Pasture on the It. ryegrass lysimeters was harvested at regular intervals, from Dec 1 till May 30 whilst the oats were harvested once only in mid-November (Nov 22). The oats treatment lysimeters were sprayed with glyphosate to kill any remaining oats activity and then the surface of each was lightly reworked to prepare a seed bed for the sowing of the second kale crop (Regal cultivar; 4 kg ha-1). An application of DAP at 200 kg ha-1 was applied (36 kg N ha-1) at sowing to help establish
the plants (Table 4.2-2). Kale that was grown after the oats harvest was cut on May 30 just prior to deconstruction of the lysimeters. All harvest material was place in a fan-forced oven and dried at 60°C. This material was stored until ground for analysis using a Retsch (Hann, Germany) cyclonic mill (<0.5 mm mesh).
After the end of the experiment in May 2014, the lysimeters were uplifted and a process of deconstruction of the soil within the lysimeters was begun on 31 May to sample soil, plant roots and tops to complete the 15N balance study. The lysimeters were inverted again using the hydraulic
lift on a tractor, the drainage base and gravel layer removed before a base plate that fitted completely within the lysimeter and with positions for legs was fitted and held in position by two cross-sectional steel bars. The lysimeter was then returned to its original position and transferred to the Field Research Centre where it was placed on a frame and the bars removed. A winch was attached to each side of the lysimeter and the frame, and then tightened progressively to force the casing down the side of the soil monolith in 10 cm increments to 40 cm, with the final increment, from 40-60 cm, collected as a single increment (Plate 4.2-5).
Soil and root mass samples were extracted and stored at 4°C for up to two weeks prior to further cleaning of the roots, drying and grinding (plant) or pulverising (soil) for analysis of total-N and 15N
content. Soil mineral-N was extracted using 2 M KCL as outlined in Blakemore et al. (1987). The exact process for the removing, weighing, drying and recording of plant, soil and stone weights is described in Appendix C.
4.2.7
15N balance measurements
Recovery of the N from the 15N-labelled urine was measured in four major components: N in
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immobilised-N (organic and inorganic N). The total mass (m) of N recovery can be represented by the following equation (4.2-5):
𝑁
15 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦 = ∑ 𝐺𝑎𝑠𝑒𝑜𝑢𝑠 𝑁15
𝑚+ 𝐷𝑟𝑎𝑖𝑛 𝑁15 𝑚+15𝑁 𝑢𝑝𝑡𝑎𝑘𝑒𝑚+ 𝑆𝑜𝑖𝑙 𝑁15 𝑚
4.2-5 where: Gaseous N is the mass of N recovered as volatilised ammonia (NH3), emitted dinitrogen
(N2) and nitrous oxide (N2O), Drain 15N is the 15N mass leached in drainage water (ammonium,
nitrate and organic-N), 15N uptake is the 15N mass held in plant harvests and retained vegetation
and roots, and soil 15N is the 15N mass still retained or immobilised in the bulk soil. The percentage
recovery of the 15N applied in urine for the four mass components was calculated as follows (4.2-6):
𝑁
15 % 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑦 = ∑𝐺𝑎𝑠 𝑁15 𝑚+𝐷𝑟𝑎𝑖𝑛 𝑁15 𝑚+15𝑁 𝑢𝑝𝑡𝑎𝑘𝑒𝑚+ 𝑆𝑜𝑖𝑙 𝑁15 𝑚 𝑁
15 𝑎𝑝𝑝𝑙𝑖𝑒𝑑 × 100
4.2-6 where: 15N% recovery is the proportion of 15N recovered in the four major components (gaseous,
liquid, plant and soil forms) from the applied 15N-labelled urine. All other terms are the same as
those in equation 4.2-5.
The 15N content of nitrous oxide (15N2O) and di-nitrogen was determined by taking an additional
gas sample (12 ml exetainer vial), 2 hours after placing the enclosure over the gas ring and once all the T2 (40 min.) N2O samples had been taken. These samples were taken in the same manner
as described in section 4.2.4. The samples were analysed using a PDZ Europa 20-22 continuous flow isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK) enabling measurement of stable isotopes of gases at both enriched and natural abundance levels. Gas samples were prepared using a TGII trace gas system using cryo-trapping and focusing to isolate the N2O/N2 species.
