Involvement of humic substances in regrowth

There appear to be interactions in the distribution system that complicate the ability to use AOC/BDOC as an independent assessment of regrowth potential. Two such complications are the limitation of the assays themselves and the potential interaction between the organic carbon concentration with the presence of disinfectants and pipe materials. To address these interactions, a series of experiments spanning several years have been conducted in model distribution systems at the Center for Biofilm Engineering (CBE) using soil-derived humics. When compared to easily utilized organics, humic substances supported the same order of magnitude of biofilm organisms. As carbon concentration was increased from 500 to 1000 to 2000 ppb, there was no increase in growth rate of the organisms, suggesting zero-order kinetics. If the system was chlorinated, there was less biomass, but growth rates were higher. In the presence of corrosion products, humic-fed systems supported more organisms than a control system fed biologically treated water. When free chlorine was maintained at a residual of about 0.2 mg/l, biofilm numbers on the surfaces were reduced. Phosphate alone did not result in fewer bacteria, while a combination of chorine and phosphate had the best results (lowest biofilm numbers). Adjustment to pH 9 was not effective. Recently completed work compared increasing levels of humic substances in the presence of free chlorine and monochloramine on biofilm growth on a number of surfaces (PVC, epoxy, cement, ductile iron). As the concentration of humic substances was increased from 0, 0.5 to 2 mg/l, there was an increase in biofilm numbers on all surfaces. This effect was the most pronounced on iron surfaces. These results illustrate that carbon compounds not measured by the BDOC or AOC tests may profoundly influence biofilm numbers. In addition, iron surfaces are at much higher risk for elevated biofilm counts in the presence of humic substances, even if disinfection is practiced. However, corrosion control may mitigate this interaction.     

Spectroscopic evidence for ternary complex formation between arsenate and ferric iron complexes of humic substances

Formation of ternary complexes between arsenic (As) oxyanions and ferric iron (Fe) complexes of humic substances (HS) is often hypothesized to represent a major mechanism for As-HS interactions under oxic conditions. However, direct evidence for this potentially important binding mechanism is still lacking. To investigate the molecular-scale interaction between arsenate, As(V), and HS in the presence of Fe(III), we reacted fulvic and humic acids with Fe(III) (1 wt %) and equilibrated the Fe(III)-HS complexes formed with As(V) at pH 7 (molar Fe/As ~10). The local (<5 ?) coordination environments of As and Fe were subsequently studied by means of X-ray absorption spectroscopy. Our results show that 4.5-12.5 μmol As(V)/g HS (25-70% of total As) was associated with Fe(III). At least 70% of this As pool was bound to Fe(III)-HS complexes via inner-sphere complexation. Results obtained from shell fits of As K-edge extended X-ray absorption fine structure (EXAFS) spectra were consistent with a monodentate binuclear ((2)C) and monodentate mononuclear ((1)V) complex stabilized by H-bonds (R(As-Fe) = 3.30 ?). The analysis of Fe K-edge EXAFS spectra revealed that Fe in Fe(III)-HS complexes was predominantly present as oligomeric Fe(III) clusters at neutral pH. Shell-fit results complied with a structural motif in which three corner-sharing Fe(O,OH)(6) octahedra linked by a single μ(3)-O bridge form a planar Fe trimer. In these complexes, the average Fe-C and Fe-Fe bond distances were 2.95 ? and 3.47 ?, respectively. Our study provides the first spectroscopic evidence for ternary complex formation between As(V) and Fe(III)-HS complexes, suggesting that this binding mechanism is of fundamental importance for the cycling of oxyanions such as As(V) in organic-rich, oxic soils and sediments.     

Radioiodination of humic substances via azocoupling with 3-[125I] iodoaniline

A new method is described for radiolabeling humic substances (HS) with iodine radioisotopes. The method radiolabels the electron-rich aromatic moieties of HS with the 3-[125I]iodobenzenediazonium ion via azocoupling. The method uses four steps: (i) 3-aminobenzenetrimethylstannane is synthesized and isolated by using a silica gel column, (ii) 3-[125I]iodoaniline is synthesized and isolated by HPLC, with radiochemical yields of up to 60%, (iii) 3-[125I]iodobenzenediazonium chloride is synthesized, and the reaction mixture from this step is used in step iv to radioiodinate HS with radiochemical yields of up to 95% (with reference to 3-[125I]iodoaniline). The advantage of this method is that it is selective radiolabeling, placing the radiolabel in a specific site (the 3-position of the phenyl ring) within HS molecules, which minimizes unwanted secondary chemical interactions. Investigations of the stability of the radiolabel and the effect of photoreductive dehalogenation showed that there was a negligible release of 125I. The production of radiolabeled HS using this method allows the sensitive detection of HS in laboratory and field studies. In addition, the method offers the possibility of using different iodine radioisotopes simultaneously in investigations using HS.     

