Pasture Recovery Period Affects Humic Substances and Oxidations of Organic Matter in Eastern Amazon

Abstract

Land management practices that overlook soil limitations and potential have led to varying degrees of degradation. This study evaluates the carbon content in chemical and oxidisable soil fractions across different pasture recovery periods, comparing them to secondary forests. The management practices assessed include the following: secondary forest (SF), perennial pasture (PP), perennial pasture recovered five years ago (P5), and perennial pasture recovered eight years ago (P8), all on Plinthosols. We analysed carbon levels in oxidisable fractions and humic substances at depths of 0–0.10 m, 0.10–0.20 m, 0.20–0.30 m, and 0.30–0.40 m. The SF and P8 areas showed the highest organic matter content within the humic fractions, compared to the PP and P5 areas. Additionally, the P8 area demonstrated an increase in the labile and moderately recalcitrant fractions of organic matter, standing out among the different fractions evaluated. The multivariate principal component analysis indicated that P8 has the greatest impact on soil quality, followed by FS, P5, and PP. The pasture recovery over the past eight years has significantly improved soil carbon accumulation, highlighting the benefits of land restoration.

1. Introduction

The Amazon biome, globally recognised as a biodiversity hotspot, is facing an urgent threat from deforestation, particularly evident in Maranhão State [1]. The rapid expansion of beef cattle production since the mid-1960s has led to the conversion of vast forested areas into pastures, significantly reducing the original extent of the Amazon forest in this region [2,3]. This extensive deforestation and subsequent land-use changes have triggered soil degradation issues. These issues are primarily attributed to inadequate management practices and the prevalent use of fire to control natural regeneration and promote pasture growth [4,5]. Consequently, only a fraction of the original forest area remains, with estimates suggesting a decline to 24–25% of its initial coverage [6,7,8].

Understanding the implications of these land-use transformations on soil quality is crucial, especially considering the vital role soil organic matter (SOM) plays in sustaining ecosystem health. Within the complex web of tropical ecosystems, SOM plays an important role, influencing essential soil attributes and fertility [9,10].

However, while studies on SOM mostly focus on carbon levels and stocks, there is a need to delve deeper into the dynamics and stability of SOM fractions to comprehensively gauge the impact of land use changes on soil quality [9,11]. Fractionation techniques, including chemical methods [12] and oxidisable approaches [13], can be used to evaluate the dynamics and stability of SOM by employing solubility in various agents. These techniques are essential and sensitive tools for detecting changes associated with different land use and management practices, as well as the plant species used [10].

Chemical fractionation provides humic substances (fulvic acids—FA, humic acids—HA, and humin—HUM), which constitute a highly reactive compartment of SOM, accounting for 70 to 90% of the soil’s carbon content. These substances participate in various chemical reactions in the soil and strongly influence soil fertility [14,15]. Oxidisable fractionation, on the other hand, yields four fractions (F1 to F4) with different degrees of oxidation, representing varying levels of nutrient availability and chemical stability [13]. Measurements of these fractions offer insights into the labile and resistant forms of carbon in the soil [9,16].

By understanding the changes in carbon within different fractions of SOM, it is possible to assess soil quality and detect potential impacts caused by specific management systems. Therefore, the present study hypothesises that pasture recovery modifies the humic substances and oxidisable fractions of SOM. The objective of this research is to evaluate the carbon contents of chemical and oxidisable soil fractions in pastures with different recovery periods and compare them to those of secondary forests.

2. Materials and Methods

2.1. Study Location

The study was conducted at the Technological Reference Unit (UTR) of Embrapa Cocais and the State University of Maranhão (UEMA) in Pindaré-Mirim/MA, situated in the western mesoregion of Maranhão State and the microregion of Pindaré. The geographic coordinates are approximately latitude 3°46′13.91″ S, longitude 45°20′46.65″ W, with an altitude of 23 m (Figure 1). The region falls under the Aw-type climate classification according to Köppen, characterised by a dry winter, with an average annual temperature ranging between 26 and 27 °C and average annual rainfall between 1900 and 2100 mm [17,18].

