Differences between Humic Substances from Riverine,Estuarine, and Marine Environments Observed by Fluorescence Spectroscopy

Sixteen samples of fulvic acids and XAD‐4 fractions of riverine, estuarine, coastal, and open ocean origin have been studied by emission and synchronous molecular fluorescence spectroscopy. Certain features of the molecular fluorescence are related to the nature, the content, and the origin of those aquatic humic substances (HS). Riverine HS appear several times richer in fluorophores than marine HS, which can be well observed by emission fluorescence spectroscopy. Synchronous‐scan spectra of fulvic acids and of XAD‐4 fractions from the aquatic environments studied, emphasized the quality differences of their fluorophores. These features are useful as tracers of humic substances related with their natural environment source or even with their anthropogenic origin.     

The Analysis of the Seasonal,Spatial, and Compositional Distribution of Humic Substances in a Subtropical Shallow Lake

The spatial and temporal distribution of humic substances in aquatic ecosystems can have important effects on ecosystem productivity, negatively impacting primary productivity while positively impacting secondary productivity. In the present investigation, a large shallow lake ecosystem was studied to determine the spatial and seasonal variation of the composition and concentration of humic substances. Concentrations of total dissolved organic matter, humic acid, and fulvic acid were found to display significant spatial distributions (1.3…13.5 mg/L, DOM; 0.1…5.4 mg/L, HA). The distribution is described by using mapping techniques and the analysis of the spatial distribution of the lake. An analysis of the seasonal variations also indicated the dependence of the occurrence of these compounds on meteorological and hydrological conditions. To identify the potential sources of these organic materials, an analysis was made of the ratio of humic and fulvic acid fractions and total DOM. It was found that areas of high DOM concentration coincided with the areas of highest HA percentage of total DOM. Furthermore using the ratio of the normalised concentrations of HA, FA, and residual DOM (< 5000 g/mol) it was found that areas dominated by each are spatially distinct. This confirms the hypothesis that in these shallow lakes, photodegradation and bacterioplankton activity will create a residence time dependent zonation of each component of the total DOM.     

Spectroscopic Characterization of Fulvic Acid Fractions of a Contaminated Groundwater

Two fulvic acid (FA) samples taken from a former gas production facility in the Southwest of Germany were characterized using advanced fluorescence techniques. Steady-state fluorescence (fluorescence excitation, synchronous fluorescence) as well as time-resolved fluorescence were applied. Distinct differences between the sample B22 FA taken within the contamination plume and the sample B53 FA taken downstream were found. Comparison with a model compound for metabolites and humic substances revealed that due to the downstream passage the characteristics of the dissolved organic matter became more humic-like. The assignment of single classes of compounds in the sample B22 FA is discussed in terms of their synchronous fluorescence spectra and fluorescence decay time distribution.     

Spectroscopic Characterization of Organic Structures and Organic‐Inorganic Interactions in Paper Mill Sludge

The organic composition and organic‐inorganic interaction in paper mill sludge (PS) solvent extracts (hexane, ethyl acetate, acetone and ethanol) and humic fractions, humic acid (HA) and humin (HU) were studied by electron paramagnetic resonance spectroscopy (EPR), proton and carbon‐13 nuclear magnetic resonance spectroscopy (¹H NMR; ¹³C NMR), Fourier‐transformed infrared spectroscopy (FTIR), and ultraviolet‐visible spectroscopy (UV‐vis). The strategy of fractionating the PS, sequentially, with organic solvents of increasing polarity is a reliable analytical procedure for humic substance sample separation because it results in more purified fractions. FTIR, ¹H NMR and ¹³C NMR results showed that hexane extract consisted mainly of aliphatic hydrocarbon structures. Their contents in the extracts decreased as the polarity of the extracting solvent increased and the content of oxygen functional groups increased. Carboxylic and carboxylate functional groups were found in the acetone extract, and ester and ether functions were predominantly found in the ethanol extract. EPR spectra revealed some Fe³⁺ complexes with rhombic structure (g₁ = 4.3; g₂ = 9.0) in the humic fractions and in all solvent extracts, except hexane. Quasi‐octahedral Fe³⁺ complexes (g = 2.3; ΔH_pp ≤ 400 G) were found in the HU fraction and in the acetone extract. The organic free radical content in the HA fraction was higher than the non‐fractionated PS sample and HU fraction.     

