Effect of the Tillage System on the Properties of Humic Acids of Soil of the Kujawy Region in Poland

The aim of this study has been to determine the properties of humic acids of soil depending on the tillage system applied. The study covered the soil where plough tillage, strip-till and ploughless tillage were used, and so the systems which differed completely in the way they affect the post-harvest residue, “plant residue management”. From averaged samples of soil humic acids (HAs) were extracted to identify their elemental composition, spectrometric properties for the UV-VIS and IR range as well as hydrophilic-hydrophobic properties. 
 
With the results one can conclude that the humic acids of the soil under plough tillage show a lower degree of humification as compared with HAs of ploughless tillage, whereas the parameters recorded for HAs of the soil with strip-till point to the similarity to HAs of soil with plough tillage and ploughless tillage. With that in mind, with some approximation, the degree of humification (maturity) of HAs can be ordered as follows: HAs with plough tillage < HAs with strip-till < HAs with ploughless tillage. Thus one can conclude that the tillage method combines two primary objectives; ensuring conditions favourable to plant growth and development and the effort to maintain the possibly highest humus stability.


Introduction
Over the recent years the concept of sustainable development has been popularised by the so-called conservation tillage (Friedrich, Derpsch, & Kassam, 2012). As reported by e.g., A. K. M. S. Islam, Saleque, Hossain, and A. K. M. A. Islam (2015), Strickland et al. (2015), and Si et al. (2018), abandoning plough tillage and introducing simplified tillage, including strip-till, results in the accumulation of organic carbon and nutrients in topsoil. In strip-till deeply-loosened strips account for one third of the field, and 60-75% of plant residue remains on its surface (Morris, Miller, Orson, & Froud-Williams, 2010). Fernandez, Sorensenb, and Villamil (2015) have demonstrated that strip-till, as compared with ploughless system, ensures a better environment for root growth and development, which is of key importance for water and nutrients uptake. As seen from the research conducted by Al-Kaisi, Douelle, and Kwaw-Mensah (2014) and by Fernandez et al. (2015), after a few years of applying strip-till, the content of organic matter in soil increases. Powlson et al. (2012) stress that simplified tillage is important not only to increase the content of organic matter in soils but also to limit the emissions of greenhouse gases. Indeed Busari, Kukal, Kaur, Bhatt, and Dulazi (2015) claim that it is necessary to apply simplified tillage as part of the soil and atmosphere protection strategy, and thus to alleviate the climate changes and to preserve the biodiversity.
Soil protection mostly involves the protection of the resources of organic matter. The key organic matter component are humus substances, including humic acids, being the most common organic compounds found in nature. Humic acids (HAs) take part in all the processes which occur in soil and affect its biological (biodiversity, growth stimulation and plant development), physical and chemical properties. Humic acids affect the soil structure and thus the level of soil aeration, soil permeability and water holding capacity. The HAs make the soils dark in colour, which facilitates soil warming. The chemical composition and the energy value of HAs make them a serious source of carbon and nitrogen as well as facilitate soil buffer properties development. There has been also demonstrated a dependence between the content of humus and the plant health status. Therefore, the amount and quality of humic acids are a major indicator of soil fertility and productivity and they depend on the habitat (climate, soil) and anthropogenic factors (tillage and plant growing, fertilisation, crop rotation) (Gonet, 1989;Dou, Zhang, & Li, 2008;Debska, Drag, & Tobiasova, 2012;Kwiatkowska-Malina, 2015;Zhang et al., 2017). Interestingly, humic acids are the main link in carbon sequestration and releasing CO 2 to the atmosphere as well as in environment detoxication (Tan, 2012;Gomes de Melo, Motta, & Santana, 2016).
The basic indicators used to evaluate the properties of humic acids are the elemental composition, including the values of atomic ratios: H/C, O/C, N/C determined from that composition as well as the degree of internal oxidation, coefficients of absorbance A 2/4 , A 2/6 , A 4/6 and ΔlogK, IR spectra and hydrophilic-hydrophobic parameters (Debska, Banach-Szott, Dziamski, & Gonet, 2010;Debska et al., 2012;Ferreira, 2013;Enev, Pospisilova, Klucakova, Liptaj, & Doskocil, 2014;Trubetskaya, Trubetskoj, & Richard, 2014;Tinoco, 2015;Rodriguez, 2016;Hayes & Swift, 2018). As seen from the literature review special role has been documented for the applying natural fertiliser (FYM, liguid manure) organic (compost, green manure) and mineral fertilisers or post-harvest residue for the properties of humic acids. Researching humic acids has demonstrated (Gonet, 1989;Kwiatkowska-Malina, 2015;Zhang et al., 2017), e.g., that FYM fertilisation results in their aliphatization, increased susceptibility to oxidation, decreased average molecular weight and in changes in optical properties, as compared with the properties of the humic acids of non-fertilised soils. The humic acids of the soils treated only with mineral fertilisers, as compared with the HAs of the soil fertilised with FYM, show a greater share of aromatic structures, a lower susceptibility to oxidation as well as higher values of absorbance at the wavelength of 465 nm. As reported by Debska (2004), fertilising soils with slurry results in the formation of humic acids with a greater share of hydrophobic fractions, a lower value of the degree of internal oxidation, a higher share of aromatic structures as compared with the HAs fertilised only with NPK.
The directions of post-harvest residue transformation are mostly determined by its chemical composition, mostly the value of the ratio of carbon to nitrogen as well as the content of easily decomposable compounds (Carvalho, Bustamante, Alcantara, Resck, & Lemos, 2009;Debska et al., 2012).
Considering the role of humic acids in the soil environment, an attempt has been made to determine the effect of various tillage systems (plough tillage, strip-till and ploughless tillage) on their properties especially a comparison of the so-called conservation crops with plough tillage.
The tillage systems compared: plough, ploughless and strip-till, differ essentially in the way they affect the post-harvest residue "the management of plant residue", the basic source of organic carbon in arable soils. Plough tillage, by reversing the soil, covers the residue a dozen or so centimetres deep. Ploughless tillage mixes the organic matter evenly with the soil layer down to 20-30 cm deep. Both systems strongly loosen and aerate the soil. After strip-till most plant residue remains on the surface of the non-tilled soil. Only narrow soil strips are loosened and aerated. The present research has assumed a hypothesis that such a strong diversification of the conditions of organic matter transformations in soil can, already after 4 years, significantly, change the properties of humic acids.
The aim of this study has been to determine the properties of humic acids of soil depending on the tillage system applied. The humic acids were assayed to identify their elemental composition, spectrometric properties for the UV-VIS and IR range as well as hydrophilic-hydrophobic properties and so the parameters which significantly determine the role of humic acids in the soil environment.
The soil (from each plot) was sampled from three layers 0-15 cm (1), 15-30 cm (2) and 30-50 cm (3), in the fourth research year after harvesting winter wheat. For each tillage system the soil was sampled from four plots; the replications. From each plot 12 samples were taken along both diagonals. All the samples from the plot were combined and carefully mixed. Having dried in room temperature, the samples were sieved. For sample assaying the symbols given in brackets were used, e.g., O1 stands for the soil sampled under plough tillage from the 0-15 cm layer.