The soil at the Technological Reference Unit (TRU) was classified as Plinthosols by the Food and Agriculture Organization (FAO) system [19] and as Plintossolo Argilúvico by the Brazilian Soil Classification System [20]. It exhibits a medium texture (

Table 1. Chemical and granulometric characterisation of the soil in the different areas and layers of Pindaré-Mirim region, Maranhão, Brazil.

Areas	Layer (m)	pH	OM	P	K	Ca	Mg	Al	H + Al
		CaCl2	g kg−3	mg dm−3	-----------------cmolc dm−3-----------------
SF	0.0–0.2	4.49	15.72	1.78	0.15	1.61	1.73	0.26	2.67
	0.2–0.4	4.18	7.59	1.74	0.14	0.97	2.21	0.91	3.65
PP	0.0–0.2	4.58	10.14	1.56	0.28	2.36	2.28	0.16	2.51
	0.2–0.4	4.25	6.85	1.37	0.29	2.42	3.45	1.37	4.97
P5	0.0–0.2	4.33	12.43	1.36	0.14	0.58	1.24	0.28	2.84
	0.2–0.4	4.15	5.04	0.92	0.10	0.38	1.33	0.62	3.09
P8	0.0–0.2	4.56	12.43	1.10	0.20	1.67	3.16	0.23	3.60
	0.2–0.4	4.38	6.05	0.95	0.23	1.25	4.22	1.04	5.96
Areas	Layer (m)	Sand		Silt		Clay
		-------------------------g kg−1------------------------
SF	0.0–0.2	746.7		135.3		118.1
	0.2–0.4	707.2		124.3		168.6
PP	0.0–0.2	579.3		343.0		77.8
	0.2–0.4	616.1		260.4		123.7
P5	0.0–0.2	745.5		103.5		150.5
	0.2–0.4	704.5		169.5		125.5
P8	0.0–0.2	741.9		120.8		137.3
	0.2–0.4	723.1		97.3		179.7

Legend: SF: secondary forest, PP: perennial pasture, P5: perennial pasture recovered five years ago, and P8: perennial pasture recovered eight years ago. pH: hydrogen potential, MO: organic matter, P: phosphorus, K: potassium, Ca: calcium, Mg: magnesium, Al: aluminium, H + Al: hydrogen and aluminium. P and K—Mehlich⁻¹ Extractor; Al, Ca, and Mg—KCl extractor 1 mol L⁻¹; H + Al—pH SMP (7.5).

) and originates from sediments of the Itapecuru formation, primarily composed of fine sandstones [17]. The terrain varies from smooth wavy to wavy, covered by Ombrophilous forest alongside secondary vegetation, prominently featuring the babassu palm (Attalea speciosa Mart.) in the “Mata dos Cocais”, a dominant feature in the Mid-North region of Maranhão state [17].

2.2. Evaluated Systems and Usage History

For the study, specific areas were chosen for assessment: secondary forest (SF), perennial pasture (PP), pasture recovered five years ago using intercropping with corn + Brachiaria brizantha (P5), and pasture recovered eight years ago using intercropping with corn + Brachiaria brizantha (P8).

Table 2. History and characteristics of the areas studied in the TRU in the region of Pindaré-Mirim, Maranhão, Brazil.

