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Measurement of gaseous losses of nitrogen from soils
Soil. Viol. &&em
Vol. 5 ,pp .133-141. Pergamon Press 1973. Printed in Great Britain
MEASUREMENT
OF GASEOUS LOSSES OF NITROGEN SOILS
FROM
J. R. BURFORD* and R. C. STEFANSON Department of Agricultural Biochemistry and Soil Science, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, South Australia 5064 (Accepted 1 March 1972) Summary-The measurement of gaseous losses of nitrogen from a red-brown earth has been investigated by direct determination of the evolved gaseous products. The results of field investigations, enclosed growth chamber experiments, and incubation experiments have indicated that the majority of the losses as N20 are due to the microbial reduction of soil nitrate at anaerobic microsites within the generally aerobic soil. The detection and measurement of N,O in the soil atmosphere by analyses for N20 in samples obtained from equilibrium diffusion reservoirs provided a satisfactory and convenient means of detecting losses of nitrogen and of observing the seasonal distributions of nitrous oxide in the soil profile. Aspects of this problem requiring further investigation before the magnitude of the total gaseous losses can be accurately assessed are indicated. INTRODUCTION THE NITROGEN economy
of Australia’s agricultural industry has for many years depended on the symbiotic fixation of nitrogen by pasture legumes (Donald, 1960). A rapid depletion of soil nitrogen reserves occurs during a cropping phase, but the contribution of gaseous losses of nitrogen to such declines and to the total losses occurring contemporaneously with nitrogen fixation are not well known. Although it has been widely assumed that nitrogen is lost in gaseous forms from apparently well aerated soils (Allison, 19.55, 1966; Broadbent and Clark, 1965; Hauck, 1968), there is little direct evidence available concerning the losses in field situations. Much more information is needed about the occurrence, nature, and magnitude of such losses. Initial work by McGarity (1961) demonstrated that samples of a South Australian redbrown earth rapidly reduced N03--N to volatile gases under anaerobic conditions. Subsequent field investigations (Burford and Millington, 1968) showed that evolved N,O could be detected in the atmosphere of this well aerated soil for a period of several months. The detection of gaseous losses of nitrogen from soils by measurement of N,O concentrations in the soil atmosphere has been reported for other soils (Arnold, 1954; Albrecht, Junge and Zakosek, 1970), and the measurement of the evolution of nitrogen gas from a redbrown earth in experiments with enclosed growth chambers (Stefanson and Greenland, 1970) has indicated that losses as N2 probably accompanied the losses as N,O observed from this soil in field experiments. The methods used to examine the evolution of N,O into the soil atmosphere have involved the gas chromatographic or infra-red spectroscopic analysis of soil atmosphere samples obtained from diffusion equilibrium reservoirs (Taylor and Abrahams, 1953) permanently installed in the soil profile. Previous studies have indicated that such reservoirs are satisfactory for examination of the composition of the soil atmosphere for soil aeration studies (Taylor and Abrahams, 1953; Yamaguchi, Howard, Hughes and Flocker, 1962; * Present address: Department
of Soil Science, University of Reading, Reading. 133
134
J. R. BURFORD
AND R. C. STEFANSON
Tackett, 1968) but there is need for some further evaluation to establish that the reservoirs provide a satisfactory means of obtaining a representative sample of the soil atmosphere for analysis in studies of the gaseous losses of nitrogen from soils. The purpose of this paper is to report the techniques used in our investigations of the composition of the soil atmosphere, the major sources of error in estimating the magnitudes of gaseous losses of nitrogen from such measurements of the evolved gases, and the probable mechanism responsible for the losses on the Urrbrae loam.
MATERIALS
AND METHODS
The diffusion equilibrium gas reservoirs used for sampling the soil atmosphere consisted of small 3 *5 x 4.1 cm dia. tinned-iron cylindrical cans. The open base of each can was covered with a cone of stainless steel mesh. The interior of each reservoir was connected with an above ground level gas sampling point (consisting of a 4-cm length of 4 mm id. copper tubing sealed with a gas chromatograph septum) by means of a 2-m length of 1 mm i.d. copper capillary tubing sealed into the top of the can. The gas reservoirs were installed at appropriate depths in the soil by placing them on a l-cm deep bed of fine, washed building sand at the bottom of a 6. l-cm dia. hole. The space between the can and the walls of the hole was packed with sand, then the soil was replaced in approximately the correct order, with frequent tamping. The gas sampling probes were similar to those described by Tackett (1968) except that the external diameter of each probe was tapered towards the tip to facilitate a good seal between probe and soil. The soil atmosphere in the diffusion equilibrium reservoirs was sampled by withdrawing a 5 ml sample from the sampling point with a 5-ml ‘TUTA’ brand disposable Penicillin syringe, fitted with a 25-gauge hypodermic needle. Before the sample for analysis was taken, a similar sample was withdrawn and discarded to flush the syringe, sampling point and copper capillary tubing. The technique used for collecting soil atmosphere samples with a probe was similar, except that the syringe and barrel were first flushed by withdrawing increments of 1 ml at l-hourly intervals. This interval was adopted to allow sufficient time for satisfactory equilibration between the soil at the probe tip and the atmosphere in the probe (Tackett, 1968). Immediately after sampling, the syringe-needle was inserted into a 2-cm thick sheet of rubber to seal it during the interval between sampling and analysis. Incubation experiments were performed in sealed 1 1. flasks fitted with high-vacuum quality ground glass joints and stopcocks. The atmosphere inside the flasks was Ar or a 20 p/, O,-in-Ar mixture. Samples of gas were analysed periodically for accumulated gaseous products by connection to a mating joint fitted to the gas sampling valve on the gas chromatograph. The sealed growth chambers used to investigate gaseous losses under controlled environmental conditions have been described by Stefanson (1970). The gas chromatographic method employed for analyses of NZ, N,O, CO,, Ar and O2 in gas samples was described by Burford (1969). The soil samples used for incubation and sealed growth chamber experiments were taken from the experimental site used in the field investigations. This was situated on the Urrbrae loam, a red-brown earth (Piper, 1936; Litchfield, 1951). The experimental site used for examining the composition of the soil atmosphere was located on two contiguous areas of the Urrbrae loam; one area had been cultivated or cropped each year for the past 20 years, and the other had been maintained under long-term annual pasture during this time. The old cropped area (designated CC) and a portion of
SOIL DENITRIFICATION
13.5
