S. Kiss, M. Simihăian (Auth.) - Improving Efficiency of Urea Fertilizers by Inhibition of Soil Urease Activity-Springer Netherlands (2002) PDF
S. Kiss, M. Simihăian (Auth.) - Improving Efficiency of Urea Fertilizers by Inhibition of Soil Urease Activity-Springer Netherlands (2002) PDF
S. Kiss, M. Simihăian (Auth.) - Improving Efficiency of Urea Fertilizers by Inhibition of Soil Urease Activity-Springer Netherlands (2002) PDF
by
S. Kiss
Babe§-Bolyai University,
Department of Plant Physiology,
Laboratory for Environmental Enzymology and Microbiology, Romania
and
M. Simih3ian
Environmental Protection Agency,
Department of Environmental Management, Romania
Preface
The purpose of our present work is to review the fundamental studies on inhibition of
soil urease activity and the applied studies on improving efficiency of urea fertilizers by
inhibition of soil urease activity. The general literature on these topics covers 65 years,
and the patent literature comprises a period of nearly 40 years.
Studies related to inhibition of soil urease activity were performed in a great number
of countries' well representing all the continents. Full texts of the papers describing
these studies were published in one of 18 languages·'.
The literature data reviewed are structured into 10 chapters, 81 subchapters, and 224
sections. The bibliographical list consists of 830 papers cited.
·In alphabetical order: Argentina, Armenia, Australia, Austria, Belgium, Belorussia, Brazil. Bulgaria, Canada,
China, Costa Rica, Cuba. Czech RepUblic, Egypt, Estonia, France, Georgia (Gruzia), Germany, Hungary,
India, Iraq, Ireland, Israel, Italy. Japan, Kazakhstan, Lithuania, Malaysia, Moldova, Netherlands, New
Zealand, Pakistan, Philippines, Poland, Romania, Russia, Saudi Arabia, Slovakia, South Africa, South
Korea, Spain, Sri Lanka. Sudan, Sweden, Thailand, Turkey, Ukraine, United Kingdom, United States of
America. Uzbekistan .
•• In alphabetical order: Afrikaans, Bulgarian, Chinese, Czech, English, French, German, Hungarian, Italian,
Japanese, Korean, Polish, Portuguese, Romanian, Russian, Spanish, Swedish, Ukrainian.
III
Acknowledgements
The authors wish to express their gratitude to Mr.Arno Flier, Publishing Editor, Biosciences
Unit, Kluwer Academic Publishers, and to his collaborator, Ms. Claire van Heukelom, for
their kind co-operation in publishing this book. We are also grateful to Professors Colleen
Sanders, Rita Moore, and Debra Taevs for their precious collegial help with revision of the
English language of our manuscript, and to Informatician Alina Veza, of the Editorial Board
of the journal Studia Universitatis Babe~-Bolyai. for her precious collegial advice in giving
the manuscript a camera-ready form. We also thank Dr. Engineer Marian Proorocu,
Director. and Economist Florica pacurar. Head of the Economics Department of the
Environmt:-'I1tal Protection Agt:-'llCY of Cluj County, for the valuable moral and material
support we received from them during the whole period of the elaboration of our
manuscri pt.
v
CONTENTS
Preface III
Acknowledgements IV
INTRODUCTION
Chapter 1. Inorganic Compounds Tested for Evaluation of Their
Inhibiting Effect on Soil Urease Activity, Urea Hydrolysis,
Ammonia Volatilization,and Nitrous Oxide Emission 5
1.1. HEAVY METAL COMPOUNDS 5
1.2. LIGHT METAL COMPOUNDS 19
1.3. SALTS OF ALKALI METALS AND ALKALINE EARTH METALS 20
1.3 .1. Effect ofAlkali Metal and Alkaline Earth Metal Salts
on Urease Activity and Urea Hydrolysis 20
1.3.2. Effect ofAlkali Metal and Alkaline Earth Metal Salts
on Ammonia Volatilization 26
1.4. BORON COMPOUNDS 30
1.5. FLUORIDES 33
1.6. ARSENIC COMPOUNDS 34
1.7. SULFUR COMPOUNDS 35
1.8. OTHER INORGANIC COMPOUNDS 42
CONCLUSIONS 359
REFERENCES 361
SUBJECT INDEX 393
INTRODUCTION
The increase of food production to meet the growing needs related to demographic
explosion is largely conditioned by the efficiency of agricultural fertilizers.
The use of urea as a nitrogen fertilizer has increased tremendously in the last 3--4
decades in both developed and developing countries. According to all predictions, the
increasing trend in fertilizer urea usage will continue. TIlliS, urea becomes gradually the
most important fertilizer in world agriculture.
The growing importance of fertilizer urea should be attributed to its advantages over
other nitrogen fertilizers. TI1ese advantages include: high nitrogen content (46%); low
cost of manufacture, transportation, storage, and distribution; ease of handling (without
fire and explosion hazard); high solubility in water; reduced COITosivity. In addition,
urea is suitable for production of compound fertilizers; it can be applied to soil in solid
state or in solution; its solution can be used as a filliar spray for some crops and also in
association with many pesticides.
In soil, urea is transformed into ammonium carbonate under the action of a
hydrolytic enzyme, the urease. Thus, urea, a neutral compound, gives rise to an alkaline
product, the ammonium carbonate. The an1I11onium cation may be retained by the
adsorptive complex of the soil, but ammonium carbonate, being an unstable compound,
decomposes producing two gases (ammonia and carbon dioxide) and water:
urease
2H~
• • 2 NH3 + c~ + H:P .
The urease in soil is of microbial origin, but a part of it may originate from plants
and animals.
In soil, urease is present as an accumulated enzyme, adsorbed on organic and
mineral soil particles and/or complexed with humic substances. Activity of the
accumulated urease is much higher than that of the urease mzymes produced by the
momentarily proliferating microorganisms.
In most soils, activity of the accumulated urease is too high, resulting in a rapid
hydrolysis of urea with concomitant rise in pH at the site of hydrolysis and liberation of
ammonia. The free ammonia may dan1age the germinating seeds and young plants and
may be lost through volatilization. The highest losses occur in calcareous soils and in
light-textured ones (low cation-exchange capacity) as well as in soils under pasture. The
high ammonium concentration and pH hinder the bacterial oxidation of nitrites into
nitrates. Consequently, the nitrites accumulate in toxic concentrations. Volatilization of
ammonia may also constitute a problem of air pollution. Moreover, ammonia volatilized
from soil may entL'f lakes and streams in the vicinity and may promote eutrophication.
It is estimated that - due to excessi ve urease activity in different soils - a significant
part (up to 70%) of the applied urea-N is lost through volatilization of an1I11onia; the
average loss is considered to be about 20 or 33%. In other words, the output of one of
five or three fertilizer urea factories is lost and, at the same time, pollutes the
environment.
2
Urea-N may be lost into the atmosphere also as nitrous oxide which contributes to
the greenhouse effect and the destruction of the stratospheric ozone layer.
Prevention of the undesirable effects of excessive urea hydrolysis in soil aims at
increasing the efficiency of urea fertilizers. Any increase in this efficiency will increase
the agronomic and economic value of the fertilizers as a means of increasing crop
production, will conserve energy and raw materials needed to manufacture the fertilizers
and will minimize the adverse effects on the environment that may result from
inefficient fertilizer use.
To prevent the excessive urea hydrolysis in soils, investigations were carried out
along four lines.
I. Urea was transforml-'d with aldehydes into compounds that are sparingly soluble
in water (ureaforms, isobutylidene diurea, crotonylidene diurea, etc.). These aldehyde
condensation products slowly decompose in soil. Urea bydrolysis is slow because of the
limited amounts of free urea. Urease activity is not inhibited, and urea remains the
substrate on which the urease will act.
2. Fertilizer urea granules were coated with hydrophobic (water-resistant) materials
(asphalt, waxes, oils, plastics, etc.) or with powders (kaolin, clay, sulfur, Si0 2 , A1 2 0 3 ,
etc.) to limit dissolving of urea. As in the first case, urea hydrolysis is slow because of
the limited amounts of dissolved urea. Urease activity is not inhibited, and urea remains
the substrate of enzyme.
3. Urea was used in association with inhibitor(s) of soil urease activity.
4. Urea was replaced: by adducts of urea with mineral or organic acids (nitric,
phosphoric, boric, oxalic or succinic acid) or with mineral salts or hydroxides of heavy
and ligllt metals (ferric sulfate, aluminium sulfate, felTic hydroxide); by complexes of
urea with stearic acid or with other unbranched, unsubstituted aliphatic compounds
containing at least 6 carbon atoms. Urea in adducts with acids or with metal-containing
compounds hydrolyzes more slowly because of the acidity and metals, respectively,
whereas hydrolysis of urea in complexes with aliphatic compounds is slower because of
the limited amounts of free urea.
To summarize: in the investigations along the first two lines, urea remains the
substrate, and urease is not inhibited; in those along the third line, urea remains the
substrate, but urease is inhibited; in those along the fourth line, the substrate is
modified, whereas the enzyme is inhibited only weakly or is not inhibited at all.
In this review work, we will deal only with the investigations aiming at inhibiting
soil urease activity (the third line of investigations).
In the first investigations this aim was exclusively theoretical: obtaining of
supplementary evidence concerning the enzymatic nature of urea hydrolysis in soil. The
first data on the effects of chemicals on soil urease activity were published by Rotini
(University of Pisa, Italy) in 1935, and by Conrad (California State University, Davis)
in 1940.
Studying hydrolysis of urea in absence and presence of antiseptics, Rotini prepared
reaction mixtures from 50 g of soil + 30 ml of 0.1 'Vo urea solution + 1 m1 of water or 5%
phenol solution. After incubation (42°C/4 hours) , the residual urea was determined. Its
amount was found to be nearly equal in the untreated and phenol-treated soil. The
conclusion was drawn that phenol stopped the growth of microorganisms but did not
lyze them and, thus, did not lead to release of urease from the microbial cells; urease
3
activity measured in the presence of phenol was due to those urease molecules that
existed in soil in a free state even before addition of phenol.
In Conrad's experiment, urea dissolved in 50% ethanol was hydrolyzed in soil at a
lower rate than the urea dissolved in distilled water; the inactivating effect of ethanol on
soil urease was only partial. Treatment of the soil with HgCh, hydroquinone or catechol
led to strong or nearly complete inhibition of soil urease activity. The inhibitions
observed prove that urea hydrolysis in soil is, really, an enzymatically catalyzed
process, thus, confimting the other evidence (e.g.. heat lability of the catalyst, i.e.. of the
urease).
The literature appeared in the 1941-1959 period does not offer information on the
inhibition of soil urease activity, excepting a finding by Kuprevich (1951), according to
which the antibiotic preparation "BIN No.?", containing usnic acid, did not influence
urease activity in soil.
Beginning with 1960 and up to now, the aim of the investigations (ID inhibition of
soil urease activity is not (mly theoretical, i.e.. to obtain supplementary evidence
concerning the enzymatic nature of urea hydrolysis in soil. Contrarily, this aim is rather
practical, Le.. to identify inorganic and organic compounds which, in agricultural
practice, may be used in form of additives to urea fertilizers as inhibitors of soil urease
activity. Accentuation of the practical aim of these investigations was determined by the
world-wide increase in use of urea fertilizers, by evidentiation of the undesirable effects
of the excessivc hydrolysis of urca in soil and, consequently, by the need to increase the
efficiency of urea fertilizers.
The number of chemical compounds tested for evaluation of their effect on soil
urease activity is impressive: more than 14,000 compounds and mixtures of compounds
were tested with this aim. A great number of compounds and mixtures of compounds,
having inhibitory effect on soil urease activity, were patented as inventions in the
U.S.A, Great Britain, France, Germany, in the former U.S.S.R., in Romania and P.R. of
China. Application of some of these patents was also examined by the European Patent
Office. The first patent in this domain was obtained by Hyson (1963) in the U.S.A.
Most patents describing inhibitors of soil urease activity were elaborated by German
and North American investigators.
Of course, all investigations aiming at identification of soil urease inhibitors should
begin with the laboratory phase. This is continued by the phase in which the
investigations arc carried out in vegetation pots and is ended by the phase of
investigations under field conditions.
Effect of the inhibitor should manifest itself not only in a significant reduction of the
urea hydrolysis rate but, consequently, also in limitation of the N losses via al11l11onia
volatilization and nitrous oxide errtission. The inhibitor should be unpolluting, free of
injurious side effects, i.e .. it should have no negative effects on processes related to soil
fertility and should not be toxic to plants, animals, and man. At the same time, the
inl1ibitor should be a stable compound that does not decompose during manufacture,
transportation, and storage of the urea fertilizers. Of course, the inl1ibitor should have a
relatively low cost, and the benefit obtained from its use in agriculture should exceed its
price at purchase and other expenses related to its use. In other words, it should be
4
agronomically, environmentally, and economically better to use than not to use the
inhibitor (see Appendix to References)".
* *
*
Due to space limitation:
a) only the pesticides patented as inhibitors of soil urease activity will be dealt
with;
h) the studies on the inhibition of soil urease activity by high urea concentrations
(substrate excess) (e.g.. Rachhpal-Singh and Nyc, 1984a; Cabrera and Kissel,
1984; Monreal et af.. 1986; MergeI' et af.. 1987; Savant et af.. 1987b; Cabrera
et al.. 1991;Thormiihler and du Preez, 1992; Zhang el af.. 1994) will not be
reviewed; and
c) the studies on the inhibition of ammonia volatilization from urea by activation
of nitrification (e.g.. Fleisher and Hagin, 1981; Praveen-Kumar and Aggarwal,
1988; Goos and de Padua Cruz, 1999) will not be considered .
• The use of soil urease inhibitors is contraindicated only on those flooded rice fields on which the water
control is poor; therefore. urea is lost in the runolrwaters from these fields more easily than the ammonium
ion that can be retained more strongly than urea by the adsorptive complex of the soil (Craswell and Vlek.
1982; Keeney and Sahrawat. 1986).
5
Comad (I940) studied the effect of HgCh (mercuric chloride, sublimate) on urease
activity in two California soils. Treating the samples of the first soil (fine sandy loam)
with HgCh· caused a significant reduction in urease activity. At the same time, heating
the samples of the moistened soil to 85°C for 48 hours led to disappearance of urease
activity. This means that in this soil the effect of heat was stronger than that of HgC12'
The other soil (loam) exhibited higher urease activity than the first soil. In samples of
the loam soil, treated with HgCh (1 <X" 4 glkg soil), urease activity was reduced almost
to Zt:ro, the reduction being more pronounced than that caused by heating the moistened
samples at 85°C. In other words, in the loam soil the effect of HgCh was stronger than
that of the heat.
Mitsui et al. (1960) added 5-g air-dry samples of a Japanese paddy soil of volcanic
ash origin to 50 ml of 1 or 0.5 M urea solution containing 0.1 % HgCh (on air-dry soil
weight basis). The mixtures were shaken for 1 or 12 hours, then analyzed for NH/.
Mixtures without HgCh were the controls. It was established that HgCh strongly
inhibited the urease activity; the degree of inhibition was 92 and 85-86% after 1 and 12
hours of shaking, respectively.
Ba'led on the finding by Yolk and Sweat (1955) that urea hydrolysis is retarded in
those Florida soils which contain copper spray residues, Yolk (1961) carried out a
laboratory experiment, using urea pellets (of 1-2-mm diameter) coated with cupric
sulfate (CUS04) dust (0.25% Cu by weight of urea). The pellets were applied on the
surface of a bare moist soil (fine sand, pH 5.6), at a rate of 112 kg N/ha. Pellets without
CUS04 served for comparison. The N losses as ammonia volatilized during 7 days were
determined. The average N losses from the CUS04-coated and uncoated urea pellets
were 32.8 and 36.2%, respectively; CUS04 did not significantly reduce NH.l
volatilization, having only a weak inhibitory effect on soil urease activity. Copper
sulfate was ineffective even when its rate was increased to 0.4% Cu by weight of urea.
Briggs and Segal (1963) obtained, from approximately 25 kg of surface forest soil
(Upper Hutt Valley, New Zealand), a crystalline urease preparation (about 12 mg),
consisting of a mixture of proteins possessing urease activity. The authors mention that
catalytic activity of the preparation was markedly inhibited by Ag+, Hg2+, and Cu 2+, but
they do not give any details on the conditions under which the inhibitory effect was
tested.
Sor et al. (1966) and Sor (1968, 1969), of the Esso Research and Engineering
Company (Delaware), described in their patented inventions that they prepared pellets
from urea, cupric sulfate and lead acetate (0.45% Cu or Pb by weight of urea, the limits
of the Cu or Pb amount being 0.01 and 10%), and a hydrophobic material (solid
hydrocarbon binder). The preferred hydrophobic material was a blend of asphalt (90%)
and microcrystalline wax (10%), representing 10% by weight of urea (the limits being 3
"The rate at which HgCb was added to this soil is not specified in the paper.
6
and 251Vo). nle urea crystals were heated up to 60-80°C, then the inhibitor was added to
the wann urea and mixed thoroughly. This warm mixture received the asphalt-
microcrystalline wax blend previously softened by heating up to 104.5°C. All the
substances were thoroughly blended and this mixture was pelletized by extruding it
from a pellet mill. Tn the pellets obtained, urea, inhibitor, and hydrophobic material
were uniformly distributed. Urea prills without inhibitor and hydrophobic material and
urea pellets without inhibitor, but with hydrophobic material were used as controls.
For evaluation of the effectiveness of copper sulfate and lead acetate, the pellets and
priUs (at an amount equivalent to rates of urea generally applied under field conditions)
were placed on the surface of soil samples (sandy loam, pH 5.5) containing 13 and 5%
moisture, respectively. During incubation at 25°C for 16 and 31 days, respectively, the
volatilized ammonia was determined cumulatively. It was found that less NH3 was lost
from the urea peliets with Cu or Pb than from the control prills and peliets. In the soil
with 13% moisture, Cu and Pb had a similar effect. For example, the cumulative NH3
loss after 16 days of incubation was about 2%, from the Cu- or Pb-containing urea
pellets and 3.5-9SVo from the control prills and pellets. In the soil with 5% moisture, Cu
inhibited more strongly than did Pb. For example, during 28 days of incubation, the Cu-
containing pellets lost via NH3 volatilization about 1.8%, the Pb-containing pellets 8%
and the control prills and pellets about 11-13% of their initial urea-N content.
Pugh and Waid (l969a,b) determined the ammonia losses from lOO-g samples of
three English soils (sandy loam, loamy sand, and clay loam) which were treated with
CUS04 (640 ppm on moist soil weigllt basis) and with urea (4,000 ppm). Samples not
treated with CUS04 served as controls. All the samples of the three soils were incubated
at 20°C for 30, 77, and 148 days, respectively. Copper sulfate caused slight delays in
NH3 loss: the half-loss time (the number of days that elapsed until the NH1loss was half
of the total loss observed in the treatment where urea had been applied alone) was
prolonged under the influence of CUS04 from 4 to 6.5 days (in sandy loam), from 11.5
to 17 days (in loamy sand), and from 34 to 40.5 days (in clay loam). But the total NH3
loss from the three soils during their whole incubation period (30, 77, and 148 days,
respectively) was reduced by CUS04 only to a slight extent (the reductions were of 2.6,
4.6, and 8.4%, respectively).
For determination of urease activity in 21 virgin and 21 cultivated volcanic ash soils,
in 16 virgin and 16 cultivated nonvolcanic ash soils, and in 30 paddy soils from Japan,
Tanabe and Ishi711wa (1969) used 10 ml of a llVo HgCb solution to inactivate the
enzyme in reaction mixtures containing 10 g of soil and 70 ml of aqueous phase.
According to Bhavanandan and Fernando (1970), copper (l,OO() ppm on soil weight
basis), added in the form of CUS04 or CU20 to samples of a tea soil from Sri Lanka,
inhibited urease activity by 30.9 and 43.7%, respectively. The inhibitory effect
increased with the rate of copper. For example, at an 8-fold rate the two forms of copper
brought about 56.2 and 87.5% inhibitions, respectively, in the urease activity.
Surprisingly, the insoluble form of copper (CU20) caused a greater inhibition than did
the soluble copper sulfate at equivalent amounts.
Bremner and Douglas (l971) evaluated, by means of the 5-hour incubation test
described by Douglas and Brenmer (1971), the effect of numerous heavy metal
compounds on the urease activity in two Iowa soils (silty clay loam and clay loam).
This test involves determination of the effect of the test compound on the anlOunt of
urea hydrolyzed by incubation of IO-g samples of soils with 1 ml of toluene, 5 ml of
7
The inhibitory effect was folubstantial only in the case of silver, mercury, gold, and
copper. Comparison of the inllibitions caused by Cu2+ and Cu+, and Pb2+ and Pb+,
respectively, showed that the valency state of these cations did not influence their
inhibitory capacity. A similar comparison of the nitrate or chloride and sulfates of Ag+
and Hg2+ indicated that the anions in these compounds did not affect soil urease activity.
In the case of HgCIz, the influence of concentration (50, 100, 200, and 300 ppm Hg)
on the urease activity in clay loam was also studied, registering the following
inhibitions: 39, 45, 56, and 61 %, respectively, i.e.. the inhibition was not complete at
the highest HgCIz concentration, either.
The effect of the preincubation of soil samples with HgCh and CUS04 (50 ppm) fi}r
3, 7, and 14 days at 30"C, before applying the 5-hour incubation test, was also dealt
with, and it wafol found that the inhibitory effect of both metallic salts decreased, to some
extent, with prolongation of the preincubation time.
8
The effect of heavy metal salts used as micronutrients on the urease activity in
Siberian soils was studied by Gamzikova and Gamzikov (1971). Samples taken from
the 0-20-cm layer of a chernozem and a grey forest soil were treated with one of five
metal salts at rates of 1 mg Mn, 0.5 mg Cu and Zn, 0.3 mg Co and Mo per 100 g soil.
The control samples received no heavy metal salt. The samples moistened to 60% of
water-holding capacity (WHC) were incubated at 2X~C for 21 days, then submitted to
urease activity determination. In the chemozem, urease activity was reduced by Cu,
stimulated by Mn and Mo, and not affected by Zn and Co. In the forest soil, Zn and Co
reduced, Mo stimulated, and Mn and Cu did not affect urease activity.
Pel'tser (1972) studied the effect of Cu(N0 3h, HgCh, and AgNO] on urea
hydrolysis in a slightly acid (pH 6.7) soddy-podzolic soil (Timiryazev Agricultural
Academy, Moscow), by using 14C-labeled urea and assaying the released 14C0 2 • The
reaction mixtures were prepared from 10 mg of urea and 15 rnl of 0.1 % solution of
metallic salt for 100 g of soil. During incubation (at 22QC), moisture content of the soil
was maintained at 60% of WHC. The results showed that complete hydrolysis of urea
took place in about 1.5 days in the control (untreated) soil, in 2 days in the Cu(N0 3 h
treatment, and in nearly 3 days in the HgCl 2 and AgN0 3 treatments.
Kozlovskaya el af. (1972) and Runkov el af. (1974) found that CUS04 used in a high
concentration (X4 mM in the reaction mixtures) strongly inhibited urease activity in peat
bog soils.
In two Sudan soils (clay and sandy loam), 1 rnl of 1% HgCl 2 solution added to 109
of soil stopped urease activity (Said, 1972).
Studying the factors affecting urea hydrolysis in three Alberta soils (a cultivated silt
loam and a virgin and a cultivated loam), Gould et af. (1973) added 5 ml of 1% HgCl 2
solution to 25-g soil samples to stop urease activity.
Lloyd and Sheaffe (1973) used HgCh at 0.1 % final concentration in reaction
mixtures for complete inhibition of urease activity in different samples of an Australian
red-yellow podzolic soil.
Andersson and Bengtsson (1975) studied samples of spruce needle mor collected in
Dalby Kronopark (located in the area of Lund, Sweden). Fresh samples, each equivalent
to 1 g dry matter, were treated with copper or/and zinc acetate solution corresponding to
0,200, 1,000,4,000, 10,000, and 25,000 ppm Cu, Zn, and Cu+Zn (on dry matter basis).
The samples were incubated at 22 QC for 1 and 5 weeks, then analyzed for determination
of their urease activity. It was found that the urease activity decreased significantly at
added metal concentrations ~ 1,000 ppm (after 1 week) and ~ 4,000 ppm (after 5
weeks). The activity could not be detected at ~ 10,000 ppm Cu+Zn and Cu and at
25,000 ppm Zn after both incubation times.
Tabatabai (1977) selected six Iowa soils with a wide range in pH, texture, organic
matter content, and urease activity for studying the effect of numerous metallic
compounds on soil urease activity, assayed by the method of Tabatabai and Brenmer
(1972). The air-dry soil samples (5 g) were treated with 1.5 rnl solution containing
either 2.5 or 25 ~moles of test compound (0.5 or 2.5 11TI10les/g soil), then 0.2 m! of
toluene, 7.5 ml of 0.05 M Tris buffer (pH 9.0), and 1 rnl of 0.2 M urea solution were
added. TIle reaction mixtures were incubated at 37<!C for 2 hours, after which time iliey
were brought to 50 ml with 2.5 M KCI solution containing 100 ppm of Ag2 S04 (as
urease inhibitor) for extracting the NH4 + that had formed. This was then analyzed by
9
steam distillation with MgO. The control reacti<m mixtures contained 1.5 ml of distilled
water instead of the test compound solution.
The results indicated that at the higher rate the heavy-metallic cations inhibited
urease activity in the six soils studied, in the following order:
Ag+ > Hg2+> Cu2+ ~ Cd2+ > Zn2 + > Sn2+ > Mn 2+.
Generally, Fe3+ and Cu 2+ had a str<mger effect than Fe2 + and Cu +, respectively. Also
at the higher rate, Ni 2+, Co2+, Pb2+, Cr3+, V4+, and Mo6+ inhibited urease activity only in
some soils. Lead nitrate was more effective than lead acetate in three of the soils, had a
similar effect as lead acetate in two soils, and was less effective than lead acetate in <me
soil. At the lower rate, the inhibitory effect of all metallic cations was weaker.
By applying the 5-hour incubation test of Douglas and Bremner (1971), Osminkina
and Mir7.aev (1979) studied the kinetics of urea hydrolysis in a calcareous grey soil
from Uzbekistan. The soil samples were treated with CUS04 at rates ranging from 0.01
to 0.1 mg Cu/g soil. The conclusion was drawn that the inhibitory effect of Cu 2+ on
urease activity of this soil corresponded to a reversible competitive inhibition,
characterized by an inhibition constant (ki) equal to 4.9 mg Cu 2+/l 00 g soil.
The English inventors Lewis and Slater (1979a,b) used ferric salts (nitrate, sulfate,
chloride), oxide and hydroxide, and ir<m ore (89.2% Fe203.H20) for obtaining fertilizer
compositions in which urea is complexed or physically mixed with a Fe compound.
Preferred are the compositions containing complexed urea together with different
amounts of free (uncomplexed) urea. The ratio between the number of Fe atoms and the
number of urea molecules should be at least 1:40. The preferred ratios range from 1:5 to
1:20. Laboratory experiments showed that these compositions applied 00 various soils
lost less arnm(mia through volatilization than treatments with urea alone. They were
applied at rates equivalent to 60-330 kglha. pH of the soils varied between 5.0 and 8.1,
and their moisture content ranged from 1.5 to 15.0%.
In a laboratory experiment Zukowska-Wieszczek (1979) added an aqueous PbO
suspension to samples taken from the 5-10-cm layer of four park soils and three street
lawn soils in Warsaw, Poland. Rates of addition were 0,10,50,100,200, and 400 ppm
Pb. Urease activity in the samples was measured after their 6-week incubation at
ambient temperature. It was found that PbO addition at each rate led to inhibition of
urease activity in each soil and the inhibition degree increased with the increasing rate
of PbO. The initially more urease-active park soils were less sL'I1sitive to inhibition than
the initially less urease-active street lawn soils. For example, 50 ppm Pb caused 20 and
81 % inhibitions in urease activity of the park soils and street lawn soils, respectively.
The inhibition became total at 200 ppm Pb in the park soils and at 100 ppm in the street
lawn soils. .
In a short report, the Russian investigators Zyrin el af. (1980) described pot
experiments in which soddy-pod7£llic, chernozemic, and peat bog soils were treated
with Cd or Pb nitrate at different rates, in the spring of 1978. The urease activity was
determined in March and June 1979. At the rate of 100 mg Cd/kg soil, the soddy-
pod7£llic soils exhibited about 50%, whereas the chemozemic and peat bog soils showed
80-90% of the urease activity measured in the untreated controls. Lead was less
inhibitory. Thus, at the rate of 2,000 mg Pb/kg soil, inhibition of urease activity was less
than 20% in the soddy-podwlic soils and 0% in the chernoz$!mic and peat bog soils.
Schinner el af. (19KO) installed experimental plots in a forest nursery on alluvial soil
in Austria. Cembra pine (Pinus cembra) seeds were sown in the plots. In May 1978,
10
when the seeds began to germinate, the plots were treated with mineral fertilizers (6 g
N, 6 g P2 0 S, and 9 g Klm2 ) with or without CUS04 (25 g/m2 ). Untreated plots served for
comparison. After 6 weeks, the soil urease activity was assayed and it was found that
the activity represented 77% in the soil treated with NPK and 48% in the soil treated
with NPK + CUS04 in comparison with that of the untreated soil (100%). This means
that CUS04, used at a high rate, intensified the depressive effect of mineral fertilizers on
the soil urease activity.
Daif and van Beusichem (1981) selected eight soils in the province of Badajoz,
Spain to obtain a wide range of properties. Samples taken from the 0-30-cm soil layer
wt.'fe allowed to air-dry, then crushed to pass a 2-mm screen and used for determination
of urease activity in reaction mixtures, to which Fe, Cu or Zn was added as the sulfate
of the bivalent cation. Rates of addition were: 0, 5, 10 or 20 Ilg Fe, Cu or Zn/g soil. The
results showed that the inhibitory effect of the metallic ions was obvious only when
they had been applied at the rate of 20 Ilg/g soil. The degree of inhibition varied from 7
to 20% depending on the soil and metallic ion. Thus, the light-textured soils were more
sensitive to inhibition than the heavier ones. Cu2+ was more inhibitory than Fe2+ or Zn 2+.
The effect of the three metallic ions (applied only at the rate of 20 Ilg/g soil) on the
microbial production of urease was also studied, but only in two soils. It was found that
these ions, although applied at a relatively high rate, did not inhibit the synthesis of
urease by soil microorganisms.
Marusina (1981) and Krasnova' (1985) described a field experiment, carried out on
1_m2 microplots installed on a soddy-podzolic soil in Russia. The experiment comprised
four variants:l. NPK-fertilized; 2. NPK-fertilized and treated with NiCb; 3. NPK-
fertilized and treated with ZnCb; 4. NPK-fertilized and treated with MnCh. The
fertilizers and heavy metal chlorides were introduced into the O-S-cm layer of the plots
at the following rates: 150 or 60 kg Nlha as ammonium nitrate, 100 or 60 kg Plha as
superphosphate, 100 or 60 kg Klha as potassium chloride; 120 mg Ni, 150 mg Zn or
750 mg Mn per kg soil. Within each variant there were plots left unsown and plots sown
with barley. During the vegetation period, urease activity was determined in the 0-5-
and S-10-cm layers. In both sown and unsown plots, urease activity decreased under the
influence of heavy metals. Ni and Mn were more inhibitory than was Zn. The inhibition
was more pronounced in the O-S-cm layer than in the 5-1 O-cm one.
According to the data published by Tu (1981a), treatment of the samples of a
Canadian clay loam soil (pH 7.2) with HgCh (70 Ilg/g soil) brought about a significant
inhibition of urease activity; degree of inhibition was 58.3% after 2 days and 29.4%
after 7 days of incubation. But in samples of a Canadian organic soil (PH 7.2), the same
amount of HgCb stimulated urease activity; the stimulation was insignificant after 7
days and significant after 14 days of incubation (Tu, 1981b). In another study
(Tu, I 990), urease activity was determined in HgClrtreated samples of a sandy loam
(pH 7.6) and an organic soil (pH 6.8) from Canada. Incubation lasted 7 and 14 days (at
2S"C). HgCh significantly inhibited urease activity of the sandy loam (67 and 81%
inl1ibitions after 7 and 14 days of incubation, respectively). In the organic soil, urease
activity was not affected by HgCl2 (after 7 days) or was significantly enhanced by it
(after 14 days). In other studies, HgCh, applied at a rate of 80 Ilg/g soil, inhibited
'N.M. Krasnova is N.M. M!ll1Isina's new name; she got it, probably. after her marriage.
11
signifil:antiy or did not affect urease activity of the sandy loam incubated for 7 days (Tu,
1992a,b).
By increasing the rate of Cu2+, Zn 2 +, and Mn2+ from 500 to 1,000 ppm in the acid
(pH 5.9) soil studied by the Japanese investigators Komai et 01. (1981), the degree of
the inhibition of urease al:tivity changed only to a small extent, nan1cly from 83 to 87%,
from 64 to 71 (Yo, and from 41 to 40%, respecti vel y.
NOT (1982) assayed urease activity in real:tion mixtures prepared from 109 of soil
(on dry weight basis), 1,200 Ilg of Ag+ or Cu2+, 5 mg of urea and distilled water, and
incubated at 37QC. Reaction mixtures without inhibitors were the controls. After
incubation- the residual urea was determined. Ag+ wmpletely inhibited urease activity
in each of the four l\1alaysian soils studied. eu 2 + had a similar effect in two of the soils
(clay loam, pH 3.8; sandy clay loam, pH 5.1) and a partial (77.5 and 91.7%) inhibitory
effect in the other two soils (sandy clay loam, pH 4.8; loamy sand, pH 5.1).
Grigoryan (1982) added 1-10 mg of Cu, Pb, Ni, Mn (in form of nitrates) or Mo (as
ammonium molybdate) to 200-g samples of flood plain meadow soils, brown forest
soils, and leached chernozems from Armenia. The heavy metalfol, especially Mn at the
lowest rate stimulated urease activity in all soilfol. But folignificant negative correlations
were registered between the higher rates of each heavy metal and urease activity in all
soils.
Krasnova (1983) prepared mixtures from samples of a s(xtdy-podzolic soil and
solution of Mn, Zn or Ni sulfate (at rates of 250-2,000 mg Mn, 25-200 mg Zn or 20-
160 mg Ni/kg foloil). The mixtures moistened to 60% ofWHC were incubated at 30!!C
for 2 weeks, then analyzed for determination of their urease activity. The activity was
inhibited in parallel with tbe rate of eal:h heavy metal. For comparing the inhibitory
effect of the three heavy metals, some data referring to four samples are specified
below: urease activity (expressed in mg of NH.1 produced by 1 g of soil in 24 hours) was
0.10 (untreated control), 0.60 (250 mg Mnlkg soi!), 0.58 (200 mg Zn/kg soil), and 0.40
(160 mg Ni/kg soi\). Thus, the metallic ions inhibited urease activity in the order: Ni >
Zn>Mn.
For studying the effect of Pb and Cd on soil urease activity, Brunner and Schim1er
(1984) used samples taken from the Ah-horizon of an alluvial soil in Tyrol, Austria. The
samples were treated with 0, 50, 200, and 1,000 Ilg ofPb (as lead acetate)/g soil or with
(), 0.5, 5, and 50 Ilg of Cd (as cadmium sulfate)/g soil. The samples were kept at 22°C
for 16 weeks, during which the water content of samples was maintained at 15-20% and
the urease activity was measured at 3-4-day intervals during the first weeks, then at 4-
week intervals. The activity oscillated in both Pb- and Cd-treated samples at each Pb
and Cd concentration: the activity increased during tbe first week, then decreased and
lat er increased again and, at week 16, approached the values found in the untreated
samples.
The Russian investigators Skvortsova et al. (l984) stated that in a soddy-podzolic
soil, treated with mercuric nitrate solution at rates of 0, t, 2.5, 10, 20, 50, 200, and 500
mg Hglkg soil, urease activity suffered a nearly lO-fold decrease under the influence of
50 mg Hg/kg soil. However, numerical data on the activity are not presented in their
paper.
Badr EI-Din et al. (1985) applied the 5-hour test of Douglas and Bremner (1971) to
study the effect of 15 heavy metal salts on urease activity in three Egyptian soils. The
salts were used at a rate of 50 ppm (soil basis). One can see from Table 2 that the
cations, in respect of their inhibitory effect, can be arranged in the following order:
Ag+ > Hg 2+> Cu+ =Cu 2+ > Co2+ > Fe2 += Fe3+ > Zn2+ > Be+ > Sn2+ > Pb2+.
Table 2 also shows that the valency state of Cu and Fe and the nature of anions had
almost no influence on the inhibitory action. Urease activity in the loamy sand was
more sensitive to inhibition than that in the silty clay.
In the case of the silty clay, longer incubations were also carried out to study the
effect of Cu(ll), Fe(III), and Co(ll) sulfates on urease activity and volatilization of
ammonia from urea. In the absence of inhibitors, urea was hydrolyzed in a single day,
but in their presence complete hydrolysis of urea required 7 days. Cu 2 + proved to be
more effective than Fe3+ or C0 2+ in retarding urea hydrolysis and reducing volatile NHJ
losses.
Dalton et al. (11.)85) studied nine soils from western Oregon and one from eastern
North Carolina. In an experiment, 25-g (dry weight basis) soil samples, after the
addition of 12.5 ml of a solution containing glucose and urea (2 mg and 114 llg/g dry
soil, respectively) with or without NiCh (0.25 llmoles/g dry soil), were preincubated at
2 7"C. Urease assays were performed at both zero time and after 3 days of preincubation.
Urease activity was detem1ined by measurement of 14C02 evolved from 14C-urea in
reaction mixtures prepared from 1 g of soil (dry weigl1t basis), 0.3 ml of 1 M 14C_urea
(3.4 llCi/nID10l) in 5 ml Hepes buffer (pH 7.1) and sufficient Hepes to bring the total
soil water volume to I ml, and then incubated at 37~C for I hour.
At zero time of preincubation, urease activity of the Ni-treated sample versus the
untreated sample did not significantly change in any of the I () soils studied. After 3 days
of preincubation, urease activity showed increased values in all soils, in both Ni-treated
and untreated samples. A comparison of the samples revealed that NiCl 2 at the rate
13
applied did not have significant effect on urease activity in the nine Oregon soils,
whereas in the North Carolina soil (a low-nickel loamy sand; total Ni content = 13
ppm), Ni at the same 0.25 ~oles/g dry soil rate caused a significant (2.5-fold)
stimulation of urease activity.
With this low-nickel soil other preincubation experiments were also carried out, all
followed by urease assays. The results showed that urease activity was highest in
samples treated with 5 ~oles Ni/g dry soil. At the concentration of 5 I!moles metal/g
dry soil, Cu2+ and C02+ (in form of chlorides) significantly inhibited urease activity, but
Mn2+ and Fe 2+ (also in form of chlorides) had no such effects. Nickel became inhibitory
at a concentration of 50 I!moles (2,940 I!g)/g dry soil.
Ushikubo et al. (1985) studied the effect of Cd, Hg, and Cr on urease activity in a
sandy loam soil (PH 6.4) and a loamy sand soil (pH 6.9) collected from a woodlot and
an uncultivated old field, respectively, near the Inland Lake Research and Study Center
of Michigan State University. Fifty-g air-dried soil samples were treated with 100 ml of
Cd, Hg or Cr salt solution containing 0.1, 0.5 or 1 mg Cd or HgIl or 10, 25 or 50 mg
Cr/l. The untreated (control) samples received 100 ml of water. The mixtures were
preincubated at room temperature for 1,2,5 or 10 days, then filtered; the residue (soil)
was air-dried, then submitted to enzymological analyses.
The urease-inhibiting effect of heavy metals showed a tendency to increase with
their concentration and with the preincubation time. The percent inhibitions of urease
activity in the two extreme cases (lowest heavy metal concentration and shortest, i.e.. 1-
day preincubation, and highest heavy metal concentration and longest, i.e.. lO-day
preincubation, respectively) were the following: 6.92 and 40.48 (Cd), 29.73 and 40.48
(Hg), and 2.78 and 19.05 (Cr), respectively, in the sandy loam; 20,00 and 46.67 (Cd and
Hg), and 20.00 and 47.62 (Cr), respectively, in the loamy sand.
In sandy loam, the untreated samples were 14 times more urease-active than
untreated samples of loamy sand, and less sensitive to inhibition than the loamy sand. In
the sandy loam, Cd and Hg, although low in concentration, were more inhibitory than
Cr, in much higher concentrations. In the loamy sand, the inhibiting effect of Cd, Hg,
and Cr was similar.
Studying an Indian clay loam soil, Yadav et al. (1986) treated soil samples with salts
(sulfates and chlorides) of heavy metals, at three rates relative to the cations (100, 500,
and 1,000 ppm on soil weight basis). The reaction mixtures, consisting of 20 g of soil, 1
ml of toluene, 5 ml of urea solution containing 200 ppm N and aqueous salt solution,
were incubated at 28!'C for 12 hours. The residual urea was then extracted and
determined. At the rate of 100 ppm, Ag+ and Hi+ caused more than 60%, whereas
Cu2 +, Zn2 +, C02 +, and Cd2+ exhibited 10-30% inhibitions of urease activity. The
inhibitions given by Pbz+, Cr.l+, Mnz+, and Fe z+ were lower than 10%. At higher
concentrations, the metallic cations had a stronger inhibitory effect. The highest (96%)
inhibition was brought about by 1,000 ppm Ag+ and Hg2+, followed by Cu2 + and Cd2+
(60% inhibition); Pb2+ showed the least effect (25% inhibition). The inhibitory effect of
cations at 1,000 ppm presented the following order:
~=H~>~>~>~>~>~>~>~>Mn2+>~~
The time necessary for complete hydrolysis of urea was also estimated in 20-g soil
samples treated with Cr3 +, Cu2+ or Hg2+ at 100,500, and 1,000 ppm and urea at 200 ppm
N, and incubated at 28!'C. In the no inhibitor control sample, urea hydrolysis was
complete in 3 days. Cr 3+ at 100 ppm did not prolong this duration. In samples treated
14
with Cr3+ at 500 and 1,000 ppm and in samples treated with Cu 2+ at the three rates,
complete hydrolysis of urea took place in 7 days. In the presence of Hg2+, complete
hydrolysis of urea required 14-2X days.
Skujins et at. (19X6) used surface (0-20 cm) samples collected from a deciduous
forest soil of sandy clay loam texture (pH 7.0), in the area of Ultuna, Sweden. After air-
drying, the samplefl were sprayed separately with CuCh and CrCh solutions at
concentrations of 50, 200, 500, and 1,000 ~g Cu or Cr/g soil. Additional water was
given up to XO% of WHC, which was maintained constant during the incubation of
samples at 20"C for 20 days before determination of their urease activity. The activity
was found to decreased logaritlunically with increasing amounts of Cu 2+ over the entire
range from 0 to 1,000 ~g Cu/g soil. The initial decrease of urease activity was more
pronounced in the Cr-treated samples. The logarithmic part of the response to Cr,
however, wafl refltricted to concentrations s 200 ~g Cr/g soil. At Cr concentrations>
200 ~g/g soil, the reflponfle to Cr was retarded toward'l a limit value of 50%.
Doelman and Haanstra (19X6) studied samples of five different soils (sand, sandy
loam, silty loam, clay, and sandy peat) collected from various areas in the Netherlands.
The field-moist samples of the five soils (55-70% of WHC) were amended with 0, 55,
150, 400, 1,000, 3,000, and X,OOO mg/kg Cd, Cr, Cu, Ni, Pb, and Zn (in form of
chlorides), then incubated at 20 QC. Urease activity was measured after 6 weeks and 18
months of incubation. The results showed that the ecological dose-50% (ED50) -
dcfincd as the heavy metal concentration (in mgikg soil), at which urease activity is half
of the initial (uninhibited) level- tended to decrease (i.e .. the inhibiting effect of heavy
metal tended to increase) from week 6 to month 1X for Cd, Cr, Cu, and Zn. For Ni and
Pb, ED50 generally did not change significantly (i.e.. the inhibiting effect stabilized) in
sandy loam, silty loam, and sandy peat, increased in sand and decreased in clay in the
period between week 6 and month 1X. The average ED50 values of Zn were, after 18
months, the lowest (varying between 100 and 3(0), which means that the inhibiting
t-'ffect of Zn on urease activity was the strongest.
Tn an experiment carried out in vegetation pots (Badura et at., 19X6), surface soil
samples taken from a Polish beech forest were treated with Zn orland Cd sulfate at rates
of 5,000 ppm Zn or Cd and 2,500 ppm Zn + 2,500 ppm Cd, respectively, and incubated
for 42 days. Urease activity, determined several times during the incubation period, did
not show any considerable differences between the Zn-, Cd-, and Zn+Cd-treated
samples and between these samples and the untreated ones.
The Chinese investigators Xue and Li (1987) compared the inhibitory effect of three
Cu(II) salts and of ZnS04 on the urease activity in a periodically water-logged paddy
soil. The reaction mixtures were prepared from 5 g of dry soil, 10 rnl of 10% urea
solution also containing the inhibitor at 20-100 ppm (on soil weight basis), and
incubated at 37"C for 48 hours. The results in Table 3 show that CUS04 was the most
effective and ZnS04 the least effective inhibitor. The degree of inhibition increased with
increasing inhibitor concentration, but did not become 100% at the highest inhibitor
concentration either. It was also established that the inhibitory effect of CUS04 was
evident even after 12 days of incubation and this effect of CUS04 showed a tendency to
decrease with increasing urea concentration (1, 2, 4,6, X, and 10%) and to increase with
increasing temperature (22, 37, and 45"C).
15
nitrate) or Zn (as oxide)/kg soil. No heavy metal was added to the control soils. The test
plant was barley. At three growth phases (germination, tillering, and ripening) of barley,
soil urease activity was determined. The results led to the conclusions that the inhibitory
effect of heavy metals increased in the order: Cd > Pb ~ Zn; the soddy-podzolic soils
were more sensitive to inhibition than was the common chernozem. For example, urease
activity in a soddy-podzolic soil decreased to 7S% under the effect of 100 mg Cd/kg soil
and to 70% when the treatments were done with 2,000 mg Pb or Zn/kg soil. The
minimum heavy metal addition for a statistically significant inhibition of urease activity
was 10 mg Cd or 500 mg Pb or 500 mg Zn/kg soil in a soddy-podzolic soil and 20 mg
Cd or 2,000 mg Pb/kg soil in the common chernozem (in which Zn even at the highest
rate applied - 2,000 mg/kg soil- had no inhibitory effect).
Kucharski and Niklewska (1992) performed a pot experiment using a brown soil of
heavy loamy sand texture (pH 7.1) from Poland. Zn (as sulfate) was added to the soil at
rates of 0, 10, 100, and 1,000 ppm. The test plant was broadbean. Soil urease activity
wa" assessed at the cutting of broadbean (at time of its flowering). The activity showed
10 and 4% increases in the treatments with 10 and 100 ppm Zn, respectively, and a
slight (3.S%) decrease at the 1,000 ppm Zn rate.
Gupta and Chaudhry (1994) studied the effect of four heavy metals on urea
hydrolysis in surface (0-15 cm) samples of a sandy loam soil collected from the farm of
the Agricultural University in Hisar, Haryana, India. The samples were amended with
urea (OJ g N/kg soil) and with Ni, Zn, Pb or Hg at a rate of 0, 0.2 or 0.4 g/kg soil, then
moistened to 60% ofWHC and incubated at 15 or 30QC. The heavy metals retarded urea
hydrolysis in the order: Hg> Zn> Ni > Pb at both temperatures. However, irrespective
of the metal added, urea was completely hydrolyzed within 7 and 14 days at 30Q C and
15~C, respectively.
As a part of a complex study on the effects of Cr(VI) on soil biological properties,
Speir et at. (1995) determined urease activity in three New Zealand soils (Egmont black
loam, Kaitoke silt loam, and Foxton loamy sand). TIle topsoil samples, each equivalent
to 100 g dry weight, were moistened then amended with 10 ml of K2 Cr 2 0 7 solutions at a
concentration range of 0-50 Ilmoles Cr(VI)/g soil or with water in the controls. Urease
activity was measured 3 days after amendment and 'again on the same soil samples 60
days after amendment. The samples were stored at room temperature (l5-22°C) over the
intervening period. In the three soils studied, the ecological dose-50% (EDSO), i. e.. the
Cr(VI) concentration or "dose" (Ilmoles/g soil), resulting in 50% inhibition of urease
activity, was 41.5,22.2, and 20.3, respectively, after 3 days and only 5.61, 9.56, and
4.30, respectively, after 60 days, indicating that the inhibitory effect of Cr(VI) increased
with time.
The effects of Cd, Zn, and Pb additions on urease activity in soddy-podzolic soils (a
clay loam, pH 4.5 and a loamy sand, pH 4.05) from the Moscow region were studied in
pot experiments and under field conditions (in microplots) by Lebedeva et at. (199S).
Rates of additions per kg soil were 0,10,15 or 20 mg Cd (as nitrate), 0, SO, 100,300,
400 or 500 mg Zn (as sulfate) or Pb (as nitrate). Before the addition of heavy metal
saits, the soils were NPK-fertilized and limed or not limed. Urease activity was assessed
in 1991 and 1992, during the growth period of test plants (carrot, beet, dill or rye). Cd
addition even at the highest rate led to no inhibition of urease activity in 1991 and to a
slight inhibition in 1992. Contrarily, Zn and Pb caused inhibition of urease activity in
both years. According to calculations, the minimum rate for a significant inhibition of
17
urease activity averaged 125 mg Zn and 90 mg Pb/kg soil. Lime (20-40 t/ha) addition
brought about a considerable increase in urease activity of both untreated and Cd-, Zn-
or Pb-treated soils. All results were similar in the two soils studied and in the pot and
microplot experiments.
Kozdr6j (1995) used a sandy loam soil (pH 4.5) from the O-lO-cm layer in a beech
forest of the sanctuary "Zloty Potok" (Poland). The 1OO-g soil samples moistened to
40% ofWHC were treated with 0 or 1 or 2 mg of Cu or Cd as chlorides. Urease activity,
determined after I, 3, 5, and 7 weeks of incubation, was inhibited to a larger extent by 2
rather than by 1 mg of heavy metal and by Cu more than Cd during the whole
incubation period, excepting the treatment with 1 mg Cd, in which urease activity was
significantly higher than in the other treatments and at weeks 3, 5, and 7 reached the
level recorded in the control samples.
Richards (1995) patented a urea fertilizer containing urease-inhibiting ferric nitrate
and urea. The fertilizer may be in the form of a liquid urea-ammonium nitrate (solution
or slurry). The molar ratio offerric nitrate-urea complex/urea in the fertilizer is 1: 1-15.
Studying the effects of heavy metals on biological properties, including urease
activity of Egyptian soils, Hernida et al. (1997) added Cu or Zn (as sulfates) at rates of
0, 0.2 or 2 ~lg/g soil to 500-g samples of a clay soil (pH 7.5) and a sandy soil (pH 7.4)
from the area of Assiut. The samples moistened to 28% ofWHC were incubated at 28 QC
and analyzed after 1, 4, and 12 weeks of incubation. It was found after each incubation
time that both Cu and Zn inhibited urease activity in both soils. At the lower rate Cu
was more inhibitory (significantly in the clay soil and insignificantly in the sandy soil)
than Zn. But at the higher rate, both Cu and Zn caused complete inhibition of urease
activity in both soils.
Kucharski (1997) summarized the results of pot experiments in which the effects of
Zn, Pb, and Cd on soil enzymes and yield of the test plant, yellow lupine, were studied.
Some of the results are reproduced in Table 4.
TABLE 4. EtTects ofZn, Ph, and Cd on soil urease and dehydrogenese activities and yield of yellow lupine"
Rate of addition Urease activity Dehydrogenase activity Lupine yield
Heavy metal
(mglkg soil) (%) (%) (%)
2 102.1 1.11.0 79.1
Zn 4 86.5 149.6 SUI
40 84.4 113.8 43.2
50 89.9 99.0 96.4
Ph 500 77.5 45.5 73.9
1000 60.9 29.6 49.7
0.5 91.0 83.8 57.7
Cd 5 68.3 51.8 0
15 71.6 34.2 0
"Adapted Irom Kucharski (1997).
lbe activities and yield are expressed as percentages of the values registered in the untreated, control soil.
It is evident from this table that at the higher rates each heavy metal greatly reduced
urease activity and, excepting Zn, dehydrogenase activity too. Phytotoxicity of Cd
exceeded that of Zn and Pb.
18
Leir6s et al. (1999) treated 400-g samples, taken from the Ah horizon (0-5 cm) of a
loamy soil developed under Atlantic oakwood climax vegetation, with CuCI 2 at rates of
0, 1,2,5, and 10 mg Cu/g soil. The samples were then moistened, and analyzed before
and after incubation (28 days at 25°C) for determination of several biochemical
parameters, including urease activity. This activity (expressed in fllUol NH3/g soil/hour)
decreased with increasing Cu rate both before incubation (19.10, 13.84,6.13,2.09, and
1.01) and after incubation (23.74, 15.77,9.55,2.32, and 0.48).
Blaise et al. (1996, 1997) described two laboratory experiments in which a loess
brown earth (pH 6.8) from cultivated fields at Dumast, Freising, Germany was used for
studying the effects of agricultural grade pyrite (consisting of pyrite, FeS2, and other
sulfides and containing 20% Fe and 22% S) on the volatilization of ammonia and
emission of nitrous oxide from urea-treated samples.
Tn the first experiment, 200-g air-dried soil samples were flooded to a depth of 2 cm,
amended with glucose (500 mg C/kg soil) for enhancing reduced conditions and then
pre incubated at 20QC for 2 weeks. After preincubation, two series of samples were
amended with urea (100 mg N/kg soil) with and without ground pyrite (equivalent to
100 mg S/kg soil) and incubated for 7 days, during which the amounts of NH3
volatilized from one series of samples and the amounts of NzO emitted from the other
series of samples were determined. The cumulative losses of N from the initially added
20 mg urca-NI200-g soil sample were thc following: 1.49 mg N as NH3 and 2.00 mg N
as N20 from the samples amended with urea only, and 0.94 mg N as NH3 and 0.91 mg
N as NzO from the samples amended with urea + pyrite, i.e., the total gaseous N loss of
3.49 mg N/sample was reduced, in the presence of pyrite, to 1.85 mg N/sample, thus the
reduction was 47.3%.
In the second experiment, 100-g air-dried soil samples were moistened to 60% of
WHC, then preincubated at 20Q C for 2 weeks. Following preincubation, the samples
were treated with urea (100 mg N/kg soil) and with 0,0.1,0.5, 1,5, and 10 g of ground
pyrite/kg soil, then submitted to incubation. For estimation of NH3 volatilization the
incubation lasted 15 days. The cumulative NH3-N loss from the initially added 10 mg
urea-N/IOO-g soil sample was 0.72 mg in the urea-alone treatment and significantly
less in the urea + pyrite treatments. The reduction in NH3 volatilization was 7-22% at
the lower rates (0.1-1 glkg soil) of pyrite, and 53 and 86% at the 5 and 109 pyrite/kg
soil, respectively.
Reduction of NH3 volatilization from urea was attributed to the acidic nature of
pyrite (to sulfuric acid formed upon oxidation of pyrite), but the role of pyrite as a
urease inhibitor is not excluded, as it is known (Bayan and Eivazi, 1999) that the iron
oxides (goethite, hematite) inhibit urease activity. To explain the reduction of N 20
emission from urca, Blaise et al. consider it most plausible that pyrite, due to the
sulfides it contains is directly involved in inhibiting at least one of the reductase
enzymes catalyzing the processes NO,' --> NO z' --> NO --> N 20, because pyrite reduced
the N 20 emission from KN0 3 also.
In a previous experiment, Blaise and Prasad (1995) used urea at a much higher rate:
100-g samples of an Indian sandy clay loam soil (pH 8.1) were amended with 1 part
urea (1,000 mg N/kg soil) blended with two parts pyrite. Soil samples amended only
with urea were the controls. All samples were then incubated under aerobic and
anaerobic conditions for 8 days, during which the volatile ammonia was determined.
19
The cumulative NH3 losses from the applied urea-N were significantly reduced by
pyrite from 27.5% (control) to 8.9% under aerobic conditions, and from 19.3% (control)
to 16.9% under anaerobic conditions.
In a greenhouse experiment, Wyszkowska et al. (2001) treated samples (3 kg/pot) of
a loamy sand soil (pH in KCI 6.6) with K2Cr207 at rates of 0, 40, 80, and 120 mg Cr/kg
soil. Nutrients were also applied (glkg soil): 0.15 N as urea, 0.1 Pas K2HP04, 0.15 K as
K2HP04 + KCl, and 0.05 Mg as MgS04.7H20. Some pots were amended with fmely
ground barley straw (4 g/kg soil) and some pots were sown with oats (25 plants/pot).
There were four variants: 1. not amended with straw and not sown; 2. amended with
straw but not sown; 3. not amended with straw but sown; and 4. amended with straw
and sown. The plants were harvested at panicle emergence stage (at day 51 of growth).
Dry matter yield of oats was determined and the soil was submitted to enzymological
and microbiological analyses.
Urease activity in soil not treated with Cr increased in the four variants in the
order: 1 < 2 < 3 < 4. Chromium inhibited urease activity in all variants. Within the same
variant the degree of inhibition increased with the increasing rate of Cr, but the residual
urease activity at the same Cr rate presented in the four variants the same order as
urease activity in the untreated soil. In other words, the amendment with straw and/or
growth of plants led to diminution of the urease-inhibiting effect of Cr.
Moreno et al. (2001) treated 100-g samples of two Italian soils (a sandy loam, pH
8.1 and a sand, pH 4.8) with 5 ml of CdS04 solutions to give a Cd concentration ranging
from 3 to 4,000 mg/kg soil. Untreated samples served as controls. The soil moisture was
adjusted to 55% of WHC, then all samples were incubated at 25°C and submitted to
several analyses, including determination of urease activity, after 3 hours, 7 and 28 days
of incubation. The analytical data were used for calculation of the ecological dose-50%
(EDso).
The EDso of urease activity, i.e., the Cd concentration (in mg/kg soil) that inhibited
urease activity by 50%, had the following values after the three incubation times:
1,538.5 (3 hours), 1,162.8 (7 days), and 1,190.5 (28 days) in the sandy loam, and
4,166.7 (3 hours), 909.1 (7 days), and no inhibition (28 days) in the sand. Thus, EDso
was lowest (and sensitivity of urease activity to Cd was highest) in both soils after 7
days of incubation. Urease activity in the sandy loam compared to urease activity in the
sand was more sensitive to Cd after both the 3-hour and 28-day incubations.
Mutatkar and Pritchett (J967) prepared mixtures from samples of two Florida soils: a
fine sandy soil (containing about 2% organic matter) and a muck (containing 62%
organic matter), by mixing the sandy soil with 8% (by weight) of muck and by adding
0, 90, 180, 360, and 720 ppm of Al in form of AI2(S04)3. The pH of the mixtures was
adjusted to 4.0, 4.8, 5.5 or 6.5 by adding either dilute HCl or NaOH. Urea was applied
at the rate of 200 ppm N (on soil mixture basis). Moisture content of mixtures was
brought to 15%. Incubation took place at 28°C; the NH4+ formed in the mixtures was
analyzed at 14-day intervals. The NH/ content increased as the pH increased. The
highest amount of NH4 + was produced in mixtures initially adjusted to pH 4.0,
regardless of the amounts of AI added. It seemed that urea was readily converted to
20
NH4 +, indicating that concentrations of AI3+ had little effect on urease activity and
ammonification processes.
Under the conditions of the 5-hour test, Bremner and Douglas (1971) established
that aluminium chloride (AICb), applied at a rate of 50 ppm Al (on soil weight basis)
did not exhibit any inhibitory effect on urease activity in two Iowa soils (silty clay loam
and clay loam) studied. At the same time, Tabatabai (1977) showed an inhibitory action
of AICh on urease activity in six soils, when the rate of AICh was 5 Ilmoles/g soil (the
inhibition varied between 12 and 50%). At the 0.5 flIDoles/g soil rate, which was applied
only to two of the soils, the degree of inhibition was 3 and 5%, respectively.
Lewis and Slater (1979 a,b) also used - besides mineral iron compounds (see page
9) - AI(N03h.9H 20 and Ah(S04)3.16H20 to obtain fertilizer compositions containing
complexed and uncomplexed urea which when applied on soils led to diminution of
ammonia volatilization from urea. The ratio between the number of Al atoms and the
number of urea molecules should be equal to at least 1:40. Compositions were also
prepared from three components: urea complexed with AI(N03)3 + urea complexed with
Fe(N03)3 + uncomplexed urea.
Badr EI-Din et al. (1985) established that in the three Egyptian soils studied (see
page 12), aluminium sulfate (50 ppm) caused 22.2, 27.2, and 25.8% inhibitions in the
urease activity assayed with the 5-hour test. Ae+ proved to be a weaker inhibitor than
Cu 2+, but a little stronger one than Co2+. AI3+ inhibited urease activity and volatilization
of ammonia from urea even during longer incubations, of at least 7 days. These effects
of Ae+ were weaker than those of Cu 2+, but more marked than those of Fe3+. It should
be mentioned that for the experiments with longer incubations only one soil (silty clay)
was used.
According to findings by Yadav et at. (1986), Ae+ added (in from of AlCb), at rates
of 100, 500, and 1,000 ppm, to samples of a clay loam soil had at each rate a weaker
inhibitory effect on urease activity than any of the heavy-metallic cations tested under
identical conditions (see page 13).
The urease-inhibited urea fertilizer patented by Richards (1995) (see also page 17)
may contain aluminium nitrate instead of ferric nitrate or both aluminium and ferric
nitrates. The molar ratio of aluminium nitrate-urea complex/urea is 1: 1-18.
In the relation between these salts and urea added to soil one can delineate two aspects:
- their effect on urease activity and urea hydrolysis in soil;
- their effect on volatilization of ammonia resulted from urea hydrolysis.
1.3.1. Effect ofAlkali Metal and Alkaline Earth Metal Salts on Urease Activity and Urea
Hydrolysis
Tomlinson (1964) mixed urea (100 ppm N) and a chemically equivalent amount ofKCl,
K2S04, KH 2 P04, CaCh, MgCl z or KF with samples of an English soil (noncalcareous
sandy loam, pH 6.5). The control was treated with urea alone. After 2 and 7 days of
incubation at lOoC, at a moisture level a little below field capacity, the NH/ released
from urea was detennined. Similar ~ + amounts were found in the salt-treated samples
as in the control. The only significant exception was the KH 2 P04 treatment, in which the
amount of N~ + after 2-day incubation (but not after 7-day incubation) was greater than
21
in the other treatments. This means that the neutral salts studied did not affect urea
hydrolysis.
Under the conditions of the 5-hour test, Bremner and Douglas (1971) established
that NaC!, Na2S04, KCl, CaC!2, and BaCh at a rate of 50 ppm cation (on soil weight
basis) did not inhibit urease activity in the two soils studied.
Tabatabai (1977) studied the influence of BaCh on urease activity in six soils,
through 2-hour incubations at 37°C. When the rate of BaCh was 5 Ilmoles/g soil, the
inhibitions were 2-3% (in three soils), 7% (in two soils), and 12% (in one soil). At a rate
of 0.5 J..l11loles/g soil, which was applied only to two of the soils, BaCh did not have any
effect on their urease activity.
Studying a leached chernozem (pH 6.6) from Armenia and a soddy-podzolic soil
(pH 4.5) from the Moscow region, Abramyan and Galstyan (1981) treated soil samples
(each 1 g) with Ca, Mg, K or Na chloride (1-12 mg cation) + 5 ml of 3% urea solution
in phosphate buffer (pH 6.7) + 0.2 ml of toluene. After incubation (30°C/24 hours), the
NH4 + released from urea was analyzed. In chemozem, CaCl2 and MgCh in low
concentrations (1-2 mg cation) stimulated and in higher concentrations (4-12 mg cation)
inhibited urease activity; KCl was stimulating and NaCI inhibitory in all the
concentrations. In podzol, the low concentrations of Ca, Mg, and Na chlorides had a
stimulating effect, whereas in higher concentrations manifested a tendency to inhibit
urease activity. KCl in all concentrations had, in this soil, too, a stimulating effect on
urease activity.
In continuation of these investigations, Abramyan (1982) worked with samples of
three Armenian soils: leached chernozem (pH 6.6), meliorated solonetz-solonchak (PH
7.6), and irrigated brown meadow soil (pH 8.1).
In an experiment, Abramyan studied the influence of the nature of anion in six
sodium salts (NaC!, Na2S04, Na2C03, Na2Si03, Na2B407, and CHrCOONa) on soil
urease activity. Each salt was added at a rate of 5 milliequivalents of Nail 00 g soil. In
each soil, the weakest inhibitory effect was produced by NaC! and Na2S04 .The most
inhibitory salts were Na2B407 (in chernozern and meadow soil) and Na2CO), Na2Si03,
and Na2B407 (in solonetz-solonchak). The other salts brought about inhibitions of
intermediary extent.
In another experiment, soil samples (100 g) were treated with the increasing
amounts of Na2C03 (1-10 milliequivalents of Na). Urease activity decreased with the
increasing rate of Na2CO], less markedly in the chernozern than in the other two soils.
For example, at 10 milliequivalents of Na, the chernozem retained 50% of its initial
urease activity, whereas urease was completely inactivated in the other two soils.
In a laboratory experiment performed by Fenn et af. (1981b), urea (at a rate
equivalent to 1,100 kg N/ha) with or without CaCh was applied on the surface of wetted
samples of a calcareous silty clay loam soil from Texas. Previously, the soil was
adjusted to 15% CaC0 3 by weight and received 1% (by wei gilt) fresh organic matter
(bluegrass clippings). CaCh was applied at chemically equivalent Ca:urea-N ratios of
0.25 and 0.50. After 3, 6, and 9 days of incubation at 32°C, the urea was extracted and
determined. The results proved that under the influence of CaCh the rate of urea
hydrolysis diminished. In samples treated with urea alone, urea was not detectable after
3 days of incubation, but in the CaCh-treated samples a significant part of the added
urea remained unhydrolyzed even after 9 days of incubation, namely 45-46% at
Ca:urea-N = 0.25 and 24-55% at Ca:urea-N = 0.50, respectively. As diminution of the
22
rate of urea hydrolysis was associated with diminution of the extractable Ca2+ content,
the authors assume the formation of some type of Ca-urea complex which is less
hydrolyzable than the uncomplexed urea.
EI-Shilmawi and EI-Shimi (198Ia) treated 5-g air-dried samples of two Egyptian
soils (alluvial clay soil, pH 7.85 and calcareous sandy loam, pH 8.43) with 15
milliequivalentsll 00 g soil of Na, K, Ca, and Mg sulfate, carbonate, and chloride, then
the samples were moistened up to 60% of WHC and incubated at 30°C for 30 days.
During incubation, urease activity was determined periodically.
The results indicated stimulating or inhibiting actions in dependence of the nature of
anions and cations from the added salts and of the nature of soils. Thus, the sulfates
stimulated urease activity of the alluvial soil in the order: Mg > Ca > K > Na. In the
calcareous soil, Mg and Ca sulfates stimulated urease activity, whereas Na and K
sulfates inhibited it. Ca and Mg carbonates highly accelerated the hydrolysis of urea in
the alluvial soil, but Na and K carbonates decreased it. In the calcareous soil, the effect
ofNa and K carbonates was stimulating, that of the MgC0 3 was inconsiderable and that
of CaC0 3 was inhibiting. The chlorides had a stimulating effect in the order K > Ca >
Mg > Na in the alluvial soil and manifested an inhibitory effect in the order Ca > Mg >
Na "" K in the calcareous soil. TIle most stimulating salts were CaCO.l in the alluvial soil
and K2CO) in the calcareous soil, whereas the most inhibiting salts were Na2CO.l in the
alluvial soil and CaCl 2 in the calcareous soil.
In other incubation experiments (30°C/30 days), EI-Shil1l1awi and EI-Shimi (1981b)
found that NaCi (at 15 milliequivalentsll 00 g soil) had a weak stimulating effect on
urease activity of the alluvial soil, and caused only inconsiderable changes in urease
activity of the calcareous soil. Depending on the soil moisture content, expressed as a
percentage of WHC, urease activity increased in the order 60 > 80 > 100 > 40% (in
samples of both soils untreated with NaCl), 60 > 80 > 40 > 100% (in the NaCI-treated
alluvial soil), and 80> 60 > 40 > 100% (in the NaCl-treated calcareous soil). At 60% of
WHC, the mixtures ofNaCI + CaCl2 (3,000 ppm), in which the Na:Ca ratio was 1:1,2:1
or 3:1, were also studied. It was established that urease activity increased in the alluvial
soil and decreased in the calcareous soil under the influence of the NaCI + CaCb
mixtures in the following order of the Na:Ca ratios: 1:1> 2:1> 3:1.
Samples of the same two soils were treated by Shehata et a1. (1982) with different
amounts of either NaCi or Na2C03 (0, 10, 20, and 25 milliequivalents/l 00 g soil). To
some samples starch (2% on soil weight basis) was also added. Moisture content in the
soil was kept at 60% ofWHC. In continuation, the experimental procedure already used
by EI-Shilmawi and EI-Shimi (l98Ia,b) was applied. NaCI at a rate of 10
milliequivalentsll 00 g soil stimulated, while at higher rates inhibited urease activity in
both soils. Na2CO.l behaved like NaCi in the alluvial soil, but in the calcareous soil it
had a stimulating d'fect inversely proportionate to its rates. In samples incubated with
added starch, the depressive effect of thc high rates of salts was attenuated, which can
be attributed to the synthesis of new urease molecules by the microorganism~ using the
starch as carbon and energy source during the incubation. This action of the starch was
more marked in the calcareous soil than in the alluvial one.
Based on the results of a laboratory experiment, in which samples of a typical
chemozem from Russia were treated with neutral salts in different amounts, Yarovenko
et a1. (1982) concluded that the salts increased soil urease activity in the order: NaCI >
23
MgCl z "" MgCl 2 + NaCI in samples without crop residues, and MgCI2 > MgCh + NaCI >
NaCI in san1ples containing residues of vetch-oats.
Frankenberger and Bingham (1982) treated samples of a California sandy clay loam
soil (pH 6.82) with four rates ofNaCI, NaZS04, and CaCl2 solutions applied to produce
electrical conductivity (Ee) readings of saturation extracts (Ee,) ranging from 2.2 to
22.4 dS/m (NaCI), from 3.8 to 20.0 dS/m (Na 2 S04), and from 2.6 to 21.6 dS/m (CaCh).
The range of Ec,. values includes threshold salinity levels associated with reduced
yields of agronomic crops. To 5-g samples (oven-dry basis) I ml of the appropiate salt
solution was added to give a moisture content of approximately 60% of WHC. The
mixtures were allowed to equilibrate at 25°C for 7 days, then were assayed for urease
activity. This activity decreased with increasing Ec,., in the following order when
compared at the same Ec,. levels: NaCI > CaCh > NaZS04. Thus, the highest Ec,. (salt
concentration) elicited an inhibition of about 21% (NaCl) , 13% (CaCh), and 4%
(Na ZS04).
In a similar study performed by McClung and Frankenberger (1985), three
California soils (clay loam, pH 8.0; sandy clay loam, pH 5.8, and sandy loam, pH 7.0)
were used. The NaCl, Na ZS04, and CaCh solutions applied at four rates produced Ec,.
values of 5, 10, 15, and 20 dS/m in each soil. The reaction mixtures, consisting of 10-g
samples (on a dry weight equivalent basis of field-moist soils) and 1 ml of the
appropiate salt solution, were allowed to equilibrate at 25°C for 7 days, then treated
with urea (200 J..lg N/g soil) in 0.5 or 1 ml solution, and incubated at 30°C for 14 days.
During incubation, the an1ffionia volatilized was determined, and after incubation, the
NH/, N0 3-, and NO z- contents in the reaction mixtures were analyzed. The results
indicated that the added salts, regardless of their type and amount, did not affect
hydrolysis of urea in any of the three soils studied. Their effect on volatilization of
ammonia from urea will be dealt with on page 30.
Of 18 Utah and California soils studied, Kumar and Wagenet (1984) selected three
(differing from each other by their urease activity and physicochemical properties, e.g.,
pH 7.2, 7.8, and 8.0, respectively) for assessing the effect of CaC0 3 addition on soil
urease activity. Ten-g soil samples were treated with 10 mg of urea-N + finely divided
amorphous CaC03 (0, 2, 4, and 8% on soil weight basis) + water up to field capacity,
then incubated at 37°C for 5 hours and, thereafter, the residual urea was determined. It
was found that addition of CaC03 decreased urease activity in each soil. The decrease
was low in the mixtures treated with 2-4% CaC0 3 , but at the 8% CaC0 3 level a
considerable decrease (68, 49, and 29%, respectively) occurred in urease activity of the
three soils studied. The authors hypothesize that the decrease in urease activity was due
to inactivation of urease by amorphous CaC03 and/or to effect of CaCO) on soil pH
and/or to direct influence of CaC0 3 on the reaction of formation of ~)ZC03 from
urea.
In another laboratory experiment, Kumar and Wagenet (1985) studied the effect of
CaCh on urea hydrolysis in two Utah soils (silty loam, pH 7.7 and fine sandy loam, pH
8.0). Soil columns were constructed of acrylic plastic tubing (7.6 em in diameter and 30
em long) fitted at both ends with porous fritted glass plates. The columns were filled
with soil to a uniform bulk density, wetted from the bottom with CaCl z carrier solution
of 1, 5 or 10 dS/m until saturated, and then reversed to a downward direction of flow.
Addition of carrier solution was maintained until a steady downward flow was achieved,
24
then a 100-ml pulse of the carrier solution containing 500 mg Nil as urea was added to
the column, followed once again by the carrier until all added N was leached. Effluent
samples were collected and analyzed for urea and NH4 +. Based on the analytical data it
was calculated from first-order kinetics that the half-life of urea increased (with
increasing CaCh concentration in carrier solution) from 9 to 47 hours in the silty loam
and from 47 to 77 hours in the fine sandy loam. This means that under the influence of
CaCh urea hydrolysis was reduced in both soils.
In an Indian clay loam studied by Yadav el al. (1986), Sr2+ and Ba2+, used in the
form of chlorides and applied at rates of 100,500, and 1,000 ppm cation (on soil basis),
caused, under the conditions specified on page 13, the following inhibitions of urease
activity: 5 and 13% (at 100 ppm), 19 and 25% (at 500 ppm), and 29 and 50% (at 1,000
ppm), respectively.
Working with a nonsaline sandy loan1 soil (pH 8.1) from Punjab, Singh and Bajwa
(1986) treated 3-kg air-dried soil samples with solutions containing 100
milliequivalents/l ofNaCl, Na 2S04, NaHC0 3 or NaCl + CaCb.2H20 (10.3:1) to produce
EC, values of approximately 10 dS/m. The salt-treated soil samples were submitted to
three wetting (with distilled water) and drying cycles and then assayed for the rate of
urea hydrolysis. The reaction mixtures were prepared from 10 g of soil + 10 ml of urea
solution (200 Ilg N/g soil) and incubated at 11°C for 1-28 days. After incubation, the
residual urea, NH/, and N0 3- contents were determined. The results obtained indicated
that the salts inhibited urea hydrolysis in the order: NaCl + CaCh > NaHC0 3 > NaCl >
Na2S04. We should note that complcte hydrolysis of urea took place in 2 days in the
control soil (not treated with salts) and in 14 days in the NaCI+CaClrtreated soil.
Urease activity determinations also showed that the least inhibitory salt was Na2S04.
The experiment conducted on three American soils by Kumar and Wagenet (1984)
(see page 23) was repeated by Kumar et al. (1988) with a sandy loam soil (PH 8.3) from
India. The reaction mixtures had the following composition: 109 of soil + 0, 2, 4 or 8%
of CaC0 3 (by weight of soil) + urea (200 ppm N by soil weight) + water up to 50% of
WHC. After different durations of incubation at 25°C, the residual urea was determined.
It was found that CaC03 markedly inhibited hydrolysis of urea. Thus, after a 3-day
incubation, the residual urea-N was 2,44,69, and 72 ppm in the samples treated with 0,
2, 4, and 8% of CaC0 3 , respectively. All the added urea was hydrolyzed in 6 days at 0
and 2% CaC03 , whereas at 4 and 8% CaC03 hydrolysis of urea was not complete until
day 12.
Ten-g san1ples of another sandy loam soil (pH 7.8; EC 0.33 dS/m) from India were
treatcd with different quantities of NaCl, CaCI 2, and MgS04 (maintaining the ratio of
Na:Ca+Mg at 1: 1) to produce EC values of 4.33 and 16.64 dS/m. After addition of 200
ppm urea-N and water up to 50% ofWHC and incubation at 25°C, the residual urea was
analyzed. The results showed that the added salts reduced the rate of urea hydrolysis. For
example, after 3 days of incubation, 5, 20, and 43 ppm of urea remained unhydrolyzed in
the samples having EC values of 0.33, 4.33, and 16.64 dS/m, respectively. Complete
hydrolysis of urea needed 6 days at EC = 0.33 and 4.33 dS/m and more than 6 but less
than 12 days at EC = 16.64 dS/m.
In comparison with urea, the fertilizer Cardonite, consisting of urea + 5.5% of Mg in
the form of dolomite, caused a significant (33%) increase in urease activity of a
chemozemic brown forest soil (pH 6.0) from Hungary. The reaction mixtures, containing
25
*.
mixture: 0.826±O.OI69 Ilmoles of urea hydrolyzedlE.lT.lminute (100%).
'Significance level (p): * 0.05: (UII: ••• 0.001.
Laura and Parshad (1991) studied the effect of NaHCO.l addition (and pH increase)
on the hydrolysis of urea in samples (20 g each on oven-dry basis) of an Indian sandy
loam soil. The samples received 0, 0.544, 0.771, 1.027, and 1.284% NaHCO.l (for
increasing pH from 7.53 up to 9.92) and O. 100. 200. and 400 J.lg N as urea/g soil. The
mixtures were moistened to 70% of WHC and incubated at laboratory temperature (20-
30°C). The periodic determination of the amounts of unhydrolyzed urea revealed that
the rate of urea hydrolysis had a perfect negative correlation (r;::: -1.00) with the amount
of NaHC0 3 added (and, thus, with pH).
In a pot experiment, Garcia and Hemandez (1996) used I-kg samples of a calcareous
soil (pH 7.98) from southeastem Spain. The samples were treated with 240 ml solutions
(60% ofWHC) of increasing NaCI and Na2S04 concentrations (0.1, 0.3, 0.6, 0.8, 1.0, and
1.3 M). The control samples received 240 ml of distilled water. At 0.1 to 0.8 M salt
concentrations, NaCI was less inhibitory on urease activity than Na2S04, but the reverse
was true at 1.0 and 1.3 M salt concentrations. However, the inhibition of urease activity
never reached 20(Vo (see also Garcia et al., 2000).
Analyzing the results of the investigations described above one can deduce that, at
unpolluting concentrations, the salts of alkali metals and alkaline earth metals are
ineffective inhibitors of urease activity in many soils or are even stimulators of this
activity in some soils.
1.3.2. Etf(xt of Alkali Metal and Alkaline Earth Metal Salts on Ammonia Volatilization
Reduction of the volatilization of ammonia from urea under the action of CaCb, KCl,
and other neutral salts was described in a series of papers (e.g., Anderson, 1962;
Tomlinson, 1964; Fenn et aI., 1981a,b, 1982; Prusinkiewicz and J6zetkowicz-Kotlarz,
1982; Rappaport and Axley, 1984; Fenn and Hossner, 1985; Gascho, 1986; Fenn, 1988)
and in two patents (Fenn, 1982, 1985). This action of CaCh is attributed to the reaction:
Thus, CaCl 2 reacts with (NH4hC0 3 formed during urea hydrolysis: the carbonate
ion from (NH4hCO.l precipitates as CaC0 3 • The resulting NH 4Cl is a weakly acidic
compound less conducive to ammonia than (NH4hC0 3 ; around the urea granules the
pH decreases reducing the volatility of ammonia.
The action of KCl is explained as follows: K+ replaces Ca2+ on the exchange sites in
the adsorptive complex of soil, then the released Ca2+ and the cr from KCl react with
(NH4hC0 3 producing, according to the reaction equation cited above, NH4Cl and
CaCO).
This mechanism of the action of CaCb and KCl is in close agreement with the
observation by Watkins et al. (1972), according to which NH3 losses from forest floor
were less from mixtures of NH4Cl and urea crystals than from urea crystals and pellets
alone.
CaCIz was more effective (to a 30-40% extent) than was KCl in reducing NH3 loss.
CaCIz was also found to be more effective than MgCb, which can be explained by the
observation that precipitation of CaC03 takes place at a less alkaline pH than that of
MgC0 3 .Mg(OH)2, forming during the reaction between MgCb and CNH4hC0 3 • The
chlorides are more effective than the sulfates of the same metals.
27
In a field experiment on a clay loam at Rothamsted, Rodgers et al. (1984) applied urea
priUs (375 kg Nlha), without or with CaCI 2, as a single dressing for fertilization of a
perelmial ryegrass ley. Ammonia volatili7A1tion losses were measured during 4 weeks after
fertilizer application. It was found that CaCh slightly reduced the NH, loss.
Fenn et at. (1987) studied the influence of plant residues and urea rate illl the
effectiveness of CaCh to inhibit anID10nia volatilization from urea. In an experiment,
samples of a calcareous silty clay loam were unamended or amended with grass clippings
(l % (m soil weight basis)', then treated on surface with urea (11, 55, and 110 g N/m2) with
or without CaCl 2 , at a Ca:urea molar ratio of 0.50, and submitted to incubation at 22°C for
12 days, during which time the volatilized NH3 was determined. In the case of samples not
amended with grass clippings, the cumulative NH3 losses from the three urea amounts were
28, 48, and 48(Yo, respectively, in treatments without CaCh addition, and 19, 5, and 1%,
respectively, in treatments with CaCl 2 addition. In the case of samples amended with grass
clippings, the cumulative NH3 loss from each urea amount was approximately 65%, and was
reduced, under the influence of CaCI 2 , to 35, 30, and 10%, respectively. Thus, incubation of
soil with grass clippings, which enhanced proliferation of microorganisms and synthesis of
new urease molecules, diminished the inhibitory effectiveness of CaCh on volatilization of
NH, from urea. At the same time, the cumulative NH3 losses, expressed as percentages of
the applied urea-N, decreased with increasing rate of urea application, when CaCl 2 had also
been added. TIlese findings were confil111ed with samples of three other soils.
In another experiment, samples of a urease-free sand were treated with Caco,
(15% on
sand weight basis) and amended with increasing amounts of grass clippings (0.01-10%).
Then, urea was applied at the same three rates as in the first experiment; the Ca:urea molar
ratio also was the same (0.50). Based on the determination of NH, volatilized during the
incubation (22°C/12-17 days), the conclusion was drawn that the inhibitory effectiveness of
CaCI 2 on NH3 volatilization progressively decreased with increasing amounts of grass
clippings added to the sand samples.
For studying volatilization of ammonia from urea and urea-MgS04 .1H 2 0 mixture
(UMM) under laboratory conditions, von Rheinbaben (1987) used three sandy and three
loess soils from Germany. The soils were first brought to 30% of their WHC, then filled into
1,250-ml pots; the weight of moist soils depending on their texture was 1,300-1,600 g/pot.
Urea or UMM was added to the soil surface at a uniform rate of 500 mg N/pot. N:Mg ratio
ofUMM varied from I :0.07 to I :0.50. The pots were kept at 25°C for 14 days, during which
the volatilized NH, was measured.
The cumulative NH3 losses from urea and UMM were compared in the six soils at a
single N:Mg ratio (1:0.21). The losses were significantly lower from UMM than from urea
in an acid loess and a sandy soil and insignificantly lower in the other soils. The influence of
N:Mg ratio on NH3 volatilization was studied in two sandy soils. Reduction of NH3
volatilization from UMM was insignificant at the N:Mg ratio of 1:0.07 and very significant
at the ratio of I :0.50 in both soils. The influence of the form (solid or liquid) of urea and
'Before addition of grass clippings, the soil was adjusted to 15% caeo" by weight.
28
UMM on NH3 volatilization was also studied (in a sandy soil). Cumulative NH3 losses were
lower when urea and UMM (N:Mg=I:0.21) were applied as solutions, not as solids on soil
surface, but the loss was insignificantly lower from the UMM solution than from the urea
solution.
Bundy and Eberle (1988) conducted field studies to determine the amount of ammonia
volatilized from several N fertilizers surface-applied on silty loam soils cultivated with
maize in Wisconsin, namely at Arlington and Lancaster. In some experiments, the N
fertilizers were urea-CaCh and urea-KCl solutions. Urea prills served for comparison. At
Arlington, the experiments were carried out in 1983 and 1984, and at Lancaster in 1984.The
fertilizers were administered at rates of 56 and 112 kg Nlha. N :Ca and N:K ratios in the two
fertilizer solutions were 1:1 and 2:1, respectively. The fertilizers were broadcast on the soil
surface after planting (mid-May) but before plant emergence. Field measurements of NH3
volatilization were made at the higher fertilizer N rate only, and lasted 8-10 days.
Use of the urea-CaCh solution significantly reduced NHJ loss relative to urea at both
locations. Thus, at Lancaster, the 19% N loss as NHJ from the applied urea was reduced to
9% in the urea-CaCh treatment. At Arlington, the corresponding N losses were 8 and 3% (in
1983) and 18 and 5% (in 1984), respectively. Ammonia volatilization from the urea-KCl
solution did not significantly differ from that observed with urea at Lancaster, but
significant reductions in NHJ loss occurred with the urea-KCl treatment at Arlington, in
both 1983 and 1984. Urea-CaCh was more effective than urea-KCI in reduction of NH3
loss.
Lightner et al. (1990) made field measurements to determine the amount of ammonia
lost through volatilization from a series of fertilizers (including prilled ammonium nitrate,
granular urea, and solutions of urea, urea-KCl, and urea-CaCh) surface-applied to a
permanent orchardgrass (Dac(y/is glomerata) sod on a silt loam soil (pH 6.0) in Indiana,
during 1982 and 1983 . Annually, each fertilizer was applied at rates of 200 kg Nlha in the
spring and 100 kg Nlha in late summer. Cationlurea-N equivalency was 0.50 KCI and 0.25
CaCh.2HzO. In both years, volatile NH3 losses from ammonium nitrate were insignificant,
but application of urea granules and of urea and urea-KCl solutions led to high cumulative
NH 3 losses that ranged from 27 to 41 % of the applied N in the spring and from 12 to 37% in
the summer. Ammonia volatilization from the urea-CaCh solution was not significantly
lower than that from urea solution in 1982, but adding CaCh to urea solution resulted in a
significant reduction ofNH 3 10sses in 1983.
Gameh et al. (1990) studied the influence of various urea-KCl mixtures on ammonia
volatilization in two soils (a silty loam and a silty clay loam) from Maryland. Soil sanlples
(each weighing 100 g) in 250-rnl flasks (in which the exposed soil surface equaled 38.5 cmz)
were treated with granular urea (control), granular urea + KCl, urea + KCI in solution and
urea coated with powdered KCl, at rates equivalent to 260 kg urealha and 260 kg KCllha.
The moistened samples were incubated at 26°C for nearly 30 days, during which the amount
of volatilized NH3 was determined. The cumulative NH3 losses showed the following
orders: urea> granular urea + KCl > solution urea + KCI > KCl-coated urea in the silty
loam, and urea ~ granular urea + KCl ~ solution urea + KCI > KCI-coated urea in the silty
29
clay loam. Thus, the only urea-KCl mixture, from which NHJ volatilization was
significantly reduced in both soils, was the KCI-coated urea.
At the International Fertilizer Development Center in Muscle Shoals, Alabama,
Christianson et al. (1995) conducted investigations to detennine if the high pH of some
commercial sources of KCl had an effect on the anunonia loss from three contrasting soils
treated with urea in pots with a surface area of 0.04 m2. Five fertilizer-grade KCl sources
ranging in pH from 6.2 to 9.5 were used, and analytical-grade KCl served for comparison.
Rate of KCl was 100 kg K20lha and that of urea was 100 kg Nlha. Urea and KCl were
applied on the soil surface as granules or in solution. Ammonia loss was measured at 2-day
intervals over 2 weeks, during which soil humidity was maintained at field capacity.
The results showed that in the urea-only treatments the cumulative NH3 losses were high
(approximately 50% of N applied) from both granular and solution urea. In two soils, the
loss was significantly reduced due to the use of KCl: the reduction was 30 and 51 %,
respectively, from granular urea and KCl and even higher from solution urea and KCl. In
the third soil, KCl was ineffective in reducing NH3 loss. pH of KCl sources had no effect on
NH] loss in any of the three soils.
In a field experiment on calcareous soil of clay loam texture in the area of Konya,
Turkey, Gezgin and Bayrakli (1995) treated the plots with urea and with urea and
phosphogypsum (PG), the rate of urea being 200 kg Nlha, while PG was applied at two rates
(1,000 kg/ha, PG 1 and 2,000 kglha, PG2) . The total volatile ammonia losses during 57 days
were: 10.6% (urea), 9.2% (urea + PG1), and 12.0% (urea + PG2), the differences between
losses being significant (p=0.05). Thus, PG at the lower rate reduced, and at the higher rate
increased, NH3 volatilization from the urea-treated soil.
