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Fulvic and humic acids isolated from groundwater: Compositional characteristics and cation binding
Journal of Contaminant Hydrology, 11 (1992) 317-330
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Elsevier Science Publishers B.V., Amsterdam
Fulvic and humic acids isolated from groundwater: Compositional characteristics and cation binding J.J. A l b e r t s a, Z. F i l i p b a n d N. H e r t k o r n c
"University of Georgia Mar&e Institute, Sapelo Island, GA 31327, USA blnstitut 3~r Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Aussenstelle Langen, Paul-Erhlieh-Strasse 29, D-6070 Langen, Germany Clnstitutj~r Okologisehe Chemie/GSF, Schulstrasse 10, D-8050 Attaching, Germany (Received August 22, 1991; revised and accepted June 3, 1992)
ABSTRACT Alberts, J.J., Filip, Z. and Hertkorn, N., 1992. Fulvic and humic acids isolated from groundwater: Compositional characteristics and cation binding. J. Contam. Hydrol., 11: 317-330. Samples of humic substances were obtained from a waterworks at Fuhrberg, Germany. The material had a bimodal molecular size distribution with ~40°/o of the total carbon in the 50,000-100,000-D (nominal molecular weight, NMW, in daltons) size fraction and ~ 50% of the carbon in the < 10,000-D (NMW) size fraction. The fulvic and humic acids isolated from the bulk humic substances were low in nitrogen content and had low H/C atomic ratios. Furthermore, the fulvic and humic acids had very similar elemental, spectral and copper binding characteristics. Over 70% of the carbon in both the fulvic and humic acids was present in aromatic or aliphatic groups, with 13C N M R analyses indicating approximately even distribution among the two types. Competitive elemental binding studies indicated that Ca 2÷ , Mg 2÷ , AI 3÷ and Fe 3+ do not effectively compete for copper binding sites on these compounds. In humic acids, these cations are predominantly bond by carboxylic groups. INTRODUCTION
Humic substances of the fulvic and humic acid type comprise a large portion of the ubiquitous organic matter reservoir in natural waters (Thurman, 1985a). They are known to complex many heavy metals, radionuclides and anthropogenic organic compounds (Alberts et al., 1986, 1989a, b; Giesy et al., 1986; Weber, 1988; Suffet and MacCarthy, 1989). Furthermore, this complexation by humic substances can enhance or retard uptake and toxicity of these compounds by organisms (Stewart, 1984; Mfiller-Wegener, 1988; McCarthy, 1989). While considerable study of humic substances has Correspondence to: J.J. Alberts, University of Georgia Marine Institute, Sapelo Island, GA 31327, USA. 0169-7722/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
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been conducted with materials isolated from soils, sediments, surface waters and estuaries (Stevenson, 1982; Christman and Gjessing, 1983; Aiken et al., 1985; Rashid, 1985; Alberts et al., 1988, 1989a; Filip and Alberts, 1988; Filip et al., 1988, 1991; Alberts and Filip, 1989) very little is known of the characteristics of these materials isolated from groundwaters (Thurman, 1985a, b), which are often the source of drinking water to large populations. In this study, we have characterized humic substances from a groundwater aquifer in Fuhrberg, Lower Saxony, Germany. This groundwater aquifer is important to the drinking water supply for the city of Hannover, Germany. The bulk humic substances from these groundwaters were examined with respect to their molecular size spectrum, while the humic and fulvic acids isolated from those humic substances were characterized with respect to their FTIR (Fourier transform infrared) and nuclear magnetic resonance (NMR) spectral characteristics and copper binding capacities (CuBC's). METHODS