Interactions of Tc(IV) with humic substances

To understand the key processes affecting 99Tc mobility in the subsurface and help with the remediation of contaminated sites, the binding constants of several humic substances (humic and fulvic acids) with Tc(IV) were determined, using a solvent extraction technique. The novelty of this paper lies in the determination of the binding constants of the complexes formed with the individual species TcO(OH)+ and TcO(OH)2(0). Binding constants were found to be 6.8 and between 3.9 and 4.3, for logβ1,-1,1 and logβ1,-2,1, respectively; these values were little modified by a change of ionic strength, in most cases, between 0.1 and 1.0 M, nor were they by the nature and origin of the humic substances. Modeling calculations based on these show TcO(OH)-HA to be the predominant complex in a system containing 20 ppm HA and in the 4-6 pH range, whereas TcO(OH)2(0) and TcO(OH)2-HA are the major species, in the pH 6-8 range.     

Binding of mercury(II) to aquatic humic substances: influence of pH and source of humic substances

Conditional distribution coefficients (K(DOM')) for Hg(II) binding to seven dissolved organic matter (DOM) isolates were measured at environmentally relevant ratios of Hg(II) to DOM. The results show that K(DOM') values for different types of samples (humic acids, fulvic acids, hydrophobic acids) isolated from diverse aquatic environments were all within 1 order of magnitude (10(22.5 +/-1.0)-10(23.5 +/- 1.0)) L kg(-1)), suggesting similar Hg(ll) binding environments, presumably involving thiol groups, for the different isolates. K(DOM') values decreased at low pHs (4) compared to values at pH 7, indicating proton competition for the strong Hg(II) binding sites. Chemical modeling of Hg(II)-DOM binding at different pH values was consistent with bidentate binding of Hg(II) by one thiol group (pK(a) = 10.3) and one other group (pK(a) = 6.3) in the DOM, which is in agreement with recent results on the structure of Hg(II)-DOM bonds obtained by extended X-ray absorption fine structure spectroscopy (EXAFS).     

Sorption of tetracycline and chlortetracycline on K- and Ca-saturated soil clays, humic substances, and clay-humic complexes

Tetracycline (TC) and chlortetracycline (CTC) are used extensively for growth promotion and therapeutic purposes in livestock production. The sorption of TC and CTC on clays, humic substances (HS), and clay-humic complexes (clay-HC) derived from two agricultural soils was quantified using dilute CaCl2 (Ca) and KCI (K) as background solutions. In all systems, the soil components sorbed > 96% of added tetracyclines. Strongest sorption was observed for clays, followed by HS, and then clay-HC. Greater sorption by the Ca systems than the K systems and decreased sorption with increasing pH suggests that cation bridging and cation exchange contribute to sorption. X-ray diffraction analysis showed that TC and CTC were sorbed in the interlayers of smectites and that the presence of HS reduced interlayer sorption of tetracyclines by smectites in clay-HC. The results indicate that tetracyclines are dominantly sorbed on soil clays and that HS in clay-HC either mask sorption sites on clay surfaces or inhibit interlayer diffusion of tetracyclines.     