For reducing N loss from urea fertilizer, Avramchuk (2000) patented a mixture
consisting of urea and PG at the ratio of 1:4 (weight per weight) and explained the reduction
of N loss through the following mechanism: Ca(HS04h of PG reacts with (NH4hC0 3
(product of urea hydrolysis) with formation of (NH4)2S04 which is more stable than
(NH4hC0 3 •
It should be mentioned that superphosphate, which contains an acid salt, Ca(H 2P0 4h.
and CaS04, reduced volatilization of ammonia from urea (e.g.. Tomlinson, 1964;
Mahendrappa and Odgen, 1973; Carrier and Bernier, 1976; TyaUi, 1982; Fan and
MacKenzie, 1993a; Sengik and Kiehl, 1995a,b). Moreover, Fenn et al. (1990) found that
Ca(H 2P0 4h enhanced the effect of CaCl 1 to reduce volatilization of NH3 from urea-treated
samples of the two Texas soil studied (a silty clay loam, pH 7.7 and a sandy clay loam, pH
4.5). Ouyang et al. (1998) demonstrated that triple superphosphate, added to urea-treated
soils, is able to reduce N10 emission, too. Samples of three Canadian soils were used: a clay
(pH 5.5), a silty clay loam (pH 6.1), and a sandy clay loam soil (pH 6.1).
The effects of superphosphate to reduce gaseous losses from urea-treated soils are
consistent with the findings that triple superphosphate reduced the rate of urea hydrolysis in
soils due to inhibition of urease activity (Fan and MacKenzie, 1993a,b; Ouyang et aI.,
1998).
30
Sor (1968, 1969) prepared pellets from urea, borax (Na2B407.1 OH 20) (0.45% by weigllt of
urea, limits of the borax amount being 0.01 and 10%) and a hydrophobic material,
preferentially the san1e asphalt -microcrystalline wax blend also used for preparation of urea
pellets with CUS04 or Pb acetate. The technology of preparation was also the same (see
page 5). Urea prills without inhibitor served for comparison. The pellets and urea prills,
applied at a practical N rate, were incubated on the surface of a sandy loam soil (containing
6.4% moisture) at 25°C for 48 days, during which the volatilized an1l110nia was estimated. A
significant part (approximately 39%) of the urea-N was lost as NH.l in 48 days from the soil
treated with urea priUs, whereas NH3 loss was only 17% when the soil received the pellets
prepared from urea, borax, and hydrophobic material.
According to the descriptions in three inventions patented for preparation of pellets from
urea + borax + hydrophobic material, Sor et al. (1968, 1971) and the Esso Company (1969)
use - in place of the asphalt-microcrystalline wax blend - other hydrophobic materials,
namely primary and secondary amines and diamines having a hydrocarbon chain of 8-22
carbon atoms, preferentially octadccylamine, CHr -{CH 2 )1,NH 2 Diamines having the
o
31
Xue and Li (1987) showed the inhibitory effect of borax on urease activity of a
periodically water-logged paddy soil. The borax, used at a concentration of 100 ppm by soil
weight, exhibited a 38.92% inhibition of urease activity. TI1e reaction mixtures were
prepared from 5 g of soil and 10 ml of 10% urea solution without or with added borax. The
incubation took place at 37°C and lasted 48 hours.
Zhan et af. (1993) described a technology for cogranu1ation of urea and borax. This urea
fertilizer contains up to 4% borax and consists of 4-6-mm granules. Fan and Ye (1995)
applied the borax-containing urea to paddy field and found that the borax acted as an
inhibitor of soil urease activity, preventing the gaseous N losses from urea.
Another use of boric acid also related to fertilizer urea will be dealt with below. For
preparing controlled-release fertilizer urea granules, Otey et al. (1984) worked out two
technologies, in one of which boric acid is also used. Pregelatinized maize (Zea mays) flour
is dispersed at 25-30°C in a solution of urea (50 ml H20/50 g urea) and concentrated
NH40H (4 mlllOO g of final dry product). Then boric acid (2 gllOO g of final product) is
mixed with the gelatinized flour-urea mixture to form a rubbery mass. Air-dried maize
starch (18 gl100 g of final product) is then added slowly with stirring which causes the
rubbery mass to break into small particles coated by starch. On a dry basis this product
contains 2% boric acid «(U5% B), 18% ungelatinized starch, and 80% urea and gelatinized
flour. Boric acid in this product serves as micronutrient; its effect on soil urease activity was
not tested.
1.5. FLUORIDES
Tomlinson (1964) studied the effect of K and Ca fluorides (KF, CaF 2) on volatilization of
ammonia from urea in two English soils (calcareous clay loan1, pH 8.1 and noncalcareous
sandy loam, pH 6.5). The soil samples were treated with 2,000 ppm urea-N and a
chemically equivalent amount of KF or CaF2 and moistened to about 40% humidity which
was less than the field capacity. Samples not treated with fluoride were the controls.
Incubation took place at 9°C and lasted 5 days. Determination of the NHJ volatilized during
the incubation gave the following percentage NHJ losses in the clay loam: 8.6 (control),
41.5 (KF), and 6.3 (CaF 2), and in the sandy loam: 32.6 (control), 52.3 (KF), and 29.3
(CaF 2 ). In other words, the soluble fluoride (KF) stimulated, while the insoluble salt (CaF 2)
inhibited NHl volatilization from urea in both soils.
Sor (1968, 1969) prepared not only urea pellets with CUS04, Pb acetate, and borax +
hydrophobic material (asphalt + microcrytalline wax) (see page 5), but also urea pellets with
NaF (0.45% by weight of urea) + asphalt-microcrytalline wax blend. In an experiment to
study the volatilization of an1l11onia from urea applied on the surface of samples of a sandy
loam soil, NaF from the urea pellets reduced the cumulative NH3 loss in 48 days to about
12%, while this loss from the control urea prills was 39%.
According to Kozlovskaya et al. (1972), urease activity in peat bog soils was not
inhibited at all by NaF, although the fluoride was applied at high concentrations (16--64 111M
in reaction mixtures).
34
In the pot experiments carried out by Gaponyuk and Kuznetsova (1984), samples taken
from the 0-20-cm layer of a soddy-podzolic soil (PH 7.(5) from Russia were treated with
NaF at rates ranging from 0.1 to 3 g F/kg soil, moistened to 60% ofWHC and preincubated
for I month, then sown with different plants. During the preincubation period, soil urease
activity was measured 20 times. Mean values of this activity showed insignificant changes
at rates of 0.1-0.7 g F/kg soil and significant increases at the 1-3 g F/kg soil rates. Thus, in
these experiments NaF exerted no inhibiting effect on soil urease activity.
Ablizova and Tomina (1997) carried out pot experiments in which samples of a dark-
chestnut soil from Kazakhstan were treated with NaF. In 1991, NaF was applied at rates of
0, 10, and 50 mg F/kg soil, before planting tomatoes. Soil urease activity was determined
several times during the vegetation period. It was found that the activity was inhibited by
the higher NaF rate in spring, by both rates in summer, and was stimulated by both rates in
autumn. In 1992, NaF was applied at higher rates (50 and 500 mg Flkg soil) and the test
plant was onion. Soil urease activity, measured in summer, showed an 11.5% increase at the
lower NaF rate and a 65% decrease at the higher NaF rate. One can state that in these
experiments, in contrast to those of Gaponyuk and Kuznetsova (1984), NaF behaved as an
inhibitor of soil urease activity and the inhibition lasted several months.
Applying the 5-hour test, Bremner and Douglas (1971) established that arsenic chloride
(AsCl 3 ), arsenic trioxide (As Z0 3), and arsenic pentoxide (As Z0 5) used at a rate of 50 ppm
(soil basis) brought about only negligible inhibitions in the urease activity of a silty clay
loam and a clay loam from Iowa: on average, the degree of inhibition by the three arsenic
compounds was 3, 4, and 3%, respectively.
Tabatabai (1977) treated 5-g samples of six Iowa soils with 1.5 ml of sodium arsenate
(Na zHAs04 ) or sodium arsenite (NaAsO z) solution (at a rate of 5 f.Ulloles As/g soil). 0.2 ml
of toluene, 7.S ml ofO.OS M Tris buffer (pH 9.0) and 1 m1 of 0.2 M urea solution and then
incubated them at 37°C for 2 hours. In two of the soils, the arsenic compounds were used at
a lower rate. too (0.5 Ilmoles As/g soil). Based on the determination of the urea remaining
unhydrolyzed during incubation, it was deduced that none of the two Na zHAs04
concentrations exerted any inhibitory effect on soil urease activity. In contrast, NaAsO z, at a
lower rate, inhibited urease activity in the two soils studied (degree of inhibition: 7 and
14%, respectively), whereas at the higher rate NaAsO z had a urease-inhibiting effect in each
of the six soils studied (degree of inhibition: 98,44,27,24,18, and 9%, respectively).
It should be added that under similar conditions sodium tungstate (Naz W04) behaved
like Na2HAs04, and selenious acid (HzSeO,l) like NaAsO z. The inhibitions caused by
HzSeO] were Sand 9% (at the lower Se rate), and 33,24,24, 19, 16, and 14% (at the higher
Se rate). But Aliev (1988) recorded increased urease activity in samples of a dark-chestnut
soil treated with sodium selenite (Na2SeO]) at rates equivalent to S, IS, and 45 kg'ha. The
increase was inversely proportional 10 the rate of selenite addition.
35
Conrad (1940) compared the antiseptic effect of carhon disu(fide (CS 2) with that of toluene
in the determination of urea hydrolysis in two California soils. The hydrolysis rate was
similar in presence of the two antiseptics. As urea hydrolysis in presence of toluene differed
only to a slight extent from that measured in absence of antiseptics, the conclusion may be
drawn that neither toluene nor CS 2affected soil urease activity.
Using the 5-hour test for studying the effect of 50 ppm of sodium sulfite (Na2S03),
sodium hisu!/ite (NaHSOI ), and lead sur/ide (PbS) on urease activity in two Iowa soils,
Bremner and Douglas (1971) recorded only negligible inhibitions due to these three sulfur
compounds (on average, < 1,4, and 3%, respectively).
The effect of sodium sulfite was also tested with other two soils: an alluvial soil and a
leached chernozen1 (Kiss and Pintea, 1987). The reaction mixtures had the following
composition: 5 g of air-dried soil + 1 ml of 0.6% urea solution + 9 ml of aqueous solution or
suspension of the compound to be tested at 2% rate by weight of urea (i.e.. 0.12 mg of
Na2S01 to 6 mg of urea). Reaction mixtures in which the solution or suspension of the test
compound was replaced by distilled water served for comparison. Incubation was carried
out at laboratory temperature. At 1-2-day intervals, drops were taken from the aqueous
phase of reaction mixtures for detecting the unhydrolyzed urea. 111e drops were placed on
chromatographic paper and, after drying, sprayed with a chromogenic reagent' to visualize
the yellow spot of urea. Na2S0, does not interfere with the detection of urea. If urea
hydrolysis is complete, no colored spot appears. The time (days) necessary for complete
urea hydrolysis is registered.
The results showed that complete hydrolysis of urea in reaction mixtures with or without
Na2S01 required the same time, namely 6 days in the alluvial soil and 9 days in the leached
chernozem. In other words, Na2S01 did not inhibit urease activity in these soils.
In a field experiment conducted on a silty clay loam soil (pH 6.0) in Alberta, Malhi and
Nyborg (1979) determined urea hydrolysis in plots treated with urea (control) or with a
mixture consisting of two parts of urea and one part of calcium sulfide (CaS) or phm,phorus
pentasu(fide (P 2SS; P4 S lO ). Urea and the urea-CaS or urea-P 2 SS mixtures were administered
at a rate of 112 kg N/ha, in bands at a depth of 5 cm. After 5 and 10 weeks, the NH4 + and
NO,- contents in soil were analyzed. The analytical data obtained indicated that after 5
weeks urea hydrolysis was complete in the control plot, but it was only 71 and 68% in the
plots treated with urea-CaS and urea-P 2 S5, respectively; after 10 weeks, urea hydrolysis
became complete in all plots. This means that the inhibitory effect of these inorganic sulfur
compounds on urease activity in the studied soil was not strong and long-lasting, although
they were applied in a considerable amount.
at the highest concentration tested (10% ATS). At the same time, ATS did not have any
effect on activity of jackbean urease.
In another experiment, with the clay loam soil, the effect of sodium thiosulfate
(NaZSZ03, reagent grade) was compared with that of three commercial ATS products,
containing impurities (free ammonia, sulfite, sulfide, sulfate). All commercial ATS products
and reagent sodium thiosulfate gave similar levels of inhibition (21-28%) in urea hydrolysis.
Data concerning the inhibitory effect of ATS on nitrification of NH4 + were also
obtained.
GODS (1985b) studied, under laboratory conditions with 20-g samples of a loamy soil,
the effect of ATS on urea hydrolysis in correlation with its effect on ammonia volatilization
from UAN. The fertilizer solutions in the form offour 0.025-rnl droplets were placed on the
soil surface approximately 2 cm apart from each other. During incubation (at 25°C/5 days),
the NH3 volatilization and, after incubation, the residual urea were determined.
ATS, at each of its rates applied (1-25% volume/volume), decreased urea hydrolysis
which was increasingly inhibited with increasing rates of ATS. At low rates (1, 2, and 5%),
ATS reduced NH3 volatilization, too. But NH3 volatilization increased somewhat at higher
ATS rates. It is possible that at higher levels of ATS there can be NH3 loss from ATS itself.
Under similar laboratory conditions, but using 40-g samples of the same soil, the effect
of ammonium polyphosphate (APP) fertilizer (10-34-0), added to UAN or to UAN + ATS
was also tested. APP was applied at a 20% rate and ATS at a 2% rate by volume ofUAN.
The solutions were applied on the soil surface in the form of a single small (0.1 ml) droplet
("spray" application) or a single large (0.5 m!) droplet ("dribble" application). After 5-day
incubation at 25°C, it was established that, at the O.I-ml droplet size, ATS, APP, and ATS +
APP did not inhibit urea hydrolysis, but reduced, to some extent, volatilization of NH3 from
UAN. At the O.5-rnl droplet size, ATS markedly slowed urea hydrolysis (from 83 to 48%),
APP decreased it to a lesser extent (from 83 to 74%), whereas the two compounds used
together decreased it up to 45%; ATS, APP, and ATS + APP significantly reduced the
volatile NHl loss, by 61, 27, and 72%, respectively.
Based on Goos' (1985a) observation, according to which ATS inhibited soil urease
activity, but did not have such an effect on jackbean urease, Goos ef al. (19800) and Goos
(1987) assume that ATS acts indirectly on soil urease, that is ATS reacts rapidly and
abiotically with Fe(OHh and MnOz from soil, forming tetrathionate anion and Fe 2+ and
Mn2+ cations which inhibit the enzyme by binding to its sulfhydryl (SH) groups:
SH S
/ / \
Urease + Mnz+ -----:>::. Urease Mn + 2H+.
\ \ /
SH S
To obtain evidence in favor of this hypothesis, Goos (1987) added Na ZS04 or NaZS203
solution to samples of a silty clay soil and, after 12 hours, removed SO/- and S20/- by
repeated extraction and centrifugation. The soil samples were then analyzed for urease
activity and Fe and Mn contents extractable with 0.1 M HCI. It was found that under the
influence of the thiosulfate treatment - as compared to the sulfate treatment - urease activity
decreased in a proportion of 40%, whereas the amounts of acidosoluble Fe and Mn
increased even in a higher proportion.
The effect of APP to reduce NH3 volatilization was attributed to its capacity to
temporarily moderate the alkalinity produced by rapid urea hydrolysis (Goos, 1985b; Goos
ef al., 1986a,b; Fairlie and Goos, 1986). This means that APP is, essentially, not an inhibitor
of soil urease activity.
Laboratory investigations related to ATS were carried out by Gascho (1986) as well. He
used a loamy sand soil (pH 6.8) from Georgia and determined the amount of NH3 evolved,
at 30°C during 7 days, from 100-g soil samples treated with UAN; UAN + ATS; UAN +
KCl; UAN + KCl + MgCl 2 ; UAN + KCl + MgCIz + CaCh; UAN + KCI + ATS, and UAN
+ KCI + MgCIz + ATS, the N:K 20:Mg:Ca:S ratio being different. Soil samples treated with
urea only or NH 4NO] only were the controls. Nitrogen was applied at the same rate in each
sample, namely 184 kg/ha (based on the area of the soil sample surface). All the solutions
were surface-applied.
No loss of NH3 was detected from the NH 4 NOdreated control, but 48% of the applied
N was lost from the urea-treated control and 20% was lost from the UAN-treated soil. ATS
added to UAN at the N:S ratio of 32: I did not reduce, but at the N:S ratio of 16: 1 reduced,
significantly the NHl loss (from 20% to 19 and 17%, respectively). In soil samples treated
with UAN + KCI + ATS at N:K 20:S ratios of 32:16:1 and 16:8:1, the NH3 loss decreased
very much (to 5 and 4%, respectively). Ammonia loss from the soil treated with UAN +
KCI decreased very remarkably only when UAN and KCl were applied at N:K20 ratios of
1:2,2:5, and 1:3, the loss being 8, 0, and 0%, respectively. Ammonia loss measured at the
N:KzO:Mg:S ratio of 16:8:1:1 and at the N:K2 0:S ratio of 16:8:1 was identical (4%). In
samples treated with UAN + KCI + MgCh + ATS or CaCh (in place of ATS), the losses
were very reduced (1-7%). Therefore it appears that a) MgCI 2, if not needed for Mg
nutrition, can be omitted as it will not bring about much additional NH3 loss reductions in
the presence of both KCI and ATS, and h) CaCl 2 can be substituted for ATS in soils not
requiring sulfur fertilizer.
The laboratory investigations were continued by Goos and Fairlie (1988) with two other
North Dakota soils (silty clay, pH 6.6 and loamy sand, pH 5.8) to study the effect of ATS on
39
hydrolysis of urea from VAN applied in the form of 50 droplets of 0.01 ml or 5 droplets of
0.1 ml or one droplet of 0.5 ml, on the surface of 100-g (oven-dry equivalent) soil samples,
the water content of which was adjusted to field capacity or to wilting point. The soil
surface area was about 50 cm2 and the soil depth was about 2 cm. TIle N rate applied, based
on the soil surface area, was about 30n kglha. The ATS solution (a high quality commercial-
grade product) was used at a single rate (5% by volume of the VAN solution). VAN, to
which water was added in place of ATS, served for comparison. All samples were incubated
at 20°C and the residual urea was determined three times during the course of urea
hydrolysis.
One can deduce from the results obtained (Figure I) that ATS inhibited urea hydrolysis
in both soils and at both water contents. Vrea hydrolysis slowed down with increasing
droplet size in both the presence and absence of ATS. The overall rate of urea hydrolysis
was somewhat slower at wilting point than at field capacity. The inhibitory effect of ATS on
urea hydrolysis was strongest with the larger droplet size and at wilting point. Comparison
of the two soils revealed that ureolytic capacity of the loamy sand was considerably weaker
than that of the silty clay. At the same time, ATS inhibited urea hydrolysis more strongly in
the loamy sand than in the silty clay.
Brenmer et al. (1986a) also carried out laboratory testings for evaluation of ATS as a
soil urease and nitrification inhibitor. Iowa soils were treated with different amounts of ATS
and then incubated at 20, 25, and 30°e. ATS, even when applied at very high rates, up to
1,000 ~lg/g soil (LOO() ppm) had no inhibitory effect on urea hydrolysis in soil.' Nitrification
was inhibited only at rates higher than 50() ~g/g soil. The conclusion that ATS has no
practical value as a soil urease and nitrification inhibitor contradicts the results of the
laboratory experimcnts of Goos (1 985a,b ), Gascho (1986), and Goos and Fairlie (1988) and
also the results of some of the other investigations conducted under field or laboratory
conditions.
Fairlie and Goos (1986) installed microplots on a clay soil (pH 6.9). Polyvinyl chloride
(PVC) cylinders 20 cm long and 12.7 cm in dianleter were forced 5 cm into the soil. The
soil surface was bare or covered with wheat residue at a rate of 2.65 tlha (3.5 g/plot). The
microplots were treated with VAN, VAN + 2.5 or 10'% ATS, VAN + 10 or 20% APP or
with VAN + 2 or 5'% ATS + 20% APP. Rale of VAN was 150 kg N/ha, ATS and APP being
additional N sources. Each of these mixed fertilizer solutions was applied at a rate of 0.817
mllmicroplot, either as a large droplet, as a spot in the center of the microplot (dribble
application), or as a fine spray evenly distributed over the soil surface. Then the ammonia
volatilized from the microplots was estimated cumulatively. The principal results were the
following.
In the case of bare or residue-covered microplots treated with VAN without ATS and
APP less NHl was lost with dribble than with spray application. The inhibitory effect of
'Similar results were obtained. when the eftect of ATS to inhibit soil urease activity was compared with that of
other compounds (see Sections 4.9.1.1 and 4.9.1.5).
40
0.01 ml 100
100 0.1 ml 0.1 ml
0.01 ml
0.01 ml + AlS
O.Sml 10
80 0.1 mI + IITS O.Sml
{
't..
IIN 80 0.5 mt + IITS "'""
N
.?;>
80
0..01 ml + ATS
~,
.@ 0.1 ml + ATS
~
1:l .
>.
"'<!" 40 O.Sml + ATS
'2::"
;:J
;:J
20
100 121i
Incu Dation time (hours., InL'ubati(m time (hours)
100
100
C 0.01 mt
0.1 ml
0.1 ml D 0.01 ml
80
# O.Sml
." O.Srnl ~
i:l
",..
."
80
~,
.?;o
8 0.01 rnl + ATS
2
."
~
0.1 ml + ATS ."
,... 40 0.01 ml + .US
0.1 mi. +
+ All! "'~" ~.T9
~
o.S mt
::> 0.5l1li + IITS
20
0
0 100 200 300 ~oo
100 150 200 Incubalion time (hulII")
IncuhHbon lim,' (hours)
Figure 1. Effect of ammonium thiosulfate (ATS) and liquid fertilizer droplet size on urea hydrolysis in two
soils.
A - Silty clay at field capacity. B - Silty clay at wilting point. C - Loamy sand at field capacity. D -- Loamy
sand at wilting point. IAdapted from Goos and Fairlie (1988), by permission of the Soil Science Society of
America, Inc.!
ATS, APP, and ATS+APP on NHJ volatilization was most evident with dribble application.
For example, after 7 days, the NH3 loss from the bare microplots treated with VAN + 2%
ATS - as compared with micToplots treated only with VAN - was reduced by 66% with
dribble application and by 40% with spray application. There was no additional advantage
to giving more than 2% ATS to UAN. Thus, when 5% ATS + 20% APP were added to
VAN, the NHJ loss after 7 days was reduced by 76% with dribble application and by 41 %
with spray application. In the case of microplots covered with wheat residue, ATS and APP
41
decreased NH3 volatilization for 14 days (dribble application) and, in general, only for the
first 4 days (spray application).
Studying three Minnesota soils (loamy sand, silt loam, and clay loam), Zadak et af.
(1987) applied a urea solution with and without ATS and, then, a lO-mm water layer on the
surface of soil columns. ATS was slightly effective in delaying urea hydrolysis in the loamy
sand, but had no effect on N loss from any of the soils.
Al-Kanani et af. (1990a) used surface (0-5 cm) samples from six eastern Canadian soils
cultivated with maize. For studying the effect of ATS on ammonia volatilization from VAN,
two levels of a 60% ATS solution (namely 1.8 and 3.6% by weight) were added to VAN
solutions. The soil samples moistened to 100% of field capacity were amended with
surface-applied VAN solutions at a rate equivalent to 147 kg N/ha, then incubated at 24°C
for lO days. The volatilized NH3 was measured daily. After incubation, the soil was
analyzed for determination of the urea-, NH/-, N0 3·-, and N02"-N contents. The results
indicated that ATS reduced the NH3 losses, the reduction ranged from 12% (a clay soil) to
23.5% (a sand soil).
Sullivan and Havlin (1992a) found that ATS significantly (p = 0.01) inhibited urea
hydrolysis in VAN-amended samples of each of the studied eight Kansas soils varying
widely in pH, cation-exchange capacity, carbonate and organic C contents, urease activity,
WHC, and clay content. The VAN + 10% ATS solution contained 1.1 M ATS-S/I. Efficacy
of inhibition was greatest for soils low in clay and organic C at high temperatures and low
soil moisture contents. Thus, the inhibition ranged from 18 to 48% in an incubation at 20°C
and -0.1 MPa soil matric potential. Inhibition of urea hydrolysis averaged 29% at 20°C and
37% at 30°C, and 28% at -0.03 MPa and 38% at -0.1 MPa. The cumulative NH3 loss was
also significantly reduced by ATS in seven of the eight soils studied (the reduction on
average was 48% in four noncalcareous soils and 22% in three calcareous soils).
Sullivan and Havlin (1992a,b,c) also demonstrated that sodium tetrathionate
(Na2S406.2H20) applied with VAN inhibited urea hydrolysis in soils to the same extent as
ATS. Since the tetrathionate inhibits, while ATS does not inhibit jackbean urease and as
ATS is oxidized by soil Fe and Mn to tetrathionate, the conclusion was drawn that soil
urease is inhibited not by ATS itself, but by tetrathionate; this is the primary urease inhibitor
which reacts with sulfllydryl (SH) groups of the enzyme. This mechanism of inhibition
differs partly from that suggested by Goos et af. (1986a) and Goos (1987) (see page 37).
In a laboratory experiment carried out in the Plant Production Institute in Prague, Czech
Republic, List'anska (1993) fertilized samples of a grey-brown podzolic soil with either
DAM 390 (a VAN solution) or N-Sol (a urea-formaldehyde solution) at a rate of 300 mg
N/kg soil, then the samples were treated with sodium thiosulfate, STS (4% by weight of
fertilizer N) and incubated at ambient temperature. During the incubation period (June 9,
1988 - May 22, 1989), the samples were analyzed monthly for NH/ and N0 3". The results
indicated that STS was a weak inhibitor of urea hydrolysis and its inhibitory effect on
nitrification was even negligible, especially in the samples fertilized with N-Sol.
Lim and Seo (1994) studied the inhibitory effects of ATS and STS on urea hydrolysis in
a Korean paddy soil. This effect of ATS was slightly lower than that of STS in glucose-
42
amended soil samples, but when glucose was not added, the effects of ATS and STS were
not significantly different.
Xue and Li (1987) prepared reaction mixtures from 5 g of paddy soil + 10 rn1 of 10% urea
solution containing 0 or 20-]()0 ppm (soil basis) of potassium ferricyanide (K3 [Fe(CN)6]) or
100 ppm of potassium permanganate (KMn04 ). After incubation (at 37°C for 48 hours), the
reaction mixtures were analyzed for NH4 + produced by urea hydrolysis. The analytical data
indicated that these compounds inhibited soil urease activity. The degree of inhibition at the
100 ppm rate was 37.52 and 24.75%, respectively.
* *
*
The following inorganic compounds will be described together with their organic
derivatives:
phosphorodiamidic acid and thiophosphorodiamidic acid in Subchapter 2.20;
phosphoryl triamide and thiophosphoryl triamide and their thermal polymers in
Subchapter 2.26; and
phosphonitrilic hexamide in Subchapter 2.27.
43
Structural formulas of the organic compounds tested as inhibitors of soil urease activity
are shown in Figure 2.
CI-H9-D-COOH Q-Hg-OOC-CH3
It should be emphasized from the very beginning that these inhibitors present only a
theoretical importance; they can not be recommended as soil urease inhibitors for field
applications because of the possibility of subsequent pollution of soil and groundwater
by mercury.
Mitsui et al. (1960) prepared reaction mixtures from 50 ml of M urea solution and 5
g of air-dried soil (control variant). In the experimental variant, sodium p-
chloromercuribenzoate (PCMB; 0.04% on dry soil weight basis) was added to the
mixture of urea solution + soil. The soil used derived from volcanic ash and was
cultivated with paddy rice. The reaction mixtures, after 1 hour of shaking, were
analyzed for NH/. The analytical results indicated that PCMB brought about a strong
(60%) inhibition of soil urease activity.
Briggs and Segal (1963) mention that activity of the crystalline urease preparation
obtained from a forest soil was markedly inhibited by PCMB (no details on the
inhibition experiment are given in their paper).
In a laboratory experiment, Moe (1967) applied PCMB, at a rate equivalent to 258
kglha, to the surface of a silt loam soil from Indiana. Due to PCMB the urea hydrolysis
rate was approximately halved, but PCMB had no significant effect on total volatile
ammonia losses from soil samples surface-treated with urea (336 kg N/ha) and
incubated at 28°C for 8 weeks, during which time soil moisture content was maintained
at 24%. Values of the total volatile NH, losses from the added urea-N were 4.68%
(untreated soil) and 3.94% (PCMB-treated soil).
Using the 5-hour test, Bremner and Douglas (1971) added 50 ppm (soil weight
basis) of phenylmercuric acetate (PMA) or PCMB to samples of three Iowa soils (silty
clay loam, loam, and clay loam). PMA, in comparison with PCMB, inhibited more
44
strongly the urease activity of the three soils. The degree of inhibition was 64-71 % (on
average, 67%) and 32-38% (on average, 35%), respectively.
The effect of the PMA rate (50, 100, 200, and 300 ppm) on urease activity in the
clay loam soil was also studied and it was established that the inhibition values
increased from 64 to 81 % with increasing PMA rate from 50 to 300 ppm.
In another experiment, samples of the three soils were preincubated with 50 ppm of
PMA or PCMB at 30°C for 3, 7, and 14 days, after which the 5-hour test was applied.
The results showed that the inhibitory effect of both organic mercury compounds on soil
urease activity markedly decreased with increased preincubation time which indicates
partial inactivation ofthese inhibitors in soil.
Some of the results concerning inhibition of urease activity in the clay loam soil
under the influence ofPCMB were also presented by Douglas and Bremner (1971).
Reexan1ining PMA, Bremner and Douglas (1973) determined not only the urea
hydrolysis but also the volatilization of an1monia from urea in five Iowa soils. PCMB
was also reexamined in one of the soils. Reaction mixtures were prepared from 109 of
air-dried soil, 1 ml of a urea solution containing 10 mg N (1,000 ppm), and 1 m1 of
water or 1 ml of a solution containing 0.5 mg inhibitor (50 ppm). Soil moisture content
was adjusted to 50% of WHC. Incubation took place at 20°e. After 3, 7, and 14 days,
the residual urea, exchangeable NIL +, and volatile NH3 were measured. The result.;;
showed that PMA and PCMB retarded urea hydrolysis and reduced NH.l volatilization.
These effects of PMA were strong in the light-textured soils and weak or inexistent in
the heavier soils. For example, during 14 days, NH3 volatilization from a sandy soil was
61.1 % of the added urea-N in the control soil and 23.5% in the PMA-treated soil, but in
the case of a heavier soil (clay loam) PMA reduced NH3 volatilization from 12.8 to
12.7%, i.e.. in this soil PMA had practically no affect on NH3 volatilization.
May and Douglas (1978) have evaluated the effect of PMA on urease activity of an
Australian sandy soil. Urease activity was determined by the method of May and
Douglas (1976). The reaction mixtures were prepared from 3 g of soil + 0.5 ml of
toluene + 12 ml of 1/15 M phosphate buffer (pH 8.8) + 3 ml of a solution containing 3
mg of urea, and were incubated at 31'C for 4 hours. After incubation the ~ + was
extracted and determined. PMA was used at four rates: 10,25,50, and 100 ppm (on soil
weight basis). At the two lower rates, PMA caused 80 and 95% inhibitions respectively,
whereas at the two higher rates, the inhibition was complete.
Shih and Souza (1978) used p-hydroxymercuribenwate (PHMB) as a urease
inhibitor to demonstrate that the 14C02 which evolved from 14C_urea in samples of a
California sandy loam soil was a product of the urease-catalyzed hydrolysis of urea. A
0.1 ml aliquot of 1.9 or 19 mM PHMB in phosphate buffer (25 mM, pH 8.5) or buffer
only (control) was added to 0.2-g soil samples. After 30 minutes, the mixtures received
14C_urea (0.01 /lCi activity) in the same buffer. Incubation took place at 23°C. 14C02
evolved during 2, 4, and 6 hours of incubation was measured. At the higher PHMB rate,
the 14C02 evolved in 2, 4, and 6 hours represented 0.38,0.47, and 0.77%, respectively,
as compared to 14C02 evolution from samples treated with urea only. These results,
corroborated with the finding that there was no 14C02 evolution when 14C_urea had been
added to autoclaved soil samples, prove that release of 14C02 from 14C_urea should be
attributed to soil urease.
Sahrawat (1979) adopted the 5-hour test of Douglas and Brenmer (1971) to evaluate
thc inhibitory effect of PMA (50 ppm) on urease activity of an Indian sandy clay loam
45
alluvial soil (PH 7.6). The inhibition caused by P.MA was 75%. But after I-week
incubation PMA inhibited urease activity to a lesser extent (62%).
Perez Mateos and Gonzalez Carcedo (1982) assayed urease activity in samples of
three Spanish soils (under vegetations dominated by poplar, gramineous, and
leguminous plants, respectively). The samples to which urea was added with or without
50 ppm (soil basis) of PMA or phenylmercuric borate (PMB) were incubated for 4
hours. It was found that the two compounds inhibited urease activity in the same soil to
a similar extent, which means that the anions (acetate and borate, respectively) did not
influence the inhibitory capacity ofPMA and PMB. Inhibition was lowest (62.38% with
PMA and 63.04% with PMB) in the soil richest in clay (the soil under leguminous
plants). In the other two soils 67.82-79.47% inhibitions were recorded. With the poplar
soil, the effect of different rates of PMA and PMB application on urease activity was
also studied and it was established that both compounds at a rate of 75 ppm gave
maximum inhibition (about 80%). Increasing the rate to 150,225, and 300 ppm did not
cause higher inhibitions. The authors drew the conclusion that in this soil the inhibition
exerted by PMA and PMB was of reversible non-competitive type.
For studying the mobility of PMA in soil, Praveen-Kumar et al. (1987) applied the
method of soil thin-layer chromatography. Samples of three Indian soils (acidic,
alkaline, and saline) were ground, sieved «0.15 mm) and coated on glass plates to
obtain thin-layers of soiL 100 11m thick. PMA was dissolved in methanol and the
solution containing 200 I1g of PMA was applied as a single spot on the soil thin-layer
plate. After volatilization of methanol, the plate was run using water as the solvent and
the solvent front was allowed to move 12 cm which took nearly 40 minutes. After
drying the plate was sprayed with a chromogenic reagent to develop color. PMA,
insoluble in cold water, was completely immobile in the thin-layers of each soil studied.
One can deduce from this observation that PMA - even if it would not cause soil
pollution - would not be an effective inhibitor of soil urease activity in agricultural
practice, because urea is very mobile and, therefore, may undergo the action of urease
from different soil depths in which urease is not inhibited since the inhibitor (PMA)
remains at the site of its application.
Studying the kinetics of urea hydrolysis in an alluvial soil (light-textured) and a
leached chernozern (heavy-textured) by determination of Michaelis constant (KM),
maximal velocity (V mIX), and inhibition constant (Ka, Simihaian and Silberg (1999)
found that PCMB exhibited stronger inhibition in alluvial soil than in chernozern; the
inhibition constant was l.8 and 2.9 mM, respectively. The conclusion was drawn that
inhibition of urease activity was competitive in both soils.
Van Der Puy et al. (l984b) patented organo boron acid compounds as inhibitors of soil
urease activity (Assignee: Allied Corporation, Morristown, New Jersey) and selected 1()
such compounds for testing their urease-inhibiting effect in a New York soil (Cazenovia
sandy loam, pH 7.2). Two tests were applied. In both tests the reaction mixtures were
prepared from 20 g of air-dried soil, 0.8 mg of test compound in 5 rnl of water, 42.8 mg
of urea in 1 ml of water, and incubated at 25°C for 3 days. In test A the soil treated with
test compound and urea was incubated, then analyzed for the remaining urea. In test B
the soil treated with test compound was incubated, then received urea and was again
46
incubated, after which the remaining urea was measured. No test compound was added
to the control reaction mixtures.
Four compounds (Figure 3) proved to be able to inhibit urease activity in both tests:
compounds I and II brought about excellent (strong) inhibitions in both tests, whereas
the inhibiting effect of compounds III and IV was partial in test A and excellent in test
B.
~V
B(OH)2 0
II
-6 B(OH)2
1'<:
: :,. . I NH2 .HO -S-OH . H2N A
II
o
3-Aminobenzeneboronic acid hemisulfate (I) 4-Hydroxyaminobenzeneboronic acid (II)
9~'
NH2 . Hel
Figure 3. Structure of four of the organo boron acid compounds patented and tested by Van Der Puy
et al. (19S4b) for inhibition of soil urease adivity.
2.3. FORMALDEHYDE
Urea pellets prepared from molten crystalline urea, 1%, (by weight) of formaldehyde
(CH 20) and 14% of blend of asphalt (90%)-rnicrocrystalline wax (10%) were used at a
practical rate and applied on the surface to samples of a sandy loam soil (pH 7.0;
moisture = 5.5%), then incubated at 25°C for 50 days. The control pellets did not
contain formaldehyde. During incubation, the ammonia volatilized was determined
cumulatively. In 50 days, the CH 2 0-containing urea pellets lost as NH3 8%, whereas the
control pellets lost about 23% of their initial N content. Formaldehyde is recommended
in amounts of (j.O I-I O'Yr, by weight of urea; the preferred rate ranges from 0.8 to 2.0'%.
For the asphalt-microcrystalline wax blend the preferred amount ranges from 3 to 20%
based on the total weight offertilizer pellets (Esso, 1966; Sor, 1968).
Verstraeten el af. (1976) performed two experiments. In the first experiment, 50-g
air-dry soil samples (pH 7.6) were treated with 100 ppm ofurea-N and 70-3,300 ppm of
CH 20, brought to 65'% ofWHC and then incubated under aerobic conditions at 30°C. In
the second experiment, 200 ppm of urea-N and 35-3,300 ppm of CH 2 0 were added to
samples of another soil (pH 5.0). These mixtures were incubated under water-logged
47
conditions. The control mixtures were prepared without CHzO. After 4(5), 7, 14, and 28
days of incubation, the mixtures were analyzed for NH4 +. It was found that under
aerobic and water-logged conditions, CHzO, even at the minimum concentration,
retarded hydrolysis of urea. This effect became more pronounced with increasing
concentrations of CH2 0, and the maximum concentration of CH 20 resulted in complete
st erilization of soil.
The effect of paraformaldehyde on urea hydrolysis was studied with an alluvial soil
and a leached chemozem (Kiss and Pintea, 1987). The reaction mixtures prepared from
5 g of air-dried soil and 10 ml of aqueous phase containing 6 mg of urea and 2% of
paraformaldehyde (on urea weight basis) were incubated at laboratory temperature.
Reaction mixtures without paraformaldehyde served for comparison. At 1-2-day
intervals the aqueous phase of reaction mixtures was analyzed for urea by means of a
chromogenic reagent (see the footnote on page 35). The results showed that
paraformaldehyde did not prolong the time necessary for complete hydrolysis of urea
since urea was completely hydrolyzed in 6 days (alluvial soil) and 9 days (leached
chemozem), irrespective of the absence or presence of paraformaldehyde.
2.4. HEXAMETHYLENETETRAMINE
N
I
1/
He CH2 CH2
~N
2
The urea derivatives tested as inhibitors of soil urease activity are specified in Figure 5.
Sor et al. (1966) patented for inhibiting soil urease activity the following urea
derivatives: methylurea, thiourea, N,N-dimethylurea, N,N' -dimethylurea, phenylurea,
t-butylurea, and n-butylurea to be applied in a proportion of 0.01 to 10%, preferably of
0.1 to 3% by weight of urea. The intimate mixtures of urea and inhibitor particles were
48
bound and coated with a hydrophobic material used in an amount ranging from 3 to
25%, preferably from 8 to 15% by weight of urea. For example, urea pellets were
prepared with addition of 0.45% methylurea or 2.44% thiourea + 13% asphalt-
microcrystalline wax blend. Incubation of these pellets, at a practical rate, on the surface
of a sandy loam soil (PH 7.5; moisture content = 6.4%) at 25 QC for 47 days led to
reduced volatilization of urea-N as ammonia. When a significant part (39.5%) of N
volatilized as NH3 from the urea priUs (no inhibitor), the NH3 losses from the
methylurea- or thiourea-containing urea pellets were 16.0 and 25.5%, respectively.
o
o s H3C, II
r-r-c-NH2
II II
H~-HN-C-N~ H;1N-C-N~ H3C""
Melhylurea Thiourea N,N·Dime1hylurea
o o
H3C- HN- c - NH--C~
II
N,N' ·Dimelhylurea
0-
~ A 11
HN-C-N H2
Phenylurea
o
H;>C, CH-CH~ HN-~- NH2
H~"" t-Bu1ylurea n-Bulyturea
NH 0 o
II II II
H;1N-C-HN-C-NH2 H~-C-NH--OH
Guanylurea Hydroxyurea
o o 0
II II II
H;>C- HN- C - NH-QH H~-C-NH--C-NH2
N·Melhyl·N'·hydroxyurea Biurel
Applying the 5-hour test, Bremner and Douglas (1971) recorded weak inhibitions in
urease activity of the three Iowa soils studied, the samples of which were treated with
urea derivatives at a rate of 50 ppm of soil. The average inhibition values were 7%
(thiourea), 4% (phenylurea), 5% (guanylurea sulfate), and 2% (hydroxyurea, biuret).
Kolyada (1970, 1973) described fertilization experiments in vegetation pots with a
soddy-podzolic soil (pH 4.4). The fertilizers (thiourea, ammonium nitrate, urea;
superphosphate; KC1) were applied at a rate of 0.1 g N, P, and K/kg dry soil. The
fertilizer treatments were the following: unfertilized control; PIC; thiourea + PK;
~N03 + PIC; 0.1 parts of thiourea-N + 0.9 parts of NH4N0 3 -N + PK; 0.1 parts of
urea-N + 0.9 parts ofNH4 N0 3-N + PK; exceptionally, only 0.1 parts ofthiourea-N (i.e..
not 0.1, but 0.01 g ofthiourea-N/kg soil) + PK. Moisture content of soil was 60% of
WHC. Oats, barley, onion, and radish were used as test plants. Urease activity was
determined at day 5 after fertilizer application and during the growing season.
The results indicated that thiourea decreased this activity only in the treatment in
which the rate of thiourea-N was 0.1 glkg soil. In this treatment with oats, at day 5 after
fertilization, urease activity was 0.45 mg NH3/g soil/24 hours, whereas in the other
treatments the corresponding activity values were between 1.00 and 1.30. During the
growing season the depressive effect of thiourea on soil urease activity has attenuated.
The effect of biuret on urease activity was the objective of a study by El-Sayed et of.
(1976). Biuret was found not to inhibit activity of pure jackbean urease, but did inhibit
the urease extracted from soil. The extract was obtained from a Giza (Egypt) soil by
shaking the soil suspension (soil:distiIIed water = 1:10) for 24 hours, followed by
filtering through Whatman No.42 paper. In the reaction mixtures containing soil extract
the concentration of urea wa<; 10 mM and that of the biuret ranged from 0.1 to 40 mM.
At concentrations up to 2 mM, biuret manifested a negligible inhibitory effect; the
inhibition increased with increasing biuret concentration and became complete at 40
mM biuret concentration. The inhibition caused by biuret in the urease activity of soil
extract was of the competitive type.
It should be mentioned that biuret can form during melting of urea for production of
priUs: at the melting temperature of urea (132.7!'C), some urea molecules are split into
NH3 and HN=C=O (isocyanic acid), which reacts with urea to form biuret:
Therefore, biuret can be present as an impurity in the urea fertilizer. If this fertilizer
contains more than 2% biuret in the case of soil application or more than 1% in the case
of foliar application, it will exhibit phytotoxicity just because of the biuret (Michaud et
af., 1978).
Gould et al. (1978) established that urease activity in samples of a silt loam soil
from Alberta was weakly inhibited by thiourea, N-methyl-N'-hydroxyurea, and
hydroxyurea, the degree of inhibition being 9, 15, and 16%, respectively. Urease
activity was measured according to the technique described by Gould et of. (1973). The
reaction mixtures consisted of 25 g of soil, 400 ppm of urea-N, 0 or 100 ppm of urea
derivative, 32% moisture (field capacity), and were incubated at 25!!C for 24 hours.
Sahrawat (1979) registered a 10% inhibition in urease activity, when thiourea was
added, at a rate of 50 ppm, to samples of a sandy clay loam soil (PH 7.6). Urease
activity was assayed under the conditions of Douglas and Bremner's (1971) 5-hour test.
so
Malhi and Nyborg (1979) conducted pot and field experiments in Alberta. In the pot
experiments, they studied the effect of thiourea on urea hydrolysis as influenced by soil
moisture levels, size, and method of application of urea and urea + thiourea pellets. Two
soils (loam, pH 7.3 and silty clay loam, pH 6.0) were used, each in an amount of 1
kg/pot. Urea and thiourea were pelleted together in a ratio of 2 parts urea and 1 part
thiourea. The size of the urea and urea + thiourea pellets was OJn or 0.21 g. The pellets
were spread uniformly over the soil surface or mixed into the soil at a rate of 224 kg
urea-N/ha (N in thiourea was not taken into consideration). The control soil was not
fertilized. h1cubation was at 20"C for 20-160 hours and urea hydrolysis was estimated
by measuring the residual urea. The results showed that at the same moisture level, size
and method of application of pellets, urea hydrolysis was significantly slower when the
soil was treated with urea + thiourea than when only urea pellets were applied. Thiourea
suppressed urea hydrolysis by about SO% for approximately 1 week. It was also
established that urea hydrolysis increased with increasing soil moisture levels and
decreased slightly with the increased size of pellets; mixing of urea into soil enhanced
the hydrolysis of urea relative to surface application.
In the field experiments on six soils (one silty clay loam, two clay loams, two loams,
and sandy loam) the effect of thiourea on urea hydrolysis was studied in relation to the
method of application of urea and urea-thiourea pellets (in these experiments, too, two
parts of urea were copelletcd with one part of thiourea). The peliets were placed in
bands (at S cm depth) or mixed into the soil 10 cm deep. The rate of urea application
was S6 kg N/ha. At day 14 after fertilization, the NH/ and NO}- contents in tl1e 0-IS-
cm soil layer were analyzed. The analytical data obtained confirmed tl1e results
registered in the pot experiments: in each soil, thiourea retarded the hydrolysis of urea
(degree of inhibition "" SO%), band placement of pellets-as compared with their mixing
into the soil- diminished the rate of urea hydrolysis.
In another field experiment, on the silty clay loam, urea and urea + thiourea (2:1)
pellets of different size (0.01, 0.21, and 2.26 or 2.S1 g) were tested. The 0.01- and 0.21-
g pellets were banded S cm deep. The larger pellets were applied in a grid pattern at
distance of 30 cm and a depth of Scm. Nitrogen was administered at a rate of 112
kg/ha, taking into account the thiourea-N, too. Urea hydrolysis was studied by
determining the NH4 + and NO} - contents in the 0-IS-cm soil layer. The results obtained
R days after fertilization are presented in Table 6 from which it is evident that thiourea
significantly reduced (by approximately SO%) the rate of urea hydrolysis. Urea
hydrol ysis also decreased with increasing pellet size.
TABLE 6. Effect of thiourea and pellet size on urea hydrolysis in a silty clay loam soil"
Fertilizer Pellet size (g) Urea hydrolysis (%)
Urea (UII 98 a
Urea + thiourea (2:1) (Ull 49 d
Urea 0.21 84 b
Urea + thiourea (2:1) 0.21 36 e
U~ 2~ ~c
Urea + thiourea (2:1) 2.51 25 f
aFrom Malhi and Nyborg (1979), by permission of Kluwer Academic Publishers.
The values are significantly different (p=0.05) when not followed by the same letter.
51
Moawad et al. (1984) treated 20-g samples of an alluvial soil (silty clay, pH 7.4)
from the Nile Delta with 400 ppm N in form of urea or thiourea, then incubated them
under favorable humidity conditions at 30"C. Untreated samples served for comparison.
Urease activity assayed after 1, 3, 7,14,21, and 28 days of incubation was always lower
in the thiourea-treated samples than in the urea-treated and untreated ones. Similarly,
ammonia volatilization from thiourea was more reduced than from urea. For example,
after 28 days of incubation, the volatile NH3 loss was 5.5% from thiourea and 1l.8%
from urea.
Studying the alluvial alkaline soils from Pakistan, Hamid and Ahmad (1987) also
dealt with the effect of thiourea on volatilization of ammonia from urea. Samples of a
calcareous soil (pH S.O) were treated with urea or with urea + 5% thiourea (on urea
weight basis) and then incubated. The NH3 volatilized during incubation was
determined. It was found that in 112 days the cumulative NH3 losses were reduced by
50% under the influence of thiourea.
The German investigators Hartbrich et al. (1978) tested the effect of thiourea on soil
urease activity in comparison with that of various derivatives of thiourea. Two testing
methods were applied.
In the short time test, a black soil with strong adsorptive capacity was used. The soil
samples (30 g) were moistened to 50% ofWHC and then mixed with 214.1 mg of urea
(I 00 mg N) with or without 4 mg of compound to be tested (4% on urea-N basis). The
mixtures were incubated at 30"C for 24 hours, during which the volatilized ammonia
was determined.
In the long time test (see also Iasche et al., 1978), a sandy soil (i.e .. a soil with weak
adsorptive capacity) was used. The 30-g soil samples were moistened to 50% of WHC
and ill1 their surface urea (214.1 mg) with or without 1 mg of test compound was
uniformly distributed. Incubation took place at 20"C. The amount of volatile NH3 was
recorded at 2-3-day intervals.
TABLE 7. Effect of thiourea and its derivatives on volatilization of ammonia from urea-treated soils"
Inhibition (%)
STT LTT
Compound
Incubation time (days)
2 4 6 8 10
Thiourea 13 21 9 0
Bis thiourea 69 85 49 35 12 0
N-Phenylthiourea 29 31 9 ()
N-Phenyl-N' -isopropylthiourea 53 10 2 ()
N-Phenyl-N'-t-butylthiourea 53 18 6 0
N-Phenyl-N'-ethyl-N'-cyclohexylthiourea 9 16 6 ()
N-Phenyl-N'-ethyl-N'-benzylthiourea 7 14 4 ()
N-Phenyl-N',N'-dibenzylthiourea 4 4 ()
Allylthiourea 19 25 IJ ()
N-2-Aminophenyl-N'-allylthiourea 26 31 19 8 ()
"From Hartbrich et al. (1978).
STT - Short time test.
LTT - Long time test.
S2
The data in Table 7 show that the degree of inhibition varied depending on the
nature of thiourea derivatives and on the incubation time. Thus, the inhibiting effect of
bisthiourea was the strongest and evident for 8 days; N-phenyl-N' ,N' -dibenzylthiourea
had a negligible inhibitory effect, detectable only during the first 2 days of incubation.
In these tests, even the thiourea was a weak inhibitor.
Using samples of brown earths from Germany, Germann-Bauer (1987) carried out
several laboratory experiments to study urea hydrolysis and ammonia volatilization
from urea under the influence of guanylthiourea (GTU):
NH S
II II
H2N-C-HN-C-N~ .
For studying urea hydrolysis, reaction mixtures were prepared from SO-g (dry
weight) samples of a loess brown earth + 20 mg of urea-N + 0, 2 or 3 mg of GTU and
submitted to incubation at 4, 8, 12 or 16QC for 1 or 2 weeks, then analyzed for residual
urea. The results indicated that GTU retarded urea hydrolysis, and this effect increased
with increasing rate of GTU and decreased with increasing incubation temperature and
time.
Ammonia volatili7ation from urea was studied with SOO-g samples of a sandy brown
earth. Two experiments were carried out. In the first experiment the reaction mixtures
were prepared from soil samples + 100 mg of urea-N + 0 or 10 mg of GTU and
incubated for 6.6 days. In the second experiment the reaction mixtures, prepared from
soil samples + SO mg ofurea-N + 0 or S mg of GTU, were incubated for 13 days. The
cumulative NH3 loss, assessed during the incubation (6.6 or 13 days, respectively), was
reduced significantly (p<O.OS) (from 17.7 to 13.3%) in the first experiment, but
insignificantly (from 31.8 to 27.3%) in the second experiment.
2.6. DITHIOCARBAMATES
r3C-CH2)~~_sJ
In
A
~aC-CH2
Diethyld~hiocarbamate
t~-""-L-j" '
Methyldithiocarbamate
A =Na+ or Zn 2+
n = Valence of A
Figure 6. Dithiocarbamates patented and tested by Hyson (1963) for inhibilion of soil urease
activity.
S
II
CH2-HN-C-S-Na
I
CH2 -HN - C - S -Na
II
S
S
II
HN-C-S-CH3
I
HN-CO-CH3
-0-
S
II
CI ~ /) HN-C-S-NH4
Figure 7. Dilhiocarbamates patented and tested by Tomlinson (1967) for inhibition of soil urease activity.
zineb reduced NH3 volatilization in parallel with its rates in four soils, irrespective of its
rates in one soil, whereas in the other four soils zineb did not reduce NH3 volatilization
at any of its rates. Maneb and the other three dithiocarbamates, tested at rates of 20 or
40 ppm with two soils, manifested a weak inhibitory effect or no inhibition, the NH3
loss in 7 days varying between 63 and 99%.
In another experiment, zineb and, separately, ChBrC-CBrCh were added to samples
of a soil, at rates of 0, 5, 20, 50, and 200 ppm. Moreover, the two compounds at the
rates mentioned before were also applied in their all possible combinations. In each
case, the urea rate was 200 ppm N. Ammonia losses during 8 days of incubation showed
a tendency to decrease with increasing rates of the two compounds. The decrease was
more marked with ChBrC-CBrCh than with zineb. Ammonia losses decreased from
7.4% (control soil without addition of zineb and C1 2BrC-CBrCIz) to a minimum value
(0.5%) in the soils treated with 0,5 or 50 ppm ofzineb + 200 ppm ofCIzBrC-CBrCl z.
In the second method wheat seedlings are used for testing the inhibitors of soil
urease activity in urea-treated soil samples. This is why this method and the results
obtained will be dealt with in Subchapter 7.2 (on pages 264-265).
55
Pugh and Waid (1969a,b) treated 100-g moist samples of three English soils (sandy
loam, loamy sand, and clay loam) with 6.66 mM of urea and 0.4 mM of sodium
diethyldithiocarbamate, (CHr CH 2 hN-CS-S-Na. The control samples received urea
only. All samples of the three soils were incubated at 20°C for 30, 77, and 148 days,
respectively, during which time the volatilized ammonia was determined. The half-loss
times of NH3 loss from the control and the diethyldithiocarbamate-treated samples were
4 and 5.5 days (soil 1), 11.5 and 14 days (soil 2), and 34 and 59.5 days (soil 3),
respectively, i.e.. sodium diethyldithiocarbamate prolonged half-loss time of NH3 in
each soil. However, the total NH3 losses from the three soils during their whole
incubation period (30, 77, and 148 days, respectively) were similar in the control and
the diethyldithiocarbamate-treated samples (51.2 and 56.8%,39.8 and 45.0%, and 84.0
and 81.9%, respectively).
Under conditions of the 5-hoUf test, Bremner and Douglas (1971) recorded 1, 3 or
7% inhibitions of urease activity in samples of three soils treated with sodium
diethyldithiocarbamate or dimethyl ammonium dimethyldithiocarbamate, (CH3hN-CS-
S-NH 2(CH3h, at a rate of 50 ppm (on soil weight basis).
Lang et al.(l976) patented ferric dimethyldithiocarbamate, [(CH3hN-CS-ShFe (the
fungicide ferbam), as an inhibitor of soil urease activity. Urea with or without 1%
ferbam (relative to urea-N) was surface-applied on soil samples, which were then
moistened to 50% of WHC and incubated at 20°C for 19 days. During incubation, the
volatile ammonia was estimated. The degree of inhibition registered after 5, 10, and 19
days of incubation was 94.7, 81.3, and 24.0%, respectively. This means that the
inhibitory effect of ferbam on the volatilization of NH3 from urea was considerable for
10 days, then decreased very much. Ferbam as an inhibitor of soil urease activity is
recommended for practice at preferred rates of 0.05-5% (relative to urea-N).
Hartbrich et al. (1978), applying the short and long time tests (see page 51),
established that all dithiocarbamates tested, which also comprised important fungicides,
inhibited ammonia volatilization from urea, but the duration of their effect did not
exceed 8 days (Table 8). The results obtained with maneb were also referred to by
Oertel et al. (1978).
Inhibition (%)
STT LTT
Compound
Incubation time (days)
2 4 8 12
Iron dimethyldithiocarbamate (ferbam) 79 100 42 12 0
Zinc dimethyldithiocarbamate (ziram) 82 74 33 14 0
Aluminium dimethyldithiocarbamate 63 82 20 3 0
Sodium di-n-propyldithiocarbamate 72 71 13 0
Sodiumn-butyldithiocarbamate 86 100 72 11 0
Zinc ethylene-I.2-bisdithiocarbamate (zineb) 67 79 17 5 0
Manganese ethylene-I,2-bisdithiocarbamate (maneb) 81 100 51 19 0
"From Ilartbrich et al. (1978).
STT - Short lime test.
L TT - Long time test.
56
In 400-g air-dried samples of an Egyptian clay loam soil treated with urea (70 ppm)
and moistened to 60% ofWHC, zineb at a rate of 3 kglha reduced only to a small extent
urea hydrolysis during the first 3 days of incubation at 25-28 D C (the unhydrolyzed urea
being 3% of the initial amount) and even this weak inhibitory effect disappeared after 7
days of incubation (Moawad et al., 1979a). In contrast, zineb decreased urease activity
in the urea-treated soil samples during the whole (35-day) incubation period
(Moawad et al., 1979b).
Tu (1980) treated samples of a Canadian sandy loam soil (pH 7.6) with maneb at
rates of 0, 100, and 200 ~glg soil and determined their urease activity after 2, 7, and 14
days of incubation. The activity was inhibited significantly (p=0.05) by maneb during
the whole incubation period. As expected, the inhibiting effect was stronger at the
higher maneb rate.
Maneb, added to samples of a clay loam soil (PH 7.2) at two rates (5 and 10 ~g/g
soil) insignificantly inhibited urease activity after 2 days of incubation and did not
influence it at all after 7 days. There was no difference between the effects of the two
maneb rates (Tu, 1981a). In contrast, in an organic soil (pH 7.2), maneb at both rates
significantly inhibited urease activity after 7 days of incubation and significantly
stimulated it after 14 days. The inhibition was stronger and the stimulation was weaker
at the higher rather than the lower rate ofmaneb (Tu, 1981b).
Hartbrich et al. (1978) also published data on testing of four carbamates and drew
the conclusion that these compounds did not show any urease-inhibiting effect.
on incubation time. For example, the inhibition was 97.5% after 4 days, 71.6% after 12
days, and 35.0% after 18 days of incubation. The preferred rates, recommended for
practice, range from 0.05 to 5% thiram relative to urea-No
Thieme et al. (1976) applied the same technique with the same incubation time, but
worked with three thiram concentrations (0.1, 0.5, and 1% relative to urea-N) and
carried out the incubation at 10o e. Determination of the volatilized ammonia indicated
that after the same incubation time inhibition of NH3 volatilization increased with
increasing thiram concentrations, but at each concentration it decreased during
incubation. For example, the inhibitions recorded in samples treated with 0.1, 0.5, and
1% thiram were 70.0, 91.2, and 94.2%, respectively, after 3 days of incubation, and 4.8,
23.4, and 42.0%, respectively, after 10 days. Thiram at 1% concentration after 18 days
of incubation caused only an 11.3% inhibition. Similar results were also presented by
Hartbrich et al. (1978), Oertel et al. (1978), and Jasche et al. (1978), with the difference
according to their data that thiram used at a rate of 1% inhibited NH3 volatilization only
for 12 days.
By applying the short and long time tests, Hartbrich et al. (1978) studied other
thiuram disulfides as well as some thiuram sulfides. Table 9 shows that the inhibitory
effect of tetramethylthiuram disulfide was weaker than that of tetramethylthiuram
sulfide. The other compounds were also weaker inhibitors and their effect was
detectable only during the first 2-8 days of incubation.
TABLE 9. Effect of thiuram disulfides and thiuram sulfides on volatilization of ammonia from urea-
treated soils·
Inhibition(%)
STT LTT
COlT(lound
Incubation time (days)
2 4 8 12 14
Tetmmethylthiuram disulfide 64 83 38 22 12 o
Dimethylthiuram disulfide 28 21 3 0
Di-n-propylthiuram disulfide 40 57 31 11 0
Diisopropylthiuram disulfide 54 78 43 20 0
Diisobutylthiumm disulfide 74 65 36 15 0
Dibenzylthiuram disulfide 31 26 0
Dipiperidylthiumm disulfide 28 35 3 0
Tetmmethylthiuram sulfide 79 93 S6 33 21 4
Dipiperidylthiumm sulfide 33 39 12 3 0
·From Hartbrich et al. (1978).
STT - Short time test.
LTT - Long time test.
2.8. XANTHATES
Brenmer and Douglas (1971) mention that, under the conditions of the 5-hour test,
potassium ethyl xanthate (KEtX) applied to three soils at a rate of 50 ppm of soil, gave
less than 4% inhibition of urease activity.
Ashworth et af. (1979) tested KEtX, cellulose xanthate (CX) (Figure 8), and a
mixture in which half of the KEtX amount was replaced by sodium trithiocarbonate
(Na 2CS). As already shown (see page 36), Na2CS] alone has a very weak inhibitory
s s
II II
CH3-CH2-0-c-S-K (cellulose chain) HC-O- C-S - Na
I
Potassium ethyl xanthate Cellulose xanthate
Figure 8. Xanthates tested by Ashworth et al. (1979) for inhibition of soil urease activity.
effect on urea hydrolysis. The tests were performed on a silty clay loam from England.
The reaction mixtures, consisting of 8 g of soil + 1 rnl of a solution containing 5 mg of
urea + 1 rnl of water or 1 ml of solution of test compound (s), were incubated at 24°C
for 5 weeks. Residual urea, NH/, and N0 3- contents were determined weekly.
Inhibition of urea hydrolysis increased in the following order:
KEtX < KEtX + Na2CS) < ex.
59
It should be emphasized that the effect of KEtX and Na2CS3 was synergistic. These
compounds inhibited nitrification more strongly than ureolysis·. Xanthates generate
CS 2, but more slowly than does Na 2 CS 3 , i.e .. xanthates are slowly acting inhibitors of
nitrification. At the same timc, their inhibitory effect on urea hydrolysis was not due to
CS 2 , because in separate tests CS 2 used in amounts stoichiometrically equivalent to
those of KEtX showed no urease inhibition.
Ashworth ef al. (1980) tested l7 xanthates as inhibitors of urease activity in a
Canadian clay loam soil. The reaction mixtures were prepared from 18 g of soil + 0.5
ml of urea solution (400 mg Nlkg soil) + 0.5 ml of water or xanthate solution (200 mg
compound/kg soil). For comparison, Na 2CS J andp-benzoquinone were used. Incubation
took place at 23°C. Residual urea was determined after 17, 24, and 41 hours of
incubation. Table 10 specifies only those xanthates which manifested an evident
inhibitory effect on soil urease activity; the percent inhibitions caused by xanthates,
Na2CS3, and p-benzoquinone are also presented.
Based on the results obtained with the xanthates specified in the table and with other
xanthates, Ashworth et al. (1980) arrived at the following conclusions: the inhibiting
effect of the xanthates of unsubstituted primary alcohols decreased with increasing size
of the hydrocarbon chain; branched-chain xanthates were less effective than those with
straight-chain; xanthates of substituted alcohols were weaker inhibitors than their
unsubstituted counterparts, except for the xanthatc of 2-nitrilo-2-propanol which,
though more effective than K isopropyl xanthate, still gave weak inhibition; the
This is why Rodgers et ai. (1987) applied the mixture ofKEtX (at 5 kg/hal and Na2CS, (at a rate equivalent
to 10 kg CSz/ha) as a nitrification inhibitor.
60
The inhibiting effect of monohydroxarnic acids on urease actIvIty in soil was the
obj ective of many studies, but this effect of dihydroxamic acids was dealt with, to our
knowledge, in a single work.
H;f--Co--NH-OH
G - c H2-CO-NH-OH
Acetohydroxamic acid
Phenylacetohydroxamic acid
H;f--GH2 - CO ---lIIH-OH
Propionohydroxamic acid Q-cO-NH-OH
Benzohydroxamic acid
H~(CH2l4 - CO--f\lH-OH
Caprohydroxamic acid
q-CO-NH-OH
H;f--(CH2ls - CO-NH-OH
Salicylohydroxamic acid
Caprylohydroxamic acid
Figure 9. Monohydroxamic acids tested by Pugh and Waid (196Qa,b) for inhibition of soil
urease activity.
61
16 soils (13 from England and three from Benin, Nigeria, and Brunei-North Borneo,
respectively), and the effect of the monohydroxamic acids on urease activity in two soils
(sandy loam and clay loam) from England (Pugh and Waid, 1969b; see also Waid,
1975).
Waid and Pugh (1967) treated moist samples (1 kg) of the acid loamy sand with
4,000 ppm of urea (weight/weight) + 0 or 200 ppm of acetohydroxamic acid (AHA),
then incubated them at constant temperature (25°C). During incubation, analyses were
carried out periodically for determination of residual urea and soil pH. AHA was
detectable for 8 days (only traces on the 8th day). In this period, urea hydrolysis was
slower and pH increase was smaller in samples treated with urea and AHA than in those
treated with urea alone. Consequently, complete hydrolysis of urea required about 14
days in AHA-treated samples and 8 days in samples without AHA addition.
In other experiments, Waid and Pugh (1967) followed volatilization of ammonia
from urea (4,000 ppm) in 100-g moist soil samples incubated at laboratory temperature
(fluctuating between 16 and 22°C), and found that this process was delayed by the
addition of AHA (200 ppm). Thus, volatilization ofNH 3 from samples without and with
AHA began after 6 and 13 days of incubation, respectively. Total urea-N losses as
volatile NH3 during 20 days of incubation were about 32 and 25%, respectively.
Pugh and Waid (1969a) also followed volatilization of NH3 from urea for studying
the effect of seven monohydroxamic acids (Figure 9) on urease activity in the acid
loamy sand. Moist 100-g soil samples, to which 4,000 ppm of urea (weight/weight) and
0,50,100,200 or 300 ppm of AHA or 200 ppm of the other six monohydroxamic acids
were added, were then incubated at laboratory temperature (14-23°C) and the amount of
volatilized NH3 was estimated during 29 days. AHA proved to be a more effective
inhibitor than the other monohydroxamic acids: half-loss time of urea-N as NH3 was
prolonged from 10 to 17.5 days in presence of 200 ppm of AHA and from 10 tol 0.5-13
days under the influence of the same concentration of the other monohydroxamic acids;
the weakest inhibitors were phenylacetohydroxamic and caprylohydroxamic acids.
Concerning total loss of urea-N in 29 days, there were no remarkable differences
between these compounds (35.3% in control soil without inhibitor, 30.2% in AHA-
treated soil and 31.3 -36.4% in soil samples treated with the other monohydroxamic
acids). By increasing the concentration of AHA from 50 to 300 ppm, half-loss time of
NH3 was prolonged from 12 to 23.5 days.
In other experiments, in which 0, 0.1, 0.2, and 0.3 mM of benzohydroxamic acid
(BHA) and 6.66 mM of urea were used per 100 g of soil and incubation was carried out
at either laboratory temperature (13-21°C) or constant (20°C) temperature, it was also
observed that half-loss time of NH3 was prolonged proportionately to the concentration
of inhibitor. Total NH3 loss decreased with increasing BHA concentrations at laboratory
temperature, but the reverse was the case at 20°e.
The relationships between the delay in NH3 loss and concentrations of urea and
AHA were also studied. The moist soil samples (100 g) were treated with 1.67, 3.33,
6.66, and 13.33 mM of urea and with 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM of AHA (1.67
mM of ureall 00 g soil is equivalent to 1,000 ppm of urea, weight/weight, and 0.2 mM
of AHA/I 00 g soil to ISO ppm of AHA, weight/weight). Incubation took place at 20°C
and lasted 29 days. One can deduce from the results presented in Figure 10 that half-loss
time of NH3 was shorter when urea was added at higher concentrations; AHA delayed
62
NH3 loss and the length of the delay was directly proportionate to its concentration at
anyone concentration of urea.
Figure 10. Relationships between the delay in ammonia loss (expressed in terms ofhaIf-loss
time) and concentration of urea and AHA. IFrom Pugh and Waid (1969a). by permission of
Pergamon Press PLC./
Pugh and Waid (1969a) also carried out an experiment for studying the effect of
AHA on the volatilization of NH3 from either crystalline or prilled urea. The crystalline
urea was mixed evenly throughout the soil or placed on the surface of the soil or at
about 2-cm depth; the prilled urea was placed on the soil surface or at about 2-cm depth.
AHA was applied in or on soil like urea. The urea rate was 4,000 ppm and that of AHA
was 0 or 200 ppm. All samples were incubated at laboratory temperature (16-23°C) for
30 days. In all treatments AHA had a delaying effect on NH3 volatilization. In the
homogeneous mixture of soil + crystalline urea + AHA, the effect of AHA was weaker
than where this compound and urea were placed together on the soil surface or at depth.
Total NH3 losses from both crystalline and prilled urea were smaller with deep than
with surface placement.
It was also established that preincubation of soil samples with 200 ppm of AHA at
25°C before addition of 4,000 ppm of urea and incubation at laboratory temperature
(14-22°C) led to diminution of the inhibitory effect of AHA on volatilization of NH3
from urea. The diminution was evident even after 2 days of preincubation, and in
samples preincubated for 5 days, the inhibitory effect of AHA became negligible. This
behavior of AHA during preincubation may have been due either to its chemical
inactivation and microbial decomposition or to the proliferation of urease-forming
microorganisms tolerant towards AHA.
StUdying the effect of AHA in 16 soils, Pugh and Waid (1969b) treated 100-g moist
samples with 4,000 ppm (6.66 mM) of urea and 300 ppm (0.4 mM) of AHA,
determining, during the incubation at 20°C, the amount of volatile NH3 • The results
indicated an underlying influence of soil properties, especially of soil texture on the
urease-inhibiting effect of AHA. Thus, AHA reduced the maximum rate of NH3 loss
63
and delayed the half-loss time in 10 light-textured soils (3 sands, 4 loamy sands, and 3
sandy loams). In contrast, AHA did not exhibit such an effect in 6 soils with high clay
content. At the same time, AHA brought about even an increase in the rate of NH3 loss
from two of these six soils.
Of the seven monohydroxamic acids tested with two soils (sandy loam and clay
loam), the most effective compound in reducing the rate of NH3 loss was AHA in the
sandy loam and BHA in the clay loam, in which the other six monohydroxamic acids,
excepting salicylohydroxamic acid, accelerated the NH3 loss. The conclusion could be
drawn that monohydroxamic acids offer promise as a means of delaying the hydrolysis
of urea only in the light-textured soils.
By applying the 5-hour test, Bremner and Douglas (1971) registered 16, 14, and
13% inhibitions in urease activity of three soils (silty clay loam, loam, and clay loam),
the samples of which were treated with 50 ppm of AHA. At the same rate, BHA
exhibited smaller inhibitions (12, 10, and 8%, respectively). In the clay loam, the
influence of different rates of AHA on urease activity was also studied and it was
established that inhibition increased from 13 to 24, 33, and 44% when AHA rate was
increased from 50 to 100,200, and 300 ppm, respectively.
Preincubation of soil samples with 50 ppm of AHA at 30°C, before applying the 5-
hour test, led to diminution and, then, to disappearance of the inhibiting capacity of
AHA in each of the three soils. For example, a 3-day preincubation of the clay loam
with AHA diminished the inhibition from 13 to 5%, whereas in samples preincubated
with AHA for 7 days the urease-inhibiting capacity of this compound was not detectable
any more. The results obtained in the preincubation experiment with the clay loam were
also presented in a paper published by Douglas and Bremner (1971).
Continuing the investigations along this line, Bremner and Douglas (1973) evaluated
the effect of AHA on both urea hydrolysis and volatilization of urea-N as ammonia in
five Iowa soils. Ten-g soil samples were treated with urea (1,000 ppm of N) + 0 or 50
ppm of AHA, and water to 50% ofWHC and incubated at 2()°C. After 3, 7, and 14 days
of incubation, the residual urea, exchangeable NH4 +, and volatilized NH3 were recorded.
It was found that AHA inhibited ureolysis and NH3 volatilization in light-textured soils
and had a weak inhibitory effect in heavy-textured soils. For example, in a sandy soil
not treated with AHA, urea was not detectable after 14 days of incubation, whereas in
AHA-treated samples of the same soil, a part (19.4%) of the initial amount of urea
remained unhydrolyzed. In this soil urea-N losses through volatilization of NH3 during
14 days were 61.1 and 25.0%, respectively. In both untreated and AHA-treated samples
of a clay loam, urea was completely hydrolyzed in 14 days, while the volatilized NH3
represented 12.8% (untreated samples) and 12.3% (treated samples) of the initial
amount ofurea-N.
May and Douglas (1978) studied the effect ofBHA on urease activity of a sandy soil
from Australia. BHA was applied at rates of 10, 25, 50, and 100 ppm (on soil weight
basis). The reaction mixtures (3 g of soil + 0.5 rnl of toluene + 12 rnl of 1115 M
phosphate buffer, pH 8.8 + 3 ml of a solution containing 3 mg of urea + BHA) were
incubated at 37°C for 4 hours, then analyzed for ~ + released from urea. Reaction
mixtures without BHA served for comparison. It was calculated from the analytical data
that BHA at its four rates inhibited urease activity in proportions of 4, 14,29, and 46%,
respectively.
64
Gould el al. (1978) and Gould (1979) prepared mixtures from 25-g samples of a silt
loam soil, urea (400 ppm of N), 0 or 100 ppm of AHA and water (up to field capacity)
and incubated them at 25°e for 24 hours. Under these conditions, AHA caused a 17%
inhibition of urease activity.
Matzel et al. (1978) conducted experiments in vegetation pots containing 8-10 kg of
acid sandy soil (pH 3.9-4.8) or black soil (loess, pH 7.2) from Germany. Urea prills
were placed on the soil surface (225 em2 ) at a rate equivalent to 200 kg Nlha and AHA
was incorporated into the 0-I-cm soil layer at a rate of 200 ppm (7% on urea weight
basis). After moistening the soils to 60% ofWHC, the pots were kept at 20-22°e, and at
days 5, 12, and 19 the 0-5-cm soil layer was analyzed for residual urea and NH4 +.
Volatilization of ammonia from urea was studied with the sandy soil in smaller pots
(1.5 kg of soil). Urea (200 kg Nlha) was surface-applied and AHA was administered
together with urea at a rate of 3% on urea weight basis. Incubation was carried out at
20-22°e for 28 days, during which time the volatile NH3 was assessed.
The analyses showed that AHA inhibited urea hydrolysis, especially in the acid
sandy soil. Thus, in this soil when not treated with AHA, urea was present after 5 days
of incubation (the residual urea was equal to 4.1 % of the initial amount), but no urea
was detectable after 12 and 19 days. In the presence of AHA, urea remained
unhydrolyzed in proportions of 38.3% (after 5 days), 13.4% (after 12 days), and 1.1 %
(after 19 days). In the loess, after the three incubation times, the residual urea
represented 31.6, 17.5, and 0%, respectively (soil not treated with AHA) and 40.7, 24.6,
and 0(%, respectively (AHA-treated soil) in comparison with the initial amount of urea.
The NH3 loss was 18.3% in the absence of AHA and 0.4% in its presence after 10
days of incubation. When the incubation was prolonged to 28 days, the effect of AHA
became negligible, as the NH3 loss was 19.3% in the absence of AHA and 18.7% in its
presence.
Matzel et al. (1978) mentioned that AHA was also tested with a series of other soils,
but the inhibitions were weaker than those specified above in the case of the acid sandy
soil (see also Matzel and Heber,1979).
By using the short and long time tests, Hartbrich et af. (1978) examined the effect of
11 monohydroxamic acids on volatilization of ammonia from urea. Caprylohydroxamic
acid and AHA were the most effective: their inhibitory effect, after 8 days of incubation,
was 25 and 6%, respectively (see also Oertel et at., 1978; Jasche et al., 1978). Very
weak inhibitory effects were exhibited by ~-dimethylaminopropionohydroxamic,
bromoacetohydroxamic, and N-phenyl-~-chloropropionohydroxamic acids which
reduced volatilization ofNHJ only by 3,5, and 8%, respectively, after I-day incubation,
and did not have any inhibitory effect after 2 days of incubation. The other hydroxamic
acids occupied, in terms of their inhibitory effect, intermediary positions in the order:
N-phenyl-n-cyclohexylarnino-AHA>chloro-AHA>~-diethylaminopropionohydroxarnic
acid>f\-ethylaminopropionohydroxarnic acid>~-methylaminopropionohydroxamic acid
>N-phenyl-n-pyrroJidino-AHA.
Strumpf et al. (l98Ib) patented seven ro-(naphthoxy)alkanohydroxarnic acids which
inhibited activity of soybean urease at lower concentrations than did AHA,
benzohydroxamic acid, and phenoxy-AHA and reconunended these compounds as
additives to fertilizer urea at preferred rates of 0.1-5% relative to urea-No Another
advantage of these compounds is their capacity to hinder the growth of soil-borne
65
phytopathogenic bacteria and fungi. At the same time, they are well tolerated by the
crops. It should, however, be emphasized that the description of invention does not
contain any data about testing of these compounds as inhibitors of soil urease activity.
Their general structural formula is presented in Figure 11.
o-(CH)n~O-NH---OH
Oj
r l A
~ .&
R = H or low alkyl
n= 1-10(R=H)
The inventors Kolc et al. (1985c) (Assignee: Allied Corporation, Morristown, New
Jersey) patented three groups of hydroxamic acid compounds as additives to urea
fertilizers for inhibition of soil urease activity. Twenty-eight such compounds are
nominalized in the patent. We mention a representative compound from each group
(Figure 12).
7-[N(2'-Nitro-2'-bromovinyl)-N-ethylaminojheptanehydroxamic acid
Fif<ure 12. Structure ofrepresentative hydroxamic acid compounds patented by Kole et al. (1985c).
But it should be mentioned that no data are presented in the description of the
invention on the inhibitory effect of these compounds on activity of soil urease or
jackbean urease.
Co-HN-OR,
I
(CH-R2ln
ICO-HN-OR,
Tested on soybean urease, these compounds proved to be more effective than AHA
and succinomonohydroxamic acid. Moreover, they suppress the growth of soil-borne
phytopathogenic bacteria and fungi and are not toxic for plants. Although they were not
tested as inhibitors of soil urease activity, based on the results obtained with soybean
urease, the inventors consider that these compounds when added to fertilizer urea will
prevent the undesirable effects of the rapid hydrolysis of urea in soil. The preferred rates
at which these compounds are recommended for use range from 0.1 to 5% relative to
urea-No
2.10. MALEIMIDES
Patenting the unsubstituted and halo-substituted alkyl, cycloalkyl, and aryl derivatives
of dichloromaleimide as inhibitors of soil urease activity, Tomlinson (1967)
nominalized four N-alkyldichloromaleimides (N-alkyl-DCMI): N-methyl-, N-ethyl-, N-
n-propyl-, and N-n-butyl-DCMI (Figure 14). Applying the first of his methods already
outlined on page 53, he treated 2.5-g samples of two soils with 2,000 ppm of urea (on
soil weight basis) and 20 ppm ofN-alkyl-DCMI, then incubated them for 3 or 7 days at
9°C. During the incubation the volatilized ammonia was measured. The results showed
that during the first 3 days volatilization ofNH3 from one soil was completely inhibited
by each N-alkyl-DCMI, but in the other soil the volatile N losses as NH3 in the presence
of the four N-alkyl-DCMI represented 26-65% relative to the NH3 losses from the
control soil to which no N-alkyl-DCMI had been added. During the whole 7-day period,
51-77% NH3 losses were registered; with the first soil the minimum loss (57%)
occurred in the N-n-butyl-DCMI-treated samples, whereas with the other soil the NH3
loss was lowest (51 %) from samples treated with N-n-propyl-DCML
Under the conditions of the 5-hour test, Bremner and Douglas (1971) studied
N-ethylmaleimide (N-ethyl-MI) which was added to samples of three soils at a rate of
50 ppm (soil basis). The inhibitions recorded in urease activity of the three soils were
28, 24, and 23%, respectively. In another experiment, soil samples were preincubated
with N-ethyl-MI at 30°C for 3, 7, and 14 days, before applying the 5-hour test. This
experiment showed that during preincubation N-ethyl-MI was partially inactivated.
67
o o o
II II II
CIC-C, CIC-C,
II ..N-R II .,N-R II
HC-C>
-R
CIC-C/ HC-C/ HC-C
II II II
o o o
Dichloromaleimide Chloromaleimide Maleimide
R=H or -CH3
-CHr-CH2-CH2-CH3
n-butyl
-0 -0 ~
~
~-naphthyl
phenyl cyclohexyl
Thus, in one of the three soils studied, inhibition of urease activity was reduced from
23% (registered in the soil not submitted to preincubation with N-ethyl-MI) to 21, 19,
and 8% in samples preincubated with N-ethyl-MI for 3, 7, and 14 days, respectively_
The inhibiting effect ofN-ethyl-MI on soil urease activity was attributed to its capacity
to react with SH groups of the enzyme molecule.
Reexamining N-ethyl-MI, Bremner and Douglas (1973) determined both urea
hydrolysis and ammonia volatilization from urea in a sandy soil. Air-dried samples (10
g) were treated with 1 ml of urea solution containing 1,000 ppm N (soil basis) and 1 rnl
of water (control soil) or I rnl of N-ethyl-MI solution (50 ppm compound), then water
was added to bring the soil moisture content to 50% of WHC, and the mixtures were
incubated at 20°C. After 3, 7, and 14 days, the amounts ofresidual urea, exchangeable
NH/, and volatilized NH3 were determined. The results indicated that N-ethyl-MI
inhibited urease activity in the sandy soil studied. Some of the results will be quoted
below. After 14 days of incubation, no urea was detectable in the control soil, whereas
the N-ethyl-MI-treated soil contained 24.2% of the initial urea amount; loss ofurea-N as
volatile NH3 was 61.1 % from the control soil and 23.6% in the presence ofN-ethyl-MI.
Based on the results obtained by Bremner and Douglas (1973) with other inhibitors
administered to heavy-textured soils, one can assume that the inhibiting effect of
N-ethyl-MI on urease activity in such soils would have been much weaker.
Hartbrich et af. (1978) tested the rnaleimides (MI), chlorornaleimides (CMI) , and
dichloromaleimides (DCMI) specified in Table 11 (see also Figure 14). Their inhibitory
68
effect on anunonia volatilization was strong for 4-8 days, but it disappeared, in general,
after 12 days. The effect of N-cyclohexyl-DCMI was the longest lasting (14 days) (see
also Oertel el af., 1978; lasche el af., 1978) and that of N-~-naphthyl-DCMI was the
least durable (4 days). It can also be deduced from the table that the influence of the
number of chlorine atoms on the degree of inhibition depends on the nature of the N-
substituent. Thus, the inhibitory effect of N-phenyl-MI, N-phenyl-CMI, and N-phenyl-
DCMI, being similar, is not influenced by the number of chlorine atoms. In contrast, the
number of chlorine atoms has an influence in the case of other maleimides as the
inhibitory effect increases in the order:
N-cyclohexyl-MI < N-cyclohexyl-CMI < N-cyclohexyl-DCMI,
or presents the following order:
N-~-naphthyl-MI ;:::; N-~-naphthyl-CMI > N-~-naphthyl-DCMI.
Inhibition (%)
STT LTT
Compound
Incubation time (days)
1 2 4 12 14 16
N-Phenyl-MI 99 98 78 9
N-Phenylchloro-MI 92 96 80 16
N-Phenyldichloro-MI 82 96 83 26 0
N-CyC\ohexyl-MI 97 96 74 26
N-Cyclohexylchloro-MI 83 99 98 47 11 0
N-Cyclohexyldichloro-MI 79 94 91 48 19 10 ()
N-~-Naphthyl-MI 97 90 68 26
N-~-Naphthylchloro-MI 92 97 94 23 0
N -~-N aphthy1dichloro. MI 49 76 47 0
Dichloro-MI 88 91 69 14
N- Methyldichloro-MI 88 100 96 23 0
N-Ethyldichloro-MI 95 95 43 18 0
N-n-Propyldichloro-MI 96 98 61 21 0
"From Hartbrich et al. (1978).
STT -- Short time test.
LTT -- Long time test.
MI - maleimide.
In respect to the three N-alkyl-DCMI studied, Table 11 shows that the inhibitory
effect ofN-methyl-DCMI was stronger than that ofN-ethyl- and N-n-propyl-DCMI. In
contrast, in one of the soils studied by Tomlinson (1967), N-n-propyl-DCMI was more
evidently inhibiting than were the other three N-alkyl-DCMI tested (see page 66).
Hartbrich el al. (1978) mentioned that, besides the compounds specified in Table 11,
other N-substituted MI, CMI, and DCMI also manifested an inhibitory effect on soil
urcase activity.
69
The structure of its tautomeric forms (dione and diol) is presented in Figure 15.
o OH
II I
,......c . . . . . ,......c~
HC NH HC N
II
HC
I
NH
II I
HC N
'c""'" , c.?'
~ 6H
Dione form Diolform
1.2-Dihydro-3.S-pyridazinedione 3.S-Dihydroxypyridazine
CI-C-CHO
II
CI-C-COOH
relative to urea-N (0.5, 1,2, and 4% in the short time test, and 0.5, 1, 1.5, and 2% in the
long time test). Determination of ammonia volatilized during 24 hours and 18 days,
respectively, indicated that mucochloric acid brought about complete inhibition of NH3
evolution during the first days of incubation, and then the inhibitory effect decreased
70
slowly. Thus, under the influence of the four mucochloric acid rates, the inhibition
persisted for 12, 14, 16, and 16 days, respectively.
Hartbrich et al. (1978) pointed out that the strong and persistent inhibitory effect of
mucochloric acid, as described in the patent, could not be confirmed in pot experiments.
The inventors Norden et 01. (1985, 1986) (Assignee: Chernische Werke Hills A.G.,
Marly, Germany) tested and patented 41 bromo-nitro compounds for inhibition of soil
urease activity. For testing, 200-g moist soil samples were treated with urea prills
(usually 0.5 g) and a bromo-nitro compound (BNC) at a rate of 0.25 or 1% by weight of
urea-N, then incubated for 4 or 7 days. Soil samples to which no BNC was added were
the controls. During incubation the ammonia volatilized was determined and expressed
as percentage of the added urea-No
All BNCs reduced NH3 volatilization. The BNCs which were found to be the
strongest inhibitors of soil urease activity are specified in Figure 17.
Tr ft Tr ft
CH3-C -CH2-0-C-NH - CH 3 CH3-CH2-C-CH2-0-C -NH -CH3
~02 1 02
2-Bromo-2-nitropropane-l,3-diol 2-Bromo-2-nitropropanol
Figure 17. Stru~ture of the strongest inhibitors among the 41 bromo-nitro compounds patented and tested by
Norden et (I/. (1985, 1986) for inhibition of soil urease activity.
Volatilization of NH3 was 52.8% from the control soil and only 0.3, 0.5, 0.9, and
2.1 % respectively, from the soil treated with these four BNCs, when they were used at
the 0.25% rate and the incubation lasted 4 days. Volatilization ofNH3 was 71.2% from
the control soil and always 0% from the soil treated with each of these four BNCs when
their rate was I % and the incubation lasted 7 days.
These inhibitors are recommended to be added to urea fertilizers at rates ranging
from about 0.05 to 15%, preferably from about 0.4 to 4% by weight ofurea-N.
It should be mentioned that before the patenting of the 41 BNCs as inhibitors of soil
urease activity by Norden et 01. (1985,1986), similar BNCs were already patented as
antibacterial and antifungal agents for use in human and veterinary medicine (Clark et
aI., 1967), for combating phytopathogenic bacteria (Clark et 01_, 1970) and
71
2.14.1. Tetrahydro-l.3.5-thiadiazine-2-thiones
Held et al. (1974) patented tetrahydro-l,3,S-thiadiazine-2-thiones (Figure 18) for
inhibition of soil urease activity. Moist soil samples treated with a mixture of urea and
test compound were incubated and during incubation the volatilized ammonia was
determined. No urea and no test compound or only urea was added to the control
samples. Data on incubation temperature and time are not given in the patent.
Derivative
CH, CH, 3,5-dimethyl-
CH,-CII, CH,-CH2 3,5-diethyl-
CH,--CH=CH2 CH,--CH=CH2 3,5-dialiyl-
C.H, C6H, 3,5-diphenyl-
C 6H'-{:H2 CH, 3-benzy1-5-methyl-
C 6 H,-{:H2 CH,--CH20H 3-benzyl-5-hydroxyethyl-
'Markert (1974) found that dazomet reduced the rate of urea hydrolysis in each of the two soils studied, but he
did not patent this compound as an inhibitor of soil urease activity.
72
Hartbrich et al. (1978), Oertel et al. (1978), and Jasche et al. (1978), applying the
long time test, obtained, after 2, 4, 8, 12, and 14 days of incubation, similar inhibitions,
namely 100, 95, 64, 28, and 11 %, respectively, under the influence of dazomet added to
urea-treated samples at a rate of 1% relative to urea-No
In Winiarski's (1990) experiments, 2-kg samples of a light-textured and a heavy-
textured Polish soil, the pH of which was adjusted to 5 (with 1 N H2 S04 ) or to 7 (with
CaC03), were treated with 328 mg of urea and 0 or 0.5, 1 or 2% of dazomet or 2% of
"thione" (tetrahydro-l,3,5-thiadiazine-2-thione) (on urea weight basis), then moistened
to 50% ofWHC and incubated at 22°C for 7 or 14 days.
Loss ofurea-N as volatile ammonia from the inhibitor-treated samples was reduced.
