The Significance of Herbicide–Humin Interactions in Sustainable Agroecosystems

Abstract

Humin, as the most stable fraction in soil organic matter, determines possibility of sustainable environmental development by influencing, among other things, the binding and migration of different chemicals in soil. The aim of this paper was to determine changes in the properties of humins after interaction with three selected active substances of herbicides differing in structure and chemical properties (pendimethalin, metazachlor, and flufenacet) and two different commercial products. In accordance with OECD 106 guidelines, humins isolated from eight different soils were saturated with herbicide compounds under study. As humin is a non-hydrolyzable organic carbon fraction, solid state research techniques (elemental analysis, NMR, FTIR, EPR, and UV-Vis) were applied. The results clearly showed that the interaction between humin and herbicides increases the concentration of oxygen-containing groups and the internal oxidation (ω) in humin. For all investigated humins, a reduction in radical concentration was observed. Radicals in humins were not completely quenched; a certain concentration of radicals with unchanged structure always remained in the samples. Other spectroscopic analyses showed no significant changes in the structure of pesticide-saturated and non-saturated humins. This suggests that sorption of the studied compounds occurs on the humins only as a result of the interaction of physical forces on the surface of the studied organic matter fraction. Thus, interaction with the studied herbicides occurs as a surface phenomenon, and the inner core remains protected by the condensed structure and/or strong binding to the clay minerals.

1. Introduction

Several distinct and often concurrent phenomena, such as chemical, biological, and photochemical degradation; transport and accumulation; volatilization; and leaching, significantly influence the fate and behavior of herbicides in the soil environment [1]. They are affected to varying extents by physical, physicochemical, biochemical, pedological, and climatic conditions [2]. It is well known that, apart from biological pathways, sorption–desorption equilibria play a crucial role in regulating pesticide availability in the soil solution, which is mainly controlled by the soil organic matter (SOM) content and composition [3,4,5]. SOM is a mixture of polymorphic substances of very high chemical reactivity, mainly humic acids, fulvic acids, and humin. The activity of these substances is the result of the presence of functional groups in their molecular structure with the most reactive carboxylic and phenolic groups, free radicals, and reactive sites of hydrophilic and hydrophobic character [6,7,8,9,10]. Polyfunctionality and polyelectrolyte character allow for them to interact with heavy metals, pesticides, and soil mineral constituents (e.g., clay minerals, oxides, and hydroxides). Humic substances with a lower molecular weight, below 5KDa, characterized by a more aliphatic structure (mainly water-soluble fulvic fraction), are of a key importance for the plant–soil system and the sorption–desorption equilibria of contaminants in soil [11]. Humic acids with a higher molecular weight, a high content of oxygen groups, and more aromatic character are the soluble fraction of humic substances only in alkaline solutions. This fraction can sorb cationic and hydrophobic contaminants mainly due to weak bonds such as hydrogen bonding, π–π interactions, and hydrophobic interactions [12,13,14,15,16]. Mentioned fractions (humic and fulvic acids) were the subjects of studies in terms of sorption–desorption phenomena of both ionic and non-ionic organic compounds used as agrochemicals in soils [17,18,19,20,21].

The fraction named humin, known as the most recalcitrant and abundant SOM fraction, is the least investigated, mainly due to its difficult extraction or isolation procedure [10,21,22,23]. Meanwhile, the available literature, although limited, indicates that humin contains redox-active moieties due to the presence of quinone moieties that play the role of redox-active centers. The interactions between humin, microorganisms, and environmental pollutants are very complex, and they include abiotic–biotic redox interactions. The type and extent of these interactions can change over time and eventually lead to the complete immobilization and incorporation of some substances into the humic matter [23]. Some recent studies have shown that the organic fraction can strongly affect the adsorption of carbofuran in soil [5]. Also, in the sorption of atrazine, 4,4′DDT, and chrysene, a stable SOM fraction consisting of biopolymers in various stages of transformation with numerous aromatic–aliphatic groups was found to be an important and interesting sorbent of organic compounds [24,25]. The above pesticides, in addition to pendimethalin, have been evaluated for non-extractable residue formation via their reaction with stable humin fractions [24]. However, to the best of our knowledge, no structural studies of the interaction between humin and commercial pesticide formulations have been reported. This analysis seems to be important, as the different constitution of commercial products (different solvent concentrations, additives, etc.) could affect interaction with soil organic matter with a different pure compound pattern form. This research provides a reliable environmental risk assessment associated with retention, translocation, and degradation levels of pesticides in soils. Moreover, this research responds to the needs of soil conservation and agricultural safety in terms of widespread climate crisis, which has resulted in numerous measures to reduce the inflow and accumulation of harmful compounds in soil to ensure their health and food quality.

Therefore, the main aim of this study was to fulfill the gap in humin studies, which further consisted of four intermediate goals. The first was to determine how the selected active substances of the herbicides flufenacet, metazachlor, and pendimethalin affect the structure of humin. The chosen active substances are commonly used in crop protection due to their high efficacy in combating specific groups of plants without affecting the physiological processes of the crop. The secondary aim was to find if the commercial formulation gives the same effect as active substances. The third aim was investigation of the influence of herbicides on humins isolated from different soil types. As it is difficult to dissolve humin to obtain a homogeneous solution for this type of study, only methods that allow for measurement in solid state were used in the investigations. Proposing methods enabling the study of humins without dissolving them was our fourth goal.

2. Materials and Methods

2.1. Humin Extraction

A detailed isolation procedure for humin is described in our previous work [22]. The method used is based on the classical separation of humin by elimination of alkali-soluble SOM fractions from the soil matrix and subsequent digestion of the remaining mineral fraction with the HF/HCl mixture until almost complete digestion of the mineral part. The whole isolation of humin takes about 9–10 weeks, depending on the SOM content.

Humin were extracted from soils collected from eight regions in Poland (more details in Ukalska-Jaruga et al. [24]). Some details about localization of sampling and main properties are presented in Appendix A as

Table A1. Localization and general information on soils [24].

Sample No	GPS Coordinates	WRB Soil Group	Cultivated Plant
1Ps	N 51°11′27.79″; E 17°02′08.24″	Gleyic/Stagnic Phaeozems	triticale
3Z	N 50°34′30.50″; E 17°55′59.81″	Rendzic Phaeozems	maize
6M	N 50°59′00.04″; E 16°56′52.48″	Gleyic/Stagnic Phaeozems	maize
7T	N 50°49′11.87″; E 16°52′39.38″	Calcic/Haplic Chernozems	sugar beets
8C	N 50°40′53.98″; E 16°55′47.78″	Gleyic/Stagnic Phaeozems	maize
9H	N 50°43′32.91″; E 23°50′05.94″	Calcic/Haplic Chernozems	wheat
10Py	N 53°09′57.87″; E 14°55′15.19″	Gleyic/Stagnic Phaeozems	sugar beets
11K	N 54°03′53.67″; E 21°21′09.66″	Gleyic/Stagnic Phaeozems	triticale

and

Table A2. Main properties of soils [24].