Areas	Area History
Secondary forest (SF)	The area in question represents the transition between the Maranhão Amazon forest and the Babassu forest, characterised by a predominance of secondary vegetation. Classified as an Open Ombrophylous forest, it stands out for the prevalent presence of the babassu palm (Attalea speciosa Mart.) in the Cocais Woods [17]. Apart from the babassu palm, there is a diverse array of vegetation, including Açaí palms (Euterpe oleracea Mart.), Bacaba (Oenocarpus spp.), Andiroba (Carapa spp.), Jatobá (Hymenaea spp.), and Embaúba (Cecropia spp.) [21]. This region serves as a reference for the natural soil conditions due to its preservation history, with an average age of over 50 years.
Perennial pasture (PP)	A pasture area initially planted with Jaraguá grass (Hyparrhenia rufa (Ness) Stapf) around 1970 remained until 1999. Subsequently, the pasture was renewed without soil correction or fertilisation, replaced by Brachiaria brizantha cv. Marandu through clearing, burning of plant residues, and broadcast seeding. This pasture is utilised for continuous grazing of beef cattle in an extensive system at a stocking rate of approximately 0.7 animal units per hectare per year. Periodic mechanised mowing is conducted to control natural regeneration.
Perennial pasture recovered five years ago (P5)	In a crop–livestock integration system (ILP), the process involved vegetation removal using a loader machine and harrowing across the entire area. Subsequently, mechanised sowing took place, combining corn DKB 175 with Brachiaria brizantha cv. Marandu. During planting, forage seeds were mixed with fertiliser, including 200 kg ha−1 of formulated 08-20-20 + Zn base fertiliser and a top dressing of 100 kg ha−1 of urea. The resulting pasture is utilised for rotational grazing of beef cattle, managing a stocking rate of 1.0 animal unit per hectare per year.
Perennial pasture recovered eight years ago (P8)	An area initially managed similarly to PP underwent recovery in 2012 within an ILP system. The process involved vegetation removal using a loader machine and harrowing across the entire area. Mechanised sowing followed, combining corn DKB 175 with Brachiaria brizantha cv. Marandu. Forage seeds were mixed with fertiliser during planting, with a fertilisation regimen of 200 kg ha−1 of formulated 08-20-20 + Zn base fertiliser and a top dressing of 100 kg ha−1 of urea. The resulting pasture is utilised for rotational grazing of beef cattle, maintaining a stocking rate of 1.0 animal unit per hectare per year.

outlines the management histories and distinctive features of these evaluated areas.

2.3. Collection of Samples and Evaluations Performed

In each land use system, five trenches measuring 1 × 1 × 0.4 m were opened, randomly placed in the areas. The samples were collected during the summer rainfall period, when the highest rates occur, collaborating so that the soil moisture levels are close to field capacity. Undeformed samples were collected from two opposite walls of the trench using a Uhland collector with volumetric rings with a known volume of stainless steel with 100 cm³ of layers of 0.0–0.10, 0.10–0.20, 0.20–0.30, and 0.30–0.40 m to determine the bulk density (BD) of the soil [22].

Deformed samples were collected at 3 equidistant points of 10 m in relation to each wall of the trench with the aid of a Dutch-type auger, totalling 12 simple samples to compose a representative sample in the same layers previously mentioned. The deformed samples were air-dried, crushed, and passed through a 2 mm mesh sieve, obtaining fine air-dried soil (FADS). The determination of total organic carbon (TOC) in FADS was conducted using the wet oxidation method. This method involves the utilisation of 0.167 mol L⁻¹ potassium dichromate solution and concentrated sulphuric acid, with the application of heat in a digester block. This methodology aligns with the process described by Mendonça and Matos [15], adapted from Yeomans and Bremner [23].

The FADS was used to obtain humic substances by extraction and separation based on the differential solubility of organic matter in a basic or acidic medium (FA and HA) and the residue (HUM), performed by the method proposed by the International Humic Substances Society (IHSS), as described by Swift [12] with adaptations by Mendonça and Matos [15]. We then separated the fractions into FA, HA, and HUM. After this step, for the different fractions, we determined the carbon contents, according to Mendonça and Matos [15]. Subsequently, we calculated the alkaline extract (AE) (AE = HA + FA) and the HA/FA and AE/HUM ratios to verify the SOM humification processes, in which the first ratio indicates carbon mobility in the soil and the degree of humification. In the second relationship, we verified the possible illuviation of organic matter in the soil [24].

The stocks of humic substances were calculated using the following equation: Est = (C × BD × e)/10, where Est represents the carbon stock in each layer expressed in Mg ha⁻¹; C represents the carbon content in the layer (g kg⁻¹); BD represents bulk density of the soil (Mg m⁻³); and e represents the thickness of the soil layer (cm).

To adequately compare the stocks between the different managements, it would be necessary to make a comparison between equal masses of soil, adjusting the values of the layers used in the calculations. However, as no differences were found in the BD (

Table 3. Bulk density of the soil (Mg m⁻³) of an Argiluvic Plinthosol under different areas in the Amazon of Maranhão.