The inhibitory effect of dazomet tended to increase with its rate and to decrease with
incubation time in both soils. The inhibition was stronger in the light than in the heavy
soil. The maximum inhibition (64.1 %) was registered in the light soil (at pH 5; 2%
dazomet) after 7-day incubation, while the minimum value (16%) was recorded in the
heavy soil (at pH 7; 0.5% dawmet) after 14-day incubation. The inhibitory effect of
thione was less pronounced at pH 5 than at pH 7 and showed a tendency to decrease
with incubation time in both soils. Thus, at pH 7, the reduction ofNH3 loss during 7 and
14 days of incubation was 31.2 and 13.6% (in the light soil) and 23.2 and 17.0% (in the
heavy soil), respectively.
The effect of dazomet on soil urease activity was studied under field conditions as
well (Tu et al., 1995). A Canadian loamy sand was treated with 0 or 56 kg of
dawmetlha ridging it into the soil to depth of 14 cm on May 10, 1978. A 3-18-24
tobacco fertilizer was applied and tobacco seedlings were planted on May 23. For
determination of urease activity, soil was sampled on May 23 (before fertilization and
planting) and on June 27. This activity was found to be insignificantly (p > 0.(5) lower
on May 23 and insignificantly higher on June 27 in the dawmet-treated soil than in the
untreated one.
2.14.2. 1.3.4-Thiadiazoline-2-thiones
In the patent of Held et al. (1976a), the 1,3,4-thiadiazoline-2-thiones as well as their
alkali metal and substituted ammonium salts are described as inhibitors of soil urease
activity. The tautomeric (thione and thiol) forms of these compounds and the derivatives
specified in the patent are presented in Figure 19.
73
Soil samples, to which a mixture of urea + test compound was added, were
moistened and incubated at 30°C for 24 hours. Samples treated with urea alone served
for comparison. During incubation, the evolved ammonia was determined. Table 13
shows the results obtained with seven derivatives, each containing an unsubstituted or
substituted mercapto (sulfhydryl; SH) group in position 5. It is evident from this table
that the degree of inhibition increased with increasing concentration of test compounds,
but not linearly. In general, the increase in inhibition was most marked when the test
compound concentration was increased from 0.5 to 1%. The alkylated derivatives were
more effective than the aralkyl (benzyl) derivative. 2,5-Dimercapto-l,3,4-thiadiazole
was more effective than its amylammonim salt, except when it was used at 0.5%
concentration. The preferred rates, at which these compounds are recommended for the
practice, range from 0.5 to 10% relative to urea-No
Hartbrich et al. (1978) presented data on the inhibitory effect of a single derivative
(2-mercapto-5-methylmercapto-l,3,4-thiadiazole) which, under the conditions of the
long time test, caused, after 2,4, and 8 days of incubation, 100,61, and 19% inhibitions,
respectively, in volatilization ofNH3 from urea-treated soil samples.
Gould et al. (1978) and Gould (1979) studied three compounds whose structure
corresponds to the general formula in Figure 19 (2,5-dimercapto-l,3,4-thiadiazole,
2-mercapto-5-amino-l,3,4-thiadiazole, and potassium salt of 5-mercapto-3-phenyl-
1,3,4-thiadiazoline-2-thione) as well as three other compounds (Figure 20), two of
which are disulfides prepared through oxidation (with H20 2) of 2-mercapto-5-amino-
1,3,4-thiadiazole and 5-mercapto-3-phenyl-l,3,4-thiadiazoline-2-thione, whereas the
third compoud is a sulfur-containing imidazole derivative.
74
The effect of these compounds was estimated by analyzing the residual urea
extracted from 25-g samples of a silt loam soil which had been treated with urea (400
ppm N, on soil weight basis), test compound (0 or 100 ppm), and water (to field
capacity), then incubated at 25°C for 24 hours. It is evident from the results obtained
(Table 14) that, of the six compounds tested, 2,5-dimercapto-l,3,4-thiadiazole had the
most pronounced inhibitory effect. Potassium salt of 5-mercapto-3-phenyl-I,3,4-
thiadiazoline-2-thione did not inhibit soil urease activity.
CsHs-N N N---N-CsH5
=~L-&-I~..,
2.2·-Di(5-amino-l.3.4-thiadiazole) d~u~ide 5.5·-Di(3-phenyl-l,3,4-thiadlazoline-2-thione) disuWlde
HC---N
I
HC" /C-SH
I
N
I
CH3
2-Mercapto-l-methylim idazole
Figure 20. Structure of three of the heterocyclic sulfur compounds tested by Gould et al. (1978) and Gould
(1979) for inhibition ofsoi! urease activity.
2,5-Dimercapto-1,3 .4-thiadiazole 46
2-Mercapto-5-amino-l,3.4-thiadiazole 27
2-Mercapto-I-methylimidazole 13
2,2'-Di(5-amino-I,3.4-thiadiazole) disulfide II
5,5 '-Di(3 -phenyl-I.3,4-thiadiazoline-2-thione) disulfide 6
5-Mercapto-3-phenyl-l,3.4-thiadiazoline-2-thione, K salt 0
"From Gould et al. (1978) and Gould (1979). by permission of the Soil Science Society of
America, Inc.
It was also established that moisture content above field capacity of the silt loam
studied had no effect on the inhibition of soil urease activity by 2,5-dimercapto-l,3,4-
thiadiazole used at 50 ppm, but the inhibition decreased somewhat at lower moisture
contents.
It should be mentioned here that, according to Gem1ann-Bauer (1987) and Amberger
(1989), the inhibition of urease activity in guanylthiourea (GTU)-treated soils (see page
52), is caused not by the GTU itself, but by 3,5-diamino-l,2,4-thiadiazole, which is
produced in soil due to abiotically catalyzed oxidation of GTU. The next, abiotically
catalyzed reaction is the reduction of this thiazole into dicyandiamide which is a potent
nitrification inhibitor (Figure 21).
- -
N - - - G -NH2 NH
NH
II
H:<N-C-NH -c-NH2
S
II I
H:<N-C, / N
I II
H:<N-C-NH -c =N
S
Guany Ithiourea 3,5-Diamino-1,2,4-thiadiazole Dicy andiam ide
Figure 21. Transformation of guanylthiourea in soil. IFrom Gennann-Bauer (1987); Amberger (I 989)./
Rhodanine-5-acetic acid
O=C---N-R
I
HOOC-CH2--HC, /G=S
I
S
R Derivative
CH, 3-methyl-
CH,-CH2 3-ethyl-
CH,-CH2-CH2 3-n-propyl-
(CH,hCH- 3-isopropyl-
CH,-CH2-CH2-CHz 3-n-butyl-
(CH,J:,-CH-CH2 3-isobutyl-
C.H" 3-cycIohexyl-
C.H 5-CHz 3-benzyl-
Figure n. Structure ofrhodanine-5-acetic acid and of its derivatives patented and tested
by Schroth e/ al. (1974) for inhibition of soil urease activity.
One can see from Table 15 that the inhibitory effect increased in the order:
rhodanine-5-acetic acid ~ l:ycloalkyl derivative < aralkyl derivative ~ alkyl
derivatives.
Rhodanine-5-acetic acid 35
3-Methylrhodanine-5-acetic acid 97
3-Ethylrhodanine-5-acetic acid 95
3-n-Propylrhodanine-5-acetic acid 92
3-Isopropylrhodanine-5-acetic acid 92
3-n-Butylrhodanine-5-acetic acid 91
3-Isobutylrhodanine-5-acetic acid 90
3-Cyclohexylrhodanine-5-acetic acid 38
3-Benzylrhodanine-5-acetic acid 93
"Adapted from Schroth et al. (1974).
Of the alkyl derivatives, the methyl derivative was the most effective. Cu, Cd, Zn,
Co, Pb, and Fe salts of 3-ethylrhodanine-5-acetic acid gave 82-93% inhibitions. In the
case of the Ni salt the inhibition was 56%, whereas the Sn and Hg salts did not inhibit
77
volatilization of NHJ from urea. The inhibitory compounds are recommended for the
practice at rates of 0.1-5%, preferably at a rate of 1% relative to urea-No
Oertel et al. (1978) found that in reaction mixtures prepared from 30-g samples of
moistened sandy soil + 214 mg of urea + I mg of surface-applied 3-methylrhodanine-5-
acetic acid (1 % relative to urea-N) and incubated at 20°C, volatilization of NH3 from
urea during 2, 4, 8, and 12 days was reduced in proportions of 95, 60, 8, and 0%,
respectively, under the influence of 3-methylrhodanine-5-acetic acid.
Muller and Forster (1980) confirmed the inhibitory effect of 3-methylrhodanine-5-
acetic acid on soil urease acivity. They worked with a humous sandy loam (PH 7.2)
from Germany. Urea (40 mg of N) was distributed uniformly in an 8-cm high soil
column (40 g) or applied on its surface. The test compound (2% on urea-N basis) was
introduced at 2 or 4 cm depth in the soil column. After moistening and incubation at
laboratory temperature for 6 days, the residual urea and NH4 + + NO) - contents were
determined. In all treatments, 3-methylrhodanine-5-acetic acid caused at least 50%
inhibitions of urea hydrolysis.
2.14.4.2-Mercaptobenzothiazole
Urease activity was not inhibited in reaction mixtures consisting of 5 g of soil + 10 ml
of aqueous phase containing 6 mg of urea and 0 or 0.12 mg of 2-mercaptobenzothiazole
(Figure 23) and incubated at laboratory temperature; in both the absence and the
2.14.5. 2- Thiocarboxamidothiazoles
Simihilian and Silberg (1996) and Simihaian et al. (1999) tested 2-thiocarboxamido-5-
aminothiazole (crysean) and 2-thiocarboxamido-5-benzamidothiazole (benzoyl cry sean)
(Figure 24) for inhibition of soil urease activity. Samples were taken from the O-lO-cm
Ht-J-ClI~s/-
s
"~NH-C-o~ 0
II -
2-Thiocarboxamido-5-aminothiazole 2-Thiocarboxamido-5-benzamidothiazole
(cry sean)
(benzoyl cry sean)
The monohydric phenols that were tested for evaluation of their inhibitory effect on soil
urease activity comprise: phenol (hydroxybenzene), a series of its derivatives, including
some derivatives of methylphcnols (cresols), as well as a-naphthol (I-naphthol) and one
6
OH OH OH
OCH' O-CH'
Phenol a-Cresol m-Cresol
OH
(J()
p-Cresol a-Naphthol
of its derivatives. Figure 25 presents the structure of phenol, cresols, and a-naphthol.
Their derivatives tested will be specified during the description of investigations.
79
Phenol 43 41 41
4-Chlorophenol 37 37 30
Other compounds" <l <I <I
"Adapted from Bremner and Douglas (1971). by permission of Pergamon Press PLC.
b2,5-Dichlorophenol, 2-nitrophenol, 4·nitrophenol, 2A-dinitrophenol, 2,4,6-trinitro-
phenol (picric acid), 4-chloro-2-nitrophenol, 4-hydroxybenzoic acid, 2-aminophenol,
4-aminophenol, 2-methoxyphenol (guaiacol), 4,6-dinitro-2-methoxyphenol.
Gould et al. (1978) tested 25-g samples of a silt loam soil with 400 ppm of urea-N, 0
or 100 ppm of phenol or a-naphthol and water to field capacity, then incubated them at
25 D C for 24 hours. Phenol exhibited a 10% inhibition, whereas a-naphthol gave a 1%
inhibition of urease activity.
Mishra and Flaig (1979) prepared mixtures from 200-g soil samples (a brown earth
and a black earth from Germany), 0 or 5 ml of urea solution containing 20 mg ofN (100
ppm in soil), 0 or 5 ml of solution containing 10 mg of2, 4-di-t-butylphenol (50 ppm in
soil) and water to 60% of WHC. After 0, 14, and 28 days of incubation at 30D C, soil
urease activity was assayed by the method of Tabatabai and Bremner (1972) (see page
8). 2,4-Di-t-butylphenol inhibited urease activity at 0 time and stimulated it after 14 and
80
28 days of incubation in the case of brown earth, and constantly stimulated it in the case
of black earth.
Xue and Li (1987) studied the effect of phenol and methylnaphthol" on urease
activity in a paddy soil. The reaction mixtures contained 5 g of soil + 10 ml of 10% urea
solution without or with 100 ppm of phenolic compound. Incubation took place at 37°C
and lasted 48 hours. Degree of inhibition was 22.35% with phenol and 30.33% with
methylnaphthol.
Rodgers (l984b) tested 5 aminocresols with three English soils (silty clay loam,
sandy clay loam, and loamy sand). Air-dried soil samples (8 g) were treated with 1.2 ml
of 0.4% aqueous urea solution (280 Ilg N/g soil) + 0.8 m1 of 60% methanol or 0.8 m1 of
aminocresol solution in 60% methanol (concentrations: 5-100 Ilg of aminocresol/g soil).
During incubation (at 30°C for about 100 hours), the amounts of residual urea and of
NH4 + were determined periodically. Table 17 presents the results obtained with one of
the three soils studied after 20 hours of incubation. One can see from this table that only
two aminocresols (4-amino-o-cresol and 4-amino-m-cresol) inhibited significantly the
urease activity; degree of inhibition increased as their concentrations increased.
TABLE 17. Inhibition of urease activity in a sandy clay loam soil by aminocresols applied at
different concentrations"
Inhibition of urease activity (%)
Compound Concentration of compound (/lWg soil)
5 10 20 50 100
2-Amino-p-cresol I I I 8 14
3- Ami no-o-cresol <I <I <I <I <1
4-Amino-o-cresol 8 26 24 53 74
4-Amino-m-cresol 9 24 21 40 62
6-Amino-m-cresol <1 <1 <1 <1 <1
"From Rodgers (1984b), by permission of Kluwer Academic Publishers.
'Positions ofthe OR and CH, groups are not specified in the paper.
81
determination of the remaining urea. According to the patent description, only eight
aminophenols were tested with the Cazenovia soil and only five of these eight
compounds were used for testing with the Plano soil.
In both soils, the strongest urease-inhibiting compounds were N-(4-hydroxyphenyl)
glycine and 4-(N-methylamino)phenol (Figure 26), both causing a 82% inhibition in the
Cazenovia soil and 61 and 55% inhibitions, respectively, in the Plano soil, whereas the
weakest inhibitor was 2-aminophenol, causing an 8% inhibition. But the degree of
inhibition by 4-aminophenol was 46% which is in contrast with the <1% inhibition
mentioned in Table 16.
H3C-H~H
N-(4-Hydroxyphenyl)glycine 4-(N-Methylami no)phenol
Figure 16. Structure of the strongest urease inhibitors among the aminophenols tested
by Kolc et al. (1985b).
These two classes of organic compounds will be dealt with together, because they
inhibit soil urease activity by similar mechanisms, which was demonstrated in the case
(JcH
Q
OH OH
Or
OH OH
O-aH ~ H
COOH COOH
4
OH
~H Q~
Phloroglucinol Protocatechuic acid Gallic acid
Q 0 CQ
0 0 0
2,6-Dichloroquinone-4-chloroimide
heated to 85°C as well as untreated samples of both soils served for comparison. The
analyses showed that in the case of the clay soil urea hydrolysis was complete in 7 days
in the untreated samples. It was about 17% in the heated samples and had much lower
values in the HQ- or CT-treated samples; in the case of the other soil, urea hydrolysis
was nearly complete in the untreated samples and almost completely inhibited in the
treated ones.
Paulson and Kurtz (l969b) carried out a laboratory experiment with a silty clay
loam soil from Illinois. Reaction mixtures were prepared from 20 g of air-dry soil +
pelleted urea or pelleted urea-stearic acid complex (200 ppm N) + p-benzoquinone (BQ)
(0 or 0.2% relative to urea-N) + water to 30% moisture (about field capacity). After 1
and 7 days of incubation at 22°C, the mixtures were analyzed for residual urea, NiLJ +,
and (N03-+N02-). The inhibitory effect of BQ on hydrolysis of pelleted urea was
evident after 1 day of incubation but not after 7 days. When the reaction mixtures
contained pelleted urea-stearic acid complex and BQ, a very small amount of urea
remained unhydrolyzed after 7 days also.
Anderson (1969,1970). assignor to the Imperial Chemical Industries. London,
patented catechol. hydroquinone, and pyrogallol. p-benzoquinone and a series of its
derivatives, p- and o-naphthoquinone and one derivative of p-naphthoquinone (Figure
30) as inhibitors of soil urease activity. The inhibitors were added to urea at rates of
0.05-5%, preferably at rates of 0.1-2% by weight of urea; the inhibitor and urea were
84
p-Benzoquinone
Rl R2 Derivative
CI H chloro-
CI CI 2,5-dichloro-
Cl CI 2,6-dichloro-
CH 3 H methyl-
CH 3 CH, 2,5-dimethyl-
CH, CH,-CHz 2-methyl-5-ethyl-
CH3-CHz CH,-CHz 2,5-diethyl-
CHz-OH CHz-OH 2,s-dihydroxymethyJ-
O-CHz-CH 3 O-CHz-CH, 2,5-diethoxy -
S--CHz-COOH H carooxymethylthio-
C6 HS H phenyl-
C.H"NH-DC-CH3 H 3 '-N-acetylaminophenyl-
p-Naphthoquinone
R Derivative
HO 5-hydroxy-
Figure 30. p-Benzoquinone and p-naphthoquinone derivatives tested by Anderson (1969, 1970).
cogranulated. Use of inhibitor mixtures was also recommended. In these mixtures the
amount of polyhydric phenol used was generally between 10 and 300% molar of the
quinone present; conveniently, substantially equimolecular quantities may be used.
For evaluation of the inhibitory effect, 2.5-g soil samples were moistened with a 3%
urea solution to approximately one third of WHC. The urea solution did or did not
contain inhibitor(s) (40-50 ppm relative to soil weight). During incubation which took
place at laboratory temperature, the amount of volatile ammonia was assayed. In the
control soil (treated with urea alone), 50% of the initial urea-N amount was lost as
volatilized NH3 in 3 days. In the presence of inhibitors, total inhibition of volatilization
required a number of days beyond the control period of 3 days. In Table 18, which
presents the results obtained with four soils, the inhibition period indicates the number
of these days. It is evident from the data in this table that catechol and hydroquinone
acted synergistically with 2,5-dimethyl-p-benzoquinone.
85
TABLE 18. Effect of catechol, hydroquinone, and 2,5-dimethyl-p-benzoquinone and of their ruixtures on
volatilization ofammonia fiom urea-treated soils"
Inhibition period (days)
Concentration
Sandy Loamy Clay Calcareous
COflllound of compound
loam sand loam loam
(ppm of soil)
pH 6.0 pH 6.5 pH 7.1 pHS.5
Catechol ICT) 50 2-3 N.D. N.D. N.D.
Hydroquinone (HQ) 50 3 N.D. N.D. N.D.
2.5-Dimethyl-p-benzo-
quinone (DBQ) 50 7 N.D. N.D. N.D.
CT+HQ 25+25 5 N.D. N.D. N.D.
CT+DBQ 25+25 10 17 J 3-4
HQ+DBQ 25+25 10-12 17 3 2-3
CT+HQ+DBQ 25+25+25 14 20 4-5 4-5
Evaluating the effect of seven polyhydric phenols and six quinooes, used at a rate of
50 ppm (soil weight basis), on the urease activity in three soils, Bremner and Douglas
(1971) obtained, by applying the 5-hour test, the results specified in Table 19. Of the
polyhydric phenols, only catechol and hydroquinone, and all of the quinooes except
TABLE 19. Effect of polyhydric phenols and Quinones on soil urease activity"
Inhibition of urease activity (%)
COflllound
Silty clay loam Loam Clay loam
Polyhydric phenols:
Catechol 77 71 73
Hydroquinone 69 63 60
Pyrogallol 6 5 2
"",0";'01 }
Phloroglucinol
Protocatechuic acid <4 <3 <2
Gallic acid
Quinones:
p-Benzoquil1one 68 61 58
2,5-Dichloro-p-benzoquinone 68 62 56
2,5-Dimethyl-p-benzoquinone 35 33 29
2,5-Dihydroxy-p-benzoquinone 7 6 6
2,6-Dichloro-p-benzoquinone 63 60 52
o-Naphthoquinone 48 42 42
a Adapted from Bremner and Douglas (1971). by permission of Pergamon Press PLC.
TABLE 20. Effect of different amounts of poly hydric phenols and quinones on soil urease activity"
Inhibition of urease activity (%)
COl1l'ound Amount of compound (ppm of soil)
50 100 200 300
Catechol 73 81 89 94
Hydroquinone 60 75 86 93
p-Benzoquinone 58 74 86 93
2,5-Dichloro-p-benzoquinone 56 75 81 87
2.6-Dichloro-p-benzoquinone 52 66 81 87
2,5-Dimethyl-p-benzoquinone 29 33 39 42
"Adapted from Bremner and Douglas (1971), by permission of Pergamon Press PLC.
In the case of the clay loam (PH 7.6), the effect of preincubation at 30°C for 7 and
14 days on the inhibitory capacity of these compounds added to 10-g soil samples at a
rate of 50 ppm was also studied. The 5-hour test was applied after preincubation. It was
found that preincubation usually led to a marked increase in the inhibitory capacity of
the methyl-substituted BQs and to a decrease in this capacity of the other BQs. A series
of BQs that were ineffective before preincubation remained ineffective after
preincubation. The inhibitory effect of dimethyl-BQs after 14 days of preincubation was
greater than that of the other BQs. Presence of a hydroxy group besides two methyl
groups eliminated the inhibitory capacity of the dimethyl-BQs. Thus, the inhibition
caused by 2,5-dimethyl-BQ increased from 27% (no preincubation) to 75% (after 7-day
88
preincubation) and to 82% (after 14-day preincubation); in contrast, the inhibitory effect
of 3-hydroxy-2,5-dimethyl-BQ remained zero after preincubation as well.
The increase in inhibiting capacity of methyl-substituted BQs during preincubation
is not attributed to their decomposition in soil with the concurrent formation of more
potent urease inhibitors, because the inhibitory effect of BQ (the only potential
decomposition product of methyl-substituted BQs shown to be an effective urease
inhibitor) was less after 7 or 14 days of preincubation than that of dimethyl-substituted
BQs. The alternative explanation is that the process whereby substituted BQs inactivate
soil urease is much slower with methyl-substituted BQs than with other substituted
BQs. This explanation is supported by the finding that the effect of methyl-substituted
BQs on soil urease activity after 3 hours of preincubation was similar to that observed
after 7 days of preincubation.
p-Benzoquinone and 10 of its derivatives were selected to study their effect on urea
hydrol ysis and ammonia volatilization from a sandy soil (pH 6.7) treated with urea. The
reaction mixtures (10 gofsoil + 10 mg N as urea + 0.5 mg of test compound + water to
50% of WHC) were incubated at 20°C for 14 days. The results of the determination of
NH3 volatilized during incubation and of residual urea and exchangeable ~ + in
incubated soil showed that the compounds that inhibited urease activity also reduced
the rate of urea hydrolysis and volatilization ofNH3 from urea. Thus, 2,3-dimethyl-BQ,
2,5-dimethyl-BQ and, 2,6-dimethyl-BQ reduced NH3 losses from 62.8 to 0.1 %. At the
same time, NH3 losses were reduced under the influence of 2 ,5-dihydroxy-BQ,
tetrahydroxy-BQ, and 2,5-diphenyl-BQ, from 62.8 only to 62.0, 62.6, and 59.4 %,
respectively.
The investigations performed by Bundy and Bremner (1973a) are also cited in detail
by Flaig (1976).
Applying the short and long time tests (see page 51), Hartbrich et al. (1976b) studied
the effect of p-benzoquinone (BQ) used at rates of 0.5, 1.0, 2.0, and 4.0% relative to
urea-N on the volatilization of ammonia from a heavy- and a light-textured soil, whose
30-g samples were treated with 100 mg ofurea-N. In both tests the inhibitory effect of
BQ on NH3 volatilization from urea increased with its amount and decreased during
incubation. For example, BQ at the four rates used caused 86, 100, 100, and 100%
inhibitions respectively, after 2-day incubation, 52, 61, 70, and 75% inhibitions
respectively, after 4 days, and 9, 19,20, and 21 % inhibitions respectively, after 8 days.
At day 10, the degree of inhibition became zero regardless of the BQ rate.
Thieme et al. (1976) dealt with the effect of hydroquinone (HQ), p-benzoquinone
(BQ), and quinhydrone (QH) and their mixtures with tetramethylthiuram disulfide
(thiram) on the volatilization of ammonia from urea-treated soil samples. Their rates
relative to urea-N were 2% (HQ) and 1 and 2% (BQ and QH). In mixtures the molar
ratio between thiram and the other compounds was 1:1 or 1:0.5 or 1:0.1, whereas the
rate of mixtures was 0.5 or 1% relative to urea-N (the rate may range from 0.01 to
10%). Incubation took place at 10°C. It resulted from the determination of the
volatilized NH3 that HQ, BQ, and QH were effective inhibitors. Thus after 4 and 12
days of incubation, they exhibited, when used at 2% rate, the following inhibitions: 99.2
and 22.6% (HQ), 97.2 and 14.8% (BQ), and 96.0 and 37.8% (QH), respectively. In
mixtures with thiram the inhibitory effect was more intense and long-lasting. For
example when thiram and the other compounds were used at 1:0.1 molar ratio, rate of
mixtures was 1% and incubation lasted 4 and 31 days, the following inhibitions were
89
registered: 100 and 52.5 1yo (HQ), 100 and 56.2% (BQ), and lOO and 68.8% (QH),
respectively.
The percent inhibitions caused by polyhydric phenols and quinones in urease
activity of a silt loam soil studied by Gould et al. (1978) and Gould (1979) are specified
in Table 22. The most effective inhibitors were p-benzoquinone and hydroquinone.
TABLE 22. Effect of polyhydric phenols and quinones on soil urease activity"
Compound Inhibition (%)
p-Benzoquinone 88
Hydroquinone 88
2.5-Dimethyl-p-benzoquinone 50
Catechol 47
2,6-Dimethyl-p-benzoquinone 45
2,6-Dichloroquinone-4-chloroimide 30
Tetrachloro-o-benzoquinone 6
Pyrogallol 2
Resorcinol 0
"From Gould et al. (1978) and Gould (l979). by permission of the Soil Science
Society of America. Inc.
Pyrogallol was a weak inhibitor, whereas resorcinol lacked inhibitory capacity. The
reaction mixtures consisted of 25 g of soil + 400 ppm of urea-N + 0 or 100 ppm of test
compound + water to field capacity and were incubated at 25°C for 24 hours.
In the experiments described by Hartbrich et al. (1978), three chloro-substituted
hydroquinones proved to be weaker inhibitors than was the parent compound. The most
effective inhibitor was 2,5-dimethyl-p-benzoquinone; its effect was evident for 14 days,
whereas the inhibitory effect of the parent compound lasted 8 days (Table 23) (see also
Oertel et at., 1978; lasche et al., 1978).
TABLE 23. Effect of polyhydric phenols and quinones on volatilization of ammonia from urea-
treated soils"
Inhibition (%)
STT LTT
Compound
Incubation time (dai:s2
2 4 8 12 14 16
Hydroquinone (HQ) 96 99 96 34 15 2 0
Chloro-HQ 93 91 69 12 0
2,5-Dichloro-HQ 69 49 17 0
Tetrachloro-HQ 72 45 15 0
p-Benzoquinone (BQ) 100 99 67 16 0
2.6-Dichloro-BQ 88 99 57 7 0
Methyl-BQ 95 96 55 16 6 0
2,5-Dimethyl-BQ 91 100 96 83 33 12 0
Tetramethyl-BQ 38 61 12 ()
Quinhydrone (QH) 96 53 12 4 0
Tetrachloro-QH 81 78 3~ 6 0
"From Hartbrich et al. (l978).
STT - Short time test.
LTT - Long time test.
90
Urease activity in a sandy soil from Australia was assayed by May and Douglas
(1978) who used reaction mixtures prepared from 3 g of soil + 0.5 ml of toluene + 12 ml
of 1/15 M phosphate buffer, pH 8.8 + 3 ml of solution containing 3 mg of urea and a
TABLE 24. Inhibition of urease activity in a sandy soil by polyhydric phenols and quinones
applied at different concentrations'
Inhibition (%)
Compound Concentration of compound (ppm of soil)
\0 25 50 100
Catechol 74 85 93 100
p-Benzoquinone 75 91 92 97
2,5-Dimethyl-p-benzoquinone b 0 14 31 45
2,5- Dimethy l-p-benzoquinone' 82 90 92 93
Hydroquinone 45 64 87 96
o-Naphthoquinone 15 26 42 51
test compound. No test compound was added to the control mixtures. Incubation took
place at 37°C and lasted 4 hours, after which the NH/ content was determined. The
compounds tested, their concentrations and the results obtained are presented in Table
24. One can see from this table that preincubation of 2,5-dimethyl-p-benzoquinone in
soil led to an increase in its inhibitory capacity. At higher concentrations (50 and 100
ppm), catechol, hydroquinone, p-benzoquinone, and preincubated 2,5-dimethyl-p-
benzoquinone manifested nearly the same inhibitory effect.
Similar results were obtained by Mulvaney and Bremner (1978) concerning
p-benzoquinone (BQ) and hydroquinone (HQ). They compared the inhibitory effect of
these two compounds on urea hydrolysis in 25 soils selected to obtain a wide range in
pH (4.6-8.0), texture (2-90% sand, 1-72% clay), organic C content (0.30-6.73%), urease
activity (4.7-84.9 Ilg of urea hydrolyzed/g soillhour at 37°C). and in other soil
properties. The reaction mixtures (5 g of air-dried soil + 2 ml of aqueous solution
containing 10 mg of urea or 10 mg of urea and 25 or 50 ~lg of BQ or HQ) were
incubated at 20°C for 24 hours, and then the residual urea was determined.
In each soil, the inhibition caused by BQ was almost identical to that brought about
by HQ. The degree of inhibition in air-dried and field-moist samples of the same soil
was the same. Simple correlation analyses showed that the inhibition of urea hydrol ysis
by BQ or HQ was very highly significantly (0.1 % level) correlated with organic C
content (r=-0.76), total N content (r=-0.74), urease activity (r=-0.70), and cation-
exchange capacity (r=-0.62), highly significantly (1 % level) correlated with sand
content (r=0.57) and significantly (5% level) with silt content (r=-0.42), clay content
(r=-0.49), and surface area (r=-0.49), but was not significantly correlated with pH
(r=0.36) or CaC03 equivalent (r=O.27). Multiple-regression analyses indicated that the
91
activity in samples of an organic soil after 7 days of incubation, but had a significant
stimulating effect after 14 days (Tu, 1981 b).
Tan (1982) studied the effect of hydroquinone (HQ) and p-benzoquinone (BQ) on
hydrolysis of urea in two Malaysian soils (sandy soil, pH 4.6 and clay soil, pH 4.7) from
rubber (Hevea brasiliensis) plantations. Reaction mixtures were prepared from 50 g of
soil + 2 ml of solution containing 0, 0.5, 1.25 or 2.5 mg of HQ or BQ (i.e.. 0, 10, 25 or
50 J1.g ofinhibitor/g soil) + 5 ml of urea solution containing 20 mg ofN (i.e.. 400 J1.g of
urea-N/g soil) + water to 50% of WHC. After 1, 2, 4, 7, and 14 days of incubation at
32°C the residual urea was determined.
In the absence of HQ and BQ urea hydrolysis was complete in 7 days in the sandy
soil and in 2 days in the clay soil. Both HQ and BQ were equally effective in retarding
urea hydrolysis. The inhibitory effect increased with their rate and was more marked in
the sandy than in the clay soil. For example after 4 days of incubation, HQ and BQ, at
the 10, 25, and 50 J1.g/g soil rates applied, exhibited the following percent inhibitions: 87
and 85, 95 and 90, and 100 and 100%, respectively, in the sandy soil, and 0, 40, and
45%, respectively, in the clay soil. The inhibitory effect decreased with the time of
incubation. For example under the action of HQ and BQ at the rate of 50 J1.glg soil, the
degree of inhibition registered after 4, 7, and 14 days of incubation was 100, 98 (96),
and 95%, respectively, in the sandy soil, and 45, 10, and 5%, respectively, in the clay
soil.
Reddy and Mishra (1983) determined urease activity and volatilization of ammonia
from urea in an alkaline sandy loam (PH 8) from India. The soil samples were treated or
not with p-benzoquinone (BQ). Urea prills were surface-applied at a rate of 100 kg Nlha
with or without 1% BQ (relative to weight of urea). Urease activity measured at 30°C
and expressed as mg of hydrolyzed urea/kg soillhour decreased from 11.8 (soil treated
with urea alone) to 5.2 (soil treated with urea + BQ). Ammonia volatilization
commenced on the day urea was applied, but from urea+BQ-treated soil no such loss
was detected for the first 4 days. In the presence of BQ, cumulative losses of urea-N as
NH3 during 16 days decreased by 60%.
Gorelik et al. (1983) prepared urea granules coated with 1, 5 or 10% of
hydroquinone (HQ) relative to urea weight. HQ was dissolved in diethyl ether. The urea
granules were introduced into the solution, then diethyl ether was allowed to evaporate
at room temperature. Three soddy podzols (sandy, sandy-loamy, and loamy texture,
respectively) and two heavier-textured soils (chemozem and sierozem) were used.
Water was added to 10-g air-dried soil samples to 50% ofWHC, then uncoated or HQ-
coated urea granules were placed on the soil surface. Rate of urea application was 1 mg
of N/g soil. Incubation was carried out at 20°C. The amounts of volatilized ammonia
and residual urea were recorded after 3, 5, 7, and 14 days of incubation.
In the very urease-active sandy-loamy podzol and chemozern, all of the added urea
was hydrolyzed in 3 and 5 days, respectively, whereas in the presence of HQ complete
hydrolysis of urea required 7 days. In the other three soils, hydrolysis of urea was
slower, it became complete in 7 days in samples treated with urea alone and in 14 days
in samples treated with urea containing 1% HQ; in the presence of higher amounts of
HQ, complete urea hydrolysis needed more than 14 days. Thus after a 14-day
incubation, cumulative losses as volatile NH3 from urea alone or with 1 and 10% HQ
were the following (in percentages of the added urea-N):
94
sierozem (2, 2, and 1%) < chernozem (3,3, and 2%) < loamy podzol (10, 10, and
3%) < sandy-loamy podzol (51,49, and 44%) < sandy podzol (75, 67, and 50%).
It is evident that HQ inhibited more effectively urea hydrolysis than ammonia
volatilization.
Rodgers et at. (1984) studied the effect of hydroquinone (HQ) over three years
(1981-1983) in field experiments on a clay loam (in 1981) and on a silty clay loam (in
1982 and 1983), covered by ryegrass leys at Rothamsted. Urea prills were applied
annually at a rate of 375 kg N/ha as a single dressing or as three equally divided
dressings. HQ was used at an annual rate of 5 kg/ha as an addition to the single dressing
in each year or to the three dressings in 1982 and 1983. Analyses were carried out for
residual urea, volatilized NH1, NH4 +, and NO)-.
Complete hydrolysis of urea always occurred in 14 days in both the absence and
presence of HQ, but HQ reduced total ammonia volatilization losses during the 4 weeks
after urea application as a single dressing in 1981 and had little effect in 1982 and 1983.
Reduction of NH3 losses was substantial: from 17 to 8 kg of NHrN/ha. However, it
should be added that never more than 5% of the applied urea-N was lost by NH3
volatilization in any year, irrespective of treatment.
In similar field experiments conducted on a silty clay loam (PH 7.5-8.0) in 1983-
1984 and on another silty clay loam (PH 7.8-7.9) in 1984-1985, both soils having been
cropped with winter oil-seed rape, Rodgers et at. (1986) used urea priUs uncoated or
coated with HQ (HQ was dissolved in acetone, then the solution was poured over urea
prills; finally, acetone was allowed to evaporate at room temperature). Three days
before sowing in August 1983 and September 1984, respectively, the seed bed was not
or was fertilized with uncoated or HQ-coated urea priUs (50 kg of N and 3 kg of
HQ/ha). In March 1984 and 1985, some plots were fertilized with urea (150 kg of Niha)
without or with HQ (10 kg/ha), whereas the other plots were treated with half of these
amounts in February 1984 and 1985 and with the other half in March 1984 and 1985.
Urea, NH4 +, and N0 3- in soil were systematically analyzed in the February-June 1984
and 1985 periods. Ammonia volatilization was determined during the first 3 weeks after
spring fertilization. The plants were harvested in July 1984 and August 1985,
respecti vel y.
Urea hydrolysis during the first 2 weeks after spring fertili7..ation was inhibited by
HQ (10 kg/ha) (degree of inhibition: 50%), but in 3 weeks urea hydrolysis became
complete. HQ significantly reduced volatilization of NH3 from urea in all treatmens in
both years. It should however be emphasized that in these experiments even the highest
losses were less than 3% of the added urea-No
The inhibitors tested by Sen and Bandyopadhyay (1986) were catechol (CT) and p-
benzoquinone (BQ). The experimental field was on a coastal saline soil (PH 7.5) in
India and was cropped with rice under submerged conditions. Prilled urea at a rate of
100 kg of Niha without or with an inhibitor (25% of CT or BQ relative to urea-N) was
applied to 3-cm deep floodwater. In a treatment, urea at the same rate, but in the form of
3-g briquettes without any inhibitor was placed at 5-cm depth in the soil after draining
off the excess floodwater from the field. In each treatment, pH and NH/content in
floodwater and the amount of ammonia volatilized were determined at 2-day intervals
for 16-18 days following fertilization.
Under the influence of inhibitors, pH and NHJ volatilization were lower for 6 days,
whereas NH4 + concentration remained lower for 8 days. Cumulative NH3 losses during
95
18 days were reduced by 30.3% due to CT and by 28.6% due to BQ. However, the most
marked reduction (71.3%) was recorded in the treatment in which urea was placed at 5-
cm soil depth.
Abdel Magid and El Mahi (1986) studied the effect of p-benzoquinone (BQ) on urea
hydrolysis in five Sudan soils (three clays, a silty clay loam, and a sandy loam). Urea
granules (at a rate of 90 kg of N/ha) were placed on the surface of 1O-g soil samples
with or without 0.05 mg of BQ/g soil. After moistening soil samples up to 50% of WHC
and incubation at 35°C for 1,3, 7, and 14 days, the residual urea was estimated. It was
found that BQ inhibited urease activity in each soil. From the analytical data obtained
after 14 days of incubation the following inhibitions could be calculated: about 60% (in
the three clay soils), about 55% (in the silty clay loam), and 100% (in the sandy loam).
Preliminary data on these investigations were published by Abdel Magid et al. (1983)
who also tested catechol, but later this compound was abandoned because its urease-
inhibiting capacity was much weaker than that of BQ. Catechol, like BQ, was applied at
the rate of 0.05 mg/g soil, whereas the rate ofurea-N was 22.5 kg/ha.
One can see from Table 25 that hydroquinone (HQ) and chlorohydroquinone
prolonged complete hydrolysis of urea in a light-textured (alluvial) soil by 4 days. In a
TABLE 25. Effect of some poly hydric phenols and tetrachloro-p-benzoquinone on soil
urease activity"
Control 6 9
Hydroquinone 10 16
Resorcinol 8 12
Pyrogallol 8 12
Chlorohydroquinone 10 14
Tetrac hI oro-p- benzoguin on e 6 9
"Adapted from Kiss and Pintea (1987).
heavier-textured soil (leached chernozem), HQ acted more markedly than did chloro-
hydroquinone, with complete hydrolysis of urea prolonged by 7 and 5 days,
respectively. In both soils, resorcinol and pyrogallol were weak inhibitors, whereas
tetrachloro-p-benzoquinone did not manifest any inhibitory effect.The reaction mixtures
(5 g of air-dried soil + 10 ml of aqueous phase containing 6 mg of urea + 0 or 0.12 mg
of test compound) were incubated at laboratory temperature (Kiss and Pintea, 1987).
The Chinese inventors Cao et al. (1987) patented the technology of hydroquinone-
containing urea fertilizer production.
Xue and Li (1987) tested HQ and seven other polyhydric phenols and quinones
(catechol, resorcinol, gallic acid, p-benzoquinone, quinhydrone, anthraquinone, and
phenanthrenequinonc; see Figures 28 and 29) on urease activity of a paddy soil. The
reaction mixtures were prepared from 5 g of dry soil + 10 ml of a 10% urea solution
(1 % solution for p-benzoquinone and anthraquinone) containing (on soil weight basis)
0, 20, 40, 60, 80, and 100 ppm test compound (only 100 ppm, with gallic acid).
Incubation took place at 37°C and lasted 48 hours.
96
The degree of inhibition depended on the nature of the test compounds and, as
expected, increased with their concentration. Thus, at the 100 ppm concentration, the
inhibitions showed the following order: HQ (68.26%), quinhydrone (64.07%), p-
benzoquinone (58.87%), catechol (55.88%), phenanthrenequinone (44.71 %), gallic acid
(26.34%), resorcinol (19.36%), and anthraquinone (5.60%). The same decreasing order
was also observed at lower concentrations, HQ, quinhydrone and p-benzoquinone
always being the most effective inhibitors.
With these three compounds and with catechol (at 100 ppm), the inhibitory effect
was followed for 12 days. The inhibition decreased during this period but remained
evident even at day 12.
In the case of HQ (at 100 ppm), the influence of urea concentration (1-10%) and that
of incubation temperature (22, 37, and 45°C) were also studied. It was established that
the urease-inhibiting effect of HQ decreased with increasing urea concentration. In
respect of the influence of temperature, the following situation was observed:
inhibition at 22°C < inhibition at 37°C;::: inhibition at 45°C.
The data in Table 26 show that the inhibiting effect of HQ increased with the
increase in pH from 4.7 to 6.6, but remained practically unchanged in the pH zone of
6.6-8.0. HQ was used at a concentration of 100 ppm.
TABLE 26. Effect of hydro quinone on soil urease activity at different pH values"
Urease activity (mg NH3-N/g dry soil)
pH Inhibition (%)
Control soil Soil treated with hydroquinone ( 100 ppm)
4.7 2.15 1.90 11.60
5.5 2.45 1.66 32.24
6.2 3.25 1.79 44.95
6.6 4.54 2.01 55.72
7.0 4.79 2.07 56.78
7.5 4.42 2.09 52.71
s.o 4.79 2.07 56.78
"From Xue and Li (1987).
Zhou et 01. (1988) studied the influence of HQ concentration and incubation time on
urease activity in a brown soil from China. Soil samples (250 g) were treated with 10 ml
ofa 10% urea solution, HQ (0, 25, 50,100 or 200 ppm on soil weight basis) and water
to 65% of WHC, then incubated at 30°C. After I, 7, 15, 30,45, 60, and 75 days of
incubation, the residual urea was determined. The results presented in Figure 31 prove
that the inhibition increased with increasing HQ concentration and decreased during
incubation. This decrease was very marked at lower HQ concentrations and less marked
at higher ones. For example, the inhibitory effect of HQ at 25 ppm after I day of
incubation (about 62%) disappeared after 45 days; at 100 ppm the disappearance of the
inhibitory effect took 75 days.
97
70
60
10 ZO 3n 40 50 60 10 80
Incubation time (days)
In a pot experiment carried out by Zhou et at. (1988), HQ added to urea, at rates of
10 and 20 mg of HQ to 0.9 g of urea-N/pot, reduced volatilization of urea-N as
ammonia, and when HQ was added at a higher rate (40 mg to 0.9 g ofurea-N/pot) NH3
volatilization remained at a level similar to that recorded in the control soil (treated with
urea alone). At the same time, the amount of residual urea-N increased with the increase
in HQ concentration.
Samples (100 g) of a soddy-podzolic soil and a calcareous chemozem were treated
with 20 mg ofurea-N + 0 or 5% hydroquinone (HQ) (relative to urea-N), moistened to
60% ofWHC and incubated at 28°C. After 6, 12,24,48, and 72 hours of incubation the
residual urea was determined. In the soddy-podzolic soil, the residual urea represented
25% (control sample) and 80% (HQ-treated sample) of the initial amount after 24 hours
of incubation, and 0 and 25%, respectively, after 48 hours. In the calcareous chernozem
no residual urea was detected in the control sample after 12 hours of incubation,
whereas in the HQ-treated samples after 12 and 24 hours a part (35 and 8%,
respectively) of the initial urea amount remained unhydrolyzed (Pisareva and Muravin,
1988; Pisareva, 1989).
Zhou el al. (1992) and Zhao el al. (1992a) compared the effect of HQ on urea
hydrolysis and ammonia volatilinltion in normal (nonfumigated) and fumigated samples
of a Chinese brown meadow soil (silty loam, pH 6.55). Fumigation was carried out with
chloroform at 30°C for 1 week. The soil samples (l00 g) were treated with 0 or 480.8
mg of urea and with 0, 0.96, 2.0, 4.81, and 9.61 mg of HQ. The mixtures were
moistened to 40% ofWHC and incubated at 30°C. Residual urea was determined after 3
and 10 days of incubation and NH/-N content and NH3 evolution were periodically
assessed during 88 days of incubation. All the experimental procedures with the
fumigated soil were performed under sterile conditions.
Urea hydrolysis in all normal and fumigated soil treatments was complete after 10
days of incubation. On day 3, urea hydrolysis in nomwl soil was inhibited by each HQ
rate (the degree of inhibition increased with increasing HQ rates, from about 20 to about
44%), but in the fumigated soil only the two higher rates of HQ were effective (degree
98
of inhibition: 21.5 and 74.0%). One can see that at the highest HQ rate, the degree of
inhibition was higher in the fumigated than in the normal soil.
In normal soil, NH/-N content during 0-10 days of incubation was higher in the
HQ-treated samples than in the untreated control, and the decreasing content was
positively related to HQ dosage. The same trend was maintained during the 10-25-day
period, but the differences between control and HQ treatments were very small. In the
25-88-day period, NH/-N content in the HQ treatments increased and exceeded that
measured in the control. The increase was positively related to HQ dosage. Ammonium-
N in the fumigated samples showed the same pattern as that of the normal soil, but its
amounts in all treatments were significantly higher than those found in the normal soil,
which was attributed to lack of microbial activity in the fumigated soil.
Volatile loss of NH3 during the first 3 days of incubation was higher in the control
than in the HQ-treated normal or fumigated soil samples. In most of the treatments the
highest loss occurred on day 6. After day 25, the loss became constant in all treatments
with only small amounts of volatile NH3 loss. Cumulative NH3 losses in 88 days were
higher in the normal soil than in the fumigated one. At the two higher rates of HQ,
cumulative losses were higher in the normal soil and lower in the fumigated soil than in
the control. Thus, the nonnal soil lost about 38 flg N/g soil from the control (urea-only
treatment) and about 50 flg N/g soil from the sample treated with urea and the highest
HQ rate. The corresponding values for the fumigated soil were about 29 and about 27
flg N/g soil. respectively.
Xu et at. (1994) preincubated soil samples treated with 0, 2.5, 5 or 15 flg HQ/g soil
for 45 days and determined their urease activity at 5-day intervals. Inhibition of urease
activity increased with the rate of HQ. In a slightly acid soil (PH 6.15), the inhibition
was maximum (- 50, 65, and 70%, respectively) on day 5, then decreased and became
negligibly low on day 45. In another, more acid soil (pH 5.58), the maximum
inhibitions (-45, 70, and 90%, respectively) were registered on day 10, then the
inhibition decreased and disappeared on day 30.
The effect of HQ on volatile losses of ammonia from urea-treated soil (pH 6.15) was
also studied, in four experimental variants: urea alone; urea + HQ; urea + wheat straw;
and urea + wheat straw + HQ. The rates of additions per 100 g of soil were: 230 mg
urea-N, 5 mg HQ, and I g straw. The volatile NH3 was measured during 30 days. The
cumulative NH3 losses in the four variants were the following: -50% (urea alone); 45%
(urea + straw); 40% (urea + straw + HQ); and 30% (urea + HQ). These data mean that
addition of straw reduced the inhibitory efficiency ofHQ.
Effect of polyphenol and quinone urease inhibitors on energy barriers of urease
activity in soils were dealt with by two research groups.
Tomar and MacKenzie (1984) investigated the effects of temperature, catechol (CT)
and p-benzoquinone (BQ) on urease activity in five Quebec soils. Reaction mixtures
were prepared from air-dry soil equivalent to 109 of oven-dry soil + 1 ml of water or 1
ml of aqueous solution containing 50 or 100 flg of CT or BQ/g soil + 1 ml of urea
solution (1 mg of N/g soil) + water to field capacity. After 3 days of incubation at 5, 10,
15,20, and 25°C, the mixtures were analyzed for residual urea, NH/, and N0 3-. The
results showed that CT and BQ reduced hydrolysis of urea in each soil. BQ was
consistently more effective than CT. The degree of inhibition increased significantly
with the amount of inhibitors applied and decreased significantly with increase in
temperature of incubation. The inhibition was strong in a loamy sand (poor in organic
99
matter) and very weak in two high clay soils (rich in organic matter) in which complete
hydrolysis of urea occurred in 3 days at 10 or 15°C and at higher temperatures.
Consequently, for these soils it was impossible to calculate thermodynamic values.
The temperature coefficient (QJO) had values ranging in general from about 1.50 to
2.60, the inhibitors manifesting a tendency to diminish the effect of temperature on urea
hydrolysis. The other thermodynamic values are presented in Table 27 from which one
can see that the inhibitors tended to increase energy of activit ion (Ea ), enthalpy of
activation (.JH\ and entropy of activation (-1S") of urease, excepting BQ in the first
soil, in which these values decreased. The effect of inhibitors was evidently influenced
TABLE 27. Energy of activation (K). enthalpy of activation (L1H'J. entropy of activation (L1S"). and free
energy of activation (L1n") of soil urease in absence and presence of catechol (eT) and p-benzoquinone
(BQ)"
Rate of
E" L1H" lIS" Llct
Soil Inhibitor inhibitor
(kcal/mo Ie) (kcalllllOle) (kcal/mole) (kcai/mole)
(I:!!l~ soi'2
0 9.2 8.6 -21.4 14.8
CT 50 10.2 9.6 -18.5 14.9
Silty clay 100 11.2 10.7 -15.2 15.0
loam 0 9.1 8.6 -21.5 14.8
BQ 50 7.7 7.1 -28.7 15.4
100 7.0 6.4 -31.5 15.5
0 6.6 6.0 -30.6 14.8
CT SO 7.S 7.0 -27.7 IS.O
100 8.S 7.0 -24.4 IS.O
Clay
0 6.9 6.3 -29.6 14.8
BQ 50 7.7 7.1 -28.8 15.4
100 7.9 7.4 -28.1 15.4
0 9.3 8.7 -22.0 15.1
CT 50 10.7 10.2 -18.1 15.4
Loamy 100 10.7 10.1 -18.5 15.5
sand 0 9.4 8.8 -21.6 15.1
BQ 50 15.8 15.2 -1.6 15.6
100 14.6 14.0 -5.8 15.7
"From Tomar and MacKenzie (1984).
by the nature of soil, but very little by their concentration. At the same time, free energy
of activation (JG*) of the urease activity had practically the same value in all soils in
both absence and presence of inhibitors, which suggests that the energetic requirement
of the urea-urease system is independent of the nature of the soils used.
The conclusion arrived at was that an increase in the activation energy of soil urease
as a result of inhibitor use is related to an increase in the effectiveness of the inhibitor.
In other words, determination of the energy of activation of urease in soil samples
treated with different compounds may prove useful in evaluating the relative
effectiveness of these compounds as soil urease inhibitors, the effectiveness being
indicated by increase in energy of activation.
In a similar study, Praveen-Kumar et al. (1990) used samples of an acidic, an
alkaline, and a saline soil from India. Eight polyphenol and quinone urease inhibitors,
namely catechol (CT), 4-methylcatechol (MeT), hydroquinone (HQ), p-benzoquinone
(BQ), p-naphthoquinone (NQ), 2-methyl-p-naphthoquinone (MNQ), 2,6-dichloro-
100
hydroquinone 0.87, 1.00, and 0.88; catechol 0.80, 0.74, and 0.46; 4-methylcatechol
0.74, 0.73, and 0.83; p-benzoquinone 0.93, 0.93, and 0.90, and 2-methyl-p-
naphthoquinone 0.54, 0.54, and 0.57, respectively. Thus, hydroquinone and p-
benzoquinone were the most mobile compounds in each soil, whereas the least mobile
compounds were 2-methyl-p-naphthoquinone (in acidic and alkaline soils) and catechol
(in saline soil). Urca was very mobile (RF1.00) in each soil.
For inhibition of urease located at different depths in soil, it is a requirement for the
inhibitor to move along with urea. This is why hydroquinone and p-benzoquinone
appear more suitable as inhibitors of soil urease than the other six compounds tested.
Zhao et al. (1991) also found that hydroquinone is mobile in soil. In their
experiment, hydroquinone applied on the surface of a soil column moved down to the
30-60-cm layer.
Finally, it should be mentioned that Zhao et al. (1992b) elaborated methods for
determination of hydroquinone and biuret in hydroquinone-containing urea fertilizers.
Some dimeric and monomeric compounds from these classes (Figure 32) were tested as
inhibitors of soil urease activity by Radel e( al. (1989). The testings were also described
in a patent by Radel and Crenshaw (1990) (Assignee: Tennessee Valley Authority,
Muscle Shoals, Alabama). Phenylphosphorodiamidate (PPDA) served as a reference
compound.
~_s-O
! !J
0," I
22'-Di1hiobis-pyridne-N-oxide
"
2-Mercaptopyndne-N-oxide
(DTPNO) (MPNO)
~ ~N'"
~_s-O ~>-s-s~)
2,2'-Dilhiobis-pyridine (Aldrithiol)
(An
2 -Mercaptopyndine 2-Mercapto-3-pyridnol
(MP) (MPOl)
~" 0-0
~ ~ 0 I
0
Figure 32. Structure of the compounds tested by Radel et al. (1989) and Radel and Crenshaw (1990)
for inhibition of soil urease activity.
102
A banded soil procedure was developed for the testings. The sample of a silt loam
soil was moistened to a water content of 20% (dry weight basis) and preincubated at
room temperature for 2 days. In the next step, plcxiglass containers (6x6x6 cm) were
one-half filled with the preincubated soil and packed to a bulk density of 1.1 glcm3 •
Urea or urea + test compound (thoroughly mixed) was distributed in a narrow band
approximately 6 cm long and about 0.5 cm wide on the surface of the soil (rates of
additions per band were 410 mg of urea and 41 mg of test compound). The containers
then were filled with soil and again packed to a bulk density of 1.1 glcm 3 , followed by
incubation at 25°C for 3 and 6 days, when the soil from each container was thoroughly
mixed and a 10-g subsample was extracted and analyzed for determination of the
unhydrolyzed urea. From the analytical data, the percent inhibition of soil urease
activity was calculated.
The results are summarized in Table 28. Two compounds (DTPNO and PPDA) were
compared in two test series.
It is evident from this table that DTPNO was nearly as effective a soil urease
inhibitor as PPDA. Strong inhibitions were produced also by MPNO, MP, and MPM,
i.e .. by compounds containing S-S or SH group and pyridine or pyrimidine moiety,
whereas the weakest inhibitors were DP and PNO, in which no S is bound to the
pyridine moiety. These findings indicate that in the monomeric compounds the key
functional group responsible for the inhibition is the mercapto group and N-oxide
function adds little to the inhibitory power, whereas in the dimeric compounds the N-
oxide moiety increases the solubility and weakens the strength of the disulfide bond,
thus producing a more active inhibitor.
The most preferable rate, at which the inhibitors are recommended as additives to
fertilizer urea, ranges from about 0.5 to about 2% relative to weight of urea.
103
HaC, T'
HaC- C -
HaC_I
HaC ..... ' N /
'=0
N
I
CI
l-Bromo-3-chloro-4.4.5,5-tetramethyl- 1,3-Dibromo-4.4,5.5-tetramethyl-
2-imidazolidinone (ABC) 2-imidazolidinone (AB)
3-Chloro-4,4-dimethyl- 3-Bromo-4.4-dimethyl-
2-oxazolidinone (9 2-oxazolidinone (8)
Figure 33. Structure of the N-halamine compounds tested by Gautney et al. (I990) for
inhibition of soil urease activity.
The same silt loam soil and banded soil procedure were used for testing as those also
applied by other collaborators of Tennessee Yalley Authority (see the preceding
subchapter). Rates of additions per soil band were 410 mg of urea and 0 or 41 mg of test
compound. The incubation was carried out at 25°C for 3 and 6 days, then the
unhydrolyzed urea was determined.
The amounts of unhydrolyzed urea were expressed as percentages of the added urea
amount. The values recorded after 3- and 6-day incubations in the different treatments
were the following: 4.9 and 1.0% (urea-only treatment); 84.7 and 53.7% (ABC); 70.6
and 15.0% (AB); 82.4 and 56.5% (I); 65.5 and 10.0% (IB); and 94.3 and 58.7%
(PPDA), respectively. In other words, the urease-inhibiting capacity increased in the
order:
IB < AB < I;::: ABC < PPDA.
The effectiveness of ABC and 1 as inhibitors of soil urease activity approached that
ofPPDA.
For use in practice, the N-halamine compounds are recommended in the most
preferred amounts of 0.5-2% relative to weight of urea.
104
The m-nitroacetanilide and 0-, m-, and p-nitroanilines, together with other (13)
compounds, all patented as nitrification inhibitors by different companies, were studied
by Bremner and Douglas (1971) for evaluation of their effect on urease activity in three
Iowa soils. The 5-hour test was applied and it was found that the urease-inhibiting effect
of m-nitroacetanilide like that of the three nitroanilines was very weak: less than 1%.
Simihiiian et al. (1992) studied three y-L-glutamyl nitroanilides (Figure 34) to
evaluate their effect on urease activity in a heavy-textured soil (leached chernozem) and
a light-textured (alluvial) soil. The free nitroanilines and aniline were also tested, and
hydroquinone served as a reference compound.
COOH COOH
H2N-!H H2N-!H
I
CH2
I
CH2
bO_HN-O b O - H N - 0 - - N02
Figure 34. Structure of the y-L-glutal1lyl nitroanilides tested by Sil1lihaian et al. (1992) for inhibition of soil
urease activity.
TABLE 29. Etfect ofy-L-glutal1lylnitroanilides and free nitroanilines on soil urease activity
as compared with the etlect of aniline and hydro quinone"
Time necessary for complete
Compound
hydrolysis of urea (days)
No (control) 11
y-L-Glutamyl m-nitroanilide 18
m-Nitroaniline 15
y-L-Glutamyl p-nitroanilide 18
p-Nitroaniline 15
y-L-GlutamyI2-l1lethoxy-p-nitroanilide 18
2-Methoxy-p-nitroaniline 15
Aniline 25
Hydroquinone 32
"From Simihiiian et al. tl992).
105
of the reaction mixtures was analyzed by means of a chromogenic reagent (see the
footnote on page 35) for detecting the unhydrolyzed urea. The time (days) necessary for
complete hydrolysis of urea was registered. Prolongation of this time, relative to
control, indicates inhibition of soil urease activity.
Surprisingly, the two soils used behaved similarly, in this experiment, in regards to
their urease activity. Therefore, the data presented in Table 29 are valid for both soils.
One can see from this table that each compound tested had an inhibitory effect on
soil urease activity. Hydroquinone was the strongest inhibitor prolonging the time
necessary for complete urea hydrolysis from II to 32 days. The three y-L-glutamyl
nitroanilides tested brought about a 7-day delay in complete urea hydrolysis. The delay
caused by equimolecular amounts of the free nitroanilines was shorter, lasting only 4
days. This means that the glutamyl moiety in glutamyl nitroanilides enhanced the
urease-inhibiting effect of nitroanilines. The inhibitory effect of aniline exceeded that of
nitroanilines. Consequently, the presence of nitro group or of nitro and methoxy groups
in nitroanilines attenuated the inhibitory effect of their aniline moiety.
This experiment was also referred to by Simihlhan et al. (1999).
The phosphoroamides constitute a very important class of the inhibitors of soil urease
activity. They are derivatives ofphosphoro(mono)amidic acid [(mono)amidophosphoric
acid]; phosphorodiamidic acid (diamidophosphoric acid); thiophosphorodiamidic acid
(diamidothiophosphoric acid); phosphoryl triamide and thiophosphoryl triamide (Figure
35).
o s
II/NH2 II/NH2
Ho--P, Ho--P,
NH2 NH2
s
II/NH2
H~-P
'NH2
2.20.1. The Patented Compounds and the First Studies on Their Inhibitory ~tfect on
Soil Urease Activity
Phosphoro(mono)arnides and phosphorodiamides as inhibitors of soil urease activity
were patented by the German investigators Hartbrich et al. (1976b), Held et al. (1976b),
and Lang et al. (1976). They studied the compounds presented in Figure 37. These
compounds were recommended to be added to urea at rates ranging from 0.01 to 50%,
the preferred rates being ()'05-5% relative to urea-N, for fertilization of light- and
medium-textured soils as well as of grasslands.
RI Ro R, R. Derivative
CH, Nih H H phenylposphorodiamidate (phenyl-PDA)
2-Cl-C,JI, Nih H H 2-chlorophenyl-PDA
4-CI-C,H4 NH, H H 4-chlorophenyl-PDA
(',H, NH-CH, CH, H phenyl-N.N·-dimethyl-PDA
J-CII,-C,H. NH-CH, CH, II m-cresyl-N,N'-dimethyl-PDA
4-CI-C,H. NH-CH, CH, H 4-chlorophenyl-N,N' -dimethyl-PDA
C,H, NH-C,H, C,H, H phenyl-N,N'-di-n-propyl-PDA
4-C1-C.H4 NH-C,H, C,H 7 H 4-chlorophenyl-N,N' -di-n-propyl-PDA
e.H5 NH-CH(CH,), CH(CH,h H phenyl-N,N'-diisopropyl-PDA
4-Cl-C,H4 NH-CH(CH 3 )o CH(CH,h H 4-chlorophenyl-N,N'-diisopropyl-PDA
C,H 5 NH-n-C4H9 n-C.H9 H phenyl-N,N'-di-n-butyl-PDA
4-CI-C,1I4 NH-n-C 4 H9 n-C4 H 9 H 4-chlorophenyl-N,N'-di-n-butyl-PDA
4-CI-C.H4 NH-C,H, C,H, H 4-chlorophenyl-N,N'-diphenyl-PDA
C,H, NH-CHo-C.H< CH,-C,H, II phenyl-N,N'-dibenzyl-PDA
C,H5 NH-C,H, C,H, H ethyl-N,N'-diphenyl-PDA
C,H, O-C,H, H H diphenylphoshoroamidate (diphenyl-PA)
4-CI-C.H4 0-4-CI-C .H 4 H H di-(4-chlorophenyl)-PA
C,H, O-C,H, CH, H diphenyl-N-methyl-PA
C 9H I9 0-C9HJ9 C,Hs C,Hs dinonyl-N ,N' -diethyl-PA
Figure 37. General structural formula of compounds patented and tested by Hartbrich et al. (1976b), Held et
al. (1976b), and Lang e/ al. (1976), with specitication of some derivatives.
107
By applying the short and long time tests (see page 51)*, Held et a1. (1976b)
obtained the results given in Tables 30 and 31. They show that both compounds
inhibited urease activity in soil at both 30 and 10°C; the inhibitory effect increased with
Diphenyl-
phospboro- 29.7 35.9 40.6 48.3 57.8 80.0 78.5 86.2 93.8
ami date
"From Held et al. (l976b).
Lang et al. (1976) found, by applying the long time test, that phenylphosphoro-
diamidate (PPDA) was more effective than were 4-chloro-PPDA and di-PPA.
Held et af. (1978) norninalized63 phosphoroarnides and specified the percent
inhibitions registered in soil urease activity under the influence of these compounds
which comprise: phosphorodiamidic acid, 5 alkyl esters, 14 phenyl esters, and 2
'In the long time test, incubation was carried out not at 20. but at lOOC.
108
TABLE 32. Effect of 63 phosphoroamides on volatilization of ammonia from urea-treated soil samples
incubated at 30°C for 24 hours"
Substituents Inhibition
COlI1'ounds Structural formula
X RorR' (%)
2 3 4 5
Pho!!horodiamidic acid HO-~O~(NH2l2 20
Phosphorodiamidic acid alkyl R-O-P(O)(NH2)2 CH, 38
esters CzHs 5
n-C,H7 3
n-C4 H9 23
n-C 6 H 13 10
Phosphorodiamidic acid phenyl X-C_H.-O-P(O)(NH2h H 100
esters 2-CI 95
3-CI 93
4-CI 100
4-Br 93
2-CH, 96
3-CH, 90
4-CH, 93
2-0CH, 99
4-COOC,H7(n) 94
3-N02 87
4-NO z 55
2-CH,-4-CI 93
2-CH,-6-CI 93
Phosphorodiamidic acid n- C JOH7-n-O-P(O)(NH2)2 93
nal!hth~1 and ~-nal!hth~1 esters ClOH7-~-O-P(Ol~H2l2 96
N-A1kyl derivatives of X-C6 H.-O-(O)(NHR·h H CH, 42
phosphorodiamidic acid phenyl H C2Hs 34
esters H n-C,H7 30
H i-C,H7 42
H n-C4 H9 34
H C6H U 0
H CH2-C6 H, 0
2-CI CH, 0
4-CI CH, 33
4-CI CzHs 23
4-CI n-C,H7 27
4-CI i-C,H7 19
4-CI n-C.H9 14
2A-CI CH, 0
2,5-CI CH, 25
2-CH, CH, 44
3-CH, CH, 26
4-CH, CH, 24
2-CH,-4-CI CH, 29
2-CH,-6-C1 CH, 71
3-NO z ClI, 32
2-0ClI, CH, 24
lO9
One can see from this table that phosphorodiamidic acid and its alkyl esters are
weak inhibitors, with their degrees of inhibition being between 3 and 38%. At the same
time, the phenyl esters (excepting the 4-N02 derivative) and the naphthyl esters of
phosphorodiarnidic acid are characterized by a strong inhibitory capacity (inhibition
degree: 87-100%). The strongest inhibitors are PPDA and 4-CI-PPDA (inhibition
degree: 100%). The ~-naphthyl ester ofphosphorodiamidic acid is a little more effective
than its a-naphthyl ester (inhibition degree: 96 and 93%, respectively). Alkylation of
NH2 groups leads to diminution or even to disappearance of the inhibitory capacity of
phosphorodiamidic acid phenyl esters. Introduction of substituents to phosphoro-
dilimidic acid phenyl esters also determines diminution of inhibitory capacity. Phenyl
esters of thiophosphorodiarnidic acid are nearly as effective as those of phospho-
rodiarnidic acid, excepting 2-CH3-6-Cl-phenylthiophosphorodiarnidate which is a weak
inhibitor when compared to 2-CH3-6-CI-PPDA.
PPDA was also found to be the most effective inhibitor in the long time test, with
1% PPDA relative to urea-N; incubation temperature: 20°C. Thus, the inhibition caused
by PPDA was 100% for 7 days, 50% after about 10 days, and 25% after about 12 days
of incubation (Held et aI., 1978). Other data published by Held et af. (1978) and
Hartbrich et af. (1978) indicate even stronger inhibitions, e.g. a 33% inhibition by 1%
PPDA after 12 days of incubation at 20°C. After the same time period of incubation at
the same temperature and PPDA concentration, Oertel et af. (1978) and Jasche et af.
(1978) registered a 94% inhibition.
These results are in agreement with those of Heiseler et af. (1980), who applied the
long time test and 1% inhibitor on urea-N basis and found that after 13 days of
incubation at 15°C urea hydrolysis was inhibited by the seven phosphorodiarnidate
(PDA) and phosphoroamidate (PA) compounds tested, in the following proportions:
PPDA (99.6%), o<resyl-PDA (59.6%), phenylthio-PDA (58.7%), di-p<resyl-PA
(15.6%), di-phenyl-PA (13.5%), di-o<resyl-PA (0%), and ethyl-PDA (0%).
110
Concerning duration of the inhibitory effect, PPDA and its chIoro derivatives and
those having a substituent in the same position (4) presented the following orders:
PPDA > 4-Cl-PPDA > 3-Cl-PPDA > 2-Cl-PPDA, and
PPDA > 4-CH 3-PPDA > 4-CI-PPDA > 4-COOC3H7 (n)-PPDA;:::: 4-Br-PPDA > 4-
NOrPPDA, respectively.
It was also established that duration of the inhibitory effect of PPDA (used at
concentrations of 0.25, 0.5, and 1% relative to urea-N) decreased with increasing
incubation temperature (from 10 to 15, 20, and 30DC). For example, PPDA, at 1%
concentration, completely inhibited volatilization of ammonia from urea for 16 days at
lODC, for 8 days at 15 DC, for 4 days at 20DC, and only for 2 days at 30 DC. At the same
concentration, PPDA was less effective at constant 20 DC temperature than under natural
conditions where the temperature varied between 10 DC (by night) and 28 DC (maximum
temperature by day) (Held et al., 1978).
TIle inhibitor and enzyme substrate, i.e.. PPDA and urea contain a similar amide
stmcture (Figure 38), which allows the attachment of PPDA to the active site of
enzyme. The hydrophilic group, O=P(NH 2h determines the recognition of receptor and,
thus, the specificity of inhibition, whereas the hydrophobic aryl residue (phenyl ring)
binds PPDA to the receptive site of the enzyme and, consequently, is decisive for the
(> 0
Figure 38. Comparison of the structure ofPPDA with that of the urea.
strength of inhibition. The inhibitor is bound with a higher affinity than urea, because
inhibition occurs even with an excess of urea. Derivatives of PPDA also act as tight-
binding inhibitors (Barth et al., 1978, 1980).
Jasche et al. (1978) and Oertel et at. (1978) underline the advantageous properties of
PPDA. PPDA has a remarkable urease-inhibiting effect at low concentrations (0.5-1 %
relative to urea-N). PPDA is not toxic for man. In soil, PPDA is degraded to nontoxic
products.
PPDA can easily be synthesized from phenol, phosphoms oxychloride, and
ammonia on a technical scale (Jasche et al., 1977, 1978; Oertel et al., 1978; see also
Kurze and Richter, 1978; Heiseler et al., 1980; Anonymous, 1985a).
PPDA is soluble in ethanol, methanol, acetone, but its solubility in water is low
(0.6643 gllOO ml of water at 25 D C) (Oertel et al., 1978).
For detennination of PPDA in urea fertilizers, soil extracts and other solutions,
colorimetric methods are available (Wenzel et al., 1981; Martens and Brenmer, 1983a).
A method based on high-performance liquid chromatography for analysis of PPDA was
also developed (Austin et al., 1984).
111
Elbe et al. (1979) patented a procedure for coating urea priUs with PPDA by means
of a mineral oil-bitumen mixture used as anticaking agent. The following example is
described in the patent: 25 kg of urea priUs are blended at 20°C, with 75 g of mineral
oil-bitumen mixture (80% of oil + 20% of bitumen), then 116 g of finely ground PPDA
(diameter of particles: 0.1 mm) is added and mixed again. The conditioned urea
fertilizer prepared contains 0.46% ofPPDA.
Kurze (1981) performed laboratory experiments, in which PPDA was used with
inhibitors more soluble in water: diammonium and calcium salts of imidodiphosphoric
acid diphenyl ester (Figure 39). Thus, solubility of the diammonium salt in water at
25°C is 46 ~100 mI, whereas that ofPPDA, as mentioned above, is only 0.6643 ~100
m!.
0-
o o
o- O-~-ONH4 _ 0-,-0\
II
- I NH NH Ca
0-
J 0 -1i - ONH4 O-o-l-!
Figure 39. Structure of diammonium and calcium salts of imidodiphosphoric acid diphenyl ester tested by
Kurze (198\).
Under the conditions of the long time test, these compounds, used at a rate of 1%
relative to urea-N, inhibited volatilization of ammonia from urea-treated soil samples to
a lesser extent than did PPDA, but acted synergistically with PPDA. For example, in an
experiment the mixture of PPDA + diammonium salt of imidodiphosphoric acid
diphenyl ester (at a molar ratio of 1: 1.5) produced a nearly complete (96%) inhibition of
the volatilization ofNH 3 from urea-treated soil samples during their 14-day incubation.
Kurze et al. (1985) evaluated, using the long time test, the inhibitory capacity of
seven heavy metal salts and the Al salt of the imidodiphosphoric acid diphenyl ester
(IPP) and of two heavy metal salts of the amidophosphoric acid monophenyl ester
(APP) on the volatilization of ammonia from urea-treated soil samples incubated for 6-
16 days. These compounds are more soluble in water than PPDA. The ~ salts of IPP
and APP served for comparison. The results obtained are reproduced in Table 33.
One can see from this table that the heavy metal salts (excepting the Fe salt) and the
Al salt of IPP were more inhibitory than was the NH4 salt. Most inhibitory were the Hg,
Cu, and Ni salts.
Cu salt of APP was more inhibitory but the Ni salt was less inhibitory than the ~
salt.
The Cu salt of APP was a stronger inhibitor than the Cu salt ofIPP, but the reverse
was true for the Ni salts.
112
TABLE 33. Effect of salts of imidodiphosphoric acid diphenyl ester (IPP) and amidophosphoric acid
monophenyl ester (APP) on volatilization of ammonia from urea-treated soil samples incubated at
200C for 6-16 da:z:s·
Inhibition (%l
Salts Incubation time (days)
6(7l 9 11(12 l 14(15l 16
Salts ofIPP
Hg 100 99.7 (99.2) 97.4 96.4
Cu 98 90 82 68 47
Ni 98 86 75 55 44
Co 94 74 61 39 17
Mn 98 81 (50) 29 17
Zn 83 36 (9.9) 6.9 4.6
Fe 74 22 (16) 4.6 1.9
Al 80 30 (16) 7.7 6.9
Nil. 72 10.4 (9.2) S.4 2.4
Salts ofAPP
Cu 100 99.5 98.6 93.4 70
Ni 96 56 36 27 8.8
NH. 98 88 51 32 10.8
·Adapted from Kurze et al. (1985).
Swerdloff et at. (1984, 1985a) and Anello et al. (1985), of the Allied Corporation
(Morristown, New Jersey), patented a great number of phosphorodiamidates and
thiophosphorodiamidates as inhibitors of soil urease activity and tested some of them
with samples of one or two soils. The soils and the testing method were the same as
those used by Kole et al. (1985b) (see page 80). Briefly, 20-g of air-dry samples of a
New York soil (Cazenovia sandy loam, pH 7.2) or a Wisconsin soil (Plano silt loam, pH
5.4) + 0.8 mg of test compound in 5 rnl of water or only 5 rnl of water + 42.8 mg of urea
in 1 ml of water were incubated at 25°C for 3 days, then analyzed for remaining urea.
Swerdloff et al. (1984) and Anello et al. (1985) patented 88 phosphorodiamidates
(PDAs) and 18 thiophosphorodiamidates (TPDAs), but testing of only 12 PDAs
(Cazenovia soil) and 6 PDAs (Plano soil) is described in the patents. The results
obtained are presented in Table 34.
The table shows that in both soils the most inhibitory PDAs were 2,2,2-
trifluoroethyl-PDA and 2,2,2-trichloroethyl-PDA, and the weakest inhibitors were
TABLE 34. Inhibition of soil urease activity by phosphorodiamidate compounds, R-O-P(O)(NH2 l2"
Inhibition (%)
R
Cazenovia soil Plano soil
2,2,2-Trifluoroethyl 97 90
2,2,2-Trichloroethyl 97 87
2-BrolJX)ethyl 85
Cyclohexyl 85
2-Chloroethyl 84
1,3-Dichloro-2-propyl 72 49
Benzyl 69 20
Allyl 65 38
3-Chloropropyl 41 22
2,2,6,6-Tetrachlorocycloheocyl 38
Methyl 34
Propyl 15
" Adapted from Swenlloff et al. (1984) and Anello et al. (1985).
113
propyl-PDA (Cazenovia soil) and benzyl-PDA (Plano soil). All PDAs tested in both
soils were more inhibitory in the Cazenovia soil than in the Plano soil. It should also be
mentioned that propyl-PDA was less inhibitory than were 1,3-dichloro-2-propyl-PDA
and 3-chloropropyl-PDA; contrarily, cyclohexyl-PDA was more inhibitory than 2,2,6,6-
tetrachlorocyclohexyl-PDA.
Swerdloff et af. (1985a) patented 74 aryl phosphorodiamidates and 18 aryl thio-
phosphorodiamidates, for inhibition of soil urease activity. However, testing of the
inhibitory effect was only described for two compounds: 4-aminophenylphosphoro-
diamidate and 3-(1', l' -dimethylethyl)-4-hydroxyphenylphosphorodiamidate (Figure
40); only the Cazenovia soil was used in testing. The two PPDAs produced 91 and 87%
inhibitions, respectively, in soil urease activity.
Figure 40. Structure of the aryl phosphorodiamidates tested by Swerdloff eI al. (1985a) for inhibition of soil
urease activily.
The use of these compounds is recommended at the following (most preferred) rates:
1-500 ppm on soil weight basis, 0.28-16.8 kglha or 0.01-20% relative to weight of urea
in liquid and solid fertilizers.
Kolc et al. (1985c) patented 10 phosphorodiarnidates and 2 thiophosphoro-
diamidates as urease inhibitors. However, it results from the patent description that none
of them were tested for evaluation of their inhibitory effect on soil urease activity.
Neither was jackbean urease used for testing of inhibitory capacity.
Matzel and Heber (1979) obtained similar results with samples of 140 other German
soils, as well. They also established that reducting the PPDA rate from 1 to 0.5%
relative to urea-N as well as replacing chemically pure PPDA (0.5%) with technical-
grade PPDA (0.5%) did not lead to a marked diminution of the inhibitory capacity of
this compound on volatilization ofNH3 from urea.
Heber et al. (1979) described experiments on microplots (700 cm2 ) located on a
permanent grassland. Urea (at a rate of 100 kg N/ha) with or without 1% PPDA was
surface-applied after each harvest (cut) in the 1974-1976 period. The volatile ammonia
was continuously assayed. Under the influence of PPDA, volatilization of NH3 began
with a delay of about 1-4 days and total NH3 losses were significantly reduced.
Muller and Forster (1980) carried out two experiments. The first was carried out
under conditions identical to those described on page 77; the inhibitor tested was
diphenylphosphoroamidate (di-PPA) which was applied, like the other inhibitors, at a
rate of 2% relative to urea-N (40 mg of urea-N!40 g of soil). It was found that when
urea was surface-applied and di-PPA was introduced at a 2 or 4 cm depth in the soil
column, urea remained unhydrolyzed during 6 days of incubation at laboratory
temperature, in proportions of about 60 and 80%, respectively. The effect of di-PPA
was weaker when urea was distributed uniformly in the soil column.
In the second experiment, urea (40 mg of N) alone or together with 2% PPDA or
with 1% di -PP A + 1% 4-chloro-PPDA was applied on the surface of soil columns (each
consisting of 40-g dry sample of a humous sandy loam, pH 7.2). The soil columns were
moistened up to 60% of WHC by adding water in three portions, namely at the
beginning of the experiment and after 24 and 48 hours. Incubation took place at
laboratory temperature and lasted 3 days. The soil was analyzed daily for residual urea,
NH/, and N0 3-. The results showed that after 3 days the residual urea represented about
60 and 80% of the initial amount in the urea + PPDA and urea + di-PPA + 4-chloro-
PPDA treatments, respectively. This proves the urease-inhibiting capacity of the
compounds tested.
Kampfe et al. (l982c) reviewed the results of 25 experiments carried out in
Bulgaria, in the former Czechoslovakia and (East) Germany, as well as in Hungary,
Poland, and Romania, within an international fertilization project, in the 1976-1978
period. The effect of PPDA on volatilization of ammonia from urea was evaluated in
Mitscherlich pots. Light- and heavy-textured soils were used in mixtures with sand (2
parts soil:l part sand) and moistened to 40% of WHC. Urea prills alone or urea prills
coated with 1% PPDA (on urea-N basis) were applied on the soil surface at a rate of 0.5
g of N/pot in 1976 and at a double rate in 1977 and 1978. TI1en the amount of NH3
volatilized in 5, 10, and 15 days was determined. One can deduce from Table 35, which
presents the mean values of NH3 volatilized at different mean air temperatures, that
PPDA manifested an inhibitory effect even at temperatures higher that 14°C, at which
NH3 losses in the absence of PPDA, were greater especially when the incubation time
was prolonged to 15 days.
Linke et at. (1982) studied volatilization of ammonia from urea and from urea-
PPDA under field conditions on three German soils (sand, loamy sand, and sandy
loam), cropped with wheat, in the 1976-1979 period. Conditioned urea (urea in the form
of prills coated with a mineral oil-bitumen mixture - 0.2% relative to weight of urea -
115
TABLE 35. Influence of air temperature on volatilization of ammonia from soils fertilized with
urea and urea-PPDN'
Ammonia losses
(% relative to applied urea-N)
Temperature (0C) Fertilizer
Incubation time (days)
5 10 15
8-10 Urea 1.7 6.8 11.4
Urea + 1% PPDA 0.5 1.3 3.9
> 14 Urea 5.4 11.5 15.2
Urea + 1% PPDA 0.6 3.4 7.8
"From Kiimpfe ef al. (1982c).
,with or without 1% PPDA on urea-N basis) was applied on soil surfaces annually twice
at the following rates: 50 + 40 kg ofurea-N with 15N label for two soils (loamy sand and
sandy loam) or 50 + 50 kg (in 1976) and 100 + 100 kg of unlabeled urea-N (in 1977-
1979) for the third soil (sand). After each fertilizer application, volatilization of NH3
was determined during 14 and 21 days. The experiments were carried out under natural
weather conditions or under conditions of simulated drought after fertilization.
According to the results obtained, the inhibitory effect of PPDA was more evident
during the first 14 days than between days 14 and 21. After the first fertilization, PPDA
had a greater effect than after the second one. The conclusion was drawn that, under
natural weather conditions, mean NH3 losses from urea were 30%, whereas losses from
urea-PPDA were not lower than 20%. Under conditions of simulated drought, urea-
PPDA was more effective than urea alone: in the presence of PPDA, more urea
remained unhydrolyzed, less NH4+ was produced, and less NH3 was lost by
volatilization.
The effect of PPDA on urea hydrolysis in a grey-brown podzolic soil from the
former Czechoslovakia was studied by List'anska (1982). Dry soil samples (350 g) were
treated with urea or with the liquid fertilizer DAM 390 (urea-ammonium nitrate) at a
rate of 300 mg N/kg soil. No PPDA or 1% PPDA (on N basis) was added to the
fertilizers. The samples were moistened (to 15-17% moisture content) and incubated at
28°C. During incubation (13 weeks) the NH/ and N03' contents were determined. The
analytical data indicated that PPDA inhibited hydrolysis of urea from both fertilizers for
2 weeks and did not retard nitrification. Similar results were published in another paper,
also (List'anska, 1984).
In a pot experiment, Byrnes et al. (1983) measured volatilization of ammonia from
flooded samples of an Alabama silt loam soil (oven-dry weight of each sample: 1,620
g). The floodwater of samples received a urea solution, containing 0.4193 g of 4.814
atom% excess urea- 15N, (equivalent approximately to 50 kg N/ha on area basis) and a
PPDA solutions to provide 1, 2 or 5% PPDA relative to weight of urea. The floodwater
depth was then adjusted to 2 cm and maintained at this level throughout the experiment.
The soil in pots was incubated at 35°C by day and at 25°C by night (12-hour days)
during 14 days. Daily analyses were performed for volatilized NH3, residual urea, NH/
and pH in the floodwater. At the end of the experiment, total N contents in floodwater
and soil were determined. The isotope ratio in total N contents and in volatilized NH3
was also determined.
Figure 41 shows that in the absence of PPDA cumulative NH3 volatilization losses
amounted to 31.4% of the added urea- 15N, while losses from the PPDA treatments were
116
3.9-5.3%. Effects of the three PPDA concentrations were not significantly different (at
p=0.05).
35r----------------------------,
- Nt, PPDA
·..····1% PPDA
-·,·,2% PPDA
._- 5% PPDA
o 2 4 6 B 10 12 14
Incubatioll time (days)
Figure 41. Cumulative ammonia volatilization losses ofurea- 15 N with time. !From Byrnes e/ al. (1983), by
pennission of the Soil Science Society of America, Inc.!
It was also found that NH.l losses from the treatment without PPDA occurred
concurrently with the rise in NH4 + concentration and pH of the floodwater during the
first few days. The final 15 N balance showed total recoveries of 96.7-99.2%. There was
no significant difference (p=0.05) between total recoveries in the absence or presence of
PPDA. Reduction of NH3 volatilization under the influence of PPDA was accompanied
by an increased recovery ofN in floodwater (algae) and in soil.
In another experiment with the same silt loam soil used in the first experiment, the
effect exerted by different amounts of PPDA in urea pellets on hydrolysis of urea was
studied. Soil samples (l00 g on oven-dry basis) placed in cylinders formed
approximately 9-cm high soil columns covered by l-cm deep floodwater. Each column
then received a single 15-mg urea pellet containing 0, 0.1, 0.25, 0.5, 1, 2, and 5% of
PPDA (relative to weight of urea). Afterwards, the soil columns were incubated in the
dark at 300C for 16 hours. Following incubation, the soil-floodwater systems were
extracted and the extracts analyzed for urea and NH4 +. It was found that PPDA at
concentrations 2: 1% in pellets completely inhibited hydrolysis of urea. The inhibition
was about 92% at 0.5% concentration of PPDA in the pellet (which corresponds to a
concentration of approximately 4 ppm of PPDA dissolved in the floodwater). At 0.25
and 0.1 % PPDA concentration in the pellets, degrees of inhibition were about 80 and
50%, respectively.
In a short report, O'Connor et al. (1983) quote a laboratory experiment carried out
for studying the influence of soil temperature (m the effectiveness of PPDA. Soil
samples treated with 1 mg of urea-N and 20 Ilg of PPDAIg soil were incubated at 5-
35°C for various time periods. PPDA prevented urea hydrolysis and associated
ammonia losses for 1,2,4,8, and 17 days at temperatures of 35,25, IS, 10, and 5°C,
117
respectively. Since surface soil temperatures during the growing season often exceed
30°C, the agronomic utility ofPPDA is very much reduced.
In another experiment, dried maize (Zea mays) leaves were moistened with a urea
solution containing 0 or 2% PPDA (relative to urea-N) and incubated at 25°C.
Ammonia losses were reduced from 82% (no PPDA) to 20% (PPDA). The same
treatments applied to bare soil resulted in cumulative NH3 losses of 26 and 12%,
respectively. These results indicate a greater potential for NH3 losses from no- or
reduced tillage systems and the role of urease inhibitors in keeping these losses to a
minimum.
Another short communication, by Ornholt and Hendrickson (1983), refers to field
experiments conducted to evaluate the ability of PPDA to inhibit urea hydrolysis and
reduce ammonia volatilization losses. A 50% urea solution (95 kg Nlha) with or without
2.2 kg of PPDAIha was applied in mid-August to bare plots and to plots amended with
oat straw. In both systems, PPDA extended the persistence of urea by only 2-3 days.
Nonetheless, PPDA reduced NH3 losses from 33 to 23% of the added urea-N on bare
plots and from 42 to 6% on straw-amended plots.
Martens and Bremner (1984a) devoted a complex study to factors influencing
effectiveness of PPDA to retard urea hydrolysis in 15 Iowa soils selected to obtain a
wide range in pH (4.6-8.0), texture (5-57% sand, 11-55% clay), organic carbon content
(0.30-6.73%), urease activity 04.2-84.9 /lg of urea hydrolyzed/hour/g soil at 37°C), and
other soil properties. The reaction mixtures, consisting of 5 g of air-dried soil + 2 ml of
aqueous solution containing 10 rug of urea without or with 1-125 (usually, 25) /lg of
PPDA, were incubated at 20°C for 1-21 days, then analyzed for residual urea.
The inhibition caused by PPDA (5 /lg!g soil) in urea hydrolysis in the 15 soils
studied, ranged, after a 7-day incubation, from 37 to 93%. The percent inhibition
correlated Significantly with organic C content (r=-O.68), total N content (r=-0,74),
cation-exchange capacity (r=-0.65), sand content (r=0.66), clay content (r=-O.64), and
surface area (r=-0.60), and insignificantly with urease activity (r=-0.29), silt content (r=-
0.46), pH (r=0.29), and CaC0 3 equivalent (r=O.03). One can see that most correlations
are negative. Multiple-regression analyses indicated that the effectiveness of PPDA to
retard urea hydrolysis in soils tends to increase with a decrease in soil organic matter
content.
Table 36 shows that the average percent inhibition increased markedly with PPDA
concentration and decreased markedly when incubation time was prolonged from 2 to
10 days.
The influence of incubation temperature on effectiveness ofPPDA (5 /lg!g soil) was
studied with six soils. The following average values of percent inhibitions were
registered at 10, 20, 30, and 40°C: 91, 90, 59, and 35%, respectively, after 3 days of
incubation, and 85, 76, 28, and I %, respectively, after 7 days. Thus, effectiveness of
PPDA decreased proportionately with an increase in temperature and this decrease
became more pronounced with prolongation of incubation time.
To study the effectiveness of PPDA, Broadbent et al. (1985) conducted a field
experiment on a silty loam soil from California. The experiment included two variants:
plots not covered and plots uniformly covered with chopped wheat straw (at a rate of
4.93 t/ha). Maize (Zea mays) was sown in all plots at a population density of 86,000
plantslha. As soon as the maize seedlings emerged, urea labeled with an excess of 15N
118
TABLE 36. Effect of different amounts of PPDA on urea hydrolysis in 15 soils after
two incubation times·
Amount ofPPDA Incubation time
Average value of inhibitions (%)
(p.gIg soil) (days)
0.2 2 60
10 18
0.5 2 68
10 24
2 76
10 30
3 2 84
10 36
5 2 91
10 45
10 2 93
10 58
25 2 96
10 71
•Adapted from Martens and Bremner (1984a), by permission of Pergamon Press PLC.
