Soil sampling and preparation
In total, soils with different land use types (Figure1) were collected from 15 sites, which were almost evenly distributed in Zhejiang Province (an area of 1,055,000 km2). Information on specific sampling points is shown in Table 1. The 15 sampling points covered six land use types, generally established in the past 5 years.
Soil samples of 0–20 cm were collected from typical fields (Long-term farmland with conventional fertilization by local farmers) in May 2018 in the second season of the rotation systems. Two samples with three replicates were taken at an interval of 1000 m in each site with same land use type, the replicates were brought back to the laboratory and mixed. Then, the mixed soil samples from each site were divided into four equal parts by the diagonal quartering method. One part was retained for a follow-up test. All samples were air-dried and separated into two parts; one was finely milled and sieved through a 2-mm mesh to determine basic physical and chemical properties, and another was broken carefully into small pieces by hands to segregate aggregates and determine Pcoll.
Aggregate separation and determination
Aggregate size distribution was determined for each soil sample using a modified wet sieving method [35]. Briefly, the second part of unground soil was first passed through an 8-mm sieve, and 50 g soil was placed carefully on the top of a nest of three sieves (2 mm, 0.26 mm, and 0.053 mm). Then, the sieves were submerged for 20 min in 2.5 L deionized water at room temperature and oscillated under water 300 times for 10 min with a 30 mm amplitude to separate aggregate fractions. Thus, four aggregates fractions were obtained on each sieve: large macroaggregates (2–8 mm), small macroaggregates (0.26–2 mm), microaggregate (0.053–0.26 mm), and (silt+clay)-sized particle (<0.053 mm) [36]. Aggregates of each size were carefully moved from the sieve into a beaker. The water used for wet sieving was left to rest for 48 h, silt and clay particles were collected, and the supernatant was used to determine total dissolved P (TDP), truly soluble P (TSP), and Pcoll content. All aggregates were oven-dried at 65 °C for 48 h, weighed, and placed in a zip lock bag. To obtain water-stable aggregates, sediment concentration was subtracted from that obtained by wet sieving because sand was not considered a component of water-dispersible aggregates [35]. Sand content was determined by the following process: 5 g of the dry aggregates obtained above were weighed, dispersed into 30 mL 5 g L–1 hexametaphosphate solution, placed into an ultrasonic cleaner, and dispersed for 30 min. The suspension was then poured through a nest of sieves. The residue left on the 0.053 mm sieve represented the sand content of each sized aggregate. After collection, sand was dried at 65 °C and weighed.
The pH of soils was determined with a glass electrode pH meter (PHS-3C, Shanghai) at a soil-to-water ratio of 1:5. Soil cation exchange capacity (CEC) was measured with ammonium acetate (12.5 mL 1M NH4OAc, 2.5 g soil) [37]. Soil and aggregates associated TP was determined by H2SO4-HClO4 digestion and evaluated by the molybdenum-blue colorimetric method [38]. Soil particle size distribution was determined by the hydrometric method according to an international soil texture classification standard. Soil- and aggregate-associated TC and TN were determined with an elemental analyzer (dry combustion with Vario MAX CNS, Elementar, Germany). Soil- and aggregate- associated Al, Fe, and Ca were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) after digestion with 5 mL HNO3 (16 M), 1 mL HClO4 (12.4 M), and 1 mL HF (23 M) for 12h. All reagents were from Sinopharm Chemical Reagent Co., Ltd.
Colloidal P was determined as described by Ilg [39]. Briefly, 10 g of unground soil was placed into a 250 mL flask, 80 mL deionized water (DDW) was added, and the sample was shaken at 160 rpm and 25°C for 24 h. The supernatant was pre-centrifuged at 3000 g for 10 min to remove coarse particles. After pre-centrifugation, the supernatant was filtered with a 1 μm microporous membrane, 5 mL of the primary filtrate was discarded, and the total filtrate was collected (sample I). This suspension included the colloidal component and the dissolved component. The filtrate was ultracentrifuged at 300,000 g for 2 h to remove colloids (Optima TL, Beckman, USA; Sample Ⅱ), and the residue at the bottom of the ultracentrifuge tube was the water-dispersible colloid. The TDP in sample I and TSP in sample II in the solution were determined after digestion with acidic potassium persulfate. The difference between TDP in Sample I and TSP in Sample Ⅱ was the concentration of Pcoll. Previous studies have shown that soil P through leaching and surface runoff is usually in soluble forms that can pass through the 0.45–1 μm filter [40, 41]; therefore, in the present study, TDP including Pcoll and TSP in aggregates was defined as the potential loss P. The TDP in the supernatant after 10 min wet sieving was considered as easy loss P.
Calculation of water-stable aggregate (WSA) size fractions
The proportion of WSA in each size fraction was obtained from equations (1) and (2), as described by Alvaro-Fuentes [42]:
[Due to technical limitations, the formulas could not be displayed here. Please see the supplementary files section to access the formulas.] (1)
(2)
where, i is the ith size fraction (2–8, 0.25–2, and 0.053–0.25 mm); dry soil aggregate (DSAi) is the oven-dried mass of total, non-dispersed aggregates collected on each sieve; Sand is the oven-dry mass of sand collected after dispersed in hexametaphosphate solution on the 0.053 mm sieve; TotalSoil is the oven-dried mass of soil (50 g) for aggregate separation.
Calculation of MWD and GMD
The mean weight diameter (MWD) and geometric mean diameter (GMD) of the aggregates can be obtained by Equations (3) and (4) [43]:
[See supp. files] (3)
(4)
where, i is the ith size fraction (2–8, 0.26–2, 0.053–0.26, and <0.053 mm) and d is the mean diameter of each size (0.053–2 mm). WSAi include 2–8, 0.26–2, 0.053–0.25, and <0.053 mm WSA and (silt+clay) sized fractions.
Contribution of aggregate associated Pcoll to TDP
To illustrate the contribution of aggregate associated Pcoll to TDP (potential loss P.), soil aggregates and Pcoll concentrations were integrated and calculated. The contribution rate (CR) of aggregate associated Pcoll to TDP was calculated using Equation (5):
[See supp. files] (5)
where, Agg_CP is the concentration of aggregate associated Pcoll (mg kg–1), TDP is the concentration total dissolved P (mg kg–1), and i is the ith size fraction (2–8, 0.26–2, 0.053–0.26, and <0.053 mm).
Statistical analysis
Microsoft Excel 2016 and Origin 8.0 were used for data processing and cartography. Data were statistically analyzed using SPSS Statistics 22.0 (SPSS Inc. Chicago, USA) software. One-way ANOVA was conducted using two samples of each site to examine differences of different variables in Table S1, S2, and 3 and Fig S1 and S2. Pearson correlation analyses were used to identify the relationship between aggregate associated Pcoll and other soil parameters. Stepwise linear regression was performed to evaluate the relationships between P indicators (Content and loss potential of Pcoll) and soil variables (pH, TP, TC, TN, C/N, Fe, Al, Ca, MWD, and GMD).