HUMIC SUBSTANCES SEMINAR III
Monday March 22 and Tuesday March 23, 1999

The Seminars had grown sufficiently to justify publication of the proceedings. A promise was made that timely manuscripts would be promptly reviewed, edited and published. Thanks to fine co-operation of the authors, this was accomplished and the book Humic Substances: Structures, Properties and Uses, G. Davies and E. A. Ghabbour (eds.) (ISBN 0 85404 704 2) was published by the Royal Society of Chemistry in December 1998.

Humic substances are ubiquitous and fascinating, and their study is important and multidisciplinary. The community of scholars seems to enjoy meeting on a regular basis at a convenient location to share the latest information on humic substances’ structures, properties and uses. The Seminars serve many useful functions, as indicated by the strong support for their continuation.

As before, timely manuscripts based on new work presented at Humic Substances Seminar III will be promptly reviewed, edited and published by The Royal Society of Chemistry. The resulting volume will contain fine current work that relates humic substance structures to their properties and applications.

Again, welcome to Humic Substances Seminar III, which we hope you will find informative, enjoyable and memorable!

Cornelius Steelink             Geoffrey Davies             Elham A. Ghabbour
Honorary Chair                 Chair                            Co-Chair
                                                                                                                March, 1999

ABSTRACTS

N. Senesi and G. Brunetti

Istituto di Chimica Agraria, University of Bari, Italy

Olive oil mill wastewater disposal is an agricultural and environmental problem for olive oil producing countries. These wastewaters have a high organic carbon content that can improve organic matter-poor soils. Fresh organic matter in these wastewaters may cause more adverse than beneficial effects on soil status and fertility. Humification is, therefore, highly recommended prior to addition to soil. Enzymatic systems that induce humification are difficult to realize in the biologically adverse conditions existing in these wastewaters. This study has tested a number of heterogeneous inorganic catalysts similar to those in soils to accelerate organic matter humification in olive oil mill wastewaters. Fresh wastewaters more digested with bentonite, iron(lll) oxides and manganese(lV) oxide with continuous stirring and air bubbling at 13-21 °C and natural pH, with no other additions. Wastewater samples were collected before and after 3 and 7 weeks of treatment. The humic acid-like fraction (HA) was isolated from each sample with modified conventional methods and analyzed for elemental and acidic functional group composition and by FTIR, ESR and fluorescence spectroscopy in the emission, excitation, and synchronous scan modes. The pH of the wastewaters increased from 5.2 to about 6. The redox potential increased from -316 mV up to +110 mV after 7 weeks of Mn(lV) oxide treatment. The O and N content and C/H and O/C ratios increased, whereas the C/N ratio decreased in HAs from 7 weeks of treatment. Carboxylic and phenolic OH group contents of HAs increased two times and one order of magnitude after 7 weeks. After 3 and especially 7 weeks of treatment, FTIR spectra showed a decrease of the aliphatic and carbonyl group absorptions and a net increase of aromatic and carboxylic group absorptions, especially in the Mn(lV) oxide treatment. With increasing time of catalytic digestion, the relative fluorescence intensity of HA decreased, the fluorescence emission maximum shifted to longer wavelength, and the intensities of excitation and synchronous scan peaks at low and intermediate wavelengths decreased relative to those of the long wavelength peaks. These effects were more evident in the HA obtained by Mn(lV) oxide catalysis. The ESR spectra of HAs and their shapes depend on the catalysts used. Organic free radical concentrations were three to four times higher than that of the original HA. In conclusion, mineral catalysts, especially Mn(lV) oxide, abiotically induce and control the humification of organic matter in fresh olive oil mill wastewaters. HA products are similar to native HAs in soils of the area.
This research has been supported by the National Research Council of Italy (CNR).

Robert L. Wershaw, Jerry A. Leenheer and Kay R. Kennedy

U.S. Geological Survey, Denver Federal Center, Denver, CO 80225

Quaking Aspen (Populus tremuloides) is the most widely distributed sylvan species in North America. In many places one finds groves that are composed almost entirely of Aspen trees with few other deciduous plants present. Such groves provide ideal sites for studying the transformation of leaf leachate to natural dissolved organic carbon compounds (DOC) because the leachate in the fall will be derived largely from fallen Aspen leaves. Solid-state ¹³C NMR spectroscopy was used to follow changes in leaf composition brought about by senescence. The NMR spectra of the leaves may be divided into spectral regions that are characteristic of lipids, carbohydrates, lignins, hydrolyzable and nonhydrolyzable tannins, and peptides. Changes in the leaf spectra brought about by senescence provide an indication of the types of reactions that take place during senescence. Senescent Aspen leaves were leached with distilled water to simulate the leaching of leaf litter by rain water. The leachates were fractionated on macroporous resins. The fractions have distinctive FTIR and ¹³C NMR spectral signatures that can be used to follow the diagenesis of plant-derived organic material to form dissolved organic carbon species (DOC) in natural water systems. Comparisons of the ¹³C NMR spectral patterns of the unfractionated leachates with those of the senescent leaves from which they were derived provides evidence of the types of degradation processes that the different leaf constituents have undergone.

Robert L. Cook^1,2 and Cooper H. Langford¹

¹Department of Chemistry, University of Calgary, Calgary AB, Canada T2N 1N4
²current address: Nova Research and Technology Centre, 2928 16th Street NE, Calgary AB, Canada

In this paper the term "biogeopolymer" will be introduced and applied to some forms of natural organic matter, most notably the humic family of substances. The concept that humic matter can be viewed as polymeric material will be developed via experimental measurements. A cross section of methods that have been used to study biogeopolymers will be reviewed, stressing the strengths and weakness of the methods. These include 1D and 2D NMR, NMR relaxation measurements, glass transitions, luminescence, light scattering, absorbance, titrimetirc studies, mild degradative studies, and non-linearities in studies of the recombination of fractions and binding capacities. The results from these methods will then be discussed in a polymeric model context. The polymeric model will be used to discuss humic structure, binding, and folding. The polymeric model will then be extended to discuss the fractal dimensionality of biogeopolymers and the possibility of their self-ordering will be considered. The model proposed will consider humics as an association of larger polymers with smaller molecular weight moieties, where conform-ation and weak forces are critical issues. The structural perspective advocated in understanding the complex mixtures known as humic, is a mesostructural one.

