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Effects of trace elements on urease activity in soils
EFFECTS
OF TRACE ELEMENTS ON ACTIVITY IN SOILS* M. A.
UREASE
TABATABAI
Department of Agronomy, Iowa State University. Ames, IA 5001 I, U.S.A. (Accepted
28 May
1976)
Summary~Disposal of sewage sludges and effluents on agricultural land is becoming a widespread practice. Most sludge samples disposed on soils contain large quantities of various trace elements. Studies of 20 trace elements commonly found in sludge samples showed that they inhibit the activity of urease in soils and that their order of effectiveness as inhibitors of urease depends on the soil. When the trace elements were compared by using 5 pmol’g- ’ soil. however, some of them showed the same order of effectiveness as urease inhibitors in the six soils studied i.e., for the monovalent > Cd” > Zn’+ > Sn*+ > Mn”, and generally, Fe3’ > and divalent ions, Ag+ 2 Hg2+ > Cu” Fe” and Cu*’ > Cu+. Other trace element ions that inhibited urease were Ni2+, Co’+. Pb’+, Ba2+, As3+, B3+ , Cr3+? A13+. V4+, Se4’, and MO”. Of the trace element ions studied, only As’+ and W6+ did not inhibit urease activity in soils. Studies on the distribution of urease activity showed that it is concentrated in surface soils and decreases with depth. Urease activity was proportional to organic C distribution in each soil profile and was significantly correlated with organic C in the surface soils studied.
INTRODUCTION
because inhibition of urease activity by trace elements added to surface soils may lead to movement of the water-soluble urea to groundwater (Doak, 1952). The objectives of the work reported here were to study the relative effectiveness of trace elements in inhibition of urease activity in soils, to determine the distribution of urease activity in a wide range of agricultural soil profiles, and to determine the relationship, if any, between urease activity and organic C in both surface and soil profile samples.
Disposal of sewage sludges and effluents on agricultural land is becoming a widespread practice. All such materials contain large quantities of various trace elements? that could be retained by and thus accumulated in soils (Berrow and Webber, 1972; Dean and Smith, 1973; Lindsay, 1973). Also, soil pollution by heavy metals is one of the major environmental problems associated with industries involved in processing of metals. Although research dealing with the chemistry of trace elements in soils and their toxicity to biological systems, including accumulation by plants, is extensive (Chaney, 1973; Kubota and Allaway, 1972; Lagerwerff, 1972; Page, 1974). little is known about the effects of these elements on biochemical reactions in soils. Knowledge of the relative effects of trace elements on urease activity is important because this enzyme is highly sensitive to trace quantities of metal ions (Shaw, 1954), and because its substrate, urea, is added to soils as a synthetic fertilizer and in animal excreta. Although the patterns of distribution of urease activity in soil profiles have been studied by many workers, the relative factors that affect the activity of urease through the profile have not been clearly established. Like other biochemical reactions in soils, however, urease activity is associated with surface soils (Hoffmann, 1959; Hofmann and Kesseba, 1962; Hofmann and Schmidt, 1953; Myers and McGarity, 1968). In studies of the effects of trace elements on urease activity in surface soils, the distribution and activity of urease in soil profiles is of real importance
MATERIALS
AND METHODS
The soils used (Table 1) were surface soils selected to include a wide range in pH, texture. organic-matter content, and urease activity. Before use, each soil was air-dried and crushed to pass a 2-mm screen. The analyses reported in Table 1 were performed as described by Tabatabai and Bremner (1969). The profile samples were selected to include a wide range of urease activity in their surface soils. The trace elements used in this work were Fisher certified reagentgrade chemicals obtained from Fisher Scientific Co., Chicago, Ill. The method of Tabatabai and Bremner (1972) was used for assay of urease activity. In testing the effects of trace elements on urease activity, 5 g soil in a 50-ml volumetric flask were treated with 1.5 ml solution containing either 2.5 or 25 pmole of trace element (0.5 or 5 pmole.g-’ soil). This solution was added dropwise to moisten the whole soil sample. After 30 min equilibration, the moist soil was treated with 0.2 ml toluene (as a bacteriostat), 7.5 ml 0.05 M tris(hydroxymethyl)aminomethane (THAM) buffer, pH 9.0, and 1 ml 0.2 M urea. The volumetric flask was stoppered, swirled to mix the contents, and incubated at 37°C. After 2 h the flask was unstoppered, and the volume was made up with 2.5 M KCI containing 100 parts/lo6 of Ag,SO,. After mixing, 20-ml of the soil
* Journal Paper No. J-8189 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Projects 1845 and 2082. t The term “trace element” is used here to refer to elements which are, when present in sufficient concentrations. toxic to living systems. 9
IO
M. A. TABATARAI Table 1. Analyses of soils Soil
Total N
Organic C (%)
PH
(%I
Clay (%)
Sand
Urease activity*
(%I
Weller
5.1
1.51
0.133
17
1
18
Nicollet
6.1
3.32
0.253
30
25
33
Webster
5.8
2.58
0.210
23
38
43
Harps
7.8
3.74
0.305
30
26
150
L"tO*
6.8
4.35
0.388
42
3
210
Okoboji
7.4
5.45
0.463
34
16
263
*
,~g Nl$,-N
re1eased.g
-1
soil.Zh
-I
.