Characterization and quantification of reversible redox sites in humic substances

Cyclic oxidation and reduction reactions using oxygen and palladium with H2, respectively, of dissolved humic and fulvic acids (HA and FA) and model quinone compounds were used to structurally characterize and quantify the electron-carrying capacity (ECC) of reversible redox sites present in humic substances. This technique was used to examine 8 quinone compounds and 14 HA and FA samples and identified 3 redox sites as a function of their stability against the Pd-catalyzed hydrogenolysis process. Six highly aliphatic HA and FA isolated from landfill leachate did not contain redox sites under any conditions; however, the other HA and FA demonstrated reversible redox properties characterized by a combination of three redox sites. On the basis of the model compound results, it is proposed that one site consists of a non-quinone structure (NQ) and the other two sites have quinone structures. The two quinone sites differ in that one group (Q1) has electron-withdrawing groups adjacent to the quinone functional group while the second group (Q2) contains either no substituents near the quinone or has nearby electron-donating groups with additional substitutents hindering hydrogenolysis through steric interactions. The reversible ECC of NQ sites ranged from 25 to 265 microequiv e- transferred/g HA or FA, representing 21-56% of the total ECC of the HA and FA when measured with the mildest reducing method (pH 8.0, pure Pd). Q1 redox sites resistant to hydrogenolysis at pH 8.0 using Pd/Al2O3 accounted for 13-58% of the total ECC and ranged from 40 to 120 microequiv e-/ g HA or FA. The most sensitive O2 reversible redox sites accounted for 8-50% of the total ECC (20-220 microequiv e-/ g HA or FA). These results directly demonstrate that HA and FA are capable of acting as reversible electron-transfer agents using different functional groups, some of which may not be quinones.     

Adsorption of humic substances on goethite: comparison between humic acids and fulvic acids

The adsorption of humic acids (HA) to goethite (at pH 3-11) and the proton co-adsorption (at pH 4.0, 5.5, and 7.0) were measured, and the results were compared to those of fulvic acids (FA). Compared to FA, the adsorption of HA is stronger and more ionic strength dependent. The adsorption of both HA and FA decreases with increasing pH. The relative change of the adsorption with pH is bigger for HA than for FA at relatively low pH. At relatively high pH, it is the opposite. Protons are released at pH 4.0 and co-adsorbed at pH 5.5 and 7.0 upon the adsorption of both HA and FA. The observed pH dependency of HA and FA adsorption is in agreement with the proton co-adsorption data. Model calculations show that the adsorbed FA particles are on average located in the Stern layer, whereas the adsorbed HA particles protrude beyond the Stern layer. The closer location to the surface of the adsorbed FA leads to stronger electrostatic interactions between the FA particles and the surface, which explains the larger amount of protons released at low pH and co-adsorbed at high pH with each mass unit of FA adsorbed than that with HA adsorbed. The model also revealsthatfor FA a mean-field (smeared-out) approximation is reasonable, but for HA a patchwise approach is more appropriate at relatively low loading.     

NMR investigation of enzymatic coupling of sulfonamide antimicrobials with humic substances

Phenoloxidases mediate the oxidative transformation of soil phenolic constituents, contributing to the formation of humic substances and the chemical incorporation of some xenobiotic organic compounds into natural organic matter. We previously demonstrated phenoloxidase-mediated covalent coupling of sulfonamide antimicrobials with model humic constituents. Here, we investigate fungal peroxidase-mediated covalent coupling of 13C-sulfamethazine and 15N-sulfapyridine to humic substances. 1H-13C heteronuclear single quantum correlation (HSQC) nuclear magnetic resonance spectroscopy provided an initial indication of peroxidase-mediated covalent binding of 13C-sulfamethazine to humic acid. To confirm the role of the sulfonamide anilinic nitrogen in coupling to humic acid and to determine the nature of the covalent linkage, we incubated 15N-sulfapyridine with humic acid and peroxidase and examined reaction products in 1H-15N heteronuclear multiple bond (HMBC) experiments. The HMBC spectra revealed the presence of Michael adducts (i.e., anilinohydroquinones, anilinoquinones) and possibly other covalent linkages. No evidence for Schiff base formation was observed. Analogous experiments with the model humic constituent catechol provided corroborating evidence for these assignments. Michael adducts are expected to exhibit greater environmental stability than imine linkages that can form between sulfonamides and 2,6-dimethoxyphenols. Because the free anilinic nitrogen is required for the bioactivity of sulfonamide antimicrobials, nucleophilic addition occurring through this moiety could result in the biochemical inactivation of these compounds.     