Sample No	pH (KCl)	TOC g kg−1	N	C/N	CaCO3 g kg−1	CEC cmol kg−1	% Particles >0.002 mm	USDA Textural Class
1Ps	7.71	13.3	1.06	12.5	1.46	28.3	16	sandy loam
3Z	7.45	24.4	2.14	11.4	3.43	50.0	41	clay
6M	7.52	21.2	1.60	13.2	1.53	33.4	22	loam
7T	5.64	41.7	3.39	12.3	0.51	53.2	24	silt loam
8C	7.39	26.1	2.03	12.8	1.03	21.6	19	silt loam
9H	7.52	39.9	2.90	13.7	3.26	52.5	21	silt loam
10Py	7.48	24.6	2.12	11.6	1.54	34.4	24	loam
11K	6.66	37.7	2.80	13.4	0.61	25.8	47	clay

. Samples were labelled as 1Ps (Psary), 3Z (Ziemnice), 6M (Magnice), 7T (Trzebnik), 8C (Ciepłowody), 9H (Hrubieszów), 10Py (Pyrzyce), and 11K (Kętrzyn), with respect to the soil sampling localizations.

2.2. Sample Preparation

Three herbicides with a high level of agricultural use as well as diverse chemical properties and molecular structures were selected for study. Among the analyzed compounds, two of them belonged to the acetamide group and one to the aniline group, namely, flufenacet, metazachlor, and pendimethalin. Their physical and chemical characterization can be found in our previous research paper [24]. The experiments on active substances were complemented by studies on commercial preparations—2 formulations representing each active substance to evaluate changes in reactivity of individual compounds in adjuvant-bound forms (

Table 1. Information about selected herbicides used in the experiments.

Active Herbicide	Formula	Commercial Product
Flufenacet (Fl) CAS 142459-58-3		Fluent—500SC (Flt) Cevino–500SC (Cen)
IUPAC: N-(4-fluorophenyl)-N-propan-2-yl-2-[[5-(trifluoromethyl)-1,3,4-thiadiazol-2yl]oxy]acetamide
Metazachlor (Me) CAS 67129-08-2		Metazanex–500SC (Mzx) Metozop–500SC (Mep)
IUPAC: 2-chloro-N-(2,6-dimethylphenyl)-N-[(1H-pyrazol-1-yl)methyl]acetamide
Pendimethalin (Pe) CAS 40487-42-1		StompAqua–455SC (Stq) Penfox–330EC (Pfx)
IUPAC: 3,4-Dimethyl-2,6-dinitro-N-(pentan-3-yl)aniline

).

The saturation of eight humin samples with each active substance and its two commercial products was carried out according to the Guidelines for the Testing of Chemicals OECD Standard 106 [26]. The sorption experiments were carried out maintaining constant carbon content in humin samples (C = 500 mg) and constant temperature conditions (20 ± 1 °C) and humidity, using nitrogen atmosphere to eliminate interference. The experimental parameters were selected based on values of the partition coefficients (K_oc) of the active substances and their absorption potential on soil components included in OCED Standard 106 annex A. The saturation process was optimized following the experimental approach outlined in the study thoroughly described in Ukalska-Jaruga et al. [24]. Based on that, metazachlor, flufenacet, and pendimethalin demonstrated different rates of sorption to humin. The sorption was most rapid within the first 6 h, with equilibrium in pesticide–HN contact being reached between 24 and 32 h. The examined active substances showed a very quick sorption within the initial hours, indicating the rapid saturation of the HN sorption sites. Therefore, the saturation process lasted no longer than 24 h due to the pesticides achieving a sorption plateau in the humin samples. Thus, Fl, Pe, and their commercial formulations (Flt, Cen, Stq, and Pfx) were dissolved in hexane in mass-to-volume ratios of 1:5, (Fl, Flt, and Cen) and 1:100 (Pe, Stq, and Pfx). Me and its preparations (Mzx and Mep) were dissolved in ethyl acetate, all at a m/v ratio of 1:1. Active substances were added to the humin to reach 100% saturation of humin with pesticides. Such prepared mixtures were agitated for 24 h; then, the solvent was decanted while the remaining hexane and ethyl acetate were evaporated under nitrogen atmosphere to the sample dry mass (after 24 and 96 h, respectively).

As an inherent part of any experiment, the main potential experimental drawbacks were identified to reduce the risk of errors and interference in the analytical process:

• Solvent Evaporation: The evaporation of hexane and ethyl acetate under a fume hood may not be fully efficient. If not completely evaporated, residual solvents could interfere with the analysis or impact the interaction between the pesticides and humin. The differing evaporation times for hexane (24 h) and ethyl acetate (96 h) might suggest that the solvents have very different evaporation rates. This discrepancy could affect the timing and effectiveness of solvent removal and introduce inconsistency in pesticide concentrations in the final samples.
• K_oc Coefficients: The experiment’s reliance on the K_oc partition coefficients is a good starting point for determining pesticide absorption, but K_oc values are general indicators that may not perfectly reflect the actual behavior of the pesticides in humin samples under the specific experimental conditions. Factors such as humin composition, moisture content, and temperature might affect sorption, leading to deviations from predicted values based on K_oc alone.
• Humin Sample Heterogeneity: Humin samples can be quite heterogeneous in terms of their chemical and physical properties; such variability might affect the reproducibility of the results. Careful sampling homogenization before saturation helps reduce the likelihood of this potential issue.
• Effect of Hexane and Ethyl Acetate on Active Substances: Hexane and ethyl acetate may not be inert solvents for all the active substances and their commercial formulations. Some chemicals might degrade, react, or change their behavior in these solvents. The effect of these solvents on the pesticides should be further validated.
• Measurement Errors/Bias: Systematic errors and random errors were eliminated by optimization of the saturation process as determined by a sorption experiment [27] and quantitative evaluation of the residual test substance in solution after saturation therefore the unpredictable variations in measurement that can increase variability in the data were eradicated.
• Instrument limitations: The tools used for measurement may have inherent limitations; therefore, the appropriate quality control standards are regularly applied and equipment used for testing is constantly maintained and supervised. The Sheward cards are maintained.

2.3. Analytical Methods

2.3.1. Elemental Analysis

The elemental composition of humin was analyzed with the CHNS Vario EL Elementar Cube analyzer (Langenselbold, Germany) in three replicates to obtain the highest result precision. The contents of C, H, N, and S were given as the absolute values, while the oxygen concentration was calculated from the mass difference (O% = 100% − C% − H% − N% − %S). In addition, the elemental composition was recalculated to the dry ash-free mass of the humin sample, and the H/C, O/C, O/H, and N/C atomic ratios were calculated to show the relationship between the elements in the humin composition. Internal oxidation (ω) [27,28] was calculated using the formula $\omega ={\displaystyle \frac{2\mathrm{O}+3\mathrm{N}-\mathrm{H}}{\mathrm{C}}}$.

2.3.2. CP MAS 13C NMR

The CP MAS ¹³C NMR spectra were acquired using a Bruker Avance III spectrometer (Bruker Inc., Karlsruhe, Germany) at 300 MHz, equipped with a 4 mm narrow MAS probe and operating in a ¹³C resonance sequence at 75.48 MHz. Humin samples were placed in a zirconium dioxide (ZrO₂) rotor (sample holder) with Kel-F caps, with a rotation frequency of 10 kHz. The spectra were obtained by collecting 4994 data points from the same number of scans with an acquisition time of 50 ms and with recycle delay of 4 s. The spectra were collected and processed using Bruker Topspin 3.6 software. Due to the very long time required to collect the spectra, measurements were only performed on the humin itself and on samples saturated with the active substances of the herbicides under investigation. Nevertheless, this part of the studies was crucial to identify the potential modulations of the humin structure, in particular by the active substances of the herbicides. The areas of characteristic functional groups were calculated by integrating appropriate parts of the spectra using Bruker Topspin 4.1.1. software.