Site description and &olation of humic substances The humic substances characterized in this investigation originated from the waterworks at Fuhrberg, Germany, where a local groundwater aquifer yields a brownish-colored water. This porous aquifer is composed of homogeneous alluvial sand, located at a depth of 30-70 m. The untreated groundwater has a pH of 6.5 and organic carbon content of 10.0 g m -3 C. Inorganic elemental concentrations (mol m -3) of the groundwater are: Na + , 1.09; K + , 0 . 0 8 ; C a 2+ , 1.80; Mg 2+, 0.37, total Fe, 280; total Mn, 20; N H 4 +, 60; C l - , 1.30; NO2-, 0.1; NO3-, 0.03; and SO42-, 1.46 (K611e, 1981). The groundwater is decolorized with ion-exchange resins, which yield a dark-brown concentrate. This concentrate was collected, filtered through paper filters, freeze-dried and used as bulk humic substances and the source of humic and fulvic acids examined in this study. Bulk humic substances were used for the molecular size fractionation experiment. However, fulvic and humic acids, which had been isolated from the bulk humic substances, were used for all subsequent analyses. These fulvic and humic acid fractions were isolated by dissolving the bulk humic substances in base (pH 12, NaOH) then acidifying to pH 1.0 (HC1). The fulvic and humic acids were separated by centrifugation, with subsequent purification of the fulvic acid fraction with XAD-8 ® macroreticular resin (Robin & Haas ~) and the humic acid fraction by dialysis (Thurman and Malcolm, 1981; Alberts et al., 1988). Both fractions were then freeze-dried.
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Molecular size fractionation
Fifty mg of bulk humic substances were dissolved in 300mL of KH2PO 4 buffer solution (0.1 M KHzPO4-0.1 M NaOH, v/v; pH = 7.4). Ten mL of solution were taken for dissolved organic carbon (DOC) analyses. The remaining solution was placed in an Amicon ® Model 404, Stirred Ultrafiltration Cell containing a X M - I O 0 ® ultrafilter. The solution volume was reduced to 100mL under nitrogen pressure (10 psi or ~ 68.95 kPa) and the solution volume of the retentate was again made to 300mL with KHzPO4 buffer solution. The ultrafiltrate, containing materials of < 100,000 D (nominal molecular weight, NMW, in daltons), were collected in 100-mL aliquots. The process was repeated until the ultrafiltrate was colorless. The retentate was collected, 25 m L of solution were taken for DOC analyses, and the remainder was acidified to pH 2 (6 N HC1) and allowed to stand overnight. The precipitate was collected by centrifugation and dialysed (1000 D) against three changes of deionized water before freeze-drying. The ultrafiltrates were combined and sequential ultrafiltration was continued as above using XM50 ~" (20 psi or --~ 137.90kPa N2) and Y M - I O ® (35 psi or ~ 2 4 1 . 3 2 k P a N2) ultrafilters to obtain molecular size fractions of 50,000-100,000 and 10,00050,000 D (NMW), respectively. The ultrafiltrate volume from the Y M - I O ® ultrafilter was so large, almost 3 L, that the organic matter was concentrated on an XAD-8 ® resin column (Thurman and Malcolm, 1981). The organic matter isolated in this manner was again dissolved in KHzPO 4 buffer and ultrafiltered, as above, over a YM-2 ~ (molecular size 2000 D NMW, 75 psi or ~ 5 1 7 . 1 1 K P a N2). The ultrafiltrate which passed the YM-2 ® filter was acidified and humic substances ( < 2000 D NMW) remaining in solution were isolated on XAD-8 ® resin. Volumes and DOC analyses of all ultrafiltrates, retentates and starting solutions were determined to allow carbon mass-balance calculations to be made for all fractions. Copper binding capacities (CuBC's)
Aliquots of humic or fulvic acids (,-~ 5 mg) were dissolved in ~ 50 mL of deionized water with the addition of 1-2 mL of 0.2 N NaOH. The solutions were made to volume with deionized water and aliquots of these solutions were pipetted into beakers. For copper binding studies, the methods of Giesy et al. (1978) were used. Briefly, 5 M NaNO3 was used as an ionic strength adjuster (2 mL/100 mL analyte) and sufficient Cu 2+ ion was added to produce a free Cu 2+ concentration between 3.2-10 -7 and 1.6.10 -4 M. The pH of the solutions were adjusted to 5.1 + 0.1 by dropwise addition of dilute HC1 to minimize hydroxyl-copper complex formation. All volume changes were
