Antioxidant capacity sources of soils under different land uses

Antioxidants (AOX) in soils originate mainly from secondary plant metabolites and are pivotal in many redox processes in environment, maintaining soil quality. Still, little is known about the influence of land uses on their accumulation in soil. The aim of the paper was to determine the content of these redox-active compounds in the extracts of A horizons of abandoned fallows, arable and woodland soils. Total antioxidant capacity (TAC) of soils under various uses and vegetation was evaluated in different soil extracts using Folin-Ciocâlteu method. The contribution of humic acids to TAC was determined and antioxidant profiles estimated using the chromatographic GC–MS method. Forest soils exhibited the highest TAC (15.5 mg g−1) and AOX contents (4.34 mg g−1), which were positively correlated with soil organic carbon content. It was estimated that humic acids contribute to over 50% of TAC in soils. The main phenolics in woodland A horizons were isovanillic and p-hydroxybenzoic acid (p-HA), while esculetin and p-HA predominated in the abandoned fallows due to the prevalence of herbaceous vegetation. Cultivated soils were the most abundant in p-HA (56.42%). In the studied topsoils, there were considerable amounts of aliphatic organic matter, which role in redox processes should be further evaluated.

Antioxidants (AOX) are defined as substances present most often in low concentrations when compared to easily oxidizable substrates that delay or inhibit their oxidation 1 .They occur naturally in soils as products of plant metabolism, including enzymes, hormones, amino acids, vitamins or polyphenols, and are specific to the land vegetation profile [2][3][4] .AOX may also originate from industrial products or wastes, which enter the soil either as leachates or as particulate matter 5,6 .Some of the low-molecular-weight antioxidants in soil, namely phenolic compounds (PC, phenolics), are the most abundant group of secondary plant metabolites exuded from the roots or grains 7,8 .The PC family covers a broad spectrum of chemical compounds, such as phenolic acids (derivatives of benzoic and cinnamic acids), flavonoids (flavones, flavanols, flavonols, isoflavones and anthocyanins), tannins, coumarins, lignans, quinones, stilbenes, curcuminoids etc 9 .They contribute to the adjustment of the plant microbiota to soil conditions, modulate its biodiversity and participate in many redox reactions, including inhibition of free radicals as well as control of plant residue biodegradation and nutrient cycling 10,11 .The PC content of soil, together with its antioxidant capacity, is considered an "antioxidant system", which was proposed as an indicator of its health and quality 11 .
In the soil environment, phenolic compounds are commonly generated during the decomposition of soil organic matter (SOM), based on the physical breakdown and biochemical transformation of complex organic molecules into simpler organic and inorganic moieties 12 .In parallel, these processes lead to the formation of humic substances (HS) in soil, containing stable semiquinone radicals 13 in their structure, most likely to be derived from the reaction of phenolic compounds with reactive radicals 14 .Several studies imply that most of the antioxidants in soil can be found among the humic substances [15][16][17][18] .This was evaluated by Ziółkowska et al 19 ., who determined the important role of phenolic compounds in the processes of organic matter transformations in meadow soils, leading to the formation of humic substances.What is more, phenolics are considered more resistant to decomposition in soils compared to other organic matter sources, and thus have often been regarded a slow carbon pool in soil dynamics models 6 .Therefore, the soil's antioxidant capacity may control the rate of breakdown of more labile organic matter pools and the recalcitrance of SOM 15 .
Despite the important role of phenolic compounds in many processes in soils, the literature on the antioxidant capacity of soils is still fragmentary and relates mostly to lands under agricultural use.The studies by Rimmer 20 demonstrated that antioxidants can be extracted from soils and their quantity varies from soil to soil, decreasing with the depth of the soil profile 14,15 .This effect was correlated with the diversity of plant residues and their • Total organic carbon (TOC) and total nitrogen (TN) were measured using a Vario Macro Cube CN analyser (Elementar Analyser System GmbH, Germany).• Soil pH was measured potentiometrically in a distilled water and 1 M KCl suspensions at the soil:water ratio of 1:5 (v/v) (Mettler Toledo, Columbus, OH, USA).• Calcium carbonate content was assessed volumetrically using Scheibler apparatus 30 ; • Particle size distribution was conducted by sieve and sedimentation method and estimated texture classes were assigned according to the USDA standard applied by the WRB classification.
The texture and the chemical composition of topsoil samples investigated in this study were given in Table 2. None of the investigated soils contained a measurable calcium carbonate level.Soil materials differed in organic carbon content, texture, and pH.

