Derivatization of Humic Compounds: An Analytical Approach for Bound Organic Residues

Abstract

Bound residues of pesticides and their metabolites are defined as being nonextractable with organic solvents, but partly extractable together with the humic matrix by NaOH or other solvents suitable to extract humic compounds. Recently, an improvement in humus extraction from soils was achieved upon derivatization of the organic matter with silylating reagents at room temperature. By this method 70–90% of the organic carbon or nitrogen either from soil or from humin became soluble in organic solvents. The extracts were analyzed by means of ¹³C NMR-spectroscopy. The spectra were well resolved with signal-separation of less than 1 ppm. The extracted humic compounds were of rather low molecular weight, ranging from 300 to 4000 to 6000 d or more.

¹⁴C-labeled residues of pesticides or other xenobiotics found to be nonextractable after exhaustive organic solvent extraction became readily dissolved along with most of the humic matrix using this derivatization procedure. Between 60–80% of ¹⁴C anilazine residues or of ¹⁴C-labeled chlorinated phenols or anilines originating from both previously solvent extracted soil samples or from humin became solubilized in organic solvents.     

Efficiency of Different Methods and Solvents for the Extraction of Polycyclic Aromatic Hydrocarbons from Soils

The efficiencies of supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), Soxhlet, and ultrasonic extraction in the analysis of polycyclic aromatic hydrocarbons (PAHs) in soils were evaluated. Solvents with different polarity were used to extract the PAHs from two soils, one with high and one with low contamination level. ASE showed good results with all solvents almost independent of the solvent polarity and the best results with acetone-toluene (1 : 1). Ultrasonic extraction with acetone-toluene for the uncontaminated soil and acetone-ethanolamine for the highly contaminated also showed good recoveries. The time-consuming Soxhlet extraction with pentane or dichloromethane was less effective. The PAH recovery from SFE was related to the soil matrix or the contamination level. The best extraction conditions (CO 2 /10% pentane) are successful for the soil with a low contamination level and a high humic acid content whereas the extractions of the highly contaminated soil gave poor results irrespective of the solvent used.     

Comparative study of methods for extraction and purification of environmental DNA from soil and sludge samples

An important prerequisite for successful construction of a metagenome library is an efficient procedure for extracting DNA from environmental samples. We compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity, and time requirement. A special focus was on methods that are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.1 g) to enable a highly parallel approach. Direct extraction procedures yielded on average 100-fold higher DNA amounts than indirect ones. A drawback of direct extraction was the small fragment sizeof approx 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the eDNA. Only the commercial kit and a direct extraction method using freeze-thaw cell lysis in combination with an in-gel patch electrophoresis with hydroxyapatite to remove humic acid substances yielded DNA, which was completely digested by all restriction enzymes. Moreover, only DNA extracted by these two procedures could be used as template for the amplification of fragments of several 16S rDNA, 18SrDNA groups under standard polymerase chain reaction conditions.     

Molecular features of organic matter in diagnostic horizons from andosols as seen by analytical pyrolysis

Andosols are usually formed from volcanic substrates, with thick A horizons high in organic carbon mainly in the form of stabilized humic fractions. The peculiar properties of these soils are, to large extent, affected by poorly crystalline materials like allophanes, imogolite and other Fe and Al oxyhydroxides that induce intense organo-mineral interactions which are considered to play a relevant role in OM protection against biodegradation as regards other soils developed under similar climatic conditions. Little is known about the molecular composition of this stabilized OM in a soil scenario often considered as an efficient C-sink in terms of C sequestration processes.Whole soil samples in addition to isolated humic and fulvic acids from organic A horizons of three soils with andic properties and one non-andic soil (Sodic Cambisol) from the island of Tenerife (Canary Islands, Spain) were analysed by double shot pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS) in order to get some insight on the molecular composition of different structural domains of progressive structural stability.Clear differences were found between soils, both in thermal desorption and pyrolysis behaviour of humic substances. In general the results suggest large yields of aliphatic (both alkyl and carbohydrate-derived pyrolysis compounds) pointing to high-performance processes of incorporation of aliphatic humic constituents in andosols probably favoured by interactions with amorphous minerals, whereas in non-andic soils the latter are comparatively less stabilized and are removed in early pyrolysis stages as if they occurred as loosely joined, thermoevaporation-released products.In fact, compared to Cambisol, humic and fulvic acids from andic soils led to comparatively richer pyrograms and compound assemblages. The lack of similar amounts of these loosely joined, mainly aliphatic structures after thermal desorption of the whole andic soils indicate an origin for humic substances based on rapid sequestration of C-forms of recent (litter or microbial) origin.     

