Mineralization and immobilization of soil and fertilizer nitrogen with nitrification inhibitors and solvents

0038-0717/92$5.00+ 0.00 Copyright 0 1992Pergamon Press Ltd

Soil Bid. Biochem. Vol. 24, No. 6, PP. 559-568, 1992 Printed in Great Britain. All rights reserved

MINERALIZATION AND IMMOBILIZATION OF SOIL AND FERTILIZER NITROGEN WITH NITRIFICATION INHIBITORS AND SOLVENTS D. M. CRAWFORD and P. M. CHALK*

School of Agriculture and Forestry, University of Melbourne, Parkville 3052, Victoria, Australia (Accepted

15 January 1992)

Summary-Rates of transformations of fertilizer N and soil N were measured in three soils labelled with (“NH&SO, in laboratory experiments of short duration (7 days). The effects of three nitrification inhibitors (N-Serve 24E, 2-ethynyl pyridine, ATC) applied to three soils in aqueous solution or emulsion, or of three solvents (water, ethanol, acetone) applied to one soil with or without an aqueous solution of N-Serve TG, on rates of nitrification, N immobilization and N mineralization were measured. Nitrification inhibitors had little or no effect on N transformation rates apart from nitrification. N immobilization rates (fertilizer or gross) and N mineralization rates (net or gross) were either not affected or only slightly affected by addition of the three inhibitors at IS pg gg ’ soil. Organic solvents inhibited nitrification and more N was immobilized and less N was mineralized in the presence of organic solvents, resulting in reduced net N mineralization or net N immobilization. The effects of organic solvents on N transformation rates were temporary and were due to the addition of an energy source rather than to inhibition of nitrilication. The work reported here indicates that nitrification inhibitors, or organic solvents used to dissolve inhibitors which are sparingly soluble in water, have only transient effects on biological transformations of N in soils in the absence of growing plants.

creased due to the inhibitor treatment from 1 to 5-8% (Juma and Paul, 1983), and part, if not all, of this N would have been included in Kjeldahl N (Bremner and Mulvaney, 1982). Also, because alkaline-hydrolysing fertilizers were used (urea, aqua ammonia), NH, fixation by soil organic matter would have occurred and may have been promoted by the inhibitor. Clay et al. (1990) concluded that NH, fixation by soil organic matter may have increased in a urea-fertilized soil due to application of the inhibitor, dicyandiamide (DCD), because increased concentrations of non-hydrolysable N were measured in this treatment. Thus, the interpretation of data can be complicated by physico-chemical fixation reactions which occur simultaneously with biological N immobilization in N fertilized soils, and which could be influenced by nitrification inhibitors due to the dependence of process rates on ammoniacal N concentration. Results obtained in a laboratory study by Guiraud et al. (1989) indicated that the amount of N applied as (NH&SO4 which could not be extracted with KC1 during the first 8 days of the experiment increased due to addition of DCD, but problems were experienced in distinguishing between effects due to NH: fixation and effects due to biological N immobilization in the soil used. The inclusion of roots in soil “N recovery data is an additional factor which may complicate the interpretation of inhibitor effects when crops are sown in the N fertilized soil. Both field and laboratory studies have shown no effect of the inhibitor, nitrapyrin, on biological N

INTRODUCTION Several authors have suggested that nitrification inhibitors increase biological immobilization of ammonium (Ashworth, 1986; Sahrawat, 1989; Hauck, 1990). It has been argued that nitrification inhibitors increase N immobilization because more N is retained in the ammonium form compared to the nitrate form, and ammonium is preferentially immobilized over nitrate by soil microorganisms (Ashworth, 1986; Sahrawat, 1989; Hauck, 1990). However, a contrary view was expressed by Chalk et al. (1990). Because N immobilization is driven by available C rather than inorganic N (Shen et al., 1984),

it seems

will increase increase

unlikely

following

in ammonium

that inhibitor

the

rate

of the process

application

concentration

due to an

per se.

Evidence for and against increased N immobilization due to the use of nitrification inhibitors has been reported. Data obtained by Juma and Paul (1983) with the inhibitor ATC (Camino-1,2,4-triazole) applied to field microplots sown to wheat are frequently cited as evidence for increased biological N immobilization (Ashworth, 1986; Sahrawat, 1989; Hauck, 1990). The recovery of 15N-labelled fertilizer in the soil (Kjeldahl method modified to include nitrite + nitrate) over the &30 cm depth in ATCtreated plots (54-59%) was higher compared to control plots (3941%). However, the recovery of fertilizer as non-exchangeable (clay-fixed) NH: in*Author for correspondence. 559

560

II.

M.

