Properties of Humic Acids in Meadow Soils Irrigated With the Slope-And Flooding System

The aim of the research has been to determine the effect of many-year irrigation of unique grasslands on the properties of humic acids defining the quality of organic matter. The research was performed based on the soil ( Albic Brunic Arenosol , the A, AE and Bsv horizons) sampled from Europe ’ s unique complex of permanent grasslands irrigated continuously for 150 years, applying the slope-and-flooding system; the Czerskie Meadows. The soil samples were assayed for the content of total organic carbon (TOC) and the particle size distribution. HAs were extracted with the Schnitzer method and analysed for the elemental composition, spectrometric parameters in the UV-VIS range, hydrophilic and hydrophobic properties and the infrared spectra were produced. The research results have shown that the HAs properties depended on the depth and the distance from the irrigation ditch. The HAs of the A horizon of the soils were identified with a lower “ degree of maturity ” , as reflected by the values of atomic ratios (H/C, O/C, O/H), absorbance coefficients, and the FT-IR spectra, as compared with the HAs of the Bsv horizon. The HAs molecules of the soils sampled furthest from the irrigation ditch were identified with a higher degree of humification, as compared with the HAs of the soils sampled within the closest distance. The results have demonstrated that many-year grassland irrigation affected the structure and the properties of humic acids.

Thanks to the studies of humic acids properties, one can determine the advancement of the processes of transformation and decomposition of organic matter which is the source of organic carbon. Due to increasing soil degradation and total organic carbon (TOC) losses due to, e.g., changes in soil use and an intensified agricultural production, the local, regional and global soil protection has become one of the key goals of the European Community policies 38 . Importantly, due to a specific biodiversity, meadow ecosystems, including the ones the research was performed in, are one of the key methods of the agricultural land use preventing the losses of organic carbon in soils. With that in mind, the aim of the present paper has been to determine the impact of many-year irrigation of unique grasslands on the properties of humic acids determining the quality of organic matter.

Materials
Soil was sampled from perennial grasslands of the unique "Czersk Meadow Complex", continuously irrigated for about 150 years with the slope-and-flooding system. The name "Czersk Meadow Complex" covers a historic term for perennial grasslands with the total area of almost 2000 hectares which used to be considered meadow and pastureland, and arranged in various parts of the Tuchola Forest 39 . For meadow irrigation, mostly the rivers of the Tuchola Forest, the Brda and the Wda River, were used. A sensation is the fact that the waters from the irrigation ditches were distributed across the meadows with a system of channels, ditches and overflow furrows creating a unique irrigation system referred to as the slope-and-flooding system.

Soil analysis
For air-dry soil samples, the following analyses were made: the content of total organic carbon (TOC) and total nitrogen (Nt). The content of organic carbon and total nitrogen were assayed with the Vario Max CN analyser provided by Elementar (Germany). The content of TOC and the content of Nt were expressed in g kg -1 of d.w. of soil 40 ; -pHin the suspension of distilled water and soil with the pH-meter MultiCal pH 540 GLP WTW 41 ;

Extraction of humic acids
Humic acids (HAs) were extracted and purified with standard methods using the following procedure:  decalcification (24 h) with 0.05 M HCl (1:10 w/v). After centrifugation, the residue was washed with distilled water till neutral;  extraction (24 h) of the remaining solid with 0.5 M NaOH (1:10 w/v), with occasional mixing, followed by centrifugation;  precipitation (24 h) of humic acids from the resulting alkaline extract with 2 M HCl to pH=2 and centrifugation;  purification of the resulting humic acids as follows: the humic acid residue was treated with a mixture of HCl/HF (950 mL H 2 O, 5 mL HCl, 5 mL HF) over a 24 h period, followed by centrifugation. The humic acid residue was treated with distilled water until a zero reaction to chloride was achieved.
The preparations were lyophilised and powdered in agate mortar. Ash content in the humic acids preparations was lower than 2%.

Characteristics of humic acids
In the separated humic acids the following were assayed:  infrared spectra (Perkin-Elmer FT-IR Spectrometer, Spectrum BX) over 400 -4400 cm -1 were produced for HAs (3 mg) in KBr (800 mg). Deconvolution was applied, with a filter making the bands of γ = 4 narrower, and using the process of smoothing, for which the length parameter was l = 80% 22 .

