Optimized isolation method of humin fraction from mineral soil material

Humic substances, including humin fraction, play a key role in the fate of organic and inorganic xenobiotics contaminating the environment. Humin is an important fraction of humic substances, which has been the least studied to date. This is due to the difficulties connected with its isolation that pose a number of methodological problems. Methods of humin fraction isolation can be divided into following main groups: (1) digestion of mineral soil components with HF/HCl followed by alkali extraction of HA and FA; (2) alkali extraction of HA and FA followed by extraction of humin by different organic solvents; and (3) alkali extraction of HA and FA followed by HF/HCl digestion of mineral soil components. Nevertheless, each of these methods has different limitations. We described in detail a useful procedure of humin isolation, in which this fraction was not extracted, but isolated from the soil by removing its soluble organic and mineral components. A modified method of HA and FA extraction with 0.1 M NaOH, according to the International Humic Substances Society, was used in the first step. Then, the mineral components in the residue were digested with the 10% HF/HCl. Unlike the procedures oriented to increase the concentration of organic matter, samples were treated several times with the HF/HCl mixture until the mineral fraction was almost completely digested. The main assumption of the method modification was to obtain the highest yield with the lowest possible ash content, but without affecting humin chemical structure. The results showed that the proposed procedure is characterized by a high efficiency and recovery and, therefore, it can be used to isolate high amounts of humin from soil.

Recommendations for isolation of humin fraction from soil material

<p>Methods of isolation of the humin fraction can be divided into two main groups: (1) extraction of humic (HA) and fulvic (FA) acids followed by extraction of humin with different organic solvents, and (2) extraction of HA and FA followed by removal of soil mineral fraction. To isolate the large amounts of humin necessary to study the interactions of this fraction with pesticides, we examined some modifications of the latter method.</p><p>The first step was to separate HA and FA according to a modified IHSS method (Swift 1996). HA and FA were extracted with 0.1 M NaOH with a 5:1 ratio of extractant to soil. 20 hours shaking was found to be more effective, but 4 hours shaking provided the advantage of being able to extract twice a day,  which ultimately shortened the procedure time.</p><p>The HA and FA free residue was then digested to remove mineral components. We used several (up to 8 weeks) digestions with 10% HF/HCl as higher concentrations of HF can result in structural alteration of the organic compounds (Hayes et al. 2017). While HF/HCl treatment can lead to hydrolysis and loss of polysaccharide and protein materials (Stevenson 1994), the advantage of using HF is the removal of paramagnetic compounds (such as Fe), which facilitates the use of spectroscopic techniques to characterize humin. In contrast to the procedures for only increasing the concentration of organic matter (Schmidt et al. 1997), the sample was digested until the mineral fraction not complexed with humin was completely digested. We tested different modes of mineral fraction digestion in 10% HF/HCl using polyethylene centrifuge bottles. Occasional shaking once a day had the same effect as continuous shaking. It takes 6 weeks to digest 200 g of pure sand in a 1000 cm<sup>3</sup> bottle, when the HF/HCL was weekly replaced. After replacing HF/HCl every 2 weeks, the digestion time of the same material increased to 8 weeks.</p><p>After treatment with HF/HCl, the residue was rinsed with 10% HCl to remove secondary minerals. The residue was washed with distilled water until the neutral pH and then dialyzed to a negative Cl<sup>−</sup> test with AgNO<sub>3</sub>. Then the humin fraction was freeze dried. </p><p> </p><p>Literature</p><p>Hayes M.H.B., Mylotte R., Swift R.S. 2017. Humin: Its Composition and Importance in Soil Organic Matter. In: Sparks D.L. (ed) Advances in Agronomy, Vol. 143, Academic Press, Burlington, 47–138.</p><p>Schmidt, M.W.I., Knicker, H., Hatcher, P.G., Kögel-Knabner, I. 1997. Improvement of 13C and 15N CPMAS NMR spectra of bulk soils, particle size fractions and organic material by treatment with 10% hydrofluoric acid. European Journal of Soil Science, 48, 319-328.</p><p>Stevenson F.J. 1994. Humus Chemistry; Genesis, Composition, Reaction. 2nd ed. John Wiley & Sons., New York.</p><p>Swift R.S. 1996. Organic matter characterization. In: Sparks, D.L., et al. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods - Soil Science Society of America, Book Series no 5,  1011-1069.</p><p> </p><p>Acknowledgements</p><p>This work was supported by the National Science Center (NCN) Poland (project No 2018/31/B/ST10/00677 “Chemical and spectroscopic properties of soil humin fraction in relation to their mutual interaction with pesticides").</p>