the pasture area (PC) were cropped to wheat in 1967 and 1968; the remaining portion of the pasture area was maintained under pasture (PP). The field experiment consisted of a randomized block experiment with a control and plus nitrogen treatment (112 kg NaNO,N/ha) replicated 8 times on each area (PP, PC and CC). Each plot contained a diffusion equilibrium reservoir in the A, horizon, A, horizon and B horizon at depths of approximately 7.5, 30 and 60 cm respectively. Wheat was sown on the plots on 17 June 1968 with a basal dressing of 253 kg/ha superphosphate and 200 g/ha MOO,. Soil moisture contents, airfilled pore-space and total pore-space were determined by sampling the soil with a PalmState modified Veihmeyer tube (State and Palm, 1962) and weighing soil cores before and after drying overnight at 105°C. A soil particle-density of 2.65 g/cm3 was assumed in calculations of air-filled and total pore-space in these samples. Soil temperatures were obtained by measuring the resistance of previously calibrated bead-type thermistors with a Wheatstone bridge. Rainfall was measured at the nearby Meteorological Station of the Waite Agricultural Research Institute. RESULTS
AND DISCUSSION
Sampling and analysis of the soil atmosphere Taylor and Abrahams (1953) have shown that a diffusion equilibrium reservoir with a small ratio of volume of reservoir to contact area with the soil is the most desirable for representative sampling of soil atmospheres. Experiments performed on the Urrbrae loam have indicated that the composition of the atmosphere inside the reservoir used in these studies is representative of the soil atmosphere in the immediate vicinity of the reservoir. However, withdrawal of volumes considerably in excess of the reservoir volume during a drying phase of the surface soil can result in the flow of soil air, relatively poor in NzO, from the surface soil (Table 1). It was possible that the soil atmosphere sampled in the immediate vicinity of the reservoir was influenced by soil disturbance during installation of the reservoirs, and to check this, a comparison was made of the composition of samples obtained from the permanently installed reservoirs and those obtained from probe samplers inserted into the soil immediately before sampling, therefore avoiding the disturbed column of soil above the volume of soil sampled. The means of the transformed N,O values were not significantly different (Table 2) and there were no significant differences in the means or variances of the other gases examined. While the error associated with the nitrous oxide measurements was large, similar results have been obtained in the routine measurements of the temporal trends in NzO concentrations in the profile of the Urrbrae loam. The Mann-Whitney U-test (Siegel, 1956) has been satisfactory in establishing differences between treatments. TABLE
1. EFFECT OF VOLUME
OF ATMOSPHERE
WITHDRAWN ANALYSED
Total volume removed
Incremental volume analysed
(ml)
(ml)
10 20 30 45 65
5-10 15-20 25-30 4@-45 6&65
FROM
RESERVOIR ON COMPOSI~ON
Composition
OF SAMPLE
of sample CO, (%)
53 51 50 48 30
78.3 78.3 78.9 78.3 78.3
0.93 0.92 0.90 0.92 0.91
20.2 20.1 20.1 20.1 20.2
0.66 0.66 0.61 0.68 0.63
136
J. R. BURFORD
AND R. C. STEFANSON
TABLE 2. COMPARISON OF NITROUS OXIDE CONCENTRATIONSIN SAMPLESOBTAINEDFROMRESERVOIR AND PROBE SAMPLES Form of data Sampler
Reservoir Probe
Untransformed (parts/lo6 N,O) Mean*
26 57
Transformed to In (parts/lo6 N,O) Mean W 3.18 3.14
0.45 1.60
* Paired measurements on 5 plots. t SE-Standard error (s/n). The variances of the transformed values were significantly different at P = 0.05.
The high variance associated with measurements of N,O concentrations can be interpreted as being an expression of the spatial heterogeneity of the soil. The soil is anaerobic at only a small number of sites within a generally aerobic soil mass (~0~ was never less than 10 per cent in the soil atmospheres sampled), and thus the environmental conditions must be regarded as only marginal for the production of N,O by denitrification when compared to production under completely anaerobic conditions. It is only for nitrous oxide that such variability is encountered and this was due to the between reservoir error as the analytical error for nitrous oxide was less than *3parts/106.1t has been found that the error associated with measurements of Na, 02, Ar and N,/Ar was quite small and the variates were normally distributed. On several occasions the coefficient of variation for the between reservoir error (sampling plus analysis) was as low as O-25 per cent for Nz and 1 per cent for N,/Ar for the A, horizon. Soil denitrification rates of anaerobic soils are known to be markedly influenced by temperature (Nommik, 19.56; Bremner and Shaw, 1958; Cooper and Smith, 1963). However, Fig. 1 shows that there was very little correspondence between fluctuations in N,O levels in the A, horizon and changes in soil temperature at this depth and that, if anything, the concentrations appear to be related to soil moisture contents. This is in agreement with the field results obtained at this site, which showed that maximum N,O concentrations in the soil atmosphere occurred during the winter when soil temperatures were lowest for the year but that soil moisture was consistently higher than at any other time in the year (Burford and Greenland, 1970). Detection and estimation of losses of nitrous oxide from soils Although the high variance associated with measurements of N,O concentrations in the soil atmosphere precludes an accurate estimation of the losses from soils, such measurements can provide good indications of the nature and magnitude of gaseous losses as NzO. For example, the N,O concentrations in the A, (7.5 cm), AZ (30 cm) and B (60 cm) horizons of the Urrbrae soil under pasture are shown in Fig. 2. In the A, horizon, N20 production occurred for short periods after rain had fallen, and the maximum concentration in this horizon was 30 parts/106. N,O was detected for 6 months in the B horizon, with a maximum concentration of 100 parts/lo6 in July-August. The N20 measured in the AZ horizon was probably mainly gas diffusing from the B horizon to the A, horizon and the atmosphere. The application of N03--N fertilizer increased the concentration of NzO throughout the whole profile, although appreciable concentrations accumulated in the B horizon without
SOIL DENITRIFICATION
July
137
1966
FIG. 1. Comparison of nitrous oxide concentrations in the Al horizon of CC area with soil temperature (at 10 cm), soil moisture contents (O-10 cm) and rainfall. the addition of N03--N. McGarity and Meyers (1968) have suggested that simultaneous leaching of nitrate and water-soluble organic materials to a heavy clay B horizon of low (air-filled) porosity could lead to conditions favouring denitrification. The maximum concentrations of N,O in the A, and B horizons were observed on 4 July and 9 August, respectively; on these sampling dates, the increases in N20 concentrations due to the application of nitrate fertilizer were found to be significant (PcO.05) by the Mann-Whitney U-test. This test was also applied to the results obtained on 4 July from the other rotations (PC and CC). It was found that, for the plots with nitrate added, the concentration in the A, horizon of the CC area (160 parts/106) was much greater (PcO.01) than the concentration in the A, horizon of the PC area, but that only moderate significance could be attached to the difference between the means of the PP and PC areas (O-05
D d_c * dz
138
J. R. BURFORD
D, can be calculated
using the equation
where D, is the diffusion porosities respectively.