2.3.3. FTIR

Oscillation spectra were performed for all humin samples, saturated with active and commercial substances using a Bruker Vertex 70 FTIR spectrometer (Bruker Inc., Karlsruhe, Germany) with KBr pellets (approximately 1 mg of sample in 400 mg of KBr). Collected spectra were presented as transmittance. For the chosen peaks, –C, –O, and –OH deformations of COOH (1190–1300 cm⁻¹); –C=C– of aromatic (1570–1677 cm⁻¹); –C=O of COOH (1677–1800 cm⁻¹); –CH₂– symmetric stretching (2796–2850 cm⁻¹); and –CH₂– asymmetric stretching (2877–2985 cm⁻¹), area integrals were calculated from absorbance spectra using OriginPro 2016 and OriginPro 9.5 software.

2.3.4. EPR

X-band EPR spectra were obtained with a Bruker Elexsys E500 spectrometer (Bruker Inc., Karlsruhe, Germany) at room temperature using a double rectangular cavity resonator dedicated to quantitative measurements. The Pahokee peat humic acid standard (1S103H) and the Leonardite humic acid standard (1S104H), extracted and distributed by the International Humic Substances Society (IHSS) (IHSS, USA), and the Bruker alanine pill were used as quantitative standards. To analyze the spectra of humin radicals, a double integration of the signals was performed using the Bruker WinEPR program (Bruker Inc., Karlsruhe, Germany). Measurements and calculations were performed for humin saturated with active and commercial substances.

2.3.5. UV-Vis

UV-Vis analysis was performed using the Varian Cary 50 Conc spectrometer. A small amount of humin sample was triturated with standard high vacuum grease using a mortar, and the homogeneous paste was applied to quartz high-precision cells (from Hellma Analytics, Hellma GmbH & Co. KG, Müllheim, Germany). The absorption coefficient at E4/E6 was calculated from the absorbance at 465 and 665 nm, respectively. Measurements and calculations were performed for all humin samples saturated with active substances and commercial formulations. Each sample was measured in five replicates to eliminate a random error and obtain the result with the lowest uncertainty. During the measurements, the same quartz cells with only the grease were used as a blank.

3. Results and Discussion

3.1. Elemental Analyses and Atomic Ratios of Investigated Samples

The interaction of pesticides (active substances and commercial preparations) with humin resulted in a change in its elemental properties. It was expected that the binding of pesticides containing nitrogen or sulfur would cause an increase in the content of these elements. However, no such dependence was found (Appendix A,

Table A3. Percentage element (molar) and ash content (weight %), atomic ratios, ω, and E4/E6 ratio for humins untreated and saturated with flufenacet and its two commercial products.

	Ash	C (SD)	N (SD)	H (SD)	O (SD)	S (SD)	H/C	O/C	O/H	C/N	ω	E4/E6 (SD)
1Ps	39.45	41.26 (0.45)	1.87 (0.02)	38.32 (0.72)	18.17 (0.98)	0.38 (0.04)	0.93	0.44	0.47	22.04	0.09	1.016 (0.005)
+FL		33.72 (0.65)	1.66 (0.06)	36.57 (0.31)	32.15 (0.88)	0.79 (0.47)	1.26	1.11	0.88	19.36	1.11	1.038 (0.003)
+Flt		38.14 (2.76)	1.84 (0.16)	32.13 (2.16)	25.65 (1.54)	2.24 (2.23)	0.84	0.67	0.8	20.68	0.65	1.064 (0.0002)
+Cen		32.55 (0.84)	1.47 (0.09)	38.13 (2.18)	27.71 (1.67)	0.13 (0)	1.17	0.85	0.73	22.08	0.67	1.053 (0.002)
3Z	22.89	39.61 (1.15)	2.12 (0.06)	40.10 (1.89)	17.93 (0.87)	0.23 (0.03)	1.01	0.45	0.45	18.66	0.05	1.019 (0.003)
+FL		38.84(1.63)	2.21 (0.09)	38.69 (1.37)	20.18(0.65)	0.12 (0.0)	1	0.52	0.52	17.54	0.21	1.017 (0.003)
+Flt		37.78 (0.26)	2.13 (0.13)	40.82 (0.32)	19.16 (0.21)	0.11 (0)	1.08	0.51	0.47	17.72	0.1	1.040 (0.001)
+Cen		40.82 (1.17)	2.27 (0.07)	36.75 (1.97)	19.22 (0.88)	0.94 (0.13)	0.9	0.47	0.52	17.95	0.21	1.036 (0.0006)
6M	38.57	38.98 (0.27)	1.99 (0.05)	38.00 (1.4)	20.36 (1.58)	0.67 (0.11)	0.97	0.52	0.54	19.62	0.22	1.0634 (0.016)
+FL		34.11(0.35)	1.75 (0.09)	38.46 (1.45)	25.28(1.29)	0.41(0.19)	1.13	0.74	0.66	19.51	0.51	1.017 (0.008)
+Flt		36.09 (0.39)	1.9 (0.01)	34.17 (0.79)	25.84 (0.72)	2.01 (0.2)	0.95	0.72	0.76	19.01	0.64	1.057 (0.0008)
+Cen		35.42 (1.07)	1.77 (0.04)	36.79 (0.46)	24.06 (0.78)	1.97 (0.2)	1.04	0.68	0.65	20.00	0.47	1.066(0.0007)
7T	41.67	30.6 (2.61)	1.79 (0.08)	38.48 (1.35)	28.96 (3.03)	0.16 (0.01)	1.26	0.95	0.75	17.12	0.81	1.069 (0.004)
+FL		29.84(0.66)	1.78 (0.02)	36.5 (0.82)	31.74 (0.38)	0.14(0.01)	1.22	1.06	0.87	16.8	1.08	1.021 (0.006)
+Flt		30.6 (0.91)	1.79 (0.08)	38.48 (0.56)	28.96 (0.71)	0.16 (0.03)	1.26	0.95	0.75	17.12	0.81	1.070 (0.001)
+Cen		29.87 (1.05)	1.66 (0.08)	36.4 (2.08)	30.89 (2.66)	1.17 (0.35)	1.22	1.03	0.85	18.00	1.02	1.057 (0.0009)
8C	28.01	42.84 (0.11)	2.3 (0.01)	37.86 (1.1)	15.92 (0.83)	1.08 (0.21)	0.88	0.37	0.42	18.65	0.02	1.059 (0.022)
+FL		38.9 (1.21)	1.77 (0.06)	34.2 (1.07)	22.69 (1.65)	2.44 (0.43)	0.88	0.58	0.66	21.92	0.42	1.018 (0.006)
+Flt		38.9 (0.23)	1.83 (0.06)	34.43 (0.46)	22.01 (0.25)	2.83 (0.36)	0.89	0.57	0.64	21.28	0.39	1.063 (0.0004)
+Cen		38.83 (2.10)	1.65(0.010)	36.15 (1.21)	22.47(1.34)	0.89 (0.02)	0.93	0.58	0.62	23.5	0.35	1.067 (0.0002)
9 H	43.31	40.14 (3.79)	1.73 (0.13)	35.65 (2.07)	21.43 (1.97)	1.06 (0.12)	0.89	0.53	0.6	23.24	0.31	1.098 (0.005)
+FL		37.99 (1.83)	1.9 (0.06)	36.26 (1.19)	22.8 (2.73)	1.05 (0.2)	0.95	0.6	0.63	20.00	0.4	1.136 (0.002)
+Flt		36.27 (2.58)	1.79 (0.13)	43.21 (2.66)	18.58 (0.2)	1.02 (0.02)	1.19	0.51	0.43	20.30	−0.02	1.096 (0.0006)
+Cen		38.81 (2.06)	1.87 (0.04)	39.65 (0.96)	19.46 (1.01)	0.21 (0.2)	1.02	0.5	0.49	20.75	0.13	1.126 (0.0008)
10Py	54.5	38.59 (1.38)	1.92 (0.12)	37.44 (2.15)	20.81 (2.96)	1.24 (0.21)	0.97	0.54	0.56	20.08	0.26	1.022 (0.0026)
+FL		36.08 (0.42)	1.87 (0.01)	35.00 (1.51)	24.23 (2.25)	2.82 (0.79)	0.97	0.67	0.69	19.33	0.53	1.044 (0.006)
+Flt		36.9 (0.05)	1.96 (0.06)	33.87 (1.19)	24.92 (1.25)	2.35 (0.12)	0.92	0.68	0.74	18.83	0.59	1.089 (0.0009)
+Cen		35.23 (0.78)	1.82 (0.09)	36.55 (1.05)	23.89 (1.79)	2.5 (0.14)	1.04	0.68	0.65	19.31	0.47	1.047 (0.0007)
11K	48.87	34.89 (0.42)	1.97 (0.05)	39.29 (1.36)	23.22 (1.01)	0.63 (0.17)	1.13	0.67	0.59	17.68	0.37	1.061 (0.009)
+FL		32.88 (1.16)	1.92 (0.07)	41.35 (0.28)	23.67 (1.13)	2.72 (0.44)	1.26	0.72	0.57	17.12	0.36	1.047 (0.007)
+Flt		31.91 (0.77)	1.92 (0.09)	40.26 (3.63)	25.04 (3.56)	0.87 (0.23)	1.26	0.78	0.62	16.63	0.49	1.077 (0.0008)
+Cen		33.7 (0.83)	1.86 (0.02)	36.88 (0.77)	23.28 (1.51)	4.28 (0.69)	1.09	0.69	0.63	18.16	0.45	1.039 (0.001)

