Role of fulvic acid on lead bioaccumulation by Chlorella kesslerii

To better understand the relationship between lead speciation and bioavailability in natural freshwaters, the interaction of lead with the freshwater alga Chlorella kesslerii was studied in the presence of the Suwannee River fulvic acid (SRFA). Special attention was paid to direct interactions of the fulvic acid on the algae, as well as potential physiological (membrane permeability and algal metabolism) influences. Lead-free ion concentration measurements were carried out using a novel ion-selective electrode. Pb uptake decreased in the presence of SRFA with respect to noncomplexed Pb, but uptake fluxes, cellular Pb, Pb bound to the transport sites, and total adsorbed Pb were all higher than predicted from Pb2+ activities, in accordance with the free ion activity model (FIAM). The discrepancies between the observed values and those predicted by the FIAM in the presence and absence of synthetic ligands increased with increasing concentration of SRFA. Several hypotheses were examined to explain the observed differences. No contributions of labile and/or hydrophobic Pb-SRFA complexes were found. Furthermore, direct biological effects, including variations in membrane permeability or algal metabolism, could not account for the observations. On the other hand, changes in the algal surface charge due to SRFA adsorption seemed to account, at least partially, for the observed increase in lead uptake in the presence of SRFA as compared to that corresponding to the same Pb2+ concentration in the presence of synthetic ligands.     

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.     

Reactions of hydroxyl radical with humic substances: bleaching, mineralization, and production of bioavailable carbon substrates

In this study, we examine the role of the hydroxyl (OH*) radical as a mechanism for the photodecomposition of chromophoric dissolved organic matter (CDOM) in sunlit surface waters. Using gamma-radiolysis of water, OH* was generated in solutions of standard humic substances in quantities comparable to those produced on time scales of days in sunlit surface waters. The second-order rate coefficients of OH* reaction with Suwannee River fulvic (SRFA; 2.7 x 10(4) s(-1) (mg of C/L)(-1)) and humic acids (SRHA; 1.9 x 10(4) s(-1) (mg of C/L)(-1)) are comparable to those observed for DOM in natural water samples and DOM isolates from other sources but decrease slightly with increasing OH* doses. OH* reactions with humic substances produced dissolved inorganic carbon (DIC) with a high efficiency of approximately 0.3 mol of CO2/mol of OH*. This efficiency stayed approximately constant from early phases of oxidation until complete mineralization of the DOM. Production rates of low molecular weight (LMW) acids including acetic, formic, malonic, and oxalic acids by reaction of SRFA and SRHA with OH* were measured using HPLC. Ratios of production rates of these acids to rates of DIC production for SRHA and for SRFA were similar to those observed upon photolysis of natural water samples. Bioassays indicated that OH* reactions with humic substances do not result in measurable formation of bioavailable carbon substrates other than the LMW acids. Bleaching of humic chromophores by OH* was relatively slow. Our results indicate that OH* reactions with humic substances are not likely to contribute significantly to observed rates of DOM photomineralization and LMW acid production in sunlit waters. They are also not likely to be a significant mechanism of photobleaching except in waters with very high OH* photoformation rates.     

Comparison of Cd(II), Cu(II), and Pb(II) biouptake by green algae in the presence of humic acid

The present study examines the role of humic acid, as a representative of dissolved organic matter, in Cd(II), Cu(II), and Pb(II) speciation and biouptake by green microalgae. Cellular and intracellular metal fractions were compared in the presence of citric and humic acids. The results demonstrated that Cd and Cu uptake in the presence of 10 mg L(-1) humic acid was consistent with that predicted from measured free metal concentrations, while Pb biouptake was higher. By comparing Cd, Cu, and Pb cellular concentrations in the absence and presence of humic acid, it was found that the influence of the increased negative algal surface charge, resulting from humic acid adsorption, on cellular metal was negligible. Moreover, the experimental results for all three metals were in good agreement with the ternary complex hypothesis. Given that metal has much higher affinity with algal sites than humic acid adsorbed to algae, the contribution of the ternary complex to metal bioavailability was negligible in the case of Cd (II) and Cu (II). In contrast, the ternary complex contributed to over 90% of total cellular metal for Pb(II), due to the comparable affinity of Pb to algal sites in comparison with humic acid adsorbed to algae. Therefore, the extension of the biotic ligand model by including the formation of the ternary complex between the metal, humic acid, and algal surface would help to avoid underestimation of Pb biouptake in the presence of humic substances by green algae Chlorella kesslerii.     

