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FAs and HAs were isolated from chemically different soils (A-horizons) by the procedure recommended by the International Humic Substances Society (IHSS) [26 ]. Selected physicochemical and chemical properties of the soils and HAs were described previously [27 (link)]. FAs were analyzed in terms of the same chemical parameters as HAs: the elemental composition (C, H, N) was determined using a CHN 2400 analyzer (Perkin Elmer). The oxygen content was calculated from the difference: O% = 100%—(C%+N%+H%) and then the atomic ratio of C/N was estimated. The carboxylic (COOH) and phenolic groups (OH) were measured via the method proposed by Dragunowa and Kucharenko [28 ]. The E2/E6 and E2/E4 indexes were determined as ratio of absorbance measured respectively at 280 and 665 nm as well as at 280 and 465 nm of FAs (40 mg dm^-3) in 0.05 M NaHCO₃ using a UV-Vis spectrometer (Jasco V-520) [29 ]. The Kumada parameter (ΔlogK) was calculated as the difference between decimal logarithm of absorbance at 400 and 600 nm: ΔlogK = logA400—logA600. However, it should be emphasized that original description and classification of the above parameter is related only to humic acids. The values for fulvic acids in this paper are only indicative. Three replicates were performed for each treatment and the results were averaged.

The isolation of HAs from soil samples was carried out by alkaline extraction according to the procedure recommended by the International Humic Substances Society [22 ]. Elemental composition of HAs was measured by using CHN 2400 analyzer (Perkin Elmer). Percent oxygen composition was calculated from the equation %O = 100%-(%H+%C+%N) and H/C, O/H, O/C, and C/N atomic ratios were calculated as well as degree of internal oxidation by Zdanow’s formula: ω = [(2O+3N)-H]/C [23 ]. The carboxyl groups (COOH) and sum of carboxylic and phenolic groups (COOH+OH) were investigated according to Dragunowa and Kucharenko [24 ]. The phenol OH groups were evaluated as the difference between the total acidity and carboxyl groups. The E₄/E₆ and E₂/E₆ parameters were calculated as the ratio of absorbance measured respectively at 465 and 665 nm as well as 280 and 665 nm of HAs (40 mg dm^-3) in a solution of 0.05M NaHCO₃ using a UV-VIS spectrometer (Jasco V-520) [25 ]. The Kumada parameter (ΔlogK) was calculated as the difference between the decimal logarithm of absorbance at 400 and 600 nm: ΔlogK = logA₄₀₀-logA₆₀₀. Three replicates were performed for each treatment and the results were averaged.

FAs were isolated from A-horizons of five chemically different soils: Haplic Fluvisol, Haplic Chernozem, Mollic Gleysol, Haplic Cambisol and Stagnic Luvisol using the procedure recommended by the International Humic Substances Society (IHSS) [88 ]. The degree of sample purity was evaluated by the analysis of ash content (A). In order to characterize the structure of FAs, the atomic ratios of H/C, O/C and O/H were determined using the CHNS/O 2400 analyzer (Perkin Elmer, Waltham, MA, USA). The internal oxidation degree (ω) was calculated with the Zdanov equation ω = [(2O + 3N) − H]/C [27 (link)]. The parameters of Q465/Q668 and Q254/Q436 expressing the humification degree and content of the compounds resistant to humification were calculated as a ratio of absorbance of aqueous FAs solution (40 mg dm⁻³) in 0.05 M NaHCO₃, respectively at 465 and 668 nm, as well as at 254 and 436 nm, using an UV-Vis spectrometer (Jasco V-520, Tokyo, Japan). The value of absorbance recorded at 280 nm was used to evaluate structure aromaticity. Three replications were performed for each measurement, and the obtained results were averaged. The content of carboxylic (COOH) and phenolic (OH) groups was determined and described earlier using the method proposed by Dragunowa and Kucharenko [35 (link)].