,

Table A4. Percentage element (molar) content with standard deviation in brackets, its ratios, ω, and E4/E6 ratio for humins untreated and saturated with metazachlor and its two commercial products.

	C (SD)	N (SD)	H (SD)	O (SD)	S (SD)	H/C	O/C	O/H	C/N	ω	E4/E6 (SD)
1Ps	41.26 (0.45)	1.87 (0.02)	38.32 (0.72)	18.17 (0.98)	0.38 (0.04)	0.93	0.44	0.47	22.04	0.09	1.016 (0.005)
+Me	32.64 (0.48)	1.72 (0.02)	33.16 (0.65)	31.01 (0.99)	1.46 (0.24)	1.02	0.95	0.94	18.94	1.04	1.053 (0.002)
+Mzx	34.2 (0.54)	1.78 (0.07)	34.39 (0.75)	28.06 (1.05)	1.57 (0.54)	1.01	0.82	0.82	19.18	0.79	1.045 (0.001)
+Mep	34.19 (0.64)	1.66 (0.03)	33.84 (1.89)	29.93 (2.57)	0.37 (0.21)	0.99	0.88	0.88	20.57	0.91	1.085 (0.001)
3Z	39.61 (1.15)	2.12 (0.06)	40.10 (1.89)	17.93 (0.87)	0.23 (0.03)	1.01	0.45	0.45	18.66	0.05	1.019 (0.003)
+Me	39.84 (1.15)	2.34 (0.09)	39.07 (2.03)	17.08 (0.91)	1.67 (0.13)	0.98	0.43	0.44	17.00	0.05	1.038 (0.002)
+Mzx	40.71 (1.13)	2.29 (0.06)	37.98 (1.39)	17.34 (0.31)	1.68 (0.45)	0.93	0.43	0.46	17.75	0.09	1.050 (0.003)
+Mep	38.35 (1.02)	2.17 (0.06)	40.9 (2.05)	18.48 (1.0)	0.1 (0)	1.07	0.48	0.45	17.66	0.07	1.035 (0.000)
6M	38.98 (0.27)	1.99 (0.05)	38.00 (1.4)	20.36 (1.58)	0.67 (0.11)	0.97	0.52	0.54	19.62	0.22	1.0634 (0.016)
+Me	35.62 (0.12)	1.97 (0.04)	39.25 (0.78)	19.73 (0.62)	3.42 (0.2)	1.1	0.55	0.5	18.04	0.17	1.088 (0.004)
+Mzx	35.08 (1.12)	1.83 (0.07)	38.83 (0.77)	22.37 (0.1)	1.89 (0.34)	1.11	0.64	0.58	19.15	0.33	1.080 (0.004)
+Mep	37.02 (0.74)	1.93 (0.03)	35.38 (0.79)	23.76 (0.67)	1.9 (0.64)	0.96	0.64	0.67	19.16	0.48	1.090 (0.001)
7T	30.6 (2.61)	1.79 (0.08)	38.48 (1.35)	28.96 (3.03)	0.16 (0.01)	1.28	0.90	0.70	19.45	0.67	1.069 (0.004)
+Me	30.49 (1.09)	1.86 (0.09)	38.95 (0.17)	25.26 (1.33)	3.44 (0.16)	1.28	0.83	0.65	16.4	0.56	1.037 (0.007)
+Mzx	29.68 (0.61)	1.74 (0.05)	37.49 (0.9)	30.8 (0.67)	0.29 (0.12)	1.26	1.04	0.82	17.03	0.99	1.093 (0.005)
+Mep	31.36 (0.27)	1.78 (0.03)	36.75 (0.64)	28.35 (0.82)	1.76 (0.17)	1.17	0.9	0.77	17.62	0.81	1.086 (0.001)
8C	42.84 (0.11)	2.3 (0.01)	37.86 (1.1)	15.92 (0.83)	1.08 (0.21)	0.88	0.37	0.42	18.65	0.02	1.059 (0.022)
+Me	40.86 (4.88)	1.54 (0.06)	39.06 (1.23)	17.66 (5.77)	2.88 (0.06)	0.96	0.43	0.45	26.55	0.02	1.055 (0.003)
+Mzx	33.74 (1.48)	1.66 (0.07)	40.00 (1.18)	24.44 (2.42)	0.16 (0.01)	1.19	0.72	0.61	20.38	0.41	1.075 (0.005)
+Mep	39.45 (0.87)	1.78 (0.03)	36.33 (0.66)	20.52 (0.47)	1.91 (0.45)	0.92	0.52	0.56	22.1	0.26	1.094 (0.000)
9H	40.14 (3.79)	1.73 (0.13)	35.65 (2.07)	21.43 (1.97)	1.06 (0.12)	0.89	0.53	0.6	23.24	0.31	1.098 (0.005)
+Me	39.33 (1.06)	1.73 (0.03)	37.86 (1.18)	18.86 (0.64)	2.22 (0.12)	0.96	0.48	0.5	22.75	0.13	1.081 (0.004)
+Mzx	38.93 (2.61)	1.9 (0.11)	39.41 (1.61)	17.92 (1.86)	1.84 (0.18)	1.01	0.46	0.45	20.44	0.06	1.124 (0.004)
+Mep	40.07 (2.23)	2.01 (0.16)	38.3 (3.38)	18.34 (1.35)	1.28 (0.31)	0.96	0.46	0.48	19.89	0.11	1.120 (0.001)
10Py	38.59 (1.38)	1.92 (0.12)	37.44 (2.15)	20.81 (2.96)	1.24 (0.21)	0.97	0.54	0.56	20.08	0.26	1.022 (0.0026)
+Me	33.12(1.2)	2.13 (0.1)	43.3 (2.3)	20.16 (1.81)	1.29 (0.82)	1.31	0.61	0.47	15.57	0.1	1.049 (0.003)
+Mzx	36.22 (1.25)	2.02 (0.06)	37.66 (1.19)	22.15 (2.21)	1.95 (0.22)	1.04	0.61	0.59	17.92	0.35	1.057 (0.004)
+Mep	29.58 (1.48)	1.62 (0.09)	45.89 (3.2)	22.81 (1.63)	0.09 (0.01)	1.55	0.77	0.50	18.22	0.16	1.093 (0.001)
11K	34.89 (0.42)	1.97 (0.05)	39.29 (1.36)	23.22 (1.01)	0.63 (0.17)	1.13	0.67	0.59	17.68	0.37	1.061 (0.009)
+Me	32.19 (1.09)	2.03 (0.09)	39.61 (2.21)	23.82 (1.8)	2.34 (0.77)	1.23	0.74	0.60	15.85	0.44	1.048 (0.006)
+Mzx	35.12 (0.55)	2.12 (0.03)	39.2 (2.48)	21.7 (3.49)	1.86 (0.65)	1.12	0.62	0.55	16.58	0.3	1.075 (0.005)
+Mep	33.73 (0.52)	2.10 (0.07)	38.10 (0.67)	23.88 (1.22)	2.19 (0.21)	1.13	0.71	0.63	16.09	0.47	1.079 (0.000)

