The present study deals with a new analytical procedure based on a cellulose diffusion membrane and immobilised tetraethylene-pentamine-hexaacetate chelator (DM-TEPHA) for an in situ differentiation of labile and inert metal species in aquatic systems. The DM-TEPHA system was prepared by placing TEPHA chelator in pre-purified cellulose bags and in situ applied immersing the system in two Brazilian rivers to study the relative lability of metal species (Cu, Pb, Fe, Mn and Ni) as a function of the time and the quantity of exchanger, respectively. The procedure is simple and enables a new perspective for understanding the complexation, transport, stability and lability of metal species in aquatic systems rich in organic matter.

This paper deals with the development and optimization of an analytical procedure using ultrafiltration and a flow-injection system, and its application in in-situ experiments to characterize the lability and availability of metal species in humic-rich hydrocolloids. The on-line system consists of a tangential flow ultrafiltration device equipped with a 3-kDa filtration membrane. The concentration of free ions in the filtrate was determined by atomic-absorption spectrometry, assuming that metals not complexed by aquatic humic substances (AHS) were separated from the complexed species (M–AHS) retained by the membrane. For optimization, exchange experiments using Cu(II) solutions and AHS solutions doped with the metal ions Ni(II), Mn(II), Fe(III), Cd(II), and Zn(II) were carried out to characterize the stability of the metal–AHS complexes. The new procedure was then applied in-situ at a tributary of the Ribeira do Iguape river (Iguape, São Paulo State, Brazil) and evaluated using the ions Fe(III) and Mn(II), which are considered to be essential constituents of aquatic systems. From the exchange between metal–natural organic matter (M–NOM) and the Cu(II) ions it was concluded that Cu(II) concentrations >485 μg L−1 were necessary to obtain maximum exchange of the complexes Mn–NOM and Fe–NOM, corresponding to 100% Mn and 8% Fe. Moreover, the new analytical procedure is simple and opens up new perspectives for understanding the complexation, transport, stability, and lability of metal species in humic-rich aquatic environments.

A new procedure was developed in this study, based on a system equipped with a cellulose membrane and a tetraethylenepentamine hexaacetate chelator (MD–TEPHA) for in situ characterization of the lability of metal species in aquatic systems. To this end, the DM–TEPHA system was prepared by adding TEPHA chelator to cellulose bags pre-purified with 1.0molL−1 of HCl and NaOH solutions. After the MD–TEPHA system was sealed, it was examined in the laboratory to evaluate the influence of complexation time (0–24h), pH (3.0, 4.0, 5.0, 6.0 and 7.0), metal ions (Cu, Cd, Fe, Mn and Ni) and concentration of organic matter (15, 30 and 60mgL−1) on the relative lability of metal species by TEPHA chelator. The results showed that Fe and Cu metals were complexed more slowly by TEPHA chelator in the MD–TEPHA system than were Cd, Ni and Mn in all pH used. It was also found that the pH strongly influences the process of metal complexation by the MD–TEPHA system. At all the pH levels, Cd, Mn and Ni showed greater complexation with TEPHA chelator (recovery of about 95–75%) than did Cu and Fe metals. Time also affects the lability of metal species complexed by aquatic humic substances (AHS); while Cd, Ni and Mn showed a faster kinetics, reaching equilibrium after about 100min, and Cu and Fe approached equilibrium after 400min. Increasing the AHS concentration decreases the lability of metal species by shifting the equilibrium to AHS–metal complexes. Our results indicate that the system under study offers an interesting alternative that can be applied to in situ experiments for differentiation of labile and inert metal species in aquatic systems.

