The 15N-labelled soil
This study was based on soil originating from the Ap-horizon (0–20 cm depth) of a 20 m2 field plot located in the B1-field at Askov Experimental Station (55o28`N, 09o06`E). During 1989 to 1993, this plot provided 15N-labelled plant materials for various studies outside the labelled plot (Thomsen 1993; Thomsen and Jensen 1994; Thomsen and Christensen 1996; Thomsen et al. 2001). In 1989, the plot grew spring barley undersown with Italian ryegrass that received K15NO3 and 15NH415NO3 (mean atom%-15N: 18.0) at a rate corresponding to 102 kg N ha− 1. In 1991, the plot was in oilseed rape and sugarbeet dressed with 15NH415NO3 (atom%-15N: 5.14) equivalent to 160 kg N ha− 1. Finally, field peas were grown in 1993 with 15NH415NO3 (atom%-15N: 10.0) corresponding to 111 kg N ha− 1 and supplemented with 14 kg N ha− 1 of K15NO3 (atom%-15N: 60.0). From 1994 to 2003, the plot grew mainly cereal crops occasionally dressed with unlabeled mineral fertilizers and with most of the plant biomass left on the plot to recirculate 15N taken up by the crops. The plot was subject to standard tillage treatments (ploughing, harrowing). The B1-field was abandoned in October 2003 and the topsoil (0–20 cm) removed from the entire 20 m2 plot and left to air-dry indoors. After sieving (< 2 cm), the soil was stored in dry condition. The soil contained 11% clay (< 2µm), 8% silt (2–20 µm), 43% fine sand (20–200 µm), and 37% coarse sand (200–2000 µm) and held 1.54% C. The atom%-15N of the soil was 0.4515 and the natural 15N abundance in a soil from a soil nearby the plot was 0.3683% (corresponding to excess atom%-15N: 0.0832). Soil pH was 5.1.
The lysimeter facility
The Askov outdoor lysimeter facility, established in 1973, consists of cylindrical glassfibre-reinforced polyester tanks, 1.5 m deep and 1 m in diameter (surface area 0.83 m2). The top 1 m of the lysimeter contains soil extracted from three soil horizons (0–20 cm, 20–40 cm and 40–100 cm) in a nearby field. During filling, repacking of the soil ensured that it regained its original bulk density (1.55 g cm− 3 for 20–40 cm and 1.23 g cm− 3 for 40–100 cm). The 20–40 cm soil layer contains 7% clay, 6% silt, 29% fine sand, 54% coarse sand and 4% organic matter; the corresponding values for the 40–100 cm soil layer are 9, 7, 31, 51 and 2%. The pH of soil layers ranged from 5.9–6.4. Below the soil column is a 50 cm layer of fine sand to facilitate collection of percolate. The percolate flows passively into 25-liter PVC containers in a subterranean gallery situated alongside the cylinders. A water trap inserted between the PVC container collecting the percolate and the tube draining the base of the cylinders prevents air from reaching the cylinder from beneath.
The set-aside grassland phase
In spring 2004, the 15N-labelled soil retrieved from the 20-m2 plot replaced the 0–25 cm topsoil in 16 of the lysimeters. After removal of the original soil, each lysimeter received 365 kg of the dried and sieved 15N-labelled soil, ensuring that the soil beneath remained undisturbed. The labelled soil, introduced in portions, reached a density of 1.75 g cm− 3. The remaining labelled soil was stored dry indoors in sealed containers for later analysis. The lysimeters were sown with a grass mixture suitable for set-aside grassland. This consisted of ryegrass (Lolium perenne L.: 16 kg ha− 1), meadow fescue (Festuca pratensis Huds.: 7 kg ha− 1) and Kentucky bluegrass (Poa pratensis L.: 5 kg ha− 1) and received unlabeled NPK mineral fertilizers corresponding to 100 kg N ha− 1, 12 kg P ha− 1 and 44 kg K ha− 1. In spring 2008, 2009 and 2012, the lysimeters received small dressings of unlabeled NPK to maintain an intact grass cover. Each dressing corresponded to 50 kg N ha− 1, 6 kg P ha− 1, and 23 kg K ha− 1. No fertilizer was applied in the other years. The grass was subject to 2–4 cuts per season during 2004–2012 with a final cut in 2015. After cutting, all plant material was returned to the cylinder in which it was grown. Table 1 gives an overview of history.
Table 1. History of soil and outline of experimental set-up in the lysimeter experiment.
1989-1993
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1993-2004
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2004-2017
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2018-2019
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Application of 15N-labelled mineral fertilizer to a 20-m2 field plot.
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Mainly cereal cropping with unlabelled mineral N fertilizer and return of crop residues. Topsoil relocated to lysimeters in 2003.