coefficient
AND R. C. STEFANSON
of Shearer,
for N,O
Millington
and Quirk (1966) :
in air and EA, E, are the air-filled
and total
150
100
I
50
r A2
FIG. 2. Nitrous oxide concentrations in the soil atmosphere of the AI, AZ and B horizons the pasture area, 1968. (Control A- - -A, plus nitrogen fertilizera-A)
of
This treatment for estimating diffusive losses has been applied to the average N,O concentrations in the atmosphere on 4 July, when the highest N20 concentrations for the year were observed in the atmosphere of the AI horizon. The data for the N-fertilized plots (Table 3) show that the calculated maximum rates of loss were quite small, especially when compared to the total amount of N03--N applied to the plots (112 kg/ha). This period of large evolution lasted for only a few days, and thus the total calculated loss of N as N,O from the surface soil was quite small.
139
SOIL DENITRIFICATION
TABLE 3. EFFECTOF POROSITY VALUESON CALCULATED DIFFUSIVELOSSES OF NITROUSOXIDE-N FROMTHE A HORIZONON 4 JULY 1968 Treatment Measurement PC +N
cc +N
32
61
161
0.44 (*0.02) 0.09 (ztO.03)
0.53 (10.03) 0.31 (ztO.04)
0.45 (10.03) 0.15 (&0.04)
0.28 0.12-0.55
0.26 0.07-0.75
PP +N Mean N,O in Ai horizon reservoir (parts/106) Porosity *(ml/ml) Total Air-filled Calculated diffusive loss (kg/ha/day) Mean 5 % Fiducial range
0.010 0~002-0~031
* Parentheses indicate 5 per cent fiducial limits.
The data for the B horizon suggest that the losses from this horizon are probably quite small. Only a very small loss is predicted for appreciable concentrations of NzO in the B horizon as the calculated rate of diffusion is small due to the long diffusion path to the surface and to the relatively small volume of pores available for diffusion. The value obtained for the diffusive loss on 9 August, when there were 100 parts/lo6 NzO in the atmosphere of the B horizon underneath the pasture, was only 0 ~008-0 *084 kg/ha/day (based on total porosity of 0.40 ml/ml and a fiducial range for the air-filled porosity of 0*05-O* 10 ml/ml). The corresponding estimate for the B horizon of the cropped soils is about half this value, as the maximum N,O concentrations were less than 55 parts/106. These results represent the highest rates of loss of N,O from the soil in 1968. As this season was one of the wettest recorded for this area and N,O losses are dependent on high soil moisture contents, the data provides estimates of the maximum rates of losses likely to be experienced from this soil. The values reported in Table 3 were obtained from plots fertilized with NaNO,; NzO concentrations and therefore losses were much lower on unfertilized plots, i.e. typical of normal agricultural practices. It seems reasonable to assume that losses as N20 on unfertilized soils would not have been important. The most important aspect of the calculations of diffusive losses is that estimates of gaseous losses can be subject to a very large error due to the air-filled porosity error. Other serious sources of error exist, e.g. the N,O values, and the uncertain prediction of diffusivities when the air-filled porosity is less than 0.10 ml/ml (Shearer et al., 1966). Also the shape of the gas and porosity gradients in the soil should be accurately known. Accurate assessment of the gaseous losses cannot be obtained from the measurements currently reported, as this will require a more precise characterization of the soil physical environment and the gas distributions associated with the losses. Detection and estimation of the losses as nitrogen gas
Complete investigation of the gaseous losses of N from soils requires the accounting for all gases involved in the losses. Previous work has indicated that Nz and NzO are probably the main species involved (Broadbent and Clark, 1968; Hauck, 1968), and in verifying this, the mechanisms and identity of the gaseous products have been investigated using sealed incubation flasks and enclosed growth chambers. Incubation experiments have shown that the majority of NO,--N and NO,--N added (200 parts/106) to samples of the Urrbrae loam was reduced rapidly to Nz and NzO under
J. R. BURFORD
140
AND R. C. STEFANSON
anaerobic conditions. When a-irradiated (5 Mrad) samples were incubated anaerobically for 53 days, only trace amounts of N,O-N (0.7 parts/IO6 w/w soil) were evolved from nitrate but appreciable amounts of N, and N,O (60 parts/106) were slowly evolved from nitrate. The chemical loss from nitrite appears to be relatively unimportant as only insignificant amounts of nitrite accumulate in the Urrbrae loam. These results indicate that biological denitrification of soil nitrate was the main mechanism responsible for N,O evolved from the Urrbrae loam. More N,O and Nz was evolved in enclosed growth chambers when soil conditions favoured biological denitrification, namely increased soil organic matter content, increased soil water content, and the presence of plants (Stefanson, 1972). Furthermore, the close association in the field experiments between N,O detected in the soil atmosphere and soil nitrate and moisture contents indicates that biological denitrification was responsible for the gaseous losses. Other gases such as NO, NO, and NH, have been reported as possibly contributing to gaseous losses of N from soils, but significant evolution of these