suspension was taken for NH,+-N analysis (Bremner and Keeney, 1966). In the controls the 1 ml of urea solution was added after addition of about 30 ml KCl-Ag,SO, reagent and before making up the volume of the incubation flask. The results of the urease activity from the trace element-treated soils were compared with those obtained with 5 g soil treated with 1.5 ml water. Percentage inhibition of urease activity by each trace element was calculated from (A - B/A) 100, where A is urease activity of untreated soil and J3 is urease activity of trace element-treated soil. Evaluation of the method for recovery of NH:-N added to the soils used in this work in the presence of the trace elements studied indicated that none of the elements interfered in the assay procedure described. All analyses reported are averages of duplicate determinations. RESULTS AND
DISCUSSION
Results (Table 2) show that, with the exception of As” and W6+, the other 18 trace elements studied inhibited the urease activity of the six soils. In studies of determination of metal ion inhibitors of the urea-urease system the pH of the incubation medium is controlled either by a pH-stat method or, more frequently, by a buffer to maintain the pH desired (Hughes et al., 1969; Katz and Cowans, 1965; Toren and Burger, 1968). Also, studies of inhibition of enzyme in soils (or in solution) are normally carried out by comparing the relative inhibition by any compound or ion using mole quantities of inhibitors and assaying the activity of the enzyme under strictly standardized conditions. The conditions required to compare the effectiveness of trace elements in inhibition of urease activity in soils were observed in the work reported. To obtain a meaningful comparison, equimolar amounts of trace elements were added and the urease activity was assayed under optimum pH and urea concentration. THAM buffer was used in the work reported in this paper because Tabatabai and Bremner (1972) found that the amount of NH:-N released in the assay procedure used was considerably higher with 0.05 M THAM buffer than with other buffers they tested. Also, Wall and Laidler (1953) found that, unlike other buffers used in studies of urease activity e.g. sodium and potassium phosphate, THAM buffer has no activating or inhibiting effect on hydrolysis of urea by jackbean urease.
THAM buffer is widely used for studies involving inhibition of purified jackbean urease by metal ions (Hanlon et ul.. 1966; Hughes rt ~1.. 1969; Katz and Cowans, 1965). The buffer used should have a minimum or no effect on the binding of trace element ions with soil proteins because the trace elements were added to the soil 30 min before urease assay, and because the stability constants of metal-soil organic matter complexes are much greater than the stability constants of metal--THAM buffer complexes (Hanlon c’t al., 1966; Stevenson and Ardakani. 1972). It is known that different metal ions exhibit quite different behavior in their ability to act as enzyme inhibitors. With urease in solutions, for example, the Ag’ ion is an extremely efficient inhibitor, but the Mn” ion is relatively very weak (Shaw, 1954). Using 50 parts/lo6 of metal ions in the absence of buffer to control the pH, Bremner and Douglas (1971) found that the inhibitory effects of metallic cations decreased in the order: Ag+ > Hg*’ > Au3+ > Cu*+, Cu+ > Co’+, Pb’+, As3+. Cr3+, Ni*’ and that Naf, K+, Ca*‘, Ba*+, Zn*+, Mn*+, A13+, and Fe3+ did not inhibit urease activity in soils. Comparison of the effectiveness of the trace elements in inhibition of soil urease activity showed that in some instances their order of effectiveness is affected by the chemical and physical properties of the soil. When the trace elements studied were compared by using 5 pmole.g-’ soil, however, some of them showed the same order of effectiveness in inhibition of urease activity in the six soils used. For the monovalent and divalent ions, the order of effectiveness was Ag+ 2 Hg*+ > Cu*+ > Cd*+ > Zn*’ > Sn’* > Mn2+ (Table 2). The results also showed that, generally, Fe3+ and Cu*+ are more effective in inhibiting urease activity than Fe*+ and Cu+, respectively, and that As5+ and W6’ do not inhibit the activity of this enzyme in soils. Other trace element ions that markedly inhibited urease activity in some soils at 5 pmole.g- ’ soil were Ni2+, Co*+, Pb2+, Ba2+. As3+, B3+. Cr3+, A13+, V4+, Se4+, and Moe+. These ions did not show any trend in their order of effectiveness. This could be due, in part, to reaction of portions of the ions added with mineral constituents of the soils, therefore causing the change in the order of their effectiveness. There was a marked decrease in the effectiveness of trace-element ions in inhibition of urease activity when 0.5 prnole’g-’ soil was compared with 5 pmole trace element. g _ ’ soil. This decrease was expected because smaller proportions of the -SH groups of
11
Effects of trace elements on urease Table 2. Effects of trace elements on urease activity in soils* wace element Valence Element state
Compound
so 4
Percentage
inhibition of wease
activity
in soil specified
Weller
Nicollet
Webster
Harps
Ixton
Okoboji
94 21
97 30
98 60
89 (46) 34 (21)
96 53
84 (31) 36 (14)
89 72 67 61 56 44 39 33 22 17 a 2
95 58 51 33 20 4 15 20 16 2 27 2
98 69 50 23 24
84 (29) 94 (24) 49 (19)
5 ( 5) 16 ( 0) 7 ( 0)
89 59 58 51 18 10 20 28 24 7 6 3
75 51 49 30 18 6 16 20 29 14 13 7
(25) (17) (13) ( 6) ( 4) ( 3) ( 7) (13) (22) ( 5) ( 0) ( 0)
3+
98 98 50 50 33
9 13 22 20 38
27 18 25 21 21
24 14 20 12 17
( 7) (11) ( 5) ( 3) ( 5)
44 27 17 17 22
18 15 29 23 19
(14) (13) (19) ( 5) ( 3)
V Se
4+
39 33
17 24
18 14
17 ( 3) 19 ( 5)
18 16
28 (13)
AS
5f
Na2XAs04
0
0
0 ( 0)
0
0 ( 0)
MC W
6+
H2Mo04 Na2W04
16 0
14 0
12 (11) 0 ( 0)
4 0
16 ( 9) 0 ( 0)
& C"
1+
Ag
Hg CU Cd zn sn Mn Fe Ni CO Pb Pb Ba
2f
&Cl2
AS B CL Al Fe
cuE1 cuso4
CdS04 ZIISO4 SnClz Ma12 F&04 NiCl..
