Site description
This study was conducted in Dinghushan National Nature Reserve (1133 ha), located in the middle part of Guangdong Province in southern China (112°30′ – 112°33′E, 23°09′ – 23°11′N). This area is characterized by a typical south subtropical monsoon climate. The mean annual temperature is 21°C, with the maximum and minimum average monthly temperature of 28.0°C in July and 12.6°C in January, respectively. Annual average relative humidity is 82%, and the mean annual rainfall is 1927 mm. Acid rain is a threat in this area with high deposition rates of 34.4 kg N ha− 1 yr− 1 and 32.6 kg S ha− 1 yr− 1 in recent decades (Liu et al. 2007), which has significantly lowered the soil pH of some forests to < 4 (Jiang et al. 2018).
In the reserve, there are three types of forests of different successional stages: the broadleaf forest, mixed pine and broadleaf forest, and pine forest. In this study, the experimental site was set up in the broadleaf forest, the most mature forest with age > 400 year. The forest is located 250–300 m above sea level and occupies approximately 600 ha. The dominant species were Castanopsis chinensis, Cryptocarya concinna, Cryptocarya chinensis, Machilus chinensis, and Schima superba (Yan et al. 2006). The bedrock is sandstone and shale belonging to the Devonian Period. Soil type is lateritic red earth (Yan et al. 2015) and is classified as an Oxisol according to the Keys to Soil Taxonomy (Soil Survey Staff 2014).
Experimental treatments and design
The acidity experiment was initiated in June 2009. Twelve plots were established and divided into four acidity treatments with three replicates each. Each treatment plot was measured 10 × 10 m2 and was surrounded by a 3 m wide buffer strip. All plots and treatments were randomly arranged. The acidity treatments were irrigated with water of different pH values: CK (control, pH 4.5), T1 (pH 4.0), T2 (pH 3.5), and T3 (pH 3.0). To reflect the real mole ratio of S:N in the region, acidic solutions were prepared by adding a mixture of H2SO4 and HNO3 in a 1:1 mole ratio to the local lake water based on the local acid rain records (Du et al. 2015). The simulated acid rain was applied to each plot below the canopy using a gasoline engine powered sprayer and sprayed twice a month. The amount applied to each plot was 40 L per application, equal to 4 mm rainfall. The H+ added in each plot was estimated as 0, 9.6, 32, 96 mol H+ ha− 1 yr− 1 in the T1, T2, T3 and T4 treatments, respectively, which was equal to about 0, 0.6, 2.0 and 6.0 times of that in the through-fall of the forest. More details on the experimental design and methods can be found in Wu et al (2016).
Sample collection and soil properties measurements
In September 2018, four cores (diameter = 5 cm) were randomly collected from the topsoil (0–10 cm) and subsoil (10–20 cm) in each plot, and combined to yield one composite sample per depth and plot. The soil sample was passed through a 2 mm sieve to remove rocks and plant roots, and then divided into two parts (field-moist soil sample and air-dried soil sample) for subsequent analysis. Fine roots (diameter ≤ 2mm) were picked and thoroughly rinsed in deionized water and dried at 60°C to constant biomass.
Soil moisture was determined by drying the field-moist soil samples at 105°C for 24 h. Soil pH values were measured using a glass electrode after shaking the samples for approximately 30 min in deionized water. The soil to water ratio was 1:2.5. Microbial biomass carbon (MBC) was measured using the chloroform fumigation extraction technique (Jenkinson 1987). The soil extractable dissolved organic carbon (DOC) was measured on the same samples used for the analysis of MBC and calculated as the K2SO4 – extractable C concentration. Total SOC was measured with the air-dried soil samples by using the K2Cr2O7 + H2SO4 oxidation method (Schollenberger 1927). Total soil N was determined by semimicro + Kjeldahl (Menefee and Overman 1940; Bremner 1960). Total Ca, Al and Fe in soil were extracted with HNO3-HF-HClO4 and measured using atomic absorption spectrophotometry with a graphite furnace. Soil exchangeable cations (Ca2+, Al3+, and Fe3+) were extracted with 0.1 M BaCl2 (soil:solution ratio of 1:50) and measured on an Agilent 5100 ICP-OES (Galí Navarro et al. 2011).
Density fractionations
Each field-moist soil sample (30 g dry mass equivalent) was separated into two operationally defined soil fractions, a light fraction (LF) and a heavy fraction (HF), following a modified density fractionation technique (Ye et al. 2018). Given that the soils used in this study were rich in clay, the LF was separated by flotation after immersing soils in NaI solution at a density of 1.85 g cm− 3 combined with a ultrasonic treatment (a total energy input of 200 J mL− 1) in order to disrupt soil aggregates. The residual soil consisted of the remaining mineral-associated organic matter (HF). The separated soil fractions were then dried in an oven at 60°C and ground to a homogenized fine powder for SOC analysis (interpreted as LF-C and HF-C).