On the origin of the optical properties of humic substances

Absorption and fluorescence spectroscopy and laser photobleaching experiments were employed to probe the origins of the optical properties of humic substances (HS). Luminescence quantum yields and the wavelengths of maximum emission were acquired for Suwannee River humic acid (SRHA) and fulvic acid (SRFA) at an extensive series of excitation wavelengths across the ultraviolet and visible. Laser irradiation at a series wavelength across the ultraviolet and visible was further employed to destroy selectively chromophores absorbing at specific wavelengths, using absorption spectroscopy to follow the absorption losses with irradiation time. The results provide unequivocal evidence that the absorption and emission spectra of these materials cannot result solely from a simple linear superposition of the spectra of numerous independent chromophores. Instead, the long wavelength absorption tail of HS (>350 nm) appears to arise from a continuum of coupled states. We propose that this behavior results from intramolecular charge-transfer interactions between hydroxy-aromatic donors and quinoid acceptors formed by the partial oxidation of lignin precursors. We further propose that these donor-acceptor interactions may be a common phenomenon, occurring within all natural hydroxy- or polyhydroxy-aromatic polymers that form appropriate acceptors upon partial oxidation. Examples of such species include lignin, polyphenols, tannins, and melanins.     

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.     

Influence of the composition of natural organic matter on Pb bioavailability to microalgae

The current work examines the effects of model allochtonous (humic substances) and autochtonous (microbial polysaccharides) natural organic matter (NOM) on Pb speciation and bioaccumulation. The results demonstrated that polysaccharides, in particular alginic acid, had complexing properties and effects on Pb bioaccumulation by the green alga Chlorella kesslerii that were similar to equivalent complexing capacity of humic substances. Pb uptake decreased in the presence of humic, alginic, and polygalacturonic acids with respect to noncomplexed Pb, but accumulated Pb was higher than predicted from measured Pb2+ concentrations or from previous results obtained in the presence of simple synthetic ligands. An improved fit between experimental observations and Pb speciation was obtained by taking into account the formation of a ternary complex at the algal surface. The contribution of the ternary complexes to Pb bioaccumulation was dependent on the relative binding constants of the Pb to the NOM and to the binding sites on the biological surface. In the presence of the humic acid, a decreased surface charge and increased membrane permeability were considered to be of secondary importance to explain the observation of increased Pb uptake with respect to that predicted on the basis of [Pb2+]. The environmental implications of the results are discussed with respect to the development of site-specific water quality criteria.     

Association with natural organic matter enhances the sunlight-mediated inactivation of MS2 coliphage by singlet oxygen

MS2 coliphage, a surrogate for human enteric viruses, is inactivated by singlet oxygen (1O2) produced via sunlight-mediated excitation of natural organic matter (NOM) in surface waters. The 1O2 concentration within a NOM macromolecule or supramolecular assembly ([1O2]internal) is orders of magnitude higher than in the bulk solution ([1O2]bulk). In close proximity of NOM, MS2 is thus exposed to an elevated 1O2 concentration ([1O2]NOM), and inactivation is likely to be enhanced as compared to the bulk solution. In experiments using a solar simulator, we determined [1O2]bulk, [1O2]internal, as well as the association of MS2 with four NOMs (Fluka humic acid, FHA; Suwannee river humic acid, SRHA; Aldrich humic acid, AHA; Pony lake fulvic acid, PLFA), and studied their effect on the MS2 inactivation rate constant, k(obs), over a range of 1-25 mg NOM/L. The k(obs) values were modeled as the sum of the inactivation rate constants in close proximity to the NOM and in the bulk solution, assuming Langmuir-type adsorption of NOM onto MS2. FHA and SRHA exhibited 13-22 fold greater adsorption equilibrium constants than AHA and PLFA. Inactivation in the bulk solution contributed between 2% (20 mg/L FHA) and 39% (5 mg/L AHA) toward the overall k(obs). Thus, even for the less adsorbing NOM, inactivation was dominated by [1O2]NOM rather than [1O2]bulk. Changes in solution chemistry to promote closer interactions between MS2 and NOM also enhanced k(obs). Addition of Mg2+ to neutralize the negative surface charge of MS2 and NOM increased k(obs) up to 4.1-fold. Similarly, lowering the solution pH closer to the isoelectric point of MS2 (pl = 3.9) enhanced k(ob), 51-fold in 5 mg/L AHA.     