Diffusion coefficients of humic substances in agarose gel and in water

Measurements of the diffusion coefficients of five different humic substances (HS) have been performed in water and in agarose hydrogels at several pH values (in the range of 3-10) and gel concentrations (in the range of 0.7-3% w/w). Fluorescence correlation spectroscopy (FCS) and classical diffusion cells were used in parallel to probe diffusion over both microscopic and mesoscopic distance scales. In general, agreement between the techniques was reasonable, which indicated that local nonhomogenities in the gel did not play an important role. Diffusion coefficients (D) in the gel were generally in the range of 0.9-2.5 x 10(-10) m2 s(-1) but were generally only 10-20% lower than in solution. At low pH values, one of the studied humic substances (a peat humic acid, PPHA) formed large aggregates that could not penetrate into the gel and therefore could not be defined by a single D value. The observed decreases of D in the gel for other HS were too large to be explained by the tortuousity and obstructive effects of the gel alone. D decreased slightly with increasing gel concentration and increased slightly with pH. Because modifications of D due to pH were similar in both the gel and the free solution, it is unlikely that complexation with the gel was greatly influenced by the pH. Rather, the main effect that appeared to decrease the diffusive flux in gels was likely small increases in the hydrodynamic radii of the humic macromolecules. An anomalous diffusion model was used to describe the FCS data in the gel. The characteristic exponent determined by fitting the autocorrelation functions with this model decreased only slightly (from 0.96 to 0.90) with increasing gel concentration providing support that HS complexation with the gel fibers was not very important. The results have important implications for our understanding of the fate and behavior of the HS and their associated pollutants and for interpreting metal speciation data obtained using gel-covered analytical sensors.     

Strong copper-binding behavior of terrestrial humic substances in seawater

In coastal areas, strong complexation of copper generally reduces its toxicity; our ability to monitor and regulate copper as a toxin therefore depends on our understanding of the sources and sinks of the copper-binding ligands. Terrestrial humic substances (HS) are well-recognized contributors to weak ligand concentrations in aquatic systems. In this work, we show that HS are likely contributors to both stronger and weaker ligand classes controlling copper speciation in coastal areas receiving typical inputs of terrestrial organic matter. We used competitive ligand exchange adsorptive cathodic stripping voltammetry (CLE-ACSV), with the added ligands benzoylacetone and salicylaldoxime, to examine copper binding by terrestrial HS in a seawater matrix, at HS and copper concentrations typical of coastal waters. Copper titration data of 1 mg/L Suwannee River humic acid (SRHA) in seawater could be modeled using conditional stability constants of 10(12.0) and 10(10.0) and total ligand concentrations of 10.4 and 199 nM for a stronger and weaker ligand, respectively. Similar results were obtained for Suwannee River fulvic acid (SRFA). Strong copper binding by SRFA in seawater was weaker than previously reported for a freshwater at similar pH, possibly indicating effects of Ca and Mg competition or ionic strength. Nevertheless,the concentrations and binding strengths of copper ligands we observed are comparable to the range reported in previous coastal speciation studies. In addition, we show that the weaker copper ligands cause internal calibration techniques to significantly underestimate the sensitivity of ACSV in the presence of HS concentrations typical of coastal waters. To address this issue, we demonstrate the use of "overload titrations", using a high enough concentration of added ligand to outcompete all natural ligands as an alternative calibration technique for analysis of coastal samples.     

Influence of humic substance adsorptive fractionation on pyrene partitioning to dissolved and mineral-associated humic substances

Changes in pyrene binding by dissolved and mineral-associated humic substances (HS) due to HS adsorptive fractionation processes were examined in model environmental systems using purified Aldrich humic acid (PAHA) and Suwannee River fulvic acid (SRFA). For PAHA, carbon-normalized pyrene binding coefficients for nonadsorbed, residual fractions (Koc(res)) were different from the original dissolved PAHA Koc value (Koc(orig)) prior to contact with the mineral suspensions. A strong positive correlation between pyrene log Koc(res) and log weight-average molecular weight (MWw) for residual PAHA fractions was observed, which was relatively independent of the specific mineral adsorbent used and hypothesized fractionation processes. A strong positive correlation between log Koc(ads) and log MWw was also found for PAHA fractions adsorbed to kaolinite at low mass fraction organic carbon levels, although the relationship was statistically different from the one found with residual PAHA fractions. The same trends and correlations found for PAHA were not observed with SRFA, suggesting that the impacts of HS adsorptive fractionation on changes in hydrophobic organic contaminants binding are also influenced by the source and other biogeochemical characteristics of HS.     

