Environ. Sci. Technol. 1999, 33, 4299-4303
Effects of Humic Acid Purification on Interactions with Hydrophobic Organic Matter: Evidence from Fluorescence Behavior REGGINAL R. ENGEBRETSON AND RAY VON WANDRUSZKA* Department of Chemistry, University of Idaho, Moscow, Idaho 83844-2343
Conventional isolation of humic materials from natural matrixes includes demineralization by treatment with HF/ HCl. The possible effect of this on the structural integrity of humic acid (HA) was investigated by comparing the interactions of two aqueous HAs, one produced by the conventional procedure (CHA) and the other by a gentler resin method (RHA), with a hydrophobic probe (pyrene). Although RHA had a substantially higher ash content, it was found that this had relatively little effect on its interaction with the probe molecule. In view of the micellar model of dissolved HAs, however, it was noted that the HF-treated HA had a greater apparent ability to sequester pyrene and isolate it from the aqueous bulk. This was revealed by the kinetics of probe fluorescence enhancement when Mg2+ was added to a solution also containing HA and by the anisotropy excitation spectra of the native humic fluorophores. The effect was ascribed to a lesser degree of conjugation in the HA demineralized with HF, producing polymers of greater flexibility that formed micelle-like structures more quickly and effectively.
Introduction The transport, bioavailability, toxicity, and final disposition of hydrophobic organic matter (HOM) in soils and natural waters are inextricably linked to the humic substance (HS) present in these matrixes (1-9). Variables such as pH, ionic strength, nature of the electrolyte, temperature, and HS concentration determine the interactions between HOM and HS (3, 10-13). Recently, these parameters have also been reported as major contributing factors in the kinetics of aquatic HOM/humic acid (HA) associations (14-16). Although numerous studies have sought to elucidate the mechanisms governing the interactions between HOM and dissolved HS, the roles of the various mechanisms suggested, including complexation (17), surface sorption (18), entrapment in structural voids (19-21), and partitioning into hydrophobic microenvironments (pseudomicelles) (22-25), are only partially understood and in some instances controversial. The structural differences among HAs (26, 27) and the profusion of chemical features present in any particular HA are impediments to the mechanistic understanding of HOM/ HA interactions. The fact that HAs are operationally defined substances implies that significant chemical differences can arise from isolation procedures as well as from innate structural variations. The spectral features obtained for HAs by fluorescence (28), NMR (29), IR (21), ESR (30), and UV/ 10.1021/es990386h CCC: $18.00 Published on Web 10/26/1999
1999 American Chemical Society
VIS (31) spectroscopy are chemically interesting and provide various insights, but they are of minimal predictive value for HOM/HA interactions. Spectral characterization of a newly isolated HA offers only limited insight into the extent and duration of the interactions of such a material with HOM. In fact, considerations of its origin are likely to be more telling in this regard. Also, molecular size and size distribution factors have proven more anticipatory of the aqueous interactions of a dissolved HA and small hydrophobic probes such as pyrene and diphenyloxazole (32, 33). The availability of International Humic Substances Society (IHSS) standards and the use of IHSS extraction and purification procedures provide some common ground for the study of the primary humic fractions of individual soil or water samples as well as a limited ability to compare these fractions among sundry HSs. As useful as these standards and procedures are, however, they provide little insight into an important practical question: how can a defined but unexplored HA be expected to interact with HOM in a natural environment? This point has proven to be unapproachable from the standpoint of an operational definition that is exclusively based on solubility. Prior to the development of a possible cataloging system for HAs that contains information about their anticipated interactions with HOM, it is vital to determine the significance of different extraction procedures (definitions) in the chemical behavior of humic materials. Information about the mechanism of aqueous HA/HOM interactions may also be derived from comparisons of the aqueous chemistry of HAs extracted by various procedures. Luminescence spectroscopy has been successfully used in the investigation of interactions between HA and polyaromatic hydrocarbons in dilute aqueous solutions (34, 35). Pyrene fluorescence is easily observable in water and has been shown to be highly sensitive to the interaction of the fluorophore with aqueous HA (36). The native fluorescence and the fluorescence anisotropy of dissolved HA itself have likewise proven useful in the study of its behavior in dilute solution (37). The present study compares the interactions of pyrene with dilute aqueous solutions of HA obtained by two different procedures from a silt loam soil. The first of these materials was isolated by the conventional method based on alkali dissolution and reprecipitation, while the other was extracted with pyrophosphate and purified by column elution and filtration (for details of both methods, vide infra). The salient difference between these two methodologies is that the latter does not use HF to solubilize the silicates invariably associated with unpurified soil HAs. In addition, however, chemical changes in HA resulting from exposure to HF have been reported (27). Comparison of HA yields and characterization by 13C NMR of the materials isolated by the two methods has been published by Hayes et al. (38). Piccolo et al. (39) have reported on the interactions of atrazine and humic fractions extracted by different methods.