Methods
From soil samples there were extracted humic acids (HAs) following the standard procedure described by Debska et al. (2010).
The humic acids separated were analysed for: In the humic acids preparations ash content was lower than 2%.

Statistical Analyses
The significance of differences of the parameters, depending on the tillage method and depth, was evaluated with Duncan's Tukey test. The effect of the tillage method on the properties of the soils was defined with cluster analysis. Data clustering was performed with the Ward method. Cluster analysis facilitates the determination of the similarities of the treatments (Euclidean distance evaluation) using their characteristics. The treatments with similar properties are located on dendrograms in homogenous groups (K. A. Gomez & A. A. Gomez, 1983). The above relationships were determined using statistical software STATISTICA MS 12.

Elemental Composition
The elemental composition is considered one of the basic features of humus substances commonly used to identify them and to draw conclusions about their structure. The key elements being part of humic acids are carbon, hydrogen, oxygen and nitrogen (Gonet & Debska, 1998;Debska et al., 2012;Tan, 2012;Gomes de Melo et al., 2016;Zhang et al., 2017). The results of the analysis of the elemental composition (in atomic percentage) of humic acids (HAs) isolated from respective layers are presented in Table 1. The highest average content of carbon (C) was found for HAs of the soil under ploughless tillage. There were demonstrated no significant differences between the content of carbon in HAs molecules under plough tillage and strip-till. The humic acids of the soil under plough tillage showed a significantly higher content of hydrogen as compared with HAs of the soil under ploughless tillage. The tillage method did not have a significant effect on the share of nitrogen and oxygen in the molecules of humic acids. Interestingly, the highest contents of carbon and the lowest contents of hydrogen were recorded for HAs isolated from the soil sampled from the 15-50 cm layers. It was also observed that HAs of the soil of the 30-50 cm layer under plough tillage and strip-till showed a slightly higher content of oxygen as compared with the content of that element in 0-30 cm layers. Note. *: layers 0-15 cm (1), 15-30 cm (2), 30-50 cm (3); **: Mean values followed by common letter are not significantly different at the 5% level.
The share of the elements in the molecules of humic acids facilitated the calculation of the values of atomic ratios.
The numerical values of atomic ratios allow for an approximate determination of the structure of the molecules of humic acids by evaluating the degree of condensation of aromatic rings (H/C ratio) and the degree of their maturity (O/C, O/H, ω) (Dou et al., 2008;Debska et al., 2012;Ferreira et al., 2013;Tinoco et al., 2015;Tan, 2012). As seen from Table 1