CRAWFORD

immobilization. For example, the application of nitrapyrin with fall-applied urea to fallow soil had no effect on the recovery of the fertilizer as organic N in the following spring (Aulakh and Rennie, 1984). Both Myrold and Tiedje (1986) and Chalk et al. (1990) found that gross N immobilization did not increase when active nitrification in individual soils fertilized with (NH&SO, was effectively inhibited by addition of nitrapyrin. In the short-term laboratory study of Chalk et a!. (1990). the absence of several factors (plant growth, losses of fertilizer N, remineralization of immobilized N and NH, fixation) simplified the interpretation of the data. However, an assessment of the general applicability of the results was not possible because only one soil and one inhibitor were used. Chalk et al. (1990) suggested that nitrification inhibitors could indirectly influence heterotrophic N immobilization when organic solvents are used to dissolve inhibitors which are sparingly soluble in water. For example, organic solvents such as acetone or ethanol have been used to dissolve the crystalline form of the inhibitor N-Serve (e.g. Goring, 1962; Gasser et al., 1967) or to dilute nitrapyrin in liquid form prior to application to the N fertilizer (e.g. Walters and Malzer, 1990). Yeomans and Bremner (1989) found that the rate of biological denitrification increased in soils treated with ethanol. The effect was still evident even after the ethanol had been allowed to volatilize, suggesting that soil organic C may also have been solubilized and made available to heterotrophic organisms by the solvent. Chalk et al. (1990) also suggested that the inhibitor itself may provide a source of C during biological degradation. The objective of the present study was to evaluate the effects of different nitrification inhibitors and solvents on N transformations in several soils. Shortterm laboratory studies were conducted to enable estimation of gross rates of immobilization of soil and fertilizer N and gross rates of mineralization of soil N. Short-term studies were considered to be appropriate in view of the generally-observed short-term effectiveness of nitrification inhibitors in the field. MATERIALS AND METHODS

Soils Three biologically-active soils were chosen on the basis of preliminary laboratory incubation tests on a range of soils to determine net N mineralization and

and P. M.

CHALK

nitrification rates. The soils, which were located at Waurn Ponds and Tarwin Lower (Victoria) and Narrabri (New South Wales), were under virgin native pasture, improved perennial pasture and cultivated bare fallow, respectively, when sampled in May 1985, and April and July 1989. Composite surface samples (O-15 cm) were air-dried, crushed to < 2 mm and thoroughly mixed. Some properties of the soils are given in Table 1.

Two experiments were conducted in the laboratory. In the first experiment, the effects of three nitritication inhibitors on N transformations in three soils were studied. The inhibitors were nitrapyrin ~2-chloro-6-(trichloromethyl) pyridinej in the liquid (emulsi~able) form (N-Serve 24E), 2-ethynyl pyridine and 4-amino-1,2,4-triazole. In the second experiment, the effects of one nitrification inhibitor, nitrapyrin, in the crystalline (technical grade) form (N-Serve TG) and three inhibitor solvents (water, ethanot, acetone) on N transformations in Waurn Ponds clay were studied. Ammonium sulphate fertilizer was applied to all treatments at 100 pg N g- I soil with the N isotopically labelled at either 19.33 atom % “N (first experiment) or 19.80 atom % “N (second experiment). The experiments were conducted in 250 ml incubation vessels containing the equivalent of 40g of oven-dry soil. The N fertilizer was applied in either 4 ml (first experiment) or 1 ml (second experiment) of aqueous solution (1 .O or 4.0 mg N ml- ‘, respectively). In the first experiment, inhibitors were applied at the rate of 15 ,ug g-’ soil in 2 ml of aqueous solution or emulsion (300 pg ml-‘). A control treatment (without inhibitor) was also included. In the second experiment, the inhibitor was applied at the rate of 10 ng g _I soil in l0ml of aqueous solution (40 pg ml-‘) which was at the limit of its solubility at 20°C (Goring, 1962). The organic solvents were applied in 1 ml of individual solutions (625 pg C g--’ soil). The inhibitor and organic solvents were applied separately jn order to achieve a precise rate of application of each, and because of potential differences in the volatility and the hydrolysis of N-Serve TG in different solvents. Also separate solutions were convenient because treatments were applied in factorial combination ( & 3 solvents, i inhibitor). All solutions were applied as uniformly as possible to the soil surface

Table I. Properties

of the sods Soil property

Soil’ Waurn Ponds c Narrabri c Tarwin 91

Great soil %mxm* R GC A

(g kg ’ soil)

DH'

CEC4 [c mol ( + ) ke. ’ sod1

Organic c

Total N

Clay

Silt

H,O xt - 33 kPa

8.0 8.2 5.9

31.4 32.4 16.0

62.7 9.9 44.3

6.34 0.95 4.13

410 360 80

200 80 30

373 389 227

‘c, clay; sl, sandy loam. ‘R, Rendzina; GC, Cirey Clay; A, Alluvial ‘soil: water, 1: 5. *CEC, cation exchange capacity

(State et al., 1968).

