Soil denitrification rates at wastewater irrigation sites receiving primary-treated and anaerobically treated meat-processing effluent

Bioresource Technology 43 (1993) 41-46

SOIL DENITRIFICATION RATES AT WASTEWATER IRRIGATION SITES RECEIVING PRIMARY-TREATED A N D ANAEROBICALLY TREATED MEAT-PROCESSING EFFLUENT J. M. Russell, R. N. C o o p e r & S. B. L i n d s e y Meat Industry Research Institute of New Zealand, PO Box 617, Hamilton, New Zealand (Received 29 July 1991; revised version received 25 February 1992; accepted 5 March 1992)

nitrogen to ammonium and then nitrate. Proteins and ammonium ions are immobilized by adsorption on to soil particles (Brady, 1974; Russell, 1982). Nitrate is usually mobile in soil systems, and nitrate that is not utilized by plants can therefore be rapidly leached to groundwater. By the process of denitrifieation nitrate can also be converted to nitrous oxide (N20)and then to nitrogen gas (N2) by microorganisms. The conditions that are required for denitrification are: an absence of oxygen (anoxic conditions), a source of metabolizable carbon and a source of nitrate (Nommick, 1956; Broadbent & Clark, 1965; Burford and Bremner, 1975; Parkin & Robinson, 1989). Conditions suitable for denitrification exist at many effluent irrigation sites. Between irrigation events the soils are aerobic and the applied nitrogen is converted to nitrate. During an irrigation event soil oxygen is displaced by the applied effluent, resulting in anoxic conditions. These anoxic conditions are further enhanced by the metabolism of organic compounds in the effluent. These same organic compounds supply a source of carbon for the denitrifying microorganisms. Denitrification can be regarded as beneficial in wastewater treatment systems. The loss of nitrogen as NzO or N2 reduces the possibility of nitrate entering groundwater. However, nitrous oxide has a complex atmospheric chemistry (Banin, 1986). It absorbs infrared radiation and therefore contributes to global warming (the 'greenhouse effect'). Furthermore, NzO is converted to nitric oxide (NO) in the stratosphere. Nitric oxide catalytically destroys ozone. Nitrous oxide has a very long lifetime in the atmosphere (> 100 years) and atmospheric concentrations are increasing (Ramanathan et al., 1985). The reasons for and sources of this increase are poorly defined (Banin, 1986). This study was designed to quantify the amounts of nitrogen that were lost from three experimental wastewater irrigation sites by deaitrifying microorganisms. Two of the sites were in pasture and had the same soil type, with one receiving primary-treated meat-processing effluent and the other receiving meat-processing effluent after it had passed through an anaerobic lagoon. The third site received primary-treated meat-

Abstract Nitrous oxide emission rates and corresponding N_,O:(N,_O+ N,_) ratios were measured at three sites receiving meat-processing effluent at an annual loading rate of approximately 1000 kg N ha -I. Two of the sites (both a Horotiu sandy loam) were in pasture. One received primary-treated effluent and the other, anaerobically treated effluent. The third site (a highly modified Maori gravelly sand) was planted in trees and received primary-treated effluent. A t all sites N_,O emission rates increased (peaked) immediately following an irrigation event. Emission of N_,O then returned to baseline levels within 24 h. Peak rates at the pasture sites were higher with primary-treated effluent (1-137g N20-N ha- i h- i) than with anaerobic effluent (1-62 g N20-N ha- t h- 9. This was attributed to the higher organic carbon concentration in primary-treated effluent and possibly to the higher soil p H (5"9 compared to 5~9 at the anaerobic effluent site). A t the forest site, peak rates were 12-240 g N,O-N ha -I h -~. Peak nitrous oxide emission rates increased with increasing surface soil temperature. It is concluded that at soil temperatures below 12°C denitrification is not an important nitrogen-removakmechanism. Key words: Slaughterhouse wastes, anaerobic treatment, denitrification, nitrous oxide, land application. INTRODUCTION