The freshwater dissolved organic matter fluorescence–total organic carbon relationship

The fluorescent properties of dissolved organic matter (DOM) enable comparisons of humic‐like (H‐L) and fulvic‐like (F‐L) fluorescence intensities with dissolved organic carbon (DOC) in aquatic systems. The fluorescence‐DOC relationship differed in gradient, i.e. the fluorescence per gram of carbon, and in the strength of the correlation coefficient. We compare the fluorescence intensity of the F‐L and H‐L fractions and DOC of freshwater DOM in north Shropshire, England, featuring a river, wetland, spring, pond and sewage DOM sources. Correlations between fluorescence and DOC varied between sample sites. Wetland water samples for the F‐L peak gave the best correlation, r = 0·756; the lowest correlation was from final treated sewage effluent, r = 0·167. The relationship between fluorescence and DOC of commercially available International Humic Substances Society standards were also examined and they generally showed a lower fluorescence per gram of carbon for the F‐L peak than the natural samples, whereas peat wetland DOM gave a greater fluorescence per gram of carbon than river DOM. Here, we propose the strength of the fluorescence–DOC correlation to be a useful tool when discriminating sources of DOM in fresh water. Copyright © 2007 John Wiley & Sons, Ltd.     

Protein‐like fluorescence intensity as a possible tool for determining river water quality

The results of a comparison between chemical water quality determinants and river water fluorescence on the River Tyne, NE England, demonstrate that tryptophan‐like fluorescence intensity shows statistically significant relationships between nitrate, phosphate, ammonia, biochemical oxygen demand (BOD) and dissolved oxygen. Tryptophan‐like fluorescence intensity at the 280 nm excitation/350 nm emission wavelength fluorescence centre correlates with both phosphate (r = 0·80) and nitrate (r = 0·87), whereas tryptophan‐like fluorescence intensity at the 220 nm excitation/350 nm emission wavelength centre correlates with BOD (r = 0·85), ammonia (r = 0·70) and dissolved oxygen (r = ?0·65). The strongest correlations are between tryptophan‐like fluorescence intensity and nitrate and phosphate, which in the Tyne catchment derive predominantly from point and diffuse source sewage inputs. The correlation between BOD and the tryptophan‐like fluorescence intensity suggests that this fluorescence centre is related to the bioavailable or labile dissolved organic matter pool. The weakest correlations are observed between tryptophan‐like fluorescence intensity and ammonia concentration and dissolved oxygen. The weaker correlation with ammonia is due to removal of the ammonia signal by wastewater treatment, and that with dissolved oxygen due to the natural aeration of the river such that this is not a good indicator of water quality. The observed correlations only hold true when treated sewage, sewerage overflows or cross connections, or agricultural organic pollutants dominate the water quality—this is not true for two sites where airport deicer (propylene glycol, which is non‐fluorescent) or landfill leachate (which contains high concentrations of humic and fulvic‐like fluorescent DOM) dominate the dissolved organic matter in the river. Mean annual tryptophan‐like fluorescence intensity agrees well with the General Water Quality Assessment as determined by the England and Wales environmental regulators, the Environment Agency. Copyright © 2004 John Wiley & Sons, Ltd.     

Influence of IHSS Standard and Reference Materials on Copper and Mercury Toxicity to Vibrio fischeri

The reduction in light emission of the marine bacterium Vibrio fischeri used in the standard Microtox^® bioassay was measured for the metals copper and mercury. The concentration at which the light emission was reduced by 50% (EC₅₀) was determined to be (3.43 ± 0.83) μmol/L for Cu²⁺ and (0.66 ± 0.01) μmol/L for Hg²⁺. The reduction of the toxicity of these metals by humic and fulvic acids were studied using IHSS Standard and Reference Materials. Copper toxicity was reduced 17...20% by the soil and peat fulvic acids and 9...20% by the aquatic fulvic acids. While there appeared to be little difference in the reduction of Cu toxicity by fulvic acids from soils, peats, or aquatic systems, Hg toxicity was reduced 3.6...7.3% by the soils and peats, while aquatic fulvic acids reduced Hg toxicity 14...16%. Soil fulvic acids appear to have significantly less capacity to reduce Hg toxicity than Cu toxicity. Humic acids had much higher reductions of Cu toxicity (44...124%) compared to the fulvic acids, with little difference between aquatic and soil or peat humic acids, 44...124% and 67...100%, respectively. However, humic and fulvic acids, regardless of origin, had approximately the same effect on Hg toxicity with 3.5...16% reduction by fulvic acids and 8...20% reduction by humic acids. Unlike the fulvic acids, no clear trend was observed relative to origin of the humic acids. There was no correlation between percent reduction of Cu or Hg toxicity by the organic compounds and copper binding capacity (CuBC), C/N ratio, or carboxyl content of the materials. Examination of natural organic matter (NOM) isolated by reverse osmosis techniques from three water sources had reductions of both Cu and Hg toxicity that were most similar to the Suwannee River and Nordic fulvic acids.     