Baoshan Xing,¹ Jingdong Mao,¹ Weiguo Hu,² Klaus Schmidt-Rohr,² Geoffrey Davies³ and Elham A. Ghabbour³

¹Department of Plant and Soil Sciences
²Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003
³Chemistry Department and the Barnett Institute, Northeastern University Boston, MA 02115

Solid-state NMR has been widely used for both qualitative and quantitative analysis of soil organic matter (SOM) and its fractions (1-5). However, no single technique is completely satisfactory due to the heterogeneous nature of SOM and/or technical difficulty (such as instrument time) associated with each technique. We examined five solid-state ¹³C NMR techniques using several humic acids of various origins and locations. The five techniques were 1) direct polarization/magic angle spinning (DPMAS), 2) conventional cross polarization/magic angle spinning (CPMAS), 3) cross polarization with total sideband suppression (CP/TOSS), 4) DPMAS corrected by CP/TOSS at two spin-lattice relaxation filter times (DPMAS-CP/T₁-TOSS), and 5) ramped amplitude CPMAS (Ramp-CPMAS). According to chemical shifts, integration results were compared between the five techniques and with elemental composition from chemical analysis. When fully relaxed, DPMAS gave the best results when compared to elemental analysis. DPMAS-CP/T₁-TOSS was also found to be a reliable, quantitative method. Ramp-CPMAS gave variable results. The integration results from CP/TOSS were consistently better than CPMAS for most chemical shift ranges of all humic acids. The limitations and advantages of these five techniques will be also presented.

References
1. C. M. Preston, Soil Sci., 1996, 161: 144.
2. J. Mao, W. Hu, K. Schmidt-Rohr, G. Davies, E. A. Ghabbour and B. Xing, in ‘Humic Substances: Structures, Properties and Uses’, G. Davies and E. A. Ghabbour (eds.), The Royal Society of Chemistry, Cambridge, 1998, p. 79.
3. B. Xing, W. B. McGill and M. J. Dudas, Environ. Sci. Technol., 1994, 28: 1929.
4. B. Xing and Z. Chen, Soil Sci., 164 (1999, in press).
5. F. J. Stevenson, ‘Humus Chemistry’, Wiley, New York, 1994.

CRYSTALLINE ALIPHATIC COMPONENT IN HUMIC SUBSTANCES DETECTED BY SOLID-STATE NMR

Weiguo Hu,¹ Jingdong Mao,² Baoshan Xing² and Klaus Schmidt-Rohr¹

¹Department of Polymer Science and Engineering,
²Department of Plant and Soil Sciences, University of Massachusetts, Amherst, MA 01003

Humic substances are a major component of soil organic matter (SOM) and exist in all soils, water, and organic-containing sediments (1). structural information on humic substances is essential to understanding their reactivity, properties, origins and development (2,3). Aliphatic components are present in nearly all humic substances. We have conducted solid-state NMR experiments to determine crystalline structures of aliphatic domains in humic substances. Crystalline domains composed of long (CH₂)_n chains have been unambiguously detected by solid-state NMR and confirmed by wide angle X-ray scattering (WAXS) in several humic substance samples, including surface soil (peat), humic acids from surface soil and coal, and humin. From the melting range of 60 ^oC to > 80 ^oC and ¹H spin diffusion experiments, a thickness of ~ 4 nm or 30 CH₂ units is deduced. The overall fraction of (CH₂)_n crystallites in these materials is up to 4%. They are expected to be resistant to environmental attack and thus inert in soils, with long residence times. The presence of crystalline and amorphous (CH₂)_n domains in soil may affect many SOM physical and chemical properties. The possible biological origin of these domains will be discussed.

References
1. M. H. B. Hayes, in ‘Humic Substances: Structures, Properties and Uses’, G. Davies and E. A. Ghabbour (eds.), The Royal Society of Chemistry, Cambridge, 1998, p. 1.
2. B. Xing, W. B. McGill and M. J. Dudas, Environ. Sci. Technol., 1994, 28: 1929.
3. F. J. Stevenson, ‘Humus Chemistry’, Wiley, New York, 1994.

Ray von Wandruszka

Department of Chemistry, University of Idaho, Moscow, ID 83844-2343.

The micellar model of aqueous humic acids (HAs) has gained a measure of acceptance in recent years, although there is still no definite proof of its correctness. The preponderance of experimental evidence, however, appears to support this model, and many, if not all, properties of dissolved HA can be rationalized through application of its tenets. Fluorescence emission, both from HAs and from added probes, has provided strong indications in favor of the micellar representation. The model that emerges for solution borne HA is one that involves both intra- and intermolecular aggregation of the humic polymer. The former entails coiling and folding, especially with high molecular weight species and at elevated temperatures, producing hydrophobic domains that can serve as sequestration sites for nonpolar species. Humic polymers of lower molecular weight have less tendency to undergo this type of rearrangement, but may aggregate in an intermolecular fashion. This is particularly true for polydisperse solutions, where it is found that fragments of different size associate strongly. The surface tension of HA solutions, particularly its dependence on pH and the presence of metal ions, varies in ways that are consistent with the model deduced from fluorescence measurements. It is especially noted that the tendency of the humic (and fulvic) polymers to partition to the surface upon addition of salts or lowering of the pH follows trends that can be explained in terms of progressive neutralization of the charged (hydrophilic) portion of the molecules. This, in turn, is followed by the formation of pseudomicellar aggregates that display reduced surface activity. Ongoing work involves the measurement of humic aggregate size evolution under changing solution conditions by quasi-elastic light scattering (QLS). The indications are that pseudomicellar structures of increasing "tightness" are formed when the ionic portions of the polymers are neutralized and/or cross-linked by metal bridges.

Changes of colloidal state in aqueous systems of humic acids

Etelka Tombácz¹ and James A. Rice²

¹Department of Colloid Chemistry, Attila József University, Aradi Vt.1, H-6720 Szeged, Hungary
²Department of Chemistry & Biochemistry, South Dakota State University, Brookings, SD 57007

HSs are chemically and structurally complex macromolecules. Their most likely structure is a self-similar fractal, cross-linked aromatic network with different functional groups and side-chains. Random polymerization processes result in HSs with different structures and conformations, and only trends in solution condition dependent intra- and intermolec-ular interactions can be predicted. Hydrophobic moieties such as long alkyl side-chains from fatty acids residues confer amphiphilic HSs character and surfactant-like properties. Thus, HSs molecules can decrease the surface tension of aqueous solutions and the interfacial tension at water-oil interfaces, or HSs micelles can form due to hydrophobic interactions. Micellar solutions can solubilize water-insoluble organic compounds. Acidic functional groups’ dissociation in aqueous HSs solutions creates an electric double layer (EDL) and Coulombic interactions affect HSs conformational changes, colloid stability and HSs aggregation. EDL counterions may penetrate the aromatic network or be expelled from it. At high pH and low ionic strengths the functional groups are fully ionized. The charged sites separate as far apart as possible, but expansion of the aromatic network is limited by chemical cross-linkages. The degree of expansion/compression of humic nanoparticles is much smaller than for linear polyelectrolytes. The expanded or collapsed aromatic network is totally or partially penetrable by water, while water-impen-etrable units can be supposed after precipitation of humic materials. In aqueous solutions, humic materials can be dissolved or precipitated, accumulate at interfaces, self-assemble, solubilize organic compounds and they exist in different colloidal states depending on the conditions. Primary factors that influence their colloidal state are pH, ionic strength, the presence of di- or multivalent metal ions, cationic organic compounds, organic liquids and solid particles. The colloidal state changes continuously with environmental conditions. We have examined the surface activity, micelle formation, solubilization, conformational changes and the fractal nature of humic materials. A theory to explain the combined effect of pH and neutral salts on the colloidal stability of HA solutions is extended to accommodate polydispersity. We intend to show characteristic trends in colloidal state changes of HSs materials and how they correlate with composition.