The separation behavior of the frequently administered pharmaceuticals sulfamethoxazol (Sulfa), carbamazepine (Carba), diclofenac (Diclo), and ibuprofen (Ibu) on different natural and synthetic ultrafiltration membranes was studied. Commercially available cattle intestine natural membranes (NM), polyethersulfone (PES), and regenerated cellulose-based (RC) flat membranes (nominal cut-off 1 kDa) have been investigated as ultrafiltration membranes in a small tangential-flow ultrafiltration unit (TF-UF). First, the nominal cut-off of the NM membranes under study was assessed at approximately 5 kDa, by using polystyrenesulfonate standards for pore-size classification at low TF-UF pressure (0.25 x 10(5) Pa). Working pressures of >1.5 x 10(5) Pa strongly increased the cut-off of NM, in contrast with that of PES and RC membranes. Sulfa, Carba, Diclo and Ibu (1 mg L(-1) each) in colloid-free aqueous solutions (400 mg L(-1) NaCl) completely permeated through NM membranes, but less through PES and RC, which had particular sorption capability towards Diclo. The drugs were routinely determined by using high-performance liquid chromatography (HPLC). Detailed TF-UF investigation of drug retention on NM in the presence of humic hydrocolloids revealed strong interactions between aquatic humic substances (HS) and Diclo and Ibu (but not with Sulfa and Carba) causing retention of up to 80% of Diclo and Ibu, probably because of their binding to macromolecular HS. The standard deviation (SD) of both drug and HS permeation through a single NM was between 2.5 (Sulfa) and 4.0% (Diclo), in contrast with the SD of permeation through separate membranes taken from different lots [SD up to 14.0% (Diclo)], presumably caused by natural variation of the studied NM. Accordingly, membrane filtration of drug-containing water samples on cattle intestines enables both analyte/matrix separations for Carba and Sulfa in the presence of humic colloids and analytical discrimination between free and colloid-bound Diclo and Ibu fractions.

Transformations of metal species (particularly Al, Ca, Fe, Mg, Mn, Zn) in ageing humic hydrocolloids were studied, applying a competitive ligand and metal exchange approach. For this purpose, metal-containing hydrocolloids, freshly collected from humic-rich German bog lake waters (Hohlohsee (HO), Black Forest; Venner Moor (VM), Muensterland; Arnsberger Wald (AW), Northrhine-Westfalia) and conventionally pre-filtered through 0.45 μm membranes, were subjected on-site to an exchange with EDTA and Cu(II) ions, respectively, as a function of time. EDTA complexes gradually formed, metal fractions exchanged by Cu(II) (as well as free Cu(II) concentrations) were operationally discriminated by means of a small time-controlled tangential-flow ultrafiltration unit (nominal cut-off: 1 kDa). Metal and DOM (dissolved organic matter) fractions obtained this way were determined off-site using instrumental methods (AAS, ICP-OES, carbon analyzer). After weeks of storage, the collected hydrocolloids were studied again by this approach. The EDTA availability of colloid-bound metals (particularly Al and Fe) exhibited different ageing trends, dependent on the sample (VM: decrease of Fe availability (98–76%), HO: increase of Fe availability (76–82%)). In contrast, the Cu(II) exchange equilibria of colloid-bound metals revealed merely low availability of Al (16–38%) and Fe (5–11%) towards Cu(II) ions, also dependent on ageing effects. In particular, the conditional copper exchange constants K ex obtained from the exchange between Cu(II) ions and available metal species (such as Ca, Mg, Mn, Zn) exhibited a strong decrease (by a factor of 2–100) during sample storage, indicating considerable non-equilibria complexation of these metal ions in the original bogwaters studied on-site.

The influence of structural features of tropical river humic substances (HS) on their capability to reduce mercury(II) in aqueous solutions was studied. The HS investigated were conventionally isolated from Rio Negro water-Amazonas State/Brazil by means of the collector XAD 8. In addition, the isolated HS were on-line fractionated by tangential-flow multistage ultrafiltration (nominal molecular-weight cut-offs: 100, 50, 30, 10, 5 kDa) and characterized by potentiometry and UV/VIS spectroscopy. The reduction of Hg(II) ions to elemental Hg by size-fractions of Rio Negro HS was assessed by cold-vapor AAS (CVAAS). UV/VIS spectrometry revealed that the fractions of high molecular-size (F1>100 kDa and F2: 50–100 kDa) have a higher aromaticity compared to the fractions of small molecular-size (F5: 5–10 kDa, F6: <5 kDa). In contrast, the potentiometric study showed different concentration of functional groups in the studied HS fractions. The reduction of Hg(II) by aquatic HS fractions at pH 5 proceeded in two steps (I, II) of slow first order kinetics (t1/2 of I: 160 min, t1/2 of II: 300 min) weakly influenced by the molecular-size, in contrast to the differing degree of Hg(II) reduction (F5>F2>>F1>F3>F4>>F6). Accordingly, Hg(II) ions were preferably reduced by HS molecules having a relatively high ratio of phenolic/carboxylic groups and a small concentration of sulfur. From these results a complex ‘competition’ between reduction and complexation of mercury(II) by aquatic HS occurring in tropical rivers such as the Rio Negro can be suggested.