gases could not be detected when samples of the Urrbrae loam were incubated aerobically or anaerobically with nitrate. Small amounts of NO were evolved from NOz--N amended samples only in the absence of oxygen. The investigations of gaseous losses in sealed incubation flasks and in enclosed growth chambers have indicated the significance of losses as nitrogen gas. Evolution of NzO in the growth chambers was always accompanied by evolution of Nz, and the ratio of N,:N,O in the evolved gases ranged from 0.06: 1 to 6: 1 where N03-N was applied to the Urrbrae soil (Stefanson, 1972). The soil environment obviously exerts a very strong influence on the ratio of the two gases, and this indicates the need for measurements of N, losses in the field. The detection and measurement of gaseous losses of nitrogen as N, in field studies has proved to be difficult. Evolved N, has to be determined in the presence of the high ‘background’ Nz content of the soil air, and therefore accurate analytical methods are required. A greater problem is that this N, content of the soil atmosphere is not constant (at 78 *08 per cent) but may vary by as much as several per cent from that in the air (Russell and Appleyard, 1915; Boynton and Compton, 1944; Yamaguchi rt al., 1962; Burford and Millington, 1968). This can be attributed to changes in the 0, and CO, content of the soil atmosphere due to soil metabolic activity and to the different solubilities of 0, and CO, in the soil water (Russell, 1961; Boyce and McCalla, 1969). Preliminary investigations into the variations in the N, concentrations in the soil atmosphere have involved the measurement of the concentrations of naturally occurring Ar in the soil atmosphere. Significant deviations from the normal level of this gas in air were observed. However, the results have indicated that the processes responsible are complex, and that further investigations are necessary before evolution of nitrogen in the soil can be measured by determinations of the N, content of the soil atmosphere. REFERENCES
ALBRECHTB., JUNGE C. and ZAKOSEKH. (1970) Der NtO-Gehalt
der Bodenluft in drei Bodenprofilen.
Z.
Pflanz. Bod. 125,205-211.
ALLISONF. E. (1955) The enigma of soil nitrogen balance sheets. Adv. Agron. 7, 213-250. ALLISONF. E. (1966) The fate of nitrogen applied to soils. Adu. Agron. l&219-258. ARNOLDP. W. (1954) Losses of nitrous oxide from soil. J. Soil Sci. 6, 116-128. BOYCEJ. S. and MCCALLAT. M. (1969) Aeration status of subtilled and plowed soils as determined by the partial vacuum method. Soil &i. 108, 241-248. BOYNTOND. and COMPTON0. E. (1944) Normal seasonal changes of oxygen and carbon dioxide percentages in gas from the larger pores of three orchard subsoils. Soil Sci. 57, 107-117.
SOIL DENITRIFICATION BREMNER
J. M. and
SHAW
K. (1958) Denitrification
in soil-II.
141 Factors affecting denitrification.
J. Agric.
Sci., Camb. 51,4&52. BROADBENT
F. E. and CLARK F. E. (1965) Denitrification. In Soil Nitrogen (W. V. Bartholomew and F. E. Clark, Eds) pp. 344359. American Society of Agronomy, Madison, Wisconsin. BURFORDJ. R. (1969) Single sample analysis of (N,-N,O-C02-A-O,) mixtures by gas chromatography. J. chromatogr. BURFORD
J. R. and
Sei. 7, 760-762. GREENLAND D. J. (1970)
Denitrification under an annual pasture. Eleventh International pp. 458-461. University of Queensland Press, St. Lucia, Queensland. BURFORD J. R. and MILLINGTON R. J. (1968) Nitrous oxide in the atmosphere of a red-brown earth. Proceedings of the Ninth International Congress of Soil Science Transactions, Adelaide, Vol. 2, pp. 505-511. COOPER G. S. and SWTH R. L. (1963) Sequence of products formed during denitrification in some diverse Western soils. Proc. Soil Sci. Sot. Am. 27, 659-662. DONALD C. M. (1960) Crop and pasture production. Aust. Inst. Agric. Sci. J. 26,134-152. HAUCK R. D. (1968) Soil and fertilizer nitrogen-a review of recent work and commentary. Proceedings of the Ninth International Congress of Soil Science Transactions, Adelaide, Vol. 2, pp. 475486. LITCHFIELD W. H. (1951) Soil survey of the Waite Agricultural Research Institute, Glen Osmond, South Australia. Divisional Report Division., Soils CSIRO Aust. NO. 2151. MCGARITY J. W. (1961) Denitrification studies on some South Australian soils. PI. Soil 14, l-21. MCGARITY J. W. and MYERS R. J. K. (1968) Denitrifying activity in solodized solonetz soils of eastern Australia. Proc. Soil Sci. Sot. Am. 32, 812-817. NOMMIKH. (1956). Investigations on denitrification in soil. Acta Agric. Stand. 6, 195-228. PIPER C. S. (1936) The red-brown earths of South Australia. Trans. R. Sot. S. Aust. 62, 53-100. RUSSELLE. J. and APPLEYARDA. (1915) The atmosphere of the soil: Its composition and the cause of variation. J. Agric. Sci., Camb. 7, l-48. RUSSELLE. W. (1961) Soil Conditions and Plant Growth, 9th Edn., Longmans & Green, London. SHEARERR. C., MILLINGTONR. J. and QUIRK J. P. (1966) Oxygen diffusion through sands in relation to capillary hysteresis-2. Quasi-steady-state diffusion of oxygen through partly saturated sands. Soil Sci. 101,432436. SIEGEL, S. (1956) Nonparametric Statistics for the Behavioural Sciences, pp. 116127, McGraw-Hill, New York. STACY H. C. T. and PALM A. W. (1962) A thin-walled tube for core sampling of soils. Aust. J. exp. Grassland
Congress,
Agric. Anim. Hub.