and

Table A5. Percentage element (molar) content with standard deviation in brackets, its ratios, ω, and E4/E6 ratio for humins untreated and saturated with pendimethalin and its two commercial products.

	C (SD)	N (SD)	H (SD)	O (SD)	S (SD)	H/C	O/C	O/H	C/N	ω	E4/E6 (SD)
1Ps	41.26 (0.45)	1.87 (0.02)	38.32 (0.72)	18.17 (0.98)	0.38 (0.04)	0.93	0.44	0.47	22.04	0.09	1.016 (0.005)
+Pe	32.57 (0.46)	1.61 (0.05)	31.46 (0.73)	33.69 (1.56)	0.67 (0.38)	0.97	1.03	1.07	20.19	1.25	1.083 (0.010)
+Stq	35.41 (0.44)	1.67 (0.06)	33.63 (1.08)	26.93 (1.58)	2.36 (0.14)	0.95	0.76	0.8	21.24	0.71	1.087 (0.001)
+Pfx	34.64 (0.19)	1.6 (0.01)	31.51 (1.02)	31.78 (1.13)	0.47 (0.11)	0.91	0.92	1.01	21.69	1.06	1.056 (0.001)
3Z	39.61 (1.15)	2.12 (0.06)	40.10 (1.89)	17.93 (0.87)	0.23 (0.03)	1.01	0.45	0.45	18.66	0.05	1.019 (0.003)
+Pe	39.22 (1.12)	2.23 (0.07)	39.19 (2.17)	19.26 (1.19)	0.1 (0.01)	1.00	0.49	0.49	17.61	0.15	1.055 (0.007)
+Stq	38.17 (0.92)	2.14 (0.05)	41.02 (1.17)	18.32 (0.29)	0.35 (0.29)	1.07	0.48	0.45	17.83	0.05	1.045 (0.001)
+Pfx	40.79 (1.18)	2.21 (0.11)	36.52 (1.84)	20.32 (0.84)	0.15 (0.02)	0.9	0.5	0.56	18.43	0.26	1.058 (0.001)
6M	38.98 (0.27)	1.99 (0.05)	38.00 (1.4)	20.36 (1.58)	0.67 (0.11)	0.97	0.52	0.54	19.62	0.22	1.0634 (0.016)
+Pe	36.89 (1.38)	1.93 (0.03)	35.43 (0.58)	23.87 (0.59)	1.87 (0.16)	0.96	0.65	0.67	19.11	0.49	1.052 (0.018)
+Stq	36.48 (0.61)	1.77 (0.03)	36.73 (1.55)	22.63 (1.46)	2.38 (0.2)	1.01	0.62	0.62	20.57	0.38	1.096 (0.001)
+Pfx	35.57 (0.51)	1.73 (0.04)	35.54 (1.01)	27.03 (0.56)	0.13 (0.01)	1.00	0.76	0.76	20.58	0.67	1.059 (0.001)
7T	30.6 (2.61)	1.79 (0.08)	38.48 (1.35)	28.96 (3.03)	0.16 (0.01)	1.28	0.9	0.7	19.45	0.67	1.069 (0.004)
+Pe	30.29 (0.95)	1.75 (0.07)	33.54 (1.68)	32.15 (1.69)	2.28 (0.67)	1.11	1.06	0.96	17.35	1.19	1.120 (0.033)
+Stq	30.49 (0.64)	1.63 (0.05)	36.38 (1.08)	30.64 (1.69)	0.86 (0.13)	1.19	1.00	0.84	18.73	0.98	1.092 (0.000)
+Pfx	29.2 (0.67)	1.58 (0.05)	38.18 (1.33)	30.89 (1.96)	0.15 (0.02)	1.31	1.06	0.81	18.5	0.97	1.092 (0.001)
8C	42.84 (0.11)	2.3 (0.01)	37.86 (1.1)	15.92 (0.83)	1.08 (0.21)	0.88	0.37	0.42	18.65	0.02	1.059 (0.022)
+Pe	37.99 (1.44)	1.57 (0.06)	34.27 (0.65)	26.01 (0.91)	0.16 (0.02)	0.9	0.68	0.76	24.13	0.59	1.060 (0.009)
+Stq	36.89 (0.36)	1.65 (0.02)	37.86 (1.02)	22.8 (1.42)	0.8 (0.05)	1.03	0.62	0.6	22.35	0.34	1.088 (0.001)
+Pfx	39.21 (2.1)	1.64 (0.01)	35.43 (1.21)	23.52 (1.35)	0.2 (0.02)	0.9	0.6	0.66	23.91	0.42	1.088 (0.000)
9H	40.14 (3.79)	1.73 (0.13)	35.65 (2.07)	21.43 (1.97)	1.06	0.89	0.53	0.6	23.24	0.31	1.098 (0.005)
+Pe	38.41 (2.05)	1.82 (0.06)	35.76 (1.1)	22.37 (1.41)	1.64 (0.08)	0.93	0.58	0.63	21.05	0.38	1.134 (0.007)
+Stq	38.53 (0.75)	1.72 (0.03)	39.66 (0.84)	16.56 (1.72)	3.53 (0.11)	1.03	0.43	0.42	22.35	−0.04	1.153 (0.001)
+Pfx	43.48 (1.71)	1.82 (0.16)	34.59 (1.28)	17.18 (1.11)	2.93 (0.54)	0.8	0.4	0.5	23.83	0.12	1.122 (0.001)
10Py	38.59 (1.38)	1.92 (0.12)	37.44 (2.15)	20.81 (2.96)	1.24 (0.21)	0.97	0.54	0.56	20.08	0.26	1.022 (0.0026)
+Pe	36.71 (1.48)	1.97 (0.11)	35.94 (3.62)	20.8 (7.48)	4.58 (2.43)	0.98	0.57	0.58	18.62	0.32	1.075 (0.012)
+Stq	34.63 (0.41)	1.73 (0.04)	37.67 (0.51)	23.24 (0.08)	2.73 (0.34)	1.09	0.67	0.62	20.02	0.4	1.063 (0.001)
+Pfx	36.67 (0.59)	1.76 (0.05)	34.44 (0.55)	23.49 (0.15)	3.64 (0.25)	0.94	0.64	0.68	20.79	0.49	1.073 (0.001)
11K	34.89 (0.42)	1.97 (0.05)	39.29 (1.36)	23.22 (1.01)	0.63 (0.17)	1.13	0.67	0.59	17.68	0.37	1.061 (0.009)
+Pe	33.4 (2.15)	1.93 (0.12)	35.86 (2.18)	26.85 (4.21)	1.96 (1.44)	1.07	0.8	0.75	17.29	0.71	1.069 (0.022)
+Stq	29.47 (0.52)	1.63 (0.03)	43.04 (2.34)	25.73 (2.0)	0.13 (0)	1.46	0.87	0.6	18.04	0.45	1.105 (0.001)
+Pfx	32.33 (0.21)	1.79 (0.03)	34.22 (2.16)	31.07 (2.14)	0.59 (0.18)	1.06	0.96	0.91	18.11	1.03	1.085 (0.001)