160
A
120
80
i 40
UreA alone
I!.
OUreA+PPDA
Z 0 -..J._-'----'-_-'---'_-'----'"----'-
<1:1'60
5
120
eo
40
o 2 4 6
Days
Figure 42. Labeled urea-N in soil samples taken fiom fertilizer band during the first week after fertilizer
application.
A - Bare soil. B - Soil + straw. Bars show ± standard deviation. IFrom Broadbent et al. (1985), by
permission of the Soil Science Society of America, Inc.!
Simpson et al. (1985) also studied the effectiveness ofPPDA under field conditions,
on an Australian clay soil (PH 8.2) cropped with flooded rice. At the stage at which the
rice plants reached 20 cm above the -11 cm deep floodwater, microplots were installed
within the rice field. They were enclosed by metal frames (54 x 54 em) inserted to
contain three rice rows and buried in the soil to a 15-cm depth so that 5 cm projected
above the floodwater surface. Prilled urea (80 kg NIha) with or without PPDA (1 %
relative to weight of urea) was administered in form of solution in the floodwater of
microplots. Some microplots received a solution of urea labeled with 15N at 1.48 atom%
excess (at a rate of 80 kg of N/ha) with or without 1% PPDA. On days 0 to 11 after
fertilization, residual urea, NIL +, and NO)' contents, pH and temperature in floodwater
as well as the volatilized ammonia and wind speed were determined. Total N and 1~ in
floodwater, soil, and plants were analyzed three times: on days 11,47 (early heading),
and 130 (maturity).
Figure 43 shows that in the absence of PPDA, urea concentration in floodwater
decreased continuously and at day 8 after fertilization it became negligible. PPDA
completely inhibited hydrolysis of urea for about 4 days (urea concentration having
been maintained at the initial value of about 80 g of N/m3 ), after which the inhibitory
effect of PPDA decreased steadily until day 11 when no urea was detectable in the
floodwater. In concordance with these results PPDA delayed the appearance and
120
,. B
"
Z l , I • 7 • • 10 '1 U
DRy"l after fertilizer application
Figure 43. Effect ofPPDA on concentration of urea CA) and ammoniacal (E) nitrogen in floodwater. (Adapted
from Simpson e/ al. (1985). by permission of the Soil Science Society of America. Inc.!
Concentration ofN0 3" in floodwater was low during the entire period of observation
and was not influenced by PPDA. In urea+PPDA treatment, pH increased slightly on
days 3 to 8, probably due to a more intense development of algae. In the urea-only
treatment, more ammonia volatilized during the first 3 days (when NH4 + concentration
in floodwater was higher) and smaller amounts of NH3 were lost on the next days.
Contrarily, in the urea+PPDA treatment, volatilization of NH3 was reduced for 6 days
and then increased. The cumulative NH3 volatilization losses during 11 days in the urea
and urea+PPDA treatments were 20.6 and 12.5% of the applied urea-N, respectively.
The greater NH3 losses in the urea-only treatment were due partly to wind speed which
was higher during the first 3 days than during the last 3 days.
Analyses of total N and 15N in floodwater, soil, and plants on day 11 after
fertilization showed that total loss of N from the applied urea-N was 40.2% in the urea-
only treatment (i.e.. N recovery was about 60%) and 18.5% in the urea+PPDA treatment
(i. e.. N recovery was over 80%). As under the conditions of these experiments no N was
lost by leaching, run-off or in form of N20, it was deduced that N was also lost as N2
(i.e .. through denitrification of nitrates resulted from oxidation of NH/ released from
urea). The loss in form of N2 represents the difference: 40.2-20.6=19.6% in the urea-
121
only treatment and 18.5-12.5=6.0% in the urea+PPDA treatment. It was concluded that
the major inhibiting effect of PPDA on soil urease activity was to reduce those N losses
which are conditioned by release of urea-N in form of NH4 + followed by oxidation of
NH4 + to N0 3- and then by denitrification ofN03- up to N 2.
In a field experiment on a Quebec silty clay loam soil, Tomar et al. (1985) studied
the effect of PPDA and chopped timothy (Phleum pratense) straw on urea hydrolysis
and ammonia volatilization from urea. Bare plots and plots on the surface of which
timothy straw (4.6 t/ha) had been spread, were fertilized with a urea solution (75 kg
N/ha) without or with PPDA (0.25,0.5, 1,2 or 3.74 kg/ha). All plots were also fertilized
with triple superphosphate and potassium chloride at rates of 63 kg of P and 100 kg of
Kiha, respectively. Residual urea, NH4 +, and (N03- + N0 2-) were extracted with 1 M
KCl solution and measured at days 0, 1,2,3,4, 8, and 14 after fertilization. Amounts of
NH3 volatilized during 1, 2, 3, 4, 14, and 17 days following fertilization were also
assessed,
PPDA at rates :s 1 kg/ha had no significant effect on urea hydrolysis. However, urea
hydrolysis was significantly retarded at 2 and 3.74 kg PPDAIha rates on days 4 and 8. A
reduction in urea hydrolysis with increased PPDA concentration was also observed
when the data were pooled across straw treatments. The inhibitory effect of PPDA was
not long-lasting: almost all the added urea had disappeared by day 14. On days 2 to 14
after fertilization, more urea was hydrolyzed in straw-amended soil than in bare soil, but
this influence of straw was significant at day 4 only.
Ammonia volatilization was negligible during the first 4 days in all treatments and
attained higher values after 14 days. Between days 15 and 17, volatilization of NH3
declined. PPDA reduced volatilization of NH3, especially at its highest rate, The straw
enhanced ammonia losses in treatment with PPDA at rates:S 1 kg/ha had no affect at
higher PPDA rates. In this experiment, volatilization NH3 losses within 17 days never
exceeded 1% of the applied urea-No
Nitrogen recoveries as residual urea-N + NH/-N + (N03' + N02')-N measured on
days 1, 2, 3, 4, 8, and 14 after fertilization gave the following values across all
treatments: 92, 89, 82, 68, 62, and 48% of the applied urea-N, respectively. This proves
that a part of urea-N was progressively incorporated into N compounds nonextractable
with 1 M KCI and/or lost mostly through nitrification-denitrification, not through NH3
volatilization.
The experiments performed by Mai and Fiedler (1986) intended to study the
influence of weather conditions on the effectiveness of PPDA, In a German spruce
forest estimated to be about 90-year-old on an acid loamy soil (PH 4.3), urea hydrolysis
was compared in plots fertilized (in 1979 and 1980) with surface-applied urea (150 kg
N/ha/year) with or without 1% PPDA. In 1979, the fertilization was followed by a
drought period, whereas in 1980 the soil was very moist at time of fertilization. In 1979,
complete hydrolysis of urea needed 17 days in urea-only treatment and 23 days when
urea and PPDA were applied. In 1980, the corresponding durations were 1 and 8 days,
respectively.
The influence of meteorological conditions on the effectiveness of PPDA was also
evidenced in the field experiments described by Fillery and De Datta (1986) and Fillery
et al. (1986a), The experimental field, on a silty clay loam (PH 5.8) in the Philippines,
was cropped with flooded rice. Urea (58 kg N/ha) alone or with PPDA (1% relative to
weight of urea) was applied to floodwater on day 18 after the transplanting of (20-day-
122
old) rice seedlings. (Triple superphosphate, 13 kg P/ha and KCl, 25 kg Klha were
incorporated into soil immediately before transplanting.)
Determinations of N~ + in floodwater and of volatile NH3 showed that PPDA
retarded accumulation of ~ + and volatilization of NH 3. Thus, NH3 volatilization
losses from urea and urea-PPDA over 8 days were 36 and 22% of the applied N,
respectively, i.e.. PPDA reduced NH3 losses by about 40%. This reduction, however,
was not attributable only to the inhibitory effect of PPDA, but also to the decline in
wind speed over the period (days 6 and 7) in which the maximum concentrations of
NH4 + were detected in the floodwater amended with urea-PPDA. Had the wind speed
increased, larger amounts ofNH3 could have volatilized and in this case PPDA may not
have reduced NH3 losses as effectively.
The investigations by Fillery et at. (I 986b ) draw attention to the importance of
timing of urea-PPDA application for inhibition of urea hydrolysis and reduction of
NH/ concentration in floodwater of rice, i.e.. for diminution of N losses caused by
volatilization of NH3. The experimental plots were installed on two soils in the
Philippines: a clay (PH 6.7) at Los Banos and a silty clay loam (PH 5.8) at Munoz. The
soils were fertilized with triple superphosphate (13 kg P/ha) and KCl (17 kg Klha)
immediately before transplanting of rice seedlings. At Los Banos, prilled urea was
administered in the dry season (90 kg N/ha) and in the wet season (60 kg N/ha) in 1982
and in the dry season (87 kg N/ha) in 1983. In this year, rnicroplots fertilized with 15 N_
labeled urea (= 5 atom% excess) were also installed. At Munoz, urea fertilization (87 kg
N/ha) was carried out in the dry season of 1983 and rnicroplots fertilized with urea- 15 N
were also installed.
Two-thirds of the urea with or without 2 or 5% PPDA (relative to weight of urea) in
1982 and with or without 1% PPDA in 1983 were incorporated into the soil or added to
floodwater immediately before or after transplanting of the seedlings or between days
18 and 26 after transplanting. The remaining third of urea and urea-PPDA was applied
later (50 days after transplanting or 5-7 days before panicle initiation). Depth of
floodwater was 4-6 cm. Daily, at the same hour, floodwater was sampled for analysis of
residual urea and NH4 +.
In both soils, PPDA inhibited urea hydrolysis and reduced accumulation of NH4 + in
floodwater only for 1 day, when it was administered together with urea immediately
before or after transplanting. The inhibition lasted 3 days, when urea-PPDA was added
between days 18 and 26 after transplanting.
In a laboratory experiment, Rao and Chai (1986b) used an alkali sandy loam soil
(pH 9.0) from India. Air-dried soil samples (50 g) were moistened with 10 ml of water
and allowed to equilibrate overnight, and then were treated with 10 ml of water or
PPDA solution,S m1 of urea solution and, finally, with 25 m1 of water to form an
aqueous surface layer. Urea was added at rates of 150, 100, 75, 50, and 37.5 ppm N,
whereas the amount of PPDA was 5% relative to weight of urea. During incubation
(35°C/20 days), the floodwater level was maintained and periodically analyzed for ~ +
and (N03- and NOz-). One can see from Figure 44 that, under the influence of PPDA,
NH4 + appeared in floodwater with a delay of 1 day (at maximum urea-N rate), of 3 days
(at 100 and 75 ppm urea-N) or of 4 days (at 50 and 37.5 ppm urea-N). Moreover, the
peak NH4 + concentration had significantly lower values in the presence than in the
absence of PPDA.
123
40.-------------------__.-__________________--.
i
,eo 30
-rrt'.1l
--. Urea+PPDA
!:l A
OJ
~
"" 20
~
.S
g
01)
.~
]
J
'S
10
4 6
Figure 44. Effect of PPDA on ammoniacal nitrogen concentration in floodwater of an alkali soil, as
influenced by incubation time and urea-N application rates (ppm): A - 150; B - 100; C - 75; D - 50;
E - 37.5. IF rom Rao and Chai (198Gb), by permission of Kluwer Academic Publishers.!
The same soil was also used to study the effect of PPDA on volatilization of
ammonia from urea. Soil samples (20 kg) placed in pots were saturated with water, then
urea (7S, SO or 37.S ppm N) or urea + PPDA (S% relative to weight of urea) were mixed
into a few centimeters of the top soil layer. Finally, the soil was flooded to 3.S cm
which was maintained throughout the 24-day incubation in greenhouse. Each day,
amounts of NH3 volatilized and of Nf4 + in floodwater were measured. The results
indicated that in urea-only treatments volatilization of NH3 began on the first day after
fertilization and continued until the 12th day, with similar trends at the three urea-N
rates. In the presence of PPDA, volatilization ofNH3 started after S days and percentage
inhibitions of NH3 volatilization from the three urea-N amounts were not significantly
different. On an average, 4S% of the applied urea-N was lost in the absence of PPDA,
but only 8.S% in its presence, the difference between the two values being very
significant.
Reviewing the investigations on N fertilizers for rice, Youngdahl et of. (1986)
present data obtained in pot experiments with rice grown under submerged conditions.
Urea without or with 1% PPDA was administered as basal fertilizer on day 21,30 or 42
after transplanting of rice seedlings. PPDA always reduced ammonia losses, regardless
of the timing of application, although the relative effectiveness varied. Thus, minimum
effectiveness was recorded when urea was applied as basal fertilizer (NH3 losses in urea
and urea-PPDA treatments were about 41 and 30% of the applied urea-N, respectively).
The maximum effectiveness of fertilization was on day 30 after transplanting (NH3
losses in urea and urea-PPDA treatments were about 28 and 10%, respectively).
A laboratory experiment performed for studying the influence of temperature on
effectiveness ofPPDA was quoted in a short communication by O'Connor et af. (1983)
(see page 116) and described in detail by O'Connor and Hendrickson (1987) who
pointed out that 20-g samples of silt loam soil (pH 7.S) from New York were used and
reiterated the conclusion that effectiveness of PPDA (20 l1g/g soil) in inhibition of
124
hydrolysis of urea (l mg N/g soil) and of ammonia volatilization from urea decreased
with increase in temperature from S to 3SoC.
Using the same soil, O'Connor and Hendrickson (1987) also performed other
laboratory experiments (A, B, C), through which the effects of urea and PPDA rates and
soil depth (of the height of soil column) on ammonia volatilization were studied. The
urea solution and then the water with or without PPDA were applied to the surface of
completely moistened soil samples. Then the mixtures were incubated at 2S C for 14-IS
D
days, during which, at 1-3-day intervals, the amount of volatilized NH3 was assessed.
In experiment A, the variable factor was the urea rate (S, 10, and 20 mg N120 g soil),
while soil depth (height of soil column) of 8 mm, corresponding to 20 g of air-dried
soil and PPDA rate (0.2 mg/20 g soil) were constant. Cumulative NH3 losses from three
urea-N amounts during incubation (14 days) were IS, 22, and 38%, respectively, in the
absence of PPDA and 7, 18, and 38%, respectively, in its presence. This means that
PPDA was most effective at the lowest urea-N rate and became ineffective at the
highest rate ofurea-N. It should be added that during the fust days of incubation PPDA
reduced volatilization ofNH3 even from the highest urea amount.
In experiment B, soil depth (height of soil column) was the variable factor (8, 16,
and 32 mm, corresponding to 20, 40, and 80 g of soil). Rate of urea (20 mg N) and that
ofPPDA (0.2 mg) were the same, irrespective of the soil weight, i.e. these rates relative
to I g of soil were I, O.S, and O.2S mg of urea-N, respectively, and 10, S, and 2.S Ilg of
PPDA, respectively. Ammonia losses decreased with an increase of soil depth. PPDA
delayed the onset of NH3 volatilization in each treatment, but reduced the cumulative
NH3 losses during incubation (IS days) only when soil depth was 16 or 32 mm.
Effectiveness of PPDA was more marked with the 32-mm deep soil sample than with
the 16-mm deep sample.
Experiment C, like experiment B, included three soil depths (8, 16, and 32 mm),
corresponding to 20, 40, and 80 g of soil, but the urea rates were 20, 40, and 80 mg of
N, respectively, and the PPDA rates were 0.2, 0.4, and 0.8 mg, respectively, i.e., the
rates relative to I g of soil were constant (1 mg of urea-N and 10 Ilg of PPDA,
respectively). The results obtained are similar to those registered in experiment B. Thus,
in the case of the 32-mm soil depth, PPDA reduced cumulative NH3 losses during IS
days from 20 to 12% of the applied urea-No
Extrapolating the results of experiments Band C to field conditions, one can assume
that conditions that promote diffusion of the surface-applied urea and PPDA more
deeply into the soil will reduce the potential for NH3 losses.
In a laboratory experiment, Hendrickson et al. (1987) studied the influence of plant
residue and PPDA on the volatilization of NH3 from urea-treated soil samples. The
same silt loam soil (pH 7.S) from New York was used as in the experiments described
above. There were three experimental variants: 20 g of air-dried soil; 0.3 g of coarsely-
ground, dried maize (Zea mays) stover; and 0.3 g of stover intimately mixed with 20 g
of soil. Each variant was treated with 1 ml of solution containing 20 mg of N as urea
and S ml of water or S ml of solution containing 0.4 mg of PPDA, and sufficient
additional water to adjust the final water potential to approximately 30 kPa. Incubation
took place at 2S C. The ammonia evolved was periodically assessed during 17 days of
D
The analytical data presented in Figure 45 prove that PPDA effectively inhibited
urea hydrolysis. Complete hydrolysis of urea occurred in 14 days in both urea-only
treatments and in 28 days in the soil treated with urea + PPDA. When urea was placed
deeply into the soil, concentration of urea in floodwater was highest not on the first, but
on the 3rd day, which shows that the upward movement of urea from the site of
placement to floodwater was relatively slow because urea is weakly adsorbed by soil
colloids.
600
0\3 7 14 21 28
I)(lV~ <tiler urea anplu.:ahon
Figure 45. Urea-N concentration in floodwater after application of urea and urea-PPDA. a -
Unfertilized control. b - Deep placement of urea. c - Surface-applied urea. d - Surface-
applied urea +2% PPDA.lFrom Phongpan et al. (1988).1
Concentration ofNa + in floodwater during the first 14 days presented the following
order of the treatments:
surface-applied urea + PPDA < deeply placed urea < surface-applied urea alone.
In all treatments, NH4 + concentration in floodwater decreased after 14 days and by
days 21 to 28 the values were approximately the same. pH and total alkalinity showed a
similar pattern. Volatilization of ammonia started on day 3 after urea application and
increased in the next period. However, the cumulative NH3 losses were small in all
treatments even after 28 days: 2.9% of the applied urea-N (surface-applied urea alone),
1.3% (deeply placed urea), and 1.2% (surface-applied urea + PPDA).
In this experiment, the eventual N losses through nitrification-denitrification were
not studied.
Based on the finding that in unsaturated soil columns, an increase of bulk density by
compacting led to a marked increase in hydrolysis of surface-applied urea (Savant et af..
1987a), Savant et al. (1988a) initiated studies concerning the influence of soil bulk
density on the effectiveness of PPDA as a soil urease inhibitor. The experiments were
carried out with clay and loam soils from the U.S.A.
In the first experiment, samples of four unsaturated soils (0.16 g water/g soil),
equivalent to 100 g of oven-dried soil, were placed into cylinders (internal diameter: 4
em; height: 6-12 cm). The soil columns were then hand-compacted to bulk densities that
ranged from 0.69 to 1.70 g/em3 • Three preweighed urea granules (about 20 mg of
128
urea/granule) not containing or containing 1% PPDA were placed on the surface of the
columns. The column systems were covered with Parafilm to prevent evaporation of
water. After incubation at 30°C, the residual urea was extracted and determined. The
results presented in Table 37 were obtained after 2-day incubation of the clay soil and
after 3-day incubation of the three other soils. It is evident from the table that inhibition
of urease activity by PPDA Significantly decreased with the increase in soil bulk
density; this effect was more apparent in the clay and clay loam soils than in the loam
and silt loam soils.
TABLE 37. Effect of soil bulk density on urease inhibition after surface application of urea
granules containing 1% PPDA in four unsaturated soils"
Average bulk density Urease inhibition (%)
(g/cmJ) Clay Loam Silt loam Clay loam
0.71(0.69-0.72) 48a 74a 78a 56 a
1.I1(O.95-1.2I) 23b 44b 35b 19b
1.47 (1.36-1.70) 19 c 36 b 38 b 4c
"From Savant et al. (1988a), by courtesy of Marcel Dekker, Inc.
Values followed by different letters in the same colwnn are statistically different (p = ()'05).
2.20.4.1. Stability o/PPDA in Solutions (Including Urea Melt) and in Solid State
Jasche et al. (1978) emphasized that to obtain urea-PPDA, addition of PPDA to urea
melt before the prilling process is not possible, because at the temperature of molten
urea PPDA undergoes a decomposition to form polyphosphates and other products
noninhibiting to urease activity. Therefore, for preparing fertilizer compositions
consisting of urea and PPDA, coating of urea prills with powdered PPDA was
recommended.
Martens and Bremner (l983a) studied the stability of PPDA in solutions. For
determination of PPDA a colorimetric method was elaborated. Water and isopropanol
were used as solvents. The solutions that contained 50 !!g PPDA/ml were refrigerated
(4-6°C) for 7 days and analyzed periodically. It was established that PPDA was stable in
isopropanol, but in aqueous solution only 43 !!g PPDA/ml could be evidenced after 7
days. Diminution of the initial concentration was attributed to partial hydrolysis of
PPDA.
131
~O.-----------------------------,
iZ 25
20 ~. ....
:....
.
....
·····0
"5
a? 15 :' l.rell-PPDA
o 3 T \4 2.\
Incubation time (days)
60r-----------------------______ ~
B
=
.9
1< 20 .··0
;l;;I
~ •. 0·· . lJrea-PPIlA
j ~
0 ::::::::..... ..:::::;:::" •• ~::.::: •• -•.....•••~~?~....•
o 3 7 \4 2\
Incubation time (day.)
Figure. 46. Apparent immobilization of nitrogen applied to unamended soil (A) and straw·
amended soil (B). IAdapted from Hendrickson e/ al. (l987), by permission of the Soil
Science Society of America, Inc.!
TABLE 38. Effect of • to ring an aqueous solution ofPPDA on its effectiveness to inhibit
urea hydrolysis in soil"
Storage Inhibition of urea hydrolysis (%)
Soil temperature Time of storage (days)
(0C) 0 7 14 21 28
Silty clay loam 20 90 88 85 81
95
30 89 84 80 75
Loam 20 92 89 86 82
97
30 91 85 82 79
"From Martens and Bremner (1984a), by permission ofPergarnon Press PLC.
132
increase in storage temperature from 20 to 30o e, which proves that PPDA underwent a
partial decomposition during the storage.
Working with phosphate buffer solutions (pH 2-12) at 25, 35, and 45°e and
developing a method based on high-performance liquid chromatography for
determination of PPDA and of its organic hydrolytic products, and using other
analytical methods, too, Austin et al. (1984) elucidated the mechanism of acidic and
basic hydrolyses of PPDA. In strongly acid solutions, hydrolytic products of PPDA are
phenylphosphoroamidate (PPA) and ammonia, whereas in strongly basic solutions
hydrolysis of PPDA results in phenol and phosphorodiamidic acid. At intermediary pH
values, both hydrolyses occur, i.e .. both PPA and phenol are formed; at pH 6, phenol
predominates over PP A (Figure 47).
+ NH3
~
Phenylphosphorodiamidate
(PPDA) o-OH
Phenol Phosphorodiamidic acid
0-
o
pH 2 II . . . . . . OH
O-P + NH3
+ H20 -
"'-OH
Phenylphosphoroamidate Phenylphosphoric acid
(PPA)
Figure 47. Mechanism of acid and basic hydrolyses ofPPDA and of acid hydrolysis ofPPA. IFrom Austin et
ai. (1984), by permission of the American Chemical Society.!
decomposition products were identical to those of the basic hydrolysis of PPDA, i.e ..
phenol and phosphorodiamidic acid. Weight loss of volatiles from a mixture of urea
melt (135°C) + 0.5, 2 or 4% PPDA was greater than from urea melted at the same
temperature. Thus, approximately 1% of the weight of mixture was lost in 20 minutes.
The loss of PPDA increased when the time of contact between urea melt and PPDA was
prolonged from 20 to 120 minutes, but remained under 10%. The loss was due to
volatilization of phenol and ammonia.
Since PPDA dissolves faster in the urea melt (140°C) than in concentrated urea
solutions and retention time for the complete urea granulation process is less than 1
minute, it was concluded was drawn that cogranulation of urea and PPDA leads to the
smallest losses when PPDA (0.5-4%) is added to the urea melt (l40°C) before
cogranulation. However, with this technology, the volatilized phenol, depending on its
concentration in the off-gas, could be an environmental problem as a source of air
pollution in the immediate area of the granulation plant.
Preparation of urea-0.5% PPDA granules is a 24% more expensive than the cost of
urea alone.
Gautney et at. (1985) evidenced the decomposition, at 25°C, of PPDA in seven fluid
fertilizers containing urea and other compounds (sources of N, P, K, and S) and being of
the following grades: 31-0-0, 36-0-0, 29-0-0-5S, 14-14-14,24-8-0,18-9-9, and 21-7-7.
In the first three fluid fertilizers, having a pH ranging from 7.08 to 7.65, PPDA
decomposed slowly, but its decomposition in the other fertilizers (pH 5.12-5.97) was
rapid. The mechanism of decomposition was basic or acidic hydrolysis as shown in
Figure 47. To minimize decomposition losses, PPDA should be added to the first three
fluid fertilizers not sooner than 1-2 days before their application on field. However,
some loss of PPDA would still occur, and it would be best to minimize the time
between PPDA addition and fertilizer application. The other four fertilizers should be
applied immediately after PPDA addition (see also Anonymous, 1983).
Gautney et al. (1983, 1984) mentioned tests which showed that dry solid PPDA and
urea-PPDA mixtures decompose very slowly to phenol at room temperature, indicating
that PPDA decomposition and evolution of phenol may be a problem during bulk
storage ofPPDA and granular urea-PPDA mixtures.
These tests were described in detail by Gautney et al. (1986). The following
preparations were studied:
-pure PPDA;
- pure PPDA to which impurities usually present in technical-grade PPDA
were added, namely (C 6HsO)zP(O)NH2 (diphenylphosphoroamidate, DPPA) and
NH4 Cl, in the proportions: 900 g ofPPDA + 100 g of DPPA or NH4 Cl; 900 g ofPPDA
+ 50 g of DPPA + 50 g ofNH 4 Cl;
- urea-PPDA mixtures, containing pure urea and pure PPDA in the proportions:
960 g of urea + 40 g of PPDA; 860 g of urea + 40 g of PPDA + 100 g of DPPA or
NH4 Cl; 860 gofurea +40 g ofPPDA + 50 gofDPPA + 50 gofNH4 Cl.
Samples (0.25 g) were weighed from each preparation, then placed in a forced draft
oven and heated at different temperatures (50-100°C) for various time periods (up to
120 hours). The effect of relative humidity (0-75%) on the decomposition of PPDA
alone at 90°C during the different times (up to 220 hours) was also studied. Then, each
sample was analyzed for residual PPDA by high-performance liquid chromatography.
134
The analyses showed that, under the influence of heat treatment, PPDA in each
preparation decomposed with the formation of phenol. The decomposition rate was
much higher in the urea-PPDA mixtures than in pure PPDA, and always increased with
the increase of temperature. The impurities (DPPA and NRtCl) alone and in
combination accelerated decomposition of pure PPDA, but did not significantly affect
decomposition of PPDA in urea-PPDA mixtures. The decomposition rate of PPDA
increased very much with an increase in relative humidity.
The decomposition of pure solid PPDA obeys first-order reaction kinetics, whereas
decomposition of PPDA in urea-PPDA mixtures obeys zero-order kinetics. The
calculations gave a value of 254 years as reaction half-life at 25°C for pure solid PPDA
and 56% decomposition per year at 25°C for PPDA in urea-PPDA mixtures. These data
suggest that the time between preparation (manufacture) and testing (field application)
ofurea-PPDA mixtures should be minimized.
The decomposition of solid PPDA at high relative humidities, where solid PPDA
liquefies, occurs by acidic and/or basic hydrolysis (see Figure 47). The mechanism of
PPDA decomposition at low relative humidities was not clarified; it was assumed on the
basis of some analytical data that in this case decomposition of PPDA proceeds
primarily via a combination hydrolysis-condensation reaction with the formation of a
diphosphorus compound containing NRt + and phenoxy (C6 HsO) substituents.
Byrnes et af. (l989a) studied the decomposition of PPDA in buffered solutions
made of KH 2P04 , H3 B03 , and CH 3COOH at concentrations of 0.024, 0.075, 0.15, and
0.30 M. The pH was adjusted to 5.0 by the addition of KOH. pH 7.0 solutions were
prepared with K2 HP04 , H3 B03 , and CH3COOK. At pH 9.0, a carbonate buffer made
from K2 C03 was used instead of acetate. Recrystallized PPDA was added to each buffer
solution to produce a concentration of approximately 25 ppm PPDA. The solutions were
kept at room temperature (- 20°C) and analyzed for PPDA at hourly intervals.
The decomposition of PPDA followed first-order reaction kinetics, in accord with
the results of similar studies performed by Austin et af. (1984) (see page 132). The rate
of PPDA decomposition increased linearly with increasing buffer concentrations.
Increasing the salt concentration shortened the half-life of PPDA to as little as one-
eighth its half-life without a buffer. PPDA was also greatly affected by the nature of
buffering salts: at pH 5.0, the effect of phosphate and acetate was strong, while borate
had no effect; at pH 7.0, phosphate and borate had little effect and acetate had no effect;
at pH 9.0, borate had a greater effect than phosphate and carbonate had an intermediary
effect.
The finding that decomposition of PPDA depends not only on pH, but also on
concentration and nature of buffering salts presents importance for studies on PPDA
decomposition in floodwaters overlying soils in which the presence of carbonate and
NH4 + due to urea hydrolysis may enhance the decomposition ofPPDA.
Following the fate of PPDA added to samples of 25 Iowa soils at a rate of 100 !!g!g
soil, Martens and Bremner (l983b) determined PPDA and inorganic N during
incubation of soil + PPDA mixtures at 20 D C for 5 days, and established that PPDA is
sorbed and decomposed in soils. The capacity of soils for sorption of PPDA was
positively and significantly correlated with clay content and surface area, but was not
significantly correlated with pH and organic matter content.
Continuing these investigations with a variety of soils, Bremner and Martens (1987)
found that the rate of decomposition of PPDA was not significantly affected when the
soils were steam-sterilized or treated with antimicrobial agents before addition of
PPDA. All these findings indicate that PPDA is decomposed in soils by nonbiological
processes. The conclusion that these processes are catalyzed by clay minerals was
supported by experiments with kaolinite, illite, and montmorillonite.
Investigations carried out at the International Fertilizer Development Center (Muscle
Shoals, Alabama) also showed that biological factors do not play any major role in the
decomposition of PPDA, since the decomposition rate was not much different in heat-
sterilized and unsterilized soils (Anonymous, 1986).
Observing that PPDA (added to urea in 1% proportion) did not exert toxic effects on
spruce, Mai and Fiedler (1986) advanced the idea that this water-soluble compound
decomposes in soil through pH-dependent hydrolysis with release of a-phosphate.
Because of the rapid decomposition of PPDA in flooded rice soils, Fillery and Vlek
(1986) express the opinion that more effective inhibitors are needed to completely
eliminate the accumulation of NH4 + in floodwater and thereby drastically reduce the
potential for ammonia volatilization after the application of urea.
Youngdahl et af. (1986) also emphasize the need for continuation of research to
identify new compounds that have a longer lasting effect than PPDA. Also, compounds
that are lower in cost should be sought.
From the findings by O'Connor et af. (1983) and O'Connor and Hendrickson (1987),
according to which in the soil studied (silt loam, pH 7.5) PPDA (20 !!g!g soil) prevented
urea hydrolysis and volatile NH3 losses for 1, 2, 4, 8, and 17 days at 35, 25, 15, 10, and
5°C, respectively, one can deduce that decomposition of PPDA in soil at low
temperatures was so slow that this compound exerted a remarkable retarding effect on
urea hydrolysis. With an increasing temperature, the rate of PPDA decomposition
increased to a large extent and at 35°C the inhibitory effect on soil urease activity was
largely diminished.
Continuing these investigations, Hendrickson and O'Connor (1987) added, to 20-g
samples of the same soil used in the investigations mentioned above, 6 rnl of water or 6
ml of a solution containing 0.4 mg of PPDA. TIle next step was incubation (more
precisely, preincubation) of samples at 5, 10, 15, 25, and 35°C for 0-28 days, after
which 1 rnl of urea solution (20 mg N) was added to each sample. TIle reaction mixtures
prepared in such a way were incubated at 25°C for 3 days, then the residual urea was
determined to evaluate the inhibitory effect ofPPDA on soil urease activity.
Preincubation of soil samples at 5-25°C gave different re~mlts from those obtained
after preincubation at 35°C.
With preincubation at 5-25°C, the inhibitory effect of PPDA decreased to 45-60% in
samples preincubated for 7 days, remained at this level in samples preincubated for 14-
21 days, then decreased again in samples preincubated for 28 days, in which the
136
inhibition was minimal (about 2S%) when preincubation took place at 2SoC and
maximum (about SO%) at preincubation temperatures ofS-lOoC.
After preincubation at 3SoC, the inhibitory effect of PPDA decreased to about 40%
in samples preincubated for 7 days, then increased markedly in samples preincubated
for 14 and 21 days, attaining about 80% in samples preincubated for 28 days.
To explain these unexpected results it is assumed that at 3SoC, during the fIrst 7 days
of preincubation, part of PPDA decomposed at a sufficiently high rate and the
decomposition products caused the increase of the inhibitory effect following
prolongation of preincubation time to 14-28 days.
In order to verify this assumption, soil samples (20 g) were preincubated with the
two hydrolytic products of the basic hydrolysis of PPDA (phenol and
phosphorodiamidic acid) as well as with two dihydric phenols (catechol and
hydroquinone). Preincubation and then incubation with urea were carried out as with
PPDA, but there were some differences, namely phenol was added to the soil samples at
the rate of 0.22 mg; preincubation temperatures were 2S and 3SoC (phenol), 10 and
2SoC (phosphorodiamidic acid), and 2SoC (catechol and hydroquinone).
Figure 48 shows that at 3SoC PPDA and phenol, applied at comparable molar rates,
brought about inhibitions to the same degree, but the inhibition caused by PPDA
appeared with a delay of about 3 days as compared to the inhibition given by phenol,
which suggests that the phenol released from PPDA did determine the increase in the
inhibitory effect of PPDA after prolongation of preincubation time. At 2SoC, PPDA was
a more effective inhibitor than phenol, but effectiveness of both compounds weakened
with prolongation of preincubation time.
100r-----------=:-~=:_:
.~·····-··~!:'!~~~.3.;;l
80 .......-- PPDA
.~/ )1'" we
\ i / r - ........
\... I PPDA
/
/\ \. I I
,,;1.. . 'ri
20 .D ..
Figure 48. Inhibition of urease activity in soil samples preincubated with PPDA (20 J.lglg soil) and phenol (11
J.lglg soil) at 25 and 35°C./From Hendrickson and O'Connor (1987), by permission ofPergamm Press PLC}
phosphorodiamidic acid did not contribute to the increase in the inhibitory effect of
PPDA in samples preincubated for 14-28 days.
Inhibitory capacity of catechol and hydroquinone on urease activity manifested a
tendency to decrease in soil samples, in which these hydroxylated phenols were
preincubated at 25°C for 28 days.
In a field experiment on a silt loam soil (PH 5.7) from Indiana, Beyrouty et al.
(1988a) have applied urea prills or urea prills coated, by means of paraffin oil, with
PPDA (urea: 200 kg Nlha; PPDA: 4 kg/ha) on the surface of microplots not covered or
covered (in a proportion of approximately 60%) by maize (Zea mays) residue left from
the previous year's crop. The unhydrolyzed urea was determined periodically during 24
days after fertilization. The results showed that PPDA retarded urea hydrolysis for 19
days in each microplot. It was deduced from this finding that there was no deactivating
interaction between maize residue and PPDA, i.e.. stability of PPDA was not reduced
by the maize residue. The PPDA molecules diffused into the residue material may even
be protected from the deactivating action of higher temperatures. It should be added
that, under similar conditions, trichloroethylphosphorodiamidate was unable to retard
urea hydrolysis which suggests some type of deactivating interaction of this compound
with the plant residue.
Results of a laboratory experiment performed by Beyrouty et al. (l988b) point out
the importance of soil pH for stability of PPDA. Samples of an Indiana soil, collected
from plots submitted to long-term liming, had pH values of 5.6, 6.5, and 7.2, and their
texture was silty clay loam, silt loam, and silty clay loam, respectively. Reaction
mixtures were prepared from 20-g air-dry soil samples, 4.8 mJ of water or 4.8 ml of urea
solution (20 mg N) with or without 0.4 mg of PPDA, and incubated at laboratory
temperature for 14 days, during which time the evolved ammonia was assessed. At pH
5.6, cumulative NH3 losses from the applied urea-N represented 5.6% in urea-only
treatment and 0.3% in the urea-PPDA treatment. At pH 6.5 the corresponding values
were 25.0 and 15.2%, respectively. At pH 7.2 the cumulative NH3 losses were similar,
31.2 and 33.1 %, respectively. It is evident from these data that the inhibitory effect of
PPDA, which was nearly total at pH 5.6, decreased very much at pH 6.5 and
disappeared at pH 7.2. Reduction or disappearance of the effect of PPDA can be
explained by partial or total decomposition of PPDA at these pH values.
Savant et af. (l988a), who proved that inhibitory effectiveness of PPDA on urea
hydrolysis decreases with increasing bulk density (see page 127), also studied the
influence of soil bulk density on stability (and decomposition) of PPDA.
Columns of an unsaturated clay soil (0.16 g water/g soil), installed in cylinders were
compacted to an average bulk density of 0.70,0.99 or 1.56 g/cm3 • One ml of aqueous
solution containing 0.5 mg of PPDA was applied to the surface (12.6 cm 2) of the
columns. After having been covered by Parafilm for preventing evaporation of water,
the columns were preincubated at 30°C for 0, 7, 14, and 30 days, then 50 mg of
powdered urea was uniformJy placed on their surface. Subsequently they were again
incubated for 16 hours, extracted, and the unhydrolyzed urea was determined.
The results confirmed the finding that the inhibitory effect of PPDA on urea
hydrolysis decreased with increasing soil bulk density. In addition, they showed that the
inhibitory effect of PPDA decreased during the 30-day preincubation at each bulk
density. It was also established that the negative slopes of the three plots presenting
percent inl1ibition versus preincubation time for the three bulk densities were nearly the
138
same, which suggests that the rate of decomposition of PPDA was not influenced very
much by soil bulk density.
Dealing with the degradation of urease inhibitors in soils, Lee and Radel (1988) pay
special attention to the literature data on degradation of PPDA, and present a brief
characterization of the new and improved methods for trace analysis of urease inhibitors
and their degradation products in soils.
***
Decomposition in soil of the PPDA derivative 3-(1 ',1 '-dimethylethyl)-4-hydroxy-
PPDA was demonstrated during studies of its effect on nitrification (Swerdloff et al..
1985a; see page 239).
For the three compounds considered the most effective, the patent also describes the
testing of their inhibitory effect on urease activity in a New York soil (Cazenovia silt
loam, pH 7.0). The testing method was the same as that used by Kolc et af. (1985b) (see
page 80).
The three compounds tested (Figure 49) produced the following percent inhibitions:
54,5, and 21, respectively. Thus, the compound having the smallest molecule was the
most effective soil urease inhibitor.
The most preferred rates, at which these compounds are recommended for practical
applications, range from 1 to 500 ppm relative to soil weight, from 0.28 to 16.8 kg/ha,
from 0.01 to 10% relative to weight of urea in liquid or solid fertilizers.
2.22. POLYPHOSPHORODIAMIDES
>o-
o
o 0 H:!N U 0
H:!N II II/NH2 -"""P- HN
H:!N/
yH 3 II NH2
C-NH-p(
-...... p--HN-(CH:!)6-NH - P
H:!N/ 'NH 2 IiJC tH3 NH2
N,N'-Bis-(diaminophosphinyl)- N,N'-Bis-(diaminophosphinyl)-
1,6-diaminohexane 1,8-diamino- p-menthane
Figure 50, Structure of the diphosphorodiamide compounds tested by Swerdloff et al. (l985c) for
inhibition of soil urease activity.
140
The inhibitions caused by the six diphosphorodiamide compounds were 86, 84, 76,
90, 81, and 70%, respectively. It is evident that the most inhibitory compound was
phosphorodiamidic acid 1,4-phenylene ester.
The most preferred amounts in which these urease inhibitors are recommended to be
used range from I to 500 ppm of soil weight, from 0.56 to 16.8 kg/ha, and from 0.1 to
20% of weight of urea in liquid and solid fertilizers.
The tested compound (Figure 51) induced 84 and 53% inhibitions in the urease
activity of the Cazenovia and Plano soils, respectively.
For use in practice, the oxirnated O-diaminophosphinyl derivatives are
recommended in the following most preferred amounts: I to 500 ppm of soil weight,
0.28 to 16.8 kg/ha, 0.01 to 20% of weight of urea in liquid and solid fertilizers.
N-(Oiaminophosphinyl)benzenesulfonamide N-(Oiaminophosphinyl)-p-loluenesulfonamide
Figure 52. Structure of the two oxidized diaminophosphinyl sulfur derivatives tested by Swerdloff et al.
(l986b. 1987) for inhibition of soil urease activity.
In their patents, Van Der Puy et al. (1984a, 1985a) nominalized 74 diamidophosphoro-
thiolates [R-S-P(O)(NH2)2l and 71 diamidothiophosphorothiolates [R-S-P(S)(NH2hl as
inhibitors of soil urease activity and tested the urease-inhibiting capacity with five S-
alkyl and one S-cycloalkyl diamidophosphorothiolates and, in comparison, with two
alkyl phosphorodiamidates [R-O-P(O)(NH2)2l. Two soils (Cazenovia silt loam, pH 7.3;
Plano silt loam, pH 5.4) were used. The testing method was the same as that applied by
Kole et af. (l985b) (see page 80), but the amount of most test compounds was reduced
from 0.8 to 0.2 mg/20 g soil. The results are reproduced in Table 39.
Table 39 shows that each DAPT compound inhibited urease activity in both soils.
The S-butyl-DAPTs were more inhibitory than the S-hexyl-DAPTs. The inhibitory
effect of S-methyl-DAPT exceeded by nearly 2.8 times that of the methyl-PDA.
The most preferred amounts recommended for practical use of diamidophosphoro-
thiolate compounds range from about 10 to about 500 ppm of soil weight and from
about 0.0 I to about 20% of weight of urea in liquid and solid fertilizers.
2.26.1. The Patented Compounds and the First Studies on Their Inhibitory Effect on
Soil Urease Activity
N-(2-Chlorocthyl)-PTA 100 77
N-(3-Brol11opropyl)-PT A 99 83
N.N-Bis-(2-chloroethyl)-PT A 30
N-Phenyl-PTA 74 19
N-( 4-Nitrophcnyl)-PTA 81 30
N-Methyl-N-( 4-nitrophenyl)-PT A 95 59
N-Methyl-N-(4-hydroxyphcnyl)-PT A 86
N-Methyl-N-( 4-mcthoxyphcnyl)-PT A 47
"Adapted from Swcrdlolf el at. (1984, 1985d) and Van der Puy el at. (1985b).
TABLE 41. Effect of some N-acyl phosphoric triamide compounds on soil urease activity"
N-(DAP)-2-chloroacetamide CHzCI-CO-NH-P(O){NHzh 83
N-(DAP)-2,2-dichloroacetamide CHCb-CO-NH-P(O)(NHz)2 73
N-(DAP)-2,2,2-trichloroacetamide CCh-CO-NH-P(O)(NH2)2 64
N-(DAP)-2,2,2-trifluoroacetamide CF3-CO-NH-P(O) (NH212 52
"Adapted from Kolc et al. (l985a).
(DAP) - (Diaminophosphinyl).
The data of Table 41 indicate that the increase in number of the CI atoms in the
acetamide moiety of the tested compounds led to a decrease in their urease-inhibiting
capacity.
The first three compounds specified in Table 41 and two other N-acyl PTA
compounds were also tested with jackbean urease and it was found that at 10-Ji M
concentration they induced 56-100% inhibitions in the activity of this urease.
144
In another patent, Kole et al. (1985c) nominalized 14 PTA and 2 TPTA compounds
as urease inhibitors, but none of them was tested for evaluating their effect on soil
urease activity. The only compound tested with jackbean urease (Figure 53) proved to
be a potent inhibitor of this urease.
N-(Diaminophosphinyl)-4-(l'-maleimido)benzamide
Figure. 53. Structure of the compound tested as a urease inhibitor by Kolc et al. (l985c).
The PTA and TPTA urease inhibitors patented by Swerdloff et al. (1984, 1985a,d),
Kole et af. (1984, 1985a,c,d), and Van Der Puy et al. (1985b) are recommended for use
in practice in the most preferred amounts ranging from about 1 to about 500 ppm of soil
weight, from 0.28 to 16.8 kglha, from 0.01 to 20% of weight of urea in liquid and solid
fertilizers.
Omilinsky et af. (1997) invented improved solvent systems which enable the
preparation of stable concentrated solutions of N-alkyl thiophosphoric triamide urease
inhibitors (including nBTPTA) for their storage, transportation, impregnation onto solid
urea fertilizers and incorporation into liquid urea fertilizer compositions. These solvent
systems are based on glycols and glycol derivatives.
Sulzer et al. (1998) patented an improved technology which makes it possible to
efficiently produce large scale commercial quantities of N-hydrocarbyl thiophosphoric
triarnides, including nBTPTA at high yields, by means of a continuous process.
Investigations performed at the National Fertilizer Development Center (Tennessee
Valley Authority, Muscle Shoals, Alabama) showed that thiophosphoryl trian1ide, H2N-
P(S)(NH 2h (TPTA) inhibits soil urease activity (Anonymous, 1985a; Radel et al.,
1987). Based on these and further investigations, TPTA was patented as an inhibitor of
soil urease by Gautney (1987) and as a dual purpose, soil urease and nitrification
inhibitor by Radel (1990).
TPTA was tested with two reference compounds (PPDA and phosphoryl trian1ide,
PTA) with two testing techniques.
In the first technique (testing in "banded configuration"), the powdered mixture of
urea and test compound (10% on urea basis) was applied in narrow bands in the soil,
and after 3- and 6-day incubations the remaining urea was extracted and determined. It
was found after 3 and 6 days that the added urea remained unhydro1yzed in the
following proportions: 72 and 33% (under the influence of TPTA) , 63 and 10%
(PPDA), and 54 and 3% (PTA), respectively.
In the second technique (testing in "well-n1ixed configuration"), solutions or
suspensions of the compounds are well-n1ixed throughout the soil, then the mixtures are
submitted to preincubation at 30°C for 0, 3, 7, 14, 21, and 36 days. Urea is added to the
preincubated mixtures and they are then incubated again, for 16 hours. The incubation is
145
capacity by capillarity. The soil samples were limed (1% on soil weight basis) to raise
pH to 7.5, then amended with urea (at a rate equivalent to 100 kg Nlba on a surface area
basis) containing 0, 0.005, 0.01, 0.05,0.1 or 0.5% nBTPTA (on urea weight basis) and
incubated at laboratory temperature (20-23°C) for 14 days, during which time the
volatile NH3 was measured.
The cumulative NH3 losses decreased with increasing nBTPTA concentrations, but
these effects of 0.1 and 0.5% nBTPTA were not significantly different. By day 14, the
losses were 32.2, 14.7, and 6.1 % of applied urea-N for the 0.005, 0.01, and 0.05%
nBTPTA, respectively, compared with a 52.0% loss from urea without nBTPTA.
In other experiments, the limed Guthrie soil and a Savannah soil (PH in H20 5.7;
clay 17%; silt 21 %; organic matter 0.9%) were used and both ammonia volatilization
and urea hydrolysis were studied. The urea rate was the same as in the first experiments,
but concentrations of nBTPTA were only 0.01, 0.05, and 0.1 %. The incubation was
carried out at laboratory temperature. The volatilized NH3 was measured during 12-14
days, and the unhydrolyzed urea was determined four times during 10 days. These
experiments were repeated using the same two soils, into which 109 of finely ground
soybean straw had been mixed in the surface 2 cm. These soils were kept moist for 30
days to establish a stable microbial population.
Ammonia volatilization losses from urea without nBTPTA ranged from 47 to 73%.
nBTPTA at all concentrations resulted in significant reductions in NH3 loss and the
losses decreased with increasing concentration of nBTPTA during the first days of
incubation. Thus, after 6-day incubation, 54.3% of urea-N had been lost from the
Savannah soil treated with urea only, whereas losses of NH3 were 9.4, 3.4, and 2.7%
from urea containing 0.01, 0.05, and 0.1% nBTPTA, respectively. As the incubation
continued, the NH 3 losses began to increase from urea-nBTPTA, but at 12-14 days, they
were still much less than those from urea alone.
The effectiveness of nBTPTA was reduced by amending the soils with straw. Thus,
NH3 losses from the straw-amended Savannah soil after 6-day incubation was 25.2, 3.5,
and 2.5% for the 0.01, 0.05, and 0.1 % nBTPTA added to urea, respectively, and 54.0%
from urea alone.
Urea hydrolysis was greatly inhibited by nBTPTA in both soils and the degree of
inhibition increased with the rate of nBTPTA. For example, after 10 days of incubation,
in the Savannah soil without straw, 96.2% of urea had been hydrolyzed, whereas
hydrolysis of urea containing 0.01, 0.05, and 0.1% nBTPTA was 66.6, 51.2, and 44.6%,
respectively. The corresponding values in the straw-amended Savannah soil were 100,
82.1, 50.8, and 34.8%, respectively. In other words, the straw amendment caused some
reductions in the urease-inhibiting capacity of nBTPTA.
Experiments were also carried out to study the effect of temperature on the
effectiveness ofnBTPTA as an inhibitor of ammonia volatilization and urea hydrolysis.
The Guthrie soil was used. The rate of urea application was the same as in the first
experiments, whereas the rate of nBTPTA was 0.01, 0.05 or 0.1 % relative to weight of
urea. The incubation was carried out in growth chambers at 18, 25, and 32°C and lasted
12 days (NH3 volatilization experiments) or 10 days (urea hydrolysis experiments).
The inhibitory effect of nBTPTA on NH3 volatilization decreased with increasing
temperature. Thus, the cumulative volatile NH3 losses at 18, 25, and 32°C in 12 days
were 42.3, 49.4, and 55.5% (urea alone); 16.8, 23.5, and 47.8% (urea + 0.01%
nBTPTA); 11.7, 17.8, and 42.4% (urea + 0.05% nBTPTA), and 4.3, 11.2, and 28.9%
148
At day 10 after transplanting, urea with or without nBTPTA was broadcast into the
floodwater. Rates of urea were 0, 25, 50, and 75 kg Nlha. The rate of nBTPTA was
constant at 0.536 kglha by addition of urea containing 1% nBTPTA by weight. The
control plots received no urea and no nBTPTA.
During 10 days after N fertilization, floodwater samples were taken twice daily (at
8-9 and 13-14 hours) from the plots that received 75 kg urea-Nlha with or without
nBTPTA and from the control plots. The residual urea, ammoniacal-N, pH, and
temperature were measured and the vapor pressure of ammonia (pNH 3) was calculated
from ammoniacal-N, pH, and temperature values.
nBTPTA delayed urea hydrolysis only for 1 day and at day 9 no unhydrolyzed urea
was detected in floodwaters of either urea- or urea+nBTPTA-treated plots.
The ammoniacal-N content increased during the first 6 days, then decreased. The
increase was higher in the urea than in the urea + nBTPTA treatments, but the decrease
was similar and at day 10 the ammoniacal-N content represented about 4% of the
applied urea-N in both treatments.
pH raised appreciably to 8.3 on the 3rd day, then gradually decreased to less than 7.0
over 10 days. nBTPTA suppressed pH rise and maintained lower values than those of
urea alone for 4 days, after which no difference in pH was observed.
Floodwater temperature was not affected by any treatment.
The values of pNH3 were negligibly low in the morning hours due to low floodwater
pH and moderate temperature. The maximum pNH3 was recorded in the early afternoon
at day 4 after N fertilization and the afternoons 3-5 days after fertilization provided the
greatest potential for NH3 loss. nBTPTA significantly reduced the pNH3 indicating that
the urea + nBTPTA treatment was less prone to NH3 volatilization loss than was that
with urea alone.
In an experimental variant, microplots (1.2 by 1.2 m) were installed within the main
plots. 15N-labeled urea (4.9 atom% excess) without or with nBTPTA at a rate of 50 kg
Nlha was broadcast into the floodwater of microplots. Analyses of floodwater, plant,
and soil for 15N at day 37 after N fertilization indicated that the losses of 15N from the
floodwater-plant-soil system were 50.17 and 49.33%, i.e., almost 50% of the applied
15N in the urea and urea + nBTPTA treatments, respectively. The losses were attributed
to ammonia volatilization in absence of nBTPTA and to denitrification in its presence.
A similar field experiment was conducted by Phongpan and Byrnes (1993) during
the 1989 dry season (March-June) on a clay soil (pH in H2 0 4.7) at the same Rice
Experiment Station in Thailand, and similar results were obtained concerning nBTPTA.
Urea applied at a single rate (100 kg Nlha) without or with 1% nBTPTA was broadcast
into the floodwater of rice plants. Total N loss during 41 days after fertilization was
26% of applied urea-N from the plots treated with urea alone and it was insignificantly
lower, 24% from the urea+nBTPTA-treated plots. Again, the losses were attributed to
ammonia volatilization from the urea-treated plots and to denitrification in plots treated
with urea + nBTPTA.
Mullen et af. (1991) prepared reaction mixtures from 10-g samples of a silt loam
soil, 40 mg of urea, 0 to 25 ).lg of nBTPTA, and 0 or 50 mg of wheat residue, and
incubated them at 25°C for 1 week. Analysis of the remaining urea indicated that
effectiveness of nBTPTA decreased with its decreasing rate. At the 25 ).lg nBTPTA rate,
80% of the added urea remained unhydrolyzed without wheat residue and only 47%
150
with residue. In another experiment, at 0.5 mg nBTPTAJkg soil, 60 and 8% of the added
urea remained unhydrolyzed after 4 and 7 days, respectively.
Mullen and Ravelle (1992) and Howard et at. (1992) evaluated the effects of
nBTPTA (1.12 kglha) on hydrolysis of urea and volatilization of ammonia from urea
and urea-ammonium nitrate (UAN), applied as solid and liquid fertilizcr, respectively,
to field microplots on a silt loam soil amended with wheat straw. The results indicated
that nBTPTA was more effective in urea- than in UAN-fertilized microplots. Thus, after
8 days, 17 and 1% of the added urea-N was recovered from the urea+nBTPTA- and
urea-treated microplots, respectively, and 3.1 and 1.3% of the added urea remained
unhydrolyzed in the UAN+nBTPTA- and UAN-treated microplots, respectively.
Ammonia volatilization from urea and UAN was reduced by 73 and 20%, respectively,
by nBTPTA over 10 days.
To study the effect ofnBTPTA on N transformations at the microsite of urea granule
placement, Christianson et al. (1993) used two contrasting soils, a silt loam (PH 5.2)
and a clay (pH 8.2). Uniform cylindrical small (12-mg) urea granules containing 0, 0.5
or 0.05% nBTPTA (weight/weight) were placed on the soil surface in plastic cups (a
single granule in exact center of each cup) for up to 6 days. Soils were then frozen in
liquid N2 and a 0.9-cm wide vertical slice was cut through the fertilizer placement site.
A section of this slice was cut into 45 squares (004 by 004 cm) and analyzed for soil pH,
extractablc ammonium, nitrate, and urea concentrations at the microsite where the
fertilizer had been placed.
In the silt loam soil, nBTPT A lowered soil pH and NH4 + concentrations at the
placement site as compared to urea alone and allowed more diffusion of urea away from
the fertilizer microsite. In the clay soil, nBTPTA was less effective in reducing NH/
concentrations from the zone of high soil pH associated with urea hydrolysis.
The conclusion was that effectiveness of nBTPTA depends on the capacity of the
soils to permit diffusion of urea and ammonium.
Watson et at. (l990a,b) carried out a field experiment at the Agricultural Research
Institute, Hillsborough, County Down, Northern Ireland, to compare the effects of mid-
summer applied urea, urea + 0.5% nBTPTA (relative to weight of urea), and calcium
ammonium nitrate (CAN) on ammonia volatilization and on herbage dry matter yield of
an established perennial ryegrass (Lolium perenne) sward on a clay loam soil (pH in
H20 6.3). The fertilizers were surface-applied, at a rate of 100 kg N/ha, to (6 by 2 m)
plots. The plots also received P and K fertilizers. No fertilizers were added to the
control plots. Ammonia volatilization was measured daily during 13 days after
fertilization.
The maximum NH.l volatilization occurred on day 1 in the urea treatment and with
an approximately 5-day delay in the urea + 0.5% nBTPTA plots. Delaying the
maximum volatilization loss increases the chance of rain falling to move the urea below
the soil surface and hence lower NHJ volatilization.
Cumulative NH3 volatilization loss expressed as a percentage of urea-N applied was
8.1 from urea, 1.89 from urea + 0.5% nBTPT A, and 0.09 from CAN. The reduction of
volatile NHjloss by nBTPTA was very significant.
Watson et al. (l994a) studied the effect of nBTPTA added to urea granules at five
rates (0, 0.01, 0.05, 0.1, 0.25, and 0.5%) on ammonia volatilization. The site was again
an established perennial ryegrass sward on a clay loam soil at the Agricultural Research
Institute, Hillsborough. As the pH of this soil was SA, it was limed (2.5 t finely ground
151
CaC03/ha) 3 weeks prior to commencing the experiment to increase the surface pH (0-
2.5 cm) to approximately 6.0. The experiment was carried out in five repetitions on
different plots (I by 2 m) and in different time periods over the 1992 growing season to
cover a range of weather conditions.
The first experimental time started at the end of April 1992 and it was repeated at
approximately 4-week intervals (noted as time periods 2-5), moving to new plots each
time. Prior to fertilizer application the grass was cut to a height of 2 cm. The plots at
each time period received 100 kg N/ha together with 8.7 kg P/ha as single
superphosphate and 67 kg KIha as muriate of potash. The plots designated for the later
time periods 3-5 received, approximately 8 weeks before their use, one basal dressing of
a compound fertilizer containing 50 kg N, 5.5 kg P, and 21 kg Klha (plots for time
periods 3 and 4) or two basal dressings (plots for time period 5). All fertilizers were
surface-applied.
Volatilization ofNH3 was measured daily for 9 days following fertilizer application
to plots for time periods 1,2,4, and 5 or for 8 days in the case of plots for time period 3.
Over all of the time periods, the mean value of the times at which maximum rate of
NH3 volatilization loss occurred was 2.59 days in the case of urea-only treatment and
increased to 3.17-4.73 days in parallel with increasing level of nBTPTA from 0.01 to
0.5% relative to weight of urea.
Over all of the time periods, the inhibition of NH3 volatilization was 50.4, 82.8,
89.0, 96.5, and 97.0% at the O.oI, 0.05, O.l, 0.25, and 0.5% nBTPTA levels,
respectively.
Over all levels of nBTPTA, inhibition of NH3 volatilization loss from plots for time
period 1 to those for time periods 2-5 was 83.8, 81.2, 77.0, 88.3, and 85.3%,
respectively. There was no significant difference in these values and no significant time
x treatment interaction, suggesting that the effectiveness of nBTPTA was not dependent
on climatic conditions.
Based on the percent inhibition values and different nBTPTA levels, it was
calculated that the optimum level of nBTPTA at the site studied would appear to be
0.1 % which would be predicted to give 93% inhibition ofNH3 volatilization loss.
Watson et al.(1994b) studied, under laboratory conditions, the influence of soil
properties on the relationship between ammonia volatilization and nBTPTA
concentration. Surface samples (0-15 cm) of 16 soils of widely differing chemical and
physical properties, collected from grassland sites in Northern and Southem Ireland,
were used.
Fresh soil samples were placed in cylindrical screw-top plastic jars (85 mm height,
80 mm diameter) to a depth of 50 mm. Urea granules (50 mg N equivalent
approximatively to 100 kg N/ha) were applied to the surface of soil samples. The urea
granules contained 0, 0.01, 0.058 or 0.28% nBTPTA. The samples were incubated at
13°C. The NH3 volatilized was measured daily for 9 days after fertilizer application.
Total NH3-N loss over 9 days (as % ofN applied) had the following mean values in
the 16 soils: 20.67 (samples treated with urea only), 15.94, 7.15, and 2.79 (samples
treated with urea + 0.01, 0.058, and 0.28% nBTPTA, respectively), indicating that the
NH3 volatilization decreased with increasing nBTPTA concentration. Similarly, the
average times of the maximum loss ofNH3 from the 16 soils were 2.99,3.86,6.43, and
6.49 days at the 0, 0.01, 0.058, and 0.28% nBTPTA levels, respectively.
152
The % inhibition of NH3 loss by nBTPTA was highly dependent on soil type; on
some soils nBTPTA was effective even at the 0.01 % concentration. nBTPTA was most
effective in reducing NH3 loss from soils with a high pH and low organic matter
content, i. e.. from soils very prone to NH3 loss from urea containing no urease inhibitor.
Simple correlation analysis proved that NH3 volatilization from samples of the 16 soils
treated with urea only was most significantly correlated with pH in H20 (r=0.896) and
pH in KCI (r=O.920).
Modeling the relationship between total NH3 loss and nBTPTA concentration
showed that the % nBTPTA required to achieve a given % decrease in NH3
volatilization was constant for all soils. For example, 0.092% nBTPTA was predicted to
lower total NH3 loss by 90% from any given soil. Little additional benefit can be
expected by using nBTPTA concentrations above 0.1 % relative to weight of urea.
Watson et al. (1998) studied the longer-term effect of repeated application of
nBTPTA-amended urea on ammonia volatilization and urease activity as well as on
herbage dry matter yield and N uptake by perennial ryegrass.
A 3-year field experiment was carried out at the Agricultural Research Institute,
Hillsborough. Plots (6 by 2 m) were installed under a three-cut silage system. The soil
received one, two or three applications of urea, CAN or urea amended with nBTPTA at
either 0.1 or 0.5% level, during each of the 1994, 1995, and 1996 growing seasons.
There were ten treatments.
In each year, the plots were fertilized three times: in March/April (120 kg Nlha, 13
kg Plha as single superphosphate, and 75.5 kg K/ha as muriate of potash); in May (100
kg N, 8.7 kg P, and 75.5 kg K/ha), and in June/July (80 kg N, 8.7 kg P and 75.5 kg
K/ha).
Ammonia volatilization was studied at the end of the first growing season (early
September 1994). Soil was collected (0-5 cm depth) from each plot. Fresh soil samples
(250 g each) were treated with urea or urea + 0.1 % nBTPTA at a rate of 50 mg N
(equivalent to -100 kg Nlha) and incubated at 15°C. The volatilized NH3 was measured
daily for 9 days after fertilizer application.
It was found that 0.1 % nBTPTA not only lowered NH3 loss but substantially
delayed the time at which maximum NH3 loss occurred; the average value over the ten
treatments was 1.6 days (urea) and 4.9 days (urea + 0.1% nBTPTA). It was concluded
that previous fertilizer application had no significant effect on the subsequent efficacy
of nBTPTA in lowering NH3 volatilization from urea.
Soil urease activity (in the 0-5-cm layer) was measured four times: at the end of the
three growing seasons (September 1994, October 1995 and 1996) and at the beginning
of the growing season of the last year (March 1996).
In September 1994 and October 1995, urease activity was significantly lower in
plots fertilized with urea + 0.5% nBTPTA than in the other plots, including those
fertilized with urea + 0.1 % nBTPTA. But in March and October 1996 the activity was
similar in all plots which indicates that 0.5% nBTPTA had no cumulative inhibitory
effect on soil urease.
Ma et al.(l995) performed a laboratory experiment to study the effects of nBTPTA
and the percolation rate on hydrolysis and movement of urea under simulated flooded
soil conditions. A silt loam soil, typical in south-west Louisiana rice fields, was used.
Soil samples (1.13 kg) were packed into PVC columns, then saturated and covered by a
2-cm floodwater layer, which was maintained during the preincubation of columns in
153
the dark at room temperature for 3 months to develop reduced soil layers. The columns
were then percolated at a rate of 0, 0.5 or 1 cm/day, the floodwater layer being replaced
by a urea solution containing 0 or 10% nBTPTA (on urea weight basis). Total urea-N
added to each colunm was 70.4 mg. In the next step, the colunms were incubated in the
dark at 30°C for 0.5, 1 or 2 days, then the floodwater was collected and the soil colunms
were sectioned into eight layers at depths of 0.5, 1, 2, 3, 5, 7.5, 10, and 12.5 em. The
soil sections were analyzed to determine residual urea.
The analytical data showed that more than 70% of the urea was hydrolyzed after 2
days of application without nBTPTA, whereas only 40% was hydrolyzed with
nBTPTA. Consequently, more urea was distributed in the soil profile in nBTPTA-
treated columns. High percolation rates caused substantial urea movement downward
into the soil when urea was not hydrolyzed.
In studying the effect of nBTPTA on ammonia volatilization from urea and on the
maize yield under the climatic conditions of the Mediterranean regions, Palazzo et al.
(1995, 1996) conducted 3-year field experiments (1989-1991) on a sandy clay soil and
on three clay soils (PH 7.87-8.25) at Metaponto and Monterotondo. Urea was surface-
applied yearly at a rate of 150 kg N/ha without or with 0.1 or 0.25% nBTPTA (relative
to weight of urea), one day after irrigation. Single superphosphate and potassium sulfate
were also applied. Ammonia volatilization was determined during 6 weeks after
fertilizer application.
Cumulative NH3 losses from urea without nBTPTA averaged 43.3% in the sandy
clay soil and 11.3% in the clay soils, whereas the average losses from the nBTPTA-
amended soils were 24.2 and 3.1% respectively. There was no significant difference
between the effects of the 0.1 and 0.25% nBTPTA concentrations.
Vittori Antisari et al. (1996) evaluated the effects of different concentrations of
nBTPTA on ammonia volatilization, urea hydrolysis, and evolution of mineral N in
surface (0-20 cm) soil samples collected at the locations Ozzano, Rimini, and Carpi. Of
the three soils, the Rimini soil has the highest pH (8.5 in H2 0), contents of sand (81 %),
organic C (2.3%), total N (2.287 glkg) and is the most urease-active. Urea was added to
the samples at a rate of 1 mg N/g dry soil. The rate of nBTPTA relative to weight of
urea ranged from 0.01 to 0.5%. The amount of the volatilized NH3 and the
concentrations ofresidual urea, exchangeable NH/, N0 2-, and N03- were determined
periodically during incubation at 23°C for 15 days.
nBTPTA reduced NH3 volatilization proportionately with its rate. The extent of
reduction was smallest in the Rimini soil.
Similar results were obtained concerning urea hydrolysis. For example, the urea
applied with 0.5% nBTPTA was hydrolyzed during 1 and 15 days of incubation in
proportions of 0 and 79.5%, respectively, in the Carpi soil and of 30 and 87%,
respectively, in the Rimini soil.
nBTPTA slowed down the formation of exchangeable ~ + due to inhibition of
urea hydrolysis and decreased the concentration of N0 2- due to reduction of NH/
formation. Both effects were weakest in the Rimini soil.
In contrast, the concentration of NO) - was increased in each soil by increasing the
nBTPTA rate and/or incubation time.
In a laboratory experiment performed by Murphy and Ferguson (1997), air-dry
samples of a silt loam soil were packed to a depth of 6 cm in acrylic chambers (3.4 cm
diameter). Urea-ammonium nitrate (DAN) solution (28% N) with or without nBTPTA
154
was placed 1.8 cm deep in the soil to simulate a subsurface band application. The rate of
N addition was 224 kglha and that ofnBTPTA was 1.12 kglha. The residual urea, NH/,
and N03 - concentrations were determined over a 14-day period. Urea-N recovery was
significantly greater and urea hydrolysis rate significantly less (p=0.05) only on day 1,
but by day 14 the urea was completely hydrolyzed and recovery ofNH/-N and that of
NO) --N became the same in both UAN-treated and UAN+nBTPTA-treated soil.
The Argentinean investigators Sainz Rozas et al. (1997) conducted a 2-year field
experiment (1994/95 and 1995/96) studying ammonia volatilization from surface-
applied urea to no-till maize as a function of urea rate (0, 70, 140, and 210 kg N/ha),
presence or not of nBTPTA and fertilization time (at seeding and 6-leaf stage). In both
years, the NH3 loss increased with an increasing rate of urea and was higher following
fertilization at 6-leaf stage tllan at planting; nBTPTA reduced the NH3 loss even when
the no-till maize was fertilized later than at planting time.
nBTPTA was submitted to a laboratory test at the South African Sugar Association
Experiment Station at Edgecombe (Anonymous, 1998). The commercial nBTPTA
Agrotain, recommended for field application at a rate of 5 1/t urea granules, was used.
The test showed that anlfllonia volatilization losses from urea applied with Agrotain
before rain could be reduced by up to 14% on bare soil and up to 10% on trash. The
strongest suppression of N losses «1 %) was, however, derived from a simulated
irrigation of 10 mm. The results also indicated that Agrotain has a limited useful life of
about 2 weeks on both bare soil and trash, so that the absence of rain or irrigation after
urea application for periods longer than 2-3 weeks would greatly reduce the N savings.
The reason for this continued loss is that the urea which was initially protected from
hydrolysis by Agrotain is still susceptible to losses after the Agrotain is depleted, unless
water additions from rain or irrigation incorporate the urea into the soil.
Gill et al. (1999) studied the effect of green manure and wheat straw amendment of
a flooded alkaline sandy loam soil from India on the effectiveness on nBTPTA to
reduce urea hydrolysis and anlfllonia volatilization from urea. Hydrolysis of the urea
applied was complete in 12, 8, and 6 days in unamended, green manure- and wheat
straw-amended soil, respectively, and as a consequence, the NHJ loss through
volatilization was enhanced by the organic amendments.
nBTPTA applied at rates of 0.5, lor 2% (on urea weight basis) was more inhibitory
on urea hydrolysis and NHJ volatilization from the unamended soil than from the soil
amended with green manure or wheat straw. For example, by applying 0.5% nBTPTA,
complete hydrolysis of urea was delayed up to 16 days in the unamended soil, whereas
in wheat straw-amended soil urea was completely hydrolyzed by the 12th day even
when it was treated with 2% nBTPTA.
***
A great number of studies, in which the effect of phosphoric triamide (PTA) and
thiophosphoric triamide (TPTA) compounds on soil urease activity, urea hydrolysis,
ammonia volatilization, and on immobilization of urea-N in soils as well as their
stability were compared with those of other inhibitors, will be dealt with in Chapter 4.
155
inhibitory effect on soil urease activity and stability in urea melt at 140°C. Besides its
reduced inhibitory capacity, this compound also has another disadvantage: its
production cost is significantly higher with respect to the two triamides as well as
PPDA (Anonymous, 1985a; Radel et al.. 1987).
The inventors Medina and Sullivan (1986, 1987) tested a series of cyclotri-
phosphazatriene (CTP AT) derivatives, also called phosphonitrilic derivatives. Some of
these compounds, namely the hexaamino derivative mentioned above and three organic
(phenoxy) derivatives (Figure 55) were found to be able to inhibit soil urease activity.
P3N3(NH2)S(OCsHs) P3N3(NH2)4(OCsHs)2
2-Phenoxy-2,4,4,6,6-pentaamino-CTPAT 2,4-Diphenoxy-2,4.6,6-tetraamino-CTPAT
2,
H N /OC 6Hs
N...... P~N
C6 HSO, II I ,.........OCsH s
/P'N PP ,
H2N NH2
P3N3(NH2b(OCsHsb
2,4,6-Triphenoxy-2,4,6-triamino-CTPAT
In testing the inhibitory effect, samples of two silt loam soils (from Alabama and
Louisiana, respectively) were used. Several procedures were applied. PPDA and PTA
served as reference compounds.
a) Small containers (6x6x6 cm) were one-half filled with soil containing 20%
moisture. Urea powder or urea powder + powdered test compound (thoroughly mixed)
was distributed in a narrow band, 6 cm long on the soil surface. The urea rate was 410
mg (6.72 mmoleslband) and equivalent to 100 kg Niha. The test compounds were used
in an equimolar amount (0.25 mmoleslband). Then the containers were filled with soil
and submitted to incubation at 25°C. After 3, 6, and 9 days, the residual urea was
determined.
It was found that after 3 days of incubation PPDA was a more effective inhibitor
than 2,2,4,4,6,6-hexaamino-CTPAT, with the unhydrolyzed urea representing 68.3 and
48.1 % of the added urea, respectively. However, after 6 days, the reverse was true, with
the corresponding values ofunhydrolyzed urea being 12.9 and 16.8% respectively. In 9
days, the urea was completely hydrolyzed in all treatments. It also established that after
both 3 and 6 days of incubation the inhibitory effect of 2,2,4,4,6,6-hexaamino-CTPAT
on soil urease activity was more marked than that of PTA.
b) This procedure is similar to the first one, but each test compound was applied at a
rate of 10% relative to urea weight (which remained 410 mglband). By using this
procedure it was found that PPDA was a more effective inhibitor than 2,2,4,4,6,6-
hexaamino-CTPAT not only after 3 days of incubation, but also after 6 days. PPDA and
the four urease-inhibiting CTPAT derivatives presented the following decreasing order
of their inhibitory capacity:
PPDA > hexaamino ~ phenoxy ~ diphenoxy > triphenoxy.
The compounds specified below were unable to inhibit soil urease activity:
P3N3C1 6 , 2,2,4,4,6,6-hexachloro-CTPAT;
P3N3(NH2h(NHCH3)4, 2,2,4,4-tetra( methylarnino)-6,6-diamino-CTPAT:
P3N3(NH2h[N (CH3 h]4, 2,2,4,4-tetra(dimethyl amino)-6,6-diamino-CTPAT;
P3N3(NHCH3)6, 2,2,4,4,6,6-hexa(methylamino)-CTP AT;
P3N3[N(CH3)2k 2,2,4,4,6,6-hexa(dimethylamino)-CTPAT;
P3N3CI4 [N(CH3hh. 2,2,4,4,-tetrachloro-6,6-di(dimethylarnino)-CTPAT;
P3N3Cls(OC6 Hs),2-phenoxy-2,4,4,6,6-pentachloro-CTPAT;
P3N3C4(OC6 Hsl2. 2,4-diphenoxy-2,4,6,6-tetrachloro-CTPAT;
P3N3Ch(OC6Hs)3,2,4,6-triphenoxy-2,4,6-trichloro-CTPAT.
c) Soil samples (100 g) were mixed with 40 ml of water, 410 mg of urea powder
without or with 41 mg of powdered test compound. The mixtures were incubated at
25°C for 1, 3, and 6 days, after which the unhydrolyzed urea was assessed. PPDA
proved again to be a more effective inhibitor than were the CTPAT derivatives tested
which showed the following order of inhibitory capacity:
phenoxy> diphenoxy > hexaarnino > triphenoxy.
One can see that the order obtained with procedure c (well-mixed system) differs
from that recorded with procedure a (banded system).
d) Moist soil samples (120 g) were well mixed with 1 ml of aqueous solution or
suspension containing 7 ~oles of test compound. The mixtures were preincubated at
30°C for 0, 3, 7, 14, and 21 days before adding urea (50 mg in 1 ml solution). After a
new mixing and incubation at 30°C for 16 hours, the residual urea was determined.
Under these conditions, the three phenoxy derivatives of CTPAT proved to be superior
157
not only to the hexaamino derivative and PTA, but also to PPDA. Thus, after 0, 7, 14,
and 21 days of preincubation, the inhibition had the following values: 22, 74, 84, and
82% (triphenoxy derivative), 75, 71, 69, and 65% (diphenoxy derivative), 95, 49, 42,
and 44% (phenoxy derivative), 46, 20,4, and 4% (hexaamino derivative), 100, 27, 4,
and 0% (PPDA), 29, 12, 0, and 0% (PTA). In other words, without preincubation,
PPDA strongly inhibited soil urease activity, but as a result of preincubation its
inhibitory effect decreased and then disappeared. Phenoxy derivatives of CTPAT
excelled in persistence of their urease-inhibiting effect.
Based on the results mentioned above, binary and tertiary mixtures of phenoxy
CTP AT derivatives were suggested in a model to predict urease inhibitions of various
intensity and duration to meet the demands of a wide variety of agroclimatic, soil, crop,
and management conditions and practices.
According to the model (Figure 56) it is possible, by using different mixtures, to
regulate the inhibition of soil urease activity between about 25 and 80% for the first 3
days after urea fertilizer application, and between 50 and 80% for periods of 3-21 days.
For use in practice, the CTPAT derivatives are recommended at a rate ranging from
about 0.01 to about 10% relative to weight of urea fertilizer.
100 A
o.---a lS"/.M 'S"IoT
¢----o
-I-
':!C·... M + 'jJo... T
.-._ •• 7S-'.M+ 25"1.T
~'~~........--"'---
----<)------------.-- ~
....._._,_._._.- _.....
....... ~.'¢-- --- --_ .. _--
....... -._.-._._._._
~
~
~ 2S
°0 '0 20
i 7S /r~"'-·"':.J(;-:;::;::~:~:~:-:-;;-=-=-~~~~--:~----<;,
~ l.'
.: " .I
~ 50 / /
!l I
~
" 2S
°DL------~----~,O-------~1~------~~~---
""yo
Figure 56. Model for prediction of the effect of different mixtures prepared from phenoxy derivatives of
cyciotriphosphazatriene on urease activity in soil.
A - Influence ofM + T compounds awlied at different rates. B - Influence of M + D + T compounds awlied
at different rates. M - 2-(Mono)phenoxy-2.4,4.6.6-pentaaminocyclotriphosphazatriene. D - 2,4-Diphenoxy-
2,4.6.6-tetraarninocyclotriphosphazatriene. T - 2,4.6-Triphenoxy-2,4.6-triaminocyclotriphosphazatriene.
IAdapted from Medina and Sullivan (1986, 1987), by permission of Tennessee Valley Authority.!
158
Savant et af. (1988b) performed investigations that are similar to those described in
the patents of Medina and Sullivan (1986, 1987). The same Alabama silt loam soil and
reference compounds (PPDA and PTA) were used. Four cyclotriphosphazatriene
(CTP AT) derivatives were studied: 2-phenoxy-2,4,4,6,6-pentaamino-CTPAT, cis-2,4-
diphenoxy-2, 4,6, 6-tetraamino-CTP AT+trans-2,4-diphenoxy-2,4, 6, 6-tetraamino-CTPAT
(cis/trans ratio 2.60), cis,trans-2,4,6-triphenoxy-2, 4,6 -triamino-CTP AT + cis,cis-2,4,6-
triphenoxy-2,4,6-triamino-CTP AT (cis/trans ratio 0.55), and 2,2,4,4,6,6-hexaamino-
CTPAT.
For testings, essentially the procedure d (see page 156) was applied. The 120-g
moist soil samples were well mixed with 7 ~moles of test compound or mixture of test
compounds in 1 ml solution or suspension and were pre incubated at 20, 30 or 40°C for
0,3,7, 14,21, and 36 days. Following preincubation, the mixtures were treated with 50
mg of urea in 1 ml solution and after a new mixing were incubated at 30°C for 16 hours
and then analyzed for residual urea.
After O-day preincubation at 30°C and 16-hour incubation at 30°C, the six test
compounds inhibited soil urease activity in the order: triphenoxy (22% inhibition) <
PTA < hexaamino < diphenoxy < phenoxy < PPDA (100% inhibition). After 14 days of
preincubation the order was: PTA = hexaamino = PPDA (0% inhibition) < phenoxy <
diphenoxy < triphenoxy (75% inhibition). After 36-day preincubation, the urease-
inhibiting order phenoxy < diphenoxy < triphenoxy was maintained. Thus, the results
obtained by Medina and Sullivan (1986, 1987) were confirmed.
After O-day preincubation, the urease-inhibiting capacity of the phenoxy and
diphenoxy derivatives was higher and that of the triphenoxy derivative was lower at 30
and 40°C than at 20°C, but the inhibition by each derivative was lowest during
incubation at 40°C.
To verify the prediction model presented in Figure 56, a separate experiment was
conducted. The (mono)phenoxy (M) derivative and its three combinations with the
diphenoxy (D) and triphenoxy (T) derivatives were used in proportions expressed as
percentages. The preincubation and incubation lasted 25 days and 16 hours,
respectively.
The urease-inhibiting capacity of the M derivative and its combinations presented
the order:
100% M > 50% M + 50% T > 33% M + 33% D + 33% T ;.::: 25% M + 75% T, after
O-day preincubation;
50% M + 50% T;.::: 33% M + 33% D + 33% T > 25% M + 75% T > 100% M, after
14 days of preincubation, and
25% M + 75% T;.::: 33% M + 33% D + 33% T ;.::: 50% M + 50% T > 100% M, after
25 days of preincubation.
It can be deduced from these orders that after shorter preincubations the urease
inhibition depended mostly on the M derivative and later on the T derivative.
The Czech investigators Minar et al. (1990) studied the effect of hexaamino-CTP AT
on soil urease activity and ammonia volatilization from soil treated with urea solution or
with the urea-ammonium nitrate liquid fertilizer DAM 390. Samples of a garden soil
(pH 7.08) were used.
In the urease activity study, 100-g soil samples were sprayed with urea solution or
DAM 390 at a rate of 100 mg N. Hexaamino-CTPAT was applied at rates of 0, 0.5, 1,
and 2% (relative to fertilizer N). The mixtures were preincubated at 20°C and after 1, 2,
159
The results of a maize pot experiment, in which Calancea et al. (1986) used labeled
hexaamino-CTPAT, P3Nl 5NH 2 )6 or P3 15 N3(NH2)6, indicated that hexaamino-CTPAT is
decomposed slowly in the soil. At the beginning the compound is deaminated which is
followed by the decomposition of the cycle (see page 351).
Simihliian (1998) and Simihaian et al. (1999) tested the effect of three phosphorylated
2-oximinophenylacetonitrile compounds (Figure 57) on soil urease activity.
Hydroquinone served as a reference compound. Weakly urease-active samples of an
alluvial soil (light-textured) and a chernozem (heavy-textured) were used.
Figure 57. Structure of the phosphOIylated 2-oximinophenylacetonitrile cOlJ1lounds tested for inhibition of
soil urease activity.
HQ A B C
Figure 58. Inhibition of soil urease activity by hydroquinone and three phosphorylated 2-
oximinophenylacetonitrile compounds.
HQ - Hydroquinone. A B, C - Compounds A, B. and C (see Figure 57). IFrom Simihllian, 1998./
Three such compounds were tested by Sirnihaian (1998) and Simihaian el al. (1999) for
evaluation of their effect on soil urease activity. Their structure is shown in Figure
59.
CHI 'CH2-0/"NH--S02-0-CH3
2-Benzenesulfonamido-2-thiono- 2-(p-Methyl}-benzenesulfonamido-2-thiono-
5,5-dimethyl-l,3.2-dioxaphosphorinane 5,5-dimethyl-l.3.2-dioxaphosphorinane
(I) (II)
CHI 'CH2-0/"NH--802-o-0-CH3
2..(p-Methoxy)-benzenesulfonamido-2-thiono-
5,5-dimethyl-l.3 ,2-dioxaphosphorinane
(III)
The reference compound, soils, and methods used were identical to those specified
in the preceding subchapter .
Figure 60 comprises the results obtained.
162
t! 50
iii. 45
E., 40
8i;'
15~ 35
-."
~.~ 30
+=025
-a
'0c,.. 20
.2 -; 15
~~
c
10
.2 5
£ 0
HQ II III
Figure 60. Inhibition of soil urease activity by hydroquinone and three 2-thiono-5,6-dimethyl-l,3,2-
dioxaphosphorinane compounds.
HQ - Hydroquinone. I, II. III - Compounds I. II. and III (see Figure 59). IFrom Simihiiian. 1998.1
It is evident from this figure that the compounds tested inhibited urease activity in
both soils. Hydroquinone was a more potent inhibitor than were compounds I, II, and III
in both soils. Compounds II and III were more inhibitory than compound I in the
alluvial soil, but these three compounds were similarly weak inhibitors in the
chemozem. The presence of p-methyl or p-methoxy substituent in benzene ring
(compounds II and III) increased the inhibitory effect, to the same extent, in the alluvial
soil, but no increased inhibitory effect was observed in the chernozem.
2.30. ANTIMETABOLITIES
These compounds were tested for their ability to suppress growth of urease-producing
soil microorganisms, i.e.. not for the inhibition of the activity of urease accumulated in
soil.
2.30.1. Sulfanilamide
Sulfanilamide (amide of sulfanilic acid; Figure 61) was tested by Pugh and Waid
(1969a,b) to evaluate its inhibitory effect on volatilization of ammonia from urea in four
soils from England. In the first experiment, 100-g moist samples of an acid loamy sand
were treated with 6.66 mM of urea, 0.582 mM (0.1 g) of sulfanilamide (SA) or 0.4 mM
of acetohydroxarnic acid (AHA) or with 0.582 mM SA + 0.4 mM AHA. Soil treated
only with urea was the control. During incubation which lasted 77 days at 20°C, the
evolved NH3 was measured. Half-loss time of NH3 increased form 11.5 days (control
163
soil) to 12.5 days (SA-treated soil), 28.5 days (AHA-treated soil), and to 38.5 days
(SA+AHA-treated soil). In other words, SA had a very weak inhibitory effect. In
contrast, SA + AHA exhibited a synergistic effect. However, total NH3 losses during the
77-day incubation period were very similar in all the samples.
In other experiments, in which three other soils were also used, amounts of soil
sample, urea, SA, and AHA were the same as in the first experiment, and the results
obtained with two of these soils (a sandy loam and a loamy sand) were also similar to
those registered in the first experiment. However, in the third soil (a sandy loam),
neither SA nor SA + AHA delayed volatilization of NH 3; in fact, they increased total
loss ofNH3 •
In the three Iowa soils studied by Bremner and Douglas (1971) under the conditions
of the 5-hour test, SA at a rate of 50 ppm (on soil basis) caused only negligible «1%)
inhibitions in urease activity.
O-S~
Pyridine-3-sulfonic acid Desthiobiotin
a.c
CI
y"'
CI
I
CI
"0
Figure 62. Structure of the antimetabolites patented and tested by Peterson and Walter (1970) for inhibition of
soil urease activity.
a) Three Iowa soils were used. Samples of field-moist soil containing 5 g of oven-
dry material, to which sufficient water was added to bring the total volume of water to 2
ml, were treated with I ml of aqueous solution containing 2 mg of urea with or without
50 or 250 f.!g of antimetabolite. An inhibitor of soil urease activity, hydroquinone (HQ),
was included for comparison. HQ was applied at the same rates as the antimetabolites.
Incubation took place at 20D e. After 24 and 48 hours, the residual urea was analyzed.
The analytical data showed that in each soil the inhibitory effect of PSA, DTB, and
OTe on urea hydrolysis was zero at their both rates and after both incubation times. In
contrast, the inhibitory effect ofHQ was strong. Thus, in the three soils incubated for 48
hours, HQ at the lower rate brought about 48, 36, and 24% inhibitions, respectively,
whereas at its higher rate the inhibitions were 68, 100, and 51 %, respectively.
b) The effect of antimetabolites and HQ on volatilization of NH3 from urea was
studied with a sandy soil. Field-moist samples (10 g oven-dry material) were treated
with 2 ml of aqueous solution containing 10 mg of urea-N with or without 0.5 mg of
antimetabolite or HQ. Water was then added to bring the total volume of water to 3 m!.
Ammonia evolved during incubation (14 days at 20De) and urea and exchangeable NH/
contents in incubated samples were determined. It was found that in contrast to HQ
which markedly retarded urea hydrolysis and greatly reduced volatilization ofurea-N as
NH3 , PSA, DTB, and OTe had no effect on these processes.
165
Thus, in the laboratory experiments of Sengik and Kiehl (l995a), peat increased these
losses by 843%. A reddish-brown podzolic clay soil from the State of Sao Paulo, Brazil
was studied. The rate of peat addition was equivalent to 10 tlha and that of urea to 72 kg
Nlha. TIle peat was mixed with the top centimeter of soil and urea in solution was
uniformly distributed on the soil surface. Volatilization of NH3 was measured over 23
days.
slurry of NHA to 7 with NH 4 0H. The effect of NHA and ANH on urea hydrolysis and
nitrification was studied with three soils: an alkaline (PH 7.7) sand, an acid (PH 5.4)
sandy loam, and an acid (pH 5.0) sand. N-(n-butyl)thiophosphoric triamide (nBTPTA)
was used as a reference compound. The reaction mixtures contained 109 of soil,S mg
of urea-N, 0 or 0.75 mg of NHA or ANH or 0.1 mg of nBTPTA, and water to field
capacity, and were incubated at 25°C for 1, 2, 7, 14,21 or 28 days. During incubation
the volatilized ammonia was measured, and after each incubation time the reaction
mixtures were analyzed for ammonium, nitrite, and nitrate.
The results showed that NHA and ANH had little effect on the rate at which
ammonium was produced by urea hydrolysis in the three soils studicd. In contrast,
nBTPTA inhibited urea hydrolysis; the inhibition was stronger in the alkaline soil than
in the two acid soils.
Ammonia volatilization from the alkaline soil was insignificantly affected by NHA
and ANH and significantly reduced by nRTPTA, whereas the NH3 loss from the two
acid soils was negligible (0-2 j.lglg soil).
The nitrate content was also very low (0-3 ~Lglg soil) in all reaction mixtures in any
timc pcriod. In the alkaline soil, but not in the two acid soils, production of nitrate was
rctarded by NHA and particularly by ANH during the second week of incubation and by
nBTPTA during the first two weeks of incubation. This effect of NHA and ANH was
attributed to inhibition of nitrification, whereas the effect of nBTPT A was explained by
inhibition of urease activity and thus, by the decreasing amount of ammonium available
for nitrification.
The conclusion drawn was that NHA and ANH did not inhibit urease activity in any
soil, but inhibited nitrification in one of the three soils studied.