Organic metal species and their size fractions in three German white wines were characterized by combining multistage ultrafiltration (MST-UF), determination of non-volatile dissolved organic carbon (NV-DOC) by a home-built carbon analyser, and metal quantification by inductively coupled plasma mass spectrometry (ICP–MS). First, NV-DOC and metal species in selected "dry" German white wines were fractionated on-line using MST-UF in the size range of >100 kDa to <1 kDa. For this purpose a 20 mL sample of the wine under study diluted 1:10 with high-purity water was processed through a cascade system of hydrophilized polyethersulfone-based flat membranes of decreasing cut-off (100, 50, 10, 5, and 3 kDa). An aliquot of the fraction <3 kDa was additionally processed through a commercial UF tube (MidGee system, cut-off: 1 kDa) to obtain low-molecular size fractions also. A home-built carbon analyser was applied to determine NV-DOC in the wines and their size fractions. The NV-DOC found in a German reference wine and its size fractions was as follows: total NV-DOC: 8.97 mg mL−1; F1 (>100 kDa), 0.15%; F2 (50–100 kDa), 0.44%; F3 (10–50 kDa), 0.74%; F4 (5–10 kDa), 0.76%; F5 (5–3 kDa), 0.7%; F*6 (3–1 kDa), 0.9%; F7 (<1 kDa), 81.6% (related to total NV-DOC). The NV-DOC recovery was 85.2%. Accordingly, most of the NV-DOC in this wine consists of low-molecular mass organic compounds of <1 kDa, presumably carboxylic acids as typical in wine. Parallel metal determinations in these wines and their fractions were performed by ICP–MS. The measurements showed that the major part of the metals investigated, up to 25 elements, were dissolved in the size fraction of <1 kDa except Ba, Sr and Pb which appeared also in other fractions. In addition, conventional UV–VIS spectroscopy was applied to characterise the studied wines and their size fractions. According to this, the UV absorbance between 254 and 280 nm of these white wines shows a parallel trend to their NV-DOC.

An improved on-site characterization of humic-rich hydrocolloids and their metal species in aquatic environments was the goal of the present approach. Both ligand exchange with extreme chelators (diethylenetetraaminepentaacetic acid (DTPA), ethylendiaminetetraacetic acid (EDTA)) and metal exchange with strongly competitive cations (Cu(II) were used on-site to characterize the conditional stability and availability of colloidal metal species in a humic-rich German bogwater lake (Venner Moor, Münsterland). A mobile time-controlled tangential-flow ultrafiltration technique (cut-off: 1 kDa) was applied to differentiate operationally between colloidal metal species and free metal ions, respectively. DOC (dissolved organic carbon) and metal determinations were carried out off-site using a home-built carbon analyzer and conventional ICP-OES (inductively-coupled plasma-optical emission spectrometry), respectively. From the metal exchange equilibria obtained on-site the kinetic and thermodynamic stability of the original metal species (Fe, Mn, Zn) could be characterized. Conditional exchange constants Kex obtained from aquatic metal species and competitive Cu(II) ions follow the order Mn > Zn > > Fe. Obviously, Mn and Zn bound to humic-rich hydrocolloids are very strongly competed by Cu(II) ions, in contrast to Fe which is scarcely exchangeable. The exchange of aquatic metal species (e.g. Fe) by DTPA/EDTA exhibited relatively slow kinetics but rather high metal availabilities, in contrast to their Cu(II) exchange.

This chapter contains sections titled: Introduction Description of Analytical Methods Sample Treatment Flame and Graphite Atomic Absorption Spectrometry (FAAS, GF-AAS) Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Inductively Coupled Plasma Isotope Dilution Mass Spectrometry (ICP-IDMS) Instrumental Neutron Activation Analysis (INAA) Total Reflection X-Ray Fluorescence Spectrometry (TXRF) Results of the Interlaboratory Study Element Concentrations in ROS Fractions and their Original Samples Relative Enrichment Factors of Elements in Separated FA and HA Fractions Quality Control of Analytical Data References

