1 Introduction

Soil is the most important element of the agricultural and natural environment. Improving our knowledge about potential impacts which could affect the natural soil processes is the responsibility of researchers from different disciplines. Humic substances—the main component of soil organic matter—constitute a condensed aromatic matrix which contains several oxygenated functional groups, mainly carboxylic (COOH), phenolic (OH), alcoholic (OH), and carbonyl (CO) groups [1,2,3,4]. Thanks to new more advanced techniques and multidisciplinary collaboration, novel methods of soil organic matter (SOM) analysis are discovered. One of the methods which in recent years have become widely used in soil science is NMR, especially CP MAS NMR [6, 7]. The first studies of humic substances by means of EPR (another kind of magnetic resonance spectroscopy) were carried out by Professor Schnitzer in the 1970s [8] and were followed by the studies done by Senesi [2,3,4,5]. Their research on the chemistry of humic substances indicates the involvement of the formation and transformation of radicals in humification processes. These semiquinone-type radicals are stabilized in polyphenolic matrices which make them inaccessible to many external factors, but, due to their existence in quinone-semiquinone-hydroquinone equilibria, the radicals are highly sensitive to various physical and chemical agents, e.g., radiation, redox reactions, acid–base reactions, etc. [3, 9]. Owing to the fact that the structure of humic substances is rich in functional groups that they can form alike simple salts, i.e., humate and metal chelate complexes [4]. Our previous study, devoted to the interaction of metal ions with humic acids at different pH values, showed that both metal ions and the pH have a bearing on radical concentration in humic acids [9,10,11].

In the present work, our aim is to demonstrate the usefulness of the EPR method for determining the structural changes occurring in the humic substances in soils in areas of damaged mountain forest. In Poland, forests, along with shelterbelts, are a form of land use second only to agricultural areas. Polish forest resources are a substantial part of the natural environment of the country. They are also a significant part of the geographic space, occupying 30.4% of the country’s area, and are mainly publicly owned (81.3%) [12]. In recent years, spruce and larch forests in Poland and Slovakia have been severely damaged by various factors (clear-cutting, wildfires, and windthrows) [13,14,15]. In the mountainous Sudety region (Poland), as in other parts of Central Europe, spruce monocultures are very common due to their artificial introduction in the nineteenth century [16, 17]. This has resulted in many problems, such as susceptibility to fungi. Millions of cubic meters of trees are infested annually by spruce bark beetles, whereby the mortality of the trees increases under extreme conditions like storms and snow. In consequence, forests are destroyed by windstorms or have to be cut down.

The above circumstances offered an opportunity to study the changes in the forest ecosystems. The previous works [7, 14, 15] presented comprehensive research on the influence of forest destruction on the structure of soil humic acids. Gonzales-Perez et al. (2004) [18] identified the general removal of external oxygen groups, yielding materials with comparatively reduced solubility, and a reduction in the chain length of alkyl compounds (such as alkanes, fatty acids, and alcohols) as the main effects of a fire on soil organic matter.

Even though the above changes can affect the matrix responsible for the stabilization of semiquinone radicals and the radicals themselves, the effect of forest removal on the structure and condensation of humic acid semiquinone radicals has not been investigated yet. Some preliminary studies were carried out by Barančiková et al. 2018 [15], but they concentrated on solely the deeper parts of soil profiles and did not cover forest clearing. The aim of our investigations is to show the usefulness of EPR spectroscopy in studies of soils from areas of forest destroyed due to various causes, with the focus on the structure and concentration of semiquinone radicals present in soil humic acids.

2 Experimental Section

The subject of the studies was humic acids extracted from the mountain soils where the forest had been removed due to different causes. The first cause was clear-cutting in the Sudety mountains in Poland, and the second and third causes were, respectively, a forest wildfire and a windthrow in the High Tatra mountains in northern Slovakia.

The soils where the mixed forest (sycamore, spruce, beech, and larch) had been clear-cut were collected from the Sudety mountains (Bialskie mountains, Sycamore Forest) in the Lower Silesia region in Poland. The reference samples were collected from the same region, but from plots with undamaged forest. The soil samples (Dystric Cambisol) were collected from plots located at altitudes 830–950 m a.s.l. in three replicated soil profiles for each stand and two horizons: Oa—the surface organic layer and A—the deeper lying mineral layer. Finally, for this area, four samples of the extracted humic acids were the subject of the studies—two reference (Oa and A horizons) and two from cleared-of-forest (Oa and A horizons).

