Humic acid model substances with pronounced redox functionality for the study of environmentally relevant interaction processes of metal ions in the presence of humic acid

Geoderma 162 (2011) 132–140

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Geoderma j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e o d e r m a

Humic acid model substances with pronounced redox functionality for the study of environmentally relevant interaction processes of metal ions in the presence of humic acid Susanne Sachs ⁎, Gert Bernhard Forschungszentrum Dresden-Rossendorf, Institute of Radiochemistry, P.O. Box 510 119, 01314 Dresden, Germany

a r t i c l e

i n f o

Article history: Received 28 April 2010 Received in revised form 20 December 2010 Accepted 23 January 2011 Available online 22 February 2011 Keywords: Humic acid Model substances Redox behavior Reducing capacity Iron(III) Uranium(VI)

a b s t r a c t Humic acid (HA) model substances with pronounced redox functionality were synthesized by oxidation of hydroquinone or catechol in the presence of glycine or glutamic acid and characterized concerning their elemental, structural and functional properties. In order to characterize the redox properties of the synthetic products, formal redox potentials and Fe(III) reducing capacities were determined and compared to purified Aldrich HA (AHA). Furthermore, the reduction of U(VI) in the presence of HA was studied. The synthetic products show elemental, functional and structural properties comparable to natural HA, however, they are characterized by high amounts of phenolic/acidic OH groups (5.3–6.6 meq/g). Furthermore, the synthetic HA show significantly higher reducing capacities for Fe3+ and [Fe(CN)6]3− at pH 3.0 (8.8–14.5 meq/g) and at pH 9.2 (27.5–36.9 meq/g), respectively, than AHA (pH 3.0: 1.2 ± 0.1 meq/g; pH 9.2: 7.2 ± 1.9 meq/g). The highest reducing capacities were obtained for HA Cat-Gly (pH 3.0: 14.5 ± 1.6 meq/g; pH 9.2: 36.9 ± 0.2 meq/g), an oxidation product from catechol and glycine, which is characterized by the lowest formal redox potential (E0⁎ = 517 ± 12 mV) of all studied HA (E0⁎ = 517–571 mV). Indications for a slight reduction of U(VI) in the presence of HA were observed, whereby, HA Cat-Gly exhibits again the highest reducing capacity (pH 6: 0.065 ± 0.002 meq/g). Using modified HA with blocked phenolic/acidic OH groups the importance of these functional groups for the redox behavior of HA was confirmed. Synthetic HA with pronounced redox functionality can be used to study the redox behavior of HA and the redox stability of metal ions in the presence of HA and furthermore, to stabilize redox-sensitive metal ions against oxidation in complexation and transport studies with HA. This contributes to a better understanding of interaction processes of metal ions with humic substances in soils, sediments and waters. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Studies of the migration behavior of radioactive and nonradioactive toxic metal ions in the environment are of high importance for a reliable risk-assessment of potential nuclear waste repositories, of facilities of the former uranium mining and milling sites, and of subsurface dumps and sites with radioactive and/or heavy metal containing inventory. Depending on the prevailing geochemical conditions, different processes and substances influence the migration of such pollutants. Humic acids (HA), organic macromolecules ubiquitous found in soils, sediments and waters, play an important role in interaction processes of metal ions. They are soluble in the pH range of natural waters and posses the ability for complex and colloid formation. HA are characterized by redox properties and can act as electron acceptors or donors in natural environments (Aeschbacher et al.,

⁎ Corresponding author. Tel.: +49 351 260 2436; fax: +49 351 260 3553. E-mail addresses: [email protected] (S. Sachs), [email protected] (G. Bernhard). 0016-7061/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.geoderma.2011.01.012

2010). Therefore, they can influence the oxidation state of metal ions and thus, their speciation and mobility (Choppin, 1999). The understanding of the redox properties of HA towards metal ions is required for the description of the influence of HA on the migration of metal ions in the environment. From literature it is known that humic substances (humic and fulvic acids) are able to reduce metal ions. Studies with regard to the reduction of Fe(III) (Chen et al., 2003; Deiana et al., 1995), Cr(VI) (Gu and Chen, 2003; Szulczewski et al., 2001), Hg(II) (Allard and Arsenie, 1991; Rocha et al., 2003), and As(V) (Palmer et al., 2006) as well as concerning the reduction of actinide ions such as Pu(VI) and Np(VI) (Choppin, 1999; Nash et al., 1981), Pu(V) (André and Choppin, 2000; Blinova et al., 2007), Pu(VI), (V) and (IV) (Marquardt et al., 2004) and Np(V) (Schmeide and Bernhard, 2009; Zeh et al., 1999) were published. The reduction of U(VI) by natural organic matter was not clearly observed (Artinger et al., 2002; Choppin, 1999; Gu and Chen, 2003; Nash et al., 1981). However, in a study of Abraham (2002), which deals with the redox stability of U(VI) in the presence of lignin, wood degradation products and unpurified natural HA, it was reported that these organic materials are able to reduce U(VI) to