Water and alkaline extraction
Water and alkaline extracts were prepared by mixing 5 g of each soil sample with 50 mL of water or 0.1 M NaOH, respectively, and agitating on a rotary shaker for 4 h (Biosan, Multi RS-60).Subsequently, the mixtures were left overnight and centrifuged at 4500 rpm for 5 min.Additionally, to assess the share of humic acids (HA) in the antioxidant potential of the extract, the alkaline extracts were acidified to pH 1 with 6 M HCl, left overnight, and the precipitated humic acids centrifuged and discarded (4500 rpm, 20 min).Therefore, three types of supernatants were obtained: water (WE), alkaline (AlE) and acidified (AcE, with HA removed).All of them were subjected to a total antioxidant capacity (TAC) assessment with the use of UV-Vis spectroscopy.Acid hydrolysis and alkaline re-hydrolysis of soil samples (AAH extracts) In addition, the extraction of phenolic compounds from soil samples described in Ziółkowska et al 24 .with some modifications, was also tested.Briefly, to 1 g of soil sample, 2 mL of ascorbic acid (1%), an aliquot of 0.0125 mM EDTA and 10 mL of 6 M HCl were added (acid hydrolysis, step I).Ascorbic acid and EDTA were utilized to protect the phenolics from degradation.It was then followed by the sample microwave extraction at 120 °C for 2 h (650 W, microwave system StartD, Milestone, Sorisole, Italy).A similar procedure was proposed by Martens 31 during the hydrolysis of ether-linked phenolic acids from soil, but the temperature was higher-160 °C.After cooling, the supernatants were transferred to the amber glass vials and kept in the fridge.The soil remnants were neutralized with ultrapure water, and 2 mL of ascorbic acid (1%), 2 mL of 0.0125 mM EDTA and 10 mL of 10 M NaOH were added, followed by 24 h of agitation (alkaline re-hydrolysis, step II).After centrifugation at 10 000 rpm for 5 min the supernatants were acidified to pH 1, using 6 M HCl.The mixtures were left overnight, and precipitated humic acids were centrifuged (4500 rpm, 20 min) and discarded.As a result, hydrolysates soluble in acid remained.The supernatants obtained in the alkaline re-hydrolysis step were combined with the solutions from acid hydrolysis and labelled as AAH.They were further analyzed for the total antioxidant capacity (TAC) on UV-Vis and the phenolic profile estimation using GC-MS.In addition, pure quartz was subjected to an identical procedure to exclude the influence of ascorbic acid on the results of the FC assay.

