Samples of two different pasture soils - Manawatu silt loam (MSL) and Manawatu fine sandy soil (MFSL), both classified as weathered fluvial recent soils (Hewitt 1998) - were used in this study. These soils were the same as those used in Chapter 3. Bulk samples (0-10 cm depth) of each soil were collected from random locations paddocks within Dairy Farm No. 1 at Massey University, Palmerston North, New Zealand. Herbage, stones, roots and other debris were removed from the soils. The field-moist soils were passed through a 2-mm sieve, homogenised by hand and then air-dried for a day. Sub-samples of each of the partially air dried soil were used to determine the soil field capacity using a pressure plate extractor at (1/10 bar) and gravimetric moisture content by drying at 105oc for 16 hours. The soil properties were presented in Table 3.1.
4.2.2 Bulk soils incubation procedure
Samples of field moist soil, equivalent to 2.5 kg of dry soil, were placed in six 3-L plastic containers for each soil type. The three main treatments used in this study were DCD at two rates (20 and 40 mg DCD kg-1 soil which were equivalent to 10 and 20 kg DCD ha-1 assuming the DCD was distributed through the top 5 cm of soil) and no DCD (control). There were two replicates of each of these main treatments. The DCD was thoroughly mixed with the soil. The application rates of DCD were calculated by assuming the soil bulk density of 1 Mg m-3. The soil moisture content was adjusted and maintained at field capacity throughout the study by monitoring the weight changes of each soil sample in the container and adding deionised water as necessary. These soil samples were incubated at 20oC. The container cover was partially lifted open to allow the exchange of air.
4.2.3 Degradation of DCD and DCD analysis
Two 10-g sub-samples of moist soil were taken from each of the incubated main treatments on day 1, followed by weekly sampling until weeks 10 and 11 for the low and high DCD additions respectively in the MFSL, and weeks 18 and 21 for the low and high DCD additions respectively in the MSL. The 10-g soil samples were mixed with deionised water (1:1 soil to water ratio), shaken for 1 hour, centrifuged at 8000 rpm for 3 minutes and filtered through “Whatman 41” filter paper. Five mL of the supernatant was transferred to a 10-mL centrifuge tube, 0.2 mL of 0.66M H2SO4 added and the
mixture left for 5 minutes. The extracted DCD was then re-centrifuged at 4300 rpm for 10 minutes. The final supernatant was kept in a 30-mL plastic bottle at room temperature until analysis for DCD (Schwarzer and Haselwandter, 1996). The DCD analysis was performed on a HPLC (Alliance, Waters 2690, Separation module) with a multi-wavelength UV detector at 210nm (Schwarzer and Haselwandter, 1996). The separation of DCD was carried out using a micro-guard column packed with Aminex Resin, heated at 60oC with a flow rate of 0.9 mL/min of 0.025 M H2SO4 as the mobile
phase. Recording and computing of data was carried out with Millenium32 Waters software. A sub-sample of soil was used for moisture determination at each sampling time by drying at 105oC for 16 hours.
4.2.4 Calculation of DCD half-life
The first-order decay rate equation as described in Saggar and Hedley (2001) was used to calculate the rate of DCD degradation in both soils. The decrease in DCD concentrations in both soils resembled first order reaction kinetics and the data were fitted to the exponential function N = N0 e-kt where, N is the amount of DCD remaining
experiment in the soil and k is the decay constant. The DCD half life is the time taken to reduce DCD concentration to half of the initial values, and was calculated as t1/2 =
0.693/k.
4.2.5 Nitrification assay and analysis of mineral N
At each of the times that the samples described in Section 4.2.3 were collected from the incubating main treatments and analysed for DCD, two further sub-samples, equivalent to 35 g of dry soil, were taken from each container of the incubating soils and were placed in each of 24 plastic cups, for each soil type. Twelve of the plastic cups of each soil type were treated with 0.5 mL of urea solution (~ 200 μg N/g dry soil), while the other 12 plastic cups of each soil types had 0.5 mL of water added. These cups acted as a control. Soil in the cups was mixed with a spatula after the addition of urea and water and the cups were covered with aluminium foil tops with pin holes. All of these 48 plastic cups were incubated aerobically at 26 oC in the dark for 2 weeks (Rodgers 1986). Moisture lost during the incubation period was maintained by adding deionised water to bring the soils up to the original weight. At the end of the 2-week incubation time, a 5 g sub-sample of soil from each cup was extracted with 2 M KCl (1:5 soil:extractant ratio) and the extract was analysed colorimetrically for mineral N by an auto analyser (Blakemore et al. 1987). Soil sub-samples from each cup were also taken for moisture determination by drying at 105 oC for 16 hours. In this study the nitrification rates were calculated as the difference between the amount of NO3--N produced in the soil treated
with urea and the amount of NO3--N produced in the soil treated with water only,
divided by 14, which was the duration in days of the incubation.
In this Chapter (and Chapters 5 and 6), soil amended with urea was incubated as a way to assess potential nitrification activity. Urea was chosen to provide the NH4+ substrate
for nitrification activity because most N added to the soil in fertiliser and animal urine is in the form of urea. In most soils urea is hydrolysed rapidly to NH4+ (e.g. Apthorp et al.
1987) and this was also the case in this experiment. An additional advantage of using urea was that hydrolysis of urea is accompanied by an increase in soil pH and this partially offset the decrease in pH from nitrification. This reduced the chance that high nitrification potentials would be masked by extremely acidic conditions developing in the incubating soils.