2,238-240.
STEFANSON R. C. (1970) Sealed growth chambers for studies of the effects of plants on the soil atmosphere. J. agric. Engng Res. 15,295-301. STEFANSON R. C. (1973) Evolution patterns of nitrous oxide and nitrogen in sealed soil-plant systems. Soil Biol. Biochem.
5,167-169.
STEFANSON R. C. and GREENLANDD. J. (1970) Measurement of nitrogen and nitrous oxide evolution from soil-plant systems using sealed growth chambers. Soil Sci. 109,203-206. TACKETTJ. L. (1968) Theory and application of gas chromatography in soil aeration research. Proc. Soil Sci. Sot. Am. 32, 346-350.
TAYLORG. S. and ABRAHAMS J. H. (1953) A diffusion-equilibrium method for obtaining soil gases under field conditions. Proc. Soil Sci. Sot. Am. 24, 279-282. YAMAGUCHIM., HOWARD F. D., HUGHESD. L. and FLOCKERW. J. (1962) An improved technique for sampling and analysis of soil atmospheres. Proc. Soil Sci. Sot. Am. 26, 512-513.
S.B.B.
5/1-K
Vol. 5 ,pp .133-141. Pergamon Press 1973. Printed in Great Britain
MEASUREMENT
OF GASEOUS LOSSES OF NITROGEN SOILS
FROM
J. R. BURFORD* and R. C. STEFANSON Department of Agricultural Biochemistry and Soil Science, Waite Agricultural Research Institute, University of Adelaide, Glen Osmond, South Australia 5064 (Accepted 1 March 1972) Summary-The measurement of gaseous losses of nitrogen from a red-brown earth has been investigated by direct determination of the evolved gaseous products. The results of field investigations, enclosed growth chamber experiments, and incubation experiments have indicated that the majority of the losses as N20 are due to the microbial reduction of soil nitrate at anaerobic microsites within the generally aerobic soil. The detection and measurement of N,O in the soil atmosphere by analyses for N20 in samples obtained from equilibrium diffusion reservoirs provided a satisfactory and convenient means of detecting losses of nitrogen and of observing the seasonal distributions of nitrous oxide in the soil profile. Aspects of this problem requiring further investigation before the magnitude of the total gaseous losses can be accurately assessed are indicated. INTRODUCTION THE NITROGEN economy
of Australia’s agricultural industry has for many years depended on the symbiotic fixation of nitrogen by pasture legumes (Donald, 1960). A rapid depletion of soil nitrogen reserves occurs during a cropping phase, but the contribution of gaseous losses of nitrogen to such declines and to the total losses occurring contemporaneously with nitrogen fixation are not well known. Although it has been widely assumed that nitrogen is lost in gaseous forms from apparently well aerated soils (Allison, 19.55, 1966; Broadbent and Clark, 1965; Hauck, 1968), there is little direct evidence available concerning the losses in field situations. Much more information is needed about the occurrence, nature, and magnitude of such losses. Initial work by McGarity (1961) demonstrated that samples of a South Australian redbrown earth rapidly reduced N03--N to volatile gases under anaerobic conditions. Subsequent field investigations (Burford and Millington, 1968) showed that evolved N,O could be detected in the atmosphere of this well aerated soil for a period of several months. The detection of gaseous losses of nitrogen from soils by measurement of N,O concentrations in the soil atmosphere has been reported for other soils (Arnold, 1954; Albrecht, Junge and Zakosek, 1970), and the measurement of the evolution of nitrogen gas from a redbrown earth in experiments with enclosed growth chambers (Stefanson and Greenland, 1970) has indicated that losses as N2 probably accompanied the losses as N,O observed from this soil in field experiments. The methods used to examine the evolution of N,O into the soil atmosphere have involved the gas chromatographic or infra-red spectroscopic analysis of soil atmosphere samples obtained from diffusion equilibrium reservoirs (Taylor and Abrahams, 1953) permanently installed in the soil profile. Previous studies have indicated that such reservoirs are satisfactory for examination of the composition of the soil atmosphere for soil aeration studies (Taylor and Abrahams, 1953; Yamaguchi, Howard, Hughes and Flocker, 1962; * Present address: Department
of Soil Science, University of Reading, Reading. 133
134
J. R. BURFORD
AND R. C. STEFANSON
Tackett, 1968) but there is need for some further evaluation to establish that the reservoirs provide a satisfactory means of obtaining a representative sample of the soil atmosphere for analysis in studies of the gaseous losses of nitrogen from soils. The purpose of this paper is to report the techniques used in our investigations of the composition of the soil atmosphere, the major sources of error in estimating the magnitudes of gaseous losses of nitrogen from such measurements of the evolved gases, and the probable mechanism responsible for the losses on the Urrbrae loam.