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U(IV). In addition, humic substances were found to enhance the reduction of U(VI) (Gu and Chen, 2003; Gu et al., 2005) and other metals like Fe(III) (Chen et al., 2003; Lovley et al., 1996; Scott et al., 1998) or Cr(VI) (Gu and Chen, 2003) by microorganisms by shuttling electrons from donors to acceptors. Due to reduction processes induced by humic substances, metal ions in lower oxidation states can be formed. These can show a strong interaction to humic colloids (Reiller, 2005; Reiller et al., 2008) which may cause an increased humic colloid-borne mobility, as known for the humic colloid-borne Np sorption and migration (Artinger et al., 2000; Schmeide and Bernhard, 2010). This demonstrates the importance of knowledge about the redox properties of humic substances and their influence on the redox speciation of metal ions, such as actinide ions. The redox behavior of humic substances is not well understood. The redox activity of humic material has been ascribed to specific structural features, e.g., the reversible quinone/hydroquinone redox pair, with semiquinone-type free radicals acting as electron-donating/ accepting species (Aeschbacher et al., 2010; Gu et al., 2005; Maurer et al., 2010; Nurmi and Tratnyek, 2002; Paul et al., 2006; Ratasuk and Nanny, 2007; Scott et al., 1998; Struyk and Sposito, 2001). Evidence for the occurrence of such structural elements has come from direct and indirect measurements, for instance from electron spin resonance spectroscopy (Paul et al., 2006; Scott et al., 1998), electrochemical measurements (Aeschbacher et al., 2010; Fimmen et al., 2007; Nurmi and Tratnyek, 2002) and fluorescence spectroscopy (Cory and McKnight, 2005; Klapper et al., 2002). The redox activity of humic substances is further attributed to the oxidation of phenolic hydroxyl groups (Helburn and MacCarthy, 1994; Matthiessen, 1995). These redox processes result in phenoxy radicals, which undergo typical subsequent reactions such as coupling reactions and tautomerizations (Helburn and MacCarthy, 1994; McDonald and Hamilton, 1973). Tautomerization of the coupled products results in regeneration of phenolic hydroxyl groups, which can be exposed to further oxidation (Helburn and MacCarthy, 1994). The successful modeling of the redox behavior of HA by the use of synthetic mixtures of phenolic compounds was reported by Helburn and MacCarthy (1994) and Matthiessen (1995). Furthermore, reduced nitrogen and sulfur containing moieties in humic substances may contribute to their redox activity (Fimmen et al., 2007; Szulczewski et al., 2001). Mostly, the reducing capacity of humic material was studied under oxygenfree conditions. However, a non-negligible reducing capacity for HA is maintained under oxic conditions, indicating that important reducing functional groups persist in humic substances (Peretyazhko and Sposito, 2006). In connection with the study of the redox behavior of humic substances and the control of the redox properties of humic materials, the application of synthetic and special chemically modified HA was described. Matthiessen (1995) synthesized a HA from hydroquinone to determine the redox capacity of humic substances as a function of pH. Perminova et al. (2005) developed quinonoid-enriched humic materials with enhanced redox properties by oxidation of phenolic fragments associated with the humic aromatic core or by polycondensation of these phenolic fragments with hydroquinone and catechol. These materials shall be used as effective redox mediators and reducing agents for remediation of soil and aquatic environments. Experimental evidence for the higher reduction potential of the quinonoid-enriched derivatives has been obtained for the reduction of Np(V) and Pu(V) (Kalmykov et al., 2008; Shcherbina et al., 2007a,b). In our previous studies various synthetic HA model substances with specific functional and structural properties were developed. These were successfully applied to investigate environmentally relevant geochemical interaction processes of HA with metal ions (Kumke et al., 2005; Křepelová et al., 2006; Mibus et al., 2007; Plaschke et al., 2002; Pompe et al., 1996, 1998, 2000; Sachs et al., 2002, 2005; Sachs and Bernhard, 2005; Schmeide et al., 2003, 2005, 2006; Schmeide and Bernhard, 2009, 2010; Seibert et al., 2001).

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The present work describes the synthesis of HA model substances with pronounced redox functionality for the study of the redox properties of HA, the redox stability of metal ions in the presence of HA and for stabilization of redox-sensitive low-valent metal ions in the presence of HA against oxidation, for instance in sorption and migration studies. Due to the fact that the redox activity of humic substances is ascribed to the redox system hydroquinone/quinone and to the oxidation of phenols, as discussed above, HA with high amounts of phenolic OH groups are synthesized by oxidation of diphenolic compounds. The obtained model substances are characterized for their elemental, functional and structural properties. In order to characterize the redox behavior of these substances, formal redox potentials and reducing capacities of the HA towards iron(III) are determined in comparison to a natural HA. The influence of HA phenolic OH groups on the reducing capacity of the synthesized HA is studied applying modified HA with blocked phenolic OH groups. Furthermore, the reduction of uranium(VI) in the presence of the synthetic HA is investigated. 2. Experimental For all syntheses and experiments Milli-Q-water (Milli-RO/MilliQ-System, Millipore, Molsheim, France) was used. 2.1. Humic materials 2.1.1. Synthesis of humic acids with pronounced redox functionality The synthesis of HA with pronounced redox functionality was based on the oxidation of diphenolic compounds in alkaline solution in the presence of α-amino acids. Potassium peroxodisulfate (K2S2O8) was used as oxidizing agent (Adhikari et al., 1985; Eller and Koch, 1920). HA type Hyd-Gly and Hyd-Glu were synthesized by oxidation of 2.5 g hydroquinone (99%, Merck, Darmstadt, Germany) in the presence of 1.70 g glycine (p.a., Merck) and 1.89 g glutamic acid monohydrate (puriss. ≥99%, Fluka, Taufkirchen, Germany), respectively. HA type Cat-Gly was prepared from 2.5 g catechol (N99%, Merck) in the presence of 1.70 g glycine. For synthesis, the starting materials were dissolved in 900 mL 0.1 M NaOH (Merck) at room temperature. Within one hour 12.5 g K2S2O8 (p.a., Merck) was charged to the reaction mixtures at 60°C. After that, the reaction mixtures were cooled down and the HA-like fractions of the oxidation products were precipitated with 2 M HCl (Merck). HA precipitates were separated by centrifugation, washed with 0.1 M HCl (Merck), purified by dialysis against Milli-Q water (dialysis tubes Thomapor®, exclusion limit MWCO b1000, Roth, Karlsruhe, Germany), and lyophilized. 2.1.2. Humic acid reference material Commercially available HA from Aldrich of natural origin was used as reference material (AHA; Aldrich, Steinheim, Germany). Before use the sodium salt of AHA was purified by repeated dissolution with NaOH in the presence of NaF and precipitation with HCl according to the method described by Kim et al. (1990). AHA batch A2/98 was applied. Its purification and characterization is described in detail in (Sachs et al., 2004). 2.1.3. Humic acids with blocked phenolic OH groups To study the influence of phenolic OH groups on the reducing capacities of HA, chemically modified HA with blocked phenolic OH groups (HA-PB) were synthesized from HA Hyd-Gly, Hyd-Glu, CatGly, and AHA in a two-step modification process according to the procedure described in Pompe et al. (2000). First, the original HA were methylated with diazomethane resulting in methyl esters of carboxylic groups and methyl ethers of phenolic and other acidic, e.g., enolic, hydroxyl groups (subsequently referred to as phenolic/acidic OH groups). Then, the methylated samples were treated with alkaline