Areas	Layers (m)
	0.00–0.10	0.10–0.20	0.20–0.30	0.30–0.40
SF	1.33 ± 0.06 ns	1.41 ± 0.03 ns	1.43 ± 0.02 ns	1.48 ± 0.01 ns
PP	1.42 ± 0.03	1.43 ± 0.02	1.43 ± 0.04	1.45 ± 0.03
P5	1.35 ± 0.03	1.48 ± 0.02	1.43 ± 0.01	1.43 ± 0.01
P8	1.37 ± 0.03	1.43 ± 0.02	1.43 ± 0.02	1.44 ± 0.03

Legend: SF: secondary forest, PP: perennial pasture, P5: perennial pasture recovered five years ago, and P8: perennial pasture recovered eight years ago. ±: Standard error, ^ns: not significant by the t test (p < 0.05).

), the stocks were calculated without using the correction for the equivalent mass. To verify trends in the accumulation or loss of C in the humic fractions in the 0–0.40 m section, the variation of the C stock (CS) of each fraction was calculated, compared to the secondary forest (reference) (ΔCS, Mg ha⁻¹ cm⁻¹).

The oxidisable fractions SOM were obtained using different degrees of oxidation through increasing concentrations of sulphuric acid (3, 6, and 9 mol L⁻¹), in which four fractions with decreasing degrees of lability, F1, F2, F3, and F4, were determined. We obtained the contents of each fraction by calculating the difference between the carbon values obtained in the concentrations of sulphuric acid and by the TOC, as described by Chan et al. [13] and adapted by Mendonça and Matos [15]. Due to the lability characteristics of the F1 fraction, the fraction is considered labile carbon (LC), and the non-labile carbon (NLC) we obtained by difference: NLC=TOC-LC [9]. Subsequently, to obtain indices that demonstrate the dynamics between the fractions obtained, we calculated the following ratios: F1/F4 and LC/TOC × 100.

2.4. Statistical Analysis

The resulting data were submitted to the Shapiro–Wilk and Bartlett normality and homogeneity tests, respectively. When these assumptions were met, the means were compared by the t-test at 5% probability. As a complementary analysis, the multivariate analysis of principal components was used, based on the Euclidean distance. Statistical analyses were performed using the R 4.0 software [25].

3. Results

3.1. Humic Substances

Differences were observed among areas regarding TOC and humic substances carbon contents (

Table 4. Total organic carbon content and humic fractions of soil organic matter in the different management systems.

Areas	TOC	FA	HA	HUM	AE	HA/FA	AE/HUM
	------------------------------g kg−1------------------------------
	0.00–0.10 m
SF	9.65 a	1.04 a	1.21 a	7.32 b	2.25 a	1.15 b	0.31 a
PP	7.27 b	0.52 c	0.66 b	5.22 c	1.18 d	1.28 b	0.23 b
P5	7.66 b	0.55 c	0.98 b	5.37 c	1.53 c	1.88 a	0.29 a
P8	9.86 a	0.76 b	1.08 a	9.13 a	1.84 b	1.42 ab	0.20 b
	0.10–0.20 m
SF	4.31 c	0.44 b	0.36 b	4.02 b	0.79 b	0.81 ab	0.21 ab
PP	5.38 a	0.35 b	0.26 b	4.24 b	0.61 b	0.76 b	0.14 c
P5	4.68 b	0.52 b	0.54 a	4.06 b	1.06 a	1.13 a	0.26 a
P8	5.63 a	0.72 a	0.35 b	5.24 a	1.07 a	0.51 b	0.20 b
	0.20–0.30 m
SF	3.75 c	0.43 b	0.26 a	3.74 a	0.69 b	0.61 b	0.19 b
PP	4.84 a	0.26 d	0.18 b	3.85 a	0.43 d	0.67 ab	0.11 c
P5	4.05 c	0.34 c	0.27 a	3.87 a	0.61 c	0.81 a	0.16 b
P8	4.49 b	0.71 a	0.26 a	3.50 a	0.97 a	0.37 c	0.28 a
	0.30–0.40 m
SF	3.79 b	0.34 b	0.17 a	3.47 a	0.52 b	0.51 b	0.15 c
PP	5.15 a	0.26 c	0.17 a	3.07 ab	0.43 c	0.67 a	0.14 c
P5	4.06 b	0.34 b	0.17 a	3.24 c	0.51 b	0.51 b	0.19 b
P8	4.81 a	0.94 a	0.17 a	3.15 b	1.11 a	0.19 d	0.36 a

Legend: SF: secondary forest, PP: perennial pasture, P5: perennial pasture recovered five years ago, and P8: perennial pasture recovered eight years ago, TOC: Total organic carbon, FA: fulvic acid, HA: humic acid, HUM: humin, EA: alkaline extract. Means followed by the same letter in the column, for each layer, do not differ from each other by t-test (p < 0.05).