A solid-phase extraction procedure for the clean-up of thiram from aqueous solutions containing high concentrations of humic substances

A simple solid-phase extraction (SPE) procedure with an octadecyl bonded phase silica (C₁₈) was developed for clean-up of the fungicide thiram from aqueous solutions containing high concentrations of humic substances, for future studies of thiram adsorption onto solid humic substances or soils. Suspensions of humic acids and soil, in aqueous 0.01 M CaCl₂ solution, were prepared and used as samples. These extracts were spiked with thiram and immediately applied to a C₁₈-SPE cartridge. Thiram was eluted with chloroform and its concentration measured by spectrophotometry at 283 nm. Non-spiked aqueous extracts (blanks) and a control sample of thiram in 0.01 M CaCl₂ aqueous solution were also prepared and submitted to the same SPE procedure. The results show that humic substances are extensively retained by the C₁₈ cartridge but are not eluted with CHCl₃. Recoveries of 100-104% were obtained for thiram in the presence of humic substances. The SPE procedure described in this work is an efficient clean-up step to remove the interference of humic substances absorbance and to be coupled to any spectrophotometric or HPLC-UV method, usually used for thiram analysis in food extracts.     

Molecular features of organic matter in diagnostic horizons from andosols as seen by analytical pyrolysis

Andosols are usually formed from volcanic substrates, with thick A horizons high in organic carbon mainly in the form of stabilized humic fractions. The peculiar properties of these soils are, to large extent, affected by poorly crystalline materials like allophanes, imogolite and other Fe and Al oxyhydroxides that induce intense organo-mineral interactions which are considered to play a relevant role in OM protection against biodegradation as regards other soils developed under similar climatic conditions. Little is known about the molecular composition of this stabilized OM in a soil scenario often considered as an efficient C-sink in terms of C sequestration processes.

Whole soil samples in addition to isolated humic and fulvic acids from organic A horizons of three soils with andic properties and one non-andic soil (Sodic Cambisol) from the island of Tenerife (Canary Islands, Spain) were analysed by double shot pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS) in order to get some insight on the molecular composition of different structural domains of progressive structural stability.

Clear differences were found between soils, both in thermal desorption and pyrolysis behaviour of humic substances. In general the results suggest large yields of aliphatic (both alkyl and carbohydrate-derived pyrolysis compounds) pointing to high-performance processes of incorporation of aliphatic humic constituents in andosols probably favoured by interactions with amorphous minerals, whereas in non-andic soils the latter are comparatively less stabilized and are removed in early pyrolysis stages as if they occurred as loosely joined, thermoevaporation-released products.

In fact, compared to Cambisol, humic and fulvic acids from andic soils led to comparatively richer pyrograms and compound assemblages. The lack of similar amounts of these loosely joined, mainly aliphatic structures after thermal desorption of the whole andic soils indicate an origin for humic substances based on rapid sequestration of C-forms of recent (litter or microbial) origin.     