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solution, causing the hydrolysis of carbonic methyl esters that were formed in the first modification step (release of carboxylic groups). Methyl ether groups of phenolic/acidic OH groups are not hydrolyzed and remain blocked (Sachs et al., 2002). 2.2. Humic acid characterization The elemental composition of the HA was determined with an elemental analyzer (Model CHNS-932, Leco, St. Joseph, MI, USA). HA ash and water content were measured thermoanalytically with the CH-analyzer RC 412 (Leco) and the thermoanalyzer STA 92 (Setaram, Lyon, France). The carboxylic group content of the studied HA was determined with the calcium acetate exchange method (Schnitzer and Khan, 1972) using an automatic titration system TPC 2000 (Schott, Hofheim, Germany). The phenolic/acidic OH group content was analyzed by a radiometric method based on the derivatization of HA functional groups with [14C]diazomethane (Bubner and Heise, 1994). The structure of the HA was studied by Fourier transform infrared spectroscopy (FTIR) using the KBr method (Spectrometer Spectrum 2000 GX, Perkin Elmer, Waltham, MA, USA). 2.3. Redox studies 2.3.1. Formal redox potentials The methodology for the determination of formal redox potentials of HA is based on the Nernst equation and was basically described by Österberg and Shirshova (1997). In the cited work the redox potentials of a HA suspension were measured as a function of pH and the formal redox potential was obtained by extrapolating these data to pH 0. Based on this approach, Mack (2002) determined formal redox potentials of HA (E0⁎) as a function of pH and HA concentration by a twofold extrapolation of measured redox potentials of HA suspensions. At first, redox potentials of separate HA suspensions with different HA concentrations were directly measured as a function of pH. The pH-dependent redox potentials at different HA concentration were extrapolated to pH 0. Following, the obtained redox potentials at pH 0 (EpH = 0) were extrapolated to a HA concentration of 0 g/L. According to this methodology recommended by Mack (2002), the formal redox potentials of HA Hyd-Gly, Hyd-Glu, Cat-Gly, and AHA were measured in the present work. HA suspensions were analyzed in a glove box under nitrogen atmosphere and exclusion of CO2 at room temperature as a function of pH (pH 1–6) and HA concentration (0.1–0.5 g/L). Pt/Ag/AgCl redox electrodes (Schott, Mainz, Germany) were applied. HA were weighed into glass bottles (5.0, 7.5, and 12.5 mg) and suspended to the final concentrations of 0.1, 0.3, and 0.5 g/L in HCl solution (1× 10−1–1 × 10−6 M, p.a., Merck; I = 0.1 M KCl, p.a., Merck) whose pH value was previously adjusted to pH 1–6. For each HA concentration an individual sample at each studied pH value was prepared (cf. Fig. S1, Supplementary data). After HCl addition, the pH value of the suspensions was measured and readjusted when necessary. After that, the bottles were equipped with the redox electrodes and the redox potential of the HA suspensions was continuously followed until a steady state was reached. During the experiments the samples were slightly shaken under exclusion of light. 2.3.2. Fe(III) reducing capacities Fe(III) reducing capacities of the studied HA were determined at pH 3.0 and 9.2 using Fe3+ and [Fe(CN)6]3−, respectively, as oxidants. The obtained values represent charge equivalents per mass unit HA transferred to Fe(III) in an one-electron transfer redox process. To assess the influence of HA phenolic OH groups on the redox behavior of HA, Fe(III) reducing capacities of unmodified and modified HA with blocked phenolic OH groups were determined and compared.

Fe(III) reducing capacities at pH 3.0 were determined according to Mack (2002). Suspensions of FeCl3 (p.a., Merck) and HA were continuously shaken ([Fe3+]0 = (8.2–8.7) × 10−3 M, [HA] = 0.12 g/L, pH 3.0, I = 0.1 M KCl) at room temperature under nitrogen atmosphere and exclusion of light. All samples were prepared from CO2free solutions. For sample preparation, 2 mL of HA stock solution (1.5 g/L) were diluted with 15 mL 1 × 10−3 M HCl (I = 0.1 M KCl). Subsequently, 2.5 mL Fe(III) stock solution (14 g/L FeCl3 in 1 × 10−2 M HCl) were added to the HA and the sample volume was filled up to 25 mL using 1 × 10−3 M HCl (I = 0.1 M KCl). Then, the pH value of the solutions was immediately controlled and adjusted to pH 3.0 using diluted NaOH (p.a., Merck) and HCl (p.a., Merck). Simultaneously, blank solutions containing HA but no Fe(III) were prepared. The pH value of the solutions was continuously checked and readjusted. The Fe(II) ions formed by reduction were periodically quantified in form of the 1,10-phenanthroline complex (Greenberg et al., 1992) by UV/ Vis spectroscopy (UV 8452, Hewlett Packard, Waldbronn, Germany) after separation of the HA and masking of Fe(III) (Mack, 2002). For HA separation, 1 mL sample solution was mixed with 0.5 mL 3.59 M H2SO4 (suprapur, Sigma, Steinheim, Germany) and immediately centrifuged at 6000 rpm for 10 min (EBA 21, Hettich Lab Technology, Tuttlingen, Germany). Subsequently, the supernatant was mixed with 0.5 mL 3.59 M H2SO4, 2.0 mL freshly prepared 2 M NH4F solution (p.a., Merck) for masking of Fe3+ ions (Tamura et al., 1974), 2 mL 1% 1,10phenanthroline chloride solution (p.a., Merck) and 2.5 mL ammonium acetate buffer solution (ammonium acetate, p.a., Sigma; acetic acid, p. a., Merck). Finally, the samples were filled to the final volume of 25 mL using Milli-Q water. In addition, a blank solution of the same composition was prepared. After 10 min, UV/Vis measurements were performed against the blank solution and the Fe(II) concentrations were obtained applying previously measured calibration curves. Fe(III) reducing capacities of HA at pH 9.2 were determined according to Matthiessen (1995) and Mack (2002). The sample preparation was performed under nitrogen atmosphere at room temperature starting from CO2-free stock solutions of borate buffer (pH 9.2; I = 0.1 M KCl), K3[Fe(CN)6] (5 × 10−3 M; p.a., Merck) and HA (200 mg/L) both in borate buffer. 20 mL of borate buffer were mixed with 5 mL K3[Fe(CN)6] and 1.25 mL HA stock solution. The samples were filled with buffer solution to a final volume of 50 mL resulting in K3[Fe(CN)6] and HA concentrations of 5 × 10−4 M and 5 mg/L, respectively. In addition, blank solutions of K3[Fe(CN)6] and HA were prepared in the same manner, however, in the absence of HA or K3[Fe(CN)6], respectively. Sample and blank solutions were stored under nitrogen atmosphere and exclusion of light. The timedependent consumption of K3[Fe(CN)6] due to the reduction by HA was regularly analyzed by UV/Vis spectroscopy as described by Matthiessen (1995). 2.3.3. Reduction of U(VI) In order to estimate the redox properties of synthetic HA Hyd-Glu and Cat-Gly towards U(VI), the redox stability of U(VI) was studied at pH 6, 8, and 9 in 0.1 M NaClO4 (p.a., Merck) in comparison to AHA. The experiments were performed at room temperature under nitrogen atmosphere and exclusion of light. According to Abraham (2002), solutions with U(VI) and HA were prepared with initial concentrations of 1 × 10−4 M and 0.4 g/L, respectively, starting from CO2-free UO2(ClO4)2 (5 × 10−3 M) and HA (5 g/L; degassed) stock solutions. The pH values of the solutions were adjusted using diluted NaOH (p.a., Merck) and HClO4 (p.a., Merck). In order to keep constant conditions, pH values were periodically checked and readjusted. After 13 weeks the samples were analyzed with regard to the reduction of U(VI) to U(IV). Redox speciation was performed by solvent extraction using 2thenoyltrifluoroacetone (TTA; p.a., Fluka) according to Bertrand and Choppin (1982). For TTA extraction, 2 mL of sample solution were acidified to pH 0.5 using degassed 5 M HClO4. Subsequently, 2 mL freshly prepared 0.5 M TTA solution in deoxygenated xylene (p.a.,