Irrigation is becoming a popular method for treating industrial effluents. Irrigation avoids the point-source discharge of wastes to surface waters and allows an economic return, through crop or animal production, to be realized. The major nutrient of concern in meat-processing wastes is nitrogen. In the untreated effluent it exists either in organic forms (proteins, amino acids, etc.) or as ammonium. When effluents are applied to soils, aerobic microorganisms convert the organic forms of Bioresource Technology 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain 41

42

J. M. Russell, R. N. Cooper, S. B. Lindsey

processing effluent but had a different soil type and was planted with trees. Direct field measurements of N20 emissions were made at these sites and total denitrification losses were estimated from laboratory measurements of the NEO:N 2 ratio. The effects of temperature, degree of effluent treatment and cover crop on denitrification rates were examined.

texture (Grange et al., 1939). In addition the area has been extensively modified and compacted during construction of an oxidation pond at this site. At the time when measurements were made, soil properties (0-17 cm layer) were: pH 4.4; N 0"38%; C 4.4% dry soil. A 680-m 2 plot was used. The area had been planted in 1985 in a variety of tree species including

METHODS

Acacia melanoxylon, Acacia dealbata, Cupressus lusitanica, Cupressus macrocarpa, Eucalyptus fraxinoides, Eucalyptus nitens, E. botryoides, E. saligna and Pinus radiata at a density of 5000 trees ha- x. Effluent irriga-

Irrigation sites All three study sites were located approximately 15 km north-west of Hamilton, New Zealand. Soils in this region are derived from showers of fine rhyolitic or andesitic volcanic ash and from pumice carried into the area by the Waikato River (Gibbs et al., 1968).

Site 1 (Pasture, primary effluent) This site was located on a Horotiu sandy loam (soil characteristics of the 0-17 cm layer were: pH 5.9; N 0.63%; C 6.4% dry soil). Two 5-m 2 plots received primary-treated meat-processing effluent every 2 weeks at a rate of 1060 mm year -x (approximately 1000 kg N ha -x year-X). Typical effluent characteristics are shown in Table 1. The cover crop on the plots was ryegrass/clover (Lolium perenne/Trifolium sp.) and the pasture was harvested with a rotary lawn mower when required. This site has been described in more detail by Russell and Cooper (1987).

Site 2 (Pasture, anaerobic effluent) This site location was the same as site 1. (Soil characteristics of the 0-17 cm layer were: pH 5"0; N 0.65%; C 6"5% dry soil.) At the same time that the plots described under site 1 received primary-treated effluent, two similar plots received the same volume (1060 mm year-~) of effluent from an anaerobic lagoon receiving primary-treated meat-processing wastes. Typical anaerobic lagoon effluent characteristics are shown in Table 1.

Site 3 (Trees, primary effluenO At this site the original soil type was a Maori gravelly sand. These soils were made artificially by the local Maori people and consist of about 25 cm blackish gravelly sand resting on an old soil of sandy-loam

tion commenced 1 year after establishment. Two years later the density was reduced to 3000 trees ha- l Sampling procedures Emission rates of N20 from the soil were measured in replicate at each of the sites throughout the year, using the enclosed chamber technique described by Ryden et al. (1979). The chambers consisted of 20-cm lengths of 20-cm-diameter pipe. One end of these pipe sections was fitted with a flange and a gas-tight lid. Each lid was fitted with a rubber gas sampling port. During a measurement the chambers were installed in the ground to a depth of 10 cm and the lids were put into place. The rate at which nitrous oxide built up in the chambers was determined by taking a 10-ml sample of the enclosed air, normally 20-60 rain after putting the lids in place. On each sampling occasion, four replicate measurements were made at each of the pasture sites (sites 1 and 2) and five or six replicates were made at the tree site (site 3). Initial atmospheric concentrations of nitrous oxide were determined on samples of air taken from near ground level at each site. The results of these control samples usually agreed well with the known atmospheric nitrous oxide concentration of 0"3 ml m -3 (Ramanathan et al., 1985). The gas samples were stored in evacuated blood-collection containers prior to analysis.