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Soil kept in lysimeters under set-aside grassland, occasionally dressed with small rates of unlabelled N fertilizer.
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Testing the lability of 15N-labelled mineral fertilizer residues by converting set-aside grassland to:
- Production grassland
- Vegetation-free fallow
- Spring barley
- Spring barley, cover crop
All treatments dressed with unlabelled mineral N fertilizer.
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In April 2017, we prepared for the subsequent test phase by sampling soil and biomass from each of the 16 lysimeters for analysis of 15N abundance. The sampling included also soil that had been stored indoors in dry condition since 2004. Soil pH measured in January 2018 averaged 5.2 and each lysimeter received a surface-application of lime corresponding to 5 t ha− 1.
Testing the lability of 15N retained in soil
We initiated the test phase in spring 2018 by establishing four treatments each with four replicates. The treatments were 1) continuation of grass vegetation but with N fertilization and harvest of cuttings, 2) vegetation-free fallow, 3) spring barley (Hordeum vulgare L.), and 4) spring barley followed by a cover crop. In 2018, the cover crop was fodder radish (Raphanus sativus L. var. oleiformis Pers.) planted after barley harvest, while in 2019 the cover crop was ryegrass undersown in the barley in spring. Lysimeters with the grass treatment remained undisturbed whereas the other treatments were subject to simulated ploughing in spring. This was done by removing the 0–25 cm soil in three successive layers. The upper third with the grass turf returned to the exposed surface of the undisturbed subsoil in the lysimeter before adding the rest of the soil. Sowing of spring barley was on 19 April in 2018 and 6 May in 2019. At the same time, all treatments (including vegetation-free fallow) received unlabeled NPK fertilizer corresponding to 150 kg N ha− 1, 40 kg Pha− 1 and 150 kg K ha− 1. During the growing seasons, the cylinders were irrigated with 100 mm in 2018 and 90 mm in 2019 (see Fig. 1).
The grass plots were cut three times in the growing season 2018 (29 May, 6 August, 24 October) and two times in 2019 (23 May, 9 July). Harvest of spring barley took place on 3 August in 2018 and 13 August in 2019. The cover crop was harvested on 21 November in 2018 and 31 October in 2019. After determination of yields and retrieval of subsamples for analysis, the cover crop biomass was returned to the cylinder in which it was grown and then covered by a net. All cylinders remained undisturbed from harvest 2018 until the next spring when they were again prepared for planting of spring barley. Samples of plant materials were dried at 60 oC and stored for analyses. Figure 1 shows monthly precipitation (including amounts of irrigation) and mean air temperature for the period January 2018 to April 2020.
Water percolating the soil column was collected individually for each replicate cylinder. Percolate collected during 13 July 2018 to 22 March 2019 was accumulated for 8 periods, while 5 periods were adopted for the leachate collected during 8 July 2019 to 1 April 2020. Representative subsamples were stored at -18oC until analysis.
Analytical methods
Plant and soil samples were dried at 60oC and milled. Ball-milled subsamples were packed in tin capsules for analysis of total N content and 15N concentration by high temperature dry combustion using a PDZ Europa ANCA-GSL elemental analyser interfaced to a PDZ Europa 20–20 continuous flow isotope ratio mass spectrometer (IRMS) at UC-Davis Stable Isotope Facility (Davis, CA, USA). Concentrations of nitrate in percolates were determined by flow colorimetry on an AutoAnalyzer III. The 15N content in the leachate was analysed after diffusion to glass filter in Teflon traps as described by Sørensen and Jensen (1991) using an elemental analyser interfaced to an IRMS.
Calculations
Experiments with 15N-labelling generally express the 15N content as an excess atom fraction 15N (Chalk et al. 2015):
atom%15N excess = %15Nsample – %15Nreference
The reference (background) value used here is 0.3663% for samples of water and plant materials and 0.3683% for soil samples. The excess atom fraction 15N was multiplied with the total N pool (in soil, plant or leachate) to calculate the amount of labelled N (excess 15N), which is also termed 15N in the following.
Diffusion samples were corrected for the abundance of N in the blank samples using the following equation (Sørensen and Jensen 1991):
Where “µg N measured” and “atom% 15N measured” is the amount of N and the enrichment measured in the enriched sample, respectively. The “µg N blank” is the amount of N measured in a blank sample. The reference value for 15N abundance was set at 0.3663%.
To facilitate interpretation, area-related results are scaled to 1 m2 by dividing measured values with the area of lysimeters (0.83 m2). Analysis of variance was carried out using the GLM procedure in SAS. Least significant differences were used to compare means with differences were considered significant at the P = 0.05 level.