Soils from the High Tatra mountains were collected from four long-term research plots (the TANAP Research Station), each covering an area of 100 ha and located at altitudes of 1040–1260 m a.s.l. and having a slope of 5–10%. Dystric Cambisol, formed from moraine material, is the dominant soil type on the plots. The slopes were affected by a windstorm and a wildfire. The soil sampling took place in differently managed areas:

  1. 1.

    a reference intact spruce forest stand (REFERENCE),

  2. 2.

    a wildfire area with extracted timber (FIRE),

  3. 3.

    a windthrow area where the fallen trees had been removed (REM),

  4. 4.

    a windthrow area where the fallen trees had been left for spontaneous succession (UNEX).

The sampling was carried out in 2013 (9 years after the windstorm and 8 years after the wildfire). Soil samples were taken from mineral soil horizon A (without the surface organic layer) at 10 m intervals along a 90 m-long transect (10 samples from each plot). Samples could not be collected from organic horizon O because of the huge damage caused by the wildfire.

Humic acid (HA) fractions were isolated from the all soil samples in accordance with the method recommended by IHSS (International Humic Substances Society) [19].

Electron paramagnetic resonance spectra were obtained using a Bruker Elexsys E500 spectrometer equipped with an NMR teslameter (ER 036TM) at room temperature. The Li/LiF (g = 2.00223) standard was used to calibrate the teslameter. To avoid any errors inherent in analysing natural substances (which can be inhomogeneous), measurements of parameter g were made in triplicate and the average was taken. Spectra were measured using a frequency counter (E 41 FC) at a microwave power of 20 mW and a modulation amplitude of 0.5 G. A double rectangular cavity resonator (ER 4105DR) operating in the TD104 mode was used for quantitative measurements. The radical species concentration standards were placed in one cavity and the analysed sample was put in the other cavity. After tuning, spectra were recorded separately for each of the cavities without changing any measurement parameters. The standards of HAs (peat and Leonardite HA extracted and distributed by the IHSS) and the Bruker alanine pill were used as spin concentration references for the calculation of the radical species concentration in the samples. Quantitative measurements were carried out in triplicate and the calculated standard deviation (SD) for every sample is given in the result tables.

The samples of humic acids were mineralized by the wet method using microwave digester. Decomposed solutions were used to determine the metal-ion content using an emission spectrophotometer ARL Model 3410 ICP (Fisons Instruments). Microwave mineralization and metal-ion concentration measurements were carried out in triplicate and the average was calculated.

Elemental analyses of HA were carried out in triplicate using a C, H, and N analyser Perkin-Elmer CHN 2400. The concordance between the parallel measurements was better than 0.3%.

The E4/E6 parameter was determined using a Varian Carry 50 Conc, with about 2 mg of each humic acid sample dissolved in 10 ml of 0.1 mol/dm3 NaOH. The average taken from four replications is presented as the result.

3 Results and Discussion

3.1 Iron Ions in Humic Acids

The EPR spectra of the humic acids isolated from the studied soils exhibit signals typical of this kind of substances: a broad signal indicating the presence of Fe ions bound to organic and inorganic substances and a narrow line signal characteristic of the semiquinone radical (Fig. 1) [3]. The EPR spectra indicate that the intensity broad line of agglomerated Fe(III) ions (g = 2.00) in the humic acid taken from mineral profiles is lower than in the humic acids taken from organic profiles. The lines at g values of 4.25 and 6 are also observed. The second at g = 6 is especially interesting as it is assigned to Fe(III) ions coordinated to nitrogen donors in heme-type protein complexes [20]; not oxygen donors as in the case of the other iron(III)-humic acid bonds. It is worth mentioning that elemental analyses confirm that humic acids characterized by peak g = 6 also contained more nitrogen in comparison with the reference (C/N in Fig. 1). Although humic acids from organic profiles contain more nitrogen, no line at g = 6 is observed in their EPR spectra. The EPR line recorded at g = 4.22–4.27 represents a typical signal from the non-heme iron(III) ion in a system of low rhombic symmetry in distorted octahedral or tetrahedral surrounding. For HA, this signal is mostly a single peak, but it could appear as a complex structure [21]. In the case study, this signal is always more intense for humic acids from the affected areas (Fig. 1). This is in agreement with Bartoll et al. 1996 [22] where burnt soils were investigated. Hence, it seems interesting that this signal intensity increase was observed by us not only as the effect of high temperature action but also for the soils changed by the forest by clear-cutting.