Analytical procedures Gas samples were analysed for N20 by injecting 0.5 ml of each sample into a gas chromatograph column containing Poropak Q (80/100 mesh) at 40"C using pure nitrogen as the carrier gas. Nitrous oxide was detected with an electron capture detector. The gas chromatograph system was calibrated by adding

Table 1. Typical characteristics of the primary-treated and anaerobically treated meat-processing wastes applied to the experimental sites

Primary

Chemical oxygen demand (g m-3) Total Kjeldahl nitrogen (g m-3) Ammonium nitrogen (g m-3) Total suspended solids (g rn-3) Total phosphorus (g m- "~) pH

Anaerobic

Mean

Range

Mean

Range

1900 115 30 640 15 7-0

530-4700 40-230 3-70 220-2100 6-34 6-0-8"5

510 115 85 160 15 7-0

100-1000 55-160 14-150 80-250 7-23 6"5-7"5

Irrigation of meat-processing effluent known amounts of medical grade nitrous oxide to air (containing 0.3 ml N20 m -3 air) and analysing these standards in the same way as field samples. The gas chromatograph response showed good linearity at nitrous oxide concentrations up to 100 ml m-3 air. The ratio of nitrous oxide to total nitrogen gases (N20+N.~) produced by the denitrifying microorganisms in soil cores was determined by the method of Tiedje (1982). Specifically, soil cores (2"5 cm diameter, 10 cm length) were taken from the study sites and transported to the laboratory. Each core was placed in a 300-ml conical flask fitted with a side arm and a sampling septum. The amount of nitrous oxide evolved was measured by taking 0.5-ml gas samples at various time intervals and monitoring the build-up of N,O in the flask. No correction or compensation was made for the small decrease in pressure that occurred each time a sample was taken. Only three or four gas samples were taken from each flask and the error introduced from not correcting for the pressure drop amounted to less than 1%. The incubations were carded out at 20 + I°C. To measure the total amount of nitrogen gases evolved by the soil microorganisms, the same experimental procedure was used except that the atmosphere above the soil cores was amended to include 5% acetylene. Acetylene blocks the formation of nitrogen gas from nitrous oxide (Yoshinari et al., 1977) and the rate of nitrous oxide increase equals the rate at which nitrate is denitrified. The rate of nitrogen gas formation was obtained by subtracting the rate of nitrous oxide increase obtained in the absence of acetylene from that obtained when acetylene was present. Ratios of N20:(N~O+N2) are reported on a mole N:mole N basis. Statistical analysis Denitrification rates were subjected to a loguormal transformation as proposed by Parkin et al. (1988) and Tiedje et al. (1989). Means and standard deviations (SD) were calculated from these transformed data and were used to assess differences between treatments. Natural logarithms were used throughout this study.

43

Linear regressions were performed between the log transformed denitrification rates and soil temperatures for each of the experimental sites and the slopes and elevations of the derived relationships were compared using the Student's t statistic and the procedures described by Zar (1984). RESULTS Soil nitrous oxide emission rates were highest immediately following an irrigation event. These high (peak) rates (Tables 2 and 3) dropped to baseline levels within 24 h of irrigation ceasing. At the pasture sites (1 and 2) baseline nitrous oxide emission rates were similar in summer and winter (0.4-1.5 g N20-N ha- ~ h- J). Similar rates were also observed on an adjacent non-irrigated site during August (0.42 g N20-N ha-~ h- ~with a SD of 0-37 g N20-N ha-~ h- 1). At the forest site (3) baseline nitrous oxide emission rates ranged between 2"6 and 13 g N20-N ha-l h-~ with the higher rates being observed in spring and summer. Peak denitrification rates showed great spatial variability at each of the sites on each sampling occasion. Table 3. Peak nitrous oxide emission rates (means and standard deviations of the natural log transformed data) at site 3 (trees/primary effluent). Units of untransformed data were g N 2 0 - N ha- t h - i