C. Liu and P. M. Huang

Department of Soil Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8, Canada

Humic acids (HAs), along with other organic colloidal materials, strongly affect a wide range of environmentally important reactions and processes (1). Both transmission (TEM) and scanning (SEM) electron microscopy have been used to determine the morphological conformation of humic and fulvic acids at various pH values and ionic strengths (2-4). In the application of TEM and SEM to image humic and fulvic acids, the sample preparation is complicated (4) and the image is obtained under vacuum. Recently, the morphological conformation of fulvic acid was observed with atomic force microscopy (AFM) (5). In the present study, atomic force microscopy of a humic acid (IHSS soil humic acid standard 1S102 H) as influenced by pH 4, 6, 8, and 10 and salt concentration (0, 0.001 and 0.01 M NaNO₃) was investigated with a NanoScope^TMIII atomic force microscope (Digital Instruments, Santa Barbara, CA) in air at room temperature (23.5 ± 0.5 C). The silicon nitride cantilever with spring constant of 0.12 N/m was used in contact mode AFM. The results show that the humic acid was basically spherical with a diameter of about 70-150 nm in the studied ranges of pH and electrolyte concentration. The spheroids aggregated into various shapes with changing solution pH and NaNO₃ concentration. The aggregation was enhanced with decreasing pH and increasing electrolyte concentration. The conformation of humic acid observed with AFM was a three-dimensional profile with high resolution under ambient conditions.

References
1. F. J. Stevenson, ‘Humus Chemistry: Genesis, Composition, Reactions’ 2nd. Edn, Wiley, New York, 1994.
2. Y. Chen and M. Schnitzer, Soil Sci. Soc. Am. J., 1976, 40: 682.
3. K. Ghosh and M. Schnitzer, Geoderma, 1982, 28: 53.
4. I. L. Stevenson and M. Schnitzer, Soil Sci., 1982, 133: 179.
5. K. Namjesnik-Dejanovic and P. A. Maurice, Coll. Surf. Sci., 1997, 120: 77.

SELF-ASSOCIATION OF SOIL HUMIC SUBSTANCES AS REVEALED BY CHANGES IN THERMAL STABILITY

P. Buurman,¹ B. van Lagen¹ and A. Piccolo²

¹Laboratory of Soil Science and Geology, Wageningen University and Research Centre, P.O. Box 37, 6700 AA Wageningen, The Netherlands,
²Dipartimento di Scienze Chimico-Agrarie, Universit di Napoli Federico II, Via Universit, 100, 80055 Portici, Italy

The thermal stabilities of soil humic extracts saturated with H, Na, Ca, and Al were followed after treatments with relatively polar organic compounds such as methanol, formic acid and acetic acid. Thermal analysis showed that while exothermic profiles of H-humates did not change upon addition of the organic compounds, exotherms of Na-humates shifted to much higher temperatures (750-820^oC) than those of controls. Substantially less dramatic was the effect on Ca humates, and no alteration in thermal profiles was observed when Al-humates were treated with polar organic compounds. These results can be explained by considering the forces holding humic molecules together. Humic components are strongly bound to each other by hydrogen bonding in H-humates, and by electrostatic bridging in Ca- and Al-humates. These binding forces were not overcome by the simple addition of polar organic molecules, and the thermal stability of the humates remained generally unchanged. In Na-humates, humic molecules are held together only by non-specific hydrophobic interactions. In Na-humates, a treatment with organic compounds less polar than water was sufficient to modify their conformational arrangement towards stronger intermolecular hydrophobic associations, thereby significantly increasing thermal stability. Our results show that humic molecules in soil may self-associate in conformations stabilized by hydrophobic forces. These findings suggest that with abundance of monovalent counterions, or of polar organic compounds such as those exuded by plant roots, organic matter stability in soil may be greatly enhanced.

MOLECULAR SIZE DISTRIBUTION OF HUMIC SUBSTANCES: A COMPARISON BETWEEN SIZE EXCLUSION CHROMATOGRAPHY AND CAPILLARY ELECTROPHORESIS

M. De Nobili,¹ G. Bragato² and A. Mori²

¹Dipartimento di Produzione Vegetale e Tecnologie Agrarie, via delle Scienze 208, 33100 Udine
²Istituto Sperimentale per la Nutrizione delle Piante, Sezione di Gorizia, Gorizia, Italy

Gel electrophoresis (1) and size exlusion chromatography (2) can both be used to determine the molecular size distribution of humic substances under experimental conditions that either suppress or minimize charge density differences. The two techniques have several common features that allow description of the behavior of macromolecules in both systems through a unified model (3) that has been generalized to non-spherical molecules (4). Comparison between results obtained by either technique can in principle provide information on interrelationships between mobilities, partition coefficients and molecular radii of HSs. In the present work we considered humic substances extracted from the A, AB, E and Bs horizons of two spodosols whose molecular weight distributions had been determined by HPLC-SEC and by capillary zone electrophoresis.

References
1. M. De Nobili and F. Fornasier, J. Soil Sci., 1996, 47: 223.
2. R. S. Cameron, R. S. Swift and A. M. Posner, J. Soil Sci., 1972, 23: 342.
3. A. G. Ogston, Trans. Faraday Soc., 1958, 54: 1754.
4. D. Roadbard and A. Crambach, Proc. Nat. Acad. Sci., 1970, 65: 970.

Characterization of humic acids: their derivatization and consecutive capillary electrophoresis (CE), pyrolysis and/or MALDI-TOF MS analysis

Josef Havel

Department of Analytical Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic

From recent studies of humic acids in this laboratory applying CE (1,2), potentiometry (3), vapor pressure osmometry, conductivity, pyrolysis and MALDI-TOF MS (4), respectively, it was found that humic acids (HA) consist of a mixture of many compounds forming oligomers and/or supramolecules. This conclusion was further examined by derivatizing HA with various reagents and then applying mostly CE and MALDI-TOF MS analysis. The results supporting the formation of supramolecules from HA fractions will be presented and discussed.