Ablizova and Tomina (1997) studied the effect of sodium and ammonium hlU11ates
on the urease activity in a dark-chestnut soil from Kazakhstan. Two experiments were
done, in 199 I and 1992, respectively. In the first experiment, sodium humate (SH) was
applied at rates of 0.5, 1, and 1.5 t/ha before the planting of tomatoes. In the second
experiment, the soil was treated with ammonium humate (A H) (0.3 and 0.6 t/ha) before
the planting of onions. Soil urease activity was measured seasonally in the first
experiment and recorded only in summer in the second experiment. The results have
shown that each rate of SH decreased urease activity in spring and summer and
increased it in autumn. The decrease was more pronounced in sumnler than in spring.
The lower rate of AH caused a 25% inhibition of urease activity, whereas the inhibition
was 42.6% at the higher AH rate.
Albiach et af. (2000) conducted as-year (1989-1994) field experiment on a
horticultural soil at Moncada, Valencia, East Spain to study the effect of organic
amendments, including a commercial humic acid (HA) product (a solution of hurnic
acids containing 20% organic matter), on soil enzyme (including urease) activities and
microbial biomass. The HA solution was applied at a rate of 100 l/ha/year on plots (4.8
by 12.0 m), which also received mineral fertilizers at aru1Ual rates of250 kg of Nih a (as
ammonium sulfate), 120 kg of P20;Jha (as calcium superphosphate), and 250 kg of
K2 0/ha (as potassium sulfate).
Soil enzyme activities and microbial biomass were determined only in the 4th and
5th years. Urease activity was not significantly affected by HA addition in any of these
years.
168
Series 1, the incubation was carried out at 24°C and lasted 69 days. In Series 2, the soil
was analyzed eight times during 3 8-day incubation.
CO 2 evolution from the treated soil increased in the order: U<U+ALS<
U+DAP+ALS during the first 25 days of incubation, then reached a steady state. The
high initial CO 2 evolution in the ALS-treatments was attributed to the stimulated
microbial activity by the sugars contained in the ALS. However, only 10.4-22.2% of the
ALS-C was evolved as CO 2 over the 69 days of incubation, indicating that microbial
degradation of ALS is a slow process.
Urea remained in the soil longer in the U+ALS treatment than in the U treatment,
proving that ALS inhibited urease activity. Urea hydrolysis was not reduced in the
U+DAP+ALS treatment, which was attributed to increased microbial production of
urease in this treatment.
The volatilized NHrN loss during the 69-day incubation, expressed as the
proportion of the added N, was significantly (p<0.05) lower from the U+ALS than from
the U treatment and insignificantly lower from the U+DAP+ALS than from the U+DAP
treatment.
Ammonium-N content after the 38-day incubation was significantly higher in the
U+ALS than in the U treatment and in the U+DAP+ALS than in the U+DAP treatment.
The reverse was true for the nitrate-N content. These results suggest that nitrification
was inhibited by ALS.
It was concluded that ALS has the potential to increase fertilizer N efficiency.
In the laboratory experiments of Xie et al. (1994), urea and ALS (PH 4.5) were
applied at several rates. Fresh samples of a Canadian clay soil equal to 30 g dry weight
were mixed with ALS (0, 25, 50, 100 or 150 glkg soil). Urea solution was added to the
mixtures (0, 500 or 1,000 mg N/kg soil). The moisture content was 70% of field
capacity. The incubation took place at 25°C and lasted 60 days.
It was found that addition of ALS enhanced the transformation ofurea-N into NH4 +-
N and organic N fraction and reduced the production of N03--N. This means that in this
experiment ALS stimulated urea hydrolysis, but inhibited nitrification.
added to the soil. In this case as well, the mixtures prepared with Acacia and inknut
extracts were more effective than the mixture prepared with tea extract.
These observations were explained by assuming that the tea waste served as
substrate for the microorganisms which proliferated and produced new amounts of
urease, whereas the polyphenols present in extracts inhibited not only the soil urease but
also the proliferation of microorganisms.
Wickremasinghe et at. (1981) presented indirect data which confirm that the tea
residues inhibit urease activity in soil. Studying four acid tea soils (PH 4.0-4.5) in Sri
Lanka, these investigators identified a negative relationship between polyphenol content
and urease activity in soils, demonstrated by the following values: 17, 24, 30, and 68 Ilg
polyphenols/g soil and 6.7, 4.5,2.5, and 1.6 Ilg urea-N hydrolyzed/g soillhour. It should
be mentioned that the tea leaf litter contains 15-20% polyphenols.
The relationship between urease activity and polyphenol content in soil was also
studied by Sivapalan et at. (1983). Samples of a red-yellow podzolic soil (clay loam, pH
4.7) from Sri Lanka were treated, in vegetation pots, with the following plant materials
rich in polyphenols: 1. tea shoot tips; 2. mature tea leaf; 3. a mixture (1:1
weight/weight) of I and 2; 4. black tea; and 5. spent tea leaf, or with plant materials
poor in polyphenols: 1. dadap (Erythrina lithosperma) leaf; 2. mana grass
(Cymbopogam c01!{ertijlorus); 3. Guatemala grass (Tripsacum (axum); and 4. tobacco
(Nicotiana tahacum) leaf. The plant material was added at a rate of 5 g/kg soil and the
addition was repeated twice at 4-month intervals, i.e. the total amount was 15 glkg soil.
All soil samples were moistened and incubated in a greenhouse for 12 months, then
analyzed for urease activity, humic matter, and polyphenol contents.
Urease activity, expressed as Ilg NH/-N/g soil/5 hours, increased in the plant
material-treated soil samples as opposed to that of the untreated control soil. The
increase in urease activity was smaller in samples treated with polyphenol-rich plant
materials (from 14.9 to 32.3-51.0, on average, 44.7) than in samples treated with
polyphenol-poor materials (from 14.9 to 60.6-138.7, on average, 97.2). The hurnic
matter and polyphenol contents showed the reverse trend, with values of 12.68-13.80
mg humic-C/g soil and 26.6-69.2 Ilg polyphenols/g soil, respectively, in samples treated
with polyphenol-rich materials, and 11.56-12.19 mg humic-C/g soil and 4.0-6.0 Ilg
polyphenols/g soil, respectively, in samples treated with polyphenol-poor materials. The
smaller increase in urease activity of soil samples treated with polyphenol-rich materials
was attributed to the polyphenols. However, calculations indicated that there were no
significant correlations between urease activity and humic matter or polyphenol content.
This suggests that urease activity is influenced by the interaction of several factors,
including the inhibitory effect of the polyphenols and the stimulatory effect of the
hurnic substances.
The conclusion drawn is that the tea soils humified with tea leaf litter have a
partially inhibited urease activity and therefore are not susceptible to anunonia losses
following urea fertilizer application.
Under the experimental conditions described above, the polyphenol-rich and -poor
plant materials did not exhibit any inhibition of nitrification. This suggests that the soil
urease is more sensitive to polyphenols than are the nitrifying microorganisms
(Sivapalan et aT. 1985).
171
buffer solutions and in a pond water: it was only 2 hours at pH 10, 12.9 days at pH 7,
and 19.2 days at pH 4 in buffer solutions, and 6.91 days in the pond water (PH 8.08). In
other words, AZ-A is very labile at alkaline pHs.
The inhibitory effect of neem cake (cake obtained after expulsion of oil from the
seeds) on nitrification has been known since the 1940's. The study of its effect on urea
hydrolysis in soil was initiated much later.
Reddy and Prasad (1975) added urea or a urea-neem cake mixture (containing by
weight 80% urea and 20% finely powdered neem cake) to 200-g air-dried samples of an
Indian sandy clay loam soil (PH 7.8). The rate ofurea-N addition was 100 ppm (on soil
basis). After thoroughly mixing the soil and fertilizer, enough water was added to bring
the moisture content of soil to field capacity. Incubation was carried out at a mean room
temperature of 29°C. At weekly intervals, the soil was analyzed for residual urea, NH/,
NO)-, and N0 2- contents. The residual urea in the urea-only treatment represented 9, 4,
3, and 0%, of the added urea after 1,2,3, and 4 weeks of incubation, respectively_ The
corresponding values in the urea-neem cake treatment were 15, 12, 8, and 0%,
respectively. In other words, neem cake had only a weak influence on urea hydrolysis.
Its inhibitory effect on nitrification was a little stronger during the first two weeks of
incubation_ Similar results were obtained when this experiment was repeated with
samples of different rice-growing soils (Thomas and Prasad, 1983).
The effects of neem cake on urea hydrolysis, urease activity, and ammonia
volatilization from urea in soil were the objectives of a series of studies performed in
India. First the pot and laboratory experiments, then the field experiments wiu be briefly
described.
Pot and Laboratory Experiments. Nair and Sharma (1979) used I-kg samples of a
silty clay loam (pH 6.9) placed in pots. Urea was applied at rates of 30 and 60 mg N/kg
soil, whereas neem cake had a proportion of 15% by weight of urea. Soil moisture was
brought to field capacity. Incubation took place at 30°C and lasted 4 weeks, during
which the residual urea, ~ +, N0 3 -, and N0 2 - contents were determined weekly. In the
urea-only treatments, 6 and 11 % of the added urea (30 and 60 mg N/kg soil,
respectively) remained unhydrolyzed after 1 week, and the hydrolysis became complete
in 2 weeks. In the urea-neem cake treatments, the corresponding values were 16 and
25% (after 1 week), 7 and 15% (after 2 weeks), 1 and 4% (after 3 weeks), and 0% (after
4 weeks). These values show that the neem cake reduced the rate of urea hydrolysis. It
has also been found that the neem cake was more inhibitory to nitrification than
ureolysis.
Reddy and Mishra (1983) studied the effect of neem cake on urease activity and
ammonia volatilization from urea in samples of an alkaline soil (sandy loam, pH 8.0).
Urea priUs were applied on the surface of moist soil, at a rate of 100 kg Nlha, without
cake or in mixture with 20% (weight/weight) powdered cake. Urease activity, expressed
in mg of hydrolyzed urea/kg soillhour at 30°C, was reduced from 11.8 (soil treated with
urea only) to 9.4 (soil treated with urea + cake). Cumulative NH3 losses in 16 days
decreased, under the influence of cake, by 31.4% as compared to losses registered in the
urea-only treatment. However, the neem cake was less effective than p-benzoquinone
used at a rate of 1% relative to weight of urea.
The inhibiting effect of neem cake on urea hydrolysis in flooded soils was also
reported by Hulagur and Shinde (1984).
173
In the pot experiments of Bopaiah and Biddappa (1987), 500-g samples of three
acid, cocoa palm (Cocos nucifera)-growing, soils (coastal sand, red sandy loam, and
laterite) were submitted to the following treatments: urea alone (250 ppm); urea mixed
with 30% neem cake powder; neem cake and coaltar mixed with urea in the presence of
kerosene (1 %); coir' dust coated with urea (at 9: 1 ratio). Soil moisture was kept at 50 %
of WHC during incubation (29°C). In each of these treatments, only trace amounts of
urea were detected after 10 days of incubation of the sandy loam and laterite soils. In
the coastal sand, unhydrolyzed urea was found, even after 10 days of incubation, in the
following amounts (in mg Nil 00 g soil): 0.68 (urea <IDly), 1.14 (urea + cake), 1.18 (urea
+ cake + tar), and only traces (urea + coir dust). In other words, neem cake did not
prove to be a potent inhibitor of urea hydrolysis in any of the three soils studied.
In the laboratory experiment performed by Sharma and Gupta (1989), 250-g air-
dried samples of an alkaline sandy soil (pH 8.2) were moistened up to and maintained at
field capacity. Urea prills at a rate of 100 kg Nlha were surface-applied. The other
treatments comprised: urea mixed with neem cake (1: 1 weight/weight); urea coated with
neem oil (urea dipped for half an hour in neem oil and then taken out and dried); urea
coated with shell-lac. During incubation (for 18 days at 35°C), the evolved ammonia
was assessed. Cumulative volatile NH3 losses in 18 days were the following: 20.4%
(urea only), 13.2% (urea + neem cake), 11.2% (urea + neem oil), and 9.1 % (urea +
shell-lac) relative to the added urea-No This means that the amendments reduced the
volatile NH3 losses from urea.
Mahajan and Tripathi (1991) collected samples from different depths (0-15, 15-30,
30-45 ,and 45-60 cm) of a silt loam soil (PH 5.9). The samples were ground and passed
through 2-mm sieve and packed in polyethylene columns (of 75-cm length and 8-cm
diameter) in the same order as they were obtained from the field. Five sources ofN (115
ppm): urea, urea supcrgranule (USG), lac-coated urea (LCU), sulfur-coated urea (SCU),
and neem cake-coated urea (NCCU) were mixed in the upper 0-10 em soil layer. Two
methods of watering were used: a) submergence (5-cm standing water was kept
constantly on the soil columns) and b) intermittent watering (the soil was irrigated to
maintain saturation). To determine leaching losses, the leachates were analyzed for urea,
NH/, and N0 3' at different intervals during 61 days of incubation and finally the
cumulative losses were calculated.
Total cumulative N leaching losses from the five N sources expressed as percentages
of the added N increased in the order:
SCU (l9.0%)<LCU (34.9%)<USG (S1.9%)<urea (62.S%)<NCCU (67.6%) under
submergence, and
USG (35J)%)<SCU (57.8%)<urea (65.5%)<LCU (76.6%)<NCCU (90.8%) under
intermittent watering.
These data show that leaching losses of N were lower under submergence (except
for USG) than under intermittent watering, and the losses were always highest from the
NCCU. Under submergence, the major form of the cumulative leaching losses ofN was
urea-N from each N source, but under intermittent watering, these losses were in form
of N0 3--N from LCU and SCU, in form of both urea-N and NH/-N from USG and
urea, and in the form ofurea-N (34.2%), NH/-N (21.5%), and N0 3 --N (35.1%) from
'Coir is a material for cordage, matting etc., consisting of prepared fibres ofthe outer husk of the coconut.
174
NCCU (which indicates that in this experiment the neem cake inhibited urea hydrolysis
and nitrification almost to the same extent).
Kubade and Mohite (1994) found that neem cake coating on urea retarded the
release of NH4 + from urea and nitrification of NH4 + to a lesser extent in samples of a
gypsum-amended saline sodic loamy soil (pH 8.8S) than in samples of a nonsaline clay
soil (pH 8.20).
In a pot experiment, Singh et af. (1996) studied the effect of various soil water
regimes on the volatilization of ammonia from prilled urea, urea supergranule (USG),
lac-coated urea (LCU), and neem oil-coated urea (NOCU). Samples (3.S kg/pot) of a
clay soil (pH 8.2) were used. Deionized water was added to the samples to bring the soil
to field capacity, SO% field capacity or saturation. Other samples were submerged under
a 3-cm water layer. The urea rate was 131.4 mg N/kg soil. The USGs of I-g weight
were placed at S or 10 cm below the surface. NOCU was obtained by mixing urea
granules and neem oil in a proportion of 1: 1 (weight/weight). The prilled urea and the
coated ureas were surface-applied. The volatile NH3 was measured during 4 weeks.
Depending on the water regimes, the cumulative NH3 losses from each N source
presented the order:
field capacity>SO% field capacity>saturated>submerged.
Depending on the nature of N sources, the following order of the cumulative NH3
losses was established:
prilled urea>USG placed at Scm >USG placed at 10 cm>LCU::::NOCO.
Consequently, in this experiment, urea granules coated with neem oil or lac were
more efficient than urea supergranules in reducing the volatile NH3 losses.
Field Experiments. To study the effect of neem cake (ID leaching losses of N from
urea, Singh and Singh (1986) used lysimeters installed in the field on a silty clay loanl.
The lysimeters had the dimensions of 120 cm in diameter and ISO cm in depth. The test
plant was wheat. The experiment was conducted over two years (1980/81 and 1981182).
The soil was fertilized with 0,60, and 120 kg urea-N/ha or with the same N rates of
urea to which neem cake was mixed in a proportion of 1: I by weight. The soil was also
fertilized with 36 kg of P (as single superphosphate) and 33 kg of Klha (as muriate of
potash). All fertilizers were mixed in the surface soil and then wheat seed~ were sown.
Soil was irrigated and the leachate was collected at 20-day intervals and analyzed for
NO]' and NH/. The soil was also analyzed for N0 3' and NH/. Total N content in the
plants was also detemlined.
In both years and at both urea-N rates, the neem cake significantly decreased the
leaching loss and downward movement of NO] -N, which was attributed to its inhibiting
effect on nitrification. At the same time, neem cake addition resulted in increased
leaching loss and downward movement ofNH/-N, which can be interpreted as lack of
its urease-inhibiting effect. However, the total (N03'-N + NH/-N) loss from the added
N was significantly reduced by the neem cake. The reduction was IS and 22% in the
first and second year, respectively, at the 60 kg urca-Nlha rate, and 33% in both years,
at the higher urea-N rate (120 kg/ha).
Agrawal et al. (1988) carried out a field experiment on microplots (l m 2), moistened
to field capacity. Urea granules (S g), urea + neem cake (I 6% weight/weigllt), urea +
neem oil (1'% weigllt/weight) or urea supergranules (S balls of I g each) were placed in
the center of microplots at IS cm depth. Soil samples were drawn from each microplot,
laterally designated as LJ (IS cm), L2 (30 cm), and L3 (4S cm) and vertically as DJ
175
(5 cm), D2 (15 cm), and D3 (25 cm) from the point offertilizer placement, after 7, 21,
and 28 days and analyzed for NH/ and N0 3-. Diminution of urea hydrolysis was
calculated for the LJD2 sample at day 7, obtaining the following values: 20.7% (urea +
neem cake), 27.9% (urea+neem oil), and 33.8% (supergranules), relative to the urea-
only treatment. It is evident that neem cake and oil inhibited urea hydrolysis, but this
process was best regulated through supergranule application. Neem cake and oil also
inhibited nitrification.
Santra et al. (1988) studied the effect of neem cake on volatilization of ammonia
from urea in a lowland rice field on an alluvial silty clay loam (PH 8.4). Urea was
applied in form ofprilled urea (90 kg Ntha) or neem cake-coated urea (90 kg Ntha) or
prilled urea (45 kg Ntha) in combination with a green manure [6-week-old dhaincba
(Sesballia aculeata) plants] incorporated into the soil at a rate of2.2 ttha for 45 kg Ntha.
After fertilization the evolved NH3 was measured. During 15 days, the cumulative
volatile NIl3 losses were highest (12.9-13.4%) in the urea-only treatment and lowest
(5.5-7.0%) in the urea + green manure treatment, and intermediary in the urea + neem
cake treatment. This means that the NIl3 losses were reduced under the influence of
neem cake.
A similar experiment was conducted by Mishra et at. (1990) on a rice field. The soil
of the plots was a silty clay loam (PH 7.6). Prilled urea, neem cake-coated urea, and
shell-lac coated-urea were applied at a rate of 90 kg urea-Ntha. In the dhaincha + urea
treatment, shoots of this plant as green manure supplied 45 kg Ntha and the rate of urea
was 45 kg Ntha. The fertilizers used as basal dressings were applied before
transplantation of rice plants. After transplanting the plots were kept flooded (5-7 cm)
until flowering stage. The prilled urea was also applied in three splits: 45 kg Ntha as
basal dressing + 22.5 kg Ntha as top dressing at tillering + 22.5 kg Ntha as top dressing
at panicle initiation. Ammonia volatilization was determined during 15 days after rice
transplanting. After harvest, the grains and straw were analyzed for total N.
The cumulative NIl3 losses, expressed in kg Ntha, presented the order:
prilled urea (19.22), green manure + urea (14.41), shell-lac-coated urea (13.47),
neem cake-coated urea (13.08).
It is evident that neem cake was most effective in reducing the volatile NIl3 losses
from urea. But the total N uptake by rice plants was highest in the treatment with split
application of urea.
of a furan ring in the karanjin molecule is the crucial structural factor for the inhibitory
activity.
Kamire and Sonar (1979) initiated a study for evaluating the effect of karanja cake
on both urea hydrolysis and nitrification in a calcareous soil (PH 8.1). Neem cake served
for comparison.
Soil samples (300 g) were uniformly mixed with 300 mg of N in the form of urea
alone and with urea replaced either by karanja or neem cake on N basis in proportions
of 25,50, 75 or 100%. All samples were moistened and incubated at 30D e for 60 days.
During incubation NH/ and N03 ' contents in soil were periodically determined. In the
presence of cakes, smaller amounts of NH4+ and N0 3' were produced than in their
absence. The rate of urea hydrolysis and nitrification decreased with an increase in the
proportion of cakes added to urea. Karanja cake was better than neem cake in delaying
the availability ofurea-N.
This order means that the neem and mahua cakes were more inhibitory than the
other coating materials. The inhibitory effect of all coating materials was stronger at
their higher rather than lower rate.
Under conditions of the 5-hour test, Bremner and Douglas (1971) established that a
series of compounds (benzene, anisole, veratrol, benzoic acid, vanillic acid, maleic acid,
citraconic acid, phthalimide, iodoacetic acid, and iodocetarnide), used at a rate of 50
ppm (on soil basis), gave less than 4% inhibition of urease activity in the three Iowa
soils studied.
For inhibiting soil urease activity, Liesegang et al. (1976) patented the compounds
which have the general formula NC-CX 2-C(O)NR]R2 , where X=H, CI, Br or J; R] and
R2 = C]-C5 alkyl, cyclohexyl or part of a heterocycle with N. Evaluation of the
inhibitory effect was performed with 30-g soil samples mixed with 214.1 mg of urea
(100 mg N) and 4 mg of test compound, then moistened to 50% ofWHC and incubated
at 30°C for 24 hours, during which time the evolved ammonia was assessed.
Satisfactory inhibitions occurred even when the rate of test compound was reduced from
4 to 0.5 mg. It was also found that these compounds are more persistent in soil than p-
benzoquinone. They are recommended as additions to urea at preferred rates of 0.05-5%
relative to urea-N for fertilization of light- and medium-textured soils, meadows and
pastures.
Applying the short and long time tests (see page 51), Hartbrich et al. (1978) found
that bis(acetylvinyl) sulfide is a weaker soil urease inhibitor than mucochloric acid.
Sahrawat (1979) studied, using the 5-hour test, the effect of several chelating
compounds [nitrilotriacetic acid (NTA) trisodium salt, ethylenediarninetetraacetic acid
(EDTA) disodium salt, tartaric, citric, and oxalic acids], used at a rate of 50 ppm (on
soil basis), on the urease activity of an Indian sandy clay loam alluvial soil. The
inhibition degree was 4% (NTA), 1% (EDTA), and 0% (other compounds). It should be
mentionated that in the red-brown earth studied by Cai et al. (1989) EDTA gave more
marked inhibitions, ranging from 1.2 to 39.6% (see page 209).
According to Liao and Raines (1982), p-phenylenediamine, as an inhibitor of soil
urease activity, is comparable to organic mercury compounds, polyhydric phenols, and
quinones as well as phosphoryl and thiophosphoryl triarnides.
In the tests described by Kiss and Pintea (1987) (see page 35), the time required for
complete hydrolysis of urea was, under the influence of p-anisidine, prolonged from 6
to 10 days in an alluvial soil and reduced from 9 to 8 days in a leached chemozem. The
reaction mixtures were prepared from 5 g of air-dried soil, 10 rnl of aqueous phase
containing 6 mg of urea and 0.12 mg of p-anisidine; incubation took place at laboratory
temperature.
Caffeine, used at concentrations of 20,40, 60, 80, and 100 ppm (on soil basis) and
nicotine at concentrations of 60, 80, and 100 ppm, manifested an inhibitory effect on
urease activity in samples of a periodically water-logged paddy soil. The reaction
mixtures, consisting of 5 g of dry soil, 10 ml of 1% urea solution with or without
caffeine or nicotine, were incubated at 37°C for 48 hours. It should be noted that in the
case of caffeine the inhibition degrees were not significantly different (48.01-48.89%) at
the 20-60 ppm concentrations and were lower (43.61%) at the two higher
concentrations, but nicotine gave maximum inhibition (45.81 %) at its highest
concentration (Xue and Li, 1987).
179
Geissler et al. (1970), of the Esso Research and Engineering Company worked, as
specified in their patented invention, with pellets prepared only from urea and CUS04,
i.e., without any hydrophobic material. Before pelletizing, the urea was melted at 130-
135°C, then CUS04 was added (in amounts of 0.01-1 0%, most preferably 0.2-4% based
on the weight of urea) to the urea melt and uniformly dispersed in the melt by stirring.
Some pellets were prepared from urea + borax or from urea + CUS04 + borax or from
urea + CUS04 + borax + sodium fluoride (NaF). Urea prills without inhibitor served for
comparison.
In an experiment conducted to study ammonia volatilization from urea, the pellets
(in an amount equivalent to rates at which urea is generally applied under field
conditions) were placed on the surface of moist soil samples. The cumulative NH3
losses measured during the incubation period (20 days) and expressed as percentages of
the added urea-N gave the following mean values (in the different treatments): 43.3%
(urea only); 26.2% (urea + 2% CUS04); 23.8% (urea + 4% borax); 20.3% (urea + 1%
CUS04 + 2% borax); 28.0% (urea + 0.6% CUS04 + 1.4% borax + 0.33% NaF). It is
evident that NH3 loss was lowest from the pellets that had been prepared by adding 1%
CUS04 + 2% borax to the urea melt. It should be mentioned that the NH3 losses did not
significantly diminish when the urea prills used had been merely coated with the same
amounts of the same inhibitors.
In another experiment, the same CUS04 amount was spread on soil samples at
intervals of 3, 10 or 17 days before the urea prills (without inhibitor) were applied to the
soil surface. After adding urea, the soil samples were submitted to incubation, during
which NH3 volatilization was assessed. The results showed that in this experiment
CUS04 had very little effect on reducing the rate of NH3 volatilization from urea.
However, this experiment was not repeated with borax or inhibitor mixtures (CUS04 +
borax and CUS04 + borax + NaF).
Huang et al. (1993) patented a long-acting urea fertilizer prepared by adding urease
inhibitors (Zn, Mn, Cu, Fe, B, and Ba) to the fertilizer composition.
Sor et al. (1971), also of the Esso Research and Engineering Company, prepared three
kinds ofCH 20-containing urea pellets:
a) 2% CH20 + 2% borax + I % hydrophobic material (Marcol 72);
b) 1% CH 20 + 1% octadecylamine (ODA);
c) 1% CH 20 + 4% CUS04 without any hydrophobic material.
Volatilization of NH3 from these pellets applied to soil samples incubated for 3 days
was lower than from the control (no inhibitor) pellets. The NH3 losses were reduced
from 2.65-3.08 to 1.28% (pellets a), from 4.95 to 3.93% (pellets b) or to 3.07% (pellets
c). It should however be mentioned that application of urea-borax-ODA pellets led to a
more marked reduction ofNH3 loss from urea.
180
Sor et al. (1971) also prepared pellets from urea + 4% borax + 1% acetaldehyde or
1% p-to1ualdehyde or 4% a-naphtha1dehyde which proved to be more effective in
reduction of NH3 loss than pellets b and c, as - under similar conditions of incubation -
the NH3 loss was reduced from 4.95% (control pellets) to 1.13% (urea-borax-
acetaldehyde pellets), 0.54% (urea-borax-tolualdehyde pellets), and 0.48% (urea-borax-
naphthaldehyde pellets).
According to the invention patented by Neumann and Richter (1976), molten urea
(1 mole) was mixed with a) 0.02 moles ofHMTA; b) 0.0238 moles ofHMTA + 0.0145
moles of CuS04.5H20; c) 5.4 g of the dehydrated product of the reaction between
1 mole ofHMTA and 1.78 moles of boric acid; d) 0.025 moles of HMTA + 0.05 moles
of phosphoric acid (80%), then the mixtures were pelletized. Another method was also
used: urea and inhibitor(s) were melted together and the resulted melt was transformed
into pellets. Pellets containing urea only were the controls.
Volatilization of NH3 from the pellets was tested with 600-g samples of an arable
soil (moisture content:::: 50% of WHC). The pellets placed on the surface of each soil
sample contained 100 mg N. During incubation (at 30°C for 10 days), the volatilized
NH3 was measured. In comparison with NH3 volatilization from control pellets (100%),
the following NH3 volatilization values were recorded (in the different treatments): 15%
(urea + HMTA), 7% (urea + HMTA + CUS04), 5.6% (urea + HMTA + boric acid), and
9% (urea + HMTA + phosphoric acid). It should be added that HMTA also has a
physical effect: it reduces aggregation of urea pellets and, thus, makes it possible to
store the fertilizer for a longer period.
Pugh and Waid (1969a,b) found that acetohydroxamic acid and sulfanilamide, when
applied together, exhibited a synergistic effect in inhibition of ammonia volatilization
from urea-treated samples of three English soils (see page 163).
As already shown in Table 18, catechol and hydroquinone acted synergistically with
2,5-dimethyl-p-benzoquinone in inhibiting volatilization of ammonia from urea-treated
soils (Anderson, 1969, 1970).
Thieme et al. (1976) proved that this inhibitory effect of hydroquinone, p-benzo-
quinone, and quinhydrone was more intense and long-lasting when were used in
combination with tetramethylthiuram disulfide (see also page 88).
181
The data in Table 42 show that hydroquinone (HQ), 1:1 mixtures of HQ + maleic
hydrazide (MH), sodium or ammonium salt of MH prolonged complete hydrolysis of
urea in a light-textured (alluvial) soil by 4 days. In a heavier-textured soil (leached
chemozem) HQ acted more markedly than did the mixtures, complete hydrolysis of
urea having been prolonged by 7 and 5 days, respectively. In both soils, MH was a weak
inhibitor. The reaction mixtures, prepared from 5 g of air-dried soil + 10 ml of aqueous
phase containing 6 mg of urea + 0 or 0.12 mg of test compound or mixture of
compounds, were incubated at laboratory temperature (Kiss and Pintea, 1987).
TABEL 42. Effect of combined use of hydroquinone and maleic hydrazide on soil
urease activity"
Time necessary for complete
Co~ound or mixture of
hydrolysis of urea (days)
compounds
Alluvial soil Leached chemozem
Control 6 9
Hydroquinone (HQ) 10 16
Maleic hydrazide (MH) 9 12
HQ + MH (1:1) 10 14
HQ + MH sodium salt (1:1) 10 14
HQ + MH ammonium salt (1:1) 10 14
"Adapted from Kiss and Pintea (1987).
Based on laboratory experiments in which the short and long time tests (see page 51)
were applied, but the inhibitors were used at four rates relative to urea-N (0.5, 1,2, and
4% in the short time test, and 0.5, 1, 1.5, and 2% in the long time test), Hartbrich et al.
(1976b) patented not only PPDA, but also its mixtures with mucocbloric acid as
inhibitors of soil urease activity. The inhibitory effect of the two compounds in mixture
was strongly synergistic, especially when the molar ratio between them was 1: 1. Thus,
this PPDA-mucocbloric acid mixture at total rates of 0.5, 1, 1.5, and 2% inhibited
ammonia volatilization from urea for 10, 14,20, and 20 days, respectively. At 1.5 and
2% rates, the inhibitions were considerable even after 24 days of incubation (81 and
85%, respectively). Under the same conditions, total inhibitions by PPDA alone and
mucocbloric acid alone lasted only 4 and 6 days, respectively. Mixtures of PPDA-
mucochloric acid were recommended for practice at rates of 0.01-20%, preferably at
0.05-5% relative to urea-No
Lang et af. (1976) found that PPDA used with thiram (in proportions of 1:2.33 and
1:9, at a total concentration of 1% relative to urea-N) or with ferbam (in a proportion of
1:4, at 1% total concentration) or with dawmet (in a proportion of 1:1, at 2% total
concentration), 4-chloro-PPDA used with thiram (in proportions of 1: 1 and 1:9, at 1%
total concentration), and di-PPA used with thiram (in proportions of 1:4 and 1:9, at 1%
total concentration) manifested a synergistic effect with these compounds in inhibition
of soil urease activity, increasing the degree and prolonging the duration of inhibition.
182
at 0.5% rate) applied in succession at 3-day intervals. The rate of urea added was 75 kg
Niha.
There were seven treatments without algicide: 1. control (only urea); 2. nBTPTA; 3.
nBTPTA repeated; 4. mixed inhibitors; 5. Cac2; 6. nBTPTA + Cac 2; and 7. mixed
inhibitors + Cac 2 , and six treatments with algicide (algicide was not added to the
repeatedly applied nBTPTA). First the algicide, then the urease inhibitors and, finally,
the urea were broadcast into the floodwater. During 10 days after urea application,
floodwater pH, ammoniacal (NH3 + NH/)-N, temperature as well as wind speed were
measured and from these data the ammonia losses were calculated.
The cumulative NH3 losses measured in treatments with and without algicide and
expressed as percentages of the applied N were the following: 19.5 and 10.4 (control),
15.8 and 14.7 (nBTPTA+CaC 2 ), 15.2 and 12.4 (Cac2 ), 13.8 and 13.l (mixed
inhibitors+CaC 2 ), 9.8 (nBTPTA repeated), 9.3 and 7.6 (nBTPTA), and 9.1 and 7.0
(mixed inhibitors), respectively.
It is evident from these numerical data that the urease inhibitor mixture was most
efficient in reducing the volatile NH3 losses; the algicide had, in each treatment, a loss-
reducing effect, but Ca~ diminished this effect of urease inhibitors, although when
used without urease inhibitors CaC 2 also reduced the NH3 loss; nBTPTA in a single
application was more effective than in multiple applications and approached the
effectiveness of mixed inhibitors.
The second experiment, described by Phongpan et al. (1995, 1997), was carried out
during the dry season of 1992. The algicide terbutryn (again at 0.2 mg/l floodwater) was
applied at 4-day intervals in all treatments. The urease inhibitors studied were nBTPTA
and PPDA used alone or in combination. Their rates (on urea weight basis) were 0.5,
1.0, and 2% when used alone, and 0.5+0.5, 1.0+0.5, and 2+0.5% when used in
combination. Urea was applied at 60 kg Niha rate. The order of the additions to the
floodwater was, as in the first experiment, algicide, urease inhibitors, and urea. The
control received only algicide and urea. Ammonia volatilization was determined during
11 days after urea application.
It was found that the rate of inhibitors used alone or in combination had no
significant effect on NH3 loss, which means that the 0.5% inhibitor rate was sufficient to
inhibit soil urease activity and thus to reduce NH3 volatilization. The cumulative NH3
losses from the added urea-N had the following mean values: 15.0% (control), 5.4%
(nBTPTA), 7.3% (PPDA), and 3.0% (nBTPTA+PPDA), which proves the high
efficiency of the combined use of the two inhibitors.
To explain the strong urease-inhibiting effect of the nBTPTA+PPDA combination,
the suggestion made by Luo et at. (1994) (see page 182) was reiterated.
A shorter report on the second experiment was published by Freney et al. (1993).
Al-Kanani et at. (1990b) studied the effect of three inhibitors: humic substance (HS)
from leonardite, boric acid (BA), and ammonium thiosulfate (ATS) and two inhibitor
combinations: HS + ATS and BA + ATS on the transformation of urea-ammonium
nitrate fertilizer in a sandy clay loam soil (PH 6.5) and a sandy soil (PH 5.9) from
Quebec.
184
TABLE 43. Effect of humic substance (HS), boric acid (BA), ammonium thiosulfate (ATS), and combinations
ofHS+ATS and BA+ATS on transformation of urea-ammonium nitrate fertilizer in two Canadian soils·
____~~~~~--~~I~nh~ib~it.io~n~(~%~)----~~~~------
San~ c1a~ loam soil Sand~ soil
Inhibitor
Ammonia Urea Nitrifi- Ammonia Urea Nitrifi-
Volatilization hydrolysis cation vo latili zatio n hydrol~sis cation
HS (1.7%) 21.1 16 29.0 15.3 5 34.2
HS (3.4%) 20.2 22 25.5 17.4 11 34.2
BA (1.8%) 13.6 IS 20.0 3.2 5 15.5
BA (3.6%) 21.1 30 23.5 15.8 12 18.7
ATS(1.8%) 31.9 22 -2.6 33.7 7 9.3
ATS(3.6%) 37.1 25 -12.0 24.7 9 -4.7
HS (1.7%)+ATS (1.8%) 38.5 26 21.5 36.3 4 18.7
BA (1.8%)+ A TS (1.8%) 35.7 28 14.5 31.1 6 12.4
"Adapted from A1-Kanani et al. (1990b).
weight of VAN solution) of BA and ATS and 1.7 and 3.4% of the 0.1%
(weight/volume) HS were used. The samples were moistened and incubated at
laboratory temperature. Ammonia volatilization, urea hydrolysis, and nitrification were
evaluated after 10 days of incubation. The results are summarized in Table 43.
These results show that the combination HS+ATS was most inhibitory on NH3
volatilization, but the strongest inhibition of urea hydrolysis and nitrification was
caused by BA and HS, respectively. Surprisingly, the effect of ATS on nitrification was
stimulatory rather than inhibitory. The combination of HS+ATS was in general, more
effective than BA+ATS. The environmentally friendly HS as an inhibitor of soil urease
and/or nitrification should be considered superior to BA and ATS.
The NH3 volatilized was measured daily during l5-day incubation at 23.1°C. After
incubation the soil was analyzed for residual urea, ~+, and (N02- + N0 3}
Cumulative NH3 losses were highest from samples treated with urea only and
decreased with each inhibitor and inhibitor combination_ The extent of this effect
depended on the nature of inhibitors as well as on form and place of the urea
application.
Following surface application of solutions and dry blends and banded application of
dry blends and tablets, the decreasing effect on NH3 volatilization is as follows:
nBTPTA:::::: PPDA > ALS > ALS+PPDA:::::: ALS+nBTPTA.
In the case of surface-applied tablets, the order was:
nBTPTA:::::: PPDA:::::: ALS > ALS+nBTPTA:::::: ALS+PPDA.
These orders indicate that, in these experiments, PPDA was always as effective in
decreasing the volatile NH3 losses as nBTPTA, but ALS exhibited a comparable
effectiveness only when it was surface-applied in urea tablets. ALS used in
combinations limited the urease-inhibiting effectiveness of PPDA and nBTPTA. The
mechanism(s) by which ALS and PPDA or nBTPTA interact has not been delineated; it
was only supposed that the soil urease reacts with the negatively charged ALS and this
reaction makes the enzyme less susceptible to the inhibitors.
It follaws from the soil analyses that recovery ofN from the inhibitor-amended urea
solutions and dry blends increased in the same order as the decrease of volatile NH3
losses. However, recovery ofN from the tablets is partially different:
nBTPTA> ALS:::::: PPDA > ALS+nBTPTA:::::: ALS+PPDA from the surface-applied
tablets, and
nBTPTA:::::: PPDA > ALS+PPDA > ALS+ nBTPTA:::::: ALS from the banded
application of tablets.
187
These studies will be grouped according to the compound which proved to be the most
efficient inhibitor in the respective comparative study. But this criterion of grouping
will not be applied for those comparative studies in which a well-known urease inhibitor
was used as a reference compound.
Laboratory Experiments. Xue and Li (1987) have found that CUS04 was a stronger
inhibitor of soil urease activity than were the plant materials (see Section 2.31.3 .3).
Sheudzhen et al. (1991) used surface (0-20 em) samples of a chernozemic soil
cultivated with rice in Russia. The samples were NPK-fertilized (N 180 kWba as urea, P
120 kWba as double superphosphate, K 60 kWba as KCI) and treated separately with Zn,
Mn, Cu, and Co (4, 4, 3, and 2 kWba, respectively, as sulfates), Mo (2 kglha as
ammonium molybdate), and B (2 kglha as boric acid). The control samples received
only NPK. Urease activity was assayed after 4 and 8 days. After both 4 and 8 days,
urease activity showed decreased values in the Zn- and Mn-treated samples and
increased values in the Mo-treated ones. Co did not influence, while Cu like B
decreased urease activity during the first 4 days and increased it later.
Sengik and Kiehl (1995b) have compared the effect of five inorganic salts (ferric
chloride, ferrous sulfate, simple and triple superphosphate, and monoammonium
phosphate) on volatilization of ammonia from peat- and urea-treated and moistened
samples of a red earth (PH 4.9) from Brazil. Peat and urea were added at rates
equivalent to 10 t and 72 kg Nlha, respectively. The ratios between the weight of salt
and that of the urea were 1:1,2:1, and 3:1. Soil samples treated only with peat and urea
were the controls. All samples were incubated for 22 days, during which the volatilized
NH3 was determined daily. Soil pH was measured periodically during 18 days.
The cumulative volatile NH3 losses were reduced by the salts at their 1:1, 2:1, and
3: 1 ratios to urea in the following proportions expressed as percentages of the NH3 loss
from the controls: 23, 52, and 70% by ferric chloride; 16, 46, and 49% by ferrous
sulfate; 14, 17, and 19% by simple superphosphate; 0, 0, and 25% by triple
superphosphate; and 0, 0, and 11 % by monoammonium phosphate.
Soil pH was also reduced by the salts in parallel with their reducing effect on NH3
volatilization.
It is evident from these data that the two iron salts were most effective in reducing
NH3 volatilization and soil pH. Their effect on soil urease activity was not evaluated.
Field Experiments. Sanikidze et al. (1987b) compared the effects of Zn and B on the
urease activity of a red earth under mandarin (Citrus nobilis) plantation in the humid
subtropical area of western Georgia (Gruzia). All experimental plots were NPK-
fertilized and limed in the 1974-1977 period. Some plots received, additionally, 4,8 or
12 kg Zn (as sulfate)lha or 3,6, and 9 kg B (as boric acid)lha, in the 1977-1982 period.
Urease activity in the 0-20-cm soil layer was measured systematically in the 1981-1983
period. For this period, the mean values of urease activity (expressed in mg NH3
produced by 1 g soil in 24 hours) were, in the different plots, the following:
188
0.3 (control) < 0.36 (12 kg Zn/ha) < 0.4 (8 kg Zn/ha) < 0.5 (4 kg Zn/ha, 3 and 9 kg
B/ha) < 0.6 (6 kg B/ha).
This order means that, at the applied concentrations, both Zn and B stimulated
urease activity, this effect of Zn being weaker than that ofB.
Laboratory Experiments. Sloan and Anderson (1995) compared the effects of CaCh and
ammonium thiosulfate (ATS) to reduce ammonia volatilization from surface (0-4 cm)
samples of two soils possessing contrasting physical and chemical properties, namely an
acid Lufkin fine sandy loam soil (pH 4.9) and a calcareous Ships clay soil (PH 7.8),
both located in Brazos County, Texas.
The air-dried soil samples (30 g) wetted to 20% moisture (Lufkin soil) or to 30%
moisture (Ships soil) were incubated at 25°C for 24 hours in order to reestablish
microbial activity, then urea was surface-applied at a rate equivalent to 200 kg N/ha
based on surface area of soil samples. CaCl 2 was used at a Ca:N equivalent weight ratio
of 0.25, whereas ATS was applied in 10% amount relative to weight of urea. The
volatilized NH3 was measured for 192 hours (Lufkin soil) and for 408 hours (Ships
soil).
Inhibition of NH3 volatilization by CaCb and ATS at 24 hours after fertilizer
application was 84.9 and 8.1 % respectively (Lufkin soil), and 42.1 and 2.6%,
respectively (Ships soil). But the inhibitory effect of CaCl 2 decreased, and that of ATS
slightly increased with time. Thus, at 48 hours the inhibition of NH3 volatilization by
eaCb and ATS was 20.9 and 9.6%, respectively (Lufkin soil), and 32.5 and 5.1%,
respectively (Ships soil). The corresponding values registered at 192 hours in the Lufkin
soil were 1.2 and 11.1 %, and those registered at 408 hours in the Ships soil were 8.1 and
1.0%. The conclusion can be drawn that eaCh was always more effective than ATS in
the Ships soil, but only during the first 48 hours in the Lufkin soil.
In another experiment, in which the soil samples were submitted to rapid drying
after fertilizer application, CaCb significantly (p<0.05) reduced NH3 volatilization from
both soils, whereas the effect of ATS was not significant in the Lufkin soil, either.
Laboratory Experiments. Sor et al. (1971) have compared ammonia volatilization from
urea granules containing an inhibitor (borax, CUS04, NH4F, formaldehyde, thiourea or
o-phosphoric acid) and octadecylamine or another hydrophobic material. The granules
were surface-applied on samples of a loamy sand soil (pH -6.4) from New Jersey. The
volatile NH3 was measured during 3-4 days. The results showed that the volatile NH3
loss was reduced most effectively by borax and least effectively by CUS04.
Bayrakli (1990) evaluated the effect of five compounds on the volatilization of
ammonia from urea-treated samples of a representative alluvial soil (PH in KCl 7.52) of
the Bafra Plain, Turkey. The effect of test compounds on the rate of urea hydrolysis was
also estimated.
189
The 40-g air-dried samples were treated with 40 mg N as urea and with nonidentical
amounts of the test compounds, namely with 2 mg of thiourea (TU), hydroquinone
(HQ) or 2,4-dinitrophenol (DNP), 20 mg of H3B03, and 100 mg of CaCI 2. The control
received only urea. All samples were moistened and incubated for 21 days (at 38 C for
Q
Laboratory Experiments. McCarty et al. (1991) prepared reaction mixtures from 5-g
air-dried samples of two Iowa soils (a silty loam and a sandy clay loam), 2 ml of water
containing 10 mg of urea or 10 mf of urea and different amounts of the compounds
compared. The mixtures were incubated at 20 Q C and, after 3 and 10 days, analyzed for
the residual urea. The compounds and their amounts were the following: sodium
thiosulfate and tetrathionate 1,000, 2,500, and 5,000 J.lglg soil; Fe 2+ (as FeS04) and
Mn2+ (as MnCl 2) 100, 250, 500, 1,000, and 2,500 J.lglg soil. Phenylphosphorodiamidate
(PPDA) was the reference compound used in an amount of 10 J.lglg soil.
Thiosulfate was found to be less inhibitory on soil urease activity than was
tetrathionate. The inhibiting effect of Fe2+ and Mn + was inexistent or very weak. As
expected, PPDA was the strongest inhibitor. Thus, at the 2,500 J.lglg soil addition, the
percent inhibitions of urease activity in the two soils after 3 and 10 days of incubation,
respectively, were: 18 and 3%, and 0 and 0% for thiosulfate; 48 and 22%, and 0 and
21 % for tetrathionate; 8 and 11 %, and 0 and 0% for Fe2+; and 10 and 10%, and 0 and
4% for Mn2+. The corresponding values for PPDA used, as mentioned above, only in the
amount of 10 J.lglg soil, were 89 and 93%, and 22 and 32%.
Using samples of three Kansas soils treated with Na2S203.5H20, Na2S406.2H20,
FeS04.7H20 or MnS04.H20 at rates of 0,0.05,0.25,2.50 or 25 mM S/kg soil, Sullivan
and Havlin (1992b) registered the following maximum urease inhibition levels: 30% for
S20/-, 29% for s40l-, 14% for Fe 2+, and 8% for Mn2+.
190
On day 10, urea was not detectable in the soil. Small amounts of urea were present
in floodwater, in which the inhibitory effect in the order PMA> HQ > NKE remained
evident. By day 20, urea disappeared from the floodwater, too.
In the urease activity experiments, the activity was measurable in soil and unfiltered
floodwater during the whole 60-day incubation period (through filtering urease was
removed with the suspended particles and colloids).
The inhibitors decreased urease activity in the order PMA > HQ > NKE, which
confirms the results of the urea hydrolysis experiments. Although this order of
inhibitors remained unchanged during prolongation of the incubation time, urease
activity increased with time in the inhibitor-treated soil. For example, urease activity
(expressed in Ilg urea-N/g soil/hour) in the nonflooded and noncropped soil was 0.993
(control), 0.896 (NKE), 0.653 (HQ), and 0.623 (PMA) on day 3, and 0.730 (control),
0.747 (NKE), 0.760 (HQ), and 0.763 (PMA) on day 60. The activity-increasing effect of
inhibitors was attributed to their microbial decomposition, during which the
microorganisms, using the inhibitors or their degradation products as carbon and energy
sources, produced new urease molecules.
Field Experiments. Malhi and Nyborg (1979) compared the effect of thiourea on urea
hydrolysis in an Alberta silty clay loam soil with that of calcium sulfide (CaS) and
phosphorus pentasulfide (P 2S5; P4 SIO ) (see page 35) as well as with that of thio-
acetamide (CHr CS-NH 2). Urea was mixed (and not copelleted) with each sulfur
compound in a ratio of 2:1. The control plots were treated only with urea and the
experimental plots with urea + sulfur compound mixtures. Both urea and mixtures were
banded at a depth of 5 cm. Rate of N application was 112 kg/ha. Analysis of the NH/
and N0 3- contents in the 0-15-cm soil layer has shown that 5 weeks after fertilization,
urea hydrolysis was complete in the control plots and partial in the experimental plots:
68% (P2SS), 71 % (CaS), 42% (thiourea), and 43% (thioacetamide). That is, thioacet-
amide was as effective as thiourea. After 8 weeks, hydrolysis of urea became complete
in all plots.
The comparative studies in which p-benzoquinone or hydroquinone was used as a reference compound have
already been referred to on pages 59,104. 160-162,164. and 181.
192
of a black soil (loess) which had been treated with urea+AHA and incubated for 19
days, no residual urea was detectable, whereas in the urea+BQ-treated samples the
residual urea represented 12.1 % of the initial urea amount. Cumulative ammonia losses
by volatilization from samples of an acid sandy soil treated with urea, urea + 3% of
AHA, and urea + 3% ofBQ during 21 days of incubation were 19.1, 18.1, and l3.2%,
respectively (see also Matzel and Heber, 1979).
Ashworth et af. (1979) mentioned that hydroquinone (HQ) at a rate of 50 mglkg soil
was a little more effective in inhibiting urease activity of a silty clay loam than was
potassium ethyl xanthate at a rate of 100 mglkg soil.
Lichko and Kiselev (1985) compared the inhibitory effect of HQ and Cu 2+ on urease
activity in samples of a grey forcst soil, a chemozem, and an alkaline light chestnut soil.
Both inhibitors were applied at rates of 50. 250. and 500 Ilg/g soil. The reaction
mixtures were incubated at 30-37°C for 1-3 hours. It was found that in each soil and at
each rate of inhibitors HQ was more effective than was Cu2+. Thus, at the 250 Ilg/g soil
rate, HQ caused 89-96% inhibitions, whereas under the influence of Cu 2+ only 45-56%
inhibitions occurred.
In the experiments of Xue and Li (1987), dihydric phenols and quinones were
stronger inhibitors of soil urease activity than the plant materials (see Section 2.31.3.6).
Studying the alkaline alluvial soils in Pakistan, Hamid and Ahmad (1987) incubated
samples of a calcareous soil (pH 8) with urea, with or without addition of 5%
hydroquinone or phenol (on urea weight basis), and determined the ammonia evolved
during incubation. Cumulative NH3 losses during 112 days were reduced by l3% in
hydroquinone-treated and by 5% in phenol-treated samples as compared to the control
soil.
Among 28 substances tested at a rate of 20 ppm (on soil basis), quinhydrone,
hydroquinone, p-benzoquinone, and caffeine were the most effective inhibitors of soil
urease activity, causing -61, 59, 39, and 48% inhibitions, respectively (Li and Xue,
19(1).
Thorrnahlen and du Preez (1991) compared the effect of seven inhibitors, namely
hydroquinone (HQ), p-benzoquinone (BQ), phenylphosphorodiamidate (PPDA),
catechol (CT), phenylmercuric acetate (PMA), thiourea (TU), and ammonium
thiosulfate (ATS) on urease activity in four widely different soils from the central
irrigation areas in South Africa.
Air-dried soil samples (500 g) were treated with 25 rnl aqueous solution containing
25 mg of urea and 0 or 25 mg inhibitor. The samples moistened to field capacity were
incubated at 30°C. During incubation, the urea-N was measured at regular time intervals
until hydrolysis of urea was completed. These data were used to calculate the urease
activity and its inhibition by each inhibitor in each soil.
On average, the inhibitions caused by the seven inhibitors studied in the urease
activity of the four soils were the following:
HQ 94%; BQ 93; PPDA 85%; CT 75%; PMA 70%; TV 30%; and ATS 19%.
The minimum time necessary for complete hydrolysis of urea was registered in the
control (non-inhibited) sample of each soil, and the prolongation of this time was
maximal in the HQ-treated samples. The minimum and maximum times (in hours) for
the four soils are specified below: 42 (control) and 374 (HQ and PMA) in soil 1; 24
(control and ATS) and 156 (HQ) in soil 2; 60 (control) and 540 (HQ) in soil 3; and 8
(control) and 192 (HQ and BQ) in soil 4.
193
TABLE 44. PTA compounds compared with PPDA and PDA in experiments described by Martens
and Bremner (1982, 1984b)
No. Compound Structural formula
I Phosphoryl triamide H2N-P(O)(NH2)2
2 N-Phenylphosphoric triamide C.H,-HN-P(O)(NH2)2
3 N-(4-Nitrophenyl)phosphoric triamide 4-02N-C.H.-HN-P(O)(NH2h
4 N-(Diaminophosphinyl)benzamide C6H,-CO-NH-P(O)(NH2)2
5 4-Chloro-N-(diaminophosphinyl)benzamide 4-Cl-C.H.-CO-NH-P(OXNH212
6 4-Fluoro-N -( diaminophosphinyl)benzamide 4-F-C6H.-CO-NH-P(O)(NH2h
7 4-Cyano-N-(diaminophosphinyl)benzamide 4-NC-C6H.-CO-NH-P(OXNH2)2
8 N-(Diaminophospinyl)benzeneacetamide C.H,-CH 2-CO-NH -P(O)(NH2)2
9 N-(Diaminophosphinyl) 3-pyridinecarlJoxamide 3-C,H.N-CO-NH-P(O)(NH212
10 3-Trifluoromethyl-N-(diaminophosphinyl)benzamide 3-F,C-C6 H.-CO-NH-P(O)(NH2)2
II N-(3-Trifluoromethylphenyl)phosphoric triamide 3-F,C-C.H.-HN-P(O)(NH2)2
Experiments performed with the aim to compare PPDA and PDA with phenyl-
mercuric acetate, catechol, hydroquinone, p-benzoquinone, and 2,5-dimethyl-p-benzo-
quinone were also described by Martens and Bremner (I 984b).
The seven Iowa soils selected for the experiments were very varied in terms of their
pH (5.0-8.0), texture (5-56% sand, 13-32% clay), organic C content (0.30-4.23%),
CaC03 equivalent (0-20.8%), urease activity (14.2-80.2 /lg of hydrolyzed urea/g
soillhour at 37°C), and their other properties. The reaction mixtures (5 g of air-dried soil
+ 2 ml of solution containing 10 mg of urea without or with 50 /lg of inhibitor) were
incubated at 20°C and analyzed for residual urea after 3, 7, and 14 days of incubation.
The analytical data indicated that PPDA was the most effective urease inhibitor in
each soil, whereas PDA was a weak inhibitor. Of the PTA compounds, 4-cyano-N-
(diaminophosphinyl)benzamide was the least effective inhibitor. It is noteworthy that
phenylation of PDA (weak inhibitor) leads to the formation of the strongest inhibitor
(PPDA). Conversely, the effect of phosphoryl triarnide was a little greater than that of
its phenyl derivative, N-phenylphosphoric triamide.
The inhibitory effectiveness of all compounds decreased with prolongation of the
incubation time .
• The comparative studies in which PPDA was used as a reference compound have already been referred to on
pages 101, 103, 144, and 189.
194
also marked; PTA was more effective than TPTA in three soils. In each soil, potassium
phosphoroamidate was the weakest inhibitor.
The effect of different concentrations of PTA, TPTA, and PPDA (2.5,5,10,25,50,
and 100 ppm relative to soil weight) on urease activity in four soils was also studied.
The inhibition increased with increasing inhibitor concentration up to 25 ppm and
tended to level off at higher concentrations. The maximum inhibition brought about by
PTA and TPTA at 100 ppm was 93% (in fine sandy loam, pH 7.7) and 86% (in silty
loam), respectively, whereas PPDA produced nearly complete inhibition at
concentrations of 10-50 ppm.
Persistence of the inhibitory effect was followed in the silty clay loam. The reaction
mixtures were incubated for 3 and 7 days, then the residual urea was assessed. After 3
days, PTA, TPTA, and PPDA caused similar inhibitions (of about 64%). Inhibition by
PTA and PPDA persisted at a level of about 15-20% even after 7 days of incubation.
The observations made by Liao and Raines (1985) that PTA was a more effective
and persistent inhibitor than was TPTA should be emphasized since in other
195
(15 g) were flooded with 30 ml of water, then amended with urea (14 mg) without or
with inhibitor (1, 2.5, 5, and 10% relative to weight of urea) and incubated at 25°C for 8
days, during which, at 2-day intervals, the residual urea was determined.
At 1% inhibitor concentration, rate of urea hydrolysis was 99% (control), 96%
(AHA), 91 % (DAPBA), 89% (nBTPTA), and 30% (PPDA) in the more urease-active
vertisol after 2 days of incubation, and 100% (control), 99% (AHA), -82% (DAPBA)
and -40% (PPDA, nBTPTA) in the less urease-active alfisol after 8 days of incubation.
Increasing concentration of AHA and DAPBA from I to 10% did little to reduce the
rate of hydrolysis of urea in either the vertisol or the alfisol. Essentially, all the urea was
hydrolyzed by day 4 in the vertisol and day 8 in the alfisol, irrespective of the
concentration of these compounds.
In contrast, increasing the concentration of PPDA and nBTPTA from I to 2.5, 5 and
10% led, at least at the 10% concentration, to a decrease in the rate of urea hydrolysis.
Thus, the rate of urea hydrolysis in the vertisol after 8 days of incubation was 100, -90,
70, and 58%, respectively, in the PPDA treatments, and 100, 100, 100, and 75%,
respectively, in the nBTPTA treatments. The corresponding values registered in the
alfisol after 8 days of incubation were: 40, 25, 17, and 9%, respectively, in the PPDA
treatments, and 28, 25, 21, and 20%, respectively, in the nBTPTA treatments.
All results indicated that in the two flooded rice soils studied PPDA was the most
effective urease inhibitor.
Wang and Douglas (1996) used surface (0-15 cm) samples of two sandy soils,
namely a solonized brown soil (PH 7.7) and a podzol (PH 5.5), both from Victoria,
Australia, for comparing the inhibitory effects of PPDA, cyc1ohexyl-PTA (CHPTA),
and nBTPTA on urea hydrolysis. The reaction mixtures, prepared from 6 g air-dried soil
and 3 ml of aqueous solution containing 0, 5, 10 or 25 J.lg inhibitor and 1 mg of urealg
soil, were incubated at 37°C for 4 hours, then analyzed for NH4 +.
The analytical data showed that each compound was more inhibitory in the brown
soil than in the podzol.
At the 5,10, and 25 J.lglg soil rates ofPPDA. CHPTA, and nBTPTA, the following
inhibitions were registered in the brown soil:
100, 100, and 100% (PPDA); 87, 100, and 100% (CHPTA); and 68, 73, and 83%
(nBTPTA).
The corresponding values for the podzol were:
79, 79, and 71% (PPDA); 78, 79, and 71% (CHPTA); and 65, 66, and 71%
(nBTPTA).
Thus, in these soils, PPDA and CHPTA were stronger inhibitors of urea hydrolysis
than was nBTPTA.
Field Experiments. Beyrouty et al. (l988a,b) conducted two field trials on a silt loam
soil (pH 5.7) (located at the Purdue University Agronomy Fann, West Lafayette,
Indiana) with the aim of studying the effect of six phosphoroamides (N,N-dimethyl-,
N,N-diethyl-, N-cyc1ohexyl- and N-benzyl-N-methyl-PTA, trichloroethyl-PDA, and
PPDA)" on urea hydrolysis and ammonia volatilization from urea-No The trials were
carried out on conventional till (CT) and no-till (NT) microplots. In the previous year,
"It should be emphasized that in these field trials N-(n-butyl)thiophosphoric triamide (nBTPTA) was not
evaluated.
197
the experimental field was cropped to maize; the maize residue covered about 60% of
the surface of the NT microplots. All microplots remained unsown and in all of them
weed control was accomplished with application of herbicides followed by hand
cultivation as needed. Urea prills (200 kg N/ha) with or without an inhibitor (4 kg/ha)
were uniformly broadcast over the soil of microplots. Each inhibitor used was coated
onto urea prills with paraffin oil. The control microplots received no fertilizer.
Trial 1 was initiated on 7 June 1983, and trial 2 on 5 July 1983. At fertilizer
application, the soil surface was moist in trial 1, and air-dry in trial 2. After fertilization,
analyses were made at 2-5-day intervals for determining residual urea and volatilized
ammonia during 24- and 20-day periods, respectively, Air and soil (5-cm depth)
temperatures had average maxima of 29 and 35°C, respectively, in trial 1, and 32 and
40°C, respectively, in trial 2. The urea prills had completely dissolved within 12 hours
(trial 1) or within 6 days (trial 2) in both CT and NT microplots. In the absence of
inhibitors, the rate of urea hydrolysis was more than twice as great in trial 1 than in trial
2, due to slower dissolution of urea prills in trial 2.
In trial 1, in the CT microplots, the most effective inhibitors were PPDA, trichloro-
ethyl-PDA and N-benzyl-N-methyl-PTA, decreasing the rate of urea hydrolysis by 68,
66, and 60%, respectively. The other compounds had limited effects on the urea
hydrolysis rate. The inhibitory effect always decreased with time and was evident for 19
days in the case of PPDA and trichloroethyl-PDA, but no inhibition was present at day
9 in the case of the other compounds. In the NT microplots, only PPDA had significant
(>70%) inhibitory effect on urea hydrolysis for 4 days, but its effectiveness declined
rapidly by day 9. However, some effect of PPDA on urea hydrolysis was observed for
as long as 19 days. Inhibition disappeared in each treatment at day 24 after fertilization.
In trial 2, in the CT microplots, none of the compounds tested significantly affected
the rate of urea hydrolysis. In the NT microplots, PPDA was the single effective
inhibitor (reduction of urea hydrolysis rate was higher than 63%).
In both trials, the weakest urease inhibitors were N,N-dimethyl- and N,N-diethyl-
PTA.
Cumulative NH3 losses in 12 days (trial 1) or in 20 days (trial 2) were smaller in the
CT microplots than in the NT ones, in both absence and presence of inhibitors. Thus,
cumulative NH3 losses from the urea-N applied without inhibitors in CT and NT
microplots were 30 and 31 %, respectively, in trial 1, and 7 and 35%, respectively, in
trial 2. In trial 1, NH3 volatilization was significantly reduced only by PPDA in both CT
microplots (degree of inhibition: 90%) and NT microplots (degree of inhibition: 61 %).
In trial 2, PPDA was again the only inhibitor which significantly reduced NH3
volatilization, but only in the NT microplots (degree of inhibition: 46%) (see also
Nelson et al., 1986).
Referring to the results obtained with two acid soils and an alkaline soil under
aerobic and water-logged conditions, Van Cleemput and Wang (1991) have drawn the
conclusion that the inhibitory effect of PPDA on urea hydrolysis and NH4 +
accumulation was more marked than that of nBTPTA in the acid soils under both
aerobic and water-logged conditions as well as in the alkaline soil under water-logged
conditions, while in the alkaline soil under aerobic conditions nBTPTA was a stronger
inhibitor than PPDA (see also Wang and Van Cleemput, 1992).
198
4.9.1. Comparative Studies on the Efff!Ct of PTA and TPTA Compounds and Other
Inhibitors on Soil Urease Activity. Urea Hydrolysis. and Ammonia Volatilization
Field Experiments. Grant et al. (1996a,b) conducted two microplot studies in 1995,
under no-till conditions on a chernozemic soil (fine sandy loam, pH 7.3) located on
Canadian prairie. The first study was initiated on May 20, 3 days after seeding of wheat,
but before emergence. The second study began on August 14, after the wheat had
headed; wheat was mowed at a height of 4 cm and the fresh residue removed. The
treatments included: urea-ammonium nitrate (VAN) solution (100 kg Nlha) , VAN +
0.25% nBTPTA (relative to fertilizer N); VAN + 10% ATS (again relative to fertilizer
N). The fertilizer was placed in a 2-cm circle on the soil surface in the center of the
microplot. After fertilization the volatile ammonia was measured for 7 days.
The cumulative NH3 losses were higher in the second study than in the first,
presumably due to higher soil and air temperatures and lower initial soil moisture levels
in August as compared to May.
nBTPTA significantly (p=0.05) reduced total NH3 losses in both studies, whereas
ATS was ineffective in the first study and less effective than nBTPTA in the second
study.
and 2 t/ha (PG 1 and PG2). KCl was applied only in the sugarbeet experiment, at rates of
540 and 1,080 kg/ha (KCn and KCI2). The N fertilizers were urea, ammonium sulfate,
and ammonium nitrate in the wheat experiment, while only urea was applied in the
sugarbeet experiment. The N fertilizer mixed with the test substance was placed on the
surface of plots in May 1993 (wheat experiment) or in July 1993 (sugarbeet
experiment). Only N fertilizer was added to the control plots.
Volatilization of ammonia was assessed up to 57 days in the wheat experiment and
up to 78 days in the sugarbeet experiment.
Total NH3 loss from ammonium sulfate and ammonium nitrate was significantly
(p<0.05) increased by the substances tested, including nBTPTA.
Loss from urea in the wheat experiment was significantly increased by PG2 from
10.6% of the added N (control) to 12.0%, whereas the loss in the other treatments was
significantly lower than in the control, with values of 9.2% (PGl), 8.6% (PRl), 8.2%
(PR2), 5.0% (nBTPTAI), and 3.9% (nBTPTA2).
In the sugarbeet experiment total NH3 loss from the added urea-N (12.6% in the
control) was significantly increased by PRI (17.2%), PGl (17.7%), and KCn (23.6%),
not affected significantly by nBTPTAI (12.6%) and PG2 (11.5%), and significantly
reduced by PR2 (11.1 %), KCl2 (10.5%), and nBTPTA2 (7.0%).
All results show that nBTPTA at the 0.5% rate was most effective in reducing the
volatile NH3 losses from urea-treated plots.
4.9.1.3. Comparison ofnBTPTA and/or TPTA. PTAs and Alkyl-PDAs with PPDA
Laboratory Experiments. Swerdloff et al. (1984, 1985d) and Van Der Puy et al. (1985b)
treated samples of a sandy loam soil with urea with or without a urease inhibitor and
incubated them at 35°C for 12 days. The rates of additions per 20 g soil were: 42.8 mg
of urea and 0.2 mg of inhibitor. The unhydrolyzed urea was determined at 2-day
intervals. Urea hydrolysis was complete in 2 days in the urea-only treatment, in 6 days
when the inhibitor was PPDA, whereas about 35 and 65% of the added urea remained
unhydrolyzed after 12 days in samples treated with N-methyl-N-(4-nitrophenyl)-PTA
and N-(2-chloroethyl)-PTA, respectively.
In a similar experiment described by Kolc et aT. (1984, 1985d), 20-g samples of a
silt loam soil were amended with 42.8 mg of urea with or without 0.2 mg of inhibitor,
then incubated at 25 or 35°C for 22 days. The residual urea content was determined at
1-2-day intervals. No urea was detected after 4-day incubation at 25°C and after 2 days
at 35°C in the soil to which only urea was added. Urea disappeared from the PPDA-
treated soil in -9 days at 25°C and -6 days at 35°C. In the N,N-diethyl-PTA-treated soil
-50 and nearly 20% of the added urea remained unhydrolyzed at 25 and 35°C,
respectively, although the incubation time was 22 days.
In another experiment performed by Kolc et al. (1984, 1985d), nBTPTA, five PTAs,
namely N-(n-butyl)-PTA, N-(sec-butyl)-PTA, N-ethyl-PTA, N-(n-octyl)-PTA, and N-
cyclohexyl-PTA, as well as PPDA at a rate of 0.2 mg were preincubated with 20-g
samples of a silt loam soil, at 25 and 35°C or in the open, for 11 days. The next
operations were: addition of urea (42.8 mg), incubation (at 25°C for 3 days), and
analysis of residual urea.
The following results were registered: nBTPTA was the most effective urease
inhibitor; the inhibitory effectiveness of each compound (except nBTPTA) decreased
during the preincubation; the decrease was more marked at 35 than at 25°C and less
200
marked in samples kept in the open; PPDA as a urease inhibitor was inferior not only to
nBTPTA, but also to the five PTAs.
Beyrouty et al. (1988a) compared the inhibitory effect of two PTAs (N-cyclohexyl-
PTA and N-benzyl N-methyl-PTA), nBTPTA, and PPDA on urea hydrolysis in a silty
clay loam soil from Indiana. The surface (0-15-cm) soil layer was sampled in two areas.
Soil pH was 5.6 in one area and 7.4 in the other area which was under a long-term
liming experiment.
The soil samples (20 g) with contrasting initial pH values of 5.6 and 7.4 were treated
with 4.8 ml of a solution containing 20 mg of urea-N with or without 0.4 mg of
inhibitor, then the reaction mixtures were incubated at 25 D e for 0, 2,4, 7, and 14 days,
after which they were analyzed for residual urea. Table 46 shows that at pH 5.6, PPDA
and nBTPTA were the most effective inhibitors of urea hydrolysis. At pH 7.4, PPDA
Field Experiments. The maize experiments conducted by Bundy and Oberle (1988)
were mentioned on page 28. Under similar conditions, nBTPTA, N,N-diethyl-PTA, and
PPDA added to urea prills and nBTPTA and PPDA added to urea-ammonium nitrate
(UAN, 28-0-0) solution were also studied. The urease inhibitors were applied at a rate
of 2% on fertilizer N basis (56 and 112 kg N/ha). Urea prills and UAN without
inhibitors were used for comparison. In these experiments also, field measurements of
ammonia volatilization were made at the higher fertilizer N rate only and lasted 8-10
days.
nBTPTA, added to urea and UAN and applied only at Arlington in 1984,
significantly reduced NH3 volatilization from urea (18%) and UAN (5%) to 1 and 2%,
respectively. Urea and urea-PPDA, which were used at both locations in both years,
gave rise to the following N losses as NH3: 8 and 1% (Arlington, 1983), 18 and 3%
(Arlington, 1984),24 and 16% (Lancaster, 1983), and 19 and 14% (Lancaster, 1984),
respectively. It is evident that PPDA was more effective at Arlington than at Lancaster.
UAN-PPDA, used only in 1984, was effective at both locations; the NH3 losses were
reduced from 5 to 1% (at Arlington) and from 16 to 7% (at Lancaster), respectively.
N,N-Diethyl-PTA, added to urea and applied at both locati(ms in 1983, was ineffective
at Arlington (NH3 loss in both presence and absence of N,N-diethyl-PTA was 8%) and
effective at Lancaster (NH3 loss was reduced from 24 to 8%).
Fertilization experiments on a silt loam under an established orchardgrass (Dactylis
glomera/a) pasture at Lancaster were also performed in 1983 and 1984. Urea prills
(67.2 kg N/ha) with or without 2% (on urea-N basis) nBTPTA (in 1984), PPDA (in both
years), N,N-diethyl-PTA (in 1983), and trichloroethyl-PDA (in 1983) were surface-
applied in mid-June following the first harvest of the orchardgrass forage. Field
measurements ofNH 3 volatilization revealed significant reductions in NH3 losses due to
the urease inhibitors. Thus, in 1983, the NH3 losses were of 19% from urea, 11 % from
urea-PPDA, 8% from urea-N,N-diethyl-PTA, and 10% from urea-trichloroethyl-PDA.
In 1984, 9% of the N was lost as NH3 from urea, 1% from urea-nBTPTA, and 6% from
urea-PPDA, which means that nBTPTA was more effective than PPDA in controlling
NH 3 10sses.
4.9.1.4. Comparison ofnBTPTA. PTAs. and Alkyl-PDAs with PPDA and Hydroquinone
(HQ)
Laboratory Experiments. Chai and Bremner (1985, 1986) and Bremner (1986)
presented experimental data on the comparison of six compounds, namely nBTPTA,
202
100
A -e- Urea
.. -- ....
.•. HQ
e:. '11
-Ir PPDA
.nBTPTA
~- ,
.,
~
::>
30
0
0 2 3 7
Incubation lime (davs)
100
11
~Urea
. • HQ
~ -tr PPDA
·il 10 "nBTPTA
~'
..
.E' ~
1l
:5 ""
20 '-.
"'.
.....•. ~.::-
Q
0
Incubalion lime (days)
Figure 66. Hydrolysis of urea as a percentage of the urea applied, under aerobic (A) and
anaerobic (B) conditions./From Wang et al. (199\a), by permission of Springer-Verlag.I
aerobic conditions. This was mainly due to the effect of these inhibitors on retarding
urea hydrolysis, resulting in a lower accumulation of NH4 + which provided a more
favorable environment for oxidation of N0 2- to N0 3- by Nitrobacter. In the control and
HQ-treated soil samples, more N0 2- accumulated, probably because the higher NH/
concentration hindered oxidation of N0 2- to N0 3-. Little N0 2- was detected under
anaerobic conditions due to lack of O2 •
Ammonia volatilization was studied with mixtures prepared from 600 g of soil +
120 mg of urea-N with or without 1% inhibitor and water to two-thirds of field capacity
or from 350 g of soil + 70 mg of urea-N with or without 1% inhibitor and sufficient
water to form a 2-cm layer on the soil surface. During incubation (at 25°C), the NH3
evolved was assessed daily for 16 days. Less NH3 volatilized under aerobic than
anaerobic conditions. In concordance with inhibition of urea hydrolysis, cumulative
NH3 loss was lowest in the nBTPTA-treated soil (3% vs. 20% in the control soil) under
aerobic conditions, and in the PPDA-treated soil (15% vs. 40% in the control soil) under
anaerobic conditions.
In another experiment, Wang et al. (1996) compared the effects of three urease
inhibitors on movement of urea and its transformation products at two soil moisture
204
levels (10 and 20%, on a soil dry weight basis). The same inhibitors and the same soil
were used as in the investigations described in the preceding paragraphs, i.e. nBTPTA,
PPDA, and HQ and a Belgian alkaline soil, respectively.
The soil sampled from the 0-20-cm depth was preincubated at 25°C at 10 and 20%
moisture contents for 5 days to restore microbial activity. Afterwasds, the soil was
packed into plastic cylinders 10-cm high and 9.5-cm in diameter. Urea (300 mg N/kg
soil) alone or together with an inhibitor (1 % relative to weight of urea) was applied 3-4
cm below the surface of soil columns which were then incubated at 25°C. After 2, 4,7,
12, and 17 days, the soil columns were sliced into 10 sections, each 1 cm thick. The
soils were analyzed to determine moisture, urea, NH4 +, and (N0 3·+ N0 2-) contents.
The results clearly showed that the effect of each inhibitor in retarding urea
hydrolysis was stronger at 10% than at 20% soil moisture level. nBTPTA, in
comparison to PPDA and HQ, was the strongest inhibitor at both moisture levels,
whereas PPDA, compared to HQ, was a weaker inhibitor at 10% moisture and stronger
at 20% moisture.
By inhibiting urea hydrolysis, the inhibitors affected movement of urea, formation
and movement ofNH/ and (N03- and NO z-) in the soil columns during incubation. It
was demonstrated that distribution of urea and its transformation products after 7 days
of incubation at 20% moisture was comparable with that observed after 17 days at 10%
moisture.
Field Experiments. Joo et al. (1989) used a Kentucky bluegrass (Poa pratensis) turf
established on a fine loamy soil (PH 7.5) to compare the effectiveness of nBTPTA,
PPDA, ATS, as well as K+ and Mg2+ in reduction of the volatilization of ammonia from
urea-treated plots. Rates of addition were: 10% N urea solution 49 kg Nlha; nBTPTA
0.5, 1, and 2%; PPDA 1,2, and 3%; ATS, K+ (as KCI), and Mg2+ (as MgCl z) 5, 15, and
25%. All percentages represent weights relative to weight ofurea-N. The control plots
were treated only with urea. The volatile NH3 was measured for 4 days.
The cumulative NH3 losses expressed as percentages of the added urea-N gave the
following values for the control plots and for those treated with the three rates of the test
compounds:
18.5 (control); lOA, 7.9, 7.2 (nBTPTA); 7.0, 6.9, 5.6 (PPDA); 14.5, 14.0, 14.7
(ATS); 17.2, 17.3, 17.6 (K+); and 15.3, 15.2, 15.9 (Mgz+).
It is evident that the effectiveness of the compounds tested for reduction of NH3
losses after fertilization with urea decreased in the order:
nBTPTA > PPDA » ATS > Mgz+ > K+.
205
TABLE 47. Effect ofdiflerent amounts ofnBTPTA and PPDA on urea hydrolysis in soil"
Inhibition of urea h:t:drol:t:sis (%2
Incubation
Amount added (/-1g!g soil)
Soil temperature
(DC) nBTPTA PPDA
5 10 5 10
Sandy loam 20 49 60 73 49 66 71
I2H 8.0 30 44 58 69 0 17 28
Sandy loam 20 80 86 87 19 26 40
I2H 8.3 30 68 SO 84 0 0 0
Loam 20 58 75 79 49 67 73
I2H 6.4 30 4 39 55 0 6 13
Silty clay loam 20 26 64 72 3 27 30
I2H6 .O 30 0 0 8 0 0 0
Average 20 53 71 78 30 47 55
30 29 45 54 0 6 10
"From Bremner and Chai (1986). by courtesy of Marcel Dekker. Inc.
were 20 and 30°C and 10 days, respectively. Four soils were used. Results of the
comparison (Table 47) prove that nBTPTA was considerably more effective than PPDA
for inhibition of urea hydrolysis at 20°C and was much more effective than PPDA at
30°C.
Beyrouty et al. (1988b) used samples of a silty loam soil (PH 6.5) from Indiana to
compare the effects of nBTPTA and PPDA on volatilization of ammonia from urea. The
experiment comprised three variants: bare soil samples (20 g); 20-g soil samples to
which 0.3 g of finely ground maize residue was added and partially incorporated below
the soil surface; and 0.3 g of maize residue alone. All samples were treated with a urea
solution (20 mg N) with or without 0.4 mg of nBTPTA or PPDA. The next step was
incubation. Ammonia volatilized during 2, 4, 8, 14, 21, and 28 days was determined.
The NH3 volatilized in 28 days was practically identical in soil only and soil + residue,
with 26-27% of the applied urea-N volatilized in the absence of inhibitors and 1-3% in
their presence. This means that in the soil studied (pH 6.5) both inhibitors were very
effective. In the variant with residue only, the NH3 volatilization loss in 28 days was
92% in the absence of inhibitors, and it was reduced by nBTPTA and PPDA to 39 and
43% respectively. The weaker effectiveness of inhibitors in this variant is explained by
the finding that urease activity in the residue was approximately 47 times greater than in
the soil on the same weight basis. It should be added that in the absence of inhibitors,
volatilization of NH3 during the first 2 days of incubation was 5 times higher from the
soil + residue variant than from the residue alone, but, as already mentioned, during 28
days the same amounts of NH3 were lost by volatilization from both variants.
Influence of redox potential on the inhibitory effectiveness of nBTPT A and PPDA
in retarding urea hydrolysis was studied by Lu et al. (1989). Samples of four rice soils
were used: a silt loam, pH 5.8 {soil 1) and a clay, pH 5.3 {soil 2) from the U.S.A., a silty
206
clay, pH 6.9 (soil 3) from China, and a silty loam, pH 8.3 (soil 4) from India. Air-dried
ground soil equivalent to 400 g of oven-dry weight and 1,600 rn1 of deionized water
were introduced into flasks. One-half of the flasks were continuously stirred and purged
with air (oxidized treatment) and the remaining set stirred and purged with argon gas
(reduced trcatment). Then the soil suspensions were preincubated in the dark at 30°C.
During preincubation, the redox potential (Eh) and pH were measured. They reached
steady state values after 19-23 days of preincubation. Afterwards, soil suspension
aliquots equivalent to 4 g of oven-dry weight were removed from the flasks and
centrifuged. The water layer was discharged while maintaining aerobic and anaerobic
conditions. respectively. The sedimented soil was treated with urea (400 /lg N/g soil)
with or without 2% (on urea weight basis) nBTPTA or PPDA. Untreated soil served for
comparison. The reaction mixtures prepared in this way were incubated at 30 D C and
after 1,3,5, 7, and 15 days they were analyzed for residual urea.
The oxidized treatment aimed at simulating the thin oxidized surface layer in
flooded soils and the oxidized zone around the roots of rice plants, whereas the reduced
zone under the soil surface was simulated by the reduced treatment.
At steady state, Eh values in suspensions of the four soils studied ranged from +630
to +730 mY in the oxidized treatment, and from -290 to -340 mY in the reduced
treatment; pH decreased in the oxidized treatment (except soil 1 in which the pH
slightly increased) and increased in the reduced treatment (except soil 4 in which the pH
remained unchanged).
The results obtained in analysis of the residual urea are presented in Table 48. In
soils treated only with urea, urea hydrolysis during 1 and 3 days was more marked in
reduced san1ples than in the oxidized ones of soils 1 and 4, whereas the opposite was
true for soils 2 and 3. Urea hydrolysis was complete in 5 days in all samples of each
soil. Both inhibitors retarded urea hydrolysis in each soil. nBTPTA was more effective
in oxidized samples and PPDA in the reduced ones. Thus, at day 7 after incubation, the
residual urea (expressed as average of the values recorded in the four soils studied) was
49 and 17% of the added urea in oxidized samples treated with nBTPTA and PPDA,
respectively. The corresponding average values registered in the reduced samples were
4% (nBTPTA) and 26% (PPDA), respectively. At day 15, urea remained unhydrolyzed
in considerable amounts only in three samples: the PPDA-treated reduced sample of soil
1, nBTPTA-treated oxidized samples of soils 3 and 4, containing -20, 44, and -54%
residual urea, respectively.
In the experiment by Bronson et al. (1989) to compare the effectiveness of nBTPTA
and PPDA on a111l110nia volatilization from urea, samples of a loamy sand soil (PH
6.9)were used. Some samples were acidified with sulfuric acid to adjust the pH to 6.5
and 6.0. The reaction mixtures contained lO g of air-dried soil + 10 mg ofurea-N + 0 or
50 ~lg of nBTPTA or PPDA. During incubation (at 25 D C for 27 days), amounts of
volatile NH3 were assessed. The results (Table 49) show that the inhibitory effect of
both compounds increased slightly when soil pH was adjusted from 6.9 to 6.5 and 6.0.
nBTPT A was more effective than PPDA. Thus, NH3 volatilization was completely
inhibited at each pH for 9 days by nBTPTA and only for 4 days by PPDA. At day 27,
the degree of inhibition by nBTPTA was between 34 and 43 at the three pHs, whereas
the inhibition by PPDA became equal to zero at each pH.
TABLE 48. Effectiveness ofnBTPTA and PPDA in retarding urea hydrolysis in four rice soils under oxidized and reduced conditions·
Soil 1 Soil 2 Soil 3 Soil 4
Residual urea (~N/g soil}
Iyi' Inhibitor'
Oxidized Reduced Oxidized Reduced Oxidized Reduced Oxidized Reduced
conditions conditions conditions conditions
nBTPTA 292 a 185 b 324 a 332 b 376 a 143 b 250 a 170 b
PPDA 292 a 271 a 349 a 391 a 307b 288 a 273 a 299 a
No 216 b 186 b 239b 304c 121 c 168 b 183 b 117 c
nBTPTA 294 a 26b 247 a 191 b 295 a 32 b 218 a 5b
3 PPDA 294 b 255 a 183 b 327 a 193 b 213 a 202 a 254a
No 30 c Ob 66c 82 c Oc 5b 13b Ob
nBTPTA 274 a 20b 170 a 32b 246 a 17 b 218 a Ob
5 PPDA 96b 238 a 133 a 182 a 120 b 101 a 142 b 254 a
No Oc Ob Ob Oc Oc Ob Oc Ob
nBTPTA 252 a 20b 52 a 18 b 260 a 18 a 217 a Ob
7 PPDA 36 b 144 a 28b 44a 80b 23 a 118 b 199 a
No Ob Oc Oc Oc Ob Ob Oc Ob
nBTPTA 25 a Oc 5a 2b 176 a Ob 214 a Oa
15 PPDA 12 b 78 a Ob 4a 4b 7a Ob 2a
No Oc Ob Ob Oc Ob Ob Ob Oa
"Adapted from Lu et aI. (1989), by courtesy of Marcel Dekker, Inc.
bIncubation time (days).
'Inhibitor added to urea (400 Ilg urea-N/g soil) at a rate of2% relative to weight ofurea-N.
No - Only 400 Ilg urea-N/g soil.
Values with the same letter are not significantly different (p=0.05).
N
o
-...J
208
TABLE 49. Inhibition of ammonia volatilization by nBTPTA and PPDA in urea-treated samples of a
loa~ sand at three initial EH values at 25°C·
Inhibition ~%l
Initial
Treatment Incubation time ~da:z:sl
pH
2 4 6 9 12 15 18 21 24 27
6.9 100 100 100 100 95 84 71 57 46 34
Urea + nBTPTA 6.5 100 100 100 100 97 87 75 64 53 42
6.0 100 100 100 100 97 89 77 66 54 43
6.9 100 100 88 53 19 8 4 2 1 0
Urea + PPDA 6.5 100 100 93 64 27 9 5 2 0 0
6.0 100 100 100 73 30 6 0 0 0 0
"From Bronson et aI. (1989), by courtesy of Marcel Dekker, Inc.
Complex investigations related to nBTPTA and PPDA were also conducted in New
South Wales, Australia, by Cai et al. (1989).
In the first experiment, air-dried samples (1 kg) of three soils were placed in pots
and flooded to a depth of 5 cm. Then nBTPTA and PPDA (at rates of 0.005,0.01,0.1,
1, and 5% of the weight of urea) were applied into the floodwater followed by the
addition of 366 mg of urea (equivalent to 80 kg N/ha). The pots were kept in a
glasshouse, in which the temperature ranged from 22 to 32°C. At zero time and after 2,
4, and 6 days, the soils were analyzed for urea.
The analytical data show that effectiveness of nBTPTA and PPDA as urease
inhibitors increased with the rate of addition. The low rates (0.005-0.1 %) only slightly
retarded the hydrolysis of urea. At higher rates (1 and 5%), both inhibitors were
effective, but the extent and duration of inhibition varied with soil type (Figure 67).
A B a
,~ \ \:
~---,~U
2 • 8
\::
'-----'-_-'-.
2
-_~.u
"Ii
~,
I
2
~~I
4 a
Timeldaysl Time (daysl Time (deysl
Figure 67. Effect of rate of addition ofnBTPTA and PPDA on urea hydrolysis in flooded soils in a
pot experiment.
A - Yellow podzolic soil, pH 6.2. B - Grey soil, pH 8.4. C - Red-brown earth, pH 5.6. U - Urea. B I -
Urea + 1% nBTPTA. B5 - Urea + 5% nBTPTA. PI - Urea + 1% PPDA. P5 - Urea + 5% PPDA.
IFrom Cai et al. (1989). by permission of Pergamon Press PLC'/
Thus, in two soils (yellow podzolic and grey soil), nBTPTA was more effective than
PPDA, whereas in the third soil (red-brown earth), only PPDA was effective. The
209
higher inhibitor rate acted always more markedly than the lower rate. During
incubation, the degree of inhibition decreased only slowly in the yellow podzolic soil
and rapidly in the two other soils.
In other experiments, carried out in bottles, the influence of a) soil moisture content
and b) growth of algae on the inhibitory effectiveness of nBTPTA as well as the
influence of c) the chelating compound ethylenediaminetetraacetic acid (EDTA) on
inhibitory effectiveness ofnBTPTA were studied.
a) Air-dried samples (15 g) of five soils received water up to field capacity (moist
samples). Thirty ml of water was added to other samples (flooded samples). The added
water contained 14 mgofurea with or without 5% nBTPTA or PPDA relative to weight
of urea. Incubation took place in the dark at 25°C. After 2, 4, and 6 days, the
unhydrolyzed urea was assayed. In the moist samples of each soil, nBTPTA inhibited
the urea hydrolysis more markedly than PPDA. In flooded samples the inhibition was
nearly complete in 6 days for 4 and 3 out of the five soils treated with PPDA and
nBTPTA, respectively. Urea hydrolysis in the flooded red-brown earth proved to be
resistant to inhibition by nBTPTA in this experiment, as well.
b) Samples (15 g) of three soils were flooded with 30 ml of water containing urea
with or without nBTPTA or PPDA in the same amounts as in experiment a. Three
treatments were applied. Some samples were incubated in the dark (at 25°C) for
preventing growth of algae (treatment D). Other samples were exposed to light under
normal daylight conditions in a glasshouse to promote growth of algae (treatment L). In
order to enhance additional algal growth, some flooded samples were exposed to light
in the glasshouse for 10 days, before urea was added with or without inhibitor and new
exposure to light as in treatment L (treatment LL). Determination of urea after 2, 4, and
6 days of incubation showed that in each soil urea hydrolysis was inhibited by PPDA
more effectively under dark than light conditions, whereas the inhibition by nBTPTA
was practically unaffected by exposure to light. This means that in the presence of light
and algae, PPDA was less effective than nBTPTA. The effectiveness of PPDA was
much lower in treatment LL than in treatment L. To explain these findings, it is
considered possible that the algae in the soils studied contain other enzymes, such as
urea carboxylase (hydrolyzing)', which catalyze decomposition of urea and that these
enzymes are not inhibited by PPDA, but are by nBTPTA
c) EDTA was studied with the red-brown earth, in which nBTPTA did not inhibit
urea hydrolysis. The reaction mixtures had the following composition: 15 g of soil +
0.67 or 2 mM of EDTA + 30 mg of urea + 0 or 1 or 5% nBTPTA (relative to weight of
urea) in 30 m1 of water. After 2 and 4 days of incubation in the dark at 25°C, the
mixtures were analyzed for residual urea. The analyses indicated that EDTA manifested
an inhibitory effect on urea hydrolysis. At 0.67 mM of EDTA, the degree of inhibition
after 2 and 4 days of incubation was 26.8 and 1.2%, respectively, whereas at 2 mM of
EDTA the corresponding values were 39.6 and 4.2%, respectively. At the same time,
EDTA improved the inhibitory capacity of nBTPTA and this effect became stronger
with the increase in concentration of the two compounds. Moreover, after a 2-day
incubation the combined effect of EDTA and nBTPTA (at each concentration) was
significantly greater than the sum of their individual effects (synergism), but after 4
days, only the combined effect of 2 mM EDTA and 5% nBTPT A (degree of inhibition:
21.6%) was significantly greater than their additive individual effects (degree of
inhibition: 4.2 + 3.2%).