Kojima湖表层水的腐殖质特征

Humic compounds are one of the major substances that affect the COD value of the surface water of Lake Kojima. The humic compounds in the lake water were studied with a spectrofluorophotometer. The 3-dimensional fluorescence spectrum suggests that two kinds of fluorescent substances exist in the lake water. One shows an fluorescence excitation maxim (Ex) at about 240 nm and an emission maxim (Em) at 413 nm. The other has an Ex at 330 nm and an Em at 418 nm. The former peaks are associated with humic acid and the latter peaks are associated with fulvic acid. These peak intensities are proportional to the COD values. The fulvic acid in the lake water may be enriched by the effluent from domestic water treatment plants.     

Spectroscopic investigation of humic substances in a tropical lake during a complete hydrological cycle

Fluorescence and UV‐VIS techniques were employed for the investigation of natural organic matter (NOM) of a tropical lake. The relationships of absorbance/dissolved organic carbon (A/DOC), fluorescence intensity/dissolved organic carbon (FI/DOC), fluorescence ratio (FR), and peak wavelength with the highest intensity (PW) were used to distinguish the pedogenic or aquagenic origin of NOM. The values of FR, PW and A₂₈₅/DOC of high waters (HW) or flooded period samples and of low waters (LW) period samples of the dry season, except for September 2002, confirm the predominance of pedogenic material. The spectra of water were similar to the standard fulvic acid (FA), and the spectra of FA from the lake were similar to the nearby soils, indicative of pedogenic predominance. The results confirm that the dissolved NOM of Patos Lagoon – MS (Brazil), in all sampling periods, predominantly consisted of humic substances (FA) of pedogenic origin.     

Association of Humic Substances: Verification of Lambert‐Beer Law

The precision UV/Vis‐absorption spectra of a naturally occurring aquatic humic substance were measured in the range of concentration 0.003 to 1.4 g/L DOC (dissolved organic carbon). Lambert‐Beer law is well obeyed at low concentrations whereas deviations have been observed for DOC concentrations above 0.1 g/L: they are interpreted in terms of self‐solvation and formation of micelles.     

The Geochemical Effects of Microbial Humic Substances Reduction

As part of a study on microbial redox alteration of humic substances we investigated the potential effect of this metabolism on the fate of heavy metals and hydrocarbons as a result of conformational alteration of the humic molecular structure due to microbial reduction. Our studies indicate that the microbial reduction of humic acids (HA) results in significant morphological and geochemical alterations. X‐ray microscopy analysis indicate that the conformational structure of the humic colloids is altered as a result of the redox change. In the reduced state, the HA appeared as small dense particles, on reoxidation, large loose aggregates were formed. In addition, spectrofluorometric studies indicated that the binding capacity of the HA for naphthalene was decreased by 10% when the HA was reduced. Similarly, the reduced HA yielded higher surface tension values at all concentrations tested which is indicative of a more hydrophilic and less hydrophobic solute. On reoxidation, the surface tension values reverted back to values similar to those obtained for the untreated oxidized HA. These data indicate that the hydrophobicity of the HA is altered on biological reduction of the HA and that this alteration is reversible. In contrast the reduced HA demonstrated a 15% higher affinity for heavy metals such as divalent cobalt than the oxidized HA. In addition to increasing the binding capacity of HA for heavy metals, the reduction of the HA also decreased the bioavailability and toxicity of bound heavy metals such as chromium. When incubated in the presence of Cr(III) and HA, cells of Escherichia coli grew much more rapidly in the presence of the reduced HA suggesting that the higher metal binding capacity of the reduced humic substances resulted in a removal of the Cr(III) from solution and hence reduced its bioavailability and toxicity. These studies demonstrate that HA redox state and reduction of humic acids by microorganisms can have a significant effect on the molecular morphology and binding constants of HA for heavy metals and hydrocarbons and also directly affects the bioavailability of these compounds in the environment.     