References:
1. D. Fetsch and J. Havel, J. Chromatogr. A, 1998, 802: 189.
2. J. Havel and D. Fetsch, in ‘Encyclopedia of Separation Science’, I. Wilson (ed.), Academic Press, London, 1999, in print.
3. P. Lubal, D. irok, D. Fetsch and J. Havel, Talanta, 1998, 47: 401.
4. J. Havel and D. Fetsch, Proceedings of IHSS-9: Humic Substances Downunder, Adelaide, September 1998.

G. Haberhauer, W. Bednar and M. H. Gerzabek

Department of Environmental Research, Austrian Research Centers, A-2444 Seibersdorf, Austria

Mass spectrometry has been used extensively in the study of humics, primarily to gain information about fragments after chemical or thermal degradation (1). Of particular interest were studies using extensive fragmentation methods such as time resolved pyrolysis - field ionization (PFI) coupled to high resolution mass spectrometry (Py-FIMS). Those experiments provided direct molecular characterizations and confirmed degradation products previously identified by GC-MS experiments. With these techniques, humic substances (HSs) were characterized by fragmentation products but no information on the mass distribution was obtained. Soft ionization techniques such as fast atom bombardment (FAB), field desorption or laser desorption are therefore of interest. Laser desorption and matrix - assisted laser desorption have been used for humics and related compounds such as lignins (2,3). Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI - TOF - MS) has been successfully applied for the mass determination of large biomolecules up to 200 KDa such as proteins and oligonucleotides (4). Thus, MALDI - TOF - MS seems to be a very promising technique for studying polymer compounds such as humic acids (HAs). We explore this mass spectrometry technique for structural characterization and determination of dynamic processes of humic and fulvic substances originating from soil. HSs obtained by different extraction procedures were investigated. Sample preparation such as matrix optimization or sample concentration are shown to be crucial for examining such compounds. Results will be presented and analysis of MALDI - TOF mass spectra of HSs from different origins will be discussed.

References
1. H. R. Schulten, in ‘Mass spectrometry of soils’, T. W. Boutton and S. Yamasaki (eds.), Marcel Dekker, New York, 1995, p. 373.
2. A. Fievre, T. Solouki, A. G. Marschall and W. T. Cooper, Energy and Fuels, 1997, 11: 554.
3. M. Remmler, A. Georgi and F. -D. Kopinke, Eur. Mass. Spectrom., 1995, 1: 403.
4. F. Hillenkamp, M. Karas, R. C. Beavis and B. T. Chait, Anal. Chem., 1991, 63: 1193A.

H. -G. Löhmannsröben, Th. Skrivanek and U. Zimmermann

Institute of Physical and Theoretical Chemistry, University of Erlangen-Nürnberg, D-91058 Erlangen, Germany

Spectroscopic research on humic substances (HSs) has always been embedded between two extreme positions. On one hand, the ubiquity and importance of HSs in the environment has prompted essentially every spectroscopic or analytical technique to be applied to study HSs. On the other hand, due to their extremely complex, diverse and heterogeneous nature, HSs elude a comprehensive characterization of their structural or physico-chemical properties. It seems to be appropriate, therefore, to evaluate to what extent modern optical spectroscopy has improved our current knowledge about structures and properties of HSs and their interactions with xenobiotics. As a starting point, results of our investigations of HSs in water with stationary and time-resolved fluorescence and absorption measurements after laser excitation will be presented. Our studies allow not only a comprehensive overview of the photophysical properties of many HSs, including total luminescence (TL) and time-resolved emission (TRE) spectra, fluorescence efficiencies, etc, but also a quantitative description of the interactions between HS and, e. g., polycyclic aromatic compounds (PAC). The influence of medium effects (pH, ionic strength) and thermodynamic properties of PAC/HSs interactions will be elucidated. In addition, our current laser desorption/laser ionization investigations of HSs in combination with ion mobility spectrometry (IMS) will be outlined. The results of our work will be evaluated in the light of recent investigations of HSs and their interactions with organic compounds reported in the literature. Promising results were obtained with modern techniques such as dynamic light scattering, polarization measurements, Raman spectroscopy (including surface-enhanced Raman spectroscopy (SERS)) and FTIR. Of particular interest are new methods combining laser excitation/ionization with ion detection schemes, such as matrix-assisted laser desorption/ ionization (MALDI). On the basis of the results so far available and in view of emerging new experimental techniques, it will be attempted to evaluate some of the many remaining challenges of HS research and promising experimental routes for future HSs investigations. It is hoped that this approach will help in our quest to understand structures, properties and the enviromental behaviour of HSs.

Gregory V. Korshin,¹ Jerry A. Leenheer,² Jean-Philippe Croué,³ Chi-Wang Li¹ and Mark M. Benjamin¹

The objective of this work was to investigate the UV absorption and fluorescence emission spectra of hydrophobic, transphilic and hydrophilic Suwannee NOM fractions obtained with the separation/isolation protocol developed by Leenheer (1,2). These samples were thoroughly characterized by FTIR, ¹³C CP-MAS NMR, pyrolysis GC-MS, elemental composition and other relevant methods. The UV and fluorescence spectra of the initial and chlorinated (Cl/DOC=2) samples were processed with several approaches. Typically, the absorption or emission bands in the spectra were approximated by one or more Gauss-shaped bands whose position, intensity and width were evaluated. To probe the presence of "hidden" features in the spectra, their first- and second-order derivatives were calculated. The differential spectra () associated with the halogen attack sites in NOM (3) were also analyzed. The "specific" absorbance (Al/DOC), width and position of the major emission and absorbance bands were well correlated with the aromaticity of samples estimated by both ¹³C NMR and pyrolysis GC/MS. No other functionality appears to significantly affect the absorbance spectra, although more detailed discussion in this regard will be provided. The chemical status of the fluorophores in Suwannee River NOM is considerably more complex. For example, in nitrogen-rich samples (e.g., hydrophilic bases), the observed high emission yield, blue shift of the emission band and a unique response to halogenation indicate substantial contribution from aromatic amino acids. The derivatives of the absorption and emission spectra of all Suwannee River fractions indicate the presence of several bands whose intensity depends on the hydrophilicity and is significantly affected by halogenation. In the UV spectra, the maxima of these bands are located at ca. 260, 300, 330 and 410 nm. In the emission spectra, the maxima of the minor bands appear to be located at ca. 410, 460, 490 and 530 nm, while the maximum of the major emission band shifts between 395 and 470 nm depending on the hydrophilicity and chemical composition of the sample. These bands have not been assigned to any specific functionality. Given the proximity of some features in the absorbance and emission bands, we suggest that they may be caused by inter- and/or intra-molecular energy transfer phenomena affected by the molecular weight and conformations of the organic molecules. Implications of these results for the intrinsic chemistry of humic substances will be discussed.

