Changes of oxygen isotope values of soil P pools associated with changes in soil pH

Site description

The Park Grass Continuous Hay experiment was established by John Lawes and Henry Gilbert at Rothamsted (Harpenden, Herts, UK) in 1856. It was established on a site which had been in permanent pasture for at least 100 years before the experiment began⁴⁴. It was started to test the effects of different combinations of mineral fertilisers and organic manures on the productivity of permanent grassland cut for hay. The treatments include, an unfertilised control against which different amounts and combinations of mineral fertilisers (including N, P, K, Na, Mg and farmyard manure/poultry manure) are compared⁴⁴. In 1903 most plots were divided into two and lime (CaCO₃) was applied to one half at 4 t ha⁻¹ every four years. In 1965 most of the plots were divided further into four sub-plots, which now receive lime every third year to maintain soil pH at 7 (sub-plot ‘a’), 6 (sub-plot ‘b’), and 5 (sub-plot ‘c’), respectively. The fourth sub-plot (‘d’) does not receive lime and its pH varies depending on the treatment but is usually around pH 4 or 5. The soil is a chromic luvisol (FAO), referred to locally as a Stagnogleyic paleo-argillic brown earth of the Batcombe series⁴⁵.

Having been designed before the pioneering research of R.A. Fisher at Rothamsted in the early 1900s, the Park Grass Experiment does not have the key statistical properties of replication and randomisation that are needed for most formal statistical analyses of data from designed experiments. Additional constraints in this study meant that samples could be collected and processed for only 6 plots with soil samples then obtained for three separate depths. Any formal statistical analysis would have to make very strong assumptions about the sources of variation, for example assessing the variation due to the main effects of the factors (fertiliser treatment, liming, depth) against the variation due to interactions amongst these factors. Any such analyses would also have low power to detect differences as statistically significant. As the aim of the study was to demonstrate the potential of the oxygen isotope ratio methodology to detect variation in P cycling responses under a range of fertiliser and pH conditions, rather than to test for differences due to these treatments, no formal statistical analysis has been performed, with observed values for the 18 treatment combinations presented to demonstrate patterns of response associated with changes in the soil system, and stimulate further study using this methodology.

Sampling and sample preparation

Our aim was to obtain a wide range of soil pH values. We therefore included treatments with triple superphosphate and either with or without application of ammonium sulphate (plots 11/1 (referred to hereafter as “+N + P”) and 7/2 (referred to hereafter as “−N + P”)) and an unfertilised control (plot 12; referred to hereafter as “−N − P”) (Table 5). Within each of the three selected treatments, soil samples were taken from the ‘a’ and ‘d’ sub-plots. Sub-plot ‘a’, which received lime to maintain soil at pH 7, is referred to as ‘+L’ and sub-plot ‘d’, which never receives lime as ‘−L’ hereafter. Lime is applied every third year and was last applied in February 2015. Sampling took place on 1 March 2016, about one and a half months before application of N fertiliser (applied 19.4.2016) but about three months after the application of the other fertilisers including P fertiliser (applied 02.12.2015) (Table 5).

A mat of partially decomposed plant material had developed as a result of the low soil pH on the surface of the +N + P − L treatment. This mat was removed prior to soil sampling. From each of the selected treatments, 18 soil cores were taken by hand in a W-sampling pattern to a depth of 30 cm using a soil corer (internal diameter 2 cm). The cores were divided into three depths: 0–10 cm, 10–20 cm, and 20–30 cm. They were bulked to give one sample per depth; a total of three samples per plot. The fresh soil samples were immediately sieved <2 mm, removing large stones, visible plant material and soil fauna and ensuring complete mixing. Three subsamples were taken from each sieved and fresh soil sample: (1) approximately 10 g for extraction of soil water, stored in tightly closed vacutainers at −20 °C, (2) approximately 50 g for soil moisture content, dried at 105 °C for 24 hours; (3) approximately 10 g for the determination of soil pH, total carbon (C), nitrogen (N) and P, air-dried, milled and sieved <2 mm. The remaining sample was stored at 4 °C prior to extraction of resin and hexanol P for δ¹⁸O_P. For the aboveground vegetation samples, vegetation was cut at ground level with scissors from the same locations as the soil cores and bulked per plot, giving six vegetation samples in total. From each sample, a small subsample (approximately 2 g) was stored in a tightly closed vacutainer at −20 °C for the extraction of plant water. The remaining vegetation samples were stored in plastic bags at −20 °C. Soil temperatures were obtained from the electronic Rothamsted archive (e-RA) of Rothamsted Research. The meteorological station is approximately 1 km from Park Grass. Soil temperatures are measured by Rothamsted Research hourly at a depth of 10, 20 and 30 cm in soil under grass.