Soil N transformations

with volumetric pipettes, and distilled water was added after the treatments were applied, to bring the soil water matric potential to -33 kPa (Table 1). The vessels were capped with Al foil pierced with a centrally-located 1 mm dia hole to allow for aeration, and were kept in the dark at 25°C. Sufficient treatments were prepared to permit destructive sampling at days 3 and 7, with four replicates for each treatment. Vessels were weighed every day to measure and replenish loss of water. Incubated samples were extracted with 100ml of 4 M KC1 with mechanical shaking for 1 h. The suspension was filtered (Whatman No. 40) under vacuum on a Biichner funnel. Filtrates were retained and stored at 4’C in sealed polythene bottles for analysis of inorganic N. The soil was leached with four 100ml aliquots of 0.05 M KC1 to remove all inorganic N. The filter paper and soil were dried at 40°C. The dried soil was separated from the filter paper, ground to < 0.17 mm, and retained in sealed vials for Kjeldahl digestion and clay fixed ammonium analysis. Soil samples which had no treatments applied were also extracted for inorganic N analysis. Analytical Soil extracts were analysed for exchangeable NH: -N and (NO; + NO<)-N by steam distillation (Keeney and Nelson, 1982). Total N was determined by a semi-micro KMnO,-reduced Fe modification of the Kjeldahl digestion method (Douglas et al., 1980). Clay fixed ammonium was determined by the method of Silva and Bremner (1966). A double distillation procedure was used to minimise cross-contamination between ‘5N-labelled distillates (Pruden et al., 1985). Ammonium distillates were dried and then converted by alkaline hypobromite (LiOBr) oxidation to N, for isotope-ratio analysis using the apparatus and procedures described by Chen et al. (1990). Isotoperatios were measured on a magnetic-deflection mass spectrometer (Sira, 10, VG Isogas) equipped with triple collectors and a dual inlet system. All data are expressed on an oven-dry soil basis. Soil water was determined by drying for 24 h at 105°C in a forced-draught oven. Nomenclature

and calculations

The nomenclature proposed by Bjarnason (1988) to identify N pools and N transformation rates was used: AL, exchangable ammonium-N, labelled; AT, exchangeable ammonium-N, total (i.e. labelled + unlabelled); CL, clay fixed ammonium, labelled; NL, (nitrite + nitrate)-N, labelled; NT, (nitrite + nitrate)N, total; ANT, sum of AT and NT; OL, organic-N, labelled; m, mineralization rate of indigenous (unlabelled) organic-N; i, immobilization rate of ammonium-N; n, nitrification rate of ammonium-N. Subscripts 1, 2 and a used with pool names denote the initial, the final and the arithmetic mean of values obtained at two consecutive sampling times, respectively.

561

with inhibitors and solvents

Labelled-N (e.g. exchangeable NH: -N) was calculated as AL = AT (x/y) where x = atom % 15N excess in AT and y = atom % 15N excess in the of the amfertilizer, using the 15N abundance monium pool in unlabelled soil to calculate atom % excess. Rates of N transformations were calculated on the basis of zero-order kinetics, i.e. constant rates between consecutive sampling times. The sampling intervals were therefore of short duration (34 days), and the experiment ran for only 7 days in order to avoid remineralization of immobilized labelled N. Nitrification rates were estimated by the rate of change in total nitrite + nitrite (i.e. n = dNT/dt). Nitrification inhibition (NI) percentages were calculated as NI (Bundy and (%) = (ncontrol- n + Inhibitor)x 100/n,,,,,,, Bremner, 1973). Net mineralization rates were estimated by the rate of change in total exchangeable ammonium + nitrite + nitrate (i.e. m - i = dANT/ dt). The rate of immobilization of fertilizer N was estimated by the rate of change of labelled organic-N (i.e. dOL/dt). An equation proposed by Shen et al. (1984) based on measurement of the rate of immobilization of fertilizer N and the means of the total and labelled exchangeable ammonium pools, was used to derive estimates of gross rates of immobilization of soil and fertilizer N (i.e. i = (dOL/dt)(AT,/AL,). Gross N mineralization rates were estimated by summation of net mineralization and gross immobilization rates. Data were analysed statistically using the analysis of variance procedures of Genstat 5 (Payne et al., 1987). Treatment means for individual N pools were compared using the least significant difference test (P < 0.05). RESULTSAND

DISCUSSION

Inhibition of nitrijication Rates of nitrification differed within and between soils, with the highest rates in the control treatment of the Waurn Ponds clay (Table 2). Rates of nitrification increased or remained constant between the first (days O-3) and second (days 3-7) sampling periods in control treatments. All of the compounds used inhibited nitrification, but the degree of inhibition differed within and between soils (Table 2). Previous laboratory studies demonstrated that nitrapyrin and ATC were effective nitrification inhibitors (Bundy and Bremner, 1973), and that 2-ethynyl pyridine was more effective than nitrapyrin (McCarty and Bremner, 1986). In the present study, 2-ethynyl pyridine had a greater inhibitory effect on nitrification compared to ATC and a greater or equal effect compared to nitrapyrin, over all soils (Table 2). The inhibitory effect of 2-ethynyl pyridine was similar between the first and second sampling periods, whereas the effectiveness of nitrapyrin and ATC were generally greater in the second period (Table 2). Myrold and Tiedje (1986)

D. M. CRAWFORD and P. M.

562 Table 2. Distribution

Soil’ Waurn

of (nitrite + nitrate)-N

Inhibitor* Ponds c

Control

NS EP ATC Narrabri

c

Control

NS EP ATC Tarwin

sl

Control

NS EP ATC Pooled standard

Day 0

CHALK

and nitrification rates in three “N-labelled nitrification inhibitors Nitrite + nitrate” -______ NT NL (pg N g ’ soil)

soils with and without

Nitrification Period (days)

Rate’ (pg N go ’ day

InhIbition’

