburgerhof soils, with statisti-
cal differences between Arezzo and Limburgerhof soils
(Table IV).
DISCUSSION
It should be noted that in this study, we were pri-
marily interested in the elucidation of the relationships
that exist between soil microbial activity and N release
from slow-release fertilizers. Regarding the effects of
the size and activity of soil microflora on UF degra-
dation, the following can be discussed. Soil microbial
activity was in the following order: Viterbo >Pisa >
Fig. 3 Nonlinear regressions of net cumulative inorganic N (Ning) in four soils (Arezzo, Limburgerhof, Pisa, and Viterbo) amended with
slow-release fertilizers (crotonylidene diurea (CDU), isobutylidene diurea (IBDU), and urea-formal dehyde (UF)) against incubation
time according to the first-order model.
NITROGEN RELEASE FROM SLOW-RELEASE FERTILIZERS 339
TABLE IV
Potentially mineralizable N in the fertilizer (N0) and mineralization rate constant (K0) in four soils amended with slow-release fertilizers
(crotonylidene diurea (CDU), isobutylidene diurea (IBDU), urea, and urea-formaldehyde (UF)) estimated by nonlinear regressions,
assuming first-order kinetics
Soil CDU IBDU Urea UF
N0K0N0K0N0K0N0K0
mg N kg−1soil d−1mg N kg−1soil d−1mg N kg−1soil d−1mg N kg−1soil d−1
Arezzo 1 025.7a) 0.002a) 235.7a 0.038a 250.4a 0.482a 158.4a 0.062a
Limburgerhof 192.6ab) 0.014a 242.0a 0.038a 222.8c 0.368a 179.0a 0.037bc
Pisa 155.4a 0.024a 246.2a 0.049a 249.6ab 0.432a 163.5a 0.053ab
Viterbo 147.3a 0.018a 170.3b 0.030a 241.3b 0.407a 131.1b 0.031c
a)Statistical significance was not reported, because data did not fit the model used.
b)Means followed by the same letter(s) within a column are not significantly different.
Arezzo >Limburgerhof. If microbial activity was the
main driving factor of UF decomposition, N release
would be expected to decrease from Viterbo to Lim-
burgerhof soils, according to the scheme above. Inte-
restingly, net cumulative Ning was the highest in Lim-
burgerhof soil, followed by Pisa and Arezzo soils. Most
importantly, it was significantly lower in Viterbo soil,
which was characterized by the largest and more ac-
tive soil microflora. These findings were additionally
supported by K0predicted for Arezzo (K0= 0.062)
and Viterbo (K0= 0.031) soils. Although the influ-
ence of microbial activity on UF degradation has been
well-documented (Benedetti, 1983; Nicolardot et al.,
1994), our results generally agree to those of Koivunen
and Horwath (2004), who did not observe a direct
link between N release from methylene urea, a slow-
release fertilizer, and soil microbial activity. This lack
of consistency could be explained as follows. A mixture
of methylene urea with different long-chain polymers
forms UF, which could be broken-down by both soil
bacteria and fungi (Alexander and Helm, 1990). To
date, different bacterial methylene urea-degrading en-
zymes have been purified (Jahns et al., 1997; Jahns and
Kaltwasser, 2000; Koivunen et al., 2004), and these
were structurally different, depending on the specif-
ic microorganisms and geographic locations. A limit of
our approach was that we neither performed enzymatic
assays, nor identified UF-degrading microorganisms.
However, it is reasonable to conclude that the degra-
dation of this fertilizer in soils was driven by the pre-
sence of specific UF-degrading microorganisms provi-
ding methylenediurea deaminase activity, and thus be
higher in soils harboring such microorganisms. There-
fore, specific enzymatic activities rather than total soil
microbial activity may better represent a predictor
of UF degradation in soils. Another important aspect
characterizing UF was that a relatively fast N release
occurred during the first 2 weeks of incubation, du-
ring which the applied N was recovered from 30% to
40%. Although this could be explained by the prese-
nce of unreacted urea in the UF, it is noteworthy that
in Pisa soil, and to some extent also in Arezzo soil,
the N released was rapidly nitrified, creating the po-
tential for N losses through NO−
3leaching or denitri-
fication. Therefore, the use of this fertilizer does not
necessarily avoid environmental problems.