Date

Soil temperature

Mean

SD

3"43 3"87 4-07 4.28 4.51 5.49 4.49 5.10 5.16 4.87 2-49 2-94 3.13

0"58 1-04 1.22 1"17 1-42 0.65 0.98 0.84 0.83 1.36 0.91 1.46 0-85

(*c) 17 Aug. 1988 12 Sep. 1988 19 Oct. 1988 22 Nov. 1988 25 Jan. 1989 2 Feb. 1989 9 Feb. 1989 22 Feb. 1989 15 Mar. 1989 29 Mar. 1989 18 May 1989 13 Jun. 1989 24 Jul. 1989

18 19 18 24 19 21 19 21 22 20 13 14 6

Table 2. Peak nitrous oxide emission rates (means and standard deviations of natural log transformed data) at site 1 (pasture/ primary effluent) and site 2 (pasture/anaerobic effluent). Units of untransformed data were g N 2 0 - N ha- i h - i

Date

20 Feb. 1990 6 Mar. 1990 20 Mar. 1990 3 Apr. 1990 18 Apr. 1990 1 May 1990 29 May 1990 26 Jun. 1990 24 Jul. 1990

Soil temperature

Site 1

(of) 27 24 23 22 17 15 15 11 14

Site 2

Mean

SD

Mean

SD

4.50 4.70 4.41 4.00 4.48 0-09 1-98 1.30 2.24

0.82 0.76 0.79 1.10 1.01 0-91 1.80 1.31 0-33

3.60 2.79 3-98 2-53 0.83 1-13 0.88 0.91 - 0.22

0.22 0.39 0.67 0.05 0"09 0.32 0.57 0-13 0-37

J. M. Russell, R. N. Cooper, S. B. Lindsey

44

The coefficients of variation of the lognormal data at site 1 ranged between 15 and 1010%, at site 2 between 2 and 168% and at site 3 between 12 and 50%. Greatest variability occurred when rates were low (May), and on most occasions coefficients of variation were between 10 and 30%. Peak denitrification rates were strongly correlated with surface soil temperatures made at the time of measurement (Fig. 1). Correlation coefficients (r) between the log transformed nitrous oxide emission data and the soil temperatures (°C) were 0"68 at pasture site 1 (36 observations on nine occasions), 0-87 at pasture site 2 (35 observations on nine occasions) and 0-51 at the forest site 3 (65 measurements on 13 occasions). At the pasture sites the slopes of the regression lines for these data were not significantly different, being 0"253 (SD = 0"046) for primary effluent application and 0.242 (SD=0.024) for anaerobic effluent application. Intercepts on the In (rate) axis were - 1.58 (SD=0-88) and - 2 . 6 4 (SD=0.46) respectively. Although the slopes were not significantly different (t = 0"21, degrees of freedom = 67), the lines had significantly different elevations (t = 4.79, df = 67). Application of primary-treated meat-processing effluent resulted in significantly higher peak nitrous oxide emission rates than application of anaerobically treated effluent. At the forest site the slope of the regression line was 0-148 (SD=0.031, 64 data points) and the intercept was 1-47 (SD = 0.58). The slope was not significantly different from the slope for primary effluent application to pasture at the 95% level (t = 1.96, df = 96), but the elevations were significantly different (t=4.24, df = 96). The ratio of N 2 0 : ( N 2 0 + N 2) at the pasture site receiving primary-treated effluent was 0"68 (SD -- 0.68, 16 df) and was 0-51 (SD = 0.22, 16 df) at the anaerobic effluent site. At the forest site (site 3) the ratio was 0.93 (SD = 0.22, 34 df). 400-