Estimation of the Total Antioxidant capacity (TAC) using Folin-Ciocâlteu method.
The Folin-Ciocâlteu (FC) reaction is an antioxidant assay based on electron transfer, which measures the reductive capacity of the sample 32,33 .The reagent utilized in the method is a mixture of sodium tungstate, sodium molybdate, lithium sulfate, bromine water, and concentrated hydrochloric and phosphoric acids.The resulting product is a blue colored complex, and the absorbance of the sample is measured within the wavelength range of 750-784 nm 34 .The method has been widely applied to determine the total phenol/polyphenol content of plant-derived food and biological samples 26 , in which the product of phenolics oxidation by the FC reagent is measured at 765 nm.In the presented paper, the assay was adopted to test the antioxidant potential of various extracts from the studied soil samples.The total antioxidant capacity of water extracts (containing readily available phenolics) was determined according to the following procedure: 5 mL of extract was mixed with 0.5 mL of FC reagent and, after 3 min, 1.5 mL of 20% Na 2 CO 3 was added 35 .The mixtures were then allowed to stand for 30 min in the dark, and in the next step, the absorbance of the solutions was measured at 765 nm using a Cary 60 UV-Vis spectrophotometer (Agilent, Santa Clara, CA, USA).The calibration curve used in the quantification was drawn up for gallic acid solutions in the concentration range 1-10 mg L −1 .For the alkaline (0.1 M NaOH before and after acidification) and AAH types of extracts 0.4 mL of the sample was taken up, 7.8 mL of water and 0.5 mL of FC reagent were added, followed by the addition of 1.5 mL of 20% Na 2 CO 3 .The rest of the procedure was analogous to the one described for water extracts.A calibration curve in the range of 0-500 mg L −1 of gallic acid equivalents was constructed (Figure S1).Bastola et al 28 .proved that gallic acid as a standard is the most appropriate in the FC assay of total phenolic content quantification compared to other conventionally used phenolic acids.

Phenolics profiles in AAH extracts
The qualitative and quantitative analysis of antioxidants present in the studied soils was conducted on the AAH extracts, as their TAC assessed by the FC method was the highest.The method described by Pachura et al 36 .was used to determine the AOX profiles.Briefly, 1 mL of soil extract was taken and 3.5 µg of caffeic acid was added as an internal standard.Subsequently, the sample was extracted three times with 1 mL aliquots of ethyl acetate.The resulting extracts were combined and subjected to solvent evaporation using a vacuum evaporator.The dry extract was dissolved in 250 µL of pyridine, 250 µL of BSTFA was added and silylation was carried out for 45 min at 60 °C.The sample was then transferred to a 1.5 mL chromatographic vial and subjected to GC-MS analysis.
GC-MS analysis was carried out using the Shimadzu GCMS QP 2020 instrument (Shimadzu, Kyoto, Japan) with a Zebron ZB-5 MSi column (30 m × 0.25 mm × 0.25 μm; Phenomenex, Torrance, CA, USA).Injection volume of 1 µL was performed at 280 °C, split 10, carrier gas flow (helium) 0.97 mL min −1 .The separation of analytes was performed using the following temperature program: 150 °C for 1 min, raised to 300 °C at a rate of 10 °C min −1 and maintained at 300 °C for 5 min.Mass analysis was performed with the following parameters: ion source temperature of 270 °C, an interface temperature of 270 °C and a scan mode of 40-800 m/z.Identification of the analytes was performed based on the NIST 20 database (National Institute of Standards and Technology).Quantification was carried out by peak area normalization to an internal standard 37,38 .

Statistical analysis
The normal distribution of the data was checked using the Shapiro-Wilk test.Basic statistical parameters such as the mean, standard deviation (SD), and standard error (SE) were calculated based on the triplicate result.The values of total antioxidant capacities (TAC) obtained for soils under different land uses, as well as the results of phenolics profiles were statistically compared using the Student's t-test (p < 0.05).Pearson's correlation was used to assess the strength of the dependence of estimated TAC values for various extract types on the TOC of the investigated horizons and the sum of individual antioxidants (SUM) identified with GC-MS in the studied soils.
All the data were processed using the software package Statistica 13.3 TIBCO Software Inc.