34 15 11 8 27 35
7
16 25 23 11 55 12
*
( 7) ( 3) ( 2) ( 4) ( 3) (13)
-1 5 wulle trace e1ement.g soil. Figure in parentheses indicates percentage of urease activity using 0.5 vmole trace element.g-l soil.
urease reacted with the trace element ions when 0.5 pmole was used. The less-than-complete inhibition of urease activity with 5 nmole Ag+ or Hg’+.g-’ soil in the soils used is of interest because these two elements are very potent inhibitors of urease activity. It seems, however, that, at this concentration (540 parts/lo6 Ag or 1000 parts/lo6 Hg), a large portion of the urease active sites was not blocked by these ions. Also, although As3+ inhibited urease activity, As5+ did not show any effect on the activity of this enzyme in the soils studied. In a study involving direct potentiometric measurement of the NH,+ produced in a urea-urease system maintained at pH 7.0 by a 0.1 M THAM buffer, Katz and Cowans (1965) showed that at 14 mM NO; demonstrates a slight competitive inhibition effect on
24 ( 9)
inhibition
the action of jackbean urease. Results reported in Table 2 Show that Pb(N0,)2 was a more effective inhibitor than lead acetate in three of the soils (Nicollet, Webster and Harps), had similar effect as lead acetate in two of the soils (Luton and Okoboji) and was less effective than lead acetate in one of the soils (Weller). The effect of NO; on urease activity in soils remains to be investigated. Sodium arsenate and sodium tungstate had no effect on the activity of urease in soils, indicating that the Na+ of the other compounds studied had no inhibitory effect at the concentration tested. The Cl- and SO:- that are associated with the metal ions used have been shown to have no effect on jackbean urease; the buffer used was made with H2S0, (Wall and Laidler, 1953).
Table 3. Urease activity of selected Iowa soil profile samples
Hagener Depth, cm o-15 15-30 30-60 60-90 90-120
oc* 0.57 0.79 0.32 0.18 0.17
UA** 23 24 22 16 9
ClSKiOIl
oc
UA
o-15 15-30 30-60 60-90 90-120
2.68 2.03 1.35 0.60 0.22
78 46 21 15 13
"OC, organic
carbon (x).
Webster OC UA 3.04 3.09 1.10 0.34 0.24
45 43 40 13 8
Hamburg oc UA 1.57 0.88 0.45 0.31 0.24
32 14 9
T_ oc 2.07 2.04 1.34 0.90 0.55
UA
SharpsburC: oc UA
75 48 25 9 7
1.80 1.19 0.56 0.34 0.25
Fayette UA oc 1.96 0.71 0.45 0.35 0.31
127, 26 6 3 2
-1 ** soil.2h-I). UA, Urease activity (pg NH&-N re1eased.g
76 30 13 4 4
Harps oc UA 4.34 3.66 1.15 0.52 0.20
390 316 112 28 4
12
M. A.
TABATABAI
Table 4. Simple correlation coefficients (r) for the relationship between nrease activity and organic carbon in surface and in profile samples of some Iowa soils Correlation
samples
Soil
Surface
0.922**
(Table 1)
samples (Table 1 plus surface of profile
Profile
samples)
0.?44**
samples
Hagener
0.678
Webster
0. s30*
Tam
0.925*
Sharpsburg
o-969**
Clarion
0.939**
Hamburg
0.990**
Fayette
0.998**
Harps
0.998*
All samples
* Significant **
(I)
samples
6 samples 14
coefficient
Significant
used
0.797*
at P < 0.05. at P < 0.01.
Studies of enzyme activity in soil-profile samples have shown that activity usually decreases with increase in sample depth and that this decrease is accompanied by a decrease in organic-matter content (Hoffmann, 1959; Myers and McGarity, 1968; Ross and Roberts, 1968; Skujins, 1967). Table 3 shows results obtained in a study of distribution of urease activity in the profiles of eight Iowa soils. The Webster, Hamburg and Harps soils were under permanent grass at the time of sampling. The other soils were from cornfields that had been cultivated before samples were taken. The data reported show that urease activity decreased markedly with depth in each of the eight profiles examined and that this decrease was associated with a decrease in organic-C content. Statistical analysis of the relationship between- urease activity and organic-C content in the surface soils and in the profile samples studied indicated that, with the exception of one profile (Hagener), urease activity was significantly correlated with organic C (Table 4). Statistical analysis of the pooled data for the eight profiles and those of Table 1 also showed that urease activity was significantly correlated with organic C (r = 0.797**). Among the many factors that may affect urease activity in soils, cropping history, soil amendments, and some environmental factors have a special influence on the activity of urease and other enzymes in soils. Considering the environmental factors, for example, Stojanovic (1959) found marked seasonal variations in urease activity in Mississippi soils.