Determination of Ca-bound C and Fe-bound C
The HF soil samples were sequentially extracted with 0.5 M Na2SO4 and the C released in the solutions was interpreted as organic C associated with Ca bridges (Xu and Yuan 1993). The difference between HF-C and the C concentrations of the residual soils after Na2SO4 extraction was calculated as Ca-bound C. Next, Fe-bound organic C was measured following a procedure adapted from Lalonde et al (2012). The residual soil after Na2SO4 extraction was added to a solution containing sodium bicarbonate and trisodium citrate in a 50 mL polycarbonate centrifuge tube and heated to 80°C in a water bath, and then sodium dithionite was added to the tube and maintained at 80°C for 15 min. After centrifugation at 3000 g for 10 min, the supernatant was separated from the solid fraction. The procedure was repeated three times and the residual soil was rinsed three times with deionized water and then oven dried at 80°C for organic C analysis, respectively. The concentrations of organic C in soil after dithionite-citrate-bicarbonate (DCB) extraction were subtracted from the Na2SO4-extracted samples to obtain Fe-associated organic C.
Determination of Fe phases
During the determination of Fe-bound C, the extracted solution was also analyzed by ICP-OES to measure the total free Fe (oxyhydr)oxides (Fed). Dry HF subsamples were separately extracted with acid ammonium oxalate or sodium pyrophosphate to measure poorly crystalline (i.e., short-range-ordered, SRO) Fe (Feo) and organically complexed and colloidal Fe (Fep), respectively. Briefly, soils were extracted in the dark using 0.175 M ammonium oxalate and 0.1 M oxalic acid at pH = 3 with a soil:solution ratio of 1:60 and shaken for 2 h. Soils were extracted by 0.1 M sodium pyrophosphate (soil:solution ratio of 1:20) at pH = 10 and the supernatant was syringe-filtered through a 0.2 µm nylon membrane filter after centrifugation at 2000 g for 15 min. The Fe concentrations were determined by ICP-OES.
Additionally, the field-moist soil samples were extracted in 0.5 M hydrochloric acid (HCl) to measure Fe(II) and Fe(III) in HCl, denoted Fe(II)HCl and Fe(III)HCl (Huang and Hall 2017). Briefly, field-moist soil samples (3 g dry mass equivalent) were immersed in a 1:10 ratio with HCl in the field, and then vortexed, shaken for 1 hr, and filtered to 0.22 µm. Concentrations of Fe(II)HCl were measured using a colorimetric ferrozine assay and corrected for Fe(III)HCl interference (Viollier et al. 2000). Field-moist soil samples were also extracted in 0.2 M sodium citrate/0.05 M ascorbic acid solutions to measure the easily reducible Fe oxides (Feca) in short-range-order minerals and organic fractions using microplate-based ferrozine methods (Huang and Hall 2017). Briefly, field-moist soil samples (1.5 g dry mass equivalent) were added to freshly prepared citrate/ascorbic solution in a 1:30 ratio, vortexed, shaken in the dark for 16 h, and centrifuged for 10 min at 10,000 g. 40 µL of extract was added to 40 µL of 10% hydroxylamine hydrochloride and 200 µL of color reagent (1 g L− 1 Ferrozine in 600 mM HEPES buffer at pH 8), the absorbance at 562 nm was then recorded at 1h. The supernatant solution was decanted to a clean HDPE bottle for dark storage at 4°C for analysis within two weeks of collection. The difference between Fed and Feca (Fed−ca) was interpreted as crystalline Fe (oxyhydr)oxides.
Statistical analyses
Data analyses were conducted using SPSS 20.0 for Windows (IBM Corporation, Armonk, New York, USA) and R version 4.0.2. Analysis of Variance (ANOVA) was used to determine the statistical significance (α = 0.05) of the acidity treatment, soil layer and their interactive effects on bulk SOC and its fractions/pools as well as mineral elements (Ca, Al and Fe including Fe oxides). Tukey's multiple comparison test (HSD) was conducted if significant effects of acid addition or soil layer were found. Pairwise relationships between soil pH and biogeochemical variables, as well as biogeochemical variables and SOC fractions/pools were assessed using Pearson correlation coefficients.
A structural equation modelling (SEM) approach was also used to test a conceptual model for the acid deposition impacts on LF-C and HF-C. The SEM analysis was performed with the IBM SPSS Amos 22.0 using the maximum likelihood estimation method. Several tests were used to assess model fit: the Chi-square (χ2)-test, comparative fit index (CFI) and root square mean error of approximation (RMSEM).