Statistical analyses
The soil properties were treated with descriptive statistics: arithmetic mean, minimum value, maximum value, standard deviation, coefficient of variation. The statistical analyses of humic acids involved the analysis of variance of the data derived from a single two-factor experiment in a randomised split-plot. The tables present the mean values from three replications. The significance of differences was evaluated using the Fisher test

Basic parameters of soils
A long-term, surface irrigation of soils with the slope-and-flooding system affected the contents of TOC and Nt in the soil samples ( Table 1). The results confirm that the expanded root system of graminaceous plants increases the content of organic matter in the AE and Bsv horizons of soils. The importance of the processes enhancing the enrichment in organic matter in the upper layers of the meadow soil profile is also stressed 44,45,46 .
Under constant meadow irrigation, in general, the highest values of the TOC/Nt ratio were reported for the soil sampled closer to the irrigation ditches, which is the key consequence below the nitrogen content in those samples. It was found that the highest TOC/Nt value was noted for the AE horizon of the soils of the highest carbon content. The soil pH values ranged from 5.3 to 7.0 and they were changing inconsiderably, which is confirmed by a very low value of the coefficient of variation (CV) ( Table 2). The soils showed a high content of the sand fraction (2.0-0.05 mm), which ranged from 86.57%-93.97% and a very low content of clay fraction (<0.002 mm) accounting for 0.32% (Table 2).

Elemental composition of humic acids
Basic indicators used in an assessment of humic acid properties include elemental composition and the values of atomic ratios calculated from the composition ( Table 3 together with the distance from the watercourse.

Spectrometric parameters of humic acids in the UV-Vis range
Based on absorbance values of humic acids in the UV-VIS range and the coefficients of absorbance A 2/4 , A 2/6 , A 4/6 , ΔlogK, the following properties can be determined: the degree of advancement of organic material humification as well as changes in the properties of the humic acids which occur due to various anthropogenic factors 24,26,27,28,29,30 . The HAs molecules were identified with a decrease in the values of A 2/4 , A 2/6 , A 4/6 , ΔlogK with depth (Table 4). Drawing on literature reports 54,55 , it was found that the humic acids of the A horizons

Analysis of the Fourier transform infrared (FTIR) spectra of humic acids
The FTIR spectra of the HAs (Fig. 1) revealed the presence of the absorption bands the ranges of which and their corresponding functional groups are given in Table 5  for the bonds C-C in the aromatic compounds (1500-1520 cm -1 ) and the bands in the range of 1460-1440 cm -1 and in 1420-1400 cm -1 was decreasing with the sampling depth. Interpreting the FTIR spectra in terms of the effect on the properties of humic acids, a considerable variation was found. Generally, the FT-IR spectra of the HAs molecules of soils showed an increase in the intensity of the band in the range of 1730-1710 cm -1 together with the distance from the watercourse.

Hydrophilic and hydrophobic nature of humic acids
The degree of organic matter humification is also related with the hydrophilic-hydrophobic properties of To acquire complete information on the differences (similarities) in the chemical composition of HAs, depending on the depth and the distance from the irrigation ditch, the cluster analysis was applied based on the elemental composition, spectrometric parameters as well as on the HIL-HOB properties dividing HAs into two groups (Fig. 3a). In the first group, similar properties were found for the HAs molecules of the Bsv horizon of the soil sampled in Podlesie and in Cegielnia, while in the second group there were identified two subgroups.
The first subgroup covered the humic acids of the A horizon of the soils sampled in Kamionna and in Podlesie.
In the second subgroup, similar properties were found for the HAs molecules of the AE horizon of the soil sampled in Podlesie and in Cegielnia as well as for the HAs of the Bsv horizon of the soils from Kamionna and the humic acids of the A horizon of the soils sampled in Cegielnia and the AE horizon of the soils sampled in Kamionna. As seen from the diagram, the soil sampling depth was the factor determining the properties of HAs.
A cluster analysis was performed based on the properties of humic acids of soils for each of the sampling sites (Fig 3b -Fig 3d). The dendrograms definitely point to the HAs isolated from the soil sampled 5 m away from the irrigation ditch in Kamionna and Cegielnia as significantly different from the soil sampled 15 and 25 m away. As for the Podlesie sampling site, the distance was not the factor which definitely determined the humic acid properties.

Conclusions
The structure of the HAs molecules corresponded to the aromatic systems coupled with the aliphatic chain containing up to 10 atoms of C. The atomic ratios, the spectrometric properties as well as the hydrophilichydrophobic properties point to a high maturity of the molecules of the humic acids of meadow soils continuously irrigated for more than 150 years. The molecules of humic acids demonstrated an increase in the degree of humification with depth. The distance from the watercourse determined the time of the water retention and, as a consequence, the humic acids of the soils sampled furthest from the irrigation ditch, in general, showed a higher degree of humification, as compared with the humic acids of the soils sampled closest.
Irrespective of the changes in the humic acid parameters, depending on the soil sampling distance or depth, the results identified that many-year grassland irrigation results in humic acids with a high degree of humification, which confirms the importance of meadow soils for carbon sequestration process.