Nitrogen release from IBDU occurred mostly du-
ring the first 28 d of incubation with a lag phase
in the first week, followed by a quick release during
days 14–28. Among parameters influencing IBDU-N
release, granule particle size, temperature, soil water
content, and pH are considered the most important.
In addition, as hydrolysis is faster in acidic condition,
N release should increase with lowering pH (Trenkel,
1997). Our results showed that N release did differ a-
mong Arezzo, Limburgerhof, and Pisa soils, which were
characterized by similar pH. However, the expected
higher N release in Viterbo soil due to its lower pH was
not observed. Conversely, IBDU degradation was found
to be higher in soils with the higher pH, where hydroly-
sis was presumed to be slower. However, the discovery
of the soil bacteria Rhodococcus erythropolis, a strain
able to degrade IBDU through the activity of a speci-
fic enzyme designated as an IBDU-amidinohydrolase,
challenged the view that IBDU degradation occurs on-
ly abiotically (Jahns and Scheep, 2001). In addition,
the optimum pH for the IBDU-amidinohydrolase acti-
vity has been shown to be in the range of 8.0 to 8.5,
which is closer to those of Arezzo, Pisa, and Limburger-
hof soils. Therefore, the biotic pattern of IBDU degra-
dation and, most importantly, the optimum pH for en-
zymes could explain our finding of higher IBDU degra-
dation in the soils with a higher pH. Finally, regarding
CDU degradation, it involves chemical hydrolysis and
microbial attack. However, throughout the incubation
period, neither the amount of the released N nor the
340 P. NARDI et al.
predicted regression coefficients differed significantly a-
mong the studied soils. Yet, our findings stand in con-
trast to previous studies reporting a faster N release
in acidic than alkaline soils (Varadachari and Goertz,
2010). Moreover, as for UF and IBDU, CDU-degrading
microorganisms have been isolated from soils (Jahns et
al., 2003). Our results indicate that both total micro-
bial activity and pH are weak indicators of CDU be-
havior in soil, which led us to speculate that the pre-
sence and activity of specific microorganisms capable
of degrading CDU may represent the key to predicting
the N release rate.
CONCLUSIONS
We did not observe significant relationships be-
tween soil microbial activity and N release from UF-
amended soils. It is possible that the latter occurs
mainly due to the activity of microorganisms able to
produce UF-degrading enzymes. Therefore, soil micro-
bial activity appears to be a poor indicator of the UF
behavior in soils. The effect of pH on IBDU degrada-
tion was of secondary importance because higher N
release in acidic conditions was not observed. Yet, N
release from CDU-amended soils was neither directly
affected by microbial activity nor by soil pH. There-
fore, although we recognize the importance of micro-
bial activity for N release from SRFs, the results of the
present study suggest that the size and the total acti-
vity of soil microflora had marginal effects on fertilizer
degradation.
ACKNOWLEDGEMENT
We thank the reviewers for the insightful comments
and suggestions.
REFERENCES
Agehara S, Warncke D D. 2005. Soil moisture and temperature
effects on nitrogen release from organic nitrogen sources. Soil
Sci Soc Am J.69: 1844–1855.
Alef K. 1995. Estimation of soil respiration. In Alef K, Nannipieri
P (eds.) Methods in Soil Microbiology and Biochemistry. A-
cademic Press, New York. pp. 464–470.
Alexander A, Helm H U. 1990. Ureaform as a slow release ferti-
lizer: A review. J Plant Nutr Soil Sci.153: 249–255.
Allen S E. 1984. Slow-release nitrogen fertilizers. In Hauck R D
(ed.) Nitrogen in Crop Production. ASA-CSSA, SSSA, Ma-
dison. pp. 195–206.