Phosphorus in plants, lime and soil

Inorganic P (Pi) from the frozen plant material was extracted with 0.3 M trichloroacetic acid (TCA) as described by Pfahler et al.³⁶ and is afterwards referred to as TCA P. The most recently available lime samples (from 2007 and 2013) and fertiliser samples (from 1986 and 2012) from the Rothamsted Sample Archive were extracted with 1 M HCl, as described below for the soil. The following soil P pools were extracted: resin, microbial, NaOH-EDTA P_i, NaOH-EDTA P_org, HCl P, and residual P. The availability of those pools decreases from resin to residual P. Resin and microbial P are considered the most labile pools, which can turnover within minutes to weeks, whereas turnover rates of HCl P are estimated to be in the order of years or even millennia³⁵. Within two weeks of sampling, resin, an approximation for available P, and microbial P were extracted from fresh soil with anion exchange membranes, conditioned with bicarbonate, with or without the addition of hexanol⁴⁶. For resin P 100 g of fresh soil, and 4 resin strips (12.5 × 12.5 cm) were shaken for 16 hours at 4 °C in 5 L ultra‐pure water (ddH₂O)⁴⁷. For microbial P, the process was the same but with the addition of 30 ml of hexanol. The recovered resin strips were washed thoroughly with ddH₂O to remove any attached soil particles. The resin strips were eluted by shaking overnight in 75 mL of 0.2 M HNO₃. As the soil to solution ratios in this extraction were elevated to enable sufficient P to be collected for analysis, a separate sequential extraction was conducted for subsequent pools using the more conventional soil to solution ratio of 1:10⁴⁸. Microbial P was extracted again from 30 g soil (dry weight equivalent) before the soil was sequentially extracted with 0.25 M NaOH – 0.05 M EDTA, targeting oxide bound inorganic and organic P, and 1 M HCl, targeting mineral P. To account for any hydrolysis of organic P or polyphosphates during the 1 M HCl step, ¹⁸O-labelled (three batches: δ¹⁸O value of 46.1‰, 14.2‰, and 22.4‰) and unlabelled 1 M HCl (two batches: δ¹⁸O value of −6.4‰ and −7.1‰) was used⁴⁹. Therefore, the soil, which was recovered from the NaOH-EDTA step, was dried at 40 °C, milled to <2 mm and divided into two equal parts. One part was extracted with ¹⁸O-labelled 1 M HCl, the other part was extracted with unlabelled 1 M HCl. The NaOH-EDTA extract was freeze-dried and homogenised afterwards with a pestle and mortar. The freeze-dried material from selected samples was used for the determination of the δ¹⁸O_P of the inorganic P as described by Tamburini et al.³⁷. Inorganic P (Pi) concentrations for all extracts were determined colourimetrically on an Aquachem 250 analyser using a molybdenum blue reaction⁵⁰, while total P in the NaOH-EDTA extracts was analysed on the same equipment following oxidation with potassium persulphate. The concentration of organic P (P_org) in the NaOH-EDTA (afterwards referred to as NaOH-EDTA P_org) extracts was calculated as the difference between total P and inorganic P concentrations in the NaOH-EDTA extracts. The Pi in the NaOH-EDTA is afterwards referred to as NaOH-EDTA Pi. Microbial P was calculated as difference between resin and hexanol P and corrected for P adsorption⁵¹.