‘1

(%I

3 7 3 7 3 7 3 7

74 98 151 87 III 78 90 89 107

0 R 21 5 II 2 3 4 9

o-3 3-7 63 3-l G-3 3-7 o-3 3-7

19 13.3 4.5 6.0 1.4 3.0 5.1 4.5

0 3 I 3 7 3 7 3 1

33 46 70 33 33 33 29 36 38

0 IO 29 I 2 2 I 3 4

o-3 3-7 &3 3-7 o-3 3.-7 &3 3-7

4.3 5.9 0.1 0 0 0 I.1 0.6

97 100 IO0 100 7s 90

0 3 7 3 7 3 7 3 7

23 36 55 31 41 23 24 30 35 3.1

0 8 16 5 9 I I 4 6 I.1

t&3 3-7 &3 3. 7 &3 3-7 &3 3 -7

4.5 4.5 2.8 2.5 0.2 0.2 2.2 I .4

37 45 96 95 51 71)

deviation

three

0 0

44 55 x2 7x 36 66 0 0

0

‘c, clay: sl, sandy loam. *Control, without inhibitor; NS, N-Serve 24E; EP. 2-ethynyl pyridine; ATC, 4-amino-l,2,4-triazole ‘NT, total (labelled + unlabelled) nitrite + mtrate; NL, labelled nitrite + nitrate. ‘n = dNT/dt. 5Nitrification inhibition (NI) percentages due to the mhiblror, where NI (%) = (n,,,,,,,, ~ nTlnhlhilor) x lOO!n,,,,,,,,

also noted a delay in the inhibition of nitrification by nitrapyrin. The inhibitors were always more effective in the Narrabri clay compared to the other soils, and generally had a greater inhibitory effect in the Tarwin sandy loam compared to the Waurn Ponds clay (Table 2). These results are consistent with the inverse relationship between concentration of soil organic C (Table 1) and inhibitor bioactivity which has been reported (Keeney, 1980). Table 3. Distribution

of (nitrite + nitrate)-N

The organic solvents, ethanol and acetone, each inhibited nitrification in Waurn Ponds clay, with acetone having a greater inhibitory effect (Table 3). Both ethanol and acetone were reported to inhibit ammonium oxidation in the activated sludge process of sewage treatment (Tomlinson ef al., 1966) and ammonium oxidation by Nitrosomonas in pure culture, with the effect being reversible (Hooper and Terry, 1973).

and nitritication rates in ‘SN-labelled Waurn Ponds clay with and wthout N-Serve TG and three solvents Nitrite + nitrate’

NTN-Serve TG Without

Day

Water (control)

0 3 I 3 7 3 I

74 94 I58 75 117 76 80

3 I 3 I 3 I

90 II8 80 98 70 77 4.9

Ethanol Acetone With

Water Ethanol Acetone

Pooled standard

(pg N go ’ ::I)

Solvent

deviation

0 7 27 3 I8

I 5 4 8 2 8 0 2 0.9

___.~__. Period (days)

Nitrificatmn Rate’ (pg N g ’ day

lx3 3-7 O-3 3-7 f&3 3-l

6.8 15.9 0.3 10.5 0.7 I.1

&3 3-7 o-3 3-l &3 3-7

5.3 72 2.2 4.3 0

‘)

InhibItIon’ (% )

96 34 90 93 23 55 68 73 100 90

I .6

‘NT, total (labelled + unlabelled) nitrite + nitrate; NL, labelled nitrite + nitrate. +I = dNT/dt. ‘Nitritication inhibition (NI) percentages due to treatment with inhibitor or organic (&“,,,I - n,,.,“,,,,) x lOw,o.,,o,.

solvent,

where

NT (%I) =

Soil N transformations

N immobilization Rates of immobilization of fertilizer N were highest in Waurn Ponds clay and lowest in Narrabri clay, and decreased between the first and second sampling periods in all treatments (Table 4). Application of inhibitors had no effect on rates of immobilization of fertilizer N in Narrabri clay, and only minor and inconsistent effects in the other two soils. Similarly, the inhibitors had only minor effects on estimated gross rates of immobilization of soil + fertilizer N (Table 4). Small amounts of applied ammonium were recovered as clay fixed ammonium in all soils, but the inhibitors had no effect on fixation of ammonium by clay minerals (Table 4). Some or all of the clay fixed ammonium would have been included in the Kjeldahl N determination of organic N, but because of the possibility of non-quantitative and variable recoveries (Bremner and Mulvaney, 1982) no attempt was made to correct OL for inclusion of CL. Because inhibitors had little or no effect on either CL or OL, the determination of CL was omitted in the second experiment. Addition of ethanol increased immobilization of fertilizer N in Waurn Ponds clay during the first sampling period but not the second period, whereas addition of acetone increased immobilization of fertilizer N during the second sampling period, but not the first period when compared to addition of water Table 4. Distribution of organic-N

563

with inhibitors and solvents

(Table 5). It appears that ethanol was more rapidly metabolized by the heterotrophs compared to acetone, and that the breakdown of ethanol was complete at day 3. If it is assumed that the efficiency of utilization of added C was only 20% (due to rapid turnover and some loss due to ethanol volatilization) and the ratio of C:N of the heterotrophs was ca 10: 1, then 625 pg C g-’ soil added as ethanol could potentially be metabolized in 3 days since an additional 14.1 pg N g-’ soil was immobilized due to ethanol addition (Table 5). The application of nitrapyrin with ethanol and acetone caused no additional immobilization of fertilizer N compared to application of organic solvents alone. The effects of ethanol and acetone on fertilizer N immobilization were confounded because the organic solvents provided not only a potential source of C for the heterotrophs but they also inhibited nitrification. However, it appears that the increases in fertilizer N immobilization due to organic solvent application were due to the provision of additional C and not due to the inhibition of nitrification, because while nitrification was inhibited by the addition of nitrapyrin with water, fertilizer N immobilization did not increase (Table 5). Treatment effects on estimated gross N immobilization were similar to those observed for fertilizer N immobilization (Table 5). The different soils used in the present studies provided variation in rates of nitrification and N immobilization, and the inhibitors used differed in

and N immobilization rates in three “N-labelled soils with and without three nitrification inhibitors Immobilization rate