Results and discussion
Total antioxidant capacity (TAC) of the investigated soils.
Results of the Folin-Ciocâlteu assay of all the tested extract types (in µg mL −1 ), expressed as the total antioxidant capacity of the investigated soils (in µg g −1 ), were collected in Table 3.The readily available, dissolved forms of phenolic compounds (aqueous extracts, WE) made up only a small part of the potential pool of antioxidants in the topsoil horizons of the studied soils.However, their role should not be marginalized, as these compounds, present by definition in low concentrations in soil solution, participate in many redox processes, including the formation of humic substances 19 .TAC in WE ranged from 5 to slightly over 21 µg g −1 for abandoned soils and farmlands to about 114 µg g −1 for forest soils.Meanwhile, alkaline extraction of soil material (AlE) resulted in 30 to even 56 times larger TAC (on average) compared to WE.It can be attributed to the high efficiency of NaOH as a solvent, which releases bound and polymerized forms of phenolics from soil 6 .The maximum TAC estimated for AlE was obtained for forest soils, achieving up to 2228.67 µg of gallic acid equivalents per g of soil, whereas the lowest value was found for cultivated soils (135 µg g −1 ).Mean TAC values, based on the AlE, point to the following decreasing order of antioxidant potential: forest soils > abandoned fallows = arable soils.Thus, the A horizon of forest soils is characterized by a higher antioxidants content than that of abandoned or cultivated soils, which is the consequence of an enhanced accumulation of fresh organic matter in the forest floor 39 .The obtained results are in contradiction to the studies of Cardelli et al 3 ., who found the higher antioxidant capacity in Mediterranean soils of grasslands rather than forests.However, the discrepancy in trends obtained between the results of Cardelli and those presented herein might result from the various climatic zones and thus, the specificity of vegetation and soil dynamics under which the AOX were accumulated.When comparing the mean TAC values for soils taken out of cultivation and farmlands, it can be elucidated that their antioxidant potential is of similar magnitude.Nevertheless, a great deal of variability within the soils of each land use and their TAC should be noted (Table 3).This can be most likely attributed to differences in the density and diversity of plant cover that is prone to mineralization and humification on various land types 40 .
Humic substances (humic acids-HA, fulvic acids-FA and humins-HU), present in the soil are the most reactive part of organic matter 41 .They are complex and heterogeneous mixtures of polydispersed materials formed by biochemical and chemical reactions during the decay and transformation of plant and microbial remains 18 .These organic molecules contain phenolic electron-donating moieties, due to which they may act as antioxidants 42 , thus significantly contributing to TAC and influencing soil quality.To determine the share of HA in the total antioxidant potential of the studied soils, the previously obtained alkaline extracts were acidified to precipitate humic acids.HA were discarded and the resultant mixtures (AcE) containing the fraction of dissolved phenols together with fulvic acids were again subjected to the FC test.Owing to that, it was possible to estimate the contribution of the HA fraction to the total antioxidant potential of the sample after alkaline extraction.It was calculated as a difference in the antioxidant capacity of the alkaline extracts and the same extracts after acidification, and expressed as a percentage share of the total antioxidant capacity of the AlE, ranging from 46 to 89% (Table 3).The highest mean HA share of TAC was determined for abandoned fallows and farmlands (65-66%), while in the case of forest soils it was equal to 53%, but the differences within the studied groups were not statistically significant.Nevertheless, the slightly lower mean value of the parameter obtained for the forest soils can be explained by a constant inflow of fresh organic matter in the organic horizons, which may increase the share of material with a low humification degree 43 .The antioxidant properties of HS were also investigated by Aeschbacher et al 42 ., who evaluated the electron-donating capacities of humic substances and correlated them with their phenolic moieties formed from higher plant precursors such as lignin and tannins.The presented results indicate that humic acids may contribute to over 50% of the TAC of soils, irrespective of the land use type.www.nature.com/scientificreports/ The last type of soil extract analyzed for the total antioxidant potential (FC assay) was obtained by the acid hydrolysis and alkaline re-hydrolysis of the soil samples (AAH) (Table 3).The average antioxidant capacity of the AAH samples was the highest for the A horizons of forest soils (15,505.50 µg g −1 ).Abandoned fallows and cultivated soils exhibited significantly lower mean TAC levels, with 10,246.67 and 6334.0 µg g −1 of soil dry mass, respectively (Fig. 1b).This is in line with the trends observed for alkaline extracts (AlE) of the tested soils.The extraction method based on acid-base hydrolysis was the most efficient one among the tested variants.It released the largest pool of compounds, capable of reacting with the FC reagent, from the studied soil materials.The measured TAC values were on average 5 to even 50 times higher than those obtained in AlE and AcE extracts.This is due to the use of very high (10 M) concentrations of sodium hydroxide and hydrochloric acid, which enforced the hydrolysis of the ester and glycosidic bonds, strongly retaining phenolic acids in the soil matrix 24 .The extraction efficiency was also enhanced by microwave radiation, which supported the acid hydrolysis and significantly shortened this step compared to the original procedure 24 .
The presented results demonstrate that various pools of antioxidants can be isolated from soils, depending on the extraction efficiency of the solvent used.The highest TAC values, measured with the FC assay, were determined in the forest A horizons, irrespective of the soil extraction type.This was attributed to the highest input of fresh organic matter in forests, which is the source of secondary plant metabolites.The measured antioxidant capacities were compiled with the total organic carbon contents of the investigated soils (Fig. 1a).Significant (p < 0.05), moderately positive correlation coefficients ranging from 0.452 for AAH extracts up to 0.586 for AcE mixtures were found (Supplementary Table S1), being in line with the findings of Rimmer and Smith 14 .The soil materials taken from abandoned fallows and farmfields were characterized by significantly lower TAC than the samples obtained from the A horizons of forest soils (Fig. 1b).
When comparing the extraction methods used in this study, it can be concluded that alkaline extraction (0.1 M NaOH) is not an efficient method for the quantification of antioxidant compounds in soil.Nevertheless, it reflects the general trend in the content of phenolic compounds in the analyzed soils.It can therefore serve as a preliminary method for the determination of the antioxidant potential of soil samples before the more efficient acid-base hydrolysis and a subsequent chromatographic identification of the extracted compounds.