Spectrometric Parameters of Humic Acids in the UV-VIS Range
The method of electron absorption has been long applied in the research of humus substances. In the UV-VIS spectra of typical humic acids, in general, there are no maximums and the spectrum assumes the shape of a monotonically decreasing line. Interpreting the spectra of humic acids, the values of absorbance at wavelengths 280, 400, 465, 600 and 665 nm are considered mostly to calculate the ratios A 2/4 , A 2/6 , A 4/6 and coefficient ΔlogK. A 2/4 , A 2/6 , A 4/6 and ΔlogK are parameters which can be used to evaluate the level of advancement of the process of humification of organic materials and the characteristics of the humus substances produced. In general humic acids with a lower molecular weight and a lower degree of humification show higher values of coefficients A 2/4 , A 2/6 , A 4/6 and ΔlogK as compared with HAs with a higher degree of "maturity" (Kumada, 1987;Debska, 2004;Enev et al., 2014;Tinoco et al., 2015;Rodriguez, Schlenger, & García-Valverde, 2016  Note. *: layers 0-15 cm (1), 15-30 cm (2), 30-50 cm (3); **: Mean values followed by common letter are not significantly different at the 5% level.
As seen from Table 2, the humic acids of variants "S" and "H", in general, revealed similar values of absorbance and absorbance ratios. The lowest values of absorbance and the highest values of the coefficients of absorbance were recorded for HAs of the soil under plough tillage, however, a significant difference was recorded between HAs of variants "O" and 'S". Interestingly, HAs from the 0-15 cm layer showed, in general, higher values of the coefficients of absorbance, as compared with HAs from 15-50 cm layers. As recorded by Kumada (1987), humic acids, based on the pattern of UV-VIS spectra, can be divided into three basic types: type A-with humic acids with a high degree of humification, for which ΔlogK assumes the values up to 0.6, type B-with values ΔlogK from 0.6 to 0.8 and type R p -with acids with the values of the coefficient from 0.8 to 1.1. The values of coefficient ΔlogK of the humic acids ranged from 0.680 to 0.766, so they can be considered one type-B.

Hydrophilic-Hydrophobic Properties of Humic Acids
Sample chromatograms of humic acids are presented in Figure 1. The humic acids, irrespective of the tillage method, showed, in general, the presence of peaks in the range 3-25 min, from which the peaks in the range from 3 to 7 min correspond to the fractions of humic acids with greater hydrophilic properties and the peaks in the range 12-25 min-hydrophobic properties. In the range corresponding to the hydrophobic fractions 3 sub-ranges were identified, divided into: HOB-1-12-16 min, HOB-2-16-20 min and HOB-3-20-25 min. Most hydrophobic are the fractions with the longest retention time (Debska et al., 2012;Trubetskaya et al., 2014), the share of which in the humic acids analysed was lowest. The share of hydrophilic fractions in the molecules of humic acids ranged from about 20 to 21% (HAs of variants "S" and "H" of 30-50 cm layer) to 23.84%-HAs of the soil under plough tillage (0-15 cm layer) ( Table 3). The total share of hydrophobic fractions did not depend on the tillage method significantly and it ranged from 76.16 (HAs of variant "O", 0-15 cm layer) to 79.72%-HAs of the soil under strip-till (30-50 cm layer). jas.ccsenet.
The share HIL/ΣHOB were lowe with a hig informatio degree of maturity.