Period

OL’ Soil’ Waurn Ponds c

Inhibitor2 Control

Day

l&3 3-l O-3 3-7 &3

GOSS6

(pg N gg’ soil day-‘)

G3 3-7

4.1 2.1 4.3 2.1 4.5 2.2 4.4 2.4

5.9 3.9 6.1 3.8 6.5 4.4 6.5 4.7

3 I

Control

3 7 3 7 3 7 3 I

8 IO 8 IO 8 II 8 II

2.5 2.6 2.5 2.9 2.6 2.9 2.6 2.9

&3 3-7 &3 3-7 c-3 3-7 o-3 3-7

2.8 0.5 2.1 0.5 2.6 0.9 2.6 0.7

2.9 0.6 2.9 0.5 2.8 I.0 2.8 0.8

3 7 3 I 3 7 3 7

I2 17 II I6 II I6 12 I6 0.8

2.2 2.2 2.0 2.2 2.1 2.3 2.2 2.5 0.09

c-3 3-7 &3 3-7 &3 3-7 &3 3-l

3.9 I.3 3.6 I.3 3.5 I.4 4.0 0.9

4.7 1.9 4.4 2.0 4.4 2.3 4.9 I.5

I

NS EP ATC Tarwin sl

0.4 0.6 0.5 0.5 0.5 0.6 0.5 0.6

Fertilizer’

ATC

EP

Narrabri c

(days)

12 21 I3 21 13 22 13 23

NS

3 I 3 7 3

(fig N g-’ s%;

Control NS EP ATC

Pooled standard deviation

3-l

‘c, clav: sl. sandv loam. ‘Control, without inhibitor; NS, N-Serve 24E; EP, 2-ethynyl pyridine; ATC, 4-amino-l,Z,Ctriazole. )OL, labelled organic-N. OL = 0 at day 0 in all treatments. ‘CL, labelled clay fixed ammonium. CL = 0 at day 0 in all treatments. SdOL/dt. 9 = (iOL/dt)(AT,/AL,) (Shen et al., 1984). Ammonium data (AT, AL) are given in Table 6.

D. M.

564 Table 5. Distribution

of organic-N

CRAWFORD

and P.

M. CHALK

and N immobilization rates in ‘sN-labelled without N-Serve TG and three solvents

Waurn Ponds clay with and

Immobilization N-ServeTG

Period

Acetone

3 1 3 7 3

9 15 21 21 10 22

&3 3-7 l&3 37 c-3 3-7

3.0 1.4 7.0 1.6 3.3 3.2

4.4 2.6 9.1 2.6 4.8 6.3

Water

3

Ethanol

3 7 3

10 16 22 27 10 24 1.4

o-3 3-7 O-3 3-7 &3 3-7

3.2 1.6 7.4 1.3 3.4 3.4

4.6 3.2 9.8 2.2 5.1 7.0

Solvent

Without

Day

Water Ethanol

With

Acetone Pooled standard

rate

OL’ (pg N g-’ soil)

deviation

Fertilizer* Gross’ (pg N g-’ soil day-‘)

(days)

‘OL, labelled organic-N. OL = 0 at day 0 in all treatments. 2dOL/dt. ‘i = (dOL/dt)(AT,/AL,) (Shen ef al., 1984). Ammonium data (AT, AL) are given in Table 7.

their ability to inhibit nitrification. If inhibitors were to promote biological N immobilization, the effect would be expected to occur quickly and to be most pronounced in soils with higher biological activities when using the more effective inhibitors. In contrast, the results of the present studies show that the inhibition of nitrification did not promote immobilization of soil or fertilizer N, confirming earlier results with nitrapyrin (Myrold and Tiedje, 1986; Chalk et al., 1990) and demonstrating that it is a finding which Table 6. Distribution

of ammonium-N

is generally applicable to different nitrification inhibitors and soils during laboratory incubation. Howwas enhanced when ever, N immobilization additional C was provided in the form of organic solvents. N mineralization Net N mineralization rates differed within and between soils, with the highest rates in Waurn Ponds clay (Table 6). Net immobilization occurred in the

and N mineralization rates in three ‘SN-labelled soils with and without nitrification inhibitors Mineralization

Ammonium’

Soil’ Waurn

Inhibitor’ Ponds c

Control

Day 0

3 NS EP ATC Narrabri

0

c

NS

3

EP

3

ATC Tarwin sl

Control

NS EP ATC Pooled standard

3 I 3 7 3 I

deviation

AT (pg N g~‘s$

Period (days)

Net4 (pg N g

three

rate

’ soil

Gross~ day ‘)