Soil water extraction

Soil water was extracted cryogenically from sealed, pre-frozen samples at the NERC Isotope Geoscience Facility, British Geological Survey, UK, following the method described by West et al.⁵². Frozen samples (approximately 10 g frozen soil) were unsealed and placed in a U-shaped vacuum tube (borosilicate glass), the sample containing side of which was immersed in liquid N to ensure complete freezing and no loss of soil water. The U tube was then evacuated to a pressure of <10⁻² mbar, removing all residual atmospherics. Once under stable vacuum, the U tube was sealed, removed from the vacuum line and the sample side of the tube placed in a furnace at 100 °C. Soil water collection was achieved by immersing the opposite side of the glass U tube in liquid nitrogen, forcing evaporating soil water to condense and collect. This set up was maintained for >1 hour to ensure complete water transfer. Soil water was collected and stored refrigerated in 1.5 ml vials with no headspace until isotope analysis.

Further soil analyses

Sieved and air-dried soil was used to determine the soil pH in water as described by Faithfull⁵³. Total carbon (TC) and total nitrogen (TN), was determined using finely ground and air-dried soil, with an elemental analyser (NA2000, Carlo Erba Instruments, Milan, Italy). Total P in the soil was determined on air-dried and milled soil via aqua regia digestion⁵⁴. Concentrations in the total P extracts were then determined by ICP-OES.

Oxygen isotopes in water and phosphate

Purification of the TCA, resin, microbial and HCl P extracts and precipitation of silver phosphate (Ag₃PO₄) followed the protocol by Tamburini et al.⁴⁹. Modifications to this method were: 1 ml of concentrated H₂SO₄ was added during the ammonium phosphomolybdate (APM) step during the purification of the TCA, resin and microbial P extracts to facilitate the precipitation of APM. Also a few silver nitrate (AgNO₃) crystals were added prior to precipitation of Ag₃PO₄ to ensure complete removal of Cl as silver chloride (AgCl). Precipitated AgCl was filtered out using 0.2 µm polycarbonate filters. Using size exclusion gel chromatography, selected freeze-dried and re-dissolved NaOH-EDTA extracts were separated in an organic and inorganic P fraction³⁷. Only the inorganic P fraction of the NaOH-EDTA extraction was purified as described above. Analysis of phosphate ¹⁸O:¹⁶O (δ¹⁸O) was undertaken by weighing approximately 300 μg of Ag₃PO₄ into a silver capsule to which a small amount of fine glassy carbon powder was added⁴⁹. The sample was converted to carbon monoxide by dropping it into a thermal conversion elemental analyser (ThermoFinnigan, Germany) at 1400 °C, the resultant CO mixed with a helium carrier gas passes through a GC column into a Delta + XL mass spectrometer (ThermoFinnigan, Germany). δ¹⁸O_P was calculated by comparison to internal Ag₃PO₄ laboratory standard, ALFA-1 (ALFA-1 = δ¹⁸O VSMOW value of 14.2‰). In the absence of an international Ag₃PO₄ reference material, we derived this value for ALFA-1 by comparison to the Ag₃PO₄ standard ‘B2207’ (Elemental Microanalysis Ltd., England), which has been measured in an inter-laboratory comparison study to have a δ¹⁸O value of 21.7‰ versus VSMOW. Samples were run in triplicate, with a typical precision σ ≤ 0.3‰.

Soil water δ¹⁸O was determined on an Isoprime Aquaprep coupled to an Isoprime 100 dual-inlet mass spectrometer (Isoprime Ltd., Cheadle, England) through a process of headspace CO₂ equilibration with water samples. The isotope ratios are reported as δ¹⁸O_H2O values versus VSMOW, based on comparison with laboratory standards calibrated against IAEA standards VSMOW and SLAP, with analytical precision typically σ ≤ 0.05‰. The theoretical equilibrium between O in soil water and O in phosphate was calculated for each depth and treatment using a modified version of the equation given by Chang and Blake²²: δ¹⁸O_P = −0.18 T + 26.3 + δ¹⁸O_H2O, where δ¹⁸O_P is the stable oxygen isotope ratio of phosphate at equilibrium in ‰, T is the temperature in degrees Celsius and δ¹⁸O_H2O is the stable oxygen isotope ratio of the soil water in ‰. The minimum and maximum soil temperatures in the 24 hours prior to the soil sample were used for calculating the equilibrium values as it can take up to 24 hours until the equilibrium is reached²². The microbial P δ¹⁸O_P was determined via mass balance using the concentrations of the resin, hexanol and microbial P and the δ¹⁸O_P values of resin and hexanol P²³.

Source: Ecology - nature.com