-7" .t= 40. Z

Site3 •

•

•

ta

t:l

4 Site 1 f D

/,4~

o

/ Site2 ,~

0

U "t"

0.4

0

5

10

15

20

25

30

Temperature (°(2)

Fig. 1. Relationship between average peak N20-N emission rate following an irrigation event and temperature at site 1 (pasture/primary effluent), site 2 (pasture/anaerobic effluent) and site 3 (trees/primary effluent). The lines are the least squares equations fitted to the data points. At site 1 In (rate) = 0.253 T- 1.58 (r= 0"68), at site 2 In(rate) = 0-242 T- 2.64 (r= 0.87) and at site 3 ln(rate)=0"148T+l-47 (r=0"51), where T is the soil temperature (°C).

DISCUSSION At wastewater irrigation sites nitrate is formed during the aerobic periods between irrigation events. When effluent is applied, the soil air is rapidly displaced by the wastewater, the soil becomes saturated and the number of anoxic sites increases (Fluhler et aL, 1976; Binstock, 1984; Grundmann & Rolston, 1987). Denitrification and nitrous oxide emission rates are at a maximum during this period. As the site drains, the number of anoxic sites decreases and background rates re-establish. A similar pattern has been reported at an agricultural site in response to rainfall and water irrigation (Sextone et al., 1985). Donnison and Cooper (1989) have suggested that irrigation sites be rested for 10-14 days before pasture is used as animal feed. For such an irrigation regime, at the primary effluent/ pasture site during the summer, with peak N20 emission rates of about 90 g N ha-l h-J and background N20 emission rates of 1 g N ha- l h- ~occurring for 13 days, total N20 losses are about 1400 g N ha-1 per irrigation cycle. The background rate accounts for 22% of this. During winter, peak rates drop to about 10 g N ha- ~h- t and the background rates account for 72% of the N20 losses. The background rate is therefore an important contributor to the overall nitrogen balance. Nitrous oxide is also an intermediate when ammonium is oxidized to nitrate (Blackmer et al., 1980; Robertson & Tiedje, 1987). The background emissions of N20 observed in this study could result from ammonia oxidation as well as from denitrification at anaerobic microsites (Fluhler et al., 1976). The peak rates occur when the soil is saturated with effluent and all of the nitrous oxide produced will be from denitrification processes. Irrigation of anaerobically treated meat-processing effluent to pasture resulted in lower peak nitrous oxide emission rates than when primary-treated effluent was used as the wastewater source (Fig. 1). Organic carbon levels in primary-treated meat-processing effluents are usually in the range 300-1000 g m-3 and 60% of this is soluble (Cooper et al., 1979). Anaerobic treatment destroys 70-90% of the organic matter (Russell & Cooper, 1983). The lower denitrification rates observed are consistent with the lower concentrations of organic carbon in the anaerobic effluent. The effluent is probably the most important source of metabolizable carbon during an irrigation event. The correlation between extractable soil carbon or mineralizable carbon with denitrification rates has been extensively studied (Burford & Bremner, 1975; Stanford et al., 1975). Recently the importance of metabolizable carbon has been demonstrated by Parkin and Robinson (1989), who developed a model for predicting denitrification rates in which the CO2 production rate is an important parameter. The CO 2 production rate is closely related to the concept of oxygen demand as used in waste treatment technology. The observed difference in NzO emission rates at the pasture sites receiving primary-treated and anae-