Selected properties of the humin fraction isolated from Chernozems and Phaeozems from various regions of Poland

<p>Humin fraction of soil organic matter is assigned to play an important role in carbon sequestration and sorption of xenobiotics. This study concerns the properties of humin (elemental composition, FTIR and SEM-EDS of humin ash) isolated from mollic horizons of eight Chernozems and Phaeozems, used as arable soils in various regions of Poland. Isolation procedure was described by Weber et al. (2021) in another abstract presented in this session. Investigated soils differed in the content of TOC, ranging from 13.3 to 41.7 g kg<sup>−1</sup>, as well as texture from loam (Magnice, Pyrzyce) through silt loam (Trzebnik, Ciepłowody, Hrubieszów) and sandy clay loam (Psary) till clay (Ziemnice, Kętrzyn). They also differed in their pH values (from 5.64 to 7.71), and CEC (from 21.6 to  53.2cmol(+)kg<sup>-1</sup>). Ash content of humin varied between 22.89%  - 54.50%, which is typical for humin originated from mineral soils (Stevenson 1994). This parameter was not correlated neither with the content of <0.002 mm fraction nor TOC content. SEM-EDS analyzes revealed that ash contained mainly Mg (3 – 14 weight%), Al (4 – 22 weight %) and Ti (10 – 25 weight%), depending on the area studied. The lowest pH as well as  the highest TOC and CEC showed Trzebnik soil. Humin from this soil indicated the lowest content of carbon (30.84 %) and the highest values of H/C ratio, which point out to the higher aliphacity of their molecules (Rice and MacCarthy 1991). High O/C ratio (0.91) calculated for humin from Trzebnik is common for more oxidized carbohydrate molecules and makes them similar to fulvic acids which are polysaccharidic in nature (Tan 2014). In contrast, the lowest TOC and CEC were determined in Ciepłowody soil. Humin molecules from this soil indicated the highest carbon content (43.12 %) and the lowest H/C ratio, what reflects the highest aromacity among investigated samples. FTIR spectra confirmed results from elemental analysis and indicated that humin from Ciepłowody and Hrubieszów was the most aromatic among all analyzed soils.</p><p> </p><p>References:</p><p>Hayes M.H.B., Mylotte R., Swift R.S. 2017. Humin: Its Composition and Importance in Soil Organic Matter. In: Sparks D.L. (ed) Advances in Agronomy, Vol. 143, Academic Press, Burlington, 47–138.</p><p>Rice J.A., MacCarthy P. 1991. Statistical evaluation of the elemental composition of humic substances. Org. Geochem, 17(5), 635-648.</p><p>Stevenson FJ. 1994. Humus chemistry: Genesis, composition, and reactions. New York: John Wiley and Sons, p 512.</p><p>Swift R.S. 1996. Organic matter characterization. In: Methods of soil analysis. Part 3. Chemical methods – SSSA Book Series no.5. Soil Science Society of America and American Society of Agronomy, pp 1011-1068.</p><p>Tan HK. 2014. Humic matter in soil and the environment, 2<sup>nd</sup> edn. CRC Press, Boca Raton, p 463.</p><p>Weber J., Jamroz E., Kocowicz A., Debicka M., Ukalska-Jaruga A., Mielnik L., Bejger R., Jerzykiewicz M., Bekier J., Ćwieląg-Piasecka I. Recommendations for isolation of humin fraction from soil material. EGU21-8315</p><p> </p><p>Acknowledgements</p><p>This work was supported by the National Science Center (NCN) Poland (project No 2018/31/B/ST10/00677 “Chemical and spectroscopic properties of soil humin fraction in relation to their mutual interaction with pesticides")</p>