). Only relatively small changes in the O/C atomic ratio were observed (an increase for the majority of samples after pesticides saturation) (

Table 2. The O/C atomic ratio calculated for humin untreated and saturated with all the investigated pesticides.

Sample	1Ps	3Z	6M	7T	8C	9H	10Py	11K
Untreated	0.44	0.45	0.52	0.90	0.37	0.53	0.54	0.67
+Me	0.95	0.43	0.55	0.83	0.43	0.48	0.61	0.74
+Mzx	0.82	0.42	0.64	1.04	0.72	0.46	0.61	0.62
+Mep	0.88	0.48	0.64	0.90	0.52	0.46	0.77	0.71
+Fl	1.11	0.52	0.74	1.06	0.58	0.60	0.67	0.72
+Flt	0.67	0.51	0.72	0.95	0.57	0.51	0.67	0.78
+Cen	0.85	0.47	0.68	1.03	0.58	0.50	0.68	0.69
+Pe	1.03	0.49	0.65	1.06	0.68	0.58	0.57	0.80
+Stq	0.76	0.48	0.62	1.00	0.62	0.43	0.67	0.87
+Ptx	0.92	0.50	0.76	1.06	0.60	0.39	0.64	0.96

). These phenomena lead to an increase in the internal oxidation parameter (ω) (Table A3, Table A4 and Table A5). The same dependencies were found for both active substances and for commercial products. Based on the elemental analysis, it can be assumed that the studied pesticides did not affect aromatic–aliphatic structure of humin, except for the increase in oxygenation of the studied system. According to Stevenson [29], this fact could be correlated with the non-biological degradation of pesticides by nucleophilic reactive groups of the types known to occur in soil humus, such as carboxyl; phenolic–, enolic–, heterocyclic–, and aliphatic–OH; amino; heterocyclic amino; imino; semiquinones; and others). Humic substances are known for their redox properties, and their activity as reducing agents in various pesticides [29,30,31]. Indeed, humin, extracted as solid fractions from paddy soils or sediments, has been shown to be involved in extracellular electron transfer (coupled with microbial reductive dehalogenation of pentachlorophenol), acting as both electron acceptor and electron donor [32]. Thus, the redox activity of humin may lead to its oxidation as a consequence of interaction with pesticides.

3.2. NMR Measurements of Unsaturated and Saturated Humin with Pesticides

¹³C CP MAS NMR spectra of untreated and pesticide-treated humin showed no significant differences (Figure 1). These observations pointed out that no new compounds/structural groupings capable of oscillation have been formed and the humins exhibit chemical inertness to the bound pesticides. The main result indicated that samples saturated with pesticides showed a similar spectral pattern to untreated humin as confirmed by the lack of new signals. However, comparison of the integrated areas of the NMR spectra revealed slight differences (

Table 3. Integral areas calculated from ¹³C CP MAS NMR spectra of untreated humin and humin saturated with active substances of investigated herbicides.

	Range of Integrated Area [ppm]
Sample	-COOH	C(Ar)-O/N	C(Aromatic)	O-C-O	CH-OH	-OCH3	-CH3
	167–188	149–167	116–151	102–116	74–91	57–73	18–57
1Ps	7.7	8.5	36.1	7.6	11.2	8.8	17.3
1Ps + Fl	8.7	8.3	32.0	5.4	9.0	7.5	22.1
1Ps + Me	10.0	8.7	30.9	5.4	8.8	7.3	20.4
1Ps + Pe	10.1	9.9	33.1	5.4	7.2	6.4	23.2
3Z	9.9	6.5	38.3	5.9	8.5	7.7	21.8
3Z + Fl	12.2	8.7	32.7	3.8	7.7	6.6	18.7
3Z + Me	10.1	8.1	31.3	6.4	9.3	8.4	19.6
3Z + Pe	11.7	8.6	29.8	5.5	8.6	7.5	18.6
6M	10.6	9.2	32.5	4.8	11.4	8.2	19.8
6M + Fl	9.8	7.7	23.6	4.2	7.3	6.5	17.8
6M + Me	7.8	7.9	31.0	7.8	11.7	9.3	16.8
6M + Pe	10.6	9.5	31.0	4.5	9.8	7.7	19.4
7T	9.7	8.5	32.4	6.2	15.6	9.8	19.1
7T + Fl	8.9	7.4	28.0	7.0	13.1	9.2	19.9
7T + Me	13.9	7.3	29.6	2.8	10.2	7.9	21.1
7T + Pe	9.5	8.3	27.7	6.7	12.7	9.2	17.3
8C	12.8	9.6	41.4	3.5	9.2	7.2	17.8
8C + Fl	7.8	8.4	32.2	6.6	10.1	8.2	16.5
8C + Me	9.4	9.8	32.5	5.3	9.7	7.5	16.4
8C + Pe	7.3	9.1	35.3	5.8	9.5	7.9	17.1
9H	13.3	9.5	52.4	2.6	6.8	5.0	16.1
9H + Fl	11.0	8.2	39.2	5.3	7.5	6.2	15.4
9H + Me	21.6	5.9	35.2	1.7	5.9	2.5	26.0
9H + Pe	13.0	10.9	35.5	0.5	5.2	4.3	18.2
10Py	6.6	7.7	34.9	8.4	14.2	9.6	15.6
10Py + Fl	9.1	8.7	31.5	5.9	11.1	8.1	17.4
10Py + Me	10.3	10.0	30.6	7.3	11.3	7.7	13.1
10Py + Pe	10.5	9.2	29.1	5.7	10.7	8.0	18.5
11K	11.1	8.7	29.3	5.6	15.1	9.8	19.9
11K + Fl	9.5	8.4	28.9	6.4	13.2	9.0	16.9
11K + Me	11.9	9.2	28.6	5.0	11.9	8.2	16.2
11K + Pe	16.6	9.4	29.1	0.2	10.8	6.5	21.3