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Fluka) were added and the samples were shaken vigorously for 10 min. The samples were centrifuged for phase separation (25 min, 4000 rpm, Megafuge 1.0, Heraeus Sepatech, Osterode, Germany). Under these conditions U(IV) species are extracted by TTA in the organic phase whereas U(VI) remains in the aqueous phase. The resulting U(VI) concentration in the aqueous phase was determined by ICP-MS analysis (Elan 6000, Perkin Elmer, Waltham, MA, USA). As HA was present in the aqueous phase, the HA was decomposed with HNO3 (Riedel de Haën, Seelze, Germany) in a microwave oven (mls 1200 mega, MLS, Leutkirch, Germany) before ICP-MS measurements. The amount of U(IV) in the sample solutions was calculated from the difference of the total uranium concentration in the sample solutions and the U(VI) concentration in the aqueous phase. The accuracy of the extraction procedure was verified by analyzing 1 × 10−4 M U(VI) test solutions. For that U(VI) test solutions were subjected to solvent extractions as described above. The aqueous extraction phases were decomposed with HNO3 in a microwave oven and subsequently analyzed by ICP-MS. The recovery of U(VI) after solvent extraction of the test solutions amounted to at least 99.4%. 3. Results and discussion 3.1. Characterization of humic acids 3.1.1. Humic acid-like oxidation products from diphenolic compounds Table 1 summarizes the elemental composition of the HA-like oxidation products from hydroquinone and catechol in comparison to those of AHA and natural HA reported in the literature (Stevenson, 1994). The synthetic products show elemental compositions that are close to those of natural HA. Compared to AHA, the synthesized products are characterized by higher nitrogen contents due to the use of amino acids as precursors. The sulfur contents of the synthetic HA are rather low. They are ascribed to the use of K2S2O8 as oxidizing agent. However, it is not clear in which form sulfur is associated to the HA molecules. The carboxylic group contents of the synthetic HA are similar to those of AHA and other natural HA. However, all HA-like oxidation products from diphenolic compounds show significantly higher phenolic/acidic OH group contents than AHA. The measured values are in the range of the upper value given for natural HA (Stevenson, 1994). Compared to the quinonoid-enriched humic materials developed by Perminova et al. (2005), which are characterized by phenolic

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OH group contents between 2.0 and 4.6 meq/g, the HA-like oxidation products of diphenolic compounds show higher phenolic OH group contents. On the basis of the phenolic/acidic OH group contents, a pronounced redox behavior can be expected for the synthetic HA. Fig. 1 shows the FTIR spectra of the synthetic products in comparison to that of AHA. The HA-like oxidation products from hydroquinone and catechol have FTIR absorption bands that are characteristic for natural HA (e.g., Stevenson, 1994). As expected, the spectra of the synthetic products show slight variations among each other due to the use of different precursor substances. Compared to AHA, the synthetic products show stronger pronounced absorption signals between 805 and 850 cm−1 pointing to substituted aromatic structural elements because of the use of hydroquinone or catechol as precursor material. In contrast to that, the synthetic products show no significant FTIR bands indicating distinct amounts of aliphatic structural elements as measured for AHA at 2850 and 2920 cm−1. In all spectra absorption bands were detected that confirm the occurrence of carboxylic groups in the HA structure (~1720 cm−1). 3.1.2. Humic acids with blocked phenolic OH groups The functional group contents of the HA after chemical modification are also summarized in Table 1. Due to the modification, all HA show significantly lower phenolic/acidic OH group contents. Their amounts were decreased for about 72 to 84%. A complete blocking of phenolic/acidic OH groups was not possible as already discussed in (Sachs et al., 2002). Furthermore, due to the modification a lowering of the carboxylic group contents is observed, which might be attributed to an incomplete hydrolysis of carbonic methyl esters (Sachs et al., 2002). However, for all HA the molar ratio of phenolic/ acidic OH to carboxylic groups is decreased by modification. This enables the application of these modified samples for the study of the influence of phenolic OH groups on the redox properties of HA. 3.2. Redox properties of the synthesized humic acid model substances 3.2.1. Direct determination of formal redox potentials Redox potentials of HA suspensions were directly measured as a function of pH and HA concentration and analyzed based on the methodology recommended by Österberg and Shirshova (1997) and Mack (2002) as described above. Exemplary, Fig. S1 (Supplementary data) shows the measured redox potentials (Eh) for HA Hyd-Gly at different pH and HA concentrations. For all HA, the Eh values at

Table 1 Characterization of synthetic humic acids in comparison to Aldrich humic acid and natural humic acids from literature. Humic acid

Hyd-Gly (batch R12/02) Hyd-Glu (batch R13/02) Cat-Gly (batch R18/02) AHA (batch A2/98) Natural HA (Stevenson, 1994) Humic acid