MATERIALS
AND METHODS
The diffusion equilibrium gas reservoirs used for sampling the soil atmosphere consisted of small 3 *5 x 4.1 cm dia. tinned-iron cylindrical cans. The open base of each can was covered with a cone of stainless steel mesh. The interior of each reservoir was connected with an above ground level gas sampling point (consisting of a 4-cm length of 4 mm id. copper tubing sealed with a gas chromatograph septum) by means of a 2-m length of 1 mm i.d. copper capillary tubing sealed into the top of the can. The gas reservoirs were installed at appropriate depths in the soil by placing them on a l-cm deep bed of fine, washed building sand at the bottom of a 6. l-cm dia. hole. The space between the can and the walls of the hole was packed with sand, then the soil was replaced in approximately the correct order, with frequent tamping. The gas sampling probes were similar to those described by Tackett (1968) except that the external diameter of each probe was tapered towards the tip to facilitate a good seal between probe and soil. The soil atmosphere in the diffusion equilibrium reservoirs was sampled by withdrawing a 5 ml sample from the sampling point with a 5-ml ‘TUTA’ brand disposable Penicillin syringe, fitted with a 25-gauge hypodermic needle. Before the sample for analysis was taken, a similar sample was withdrawn and discarded to flush the syringe, sampling point and copper capillary tubing. The technique used for collecting soil atmosphere samples with a probe was similar, except that the syringe and barrel were first flushed by withdrawing increments of 1 ml at l-hourly intervals. This interval was adopted to allow sufficient time for satisfactory equilibration between the soil at the probe tip and the atmosphere in the probe (Tackett, 1968). Immediately after sampling, the syringe-needle was inserted into a 2-cm thick sheet of rubber to seal it during the interval between sampling and analysis. Incubation experiments were performed in sealed 1 1. flasks fitted with high-vacuum quality ground glass joints and stopcocks. The atmosphere inside the flasks was Ar or a 20 p/, O,-in-Ar mixture. Samples of gas were analysed periodically for accumulated gaseous products by connection to a mating joint fitted to the gas sampling valve on the gas chromatograph. The sealed growth chambers used to investigate gaseous losses under controlled environmental conditions have been described by Stefanson (1970). The gas chromatographic method employed for analyses of NZ, N,O, CO,, Ar and O2 in gas samples was described by Burford (1969). The soil samples used for incubation and sealed growth chamber experiments were taken from the experimental site used in the field investigations. This was situated on the Urrbrae loam, a red-brown earth (Piper, 1936; Litchfield, 1951). The experimental site used for examining the composition of the soil atmosphere was located on two contiguous areas of the Urrbrae loam; one area had been cultivated or cropped each year for the past 20 years, and the other had been maintained under long-term annual pasture during this time. The old cropped area (designated CC) and a portion of
SOIL DENITRIFICATION
13.5
the pasture area (PC) were cropped to wheat in 1967 and 1968; the remaining portion of the pasture area was maintained under pasture (PP). The field experiment consisted of a randomized block experiment with a control and plus nitrogen treatment (112 kg NaNO,N/ha) replicated 8 times on each area (PP, PC and CC). Each plot contained a diffusion equilibrium reservoir in the A, horizon, A, horizon and B horizon at depths of approximately 7.5, 30 and 60 cm respectively. Wheat was sown on the plots on 17 June 1968 with a basal dressing of 253 kg/ha superphosphate and 200 g/ha MOO,. Soil moisture contents, airfilled pore-space and total pore-space were determined by sampling the soil with a PalmState modified Veihmeyer tube (State and Palm, 1962) and weighing soil cores before and after drying overnight at 105°C. A soil particle-density of 2.65 g/cm3 was assumed in calculations of air-filled and total pore-space in these samples. Soil temperatures were obtained by measuring the resistance of previously calibrated bead-type thermistors with a Wheatstone bridge. Rainfall was measured at the nearby Meteorological Station of the Waite Agricultural Research Institute. RESULTS
AND DISCUSSION
Sampling and analysis of the soil atmosphere Taylor and Abrahams (1953) have shown that a diffusion equilibrium reservoir with a small ratio of volume of reservoir to contact area with the soil is the most desirable for representative sampling of soil atmospheres. Experiments performed on the Urrbrae loam have indicated that the composition of the atmosphere inside the reservoir used in these studies is representative of the soil atmosphere in the immediate vicinity of the reservoir. However, withdrawal of volumes considerably in excess of the reservoir volume during a drying phase of the surface soil can result in the flow of soil air, relatively poor in NzO, from the surface soil (Table 1). It was possible that the soil atmosphere sampled in the immediate vicinity of the reservoir was influenced by soil disturbance during installation of the reservoirs, and to check this, a comparison was made of the composition of samples obtained from the permanently installed reservoirs and those obtained from probe samplers inserted into the soil immediately before sampling, therefore avoiding the disturbed column of soil above the volume of soil sampled. The means of the transformed N,O values were not significantly different (Table 2) and there were no significant differences in the means or variances of the other gases examined. While the error associated with the nitrous oxide measurements was large, similar results have been obtained in the routine measurements of the temporal trends in NzO concentrations in the profile of the Urrbrae loam. The Mann-Whitney U-test (Siegel, 1956) has been satisfactory in establishing differences between treatments. TABLE
1. EFFECT OF VOLUME
OF ATMOSPHERE
WITHDRAWN ANALYSED
Total volume removed
Incremental volume analysed
(ml)
(ml)
10 20 30 45 65
5-10 15-20 25-30 4@-45 6&65
FROM
RESERVOIR ON COMPOSI~ON
Composition
OF SAMPLE
of sample CO, (%)
53 51 50 48 30
78.3 78.3 78.9 78.3 78.3
0.93 0.92 0.90 0.92 0.91
20.2 20.1 20.1 20.1 20.2
0.66 0.66 0.61 0.68 0.63
136
J. R. BURFORD
AND R. C. STEFANSON
TABLE 2. COMPARISON OF NITROUS OXIDE CONCENTRATIONSIN SAMPLESOBTAINEDFROMRESERVOIR AND PROBE SAMPLES Form of data Sampler
Reservoir Probe
Untransformed (parts/lo6 N,O) Mean*
26 57
Transformed to In (parts/lo6 N,O) Mean W 3.18 3.14
0.45 1.60
* Paired measurements on 5 plots. t SE-Standard error (s/n). The variances of the transformed values were significantly different at P = 0.05.