Toxicity and internalization of CuO nanoparticles to prokaryotic alga Microcystis aeruginosa as affected by dissolved organic matter

This is the first study investigating the toxicity of nanoparticles (NPs) to algae in the presence of dissolved organic matter (DOM). Suwannee river fulvic acid (SRFA), a type of DOM, could significantly increase the toxicity of CuO NPs to prokaryotic alga Microcystis aeruginosa. Internalization of CuO NPs was observed for the first time in the intact algal cells using high resolution transmission electron microscopy (HRTEM), and the cell uptake was enhanced by SRFA. A fast Fourier transformation (FFT)/inversed FFT (IFFT) process revealed that a main form of intracellular NPs was Cu(2)O, and an intracellular environment may reduce CuO into Cu(2)O. The internalization behavior alone did not seem to pose a hazard to membrane integrity as shown from the flow cytometry data. Elevated CuO nanotoxicity by SRFA was related to a combination of a lesser degree of aggregation, higher Cu(2+) release, and enhanced internalization of CuO NPs.     

Fluorescence quenching and luminescence sensitization in complexes of Tb3+ and Eu3+ with humic substances

Intrinsic fluorescence quenching of humic substances (HS) and the sensitization of Ln3+ luminescence (Ln3+ = Tb3+, Eu3+) in HS complexes were investigated. Both measurements yielded complementary information on the complexation of metals by HS. Large differences between fulvic acids (FA) and humic acids (HA) were found. From time-resolved luminescence measurements it is concluded that a combination of energy transfer and energy back transfer between HS and Ln3+ is responsible for the observed luminescence decay characteristics. In the case of Eu3+, an additional participation of charge-transfer states is suggested. A new concept for the evaluation of the sensitized luminescence decays of Ln3+ was adapted.     

Mutual effects of Pb(II) and humic acid adsorption on multiwalled carbon nanotubes/polyacrylamide composites from aqueous solutions

This paper examines the adsorption of Pb(II) and a natural organic macromolecular compound (humic acid, HA) on polyacrylamide (PAAM) -grafted multiwalled carbon nanotubes (denoted as MWCNTs/PAAM), prepared by an N(2)-plasma-induced grafting technique. The mutual effects of HA/Pb(II) on Pb(II) and HA adsorption on MWCNTs/PAAM, as well as the effects of pH, ionic strength, HA/Pb(II) concentrations, and the addition sequences of HA/Pb(II) were investigated. The results indicated that Pb(II) and HA adsorption were strongly dependent on pH and ionic strength. The presence of HA led to a strong increase in Pb(II) adsorption at low pH and a decrease at high pH, whereas the presence of Pb(II) led to an increase in HA adsorption. The adsorbed HA contributed to modification of adsorbent surface properties and partial complexation of Pb(II) with the adsorbed HA. Different effects of HA/Pb(II) concentrations and addition sequences on Pb(II) and HA adsorption were observed, indicating different adsorption mechanisms. After adsorption of HA on MWCNTs/PAAM, the adsorption capacity for Pb(II) was enhanced at pH 5.0; the adsorption capacity for HA was also enhanced after Pb(II) adsorption on MWCNTs/PAAM. These results are important for estimating and optimizing the removal of metal ions and organic substances by use of MWCNT/PAAM composites.     

Reversed-phase chromatography fractionation tailored to mass spectral characterization of humic substances

Large-scale structural characterization of humic substances via mass spectrometry requires reduction of complexity within nominal mass and separation of isomers, i.e., prefractionation. Humic substances (here loosely defined to encompass all humic, humic-like, and humic-containing material) are notoriously difficult to fractionate. Equally challenging is deriving information on whether and howfractionation has occurred. Here, reversed-phase high-performance liquid chromatography was used to induce tailored fractionation of Suwannee River fulvic acid (SRFA) within nominal mass. The fractionation was optimized on synthetic standards thatdiffered in polarity and had elemental formulas similar to SRFA. Fractions were analyzed via electrospray ionization ion-cyclotron resonance mass spectrometry. Kendrick and Van Krevelen comparisons showed that fractionation occurred as predicted based on known molecular formula patterns.     