Phenolic profiles in soils under various land use.
The quantitative and qualitative results of the GC-MS analysis of the studied AAH extracts within each of the analyzed land use groups are presented in Table 4 and Supplementary Figure S2.In the combined mixtures from acid hydrolysis and alkaline re-hydrolysis of soil samples, the following nine compounds were identified: p-Hydroxybenzoic acid (p-HA), Suberic acid (SA), Isovanillic acid (IVA), Homovanillic acid (HVA), Azelaic acid (AZA), Protocatechuic acid (PA), Syringic acid (SYR), Esculetin (ESC) and p-Coumaric acid (p-CO).Most of them have literaturally reported antioxidant capacities, although not all the compounds belong to the phenolics group.One of such chemicals is suberic (octanedioic) acid, commonly present in the bark of some tree species, such as birch 44 , exhibiting a significant share in the studied soil extracts.It is a fatty-acid derivative originating from plant biomacromolecules (cutin and suberin), classified as a recalcitrant aliphatic organic matter fraction 45 .Due to its insoluble and non-hydrolysable nature, it tends to accumulate in soil, enriching SOM fractions.Another representative of this group found in the studied topsoils was azelaic acid.This 9-carbon atom dicarboxylic acid is produced by a variety of plants, commonly wheat species, in response to biotic and abiotic stress conditions 46 , therefore having antioxidant properties.
Among the phenolic compounds identified in soil extracts, there were representatives of vanilyl (IVA, HAV), syringyl (SYR), as well as hydroxycinnamic (p-CO, ESC) acid derivatives (Table 4, Figure S2).These three groups of phenolic compounds content and ratios in lignins are plant species-specific 19,43,47 , which was reflected in their mutual percentage share in the soils under various land uses (Fig. 2).Although the studies on the phenolic profiles www.nature.com/scientificreports/ of soil are still limited, the results obtained here are in accordance with the available literature findings 14,18,19 , where the same or similar compounds belonging to the three PCs groups were reported.
There was considerable variation in the content of the respective phenolic and non-phenolic compounds identified in the AAH soil extracts.In general, the highest contents of antioxidants were found in the forest soils (Table 4), achieving an average of 4341.07 µg g −1 of soil dry mass, which was over 17 and 47 times higher than its mean value for farmlands (248.70 µg g −1 d.m.) and fallows (92.77 µg g −1 d.m.), respectively.When compared with the literature, the sum of compounds determined in forest soils was in the same range as the content of phenolics in the plant material of meadows (3204.72-5736.45µg g −1 d.m.) 19 .What is more, the sum of compounds found in the topsoils of investigated fallows under meadow vegetation was significantly lower than the amounts estimated by Ziółkowska et al 24 .This might be due to the differences in the chemical composition of the extracts, which is highly plant-specific and may also arise from the specificity of the chromatographic method utilized in the studies (GC-MS vs LC-MS).
It is also worth emphasizing that the sum of compounds in AAH extracts estimated with the GC-MS technique (SUM) was much lower than the total antioxidant capacity (TAC) assessed in the FC test (Table 3), although the main trends observed within the land use groups were maintained.This corroborates the observations made by other authors 28,33,34,48 , who claim that the FC method is useful in assessing the total antioxidant potential of the sample (including the share of non-phenolic redox-active compounds) rather than solely the total phenolic compounds content.Therefore, FC assay should be coupled with the phenolic profiling to verify the actual contribution of PCs in the antioxidant properties of soils 49 .