). In the SF and P8 areas, the highest TOC levels were found in the surface layer (0.00–0.10 m). Deeper soil layers (0.10–0.20 and 0.30–0.40 m) exhibited higher values in the P8 area, while in the PP area, elevated values were observed between depths of 0.10–0.20 and 0.30–0.40 m.

In all layers, regardless of the area, the HUM fraction predominated, accounting for 71.68% to 89.8% of total C (Table 4). In the surface layer (0.00–0.10 m), higher levels of HA fractions (9.84% to 12.61% of total C) were observed compared to the FA fraction (6.92% to 10.86%). Conversely, in deeper layers, the FA fraction ranged from 6.06% to 22.07%, and HA from 3.99% to 10.55% of total C.

At 0.00–0.10 m, the SF area showed greater participation of FA and HA fractions, followed by P8 and P5 areas. Higher HUM levels were found in P8, primarily in the first two layers, followed by SF in the last two layers (Table 4). The P8 area exhibited higher FA values, notably between 39.36% and 63.41% in layers 0.10–0.20 to 0.30–0.40 m, surpassing SF. P5 displayed the highest HA levels at 0.10–0.20 and 0.20–0.30 m, with no differences in the last layer between areas.

The highest AE values were observed in SF (0.00–0.10 m), P5 (0.10–0.20 m), and P8 (0.10–0.20 and 0.30–0.40 m) (Table 4). The HA/FA ratio was notably higher in P5 across layers. SF, P5 (0.00–0.10 m and 0.10–0.20 m), and P8 (0.20–0.30 and 0.30–0.40 m) displayed the highest AE/HUM ratio.

Examining the variation in humic substances’ C stock (0.00–0.40 m), positive values for ΔCS-FA (0.29) and ΔCS-HUM (0.82) were found in P8, indicating an increase by 199.86% and 187.76%, respectively, compared to PP. Conversely, a negative variation for ΔCS-HA was noted in all pasture areas, with P5 (−0.01) displaying the least negative change (Figure 2).

3.2. Oxidisable Fractions

Differences in C contents of oxidizable fractions and their relationships were observed among the studied layers and areas (

Table 5. Carbon contents of oxidisable fractions, non-labile carbon (NLC), labile carbon/total organic carbon ratio (LC/TOC), and between oxidisable fractions (F1/F4) in different areas in the Amazon of Maranhão.

Areas	F1	F2	F3	F4	F1/F4	NLC	LC/TOC
	------------------------g kg−1-------------------------					g kg−1	%
	0.00–0.10 m
SF	3.24 b	0.76 a	1.35 b	4.30 a	0.76 b	6.41 a	33.61 b
PP	2.01 d	0.31 b	1.16 ab	3.79 a	0.54 c	5.26 b	27.71 d
P5	2.31 c	0.75 a	0.94 c	3.66 a	0.63 bc	5.35 b	30.18 c
P8	3.63 a	0.59 a	1.78 a	3.86 a	0.96 a	6.23 a	36.89 a
	0.10–0.20 m
SF	1.27 b	0.76 a	0.97 a	1.31 c	0.99 b	3.04 b	29.55 bc
PP	1.41 b	0.47 a	0.51 b	3.00 a	0.48 c	3.97 a	26.16 c
P5	1.97 a	0.55 a	0.63 b	1.53 c	1.33 a	2.71 b	42.18 a
P8	2.01 a	0.51 a	0.57 b	2.54 b	0.80 b	3.62 a	35.80 b
	0.20–0.30 m
SF	1.02 c	0.67 ab	0.56 b	1.50 b	0.70 b	2.72 b	27.37 c
PP	1.38 b	0.81 a	0.71 ab	1.94 a	0.76 b	3.46 a	28.61 bc
P5	1.40 b	0.62 ab	0.48 b	1.55 ab	0.91 b	2.65 b	34.73 ab
P8	1.82 a	0.36 b	0.94 a	1.38 b	1.36 a	2.68 b	40.46 a
	0.30–0.40 m
SF	1.00 c	0.53 ab	0.42 bc	1.75 b	0.73 b	2.79 b	27.13 b
PP	1.43 b	0.31 b	0.30 c	3.10 a	0.46 b	3.71 b	27.88 b
P5	1.25 bc	0.58 ab	0.68 ab	1.55 b	0.85 ab	2.81 b	30.82 ab
P8	1.80 a	0.64 a	0.93 a	1.58 b	1.18 a	3.02 a	37.55 a