These investigations were also referred to by Freney et al. (1989).
Byrnes and Amberger (1989) compared the urease inhibition by nBTPTA and
PPDA in flooded, unplanted samples of a silty clay loam soil (pH in H20 5.9). The 300-
g samples were placed in plastic containers 10.8 cm in diameter and 6 cm deep, and
were flooded and puddled to provide a soil depth of about 3 cm and 2.5 cm of
floodwater. The rates of additions to floodwater were: 75 mg of urea, 0, 0.5, I, 2, and
5% nBTPT A or PPDA relative to weight of urea. The samples were incubated in a
greenhouse under normal daylight conditions.
Daily analysis of urea in floodwater showed that urea disappeared in 5 days from the
control sample and in 5-8 days from the samples treated with 0.5-5% PPDA. Urea was
still detectable in all nBTPTA-treated samples even at day l3; the amount of
unhydrolyzed urea was proportionate to the rate of nBTPT A. Even at its highest rate
(5%), PPDA was less inhibitory on urea hydrolysis than was nBTPTA at its lowest rate
(0.5%).
In a pot experiment with flooded rice under normal daylight conditions, it was also
found (Byrnes et aI., 1989b) that nBTPTA was a stronger inhibitor of urea hydrolysis in
soil than PPDA (see page 285).
Bhupinderpal-Singh et al. (1992) used surface (0-15 cm) samples of two
representative soils from semiarid regions of northwestern India (a silty loam, pH 8.3
and a sandy loam, pH 8.4). Soil samples (200 g) were treated with urea (40 mg N) and 1
mg of nBTPT A or PPDA. The control received only urea. The mixtures moistened to
field capacity were incubated at 35°C. The unhydrolyzed urea was estimated after 1,2,
5, and 9 days of incubation.
Urea hydrolysis was complete in 2 days (control), 5 days (PPDA), and 9 days
(nBTPTA). After 1 day, rates of urea hydrolysis in the silty loam and sandy loam were
97 and 55% (control), 49 and 41% (PPDA), and 43 and 15% (nBTPTA), respectively.
Thus, nBTPTA was superior to PPDA in retarding urea hydrolysis in the soils studied.
Joo et af. (1992) transplanted sod from an established Kentucky bluegrass turf (on a
fine loamy soil, pH 7.5) into plastic pots (21 cm diameter, 16 em height). The
transplanted sod was allowed to grow in the greenhouse for -3 months before receiving
10% N urea solution (49 kg N/ha) with nBTPTA (0.125, 0.25, 0.5, and 1% of the
weight of urea-N) or PPDA (0.5, 1, and 2% of the weight of urea-N)o Only urea was
added to the control pots. The volatilized ammonia was determined daily for 7 days.
The cumulative NH3 loss from the control pots (49.9% of the added urea-N) was
reduced by the four rates of nBTPTA to 29.0, 24.6, 22.8, and 20.4%, and by the three
rates ofPPDA to 32.8, 26.7, and 24.2%.
These numerical data prove that nBTPT A was a more effective inhibitor than PPDA
in reducing the volatile NH3 loss from urea, and the effectiveness of both inhibitors
increased with their rates.
were or were not coated with paraffin oil (0.8% relative to weight of urea) and with
powdered nBTPTA (0.5 or 2 kg/lOO kg N) or PPDA (2 kg/lOO kg N). After fertilizer
application, the NH3 volatilized during 18 days (CT microplots) <r 15 days (NT
microplots) was assessed. Cumulative NH3 losses from urea applied on CT microplots
were the following (in kg Nlha): 6.7 (no inhibitor), 5.7 (PPDA), and 2.0 (0.5 and 2%
nBTPTA). The corresponding values for the NT plots were 13.3 (no inhibitor), 5.7
(PPDA), and 4.0 (both nBTPTA rates). Thus, less NH3 was lost from the CT microplots
than from the NT microplots. nBTPTA at each rate was more effective than PPDA.
Buresh et al. (1988a) described experiments in the 1986 dry season for comparing
the urease-inhibitory effectiveness of nBTPTA and PPDA on two flooded rice fields in
the Philippines. One field on a clay soil is located at Pila, the other on a silty clay soil at
Munoz. Urea was applied at three rates (35, 70, and 140 kg N/ha) with or without 0.9%
nBTPTA or 2% PPDA relative to weight of urea. Two-thirds of each rate of urea with
or without inhibitor was broadcast into the 5-cm deep floodwater at day 18 after
transplanting rice seedlings and one-third at day 5-10 after panicle initiation. The
control plots were not fertilized. Following each urea application, the floodwater was
analyzed daily for 8-10 days for residual urea and ~ +; vapor pressure of NH3 (pNH3)
was also assessed.
Analysis of urea has shown that after administration of the first urea amount
nBTPTA was a more effective inhibitor of urea hydrolysis than PPDA, especially at the
Munoz rice field. For example, at day 7 after application of the first urea amount (23,
47, and 93 kg Nlha, respectively), residual urea represented 21, 31, and 31 % of the urea
applied with nBTPTA, and 7, 13, and 0% from the urea applied with PPDA at Munoz,
whereas at Pila the corresponding values were 2, 2, and 9% (nBTPTA) and 1, 1, and 0%
(PPDA), respectively. In contrast, after the second application of urea, PPDA proved to
be more effective than nBTPTA at both Pila and Munoz.
Determination of the ~ + content gave results concordant with those obtained by
analysis of residual urea. Thus, after the first administration of urea, nBTPTA prevented
the accumulation ofNH4+ in floodwater for 10 days, whereas PPDA tended to delay and
not to eliminate accumulation ofNH/. These findings were valid for each urea rate and
for both rice fields. After the second application of urea, nBTPTA reduced less
markedly the accumulation of~+ in floodwater in comparison with PPDA.
Variation of pNH3 followed trends similar to those of~+.
4.9.2. Comparative Studies on the Effect of nBTPTA and Other Inhibitors on the
Immobilization of Urea-N in Soils
In a variant of the pot experiment performed by Cai et al. (1989) and referred to on page
208, the effect of nBTPTA on distribution of urea-N in upper layers of a flooded grey
soil and the effect of both nBTPTA and PPDA on immobilization of urea-N into
organic matter of two flooded soils (grey soil and yellow podzolic soil) were studied.
nBTPTA or PPDA at a rate of 5% of the urea weight was added to the floodwater of the
I-kg soil samples and followed immediately by 366 mg of J5N-labeled urea (5 atom%
excess 15N). The next step was incubation in a glasshouse. After 0, 1,2,4,6, 8, and 35
days, total N and 15N were determined in the 0-1-, 1-2-, and 2-4.5-cm layers for
establishing the depth of penetration of urea into the soil. For assessing 15N immobilized
into organic matter, total N and 15N were determined after extraction with a eaCh
solution to remove mineral N.
212
The bulk of the added N was recovered in the O-l-cm layer, although appreciable N
had diffused to the 2-4.5-cm layer. Considerably more N was retained in the soil treated
with nBTPTA than in the soil receiving urea only. Thus, after 35 days of incubation the
0---4.5-cm soil layer contained 47.4% of the added 15N in the urea-only treatment and
74.7% in the urea + 5% nBTPTA treatment.
Both inhibitors increased immobilization of added urea_ 15 N into organic matter of
both soils studied. After 35 days of incubation, the following percentages of the added
urea- 15N were immobilized in the urea, urea + 5% nBTPTA, and urea + 5% PPDA
treatments: 37.5, 46.3, and 47.5%. respectively (in the grey soil), and 43.8, 47.2, and
49.8%, respectively (in the yellow podzolic soil). Increased immobilization should
reduce losses of applied N and may increase the efficiency of fertilizer N.
Wang et al. (1991b) studied the immobilization of urea-N in samples of a Belgian
loam soil treated or not treated with air-dried, ground barley straw and nBTPTA, PPDA,
and hydroquinone (HQ). The reaction mixtures consisted of 20 g of soil + 0, 1 or 2%
straw + 4 mg ofurea-N, either unlabeled or labeled with 15N (5.297 atom% excess 15N;
i.e .•. 10.594 mg 15Nlkg soil) and 1% of inhibitor (based on weight of urea). The soil
moisture was brought up to two-thirds of field capacity. Then, the reaction mixtures
were incubated at 25°C for 15 days (mixtures with unlabeled urea) or for 20 days
(mixtures with labeled urea). Urea and inorganic N, including NH/, N0 3 -, and N0 2 -
were periodically extracted with 1 M KCl solution and determined. The reaction
mixtures with labeled urea were washed twice with 2 M KCl solution to remove the
mineral N. Then the mineral 15N and the 15N remaining in the soil organic fraction were
determined.
Analysis of residual urea showed that addition of straw to the soil caused an
extremely rapid urea hydrolysis and decreased the inhibitory effect of each test
compound. HQ and PPDA were more affected by straw than was nBTPTA. The order
of their inhibitory capacity, HQ < PPDA < nBTPTA, was the same in both the absence
and presence of straw. The inhibitors, especially nBTPTA reduced the formation of
NH4 +. Much less NH4 + was formed in the soil treated with 2% straw, due to
immobilization in microbial biomass and chemical fixation by humus. From the 9th day
on, a little more N0 3- + N0 2- was found in mixtures with 1% straw + inhibitor. This
means that the inhibitors slightly decreased the N immobilization. However, this effect
was less clear when 2% straw was used, since all reaction mixtures showed a low level
ofN0 3 - + N0 2-.
In contrast to the mixtures amended with 1% straw in which the inhibitors slightly
decreased immobilization ofurea-N, immobilization of 15N into soil organic matter was
enhanced and the amount of mineral 15N decreased in the unamended mixtures (Table
50). The immobilizing effect of the inhibitors increased in the order: PPDA < HQ <
nBTPTA. The order was the same for the recovery of 15N under the influence of
inhibitors.
These investigations were also referred to in a communication presented in 1990 at
an International Symposium held in Vienna (Van Cleemput et al.• 1991).
213
TABLE 50. Effect of urease inhibitors on immobilization ofurea_ 15 N in soil samples incubated at 25°C
for 20 days·
Distribution of 15Nb
Treatment ISN, Il Nm IlN/'N f x 100 IlNm/IlNfX 100 Recovery
(m!ikg soil) (m!ikg soil) (%) (%) (%)
Urea' 4.2 5.1 39.4 48.0 87.4
Urea + nBTPTA 7.4 3.2 69.4 30.2 99.7
Urea + PPDA 4.8 4.9 44.9 46.3 91.1
Urea + HQ' 5.2 4.6 49.4 43.5 93.0
Q Adapted from Wang et al. (l99Jb), by permission of Springer-Verlag.
b 15N, _ I5 N in the soil organic fraction. 15 Nm _ 15 N in the soil extract (mineral 15N). 15 Nr _ 15N initially
4.9.3. Comparative Studies on the Stability of PTA and TPTA Compounds and Other
Inhibitors
part of the TPTA is decomposed via two partial hydrolysis reactions (Figure 68). The
other decomposition products and reactions were not identified.
H~-P
s
II ......NH 2
'NH2
Thiophosphoryl triamide
(TPTA)
+ H:P
- S
II ......NH 2
NH40-P,
NH2
Ammonium thiophosphorodiamidate
(ATPDA)
ATPDA
s
II.,....NH2
NH40-P
' NH2
+ H:P
- NH4
s
NH4o-....11
i fP - NH2
Diammonium thiophosphoroamidate
(DATPA)
Figure 68. Solid-state decoJ1llosition of thiophosphoryl triarnide via partial hydrolysis reactions.
(Adapted from Radel et al. (1987). by permission ofTennessee Valley Authority.!
The possibility for using TPTA in fluid fertilizers was also studied. This possibility
was substantiated by observations that TPTA is very soluble and, additionally, very
stable for up to 30 minutes in 75 and 87% urea solutions at 90 and 99°C, respectively.
The fluid fertilizers studied comprised: urea-ammonium nitrate suspension (UAN,
250
~ur..
--
aolut1OD
pH 8.54
191
DB
31-0-0
H2O pH S.lO
ua pH 7.24 !l3
)6-0-0
pH 7.)6 68
I 43
t
Figure 69. Stability ofTPTA in water and fluid fertilizers at 25°C. !From Anonymous (1987), Radel
et al. (1987), Gautney (1987), by permission of Tennessee Valley Authority.!
36-0-0) and solution (UAN, 31-0-0), and 40% urea solution. TPTA in water served for
comparison. In each case concentration of TPTA was 1% (weight/weight). Figure 69
shows that the first-order reaction half-life for decomposition of TPTA increases with
215
the pH of fluid fertilizers. As these half-life values are high (43-191 days), there is
sufficient time for adding TPT A to fluid fertilizers and for their application on the soil.
Decomposition of TPTA in solution phase is the result of a single hydrolytic
reaction at slightly alkaline pHs, the only decomposition product being the ammonium
thiophosphorodiamidate (ATPDA).
Stability of nBTPTA in aqueous solutions was studied by Bremner and Chai (1986).
nBTPTA was not directly analyzed. An indirect method was applied: diminution of the
inhibitory effectiveness of nBTPTA solution for urease activity in soils was evaluated
after its storage, because diminution of effectiveness indicates decomposition of
nBTPTA. The aqueous solution, containing 50 fig of nBTPTAlml, was stored at 5, lO,
20, and 30°C for 0, 3, 7, 21, and 28 days, and then 1 ml of stored solution and 1 ml of a
urea solution (10 mg of urea) were added to 5-g air-dried samples of three Iowa soils.
After incubation of the reaction mixtures (20°C for 2 days), the residual urea was
assessed and percent inhibition calculated.
The results showed that storage of the nBTPTA solution at 5°C for 3 days did not
significantly affect its ability to inhibit urease activity in soils, i.e., nBTPTA did not
decompose under these conditions. With increasing temperature and prolongation of
storage time, the inhibitory effectiveness of nBTPTA gradually decreased, i.e., more
and more nBTPTA molecules were decomposed. For example, the inhibitions, that the
fresh nBTPTA solution and the solution stored at 30°C for 28 days exhibited in the
urease activity of the three soils studied, were: 98 and 71 %, 95 and 61 %, and 69 and
47%, respectively.
Douglass and Hendrickson (1991), who developed a sensitive high-performance
liquid chromatography method for direct analysis of nBTPTA and its oxygen analogue
(nBPTA), studied stability of these compounds in solutions. lO,5 M nBTPTA solutions
were prepared in deionized water or 20% Hoagland's nutrient solution (adjusted to
either pH 5.5 or 7.5 with H2S04 or KOH, respectively). Similar solutions were prepared
to a final concentration of 40% (volume/volume) methanol. The solutions were stored at
25,4, and -20°C for 3, 7, and 14 days prior to analyses. The results obtained after 14-
day storage are summarized in Table 51.
TABLE 51. Stability of nBTPT A in 10-5 M solutions following storage for 14 days
at various temperatures
Q
One can see from Table 51 that solutions of nBTPT A in water and nutrient solutions
can be stored without decomposition for at least 2 weeks at room temperature or when
refrigerated. Considerable nBTPTA decomposition occurred, however, when the
solutions were stored at -20°C (i.e., in frozen state), especially if they were acidic (pH
216
5.5). The decomposition at _20DC was completely eliminated by adding methanol to the
solutions to prevent freezing. However, for storage at 25 and 4 DC, addition of methanol
to the solutions is not recommended, since at these temperatures methanol enhances
decomposition of nBTPTA in non-alkaline solutions.
Similar results were obtained with solutions of nBTPTA.
4.9.3.2. Comparative Studies on the Stability Qf PTA. TPTA, nBTPTA, and Other
Inhibitors in Soils
In the field experiment conducted by Beyrouty et al. (1988a, b) and referred to on page
196, N-benzyl-N-methylphosphoric triamide (in contrast to PPDA which retarded urea
hydrolysis for 19 days in rnicroplots covered in a proportion of about 60% by maize
residue) was not able to retard urea hydrolysis under similar conditions. This finding
was interpreted as evidence of a deactivating action of plant residue on this
phosphorotriarnide.
The mechanism through which nBTPTA is decomposed in soil was studied by Chai
et al. (1988), Byrnes and Christianson (1988), and McCarty et al. (1989). These
investigators found that a) nBTPTA is a strong inhibitor of soil urease activity, but a
very poor inhibitor of ureases of plant (jackbean) and bacterial (Bacillus pasteurii)
origin; b) aqueous extracts from soils previously treated and incubated aerobically with
nBTPTA inhibited activity of jackbean urease; c) nBTPTA is rapidly converted in soil
(largely abiotically) to a compound that is much more effective than the parent
compound for inhibition ofjackbean urease activity; d) data from 31p nuclear magnetic
resonance and infrared spectroscopy of unfractionated soil extracts indicated that the
decomposition product of nBTPTA is its oxygen analogue, i.e.. N-(n-butyl)phosphoric
triamide (nBPTA). This means that the mechanism through which nBTPTA is
converted in soil to nBPTA is oxidative' desulfuration:
Creason et al. (1990) extracted this compound with water from samples of a silt
loam soil (PH 6.9) previously treated with nBTPTA (5 mglg soil) and incubated in
loosely capped bottles (for access of air) at 25 DC for 24 hours. Then, the compound was
purified by high-performance liquid chromatography fractionation and identified by
mass spectroscopy as nBPTA. The purified nBPTA was shown to be highly active,
giving 50% inhibition of jackbean urease at concentrations between 10 and 100 mM.
This urease-inhibiting ability was almost equivalent to that of PPDA. The further fate of
nBPTA in soil was not studied.
'The oxydative mechanism is also supported by the results of an experiment of Luo et al. (1994). Air-dried
samples (15 g) of an Australian vertisol (pH 7.6), placed in 120-ml glass bottles, were flooded with 30 ml of
water, then nBTPTA (1% relative to weight of urea) and urea (14 rng) were added and the mixtures were
oxygenated by bubbling O2 into the floodwater for 3 hours or by adding 1 ml of 30% H20 2 • Mixtures
without nBTPTA, O2, and H20 z were the controls. All mixtures were incubated at 25°C for 8 days, during
which at 2-day intervals the residual urea was determined.
It was found that rate of urea hydrolysis in the nBTPTA-treated mixtures was significantly (p<0.05)
slowed by O2 and very significantly (p<O.OO 1) by HzO z, indicating that at least a part of the nBTPTA was
oxidized to nBPTA, which was more inhibitory than the parent compound.
217
The aim of other laboratory studies was to compare the effects of nBTPTA and
nBPTA on hydrolysis of urea in samples of 24 soils obtained from different sites
throughout the U.S.A. The reaction mixtures, containing 1 mg ofurea-N and 0 or 10 flg
of inhibitor/g soil, were incubated at 25°C for periods up to 10 days. Both compounds
significantly extended the persistence of urea in all soils, but inhibition of urea
hydrolysis with nBTPTA was more persistent than that with nBPTA in nearly every
soil. The average half-life of urea was more than 8 days for nBTPTA, 6 days for
nBPTA, and less than 2 days without inhibitors. The superior effectiveness of nBTPTA
can be explained by its greater stability. Although nBPTA is the active inhibitor,
application of nBTPTA - due to the subsequent generation of nBPTA - is more
effective in most soils than direct application of nBPTA.
This conclusion gained further support in a laboratory experiment described by
Hendrickson and Douglass (1993). nBTPTA and nBPTA were applied to an acid (PH
4.9) silt loam soil (soil A) and to same soil that had been neutralized (PH 7.1) by long-
term liming (soil B) or by recent application of Ca(OHh (soil C). In addition to the
effect of the two inhibitors on urea hydrolysis, their stability in soil was also studied.
The reaction mixtures contained 2 g of soil A, B or C, 0 or 2 mg ofurea-N/g soil and
o or 10 flg of nBTPTA or nBPTAIg soil. Additionally, reaction mixtures were prepared
from 2 g of soil B, 0 or 2 mg of urea-N/g soil and O. 1 or 100 flg of nBTPTA or
nBPTAIg soil. All reaction mixtures were incubated at 25°C for 14 days and analyzed
for remaining urea several times during the incubation. The results showed that the
inhibition of urea hydrolysis was weak in soil A and strong in soils B and C. nBTPTA
was more inhibitory than nBPTA in each soil and also in the soil B amended with 1 or
100 flg nBTPTA or nBPTAIg soil.
The reaction mixtures were also analyzed also for quantitative determination (by
HPLC) of the inhibitors, including nBPTA formed from nBTPTA during incubation of
the reaction mixtures.
In soil A, B, and C, to which nBTPTA or nBPTA was applied at the rate of 10 flglg
soil, nBTPTA, nBPTA formed from nBTPTA during incubation and nBPTA applied
disappeared in 8, 8, and 2 days, respectively (soil A), in 14, 14, and 8 days, respectively
(soil B), and in 10, 10, and 8 days, respectively (soil C).
In soil B, depending on the rate of nBTPTA or nBPTA application (1, 10, and 100
flglg soil), nBTPTA disappeared in 3, 14, and more than 14 days, respectively, nBPTA
formed from nBTPTA disappeared in 8, 14, and more than 14 days, respectively, while
the disappearance of the applied nBPTA occurred in 2,8, and 8 days, respectively.
It is evident from these data that both nBTPTA and nBPTA were less persistent
(and, thus, less urease-inhibitory) in the acid soil A than in the neutral soils B and C.
Therefore, a primary role can be attributed to soil pH or pH-associated characteristics in
controlling persistence of both inhibitors. It is also evident that the nBPTA formed from
nBTPTA during incubation was more persistent (and consequently nBTPTA was more
urease-inhibitory) than the nBPTA applied directly to soil.
Christianson et af. (1990) compared the effects of the two thio and two oxygen
analogues, nBTPTA and nBPTA, CHTPTA and CHPTA on volatilization of ammonia
from urea and hydrolysis of urea in soil samples. The same Guthrie soil and methods
were used as those used in the laboratory experiments of Carmona et al. (1988, 1990)
(see page 146). The rate of urea addition was again 100 kg N/ha on a surface area basis,
219
but rates of inhibitors were only 0.01 and 0.1% relative to weight of urea. Ammonia
volatilization and urea hydrolysis were assessed several times during the incubation of
TABLE 52. Cumulative ammonia volatilization from urea during 14 days and urea
hydrolysis during 10 days as influenced by urease inhibitors"
N volatilized (%) Urea hydrolysis (%)
Inhibitor Inhibitor rate (% relative to weight of urea)
0.1 0.01 0.1 0.01
No (urea alone) 47 47 87.0 a 87.0 a
nBTPTA 7 28 65.4 be 84.7 a
nBPTA 7 15 71.3 b 77.5 ab
CHTPTA 16 30 56.6c 78.0 ab
CHPTA 10 14 60.0c 60.5 b
"Adapted from Christianson et al. (1990).
Numbers followed by the same letter are not significantly different at p=0.05.
14 and 10 days, respectively. The results obtained at the end of the incubation periods
are summarized in Table 52.
It is evident from this table that the inhibitory effect of the four triarnide compounds
studied was higher at their 0.1 % than at their 0.01% rate.
At the 0.1 % rate, NH3 volatilization was inhibited to the same extent by nBTPTA
and its oxygen analogue, and to a smaller extent by CHTPTA than by its oxygen
analogue. At the 0.01 % rate, the thio analogues were less inhibitory than the oxygen
analogues.
The inhibition of urea hydrolysis was stronger by the thio analogues at the 0.1 % rate
and weaker at the 0.01% rate in comparison with the oxygen analogues, but the
differences in inhibition degrees were not significant at p=0.05. The inhibiting effect of
the four compounds on urea hydrolysis increased, although insignificantly, in the
following orders: nBPTA < nBTPTA < CHPTA < CHTPTA (at 0.1 %), and nBTPTA <
CHTPTA < nBPTA < CHPTA (at 0.01 %).
A comparative study on the mobility of these four triarnides and urea was also done
(Christianson and Howard, 1994), by means of soil thin-layer chromatography (TLC).
Five soils were used, from which TLC plates were prepared. Each triamide (0.5 mg
dissolved in methanol) and urea were applied as separate spots on each plate, then the
plates were placed in a developing tank containing water and wetting front was allowed
to advance 10 cm. The plates were then removed, dried at 60°C, cooled and placed in an
iodine developing chamber overnight. The triamides and urea were readily discerned as
golden-brown spots.
nBTPTA moved on plates of all soils at a rate comparable with that of urea and
although no nBTPTA remained at the starting point, it exhibited significant trailing.
Contrarily, the other thio analogue, CHTPTA was very immobile and most of the
compound remained at the starting point. On some plates however a narrow, faint streak
was visible which emanated from the CHTPTA spot and migrated distances similar to
those of urea. The two oxygen analogues, nBPTA and CHPTA moved as compact spots
at rates similar to that of urea.
220
The Rr values of urea and triamides on the plates prepared from the five soils varied
between 0.68 and 0.86 (urea), 0.71 and 0.88 (nBTPTA), 0.67 and 0.79 (nBPTA), 0.64
and 0.75 (CHPTA) or were invariably 0 (CHTPTA).
The trailing spot of nBTPTA eluted from the plates produced a significant inhibition
of jackbean urease, while the inhibitory effect of the eluted faint streak above the
CHTPTA spot was very weak on jackbean urease. These findings indicate that in soil a
partial, but rapid conversion of nBTPTA to its more urease-inhibiting oxygen analogue,
nBPTA took place, while conversion of CHTPTA to a more active inhibitor was very
limited. Immobility and limited conversion of CHTPTA in soil are disadvantageous
properties of this inhibitor.
The investigations performed by Keerthisinghe and Freney (1994) make it possible
to compare the urease-inhibiting effectiveness of nBTPTA and TPTA with that of their
oxygen analogues, nBPTA and PTA, respectively. Flooded samples of two Australian
soils were amended with urea and inhibitors (at a rate of 1% relative to weight of urea),
then incubated for 12 days and analyzed for remaining urea every 2 days.
The conclusion which may be drawn from the results concerning comparative
efficiency of the two pairs ofthio and oxygen analogues is that nBTPTA was a stronger
inhibitor than nBPTA and that TPTA was superior to PTA.
These investigations, in which the effect of an algal inhibitor on the effectiveness of
urease inhibitors was also studied, are referred to in Chapter 6, on page 249.
By using the procedure a (see page 156), Medina and Sullivan (1986, 1987) found that
the inhibitory effect of 2,2,4,4,6,6-hexaarnino-CTPAT on soil urease activity was more
marked not only than that of phosphoryl triarnide, but also than that of acetohydroxamic
acid, hydroxyurea, thiourea, and ammonium thiocyanate.
221
First, the effect of nitrification inhibitors on soil urease activity, then the urease
inhibitors also possessing nitrification-inhibiting capacity will be dealt with.
5.1.1. Effect of Nitrapyrin and Other Nitrification Inhibitors, Except Azide and
Dicyandiamide, on Soil Urease Activity
Aiming at evaluating the effect of nitrification inhibitors on urease activity in three Iowa
soils, by applying the 5-hour test, Bremner and Douglas (1971) studied 17 compounds
(Figure 70) that were patented as nitrification inhibitors by different companies. Each
compound was used at a rate of 50 ppm (on soil basis). Inhibition of soil urease activity
was very weak: 1-2% with CL-1580 and less than 1% with the other compounds. The
finding that nitrapyrin did not inhibit soil urease activity is in concordance with the
observation of Goring (1962), according to which this nitrification inhibitor, added at
rates of 0.05-10 ppm to 50-g samples of a California sandy loam soil (pH 7.3) treated
with urea (200 ppm N), did not prevent the conversion of urea-N to Nl4+ during
incubation (4,8 or 12 weeks at 21°C).
Bundy and Bremner (l974b) studied three nitrification inhibitors: nitrapyrin, CL-
1580 (see Figure 70), and 4-amino-l,2,4-triazole (Figure 71) in form of hydrochloride
(ATC), determining their effects on urea hydrolysis, ammonia volatilization, and
nitrification. Ten-g samples of three Iowa soils (two clay loams and a sandy clay loam)
were treated with 1 ml of urea solution containing 4 mg of N and with 2 ml of water or
2 ml of aqueous solution containing 100 f.lg of inhibitor. Water was then added to bring
the soil moisture content to 60% of WHC. During incubation (at 30°C), the NH3
volatilized was determined. After various times, the unhydrolyzed urea, NH/, N02',
and N0 3', were analyzed.
Nitrapyrin did not have any effect on urea hydrolysis. CL-1580 and ATC reduced
the amount of hydrolyzed urea by 2-8% during the first 12 and 24 hours, but in 3 days
hydrolysis of urea became complete in both absence and presence of inhibitors.
Volatilization ofNH3 has intensified under the influence of inhibitors in the order:
nitrapyrin> ATC > CL-1580,
as they inhibited nitrification of NH/ (and accumulation of N0 2' and N0 3") in this
order:
Due to low water solubility ofnitrapyrin and CL-1580, the effect of higher inhibitor
rates could be studied only in the case of ATC. It was established that application of 250
and 500 f.lg of ATC/1O g soil did not have any significant effect on urea hydrolysis and
volatilization of urea-N as NH3 but appreciably increased the percentage inhibition of
nitrification.
In the laboratory and field experiments conducted by Ashworth et al. (1977) and
already mentioned on page 36, the effect of nitrapyrin (1.25 kglha) on urea hydrolysis
·Other investigators (e.g.. Knop, 1982) also found that nitrapyrin increased the volatile NH3 losses from
different urea-treated soils which indicates that nitrapyrin did not inhibit urease activity of these soils.
N
N
N
NH2
IN N.J..,.N
CI,c-O-CI H,c~.,)LNH2
N N
H,N~N..Jl- CCls
2-Chloro-6-(trichlorometl1yl)pyridine 2-Amino-4-chloro-6-metl1ylpyrimidine 2,4-Diamino-6-trichloromethyl os-triazine
(nitrapyrin; N-Serve) (AM) (CL-1580)
q-NH2 NH C I - Q -NH 2
P- 2
CI CI
o-Chloroaniline m-Chloroaniline p-Chloroaniline
?-NH-OC-C H 3 ?-NH-OC-CH3
~N)LN 2
0H ~.,)l-CI
o N
CI NO.
2-Aminopyridine 2-Chloropyridine 3-Chloroacetanilide m-Nitroacetanilide
OH
HSC ~ N"CH3
I
HC=C-C-CH3
'N-NO
H,c"
o-
6 H3
- 'NO
N-Nitrosodimetl1ylamine N-Metl1yl-N-nitrosoaniline 2-Metl1yl-3-butyn-2-o1
Figure 70. Structure of nitrification inhibitors tested by Bremner and Douglas (1971) for evaluation of their effect on soil urease activity.
223
was also studied, and it was found that nitrapyrin, like Na 2 CS 3 , (NH4hCS 3 , and CS 2 , had
no effect on urea hydrolysis.
A H:tJ-N--CH B
I II
HC~/N
Khadzhiev (1985) reported that ATC added to irrigated grey soils under cotton
plantations in Uzbekistan effectively inhibited nitrification and, contrarily, increased
urease activity.
Raguotis and Shleinys (1986) introduced urea (90 or 180 kglba) with or without
nitrapyrin (1 or 2% relative to weight of urea) under the canopy in a pine forest.
Following fertilization, the volatilized ammonia was measured during 41 days.
Nitrapyrin at 1% rate did not reduce the volatile NH3 losses, but when it was applied
at 2% rate 14 and 25% reductions occurred in the cumulative NH3 losses from 90 and
180 kg urealha, respectively. This experiment carried out in 1980 was repeated in 1981,
when nitrapyrin had, even at its 2% rate, no significant reducing effect on the volatile
NH310sses.
Simon and Bergerova (1986) determined urease activity in samples of two soils
(chernozern, pH 7.4 and pararendzina, pH 7.8) from Slovakia. The samples were
previously incubated with or without urea (or ammonium sulfate) and with or without
nitrapyrin (2% relative to fertilizer N) at 28°C for 14 days. The results showed that
urease activity was slightly inhibited in samples previously treated with nitrapyrin
without fertilizers, but it was practically not affected in samples previously treated with
nitrapyrin + urea or nitrapyrin + (N~)2S04.
In a pot experiment, Mekhtiev et al. (1988) fertilized samples of a Moldavian soil
with P and K (each at a rate of 60 kglha) and urea (180 kg NIha) with or without
nitrapyrin (0.5 or 1% relative to urea-N). The control soil was fertilized only with P and
K. There were variants unsown and sown with a mixture of vetch + oats. Urease activity
assayed after three incubation times' gave the following mean values (mg NH3/g soil):
2.4 (control), 0.7 (urea), and 1.0 (urea + nitrapyrin) in the unsown variants, and 2.7
(control), 3.0 (urea), and 4.1 (urea + nitrapyrin) in the sown variants. This means that
nitrapyrin attenuated the negative effect of the high urea rate on urease activity in the
unsown soil and intensified the positive effect of urea on urease activity in the sown
soil. In other words, nitrapyrin administered together with urea did not act inhibitorily
on soil urease activity.
All investigations mentioned above indicate that nitrapyrin, used at concentrations
efficient for inhibition of nitrification, does not inhibit urease activity in soil. This
conclusion should be compared with the result described by Reddy and Prasad (1975).
These investigators treated 200-g air-dried samples of a sandy clay loam soil (PH 7.8)
from India with urea (100 ppm N) with or without sulfathiazole (ST) or nitrapyrin (1 %
relative to urea-N). Then the samples were moistened to field capacity and incubated at
room temperature of 29°C for 4 weeks. At weekly intervals, the samples were analyzed
for residual urea, NH/, N02-, and N03-. ST had slight influence on urea hydrolysis.
Concerning nitrapyrin (N-Serve), the authors write: ''N-Serve showed considerable
retarding influence on the hydrolysis of urea in the present investigation but this needs
confirmation by other workers". But we should reiterate that this result was not
confirmed in other investigations.
The effect of ST on hydrolysis and nitrification of urea was also studied by
Muthuswamy et al. (1975) in pot experiments with red, black, and alluvial soils from
India. When urea granules coated with tar (from different coals) and ST were used
instead of uncoated urea, formation of both NH4 + and N03- was reduced and,
consequently, the N loss was decreased.
We mention here that the isomer of ATC, 3-arnino-l,2,4-triazole (the herbicide
amitrole; Figure 71) was also evaluated as an inhibitor of soil urease activity.
Gauthier et of. (1976) used three clay soils from Quebec. The soil samples were
arranged in pots (45 cm high x 23 cm diameter), each preserving the natural layering.
Soybeans (Glycine max) were planted in each pot and, after 3 weeks, the soil received
10 ml of 2.10-3 , 2.10-4, 2.10-5 or 2.10-6 M amitrole solution. The control soil was not
treated with arnitrole. After 1 week, samples were collected from the pots, 2.5 cm below
the surface (S samples) and 5 cm from the bottom, i.e. 40 cm below surface (deep, D,
samples), and their urease activity was assayed. The results showed that amitrole
inhibited soil urease activity. There was a linear relationship between percentage
inhibition and arnitrole concentration. The inhibition was stronger in the S than in the D
samples. Thus, arnitrole applied at 2.10-3 and 2.10-4 M concentrations broUght about
complete inhibition of urease activity in Sand D samples of two soils and inhibitions of
82 and 74% in S samples and of 68 and 63% in D samples of the third soil. Amitrole at
2.10-5 and 2.10-6 M concentrations reduced urease activity by 56-38% in S samples and
by 63-17% in D samples of the three soils studied. The only exception was sample D of
the third soil in which urease activity was not inhibited by the lowest amitrole
concentration. Further observations were that amitrole also manifested nematocidal
effect and, unfortunately, inhibited nodulation on soybean roots.
The effect of amitrole on urea hydrolysis in soil was also studied by Vlek et al.
(1980). Prilled urea alone (at a rate of 100 kg N/ha) or in a mixture with arnitrole (2%
on urea weight basis) was incorporated into a silt loam soil placed in pots and then
flooded (depth of floodwater = 5 cm). During incubation (10 days at 35°C day and 25°C
night), the floodwater was analyzed daily for residual urea. After 0 and 1-6 days, the
following values were registered: 140, 71, 29, 5, 1, 0, and 0 mg of urea-Nil,
reo;pectively, in the control soil (not treated with amitrole), and 137, 67, 22, 5, 2, 1, and
omg of urea-Nil, respectively, in the amitrole-treated soil. It is evident that in these pot
226
N--C-CCla
II II
CHa-CH2-o--C, ..,.....N
S
1982, and 1983. In all treatments, urea was completely hydrolyzed in 7-14 days after
injection; the two nitrification inhibitors did not slow the rate of urea hydrolysis.
This finding is in concordance with the results obtained by Abdullatif and Stroehlein
(1990) in a study with three Arizona soils (sandy loam, pH 7.2, loam, pH 7.8, and
loamy sand, pH 7.7). Soil samples (50 g), treated with urea only (at a rate equivalent to
224 kg N/ha) or with urea and etridiazole (1.27 kglha), were incubated at 33°C and their
pH was measured at intervals of 0, 1, 2, 3, 4, 6, 8, and 16 days. Changes in pH were
similar in untreated and etridiazole-treated samples which was interpreted as evidence
proving that etridiazole did not affect urea hydrolysis, did not reduce urease activity. In
another experiment with the same three soils, etridiazole was found to inhibit
nitrification of urea and ammonium sulfate for 10-15 days.
Bremner (1986) mentioned that 2-ethynylpyridine and phenylacetylene (Figure 73),
which proved to be the most effective nitrification inhibitors among 15 monosubstituted
(RC=CH) and 6 disubstituted (RC=CR) acetylenes tested in different <NH4hS04-
treated Iowa soils, had no significant effect on urea hydrolysis in soil. In another study
O-C5CH
N
B
o-C=CH
Figure 73. Structure of2-ethynylpyridine (A) and phenylacetylene (B).
r T CH3
"'-N. . . N
ICONH2
Figure 74. Structure of3-methylpyrazole-l-carboxamide.
74) added to 100-g samples of three soils (soddy podzol, common chernozem, and
calcareous chemozem) had no effect on urea hydrolysis but inhibited nitrification of
NH4 + released from urea. The samples received 20 mg of urea-N with or without 2%
MPC (on urea-N basis) and water to 60% of WHC and were incubated at 28°C. Their
residual urea content was determined after 6-72 hours of incubation. For NH4 + and
N0 3 -, the samples were analyzed after 15,30, and 45 days of incubation.
Similar results were registered by McCarty and Bremner (1990b), who treated
samples of three Iowa soils with MPC at a rate of 10 or 50 ~glg soil, and also by Popov
et al. (1990), who carried out a pot experiment using a grey forest soil, fertilized with
NPK, treated with MPC (1.6 ~glg soil) and sown with maize.
Shcherbakov and Stakhurlova (1990) studied the effect of MPC on nitrification and
urease activity in microplots on a leached chernozem. Urea was applied with or without
MPC in bands at 10-cm depth. Urea rate was equivalent to 90 kg of Nlha, whereas that
of MPC was 3% relative to urea-No The soil was seeded to cucumber. MPC inhibited
nitrification for approximatively 4 weeks. Contrarily, urease activity in the period of 8-
32 days after fertilization showed higher values in the urea + MPC treatment than in the
urea-only treatment. The increased urease activity, which should be ascribed to
microbial synthesis of new urease molecules, indicates that MPC did not inhibit activity
and microbial synthesis of urease.
In Kucharski's (1991) experiment, 50-g air-dry samples of a loamy sand soil (PH
6.7) from Poland were amended with urea (10 mg N) and nitrapyrin, A TC, and MPC at
rates of 0,0.35,0.70, 1.05,2.10, and 3.15% relative to urea-No The soil moisture was
kept at 60% ofWHC during the incubation (120 days) at 20°C. Analyses ofNH4 +-N and
N0 3--N at days 10,20,30,60,90, and 120 showed that none of the three nitrification
inhibitors at any rate inhibited hydrolysis of urea.
In the pot experiments of Khabirov et al. (1992) (see page 58), besides thiram, five
pyridine derivatives, all being nitrification inhibitors (Figure 75), were also tested for
evaluation of their effect on urease activity in a leached chernozem. The experimental
conditions were identical to those under which thiram was tested.
228
CH3
'''~'" N
2,4,6-Trimethylpyridine 2-n-Propyl-3,5-c1ethylpyridine
(C) (0)
HaC- H
2-Pheny~3,5-diethylpyridine
(E)
Figure 75. Structure of the nitrification inhibitors tested by Khabirov et al. (1992) for evaluation of their effect
on soil urease activity.
which in absence ofKN 3 did not produce N0 2- from urea in appreciable amounts, KN3
strongly inhibited nitrification, whereas in the other two soils, which in absence of KN 3
accumulated more N0 2-, KN3 did not have any significant inhibitory effect on
nitrification ofurea-N as it did not decrease accumulation and maximum concentrations
of N0 2-. It is assumed that the lack of inhibitory effect on nitrification is due to
decomposition ofKN3 by its reaction with N0 2-:
In addition, in acidic soils KN3 may hydrolyze with formation of volatile hydrazoic
acid':
NH
II
Ca=N-C:=N H2N-C-NH -C=N
NH a NH
II II II
H2N-C-NH -C-NH2 H2N- C- NH 2
Guanylurea Guanidine
Figure 76. Structure of calcium cyanamide and of products resulting from its transformations in soils.
Finally, urea is hydrolyzed and the released ~ + can be nitrified. DCD, produced
by chemical synthesis (didin) and added to soil, acts, at the beginning, as a nitrification
inhibitor. Then it is slowly decomposed and mineralized with formation of plant-
'Hydrazoic acid is not only volatile, but it is also poisonous and spontaneously explosive. Its explosion hazard
increases in the presence ofHg and Ag salts. Thus, mercuric azide formed in soil is detonated even under the
action ofa fiiction (Rozicky and Bartha, 1981).
•• Guanylthiourea can also give rise in soil to dicyandiamide (see page 75).
230
than did the neem cake- and lac-coatings. The loss was lowest from the urea
supergranule.
Some of these investigations are also referred to by Sahrawat (1989) and Prasad and
Power (1995).
Percent inhibition of nitrification increased with the rate of pyrite and decreased
with incubation time. After 30 days of incubation, inhibition of nitrification was 40.3%
at the highest pyrite rate and 55.9% in the DCD treatment.
5.2.1.3. Fluorides
Sodium fluoride, which was patented as an inhibitor of soil urease actlVlty (see
Subchapter 1.5), is also able to inhibit nitrification in soil (Baumgartner and Otlow,
1985).
235
urea with 2.5-7.5% TU-N either increased or had no effect on NH3 volatilization,
because nitrification was inhibited but rate of urea hydrolysis was not reduced.
Amending urea with 11-33% TU-N reduced NH3 volatilization between 11 and 94%,
because urea hydrolysis rate was reduced.
Germann-Bauer (1987), who proved that guanyithiourea (GTU) inhibits soil urease
activity (see page 52), also established that GTU is a stronger inhibitor of nitrification
than of the soil urease activity. One of the abiotically catalyzed transformations of GTU
in soil is dicyandiamide, a potent nitrification inhibitor (see also Amberger, 1989).
5.2.2.4. Xanthates
Ashworth et of. (1979) found that potassium ethyl xanthate and cellulose xanthate
inhibited nitrification more markedly than ureolysis (see page 58). The data published
by Ashworth et 01. (1980) confirm the inhibitory effect of some xanthates on both
urease activity and nitrification.
TABLE 54. Effect of eight urease inhibitors on nitrification in soils, as compared to that of three nitrification
inhibitors"
Inhibition of nitrification (%)
Amount of
Clay Clay Sandy
Compound compound
loam loam clay loam Average
(flglg soil)
~H 7.6 EH7.3 EH7.2
Urease inhibitors:
Catechol !O 3 0 5 4
50 4 8 20 II
Hydroquinone 10 2 7 11 7
50 4 54 71 43
p-Benzoquinone (BQ) 10 0 4 8 4
50 3 56 77 45
2,3-Dimethyl-BQ to 2 8 4
50 3 43 72 39
2,5-Dimethyl-BQ 10 2 10 17 10
50 30 83 96 70
2,6-Dimethyl-BQ 10 I 6 13 7
50 27 87 94 69
2,S-Dichloro-BQ 10 2 4 7 4
50 3 32 52 29
2,6-Dichloro-BQ !O 2 3 16 7
50 3 38 46 29
Nitrification inhibitors:
Nitrapyrin to 69 85 96 83
2-Amino-4-chloro-6-methylpyrimidine (AM) 10 1 25 68 31
Sultathiazole (ST) 10 1 24 40 22
"Adapted from Bundy and Bremner (1974a), by permission of Pergamon Press PLC.
Table 54 shows that the eight urease inhibitors tested had little effect on nitrification
when applied at the rate of 10 Ilg/g soil. but at the rate of 50 Ilg/g soil some of them,
especially 2,5-dimethyl-p-benzoquinone and 2,6-dimethyl-p-benzoquinone, markedly
inhibited nitrification. However, none of them inhibited nitrification as effectively as
nitrapyrin. Degree of inhibition was higher with the light-textured soil than with the
clay loams.
Using samples of four Indian soils (alluvial, black, red, and laterite), Sachdev et al.
(1977) prepared reaction mixtures from 10 g of air-dried soil + 10 g of quartz sand + 1
ml of a solution containing 2 mg of N as (NH4hS04 or urea with or without 500 Ilg of
2,5-dimethyl-p-benzoquinone + water to 60% of WHC. The mixtures were incubated
aerobically at 30°C for 14 days, after which the NH/-N and N0 3--N contents were
determined. It was established that 2,5-dimethyl-p-benzoquinone strongly inhibited
238
nitrification of both (NH 4hS04 and urea; degree of inhibition was 91.7-98.0% and 92.2-
97.3%, respectively.
Studying a great number of polyhydric phenols and quinones, Mishra and Flaig
(1979) and Mishra et al. (1980) found that some of these compounds inhibit both soil
urease activity and nitrification (see pages 91-92).
Rodgers (1984a) mentioned that the 2,6-dimethyl-p-benzoquinone concentrations
needed to inhibit nitrification in soil are 5-10 Jlg/g soil.
Hera et al. (1986) compared the effects of hydroquinone (HQ) and two nitrification
inhibitors, nitrapyrin and ATC, on the formation of NO)- in urea-treated samples of a
chemozernic soil. The three compounds were applied at rates of I, 2, and 10 mglkg soil.
The experiment lasted 200 days, during which the NO)- content was determined
periodically.
By inhibiting urea hydrolysis, HQ reduced the formation of NO)-. But the effects of
nitrapyrin and ATC were stronger. With each compound, the relation between dose and
effect was directly proportionate. In comparison with the soil treated with urea only, HQ
reduced the formation of NO)- for 14 days when applied at rates of 1 and 2 mglkg soil
and for 28 days when its rate was 10 mglkg soil. After this period, nitrification showed
a trend for recovery_ But, even after 200 days, the amount of NO)- remained smaller in
the soil treated with urea and an inhibitor than in the soil treated with urea only.
Ten-g air-dried samples of a Belgian loam soil were used by Wang et al. (1990) for
evaluating the effect of HQ, PPDA, and nBTPTA on nitrification of NH/ and N0 2 -.
One ml ofa solution containing 2 mg ofNH/-N as (NH4hS04 or 0.1 mg of N0 2 --N as
NaN0 2 and I ml of a solution containing 0.04 g of urease inhibitor were added to the
soil samples; the resulting moisture content was two-thirds of field capacity, assuring
aerobic conditions. During incubation at 25 D C, the samples were analyzed periodically
for NH/, NO)-, and N0 2-.
HQ brought about a 2-day delay in oxidation of the added NH4 +. HQ delayed the
oxidation of soil-derived + added N0 2- similarly by 2 days. Thesefmdings suggest that
HQ inhibited oxidation of NH/ to N0 2 (including oxidation of soil NH/ to soil-
°
derived N0 2 -) and had little or no effect on the oxidation of N0 2 - to NO)-. PPDA and
nBTPTA had no effect on nitrification since in both absence and presence of these
urease inhibitors complete oxidation of added NH4 + and that of the added N0 2 - occurred
in 5 and 9 days, respectively.
A part of these investigations, namely those referring to HQ are also described in
another paper whose co-author is Wang (Zhou et aI., 1992).
Xu et al. (1994) presented evidence that HQ is an inhibitor of both soil urease
activity (see page 98) and nitrification. In one of their experiments, samples of an acid
soil (pH 5.58) were treated with (NH4hS04 or urea (50 mg N/lOO g soil) with or
without 2 mg HQ/IOO g soil. HQ inhibited nitrification of both ammonium sulfate and
urea_ The inhibition was 92.5 and 92.1 % respectively, after I-week incubation and 78.5
and 84.1 %, respectively, after 2 weeks.
Figure 33. The same compounds, but another silt loam soil, were used for testing their
nitrification-inhibiting ability.
Soil samples (384 g) wetted to field capacity were amended with 410 mg of urea and
o or 41 mg of N-halamine compound. Dicyandiamide (DCD) served as a reference
compound. The samples were then incubated at 25°C for 5 weeks and analyzed weekly
for N0 3--N, N02--N, and urea-No
Compounds ABC, AB, and IE were stronger nitrification inhibitors, and compound I
was a weaker one than DCD. The inhibition caused by compounds ABC, AB, and IE
was 100% during 4 weeks and 99.6, 98.3, and 99.6%, respectively, after 5 weeks. The
corresponding values recorded after week I to week 5 were: 78.2, 88.6, 87.4,87.4, and
90.0%, respectively, for DCD, and 74.4, 68.9, 4.9, -2.8, and -1.5%, respectively, for
compound I.
Compounds ABC and IE were identically potent inhibitors of nitrification, but
compound ABC was a stronger urease inhibitor than IB. Therefore, compound ABC
(l-bromo-3-cWoro-4,4,5,5-tetramethyl-2-imidazolidinone) may be considered the best
dual purpose inhibitor among the N-halamine compounds studied.
5.2.2.9. Phosphorodiamides
One of the phosphoroamides patented and tested as urease inhibitors by Swerdloff et al.
(1985a), namely 3-(1 ',1 ,-dimethylethyl)-4-hydroxy-PPDA (see page 113 and Figure 40)
was also tested for evaluating its effect on nitrification. Air-dried samples (20 g) of a
New York soil (Cazenovia silt loam, pH 7.3) were treated with 0 or 0.8 mg of test
compound in 5 ml of water and, immediately or after 14 days of preincubation, with 6
mg of diammonium phosphate in I ml of water. Then all samples were incubated at
25°C for 14 days, followed by their analysis for nitrate content.
Inhibition of nitrification was found to be 39% in non-preincubated soil samples and
0% in the preincubated ones. This finding indicates that the test compound was
degraded in soil during the 14 days of preincubation.
Bremner et al. (l986b) studied the effect of nine phosphoroamides, including
trichloroethyl-PDA and nBTPTA, on nitrification ofNH/. Three Iowa soils were used
(see Table 54). Nitrapyrin and etridiazole served as reference compounds. The reaction
mixtures (lOg of air-dried soil + 1 ml of a solution containing 2 mg of N as (NH4)2S04
+ 3 ml of water or aqueous solution containing 0.05, 0.10, 0.50 or 1.0 mg of
phosphoroamide or 0.05 mg ofnitrapyrin or etridiazole) were incubated at 20°C for 21
days or at 30°C for 14 days and analyzed for (N03- + N02-)-N.
Nitrification was inhibited only by trichloroethyl-PDA at each of its rates and by
nBTPT A at rates of 50 and 100 Ilg/g soil. The inhibitory effect increased with
increasing inhibitor rate and was more marked in the light-textured soil than in the
heavier-textured ones but less marked at 30°C than at 20°C (see also Bremner, 1986).
It should be emphasized that trichloroethyl-PDA and nBTPTA, even at their highest
rate, were much weaker nitrification inhibitors than were nitrapyrin and etridiazole used
at a rate of only 5 Ilg/g soil.
samples even after 5 weeks. Thus, the order of urease inhibition was: nBTPTA »
TPTA» DCD.
The NH/-NIN0 3--N ratio in the urea+inhibitor-treated samples had, after 1-,4-, and
5-week incubation, the following values: 7.0, 106.1, and 28.2 (TPTA); 3.4, 0.24, and
0.15 (nBTPTA); and 78.2, 5.7, and 7.2 (DCD). It was emphasized that the time of
maximum ratio can be varied by varying the amount of TPTA for maintaining proper
ammonium/nitrate nutrition of agricultural plants. Therefore, TPTA can be designated
as an ammonium/nitrate ratio control agent (Radel et al., 1992).
In the experiment performed by Bronson and Mosier (1994), 20-g fresh samples
taken from the 0-15-cm layer of two native shortgrass prairie soils from northeastern
Colorado (a fine sandy loam, pH 6.1 and a sandy clay loam, pH 5.7) were treated with
NH4 Cl at a rate of 25 flg N/g soil and with 0, 5, and 25 flg nBTPTA or PPDAIg soil.
Percent inhibitions of nitrification registered after 6 days of incubation at 28°C at the 5
and 25 flglg inhibitor rates were 0 and 13 (nBTPTA) and 3 and 12 (PPDA) in the fine
sandy loam and 6 and 17 (nBTPTA) and 7 and 12 (PPDA) in the sandy clay loam. The
oxygen analogue of nBTPTA was also tested in the fine sandy loam at a single rate (25
flglg soil), and it was found that its nitrification-inhibiting effect was 13% of that of the
nBTPTA.
In the laboratory experiments of Vittori Antisari et al. (1996), concentration of
nitrite was decreased and that of nitrate was increased by nBTPTA during incubation of
urea-treated samples of three Italian soils (see also page 153).
For improving the storage and/or fertilizer properties of urea, Richter et aT. (1978)
patented a technology for coating the urea prills with distillation residue of synthetic
fatty acids manufactured by oxidation of paraffins and for amending the coated prills
with biologically active compounds, including urease and nitrification inhibitors. But
application of the technology for urease and nitrification inhibitors is not exemplified, in
the patent description, by any nominalized urease or nitrification inhibitor.
Amberger (1989) described an experiment in which ammonium thiosulfate (ATS)
(nitrification and urease inhibitor) used with dicyandiamide (DCD) prevented the rapid
degradation of DCD and, thus, prolonged the nitrification-inhibiting effect. Amberger
(1989) also recommended the combined use of guanylthiourea (nitrification and urease
inhibitor) and DCD with the same aim to prolong inhibition of nitrification. According
to the data published by Amberger and Gutser (1984), thiourea (TO) (nitrification and
urease inhibitor) markedly reduced decomposition of DCD. Soil samples (50 g each)
were amended with 1.5 mg DCD and 0 or 0.5 or 1.0 mg TU and incubated at 15°C for 3
weeks. The residual DCD content was 0.66 mg (no TO), 0.80 mg (0.5 mg TU), and 0.87
mg (l.OmgTO).
Goos et al. (1990) and Goos and Johnson (1992) conducted laboratory, microplot,
and field experiments to compare the effects of ATS, DCD, and ATS+DCD mixtures on
nitrification of urea and urea-ammonium nitrate (UAN) in North Dakota soils.
In the laboratory experiment, a silt clay (PH 6.6) and a loam (PH 6.9) were studied.
Urea with or without inhibitor or inhibitor mixture was applied in solutions containing
186 g Nil. Rate of ATS was 8.7% (volume/volume) and that of DCD was 0.5, 1, 2 or
5% ofN as DCD-N. A droplet (0.1 ml) of fertilizer solution was placed on the surface
of 50-g soil samples. Incubation took place at 25°C (silty clay soil) or at 20°C (loam
soil) and lasted 17 days. The ammonium-N and (nitrite+nitrate)-N contents determined
after incubation served for calculation of the percent inhibition of nitrification. Similar
results were obtained in the two soils studied. The following average inhibitions were
registered: 68% (ATS alone), 48-92% (DCD alone at rates of 0.5-5%), and 76-94%
(ATS+DCD 0.5-5%). Thus, the inhibition was greater with ATS + DCD than with DCD
alone. It should also be mentioned that some nitrite accumulated when ATS was added,
but little or no nitrite accumulated when both ATS and DCD were present in the
fertilizer solution.
The microplots were installed, in the spring or autumn of 1988 or in the spring of
1989, in the field at 11 sites on different soils commonly used for wheat production. The
microplots were "buried bags". Top soil samples (300 g each) packed in nylon mesh
bags were amended with a droplet (0.25 ml) of fertilizer solution; then other 300-g soil
samples were added, and the bags were buried in holes having 10 cm in diameter. The
fertilizer solution prepared from urea Of from urea + ATS or DCD or ATS+DCD
contained 186 g N/l. Rate of ATS was 10% (volume/volume) and that ofDCD was 2%
of N as DCD-N. Soils were taken from the bags 4 or 8 or more weeks after fertilizer
application for determination of ammonium-N contents. The results showed that ATS
added to urea solution significantly (p:SO.lO) increased residual ammonium contents
244
over urea alone at six of II sites. ATS was usually a less effective nitrification inhibitor
than was DCD, and ATS+DCD outperformed DCD at only one of 11 sites.
Three field experiments were carried out in the spring of 1990. Size of the plots was
2.5 x 9 m. The fertilizer, UAN solution, was applied at a rate of 75 kg N/ha. Rate of
ATS was 10% (volume/volume) and that of DCD was 2% of N as DCD-N. The
fertilizer solutions were banded 10 cm deep on 35-cm centers of the plots. The soils
were sampled 1 day, 2, 4, and 8 weeks after fertilizer application. The I-day, 2- and 4-
week samples were analyzed for (urea+ammonium)-N and the 8-week samples for
ammonium-No In all three field experiments, ATS added to UAN increased residual
ammonium contents. Again, ATS was less effective than DCD, and no ATS+DCD
synergism was observed.
Sutton et al. (1991) patented a homogeneous granular fertilizer composed of urea
(90-98% by weight), DCD (1.4-3.0%), and ATS (0.4-1.0%), and, optionally, of a
phosphate, most preferably, of an ammonium polyphosphate (APP) (0.3-1.0%). Besides
the technology for manufacturing of urea+DCD+ATS and urea+DCD+ATS+APP,
testings of urea+DCD+ ATS are also described in the patent. The testings were carried
out on maize fields (see page 262) and on turf (see page 313).
In the field experiment conducted by Clay et al. (1990), urea alone or urea with
nBTPT A or DCD or nBTPTA + DCD were surface-applied on soil covered by dried
maize leaves (2-4 cm in diameter) and on bare soil (a sandy loam from Minnesota).
Rate of additions per m2 was: 160 g of urea-N, 0.8 g of nBTPTA and/or 2 g of DCD.
Ammonia volatilization was measured during the first 4 days after fertilization.
Volatile NH3 losses in the same treatment were lower from the residue-covered than
from the bare soil. nBTPT A reduced NH3 volatilization by 100 times over urea only,
and the effect was even stronger when urea was applied with nBTPTA + DCD. DCD
alone had no effect on NH3 volatilization from the residue-covered soil but increased
the volatile NH3 loss from the bare soil.
In a laboratory experiment with the same soil and treatments as in the field
experiment, ammonia volatilization was measured during 8 days of incubation at 35°C.
Volatilization of NH3 presented the order: control (urea only);::: DCD > nBTPTA >
nBTPTA+DCD.
Weston et af. (1994) patented a liquid fertilizer, namely an aqueous solution of urea,
ammonium nitrate, nBTPTA, and DCD, and, optionally, a clay as a suspending agent.
This fertilizer contains 24-32% (preferably 26-32%) of urea, 34-42% (preferably 36-
42%) of ammonium nitrate, 0.01-0.4% (preferably 0.02-0.3%) of nBTPTA , and 0.01-
2% (preferably 0.03-1.5%) of DCD. The nBTPTA to DCD ratio should exceed 0.01,
should preferably be between 0.02 and 8.0 and most preferably between 0.05 and 1.0.
nBTPTA as a concentrated solution in an N-alkyl-2-pyrrolidone, preferably N-methyl-
2-pyrrolidone, is incorporated into a urea-ammonium nitrate (UAN) solution or
suspension, whereas DCD is added to UAN as a solid, a suspension or in dissolved form
along with the nBTPTA.
In the laboratory experiments of Chen et aZ. (1995) and in the pot experiments of
Chen et al. (1998), hydroquinone (HQ) was used in combination with either DCD or
acetylene provided by encapsulated calcium carbide (CaC 2). Besides hydrolysis and
nitrification of urea, N 20 emission was also studied.
In the laboratory experiments, 250-g samples of a Chinese soil (pH 6.66) were
treated with: 1. urea only (1.2 g); 2. urea + 0.3% HQ (on urea weight basis); 3. urea +
245
0.3% HQ + 3% DCD; and 4. urea + 0.3% HQ + 20% CaC z• The samples were wetted to
22% soil moisture content or were water-logged. Incubation took place at 30°C.
The ammonium-N content in the wet and water-logged soils was determined
periodically during 90 days, and this content was also measured periodically in the
floodwater during 60 days. The following orders were established in the different
treatments: control (urea only) ~ HQ+CaC 2 < HQ < HQ+DCD in the wet soil and
control < HQ < HQ+CaCz < HQ+DCD in the water-logged soil. The floodwater
ammonium-N contents were not significantly different between the treatments.
The nitrate-N content in the wet soil and floodwater was determined periodically
during 90 and 21 days, respectively. In both cases, the following order was established:
HQ+DCD < HQ+CaCz < HQ < control.
The ammonia volatilized from the wet and water-logged soils was measured during
90 and 28 days, respectively. The NH3 loss from the wet soil presented the order:
HQ+DCD < HQ < HQ+CaC z ~ controL and an almost reverse order was established for
the water-logged soil: control < HQ < HQ+DCD < HQ+CaC z.
NzO emission from the wet and water-logged soils was detectable during the first 14
and 28 days, respectively, in the same order: HQ+DCD < HQ < control < HQ+CaC z•
In the pot experiments, 2.5-kg samples of a sandy loam soil (PH 7.2) were submitted
to the same four treatments as were the soil samples in the laboratory experiments.
Rates of additions were: urea 0.58 g N/kg soil, HQ 0.3%, DCD 8%, and CaCz 20%.
There were also pots to which no urea and no inhibitor were added. The soil moisture
content was maintained at 20% (about field capacity). Thirteen spring wheat plants were
grown in each pot. The experiments were carried out at 22°C.
The amounts of ammonium-N, nitrate-N, and NzO were measured periodically
during 56 days. The ammonium-N content showed the order: control ~ HQ ~ HQ+CaC 2
«HQ+DCD, whereas the reverse of this order was valid for the nitrate-N content. NzO
was emitted in the order: HQ+DCD < HQ+CaC z < HQ < control.
The conclusion drawn from both laboratory and pot experiments was that HQ+DCD
was an effective combination for inhibition of hydrolysis and nitrification of urea and
reducing ofNzO emission.
This conclusion was confirmed by results of another pot experiment in which
labeled urea (urea_ 15 N) was applied and the test plant remained spring wheat (Xu et al.,
2(00).
Grego et al. (1995a,b) conducted a field experiment on a loam soil (PH 7.0) at the
Experiment Station of the Pisa University. Urea (60 and 120 kg Nlha) unamended or
amended with 0.25% nBTPTA or with 0.25% nBTPTA + 4.5% DCD (on urea weight
basis) was surface-applied on plots (2 by 5 m) installed in a wheat field. Soil was
sampled from the O-15-cm layer for analysis of residual urea, exchangeable NH/-N,
and N03--N contents at days 5,12, and 19 after fertilizer application.
The amount of residual urea presented the orders: nBTPTA+DCD < control (urea
only) < nBTPTA (at the urea rate of 60 kg Nlha), and control < nBTPTA+DCD <
nBTPTA (at the urea rate of 120 kg Nlha) at day 5 and was very low and negligibly low
at days 12 and 19, respectively (at both urea rates). These findings indicate that a)
nBTPTA efficiently inhibited urease activity only for 5 days and b) DCD enhanced
hydrolysis of urea. Contrarily, the effect of DCD in inhibiting nitrification was evident
even at day 19, at which the exchangeable ammonium-N content was highest and the
nitrate-N content was lowest in the nBTPTA+DCD treatment at both urea rates.
246
Grego et al. (1995b) described a similar wheat field experiment on a sandy loam soil
(PH 6.3) at the Experiment Station of the Bologna University. Rates of additions were:
urea-N 120 kgtha, nBTPTA 0.25%, and nBTPTA 0.25% + DCD 4.5% relative to
weight of urea. As in the Pisa experiment, the fertilizer was surface-applied on plots (2
by 5 m). Soil was sampled from the 0-5-cm layer at days 2, 5, and 12 after fertilizer
application. The samples were analyzed as were the Pisa soil samples.
The results were not different from those registered in the Pisa experiment.
Palazzo et al. (1996) conducted experiments in wheat fields on a silty clay soil (PH
8.11) and a sandy clay soil (PH 8.12) at Metaponto (southern Italy). Ammonia
volatilization was studied in the following treatments: control (urea only, 120-150 kg
Nlha), urea + 0.25% nBTPTA, and urea + 0.25% nBTPTA + 4.5% DCD. The volatile
NH3 was measured during 6 weeks after fertilization.
The cumulative NH3 losses from the urea-N added to the silty clay soil were: 7.6%
(control), 3.3% (nBTPTA), and 8.1% (nBTPTA+DCD), whereas the losses from the
sandy clay soil were: 16.1% (control), 5.0% (nBTPTA), and 10.0% (nBTPTA+DCD).
Thus, nBTPTA applied without DCD markedly reduced the NH3 losses, but DCD
enhanced volatilization ofNH 3 from the silty clay and hindered the effect of nBTPTA in
the sandy clay. Nevertheless, the grain yield (tlha) increased from 4.49 (control) to 4.50
(nBTPTA) and 4.67 (nBTPTA+DCD).
Based on the finding that nBTPTA decreased the concentration of nitrite but
increased that of nitrate during incubation of urea-treated soil samples, Vittori Antisari
et al. (1996) also suggested the combined use of urease and nitrification inhibitors.
Hong and Chen (1997) patented a slow-release granular urea fertilizer to which both
urease and nitrification inhibitors are also added. Heavy metal salts [FeS04, ZnS04,
MnS0 4, ~hMo04]' boric acid, sodium borate, hydroquinone are among the applied
urease inhibitors, whereas thiourea and DCD are the preferred nitrification inhibitors.
The average amount of urease + nitrification inhibitors is about 0.5% relative to weight
of urea.
Montemurro et al. (1998) studied nBTPTA, DCD, and nBTPTA+DCD applied with
urea on a clay soil (PH 7.8). The experiment was carried out in a cold greenhouse at
Metaponto. The best plant was lettuce. Before planting, the soil was fertilized with 20
kg Plha as superphosphate and 40 kg Klha as K2 S0 4 • On 14 December 1995, 3-week-
old seedlings were transplanted and the soil was irrigated. On 15 January 1996, at the
stage of four leaves, urea (60 kg Nlha) was incorporated into top 20 cm of soil in four
treatments: urea only (control), urea amended with 0.25% nBTPTA or 4.0% DCD or
0.25% nBTPTA+4.0% DCD (percentages mean weight relative to weight of urea). The
volatile ammonia was measured up to the harvest time (2 April 1996). Ammonium-N
and nitratc-N were determined in the 0-20-cm soil layer at days 8, 19,33, and 59 after N
fertilization and at harvest.
The cumulative NH3 losses expressed in kg Nlha were the following: 11.3 (control),
2.7 (nBTPTA), 8.3 (DCD), and 7.4 (nBTPTA+DCD). Thus, nBTPTA alone was more
effective than in combination with DCD. During the whole experimental period, the
ammonium-N content was higher and the nitrate-N content was lower in the DCD and
nBTPTA+DCD treatments than in the nBTPTA treatment and control, which means
that DCD inhibited nitrification for more than 2 months.
247
In the pot experiments conducted by Vlek et al (1980) and referred to on page 60, the
effect of phenylphosphorodiamidate (PPDA) used alone and in combination with the
herbicide (algicide) simazine was also studied. Urea prills (100 kg of N/ha) with and
without 1 or 2% PPDA (relative to urea weight) were puddled into soil covered by 5-cm
deep floodwater. In other pots, only urea was incorporated into the soil, whereas PPDA
with or without simazine was administered directly into the floodwater (rate of PPDA
was 1% relative to weight of urea and that of simazine was 3 mg/l floodwater). During
incubation at 35°C day and at 25°C night for 10 days, the daily analyses for residual
urea and NH/ + N0 3- from floodwater showed that complete hydrolysis of urea
occurred in 3 days in the absence of PPDA, in 6 days when PPDA had been added to
floodwater and in 6-8 days when PPDA with urea had been incorporated into the soil.
Inhibition of urease activity was strong only during the first 3 days of incubation,
and the degree of inhibition was higher with PPDA incorporated into soil than with
PPDA added to floodwater. This is explained by the finding that urease activity in
floodwater was negligibly low in comparison to that of soil. PPDA was more effective
at 2 than at 1% amount. Inhibition of urease activity was accompanied by diminution of
NH4+ concentration in floodwater; thus, susceptibility of NH3 to volatilization was
reduced. Simazine administered in combination with PPDA had little additional effect.
Moreover, simazine may even have a negative effect by preventing development ofN2-
fixing blue-green algae (cyanobacteria) (see also Vlek and Craswell, 1981).
But in other pot experiments, the algicide added to floodwater in combination with
PPDA delayed the decomposition of PPDA, prolonged its urease-inhibiting effect,
which means that, from this viewpoint, the algicide had a positive effect. According to
the first data published on these investigations, CUS04, acting as an algicide, decreased
the decomposition of PPDA and maintained urea in the floodwater for a longer time
(Anonymous, 1986). These investigations were developed and published in detail by
Byrnes et al. (1989a, b).
Byrnes et al. (1989a) used two soil systems: 300-g samples of a silt loam (PH in
H20 5.2) and mixtures of 60 g silt loam + 260 g coarse washed sand. Urease activity is
high in the soil and low in the soil+sand mixture due to the sand. The samples of soil
and soil+sand mixture were flooded and kept flooded at 3-cm depth for 3 weeks. Then
an acidifying agent or sodium acetate or algicide + urea + PPDA were added to the
floodwater in the following amounts per pot: 600 mg of Ab(S04}3.16H20; 126 mg of
Ca~(P04bH20; 62 mg ofH300 3; 136 mg of CH3COONa; 16 mg of CuS04.5H20 (as
algicide); 35 mg ofurea-N (-200 ppm urea-N); 9% PPDA (on the weight of urea) (-40
ppm PPDA). The control soil and soil+sand mixture received only urea. Daytime pH,
concentrations of PPDA, urea, and ammonium-N were determined daily for 26 days
after addition of urea.
The average daytime (afternoon) pH of the floodwater, which was -9 and 8 in the
control soil and soil+sand mixture, respectively, was reduced by the acidifying agents,
namely to 4.3 and 4.2 by Alz(S04)3, to 8.2 and 5.1 by Ca~(P04)2' and to 8.2 and 7.1 by
H3B03, respectively. The average daytime pH remained 9 and increased to 9.1 in the
floodwater of the CH3 COONa-treated soil and soil+sand mixture, respectively.
The pH below 5 caused rapid decomposition of PPDA, and PPDA was essentially
undetectable in 3 or 4 days. In the less urease-active soil+sand mixtures, the acidifying
248
agents, except Alz(S04)3, did not diminish the floodwater pH below 5. Thus, PPDA
remained in the floodwater for 9-12 days.
At the very alkaline floodwater pH resulting from addition of CH3COONa, the
decomposition of PPDA was, as at pH below 5, very rapid in both soil and soil+sand
mixture.
The algae are responsible for the very high daytime pH (9-10) of paddy floodwaters
because of algal uptake of HC0 3' during photosynthesis. By inhibiting the growth of
algae, CUS04 reduced the average daytime floodwater pH to -8 in soil and to -6 in
soil+sand mixture. Thus, effectiveness of PPDA was prolonged for about 3 days more
than without the algicide.
The conclusion concerning fate of PPDA in floodwater was that at daytime pHs of
approximately 9 the half-life of PPDA was only 10 hours; at neutral or slightly acidic
pHs the half-life could be extended to about 25 hours with the soil and up to about 90
hours with the soil+sand mixture.
Inasmuch as the longevity of PPDA in floodwater of samples submitted to different
treatments was different, disappearance of urea took place during different incubation
times: in 5 days (urea-only treatment), 6 days (PPDA), 7 days (PPDA + CH3COONa),
11 days (PPDA + CUS04), and 12 days (PPDA + H3B03)'
The relatively long-lasting effect of PPDA + H3B03 is attributed not only to the
acidifying effect of H3B03 but also to its urease-inhibiting capacity in addition to that of
PPDA.
In the pot experiment carried out with flooded rice, Byrnes et al. (1989b) studied the
influence of Cu-chelate (commercial algicide; tradename: Cutrine Plus) on the
inhibitory effectiveness of nBTPTA and PPDA. In the case of PPDA, the influence of
an acidifying agent, Alz(S04)3, was also studied.
A silt loam (brown earth from loess; pH 6.5) was used. The soil (7.22 kg dry
weight/pot) was flooded for 3 weeks, then fertilized with 0.43 g of P [Ca(H2P04)2.H20]
and 1.00 g of K and 0.22 g of S (K2S04)/pot, incorporated in the top 8 cm of soil. Two
25-day-old IR36 rice plants were transplanted in each pot. The treatments comprised:
control (no N added), urea alone, urea + nBTPTA with or withoutCu-chelate, urea +
PPDA with or withoutCu-chelate or Alz(S04)3 or Cu-chelate + Al 2(S04)3, added to
floodwater. Rates of additions were: 300 mg of N/pot as 1~-labeled urea (4.67 atom%
excess 15N) at 15 days after transplanting (DAT) and 100 mg ofN/pot as unlabeled urea
at 42 DAT; 13 mg of urease inhibitor/pot (20 glkg urea); 1 mg of Cu/kg soil as Cu-
chelate; 0.1 g of Al2(S04)3.16H20/pot. Each treatment was performed either with water
percolation of 5 mm/day through the soil or without percolation. Floodwater was
maintained at 3 em depth. Urea and NH4 + in floodwater were analyzed daily for 20 days
after fertilization, and pH was measured every afternoon for 10 days after fertilization.
Plants were harvested at 42 DAT and at maturity (119 DAT) to determine their total N
and 15N contents and yields. Total N and 15N contents in soil + roots were also
measured.
Complete hydrolysis of urea occurred in 2 days in absence of inhibitors, in 14 and 4
days in the urea+nBTPTA and urea+PPDA treatments, respectively. This means that
nBTPTA was more effective than PPDA. There was no difference in urea disappearance
when nBTPTA was used with Cu-chelate compared with nBTPTA itself. In the urea
+PPDA+Cu-chelate and urea+PPDA+Ah(S04)3 treatments, urea disappeared in 7 and 5
days, respectively, which shows that Cu-chelate improved more markedly the urease
249
inhibition by PPDA than did Ah(S04h. There was no significant difference in the rate
of urea disappearance between Cu-chelate with PPDA and Cu-chelate+AI2 (S04h with
PPDA.
In the urea+nBTPTA treatment, NH/ concentration in floodwater was very low
throughout the 20 days following fertilization; urea disappeared from the floodwater
mainly by movement into the soil, and urea hydrolysis was so slow that adsorption and
immobilization by algae allowed no accumulation of NH4 + in the floodwater. Addition
of PPDA decreased the peak concentration of NH4 + in floodwater to about one-half that
of the urea-only treatment, and Cu-chelate and Alz(S04)3 reduced it further.
Addition ofCu-chelate to nBTPTA resulted in reduction of floodwater pH by about
one-half pH unit. In the urea+PPDA treatment, the lower daytime pH was maintained
more effectively by Cu-chelate than by Alz(S04)3. Even though the pH reductions were
not great, they decreased decomposition of PPDA by basic hydrolysis and, thus,
extended its inhibitory effectiveness.
In the laboratory experiments of Keerthisinghe and Freney (1994), the urease
inhibitors tested were thiophosphoryl triarnide (TPTA), N-(n-butyl)thiophosphoric
triarnide (nBTPTA) and their oxygen analogues PTA and nBPTA, as well as
cyclohexyl-PTA (CHPTA). The algicide was terbutryn [2-(t-butylarnino)-4-(ethyl-
arnino)-6-(methylthio)-s-triazine]. Two Australian clay soils (PH 5.8 and 6.7,
respectively) cultivated with rice were studied. Air-dried soil samples (15 g) were
placed in 120-ml glass bottles, and the soils were flooded by addition of 30 ml distilled
water. Terbutryn (0.2 mg active ingredientll) was added with the water to half of the
bottles, which were kept in the dark, and water only to the other half, which were kept
in the light. All samples were maintained at 25°C. After 3 weeks, a profuse growth of
algae was noted in the samples kept under light, whereas no algae were visible in the
samples kept in the dark. At this stage, the urease inhibitors were added to the samples
at a rate of 1% of the weight of urea (14 mg urea per bottle). The samples were then
incubated for 12 days and analyzed every 2 days.
Results of the analysis of residual urea showed that elimination of the algal growth
led to a) diminution of the urease-inhibiting effect of TPTA and nBTPTA (due,
probably, to lack of O2 produced by the algae, the thio analogues were not oxidized into
the more urease-inhibitory oxygen analogues) and b) to increased urease-inhibiting
capacity of the oxygen analogues and CHPTA in both soils. In the absence of algae, the
effectiveness of the inhibitors was CHPTA> nBPTA>PTA>nBTPTA>TPTA.
The conclusion was drawn that CHPTA and nBPTA in conjunction with an algicide
have the potential to reduce considerably ammonia loss from flooded rice soils.
In the rice field experiment described by Chaiwanakupt et af. (1996) and Phongpan et
af. (1997), the algicide terbutryn reduced the volatile ammonia losses to a greater extent
than did the nitrification inhibitor acetylene provided by calcium carbide. In addition,
terbutryn enhanced, while calcium carbide diminished, the effect of urease inhibitors to
reduce the NH3 losses (see page 182).
Freney et af. (1995) and Phongpan et af. (1997) described an experiment which was
carried out in a rice field located on a clay soil (PH 5.1) in the Central Plain region of
Thailand during the dry season of 1993. The urease inhibitors tested were N-(n-
250
that addition of ATS to UAN had little effect on the fertilizer efficiency of UAN
applications to no-till maize.
Using samples of four Iowa soils and applying ATS concentrations ranging from
100 to 5.000 J.lglg soil. Bremner et af. (1990) and McCarty et af. (1990) found that ATS
had an adverse effect on germination of maize and wheat seeds when applied at the rate
of 2.500 or 5,000 J.lglg soil and caused a dramatic reduction of early growth of maize
and wheat seedlings when applied at rates:::: 1,000 J.lglg soil.
McCarty et at. (1991) studied the effect of sodium thiosulfate, tetrathionate, sulfite,
and sulfate on growth of maize and wheat seedlings in 7 days. Samples of two Iowa
soils were used. Rates of salt additions were: 1,000. 2,500, and 5,000 J.lg anion/g soil.
Sodium thiosulfate was more toxic than sodium tetrathionate, whereas sodium sulfite
and sulfate had no adverse effect on seedling growth.
Graziano (1990) conducted field experiments in 1988 in the Po Valley near
Mantova, Italy, on a clay loam soil (pH 7.85) for evaluating the efficiency of ATS as an
additive to UAN fertilizer for maize. Before seeding, the ploughed soil of plots (6 by 6
m) was sprayed with a mix of UAN + ammonium polyphosphate + KCI ± ATS. Rates
of additions per hectare were: 300, 200, and 150 kg N, 65 kg P, 166 kg K, 100 kg of a
60% ATS solution. Nand ATS were also applied as top dressings at rates of 100 or 50
kg Nlha and 50 kg ATS solutionlha. There were five pairs of treatments, each pair
comprising plots treated with UAN and with UAN+ATS. Grain yields were always
higher in the UAN+ATS-treated plots than in the corresponding plots treated with UAN
without ATS, but the yield differences were not significant at p < 0.05.
But in other field experiments, Graziano and Parente (1996) recorded significant
maize grain increases by using ATS. These experiments were carried out during the
growth seasons of 1992, 1993, and 1994, at Spilimbergo, on. a stony sandy loam soil
(pH 7.8) typical of the northeastern Italian plain. In each year, all plots (75 m 2) were
fertilized with 35 kg Plha, 125 kg K/ha, 250 kg N as UAN or 239.7 kg N as UAN +
10.3 kg N as ATSlha (10% by weight ATS to UAN) and irrigated.
Grain yields (tlha) in the plots treated with UAN and with UAN + ATS were: 6.2
and 8.1 (in 1992), 10.5 and 12.2 (in 1993), and 9.5 and 11.1 (in 1994), respectively. N
uptake by plants was also higher in the UAN+ATS-treated plots than in those treated
with UAN without ATS. The increases in grain yields and N uptakes were significant
(p<O.OI or p<O.05) in 1992 and 1993 but not in 1994. The conclusion was drawn that
adding 10% by weight ATS to UAN seems to be a beneficial practice for maize
production in the northeastern plain of Italy.
growing season, no fertilizers were applied. Grain yields were significantly higher in the
urea+HQ treatment than in that with urea alone. For example, the yield increase in 1985
was 6.5-8.5% in Changtu county and 6.9% in Tieling county.
In the field experiments of Zhao et af. (1993b), 10-m2 plots on a silty loam soil (PH
6.6) were submitted to the following treatments: control (no urea and no HQ); urea
alone (138 kg N/ha); HQ alone ( 3 kg/ha); urea (138 kg Nlha) + 0.9, 1.5, 3.0 or 9.0 kg
HQlha. All plots were treated with 22.2 kg/ha of P as ~H2P04 and then sown with
maize.
As expected, the maize yield was lowest in the control plots. HQ alone increased
insignificantly, whereas urea alone increased significantly the yield. Addition of HQ to
urea led to further yield increases. The yield was highest (7.8 tlha) in plots treated with
urea + 1.5 kg HQlha. The N content in grains also increased in the urea + HQ
treatments. The highest N content in grains (110.88 kg/ha) was recorded in the
treatment with urea + 3 kg HQlha.
See also Table 61.
TABLE 55. Effects of nitrogen fertilizers, timothy straw, and PPDA on dIY matter yield
of silage maize"
Timothy straw PPDA DIY matter yield
Fertilizer
(kg/ha) (kg/ha) (tlha)
Unfertilized control 0 0 8.49d
4600 0 8.94 bed
NH4N03 0 0 9.46 abed
4600 0 10.30 a
Urea 0 0 9.72 abed
4600 0 9.99 ab
0 0.25 9.17 abed
4600 0.25 10.01 ab
0 0.50 9.75 abed
4600 0.50 10.09 ab
0 1.00 10.44 a
4600 1.00 10.17 ab
0 2.00 9.19 abed
4600 2.00 9.78 abc
0 3.74 8.68 cd
4600 3.74 9.63 abed
Fvalue 2.35"
Coefficient of variation 7.87
"From Tomaretal. (1985).
Values followed by the same letter or no letter are not significantly different (p=O.OS).
"Significant (p=O.OS).
surface- and dribble-applied. Rates of additions per hectare were: 134 kg N as urea +
2.7 kg inhibitor and 202 kg N as urea + 4 kg inhibitor; 134 kg N as VAN + 2.24 kg
inhibitor and 202 kg N as VAN + 2.24 kg inhibitor.
TPDA significantly increased grain yield on one soil which had been fertilized with
surface-applied urea and dribble-applied VAN at the 202 kg N/ha rate. DPTA had a
significant increasing effect on grain yield on the second soil fertilized with dribble-
applied VAN at the 202 kg N/ha rate. The inhibitors had yield-increasing effect on the
third soil.
The maize experiments conducted by Bundy and Oberle (1988) are referred to on
page 28. In these experiments, the effect of PPDA on grain yield and N uptake was also
evaluated. PPDA was added to urea prills and VAN solution at a rate of 2% on fertilizer
N basis (56 and 112 kg N/ha). The PPDA-containing fertilizers were surface-applied on
silt loarns at Arlington and Lancaster in 1983 and 1984. PPDA had no significant effects
on grain yield and N uptake, except in a single case: at the lower fertilizer N rate, urea-
PPDA VS. urea significantly increased N uptake (without increasing grain yield) at
Lancaster in 1984.
In pot experiments of Winiarski (1990), 1% PPDA added to urea fertilizer brought
about the increase of dry matter yield of maize by about 6%; the N uptake by plants also
increased.
The experiments in which Bremner and Krogrneier (1989) found that PPDA added
to soil samples completely eliminated the adverse effect of urea on germination of seeds
of four plant species, including maize, are dealt with on page 276. See also Table 61.
256
In the 1983 experiments, plots sited on a silty clay loam (pH 5.9) and a silt loam (PH
5.7) were installed. The sites were cropped to maize in 1982. The plots on silty clay
loam were tilled (conventionally tilled, CT plots), whereas those on silt loam were left
untilled except for chopping the stalks (no-tilled, NT plots). At day 10 (first soil) or at
day 21 (second soil) after sowing (at a population density of 64,000 seedslha), urea
(prills and solution) and UAN solution, at a rate of 84 kg Nlha, with or without a
phosphoroamide, were broadcast on the plots. UAN solution was also applied in bands
on the soil surface. Each of the six phosphoroamides tested (N,N-dimethyl-, N,N-
diethyl-, N-cyclohexyl- or N-benzyl-N-methylphosphoric triamide or trichloroethyl-
phosphorodiamidate, TPDA, or PPDA) was adhered to urea prills by first coating the
prills with paraffin oil (0.8% relative to urea weight) by mixing, then adding powdered
phosphoroamide at 2 kg/100 kg N and mixing. In the case of urea and UAN solution,
the phosphoroamides were dissolved and applied at a rate of 2.24 kglha.
The 1984 experiments were also carried out on two soils. The silty clay loam (PH
5.9) had been cropped to maize the previous year and was divided into two areas, CT
and NT plots. The NT plots, also in these experiences, were left untilled except for
chopping the stalks. The plots on the other soil (silt loam, pH 5.3), on which the
previous crop was winter wheat, were left untilled (NT plots). At day 15 (first soil) and
at day 12 (second soil) after sowing (60,000 seeds/ha), urea prills and UAN solution
with or without an inhibitor were broadcast on plots, at rates of 60, 120, and 180 kg
Nlha. Two inhibitors were tested: nBTPTA and PPDA. The rate of nBTPTA was 2 or
0.5 kg/IOO kg N (as coating on urea priUs) or 2.24 and 0.56 kglha (dissolved in UAN
solution). Rates of PPDA were 2 kg/100 kg N (on urea prills) and 2.24 kglha (in UAN
solution).
Fertilizer application was followed by a rainy period in 1983 but by a dry one in
1984.
In 1983 grain yield and N content in grain did not significantly increase under the
influence of inhibitors. In 1984 the inhibitors applied as coatings on urea prills led to
significant grain yield increases in the NT plots on both soils. Thus, average of grain
yields recorded at the three N rates was 7.46 tlha in urea-only treatment and 7.95 tlha in
urea + PPDA treatment on the silty clay loam soil; on the other soil, nBTPTA, at the
higher rate, increased the mean grain yield from 5.14 to 5.99 tlha. In addition, grain N
levels in the NT plots on silty loam were higher from urea with the higher nBTPTA rate
than those from urea alone. Urease inhibitors did not increase grain yield when applied
on CT maize or when added to UAN solution.
The conclusion was drawn that urease inhibitors will be most beneficial under the
following conditions: urea is applied on soil surface with large amounts of plant residue
cover; there is adequate moisture on soil surface to promote urea hydrolysis and
ammonia volatilization; the precipitations in the first 10 days after urea application are
insufficient to leach urea into the soil (see also Nelson et al.. 1986).
Leis et al. (1989) and Varsa et al. (1989) conducted seven experiments on no-till
maize fields on silt loam soils, at two locations (Belleville and Carbondale) in southern
Illinois in 1985, 1987, and 1988. nBTPTA was used in form of coating on urea
granules, copelleted with urea, and in urea and UAN solutions.
In 1985 urea coated with 0.92% nBTPTA was used at rates of 84 and 168 kg Nlha.
In 1987 and 1988, the urea-nBTPTA pellets containing 0.25 or 0.50% nBTPTA and
urea-nBTPTA and UAN-nBTPTA solutions containing 2.24 kg nBTPTAlha were used
258
at a single rate of urea, namely 134 kg N/ha. The controls received urea and UAN
without nBTPT A. In all years, the N fertilizers were broadcast or dribbled.
In six of the seven experiments, nBTPTA significantly increased the grain yield.
Lack of a yield response from nBTPT A was associated with a rain event of major
proportions soon after fertilizer application. nBTPT A was more effective with dribbled
than with broadcast urea; at 0.5% than at 0.25% nBTPT A content in urea pellets; and in
urea solution than in UAN solution.
The investigations were continued at both Belleville and Carbondale, and the results
obtained in 1990 were reported by Varsa (1991).
Granular urea and urea cogranulated with 0.33% nBTPTA, UAN solution with and
without 1.12 kg nBTPTAlha as well as granular ammonium nitrate (AN) were used at
rates of90, 134, and 180 kg N/ha. The fertilizers were all broadcast-applied.
At both locations, use urea cogranulated with nBTPT A resulted in significant grain
yield increases as compared with urea alone; addition of nBTPTA to UAN had no
significant increasing effect on grain yields; the significantly highest yields were
obtained with AN.