Adsorbed and Bound Residues in Fulvic Acid Fractions of a Contaminated Groundwater – Isolation:, Chromatographic and Spectroscopic Characterization

Humic substances (HS) were isolated from two contaminated groundwater samples (B22 and B53) from a site of a former gas facility. The isolation yielded almost only the fulvic acid fractions (FA). For characterization spectroscopic (UV, fluorescence) and chromatographic techniques (hydrophobic interaction chromatography – HIC as well as size-exclusion chromatography – SEC) were applied. The sample designated B22 FA was collected from the contamination plume whereas the sample B53 FA was collected downstream. Distinct differences were exhibited by these samples. The UV and fluorescence spectra as well as the HIC and SEC chromatograms of the B53 FA sample resemble those of the FA fraction obtained from natural water (groundwater, bog). The HIC and SEC chromatograms reveal the presence of organic compounds in B22 FA which can be derived from coal tar contaminants or their metabolites. Some of the compounds can be extracted from the FA fraction with non-polar organic solvents indicating adsorptive forces between the contaminants and the FA fraction.     

Acid Rain and Natural Organic Matter (NOM)

Twentyfive years of research on the effects of acid rain on rivers and lakes has, to a very small extent, documented changes in the nature and properties of natural organic matter (NOM). In Western Norway, a "whole-watershed-artificial-acidification-experiment" took place in the period 1988–1996. The goals of this long-term experiment were to study the role of NOM in acidification of surface water and the effects of acid precipitation on the quality and properties of NOM. In the HUMEX project (Humic Lake Acidification Experiment) one half of a lake and the corresponding catchment was artificially acidified with H₂SO₄ and NH₄NO₃ over a period of 5 years. The other half of the lake and catchment served as a control. In addition to monitoring of the general chemical composition of the water from the two lake halves, a number of other chemical and biological characteristics were studied. Here, we report the results related to changes in the nature and chemical properties of NOM. During the first few years of acidification, a significantly lower concentration of NOM was recorded in the acidified half of the catchment, compared with the control. However, statistical analyses of all data (covering a 2-years pre-treatment period and 5 years of treatment) related to the concentration of NOM (TOC, colour, and UV absorbance) did not suggest any significant effect on the quantity of NOM. This apparent discrepancy between the initial decrease in the concentration of NOM and no effect when the whole 5-years period is considered, may be due to the results of two different simultaneous processes. The results suggest that there first was a reduction of TOC and colour, as a consequence of the acidification, followed by an increase, perhaps owing to increased fertilisation (nitrogen) and in addition to a general temperature increase during the period. In addition, short-term studies of the aquatic surface microlayers, lipophilicity of the NOM, content of organic sulfur, and molecular size indicate differences in the quality of the NOM between the two lake halves, which could affect light absorption.     

Photochemical activity and fluorescence of electrophoretic fractions of aquatic humic matter

A combination of sephadex gel-chromatography and polyacrylamide gel electrophoresis was used to obtain stable electrophoretic fractions with different molecular sizes from a preparation of humic substances isolated from river water by reverse osmosis. The photochemical activity of humic substances and their fractions was determined. For the first time it is shown that low-molecular fractions of aquatic humic substances, when irradiated by polychromatic light, can intensely fluoresce and initiate the formation of singlet O₂, effectively destroying model compound furfural. The photochemical activity and intensity of fluorescence of high-molecular fractions is insignificant. The obtained data will help to extend the current knowledge in the field of natural water hydrochemistry about the ways of its natural self-purification from anthropogenic pollutants.     

Interactions of Chlorine with Natural Organic Matter and Formation of Intermediates: Evidence by Differential Spectroscopy

This communication describes the use of differential absorbance spectroscopy to explore the intermediates formed during halogenation of natural organic matter (NOM). The differential spectra of chlorinated NOM comprise two contributions. The primary component is negative and has a peak near 270 nm. The shape of this band is independent of chlorine dose, and its intensity increases monotonically with Cl₂ dose. The second component is positive, with a well‐defined peak near 280 nm and another, broader band in the 340...380 nm range. The second component is noticeable at low chlorine concentrations but disappears with increasing Cl₂ dose. We attribute this component to aromatic chlorinated intermediates formed prior to the release of identifiable smaller species such as haloacetic acids. We believe that this component of the differential spectra can be used to probe the identity, formation and breakdown of the halogenated intermediates.     