Irrigation of meat-processing effluent robically-treated effluent could also be a pH effect. Between pH 4.4 and 8.2 increasing pH values increase the total losses of nitrogen by denitrifying bacteria (Nommick, 1956; Broadbent & Clark, 1965). The higher soil pH at the pasture primary effluent site (pH 5"9) compared to the anaerobic effluent site (pH 5"0) may have influenced the observed denitrification rates. However, the proportion of total gaseous emissions (N20 + N2) that are in the form of nitrous oxide would be expected to be lower for the higher pH soil (Christensen, 1985; Weier & GiUiam, 1986). In the present study the ratios were similar at the primary-treated site (0.51) and the anaerobic site (0"68). The effect of soil pH on the observed denitrification rates is therefore inconclusive. The pH, effluent organic carbon and denitrification relationships observed at the two pasture sites are consistent with known theory. Where ammonium is converted to nitrate, alkalinity is consumed and the pH decreases. During denitrification, alkalinity is produced and the pH will increase. The amount of metabolizable carbon added to the soil at the pasture site receiving primary-treated effluent was greater than that at the site receiving anaerobic effluent. Therefore, denitrification rates would have been enhanced and the soil pH would have been higher at the site receiving primary-treated effluent. Changes in soil pH of this type with primary-treated effluent application compared to anaerobic effluent application have been observed with this soil type in small-scale irrigation trials (Russell, 1986). Further research and monitoring is required to separate the effects of organic carbon and pH on the denitrification process. At the sites used in this study, effluent-nitrogen loading rates were approximately 1000 kg N ha-l year-1. About 60% of this nitrogen can be harvested as pasture plant material (Russell & Cooper, 1987), Nitrogen uptake by forests is likely to be lower than that of pasture systems (Anon., 1990). A continuous total denitrification rate of 46 g N ha-t h - t is required if contamination of groundwater by nitrate is to be avoided. These rates were achieved only for limited periods of time immediately following irrigation events during summer periods (Fig. 1). During winter, when soil temperatures dropped below 12°C, denitrification was only a minor removal mechanism for nitrogen. At this time plant uptake would also be minimal, and during periods of excess rainfall or additional irrigation, leaching of nitrate is likely to occur. Therefore, winter application rates of effluent should be reduced as much as possible.

ACKNOWLEDGEMENTS This work was made possible by funding from the New Zealand Foundation for Research, Science and Technology and from the New Zealand Meat Research and Development Council.

45

REFERENCES Anon. (1990). Natural Systems for Wastewater Treatment, Water Pollution Control Federation Manual of Practice FD-16. Water Pollution Control Federation, Alexandria, Virginia. Banin, A. (1986). Global budget of N20: the role of soils and their change. Sci. TotalEnviron., 55, 27-38. Binstock, D. A. (1984). Potential denitrification in an acid forest soil: dependence on wetting and drying. Soil Biol. Biochem., 16, 287-8. Biackmer, A. M., Bremner, J. M. & Schmidt, E. L. (1980). Production of nitrous oxide by ammonia-oxidizing chemautotrophic microorganisms in soil. Appl. Environ. MicrobioL, 40, 1060-5. Brady, N. C. (1974). The Nature and Properties of Soils, (8th edn). Macmillan, New York. Broadbent, F. E. & Clark, E (1965). Denitrification. In Soil Nitrogen, (eds W. V. Bartholomew & F. E. Clark.) American Society of Agronomy, Madison, pp. 344-59. Burford, J. R. & Bremner, J. M. (1975). Relationships between the denitrification capacities of soils and total, water-soluble and readily decomposable soil organic matter. SoilBiol. Biochem., 7, 389-94. Christensen, S. (1985). Denitrification in an acid soil: effects of slurry and potassium nitrate on the evolution of nitrous oxide and on nitrate-reducing bacteria. Soil Biol. Biochem., 17,757-64. Cooper, R. N., Heddle, J. E & Russell, J. M. (1979). Characteristics and treatment of slaughterhouse effluents in New Zealand. Prog. Water Tech., I l, 55-68. Donnison, A. M. & Cooper, R. N. (1989). Faecal coliform decline on pasture irrigated with primary-treated meatprocessing effluent. N.Z.J. Agric. Res., 32, 105-12. Fluhler, H., Stolzy, L. H. & Ardakani, M. S. (1976). A statistical approach to define soil aeration in respect to denitrification. SoilSci., 122, 115-23. Gibbs, H. S., Cowie, J. D. & Pullar, W. A. (1968). Soils of North Island. In Soils of New Zealand Part 1, N.Z. Soil Bureau Bulletin no. 26 (1). Government Printer, Wellington, New Zealand, pp. 48-66. Grange, L. 1., Taylor, N. H., Sutherland, C. E, Dixon, J. K., Hodgson, L. & Seelye, E T. (1939). Soils and Agriculture of Part of Waipa County. Department of Scientific and Industrial Research Bulletin no. 76. Government Printer, Wellington, New Zealand. Grundmann, G. & Rolston, D. E. (1987). A water function approximation to degree of anaerobiosis associated with denitrification. Soil Sci., 144, 437-41. Nommick, H. (1956). Investigations on denitrification in soil. Acta Agric. Scand., 6, 195-228. Parkin, T. B. & Robinson, J. A. (1989). Stochastic models of soil denitrification. Appl. Environ. Microbiol., 55, 72-7. Parkin, T. B., Meisinger, J. J., Chester, S. T., Starr, J. L. & Robinson, J. A. ( 1988). Evaluation of statistical estimation methods for lognormally distributed variables. Soil Sci. Soc. Am. J., 52, 323-9. Ramanathan, V., Cicerone, R. J., Singh, H. B. & Kiehl, J. T. (1985). Trace gas trends and their potential role in climate change. J. Geophys. Res., 90, D5547-66. Robertson, G. E & Tiedje, J. M. (1987). Nitrous oxide sources in aerobic soils: nitrification, denitrification and other biological processes. Soil Biol. Biochem., 19, 187-93. Russell, J. M. (1982). Interaction of slaughterhouse effluent protein with three New Zealand soils. N.J.Z. Agric. Res., 25, 21-6. Russell, J. M. (1986). Irrigation of primary-treated and anaerobically treated meat-processing wastes onto pasture: lysimeter trials. Agric. Wastes, 18, 257-68.