Varsa and Ebelhar (1992) reported on investigations conducted at Belleville and
Dixon Springs in 1991. The studied fertilizers were: urea, urea+0.3% nBTPTA. UAN,
AN as well as Super Urea and Super N. At Dixon Springs UAN with 1.12 kg
nBTPT A/ha was also used. All were broadcast and dribbled at rates of 90 and 180 kg
N/ha.
At Belleville the grain yields presented the orders: AN > urea-nBTPTA:::; urea>
UAN:::; Super N :::; Super Urea (broadcast fertilizers), and AN > urea-nBTPTA > urea:::;
Super N:::; UAN :::; Super Urea (dribbled fertilizers). The dribbled urea- nBTPTA
performed better than the broadcast urea-nBTPTA.
At Dixon Springs the orders were: AN > UAN-nBTPT A:::; Super Urea:::; urea :::;
Super N:::; urea-nBTPTA:::; UAN (broadcast fertilizers), and urea-nBTPTA > AN >
UAN-nBTPTA > UAN:::; Super N :::; urea:::; Super Urea (dribbled fertilizers). Thus,
urea-nBTPTA was more effective than AN. Urea-nBTPTA and UAN-nBTPTA gave
better results when dribbled than when broadcast. The active ingredients in Super Urea
and Super N were not as effective as nBTPTA in increasing grain yields.
Goodroad and Wilson (1989) conducted field experiments in the Piedmont region of
Georgia using urea fertilizer with or without nBTPTA. Grain yields of irrigated maize
were higher when urea+nBTPTA was used.
Bronson et al. (1990) also found that nBTPT A can increase the N use efficiency of
urea surface-applied to no-till maize.
In a short report on 2-year (1989-1990) field experiments carried out in North
Carolina, Baird and Ngueguim (1991) pointed out that in 1989 urea, as compared with
nBTPTA-amended urea, produced more grain yield, total dry matter, and total N
uptake, but in 1990 the nBTPT A-amended urea performed more efficiently than urea
alone with respect to grain yield, grain N uptake, and total above-ground dry matter
uptake ofN.
Schlegel (l99Ia,b) conducted field experiments on silt loam soils in west-central
Kansas, over 3 years (1988-1990), for evaluating the effect of nBTPTA to reduce the
damage caused to maize by ammonia released from urea under the action of soil urease.
Size of plots was 3 by 9 m. UAN solution (0, 12.5, 25, 50, and 100 kg N/ha) and
nBTPT A (0 and 1.12 kglha) were applied in dribble band with seed at planting. The
259
addition of nBTPTA allowed the rate ofN to be doubled without reducing emergence or
early plant growth and reduced delays in emergence by 50%. Thus, nBTPT A was
effective in reducing the phytotoxic effects of urea fertilizer of maize.
In field studies by Mullen et al. (1991) and Howard et af. (1992), 168 kg N/ha as
solid urea or liquid UAN with or without 1.12 kg nBTPTA/ha was broadcast on plots
planted to no-till maize on a silt loam soil with maize stubble or wheat residue cover.
Plots with injected UAN were the control.
Grain yields with urea+nBTPTA were not different from injected UAN yields for
both plant residues (approximately 5,895 kglha). The yields with urea alone were
significantly lower at approximately 4,421 kglha. For UAN broadcast in maize stubble,
the yields were 5,330 and 4,640 kglha with and without nBTPTA, respectively. For
UAN broadcast in wheat residue, the yields dropped to 4,703 and 3,198 kglha with and
without nBTPTA, respectively. Comparison of yield values makes it evident that
nBTPTA was more efficient in the urea than in the broadcast UAN treatments.
Fox and Piekielek (1993) studied the effect of urea-nBTPTA and UAN-nBTPTA on
no-till maize in three field experiments carried out on silt loam soils in central
Pennsylvania in 1989, 1990, and 1991. Rates offertilizer additions were: 56, 112, and
168 kg N/ha. The urea-nBTPTA contained 0.5% nBTPTA by weight. The nBTPTA
concentration in UAN was adjusted so that 1.12 kg nBTPTA/ha was applied at all N
rates. Urea treatments were broadcast at planting and applied as surface band at
sidedress. UAN was sprayed at planting, broadcast at planting, banded at sidedress, and
injected at sidedress.
All average grain yield was higher in the urea-nBTPTA than in the urea-only
treatment. The increase was significant at p=0.01 when urea and urea-nBTPTA were
broadcast at planting and at p=0.05 when they were surface-banded at sidedress. UAN-
nBTPTA compared with UAN increased significantly (p-0.1) the grain yield only when
these fertilizers were applied by spraying at planting.
Murphy and Ferguson (1992, 1997) evaluated, under field conditions, the effect of
nBTPTA-amended and unamended urea and UAN solution (28% N) on ridge-till,
irrigated maize. The investigations were carried out on silt loam soil at the University of
Nebraska South Central Research and Extension Center located near Clay Center over 3
years (1990-1992). Rate of N application was 112 or 224 kg/ha and that of nBTPTA
1.12 kglha. Methods of application for urea and UAN were broadcast and surface band.
Additionally, the subsurface band (knifed) method was applied for UAN.
It was found that the climatic conditions are the primary factors influencing the
effectiveness of nBTPTA to reduce rate of urea hydrolysis and ammonia volatilization
from urea and to increase the maize yield.
Precipitation soon (during the first 14 days) after fertilization in 1990 and 1991
resulted in little or no benefit from the use of nBTPTA, as precipitation moved urea into
the soil, reducing the potential for NH3 volatilization. Limited precipitation and low
humidity for an extended period following fertilization in 1992 resulted in an
approximately 3,600 kglha increase in grain yield when nBTPTA was applied with urea
(averaged over rates and application methods), but there was no yield increase when
nBTPTA was applied with UAN. No differences in yield were observed between
broadcast and surface band methods. In 1991 (but not in 1990 and 1992), nBTPTA in
the UAN applied by the knifed method led to a yield decrease of 2,156 kglha, perhaps
260
indicating that nBTPTA slowed the hydrolysis rate of urea too much than when VAN
was applied by the other methods.
The conclusion was drawn that in some years in south-central Nebraska the use of a
urease inhibitor such as nBTPTA will help protect surface-applied urea from volatile
NH 3 10ss.
Lamond et al. (1993, 1994) reported on field experiments conducted in 1993 and
1994 on no-till maize sites in Kansas. N fertilizers were surface-broadcast just prior to
or shortly after planting. In 1993 two continuous maize sites and two maize sites after
soybeans were studied. In 1994 four other maize sites were established. In both years,
ammonium nitrate and urea with nBTPTA performed better than urea and VAN. Thus,
in 1993, at three sites, urea + nBTPTA produced significantly higher grain yields than
urea and VAN.
In the 3-year field experiments of Palazzo et al. (1995, 1996) (see page 153),
nBTPTA addition to urea resulted in maize grain yield increases ranging from 11.0 to
30.6%. nBTPTA at a concentration of 0.1 % (on urea weight basis) was as efficient as at
its 0.25% concentration.
See also Table 61.
* *
*
Results of 78 field experiments conducted from 1984 to 1989 across the V.S.A. for
evaluation of the effect of nBTPTA on maize were summarized by Hendrickson (1992).
nBTPTA was applied with urea at rates of 0.25 to 1.0% (weight/weight) and with VAN
solution at rates of 0.56 to 2.24 kglha. On overall average, nBTPTA increased grain
yields by 4.3 bu/acre when applied with urea and by 1.6 bulacre when applied with
VAN. Average responses to nBTPTA were greater on sites responsive to N (6.6 bulacre
for urea and 2.7 bulacre for VAN). Results from 21 experiments employing multiple N
rates showed that maximum grain yields could be obtained using an average of 83 kglha
less N when nBTPTA was included with surface-applied urea.
Varsa et al. (1993) summarized the results from 7 years of experiments on no-till
fields at two locations in southern Illinois (Belleville and Carbondale). nBTPTA
addition to broadcast urea, when evaluated across N rates and locations, gave grain
yield increases averaging 8.4 bulacre in 13 experiments. For dribbled urea the response
to nBTPTA was 12.0 bu/acre for 9 experiments across the two locations. Grain yield
responses to nBTPTA added to VAN solutions were much smaller. In 8 experiments
broadcast VAN-nBTPT A resulted in an average grain yield increase of 2.3 bulacre
across the two locations. In 13 experiments, in which dribbled VAN-nBTPTA was
evaluated, yield increases of 3.3 bu/acre were obtained.
The manufacturer of the commercial nBTPTA under the registered trade product
name of Agrotain has elaborated a collection of 38 summaries of reports presenting
results obtained with Agrotain in experiments carried out on maize fields at a great
number oflocations in the U.S.A., namely in 19 states (IMC, 1995).
soil-sand mixture (1:1). After sowing, 100 ml of urea solution (1.2 g N) with or without
2% hexaamino-CTPAT (relative to weight of urea) was applied on the surface of soil. In
the field experiment, the fertilizer was DAM 390 at rates of 100 and 200 kg Nlha,
whereas the rate of hexaamino-CTP AT was I % relative to fertilizer N. In both
experiments, the over-ground parts of 43-day-old plants were analyzed for fresh and dry
weights and N, P, Ca, and Mg contents.
In the pot experiment, fresh and dry weights and N contents were higher, and P, K,
Ca, and Mg contents were lower, in the urea-hexaamino-CTPAT treatment than in the
urea-only treatment. The weight increase was about 10%. Similar results were
registered in the field experiment, but Ca and Mg contents were higher in plants
growing in plots received DAM 390 + hexaamino-CTPAT.
See also Table 61.
In separate field experiments, 15N-labeled urea (at 33 atom% excess of 15N) was
used, and it was found that ALS resulted in significantly less 15N immobilized in soils.
nBTPTA. 3. urea + DCD, and 4. urea + nBTPTA + DCD at rates of 0, 67, l34, and 202
kg N/ha. The following grain yields (bu/acre) were regictered with the four fertilizers:
143 (no N added), 174, 188, 174, and 176 (at 67 kg Nlha), 190, 192, 183, and 202 (at
l34 kg Nlha), and 196, 198, 183, and 186 (at 202 kg Nlha), respectively. Thus,
urea+nBTPTA was most efficient at the lowest and highest N rates and
urea+nBTPTA+DCD at the medium N rate, but the differences between the yields were
not significant at p=0.05.
Varsa and Jan (1997) conducted experiments in 1992 and 1994 to 1996 at
Carbondale, Kansas, to evaluate the effect of nBTPTA and DCD alone and in
combination on no-till maize. The urea rates were 90, 135, and 180 kg Nlha. nBTPTA
and DCD were incubated alone with urea at concentrations of 0.14 and 2.2%,
respectively. Combination included nBTPTA at 0.14% and DCD at 0.55 and 1.1%.
When a response to inhibitors was obtained, usually nBTPTA alone was the highest or
equal to urea treatments that included a combination of nBTPT A and DCD. Urea plus
DCD alone never increased the maize yield over urea. Inhibitor response failures were
usually associated with seasons of severe drought or when high N levels remained in the
soil from the previous crop.
solution were spring topdress on winter wheat. None of the treatments significantly
increased yield in any experiment, or consistently increased mid-season N uptake.
Bremner et af. (1990) and McCarty et af. (1990,1991) found that the adverse effect
of ATS, sodium thiosulfate, and tetrathionate on germination of maize and wheat seeds
and growth of maize and wheat seedlings is similar (see page 253).
not treated or treated with inhibitor(s) was distributed along the bottom of the drills.
Then wheat seeds were placed on the bottom of drills and covered with the soil. The
treatment of urea consisted of mixing urea granules or prills with powdered inhibitor(s).
Urea was applied at rates of 16.8 and 33.6 kg N/ha (in one soil), 44.8 or 56 kg N/ha
(four soils). The inhibitors tested were: zineb 1% (on urea weight basis) + Br3C-CBr3
1% (one soil); zineb 0.5% and zineb 0.5% + ClzBrC-CBrC12 1% (four soils). The
control soils in pots were not treated with either urea or inhibitor(s). After sowing, all
pots were stored in the dark at lO°C. The shoots emerging above the soil surface were
counted every second or third day for 35 days.
One can deduce from the results tabulated in the patent that urea at low rate (16.8 kg
N/ha) with or without zineb + Br3C-CBr3 retarded emerging of shoots, but at day 35 the
number of shoots emerged from these treated soil samples did not differ from that
registered in the control soil. In the soil samples treated with 33.6 kg urea-N/ha, the
number of emerged seedlings was lower than in the control soil during the whole 35-
day period. This effect of urea was attenuated. to some extent, by the zineb + Br3C-
CBr3 mixture. At the rates of 44.8 and 56 kg N/ha, urea strongly inhibited emerging of
shoots. Thus, at day 35 the number of emerged seedlings in the four soils treated with
urea at these rates varied between 5 and 25%, whereas 84-92% of the seeds produced
shoots in the control soils. Zineb (0.5%) and zineb (O.5%) + C1 2BrC-CBrClz {l %}
attenuated somewhat the effect of urea, increasing the number of emerged shoots to 13-
26%. The action of zineb did not differ from that of the zineb + ChBrC-CBrCh
mixture. This means that CI 2BrC-CBrClz, which, when tested with the first method
(page 54), was more effective than zineb in reducing volatile ammonia losses, had no
inhibitory effect on soil urease activity in the brairding experiments.
TABLE 56. Effect of urea and urease inhibitors on wheat grain yield"
Yield (g dry matter/potl
Treatment Urea-N awlication rate (Wm of soil)
13.4 53.6
Control (urea with no inhibitor) 3.00 ab 0 c
Catechol (solid) 2.87 ab 3.14 ab
Catechol (solution) 2.32 b 0 c
p-Benzoquinone 2.89 ab 4.16 a
2,5-Dimethyl-p-benzoquinone 2.86 ab 3.84 a
"From May and Douglas (1978).
"when no urea or inhibitor was applied, a yield of 1.29 g/pot was obtained.
Yields not followed by the same letter are significantly different (p=O.05).
267
catechol in solution, soil urease was inhibited, NH4 + in phytotoxic concentration was not
produced and, thus, the plants could grow. Inefficiency of catechol in solution is
attributed to its migration from the soil zone near the germinating seeds.
At the 13.4 ppm urea-N rate, little increase in N uptake by plants occurred when
urea was applied with an inhibitor as compared to urea applied alone. In treatments
where 53.6 ppm urea-N + either catechol (solid), p-benzoquinone or 2,5-dimethyl-p-
benzoquinone was added to soil, N recoveries by plants were 60, 57, and 72%,
respectively.
Mishra et al. (1980) also used wheat as a test plant. Samples (300 g) of a black earth
from Germany were mixed with sand (100 g), then treated with 5 ml of solutions of the
compounds specified in Table 57 (to obtain concentrations of 20 and 50 ppm in soil),
moistened to 60% of WHC and sown with 100 wheat seeds. Germination and, then,
growth took place at 20°C. The number of germinated seeds was determined after 4
days, the height of plantlets was measured after 8 days, and the dry weight of roots and
shoots was recorded aftcr 21 days. One can deduce from this table that none of the
compounds affected germination of wheat seeds. After 8 days, the plantlets were shorter
with the addition of 2-methyl-p-naphthoquinone and 4,6-di-t-butylcatechol at 50 ppm,
but this adverse effect disappeared later and, thus, after 21 days of growth dry weight of
plants was practically the same in the control and in all treatments.
In a short report, Edwards (1982) described a field experiment, in which winter
wheat was fertilized with prilled JSN-labeled urea (l00 kg N/ha) with or without
addition of p-benzoquinone (BQ) at a rate of 2.5% relative to urea-No Neither urea nor
BQ was added to the control. Under these conditions, BQ increased the uptake of urea-
N by an average of 7 kg/ha reduced plant uptake ofN from soil by 34% compared to the
unfertilized control and by 57% compared to the treatment with urea alone. Grain yields
were increased by \,280 kglha from the urea + BQ treatment and by 2,260 kg/ha from
the application of urea alone. It is assumed that the response difference represents the
summation of the negative effect of BQ on the mineralization of soil organic N and the
positive effect of urea on the release of complexed soil organic N.
268
Under conditions identical to those described on page 264, Rao and Ghai (l986a)
also studied the effect of catechol (CT) and hydroquinone (HQ), used at a rate of 10%
relative to weight of urea, and established that percentage of germinated wheat seeds,
estimated at day 15 after sowing, was not affected by CT but was reduced by HQ. A
similar situation was observed concerning dry matter of 21-day-old plants. Contrarily, at
day 35 after sowing, dry matter of plants in both CT and HQ treatments did not
significantly differ from that measured in the control plants (treated with urea alone),
whereas at the end of the growing season (125 days), grain yield was highest in the urea
+ HQ treatment, followed by the urea + CT treatment and exceeding by 20.0 and 14.8%,
respectively, the yield of the control plants. N content in grain also increased in the
order:
control (urea) < urea + CT < urea + HQ.
Dry weight of straw showed the order:
urea + CT < control (urea) < urea + HQ.
Accumulation of N in straw was not influenced by CT and HQ.
The explanation for the more favorable effect of HQ than of the CT is that HQ
moves together with urea, which means that it moves more rapidly as compared to the
movement of CT.
Hera et al. (1986) compared the effects of hydroquinone (HQ), nitrapyrin, and 4-
arnino-1,2,4-triazole hydrochloride (ATC) on the yield of wheat cultivated on a
chemozemic soil in vegetation pots. Urea (0.5 glpot) was applied only in autumn at
sowing time or in both autumn and spring (0.5 + 0.5 glpot). The inhibitors were used
together with urea at a rate of 5 mglpot. The controls received no urea and/or no
inhibitor. On 1st of June, some pots were submitted to simulated rainfalls (50 mm) for
creating excessive moisture, i.e.. conditions favorable for leaching of nitrates. The soil
in the other pots was maintained at optimum moisture content. Periodical analysis of
nitrates formed in soil showed that the inhibitors delayed the appearance of the
maximum nitrate amount from the time of stalk shooting (control) to times of
inspication (HQ), flowering (nitrapyrin), and maturation (ATC). The wheat yield
increased due to inhibitors in the following order: HQ>nitrapyrin>ATC. These
differences between the three compounds were more marked under conditions of
excessive moisture, although under these conditions, HQ, nitrapyrin, and ATC reduced
the losses through leaching of nitrates by 42.4, 52.9, and 61.2%, respectively, i.e., in an
order opposite that of their yield-enhancing effect.
The results obtained in pot experiments carried out by Zhou et al. (1988) for
studying the effect of HQ on spring wheat plants sown in a brown soil from China are
presented in Table 58. They show that the optimum amount of HQ for increasing plant
uptake ofurea-N was 10 mg with 0.9 g ofurea-N, whereas HQ at the rate of 40 mglpot
reduced uptake of N from urea. Grain yield was also highest at 10 mg of HQ/pot which
led to a significant (l 0.23 %) increase as compared to the control treated with urea
alone.
In another laboratory experiment, in which urea labeled with 15N was applied
together with HQ at rates of 0, 10, 20 or 40 ppm (on soil basis), the spring wheat plants
growing in soil treated with 0, 10, and 20 ppm of HQ took up more N from urea than
from soil (the ratio of N taken up from soil to N taken up from urea < 1) during the
whole period of experiment (90 days). At 40 ppm of HQ, this ratio was also smaller
269
TABLE 58. Effect of hydroquinone, applied at various rates, on uptake ofurea-N by spring wheat plants"
Rate of Rate of urea-N Urea-N taken up by Gaseous loss of Residual urea-N
hydroquinone plants urea-N in soil
(mg/pot) (g/po!) (g/pot) (%) (g/pot) (%) (g/pot) (%)
0 0.9 0.4403 48.92 0.4078 45.31 0.0519 5.77
10 0.9 0.4733 52.59 0.3655 40.61 0.0612 6.80
20 0.9 0.4678 51.98 0.3199 35.54 0.1123 12.48
40 0.9 0.3167 35.19 0.4106 45.62 0.1727 19.19
"From Zhou el al. (1988).
than 1 during the first 60 days; thereafter, it became greater than 1. But at all HQ rates,
utilization ofN from soil increased linearly with time.
It was also established that HQ applied at rates of 5, 10, 20, and 40 mg/pot did not
accumulate in soil, stems and grains of wheat (sec also Zhao et al.. 1991, 1992a; Zhou
et aI., 1992).
Xue et al. (1991) amended wheat fields with 15 kg of urea + 10 kg of KCl + 0 or 50,
100 or 150 g of hydroquinonc (HQ) or quinhydrone (QH). All rotes refer to an area of 1
mu' (1/15 ha). The groin yicld increased with increasing inhibitor rate. The increases
were 6.8, 14.1, and 19.8% (HQ), and 11.8, 16.9, and 22.8% (QH). The yield increases
were accompanied by increases in coefficient of plant utilization of N from urea, from
2.8 to 5.3% in the HQ treatments, and from 2.4 to 6.1 % in the QH treatments.
See also Table 61.
'Mu is a traditional Chinese unit of land surface area which in modem China is reckoned to be 1/15 ha.
"In the balance studies, the labeled NH 4 N0 3 and urea contained 52.2 and 50.6 atom% excess lIN,
respectively.
"'Water to the soil mass was supplied through a strip of glass fibre cloth, which was embedded in the soil
and. passing through the bottom of the pot, immersed in a subjacent water container.
270
One can see from Table 59 that, when the fertilizers were immediately washed into
the soil, the grain yield increased, though insignificantly, in order:
urea < urea+ I % PPDA < NH4N0 3,
whereas the straw yield was highest in the urea treatment; more N was taken up by
plants from NH4 N0 3 than from urea, and PPDA did not enhance plant uptake of N.
TABLE 59. Wheat yield and nitrogen uptake from fertilizers under different conditions of their application"
Wheat yield N uptake (% of the
(g/pot) applied N with the
Fertilizer Conditions of application
second dose)
Grain Straw Grain Straw Total
Experiments performed in 1976
NH 4NO, Application at a plant growth 41.6a 57.2 ab 49 15* Ma
Urea height or 25 em; immediately 40.7 a 60.6 b 43 a 15 58 b
Urea + 1% PPDA washed into the soil 41.3 a 57.5 ab 44 a 14 58 b
NH4NO, Application at a plant growth 43.6 a 53.1 a 57 14 71
Urea height of 40 cm; immediately 40.2 a 53.6 a 52 b 13 65 a
Urea + 1% PPDA washed into the soil 41.1 a 44.9 53 b 12 65 a
Experimel1lS performed in 1977
NH 4 NO J Application to a moist soil surface 32.5 a 39.3 a 58 a 15 73
Urea at a plant growth height of 25 cm; 30.1 a 38.2 a 40 II 51
Urea + 1% PPDA washed into the soil after 14 days 34.4 41.6a 54 be 13 67 a
NH 4 NO J Application to a dry soil surface 21.6 b 28.3 b 56 ab II 67 a
Urea at a plant growth height of 40 cm; 22.7b 29.3 b 47 d 9 56
Urea + 1% PPDA washed into the soil after 14 days 24.5 b 32.5 51 cd II 62
"Adapted from Matzel et al. (1979a,b).
The same letter indicates insignificant differences (p=O.05).
"No statistical evaluation.
Grain yield was not influenced significantly by timing offertilizer application (at 25- or
40-cm high plants), but total N uptake was higher when the fertilizers were
administered for 25-cm high plants than for 40-cm high ones. When the fertilizers were
not washed into the soil immediately but only at day 14 after their surface application,
the grain and straw yields decrcased, especially following fertilizer application on the
dry soil surface, at 40-cm high plants. The plants took up less N from urea than from
NH4N0 3 or urea+ I % PPDA; the uptake was minimal when urea was applied on moist
soil surface. PPDA increased both crop yield and plant uptake of N from urea; this
effect was more consistent with urea application on moist soil surface.
Balance offertilizer N at maturity stage of wheat in 1977 (Table 60) shows that the
plants took up by 11 and 5% more N from NH4N0 3 than from urea and urea+ I %
PPDA. respectively. This means that PPDA increased the uptake of N from urea. At the
same time, PPDA enhanced immobilization of urea-N in soil and reduced the N deficit
from 32 to 22%, i.e., to the same level as that recorded in the NH4N0 3 treatment. In
other words, application of urea alone led to a 10% loss by NH3 volatilization, but this
loss could be prevented by adding PPDA to urea.
271
Kampfe et al. (l982b) reviewed the results of 136 winter cereal (wheat and rye)
experiments carried out under field conditions, on mostly sandy soils as well as on
loamy sands, in the 1974-1979 period. In the review, the wheat and rye are evaluated
together as winter cereals.
Conditioned urea with or without 0.5 or 1% PPDA (on urea-N basis) and lime
ammonium nitrate were the fertilizers tested. They were surface-applied and not worked
into the soil.
Grain and straw yields and N uptake by plants on 9-17 _m2 plots were determined.
In 1974 and 1975, at the beginning of the growing season, 50 kg of N/ha were
applied. In the 1976-1979 period, the same amount of 50 kg of Niha was administered
at the beginning of the growing season, but later a second amount (40 kg ofNIha) was
added. The plots were also fertilized with 30 kg of P and 100 kg of Klha at the
beginning of growing season in each year.
For evaluation of the effect of N fertilizers on crop yield and N uptake by plants,
only the results of 21 winter wheat and 80 winter rye experiments conducted in the
1976-1979 period were taken into consideration, but for evaluation of the relationship
between crop yield and the amount of rainfall during the first 5 days after fertilization
the results registered in 1974 and 1975 were also taken into account.
It was established that the grain yield was significantly smaller in the treatment with
conditioned urea than in that with lime ammonium nitrate, when fertilization was
followed by dry weather. In this case, PPDA reduced the grain yield losses. Reduction
of these losses under the influence of 1% PPDA was total in loamy sands and about
50% in sands; at the 0.5% PPDA rate the effect of 1% PPDA was attained only in the
sands. PPDA also improved N uptake by plants. Straw yields were nearly the same in
all treatments.
When it rained during the first 5 days after fertilizer application, urea with or
without PPDA acted more efficiently on grain yield and N uptake than did lime
ammonium nitrate. The fertilizers did not exhibit significantly different effects on straw
yield.
One can deduce from Figure 77, which presents the relationship between grain yield
and amount of rainfall during the first 5 days after fertilization, that the yield losses
272
2,0
'iii'
i":~i
7'
'[i! -1.0
CJ
- 2.0 ""r--T'" i I I , i ,
o 5 10 15 20 25 )J 35
Rainfalls (mm)
Figure 77. Influence of rainfalls on the difference between cereal grain yield recorded in the
treatment with lime ammoniwn nitrate and grain yields obtained in the treatment with urea with or
without 0.5 or 1% PPDA addition.
a - Conditioned urea. b - Conditioned urea + 0.5% PPDA. c - Conditioned urea + 1% PPDA. /From
Kiimpfe e/ al. (l982b)./
were great whert it did not rain, especially in the urea-only treatrnertt. With increasing
amounts of rainfall, both urea+0.5% PPDA and urea+ 1% PPDA became more efficient
than lime ammonium nitrate, but at rainfalls ~ 30 mm only urea+ 1% PPDA preserved
the efficiency.
The results obtained in the winter wheat experimertts concerning the grain yield-
increasing effect of 1% PPDA addition to urea fertilizer were also referred to by Linke
and Kampfe (1987) in their communication presented at the International Fair
Symposium held in Leipzig.
The first results obtained in experiments carried out within an international
collaboration in Bulgaria, the former Czechoslovakia, (East) Germany, Hungary,
Poland, Romania, and the former U.S.S.R. for studying the efficiency of urea+PPDA
were published by Koren'kov et al. (1980). The experiments started in 1976. Soils in
Mitscherlich pots were surface-treated with urea (0.5 g N/pot) with or without 1%
PPDA relative to urea-No The test plants were spring wheat (on differertt soils, in all
countries, except in Romania) and oats (on a chestnut chernozem in Romania).
Generalization of the results of all experiments led to the conclusion that PPDA did not
significantly increase the crop yield but enhanced, to some extent, plant uptake of N
from urea.
The investigations were continued in 1977 and 1978, and their results were
published by Kampfe et al. (1982c). In these years, rate ofurea-N was increased to 1
glpot, whereas that of PPDA remained at 1% on urea-N basis. If in 1976 wheat (grain
and straw) yield and plant utilization offertilizer N in urea treatmertt (100%) increased
to only 102% in the urea+PPDA treatment, in 1977 and 1978 PPDA increased crop
yield by 8-12% and plant utilization ofurea-N by 9-18%.
Rao and Ghai (1986a) studied, under conditions identical to those shown on pages
264 and 268, the effect of PPDA used in a proportion of 10% relative to urea weight
(40, 80, and 120 mg N/kg soil). The principal results are specified below.
Germination of wheat seeds, evaluated at day 15 after sowing, was not affected by
PPDA. Dry matter of plants after 21 and 35 days of growth was higher in treatments
with urea + PPDA at three rates than in the control plants treated with urea alone (on
273
average, by 13.1 and 38.7%, respectively). PPDA increased grain yield by 25.1% and
decreased straw yield, and, in such a way, the total yield remained at a level similar to
that of the control plants. Under the influence of PPDA, N content in grain also
increased (by 25.8%), but that in straw remained unchanged; therefore, the total N
content (grain + straw) exceeded the value recorded in control plants only by 13.8%.
In the field experiments described by Schlegel et af. (1987), PPDA had no
significant effect on crop yield. Urea prills, urea solution, and urea-ammonium nitrate
solution with or without PPDA were applied on surface of two Indiana soils (silt loam
and loam), at a rate of 34 or 67 kg NIha, and 0.56 or 2.24 kg PPDAlha, then
immediately sown with winter wheat. Grain yield and N content in grains were not
significantly different in soils N-fertilized with and without PPDA.
In field experiments (Winiarski, 1990), PPDA added at a rate of 1% to urea fertilizer
had no significant effect on spring wheat yields. However, PPDA influenced positively
the N uptake by plants.
In a 3-year field trial on 16-m2 plots installed on a brown soil of loamy sand texture
(PH 6.3-6.4), Kucharski (1992) compared the effects of urea and urea+PPDA on winter
wheat. The plots were fertilized yearly with 35 kg Plha as triple superphosphate and 83
kg Klha as KCI in a single application, and with 120 kg Nlha as urea with or without
1% PPDA in single and divided applications (120 kg Nlha and 60 + 60 kg Nlha,
respectively). The results indicated that PPDA in both single and divided applications of
urea+PPDA exerted no significant effect on grain and straw yield, on uptake and
utilization ofN by the wheat plants.
It is mentioned in a short report by Yeomans and Cerrato (1993) that in growth
chamber experiments the wheat plants treated with urea and urea + PPDA had lower
yields than those treated with nitrate and nitrate + PPDA.
Czapla and Hurniecki (1998a,b) studied the effect of PPDA on spring wheat in 3-
year field experiments (1987-1989) on a light-textured soil (pH 5.7-5.9). Before sowing,
all plots (20 m2 each) were fertilized with 32 kg Plha as triple superphosphate and 100
kg Klha as KCl. For soil application, urea was used at rates of 0, 50, 100, and 150 kg
Nlha, whereas for foliar application rates of urea were 0, 25, 50, and 75 kg Nlha. PPDA
was used at a rate of 1% relative to weight of urea.
Grain and straw yield and N content data allowed the researchers to draw the
conclusion that PPDA in both soil and foliar applications with urea had no significant
effect of wheat yield. When applied with urea to soil, PPDA caused a several percent
increase in plant utilization of N from urea. In foliar application, PPDA increased the N
content in grain and decreased it in straw.
See also Table 61.
* *
*
Triticum durum was the test plant in a field experiment in which Katyal et af. (1987)
studied the effect of timing of urea, urea-PPDA, and urea-DCD applications relative to
irrigation on the fertilizer urea-N use efficiency. The experiment was carried out on a
coarse-textured alkaline (PH 7.9) loamy sand soil in the 198211983 growing season, at
the research farm of the Punjab Agricultural University, Ludhiana, India. The fertilizers
were 15N-Iabeled: urea alone C5N excess% 4.836), urea-PPDA (10 g PPDA/kg urea; 15N
excess% 4.733), urea-DCD (100 g DCD-N/kg N in the final product; 15N excess%
274
4.273). KNO) C5N excess% 4.085) was the reference fertilizer. The labeled fertilizer
were applied on microplots (1.2 by 1.2 m) installed within the large plots (5.5 by 2 m).
No N fertilizers was added to the control plots.
Rate of N fertilizers was 120 kg N/ha, applied in two equal splits - half basal and
half topdressed. For basal application, the fertilizers were broadcast and incorporated
into the moist seedbed before sowing; for topdressing, the fertilizers were surface-
applied on the dry soil within a half hour before the first irrigation or on the wet soil 20
hours after the first irrigation, which took place about 3 weeks after sowing.
Before the basal N fertilization, superphosphate (22 kg P/ha) and ZnS04 (10 kg
Zn/ha) were broadcast on all plots. The K that was added through KN0 3 was balanced
across all plots by adding equivalent amounts of K as KCl.
At maturity, besides estimation of grain yield, the plants and soil were analyzed for
total Nand 15N.
KN0 3 gave the highest grain yield irrespective of the timing of topdressing (before
or after irrigation). In the urea, urea-PPDA, and urea-DCD treatments, the grain yields
were significantly higher (p=0.05) when they were topdressed before irrigation than
when they were applied after irrigation. In the case of topdressings before irrigation, the
grain yield in the urea-PPDA treatment was significantly higher than that in the urea-
DCD treatment and not significantly different from that registered in the urea-only
treatment. The grain yield obtained with urea-PPDA topdressed after irrigation was
significantly higher than that with urea or urea-DCD also topdressed after irrigation;
following topdressings after irrigation, urea-PPDA yielded 400 kg/ha more than urea
alone.
15N loss was lowest in the KN0 3 treatment and had the following values for the
other fertilizers topdressed before and after irrigation: 15.8 and 42.2% (urea), 32.9 and
33.2% (urea-PPDA), and l3.7 and 46.2% (urea-DCD), respectively.
The highest grain yield and the lowest N loss in the KNO) treatment indicate that
leaching and denitrification were not significant loss mechanisms. The higher grain
yield and the lower N loss with urea-PPDA compared to yield and N loss with urea and
urea-DeD suggest that volatilization of ammonia released from urea was the major N
loss mechanism.
In the 1983/1984 experiments, plots on a silt loam (PH 6.3) and a loam (PH 6.3)
were fertilized by broadcast urea prills or UAN solution either in autumn (after sowing)
or in spring at rates of 34, 50, and 67 kg N/ha. The inhibitors tested were cyclohexyl-
PTA, trichloroethyl-PDA, and PPDA at rates of2 kg/I 00 kg N (in prills) and 2.24 kg/ha
(in UAN solution).
In all experiments, the inhibitors did not significantly affect grain yield and N
content. There were only two exceptions, both in the 1983/1984 experiments. The first
instance was in the autumn experiment on silt loam where grain N content was higher
with urea (50 kg N/ha) plus PPDA than urea alone (21.0 and 19.0 g/kg, respectively).
The second instance was in the spring experiment on loam where grain yield was higher
with UAN (34 kg N/ha) plus PPDA than UAN alone (4.09 and 3.46 t/ha, respectively).
The general inefficiency of inhibitors in the wheat experiments was attributed to
precipitation that fell on the first days after fertilizer application; the rainfall leached the
~urface-applied urea into the soil, preventing volatilization of ammonia.
The effect of nBTPTA on wheat was also studied in a greenhouse experiment
conducted at the Headquarters of the International Fertilizer Development Center
(Muscle Shoals, Alabama). Urea with 0.1 % nBTPTA, surface-applied on a wheat soil,
led to a 65% increase in urea-N uptake by the plants and to a 16% increase in grain
yield over those achieved with urea alone (Carmona et al.. 1988; Christianson and Vlek,
1991 ).
Bremner and Krogrneier (1988) studied the ability of 10 urease inhibitors to prevent
or reduce the adverse effects of urea on seed germination, seedling growth, and early
plant growth, these effects being caused by ammonia released from urea under the
action of soil urease. The inhibitors studied were: nBTPTA, PPDA, four phosphoric
triamides [phosphoryl triamide, phenylphosphoric triamide, N-(diaminophosphinyl)
benzamide, 4-fluoro-N-(diaminophosphinyl)benzamide], phenylmercuric acetate,
catechol, hydroquinone, and p-benzoquinone. Wheat, barley, oats, rye, sorghum, and
alfalfa served as test plants. Three Iowa soils were used.
In the germination test, air-dried soil samples (40 g) placed in Petri dishes (1.5 x 10
cm) were moistened with 10 ml of water or 10 ml of a solution containing 100 mg of
urea with or without 1-100 Ilg of inhibitor, then sown with 100 seeds and incubated in
the dark, at 20 0 e for 7 days, after which time the germinated seeds were counted.
In the soils not treated with urea, the seeds germinated in high proportions. In the
urea-treated soils, no seed germinated which proves toxicity of urea. In the presence of
both urea and inhibitors, the seeds germinated in the same number as in the untreated
soils or in a smaller number. In other words, the urease inhibitors eliminated or reduced
the adverse effect of urea on germination. The most effective inhibitor was nBTPTA,
followed by PPDA. Thus, the adverse effect of urea on germination of seeds (wheat,
barley, oats, and sorghum) was eliminated by nBTPT A used in a concentration as low
as 0.001 % relative to weight of urea (in two soils) or at 0.005% concentration (in one
soil).
The procedure used for studying seedling growth was similar to that used in the
germination test, but only 15 seeds were sown instead of 100 seeds. Shoot length was
measured after 7 days. No seedling growth occurred in soils treated with urea only.
Again, nBTPTA proved to be the most effective compound, eliminating even at 0.005-
0.01 % concentrations the adverse effect of urea.
276
For studying early plant growth, the following procedure was applied. Air-dried soil
samples (200 g) placed in small pots were moistened with 10 ml of a solution
containing 10 mg ofK2S04, 10 mg of NaH 2P0 4, and 50 mg of (NH4hS04, then 3 seeds
(only wheat or sorghum seeds) as well as 3 granules, each containing 25 mg of urea
with or without nBTPTA or PPDA (0.001, 0.01 or 0.1% on urca weight basis) were
placed 2 cm below the soil surface. Finally, 20 ml of water were added and, then, the
pots were introduced to a growth chamber (22°C). After 21 days of growth, dry matter
of plants was determined. It was established that nBTPT A, even at 0.0 I %
concentration, markedly reduced the adverse effect of urea on early plant growth. PPDA
acted less markedly.
However, PPDA was found very effective in a similar experiment in which Bremner
and Krogmeier (1989) studied the adverse effect of purified urea and fertilizer urea on
seed germination and elimination of this effect by PPDA. Samples of four Iowa soils
and seeds of wheat, maize, barley, and rye were used. Rates of addition per 40 g of air-
dried soil were 100 mg of urea and 1 mg of PPDA. As the adverse effect of fertilizer
urea was not significantly different from that of purified urea, it was deduced that the
effect of fertilizer urea was not due to impurities (biuret, cyanuric acid) but to ammonia
formed through hydrolysis of urea by soil urease. This deduction was supported by the
findings that biuret and cyanuric acid alone had little, if any, effect on germination in
soil when compared with urea, and the adverse effect of urea was completely eliminated
by PPDA. The results were similar with the four soils and seeds of the four plant species
studied.
In continuation of these investigations, Krogmeier et al. (l989a) performed other
experiments to give answer on the following question: does inhibition of soil urease
activity by nBTPT A or PPDA lead to accumulation of urea in plants grown in soil
fertilized with urea because, as known, urea may cause leaf tip necrosis?
For these experiments wheat and sorghum were grown on two Iowa soils.
In the first experiment, air-dried soil samples (500 g) in pots were moistened with 25
ml of a solution containing 20 mg of K2S04 and 20 mg of NaH2 P0 4, then sown with 15
seeds at 2-cm depth and treated with 50 ml of a solution containing 0.5 g of urea with or
without 0.05, 0.5 or 5 mg of nBTPT A or PPDA. The pots were then placed in a growth
chamber (22°C). After 21 days of growth, dry weight of plants was determined. Leaf tip
necrosis was assessed by separating the necrotic portions of the plant shoots from the
nonnecrotic portions and determining their dry weight. Urea content in both necrotic
and nonnecrotic portions was also assayed.
Dry weight of plants of both species was smallest, i.e., the growth was weakest, in
soils treated with urea without inhibitors. These plants did not manifest any symptoms
of leaf tip necrosis and contained only negligible amounts of urea. The weak growth
was due to ammonia which could be released from urea since soil urease had not been
inhibited. The plants grew incomparably better when urea and inhibitor were applied
together, but leaf tip necrosis occurred and urea content increased in necrotic portions
and remained negligibly low in nonnecrotic portions. Severity of necrosis and urea
content increased with increasing inhibitor conccntration and were much more marked
with the stronger inhibitor, i.e., with nBTPTA than with PPDA. Dry weight of necrotic
portion highly correlated (r=0.99) with their urea content. It results from these
observations that urea, which could not be hydrolyzed because of the presence of the
277
soil urease inhibitors, had accumulated in toxic concentrations in leaf tips and induced
necrosis.
In the second experiment, non-autoclaved and autoclaved soil samples were used
and the test plant was wheat. Autoclaving (at 120D e for 2 hours) led to disappearance of
urease activity in soil. The procedure was the same as in the first experiment, but urea
was applied at several rates (0, 0.0625, 0.125, 0.25, and 0.5 glpot) with or without
nBTPTA or PPDA at a single rate (2.5 mg). Analyses of plants after 21 days of growth
showed that in the absence of inhibitors the necrosis appeared and urea accumulated
only in plants grown in the autoclaved soil treated with 0.25 and 0.5 g of urea/pot. In the
presence of inhibitors, the necrosis occurred and accumulation of urea in necrotic
portions of leaves also took place at lower urea rates. These effects were more marked
in autoclaved soil than in the non-autoclaved soil and with nBTPTA than with PPDA.
In the third experiment, the effect of nBTPTA and PPDA on urease activity in wheat
and sorghum shoots was studied. The procedure of experiment 1 was applied, but the
soil samples were not treated with urea, and, after 21 days of growth, the shoots were
immediately weighed and analyzed for urease activity. The results indicated that
nBTPTA and PPDA did not significantly decrease, at any of their rates, urease activity
in shoots.
The conclusion is that nBTPTA and PPDA applied together with urea led, through
inhibition of urease activity, of urea hydrolysis in soil, to manifestation of the
phytotoxic effects of urea. However, these phenomena were observed only when the
soil concentrations of nBTPTA and PPDA markedly exceeded those likely to exist
when these compounds are used as fertilizer amendments to reduce problems
encountered in use of urea as a fertilizer.
The potential of nBTPTA and PPDA for inducing phytotoxicity should not preclude
their use to eliminate the adverse effects of urea fertilizer on seed germination and
seedling growth because the ammonia produced through hydrolysis of urea fertilizer by
soil urease is much more detrimental to plant growth than is urea accumulation induced
by urease inhibitors.
In the field experiments conducted by Goodroad and Wilson (1989) in Georgia, late
winter topdress application ofurea+nBTPTA to winter wheat resulted in increased plant
N content and also in increased grain yield when N was limiting.
Bremner and Krogmeier (1990) evaluated the effect of 23 urease inhibitors on the
germination of wheat, maize, barley, sorghum, and alfalfa seeds in soil samples to
which no urea was added. Three Iowa soils (loam, pH 6.9, clay loam 7.5, and silty clay,
pH 7.7) were studied. Rates of inhibitor addition were: 0, 50, 250, 500, and 2,500 J-lglg
soil. The urease inhibitors are nominalized. and their effect on germination is specified
in Table 61.
One can see from this table that 2,5-dimethyl-l,4-benzoquinone exhibited the
strongest adverse effect on germination and that wheat and sorghum seeds were most
sensitive to this urease inhibitor.
In the wheat field experiment of Gezgin and Bayrakli (1995), nBTPTA added to
urea at rates of 0.25 and 0.50% relative to weight ofurea-N (200 kg urea-N/ha) greatly
reduced the volatile ammonia losses (see page 198) and increased grain yield (t/ha)
from 3.763 (urea only) to 4.443 (urea+0.25% nBTPTA) and 4.313 (urea+0.50%
nBTPTA), but had no effect on the protein content in grain.
278
Wang et at. (1995) studied the effect of nBTPTA on spring wheat under growth
chamber conditions. Five-kg samples of a black chernozemic soil from Manitoba were
placed in plastic pots (20 cm high with a 20-cm diameter). Urea was applied at rates
equivalent to 0,40, 80, and 120 kg Nlha, with or without 0.15 or 0.25% nBTPTA by
weight of urea, and was seed-placed or surface dribble-banded. Fifteen wheat seeds
were sown in each pot. The soil moisture was cycled between 70 and 100% of field
capacity by watering.
Seedling emergence was significantly decreased by seed-placed urea at its rates of
80 and 120 kg Nlha but not by surface-dribbled urea. The damage caused by seed-
placed urea was reduced by nBTPTA. There was no significant difference between the
rates of 0.15 and 0.25% nBTPTA. Vegetative yield measured at heading increased with
application of urea, but no difference in vegetative yield occurred due to urea placement
or the use of nBTPTA. N accumulation in plants increased with increasing urea rate,
and the increase was higher when urea was seed-placed than surface-dribbled. nBTPTA
279
The results obtained with neem cake-coated urea applied by Sharma and Prasad
(1996) in a 2-year maize-wheat rotation are referred to on page 262.
Zhao et al. (1993a) performed a pot trial for studying the fate of HQ and urea in a
soil-rice system. Samples of a brown soil of silty loam texture (PH 6.4) were amended
with 15N_urea (atom excess, 5.56%) at a rate of 61.33 !lg N/g soil with or without 14C_
HQ (having a specific activity of 235.727 x 104 Bq/mg) at a rate of 533.2 ng/g soil.
Thus, the initial ratio (weight/weight) of 14C to 15N was 1: 115. Then rice seedlings were
transplanted into the soil samples. During the growth period and after harvesting, the
aboveground parts of rice plants and the soil were analyzed for 14C_HQ and other 14C_
materials as well as for total N and 15N.
The results showed that the rice plants absorbed only a minor part (0.25%) of the
amount of 14C_HQ added to soil. The 14C_HQ content was 37 nglg refined grain and 47
nglg straw. Most of the absorbed 14C_HQ was degraded or converted by the plant tissues
into other forms of 14C-materials, the content of which was 396 nglg refined grain and
116 nglg straw. In soil, 14C_HQ was gradually decomposed. Thus, after 3 months of rice
growth, 1 g of soil at different depths in the 0-20-cm layer contained, on average, 220
ng 14C-materials and 71 ng 14C_HQ which is lower than the permissible ecological value
(2,000 nglg soil) and the permissible healthy value (200 nglg soil) of the 14C_HQ.
The initial 14C:15N ratio of 1:115 changed to 1:72 in soil, 1:1,160 in straw and
1:3299 in grain, i.e., the rice plants absorbed relatively more 15N and there was more
14C-material left in the soil. Nevertheless, in this experiment the aboveground plant
biomass was not significantly different in the urea and urea + HQ treatments.
The conclusion was drawn that applying appropriate concentration of HQ with urea
as a urease inhibitor will not produce harmful effects on human health and the
environment. This conclusion is consistent with the opinion of Zhao et at. (1988, 1989),
based on results obtained with unlabeled urea and HQ: the mipor amount ofHQ applied
with urea fertilizer will not accumulate in soil and plant and will not enter the
underground water and atmosphere and, therefore, will not induce pollution of
environment and poisoning of food chain.
In the experiment of Fan and Ye (1995), HQ applied with urea enhanced the
efficiency of utilization of fertilizer N by rice plants, as it prevented the evaporation loss
of N in the paddy field.
transplanting and the second broadcast without incorporation at maximum tillering (30
days after transplanting). The basal urea was 15N-labeled. Two sets of split-applied
treatment were made, one with only the basal-incorporated portion 15N-labeled and the
other with only the broadcast or delayed application 15N-labeled. The labeled urea
contained 4.2 atom% excess 15N and was tableted with or without PPDA in a press to
produce pellets 2.4 mm in diameter and 10-15 mg in weight.
The soil in each treatment was kept flooded with 3 cm of water throughout the 77-
day experiment. The rice crop was harvested prematurely because poor light conditions
caused erratic heading and grain formation. Dry weight of the entire aerial portions of
the plants was determined. Total N and 15N contents in aerial parts as well as in soil and
roots were also assessed.
The results obtained (Table 62) are surprising: under the influence of PPDA, the dry
matter production decreased significantly for both basal and split applications in
contrast with the significantly increased plant recovery of applied 15N. The increase was
10-15%. The highest increase was registered in the treatment in which the urea applied
at maximum tillering stage was labeled (41.0% of 15N recovered by plants without
PPDA and 66.7% with PPDA); in this treatment, the soil and roots recovered 36.4 and
34.0% of 15N. Thus, in the presence of PPDA, the recovery of 15N in the plant-soil
system was 100%. The great effectiveness of PPDA at maximum tillering stage was
probably caused by the large capacity of the root system at this growth stage to take up
N, together with the poor competitive position of the algae which were shaded by the
rice plants.
TABLE 62. Effect ofPPDA on dry matter production by rice plants and on recovery of 15N in
a ~t e:seeriment"
I~N recove!1 (%J
Dry matter
Treatment Plants Soil and
(glpot) Total
(aerial ~rts) roots
Control 35.3 d
Basal urea 44.4 ab 13.4 d 33.1 d 46.5 c
Basal urea + PPDA 36.8 bc 23.8 c 42.3 c 66.1 d
Split application
First split 15N-labeled'
Urea 46.0 a 15.8 d 51.2 b 67.0 d
Urea + PPDA 40.8 bc 24.5 c 61.7 a 86.1 b
Second split liN-labeled
Urea 46.0 a 41.0 b 36.4 d 77.4c
Urea + PPDA 40.8 bc 66.7 a 34.0 d 100.7 a
"From Byrnes et al. (1983). by permission of the Soil Science Society of America, Inc.
Data in the same column not followed by the same letters are significantly different
(p=0.05).
'Dry matter yields for the split applications with 15N-labeling at different times were
averaged.
In the experiment of Simpson et al. (1985), the microplots cropped with flooded rice
were fertilized with urea (80 kg Nlha) labeled with 15N (at 1.48 atom% excess 15N) with
or without 1% PPDA (relative to weight of urea) addition. Urea and urea-PPDA were
applied at the stage when the plants reached 20 cm above the floodwater (see page 119).
At early heading stage (at day 47 after fertilization), the plants (shoots and roots)
contained significantly more 15N in the urea-PPDA treatment (47.6% ofthe 15N applied)
than in the urea-only treatment (28.2%). This effect persisted at maturity. Thus, at this
stage, 15N contents in grain and straw in the urea-PPDA treatment exceeded those
recorded in the urea-only treatment by 53 and 56%, respectively. However, there was
no increase in grain yield. This is explained by the property of the rice variety used
(Inga) not to respond well to N fertilizers applied at early tillering. Consequently, for
studying the effect of PPDA on grain yield, it is necessary to use more responsive rice
varieties.
The rice variety IR36, used by Fillery et al. (1986b) in the investigations mentioned
on page 122, responded differently to PPDA depending on locality and season. At Los
Banos, in both dry and wet seasons of 1982, PPDA had no increasing effect on plant N
uptake and grain yield. Neither did PPDA enhance the grain yield at Munoz. Contrarily,
application ofPPDA significantly increased the grain yield (from 4.7 to 5.2 tlha) in the
experiment conducted at Los Banos in the dry season of 1983, in which two-thirds of
urea and urea-PPDA were administered at day 26 and the remaining third at day 50 after
transplanting. In the experiment, the plants took up more N from urea-PPDA than from
urea, but the difference was only significant at day 60 after transplanting; 15N recovered
in plant and soil at grain harvest was 77.7% in the urea-PPDA treatment and only 65.8%
in the urea treatment. At Munoz, 15N recovery (in plant and soil) was 73.6% (urea
treatment) and 83.4% (urea-PPDA) at day 14 after fertilization and similar for both
treatments at grain harvest. As mentioned above, the grain yield was also similar.
Rao and Ghai (l986b) described a pot experiment aimed at studying the effect of
PPDA on rice grown on an alkali soil (sandy loam, pH 9.0). Soil samples (20 kg/pot), to
which 20 ppm P (as single superphosphate) and 12.5 ppm ZnS04 had been added, were
saturated with water, following which urea with or without PPDA was surface-mixed
and the pot watered to maintain 3.5 cm of standing water. Then four 33-day-old rice
seedlings were transplanted into each pot.
Urea-N (at a total rate of 150 ppm in all treatments) was applied by three modes:
M-1: one-half of N at transplanting and one-fourth each at weeks 3 and 6 after
transplanting;
M-2: one-third ofN each at transplanting and weeks 3 and 6 after transplanting;
M-3: one-fourth ofN each at transplanting and weeks 3,6, and 9 after transplanting.
PPDA was used at a rate of 5% relative to weight of urea.
At weeks 6 and 9 after transplanting and at maturity, the plant dry weight, N uptake
and N uptake efficiency were recorded.
At weeks 6 and 9, in urea-only treatments, dry matter, N uptake, and N uptake
efficiency in the three modes of application had the following order: M-2> M-1 > M-3.
At maturity, the three modes of application presented the orders: M-2 ~ M-3 > M-1
(grain yield); M-2 = M-1 ~ M-3 (straw yield); M-3 > M-2 ~ M-l (N in grain); M-l ~ M-
3 ~ M-2 (N in straw); M-3 > M-2 ~ M-l (N uptake efficiency). Thus, taking into
account the grain yield and N uptake efficiency at maturity, M-3 (Le., split application
284
of urea in four equal amounts) proved to be the most efficient mode of application and
M-1 the least efficient one.
At week 6, PPDA increased plant dry matter, N uptake and N uptake efficiency in
each mode of application. At week 9 and at maturity, the effect of PPDA remained
positive in M-3 and M-1, but became negative, depressive, though insignificantly, in M-
2. Under the influence ofPPDA, in the most efficient M-3, the grain yield and N uptake
efficiency at maturity increased by 14.3 and 17.0%, respectively. Therefore, a saving of
fertilizer can be achieved by applying urea and PPDA as in M-3.
In the field experiments of Buresh (1987) (see page 126), the rice grain yields were
not significantly different depending on the nature offertilizers (urea, urea-PPDA, urea
phosphate, urea-urea phosphate) applied at 30 kg Nlha rate and the methods of their
application (into soil or floodwater).
But in two other rice experiments on a clay soil at Pila, Buresh et al. (1988b,c)
found that PPDA, used at a rate of 2% relative to weight of urea, was efficient in
increasing N content in grains. One of the experiments was conducted in the 1985 dry
season (see page 126). Urea rates were 30, 60, and 120 kg N/ha. Two-thirds of the urea
was applied at day 18 after transplanting of seedlings and one-third at days 5-10 after
panicle initiation. In the other experiment, conducted in the 1986 dry season, urea rates
were 40, 80, and 120 kg Nlha, one half of which was administered at day 16 after
transplanting and the other half at days 5-10 after panicle initiation. In both
experiments, urea and urea-PPDA were broadcast into 5-cm standing floodwater.
Within the plots there were microplots (80 by 80 cm) installed. Each microplot was
surrounded by 30-cm deep border made of painted, galvanized metal that had been
pushed approximately 20 cm into the soil. Urea broadcast (with or without PPDA) into
the microplots was labeled with 5 atom% 15N. At rice maturity, grain yield was recorded
and total Nand 15N in grain, straw, and soil+root were determined. The unrecovered 15N
was assumed to represent total gaseous N loss, because runoff loss was prevented by the
metal border of microplots and leaching loss was negligible (as the fraction of added
15N recovered in the 15-30-cm soil layer was consistently less than 2%).
PPDA significantly reduced N losses from the higher urea-N rates but did not
eliminate them. Thus, in the first experiment, the N losses from the 30, 60, and 120 kg
urea-Nlha rates were reduced from 5 to 3 kg Nlha, from 14 to 10 kg Nlha and from 34
to 16 kg Nlha, respectively. In the second experiment, N losses from 40,80, and 120 kg
urea-Nlha in the absence and presence ofPPDA were 5 and 6, 27 and 16, and 44 and 30
kg Nlha, respectively.
Reduction of N losses was not associated by a significant increase in grain yield.
Thus, mean yields for the three urea-N rates were 6.0 tlha (urea-only treatments) and 6.2
tlha (urea-PPDA treatments) in the first experiment; the corresponding values in the
second experiment were 6.0 and 6.3 tlha, respectively. But significant increases
occurred in the N content of grains. For example, in the first experiment at the 120 kg
urea-Nlha rate, the grains contained 33% (urea treatment) and 44% (urea-PPDA
treatment) of the applied N. In the second experiment, the corresponding values were 23
and 32%, respectively.
Elimination of gaseous N loss could increased grain yield by a maximum of 6 and
8% in the first and second experiment, respectively. These percentages corresponded to
0.4 and 0.5 tlha increases in grain yield, respectively.
285
But in other rice field experiments, in which unamended and PPDA-amended urea
were broadcast to floodwater, amendment of urea with PPDA did not increase either
yields or grain uptake of N compared with unamended urea (Snitwongse et aI., 1988;
Raju et al. , 1989; Satrusajang et al., 1991).
In the rice field experiment carried out in Fuzhu, China, prilled urea was applied
alone (control) and with a urease inhibitor (nBTPTA) or with a nitrification inhibitor (2-
ethynylpyridine, 2EP) or with both inhibitors. The following grain yields (tlha) were
obtained: 4.74 (control), 5.30 (nBTPTA), 5.15 (2EP), and 5.45 (nBTPTA + 2EP). The
grain N contents (%) showed the same order: 0.947 (control), 0.979 (nBTPTA), 0.968
(2EP), and 1.012 (nBTPTA + 2EP). All increases were significant (p=0.05). The best
results were obtained by combined use of the two inhibitors (Freney et aI., 1989).
In the experiments of Khanif and Husin (1992), urea + hydroquinone (HQ), as
compared to urea alone, brought about only an insignificant increase in rice yield, but
when HQ was used in combination with DCD, the plant uptake ofN from urea fertilizer
significantly increased.
In the rice field experiments conducted by Chaiwanakupt et al. (1996) and
Phongpan et al. (1995, 1997) in Thailand (see page 182), the grain yields increased in
parallel with the reduction of volatile ammonia losses. In the experiment carried out
during the 1991 wet season, the highest yield was recorded in the treatment with
algicide + mixed urease inhibitors (4.66 tlha), which is significantly higher than that
obtained in the control treated with algicide + urea (4.00 tlha). nBTPTA in single
application without algicide increased the yield (4.22 tlha) in comparison with that of
control (urea only) (3.57 tlha), but when nBTPTA was applied with algicide, it reduced
the yield from 4.00 to 3.87 tlha.
In the experiment, which was carried out during the 1992 dry season and in which
algicide was applied to all treatments, the following grain yields (tlha) were obtained:
3.6 (control), 3.7 (nBTPTA), 4.0 (PPDA), and 4.1 (nBTPTA+PPDA). In other words,
the nBTPTA +PPDA combination was most efficient in increasing the grain yield.
In the field experiment described by Freney et al. (1995) and Phongpan et al. (1997)
(see page 249), the rice grain yield of3.14 tlha in the urea-only treatment was increased
to 3.17-3.81 tlha in the treatments with inhibitors. The increase was not significant
when urease inhibitor was not used and significant (P<0.05) when the urease inhibitors
cyclohexyl-PTA and N-(n-butyl)-PTA with or without the nitrification inhibitor
phenylacetylene were applied together with the algicides copper sulfate and terbutryn.
At the same time, addition of CHPTA and nBPTA in combination with phenylacetylene
and algicides resulted in a 2- or 3-fold increase of applied urea-N in the grain.
were applied twice in each year. Finally, the grain yield and total N contents in green
matter and grain were assessed.
The mean values of grain yields obtained in the whole 1978-1980 period, in the N
fertilizer treatments, and expressed as percentage of those registered in the lime
ammonium nitrate treatments (100%) were the following in the barley and rye
experiments: 99 and 95% (urea), 104 and 100% (urea + 0.5% PPDA), and 103 and
106% (urea + 1% PPDA ), respectively. These data show that urea and urea + 0.5%
PPDA were more efficient, whereas urea+ 1% PPDA was less efficient, in the barley
than in the rye experiments.
These experiments also proved that, when fertilizer application is followed by dry
weather, the grain yield is significantly lower in urea treatment than with lime
ammonium nitrate, and PPDA improves the fertilizing efficiency of urea.
It is evident from Table 63 that the effect of urea on grain yield is greatly influenced
by soil texture. In sandy soils, urea was less efficient, whereas in loamy soils its
TABLE 63. Effect of nitrogen fertilizers on grain yield of winter cereals in experiments on large surfaces,
as influenced by soil texture (mean values for the 1978-1980 period)"
Sands Slightly loamed Sandy loams and
sands and loamy loams
Fertilizer sands
(13 experiments) (20 experiments) (10 experiments)
dtlha % dtlha % dtlha %
Unfertilized control 20.2 61 26.8 68 33.5 75
Lime ammonium nitrate 33.3 100 39.1 100 44.7 100
Conditioned urea 31.0 93 38.1 97 44.7 100
Conditioned urea + 0.5% PPDA 33.2 100 40.5 103 45.3 101
Conditioned urea + 1% PPDA 35.1 105 40.5 103 47.6 106
LSD 10% 2.8 8 2.7 7 3.4 8
LSD 20% 2.2 7 2.1 5 2.6 6
a From Kiimpfe et al. (1983).
efficiency was equal to that of lime ammonium nitrate. Under the action of PPDA,
especially of its 1% amount, urea became a fertilizer as efficient or even more efficient
as lime ammonium nitrate in all soils.
The higher efficiency of urea in the barley experiments than in those with rye is
explained by a greater number of sandy soils used in the rye experiments and not by a
differentiated urea action determined by the species of cereals, because the sandy soils
lost more urea-N as volatile ammonia than did the other soils.
The effects exerted by weather conditions and soil texture on total N contents in
green matter and grain were similar to those exerted by them on grain yield.
In conclusion, the experiments carried out on large surfaces confirmed the results
obtained in microplots and plots in respect of the fertilizing efficiency of urea-
PPDA.
The experiment in which Bremner and Krogmeier (1989) found that PPDA added to
soil samples completely eliminated the adverse effect of urea on germination of seeds of
four plant species, including barley, is dealt with on page 276. See also Table 61.
291
in soil, isotopic composition ofN in soil and plants. Crop yields were also assessed, and
the N balance was calculated.
In the soil treated with urea alone, the plants took up 51 % of the applied urea-N,
19% remained in soil, and 30% was lost through volatilization. The corresponding
values recorded in the other treatments were the following: urea + nitrapyrin: 54, 21,
and 25%; urea + nitrapyrin + HQ: 57, 23, and 20%; urea + ATC: 53,23, and 24%; urea
+ ATC + HQ: 54, 24, and 22%, respectively. Thus, the most favorable effect on N
uptake by plants and reduction of volatile N losses was exerted by the mixture of
nitrapyrin and HQ. Grain yield was also highest in the urea + nitrapyrin + HQ treatment
(see also Muravin, 1989).
Pisareva and Muravin (1988) and Pisareva (1989) described pot experiments in
which the effect of HQ applied together with the nitrification inhibitor, 3-
methylpyrazole-l-carboxarnide (MPC) and with 15N-labeled urea (25 atom% excess)
(urea + MPC + HQ) on barley plants was compared with those of urea + MPC and urea
alone. Two soils were used: a soddy-podzolic soil (PH 5.5) and a calcareous chernozern
(pH 7.8). Both received PK as basal fertilizers. Rates of application per kg of soil were:
P2 0 S: 50 mg; K2 0: 75 mg; urea-N: 100 mg; MPC: 2 mg; HQ: 5 mg. The experiments
were initiated in 1985 and repeated in 1986. Grain yields on the podzolic soil in 1986
and 00 the chemozern in both 1985 and 1986 were highest in the urea + MPC + HQ
treatment (the highest grain yield on podzolic soil in 1985 was recorded in the urea +
MPC treatment). But N contents in grains were always lower in the urea + MPC + HQ
treatment than in those with urea + MPC and urea alone. HQ decreased plant uptake of
N from soil (podzol in 1985; chernozem in both years) or increased it (podzol in 1986).
surface application, the fertilizers were washed into the soil with 200 ml of water. Dry
matter and total N contents in grains and straw were determined.
The results showed that in both soils and both years the grain and straw yields as
well as the total N contents increased, under the influence of the applied fertilizers, in
the following order:
urea < urea+ I % PPDA < NH4 N0 3 •
Utilization of N from urea, urea+ I % PPDA, and NH4N0 3 had the mean values of
30, 70, and 90%, respectively. In the variant in which urea was mixed into the soil
before sowing of oats, crop yields and utilization of N corresponded, in most cases, to
the values registered in the urea+ I % PPDA treatments. The rate of 0.8 g N/pot applied
after emergence of oat seedlings was more efficient than the same amount applied at 25-
cm high plants, which was explained by higher temperatures in the period when the
plants reached this height. The same N rate in divided application (0.4 + 0.4 g N/pot)
did not lead to increased oat yield.
In 1977 a similar pot experiment was carried out by Matzel et al. (1979b). Two soils
(a sandy loam and a sand) were used and oats served as test plants. In a variant, 15N_
labeled urea (0.8 g N/pot) without PPDA was mixed into the soil (6 kg) before sowing.
In the other variants, urea was surface-applied and washed into the soil with 200 ml of
water after 14 days. We specify these variants: labeled urea (0.8 g N/pot) with or
without 1% PPDA was applied immediately after emergence of seedlings; the same rate
of labeled urea with or without 1% PPDA was applied at 25-cm high plants; unlabeled
urea (0.4 g N/pot) with or without 1% PPDA was administered at emergence of
seedlings, whereas the labeled urea (also 0.4 g N/pot) with or without 1% PPDA was
applied at 25-cm high plants.
Analyses of total N and 15N in plants indicated that in both soils the crop yield (grain
and straw) and plant uptake of N were highest in the variant in which urea without
PPDA had been mixed into the soil. In the other variants, PPDA increased both crop
yield and uptake of N from urea. Under the influence of PPDA, the plants took up, on
an average, II % more N from urea.
7.5.6. Effect of' Phosphoric Triamide (PTA) and Thiophosphoric Triamide (TPTA)
Compounds
Bremner and Krogmeier (1988) used oats among the test plants on which they studied
the effect of 10 inhibitors, including four PTAs and one TPTA (see page 275).
drought began after addition of the first or second N rate and lasted 14 days.
Unfertilized plots served as controls.
TABLE 64. Effect of nitrogen fertilizers on grain yield of winter rye (mean
values for the 1977-1979 period)"
Grain yield ofwinter rye
Fertilizer
dtlha %
Unfertilized control 20.8 53
Lime ammonium nitrate 39.0 100
Conditioned urea 36.8 94
Conditioned urea + 0.5% PPDA 38.6 99
Conditioned urea + 1% PPDA 38.7 99
LSD 5% 2.0 5
"Adapted from Kiimpfe et al. (1982a).
Table 64 shows that diminution of grain yield in urea treatment in comparison with
the lime ammonium nitrate treatment could be prevented by using urea+PPDA. There
was no significant difference between the effects of the 0.5 and 1% PPDA rates.
The 14-day simulated drought after fertilization led to a decrease in grain yield as
compared to that recorded under natural weather conditions. In the 1977-1979 period,
the yield decreased, under the influence of simulated drought, to 96% (in the lime
ammonium nitrate treatment), 89% (urea), 92% (urea + 0.5% PPDA), and 94% (urea
+1%PPDA).
Total N contents in green matter of plants were determined twice, namely at days 14
and 28 after application of each of the two N fertilizer rates. During the first 14 days,
PPDA retarded N uptake by the plants, but at day 28 after N application there were no
significant differences between the lime ammonium nitrate treatment and the urea + 0.5
and 1% PPDA treatments as concerns total N contents in green matter. In the 1977-1979
period, neither were these contents influenced by simulated drought.
The results of the 136 field experiments reviewed by Kiimpfe et at. (1982b) (see
pages 271-272) are valid not only for winter wheat but also for winter rye. The results
of the 43 experiments on large surfaces (Kiimpfe et at.. 1983) refer not only to winter
barley but also to winter rye (see page 289).
The experiment in which Bremner and Krogmeier (1989) found that PPDA added to
soil samples completely eliminated the adverse effect of urea on germination of seeds of
four plants, including rye, is dealt with on page 276.
carried out in the 1986-1988 period on a silt loam and in the 1987-1989 period on a silty
clay loam. Urea-ammoniumnitrate (UAN) solution was the N fertilizer at rates of 0, 56,
and 112 kg N/ha. Ammonium thiosulfate (ATS) was added to UAN at a rate of 10%
(volume/volume). Placement methods were: surface broadcast, dribble surface band on
51-cm centers, and knifed-injected 15-18 cm deep on 51-cm centers.
Grain yields and N concentrations of leaf tissue and grain were consistently and
significantly increased by N fertilization of both soils. Knifed placement of UAN was
superior to broadcast and dribble application. The results obtained with ATS added to
UAN were inconsistent. For example, in 1986 the broadcast UAN + ATS, as compared
to broadcast UAN, produced a 5% grain yield increase (at 56 kg N/ha) and an 11.7%
increase (at 112 kg N/ha); in 1989 ATS broadcast with UAN induced an 11% grain
yield decrease (at 56 kg N/ha) and a 3.5% increase (at 112 kg N/ha). On overall
average, the grain yield increase due to ATS was 4.5% on silt loam, and 1.2% on the
silty clay loam, both increases being unsignificant at p=0.05.
7. 7. 4. Effect ofPhosphorodiamides
A pot experiment in which the effect ofphenylphosphorodiarnidate (PPDA) on sorghum
was studied, is mentioned in a report presented by the International Fertilizer
Development Center (IFDC, 1981). 15N-Labeled urea was used in form of large
granules (supergranules of 0.25-1 g) point-placed at 10-cm depth in soil (clay) or as
prills incorporated into the top 2 cm of soil or as prills with or without 2% PPDA
applied on soil surface. The plants were watered by simulated rainfalls (245 or 465
mm). Depending on the fertilizer and water managements, the plants took up 15N in the
following proportions: 47.2% (from surface-applied urea), 52.5% (from surface-applied
urea-PPDA), 56.5% (from urea incorporated into the soil), and 63.5% (from
supergranules) under the conditions of 245-mm simulated rainfalls. The corresponding
values in the case of more abundant (465 mm) simulated rainfalls were: 52.3, 54.7,
59.4, and 70.6%, respectively. Thus, PPDA increased, to some extent, efficiency of the
surface-applied urea. The supergranules point-placed at 10-cm depth were more
efficient than was the surface-applied urea-PPDA, but an eventual enhancing of
efficiency by PPDA added to supergranules was not studied. The data cited also show
that incorporation of urea prills is more advantageous than their surface application, and
each form of urea was more efficient with more abundant simulated rainfalls.
These investigations, performed at IFDC Headquarters (Muscle Shoals, Alabama),
were described, with more details, but with similar conclusions, by Buresh et al. (1984).
See also Table 61.
Krogmeier et al. (1989a) studied the relation between the urea-caused leaf tip necrosis
and the soil urease-inhibiting effect of nBTPTA and PPDA (see page 276). See also
Table 61.
In the field experiments conducted by Goodroad and Wilson (1989) in Georgia, the
effects of urea and urea-nBTPTA on the grain yields of sorghum were not apparently
different, because drought conditions were most likely to reduce yields.
Lamond et at. (1993, 1994, 1998) evaluated the effect of nBTPTA on no-till and
conventional-till continuous grain sorghum in Kansas. The N sources [urea, urea +
nBTPTA (Agrotain), and ammonium nitrate (AN)] were surface-broadcast and were
incorporated in the conventional tillage system.
All N sources performed similarly in the conventional tillage, but AN and
urea+nBTPTA often outperformed urea in no-till. For example, urea, urea+nBTPTA,
and AN, applied at rates of 0, 56, 112, and 168 kg N/ha on the no-till sorghum fields at
Belleville, produced the following grain yields (bu/acre): 76 (no N added), 102, 100,
and 107 (at 56 kg N/ha), 116, 118, and 129 (at 112 kg NIha), and 127, 129, and 128 (at
168 kg N/ha), respectively, in 1993, and 37 (no N added), 85, 88, and 90 (at 56 kg
N/ha), 107, 116, and 108 (at 112 kg N/ha), and 112, 118, and 118 (at 168 kg N/ha),
respectively, in 1994. Thus, the results indicated that the use of nBTPTA can improve
efficiency of urea when surface-broadcast in no-till production system.
partially not confirmed through the field experiments conducted by Gascho and Burton
(1987), in Georgia, on a loamy sand (pH 4.7) covered with an established sod of Tifton
44 hybrid bermudagrass. Eleven days before starting the experiments, the soil was
limed with 3.36 tlha of broadcast agricultural-grade dolomite, which resulted in the
increase of pH to 6.2 in the 0-5-cm soil layer but only to 5.0 in the 5-10-cm layer.
Three experiments were carried out over two years. N was applied at a rate of 336
kglha/year in the first and third experiment and 168 kglhalyear in the second one. In
each experiment, VAN served as N source. In the second experiment, urea alone and
NH 4 NO} alone were also used. ATS was added to VAN at a rate of 5%
(volume/volume). Besides the treatments with VAN and VAN + ATS, there were other
treatments, namely VAN + ATS + KCI, VAN + ATS + KCl + ammonium
polyphosphate (APP), and UAN + ATS + KCI + APP + CaCb. Solutions were applied
as a dribble on a spacing of 15 cm.
Four parameters were estimated: dry matter yield, percent N content and N removed
in dry matter, and relative N efficiency, i.e.. (N removed in dry matterlN removed in dry
matter when VAN was applied alone) x 100.
The estimations showed that in experiments 1 and 2 ATS added to VAN had a
favorable, statistically significant or insignificant effect on these paran1eters. In all
experiments, KCl, APP, and CaCl 2 added to VAN with or without ATS as well as ATS
added to UAN in experiment 3 did not influence the parameters or did diminish them,
but the diminutions were, in general, insignificant. Dry matter yield was significantly
higher with NH 4 N0 3 than with urea, but in the VAN treatment the dry matter yield did
not significantly differ from yields obtained with NH4 N0 3 alone and urea alone. At the
same time, in respect of the other parameters, differences between the VAN, NH4 N0 3 ,
and urea treatments were not significant. The authors consider that these results are
related to the reduced NH3 volatilization which was due, probably, to soil pH remaining
acid even after dolomite broadcast.
In a short report, Lamond and Bonczkowski (1989) summarized the results of 2-year
field experiments (1987 and 1988) initiated to evaluate the effect of VAN (at rates of 0,
67, and 134 kg Nlha) applied with or without ATS on bromegrass. Addition of ATS to
VAN produced inconsistent results but in some cases increased the yields.
In the field and greenhouse experiments conducted by Sloan and Anderson (1987,
1998) and referred to in Section 7.8.1, ATS added to urea at a rate of 10%
(weight/weight) was also tested. In the field experiments, urea+ATS performed no
better than urea alone. In the greenhouse experiments, in which two soils were studied
under a high and a low rainfall regime, ATS did not affect bermudagrass N use
efficiency for either soil or rainfall regime. On both soils, bermudagrass dry matter
yields in the urea+ATS and urea treatments were not significantly different under low
rainfall regime, but under the high rainfall regime ATS significantly decreased the
yields.
7. R. 5. Effect ofDithiocarbamates
Hyson (1963), who patented dithiocarbamates as inhibitors of soil urease activity (see
page 52), pointed out that the dithiocarbamate-amended urea fertilizer was applied by
broadcasting to areas of bermudagrass at a rate of 28-560 kglha with outstanding
beneficial results.
In the field experiments carried out in England, the same fertilizers and inhibitors
were applied on grasslands as in the pot experiments. Rate of fertilizer application was
301
50 or 100 kg N/ha, and that of inhibitors was 1% relative to urea-N. The inhibitors
increased the yields, but efficiency of urea + inhibitor seldom attained the efficiency of
AN. Similar results were obtained in field experiments performed in South Africa where
the fertilizers and inhibitors were applied to lovegrass plots.
Calancea et al. (1977) carried out pot and field experiments for studying the effect
of hydroquinone (HQ) and p-benzoquinone (BQ) on Italian ryegrass sown on a
chemozemic soil. The pots contained 1 kg of soil to which 1 kg of sand was mixed.
Each of the plots had a 30-m2 area.
In the pot experiments, in which urea-N rates were 200,600, and 1,800 mg/pot at a
constant HQ rate (30 mg/pot), ryegrass yield (dry matter of herbage), total N uptake by
plants, and coefficient of N utilization from urea tended to increase with increasing
urea-N rate. When urea-N rate was constant (200 mg/pot) and HQ rates were different
(60,90, and 120 mg/pot), the highest coefficient ofN utilization was recorded at the 90
mg/pot HQ rate, although yield and total N uptake showed a trend to increase with HQ
rate. These effects of HQ were very marked at the first harvest (cut) and less evident at
the second and third cuts.
Under field conditions, HQ was more efficient when urea was applied as a single
dressing (300 kg N/ha) than when it was administered as two or three divided dressings
(2 x 150 kg N or 3 x 75 kg N/ha). Thus, the coefficient of N utilization from urea
increased, due to HQ, by 18.4, 12.0, and 7.0%, respectively.
Of six treatments (100, 200, and 400 kg urea-N/ha + 0 or 1 kg BQ/ha), the best
results concerning coefficient of N utilization from urea were obtained with 200 kg
urea-N/ha + 1 kg BQ/ha.
In other pot experiments on the same chernozemic soil, Calancea et at. (1982) found
that HQ enhanced the uptake of N by ryegrass plants from both urea and glycoluril
(acetyleneurea; a slow-release N fertilizer), and this effect ofHQ was more marked with
glycoluril than with urea.
In the pot experiments described by Hera et al.( 1980), samples of a leached
chernozem and a brown forest soil were used. The soil (1 kg) and sand (0.5 kg)
mixtures were treated with 150 mg of urea, labeled with 15N (5.225 atom% excess), and
with 3 mg of HQ, then sown with ryegrass. Untreated mixtures and mixtures treated
with urea alone were the controls. After 6 weeks of ryegrass growth, it was established
that on the chernozem the yields increased by 57% in the urea + HQ treatment and by
31 % in the urea treatment as compared to the untreated control. The corresponding
values registered on the forest soil were 51 and 25%, respectively. Analysis of 15N in
plants indicated that uptake of urea-N was higher by 19% (chernozem) or by 23%
(forest soil) when urea was applied together with HQ than when urea was used alone.
All increases were statistically very significant.
A field experiment was also conducted on 30-m2 plots on the leached chernozem.
Urea was applied at rates of75, 150, and 300 kg N/ha with or without 2% HQ (on urea
weight basis). Under the influence of HQ, the ryegrass yield and N uptake from urea
increased by 30-37 and 15-20%, respectively.
The effect ofHQ on N uptake from urea and soil by ryegrass plants was also studied
in a pseudogleic podzolic soil (Calancea and Kiss, 1984). The pots contained mixtures
from 5 kg of soil and 1 kg of sand. Urea, 15N-Iabeled (5.225 atom% excess), was added
to the pots at a rate of 200 mg of N with or without 30, 60, 90, and 120 mg of HQ/pot.
These amounts are equivalent to 100 kg of urea-N/ha and to 15, 30, 45, and 60 kg of
302
HQ/ha. The soil-sand mixtures were sown with ryegrass, then moistened and kept at 65-
70% of WHC in greenhouse for 90 days, during which the herbage was harvested (cut)
three times, then analyzed for total Nand I'N contents.
One can see from Figure 78 that the ratio of N uptake from soil to N uptake from
urea was lower in the HQ-treated soil than in the untreated control during the whole
experimental period. The ratio increased linearly with the age of plants, and there was a
s,o
E
g JD
i
~ 2Sl
E
g
.l!
i 1.0
z
Figure 78. Elleet of hydroquinonc (IIQ) on the ratio of N uptake Irom soil to N uptake from urea by
ryegrass plants.
a - Control (no HQ), b- 30 mg HQ/pot. e - (i0 mg HQ/pot. d - 90 mg HQ/pot. e- 120 mg HQ/pot.
IFrom Calancea and Kiss (1984)./
parallelism between the increase of this ratio and the increase in the rate of HQ. The
ratio was higher than I, i.e., the plants took up more N from soil than from urea, except
in the lO-day-old plants growing in pots treated with 30 and 60 mg of HQ. These young
plants took up more N from urea than from soil, especially at 30 mg of HQ/pot. In
addition, of all treatments, that with 30 mg of HQ resulted in the lowest ratio between N
uptake from soil and N uptake from urea. Thus, the optimum rate for stimulation of N
uptake from urea was 30 mg of HQ/pot, i.e., 15 kg ofHQ/ha.
Gorelik et al. (1983) studied, in the 1979-1981 period, the effeet of hydroquinone
(HQ) on crop yield and coefficient of utilization ofurea-N in vegetation pots, eaeh filled
with 2.5 kg of soil. Details on the experiments are given below:
Rates of the urea and HQ, divided into 5 equal parts, were applied at intervals of
about 2 weeks on the soil surface immediately after each harvest (cut). Dry weight and
total N content of plants were determined.
The results showed that in the loamy-sandy podzol fertilized with urea at the higher
rate (1.5 g N/pot) and in sierozem, at both urea rates, HQ did not significantly affect the
efficiency of urea. When the loamy-sandy podzol was fertilized with urea at the lower
rate (0.75 g N/pot), the yields increased significantly under the influence of both HQ
rates. whereas the coefficient of utilization of urea-N by plants increased from 41 % (no
HQ) to 49% (1 % HQ). and 52% (5% HQ). In the sandy podzol under orchardgrass. HQ
increased significantly the yield only when urea was applied at the lower rate (0.38 g
N/pot) and HQ at the higher rate (5%); coefficient of utilization ofurea-N (35% in the
no HQ treatment) increased. under the influence of 1 and 5% HQ. to 40 and 48%,
respectively. In the sandy podzol under ryegrass. HQ was efficient in yield-increasing at
both urea rates; the coefficient of utilization of urea-N, in presence of 0, 1, 2, and 5%
HQ, had the following values: 40, 40. 45, and 48%, respectively, when urea rate was
0.38 g N/pot, and 36,41,44, and 49%, respectively. when urea rate was 0.75 g N/pot. It
appears evident from these results that HQ acted more efficiently in a light-textured
than in a heavier-textured soil.
Rodgers et al. (1984) studied the effect of HQ on the efficiency of urea in
experiments on ryegrass leys at Rotharnsted. HQ, added at a rate of 5 kglha to the
annual rate of 375 kg of urea-Niha applied as a single dressing in the 1981-1983 period,
increased the crop yield and N uptake by plants in 1981 and 1983, but did not affect
them in 1982. When HQ was added at a rate of 5 kglha to 375 kg of urea-Niha applied
annually in three equally divided dressings in 1982 and 1983, its effect on crop yield
and N uptake was depressive in 1982 and stimulating in 1983. HQ did not affect the
K/(Ca + Mg) ratio in the ryegrass plants.
The N fertilized used were: conditioned urea (prilled urea coated with mineral oil-
bitumen mixture), conditioned urea + 0.5 and 1% PPDA (on urea-N basis), and lime
ammonium nitrate. They were applied twice, at rates of 100 and 60 kg Nlha,
respectively. TIle plots were also fertilized with P and K (35 kg P and 100 kg K/ha). The
control plots received no fertilizers.
Drought was simulated by means of plastic tents (6 by 4.5 m) put up over the plots.
The simulated drought began after addition of both N rates and lasted 14 days. Dry
matter content of ryegrass hay and total N content in green matter of plants were
determined.
The data in Table 66 show that diminution of hay yield in urea treatment in
comparison with the lime an1monium nitrate treatment could be partially or completely
prevented by using urea+PPDA. The effects of the 0.5 and 1% PPDA rates were not
significantly different.
TABLE 66. Effect of nitrogen fertilizers on dry matter content of rye grass hay (mean values for
the 1977-1979 period)"
Dry matter content of ryegrass hay from cuts I + 2
Fertilizer
dvha 0/0
Unfertilized control 57.4 62
Lime ammonium nitrate 92.3 100
Co nditi on ed urea 87.6 95
Conditioned urea + 0.5% PPDA 93.7 102
Conditioned urea + 1% PPDA 90.7 98
LSD 5% 6.5 7
"Adapted from Karnpfeetal. (1982a).
The 14-day simulated drought after fertilization led to no significant changes in the
hay yields from cuts 1+2 as compared to that recorded under natural weather conditions.
Total N contents in green matter of plants were determined twice, namely at days 14
and 21 after application of each of the two N fertilizer rates. During the fust 14 days,
PPDA retarded N uptake by the plants. But at day 21 after N application, there were no
significant differences between the lime ammonium nitrate treatment and the urea + 0.5
and 1% PPDA treatments as concerns total N contents in green matter of plants. In the
1977-1979 period, neither were these contents influenced by simulated drought.
The experiments of Gorelik et at. (1983) for studying the effect of hydroquinone
(HQ) on orchardgrass and perennial ryegrass (see page 3(2) also comprised treatments
with PPDA used at a rate of 1% relative to weight of urea. Thus, in a heavy-textured
sierozem, the effect of urea-PPDA on crop yield (dry matter of aboveground parts of
orchardgrass) and on plant utilization ofurea-N did not differ from that of urea alone. In
the other two soils studied (loamy sandy podzol and sandy podzol), PPDA significantly
increased yields of orchardgrass and ryegrass, and the coefficient of plant utilization of
urea-N showed, under the influence ofPPDA, 4-13% increases. In comparison with HQ
also used at 1% rate, PPDA was more efficient.
Linke et at. (1983) synthesized the results of 69 field experiments on natural
grasslands in the 1974-1979 period. The same fertilizers were used, the same aspects
were studied, and the same methods were applied as in the cereal experiments (Karnpfe
et 01., 1982b) (see pages 271-272) and, essentially, similar results were obtained. The N
305
fertilizers were administered, for the first emergence of plants, at a rate of 60 kg N/ha
(in 1974 and 1975) or 100 kg N/ha (in the 1976-1979 period), and for the second
emergence at a rate of 60 kg N/ha (in each year). At the beginning of growing season in
each year, 30 kg P/ha and 100 kg Klha were also administered. The plots, from which
plants were collected for determination of hay yield (dry matter) and total N content,
were of 15 m2 •
Figure 79 presents the results concerning the influence of the amount of rainfall
during the first 5 days after fertilization on the hay yield. In the drought period, the hay
yield was much lower in the urea treatment than in that with lime ammonium nitrate.
PPDA improved, to some extent, the efficiency of urea. Between some limits of the
rainfall amounts, all urea fertilizers were more efficient than lime ammonium nitrate. In
comparison with the results obtained in the cereal experiments (see Figure 77), the 0.5%
PPDA rate had a weaker effect, as the yield recorded in the urea+0.5% PPDA treatment
was nearly the same as in the urea-only treatment. PPDA increased evidently the
/-- ......,
2.5
/ '
I "
I \.
I ,
/ \.
I .~ ,
(ij'
S
10
/ .~\
~ / ~.
j 0.0 ----r-
E :::.:---",.."
~
0 -1.0
'- a
o
-2.0
i i i
o 5 10 15 20 25 30
Rainfalls (mm)
Figure 79. Influence of rainfalls on the difference between hay yield (dry matter) recorded in the
treatment with lime ammonium nitrate and hay yields obtained in the treatments with urea with or
without 0.5 or 1% PPDA addition.
a - Conditioned urea. b - Conditioned urea + 0.5% PPDA. c - Conditioned urea + 1% PPDA.
/From Linke et al. (1983)./
efficiency of urea when its rate was I %. However, even the effect of this PPDA amount
disappeared at rainfalls ~ 27 IllIll. whereas in the cereal experiments the effect persisted
at rainfalls of about 33 mm.
The yield-increasing effect of 1% PPDA addition to urea fertilizer applied in these
field experiments on natural grasslands was also pointed out by Linke and Kampfe
(1987) in their communication presented at the International Fair Symposium held in
Leipzig.
In the field experiment carried out on a grass ley in 1984 and 1985 (Rodgers et aI.,
1987; see page 125), the dry grass yields from three cuts (t/ha) in the urea and
urea+PPDA treatments were 7.96 and 8.97, respectively (single application of 375 kg
306
urea-Nlha), and 8.50 and 8.58, respectively (divided applications of urea: 3 x 125 kg
Nlha), in 1984. In 1985 the corresponding values were 9.11 and 11.36, and 12.46 and
12.32, respectively. In both years, the yield-increasing effect of PPDA was great only in
the single application of urea. Similar results were obtained concerning N uptake by
plants.
Joo and Christians (1986) conducted experiments in 1984 at the Iowa State
University Turfgrass Research area north of Ames. The treatments of plots (2.32 m 2)
installed on blueglass turf on a fine loamy soil (PH 7.5) included urea solution (at 0,49,
and 98 kg Nlha) amended or not amended with 1 and 2% PPDA and 25% Mg2+ as
MgCb.6H20 (all percentages mean weights relative to weight ofurea-N). The fertilizer
solutions were surface-applied monthly in June through September. Clipping yields
were determined weekly for 5 weeks after each treatment.
The average fresh weight of clippings over a 5-week period in plots treated with 2%
PPDA increased 28% at the lower N rate in August, 31 % at the lower N rate and 20% at
the higher N rate in September. On overall average, 2% PPDA significantly (p<O.OI)
increased the clipping yields, whereas the effect of 1% PPDA was not significant
(p>0.05). The MgCl 2 treatment showed a 13 to 25% increase of the fresh clipping yield
at the lower N rate in August and September but a 20% decrease at the higher N rate in
August.
PPDA reduced foliar bum significantly at the lower N rate in September, but the
decreasing effect was not consistent in each month. In the heat of July, the MgCb
treatment resulted in greater foliar bum at the higher N rate, but it reduced bum at both
N rates under the relatively wet conditions and cool temperatures of September.