) in the content of carboxyl groups. With few exceptions, the integrated areas of the chemical shifts in COOH groups were higher in the herbicides treated with samples than in pure humin. These observations converge the results of elemental analysis, confirming the higher O/C ratio in the samples investigated. According to Song et al. [10], such a difference is an indicator of organic matter oxidation processes.

Interesting observations were also made in the regions attributed to the following hydrocarbons: 116–151 ppm aromatic C; 102–116 ppm O-C-O, anomeric, and carbonyl C; and 74–91 ppm CH-OH, carbohydrates, or higher alcohols. A decrease in the intensity of most of these bands was observed after saturation of humin with herbicides. This was accompanied by the broadening of lines attributed to aliphatic carbons in the range of 18–57 ppm. This may indicate that the sorbed pesticide compounds studied play a key role in the interaction with the humin fraction and exhibit the tendency to incorporate into its structure, modifying the mutual proportion of different carbohydrate moieties. For the majority of the treated samples, an increase in carboxylic group areas was also observed, in agreement with the results of elemental analysis (Table 2), which may confirm the oxidation of humin.

3.3. Functional Groups Analysis of Investigated Humin

All the FTIR spectra of the humin studied showed a similar pattern, typical of humic substances [6], with aliphatic (2877–2985 cm⁻¹) and aromatic (1570–1677 cm⁻¹) stretching as the best representative of the hydrocarbon structure and carboxylic groups (1677–1800 cm⁻¹). This is well documented in Figure 2, which shows sample 6M untreated and treated with the active substances (6M + Me) and its commercial products (Mzx and Mep). Some other studies on the sorption of pesticides (atrazine and metsulfuron–methyl) by SOM pointed to the formation of new interactions, as evidenced by the stretching of the N-H vibration band towards lower wavenumbers [33]. In the case of the herbicides investigated, no band shifts were observed, and no new band appeared. However, some small but evident changes in the intensity of the analyzed bands were observed (

Table 4. Integral areas [%] calculated from absorbance FTIR spectra of humin and humin saturated with investigated herbicides.

Sample	Range of Integrated Areas [cm−1]
	-C-O; -OH (COOH)	(Ar)C=C	-C=O (COOH)	sCH2	asCH2
	1190–1300	1570–1677	1677–1800	2796–2850	2877–2985
1Ps	5.22	8.60	5.58	1.07	4.03
1Ps + Fl	3.95	6.62	3.61	0.54	2.59
1Ps + Me	3.51	6.40	4.32	1.02	3.40
1Ps + Pe	3.74	7.32	4.18	0.37	1.79
3Z	6.72	9.17	6.64	1.16	3.87
3Z + Fl	5.21	8.05	6.14	1.31	4.31
3Z + Me	5.97	8.51	5.96	1.24	3.96
3Z + Pe	5.43	7.89	6.21	1.30	4.08
6M	4.80	8.47	5.08	0.54	3.10
6M + Fl	4.52	7.81	4.83	1.03	3.71
6M + Me	4.38	7.45	4.40	0.69	3.06
6M + Pe	2.21	6.89	3.91	0.92	3.35
7T	3.64	6.86	3.82	0.76	3.12
7T + Fl	4.02	7.81	4.26	0.47	2.11
7T + Me	3.44	6.61	3.96	0.89	3.36
7T + Pe	4.07	7.46	4.59	0.94	3.64
8C	6.32	9.04	6.11	1.03	3.67
8C + Fl	4.74	7.91	4.62	0.78	2.79
8C + Me	4.62	7.63	4.49	1.07	3.85
8C + Pe	5.07	8.92	5.42	0.66	2.56
9H	4.98	8.68	5.75	0.72	2.73
9H + Fl	4.69	8.20	5.63	0.79	2.90
9H + Me	4.61	7.82	5.15	1.21	3.63
9H + Pe	3.36	8.57	5.91	0.39	2.49
10Py	3.12	6.23	3.20	0.95	3.02
10Py + Fl	3.53	6.79	3.45	0.26	1.99
10Py + Me	2.99	6.09	3.63	0.79	2.89
10Py + Pe	3.64	7.05	4.32	0.44	2.61
11K	4.77	8.20	4.92	0.96	3.79
11K + Fl	4.38	7.48	4.52	0.55	2.87
11K + Me	3.34	6.44	3.52	0.89	3.31
11K + Pe	4.32	7.58	4.48	0.56	3.02

), such as the decrease in the proportion of aromatic structures in the humin. This correlates well with the decrease in aromatic C-integral areas observed in the ¹³C CP MAS NMR spectra (Table 3). In most cases, a decrease in signal intensities was observed in favor of a broad group of hydrogen bonding bands (3200–3700 cm⁻¹). Since all of the investigated pesticides have a relatively high polar surface area [24] and are hydrogen bond acceptors, the formation of hydrogen bonds is a very likely mechanism of interaction between the aforementioned herbicide active substances and electronegative donor oxygen groups present in the studied humin. Nevertheless, it must be considered that humin may also react with Fl, Me, and Pe via hydrophobic retention, taking into account their non-polar regions and the lipophilicity of the studied herbicides.

3.4. UV-Vis Studies

Comparison of the UV-Vis analysis results for pure humin—both untreated and saturated with active substances and commercial herbicides—revealed small changes in the E4/E6 parameter (Appendix A Table A3, Table A4 and Table A5). Measurements of electron spectra of humic substances in the solid state are not a very common, so it is difficult to say how significant the observed changes are based on the literature. However, a tendency for E4/E6 to increase was observed for most of the humin after sorption of the studied herbicides, indicating a slight change in the molar mass of the tested humin supporting a decrease in the degree of condensation and aromaticity of the humic fraction [34].