Organic matter, however, seems to have a special influence on urease activity in soils, as evident from the correlation coefficients of the relationship between urease activity and organic C reported in Table 4. REFERENCES
BERROWM. C. and WEBBERJ. (1972) Trace elements in sewage sludges. J. Sci. Fd Agric. 23, 93-100. BR~MNERJ. M. and DOUGLASL. A. (1971) Inhibition of urease activity in soils. Soil Biol. Biachem. 3, 297-307. BREMNERJ. M. and KEENEYD. R. (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils-3. Exchangeable ~monium, nitrate, and nitrite by extraction-dist~liation methods. Pror. Soil Sci. Sm. Am 30, 577-582. CHANEYF. C. (1973) Crop and food chain effects of toxic elements in sludges and effluents. In Recq’cling Municipal Slurlges and Effluents on Land, pp. 129-141. National Assoc. of State Universities and Land Grant Colleges, Washington D. C. DEAN R. B. and SMITHJR. J. E. (1973) The properties of sludges. In Recycling Municipal Sludges and EfPurnts on Land, pp. 39-47. National Assoc. State Univeysities and Land Grant Colleges. Washington D. C. DOAK B. W. (1952) Some chemical changes in the nitrogenous constituents of urine when voided on pasture. 1. Agric. Sci. 42, 162-l 71.
HANLON D. P., WATT D. S. and WESTHEADE.
W. (1966) The interaction of divalent metal ions with tris buffer in dilute solution. Anal. ~ior~~~. 16. 225-233. HOFFMANNG. (1959) Verteilung und herkunft eiginer enzyme in boden. Z. ~~Eril~~r. Diing. Bodenk. 85. 97-104.
Eflects of trace elements HOFMANN E. and KESSEBA A. (1962) Untersuchungen uber enzyme in Agytichen boden. Z. PjErniihr. Diing. Bodenk. 99, 9-20. HOFMANN E. and SCHMIDT W. (1953) Uber das Enzymsystern unserer Kulturboden. II. Urease. Biochem. Z. 324, 125-127. HUGHES R. B., SIDNEY A. K. and STUBBINS S. E. (1969) Inhibition of urease by metal ions. Enzymologia 36, 332~m334. KATZ S. A. and COWANS J. A. (1965) Direct potentiometric study of urea-urease system. Biochem. Biophys. Acra 107. 605.-608. KUBOTA J. and ALLAWAY W. H. (1972) Geographic distribution of trace element problems. In Micronutrients in Agriculture (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, Eds) pp. 52>554. Soil Sci. Sot. Am. Madison, Wisconsin. LAGERWERFF J. V. (1972) Lead, mercury, and cadmium as environmental contaminants. In Micronutrients in Agriculture (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, Eds) pp. 593-636. Soil Sci. Sot. Am. Madison, Wisconsin. LINDSAY W. L. (1973) Inorganic reactions of sewage wastes with soils. In Recycling Municipal Sludges and EfPuents .. on Land. pp. 91-96 National Assoc. State Universities and Land Grant Colleges, Washington, D.C. MYERS M. G. and MCGARITY J. W. (1968) The urease activity in profiles of five great soil groups from Northern New South Wales. PI. Soil 28, 25-36. PAGE A. L. (1974) Fate and Effects of Trace Elements in Sewage Sludge when Applied to Agricultural Land: A
on urease
13
Literature Review. Environmental Protection Agency, Cincinnati, Ohio. Ross D. J. and ROBERTS H. S. (1968) A study of activities of enzymes hydrolysing sucrose and starch and of oxygen uptake in a sequence of soils under tussock grassland. J. Soil Sci. 19, 186-196. SHAW W. H. R. (1954) The inhibition of urease by various metal ions. J. Am. them. Sot. 76, 2160-2163. SKUJINS J. J. (1967) Enzymes in soil. In Soil Biochemistry (A. D. McLaren and G. H. Peterson, Eds) pp. 371414. Marcel Dekker, New York. STEVENSONF. J. and ARDAKANI M. S. (1972) Organic matter reactions involving micronutrients in soils. In Micronutrirnts in Agriculture (J. J. Mortvedt. P. M. Giordano, and W. L. Lindsay. Eds) pp. 79-114. Soil Sci. Sot. Am.. Madison. Wisconsin. STOJANOVIC B. J. (1959) Hydrolysis of urea in soil as affected by season and added urease. Soil Sci. 88, 251-255. TABATABAI M. A. and BREMNERJ. M. (1969) Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol. Biochem. 1, 301-307. TABATABAI M. A. and BREMNER J. M. (1972) Assay of urease activity in soils. Soil Biol. B&hem. 4, 479487. TOREN E. C. and BURGER F. J. (1968) Trace determination of metal ion inhibitors of the urea-urease system by a nH-stat kinetic method. Mikrochim Acta 5. 1049- 1058. WILL M. C. and LAIDLER K. J. (1953) The molecular kinetics of the urea-urease system-IV. The reaction in an inert buffer. Arch. Biochem. Biophys. 43. 299-306.