Materials and Methods Chemicals. Pyrene (98%) was obtained from Sigma, recrystallized from absolute ethanol, and sublimed onto a coldfinger. A 0.02 M pyrene stock solution was prepared in absolute ethanol and stored in the dark at room temperature. It was used to prepare aqueous solution by placing appropriate amounts of the ethanolic pyrene in deionized water and sonicating for at least 1 h. DAX-8 resin was obtained from Alltech and cleaned as recommended by Hayes et al. (38). A pH 9 Tris buffer [tris(hydroxymethyl)aminomethane] was selected for the SEC separations because of its efficiency VOL. 33, NO. 23, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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in eliminating electrostatic gel/solute interactions. This was obtained from Sigma and prepared as recommended by Cameron et al. (40). Analytical grade MgCl2 was obtained from J. T.Baker and used as received. HF, HCl, NaOH, Na4P2O7, H3PO4 (analytical grades), Spectra/Por 6 dialysis tubing (approximately 1000 Da molecular mass cutoff), and 0.2-µm membranes were obtained from Fisher and used as received. Glycerol (99.5+%, spectrophotometric grade) was obtained from Aldrich and used as received. Water used for all solutions was deionized and treated with a 0.22-µm Millipore filter system to a resistivity of 18 MΩ‚cm. Humic Acids. The top 30 cm of a Latahco silt loam soil (LSLS; Argiaquic Xeric Argialbolls) maintained as pasture for at least 20 years was collected, air-dried, and crushed to pass a 2.0-mm sieve (25). The collected soil contained 41.5 g of organic C, 159 g of clay, 121 g of silt, and 3.9 g of total N/kg of soil. Organic C was determined by the Walter-Black method (41), and total N was determined by combustion (LEGO CHN600 determinator). Humic acid extraction was carried out according to the International Humic Substances Society (IHSS) procedure, which involves two cycles of HCl treatment for the removal of acid-soluble materials; extraction of the residue with 0.1 M NaOH; deashing with 0.1 M HCl/0.3 M HF; purification by dialysis; and isolation by freeze-drying. Details of the process can be found in the product literature provided by the IHSS (42). The material thus obtained will henceforth be designated CHA. Isolation of HA by the alternative route began with the extraction of LSLS by the exhaustive sequential procedure developed by Clapp and Hayes (43). This involved extraction of the soil with 0.1 M Na4P2O7 at pH 7, pH 10.6, and pH 12.6; NaOH extraction at pH 12.6; and DMSO extraction. A resins-in-tandem separation procedure was then implemented to recover the HA. The entire process as published (38, 43) involves elution through tandem XAD-8 and XAD-4 columns, followed by elution through an IR-120 column. The HA fraction of the humic material, however, is recovered from XAD-8 alone in a process that includes back elution of the column, precipitation of the eluted material, and finally its re-dissolution. In the present work, the XAD-8 stationary phase was replaced by the equivalent DAX-8 phase, since the former is no longer commercially available. Flow rates were reduced to ca. 2.5 mL/min from the reported 40 mL/min to be commensurate with the column dimensions of 2.5 × 48 cm (diameter × length). Because of the relatively small DAX-8 resin column used, five loadings and back elutions were required to obtain sufficient HA for this study. No attempt was made to recover the “neutral” HA fraction, which is soluble in ethanol but insoluble in water. The material isolated by this method will henceforth be referred to as RHA. In view of the polydispersity of the HA solutes and their difference in ash content (vide infra), it was considered that solutions of similar UV/Vis absorbances could be more validly compared than those prepared strictly by weight. The CHA stock solution used therefore contained 100 ppm of the HA (without correction for ash content), while the RHA stock solution had quantitatively the same absorption spectrum ((1%) in the 250-600-nm range. CHA and RHA stock solutions containing 1.0 × 10-7 M pyrene were prepared with minimum sonication in 1.0 × 10-4 M NaOH and allowed to stand at room temperature, in the dark, for at least 2 weeks. Working solutions, prepared by dilution of the stock solutions with 1.0 × 10-7 M aqueous pyrene, were used immediately. The pH of the final solutions was in the range of 7.0-7.8. Procedures and Instrumentation. Size exclusion chromatography (SEC) measurements were made using a SigmaChrom GFC-1300 gel filtration HPLC column (13.25 mL; 300 × 7.5 mm) supplied by Supelco and a Waters 510 HPLC pump operating at 0.5 mL min-1 and a back pressure of 500 psi. Detection was with a Waters 411 absorbance detector 4300