Linda B. McGown and Joseph D. Hewitt

Department of Chemistry, Duke University, Durham, NC 27708

Humic substances (HSs) are ubiquitous in nature and play a key role in the storage and transport of many environmental contaminants. The goal of our research is to gain insight into the structure of HSs and their interactions with organic and metal species. Our approach is to analyze HSs by capillary electrophoresis (CE) with fluorescence lifetime detection. Preliminary CE separation results with a 1000 mg/L Suwanee River Humic Acid (HA) sample in borate buffer and 30 kv separation voltage show one broad peak with lifetimes in the range 2-3 ns. Work also is in progress on a related Suwanee River Fulvic Acid sample. Efforts are underway to improve the separation of the humic substances; this will help to elucidate the contribution of each of the various components in the humic matrix to the observed fluorescence lifetime in batch solutions.

Monika Takács and James J. Alberts

University of Georgia Marine Institute, Sapelo Island, GA 31327

Humic and fulvic acids were isolated by base extraction and acidification of samples from two Hungarian peat profiles (Pötréte and Zalaszentmihály). Elemental analysis, UV-VIS, FTIR, fluorescence (emission, excitation, synchronous scan and total luminescence) spectra, and copper complexation capacities were used to characterize the humic substances. In addition, laboratory studies of the changes in spectral and metal complexing capacities were undertaken following photo-reaction of the humic materials by exposure to a medium pressure Hg-arc lamp. humic acids (HAs) from the deeper layer had more aliphatic character, especially in the case of the Zalaszentmihaly peats. The HAs isolated from the upper layers of the profiles were enriched in nitrogen compared to the HA from deeper in the profile. Calculated changes in the composition of these HAs also showed an enrichment in oxygen in materials at the surface. This result is supported by the FTIR spectra, which show an increase of carboxylic groups in HAs from the upper layers of the peat. The fluorescence emission spectra of all samples showed one dominant broad peak with a maximum around 455-465 nm. The synchronous scan spectra had several peaks and two distinct peaks were observed for the total luminescence spectra. After photo-reaction, the emission peak maxima shifted to shorter wavelengths. The number of peaks observed in the synchronous scan spectra increased and also exhibited a shift towards the blue region of the spectrum. The ratios of the relative fluorescence intensities of the two main peaks (synchronous scan spectra) decreased after the photo-reaction. Photo-reaction also decreased the copper complexation capacity of the humic acids. All the observations indicate that photo-reaction changes not only the quantity but also the quality of the fluorescence moities in HAs. A possible explanation for the changes observed in the humic acids isolated from the peat profiles is oxidation resulting from the fact that surface layers of the profiles are no longer under permanent water influence. Thus the peats are exposed to a higher oxygen tension than those that are permanently submerged, leading to a greater potential for oxidation processes to alter the humic acids. The potential changes to humic substances as a result of photo-oxidation can also lead to increased oxidation of the HAs and this process deserves further examination.

Masakazu Aoyama

Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan

The fluorescence of humic substances has been considered to be a general phenomenon. However, it was suggested from high performance size exclusion chromatography (HPSEC) that in compost- and soil-derived humic acids (HAs) the fluorescent substances were different from the humic substances and consisted of different components (1). In this study, further attempts were made to separate the fluorescent substances from the humic substances in the HA fraction using HPSEC and Sephadex gel chromatography. HPSEC was performed on a compost HA and seven soil HAs using Asahipack GS-220HQ and GS-320HQ columns (Asahi Chemical Industry) linked in series. The columns had higher resolving power than that used in the previous study (1). The eluent was 0.05 M phosphate buffer (pH 8.0) containing 0.3 M NaCl mixed with CH₃CN in the volume ratio of 4:1 (1). Humic substances (HSs) peaks detected by the absorbance at 280 nm appeared between V₀ and the V₀+V_i, but the elution volume varied with the HA samples used. In contrast, when detected at the near-optimum excitation and emission wavelengths (excitation wavelength = 460 nm, emission wavelength = 520 nm) of soil HAs, the fluorescent substances were eluted considerably later than the humic substances and resolved into as many as 15 sharp peaks or more, except for the compost HA. Each fluorescent peak had the same elution volume irrespective of the soil HA samples used, although the peak intensity varied considerably. When the fluorescent substances eluted from the columns were collected using a fraction collector and subjected to HPSEC again, the fractionated and original samples gave corresponding elution patterns. These results provide further evidence that the fluorescent substances contained in the HA fraction are different from the humic substances and consist of different components. Sephadex gel chromatography was also examined to separate the fluorescent substances from the humic substances in HAs. When a Sephadex G-25 column (37 mm I. D. x 500 mm) was used with distilled water as an eluent for the gel chromatography of soil HA, two peaks appeared at the V₀ and the V₀+V_i. HPSEC revealed that the humic substances were mostly recovered in the former peak, with the fluorescent substances in the latter peak. Thus, the gel chromatographic method enables collection of the fluorescent substances-rich fraction for the characterization of the fluorescent substances in the HA fraction.

References
1. M. Aoyama, Abstracts, 9th International Meeting of the International Humic Substances Society, Adelaide, 1998, p. 30.

X-ray absorption studies of humic substances: perspectiveS and limitations. Analysis from the standpoint of physical realities

Anatoly I. Frenkel¹ and Gregory V. Korshin²

¹ Materials Research Laboratory, University of Illinois at Urbana-Champaign (mailing address: Building 510 E, Brookhaven National Laboratory, Upton, NY 11973)
² Department of Civil and Environmental Engineering, University of Washington, Seattle, WA 98195-2700

The structural characterization of humic substances (HSs) and metal complexation sites associated with them has been a challenge since these compounds and their solutions lack long-range order prerequisite for conventional X-ray diffraction, while diffuse X-ray scattering can be used only for very high concentrations of the target element. By contrast, X-ray absorption techniques are unique in their ability to examine the structure of highly disordered systems. In extended X-ray absorption fine structure (EXAFS) experiments, the photoelectrons excited by X-ray absorption of the target atom (e.g., copper) are used to probe the local structure at distances up to ca. 10 Å from the target. The coherence between the photoelectron and the core hole in the target atom lasts only ca. 10^-15 sec, thus providing a "snapshot" of the local chemical environment. An important feature of the EXAFS technique is its atomic specificity, which allows identification of the atoms surrounding the target. Due to the high cross-sections typical in X-ray absorption experiments that employ synchrotron sources, the EXAFS method can be used to study the structure of metal-HS complexes at concentrations several orders of magnitude smaller than those needed for X-ray scattering. In this presentation we will use the results of EXAFS studies of Cu²⁺-HS complexation (1) to demonstrate the advantages and limitations of modeling the EXAFS spectra using theoretical FEFF calculations (2). The FEFF approach will be compared with other EXAFS data analysis methods. The presentation will explore the limits of the EXAFS technique in studies of very diluted solutions. We will emphasize the necessity to employ correct error estimation procedures to obtain reliable structural parameters. It will be demonstrated that in the case of Cu²⁺-HS complexes, the analysis can be unambiguously performed only for the atoms found in the first complexation shell. Their chemical nature and structural properties can be probed with high precision. The method of X-ray absorption near edge structure (XANES) will be also examined. Its data contain information about the coordination geometry and the oxidation state of the target. In agreement with the literature (1,3), our XANES results for Cu²⁺-HS complexes indicate a tetragonal distortion of the octahedral Cu-binding site in HS.