Isolation of the humin fraction from soil: preliminary comments

<p>The organic matter is the most important component of soil material, which determines most soil properties. Among humic substances, humin fraction has been the least studied to date, although it usually constitutes over half of their composition. This is probably due to the fact, that humin fraction has highly hydrophobic properties and is insoluble at all pH values, which makes its isolation much more difficult, compared to humic (HA) and fulvic (FA) acid fractions. In addition, humin fraction forms very stable humic-clay complexes with mineral part of the soil (Stevenson 1994), which cannot be destructed during humic substances extraction. According to the literature, the methods of humin fraction isolation can be divided into two main groups: (1) extraction by different organic solvents, and (2) isolation by extraction of HA and FA followed by digestion of mineral soil components. Nevertheless, each of these methods has different limitations.</p><p>We investigated some modifications of the latter method, obtaining humin fraction from eight mollic horizons of Chernozems and Phaeozems, which differed in their physico-chemical properties.</p><p>The first step was to separate HA and FA according to IHSS method described by Swift (1996), however we adopted different shaking procedure. To asses differences, each supernatant obtained was analyzed for the carbon content concentration, which corresponded to HA and FA extracted.</p><p>HA and FA free residue was then digested to reduce the content of mineral components. We used several digestion with 10% HF/HCl , as higher concentrations of HF can result in structural alteration of the organic compounds (Hayes et al. 2017). To find the optimal time of the procedure, the ash content was determined following each digestion stage. After the HF/HCl treatment, the residue was rinsed with 10% HCl to eliminate secondary minerals. The residue was washed with distilled water until the neutral pH, then transferred to dialysis membranes and dialyzed with distilled water until a negative Cl<sup>−</sup> test with AgNO<sub>3</sub>. Afterwards the humin fraction was freeze dried. </p><p>Finally, obtained humin fraction contained various ash content, ranged from 6 to 30%, depending on the soil. The conducted test indicated that: (1) the concentration of carbon in supernatant considerably increased as shaking time was extended to 20 hours, and (2) longer than 4 weeks digestion with HF/HCl did not affect the reduction of the ash content of the humin fraction obtained.    </p><p> </p><p>Literature</p><p>Hayes M.H.B., Mylotte R., Swift R.S. 2017. Humin: Its Composition and Importance in Soil Organic Matter. In: Sparks D.L. (ed) Advances in Agronomy, Vol. 143, Academic Press, Burlington, 47–138.</p><p>Stevenson F.J. 1994. Humus Chemistry; Genesis, Composition, Reaction. 2nd ed. John Wiley & Sons., New York.</p><p>Swift R.S. 1996. Organic matter characterization. In: Sparks, D.L., et al. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods - Soil Science Society of America, Book Series no 5,  1011-1069.</p><p> </p><p>Acknowledgements</p><p>This work was supported by the National Science Center (NCN) Poland (project No 2018/31/B/ST10/00677 “Chemical and spectroscopic properties of soil humin fraction in relation to their mutual interaction with pesticides").</p>

Interaction of soil humin fraction with pesticides - a review

<p>The use of pesticides significantly influences the efficiency of agriculture production, but at the same time, their extensive and widespread use, raises serious concerns regarding the release of harmful substances into the environment [1,2]. The fate of pesticides in soil depends on many factors related mainly to the physico-chemical properties of these compounds as well as content and quality of organic matter [3]. Humin as the predominant fraction of organic matter, may significantly determine the behavior and transformations of pesticides in soil [5]. Therefore, the aim of this review was to present the state of the art of humin-pesticides mutual interactions.</p><p>Sorption-related studies showed that humin has dissimilar binding strengths with pesticides [4,5]. According to Pignatello [7], the sorption selectivity by humin has a number of potential causes: (1) preference for particular microdomains within fractions that are envisioned to segregate on the basis of functional group identity (aromatic, paraffinic, carbohydrate domains); (2) preference based on strong functional group interactions, such as hydrogen bonding and (3) preference based on the nature of the thermodynamic physical state of humin, namely the configurations and conformations of the molecules and strands at microstructural level.</p><p>Moreover, humin exhibits potentially different accumulation capacities for xenobiotics. Wang et al. [9] explained these relations with the limited accessibility to microporous domains of humin matrices for the larger-molecular-weight particles. The authors [9] observed a lower adsorbed mass of spatially developed compounds compared to compounds with small diameters. This process is probably most likely related to the structural rearrangement of the humin matrix under slow diffusion into microporous domains pronounced with the adsorption of large molecular weight compounds. Additionally, Pignatello [7] as well as Schaumann [4,5] indicated that the humin surface is covered with various polar and non-polar functionalities, which may efficiently interact with pesticides by van der Waals forces, hydrophobic attraction, hydrogen bonding, charge transfer or ligand exchange processes. Nevertheless, the chemical properties of pesticides as well as their coexistence with other chemical compounds i.e.: surfactants, coagulants, decomposition inhibitors and others [8] can modify the interactions of pesticides with humin in natural soil environment.</p><p>Literature:</p><p>[1] FAO, ITPS Global Assessment of the Impact of Plant Protection Products on Soil Functions and Soil Ecosystems. FAO, Rome 2017, 40 pp.</p><p>[2] Silva, V.; Mol, H.; Zomer, P.; Tienstra, M.; Ritsema, C.J.; Geissena, V. Sci. Total. Environ.  2019, 653, 1532–1545.</p><p>[3] Stolte, J.; Tesfai, M.; Øygarden, L.; Kværnø, S.; Keizer, J.; Verheijen, F.; et al. Soil Threats in Europe: Status, Methods, Drivers and Effects on Ecosystem 4 Services, 2016, Report</p><p>[4] Stevenson F. 1994, John Wiley & Sons, New York</p><p>[5] Schaumann G. 2006a, J Plant Nutr Soil Sci 169:145–156</p><p>[6] Schaumann G. 2006b, J Plant Nutr Soil Sci 169:157–167</p><p>[7] Pignatello J. 2012,  J Soils Sediments 12:1241–1256</p><p>[8] Ehlers, G.; Loibner, A. 2006, Environ. Pollut. 141, 494-512</p><p>[9] Wang X, Guo X, Yang Y, Tao S, Xing B. 2011, Environ Sci Technol 45:2124–2130</p><p> </p><p><em>Acknowledgement: The studies were supported from the National Science Centre project no. 2018/31/B/ST10/00677 “Chemical and spectroscopic properties of soil humin fraction in relation to their mutual interaction with pesticides”</em></p>