土壤胡敏酸的^13C核磁共振研究

胡敏酸(HA)是土壤有机质的重要组分,其结构性质与土壤形成和肥力特性有密切关系,长期以来一直是土壤化学研究的难点和重点之一。NMR是研究HA结构的有效手段。自Bar-ton和Schnitzer(1963)首次用~1H NMR研究土壤有机质以来,国外已先后应用~1H、~(13)C NMR和CP-MAS-~(13)C NMR等对土壤及其它来源的HA进行了许多研究,并取得引人注目的进展。但在国内,这方面研究还刚刚开始。本文应用~(13)C NMR方法对东北几种主要耕作土壤以及泥炭和猪粪的HA进行了结构表征,为土壤腐殖酸化学的基础研究提供了资料。     

Extraction of humic acids in two-phase aqueous polymeric systems

Extraction of humic acids isolated from various soils was studied in polyethylene glycol(NH₄)₂SO₄, polyethylene glycol-dextran, and polyethylene glycol-dextran sodium salt two-phase aqueous polymeric systems. It was shown that, in all systems, humic acids are extracted into the polyethylene glycolrich phase. It was studied how the composition of the extraction system, molecular weight of the polymer, and the sample nature affect the distribution ratios of humic acids.     

Hyphenated techniques of thermal analysis for characterisation of soil humic substances

Our aim was to investigate the thermal behaviour of humic substances extracted from temperate and tropical soils by means of hyphenated techniques of thermal analysis (e. g. simultaneous thermal analysis DSC/TG coupled with mass spectrometry, MS, for the analysis of evolved gas, EGA) in order (i) to verify whether the chemical composition of isolated humic substances also reflected the differences in microbial parameters previously measured in related soil samples and (ii) to identify suitable indices of thermal stability. Our results show that the investigation of humic substances by thermal methods can provide information on soil organic matter dynamics. Differences in thermal behaviour between the two groups of soils were found. The indices of thermal stability here proposed, IR (index of thermal recalcitrance), and ID (index of thermal decomposability) clearly showed that in humic substances from tropical soils the thermally recalcitrant organic fraction dominated, whilst in temperate humic substances the opposite held. This agrees with previous results on the microbial dynamics and organic matter turnover of the respective soils and indicates that these indices of thermal stability could represent a useful tool in soil environmental quality investigations.     

Anodic stripping voltammetric determination of lead and cadmium in soil extracts

Two single-step extraction procedures validated by BCR, extraction with 0.43 mol dm⁻³ acetic acid and 0.05 mol dm⁻³ EDTA, are used for assessing bioavailable species of Pb and Cd in soils. After an extensive study of interferences of humic substances and using standard additions procedures, the extractable contents of Pb and Cd have been determined by DPASV directly in soil extracts with a good reproducibility (RSD varied from 1.0 to 7.9 %). Pb(II) and Cd(II) were released from complex in EDTA extracts by pH adjustment to 1 with 1 mol dm⁻³ HCl. The results were compared with those obtained with atomic absorption spectrometry and indicated reasonable agreement of both methods. Exchangeable forms represented 0.2 to 3.3 % and 13.0 to 63.6 % of total content of Pb and Cd in soils, respectively. EDTA extraction released from soils 7.9 to 29.9 % and 33.5 to 59.6 % of total content of Pb and Cd, respectively.     

Stability and Mobility of Metal-Humic Complexes Isolated from Different Soils

The contamination of potable water aquifers by heavy metals is one of the most severe environmental threats. For the transport of heavy metals from various types of contaminated sites into the ground water and also into surface water aquifers, humic substances (HS) are recognized to be of main importance. Dissolved in natural waters humic substances are readily complexed with a variety of metal ions. Therefore, humic substances are of cardinal importance for the migration and, consequently, the pollution of ground waters with heavy metals. Our paper presents the results of a comprehensive comparison of several isolated humic acids of soils of different origin (different geochemical milieu) and their metal complexes. Two polluted sites in Germany, which differ in their geochemical milieu (pH-value) were selected. The aim of our experiments was to describe the properties of terrestrial humic substances depending on their origin and genesis as well as the effects of the transport of humic substance-bound metals into the water-unsaturated soil zone. After determination of heavy metals in the soils by photon activation analysis the activated soil was used as an inherent tracer in batch experiments with the isolated humic acid. After adsorption of the loaded humic acid on an XAD-8 resin column, the partition of metals mobilized by humic acids could be quantified. There are correlations of the formation of metal-humic complexes with the soil pedogenes, with the pH-value as well as with the humic acid concentration.     