Benedetti A. 1983. Soil Biological Fertility and Slow-Release Fer-
tilizers (in Italian). Institute Sperimentale por la Nutritione
delle Plante, Rome.
Benedetti A, Alianiello F, Dell’Abate M T. 1994. A modified Sta-
nford and Smith method for the study of the mineralization
of nitrogen from organic materials. In Neeteson J J, Has-
sink J (eds.) Nitrogen Mineralization in Agricultural Soils.
AB-DLO Thema’s, Haren. pp. 127–132.
Engelsjord M E, Fostad O, Singh B R. 1997. Effects of tem-
perature on nutrient release from slow-release fertilizers. I.
Commercial and experimental products. Nutr Cycling Agroe-
cosyst.46: 179–187.
Fan X H, Li Y C. 2010. Nitrogen release from slow-release fertili-
zers as affected by soil type and temperature. Soil Sci Soc
Am J.74: 1635–1641.
Fan X H, Li Y C, Alva A K. 2011. Effects of temperature and
soil type on ammonia volatilization from slow-release nitro-
gen fertilizers. Commun Soil Sci Plan.42: 1111–1122.
Food and Agriculture Organization of the United Nations
(FAO). 2015. World Reference Base for Soil Resources 2014.
International Soil Classification System for Naming Soils and
Creating Legends for Soil Maps. Update 2015. World Soil Re-
sources Report No. 106. FAO, Rome.
Guertal E A. 2009. Slow-release nitrogen fertilizers in vegetable
production: A review. HortTechnology.19: 16–19.
Jahns T, Ewen H, Kaltwasser H. 2003. Biodegradability of urea-
aldehyde condensation products. J Polym Environ.11: 155–
159.
Jahns T, Kaltwasser H. 2000. Mechanism of microbial degrada-
tion of slow-release fertilizers. J Polym Environ.8: 11–16.
Jahns T, Schepp R, Kaltwasser H. 1997. Purification and chara-
cterization of an enzyme from a strain of Ochrobactrum
anthropi that degrades condensation products of urea and
formaldehyde (Ureaform). Can J Microbiol.43: 1111–1117.
Jahns T, Schepp R. 2001. Isobutylidenediurea degradation by
Rhodococcus erythropolis.Biodegradation.12: 317–323.
Koivunen M E, Horwath W R. 2004. Effect of management histo-
ry and temperature on the mineralization of methylene urea
in soil. Nutr Cycl Agroecosys.68: 25–35.
Koivunen M E, Morisseau C, Horwath W R. Hammock B
D. 2004. Isolation of a strain of Agrobacterium tumefaci-
ens (Rhizobium radiobacter) utilizing methylene urea (ure-
aformaldehyde) as nitrogen source. Can J Microbiol.50:
167–174.
Nicolardot B, Fauvet G, Cheneby D. 1994. Carbon and nitrogen
cycling through soil microbial biomass at various tempera-
tures. Soil Biol Biochem.26: 253–261.
Trenkel M E. 1997. Controlled-release and Stabilized Fertilizers
in Agriculture. International Fertilizer Association, Paris.
Trinchera A, Nardi P, Benedetti A. 2010. Influence of biologi-
cal fertility of soil on methylenurea biodegradation. In In-
ternational Scientific of ferlizers (ed.) Proceedings of 18th
Symposium of the International Scientific Centre of Fertili-
zers. International Scientific Center of Fertilizers, Rome. pp.
506–510.
Vance E D, Brookes P C, Jenkinson D S. 1987. An extraction
method for measuring soil microbial biomass C. Soil Biol
Biochem.19: 703–707.
Varadachari C, Goertz H M. 2010. Slow-release and controlled-
release nitrogen fertilizers. In Singh B (ed.) ING Bulletins on
Regional Assessment of Reactive Nitrogen. Bulletin No.11.
Scon-Ing, New Delhi. pp. 1–42.
von Ende C N. 1993. Repeated-measures analysis: Growth and
other time-dependent measures. In Scheiner S M, Gurevitch
J (eds.) Design and Analysis of Ecological Experiments.
Chapman & Hall, London. pp. 113–137.