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J. M. Russell, R. N. Cooper, S. B. Lindsey

Russell, J. M. & Cooper, R. N. (1983). Lagoons in the export meat industry. In Proceedings of the 15th New Zealand Biotechnology Conference, May 1983, Massey University, Palmerston North, New Zealand, pp. 203-17. Russell, J. M. & Cooper, R. N. (1987). Irrigation of pasture with meat-processing plant effluent. In Proceedings of the 42nd Industrial Waste Conference, May 1987, Purdue University, West Lafayette, Indiana. Lewis Publishers, Chelsea, Michigan, pp. 491-7. Ryden, J. C., Lund, L. J., Letey, J. & Focht, D. D. (1979). Direct measurement of denitrification losses from soils, llDevelopment and application of field methods. Soil Sci. Soc. Am.J., 43, 110-18. Sextone, A. J., Parkin, T. B. & Tiedje, J. M. (1985). Temporal response of soil denitrification rates to rainfall and irrigation. Soil Sci. Soc. Am. J., 49, 99-103.

Stanford, G., Vander Pol, R. A. & Dzienia, S. (1975). Denitrification rates in relation to total and extractable soil carbon. Soil Sci. Soc. Am. J., 39,284-9. Tiedje, J. M. (1982). Denitrification. Agronomy, 9, 1011-26. Tiedje, J. M., Simkins, S. & Groffman, P. M. (1989). Perspectives on measurement of denitrification in the field including recommended protocols for acetylene based methods. Plant and Soil 115, 261-84. Weier, K. L. & Gilliam, J. W. (1986). Effect of acidity on denitrification and nitrous oxide evolution from Atlantic Coastal Plain soils. SoilSci. Soc. Am. J., 50, 1202-5. Yoshinari, T., Hynes, R. & Knowles, R. (1977). Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil. Soil Biol. Biochem., 9, 177-83. Zar, J. H. (1984). Biostatistical Analysis. Prentice-Hall, Engeiwood Cliffs, New Jersey.