114 137 88 137 122 148 147 145 131

100 75 47 77 66 80 69 78 64

&3 3-l o-3 3-7 g-3 3-7 l&3 3-7

15.7 1.0 12.3 2.2 12.9 2.8 15.4 1.1

21.6 4.9 18.4 6.0 19.4 7.2 21.9 5.8

103 87 64 98 100 99 101 9s 98

100 81 58 88 86 90 85 86 84

&3 3-7 G-3 3-7 c-3 3-l O-3 3-l

-0.9 0.1 -1.5 0.5 - 1.4 -0.2 - 1.6 1.5

2.0 0.7 I .4 1.0 1.4 0.8 1.2 2.3

105 116 109 122 122 130 I41 125 130 2.3

100 81 68 83 70 88 83 85 79 2.1

G-3 3-l c-3 3-l O-3 3-7 o-3 3-7

8.2 2.8 8.5 2.6 8.7 3.0 9.0 2.5

12.9 4.8 12.9 4.6 13.1 5.2 13.9 3.9

‘c, clay; sl, sandy loam. *Control, without inhibitor; NS, N-Serve 24E; EP, 2-ethynyl pyridine; ATC, 4-amino-1,2,4-triazole. ‘AT, total (labelled + unlabelled) ammonium; AL, labelled ammonium. ‘m - i = dANT/dt, where ANT = AT + NT (Table 2). Negative values indicate net N immobilization ‘m = net mineralization (m - i) + gross immobilization (i) (Table 4).

Soil

N

transformations

with inhibitors and solvents

Narrabri clay during the days &3 sampling period. Net mineralization rates were higher in the first compared to the second sampling period, which is consistent with the well known but transient increase in biological activity following the rewetting of airdry soil. Application of inhibitors had no effect on net mineralization or net immobilization rates in Tarwin sandy loam or Narrabri clay, whereas nitrapyrin and 2-ethynyl pyridine had a slight depressing effect on net mineralization rates in Waurn Ponds clay during the first sampling period (Table 6). However, cumulative net N mineralization in Waurn Ponds clay (days c-7) was little affected by inhibitor application. There were no marked treatment effects on gross N mineralization rates estimated by summation of net mineralization and gross immobilization rates (Table 6) since these rates were generally not affected by applications of inhibitors. Net and gross N mineralization rates in Waurn Ponds clay were not affected by application of technical grade nitrapyrin (Table 7). Net N immobilization occurred in Waurn Ponds clay treated with ethanol or acetone during the first and second sampling periods, respectively, both in the presence and absence of nitrapyrin (Table 7). Net N immobilization occurred because the addition of the organic solvents increased gross N immobilization rates during these respective sampling periods (Table 5). Net N immobilization in the ethanol treatment (days O-3) was followed by net N mineralization (days 3-7) again indicating that ethanol metabolism was complete in the first 3 days. Gross N mineralization rates without nitrapyrin also decreased during the first and second sampling periods due to the addition of ethanol and acetone, respectively, suggesting a competitive interaction whereby the mineralization of indigenous organic C (and hence indigenous organic N) was reduced due to the addition of a readily mineralizable organic C substrate. The effect of the organic solvents on both net and gross N mineralization during days O-3 was

Table 7. Distribution

attenuated by the addition of nitrapyrin, but there is no apparent explanation for this effect. Thus addition of organic solvents both increased gross N immobilization rates and decreased gross N mineralization rates, and therefore the measured net effect reflected both of these changes which resulted in either decreased net N mineralization or net N immobilization. The data illustrate well the need to estimate gross rates of N transformations in order to understand mechanisms leading to changes in soil inorganic N, a need which has recently been emphasized by several authors (Bjarnason, 1988; Guiraud et al., 1989; Chalk et al., 1990). The data also have important implications with respect to the use of nitrification inhibitors for in situ measurement of nitrifcation rates in soils and sediments. The technique is based on the measurement of differences in ammonium and nitrate concentrations between treatments with and without addition of a nitrification inhibitor (Henriksen, 1980; Hall, 1984). The method not only requires that the inhibitor be completely effective, but that rates of N immobilization, N mineralization and other N transformations are not affected by the inhibitor. Thus, an organic solvent such as acetone cannot be used to dissolve an inhibitor such as N-Serve TG (e.g. Henriksen, 1980; Hall, 1984) when inhibitors are used to estimate in situ nitrification rates. Models of gross N transformations Estimates of gross N immobilization rates using models based on measurement of the size of the labelled and total NH: pools (Kirkham and Bartholomew, 1954; Shen et al., 1984) were similar (Table 8). Estimates of i based on measurement of the size of both the labelled organic-N and NH: pools (Shen et al., 1984; Guiraud et al., 1989) were also similar (Table 8). However, the data show that equations based only on measurement of the NH: pool underestimated i during both the first and

of ammonium-N and N mineralization rates in ‘SN-labelled Waurn and without N-Serve TG and three solvents Ammonium’

N-Serve

TG

Without

Solvent Water

Ethanol Acetone With

Water Ethanol Acetone

Pooled standard

505

deviation

Day

Mineralization

AT (pg N g-’ s:l?