Generally, in investigated abandoned fallow soils the highest share in the sum of identified compounds constituted p-hydroxybenzoic acid (24.75%) and coumarin derivative esculetin with p-coumaric acid, comprising a total of 28.01% (Fig. 2).The highest p-HA share coincides with the studies of Rimmer and Abbott 20 , who found that it was one of the dominant phenolics in the subset of surface soil samples under various land uses, including semi-natural soils and pastures.Esculetin (21.60% of the SUM) is a polyphenol present in many medicinal herbal plants, such as various Artemisia species 50,51 , which were also found in the vegetation profile of the studied fallows (Table 1).It has been reported to exert strong anti-proliferative and antioxidant activities 50 .In the undisturbed ecosystem of abandoned fallows, there was also an accumulation of aliphatic organic matter fraction (sum of SA and AZA) observed, which contributed up to 27.81% of the identified compounds.Isovanillic acid amounted to 12.83%, whilst syringic and protocatechoic acids constituted less than 5% of the SUM.www.nature.com/scientificreports/ In the cultivated soils, similarly to fallows, p-hydroxybenzoic acid was dominant, constituting 56.42% of the compounds sum.This is in agreement with the findings of several studies performed on arable lands, where p-HA was identified as the most abundant phenolic compound in the field soil samples 2,52,53 .Also, Zhou et al 54 .noticed that p-HA was one of the main PCs in rhizospheric soil under a continuous mono-cropping system.The greatest concentration of p-HA in the arable soils of this study can be attributed to its production by microbial synthesis 55 .This is due to the regular input of post-harvest residues on soil surface, which enhances microbial activity and results in the overall highest share of p-hydroxybenzoic acid 31 in SUM.Isovanillic acid constituted 15.97% of SUM.It is a product of the microbial degradation of lignin, presumably present in woody post-harvest residues in investigated arable soils.Aliphatic acids (SA and AZA) were in the minority (11.91% of SUM), contrary to their content in fallow or forest soils.Similarly, esculetin abundance was decreased in farmland soils (8.42%), which is in line with the very limited and random occurrence of herbal-type vegetation on cultivated plots.
According to the results obtained, isovanillic acid was the predominant phenolic in the extracts of forest A horizons, constituting 38.86% of the extracted compounds pool (Fig. 2).This is in line with the studies of Rimmer and Abbott 20 , who observed the highest concentration of IVA in woodland soils.Also, Dębska and Banach-Szott 56 demonstrated considerably higher amounts of vanilyl compounds in the mineral horizons of forest soils, with respect to syringyl and cinnamyl derivatives.p-HA share was much lower here (13.99%), compared to fallow and cultivated soils.Due to the literature, IVA and p-HA are common products of lignin biodegradation 57 in conifer forests.They are products of veratric and p-anisic acid demethylation and hydroxylation of the latter by the specific bacteria strains growing on the wood flour 58 .Their concentration considerably increases in litters with biodegradation progress 59 .In the extracts of forest soils there were also significant amounts of azelaic and suberic acids, reaching on average 24.74 and 11.64% of the chemicals sum, respectively.They originate from the cuticle that seals the aerial epidermis, and suberin that is present in the periderm of barks and underground organs of trees 60 .Hydroxycinnamic acid derivative p-coumaric acid and esculetin were in the minority (3.65% and 5.62%, respectively).
Considering the mutual share of the compounds identified in the AAH extracts (Fig. 3), it can be concluded that the vegetation, characteristic of the land use type, may be the predominant factor influencing the phenolics profile in the studied soils.However, more extensive research is necessary to fully verify the synergistic effect of land use and soil typology on the antioxidants content of soils.p-Hydroxybenxoic acid, isovanillic acid and esculetin were the most abundant phenolics in the studied A horizons of soils in the temperate climate zone.However, one should not underestimate the significant share of aliphatic organic matter constituents (suberic and azelaic acids) in the studied topsoils.Therefore, further studies to clarify their role in the antioxidant capacity of soils should be evaluated.