OF TRACE ELEMENTS ON ACTIVITY IN SOILS* M. A.
UREASE
TABATABAI
Department of Agronomy, Iowa State University. Ames, IA 5001 I, U.S.A. (Accepted
28 May
1976)
Summary~Disposal of sewage sludges and effluents on agricultural land is becoming a widespread practice. Most sludge samples disposed on soils contain large quantities of various trace elements. Studies of 20 trace elements commonly found in sludge samples showed that they inhibit the activity of urease in soils and that their order of effectiveness as inhibitors of urease depends on the soil. When the trace elements were compared by using 5 pmol’g- ’ soil. however, some of them showed the same order of effectiveness as urease inhibitors in the six soils studied i.e., for the monovalent > Cd” > Zn’+ > Sn*+ > Mn”, and generally, Fe3’ > and divalent ions, Ag+ 2 Hg2+ > Cu” Fe” and Cu*’ > Cu+. Other trace element ions that inhibited urease were Ni2+, Co’+. Pb’+, Ba2+, As3+, B3+ , Cr3+? A13+. V4+, Se4’, and MO”. Of the trace element ions studied, only As’+ and W6+ did not inhibit urease activity in soils. Studies on the distribution of urease activity showed that it is concentrated in surface soils and decreases with depth. Urease activity was proportional to organic C distribution in each soil profile and was significantly correlated with organic C in the surface soils studied.
INTRODUCTION
because inhibition of urease activity by trace elements added to surface soils may lead to movement of the water-soluble urea to groundwater (Doak, 1952). The objectives of the work reported here were to study the relative effectiveness of trace elements in inhibition of urease activity in soils, to determine the distribution of urease activity in a wide range of agricultural soil profiles, and to determine the relationship, if any, between urease activity and organic C in both surface and soil profile samples.
Disposal of sewage sludges and effluents on agricultural land is becoming a widespread practice. All such materials contain large quantities of various trace elements? that could be retained by and thus accumulated in soils (Berrow and Webber, 1972; Dean and Smith, 1973; Lindsay, 1973). Also, soil pollution by heavy metals is one of the major environmental problems associated with industries involved in processing of metals. Although research dealing with the chemistry of trace elements in soils and their toxicity to biological systems, including accumulation by plants, is extensive (Chaney, 1973; Kubota and Allaway, 1972; Lagerwerff, 1972; Page, 1974). little is known about the effects of these elements on biochemical reactions in soils. Knowledge of the relative effects of trace elements on urease activity is important because this enzyme is highly sensitive to trace quantities of metal ions (Shaw, 1954), and because its substrate, urea, is added to soils as a synthetic fertilizer and in animal excreta. Although the patterns of distribution of urease activity in soil profiles have been studied by many workers, the relative factors that affect the activity of urease through the profile have not been clearly established. Like other biochemical reactions in soils, however, urease activity is associated with surface soils (Hoffmann, 1959; Hofmann and Kesseba, 1962; Hofmann and Schmidt, 1953; Myers and McGarity, 1968). In studies of the effects of trace elements on urease activity in surface soils, the distribution and activity of urease in soil profiles is of real importance
MATERIALS
AND METHODS
The soils used (Table 1) were surface soils selected to include a wide range in pH, texture. organic-matter content, and urease activity. Before use, each soil was air-dried and crushed to pass a 2-mm screen. The analyses reported in Table 1 were performed as described by Tabatabai and Bremner (1969). The profile samples were selected to include a wide range of urease activity in their surface soils. The trace elements used in this work were Fisher certified reagentgrade chemicals obtained from Fisher Scientific Co., Chicago, Ill. The method of Tabatabai and Bremner (1972) was used for assay of urease activity. In testing the effects of trace elements on urease activity, 5 g soil in a 50-ml volumetric flask were treated with 1.5 ml solution containing either 2.5 or 25 pmole of trace element (0.5 or 5 pmole.g-’ soil). This solution was added dropwise to moisten the whole soil sample. After 30 min equilibration, the moist soil was treated with 0.2 ml toluene (as a bacteriostat), 7.5 ml 0.05 M tris(hydroxymethyl)aminomethane (THAM) buffer, pH 9.0, and 1 ml 0.2 M urea. The volumetric flask was stoppered, swirled to mix the contents, and incubated at 37°C. After 2 h the flask was unstoppered, and the volume was made up with 2.5 M KCI containing 100 parts/lo6 of Ag,SO,. After mixing, 20-ml of the soil
* Journal Paper No. J-8189 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Projects 1845 and 2082. t The term “trace element” is used here to refer to elements which are, when present in sufficient concentrations. toxic to living systems. 9
IO
M. A. TABATARAI Table 1. Analyses of soils Soil
Total N
Organic C (%)
PH
(%I
Clay (%)
Sand
Urease activity*
(%I
Weller
5.1
1.51
0.133
17
1
18
Nicollet
6.1
3.32
0.253
30
25
33
Webster
5.8
2.58
0.210
23
38
43
Harps
7.8
3.74
0.305
30
26
150
L"tO*
6.8
4.35
0.388
42
3
210
Okoboji
7.4
5.45
0.463
34
16
263
*
,~g Nl$,-N
re1eased.g
-1
soil.Zh
-I
.