Legend: SF: secondary forest, PP: perennial pasture, P5: perennial pasture recovered five years ago, and P8: perennial pasture recovered eight years ago. Means followed by the same letter in the column for each layer, do not differ from each other based on the t-test (p < 0.05).

). Overall, irrespective of area or layer, the study revealed higher carbon content in the most recalcitrant fractions (F3 and F4), accounting for 46.22% to 68.11% of the total, compared to the more labile fractions (F1 and F2), which ranged from 31.89% to 53.78%. This trend was supported by high NLC levels and low values for F1/F4 and LC/TOC ratios.

Our research showed an increase in F1 fraction carbon content ranging from 12.22% to 79.41% in the P8 area across all layers compared to the forest area. Similar patterns were observed for the F3 fraction, although it did not significantly differ from P5 and PP areas in some layers (Table 5).

Focussing on specific fractions: The F2 fraction displayed the highest carbon contents in SF, P5 (0.00–0.10 m), P8 (0.00–0.10 and 0.30–0.40 m), and PP (0.20–0.30 m) areas, with no notable differences in the 0.10–0.20 m layer. The F4 fraction showed no differences between areas in the 0.00–0.10 m layer. In other layers, PP displayed the highest C levels, comparable to P5 in the 0.20–0.30 m layer.

Regarding ratios, P8 (0.00–0.10, 0.20–0.30, and 0.30–0.40 m layers) and P5 (0.10–0.20 m layer) showed higher F1/F4 and LC/TOC ratios, ranging from 78.81% to 176.96% and 33.14% to 61.24%, respectively, compared to PP (Table 5). NLC peaked in SF (0.00–0.10 m layer), P8 (0.00–0.10, 0.10–0.20, and 0.30–0.40 m layers), and PP (0.10–0.20 to 0.20–0.30 m layers).

3.3. Multivariate Analysis

Principal component analysis revealed that PC1 and PC2 accounted for over 90% of the total data variation, with PC1 explaining 52.3% and PC2 explaining 42.7% (Figure 3 and

Table 6. Weight coefficients (eigenvectors), eigenvalues, variance explained, and correlation of variables by each principal component (PC1 and PC2) of the different soil organic matter fractions under different areas in the Amazônia Maranhense.

Variance Component	Principal Component
	1	2
Eigenvalues	4.706	3.841
Proportion (%)	52.285	42.682
Cumulative proportion (%)	52.285	94.966
Variables
TOC	0.718	0.695
F1	0.931	0.120
F2	0.005	−0.924
F3	0.990	0.103
F4	−0.379	0.919
CNL	0.048	0.931
FA	0.996	−0.059
HA	0.488	−0.871
HUM	0.984	0.080

). Notably, variables such as TOC, F1, F3, FA, and HUM contributed significantly to PC1, while F2, F4, NLC, and HA had higher correlations with PC2 (>0.70) (Figure 3 and Table 6).

The areas are segregated into distinct quadrants, illustrating an increasing impact on soil quality in the order P8 > SF > P5 > PP (Figure 3). Upon examining the components, PC1 shows positive associations with P8 and SF, but negative associations with PP and P5. PC2 displays positive correlations with PP and P8, while negative correlations are observed for P5 and SF. The areas closer to the vectors contribute significantly to these patterns, aligning with individual analysis results.

SF exhibited the highest values for HA and the moderately labile fraction (F2). PP displayed the highest levels of the recalcitrant fraction (F4). P5 showcased the highest values for the moderately labile fraction (F2). In the P8 area, strong correlations were found for attributes like TOC, oxidisable fractions of greater lability (F1, F3), and humic substances (FA and HUM).