Conclusions
The presented results verify and significantly complement the existing literature findings on the phenolic and non-phenolic compounds content in soils in temperate climate zone.The highest total antioxidant capacity was assigned to woodland A horizons.Cultivated soils and abandoned fallows were characterized by similar and much lower TAC values than the forest soils, regardless of the extract type.Acid hydrolysis combined with alkaline re-hydrolysis released the biggest pool of redox-active compounds, from soil materials among the tested extraction variants.The antioxidant capacity of the studied soils was positively correlated with their The estimated phenolic profiles in soils were vegetation specific.The major compounds identified in soils were p-hydroxybenzoic acid, isovanillic acid and esculetin.p-HA dominated in soils taken out of cultivation, whereas IVA prevailed in forest A horizons.Herbaceous plants in abandoned fallows were presumably the source of esculetin, which together with p-HA were the main PCs in set-aside soils.There were also considerable amounts of aliphatic fatty acids (azelaic and suberic acids) found in the studied topsoils, whose role in redox processes should be further evaluated.

Figure 1 .
Figure 1.Dependence of organic carbon content (TOC) in the A horizon of the studied soils on their antioxidant potential (TAC), estimated in AAH extracts (a).Summary of the mean TAC concentrations in the soil materials from surface horizons of abandoned fallows (O), farmlands (P), and forest (L) soils (b).Results are expressed as mean values ± standard deviation (n = 3).Letters a and b indicate significant differences between mean TAC values for each extract type, within studied land type groups (p < 0.05).

Figure 2 .
Figure 2. The mean percentage share of individual compounds identified in AAH extracts from investigated abandoned fallows (a), farmlands (b), and forest soils (c).

Figure 3 .
Figure 3.The comparison of mean percentage shares of individual compounds identified in AAH extracts from investigated abandoned fallows, farmlands, and forest soils.Results are expressed as mean values ± standard deviation (n = 3).

Table 1 .
Soil location, land use and classification.

Table 2 .
Selected physicochemical properties of topsoils (A horizons) under study.Results are expressed as the mean values ± standard deviation (n = 3).

Table 3 .
Summary of the total antioxidant capacity (TAC) of water extracts (WE), alkaline extracts before (AlE) and after acidification (AcE) and combined hydrolysates (AAH), determined by the FC method, expressed in µg of gallic acid per g of investigated soil dry mass.Results are expressed as mean values ± standard deviation (n = 3).Letters a and b indicate significant differences between mean TAC values for each extract type, within studied land type groups (p < 0.05).Significant values are in bold.

Table 4 .
Qualitative and quantitative (in µg g −1 of dry soil mass) characteristic of individual compounds in the AAH extracts of abandoned soils, farmlands, and forest soils.*SE-standard error.