Quantification of the interaction of Tc with dissolved boom clay humic substances

Technetium-99 (Tc), a fission product of uranium-238, is an important radionuclide because of its long half-life and its high yield in radioactive waste. To elucidate the Tc geochemical behavior in reducing environments relevant to geological disposal and in the presence of humic substances (HS), experiments were set up that resulted forthe first time in the determination of an interaction constant for Tc with dissolved humic substances. A number of lab-scale Boom Clay (a possible geological underground High-Level Radioactive Waste storage site in Mol, Belgium) batch experiments were set up, combining both different initial Tc(VII) concentrations and different solid/liquid ratios. On these batches several sequential extraction steps with HS-free synthetic Boom Clay water were performed. Equilibration times were fixed at 1 week for each extraction step. Tc(VII) was found to be readily reduced to Tc(IV) by the solid Boom Clay phase. This solid phase was able to sorb Tc(IV) to a very large extent (log Kd approximately 2.5-4.0), and two sorption sinks (one of which is humic substances) were detected. In solution, Tc(IV) was mainly associated with HS. Concentrations in solution were found up to the order of 2 x 10(-6) M. The results were quantitatively described as a competition for Tc(IV) between the solid phase and the dissolved HS (Schubert-like approach). It was concluded that a hydrophobic sorption of uncharged Tc(IV) species in solution would act as the dominating interaction mechanism with HS, with an interaction constant log K(HS) = 5.3 +/- 0.3.     

Utilization and transformation of aquatic humic substances by autochthonous microorganisms

Aquatic humic substances (HS) from a bog lake water, a riverwater, and a groundwater were isolated after enrichment on XAD 8 columns and added to a Czapek-Dox nutrient broth which was used either in full strength or without glucose and/or NaNO3. The individual flasks were inoculated with natural microbial populations of corresponding water samples or with a Pseudomonas fluorescens strain isolated from groundwater. The presence of HS resulted in an increase of bacterial numbers in nearly all cultures incubated for 3 weeks at 25 degrees C on a shaker. HS reisolated from cultures without glucose or NaNO3 showed no or only minor quantitative differences as compared to those from sterile controls. In full strength nutrient broth up to 27% of HS were utilized. Data obtained by spectroscopic methods (UV/vis/FTIR) and elemental analysis indicated a decrease in particle size and a loss in aromaticity and aliphatic carbon in HS reisolated from the microbial cultures. Simultaneously an increase in the N content of HS was observed, which probably originated from some constituents of microbial biomass such as proteins and amino sugars. The NMR data also documented that significant transformations of HS occurred in the individual microbial cultures. After incubation, increased amounts of aromatic acids were detected in some liquid media and residual HS by GC/MS or capillary electrophoresis. 1H NMR spectroscopy was less effective in indicating structural differences in the HS than 13C NMR but revealed considerable detail of the microbial degradation of riverine HS, when limited sample was available. The newly developed NMR increment analysis provided substantial detail of aromatic structures in a microbially altered HS. The microbial degradation of HS strongly depended on the composition of the HS, the species selection of the microorganisms, and to a lesser extent on the culture conditions. For any series of identical inoculum and HS, full broth media initiated the most extensive alteration of HS.     

Antimony(III) binding to humic substances: influence of pH and type of humic acid

Conditional distribution coefficients (Dom) for Sb(III) binding to three commercial humic acids (terrestrial, coal, and aquatic) were measured at environmentally relevant Sb(III)/DOC ratios and as a function of pH using an equilibrium dialysis method. Maximum binding of Sb(III) was observed around pH 6 for two of the humic acids. The third humic acid showed constant Dom values up to pH 6 and decreasing Dom values for pH > 6. Sb(III)/DOC ratio was found to be important for Dom (20 times higher Dom for 60 times lower Sb(III)/DOC ratio). Moreover, Dom depends on the individual humic acid, suggesting that different functional groups are involved and/or different degrees of stabilization by chelation or H-bridges. Chemical modeling of Sb(III)-humics binding at different pH values is consistent with two binding sites involving (i) a phenolic entity forming a neutral complex and (ii) a carboxylic entity forming a negatively charged complex. Under environmentally relevant conditions, over 30% of total Sb(III) may be bound to natural organic matter.