Fig. 1
figure 1

EPR spectra of humic acids extracted from soils (Oe and A horizons) from areas cleared-of-forest and from reference soils in East Sudety mountains

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3.2 Semiquinone Radicals

The EPR studies of the humic acids extracted from the soils where forests had been destroyed or clear-cut indicate a change in the structure and concentration of the semiquinone radical in comparison with the reference samples. All the samples of the humic acids extracted from the soils of the disturbed plots exhibit lower semiquinone-type radical concentrations than the reference samples (Tables 1, 2). The change in radical concentration indicates a modification in the humification process occurring in the soils after the disturbances. During composting, the highest radical concentrations are observed at the peak of the humification processes [23]. The decrease in radical content could be the result of several processes, like the shifting of the hydroquinone–semiquinone–quinone equilibria towards diamagnetic forms (hydroquinones or quinones). The changes in the equilibria could arise from pH variation or due to redox reactions. These processes can take place concurrently with the transformation of organic matter towards less aromatic structures, e.g., the reaction of the unpaired electron of a semiquinone with smaller molecules incorporated upon an environmental change. Finally, diamagnetic structures with a more aliphatic structure are formed. The decrease in semiquinone radical concentration could also be the result of the antiferromagnetic interaction of the radical with some metal ions (d-block like Cu(II), Fe(III), and Mn(II)) [9, 24]. However, in the present case, no significant changes in metal-ion concentrations were observed. Concentrations of several metal ions (Cu(II), Fe(III), Mn(II), Ca(II), and Mg(II)) were measured for all the studied humic acids, but no significant differences between the samples from the disturbed plots and the reference samples were found.

Table 1 Radical concentration, g parameter, atomic H/C ratio, and E4/E6 ratio calculated for humic acids extracted from East Sudety mountains soils (Oa and A horizons) from areas cleared-of-forest and from reference area
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Table 2 Radical concentration, g parameter, atomic H/C ratio, and E4/E6 ratio calculated for humic acids extracted from Tatra mountains soils (A horizons) from areas where forest had been destroyed by wildfire and windstorm
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To identify the cause of the decrease in radical concentration, the results were compared with the g parameter calculated from the EPR spectra. The lower radical content in the humic acids occurs in the same samples for which the g parameter was the highest (Tables 1, 2). For radicals originated in SOM and other natural materials (composts and peats), an increase in the g parameter value indicates lower condensation of the studied samples [23]. This conclusion can also be drawn from the additional analyses of the E4/E6 parameter and the H/C ratio which were carried out. The values of E4/E6 and H/C are the lowest for the reference samples and higher for the humic acids from the areas where forest had been removed by clear-cutting or destroyed by a fire or a windstorm. According to Chen et al. 1977, the lower E4/E6 ratio is attributed to a more complex structure of the humic substances [25]. This parameter indicates not only the aromaticity and condensation of the structure, but also the particle/molecular size of the humic acid [25]. Owing to the higher molecular size of the reference humic acids, the core of their matrix is shielded against external factors, whereby their whole structure is more stable. Moreover, the values of the g parameter, E4/E6 and H/C strongly suggest that the decrease in radical concentration in the humic acids from the soils exposed to the calamities is rather the result of a reduction in aromaticity and the incorporation of more oxygen-containing groups into the NOM structure. The matrix of the humic acid extracted from the environmentally affected areas has a more open and more reactive structure, whereby this organic matter is less stable than before the calamities.

More distinct differences are observed for the humic acid from the soils in the East Sudety mountains and especially from organic horizons (Oa). For this soil type, the concentration of radicals in the area cleared-of-forest was twice lower than for the reference sample. The same set of samples exhibit clear differences also in the case of g parameter and E4/E6 values. The more distinct changes occurring in organic horizons Oa are understandable, since the upper layers of soil profiles are more exposed to external factors. Even if soil from any of these horizons cannot be collected, as in the case of the soil affected by the forest wildfire, it is essential that at least small changes can be observed in the deeper (mineral) soil horizons.

4 Conclusion

The presented results show that significant changes have taken place in the structure of the humic acids in the soils affected by forest destruction. Regardless of the cause of forest removal: clear-cutting, the wildfire or the windstorm, the effect on the soil humic acids was the same. The elemental analysis and the spectroscopic data (UV–Vis, EPR) indicate a higher content of aliphatic moieties and, hence, a lower degree of humification (a higher g parameter) of the organic matter extracted from the affected plots in comparison with the reference ones. As a result of the slowing down of the humification processes, the SOM extracted from the spruce forest on the disturbed plots can be more susceptible to oxidation than the SOM taken from the reference plots. Thus, the humic acids from both the studied horizons (Oa and A) are less stable and can affect soil aggregates. Low molecular fractions are more easily dissolved and transported to the deeper parts of the soil profile. As a result, the leaching of nutrients from the upper soil horizons (Oa and A) to the deeper parts can occur.

In conclusion, we suggest that humic acids are suitable indicators for tracking changes in the humified organic matter in the organic and mineral parts of forest soil under different management regimes in disturbed areas. Principally, the EPR method is an appropriate instrument which, through the detailed quantitative and qualitative determination of different carbon types, makes it possible to track changes in the inner chemical structure of humic acids. Our investigations have shown EPR spectroscopy to be suitable for soil degradation studies.