The binding and availability of metals (Al, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Pb, Zn) in therapeutically applied peat (Grosses Gifhorner Moor, Sassenburg/North Germany) was characterized by means of a versatile extraction approach. Aqueous extracts of peat were obtained by a standardized batch equilibrium procedure using high-purity water (pH 4.5 and 5.0), 0.01 mol l(-1) calcium chloride solution, 0.01 mol l(-1) ethylenediaminetetraacetic acid (EDTA) and 0.01 mol l(-1) diethylenetriamine pentaacetic acid (DTPA) solution as metal extractants. In addition, the availability of peat-bound metal species was kinetically studied by collecting aliquots of extracts after different periods of extraction time (5, 10, 15, 30, 60 and 120 min). Metal determinations were performed by atomic spectrometry methods (AAS, ICP-OES) and dissolved organic matter (DOM) was characterized by UV/Vis measurements at 254 and 436 nm, respectively. Of the extractants studied Ca, Mg and Mn were the most available metals, in contrast to peat-bound Fe and Al. The relative standard deviation s(r) of the developed extraction procedures was mostly in the range of 4 to 20%, depending on the metal and its concentration in peat. A pH increase favored the extraction of metals and DOM from peat revealing complex extraction kinetics. Moreover, a competitive exchange between peat-bound metal species and added Cu(II) ions showed that >100 mg of Cu(II) per 50 g wet peat was necessary to exchange the maximum of bound metals (e.g. 21.8% of Al, 3.9% of Fe, 79.0% of Mn, 81.9% of Sr, related to their total content).

Humic-rich hydrocolloids and their metal species (e.g., Al, Fe, Ca, Cu, Fe, Mg, Mn, Pb, Zn) in selected bogwaters of German origin (Black Forest (HO), Muensterland (VM), Arnsberger Wald (AW)) were size classified in the subparticulate and macromolecular range by on-site multistage ultrafiltration (MST-UF). For this purpose two MST-UF cascades, equipped with up to eight conventional flat membranes (0.45, 0.22, 0.1 micron (Millipore); 100, 50, 10, 5, 3, and/or 1 kDa (Gelman PallFiltron OMEGA), were coupled and used for an online fractionation of hydrocolloidal matter immediately after water sample collection. Quantification of dissolved organic carbon (DOC) and metals was carried out off-site by conventional laboratory methods (carbon analyzer, atomic spectrometry). The size distribution of some humic-rich hydrocolloids (e.g. in HO) was exhibited to be surprisingly stable even over storage periods of 1-4 weeks. On the other side, their size distribution (e.g. in VM) considerably varied during the collection period. The natural metal loadings (e.g. Al, Fe, Mn, Zn) of hydrocolloids showed characteristic size distributions and, mostly, strong metal enrichment in subparticulate and macromolecular fractions. Further results of on-site classification of hydrocolloids, obtained by parallel single-stage ultrafiltration (P-UF) with the same membranes, were only comparable to those of MST-UF in the case of low DOC concentration.

Two-dimensional phase sensitive C,H correlation spectra were successfully applied to the quantification of carbohydrate substructures in aquatic humic substance (HS) fractions obtained by tangential flow multistage ultrafiltration (TFMSTUF) of a selected bog water HS (HO13, German Research Program DFG-ROSIG) as well as a river HS (Suwannee River Fulvic Acid Reference of the International Humic Substances Society, IHSS). It turns out that after size fractionation the HS samples give very well resolved C,H-correlation spectra which offer a great potential for substructure quantification. Details of the combined substructure quantification technique, novel in HS characterization, are presented. The results of the combined procedure point out that carbohydrate moieties predominantly occur in higher molecular mass fractions (> 10 kDa) of isolated HS.

Humic-rich hydrocolloids and their metal loading in selected German bog-waters have been characterized by a novel on-site approach. By use of an on-line multistage ultrafiltration (MST-UF) unit equipped with conventional polyethersulfone (PES)-based flat membranes (nominal cut-off 0.45, 0.22, and 0.1 µm, or 100, 50, 10, 5, 3 kDa) the hydrocolloids could be fractionated on-site in both sub-particulate and macromolecular size ranges. Characterization (dissolved organic carbon (DOC), metals) of the colloid fractions obtained this way was performed off-site by use of conventional instrumental methods (carbon analyzer, AAS, ICP–OES, and TXRF (total reflection X-ray fluorescence)). Major DOC fractions of the hydrocolloids studied were found to be in the size range <5 kDa. The assessed metals (Al, Cu, Fe, Mn, Pb, and Zn) were, however, predominantly enriched in the macromolecular and sub-particulate range, depending on the metal and the sample, respectively. In addition, metal species bound to these hydrocolloids were kinetically characterized on-site by use of competitive ligand (EDTA (ethylenediaminetetraacetate)) and metal (Cu(II)) exchange; the EDTA complexes formed and the metal ions exchanged were separated by means of a small time-controlled tangential-flow UF unit (cut-off 1 kDa). Bound metal fractions, in particular Al and Fe, reacted only slowly (500 to 1000 min) with EDTA; the conditional availability was 60–99%, depending on the hydrocolloid. In contrast, the Cu(II) exchange of colloid-bound metal species approached equilibrium within 5–10 min, with characteristic exchange constants, Kex, of the order of 0.01 to 90 for the metals (Fe<Al<Zn<Mn≈Ca≈Mg). The results were clearly dependent on the water investigated. Thus detailed information on the conditional kinetic and thermodynamic stability of colloid-bound metal species could be obtained from competitive EDTA and Cu(II) exchange under on-site conditions.