Concentrations of 15N%-N2O were interpolated between weeks and this allowed calculation of the
proportion of N2O derived from the applied urine for the unmeasured as well as measured collection
of 15N%-N2O. Total 15N%-N2O evolved between the twice weekly samplings was derived from
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had declined to negligible levels) and calculated using the formula from Cabrera and Kissel (1989) to produce the following equation (4.2-7):
𝑁2𝑂 − 𝑁 𝑇𝑜𝑡15 = ∑ ( 𝑁15% 𝑎−15%𝑁𝑏)× 𝑁2𝑂𝑓𝑙𝑢𝑥 × 𝐶𝐹 𝑁𝑐− % 15 𝑁 𝑏 % 15 4.2-7 where: 𝑇𝑜𝑡15𝑁2𝑂 − 𝑁 = the total nitrous oxide loss derived from the applied 15N-enriched urine over the sampling period (kg N2O-N ha-1), 15%𝑁𝑎 is the daily average atom% 15N abundance of nitrous oxide, 15%𝑁𝑏 is the atom% 15N natural abundance of nitrous oxide (0.3663%), N2Oflux is the hourly nitrous oxide emission (mg N-N2O h-1m-2) (from equation 4.2-7), CF is the conversion factor (0.24)
to convert units for mg N2O-N m2 h-1 to kg N2O-N ha-1 day-1, and 15%𝑁𝑐 is the 15N-enriched content (%) of the applied urine or water (controls).
Calculations of the 15N enrichment in di-nitrogen gas (N2) were carried out using essentially the
same equations as those for 15N-N2O determination but were more problematic due to the relatively
small differences created by a high background of N2 concentration (i.e. N2 =~80% of atmospheric
gases) and a relatively low initial 15N enrichment (~9%). More comprehensive measurements of
N2 denitrification losses would require increasing the 15N enrichment to ~40% (Clough et al. 2001)
but the cost of doing so in this study was prohibitive. Measurements were linearly interpolated between weeks and the rate of 15N2 evolved was also assumed to be linear (Clough et al. 2001).
Total 15N-N2 was measured over 15 weeks and calculated using the following equation (4.2-8):
𝑁2 𝑇𝑜𝑡15 = ∑ 𝑁2%× ( 𝑁15% 𝑎−15%𝑁𝑏)× 𝑉 × 𝑃 × 𝑀𝑊𝑁× 𝐶𝐹 ( 𝑁𝑐15% −15%𝑁𝑏) × 𝑅 × 𝑇 × 𝑆𝐴 4.2-8 where: 𝑁2
𝑇𝑜𝑡15 = total N loss over monitoring period as di-nitrogen gas derived from the 15N-enriched labelled urine (kg N ha-1),
𝑁2% = Percentage of air in enclosure present as di-nitrogen,
𝑁𝑎 %
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%
15 = is the atom% 15N natural abundance of di-nitrogen (0.3663%),
𝑁𝑐 %
15 = is the atom% 15N abundance of the applied urine,
V = enclosure headspace volume (L),
P = atmospheric pressure (nominally assumed at 1 atmosphere),
CF = conversion factor (7 x 10-4) for days to weeks (days x7) L to L (L/106) N2 and dm2 to m2
(dm2 x102),
MWN= molecular weight of N in N2 (28.0 g mol-1),
R = universal gas constant (0.0821 L atm mol-1 K-1),
T = temperature (K) at midday-to-2 pm for each measurement time, and
SA = surface area (m2) of the lysimeter.
Measurements of the 15N proportion of the ammonium and nitrate content of the leachates was
undertaken using a selection of samples covering the breakthrough curve of concentration of both ions after FIA results were obtained. The 15N present in the samples was concentrated on 7 mm
glass fibre disks using the method described by Brooks et al. (1989) before combustion at 1000°C using a PDZ Europa 20-20 stable isotope mass spectrometer (Sercon Ltd., Cheshire, UK). Further details are outlined in Appendix C. The 15N content in organic-N was determined in a selected
range of drainage water samples after oxidation to nitrate using the method as described by Cabrera and Beare (1993). The proportion of 15N present as NH4+ and NO3- in the leachate was
interpolated between sampling points and N drainage losses calculated using the following equation (4.2-9):
𝑁
𝑇𝑜𝑡15 = ∑15%𝑁 × 𝑁𝑑𝑟𝑛
4.2-9 where: 𝑇𝑜𝑡15𝑁 = The total-15N leaching loss as mineral-N, ammonium or nitrate summed over the drainage period, 15%𝑁 is the actual or interpolated 15N fraction of the ammonium and/or nitrate ion concentration, and Ndrnis the N leaching loss for each drainage collection (equation 4.2-4).
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4.2.8
Statistical analysis
Statistical analysis of the data was conducted in two stages: crop-by-N-rate interactions between treatments was conducted using an orthogonal non-block ANOVA procedure in Genstat 9.0 (Lawes Agricultural Trust 2007), whilst the testing of the effect of DCD on both crop types was done using a standard t-test. Residuals were tested for normalisation of data.
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