suspension was taken for NH,+-N analysis (Bremner and Keeney, 1966). In the controls the 1 ml of urea solution was added after addition of about 30 ml KCl-Ag,SO, reagent and before making up the volume of the incubation flask. The results of the urease activity from the trace element-treated soils were compared with those obtained with 5 g soil treated with 1.5 ml water. Percentage inhibition of urease activity by each trace element was calculated from (A - B/A) 100, where A is urease activity of untreated soil and J3 is urease activity of trace element-treated soil. Evaluation of the method for recovery of NH:-N added to the soils used in this work in the presence of the trace elements studied indicated that none of the elements interfered in the assay procedure described. All analyses reported are averages of duplicate determinations. RESULTS AND
DISCUSSION
Results (Table 2) show that, with the exception of As” and W6+, the other 18 trace elements studied inhibited the urease activity of the six soils. In studies of determination of metal ion inhibitors of the urea-urease system the pH of the incubation medium is controlled either by a pH-stat method or, more frequently, by a buffer to maintain the pH desired (Hughes et al., 1969; Katz and Cowans, 1965; Toren and Burger, 1968). Also, studies of inhibition of enzyme in soils (or in solution) are normally carried out by comparing the relative inhibition by any compound or ion using mole quantities of inhibitors and assaying the activity of the enzyme under strictly standardized conditions. The conditions required to compare the effectiveness of trace elements in inhibition of urease activity in soils were observed in the work reported. To obtain a meaningful comparison, equimolar amounts of trace elements were added and the urease activity was assayed under optimum pH and urea concentration. THAM buffer was used in the work reported in this paper because Tabatabai and Bremner (1972) found that the amount of NH:-N released in the assay procedure used was considerably higher with 0.05 M THAM buffer than with other buffers they tested. Also, Wall and Laidler (1953) found that, unlike other buffers used in studies of urease activity e.g. sodium and potassium phosphate, THAM buffer has no activating or inhibiting effect on hydrolysis of urea by jackbean urease.
THAM buffer is widely used for studies involving inhibition of purified jackbean urease by metal ions (Hanlon et ul.. 1966; Hughes rt ~1.. 1969; Katz and Cowans, 1965). The buffer used should have a minimum or no effect on the binding of trace element ions with soil proteins because the trace elements were added to the soil 30 min before urease assay, and because the stability constants of metal-soil organic matter complexes are much greater than the stability constants of metal--THAM buffer complexes (Hanlon c’t al., 1966; Stevenson and Ardakani. 1972). It is known that different metal ions exhibit quite different behavior in their ability to act as enzyme inhibitors. With urease in solutions, for example, the Ag’ ion is an extremely efficient inhibitor, but the Mn” ion is relatively very weak (Shaw, 1954). Using 50 parts/lo6 of metal ions in the absence of buffer to control the pH, Bremner and Douglas (1971) found that the inhibitory effects of metallic cations decreased in the order: Ag+ > Hg*’ > Au3+ > Cu*+, Cu+ > Co’+, Pb’+, As3+. Cr3+, Ni*’ and that Naf, K+, Ca*‘, Ba*+, Zn*+, Mn*+, A13+, and Fe3+ did not inhibit urease activity in soils. Comparison of the effectiveness of the trace elements in inhibition of soil urease activity showed that in some instances their order of effectiveness is affected by the chemical and physical properties of the soil. When the trace elements studied were compared by using 5 pmole.g-’ soil, however, some of them showed the same order of effectiveness in inhibition of urease activity in the six soils used. For the monovalent and divalent ions, the order of effectiveness was Ag+ 2 Hg*+ > Cu*+ > Cd*+ > Zn*’ > Sn’* > Mn2+ (Table 2). The results also showed that, generally, Fe3+ and Cu*+ are more effective in inhibiting urease activity than Fe*+ and Cu+, respectively, and that As5+ and W6’ do not inhibit the activity of this enzyme in soils. Other trace element ions that markedly inhibited urease activity in some soils at 5 pmole.g- ’ soil were Ni2+, Co*+, Pb2+, Ba2+. As3+, B3+. Cr3+, A13+, V4+, Se4+, and Moe+. These ions did not show any trend in their order of effectiveness. This could be due, in part, to reaction of portions of the ions added with mineral constituents of the soils, therefore causing the change in the order of their effectiveness. There was a marked decrease in the effectiveness of trace-element ions in inhibition of urease activity when 0.5 prnole’g-’ soil was compared with 5 pmole trace element. g _ ’ soil. This decrease was expected because smaller proportions of the -SH groups of
11
Effects of trace elements on urease Table 2. Effects of trace elements on urease activity in soils* wace element Valence Element state
Compound
so 4
Percentage
inhibition of wease
activity
in soil specified
Weller
Nicollet
Webster
Harps
Ixton
Okoboji
94 21
97 30
98 60
89 (46) 34 (21)
96 53
84 (31) 36 (14)
89 72 67 61 56 44 39 33 22 17 a 2
95 58 51 33 20 4 15 20 16 2 27 2
98 69 50 23 24
84 (29) 94 (24) 49 (19)
5 ( 5) 16 ( 0) 7 ( 0)
89 59 58 51 18 10 20 28 24 7 6 3
75 51 49 30 18 6 16 20 29 14 13 7
(25) (17) (13) ( 6) ( 4) ( 3) ( 7) (13) (22) ( 5) ( 0) ( 0)
3+
98 98 50 50 33
9 13 22 20 38
27 18 25 21 21
24 14 20 12 17
( 7) (11) ( 5) ( 3) ( 5)
44 27 17 17 22
18 15 29 23 19
(14) (13) (19) ( 5) ( 3)
V Se
4+
39 33
17 24
18 14
17 ( 3) 19 ( 5)
18 16
28 (13)
AS
5f
Na2XAs04
0
0
0 ( 0)
0
0 ( 0)
MC W
6+
H2Mo04 Na2W04
16 0
14 0
12 (11) 0 ( 0)
4 0
16 ( 9) 0 ( 0)
& C"
1+
Ag
Hg CU Cd zn sn Mn Fe Ni CO Pb Pb Ba
2f
&Cl2
AS B CL Al Fe
cuE1 cuso4
CdS04 ZIISO4 SnClz Ma12 F&04 NiCl..