4. Discussion

The similarity in TOC content between the P8 and SF areas in the surface layers, as well as the prevalence of PP and P8 areas in the deeper layers, might be connected to the gradual carbon deposition in the soil of secondary forests facing water stress conditions [26]. Secondary forests are susceptible to water stress, resulting in altered carbon balance and reduced growth rates during dry periods [26], which are frequent in the state of Maranhão due to its low rainfall periods [17].

Bastos et al. [27] found no differences in TOC content in a Plinthosol in the State of Rondônia when compared to pasture and forest areas. Similar patterns were also noted by Reis et al. [5] and Santos et al. [28] for Plinthosol in the Maranhão state.

Pastures cultivated in the Amazon biome can deposit an average of 18.90 Mg ha⁻¹ of dry biomass on the soil surface [29], resulting in an input of over 7 Mg ha⁻¹ of C on the soil surface [29,30]. Below the surface, tropical grasses can produce about 10 Mg ha⁻¹ of dry root biomass, with approximately 21% and 12% of root and shoot biomass, respectively, converting into TOC [31,32].

Sarto et al. [33] observed that after eight years of pasture establishment, C levels similar to those found in forest areas were present in the surface layer of the soil. This pattern can be attributed to several factors, such as higher residual deposition of aerial and root plant parts, contributions from animal residues enhancing biological activity, and the higher C/N ratio of grasses. In the deeper layers, the improved results in the P8 and PP areas can be attributed to the capacity of Brachiaria brizantha cv. Marandu to form a voluminous, fast-growing, and well-developed root system. Due to constant renewal through grazing, dead roots are decomposed by soil microorganisms, releasing nutrients, and promoting the increase of organic compounds [34,35].

Pasture recovery eight years ago contributed beneficially to the increase of the HUM fraction compared to the PP area. This pattern is evident through the analysis of ΔCS-HUM and the principal component analysis (Figure 2 and Figure 3). This increase may have been facilitated by the preservation of HUM inherited from the forest, which was protected from biological activity during the initial development of the pasture. The higher biomass input in the form of easier-to-degrade material for microorganisms could explain this pattern [34,36]. The higher levels of C in the HUM fraction, across all areas and layers, as confirmed by the low values of the EA/HUM ratio, are common in regions with tropical climates. In these regions, the dominance of the most stable and insoluble fraction is a result of intense residue mineralisation and edaphic limitations on biological activity, which reduce the humification process, indicating the strong stability of SOM [14,37,38].

The predominance of the HUM fraction in all areas and layers can be associated with its recalcitrance, low solubility, and association with iron oxides [39], which are abundant in Plinthosols [20]. Other factors that contribute to the predominance of the HUM fraction include the aliphatic chemical nature of the molecule, derived from highly resistant macromolecules from plants and/or microorganisms [40], as well as the high molecular weight and greater stability compared to other fractions, resulting in greater protection of fixed carbon. The HUM fraction is considered the most significant reserve of organic carbon in the soil [36].

Regarding the Alkali-soluble fractions (FA and HA), which showed better results in the recovered pasture areas (5 and 8 years) compared to the perennial pasture, this difference may be due to the lower deposition of residues in the PP area. This decrease in crop productivity after 20 years of establishing perennial pasture, along with inadequate soil management practices, could be responsible for this result [41]. The recovery of pasture areas (5 and 8 years) led to an increase in HA fraction levels. Although the ΔCS-HA values were negative, they were still better compared to the PP area (Figure 2). The principal component analysis also highlighted their greater contribution to the PC2 axis (Figure 3), suggesting greater stabilisation of SOM with higher carbon content in a humified form [36].

The high C contents of the FA fraction in the P8 area, as well as the positive values in the ΔCS-FA (Figure 2), can be attributed to the greater deposition of residues from aerial and root biomass, as well as animal residues, which contribute to the accumulation of decomposable material and the formation of this fraction. In the deeper layers, these results can be attributed to the greater mobility and less condensation of the FA fraction compared to HA, allowing it to be present in higher proportions. The predominance of the FA fraction compared to HA, particularly in the deeper layers, was observed across all areas, as indicated by the low values in the HA/FA ratio. This pattern suggests limited SOM evolution due to edaphic reasons, possibly due to soil wetting and drying cycles and/or water table movement, as well as recent inputs of SOM [14,24].