References
1. G. V. Korshin, A. I. Frenkel and E. A. Stern, Environ. Sci. Technol., 1998, 32: 2699.
2. S. I. Zabinsky, J. J. Rehr, A. Ankudinov, R. C. Albers and M. J. Eller, Physical Review B, 1995, 52: 2995.
3. K. Xia, W. Bleam and P. A. Helmke, Geochim. Cosmochim. Acta, 1997, 61: 2211.

Paul R. Bloom,¹ Kang Xia,² William F. Bleam,³ Chung-Ming Lin¹ and Edward A. Nater¹

¹Department of Soil, Water & Climate, University of Minnesota, St. Paul MN 55108
²Department of Agronomy, Kansas State University, Manhattan KS 66506
³Department of Soil Science, University of Wisconsin, Madison WI 53706

Atmospheric deposition of anthropogenic Hg has resulted in a rise in Hg concentrations in surface waters of forest and wetland ecosystems at northern latitudes, and fish frequently accumulate Hg at levels above health recommendations. The bonding of Hg(II) in soil and aquatic organic matter is an important factor controlling the transport and transformations of Hg in these ecosystems. In a previous study using competitive Br^- complexation and x-ray spectroscopy, we found that Hg(II) is bonded very strongly to soil organic matter (SOM) at RSH sites. From data for reduced S concentrations determined by x-ray absorption near-edge spectroscopy (XANES), we determined model dependent surface complexation formation constants, at native Hg concentrations, consistent with solution complexation constants for mercaptoacetic acid. The present study used Hg extended x-ray absorption fine structure (EXAFS) to investigate the change in the contribution of O sites to bonding as Hg(II) as loading was increased to greater than the concentration of reduced S in SOM. A peat soil obtained from the edge of a fen in the Marcell Experimental forest in Northern Minnesota was air dried, sieved, washed with 1 M HNO₃ followed by distilled water, then freeze dried. Total S and C were determined and the relative quantity of organic S in different oxidation states was determined by XANES spectroscopy. Mercury(II) as Hg(NO₃)₂ was added to aqueous suspensions of soil to yield mole ratios of Hg(II) to reduced S from 0.5:1 to 10:1. After 24 h the solution was decanted and the Hg(II)-soil complex was freeze dried. Samples of the complex were analyzed for Hg content. The nature of bonding of the Hg(II) bonding environment was determined by Hg-EXAFS. At a Hg:reduced S ratio of 0.25 the EXAFS radial structure functions show a 2-coordinate environment involving one reduced S atom and one O atom. At this loading the two peaks were well resolved and interatomic distances corresponded to the bond lengths for Hg-O and Hg-S bonds. At a ratio of 0.81:1 the S and O peaks were both broadened but the relative magnitudes of the two peaks was unchanged. At a ratio of 8.5:1 the O peak was predominant and the S peak was hardly visible. At this ratio there is only enough reduced sulfur to bond with less that 12% of the added Hg. The EXAFS spectra at ratios of 0.81 or less are similar to the results we obtained for a complex of Hg(II) with humic acid extracted from this same soil. When Hg(II) is added greatly in excess of the reduced S, the bonding of the additional Hg(II) is to O sites. At a ratio of 8.5:1, the quantity of Hg in the SOM is more that 10,000 greater that for in situ SOM. This accounts for the low binding constants found in laboratory studies by previous workers who used high Hg(II) additions. In natural systems, the binding constants are many orders of magnitude greater because Hg(II) binding is dominated by bonding to reduced S.

Sandeep Bhandari,¹ Dula Amarasiriwardena¹ and Baoshan Xing²

¹School of Natural Science, Hampshire College, Amherst, MA 01002
²Department of Plant and Soil Sciences, University of Massachusetts, Amherst, MA 01003

Humic acids (HAs) are naturally occurring heterogeneous macromolecules in the soil and aquatic environments. They have affinity for inorganic and organic solutes and thus play an important role in the uptake and transportation of nutrients and contaminants in surface and subsurface soil environments (1-3). Previously, high performance size exclusion chromatography (SEC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) has been used for identification of trace metal-bound HA molecular fractions in water (4) and in soil HA (5). The main focus of this investigation is to expand our knowledge about the metal complexation properties of humic acids, to obtain further insight about the speciation of the metal-HA complexes using SEC-ICPMS, and to elucidate the distinct affinities that HAs show towards different metals. HAs extracted from five different soils with moderate to low soil organic matter were investigated. HA samples were characterized for their functionality by diffuse reflectance infrared spectroscopy (DRIFT) and UV-visible spectroscopy. Total trace metal (Al, As, Cu, Mn, Pb and Zn) content in each HA was determined by ICP-MS after nitric acid digestion. The chromatographic profiles of those trace metals bound to various soil HA molecular weight fractions were determined by SEC-ICPMS. HA samples were equilibrated with excess heavy metal solutions at room temperature and allowed to competitively replace native heavy metal cations. The isolated solid HA product was water washed, freeze-dried, and subsequently dissolved in 0.05 M NaHCO₃ solution, and trace metal chromatographic profiles were obtained. The results of the SEC-ICPMS investigation of the affinities of the complexation sites in HA-metal binding molecular fractions will be presented.

References
1. F. J. Stevenson, ‘Humus Chemistry: Genesis, Composition, and Reactions’, 2nd. Edn., Wiley, New York, 1994.
2. P. MacCarthy, C. E. Clapp, R. L. Malcolm and R. R. Bloom, ‘Humic Substances in Soil and Crop Sciences: Selected Readings’, American Society of Agronomy, Madison, Wisconsin, 1990.
3. N. Senesi, Trans. World Congr. Soil, 1994, 39: 384.
4. L. Rottmann and K. G. Heumann, Anal. Chem., 1994, 66: 3709.
5. P. Ruiz-Haas, D. Amarasiriwardena and B. Xing, in ‘Humic Substances: Structures, Properties and Uses’, G. Davies and E. A. Ghabbour (eds.), The Royal Society of Chemistry, Cambridge, 1998, p. 147.