Degradation and Transformation of Aquatic Humic Substances by Laccase-producing Fungi Cladosporium cladosporioides and Polyporus versicolor

A phenoloxidase-producing fungus – Cladosporium cladosporioides – was isolated from water of a bog lake. At high concentrations of carbon (333 mM) and nitrogen (12 mM), the fungus demonstrated the formation of a laccase and a high ligninolytic activity. After addition of riverine or groundwater humic substances into culture media (ca. 1 mg mL^–1), the fungus showed the ability of degrading (utilizing) of about 60% of these substances. However, the exact quantification of the degree of degradation was difficult because of adsorption of humic matter on fungal mycelium. Reisolated humic substances were lower in aromatic and higher in aliphatic structures. A crude enzyme from the C. cladosporioides culture showed only low activity in decolorizing humic substances, whereas decolorization up to 50% was observed when using a laccase preparation from Polyporus versicolor, and especially in the presence of a redox mediator.     

Chemical and spectroscopic characterization of humic substances from sediment and riparian soil of a highly polluted urban river (Suquía River,Córdoba,Argentina)

The Suquia River,the largest urban river in Cordoba(Argentina),has been severely polluted for decades.Actions must be taken to restore its environmental quality by managing riparian zones for increased water-self purification.The current study aimed to characterize organic matter(OM) dynamics and humic substances(HS) spectrochemical properties along the lower-middle basin of the Suquia River.Riparian soil(0-20 cm) and sediment(0-10 cm) samples were collected from a reference location(S1)and four polluted sites(S2-S5) during a low-flow period.The contents of soil and sedimentary OM and HS fractions were analyzed by wet oxidation,as well as HS Fourier transform infrared(FT-IR) and ultraviolet-visible(UV-Vis) spectrochemical properties.The OM and HS fractions from riparian soil were high upstream of Cordoba City(S1 and S2,50.2-50.4 g/kg OM) and within a 50 km downstream location(S5,30.9 g/kg OM) owing to a surplus of fresh plant biomass-carbon(C) inputs.Highly heterogeneous sediment samples did not show any significant differences among sites(P 0.05).The lowest values of the ratio of absorbances at 465 and 665 nm(E4/E6)(1.78) and the Δ log K(0.15) coefficient(a measure of HS maturity degree) were obtained downstream of Cordoba City,for both riparian soil and sediment,indicating that HS were enriched by more condensed aromatic structures within highly degraded portions of the river.All samples exhibited similar IR spectra,implying overlapping recalcitrant-C structures at the functional group level,but with different absorbance intensity.Data from the current study constitute a baseline for understanding the chemical nature of HS from sediment and riparian soil along the Suquia River and can be used as a reference for future studies tracking OM compositional changes over time.     

Anwendung einer enzymatischen Methode zur Charakterisierung von Huminstoffen Enzymatic Characterization of Humic Substances

A method is described for the specific hydrolysis of amino acids from humic substances by using the proteolytic enzyme “Pronase E”. The amino acids are analyzed by a fluorescence detector after precolumn derivatization and HPLC-separation. The pattern and the concentration of amino acids after the enzymatic hydrolysis are set up for humic substances of different origin. Due to the results the enzymatic hydrolysis is suitable for the characterization of humic substances with a nitrogen content higher than 3%. As shown in a kinetic study of the hydrolysis of a fulvic acid the influence of humic substances on the enzyme activity increases in case of lower nitrogen content. Due to the specificity of the proteolytic enzyme the operationally defined method is limited as shown by a protein of a known amino acid content. The amino acids leucine, valine, tyrosine and histidine are released to the highest extent. The determined total extent for the hydrolysis of Insulin B is 26.34% and for Casein 38.37%. By comparison of the enzymatic hydrolysis with the classic acid hydrolysis the advantages of the biochemical method are the simple experimental handling and the mild reaction conditions. Amino acids which are very unstable like glutamine and asparagine can only be analyzed by the enzymatic hydrolysis.