FTIR-S
The spectr absorption spectra in The intens method, w layer of th and a lowe cm layer t recorded fo a higher in of the soil significant 30-50 cmlower-fo compared were noted revealed a and 1130 intensity o bands 296 depth.    Vol. 12, No. 8;2020 Changes in the intensity of absorption bands show that the molecules of HAs from the deepest layer (30-50 cm) are identified with a higher degree of oxidation (a higher intensity of the band pointing to the presence of carbonyl groups), a lower share of aliphatic structures (bands 2960-2920 and 2850 cm -1 ) and simple aromatic structures (1520-1500 and 1420-1400 cm -1 ) as compared with HAs of topsoil. As reported by e.g., Carvalho et al. (2009), Gonet and Debska (1998), it can signify a lower "degree of maturity" of the HAs isolated from topsoil, due to the inflow of fresh organic matter (post-harvest residue). As seen from the results, the tillage method affects the structure of humic acids distributed in the soil profile. For plough tillage and strip-till the changes in the structure of humic acids occur between 0-30 layer and 30-50 cm layer and, as for ploughless tillage, the differences in the pattern of spectra between the HAs of each layer were noted (0-15; 15-30 and 30-50 cm). The dependencies are confirmed by the results of the parameters discussed earlier. With the results one can conclude that the humic acids of the soil under plough tillage show a lower degree of humification as compared with HAs of ploughless tillage, whereas the parameters recorded for the HAs of the soil with strip-till point to the similarity to HAs of soil with plough tillage and ploughless tillage. As provided in the introduction, abandoning plough tillage and introducing simplified tillage, including strip-till, results in the accumulation of organic carbon (Islam et al., 2015;Strickland et al., 2015;Al-Kaisi et al., 2014;Fernandez et al., 2015). Applying ploughless tillage and, to some extent, strip-till decreases the intensity of mineralization not only of fresh organic matter but also humus substances, as well as slows down the processes of humification of fresh organic matter. As a result, HAs of soil with ploughless tillage demonstrated the highest degree of maturity, and thus stability, which is of great importance for carbon sequestration process.
To obtain complete information on the differences (similarities) of humic acids depending on the tillage method, the cluster analysis was applied. The cluster analysis results the HAs isolated from the 0-15 cm and 15-30 cm layers are given in Figure 3 and involved the parameters discussed (elemental composition, UV-VIS parameters and hydrophilic, hydrophobic properties). The dendrograms obtained show clearly that the properties of humic acids are determined by the tillage method. Humic acids under ploughless tillage (H1, H2) and strip-till (S1, S2) constituted one group; the HAs of the plough tillage system (O1, O2) were found outside it. Those dependencies referred to both the humic acids isolated from the 0-15 and 15 -30 cm layer. Figure 3. Cluster analysis of humic acids determined based on the following parameters: elemental composition, A 2/4 , A 2/6 , A 4/6 , ΔlogK, HIL/ΣHOB Fernandez et al. (2015) show that strip-till is a method of tillage and plant cultivation facilitating the accomplishment of the basic objectives of contemporary agriculture. Similarly as other zone tillage methods (Zibilske & Bradford, 2007;Zhu, Hu, Yang, Zhan, & Zhang, 2014;Williams et al., 2016), it creates favourable conditions for plant growth and yielding and, at the same time, enhances the soil properties, including organic carbon management.
The quality parameters for HAs of soil with strip-till point to an increase in their degree of maturity (stability) as compared with HAs of the soil under plough tillage and their higher similarity to HAs of soil under ploughless tillage (Figure 3). Thus one can conclude the strip-till that tillage method combines two primary objectives; ensuring conditions favourable to plant growth and development and the effort to maintain the possibly highest humus stability.

Conclusions
The results have demonstrated that the tillage system determines the properties of humic acids.
The HAs isolated from the samples of the soil with ploughless tillage, as compared with the HAs of the soil under plough tillage showed a higher content of carbon and a lower content of hydrogen and, as a result, a lower value of H/C and higher values of O/H, the degree of internal oxidation and a higher intensity of the band 1730-1710 cm -1 related to the presence of carbonyl groups; a higher share of hydrophobic fractions and a lower share of hydrophilic fractions and, as a result, a lower value of the ratio HIL/ΣHOB.
The HAs of the soil with the strip-till applied, as compared with the HAs of the soil under plough tillage, demonstrated a higher value of internal oxidation, lower values of the ratios: A 2/6 , A 4/6 and coefficient ΔlogK; a lower value of HIL/ΣHOB; a higher share of carbonyl groups and a lower-1520-1500, 1420-1400 and 1130 cm -1 bands (correspondingly as the ploughless method).
The dependencies and the application of the cluster analysis have shown that the degree of maturity (stability) of HAs can be ordered as follows: HAs with plough tillage < HAs with strip-till < HAs with ploughless tillage.