James J. Alberts, Monika Takács and Mala Pattanayek

University of Georgia Marine Institute, Sapelo Island, GA 31327

The impact of copper(II) and mercury(II) on the physiological response of the phosphorescent bacterium Vibrio fischeri was investigated in the presence and absence of natural organic matter (NOM). The organic matter included two aquatic fulvic acids and two aquatic humic acids from the IHSS Standard and Reference materials collection, and a natural organic matter isolated by ultrafiltration from a stream on Sapelo Island, GA. Both metals greatly reduced the physiological response of the bacteria when challenged with uncomplexed metals. The resulting EC₅₀ concentrations were 3.46 µmol Cu(II)/L and 0.65 µmol Hg(II)/L. Complexation of the metals with organic matter (10 mg C/L) greatly reduced the apparent toxic effect by as much as 77% and 39%, respectively. Humic acids caused the greatest reduction on the effect of Cu (58-77%) as opposed to 29-36% reduction by fulvic acids. Humic and fulvic acids had about the same effect on reducing apparent Hg toxicity, 24-39% and 30-37%, respectively. The NOM sample isolated by ultrafiltration was intermediate between the two types of organic matter with respect to Cu (58% reduction) and was within the range of both with respect to Hg (32% reduction). to determine possible mechanisms for the observed toxicity reductions, the copper binding capacities (CuBC) of the organic matter samples were determined. Reduced effect on physiological response was only weakly correlated to CuBC (r² = 0.52). However, it was significantly correlated (r² = 0.91) with the atomic C/N ratio of the organic matter. The correlation was negative, indicating that increased nitrogen content of the organic matter had an increased effect in reducing negative physiological response in the organisms when they were challenged with copper and mercury.

APPLICATION OF SOLID PHASE MICROEXTRACTION TO STUDY SORPTION PHENOMENA ON HUMIC ORGANIC MATTER

Juergen Poerschmann and Frank-Dieter Kopinke

Center for Environmental Research, Permoserstr. 15, 04318 Leipzig, Germany

Solid phase microextraction (SPME) has gained wide acceptance as a simple, time- efficient and solvent-free sample preparation technique in analytical laboratories. This presentation describes the application of SPME to study sorption phenomena of primary organic and organometallic pollutants with humic organic matter (HOM). The basic idea is based upon the fact that only the freely dissolved fraction of the sorbate can be taken up by the fiber rather than the fraction bound to the HOM. This freely dissolved fraction can be desorbed in the hot GC splitless injector or in an SPME-HPLC interface. In comparison to the traditional methods for studying sorption processes such as dialysis, reverse phase HPLC and fluorescence quenching, the SPME technique is more precise, simpler and less time-consuming. The sorption can be measured both for dissolved and particulate HOM. By means of SPME results, partition coefficients based upon organic carbon (K_OC) can be determined. The results indicate that hydrophobic, nonpolar interactions play the key role in sorption processes on HOM for PAHs and PCBs. Polar interactions contribute to the sorption of nitroaromatic compounds on HOM, especially on particulate HOM. The combination of SPME with the selective and sensitive negative chemical ionisation technique (SPME-GC/NCIMS) allows detection of sub-ppt trace levels of higher chlorinated PCBs and pesticides. Total analyte concentrations, composed of a freely dissolved fraction and a fraction that is bound to HOM, can be obtained by calibration with deuterated standards, whereas an external calibration gives the freely dissolved portion (1).

References
1. J. Poerschmann, Zh. Zhang, F. -D. Kopinke and J. Pawliszyn, Anal. Chem., 1997, 69: 597.

Humasorb-cs: A Humic Acid-based Adsorbent to Remove Organic and Inorganic Contaminants

H.G. Sanjay, Amjad Fataftah, Daman Walia and Kailash Srivastava

ARCTECH, Inc., 14100 Park Meadow Dr., Chantilly, VA 20151

Humic acid is a naturally occurring material with the following desirable properties: high cation exchange capacity, ability to chelate metals and ability to sorb organics. HUMASORB-CS, a new material developed by cross-linking and immobilizing a coal-derived humic acid, exhibits all the properties of the original humic acid. HUMASORB-CS is a water insoluble adsorbent that is stable over a wide pH range. HUMASORB-CS has been evaluated to remove single and mixtures of contaminants both in batch and column tests. The contaminants include metals, radionuclide surrogates, oxo-anions and organic compounds. The results demonstrate that HUMASORB-CS can be used for removal of multiple types of contaminants in a single-step process. HUMASORB-CS is also being evaluated for contaminant removal under simulated barrier conditions at pressures of 10 psig and 100 psig to simulate barrier installation depths of approximately 10 feet and 100 feet. HUMASORB-CS was compared to Zero Valent Iron (ZVI) for effectiveness in removing chlorinated organic contaminants from groundwater. The results show that HUMASORB-CS not only adsorbs the chlorinated organic contaminants, but it also potentially reduces them in the same way as ZVI. These studies show that the reaction of TCE with HUMASORB-CS appears to follow a mechanism similar to that with ZVI (reductive dehalogenation). The destruction of TCE by reductive dehalogenation was confirmed by GC-MS, IC and H-NMR analysis. HUMASORB-CS has also been evaluated in treatability and demonstration tests using actual waste streams from a number of sites. HUMASORB has been evaluated using contaminated water from DOE sites including the Savannah River Site, Idaho National Engineering and Environmental Laboratory (INEEL) and Rocky Flats. In addition, HUMASORB-CS has been successfully evaluated using contaminated water from industrial sites including the Berkeley Pit in Montana and the Iron Mountain Site in California. Column and batch test results that show the ability of this material to remove mixtures of contaminants, the ability to work under simulated barrier conditions, and the ability to degrade chlorinated hydrocarbons will be discussed. In addition, the stability test results from the development stages of HUMASORB-CS will be discussed. Results from the demonstration of HUMASORB-CS on Berkeley Pit water in Montana will also be presented.

References
1. H. G. Sanjay, D. Walia and K. Srivastava, US Patent in process.
2. H. G. Sanjay et al. "Development of HUMASORB, A Lignite Derived Humic Acid for Removal of Metals and Organic Contaminants from Groundwater", Proceedings of the Environmental Technology Through Industry Partnership Conference, Volume II, October 1995, p. 411.