34 15 11 8 27 35
7
16 25 23 11 55 12
*
( 7) ( 3) ( 2) ( 4) ( 3) (13)
-1 5 wulle trace e1ement.g soil. Figure in parentheses indicates percentage of urease activity using 0.5 vmole trace element.g-l soil.
urease reacted with the trace element ions when 0.5 pmole was used. The less-than-complete inhibition of urease activity with 5 nmole Ag+ or Hg’+.g-’ soil in the soils used is of interest because these two elements are very potent inhibitors of urease activity. It seems, however, that, at this concentration (540 parts/lo6 Ag or 1000 parts/lo6 Hg), a large portion of the urease active sites was not blocked by these ions. Also, although As3+ inhibited urease activity, As5+ did not show any effect on the activity of this enzyme in the soils studied. In a study involving direct potentiometric measurement of the NH,+ produced in a urea-urease system maintained at pH 7.0 by a 0.1 M THAM buffer, Katz and Cowans (1965) showed that at 14 mM NO; demonstrates a slight competitive inhibition effect on
24 ( 9)
inhibition
the action of jackbean urease. Results reported in Table 2 Show that Pb(N0,)2 was a more effective inhibitor than lead acetate in three of the soils (Nicollet, Webster and Harps), had similar effect as lead acetate in two of the soils (Luton and Okoboji) and was less effective than lead acetate in one of the soils (Weller). The effect of NO; on urease activity in soils remains to be investigated. Sodium arsenate and sodium tungstate had no effect on the activity of urease in soils, indicating that the Na+ of the other compounds studied had no inhibitory effect at the concentration tested. The Cl- and SO:- that are associated with the metal ions used have been shown to have no effect on jackbean urease; the buffer used was made with H2S0, (Wall and Laidler, 1953).
Table 3. Urease activity of selected Iowa soil profile samples
Hagener Depth, cm o-15 15-30 30-60 60-90 90-120
oc* 0.57 0.79 0.32 0.18 0.17
UA** 23 24 22 16 9
ClSKiOIl
oc
UA
o-15 15-30 30-60 60-90 90-120
2.68 2.03 1.35 0.60 0.22
78 46 21 15 13
"OC, organic
carbon (x).
Webster OC UA 3.04 3.09 1.10 0.34 0.24
45 43 40 13 8
Hamburg oc UA 1.57 0.88 0.45 0.31 0.24
32 14 9
T_ oc 2.07 2.04 1.34 0.90 0.55
UA
SharpsburC: oc UA
75 48 25 9 7
1.80 1.19 0.56 0.34 0.25
Fayette UA oc 1.96 0.71 0.45 0.35 0.31
127, 26 6 3 2
-1 ** soil.2h-I). UA, Urease activity (pg NH&-N re1eased.g
76 30 13 4 4
Harps oc UA 4.34 3.66 1.15 0.52 0.20
390 316 112 28 4
12
M. A.
TABATABAI
Table 4. Simple correlation coefficients (r) for the relationship between nrease activity and organic carbon in surface and in profile samples of some Iowa soils Correlation
samples
Soil
Surface
0.922**
(Table 1)
samples (Table 1 plus surface of profile
Profile
samples)
0.?44**
samples
Hagener
0.678
Webster
0. s30*
Tam
0.925*
Sharpsburg
o-969**
Clarion
0.939**
Hamburg
0.990**
Fayette
0.998**
Harps
0.998*
All samples
* Significant **
(I)
samples
6 samples 14
coefficient
Significant
used
0.797*
at P < 0.05. at P < 0.01.
Studies of enzyme activity in soil-profile samples have shown that activity usually decreases with increase in sample depth and that this decrease is accompanied by a decrease in organic-matter content (Hoffmann, 1959; Myers and McGarity, 1968; Ross and Roberts, 1968; Skujins, 1967). Table 3 shows results obtained in a study of distribution of urease activity in the profiles of eight Iowa soils. The Webster, Hamburg and Harps soils were under permanent grass at the time of sampling. The other soils were from cornfields that had been cultivated before samples were taken. The data reported show that urease activity decreased markedly with depth in each of the eight profiles examined and that this decrease was associated with a decrease in organic-C content. Statistical analysis of the relationship between- urease activity and organic-C content in the surface soils and in the profile samples studied indicated that, with the exception of one profile (Hagener), urease activity was significantly correlated with organic C (Table 4). Statistical analysis of the pooled data for the eight profiles and those of Table 1 also showed that urease activity was significantly correlated with organic C (r = 0.797**). Among the many factors that may affect urease activity in soils, cropping history, soil amendments, and some environmental factors have a special influence on the activity of urease and other enzymes in soils. Considering the environmental factors, for example, Stojanovic (1959) found marked seasonal variations in urease activity in Mississippi soils.