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FIGURE 1. Change of pyrene fluorescence intensity (ex. 240 nm, em. 372.5 nm) in 20 ppm CHA (0) and RHA ([) after addition of MgCl2 (to 0.02 M Mg2+). set at 254 nm and recorded on a Hewlett-Packard 3390A integrator. The 100 ppm HA stock solutions were used for the SEC measurements with a sample injection volume of 10 µL. Absorbance values were measured with a Hitachi U-3000 UV/VIS spectrophotometer. Fluorescence spectra and anisotropies were measured with a SLM AMINCO 8100 fluorescence spectrophotometer equipped with T-optics (two orthogonal emission channels) and a thermostated cuvette holder. Pyrene fluorescence intensities were obtained at 240 nm excitation and 372.5 nm emission with a band-pass of 4 nm for each. Atomic absorption measurements were taken with a Buck Scientific model 200A atomic absorption spectrophotometer using an acetylene/air flame. All individual measurements were performed in triplicate, and the results shown had relative standard deviations of 5% or better.
Results and Discussion The ash content of the HF-treated CHA was a negligible 0.08%, but the resin method left a relatively high 32% ash in RHA (both on oven-dried basis). It was not possible to substantially lower the latter value by a second processing through the DAX-8 column under the conditions described by Malcolm and MacCarthy (44). The RHA ash was red in color and insoluble in 1.0 M HCl or 1.0 M NaOH but entirely soluble in 1.0 M HF, indicating that it was comprised of silicates. Qualitative atomic absorption analysis at of the ash dissolved in 1.0 M HF showed a presence of iron. The UV/VIS spectra (250-600 nm) of CHA and RHA were found to be virtually identical (data not shown). Figure 1 shows the changes in pyrene probe fluorescence in CHA and RHA solutions for 1 h following the addition of sufficient MgCl2 to give a total added Mg2+ concentration of 0.02 M. The first question that must be considered is whether the lesser rate of pyrene fluorescence increase in the RHA solution is the result of chemical differences between the HAs or a consequence of the high ash content of RHA. Increased fluorescence of hydrophobic, polyaromatic probes in the presence of some dissolved HAs upon the addition of metal ions is well-known (25). In general (dependent on the relative importance given to static and dynamic mechanisms in the quenching of pyrene fluorescence by dissolved HA), the enhancement of pyrene fluorescence upon the addition Mg2+ has been ascribed to one of two mechanisms: (i) the desorption of the statically quenched fluorophore by competitive sorption of the metal cations to the binding/ quenching sites of HA (12), or (ii) the sequestration of pyrene in the hydrophobic interiors of micelle-like HA structures (pseudomicelles). The formation of these structures is facilitated by charge neutralization of anionic HA sites and metal ion bridging between such groups (13, 15, 16). As described in a recent review (28), it is thought that the confinement of the probe in the relatively nonpolar pseudomicelles minimizes its encounters with more polar,
potentially quenching groups on the HA polymer. The probe desorption mechanism cannot be reconciled with the results at hand irrespective of the question whether silicates or structural differences in the HAs caused the different behavior illustrated in Figure 1. Silicates can be either polyanionic or neutral in solution. In the former case, they are unlikely to be associated with HA under basic conditions, since it is rich in carboxyl and hydroxyl groups. The association of neutral silicates with HA is most likely due to entanglement and entrapment in the humic macromolecule. In either case, it is improbable that the interaction of Mg2+ with the carboxyl and hydroxyl sites of humic acid would be affected by the presence of silicates. Use of MgCl2 in large excess of the amount required for maximum response of the pyrene probe with CHA under the solution conditions in question eliminated Mg2+ consumption by silicate as a cause of the reduced pyrene fluorescence enhancement in RHA solutions (Figure 1). This conclusion was verified by the observation that the addition of larger amounts of MgCl2 to the RHA solution did not cause a greater increase in pyrene fluorescence but rather a small decrease (data no shown). This effect should be ascribed to the increased presence of chloride ion, which slightly quenches pyrene fluorescence. A second consideration argues against silicates being the cause of the lesser increase of pyrene fluorescence after the addition of Mg2+ to the RHA solution. Figure 1 shows that the shapes of the time response of pyrene fluorescence following the addition of Mg2+ to the RHA solution is virtually identical to that for the CHA solution, suggesting that the mechanisms in the two cases are alike. Last, if the silicates in RHA had a quenching effect on pyrene fluorescence or affected the accessibility of quenching/ binding sites, then the initial pyrene fluorescence intensities in RHA and CHA solutions would be expected to be different. In fact, they are the same. Analysis of the pyrene fluorescence behavior shown in Figure 1 is straightforward when a pseudomicellar mechanism of probe sequestration is invoked. In this scenario, the presence of anionic or