Humic Substances and pH effects on clay dispersion and the hydraulic conductivity of soils

Yona Chen and Jorge Tarchitzky

Department of Soil and Water Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Humic substances (HSs) are important factors in soil particle associations. Little is known, however, about the mechanisms of clay-HSs interaction, and to date their effects on aggregation at various pH levels have not been adequately studied. Studies on flocculation and dispersion characteristics of homoionic montmorillonite were performed as a function of exchangeable cation, humic acid (HA) and fulvic acid (FA) concentration and pH. A matrix of experimental conditions was employed and the corresponding flocculation values (FV) were measured. Edge-face (E-F) and edge-edge (E-E) interactions at various pH levels, as well as heteroflocculation and its response to pH and concentration of HSs will be discussed. The concept and experimental approach that evolved from the first stage of this research were further employed for a study of the effects of humic-like substances originating from treated wastewater on clay dispersion and hydraulic conductivity of soils. Recycled wastewater effluent is an important source of irrigation water in arid and semi-arid regions. wastewater effluents generally contain high concentrations of dissolved solids, both organic and inorganic. Maximum FV values were exhibited by Na-montmorillonite at the highest dissolved organic matter (DOM) concentration. Soil packed in columns and treated with wastewater effluent exhibited a sharp decrease in hydraulic conductivity (HC) to only 20% of its initial value. The reduction in HC is likely the result of decreased soil-pore size, which reflects two processes: (i) retention of part of the DOM during water percolation; and (ii) a change in pore-size distribution due to swelling and dispersion of clay particles. The latter could result from a higher percentage of adsorbed sodium combined with the presence of humic substances from the wastewater effluent.

STIMULATION OF PLANT GROWTH BY HUMIC SUBSTANCES: EFFECTS ON IRON AVAILABILITY

Y. Chen,¹ C. E. Clapp² and H. Magen¹

¹Department of Soil and Water, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, POB 12, Rehovot 76100, Israel
²USDA-ARS and Department of Soil, Water, and Climate, College of Agricultural, Food and Environmental Sciences, University of Minnesota, St. Paul, MN 55108

Stimulatory effects of humic substances (HSs) on plant growth have been observed and widely documented. Studies have often shown positive effects on seed germination, root initiation and total plant biomass. The consistency of these observations has been uncertain, predominantly due to the lack of understanding of the plant growth promotion mechanism. Often these effects were attributed to plant growth hormones and the term "hormone-like activity" was used to describe the plant growth stimulation. Yet investigators were unable to prove that plant growth regulators are present in HS preparations. An alternative hypothesis suggests that growth enhancement of plants grown in nutrient solution containing HSs is the result of improved micronutrient availability; Fe in particular has been postulated and tested in the present study. Nutrient solutions containing N, P, K, Ca, Mg, S, B, Mo, Cu, Mn, Zn and Fe at concentrations considered to be optimal for plant growth were tested for solubility of Fe, Zn and Mn seven days after preparation. In addition to control solutions at pH 5, 6, 7 and 7.5, a 50 mg/L preparation of a leonardite humic acid (HA) was tested for Fe, Zn and Mn solubility. The HA greatly enhanced the maintenance in solution of Fe and Zn, especially at pH 7 and 7.5 and did not affect Mn solubility. Plant growth experiments were performed on both dicotyledonous plants (melon) and monocotyledonous plants (creeping bentgrass) due to the major difference in their Fe uptake mechanisms. Plants grown in the absence of Fe exhibited severe Fe deficiency that could only partially be corrected with the addition of Fe salts. The addition of only HA or FA did not result in a growth enhancement or a remedy of the Fe deficiency, suggesting that no plant growth hormones were present in these preparations. However, addition of Fe, Zn and either HA or FA resulted in healthy, chlorophyll-rich plants and enhanced growth, thereby proving that improved Fe, and possibly Zn nutrition, is a major mechanism of plant growth stimulation by HSs.

References
1. Y. Chen and T. Aviad, In ‘Humic Substances in Soil and Crop Sciences: Selected Readings’, P. MacCarthy et al. (eds.), Am. Soc. Agron, Madison, WI, 1990, p. 161.
2. Y. Chen, In ‘Humic Substances in Terrestrial Ecosystems’, A. Piccolo (ed.), Elsevier, Amsterdam, 1996, p. 507.
3. C. E. Clapp, R. Liu, V. W. Cline, Y. Chen and M. H. B. Hayes, In ‘Humic Substances: Structures, Properties, and Uses’, G. Davies and E. A. Ghabbour (eds.), Royal Society of Chemistry, Cambridge, 1998, p. 227.
4. S. Nardi, G. Concheri and G. Dell’Agnola, In ‘Humic Substances in Terrestrial Ecosystems’, A. Piccolo (ed.), Elsevier, Amsterdam, 1996, p. 361.

USING ACTIVATED HUMIC ACIDS IN THE DETOXIFICATION OF SOILS CONTAMINATED WITH POLYCHLORINATED BIPHENYLS

Alexander Shulgin,¹ Alexander Shapovalov,¹ Cicilia Bobovnikov,² Galena Pleskachevski,³ Yuriy Putsykin⁴ and Andrew J. Eckles, III⁵

¹Special Bio-Physical Technology Institute of Moscow, Moscow, Russia
²Science-Industrial Complex "Typhoon", City of Obnisk, Russia
³Center of Epidemiology, Health Department, City of Serpukhov, Russia
⁴Research Institute of Plant Protection Chemicals, Moscow, Russia
⁵Stable Earth Technology and Science Coordinators International, 4020 Landheer Drive, Louisville, KY 40299

The use of polychlorinated biphenyls (PCB) for 25 years by the transformer manufacturing factory in Serpukhov, Russia has resulted in significant contamination of the environment of the whole city. In spite of the fact that the use of PCB has been prohibited since 1988, the level of contamination in local soils and in produce grown on those soils is found to be very high. In some of the agricultural soils, the PCB levels reach several tens of mg/kg. This is an extremely serious problem for the Southern areas of the city: its agricultural cooperatives and private gardens supply the city markets with fresh vegetables, fruits, lettuce and other fresh greens. A new technology based on the in-situ use of a unique "Activated Humic Acid" (AHA) for the remediation of PCB contaminated soils was tested and the content of PCB in the agricultural produce was measured. The data indicate that the PCB levels both in the soils and in the agricultural products were significantly lowered. The results of this study indicate that AHA can offer a practical solution for the remediation of PCB contaminated soils. This report covers only a 60 day field study, and some residual PCB contamination remained in the treated soils after the 60 days. A follow-on study conducted twelve months later found that PCBs were not detected in the soils that had been treated with AHA. The tests used would have detected any remaining PCBs at levels as low as 1 ppm. AHA will be produced by Stable Earth Technology, a US company, and available in the US under the tradename "Stabilite."