Hyd-Gly (batch R12/02) Hyd-Gly-PB (batch R19/02) Hyd-Glu (batch R13/02) Hyd-Glu-PB (batch R20/02) Cat-Gly (batch R18/02) Cat-Gly-PB (batch R22/02) AHA (batch A2/98) AHA-PB (batch M173) Natural HA (Stevenson, 1994) a b

Elemental composition a

C (%)

H

54.2 ± 0.2 53.7 ± 0.1 48.8 ± 0.1 58.6 ± 0.1 53.8–58.7

2.3 ± 0.2 2.3 ± 0.1 2.8 ± 0.2 3.0 ± 0.1 3.2–6.2

(%)

N (%)

S (%)

Ob (%)

Ash (%)

Moisture (%)

5.3 ± 0.1 2.0 ± 0.1 5.1 ± 0.1 0.8 ± 0.1 0.8–4.3

0.4 ± 0.1 0.2 ± 0.1 0.9 ± 0.1 3.8 ± 0.1 0.1–1.5

26.6 ± 0.2 28.2 ± 0.1 31.1 ± 0.2 23.5 ± 0.1 32.8–38.3

1.3 1.6 1.7 2.4

9.9 11.9 9.5 7.9

Functional groups COOH (meq/g)

Phenolic/acidic OH (meq/g)

Phenolic/acidic OH:COOH

4.30 ± 0.14 2.99 ± 0.10 3.65 ± 0.14 2.67 ± 0.03 4.16 ± 0.04 2.88 4.49 ± 0.14 2.67 ± 0.01 1.5–5.7

5.3 ± 0.7 1.5 ± 0.1 5.8 ± 0.2 1.4 ± 0.1 6.6 ± 0.7 1.3 ± 0.1 3.1 ± 0.1 0.5 2.1–5.7

1.23 0.50 1.59 0.52 1.59 0.45 0.69 0.19

Corrected for the moisture content of the humic acid. The oxygen content was calculated from the difference to 100% under consideration of the ash and moisture content of the humic acid.

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Österberg and Shirshova, 1997; Szilágyi, 1973). The obtained pH dependencies are similar for all studied HA indicating comparable redox processes. They are comparable to those reported by Mack (2002) for AHA and lignin (see above), however, somewhat higher than those discussed by Österberg and Shirshova (1997) with −(42– 44) mV/pH. From the experimental results, the HA can be ordered as follows with regard to their E0⁎ values: AHA ≥ HA Hyd-Glu N HA Hyd-Gly N HA Cat-Gly. Based on that it is expected that HA Cat-Gly shows the highest metal ion reducing strength, whereas AHA should be characterized by the lowest.

Fig. 1. FTIR spectra of synthetic humic acid type Hyd-Gly, Hyd-Glu and Cat-Gly in comparison to AHA.

different HA concentrations were extrapolated to pH 0. The resulting redox potentials at pH 0 (EpH = 0) and their corresponding pH dependencies (ΔE/ΔpH) are summarized in Table S1 (Supplementary data). For all studied HA no dependence of EpH = 0 from the HA concentration was observed. Thus, the mean values of EpH = 0 and ΔE/ ΔpH were calculated. The resulting formal redox potentials (E0⁎) of the HA at pH 0 and their pH dependencies (ΔE/ΔpH) are summarized in Table 2. The given uncertainties represent standard deviations (3σ) of the mean values. E0⁎ of AHA determined in this work agrees with that of AHA 0⁎ (E = 578 ± 16 mV; ΔE/ΔpH = − 57 ± 10 mV/pH) and lignin (E0⁎ = 579 ± 6 mV; ΔE/ΔpH = −54 ± 1 mV/pH) reported by Mack (2002). Compared to AHA, HA Hyd-Glu shows a similar redox potential, whereas those of HA Hyd-Gly and Cat-Gly are slightly lower. The obtained data for HA Hyd-Glu and AHA are somewhat higher than the apparent standard redox potential of 528 mV (pH 0 and I = 0.1 M NaCl) determined for a soil HA by Österberg and Shirshova (1997) and lower than the normal potential reported by Szilágyi (1973) for peat water HA (~ 700 mV). E0⁎ of HA Hyd-Gly and Cat-Gly is comparable to the value of Österberg and Shirshova (1997). The observed dissimilarities in the redox potentials can be explained by the different nature of the humic substances, the different methods used for their determination and probably by differences of the initial redox states of the HA which depend on their history, e.g., origin, sampling, isolation, and purification. All HA studied in this work were subjected to a comparable preparation procedure (synthesis or purification under alkaline conditions, acidic precipitation, purification, lyophilization). Thus, their initial redox states are comparable. However, these can be different from those of the HA cited above. In the present work, the studied HA were used without further preparation. Thus, the obtained E0⁎ values are caused by the native reducing capacity of the HA which is attributed to its redox-active functional groups (Peretyazhko and Sposito, 2006). With increasing pH values all HA show decreasing redox potentials, as already discussed in the literature for HA and redoxactive components in HA (e.g., Helburn and MacCarthy, 1994;

Table 2 Formal redox potentials (E0⁎) as well as their pH dependencies (ΔE/ΔpH) for the studied humic materials (I = 0.1 M KCl). The uncertainties represent 3σ. Humic acid

E0⁎ (mV)

ΔE/ΔpH (mV/pH)