neutral silicates would have little affect on the evolution of pyrene fluorescence after the introduction of excess Mg2+ to the HA solutions. In both CHA and RHA, the cation promotes the formation of hydrophobic domains that serves as sequestering sites for pyrene. The formation of these domains occurs at a relatively slow pace after the metal ion is introduced, causing the gradual increase in pyrene fluorescence in both HA solutions. In terms of the pseudomicelle mechanism, the difference in fluorescence enhancement shown in Figure 1 can be understood by considering the effect of the HF treatment experienced by CHA. The model dictates that pseudomicellar organization has a significant intramolecular component, and it therefore stands to reason that the ability of HA to sequester pyrene depends on the molecular flexibility of the humic polymers. The harsh de-ashing procedure used for CHA (shaking for 12 h with 0.1 M HCl/0.3 M HF) is virtually certain to increase the degree of saturation in the HA polymers, thereby destroying conjugated systems. This is consistent with the observation that CHA has lower native fluorescence than RHA (Figure 2). In this view, CHA has more alkyl links between remaining aromatic moieties, producing more flexible polymer chains. This, as discussed above, promotes pseudomicelle formation. A similar effect was noted in a previous study (25) in which conventional HA extraction was used. A portion of the products was subjected to HF demineralization, while another portion was not. In the latter case, it was also noted that the HA solutions gave less pyrene fluorescence enhancement upon addition of Mg2+. Size exclusion chromatograms of RHA and CHA (Figure 3) showed that CHA contained a more polydisperse population of larger humic acid molecules. This is indicated by the broadness of the main CHA peak and its
FIGURE 2. Fluorescence emission spectra (ex. 340 nm) of CHA (s) and RHA (- - -).
FIGURE 3. Size exclusion chromatograms of CHA and RHA on a SigmaChrom GFC-1300 column eluted with a pH 9 Tris buffer. shift toward shorter elution times relative to the corresponding RHA peak. The presence of larger molecules in CHA demonstrates that the HF treatment did not result in fragmentation of the HA polymer. Barring the unlikely possibility of condensation during CHA extraction, either the larger humic molecules of LSLS were lost during extraction of RHA or their presence in CHA is merely a manifestation of greater molecular flexibility. De Nobili et al. (45) have reported that this can occur when more flexible macromolecules elute in SEC before “stiffer” ones of comparable molecular weight do. In either event, the presence of these larger and/or more flexible molecules in CHA is consistent with more efficient pseudomicelle formation and increased probe fluorescence when the HA is dissolved in near-neutral aqueous solution. In the SEC elution process itself, it is unlikely that the solute is highly aggregated, in view of the high pH Tris buffer used as the mobile phase. Effects of Temperature on Pyrene Fluorescence in Aqueous Solution of RHA and CHA. In general, fluorescence quantum efficiency decreases with increasing solution temperature. Elevated temperature increases the frequency of molecular collisions and the probability of dynamic quenching. The fluorescence of a pyrene probe in the presence of some humic acids, however, has been found to increase with temperature (25). Analysis based on pseudomicellar behavior VOL. 33, NO. 23, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Response of pyrene fluorescence intensity (ex. 240 nm, em. 372.5 nm) (0) to a temperature ramp (s) in 10 ppm RHA (A) and CHA (B). showed that the increase of pyrene fluorescence with temperature in HA solution can be related to the decrease of the dielectric constant of water with increasing temperature. As water becomes a poorer solvent, humic polymers are thought to coil up and “squeeze” out the solvating water. Such dehydration has been offered in explanation of the well-known cloud-point behavior of nonionic surfactants (46). Increased pyrene fluorescence intensity with increased temperature in the presence of some humic acids thus may be ascribed to intramolecular micellar behavior analogous to clouding. Figure 4 shows the time response of pyrene fluorescence intensity to temperature changes of RHA and CHA solutions, respectively. It is worth noting that in both cases there is an initial, significant drop in pyrene fluorescence intensity with increasing temperature. The drop is to be expected in light of the predictably slow nature of HA conformational changes through dehydration. Because of this, the first effect in both solutions is the more commonly observed decrease in fluorescence intensity caused by an increase in dynamic quenching. Following this initial stage, the much greater increase in pyrene fluorescence in the CHA solution is again anticipated for a HA with more flexible and/or larger polymers. Speaking more directly to the greater apparent flexibility