Investigation of distribution of soil antimony using sequential extraction and antimony complexed to soil-derived humic acids molar mass fractions extracted from various depths in a shooting range soil

Trace metal contamination from bullet fragments in shooting ranges is a major environmental concern. In particular, trace metals such as lead, antimony, and copper are toxic and have the potential to enter groundwater supplies and to be absorbed by plants. Soil humic acids can play a critical role in mobilizing some of these released metals through complexation. The purpose of this study is to investigate the antimony complexed to soil-derived humic molar mass fractions extracted from various depths in a shooting range soil and to examine the distribution of antimony in various fractions of shooting range soils using sequential chemical extraction approach. The surface soil and soil core samples from a local shooting range were collected. Soil-derived humic acids were extracted from different depths of the top soil layer and characterized by various spectroscopic methods. Results of sequential chemical extraction demonstrated that Sb was found in shooting range in the upper 30 cm depth of the soil core. Highly elevated Sb is present in the exchangeable and ammonium acetate extracted fractions. Antimony is also present in the residual fraction in both surface and core soil samples, but is most likely present in a lithic phase which may not be readily bio-available. Leached antimony complexed to soil humic acid molar mass fractions was determined by size exclusion chromatography coupled to inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The results demonstrate that Sb is ‘tightly’ bound to humic acid mass molar mass fractions and confined in the top 10 cm of soil-derived humic acids.     

"In-gel patch electrophoresis:" a new method for environmental DNA purification

Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple, and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and polymerase chain reaction (PCR) amplifications.     

Concentration levels of rare-earth elements and thorium in plants from the Morro do Ferro environment as an indicator for the biological availability of transuranium elements

Plants and soils from a natural thorium and rare-earth element occurrence (Morro do Ferro, Brazil) were analyzed by alpha spectrometry (Th) and ICP-AES (REE), after pre-concentration of the elements by solvent extraction, co-precipitation and ion exchange procedures. Leaching experiments with humic acid solutions and different soils were performed to estimate the fraction of elements biologically available. High concentrations of the light rare-earth elements (LREE) and of Th, reaching some hundreds of g/g-ash, were measured in plant leaves from the areas of the highest concentration of these elements in soil and in near-surface waters. Chondrite normalized REE plots of plant leaves and corresponding soils are very similar, suggesting that there is no significant fractionation between the REE during uptake from the soil solution and incorporation into the leaves. However, Ce-depletion was observed for some plant species, increasing forSolanum ciliatum in the sequence: leaves<fruits<seeds. Soil to plant concentration ratios (CR's) for Th and the REE, based on the total concentration of these elements in soils, are in the range of 10^–3 to 10^–2. Leaching experiments confirmed the importance of humic acid complexation for the bio-uptake of Th and REE and further showed that only a very small fraction of these elements in soil is leachable. The implications of these results on the calculated CR's will be discussed.     

Sequential extraction and thermal desorption of mercury from contaminated soil and tailings from Mongolia

Mercury forms in contaminated environmental samples were studied by means of sequential extraction and thermal desorption from the solid phase. The sequential extraction procedure involved the following fractions: water soluble mercury, mercury extracted in acidic conditions, mercury bound to humic substances, elemental Hg and mercury bound to complexes, HgS, and residual mercury. In addition to sequential extraction, the distribution of mercury species as a function of soil particles size was studied. The thermal desorption method is based on the thermal decomposition or desorption of Hg compounds at different temperatures. The following four species were observed: Hg⁰, HgCl₂, HgS and Hg^(II) bound to humic acids. The Hg release curves from artificial soils and real samples were obtained and their applicability to the speciation analysis was considered.      