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recorded. Samples were covered and allowed to stand overnight ( ~ 18 h) at room temperature (19.6_+2.7°C). The pH was again measured before determining the free Cu 2+ ion concentration (procedure described below). Analyses were conducted in triplicate and standard curves were prepared immediately prior to estimating the Cu 2+ ion concentrations. Experiments to determine the effect of competitive ions on CuBC's were conducted as above, except that the competing ion was added with the ionic strength adjustor. The solutions containing the humic or fulvic acids and competing ions were allowed to equilibrate overnight before the Cu 2+ ion solution was added and the pH was again adjusted to 5.1. The solutions were once again equilibrated overnight before free Cu 2+ concentrations were determined. In all competitive ion studies, humic and fulvic acid samples not containing competing ions were analyzed in parallel with the competition studies so that differences due to daily electrode variations were eliminated. Following free Cu 2+ ion determinations, the humic acid samples were dialysed against deionized water for three days, freeze-dried and retained for F T I R spectral analyses.
Instrumental analyses F T I R spectra of all isolates and starting materials were collected from 2% KBr disks using a Bruker ® Model 48 F T I R Spectrophotometer. Ultraviolet/ visible spectra were collected with a Carlo-Erba ® Recording Spectrophotometer (Model Spectrocomp 601). Solid-state 13C N M R spectra were obtained by the methods of cross-polarization and magic angle spinning (CP-MAS) on a Bruker ® Model MSL 200 spectrometer, operated at 50.3 MHz. Typically, 12,000 scans were acquired with a pulse width of 5/~s and an acquisition time of 25.6 ms; a 4.5-/ts proton preparation pulse, followed by a cross-polarization contact time of 1 ms and a pulse delay of 4 s were used. The samples were spun at 6 kHz. Free Cu 2+ ion concentrations were determined with an Orion :R~ Model 942900 copper-selective ion electrode and an Orion '~R;Model 900200 Double Junction Reference Electrode. Elemental analyses (C, H, N and O by difference) were conducted with a Carlo-Erba ~: C H N Elemental Analyzer (Model EA 1108). DOC analyses were performed with an Astro ® Model 1850 DOC Analyzer. RESULTS AND DISCUSSION
Molecular size distribution The ultrafilter fractionation of the bulk humic substances resulted in 58% recovery of the organic carbon in the original sample. This was significantly
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lower than recoveries for humic acids isolated from estuarine sources, which normally had carbon recoveries of 96-100% (J.J. Alberts, unpublished data, 1990). Since there was little evidence of loss of humic acid carbon on the ultrafilters from the estuarine samples, it must be inferred that the losses of the groundwater humic substances were due to small molecules, which were isolated on the initial ion-exchange resin, but were subsequently lost during the isolation of the < 2000-D (NMW) fraction on the XAD-8 ® resin, perhaps by nonreversible adsorption to the XAD resin. The carbon which could be accounted for was bimodal in distribution, with ~ 6 0 % in the 50,000-100,000-D (NMW) size fraction. Another 20% of the carbon was in the 2,000-10,000-D (NMW) fraction. Less than 1% of the carbon was in the > 100,000-D (NMW) fraction and 7% was in the 10,00050,000-D (NMW) fraction. Ten percent of the carbon was in the < 2000-D fraction (NMW), but this number is an underestimate if the unrecovered carbon was small molecular size material. The latter would agree with studies which show that groundwater humic substances are of relatively small molecular size (Thurman, 1985b).