Hyd-Gly Hyd-Glu Cat-Gly AHA

535 ± 12 565 ± 12 517 ± 12 571 ± 9

−65 ± 3 −64 ± 12 −57 ± 12 −65 ± 2

3.2.2. Fe(III) reducing capacities of unmodified and modified humic acids Table 3 summarizes the measured Fe(III) reducing capacities of the synthetic HA in comparison to AHA. The reducing capacities were determined from the Fe(II) concentration or the [Fe(CN)6]3− consumption in the corresponding solutions after about 3 weeks of equilibration. The different magnitudes of the reducing capacities obtained at pH 3.0 and 9.2 (factor 2.5–6.8) are attributed to different reaction mechanisms contributing to the reduction of Fe3+ and [Fe(CN)6]3− (McDonald and Hamilton, 1973) and to the pH dependent behavior of the humic material. As expected from the E0⁎ value and the high phenolic/acidic OH group content, HA Cat-Gly shows the highest reducing strength for Fe3+ and [Fe(CN)6]3− of all studied HA. At pH 3.0 and 9.2 the reducing capacities of HA Cat-Gly are about 12 and 5 times, respectively, higher than those of AHA. HA Hyd-Gly and Hyd-Glu show also significantly higher reducing capacities than AHA. From that it can be concluded that the oxidation products of diphenolic compounds are characterized by more pronounced redox functionalities towards Fe(III), thus, showing a more pronounced redox behavior than AHA. Compared to literature data (Mack, 2002), the reducing capacities of HA Hyd-Gly and Hyd-Glu at pH 3.0 resemble to those reported for synthetic polymerization products of 2,5-dihydroxybenzoic acid (12.0 ± 0.3 meq/g), 3,4,5-trihydroxybenzoic acid (11.4 ± 0.3 meq/ g), and 3,4-dihydroxycinnamic acid (9.2 ± 0.2 meq/g), whereas the reducing capacity of HA Cat-Gly is higher. The synthetic HA were prepared under oxidizing conditions. That means, the initial redox state of the synthetic products is rather oxidative than reductive. Consequently, the measured reducing capacities correspond to the non-negligible or native reducing capacity of HA already mentioned above. Hence, the high reducing capacities of the synthetic HA point to important reducing functional groups in these HA. The [Fe(CN)6]3− reducing capacities of HA Hyd-Gly, Hyd-Glu and Cat-Gly determined at pH 9.2 are distinctly higher than those reported by Matthiessen (1995) for natural HA from different origin as well as for phenol, which were about 2 meq/g. Furthermore, they are also higher than those measured by Mack (2002) for lignin (7.2 ± 0.2 meq/ g) and Aldrich HA (5.9 ± 0.5 meq/g). The reducing behavior of a synthetic HA-like product obtained by oxidation of hydroquinone was

Table 3 Fe(III) reducing capacities of unmodified (Hyd-Gly, Hyd-Glu, Cat-Gly, AHA) and modified (Hyd-Gly-PB, Hyd-Glu-PB, Cat-Gly-PB, AHA-PB) humic materials (I = 0.1 M KCl). Humic acid

Hyd-Gly Hyd-Gly-PB Hyd-Glu Hyd-Glu-PB Cat-Gly Cat-Gly-PB AHA AHA-PB a

After ~ 3 weeks reaction time.

Fe(III) reducing capacitya (meq/g) pH 3.0

pH 9.2

8.8 ± 0.2 3.0 ± 0.2 10.7 ± 0.2 2.6 ± 0.1 14.5 ± 1.6 2.7 ± 0.2 1.2 ± 0.1 0.5 ± 0.1

27.5 ± 2.7 10.1 ± 1.8 33.6 ± 4.0 12.0 ± 0.8 36.9 ± 0.2 13.4 ± 0.1 7.2 ± 1.9 3.4 ± 1.4

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studied by Matthiessen (1995) between pH 4 and 10.5. A [Fe(CN)6]3− reducing capacity of about 21 meq/g was measured by redox titration at pH 9, which is also lower than those measured for the HA-like oxidation products studied in the present work. A direct comparison of the [Fe(CN)6]3− reducing capacities measured in this work with those reported by Perminova et al. (2005) for quinonoid-enriched humic materials is not possible because of the different pH values used for their determination. Perminova et al. (2005) reported [Fe (CN)6]3− reducing capacities between 1.1 and 4.0 meq/g at pH 6. With increasing pH from 6 to 9, Matthiessen (1995) observed an increase of the [Fe(CN)6]3− reducing capacity of a HA-like hydroquinone oxidation product from about 5 to 21 meq/g, which represent an increase for about factor 4. Assuming a comparable pH dependence of the reducing capacities of the quinonoid-enriched humic materials developed by Perminova et al. (2005), reducing capacities of about 4 to 16 meq/g can be expected for these materials at pH 9. These estimated values are lower than those measured in this work for the HA-like oxidation products of diphenolic compounds. The observed differences in the reducing capacities can be explained by different structural and functional properties of the humic material, however, most probably by their different phenolic OH group contents (see below and Fig. S2; Supplementary data). Furthermore, the [Fe(CN)6]3− reducing capacities reported by Matthiessen (1995) and Mack (2002) were measured after 24 h equilibration time, whereas the data reported in this work were obtained after about 3 weeks. Hence, it can be assumed that the reducing capacities measured in this work include a higher part of irreversible redox processes such as coupling reactions of phenoxy radicals and tautomerizations of the coupled products resulting in the regeneration of phenolic OH groups (Helburn and MacCarthy, 1994). Using mediated electrochemical reduction, Aeschbacher et al. (2010) measured an electron accepting capacity for AHA (0.923 ± 0.06 meq/g at Eh = −0.49 V) which represents the number of electrons reductively transferred to the HA at a given potential. In the present work, Fe(III) reducing capacities were measured which represent the reverse process, i.e., the release of electrons from HA to a metal ion. For that, HA in their native form without further previous preparation, e.g., prereduction, were applied. In spite of that, both the Fe(III) reducing capacities of AHA determined at pH 3.0 and 9.2 (cf. Table 3) are higher than the electron accepting capacity for AHA determined by Aeschbacher et al. (2010). This indicates that the original HA contain native redox-active functional groups. In addition, it could be an indication for irreversible redox processes as mentioned above that contribute to the reduction of Fe(III) under the studied conditions. Based on the performed studies, a differentiation between reversible and irreversible redox-sensitive functional groups of the HA contributing to the Fe(III) reducing capacities is not possible. The electrochemical methods proposed by Aeschbacher et al. (2010) would offer the possibility to measure these. Referring the measured reducing capacities of the HA to their phenolic/acidic OH group contents (cf. Table 1) it is possible to draw conclusions concerning the type of the redox-active, i.e., electron transferring, functional groups (Mack, 2002). Fig. 2 shows the reducing capacities of the HA after 3 weeks of equilibration in comparison to the phenolic/acidic OH group contents of the HA. At pH 3.0 and 9.2 all synthetic HA show reducing capacities that are higher than their phenolic/acidic OH group contents. This indicates that there are additional functional groups or redox processes other than the single oxidation of phenolic OH groups contributing to the reduction of Fe(III). For AHA this fact applies only for the redox process at pH 9.2. At pH 3 the Fe(III) reducing capacity of AHA is lower than its amount of phenolic/acidic OH groups indicating that not all of these functional groups are involved in the reduction of Fe(III). From literature it is known, that redox reactions of phenols with oneelectron-transfer oxidizing agents such as Fe3+ and [Fe(CN)6]3− are complex, consisting of numerous dimerization and oxidative coupling processes (e.g., Helburn and MacCarthy, 1994; McDonald and

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Fig. 2. Fe(III) reducing capacities (RC) of the studied humic acids at pH 3.0 and 9.2 after 3 weeks of equilibration in comparison to their phenolic/acidic OH group contents.