Fractions of soil organic matter in the vineyards of altitude regions in Santa Catarina

The implementation of agricultural systems such as viticulture can quantitatively and qualitatively affect the contents of soil organic matter (SOM). These changes may modify the edaphic features of the soil as well as the soil quality. The objective of this study was to evaluate the chemical and physical fraction of SOMand to analyze changes in the carbon stock and C management index in areas of implanted vineyards in altitude regions of Santa Catarina. Four regions were selected: Region I (Urubici); Region II (San Joaquim); Region III (Campos Novos) and Region IV (Água Doce). In each region, we selected vineyards implanted between 2001 and 2005 as well as surrounding forested areas. Disturbed and undisturbed samples were collected from the 0-5, 5-10, and 10-20 cm layers of the soil. Samples were prepared in the laboratory to obtain air-dried soft soil, which was then used for the analysis of several parameters, namely total organic carbon (TOC), carbon stock,and chemical fractionation of the soil. The chemical fractionation was then used to determine carbon content in the fulvic acid fraction (C-FAF), humic acid fraction (C-HAF), and humin fraction (C-HUM). We also analyzed particle size, quantified the levels of particulate carbon (COp) and carbon associated with clay and silt (COam), and calculated the carbon management index (CMI). We evaluated normality and homogeneity for all data. The results were evaluated with an analysis of variance and subsequent F-test. Mean values were compared using a 5% Student’s t-test and subsequently submitted to a Tukey’s test. The highest TOC levels were observed in Region II in the 0-5 cm layer in both vineyard and forested areas. Vineyard areas exhibited lower values of TOC, Cop, and COam compared to forested areas indicating that the management adopted in these areas contributed to the reduction of these fractions. Forested areas exhibited a higher proportion of Cop compared to vineyard areas. The humin fraction represented the largest portion of the TOC and comprised the highest values in both forested and vineyard areas. The carbon management index indicated a low contribution of vineyard areas or a reduction in carbon storage in their soils.

SPATIALIZATION OF FRACTIONS OF ORGANIC MATTER IN SOIL IN AN AGROFORESTRY SYSTEM IN THE ATLANTIC FOREST, BRAZIL

ABSTRACT This study aimed to spatialize fractions of organic matter of soil in an agroforestry system (AFS) located in the Atlantic Forest in Brazil. Thirty-one soil samples were collected at depths of 0-10, 10-20 and 20-40 cm from georeferenced collection points. We determined total organic carbon (TOC), particulate carbon (COp), carbon associated with clay and silt (COam), carbon content in the fulvic acid fraction (C-FAF), humic acid fraction (C-HAF) and humin fraction (C-HUM). Semivariogram analysis and model adjustment were carried out using ArcGIS 10.2 software. Subsequently, spatial interpolation was performed using Ordinary Kriging. We observed spatial dependence for all variables except for TOC and COp at the 0-10 cm depth, which presented a pure nugget effect. It was possible to observe modifications in the distribution of humic substances in the study area. The results from this study are similar to those of other studies conducted in naive areas in the Atlantic Forest, demonstrating the benefits of using the agroforestry system.