Araújo et al. [42], in their study on the impact of converting the Amazon forest into pasture in the State of Acre, Brazil, found similar behaviour of humic substance fractions as observed in this study. The authors observed higher levels of TOC in pasture areas with a predominance of the HUM fraction and greater formation of FA compared to HA at depth.

The analysis of oxidisable organic carbon fractionation confirms the predominance of the most recalcitrant forms of C (F3 and F4) and NLC, which aligns with the predominance of the HUM fraction observed. Additionally, low values of the F1/F4 and LC/TOC ratios indicate that even the low levels of C in the soil are largely associated with compounds of greater stability and interaction with the mineral fraction of the soil. This limits microbial attack, resulting in a higher molecular weight and a longer residence time of these products in the soil. [9,35,43].

The recovery of pasture areas eight years ago led to increases in C levels in the labile (F1), moderately recalcitrant (F3), and NCL fractions compared to the perennial pasture area. These increases can be attributed to greater initial contributions and plant vigour, resulting in higher biomass inputs on the surface through shoot deposition and in the soil through the root system [41,43]. These increases reflect the soil’s capacity for C recovery, and in some cases, the quantified values were higher than those found in the forest area. The accumulation of C in the P8 area for the F1, F3, and NCL fractions, as well as the F1/F4 ratio, indicates that pasture recovery contributes to increased carbon storage in the soil, reducing emissions to the atmosphere. The results observed for labile fractions resulting from pasture recovery demonstrate the benefits of the implemented soil changes, indicating changes in soil attributes caused by management shifts [44,45]. Similar findings have been observed in other studies that reported changes in soil attributes due to management changes, as well as for moderately recalcitrant and non-labile fractions, demonstrating their contribution to soil organic carbon stabilisation [43,46].

Xu et al. [44] also observed increases in the labile fraction (F1) of soil C levels, which were associated with improvements in soil structure and nutrient content, as well as increases in carbon reservoirs and biological activity. Chemical transformations undergone by SOM, combined with the physical protection of C, contribute to the accumulation of recalcitrant fractions, leading to greater C stability and enhancing soil quality [46].

The improved quality of the soil in the pasture areas that underwent recovery can be attributed to several factors. Firstly, the establishment of perennial pasture led to increased biomass input, both above and below ground, through plant residues and root systems. This increased organic material contributed to the accumulation of labile fractions (F1 and FA) and promoted nutrient cycling and biological activity in the soil [44,45]. Secondly, the recovery process likely involved changes in soil management practices, such as reduced soil disturbance and improved soil cover, which enhanced the protection and stabilisation of SOM [9]. These changes favour the formation and preservation of recalcitrant fractions (F3 and HUM), which are more resistant to decomposition and have longer residence times in the soil [36].

Overall, the findings support the positive impact of pasture recovery on SOM fractions and highlight the potential of this management strategy for soil conservation and improvement in the Amazon region of Maranhão. The results also emphasise the importance of natural regeneration processes, as exemplified by the secondary forest, in promoting soil quality and SOM accumulation.

In contrast, the perennial pasture area showed the lowest soil quality among the studied areas. Continuous pasture management practices, such as grazing and soil compaction, can lead to reduced SOM inputs and increased decomposition rates, resulting in lower levels of labile and recalcitrant fractions. The lower soil quality in the perennial pasture area highlights the need for sustainable land management practices, such as pasture recovery, to improve soil health and preserve SOM stocks.

5. Conclusions

The pasture recovery process carried out eight years ago led to significant improvements in both labile (F1 and FA) and recalcitrant (F3 and HUM) fractions of soil organic matter across the evaluated layers. This management approach showed superior results compared to perennial pastures, secondary forests, and pastures recovered five years ago.

Principal component analysis ranked the study areas by soil quality in descending order: perennial pasture recovered eight years ago, secondary forest, perennial pasture recovered five years ago, and perennial pasture. This ranking reflects the positive impacts on various soil organic matter fractions and demonstrates the lowest impacts provided by the different management practices.