of CHA is fact that the probe fluorescence from the solution of CHA much more rapidly tracks with the temperature program. This is especially evident during the cooling of the solutions. The ability of a macromolecular structure such as CHA to coil and uncoil with relative ease suggests the existence of considerable sectional motion. Fluorescence Anisotropy of RHA and CHA in Glycerol/ Water Solutions. When a fluorophore dissolved in a viscous liquid is irradiated with polarized light, the fluorescence is partially polarized (47). The intrinsic anisotropy calculated from the polarized intensities in such a medium is a measure of the angle between the excitation and emission dipoles of the fluorophore in question. In nonviscous solution there is a further loss of polarization (lowering of anisotropy) through rotational diffusion, which is the main source of extrinsic depolarization. This dynamic loss of polarization becomes part of the observed anisotropy, which is therefore expressed 4302
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FIGURE 5. Fluorescence anisotropy excitation spectrum of RHA (A) and CHA (B) at different temperatures in glycerol/water: 20 °C ([); -5 °C (0); the difference between the anisotropy values at the two temperatures is indicated by [; em 550 nm. as the sum of these two (intrinsic and extrinsic) components. The anisotropy excitation spectra of native CHA and RHA fluorescence in glycerol/water (80/20) solutions at -5 and 20 °C are presented in Figure 5. At the lower temperature, the viscosity of the medium is such that little dynamic depolarization occurs during the lifetime of the excited state, causing the intrinsic component of the measured anisotropy to dominate. At the higher temperature, rotational diffusion of the fluorophore (largely through sectional motion in HA) is increased, producing lower anisotropy values. The difference between the two anisotropy curves in Figure 5, therefore, gives an indication of the difference of sectional motion of the various fluorophore groups located on the humic polymer. In the absence of sectional motion, increased rotational diffusion due to increased temperature affects the entire molecule in the same way. In that case, the loss of anisotropy due to heating would be linear with wavelength. Distinctly different losses in anisotropy at different excitation wavelengths, however, would be expected if different portions of the macromolecule can move independently of the entire molecule. The anisotropy excitation spectra of RHA and CHA indicate the greater possibility for sectional motion on the part of CHA. This is again consistent with a lower degree of conjugation/aromaticity in CHA and its attendant ability to enhance the fluorescence of the pyrene probe through the formation of pseudomicelles. Comparison of RHA and CHA anisotropies at 450 nm as a function of time following the addition of Mg2+ leads once again to the conclusion that CHA is less conjugated and more flexible than RHA (Figure 6). The initial increase in anisotropy shown here is ascribed to the restriction of sectional motion when Mg2+ is added (which is greater for CHA), while the subsequent decline in anisotropy is consistent with the formation of smaller and
FIGURE 6. Change of HA fluorescence anisotropy in glycerol/water at 20 °C after addition of MgCl2 (0.01 M added Mg2+); CHA (0); RHA ([). tighter structures through coiling and contraction of the humic polymers. There is little doubt that prolonged treatment of humic material with HF/HCl, as is the case for CHA, leads to a substantial reduction of unsaturation. This circumstance, which follows a priori from chemical principles, manifests itself in the enhanced flexibility of the more aliphatic polymers that are produced. This type of chemical modification necessarily yields a product that is significantly different from the original material, especially with regard to its interactions with hydrophobic species in aqueous media. The procedure used to isolate RHA gave, at least in the case of LSLS, a material that retained a significant proportion of silicates. It appeared, however, that this had only a minor effect on its associative behavior. This, in turn, suggests that laboratory studies of wholly or partially demineralized HAs (provided they were not chemically altered in the manner of CHA) are a better indicator of their environmental behavior than may have been anticipated. It is also clear that a definitive parameter describing microorganization of dissolved HAs must be included in any cataloguing system used to predict their interactions with HOM.
Acknowledgments The authors gratefully acknowledge financial support by the NSF EPSCoR program and the EPA (R82-2832-010).
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Received for review April 5, 1999. Revised manuscript received September 23, 1999. Accepted September 24, 1999. ES990386H VOL. 33, NO. 23, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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