The interpretation of the results of pyrolysis studies of humic substances is fraught with difficulties if only a single analytical technique is employed for substructure analyses. In this paper we have carried out investigations into humic substances utilising the additional information from the combination of gas chromatography/infrared spectroscopy and gas chromatography/mass spectrometry detection following Curie-point pyrolysis at five different temperatures. The results were compared to those obtained from conventional flash pyrolysis. The aim was to ensure the validation of the results of the compounds identified by at least one other spectroscopic technique. Some 30 substances have been positively identified by infrared spectroscopy, although their appearance is dependent on pyrolysis method and temperature. Most assignments were validated by mass spectrometry or the substance class confirmed.

Two-dimensional phase sensitive C-13,H-1 correlation spectra were applied to the investigation of substructures in size fractions obtained by tangential flow multistage ultrafiltration (MST-UF) of humic substances (HS) Suwannee River Fulvic Acid Reference (HS SR FA) and Suwannee River Humic Acid Reference (HS SR HA), purchased from the International Humic Substances Society (IHSS). After size fractionation with MST-UF the HS samples give well resolved two-dimensional C-13,H-1-correlated NMR spectra which offer a great potential for substructure elucidation and even quantification. It is shown that low molecular size lignin moieties undergo demethylation of the methoxy groups, accompanied by removal of the phenylpropane side chains and subsequent reaggregation of the aromatic rings. These findings provide insight into the processes of lignin degradation. Only the fraction > 100 kDa contains macromolecules that have spin-spin relaxation times too short for investigations employing NMR multipulse sequences.

The conditional metal availability and the kinetic stability of humic substance-metal species in humic-rich waters (e.g. bog water) was characterized by means of EDTA exchange. For this purpose a combined procedure consisting of time-controlled ligand exchange by EDTA, species differentiation by a fast single-stage tangential-flow ultrafiltration (TF-UF) technique (cut-off 1 kDa) and sensitive atomic spectrometry methods (e.g. AAS, ICP-OES, TXRF) was developed. The kinetics and the yield of the EDTA exchange served as operational parameters for assessing the kinetic stability and EDTA availability of HS–metal species, respectively. Considerable fractions of natural HS–metal species studied were shown to be EDTA-inert (e.g. 31% of the total Fe, 44% of the total Al) even after long reaction times (48 h), in contrast to artificial ones formed in solutions of isolated HS. Moreover, the conditional thermodynamic stability of HS–metal complexes formed by successive loading of an aquatic reference HS (HO14) with a number of heavy metal ions (e.g. Cr(III), Cu(II), Fe(III), Mn(II), Zn(II)) was also evaluated discriminating the free metal concentrations by means of TF-UF. In addition, from the loading isotherms obtained conditional complexation capacities could be derived for the studied HS exhibiting the order Fe(III)>>Cu(II)>Cr(III)>Co(II)>Mn(II).

This study exhibits that size fractionation of humic substances (HS) and their metal complexes by ultrafiltration is an efficient procedure for simultaneous determination of stability constants. Using sequential-stage ultrafiltration and a radiotracer technique the HS–Cu and HS–Zn complexes studied can gently be size-fractionated and their free metal fractions simply be discriminated. The conditional stability constants Ki obtained for size fractions of these HS metal complexes exhibit a clear molecular size dependence. Accordingly, the highest Ki values (6.6 for Zn and 6.4 for Cu) are found in the HS fractions of >105 kDa. Moreover, the overall stability constants K found for Cu (log K=5.5) and Zn complexes (log K=4.5) of the aquatic HS complexes studied are quite comparable to those reported in the literature.

Binding of metals in typical bath peat samples (“Großes Gifhorner Moor”, Sassenburg/North Germany) and their aqueous extracts was characterized by means of a multi-method approach. For that purpose a sequential extraction procedure based on peat-filled chromatography columns was developed. Water-soluble metal and DOM (dissolved organic matter) fractions were subdivided by use of a stepwise increased pH gradient (pH 3.8–5), finally by the chelator EDTA and 0.1 mol L–1 hydrochloric acid. Metal fractions very strongly bound to peat were assessed by an aqua regia extraction.