Hamilton, 1973; Musso, 1967). The reducing capacities which are higher than those which would be expected based on the HA phenolic/acidic OH group content, might be explained with a higher concentration of phenoxy radicals compared to the HA phenolic/ acidic OH group concentration and an increased oxidative coupling as discussed by Helburn and MacCarthy (1994). As already mentioned, the dimerization of phenoxy radicals is irreversible. Tautomerization of the coupled products results in regeneration of phenolic OH groups, which are capable of further oxidation. This indicates again the contribution of irreversible reactions to the redox process. The differences in the redox behavior of the synthetic HA and AHA can be explained by structural and/or functional dissimilarities of the humic matter, most likely by their different amounts of phenolic/ acidic OH groups which contribute to irreversible redox processes. By correlation of the Fe(III) reducing capacities at pH 3.0 and 9.2 with the phenolic/acidic OH group content of the HA a linear correlation is found, which indicates the dominant role of phenolic/acidic OH groups for the redox behavior (R2 = 0.99 for pH 3.0 and 9.2; Supplementary data Fig. S2). Differences in the correlation functions are due to the different reaction mechanisms contributing to the reduction of Fe3+ and [Fe(CN)6]3− (McDonald and Hamilton, 1973) and to the pH dependent behavior of the humic material. In order to proof that the high amounts of phenolic/acidic OH groups of the synthesized HA cause their strong reducing capacities, Fe(III) reducing capacities of the modified HA with blocked phenolic/acidic OH groups were determined and compared to those of the corresponding unmodified HA. The obtained data are summarized in Table 3. The blocking of phenolic/acidic OH groups inhibits the reduction of Fe3+ and [Fe(CN)6]3− for all HA resulting in a significant decrease of the reducing capacities. At pH 3.0 the reducing capacities of the synthetic HA are decreased for 66 to 81% which is comparable to the decrease of the phenolic/acidic OH group contents (72–80%). This correlation indicates the significance of phenolic/acidic OH groups for the reduction of Fe(III). Comparable results were reported by Mack (2002) for an unmodified and modified oxidation product of 3,4,5-trihydroxybenzoic acid. The decrease of the Fe(III) reducing capacity of AHA (58%) is lower than its decrease of the phenolic/acidic OH group content (84%). However, the role of phenolic/acidic OH groups for the reduction of Fe(III) becomes also clear from these data. Even though slightly less, a decline of the reducing capacities of the HA due to the modification is also observed at pH 9.2, pointing again to the importance of HA phenolic/acidic OH groups for the redox properties of the studied HA. The residual reducing capacities after the blocking of phenolic/acidic OH groups can be attributed to the fact that it was not possible to modify all phenolic/acidic OH groups (cf. Table 1) and furthermore, to other structural elements of the HA

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contributing to the reduction of Fe(III). Based on these results it can be concluded that the high phenolic/acidic OH group contents of the oxidation products of diphenolic compounds cause their stronger reducing capacities towards Fe3+ and [Fe(CN)6]3− compared to AHA. However, it remains unclear if the reducing phenolic OH groups in the studied HA have its origin in hydroquinone-like structures. The highest reducing capacities were found for HA Cat-Gly, synthesized from catechol, where the phenolic OH groups are substituted in oposition in contrast to hydroquinone with p-substituted phenolic OH groups. The present results agree with literature discussions where the reducing properties of HA were attributed amongst others to the oxidation of phenolic OH groups (Helburn and MacCarthy, 1994; Deiana et al., 1995; Matthiessen, 1995; Schmeide and Bernhard, 2009). Furthermore, they are in agreement with a statement of Chen et al. (2003) who ascribed differences in the Fe(III) redox behavior of various natural organic matter to dissimilarities in the amount of specific electron-donating functional groups, including polyphenolic and phenolic OH groups. 3.2.3. Reduction of U(VI) in the presence of humic acids with pronounced redox functionality The reduction of U(VI) in the presence of HA Hyd-Glu and Cat-Gly was studied and compared to AHA. Comparing the redox potential of the redox couple U(VI)/U(IV) (−70 ± 80 mV, Allard et al., 1980) at pH 8 with those of HA Hyd-Glu (~53 mV) and Cat-Gly (~61 mV) at the same pH value, it becomes clear that they are close together within the range of their uncertainties. Based on thermodynamic data, Reiller (2005) prognosticated that a reduction of U(VI) in the presence of HA would be possible at pH 7, when Eh ≤ 80 mV/SHE. Taking these facts and the measured redox potentials of HA as a function of pH into account (cf. Table 2), a weak reduction of U(VI) by HA cannot be excluded under the applied experimental conditions. Fig. 3 shows the percentage of U(VI) in the HA solutions after 13 weeks of equilibration of an initial U(VI) humate solution. The main part of uranium occurs in form of U(VI) in the HA solutions. Only a very low amount of U(IV) (cf. Fig. 3a, difference to 100%) was detectable by solvent extraction pointing to only a slight reduction of U(VI). Comparing the percentage of U(VI) as a function of pH it can be seen that it slightly increases with increasing pH, simultaneously, the percentage of U(IV) decreases. This indicates a slight pH dependence of the reduction of U(VI) to U(IV) in the presence of HA. Due to the very complex processes occurring in solution, such as changes of the uranium speciation and of the redox potentials as a function of pH, the reason for the observed pH dependence of the U(VI) reduction is not clear up to now. Assuming a transfer of two electrons from the HA to U(VI), U(VI) reducing capacities were calculated. The obtained data are summarized in Fig. 3b in comparison to the phenolic/acidic OH group contents of the HA. The U(VI) reducing capacities of the HA are significantly lower than their phenolic/acidic OH group contents. As expected based on E0⁎ and the measured Fe(III) reducing capacities, HA Cat-Gly shows the highest reducing strength for U(VI). The U(VI) reducing capacities of HA Hyd-Glu and AHA are comparable. The reason for that is still not clear. Compared to the reduction of [Fe (CN)6]3− and Np(V) (Schmeide and Bernhard, 2009) by the same humic materials at pH 9.2 and pH 9.0, respectively, the U(VI) reducing capacities are distinctly lower. This is ascribed to the lower redox potential of the redox couple U(VI)/U(IV) (e.g., Eh (pH 9): ~–100 mV, deduced from Allard et al., 1980; see also data compiled by Takeno, 2005) compared to that of [Fe(CN)6]3−/[Fe(CN)6]2− (Eh = 360 mV; Helburn and MacCarthy, 1994) and Np(V)/Np(IV) (e.g., Eh (pH 9): ~ 200 mV, deduced from Allard et al., 1980; see also data compiled by Takeno, 2005). The obtained U(VI) reducing capacities are also significantly lower than those observed for U(VI) in the presence of HA and microorganisms as measured, for instance by Gu and Chen (2003), with a value of about 3.5 meq/g at pH 8.1 and an initial HA concentration of 100 mg C/L (estimated based on data given in Gu and