0 3 7 3 7 3 7

II4 I35 87 93 64 154 126

3 7 3 7 3 7

142 I25 102 95 142 II7 2.3

100 74 46 58 37 81 60 76 61 63 53 70 58 2.1

Period (days)

Net’ (pg N g

Ponds clay with rate Gross’ day

’ soil

&3 3-7 c-3 3-7 &3 3-7

14.0 3.9 -6.7 3.4 14.2 -5.9

18.3 6.6 2.5 5.9 19.0 0.4

O-3 3-7 O-3 3-7 &3 3-7

14.6 3.1 - 1.9 2.7 8.2 -4.5

19.2 6.2 7.9 4.9 13.3 2.5

‘AT, total (labelled + unlabelled) ammonium; AL, labelled ammonium. ‘m - i = dANT/dt, where ANT = AT + NT (Table 3). Negative values indicate ‘m = net mineralization (m - i) + gross immobilization (i) (Table 5).

net N immobilization

‘)

566

D. M. CRAWFORD Table8.Comparison

and P.M. CHALK

of estimates of N transformation rates in ‘SN-labclled Waurn Tarwin sandy loam without addition of nitrification inhibitor

Ponds clay and

Rate&g N g-' soil day-‘) _

Waurn N transformation “N immobilization Gross

immobilization

Net mineralization Gross mineralization

Ponds clay

Tarwin sandy loam

Equation’

days O-3

days 3-7

days O-3

dOL;dt

4.1

2.1

3.9

1.3

4.1 4.0 5.9 5.8

-0.7 -0.7 3.9 3.9

3.4 3.4 4.7 4.7

0.3 0.2 1.9 1.9

iI i, i> & dANT:di IA, m, m,

days 3--7

15.7

I.0

8.2

2.8

19.7 19.7 19.4

0.4 0.4 0.4

II.6

3.1 3.1 3.1

Il.6 Il.5

‘i, = [( --dAT/dt)log (AL,JAL,)/log(AT,/AT,)] - n (Kirkham and Bartholomew, 1954): I: = [( -dAL/dt)(AT,/AL,)] - n (Shen rl al, 1984); i, = (dOL/dt)(AT,;AL,) (Shen ef al., 19X4); L = (dOL/dt)!(AL/AT), (Guiraud er a/., 1989); m, = (-dAT/dt)log(AL,AT,/AL,AT,)jlog(AT,/AT,) (Kirkham and Bartholomew, 1954); rn: = (dAT/dt) - (dAL/dt)(AT*~AL~) (Shen PI al.. 1984); m, = (dATidt) - (dAL~dt)~(AL;AT)~ (Tiedje ef af., 1981).

second sampling periods because the rates were either equal to or less than the measured rates of immobilization of fertilizer N alone. Immobilization rates can be underestimated if remineralization of immobilized labelled N occurs, but remineralization is unlikely to be a significant factor in the first week of incubation under aerobic conditions (Bjarnason, 1988). In contrast, Shen et al. (1984) found that higher estimates of i were sometimes obtained using only NH; pool data compared to those based on a combination of NH,+ and OL data, whereas Chalk et al. (1990) found similar estimates using models based on different N pools. Shen et al. (1984) considered that estimates of i obtained using only NH: data were less accurate compared to estimates based on both OL and NH,i data, because the rates of all processes which deplete the NH,f pool (e.g. nitrification, NH, volatilization) must be subtracted in the former model to avoid overestimation of i. Thus it is essential that the rates of such processes are accurately measured. For example, nitrification may be more accurately determined using models based on total and labelled NO,-pools following addition of “NO; at the beginning of incubation (Koike and Hattori, 1978; Barraclough, 1991) compared to the simple measurement of dNT/dt. Although there is no apparent explanation for the underestimation of i when using only the NH,’ pool data in the present study, the results emphasize the need to sample frequently, particularly when N transformation rates may change rapidly such as following the rewetting of air-dry soil, because accuracy depends on the constancy of all N transformation rates during the sampling period. Estimates of gross N mineralization rates using models based on measurement of the size of the labelled and total NH,* pools (Kirkham and Bartholomew, 1954; Shen et al., 1984: Tiedje ef al., 1981) were similar within soils and sampling periods (Table 8). Estimates of m, and m2 can be obtained either by summation of the measured net mineraliz-

ation rate and i, and i,, respectively, or by the mathematical expressions shown in the footnotes (Table 8). Similar estimates of gross mineralization between M, and those based on mean data (m, and m3) indicate that arithmetic mean data can closely approximate the true means of continuously changing AL and AT pools when sampling is frequent (Bjarnason, 1988). Nevertheless, because i was underestimated using models based on exchangeable NH,+ pool data, m was also underestimated using such data. GENERAL

DISCUSSION

Hauck (1990) claimed that evidence from laboratory, glasshouse and field studies, almost without exception, indicate that heterotrophic immobilj~tion of added ammonium is increased in the presence of a nitrification inhibitor. However, the present laboratory investigation has provided direct evidence, based on several different inhibitors and soils, that nitrification inhibitors do not cause increased bioiogical immobiIization of either fertilizer ammonium or gross N immobilization by retaining a higher concentration of N in the ammonium form. The present study has demonstrated, however, that increased biological N immobilization will occur if an inhibitor is dissolved in an organic solvent which provides an additional and easily decomposabIe C source. Also, being an organic compound, the inhibitor may itself constitute a source of C for the heterotrophs. The agronomic significance of such effects can be estimated. For example, the C : N ratios of the inhibitors ATC, DCD and nitrapyrin (N-Serve TG) in aqueous solution are 0.4, 0.4 and 5. I. respectively, and increased net N mineralization would immediately result from decomposition of organic compounds with such low C : N ratios. For nitrapyrin (N-Serve TG) dissolved in ethanol (29 g nitrapyrin 100 g-’ ethanol at 20°C; Goring, 1962) the C:N ratio is 3.5, and net immobilization would be likely to occur