3.5. Radical Structures in Humin

The most sensitive method for monitoring herbicide interactions with humin was EPR spectroscopy. The same radical signal with a g parameter of 2.0029–2.0031 was recorded in all the untreated humin and humin saturated with pesticides (both active substances and preparations) tested, indicating the very similar structure of the radicals in all the humin investigated and their predominantly aromatic structure. The g-parameter values of the investigated humin were lower than those typically observed for HA and FA [35,36], which is not surprising given the different structures within the humin fraction. This is indicative of an unpaired electron located in the more aromatic moieties [37]. A reduction in radical concentration (RC) was observed after treatment of humin with active pesticide substances and commercial preparations (Figure 3). Thiadiazoles, as well as various other heterocycles, have been shown to be potent radical scavengers and are known to have radical scavenging properties [38]. So far, the scavenging activity has been investigated against small reactive oxygen species [39]. Similar studies, but based on the interaction of permanent radicals in humic acids with groups of pesticides, were investigated by Senesi et al. [3,31,40]. Two opposite effects—an increase or decrease in radical concentration—were observed, depending on the type of chemical used as a pesticide. Thus, s-triazine derivatives caused an increase in radical concentration. This result was explained by assumption that electrons are removed from the electron-rich donating amine and/or heterocyclic nitrogen atoms of the triazine molecule by electron-deficient quinone-like structures in the humic acids through single electron donor–acceptor processes involving semiquinone free radical intermediates [3]. However, the opposite effect was observed for carbamates, whose interaction with humic acids led to a decrease in the concentration of semiquinone radicals, via a hydrogen atom transfer reaction [41]. The above mechanisms involve carbonyl, quinone, and carboxyl groups of humic substances leading to hydrolyzable (probably anil, a Schiff base, and anilinoquinone) or non-hydrolyzable (probably heterocyclic rings or ethers) bond forms. The electron withdrawal ability of nitro (and other) groups may also prevent radical formation [42].

The occurrence of stable free radicals in humic substances, including humin fraction, further implicates organic matter in the chemical transformation of herbicides [43]. The phenomena described are particularly possible due to the high content of oxygen-containing groups in humic acids. However, humin contains redox-active moieties, and in addition to the results presented here, previous electron spin resonance studies also suggested that quinone moieties in humin are the redox-active centers [33]. Therefore, it can be concluded that humin may play an important role in mediating electron transfer reactions between SOM and pesticides in soil.

The concentrations of radicals determined from EPR spectra (

Table 5. Radical concentrations (RCs) with standard deviation in brackets of untreated humin and humin treated with herbicides and its commercial products.

Humin	Radical Concentration (SD) [×1016spin/g]
	1Ps	3Z	6M	7T	8C	9H	10Py	11K
Untreated	8.32 (0.42)	9.14 (0.50)	12.45 (0.2)	12.17 (0.3)	11.67 (0.3)	21.3 (0.5)	7.59 (0.3)	16.18 (0.5)
+Me	4.54 (0.11)	4.16 (0.2)	3.59 (0.3)	3.96 (0.2)	5.48 (0.6)	13.07 (0.2)	2.19 (0.7)	3.27 (0.2)
+Mzx	2.46 (0.11)	5.25 (0.13)	4.44 (0.2)	4.36 (0.1)	4.24 (0.33)	18.7 (0.1)	1.43 (0.5)	4.05 (0.11)
+Mep	3.72 (0.05)	3.53 (0.12)	5.34 (0.11)	3.09 (0.15)	4.96 (0.4)	10.57 (0.7)	2.17 (0.2)	2.84 (0.22)
+Fl	3.82 (0.2)	5.99 (0.22)	3.49 (0.12)	3.24 (0.1)	3.69 (0.2)	17.28 (0.2)	3.74 (0.2)	3.43 (0.3)
+Flt	5.17 (0.09)	5.38 (0.2)	4.71 (0.3)	3.74 (0.4)	7.7 (0.1)	15.44 (0.1)	3.07 (0.1)	3.14 (0.4)
+Cen	4.88 (0.11)	3.71 (0.14)	4.59 (0.4)	4.38 (0.2)	5.29 (0.2)	12.64 (0.2)	3.00 (0.4)	3.39 (0.4)
+Pe	4.77 (0.03)	4.77 (0.15)	4.64 (0.2)	6.61 (0.2)	6.99 (0.22)	15.29 (0.4)	3.01 (0.3)	3.56 (0.10)
+Stq	5.36 (0.04)	5.96 (0.22)	3.71 (0.2)	3.68 (0.5)	5.13 (0.14)	14.77 (0.6)	2.69 (0.10	3.26 (0.2)
+Ptx	5.80 (0.1)	5.86 (0.2)	4.99 (0.2)	6.00 (0.4)	6.62 (0.13)	17.25 (0.1)	4.16 (0.02)	3.60 (0.08)

) were reduced by the sorption of all herbicides tested. Interestingly, only a partial reduction of the radical concentration was observed, not their complete quenching, as is known for interactions of humic acids with, e.g., copper ions [36]. This suggests that the herbicide molecules may not be able to penetrate deep into the humin structure, probably due to the humin strong binding to the surface of the clay minerals. According to some previous studies, particles of humin may create micelles of a polymeric nature, where the basic structure consists of an aromatic ring of phenyl type bridged by -O-, -NH-, -N=, -S-, and other functional groups that contain both free -OH and -COOH groups and the double linkages of quinones [21]. Therefore, humin structure is dominated by the condensed strong hydrophobic core with numerous aliphatic side chains, enabling different magnitudes of pesticide sorption [24]. This is in line with the studies of Pham et al. [44], who concluded that humin serves as extracellular electron mediator in multiple reactions, where quinone and sulfur-containing moieties are suggested as redox-active centers. In such a case, the reactions would take place in the surface layers, while part of the radicals in the core of the stable humin matrix would be inaccessible to the tested compounds, possibly also due to the steric hindrance. Thus, the observed oxidation of humin, as a result of their interaction with the tested herbicides, could be a surface phenomenon, as the inner region of the humin matrix would be protected by the humin condensed structure [21].

4. Conclusions

The conducted research indicates that the interaction of humin with pesticides (both active substances and commercial products) resulted in a decrease in the proportion of aromatic groups and an increase in the O/C ratio of humin and internal oxidation (ω). Based on the change in intensity and the absence of band shifts in the recorded NMR and infrared spectra of humin after their saturation with the studied herbicides, it is postulated that no significant chemical changes occurred in the structures of humin. However, physical sorption processes are not excluded especially on the surface of highly charged humin moieties. The increase in the proportion of oxygen-containing functional groups suggests that hydrogen bonding could be an important and favored mechanism of incorporation of the tested herbicides on the surface region of humin. In addition, the observed decrease in the content of aromatic semiquinone radicals in humin as a result of their interaction with tested herbicides highlights an important role of this fraction as a redox-active center in SOM, with potential to participate in non-biological degradation of pesticides in soils. The results obtained also suggest that the interaction of humin with the studied herbicides may occur as a surface phenomenon, and the inner region of the humin matrix could possibly be protected by the condensed humin structure and/or strong binding to the clay minerals. This could be proof of humin as the most recalcitrant SOM fraction, which is essential to predict the transformation and migration of organic pollutants. Such information may significantly contribute to the risk assessment of surface and groundwater contamination by the tested pesticides and to optimize the sustainable remediation treatment strategies in soils. However, to more adequately predict the real-field scenario, our future directions will focus on the comparison of the sorption affinity of the aforementioned compounds towards the individual SOM fractions in the field.