The evaluation of different sorbents for the preconcentration of phenoxyacetic acid herbicides and their metabolites from soils

A procedure using alkaline extraction, solid-phase extraction (SPE) and HPLC is developed to analyze the polar herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) together with their main metabolites in soils. An ion-pairing HPLC method is used for the determination as it permits the baseline separation of these highly polar herbicides and their main metabolites. The use of a highly cross-linked polystyrene-divinylbenzene sorbent (PS-DVB) gives the best results for the analysis of these compounds. This sorbent allows the direct preconcentration of the analytes at the high pH values obtained after quantitative alkaline extraction of the herbicides from soil samples. Different parameters are evaluated for the SPE preconcentration step. The high polarity of the main analytes of interest (2,4-D and MCPA) makes it necessary to work at low flow rates (< or =0.5 mL min(-1)) in order for these compounds to be retained by the PS-DVB sorbent. A two stage desorption from the SPE sorbent is required to obtain the analytes in solvents that are appropriate for HPLC determination. A first desorption with a 50:50 methanol:water mixture elutes the most polar analytes (2,4-D, MCPA and 2CP). The second elution step with methanol permits the analysis of the other phenol derivatives. The humic and fulvic substances present in the soil are not efficiently retained by PS-DVB sorbents at alkaline pH's and so do not interfere in the analysis. This method has been successfully applied in the analysis of soil samples from a golf course treated with a commercial product containing esters of 2,4-D and MCPA as the active components.     

Pyrolysis—gas chromatography—mass spectrometry of a low organic matter calcareous soil

A modification to the general method of pyrolysis-gas chromatography of soils which improves the reproducibility of the technique is described. It has been applied to the study of a calcareous soil, low in organic matter and varations have been studied in programs from soil at different depths as well as from the corresponding humic acids, fulvic acids and extraction residues.Most of the major peaks have identified by gas chromatography-mass spectrometry: they correspond to those cited in earlier works. Data are presented on the characteristics of the pyrolysis products of this soil in the relation to their probobic origin. The evolution of organic matter through various pyrolysis relations is also discussed.     

The association between selenium and humic substances in forested ecosystems—laboratory evidence

In the soils and aquatic systems of coniferous forests, selenium is usually associated with humic substances. To clarify further some of the mechanisms involved, labelled and unlabelled selenite were added to two forest floors and to a brownwater lake. Sequential extraction procedures and chromatographic methods were used to evaluate the resulting association between selenium and humic substances. It was observed that the forest floors fixed most of the added selenite by means of microbial reductive incorporation and that selenium was preferentially incorporated into lowmolecular-weight fractions of the humic substances. By contrast, selenium reduction was much slower in the brown-water lake and instead, inorganic complexation of selenite to metal–humic complexes was important during the experiment, provided that the concentrations of competing ligands were low.     

Fractionation analysis of trace metals in humic substances of soils irrigated with wastewater in Central Mexico by particle induced X-ray emission

The Mezquital Valley in Central Mexico has received wastewater from Mexico City for nearly 100 years. Wastewater brings in organic matter and nutrients but also trace metals. Humic substances, the main components of organic soil matter, are responsible for retaining and regulating the mobility of trace metals in soils. In this study, humic substances were extracted from the soil and separated into distinct fractions (humic acids, fulvic acids and humins). The particle induced X-ray emission (PIXE) technique was applied to determine the metal content in bulk soil as well as in humic acids and fulvic acids not soluble in H₃PO₄. In order to assess whether the long-term input of organic matter and metals modifies the metal association with these humic substances, parcels irrigated for three time periods (5, 47 and 89 years) were selected for this study. It was observed that metals such as Zn and Cu are mainly associated with the humic acids. Fulvic acids retain mainly Cr while Pb is distributed among humic and fulvic acids. It was also observed that in general, metal retention by humic substances increases with irrigation time. Depth also affects metal association with the humic substances.