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.     

Complexation with dissolved organic matter and solubility control of heavy metals in a sandy soil

The complexation of heavy metals with dissolved organic matter (DOM) in the environment influences the solubility and mobility of these metals. In this paper, we measured the complexation of Cu, Cd, Zn, Ni, and Pb with DOM in the soil solution at pH 3.7-6.1 using a Donnan membrane technique. The results show that the DOM-complexed species is generally more significant for Cu and Pb than for Cd, Zn, and Ni. The ability of two advanced models for ion binding to humic substances, e.g., model VI and NICA-Donnan, in the simulation of metal binding to natural DOM was assessed by comparing the model predictions with the measurements. Using the default parameters of fulvic and humic acid, the predicted concentrations of free metal ions from the solution speciation calculation using the two models are mostly within 1 order of magnitude difference from the measured concentrations, except for Ni and Pb in a few samples. Furthermore, the solid-solution partitioning of the metals was simulated using a multisurface model, in which metal binding to soil organic matter, dissolved organic matter, clay, and iron hydroxides was accounted for using adsorption and cation exchange models (NICA-Donnan, Donnan, DDL, CD-MUSIC). The model estimation of the dissolved concentration of the metals is mostly within 1 order of magnitude difference from those measured except for Ni in some samples and Pb. The solubility of the metals depends mainly on the metal loading over soil sorbents, pH, and the concentration of inorganic ligands and DOM in the soil solution.     

Aminated polyacrylonitrile fibers for humic acid adsorption: behaviors and mechanisms

Aminated polyacrylonitrile fibers (APANFs) were prepared by surface modification of polyacrylonitrile fibers (PANFs) with diethylenetriamine in a solution, and the APANFs were studied as an adsorbent for humic acid removal in a series of batch adsorption experiments. The surface modification reaction introduced the amine groups on the surface of the fibers, and the APANFs had a zero point of zeta potentials at pH 8.1, in contrast with pH 3.5 for the PANFs. Adsorption experiments indicated that the APANFs were very effective in removing humic acid from aqueous solutions in the pH range of 2-10, whereas the PANFs did not adsorb humic acid at all under the same conditions. It was found that both electrostatic interaction and surface complexation mechanisms played important roles in humic acid adsorption on the APANFs, although the relative importance of each of the adsorption mechanisms varied with solution pH values. With the advantages of large specific surface areas and enhanced reactive surface properties, the APANFs have great potentials in water treatment for the removal of humic substances and other polarized or electrically charged species.     

Specific UV absorbance of Aldrich humic acid: changes during transport in aquifer sediment

This study examined the transport behavior of Aldrich humic acid (AHA) in low natural organic carbon content sediment contaminated with tetrachloroethene (PCE), for comparison to a nonionic surfactant mixture previously examined in the same system. Tracking of individual molecular weight (MW) fractions of AHA was attempted by UV absorbance, followed by conversion to mass of carbon using specific ultraviolet absorbance (SUVA) (UV absorbance per mass of carbon) measurements. The analysis required determination of variations of SUVA with MW, which showed a maximum at 10 000 Daltons. Furthermore, SUVAs of AHA MW fractions greater than about 10 000 Daltons increased following AHA interaction with sediment in batch experiments, and this was associated with AHA-driven leaching of cations from the sediment. AHA transport was examined in a series of three columns representing the up-gradient, residual-zone, and down-gradient portions of a DNAPL contaminated site. SUVAs of larger MW AHA fractions cycled through decreased, increased, and eventual return to influent values during the early, intermediate, and final stages of breakthrough, respectively. These variations were attributable to a combination of preferential adsorption of low MW fractions of the AHA during early breakthrough and AHA-driven leaching of sediment cations during intermediate breakthrough, with eventual exhaustion of sediment cation complexation during the final stage of breakthrough. The complex variations in SUVA precluded accurate conversion of measured UV absorbance to carbon mass. However, the effect of AHA loss to sediment on the solubilizing capacity of the AHA solution was indicated by the breakthrough behavior of AHA-solubilized PCE, which showed that AHA loss from the aqueous phase during transport in this system did not decrease the solubilizing capacity of the AHA mixture.     