'.0<
Figure 80. Effect of nBTPTA on clipping yield of bluegrass in plots fertilized with liquid urea. /From
Joo et al. (1987),/
79 (PPDA), 80.7 (nBTPTA), 75.7 (ATS), 73 (KJ, and 72.3 (MiJ in 1986. In 1985 the
differences among amendments were small due to a very dry period in early summer
that restricted growth. In 1986 nBTPTA, PPDA, and ATS significantly increased the
yield, but ATS was less efficient than nBTPTA and PPDA. K+ and Mi+ had no
significant effect on the yield.
The investigations on the effect of urease inhibitors on the bluegrass turf were
continued (Joo et al., 1991 b, 1992, 1993).
Joo et al. (1991b, 1993) described the investigations carried out in 1987. The 2.32-
m 2 plots were treated with liquid urea (49 kg N/ha) amended with nBTPTA at rates of 0
(urea alone), 0.25, and 0.5% of the weight of urea-No Urea labeled with 5% 1~ was
applied at the center of each plot to an area measuring 0.2 m 2 • The rest of the plot
received the same rate of urea-N minus the 15N label. Clippings were collected weekly
at a 5-cm mowing height for 5 weeks; then shoots, thatch, and soil (0-7.5- and 7.5-
15-cm layers) were sampled and analyzed for total N and 15N.
During the 5-week experimental period, the N recovered in the five clippings
represented 7.5% of the added urea-N in the urea-only treatment and 7.9 and 8.1% in
the treatments with urea+0.25% nBTPTA and urea+0.5% nBTPTA, respectively. Total
N recovery (i.e.. recovery in clippings, shoots, thatch, and soil) was 28.8% (urea), 45%
(urea+0.25% nBTPTA), and 36.1% (urea+0.5% nBTPTA). Thus, N recovery in
clippings was highest with 0.5% nBTPTA and total N recovery with 0.25% nBTPTA.
The field experiments carried out in 1988 and 1989 were described by Joo et al.
(1992). Granular (and not liquid) urea was applied to plots (1.92 by 1.92 m) at rates of
0,49, and 73.5 kg N/ha (in 1988) and at rates of 0,24.5, and 49 kg N/ha (in 1989). In
these years, the averaged dried clipping weights of bluegrass were not significantly
different in the urea-only and urea+nBTPTA treatments, irrespective of the rates of urea
and nBTPTA. These findings are in contrast to the results of laboratory experiments of
Joo et al. (1992), as in these experiments nBTPTA significantly decreased loss of urea-
N through ammonia volatilization (see page 210). It was assumed that lack of yield
response to nBTPTA was due to other loss mechanisms and immobilization of urea-N
in the soil-bluegrass plant system.
308
The orchardgrass pasture experiments conducted by Bundy and Oberle (1988) are
referred to on page 201. In these experiments, urea (67.2 kg N/ha) with or without
urease inhibitors (2%) was surface-applied at Lancaster in 1983 and/or 1984, and,
besides ammonia volatilization, the orchardgrass dry matter yield and N uptake were
also assessed. In 1983 the significant reductions in NH3 losses due to addition of PPDA,
diethyl-PDA, and trichloroethyl-PDA to urea prills resulted in significantly higher dry
matter yields. However, only the urea-PPDA treatment showed significantly higher N
uptake than urea. In 1984 nBTPTA, which was more effective than PPDA in reducing
NH3 losses from urea, increased significantly (at p=0.1 level, but not at p=O.05 level)
the yield and N uptake. The effect of PPDA on yield and N uptake was insignificant
even at the p=O.llevel.
For comparing the effects of nBTPTA, PPDA, and hydroquinone (HQ) on perennial
ryegrass, Li et af. (1993) carried out a greenhouse experiment. Air-dried soil samples (1
kg) were placed in plastic pots (with a height of 12 cm and a diameter of 15 cm). The
soil moisture content was brought up to 20% on weight basis and kept constant by a
spray of water each day. Ryegrass seeds (200) were broadcast evenly in each pot and
covered with a thin layer of soil. Two days after germination, 100 mg urea-N labeled
with 15N, amended or not amended with urease inhibitors in solution was added to each
pot (rate of inhibitors is not mentioned in the paper). At days 15 and 30 after
fertilization, the grass shoots were harvested and their dry weight, total N and 15N
contents determined. The roots and soil were also analyzed for total N and 15N after
both harvests.
The results indicated that the dry matter weights of grass shoots were not
significantly different in the urea-only and urea+nBTPTA or urea+PPDA treatments at
any harvest. In contrast, the yields obtained at the first harvest were insignificantly
higher and those registered at the second harvest were significantly lower in the
urea+HQ than in the urea-only treatment. During the first 15 days of growth, the plant
uptake of N from urea was not significantly different among the treatments, but during
the second 15-day period of growth the urea+nBTPTA treatment, in comparison with
the other treatments, induced a significant (10%) increase in plant uptake ofurea-N.
In the field experiment of Watson et at. (1990a,b) (see page 150), the ryegrass plants
were harvested approximately 7 weeks after surface application of urea, urea+0.5%
nBTPTA, and calcium ammonium nitrate (CAN) at a rate of 100 kg N/ha. Herbage dry
matter yield of ryegrass (t/ha) was 0.644 (in unfertilized plots), 2.991 (in urea plots),
3.255 (in urea+0.5% nBTPTA plots), and 3.278 (in CAN plots). It is evident that the
yield performance ofurea+0.5% nBTPTA was comparable to that of CAN.
Watson et af. (1994a) studied the effect of different levels of nBTPTA in urea
granules on the herbage dry matter yield of ryegrass. A field experiment was carried out
in parallel with that for studying ammonia volatilization (see page 150). But the plots
were a little smaller (1.5 by 1.5 m), and unfertilized and CAN-fertilized plots were also
used. The ryegrass plants were harvested after 6 weeks of regrowth in plots installed for
each of the time periods I to 5. The dry matter yield (t/ha) in the differently treated plots
had the following average values: 2.02 (unfertilized), 4.57 (urea), 4.75-4.98
(urea+nBTPTA), and 4.82 (CAN). In the urea+nBTPTA-treated plots, the highest
yields: 4.98 and 4.87 t/ha were recorded at the 0.05 and 0.01% nBTPTA levels,
respectively, and the lowest ones: 4.75 and 4.77 tlha at the 0.1 and 0.5% nBTPTA
309
levels, respectively. In other words, the lower levels of nBTPTA affected more
positively the dry matter yield than did its higher levels.
As the growing season progressed, the yield decreased from 7.47 tlha (in time period
1) to 1.93 tlha (in time period 5).
In another experiment carried out in parallel with that mentioned above, 15N-labeled
urea granules (at approximately 18 atom% excess 15N) with or without nBTPTA (at five
levels) were applied to microplots (50 by 50 cm). Total N and 1~ in soil and plant were
determined and the 15N recovery was calculated.
nBTPTA increased inSignificantly the 15N recovery in soil at all depths down to 15
cm, but caused a significant increase in percent N derived from urea in the shoot and the
shoot recovery of 15N. Total 15N recovery in the soil-plant system was increased by up
to 17% by incorporating nBTPTA into urea granules. All these increasing effects of
nBTPTA were similar at its five levels.
In these experiments, the contents of macronutrients (P,K, Na, Ca, Mg, S) in dry
matter were not significantly different between the N treatments, and there were no
visual signs of phytotoxicity (leaf tip scorch) on any treatment.
A 3-year study was initiated by Joost (1995) in 1992 to compare the effectiveness of
ammonium nitrate, urea, and urea+O.l4% nBTPTA applied to caucasian bluestem in
Missouri. Rates offertilizers were 0, 78, 156, and 234 kg NlhaJyear, as either a single or
in two equal split applications. Forage dry matter yield and total N uptake of plants
were significantly lower in response to urea than either NH4N0 3 or urea+nBTPTA.
Applying half of the total N in the spring and half after the first harvest resulted in
significantly higher July dry matter yield and crude protein content in 1992 and 1993,
but regrowth was insufficient to produce a July harvest in 1994 due to drought
conditions.
Lamond et al. (1996) conducted field experiments on established bromegrass.
Nitrogen fertilizers (ammonium nitrate, urea, urea+nBTPTA, urea-ammonium nitrate
solution) were applied at rates of 0, 50, and 100 kg Nlha in mid to late March.
Ammonium nitrate and urea+nBTPTA consistently produced higher yield and often
higher forage protein levels than urea or urea-ammonium nitrate.
Watson and Miller (1996) studied the short -term plant physiological effects not only
of nBTPTA-amended urea but also of nBTPTA used without urea. The test plant was
perennial ryegrass. Four experiments were carried out.
In Experiment 1, field-moist samples (380 g at 21 % H2 0, weight/weight) of a clay
loam soil (pH 5.7), collected from under a permanent grassland sward at Baillies Mills,
County Down, Northern Ireland, were placed in pots (10 cm diameter, 9 cm depth). A
solution containing 20 mg N (as NH4 N0 3 ), 10.3 mg P (as KH 2 P0 4 ), and 39.3 mg K (13
mg K as KH 2 P0 4 and 26.3 mg K as K2 S04 ) was added to the soil in each pot; then four
I-week-old ryegrass seedlings were planted into the soil. The soil was maintained at
80% field capacity with distilled water. The plants grew under natural lighting
conditions in a greenhouse. After 6 weeks of growth, the soil was again fertilized as
before the planting. After 4 weeks, only 20 mg of NH4 N0 3 was added, and after a
further 4-week period, 10.3 mg P, 39.2 mg K, and 15N-labeled urea granules (the 15N
enrichment being 18 atom% excess 15N) at a rate of 78.5 mg N/pot (equivalent to 100
kg Nlha) were uniformly applied to the soil surface. The urea granules contained no or
0.01,0.1 or 0.5% nBTPTA (weight/weight). At intervals of 0.75, 1.75,4,7, and 10 days
after urea fertilization, the content of each pot was separated into three fractions: shoot,
310
root, and soil. The analyses to which the three factions were submitted and some of the
results obtained are briefly summarized below.
Urease activity in shoot significantly (p<0.05) decreased in the urea treatment over
the 10-day period. In the urea+nBTPTA treatments, shoot urease activity was strongly
reduced at day 0.75 compared with the urea treatment. Thus, during 0.75 days in the
urea+0.5% nBTPTA treatment, shoot urease activity was reduced by 90.2% compared
with 0.15% in the urea treatment. The time taken for shoot urease activity in the
urea+0.01, 0.1, and 0.5% nBTPTA treatments to return to that in the urea treatment was
4,7, and 10 days, respectively. Urease activity, which was higher in root than in shoot,
was not affected by nBTPTA.
15N recovery of urea in the shoot at days 7 and 10 was significantly lower (P<O.Ol)
than that ofurea+nBTPTA. 15N recovery in root was similar to that in shoot. At day 10
total 15N recovery (shoot + root + soil) of urea was significantly lower (P<0.05) than
that ofurea+nBTPTA.
After l.75 days, the residual urea-N in soil was 1.3 and 62% of the N applied in the
urea and urea+0.5% nBTPTA treatments, respectively. However, the effect of nBTPTA
was short-lived. Thus, after 4 days, less than 1% ofN applied was as urea with 0.01 and
0.1 % nBTPTA and only 6.8% with 0.5% nBTPTA. Urea and mineral N levels in the
shoot and root were very low or negligible during the whole 10-day period.
There was no significant difference in soluble protein and water-soluble
carbohydrate contents in shoot and root between urea and urea+nBTPTA. However,
nBTPT A produced significant changes in composition of free amino acids in shoot and
root, suggesting a difference in metabolism.
Experiment 2 was a field experiment which ran concurrently with Experiment 1.
The site was an established perennial ryegrass-white clover sward on a clay loam soil
(pH 6.3) at the Agricultural Research Institute, Hillsborough. The plots (1.5 by 1 m)
received 100 kg N/ha as unlabeled urea, urea+O.1 or 0.5% nBTPTA or calcium
ammonium nitrate as well as P and K fertilizers. The plots were harvested at intervals of
2, 4, 8, 14, and 30 days after N fertilization. The same analyses were made with
generally similar results as in Experiment 1.
However, in the field experiment, contrary to the pot experiment, substantial levels
of urea were measured in the shoot at days 2 and 4 following application of urea+O.l
and 0.5% nBTPTA.
In Experiment 3, nBTPTA was applied alone in a pot experiment similar to
Experiment 1. After 4-week growth of ryegrass seedlings, the soil in pot was treated
with 40 rn1 water or 40 ml aqueous solution containing 0.055, 0.55 or 2.77 !1g
nBTPTA/g oven-dry soil. Urease activity was determined in the three fractions (shoot,
root, and soil) at days 0.75,1.75,4,7, and 10 after nBTPTA application.
Urease activity in each fraction was significantly reduced by all concentrations of
nBTPTA compared to control (no nBTPTA) at day 0.75 and appeared recovered to that
of control by day 10.
In Experiment 4, the effect of urea and urea+nBTPTA on leaf tip necrosis was
studied in a pot experiment similar to Experiment 1. After 4-week growth of ryegrass
seedlings, the soil in pots was N-fertilized at rates of 0,23.9,47.8, and 95.6 mg N/pot as
urea amended with nBTPTA at 0.1 or 0.5% (weight/weight) applied in 15 rn1 solution.
Leaf tip necrosis (scorch) on living leaves was measured 15 days after fertilizer
application.
311
Leaf tip necrosis increased with increasing urea application rate and nBTPTA
concentration. Thus, increasing the urea-N applied from 23.9 to 95.6 mg N/pot
(equivalent to an increase from 50 to 200 kg Nlha), in the absence of nBTPTA, resulted
in a significant increase in the average tip damage per leaf from 0.92 to 3.04 mm.
Increasing the concentration of nBTPTA to 0.5% led to markedly increased leaf tip
necrosis, particularly at the high urea-N application rate. New developing leaves
showed no visual sign of tip necrosis.
The effect of nBTPTA-amended urea to cause some leaf tip necrosis, like its effect
to inhibit shoot urease activity, was transient and short-lived. Therefore, the conclusion
was drawn that the benefit of nBTPTA in reducing ammonia volatilization from urea
and, thus, in increasing dry matter production of ryegrass would appear to far outweigh
any of the observed negative, short-term effects.
In the 3-year field experiment conducted by Watson et af. (1998) (see page 152),
approximately 6 weeks after each fertilizer application, the central part of each plot was
harvested, and the herbage dry matter yield and N uptake by perennial ryegrass were
determined.
The results showed that repeated application of nBTPTA on the same soil exerted no
adverse effect on grass production.
Total yields of the three harvests in the PU, NCCU, and NOCU treatments with the
60 and 120 kg Nlha/year had the following average values for biomass yields (tlha):
44.9 (PU), 58.4 (NCCU), and 58.6 (NOCU), and for essential oil yields (llha): 305.3
(PU), 413.1 (NCCU), and 407.3 (NOCU). Both biomass and essential oil yields and N
use efficiency of lemongrass were significantly (p<0.05) higher with coated ureas than
with uncoated urea, while the difference between the effects of the two coated ureas was
not significant. The contents of citral and geraniol in essential oil was not influenced by
rate and nature of N fertilizers.
TABLE 67. Effect of bydroquinone and nitrapyrin on ryegrass yield and coefficient of N utilization
from urea"
Average Total N Coefficient ofN Difference
Treatment" herbage yield uptake utilization from urea
~~~t2 ~m~~t) ~%2 ~%2
No (control) 5.64 98.7
Urea 9.03 157.4 37.2
Hydroquinone (HQ) 6.56 114.2
Urea + HQ 9.76 180.3 50.7 +13.5
Urea + nitrapyrin (NP) 9.77 184.9 45.9 + 8.7
Urea + HQ+NP 10.05 189.0 51.4 +14.2
"From Calancea el ai. (1977); see also Hera and Eliade (1981).
b Rates of additions per pot were: urea 100 mg N; HQ 2 mg; nitrapyrin 2 mg.
coefficient of N utilization from urea were registered when HQ and nitrapyrin were
applied together.
For studying the effect of BQ, the soil in pots was treated with 1 g of urea + or
0.01,0.1 or 1% BQ (relative to urea-N) or 0.1% BQ + 0.1% sulfathiazole. The urea +
°
0.01 % BQ treatment led to increased yield, N uptake by plants and coefficient of N
utilization from urea, but BQ at higher rates and, especially BQ + sulfathiazole, proved
to be phytotoxic.
The urea+DCD+ATS fertilizer patented by Sutton et al. (1991) (see page 244) was
applied on turf at rates of 40,80, 120,and 160 kg ofurea-Nlha. For comparison, urea (at
the same four rates) as well as ammonium nitrate, commercial fertilizers Sulfur Kote
and Osmocote (at 80 kg Nlha) were used. Color ratings were estimated for 78 days after
fertilizer application, and it was found that the patented fertilizer provided a greener turf
over a longer period of time than did the other fertilizers.
The legume species studied include alfalfa (Medicago sativa), broadbean (Vicia [aba),
common bean (Phaseolus vulgaris), pea (Pisum sativum), red clover (Trifolium
pratense), soybean (Glycine max), and yellow lupine (Lupinus luteus).
The effect of PMA on germination of alfalfa seeds was also studied by Bremner and
Krogmeier (1990) (see page 277 and Table 61).
amounts of urea rather than formation of toxic amounts of ammonia in leaves and b) is
accordingly increased rather than decreased by addition of a urease inhibitor such as
phenylphosphorodiamidate (PPDA) to the urea fertilizer applied. It was also proved by
Krogmeier et al. (199Ia,b) that the leaves of soybean plants grown on media deficient
in Ni (which is an essential component of the urease molecule) have a lower urease
activity and, consequently, these plants are more susceptible to leaf tip necrosis when
they are foliar-fertilized with urea. Their susceptibility would be even higher after foliar
application of urea with a urease inhibitor.
Nevertheless, the potential of urease inhibitors for inducing phytotoxicity should not
preclude their use as additives to urea fertilizers, because the ammonia produced
through hydrolysis of urea by soil urease is much more detrimental to plant growth than
is urea accumulation induced by them (Krogmeier et al., 1989a; see also page 276).
The effect ofphosphorodiamidic acid, PPDA, and 4-chloro-PPDA on germination of
alfalfa seeds was studied by Bremner and Krogmeier (1990) (see page 277 and Table
61).
Many plants were studied: cotton (Gossypium hirsutum), cucumber (Cucumis sativus),
geranium (Pelargonium graveolens), Japanese mint (Mentha arvensis), lettuce (Lactuca
sativa), oilseed-rape (Brassica napus), onion (Allium cepa), potato (Solanum
tuberosum), pumpkin (Cucurbita pepo), radish (Raphanus sativus), sugarbeet (Beta
vulgaris cv. altissima), sugarcane (Saccharum ojJicinarum), sweet potato (Ipomoea
batatas), tobacco (Nicotiana tabacum).
p=0.05) in refined sugar yields (t/ha), namely to changes from 7.61 (urea alone) to 8.14
(urea + KCl at the lower rate) and to 7.38 (urea + KCl at the higher rate).
Hallmark et al. (1996, 1998) carried out investigations to determine whether the
product N-HIB Ca (composed of 12% Ca as CaClz, 1.5% Mg as MgClz, and a urease
inhibitor) can increase N use efficiency and sugarcane yields. The results showed that
adding 45 kg Calha as liquid N-HIB Ca to 135 kg N/ha of liquid urea increased sugar
yields by 2.95 tlha across four years (1991-1994). This compares with a 2.25 tlha sugar
response from increasing N from 135 to 200 kg N/ha across four years where calcium
was not added.
7. J O. 3. Effect of Fluorides
In the pot experiments of Gaponyuk and Kuznetsova (1984), urease activity was not
inhibited in soil samples treated with NaF at rates of 0.1-3 g F/kg soil (see page 34), but
root growth of cucumber and pumpkin plantlets was strongly reduced at rates ~ 1.5 g
F/kg soil.
experimental plots. For example, in the 1984-1985 experiment, in plots fertilized with
50 kg ofurea-N + 0 or 3 kg ofHQlha applied to seedbed and with 75 + 75 kg ofurea-N
+ 0 or 5 + 5 kg of HQlha given as spring dressings, the following yield data were
recorded: seed yield (tlha at 90% dry matter): 2.40 (urea alone) and 2.72 (urea + HQ);
oil yield (tlha): 0.83 and 1.01; protein yield (tlha): 0.45 and 0.52, respectively.
Arziani et al. (1985) proved that the axenic cultures of pumpkin seedlings take up
the exogenous 14C_HQ and inactivate it by glycosylation and conjugation with peptides,
glycosylation being the principal inactivating reaction.
Sweet potato fields were amended by Xue et at. (1991) with 10 kg of urea + 5 kg of
KCl + 0, 50, 100 or 150 g of HQ or quinhydrone (QH) per mu (1/15 ha). The yield
increases were 17.6, 25.3, and 31.3%, and 17.3,31.2, and 40.2% in the treatments with
the three increasing rates ofHQ and QH, respectively.
but the nitrate content in plants (mglg fresh weight), which was 2.182 in the urea-only
treatment, was reduced insignificantly to 2.143 by nBTPTA and significantly to 1.650
and 1.736 by DCD and nBTPTA+DCD, respectively.
321
A primordial requirement with which the inhibitors of soil urease actlVlty should
comply is the specificity: they should inhibit urease activity effectively without
exhibiting negative side effects on the soil life; they should not affect the other
enzymatic activities, the microorganisms and microbial processes playing an important
role in the biological cycles of elements and in soil fertility.
more concentrated salt solutions. The increasing effect of Na2S04 was more marked
than that of NaCl. At the same time, both salts inhibited ~-glucosidase, BAA-
hydrolyzing protease, and phosphatase activities. NaCI was more inhibitory than
Na2S04. The degree of inhibition increased with increasing salt concentrations. The
inhibition caused by NaCl reached 40% (~-glucosidase), 56% (BAA-hydrolyzing
protease), and 75% (phosphatase).
days of incubation. Amylase activity determined after I and 3 days of incubation was
not significantly affected by either 5 or 10 Ilg maneb/g soil (Tu, 1982).
From the results presented in Table 69, one can see that urease activity decreased
greatly in the presence of HQ. In the leached chernozem, there was a parallelism
between the activity decrease and the rate of HQ. In the alluvial soil, the strongest
inhibition was produced, surprisingly, not by the highest HQ rate (0.120 mglg soil) but
by the 0.048 mg rate.
Actual dehydrogenase activity showed a trend to decrease with increasing HQ rate
in both soils. The HQ rates of 0.012-0.048 mglg soil enhanced potential dehydrogenase
activity in the leached chemozem but had an opposite effect in the alluvial soil. At the
same time, the 0.120 mg HQ rate markedly diminished potential dehydrogenase activity
of both soils. HQ did not bring about any changes in catalase activity of the two soils
studied. Invertase activity measured in the presence of buffer solution remained
unchanged in the HQ-treated samples of the leached chernozern but decreased, to some
extent, in similarly treated samples of the alluvial soil. When invertase activity was
measured without buffer, the activity values were a little lower in the HQ-treated
samples of chernozern than in the untreated sample, but the activity was practically
identical in all samples of the alluvial soil. Phosphatase activity in the chemozem was
not significantly influenced by any of the HQ amounts applied. In the alluvial soil, the
0.012-0.048 mg HQ rates did not affect phosphatase activity, while the highest rate
slightly increased it.
The conclusion can be drawn that, besides the inhibitory effect on urease activity,
HQ also manifested inhibitory or stimulatory effects on other enzymatic activities,
depending on its rate, soil type, and experimental conditions. However, all these side
effects of HQ were less marked than its strong inhibitory effect on soil urease activity.
Reddy and Chhonkar (1990a,b) studied the effect of three dihydric phenols and five
quinones on the nitrate reductase activity in a sandy clay loam (PH 7.6). Reddy and
Chhonkar (1990a) conducted a pot experiment in which hydroquinone, added to 3-kg
soil samples at a rate of 2 mglkg soil, decreased nitrate reductase activity. Reddy and
Chhonkar (1990b) determined the inhibition constant of the eight compounds, each
having been applied at rates of 1 and 2 !1g1g soil. The constants ranged from 5.3 to
12.7 x 10.9 M and their values (x 109 M) presented the following descending order:
catechol (12.7), l,4-benzoquinone (9.7), hydroquinone (9.6), l,4-naphthoquinone (9.5),
4-methy1catechol (8.9), 2-methyl-l,4-naphthoquinone (7.2), 2,6-dibromoquinone-4-
chloroimide (5.4), and 2,6-dichloroquinone-4-chloroimide (5.3).
Zhao and Zhou (1991) and Zhao et al. (1991) performed a long-term laboratory
experiment to study the effect of HQ applied with urea on several enzyme activities in a
meadow brown soil (silty loam, pH 6.55). Soil samples (100 g) were submitted to the
following treatments: no urea and no HQ; urea alone (480.8 mg); urea + HQ (0.96, 2.0,
4.81 or 9.61 mg). The moisture content was adjusted to 40% of WHC. Incubation took
place at 30°C. After 3, 10,25,55, and 88 days of incubation, subsamples were taken to
run specific enzyme assays.
The results showed that HQ had no effect on invertase activity but temporarily
promoted or inhibited the activities of polyphenol oxidase, dehydrogenase, protease,
and phosphatase through its direct effect on these enzymes or indirectly through causing
a delay in urea hydrolysis and changes in soil microbial activity. With time, the
promoting and inhibitory effects gradually decreased with no significant difference at
the end of incubation.
TABLE 69. Effect of hydro quinone on enzymatic activities and respiration of soil"
En~matic activities" Res2iration'
Hydroquinone
Soil Deh:tdrogenase Invertase With added Without added
(J.lglg soil) Urease Catalase Phosphatase
Actual Potential With buffer Without buffer glucose glucose
0 2.30 0.30 0.40 42.25 1.10 0.74 2.05 80.90 16.97
0.012 0.75 0.17 0.66 43.97 1.13 0.65 2.22 90.71 21.48
Leached
0.024 0.71 0.27 0.44 43.97 1.09 0.66 2.09 87.79 17.77
chernozem 2.19
0.048 0.52 0.25 0.50 45.69 1.18 0.47 81.69 9.54
0.120 0.27 0.09 0.31 43.28 1.13 0.60 2.19 63.12 10.07
0 1.89 0.42 0.89 40.54 1.05 0.64 0.85 55.44 30.58
0.012 0.70 0.34 0.71 38.48 0.71 0.61 0.84 77.44 19.14
Alluvial soil 0.024 0.63 0.37 0.67 38.13 0.56 0.65 0.87 77.88 21.98
0.048 0.00 0.30 0.85 40.88 0.74 0.63 0.93 102.08 26.62
0.120 0.63 0.22 0.47 38.82 0.72 0.61 1.05 206.58 26.84
"From Radulescu et al. (1984).
bEnzymatic activities are expressed as follows: urease in rng of ammonia produced by 5 g of soil during a 24-hour incubation at 37°C; dehydrogenase activity
in rng oftriphenylforrnazan/3 g of soill24 hours at 37°C; catalase in rng ofH20 212.5 g of soil! 1 hour at 20°C; invertase in difference of optical rotation (ilu°)/lO
g ofsoil/24 hours at 37°C; phosphatase in rng ofphenoll2.5 g ofsoill2 hours at 37°C.
'Respiration is expressed as rng of CO,!50 g of soil!7 days at 20°C.
w
N
-...I
328
* *
*
Hickisch (1981) described the effect of 15 urease inhibitors (without giving their
names) on urease and dehydrogenase activities in a sandy soil. The fresh soil samples
(250 g in a 5-cm layer) were treated with standard (practical) dose of inhibitors, then
incubated at 25°C for 3 days. Untreated samples served for comparison. Incubation was
followed by determination of the enzyme activities. As expected, each of the 15
compounds inhibited urease activity; degree of inhibition varied between 23 and 80%
(average: 50.6%). Dehydrogenase activity was inhibited by 7 urease inhibitors (degree
of inhibition: 1-17%; average: 6.1%), stimulated by other 7 urease inhibitors (degree of
stimulation: 2-11 %; average: 5.1 %), and unaffected by one urease inhibitor.
Kozdr6j (1995) determined urease activity in 200-g samples of a forest soil treated
with I or 2 mg Cu or Cd (as sulfates) (see page 17) and also the numbers of bacteria and
fungi tolerant to 0.1 or I mg of Cu or Cdil nutrient medium. Urease activity did not
correlate with the number of tolerant bacteria, but it correlated significantly and
positively with the number of fungi tolerant to 0.1 mg Cd when the soil sample was
treated with 1 mg Cd. The correlation remained significant but became negative when
the soil sample received 2 mg Cd.
In the complex study of the effects of Cr(VI) on soil biological properties, Speir et
af. (1995) determined urease, phosphatase, and arylsulfatase activities (see pages 16 and
322) and other parameters, including microbial biomass C, and it was found that
sensitivity of biomass C to Cr(VI) was similar to that arylsulfatase activity and higher
than those of phosphatase and urease activities.
Hemida et af. (1997) amended samples of a clay soil and a sandy soil with 0, 200 or
2,000 Ilg Cu or ZnJg soil. After L 4, and 12 weeks of incubation, the soils were
analyzed enzymologically (see pages 17 and 322) and also microbiologically.
The results obtained after 12 weeks will be summarized below. The number of
glucophilic fungi was not affected significantly (p=0.05) by Cu and Zn in the clay soil,
but it was increased in the sandy soil amended with 2,000 Ilg ZnJg soil. The number of
thermophilic and thermotolerant fungi was not affected by Cu and Zn in either clay or
sandy soil. The number of cellulose-decomposing fungi was reduced by both rates of
Cu and Zn in the clay soil and not affected in the sandy soil. The number of bacteria did
not suffer significant changes due to Cu and Zn in any soil. The number of
actinomycetes did not change significantly in the clay soil, but it was increased in the
sandy soil at both 200 and 2,000 Ilg ZnJg soil rates.
Potassium dichromate applied at rates of 80 and 120 mg Cr/kg soil, in the pot
experiments of Wyszkowska et al. (2001) (see page 19), adversely affected growth of
Azotobacter sp. and actinomycetes but stimulated the proliferation of oligotrophic,
copiotrophic, anl!llonifying, and Nrfixing bacteria.
(p=0.05) the number of these bacteria at both rates. Number of fungi, determined after 1
and 2 weeks of incubation was significantly decreased by both rates of maneb for 1
week, but no significant effect was recorded after 2 weeks.
Lower maneb rates (5 and 10 Ilg/g soil) were applied by Tu (l981a) to samples of a
clay loam soil (pH 7.2). The incubation lasted 2 or 7 days. After 2 days, decreased
number of bacteria was found at the higher maneb rate and decreased number of fungi
at both maneb rates. After 7 days, no significant effect of maneb was recorded. The
number of non-symbiotic Nrfixing bacteria was not significantly affected by maneb
(except in a single case: the number of these bacteria was higher at the lower maneb rate
after 7 days of incubation).
be mentioned that comparison of the plots that received urea alone with the untreated
control plots revealed that urea addition led to a significant increase in counts of soil
microorganisms from all groups studied.
In the wheat experiment carried out under field conditions by Kucharski (1992) (see
page 273), PPDA had no visible effect on total number of heterotrophic soil bacteria,
number of Azotobacter cells, actinomycetes, proteolytic, and cellulolytic micro-
organisms, but slightly stimulated the proliferation of fungi.
In the laboratory experiments of Kucharski (1994) (see page 129), the results
obtained concerning the effect of PPDA on the munber of heterotrophic soil bacteria,
actinomycetes, proteolytic and ammonifying microorganisms, and fungi indicated no
regularity: PPDA had no effect or exerted an inhibitory or stimulatory effect depending
on duration (2 up to 25 days) and/or temperature (l0, 20 or 30°C) at which the urea-
and urea+PPDA-treated samples were incubated.
activities was studied by Builov et al. (1979) (see Section 8.1.3). Numbers of
heterotrophic bacteria were determined I, 2, and 3 years after ALS application to the
solonetz soil and 1 year after ALS application to the compact meadow soil. The
numbers (x 106/g soil) in the solonetz soil, untreated and treated with 1.3 and 1.7%
ALS, were the following: 18.2,31.8, and 54.3 (after 1 year), 6.8, 70, and 64 (after 2
years), and 126, 157, and 148 (after 3 years), respectively. The numbers recorded 1 year
after ALS application to the compact meadow soil were: 57.6 (untreated), 77.7 (treated
with 0.3% ALS), and 57.7 (treated with 1% ALS). Thus, in the solonetz soil the 1.7%
ALS rate was more efficient than the 1.3% rate 1 year after its application, but the
reverse was true 2 and 3 years after ALS application.
* *
*
Muller and Hickisch (1979) studied three urease inhibitors, marked by A, B, and C
(as they were not nominalized). Two experiments were carried out, both with a black
earth from Germany.
In the first experiment, fresh soil samples (50 g) were treated only with an inhibitor
at a rate of 1% relative to weight of urea-N if urea had been applied at a rate of 500 kg
N/ha. The next steps were incubation at 25°C for 7 days and, then, determination of the
counts of all bacteria, proteolytic, cellulolytic, and nitrifying bacteria as well as total
fungal mycelial mass and mycelial mass of penicillia, mucoraceae, and fusaria.
The microbiological analyses showed that inhibitor A significantly decreased the
count of proteolytic bacteria and the mycelial mass of mucoraceae and insignificantly
the count and mass of the other microorganisms, except mycelial mass of penicillia
which increased and count ofnitrifiers which remained unchanged. Under the influence
of inhibitor B, there was an insignificant decrease in the counts of bacteria and a
significant one in mycelial mass of all fungi. Inhibitor C did not bring about any
significant changes in bacterial counts and fungal biomass.
The second experiment was carried out in pots, each containing 5 kg of soil.
Inhibitors A and C were applied with or without urea four times, namely before
incubation and after 4, 8, and 12 weeks of incubation at 18°C. Each of the four
applications consisted of 0 or 200 kg urea-N/ha + I % inhibitor relative to N. During
incubation, total count of bacteria was determined periodically (in total, 20 times).
Inhibitor A had, also in this experiment, a decreasing effect on total bacterial count.
This effect became more and more pronounced during the incubation but it was
attenuated when urea was also applied. Inhibitor C led to an increase in total bacterial
count, and the increase was enhanced by urea application. In conclusion, inhibitor A
does not and inhibitor C does correspond for use in practice as a soil urease inhibitor.
Hickisch (1981) studied the effect of 15 urease inhibitors on counts of bacteria and
fungi in a sandy soil, under experimental conditions identical to those mentioned on
page 329. The results showed that growth of bacteria was inhibited by 10 urease
inhibitors (5-70% inhibitions; average: 24.8%) and stimulated by 5 urease inhibitors (2-
33% stimulations; average: l3.8%). Nine urease inhibitors inhibited and six stimulated
the growth of fungi; the degree of inhibition was 2-82% (average: 12.0%) and the
stimulation ranged from 4 to 36% (average: 10.0%). It was emphasized that under field
conditions even a 60% inhibition of microbial growth in the inhibitor-affected soil
334
higher in both BQ-treated samples (103 and 126 J1l O2 consumedlg soil, respectively)
than in the untreated control (75 III O2 consumed/g soil). In other words, BQ stimulated
the soil respiration.
same in each soil treated with alanine with or without addition of phosphoroamides.
This means that the nine phosphoroamides tested did not affect mineralization of
alanine in any of the three soils studied (see also Bremner, 1986).
Banerjee et ai. (1997, 1999) conducted field experiments to evaluate the effects of
conventional and zero tillage, urea, and urea + nBTPTA [N-(n-butyl)thiophosphoric
triamide] fertilization on soil properties (see Section 8.1.12), including potential N
mineralization (No). The conclusion was drawn that No was not significantly affected by
either urea with or without nBTPTA or tillage system.
TABLE 70. Effect of various urease inhibitors on denitrification of nitrate in a cla~ loam soil"
N03·-Nlost N I2roduced !Jlg/g soiQ
COIIllound
(/tg!1l soil) NOi-N Nz()'N Nz-N (NOz·+NzO+Nz!;N
None 109 0 34 74 108
Na p-chloromercuribenzoate (PCMB) 151 85 8 58 151
Catechol (CT) 106 0 34 71 105
Hydroquinone (HQ) 122 3 62 59 124
p-Benzoquinone (BQ) 121 0 61 61 122
2,5-Dimethyl-BQ 82 22 2 59 83
2,6-Dimethyl-BQ 85 31 3 52 86
2,5-Dichloro-BQ 106 0 35 72 107
2,6-Dichloro-BQ 115 0 42 75 117
Phenylphosphorodiamidate (PPDA) 107 0 15 93 108
Phosphoryl triamide (PTA) 126 0 26 100 126
N-Phenylphosphoric triamide (PPTA) 97 0 32 66 98
N-(Diaminophosphinyl)benzamide
(DAPBA) 128 0 9 120 129
4-Fluoro-N-(diaminophosphinyl)
benzamide (4-F-DAPBA) 121 0 21 102 123
N-(Diaminophosphinyl)
benzeneacetarnide (DAPBAA) 126 0 0 125 125
"From Yeomans and Bremner (1986), by courtesy of Marcel Dekker, Inc.
ratio simulated flooded soil conditions. The bottles containing the mixtures were closed
and incubated at 25°C up to 20 days, during which time N03' and N02- contents in
mixtures were periodically determined. Only a small decrease of N03- content was
noted in both presence and absence of urease inhibitors. To show whether the low
denitrification was related to lack of carbon source, 0.03 mg of glucose was added to
each mixture after 20 days of incubation. Addition of glucose resulted in a rapid
decrease ofN03- content, irrespective of the presence of urease inhibitors, which clearly
shows that the original soil did not contain enough readily available carbon for
denitrification and that the urease inhibitors tested did not influence denitrification.
In the second experiment, 1% (on soil weight basis) of ground barley straw was
added to the soil samples. In continuation, the procedure was the same as in the first
experiment. In these mixtures, the loss of N03--N was rapid and, after 2 days of
incubation, reduction of N0 2- and N03- was almost complete in both absence and
presence of urease inhibitors. However, during the first 24 hours, HQ retarded
denitrification by about 20%.
In the third experiment (see also Zhou et al.. 1992), only the HQ was studied. Its
rates were 0, 40, 200, and 400 flgllO g soil. No or 1% barley straw was added to the
soil. Denitrification of N03- was followed by determination of N20. None of the HQ
rates affected emission ofN20 in the no-straw treatments. Thus, in 3 days about 5% of
the added N0 3--N was lost as N 20 from both untreated and HQ-treated soil samples.
When soil + 1% straw mixtures were used, HQ reduced, during the first day of
incubation, the emission ofN20 from 74.75% (no HQ) to 62.50% (40 flg HQ/lO g soil),
52.25% (200 flg HQ), and 36.75% (400 flg HQ). However, this inhibiting effect of HQ
was short-lived, as after 3 days of incubation the N20 loss was always about 98%.
The last experiment differed from the third by using less straw (0.1 %) and assessing
denitrification through analysis ofN03- and N02-. After 5 days of incubation, about half
of the added nitrate was lost in the treatments with 0, 40, and 200 flg HQ/IO g soil, but
only about 20% when the rate ofHQ was 400 flgllO g soil.
major effect of PPDA was not so much to prevent ammonia volatilization but to reduce
the losses caused by denitrification to N2 of the nitrates derived from urea-N (see page
119).
Six phosphoroamides were tested by Yeomans and Bremner (1986) (see page 337
and Table 70), nine by Bremner et al. (1986b) (see page 337), and two by Wang et al.
(1991c) (see page 338).
(Tu, 1990). However, HgCh added to samples of the sandy loam soil (PH 7.6) at a
higher rate (80 Ilg/g soil) significantly decreased the ATP content after both incubation
times (Tu, 1992a,b).
The heavy metal salts tested by Kandeler et al. (1990) (see page 15) decreased the
ATP content in both Austrian soils studied. The decrease was -60% in a sandy loam
and -30% in a clay loam.
Besides urease and dehydrogenase activities, the ATP content was also determined
in CdS04 -treated samples of two Italian soils (a sandy loam and a sand) studied by
Morano et at. (2001) (see pages 19 and 322). Sensitivity of the microbial ATP
production to Cd in the sandy loam was lower after 7 days than after 3 hours or 28 days
of incubation, whereas this sensitivity in the sand decreased continuously during the
incubation.
The urease, present and active in soil, may cause errors in analysis of urea and/or
ammonium, because, acting on urea, the urease splits it and, thus, decreases the real
amount of urea and, by producing NH/, increases the real amount of NH/.
Consequently, it is necessary to inhibit urease, to stop the reaction catalyzed by urease
in soil samples to be analyzed. Only these conditions assure obtaining accurate data on
the amount of urea and/or NH4 + in soil samples submitted to analyses. In other words,
inhibition of urease activity constitutes a step in these analyses.
Table 71 specifies the inhibitors and methods of their application and refers to the
investigators who elaborated the methods of application of urease inhibitors for soil
analyses.
The inhibitor preferred by majority of the investigators for analysis of urea and/or
NH4+ in urea-treated soil is phenylmercuric acetate (5 llg per ml of 2 M KCI solution).
TABLE 71. Compounds used as inhibitors of urease activity in soil samples analyzed for determination of
their content in urea and/or NH/
Compound Method of application Reference
2 3
60 mg ofCuS04 is added to a suspension of Yolk (1966)
60 g of soil in 250 ml of solution made
alkaline with MgO.
0.01 N HCl The soil in an amount equivalent to 10 g of Paulson and Kurtz (1969a)
oven-dry matter is extracted with 100 ml of
IN KCl: 0.01 N HCl solution.
0.1 NHCl It serves as an extractant and inhibitor of Yolk (1970)
soil urease activity.
HgCh The reaction mixture, prepared from 10 g of Tanabe and Ishizawa (1969)
air-dried soil, 2-4 ml of toluene, 60 ml of
0.2 M phosphate buffer (pH 6.7), and 10 ml
of 10% urea solution, and incubated, is
treated with 10 ml of 1% HgCIz solution
and brought to 200 ml by adding a solution
containing 20 g of KCI.
HgCh The aqueous suspension containing 25 g of Gould et al. (1973)
soil is treated with 0.2 g of CaCh, 0.2 g of
decolorizing caIbon, and 5 ml of 1% HgCh
solution, then enough distilled water is
added to dilute to a total water volume of
100 ml.
To 10 g of soil in 20 ml of aqueous Uoyd and Sheaffe (1973)
suspension is added a HgCh solution with a
final concentration of 0.1 %; KCI is used as
an extractant at a rate of 0.8 glkg soil.
40 ml of2.5 M KCI solution containing 100 Tabatabai and Bremner (1972) (see
Ilg of Ag;,SOJml is added to 5 g of soil in also Tabatabai,1982, 1994)
10 ml of aqueous suspension.
344
TABLE 7L -continued-
2 3
The reaction mixture, prepared from I g of Bums et al. (1978),
air-dried soil, I ml of 0.2% NaN 3 solution, Lethbridge et al. (1980)
2 ml of 0.5 M Tris-maleate buffer (pH 7.0),
and I ml of 6 M urea solution, and
incubated, is treated with 0.5 ml of 10 mM
Ag2SO. solution.
The reaction mixture, prepared from 5 cm.! Schinner and Pfitscher (1978)
of air-dried soil, 0.7 ml of toluene, 9 ml of
0.05 M Tris buffer (pH 9.0), I ml of 0.2 M
urea solution in Tris buffer, and incubated,
is treated with about 35 ml of 2.5 M KCI
solution containing 0.01% AgN03 and
brought with distilled water to 50 ml.
Phenylmercuric 100 ml of2 M KCI solution containing 5 I1g Douglas and Bremner (1970) (see
acetate (PMA) of PMA/ml is added to 109 of soil. also Bremner, 1982;
Tabatabai, 1982, 1994)
20 g of soil is extracted with 2 M Na2S0. Singh et al. (1984)
solution containing 5 j1g of PM AIm I.
For determination of urea and NH.+ in Fillery et al. (1984)
floodwater from rice fields, 18 ml of
floodwater is treated with KCI to clarify the
solution and with PMA to inhibit urease
activity (final concentration: 2 M KCI and 5
j1g PMAlml).
For conservation of urea and NH4 + in Simpson et al. (1985)
floodwater from rice fields, I ml of 1%
H.lP04 and I ml of 1.5 mM PMA solution
are added to 8 ml of floodwater.
The mixture containing 5 g of soil and 2.5 Liao and Raines (1985)
ml of aqueous phase is extracted with 22.5
ml of I N K2S04 solution containing 10 I1g
of PM AIm I.
p-Hydroxymercuri- To 0.2 g of soil is added 0.1 ml of 19 mM Shih and Souza (1978)
benzoate (PHMB) PHMB solution in 25 mM phosphate buffer
Thiourea (TU) (pH 8.5) or 0.1 ml of 2 M TV solution in 50
mM phosphate buffer (pH 7.0).
Phenylmercuric The reaction mixture, prepared from 25 g of Perez Mateos and Gonzalez
borate (PMB) soil, 45 ml of 50 mM Tris buffer (pH 9.0) Carcedo (1988)
and 5 ml of 1-20 mM urea solution, and
incubated, is treated with 175 ml of 2.5 M
KCI solution containing 200 111 ofPMB/1.
Dimethyl-p-benzo- For measuring pH in suspensions of soil- Rachhpal-Singh and Nye (I 984b)
quinone (DBQ)O 0.0 I M CaCh solution 1:5, DBQ is added to
the CaCh solution at a rate of I 0 11g/1.
Hydroquinone (HQ) To I g of soil in 5 ml of aqueous suspension Lichko and Kiselev (1985, 1986)
is added 2.0-2.5 ml of I mM HQ solution.
Phenylphosphoro- I N KCI solution containing 10 I1g of Savant etal. (l987a,b, 1988b)
diamidate (PPDA) PPDAlml is used as an extractant and
urease inhibitor.
The soil sample (100-120 g) is extracted Medina and Sullivan (1986, 1987)
with 250 ml of 2 M KCI solution containing
5 j1g of PPDAlml.
The soil is extracted with 2 N KCI solution Christianson et al. (1990)
containing 0,01 g of PPDA/I. The
soil:solution ratio is 1:3.
'Position of the two methyl groups is not specified by Rachhpal and Nye (1984b).
345
Chapter 10. Urease Inhibitors Used with Another Purpose than Inhibition of Soil
Urease Activity
10.1.3. Hexamethylenetetramine
As shown on page 47, hexamethylenetetramine (HMTA) was patented as an inhibitor of
soil urease activity by Neumann and Richter (1976). In winter wheat field experiments
conducted by Verstraeten and Livens (1975, 1977), HMTA was found to be a slow-
release N fertilizer. HMTA displayed a fairly high N efficiency and reduced the
incidence of mildew disease (Erysiphe graminis). HMTA is gradually mineralized in
soils under both aerobic conditions (Verstraeten, 1977) and water-logged conditions
(Taslim and Verstraeten, 1977). Mineralization of HMTA added to samples of seven
Belgian soils in amounts of 100 to 200 ppm N and incubated under aerobic conditions at
10 or 30°C for 4 weeks ranged between 60 and 90% compared with urea mineralization.
Under water-logged conditions, a clay loam soil was studied. In one of the experiments,
rate ofHMTA or urea addition was 100 ppm N, the incubation took place at 30°C and
lasted 45 days. Release of ammonium from HMTA was highest at day 15 and
represented -50% of the added HMTA-N, whereas -90% of the added urea-N was
hydrolyzed during 3 days of incubation. In other words, HMTA is mineralizable also
under anaerobic conditions, but this process is slower than hydrolysis of urea.
In a 3-year (1991-1993) field experiment conducted by Borovskii and Yanishevskii
(1994), HMTA, applied as a nitrogen fertilizer for vegetable crops on an alluvial
meadow soil and a soddy-podzolic soil in the Moscow region, produced high crop
yields. Moreover, the crops produced with HMTA were of an excellent quality as their
nitrate content was smaller than that of crops obtained with urea or ammonium nitrate.
The three fertilizers were administered at the same yearly rate: 140 kg Nlha in 1991 and
120 kg Nlha in 1992 and 1993.
Based on the results of pot experiments, Kolyada (1970, 1973) drew the conclusion
that thiourea was an efficient N fertilizer when applied 8-16 days before planting the
soil to barley, oats or radish. During this period, thiourea lost phytotoxicity due to its
decomposition.
In a laboratory experiment, Sahrawat (1981) proved that biuret was mineralized
under both water-logged and aerobic conditions. In soil samples amended with 100 mg
biuretlkg soil and incubated at 30°C for 5 weeks, the biuret-N was mineralized to
NH4 +-N in a proportion of 48.3% under water-logged conditions and to N0 3·-N in a
proportion of 18.3% under aerobic conditions. Thus, biuret may be considered a slow-
release N fertilizer.
10.1. 5. Phosphoroamides
Phosphoryl triamide [P(O)(NH 2)3; PTA] and some other phosphoroamides were
patented as NP fertilizers by Pellegrini (1965, 1968), Wanek et al. (1966), Fiedler et al.
(1974b) and studied by other investigators as well.
Pellegrini (1965) conducted a field experiment to evaluate the efficiency of PTA as
a NP fertilizer. The test plant was winter wheat. The plots were installed on a silt loam
soil (PH 8.4). Urea and Ca(H 2P04)2 served for comparison. Rates of additions were
191.4 kg P20s/ha and 102 kg N/ha. All fertilizers were surface-applied in mid-February
at the end of the emergence of young plants. The grain yields in the different treatments
presented the order: Ca(H2P04h < urea+ Ca(H 2P04)2 ::::: urea « PTA.
Based on the patent of Wanek et at. (1966), Ambroz et al. (1970) compared the
nitrification of PTA with that of (NH4)2HP04. These compounds as sole N sources were
added to a liquid nutrient medium at rates of 0.01, 0.02, and 0.04% N. The medium (20
ml) was inoculated with 0.5 g soil (rendzina, pH 7) and incubated at 28°C for 14 days,
during which the N03·-N content was determined at 3-day intervals. The results showed
that the two compounds were nitrified to the same extent.
Pellegrini (1968) commented on his own patent (Pellegrini, 1965) by pointing out
two disadvantageous properties of PTA: very different mobility in different soils and
weak stability on storage when exposed to air. In his new patent six
alkylphosphoroamides, namely two phosphoromonoamidate (PA) and four phosphoro-
diamidate (PDA) compounds are nominalized as NP fertilizers: dimethyl-PA, methyl-
ethyl-PA, methyl-PDA, ethyl-PDA, n-butyl-PDA, and 2-ethyl-n-hexyl-PDA. All these
compounds labeled with 32p showed a better mobility in the nine soils studied as
compared with the mobility of Ca(H 2P0 4 )2 or with that of PTA. In a pot experiment,
they were tested as P sources for common beans at a rate of 50 kg P20 5/ha, and it was
found that they performed better than Ca(H2P0 4 h.
Wakefield et al. (1971) carried out greenhouse experiments. In one of the
experiments, thiophosphoryl triamide [P(S)(NH 2)3; TPTA] was tested as a source of N
and P for two successive crops of maize on a silt loam limed to pH 6.4. The uptake ofN
and P from TPTA was the same as that from ammonium nitrate and superphosphate.
The yield of dry forage was less from TPTA, however, especially at the high application
rates because of a slight toxicity to the first crop. This toxicity soon disappeared, and no
abnormalities were observed in the second crop. It was found in another experiment that
three derivatives of TPTA, sodium diamidothiophosphate [NaOP(S)(NH 2)2], diam-
monium monoamidothiophosphate [(~OhP(S)NH2]' and diammonium thiophosphate
[(~O)2P(S)OH], were also effective Nand P sources, but condensation products of
349
the pyrolysis of TPT A were less effective and initially toxic. These investigations were
also referred to by Sheridan (1970).
Fiedler et al. (l974a) conducted pot experiments to evaluate ethylphosphoro-
diamidate (EPDA) - C2HsOP(O)(NH2)2; phenylphosphorodiamidate (PPDA) -:-
C6HsOP(O)(NH2h; diethylphosphoroamidate (DEPA) - (C2HsO)2P(O)NH2; and
dimethylthiophosphoroamidate (DMTP A) - (CH30)2P(S)NH2 as NP fertilizers and
triethylphosphate - (C 2HsO)}P(O) as a P fertilizer. Diammonium phosphate (DAP) -
(NH4)2HP04 was used as a reference NP fertilizer. The test plants were Italian rye grass,
oats, and mustard. Three soils were used: a neutral loess loam, and acid and a neutral
heath sand. The pots contained 3 kg loam + 4 kg quartz sand, 3.5 kg sand + 4 kg quartz
sand, and 3 kg sand + 3 kg quartz sand, respectively. Rates of P and N additions/pot
were: 120 mg P + 1,200 mg N (P:N=I:lO) or 240 mg P + 1,200 mg N (N:P=I:5). To
achieve these ratios, urea or ammonium nitrate was also added with the test compounds.
Each pot also received K (0.66 g), Mg (0.1 g), and micronutrients (Mo, Cu, Zn, B, and
Mo). The crop yields and P and N contents in plants were determined, and the
phytotoxicity of the compounds tested was also evaluated.
The results led to the following conclusions: a) EPDA exhibited the best fertilizer
effect; b) with increasing degree of esterification, replacement of ethyl group by the
phenyl group and of the PO group by the PS group, the compounds became less
effective in supplying the plants with physiologically active P and their phytotoxic
effect increased; c) if homogeneously distributed in acid to neutral, non-calcareous soil,
the effect of EPDA was comparable with that of DAP, whereas in calcareous soils
EPDA performed better than DAP due to its better mobility.
Fiedler et al. (1974b) patented several alkylphosphoric triamides (alkyl-PTAs) as
NP fertilizers and tested them in pot experiments.
In one of the experiments, the test plant was Italian ryegrass grown on three soils (a
loess loam, pH 7.0, a sand, pH 5.5, and another sand, pH 7.0). The compounds tested
were: dimethyl-PTA - (CH3hNP(O)(NH2h; diethyl-PTA - (C 2HshNP(O)(NH2)2; and
bisdimethyl-PTA - [(GI3hN)zP(O)NH2' Diammonium phosphate (DAP) - (NH4hHP04
was the reference NP fertilizer. All fertilizers were surface-applied. The crop yields
obtained with these three alkyl-PT As were comparable with that produced with DAP on
the loam and sand (pH 7.0). In the other sand (pH 5.5), the alkyl-PTAs performed a
little better than DAP. Imidodiphosphoric tetraamide - [(NH2)2POhNH was also tested
with Italian rye grass on the loam and sand (pH 5.5). This compound increased the yield
on the loam and decreased it on the sand in comparison with the yield registered with
DAP.
In another experiment, dimethyl-PTA and bisdimethyl-PT A were tested with oats
grown on a loess loam (pH 6.3). The grain yields had the following relative values:
100% (DAP), 151% (dimethy-PTA), and 140.5% (bisdimethyl-PTA). The P content of
grains was also increased by both PTA compounds.
Some alkyl-PTAs, e.g., bisdiethyl-PTA - [(CzHS)2NhP(O)NH2 and hexamethyl-PTA
- [(Oi,hNhP(O) were much less effective than DAP and very toxic and, therefore,
excluded as NP fertilizers. Thus, the effectiveness of alkyl-PTA compounds as NP
fertilizers and their toxicity are determined by the number and kind of their alkyl
groups.
The studies on the fertilizer effect of PTA compounds on oats, briefly described in
the patent of Fiedler et al. (1974b), were described in details by Fiedler et al. (1975).
350
chemical composition of beans, with the exception of a slight decrease in the lipid
content.
At the Tennessee Valley Authority, Muscle Shoals, Alabama, HA-CTPAT was
studied initially, more precisely before discovering its capacity to inhibit soil urease
activity (Medina and Sullivan, 1986, 1987) as a potential NP fertilizer (Anonymous,
1985b).
In a pot experiment, Calancea et al. (1986) used HA-CTPAT labeled at the level of
NH2 groups or at the cycle, P3N3C5NH2)6 or P3 15 N3(NH2)6. Maize was sown in a weakly
podzolized soil (1,4, and 7 kg soil/pot). Rates of HA-CTPAT addition were 50, 100,
200, 300, and 400 mg N/pot. Beginning with the 12th day of growth up to the 69th day,
the plants were systematically sampled for determination of their dry weight and total N
and 15N contents.
It was found that in the soil treated with HA-CTPAT the plants grew slowly and
took up less 1~ during the first 45 days, but in the 46-69-day period they grew
vigorously and took up more 15N.
In the next growing season, the experiment was repeated by submitting the same soil
samples to the same treatments as in the first experiment. The maize plants in this
second culture were examined like those of the first culture.
It became evident that plant growth and uptake of N were more marked in the
second culture than in the first and significantly correlated with the rate ofHA-CTPAT.
Of the 15N taken up, 70-80% represented the NH2-N and 20-30% the cyclic-No But the
plants always took up more N from the soil reserve than from HA-CTPAT. One can
deduce from these findings that HA-CTPAT is decomposed slowly in the soil; at the
beginning, the compound is deaminated which is followed by decomposition of the
cycle. Nitrogen (and phosphorus) are released in plant-available forms.
This conclusion was confirmed in a newer pot experiment using the two 15N-labeled
forms of HA-CTPAT (Calancea and Chiriac, 1993). Total N content in the maize plants
(100%) originated from the soil reserve and HA-CTPAT in proportions of
67.50±10.94% and 32.50±10.94%, respectively. Of the total N, N from 1~2 and
cyclic- 15N represented 20.58±7.56% and 11.92±3.87%, respectively.
Italian ryegrass was the test plant in another pot experiment (Calancea et al., 1990).
The effect ofHA-CTPAT on dry matter yield was compared with that of urea. Rates of
additions/pot containing 2 kg of alluvial podzolized clay, pH 5.8 mixed with 1 kg of
sand were 0,50, 100,200,300,400, and 500 mg N. In the urea treatment, Ca(H2P04)2
(100 mg P/pot) was also added.
Dry matter yields increased with increasing rate of both fertilizers, but the increase
was higher with urea than with HA-CTPAT at rates of 50-400 mg N/pot, and the same
increase was recorded with urea and HA-CTPAT at the highest N rate. When HA-
CTP AT was applied at lower rates, the plants took up N mostly from the soil reserve but
at its higher rates the plant uptake of N from this fertilizer was more pronounced than
from urea.
One-l slurries of cattle and swine wastes (1: 1 g:g feces to urine) were treated with
PPDA or CHPTA and incubated at ambient temperature (22-25°C). No inhibitor was
added to the controls. During incubation, all slurries were analyzed periodically for urea
and ammonia. Different amounts of inhibitors were applied per 1 slurry: 10 mg PPDA,
10 and 40 mg CHPTA, and 10 mg CHPTA weekly - incubation time: 28 days (both
cattle and swine wastes); 10,40, and 100 mg PPDA applied weekly for 7 weeks -
incubation time: 70 days (cattle waste) and 84 days (swine waste).
With cattle waste (3.3 g ureall) and swine waste (4.8 g urea/l), both inhibitors at 10
mg/l slurry prevented hydrolysis of urea for 4-11 days, and then a gradual hydrolysis
occurred until complete at day 28. Hydrolysis of urea in untreated cattle and swine
wastes (controls) was complete within 1 day. Addition of inhibitors once per week was
the most effective method of preventing urea hydrolysis. Weekly addition of 10,40 or
100 mg PPDAIl cattle waste slurry (5.6 g urea/I) prevented 38, 48, and 70% of the urea
from being hydrolyzed after 28 days, respectively. With swine waste slurry (2.5 g
ureall), these PPDA concentrations prevented 72, 92, and 92% of the urea from being
hydrolyzed after 28 days, respectively. As additions of PPDA were stopped after 7
weeks, all urea at the three PPDA concentrations was hydrolyzed after 70 and 84 days
for the cattle and swine wastes, respectively.
The conclusion was drawn that use of the inhibitors makes possible a significant
control of ammonia emission from livestock wastes and an increase in fertilizer value of
wastes by improving the N to P ratio for plant growth.
Sotomura et al. (2000) patented a deodorant composition comprising extracts from
plants (especially pine or green tea), a urease inhibitor (e.g., thiourea, boric acid,
p-benzoquinone or tannic acid), a lipase, and a lower alcohol such as methanol, ethanol
or propanol. The composition is durable and effective for removing odorous gases
(especially, ammonia, trimethylamine, butyric acid or propionic acid) from pet manure,
etc.
Baintner (1964) and Tang! and Baintner (1969) used acetohydroxamic acid (AHA) to
reduce susceptibility of goats to ammonia poisoning induced by the feeding of large
quantities of urea. Larger amounts of urea were tolerated by the animals when urea was
fed with AHA than when it was fed alone. Thus, the goats to which the lethal dose of
urea (150 g) was added with AHA did not die. It was also found that in the presence of
AHA only a small proportion of urea-N was absorbed as ammonia; this suggested that
AHA inhibited the activity of rumen urease. The inhibiting effect of AHA on rumen
urease was also directly demonstrated in in vitro experiments. In other experiments,
sheep with fistulated rumen were used. When urea in a dose of 30 g without AHA was
infused into the rumen, the animals showed symptoms of severe poisoning or died. No
symptoms of poisoning were observed when 30 g of urea was introduced with 13 g of
AHA. In this case, hydrolysis of urea was retarded from 4 hours 15 minutes to 13 hours
15 minutes. Finally, it was emphasized that only pure AHA should be used because
hydroxylamine present as an impurity has poisoning and even lethal effect as it causes
methaemoglobinemia. The pure AHA is a costly product. Therefore, the perspective for
using AHA as an additive to fodder of ruminants is questionable on economic grounds.
354
urea + CVA. Reaction mixtures, prepared from rumen fluid and urea (53 mg%) or urea
+ CUA (4 mg%), were incubated for 6 hours and analyzed for urea and ammonia at 1-
hour intervals. In the absence of CVA, urea was completely hydrolyzed in 4 and 5 hours
in rumen fluid of the non-adapted and adapted animals, respectively. In reaction
mixtures with urea+CVA, urea at concentrations of 8.6 and 3.4 mg% remained
unhydrolyzed in the rumen fluid of the non-adapted and adapted animals, respectively,
after 4 hours of incubation. In other words, CUA was more inhibitory in the rumen of
non-adapted than adapted animals.
There are reports showing that Yucca schidigera extracts, containing sarsaponin and
sarsaponin fractions, improved performance and health in ruminants by inclusion of
these extracts in fodders at rates of 100-250 glt fodder, and the effect was attributed to
inhibition of urease activity (e.g., Ellenberger et al., 1984). Killeen et al. (1994) studied
the effect of a commercial Yucca schidigera extract on urease of a bacterium (Bacillus
pasteurii) and on the ~-galactosidase of the fungus Aspergillus oryzae. It was found that
the effect of the extract on urease was not specific as ~-galactosidase was also inhibited.
Another finding was that the urease-inhibiting effect of the extract was much too low to
account for the in vivo effects of the Y. schidigera extracts at fodder inclusion levels as
little as 100 giL
Ludden ef al. (2000a,b) conducted in vitro and in vivo experiments using N-(n-
butyl)thiophosphoric triamide (nBTPTA) as a urease inhibitor.
The in vitro experiments were carried out with steer rumen fluid. The reaction
mixtures, prepared in test tubes, contained rumen fluid, ground fescue hay or ground
fescue hay and ground maize in 1:1 mixture, urea with and without nBTPTA and were
incubated at 39°C for 6 or 48 hours. During and after incubation, they were analyzed for
urea, ammonia, and volatile fatty acid (VFA) contents and fiber digestibility (FD).
nBTPTA decreased rate of urea hydrolysis and, consequently, formation of ammonia.
Total VFA concentration was not affected, but acetate/propionate ratio and FD were
decreased by nBTPTA. The conclusion drawn from the in vitro experiments was that
nBTPTA can be used to decrease the rate of ammonia release from dietary urea and
offers a way to improve urea-N utilization in ruminants.
In the in vivo experiments, ruminally cannulated lamb wethers were used to
investigate the chronic effect of nBTPTA on ruminal N metabolism and N balance. In
one of the experiments, the animals were given into the rumen 0 or 0.125 up to 4 g
nBTPTA daily and fed a cracked maizelcotton seed hull diet containing 2% urea twice
daily at 2.5% of initial body weight for 15 days. nBTPTA inhibited ruminal urease
activity and thus decreased the rate of ammonia formation, but this effect of nBTPTA
diminished as the experiment progressed. On day 15, no differences were found in the
VFA concentration and in FD between the treatments with and without nBTPTA.
However, nBTPTA increased urinary N excretion and thus decreased N retention. In
contrast to the results obtained in the short-term in vitro experiments, the results of the
in vivo experiments indicated that the nunen microbiota was able to adapt to chronic
nBTPTA administration, thereby limiting its practical use in improving the utilization of
dietary urea.
356
The bacteria producing urease implicated in the genesis of some human diseases infect
the urinary tract (containing substantial amounts of urea) or the gastrointestinal tract
(containing limited amounts of urea - its concentration in normal stomach is of about 3
rnM).
The urease of urophathogenic bacteria is directly associated with the formation of
infection stones [urinary and renal stones (calculi); urolithiasis and nephrolithiasis] and
contributes to the pathogenesis of acute pyelonephritis and urinary catheter encrustation.
Urease of the bacteria infecting the gastrointestinal tract generates gastritis and
peptic ulcer and contributes to the pathogenesis of hyperammonemia, hepatic
encephalopathy, and hepatic coma.
Some of these diseases also affect animals.
The infection stones are a mixture of struvite (MgNH4P04.6H20) and carbonate
apatite [Ca\O(P04)6.C03]. The ammonia released by bacterial urease-catalyzed
hydrolysis of urea increases the pH from 6.5 to 9.0, at which the normally soluble
bivalent cations become supersaturated and crystallize forming the stones. In humans,
two urinary tract bacteria, Proteus mirabilis (P. mirabilis) and Ureaplasma urealyticum
(U urealyticum), are the most common bacteria implicated in stone formation. Other
Proteus species (e.g., P. morganii) and bacteria belonging to other genera such as
Klebsiella. Morganella, Providencia are also implicated in stone formation.
In dogs, struvite stone formation is associated with Staphylococcus aureus. Bovine
pyelonephritis is caused by Corynebacterium renale that possesses a potent urease.
In humans, the bacterium implicated in development of gastritis and peptic
ulceration is Helicobacter pylori (H pylori) (formerly Campylobacter pylori and C.
pyloridis). It is an acid-sensitive bacterium which multiplies in a pH range of 6.9 to 8.0.
H pylori is uniqely adapted for surviving in the highly acidic environment of human
TABLE 72. COJ1llounds patented as inhibitors of bacterial urease implicated in the genesis of some human
diseases
Urease inhibited/ Disease
COIl1lound(s) Reference
treated and prevented
[[(4-Aminophenyl)sulfonyl)aroino)phenyl P. mirabilis urease! Alaimo et al. (1980)
phosphorodiamidates Urinary tract infections
8-[(4-Aminophenyl)sulfonyl)amino-2- P. mirabilis urease! Alaimo and Millner
naphthalenyl phosphorodiamidate Urinary stones (1980)
N-(Diaminophosphinyl)arylcatboxarnides P. morgan;; urease! Bayless and Millner
Urinary stones (1980a)
Phosphorotriamides P. mirabilis urease! Bayless and Millner
Urinary stones (1980b)
Hydroxamic acid glycoside derivatives Fecal urease! Ito et al. (1994)
Hyperamrnonemia
2-Benzamido-3-carbostyrylpropanoic acids and H. pylori urease! Yamazaki and
their salts (rebamipide) Gastric IlUICOSal disoniers Oosaka (1995)
2-[4-(3-Methoxypropoxy)-3 -methylpyridin-2-yl)- H. pylori urease! Tsuchiya et al.
methylsulfinyl-IH-benzimidazole Hyperamrnonemia, (l995a)
hepatic encephalopathy
Proanthocyanidins (e.g.• from pine bark or grape H. pylori ureaselUlcer (rat Salo (2000)
seed) + catechin and epicatechin model)
357
stomach. It produces a potent urease. Due to a very high affmity for urea, the H pylori
urease is able to hydrolyze the limited amounts of urea in stomach and to produce a
"cloud" of ammonia that protects the bacterium from stomach acid and enables it to
colonize and damage the gastric mucosa. Being adjacent to mucosa, H pylori becomes
able to scavange urea from the blood. Thus, urease in an important virulence factor for
Hpylori.
As the bacterial urease is implicated in the genesis of the diseases mentioned, for
their treatment and prevention inhibitors of urease activity were used (Rosenstein and
Hamilton-Miller, 1984; Mobley and Hausinger, 1989; Park et al., 1996).
First, the compounds patented as inhibitors of bacterial urease implicated in the
genesis of some human diseases are presented (Table 72). Then examples are cited from
TABLE 73. Studies on the inhibition of bacterial urease and therapeutic and prophylactic effect of urease
inhibitors in some human diseases
Urease inhibited/Disease treated
Inhibitor(s) Reference
and prevented
Acetohydroxamic acid (AHA) Hyperammonemia Fishbein et al. (1965)
AHA Mucosal and fecal urease Aoyagi and Summerskill
(1966)
AHA Hyperammonemia Summerskill et al. (1967)
AHA Renal stones Griffith et al. (1979)
AHA Renal stones Martelli et af. (1981)
AHA Renal stones Williams et al. (1984)
AHA Urinary catheter encrustation Bums and Gauthier (1984)
AHA Urinary stones Griffith et al. (1988)
AHA H. pylori urease Goldie el al. (1991)
Hydroxamic acid derivatives (HADs) Urease of T -strain mycoplasmas Ford (1973)
(u. urealyticum)
HADs Urinary stones Kobashi et al. (1980)
HADs Urinary stones Munakata et al. (1980)
HADs H. pylori urease/Gastritis, Odake el al. (1994)
peptic ulcer
HADs Urinary stones Abou-Sier et al. (1995)
Hydroxyurea (HU) Urinary stones Carmignoni et al. (1980)
HU Renal stones Martelli et af. (1981)
N-(Diaminophosphinyl)-4-tluoro- Urinary stones Millner et al. (1982)
benzamide (tlurofamide)
Flurofamide H. pylori urease Kohler et al. (1995)
N-Acyl phosphoric triamides Urinary stones Takabe et al. (1984)
4-Substituted H. pylori urease Faruci el al. (1995)
phenylphosphorodiamidates
Proton pump inhibitors (omeprazole, H. pylori urease, P. mirabilis Tsuchiya et al. (l995b)
lansoprazole. rabeprazole) urease/Gastritis, peptic ulcer
Omeprazole H. pylori urease Bugnilo et af. (1993)
Lansoprazole, omeprazole H. pylori urease Nagata et al. (1993)
Omeprazole, lansoprazole H. pylori urease KOhler et al. (1995)
Rabeprazole H. pylori urease Park et al. (1996)
Ebrotidine (antiulcer drug) H. pylori urease Pietrowski et al. (1995)
Ecobet (antiulcer drug) H. pylori urease Ito et al. (1998)
Ethylalcoholic herb extracts H. pylori urease Imamura et al. (1995)
Peptides (a 24-mer and a 6-mer H. pylori urease Houimel et al. (1999)
peptide)
358
a great number of experimental and clinical studies using different compounds for
inhibition of bacterial urease and thus for treatment and prevention of urease-induced
human diseases (Table 73).
CONCLUSIONS
The investigation performed with the aim to increase the efficiency of urea fertilizers
led to identification of many chemical compounds able to inhibit urease activity in soils.
A great part of these compounds were tested not only in laboratory and vegetation
pots but also under field conditions, on experimental plots. A small number of
compounds were also tested on large agricultural areas.
The investigations were multidisciplinary. Taking part in the investigations were the
chemists who synthesized these compounds, the microbiologists and biochemists who
tested the compounds in laboratory, the agronomists who conducted the experiments in
greenhouse and experimental fields.
N-(n-Butyl)thiophosphoric triamide, phenylphosphorodiamidate, and hydroquinone
can be considered as the most thoroughly studied soil urease inhibitors.
At present, only two urease inhibitors have gained commercial importance.
Hydroquinone is being used on large agricultural areas in China since the 1990s. The
other urease inhibitor, N-(n-butyl)thiophosphoric triamide, was introduced under the
registered trade product name of Agrotain to the United States agricultural market by
IMC-Agrico Company (Bannockbum, Illinois) in spring 1996.
For the future, it is necessary to continue and even to intensify the concerted efforts
of investigators because a) other soil urease inhibitors; b) dual (both urease and
nitrification) inhibitors; c) urease inhibitors in combined use with nitrification
inhibitors; and d) urease inhibitors in natural products also present perspectives to have
commercial importance and to exhibit more advantageous properties than the inhibitors
tested in the past. Research of these compounds and their metabolites as well as
development of large scale production processes deserve further attention as the
increased use of urease (and nitrification) inhibitors in world agriculture will lead to
increased world food production and, even more significantly, to more efficient
environmental protection, both effects being of major importance for the future of
mankind.
361
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390
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Appendix
The review papers listed below were also valuable sources of information for elaboration of the Introduction
and Chapters 1-9 of this book.
Beaton, J.D. (1978) Urea: Its popularity grows as a dry source of nitrogen, Crops Soils Mag., 30 (6), 11-14.
Bremner, lM. (1990) Problems in the use of urea as a nitrogen fertilizer, Soil Use Manage. 6 (2), 70-71.
Bremner, 1.M. (1995) Recent research on problems in the use of urea as a nitrogen fertilizer, Fert. Res. 42, 321-329.
Bremner, 1.M. and Hauck, R.D. (1974) Perspectives in soil and fertilizer nitrogen research, Trans. 10th Int. Congr.
Soil Sci. (Moscow, 1974) 9, 13-29.
Bremner, 1.M. and Mulvaney, R.L. (1978) Urease activity in soils, in R.G. Bums (ed.), Soil Enzymes, Acad.
Press, London, pp.149-196,
Buresh, R.J. and Baanante, CA. (1993) Potential economic benefits of modifications to urea that increase yield
through reduction in nitrogen losses, Agron. J 85,947-954.
Byrnes, B.H. and Freney, J.R. (1995) Recent developments on the use of urease inhibitors in the tropics, Fert. Res.
42,251-259.
Christianson, C.D. and Schultz, JJ. (1989) Potential for alternative nitrogen fertilizers in developing country
agriculture, Proc. AKro-Econ. Meeting Int. Fert. Ind. Assoc. (Budapest, 1989), pp. 24-72.
Faurie, G. and Bardin, R. (1979) Volatilization of ammonia. I. Influence of the nature of soil and nitrogen
compounds, Ann. Agron. 30, 363-385 (in French).
Fertiliser Association ofIndia (1977) Fertiliser urea- its efficient use, Fert. News 22 (9), 3-18.
Gasser, J.K.R. (1964) Urea as a fertilizer, Soils Fert. 27, 175-180.
Gorelik, L.A. and Gritsevich, Yu.G. (1984) Inhibitors of soil urease activity, Agrokhimtya (2), 103-126 (in Russian).
Gould, W.O., Hagedorn, C., and McCready, R.G.L. (1986) Urea transformations and fertilizer efficiency in soil,
Adv. Agron. 40, 209-238.
Hargrove, W.L. (1988) Soil, environmental, and management factors influencing ammonia volatilization under
field conditions, in B.R. Bock and D.E. Kissel (eds.), Ammonia Volatilization jrom Urea Fertilizer.s, Natl. Fert.
Dev. Center. Muscle Shoals, Alabama, pp. 17-36.
Harre, E.A. and Bridges, J.D. (1988) Importance of urea fertilizers, in B.R. Bock and D.E. Kissel (eds.), Ammonia
Volati/izationjrom Urea Fertilizer.s, Natl. Fert. Dev. Center, Muscle Shoals, Alabama, pp. 1-15.
Hauck, R.D. (1972) Synthetic slow-release fertilizers and fertilizer amendments, in CA.L Goring and 1.W.
Hamaker (eds.), Organic Chemicals in the Soil Environment, M. Dekker, New York, pp. 633-690.
Hauck, R.D. (1983) Agronomic and technological approaches to minimizing gaseous nitrogen losses from
croplands, in J.R. Freney and J.R. Simpson (eds.), Gaseous Loss of Nitrogen from Plant-Soil Systems, M.
Nijhofl7Dr. W. Junk, The Hague, pp. 285-312.
Hauck, R.D. (1984) Technological approaches to improving the efficiency of nitrogen fertilizer use by crop plants,
in R.D. Hauck (ed.), Nitrogen in Crop Production, Am Soc. Agron.-Crop Sci. Soc. Am-Soil Sci. Soc. Am,
Madison, pp. 551-560.
Hauck, R.D. (1985) Slow-release and bioinhibitor-amended nitrogen fertilizers, in O.P. Engelstad (ed.),
Fertilizer Technology and U~e (3rd Edition), Soil Sci. Soc. Am., Madison, W. 293-322.
391
Kiss, S., Driigan-Bularda, M., and Riidulescu, D. (1975) Biological significance of enzymes accumulated in
soil, Adv. Agron. 27, 25-87.
Kiss, S., Pllljca, D., Driigan-Bularda, M., Zborovschi, E., and Cri§llIl, R. (1991) Inhibition of soil urease
activity for increasing the efficiency of urea fertilizer, Environ. Enzymol. (Bucharest) 1, pp. 93-288 (in
Romanian).
KoelJiker, J.K. and Kissel, D.E. (1988) Chemical equilibria affecting ammonia volatilization, in B.R. Bock
and D.E. Kissel (eds.), Ammonia Volatilization from Urea Fertilizers, Nat!. Fert. Dev. Center, Muscle
Shoals, Alabama, pp. 37-52.
Ladd, J.N. and Jackson, R.B. (1982) Biochemistry of ammonification, in FJ. Stevenson (ed.), Nitrogen in
Agricultural Soils, Am. Soc. Agron.-Crop Sci. Soc. Am.-Soil Sci. Soc. Am., Madison, pp. 173-228.
Matzel, W., Ackermann, W., Brinschwitz, W., Buchler, D., Hannusch, L. Heymann, W., Kretschmar, M.,
Uppold, H., Runge, P., and Teske, W. (1974) Effective application of urea as a fertilizer in agriculture,
Fortschrittsber. Landw. Nahrungsguterw. (Berlin) 11. (7-8), 1-68 (in German).
Medina, R. and Radel, R.J. (1988) Mechanisms of urease inhibition, in B.R. Bock and D.E. Kissel (eds.),
Ammonia Volatilizationfrom Urea Fertilizers, Natl. Fer!. Dev. Center, Muscle Shoals, Alabama, pp. 137-
174.
Mulvaney, R.L. and Bremner, J.M. (1981) Control of urea transformations in soils, in E.A. Paul and J.N. Ladd
(eds.), Soil BiochemiStry, Vol. 5, M. Dekker, New York, pp. 153-196.
Nelson, D.W. (1982) Gaseous losses of nitrogen other than through denitrification, in F.J. Stevenson (ed.),
Nitrogen in Agricultural Soils, Am. Soc. Agron.-Crop Sci. Soc. Am.-Soil Sci. Soc. Am., Madison, pp.
327-363.
Radel, R.J., Gautney, J., and Peters, G.E. (1988) Urease inhibitor developments, in B.R. Bock and D.E. Kissel
(eds.), Ammonia Volatilization from Urea Fertilizers, Natl. Fert. Dev. Center, Muscle Shoals, Alabama,
pp.I11-136.
Sahrawat, K.L. (1979) Nitrogen losses in rice soils, Fert. News 24 (12), 38-48.
Sahrawat, K.L. (1980) Control of urea hydrolysis and nitrification in soil by chemicals - prospects and
problems, Plant SoilS7, 335-352.
Scharf; P.C. and Alley, M.M. (1988) Nitrogen loss pathways and nitrogen loss inhibitors: A review, J. Fert.
Issues S (4), 109-125.
Sullivan D.M. and Havlin, J.L. (1988) Agronomic use of ammonium thiosulfate to improve fertilizer
efficiency, J. Fert. Issues S (2), 37-44.
Terman, G.L. (1979) Volatilization losses of nitrogen as ammonia from surface-applied fertilizers, organic
amendments, and crop residues, Adv. Agron. 31, 189-223.
Tisdale, S.L., Nelson, W.L., and Beaton, J.D. (1985) Soil Fertility and Fertilizers (4th Edition), Macmillian,
New York, pp. 161-168 and 177-178.
Trenkel, M.E. (1997) Improving Fertilizer Use Efficiency. Controlled-Release and Stabilized Fertilizers in
Agriculture, In!. F ert. Ind. Assoc. (IFA), Paris.
Tucker. T.C. and Westerman, R.L. (1989) Gaseous losses of nitrogen from desert region soils, Arid Soil Res.
Rehabil. 3, 267-280.
Voss, R.D. (1984) Potential for use of urease inhibitors, in R.D. Hauck (ed.), Nitrogen in Crop Production,
Am. Soc. Agron.-Crop Sci. Soc. Am.-Soil Sci. Soc. Am., Madison, pp. 571-577.
Watson, C.J., Stevens, R.J., Garrett, M.K., and McMurray, C.H. (1990) Efficiency and future potential of urea
for tempemture gmssland, Fert. Res. 26,341-357.
Yeomans, J.e. (1991) Inhibition of nitrogen transfurmations in soils. Potentials and limitations for agriculture,
Trends Soil Sci. 1, 127-158.
393
SUBJECT INDEX
Cupric (continued) D
- ions 5, 8, 9, 11, 13, 14,20,53, 192, Dactylis glomerata 28, 201, 297
233 Dadap 170
- nitrate 8, 11, 15 DAM 390 41, 115, 159,261
- oxide 6 Dazomet 71, 72, 128, 181, 195,236,
- sulfate 5-10,12-15,17,30,33,177, 316,324,331
179,180,187,188,190,242,247, Dehydrogenase 17,321-326,328,329,
248,250,288,318,330,343 342
Cuprous chloride 7 -, actual 326, 327
- sulfate 12 -, potential 326, 327
Cutrine Plus 248 Denitrification 120, 121, 127, 149, 168,
Cyanamide 229, 230 274,337-340
Cyanobacteria 247 Desthiobiotin 163-165
4-Cyano-N-(diaminophosphinyl) benz- Desulfuration 216, 217
amide 193, 278 Dhaincha 175
Cyanoguanidine 229 Dialkyldithiocarbamates 52
Cyanuric acid 276, 354, 355 3,5-DiaUyltetrahydro-l ,3,5-thiadiazine-
S-Cycloalkyldiamidophosphoro- 2-thione 71
thiolates 141 Diamidophosphoric acid 105
N-Cyclohexylchioromaleimide 68 - - phenyl ester 105
S-Cyclohexyldiamidophosphoro- Diamidophosphorothiolate compounds
thiolate 141 141, 142
N-Cyclohexyldichloromaleimide 68 Diamidothiophosphoric acid 105
N-Cyclohexylmaleimide 68 Diamidothiophosphorothiolate com-
N-Cyclohexylphosphoric triamide 196, pounds 141
199-202,217-220,249,250,257, N-(Diaminophosphinyl)arylcarbox-
275,279,288,336,352,353 amides 356
Cyclohexylphosphorodiamidate 112, N-(Diaminophosphinyl)benzamide
113 182, 193, 195, 196,275,278,337,
3-Cyclohexylrhodanine-5-acetic acid 338
76 N-(Diaminophosphinyl)benzene-
N-Cyclohexylthiophosphoric triamide acetamide 193,278,337,338
143,217-220 N-(Diaminophosphinyl)benzenesulfon-
Cyclophosphazane compounds 350 amide 141
Cyclophosphazanic acids, potassium N-(Diaminophosphinyl)-2-chloroacet-
salts of350 amide 143,240
Cyclotetraphosphazatetraene deri- O-Diaminophosphinyl derivatives of
vatives 159 oximes 140
Cyclotriphosphazatriene derivatives N-(Diaminophosphinyl)-2,2-dichloro-
155-160,220,260,278,291,297, acetamide 143,240
315,318,332,335 N-(Diaminophosphinyl)-4-fluorobenz-
Cymbopogam confertiflorus 170 amide 357
Cymbopogon flexuosus 297 N-(Diaminophosphinyl)-4-( l' -male-
- - var. flexuosus 311 imido)benzamide 144
- winterianus 231 N-(Diaminophosphinyl)-4-methoxy-
Cynodon dactylon 297 benzamide 240
401
Starch 22 2,2,6,6-Tetrachlorocyc1ohexylphos-
Stearamine 31 phorodiamidate 112, 113
Stearic acid 2, 83 1,1,2,2-Tetrachloro-l ,2-dibromoethane
Stenotaphrum secundatum 297 53,54,256
Strontium chloride 24 2,2,4,4-Tetrachloro-6,6-di(dimethyl-
Struvite 356 amino )cyc1otriphosphazatriene 156
Subtropical plants 231 Tetrachlorohydroquinone 89
Succinic acid 2 Tetrachloroquinhydrone 89
Succinomonohydroxamic acid 66 2,2,4,4-Tetra(dimethylamino)-6,6-di-
Sugarbeet 198, 199,315,317,318 aminocyc1otriphosphazatriene 156
Sugarcane 30,315,316,318 Tetrafluoro-p-benzoquinone 87
Sulfanilamide 162, 163, 180 Tetrahydro-l,3,5-thiadiazine-2-thiones
Sulfanilic acid, amide of 162 71, 72, 236
Sulfates 7, 9, 13, 26, 37, 38, 358 1,2,5,8-Tetrahydroxyanthraquinone 91
Sulfathiazole 222, 225, 237, 312, 313 Tetrahydroxy-p-benzoquinone 87, 88
Sulfide 37 Tetramethoxy-p-benzoquinone 87
- minerals 358 Tetramethyl-p-benzoquinone 87, 89
Sulfite 37 Tetramethylthiuram disulfide 56,57,
Sulfur 2, 143, 173, 176, 252, 263, 318 88,180,236,314,324,331,341
- Cote 313 Tetranortriterpenoid isomers 171
Sulfuric acid 18, 72 Tetraphosphorodiamide 139
Supergranules 129, 173-175,232,233, Tetrathionate 41
296 - anion 37
SuperN 258 Thermodynamic parameters/values 99,
Superphosphate 10,29,32,49, 121, 100
122,148,151-153,167,174,182, 1,3,4-Thiadiazoline-2-thiones 72, 73,
187,234,246,250,264,273,274, 358
283,291,311,347,348,350 -, thiol form of 72, 73
Super Urea 258 -, thione form of 72, 73
Sweet potato 315, 317 Thioacetamide 191
Synergism/synergistic 59, 84, Ill, 163, Thiobacillus ferrooxidans 358
180-182,210,242,244 - thioparus 358
2-Thiocarboxamido-5-aminothiazole
T 77
Tailings 358 2-Thiocarboxamido-5-benzamido-
Tamarind 177 thiazole 77
Tamarindus indica 177 2-Thiocarboxamidothiazoles 77
Tannic acid 353 Thione 72, 128, 195
Tannins 166, 169 2-Thiono-5,6-dimethyl-l ,3,2-dioxa-
Tartaric acid 178 phosphorinane compounds 161,
Tea 169, 170, 177,241,353 162
Temperature coefficient 99 Thiophosphoric triamide compounds
Terbutryn 182, 183,249,250,288 143,198,213,256,274,285,291,
Terminalia chebula 169 294-296,306,315,318,328,332,
Terradiazole 226 335
Tetrachloro-o-benzoquinone 89 Thiophosphoric triamides 142, 144
Tetrachloro-p-benzoquinone 87, 95 - -, N-acy1143
416
Urea (continued) W
- hydrolysis 1-3,5,6,8,9, 12 13, 16, Waste rocks 358
20-26,29,35-45,47,50,52,56, Waxes 2, 5, 6,30, 33,46,47, 182
58-61,63,64,66,67,69, 71, 72, Wheat 54, 98, 117, 129, 149, 150, 154,
74,77,79,81,83,84,86,88,90- 198,199,230,243,245,246,253,
95,97-100,105,109,115-119, 257,259,262-280,291,295,310,
121,122,124-127,129-131,134, 313,328,332,347,348
135,137,146-150,152-154,156,
160,163-165,167-170,172-178, X
181, 182, 184, 188-192, 195-212, Xanthates 58-60, 236, 250
216-219,221,223,225-236,238, -, branched-chain 59
244,245,247-249,252,257,259, -, straight-chain 59
260,264,276,277,279,282,285, Xylanase 321
315,326,331,347,352-354,356
- phosphate 126,284 Y
Urea-ammonium nitrate 17, 36-41, Yellow lupine 313
115, 145, 150, 153, 154, 158, 183, Yields, crop/plant 17, 19,23, 144, 150,
184,198,201,214,243,244,252- 152,153,246,248,251-275,277,
260,263,265,273-275,296,298, 279-309,311-318,347-351
299,309,352 Yucca schidigera 171,262,355
Urea-calcium nitrate 298
Urea-formaldehyde 41 Z
Ureaforms 2 Zea mays 33,117,124,137,148,171,
Ureaplasma urealyticum 356, 357 251
Urease 1-26,29,32-39,41-49.51-83. Zinc 8, 9, 11. 13, 15-17, 179, 188,233.
85-93,95-107,110,112-114,117, 313,322,329,330,336,349
118.121,127-130,134-148,152- - acetate 8
167,169-172,174,177,179-183, - chloride 7, 10, 12-14
185,187.189-201.205,208,210, - d~methyldithiocarbamate 55
211.213,215-243,245-251,258, - ethylene-l ,2-bisdithiocarbamate 54,
261-267,275-277,280,281.287, 55
288,291-293,297,300,310-313, - ions 53, 233
315,316,318,321-331,333-343, - oxide 16
347,351-359 - salts 76. 112
Usnic acid 3 - stearate 52
- sulfate 10, 11, 13-17, 187, 246, 264,
V 274,283,313
Vanadium 15,336 Zineb 53-56, 265
Vanadium (II) ions 9 Ziram 55
Vanillic acid 178
Vegetable crops 347
Veratro1178
Verdure density 298
Vetch 23,224
Viciafaba 313