Organic matter, however, seems to have a special influence on urease activity in soils, as evident from the correlation coefficients of the relationship between urease activity and organic C reported in Table 4. REFERENCES
BERROWM. C. and WEBBERJ. (1972) Trace elements in sewage sludges. J. Sci. Fd Agric. 23, 93-100. BR~MNERJ. M. and DOUGLASL. A. (1971) Inhibition of urease activity in soils. Soil Biol. Biachem. 3, 297-307. BREMNERJ. M. and KEENEYD. R. (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils-3. Exchangeable ~monium, nitrate, and nitrite by extraction-dist~liation methods. Pror. Soil Sci. Sm. Am 30, 577-582. CHANEYF. C. (1973) Crop and food chain effects of toxic elements in sludges and effluents. In Recq’cling Municipal Slurlges and Effluents on Land, pp. 129-141. National Assoc. of State Universities and Land Grant Colleges, Washington D. C. DEAN R. B. and SMITHJR. J. E. (1973) The properties of sludges. In Recycling Municipal Sludges and EfPurnts on Land, pp. 39-47. National Assoc. State Univeysities and Land Grant Colleges. Washington D. C. DOAK B. W. (1952) Some chemical changes in the nitrogenous constituents of urine when voided on pasture. 1. Agric. Sci. 42, 162-l 71.
HANLON D. P., WATT D. S. and WESTHEADE.
W. (1966) The interaction of divalent metal ions with tris buffer in dilute solution. Anal. ~ior~~~. 16. 225-233. HOFFMANNG. (1959) Verteilung und herkunft eiginer enzyme in boden. Z. ~~Eril~~r. Diing. Bodenk. 85. 97-104.
Eflects of trace elements HOFMANN E. and KESSEBA A. (1962) Untersuchungen uber enzyme in Agytichen boden. Z. PjErniihr. Diing. Bodenk. 99, 9-20. HOFMANN E. and SCHMIDT W. (1953) Uber das Enzymsystern unserer Kulturboden. II. Urease. Biochem. Z. 324, 125-127. HUGHES R. B., SIDNEY A. K. and STUBBINS S. E. (1969) Inhibition of urease by metal ions. Enzymologia 36, 332~m334. KATZ S. A. and COWANS J. A. (1965) Direct potentiometric study of urea-urease system. Biochem. Biophys. Acra 107. 605.-608. KUBOTA J. and ALLAWAY W. H. (1972) Geographic distribution of trace element problems. In Micronutrients in Agriculture (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, Eds) pp. 52>554. Soil Sci. Sot. Am. Madison, Wisconsin. LAGERWERFF J. V. (1972) Lead, mercury, and cadmium as environmental contaminants. In Micronutrients in Agriculture (J. J. Mortvedt, P. M. Giordano, and W. L. Lindsay, Eds) pp. 593-636. Soil Sci. Sot. Am. Madison, Wisconsin. LINDSAY W. L. (1973) Inorganic reactions of sewage wastes with soils. In Recycling Municipal Sludges and EfPuents .. on Land. pp. 91-96 National Assoc. State Universities and Land Grant Colleges, Washington, D.C. MYERS M. G. and MCGARITY J. W. (1968) The urease activity in profiles of five great soil groups from Northern New South Wales. PI. Soil 28, 25-36. PAGE A. L. (1974) Fate and Effects of Trace Elements in Sewage Sludge when Applied to Agricultural Land: A
on urease
13
Literature Review. Environmental Protection Agency, Cincinnati, Ohio. Ross D. J. and ROBERTS H. S. (1968) A study of activities of enzymes hydrolysing sucrose and starch and of oxygen uptake in a sequence of soils under tussock grassland. J. Soil Sci. 19, 186-196. SHAW W. H. R. (1954) The inhibition of urease by various metal ions. J. Am. them. Sot. 76, 2160-2163. SKUJINS J. J. (1967) Enzymes in soil. In Soil Biochemistry (A. D. McLaren and G. H. Peterson, Eds) pp. 371414. Marcel Dekker, New York. STEVENSONF. J. and ARDAKANI M. S. (1972) Organic matter reactions involving micronutrients in soils. In Micronutrirnts in Agriculture (J. J. Mortvedt. P. M. Giordano, and W. L. Lindsay. Eds) pp. 79-114. Soil Sci. Sot. Am.. Madison. Wisconsin. STOJANOVIC B. J. (1959) Hydrolysis of urea in soil as affected by season and added urease. Soil Sci. 88, 251-255. TABATABAI M. A. and BREMNERJ. M. (1969) Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol. Biochem. 1, 301-307. TABATABAI M. A. and BREMNER J. M. (1972) Assay of urease activity in soils. Soil Biol. B&hem. 4, 479487. TOREN E. C. and BURGER F. J. (1968) Trace determination of metal ion inhibitors of the urea-urease system by a nH-stat kinetic method. Mikrochim Acta 5. 1049- 1058. WILL M. C. and LAIDLER K. J. (1953) The molecular kinetics of the urea-urease system-IV. The reaction in an inert buffer. Arch. Biochem. Biophys. 43. 299-306.