Humic substances in soils: are they really chemically distinct?

Humic substances (HS) are an operationally defined fraction of soil organic matter, and they represent the largest pool of recalcitrant organic carbon in the terrestrial environment. It has traditionally been thought that extractable HS consist of novel categories of cross-linked macromolecular structures. In this study, advanced nuclear magnetic resonance approaches were used to study the major components (proteins, carbohydrates, aliphatic biopolymers, and lignin) that are known to be present in HS, and to identify their fingerprints in humic mixtures. Theoretically, once all known components have been identified, the remaining signals should be from materials with novel structures, themselves forming a distinct chemical category of humic materials. Surprisingly, nearly all of the NMR signals in traditional HS fractions could be assigned to intact and degrading biopolymers. We therefore suggest that the vast majority of operationally defined humic material in soils is a very complex mixture of microbial and plant biopolymers and their degradation products but not a distinct chemical category. It is important to note this work in no way rules out the existence of a distinct category of humic macromolecules, either at low abundance in the soluble fraction from young soils, in diagenetically evolved samples (for example lignites, etc.), or in the nonextractable humin fraction.     

Humic substances and crude oil induce cytochrome P450 1A expression in the Amazonian fish species Colossoma macropomum (Tambaqui)

Cytochrome P450 1A (CYP1A) induction is used widely as a biomarker of exposure to pollutants, such as petroleum hydrocarbons, yet CYP1A inducibility has been characterized in few tropical fish. Using Western blot analysis, catalytic assay, and immunohistochemistry, we evaluated CYP1A induction in an Amazonian fish (tambaqui; Colossoma macropomum) acclimated to humic substances (HS) and acutely exposed to crude oil. HS are ubiquitous in Amazonian waters, and they are known to affect the bioavailability of pollutants. CYP1A activity was also measured in fish exposed for 10 days to a range of concentrations of HS from both natural and commercial sources. Crude oil induced CYP1A expression in tambaqui, as expected. Exposure to both HS and crude oil resulted in greater levels of CYP1A expression relative to that in fish exposed to petroleum alone. Interestingly, CYP1A induction was also observed in fish exposed to HS alone. Induction by HS was concentration-dependent, and activity was higher in fish exposed to HS from the commercial source than in fish exposed to the HS from the natural source. The use of CYP1A as a biomarker of exposure to pollutants such as petroleum hydrocarbons in fish living in environments rich in humic substances should be considered with caution given that HS themselves induce CYP1A expression. Our results suggest that there may be as yet unknown CYP1A inducing components (aryl hydrocarbon receptor agonists) in humic substances.     

Evidence for strong but dynamic iron-humic colloidal associations in humic-rich coastal waters

This study investigated the physicochemical forms of dissolved iron in the coastal plume (salinity = 28-35) of a small river draining a peat-rich catchment. Speciation information was obtained through a combination of fractionation by crossflow filtration (CFF) along with voltammetric detection of either naturally occurring iron-humic complexes (July survey) or known, synthetic complexes (September survey) formed by titrating the samples with the competing ligand 2-(2-thiazolylazo)-p-cresol (TAC). The majority of colloidal iron (>5000 Da) was present as iron-humic complexes supplied by the river and showing uniform conditional stability constants throughout the plume (log K′(Fe′HS) = 11.3 ± 0.1, i.e. log K(Fe?+HS) = 21.3 ± 0.1). Noncolloidal or soluble iron was strongly complexed to ligands of marine origin with log K′(Fe′HS) = 11.9 ± 0.1. Equilibrium of the total iron pool with the added TAC ligand was achieved in all but the highest salinity sample, albeit more slowly for colloidal than for soluble iron. In addition, measurements of humic like fluorescence suggested that the conformation of colloids could change over time as a result of dissociation of the iron-humic associations. These results are consistent with the concept that iron in coastal waters is strongly but reversibly bound to humic substances and therefore may be available for complexation by siderophore-type ligands released by microorganisms.     

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.