The high variance associated with measurements of N,O concentrations can be interpreted as being an expression of the spatial heterogeneity of the soil. The soil is anaerobic at only a small number of sites within a generally aerobic soil mass (~0~ was never less than 10 per cent in the soil atmospheres sampled), and thus the environmental conditions must be regarded as only marginal for the production of N,O by denitrification when compared to production under completely anaerobic conditions. It is only for nitrous oxide that such variability is encountered and this was due to the between reservoir error as the analytical error for nitrous oxide was less than *3parts/106.1t has been found that the error associated with measurements of Na, 02, Ar and N,/Ar was quite small and the variates were normally distributed. On several occasions the coefficient of variation for the between reservoir error (sampling plus analysis) was as low as O-25 per cent for Nz and 1 per cent for N,/Ar for the A, horizon. Soil denitrification rates of anaerobic soils are known to be markedly influenced by temperature (Nommik, 19.56; Bremner and Shaw, 1958; Cooper and Smith, 1963). However, Fig. 1 shows that there was very little correspondence between fluctuations in N,O levels in the A, horizon and changes in soil temperature at this depth and that, if anything, the concentrations appear to be related to soil moisture contents. This is in agreement with the field results obtained at this site, which showed that maximum N,O concentrations in the soil atmosphere occurred during the winter when soil temperatures were lowest for the year but that soil moisture was consistently higher than at any other time in the year (Burford and Greenland, 1970). Detection and estimation of losses of nitrous oxide from soils Although the high variance associated with measurements of N,O concentrations in the soil atmosphere precludes an accurate estimation of the losses from soils, such measurements can provide good indications of the nature and magnitude of gaseous losses as NzO. For example, the N,O concentrations in the A, (7.5 cm), AZ (30 cm) and B (60 cm) horizons of the Urrbrae soil under pasture are shown in Fig. 2. In the A, horizon, N20 production occurred for short periods after rain had fallen, and the maximum concentration in this horizon was 30 parts/106. N,O was detected for 6 months in the B horizon, with a maximum concentration of 100 parts/lo6 in July-August. The N20 measured in the AZ horizon was probably mainly gas diffusing from the B horizon to the A, horizon and the atmosphere. The application of N03--N fertilizer increased the concentration of NzO throughout the whole profile, although appreciable concentrations accumulated in the B horizon without
SOIL DENITRIFICATION
July
137
1966
FIG. 1. Comparison of nitrous oxide concentrations in the Al horizon of CC area with soil temperature (at 10 cm), soil moisture contents (O-10 cm) and rainfall. the addition of N03--N. McGarity and Meyers (1968) have suggested that simultaneous leaching of nitrate and water-soluble organic materials to a heavy clay B horizon of low (air-filled) porosity could lead to conditions favouring denitrification. The maximum concentrations of N,O in the A, and B horizons were observed on 4 July and 9 August, respectively; on these sampling dates, the increases in N20 concentrations due to the application of nitrate fertilizer were found to be significant (PcO.05) by the Mann-Whitney U-test. This test was also applied to the results obtained on 4 July from the other rotations (PC and CC). It was found that, for the plots with nitrate added, the concentration in the A, horizon of the CC area (160 parts/106) was much greater (PcO.01) than the concentration in the A, horizon of the PC area, but that only moderate significance could be attached to the difference between the means of the PP and PC areas (O-05
D d_c * dz
138
J. R. BURFORD
D, can be calculated
using the equation
where D, is the diffusion porosities respectively.
coefficient
AND R. C. STEFANSON
of Shearer,
for N,O
Millington
and Quirk (1966) :
in air and EA, E, are the air-filled
and total
150
100
I
50
r A2
FIG. 2. Nitrous oxide concentrations in the soil atmosphere of the AI, AZ and B horizons the pasture area, 1968. (Control A- - -A, plus nitrogen fertilizera-A)
of
This treatment for estimating diffusive losses has been applied to the average N,O concentrations in the atmosphere on 4 July, when the highest N20 concentrations for the year were observed in the atmosphere of the AI horizon. The data for the N-fertilized plots (Table 3) show that the calculated maximum rates of loss were quite small, especially when compared to the total amount of N03--N applied to the plots (112 kg/ha). This period of large evolution lasted for only a few days, and thus the total calculated loss of N as N,O from the surface soil was quite small.
139
SOIL DENITRIFICATION
TABLE 3. EFFECTOF POROSITY VALUESON CALCULATED DIFFUSIVELOSSES OF NITROUSOXIDE-N FROMTHE A HORIZONON 4 JULY 1968 Treatment Measurement PC +N
cc +N
32
61
161
0.44 (*0.02) 0.09 (ztO.03)
0.53 (10.03) 0.31 (ztO.04)
0.45 (10.03) 0.15 (&0.04)
0.28 0.12-0.55
0.26 0.07-0.75
PP +N Mean N,O in Ai horizon reservoir (parts/106) Porosity *(ml/ml) Total Air-filled Calculated diffusive loss (kg/ha/day) Mean 5 % Fiducial range
0.010 0~002-0~031
* Parentheses indicate 5 per cent fiducial limits.
The data for the B horizon suggest that the losses from this horizon are probably quite small. Only a very small loss is predicted for appreciable concentrations of NzO in the B horizon as the calculated rate of diffusion is small due to the long diffusion path to the surface and to the relatively small volume of pores available for diffusion. The value obtained for the diffusive loss on 9 August, when there were 100 parts/lo6 NzO in the atmosphere of the B horizon underneath the pasture, was only 0 ~008-0 *084 kg/ha/day (based on total porosity of 0.40 ml/ml and a fiducial range for the air-filled porosity of 0*05-O* 10 ml/ml). The corresponding estimate for the B horizon of the cropped soils is about half this value, as the maximum N,O concentrations were less than 55 parts/106. These results represent the highest rates of loss of N,O from the soil in 1968. As this season was one of the wettest recorded for this area and N,O losses are dependent on high soil moisture contents, the data provides estimates of the maximum rates of losses likely to be experienced from this soil. The values reported in Table 3 were obtained from plots fertilized with NaNO,; NzO concentrations and therefore losses were much lower on unfertilized plots, i.e. typical of normal agricultural practices. It seems reasonable to assume that losses as N20 on unfertilized soils would not have been important. The most important aspect of the calculations of diffusive losses is that estimates of gaseous losses can be subject to a very large error due to the air-filled porosity error. Other serious sources of error exist, e.g. the N,O values, and the uncertain prediction of diffusivities when the air-filled porosity is less than 0.10 ml/ml (Shearer et al., 1966). Also the shape of the gas and porosity gradients in the soil should be accurately known. Accurate assessment of the gaseous losses cannot be obtained from the measurements currently reported, as this will require a more precise characterization of the soil physical environment and the gas distributions associated with the losses. Detection and estimation of the losses as nitrogen gas