Soil N transformations

with inhibitors and solvents

until decomposition was complete. However, at normal application rates of nitrapyrin of 0.5-1.0 kg ha-’ (Touchton and Boswell, 1980) the effect of adding 2.1 kg C ha-’ in the solvent + inhibitor would be transient, and net N immobilization would be quickly followed by net N mineralization as shown in the present study. Although not shown in the present study, nitrification inhibitors may cause increased fixation of fertilizer NH: by clay minerals, or delay the release of recently-fixed NH: (Juma and Paul, 1983; Aulakh and Rennie, 1984). Nitrification inhibitors may also cause increased NH, fixation by soil organic matter when alkaline-hydrolysing N fertilizers are used, but some of this fixed N will be remineralized. Since only small proportions of fertilizer N are usually fixed by these physico-chemical pathways, the proportional increase due to the inhibitor would be correspondingly small and of little agronomic significance. In laboratory experiments, nitrification inhibitors such as nitrapyrin have been shown to increase loss of fertilizer N via NH, volatilization (e.g. Magalhaes and Chalk, 1987) but NH, volatilization will be minimal if the N fertilizer is well covered by the soil. Thus there is little apparent cause for the concern expressed by Hauck (1990) that nitrification inhibitors may have potential adverse environmental effects through increasing fixation or loss of fertilizer N. On the contrary, nitrification inhibitors may have beneficial effects. For example, nitrapyrin prevented or reduced emissions of gaseous forms of N (N2, N,O, NO,) during nitrification in N fertilized soil (Smith and Chalk, 1980; Magalhaes et al., 1984; Magalhaes and Chalk, 1987). Also, if formation of nitrate is retarded, the potential for N losses due to nitrate leaching or formation of N, and N,O via biological denitrification will be reduced. Leaching of nitrate can result in pollution of groundwater, and the process is regarded as a major contributing factor to accelerated rates of soil acidification in some agricultural systems (Helyar and Porter, 1989). The gaseous forms of N, N,O and NO,, are naturally-occurring radiatively-active trace atmospheric constituents (greenhouse gases) whose concentrations are increasing with time on regional and global scales (Bouwman, 1989). Thus, if N losses are reduced by the use of nitrification inhibitors, positive effects on issues of environmental concern may ensue, and the potential for plant uptake of fertilizer and soil N may increase. It is possible that the effect of nitrification inhibitors on N immobilization may be exerted indirectly through influencing uptake of N by a crop. However, it has been observed that whereas an inhibitor may cause increased amounts of fertilizer N to enter the soil organic N pool (which includes plant roots), this has been accompanied either by no increase in uptake of fertilizer N by the crop (Juma and Paul, 1983; Bronson et al., 1991) or by decreased uptake of both total and fertilizer N (Walters and Malzer, 1990; Clay

561

et al., 1990). Such results indicate that factors directly associated with the inhibitor, the fertilizer or the crop rather than indirect effects on N uptake must be responsible for increased incorporation of fertilizer N into the soil organic N pool, and point to the need for further research to understand the effect of nitrification inhibitors on N transformations in the soil-plant system. Acknowledgements-The senior author thanks the State Chemistry Laboratory, Department of Agriculture, Victoria, for study leave to allow this work to be undertaken. We thank Dow Chemical (Australia) P/L for gifts of N-Serve 24E and N-Serve TG, Dr J. R Freney for providing 2-ethynyl pyridine and the Narrabri soil, Mr R. Teo for technical assistance and Dr R. Jarrett for advice on statistical analysis.

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Bundy L. G. and Bremner J. M. (1973) Inhibition of nitrification in soils. Soil Science Society of America Proceedings 37, 396398. Chalk P. M., Victoria R. L., Muraoka T. and Piccolo M. C. (1990) Effect of a nitrification inhibitor on immobilization and mineralization of soil and fertilizer nitrogen. Soil Biology & Biochemistry 22, 533-538. Chen D., Chalk P. M. and Freney J. R. (1990) Release of dinitrogen from nitrite and sulphamic acid for isotope ratio analysis of soil extracts containing nitrogen-15 labelled nitrite and nitrate. Analyst 115, 3655370.Clay D. E.. Malzer G. L. and Anderson J. L. (1990) Tillage and dicyandiamide influence on nitrogen fertilizer immobilization, remineralization, and utilization by maize (Zea mays L.). Biology and Fertility of Soils 9, 220-225. Douglas L. A., Riazi A. and Smith C. J. (1980) A semimicro method for determining total nitrogen in soils and plant material containing nitrite and nitrate. Soil Science Society of America Journal 44, 431433. Gasser J. K. R., Greenland D. J. and Rawson R. A. G. (1967) Measurement of losses from fertilizer nitrogen during incubation in acid sandy soils and during subsequent growth of ryegrass, using ‘SN-labelled fertilizers. Journal of Soil Science 18, 2899300.

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568

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