# Soil Quality in Rehabilitated Coal Mining Areas
## 2 Materials and Methods
### Samplings and Analysis  
Eight years after the plot establishment, three composite samples (0-0.15 m depth) were obtained along the experimental plots. Each of them included five sampled points, following an imaginary zig-zag line from top to bottom of the plot. The analytical methods used for SOC evaluation were: dry combustion in an analyzer (TruSpec(r) Micro Elemental Series from Leco, St. Joseph, MO, USA), based on the Dumas method [30] and after the removal of carbonates [31]; calcination at 560 \({}^{\circ}\)C [32]; wet oxidation [33]; and thermogravimetric [34] using a TGA/SDTA851 apparatus from Mettler-Toledo (Cornella, Spain). Calcium carbonate equivalent (CaCO\({}_{3}\) eq) was quantified following the calcimeter method [35] and using a Bernard calcimeter from Pobel (Reference 10 CABE6256, Madrid, Spain). The carbon content of soil microbial biomass was determined by the fumigation-extraction UNE 77310-2 method [36]. For this analysis, two composite samples from each plot were used. Analyses were done in triplicate for each composite sample. The implemented test for water-stable aggregates (WSA) was the single-sieve (0.25 mm) method [37]. The bulk density of each plot was obtained by the excavation method [38]. The hole was covered by a plastic bag and filled with water to determine the volume of the excavated sample. Coarse fragments within the sample were screened out and weighed. The volume of coarse mineral fragments was determined from dry mass, assuming a particle density of 2.65 Mg m\({}^{-3}\). Bulk densities of mineral soil samples were calculated as the mass of dry, coarse fragment-free soil per volume of field-moist soil, where volume was also calculated on a coarse fragment-free basis. Micromorphology techniques were used for pore, aggregate and SOM distribution studies. A prism (6 cm depth \(\times\) 9 cm thick \(\times\) 19 cm long) was obtained from a representative surface of plots 4-F, 5-F, 8-F, and 9-F. Vertically thin sections (5 cm wide \(\times\) 13 cm long \(\times\) 30 \(\upmu\)m thick) were prepared. Their description was done using an Olympus BX51 polarizing microscope (Evident, Tokyo, Japan) and according to the terminology and concepts developed by [PERSON] [39; 40]. The percentage of the area (from the total thin section of 65 cm\({}^{2}\)) occupied by pores and aggregates was obtained. The procedure started with the selection of 30 sectors, 1.75 \(\times\) 1.3 cm in size, per thin section. Each observed sector was photographed under plain-polarized light (PPL) and cross-polarized light (XPL) with a resolution of 3072 \(\times\) 2304 pixels. \begin{table}
\begin{tabular}{c c} \hline \hline
**Reference** & **Substrate Characteristics** \\ \hline wSnP 1:3:1 & Substrate was created with sludge (wS) but without pig slurry on the surface (nP). It was obtained by mixing \\  & 1 volume of sludge, 3 volumes of coarse sand material, and 1 volume of silt material (1:3:1) (Tables 2 and 3). Three experimental plots (1-F, 3-F, and 5-F) were set up. \\ \hline wSwP 1:3:1 & Substrate was created with sludge (wS), and pig slurry (wP) was spread over the surface at a rate of 90 m\({}^{3}\) ha\({}^{-1}\). The ratio of materials (1:3:1) was similar to wSnP plots. Three experimental plots (2-F, 4-F and 6-F) were set up. \\ \hline nSwP 1:1 & Plot (7-F) without sludge (nS). Substrate (1:1) was created by mixing 1 volume of coarse sand and 1 volume of \\  & treated-silt material. Pig slurry was spread over the surface as in wSwP plots. \\ \hline nSwP 1:2 & Plot (8-F) without sludge (nS). Substrate (1:2) was created by mixing 2 volumes of coarse sand and 1 volume of \\  & treated-silt material. Pig slurry was spread over the surface as in wSwP plots. \\ \hline nSnP 1:1 & Plot (9-F) without sludge (nS) and without pig slurry applied over the surface (nP). Substrate (1:1) was created as \\  & in nSwP 1:2. \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Description of substrate characteristics for the different referenced field plots.