Comparison of different methods for the determination of total organic carbon and humic substances in Brazilian soils

ABSTRACTAiming to compare three different methods for the determination of organic carbon (OC) in the soil and fractions of humic substances, seventeen Brazilian soil samples of different classes and textures were evaluated. Amounts of OC in the soil samples and the humic fractions were measured by the dichromate-oxidation method, with and without external heating in a digestion block at 130 °C for 30 min; by the loss-on-ignition method at 450 °C during 5 h and at 600 °C during 6 h; and by the dry combustion method. Dry combustion was used as reference in order to measure the efficiency of the other methods. Soil OC measured by the dichromate-oxidation method with external heating had the highest efficiency and the best results comparing to the reference method. When external heating was not used, the mean recovery efficiency dropped to 71%. The amount of OC was overestimated by the loss-on-ignition methods. Regression equations obtained between total OC contents of the reference method and those of the other methods showed relatively good adjustment, but all intercepts were different from zero (p < 0.01), which suggests that more accuracy can be obtained using not one single correction factor, but considering also the intercept. The Walkley-Black method underestimated the OC contents of the humic fractions, which was associated with the partial oxidation of the humin fraction. Better results were obtained when external heating was used. For the organic matter fractions, the OC in the humic and fulvic acid fractions can be determined without external heating if the reference method is not available, but the humin fraction requires the external heating.

Late Pleistocene and Holocene Vegetation, Climate Dynamics, and Amazonian Taxa in the Atlantic Forest, Linhares, SE Brazil

Analysis of biological proxies in lake sediment and geochemical analysis of soil profiles reveal natural vegetation dynamics, with climate inferences, since the late Pleistocene in a fragment of the pristine lowland Atlantic Forest of southeastern Brazil. Carbon isotopes from soil organic matter and 14C ages from the humin fraction indicate the dominance of C3 plants since ∼17,000 cal BP. Palynological analysis of a sediment core indicates the presence of Atlantic Forest vegetation since 7700 cal BP. Changes in the relative abundance of tree ferns and palms suggest the predominance of a humid period from ∼7000–4000 cal BP and establishment of the modern seasonal climate at ∼4000 cal BP. Data indicate maintenance of the regional forest coverage since the late Pleistocene, corroborating previous suggestions that this region was a forest refuge during less humid periods of the late Pleistocene and Holocene. Some plant taxa with currently divided distributions between Amazonia and the Atlantic Forest colonized the region since at least 7500 cal BP, indicating an earlier connection between Amazonia and Atlantic Forest.

Distribution of organic carbon in the stable soil humic fractions as affected by tillage management

Soil humus comprises a large and stable pool of soil organic matter (SOM); hence a better understanding of the fate of C in soil humic fractions can provide valuable information for the development of alternative tillage practices that will lead to long-term soil C sequestration. We used δ13C techniques to investigate the effects of tillage on the dynamics of native (C3–C) and corn derived C (C4–C) in fulvic acid (FA), humic acid (HA) and humin fractions. Humic substances were extracted from soils cropped to corn for 11 yr and managed under either conventional (CT) or no-tillage (NT), and from a conventionally tilled soil under > 55 yr of tobacco/rye rotation. No-tillage resulted in higher proportions of C4–C in the upper 5 cm and generally lower C4–C proportions below 5 cm than CT. Up to 31, 27 and 34% of C4–C were assimilated into FA, HA and humin fractions, respectively, indicating that even the humin fraction, often described as passive, old or resistant, acted as a sink of recently added C, and that it is heterogeneous with some of its components being young. Recovery of large proportions of C3–C in the humic fractions demonstrated their importance in the long-term stabilization of SOM. Within each sampling depth, there were no unique differences in the distribution of C3–C among the three humic fractions, suggesting similar turnover of C3–C in all the fractions. Therefore, there was no unique active fraction corresponding with the concept of C pools with defined turnover characteristics used in models of SOM turnover. Key words: Soil humic fractions, corn derived C, native C, δ13C techniques, tillage practices