(a)

(b)

Fig. 3. a) Percentage of U(VI) in solutions of humic acid Cat-Gly, Hyd-Glu and AHA at pH 6, 8, and 9 after 13 weeks equilibration time ([U]tot = 1 × 10−4 M, [HA] = 0.4 g/L, I = 0.1 M NaClO4). b) U(VI) reducing capacity (RC) of humic acid Cat-Gly, Hyd-Glu and AHA at pH 6, 8, and 9 after 13 weeks equilibration time in comparison to their phenolic/ acidic OH group contents.

Chen, 2003). The U(VI) reduction by HA was already studied by Abraham (2002), however, applying unpurified AHA. At pH 8.4 after four weeks of equilibration, Abraham (2002) measured a reducing capacity of (4.26 ± 0.44) × 10−3 meq/g. This value is lower than those obtained in the present work. This could be due to the fact that the used unpurified AHA contains a significant fraction of inorganic components including iron (Aldrich HA unpurified: 12 mg Fe/g according to Kim et al., 1990) which can influence the redox behavior of the organic material. Furthermore, a shorter equilibration time was applied. Gu and Chen (2003) did not observe an abiotic reduction of U(VI) by natural organic matter, however, its occurrence was not fully excluded, because of the presence of small amounts of nitrate in the reactant solutions used. During the HA-mediated reduction of U(VI) to U(IV) meta-stable U(V) should occur as an intermediate. However, under the applied experimental conditions a spectroscopic proof for trace amounts of U(V) as discussed by Steudtner et al. (2006) and Grossmann et al. (2009) for U(VI) redox processes in biological, organic and inorganic systems, is not possible. In the present work the reduction of U(VI) in the presence of HA was studied applying HA prepared under oxidative conditions. Under reducing conditions, e.g., in deep geological formations, or in the presence of microorganisms, the redox state of the HA can be much more reductive. Hence, HA can play a more important role in the reduction of U(VI).

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4. Conclusions HA-alike model substances were synthesized based on the oxidation of diphenolic compounds in the presence of α-amino acids. The synthetic products are characterized by elemental, functional and structural properties comparable to natural HA. However, they exhibit high phenolic/acidic OH group contents that range at the upper limit cited for natural HA (Stevenson, 1994). For all synthetic products formal redox potentials were measured which are comparable or somewhat lower than that determined for purified HA from Aldrich. The pH dependence of the formal redox potentials is similar for all studied humic materials. Although the HA-alike products were prepared under oxidative conditions, they are characterized by a pronounced reduction capability which can be attributed to their non-negligible native reducing capacity which is maintained even under oxidizing conditions (Peretyazhko and Sposito, 2006). Fe(III) reducing capacities were measured which are significantly higher than those of HA from Aldrich. Furthermore, indications for a slight reduction of U(VI) in the presence of HA were observed, especially in the presence of the HA-like oxidation product from catechol and glycine. Applying modified HA with blocked phenolic/acidic OH groups evidence for the significant role of phenolic/acidic OH groups for the redox properties of HA was given, confirming literature data (e.g., Deiana et al., 1995; Helburn and MacCarthy, 1994; Matthiessen, 1995; Schmeide and Bernhard, 2009). This study shows that it is possible to synthesize HA model substances with pronounced redox properties even under oxidizing conditions. These substances offer the possibility to perform basic studies with regard to environmentally-relevant interaction processes of humic materials, for instance, studies concerning the redox behavior of humic substances and the redox stability of metal ions in the presence of HA. The synthesized HA represent model substances with high native reducing capacities that exceed those of natural HA considered in this work. It is conceivable to further increase the reducing capacities of the synthetic HA by electrochemical reduction of the humic material as proposed by Aeschbacher et al. (2010); however, this was not performed in the present work. Applying these synthetic products, the influence of natural humic matter on redox processes of metal ions which can appear under natural conditions, for instance in soils or deep geologic formations, can be assessed. From literature it is known that reoxidation processes of metal ions in lower oxidation states in the presence of humic material can occur already at very low oxygen concentrations, e.g., reoxidation of U(IV) and Np(IV) to U(VI) and Np(V), respectively (Gu et al., 2005; Schmeide and Bernhard, 2009). The application of synthetic HA model substances with pronounced redox functionality instead of natural HA with less developed redox behavior, enables the stabilization of low oxidation states of redox-sensitive metal ions, such as actinide ions, e.g., in complexation, sorption and migration studies with concern to the influence of HA on geochemical interaction processes of metal ions in the environment. The ability of a synthetic HA model substance with pronounced redox functionality to maintain effectively Np in the tetravalent oxidation state was demonstrated by Schmeide and Bernhard (2009 and 2010). The specific use of synthetic HA model substances contributes to a more reliable modeling of geochemical interaction processes of metal ions in the environment which enables a trustworthy risk-assessment for potential nuclear waste repositories and contaminated sites with radioactive and toxic heavy metal inventory. Acknowledgements This study was supported by the German Federal Ministry of Economics and Technology (BMWi) under contract No. 02 E 9299 and by the EC Commission under contract No. FIKW-CT-2001-00128. The authors thank R. Ruske, M. Meyer, and S. Heller for the preparation

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