Complete investigation of the gaseous losses of N from soils requires the accounting for all gases involved in the losses. Previous work has indicated that Nz and NzO are probably the main species involved (Broadbent and Clark, 1968; Hauck, 1968), and in verifying this, the mechanisms and identity of the gaseous products have been investigated using sealed incubation flasks and enclosed growth chambers. Incubation experiments have shown that the majority of NO,--N and NO,--N added (200 parts/106) to samples of the Urrbrae loam was reduced rapidly to Nz and NzO under
J. R. BURFORD
140
AND R. C. STEFANSON
anaerobic conditions. When a-irradiated (5 Mrad) samples were incubated anaerobically for 53 days, only trace amounts of N,O-N (0.7 parts/IO6 w/w soil) were evolved from nitrate but appreciable amounts of N, and N,O (60 parts/106) were slowly evolved from nitrate. The chemical loss from nitrite appears to be relatively unimportant as only insignificant amounts of nitrite accumulate in the Urrbrae loam. These results indicate that biological denitrification of soil nitrate was the main mechanism responsible for N,O evolved from the Urrbrae loam. More N,O and Nz was evolved in enclosed growth chambers when soil conditions favoured biological denitrification, namely increased soil organic matter content, increased soil water content, and the presence of plants (Stefanson, 1972). Furthermore, the close association in the field experiments between N,O detected in the soil atmosphere and soil nitrate and moisture contents indicates that biological denitrification was responsible for the gaseous losses. Other gases such as NO, NO, and NH, have been reported as possibly contributing to gaseous losses of N from soils, but significant evolution of these gases could not be detected when samples of the Urrbrae loam were incubated aerobically or anaerobically with nitrate. Small amounts of NO were evolved from NOz--N amended samples only in the absence of oxygen. The investigations of gaseous losses in sealed incubation flasks and in enclosed growth chambers have indicated the significance of losses as nitrogen gas. Evolution of NzO in the growth chambers was always accompanied by evolution of Nz, and the ratio of N,:N,O in the evolved gases ranged from 0.06: 1 to 6: 1 where N03-N was applied to the Urrbrae soil (Stefanson, 1972). The soil environment obviously exerts a very strong influence on the ratio of the two gases, and this indicates the need for measurements of N, losses in the field. The detection and measurement of gaseous losses of nitrogen as N, in field studies has proved to be difficult. Evolved N, has to be determined in the presence of the high ‘background’ Nz content of the soil air, and therefore accurate analytical methods are required. A greater problem is that this N, content of the soil atmosphere is not constant (at 78 *08 per cent) but may vary by as much as several per cent from that in the air (Russell and Appleyard, 1915; Boynton and Compton, 1944; Yamaguchi rt al., 1962; Burford and Millington, 1968). This can be attributed to changes in the 0, and CO, content of the soil atmosphere due to soil metabolic activity and to the different solubilities of 0, and CO, in the soil water (Russell, 1961; Boyce and McCalla, 1969). Preliminary investigations into the variations in the N, concentrations in the soil atmosphere have involved the measurement of the concentrations of naturally occurring Ar in the soil atmosphere. Significant deviations from the normal level of this gas in air were observed. However, the results have indicated that the processes responsible are complex, and that further investigations are necessary before evolution of nitrogen in the soil can be measured by determinations of the N, content of the soil atmosphere. REFERENCES
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SHAW
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F. E. and CLARK F. E. (1965) Denitrification. In Soil Nitrogen (W. V. Bartholomew and F. E. Clark, Eds) pp. 344359. American Society of Agronomy, Madison, Wisconsin. BURFORDJ. R. (1969) Single sample analysis of (N,-N,O-C02-A-O,) mixtures by gas chromatography. J. chromatogr. BURFORD
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Denitrification under an annual pasture. Eleventh International pp. 458-461. University of Queensland Press, St. Lucia, Queensland. BURFORD J. R. and MILLINGTON R. J. (1968) Nitrous oxide in the atmosphere of a red-brown earth. Proceedings of the Ninth International Congress of Soil Science Transactions, Adelaide, Vol. 2, pp. 505-511. COOPER G. S. and SWTH R. L. (1963) Sequence of products formed during denitrification in some diverse Western soils. Proc. Soil Sci. Sot. Am. 27, 659-662. DONALD C. M. (1960) Crop and pasture production. Aust. Inst. Agric. Sci. J. 26,134-152. HAUCK R. D. (1968) Soil and fertilizer nitrogen-a review of recent work and commentary. Proceedings of the Ninth International Congress of Soil Science Transactions, Adelaide, Vol. 2, pp. 475486. LITCHFIELD W. H. (1951) Soil survey of the Waite Agricultural Research Institute, Glen Osmond, South Australia. Divisional Report Division., Soils CSIRO Aust. NO. 2151. MCGARITY J. W. (1961) Denitrification studies on some South Australian soils. PI. Soil 14, l-21. MCGARITY J. W. and MYERS R. J. K. (1968) Denitrifying activity in solodized solonetz soils of eastern Australia. Proc. Soil Sci. Sot. Am. 32, 812-817. NOMMIKH. (1956). Investigations on denitrification in soil. Acta Agric. Stand. 6, 195-228. PIPER C. S. (1936) The red-brown earths of South Australia. Trans. R. Sot. S. Aust. 62, 53-100. RUSSELLE. J. and APPLEYARDA. (1915) The atmosphere of the soil: Its composition and the cause of variation. J. Agric. Sci., Camb. 7, l-48. RUSSELLE. W. (1961) Soil Conditions and Plant Growth, 9th Edn., Longmans & Green, London. SHEARERR. C., MILLINGTONR. J. and QUIRK J. P. (1966) Oxygen diffusion through sands in relation to capillary hysteresis-2. Quasi-steady-state diffusion of oxygen through partly saturated sands. Soil Sci. 101,432436. SIEGEL, S. (1956) Nonparametric Statistics for the Behavioural Sciences, pp. 116127, McGraw-Hill, New York. STACY H. C. T. and PALM A. W. (1962) A thin-walled tube for core sampling of soils. Aust. J. exp. Grassland
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S.B.B.
5/1-K