Elemental analyses The fulvic and humic acids isolated from the groundwater had very similar elemental compositions (Table 1). Both were low in nitrogen content. The fulvic acids had nitrogen contents similar to fulvic acids recovered from some groundwater samples from the U.S.A., but the humic acids were significantly lower in nitrogen content as compared to groundwaters in the U.S.A. (Thurman, 1985b). Accordingly, the atomic ratios of N/C in the humic acids reported here are significantly less than those of the U.S. groundwaters, while the fulvic acids have similar values. The atomic ratios of H/C of the humic acids presented here are similar to the U.S. groundwater material, but the fulvic acids from the Fuhrberg groundwater are low relative to groundwater fulvic acids as reported by T h u r m a n (1985b). The low H/C ratios in the fulvic and humic acids from Fuhrberg groundwater indicates considerable unsaturated character in both. The UV/visible spectra of both the fulvic and humic acids were featureless and decreased monotonically with increasing wave number. The ratio of light absorbance at 465 nm to that at 665 nm (E4/E6, Table 1) indicates that the fulvic acids are either composed of smaller molecules than the humic acids, or that they have a lesser degree of conjugation. The former conclusion would agree with the ultrafiltration results reported above. The E4/E6 ratio for the humic acids is high relative to soil humic acids (Stevenson, 1982), but the E4/E6 for both the fulvic and humic acids agree with values reported for groundwaters by T h u r m a n (1985b).
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TABLE I Elemental composition *j , E4/E 6 ratios and copper binding capacities (CuBC's) of groundwater humic and fulvic acids Sample
Ash (wt%)
C (wt%)
H (wt%)
N (wt%)
Fulvic acids Humic acids
1.51 2.26
54.74_+ 1.31 54.43 + 0.15
4.23 + 0.11 4.61 + 0.13
1.02 + 0.08 t.69 _+0.10
Fulvic acids Humic acids
E4/E 6
H/C (*2)
N/C 1,2,
l 1.7 7.6
0.93 + 0.02 1.01 +_0.03
0.016 _+0.001 0.026 +_0.002
Competing ions *~
Fulvic acids Humic acids
CuBC .3
AI 3+
Fe 3+
Ca 2+
Mg 2-
0.311 + 0.005 0.316_+ 0.0
90 84
86 80
100 100
100 100
*~All values corrected for ash content of humic and fulvic acids. *2Atomic ratios. *3Copper binding capacity as/~g-at. Cu 2+/mg humic or fulvic acids. *4Reported as percent of original CuBC remaining after equilibration with the competing ions.
While the groundwater fulvic and humic acids characterized here have many similarities to those from groundwaters in the U.S.A., there are some significant differences. These differences may be the result of fossil plant material inputs into the groundwater humic substances of this region of Germany (Filip and Smed-Hildmann, 1992). F T I R spectra The F T I R spectra of the fulvic and humic acids from this groundwater are relatively uncomplex and dominated by absorbances due to aromatic, aliphatic and carboxylic groups (Fig. 1). The spectra are very similar to each other and dominated by a large and complex band of peaks at the 3000-3400cm -~ region, which can be attributed to hydrogen bonding and O - H stretching. The presence of aliphatic groups is evidenced by the weak doublet between 2850 and 2970 cm -~ . The peak at 1618 cm-J and broad band around 1400 cm -~ , represents the C = C stretching of substituted aromatic rings. The absorption in these regions relative to the aliphatic doublet at 28502970 cm-1 appears somewhat stronger in the humic acids than in the fulvic acids, thus indicating that the humic acids may have greater aromatic character than the fulvic acids. If this is true, then the higher E4/E 6 ratio for the fulvic acids is likely to be due to the presence of smaller molecules.
FULVIC AND HUMIC ACIDS ISOLATED FROM GROUNDWATER
LU o z < tn cr
I
hi
t
323
i#
O co 1:13 ,<
4000
3500
3000
2500
2000
WAVENUMBER
1500
1000
500
c m -1
Fig. 1. FTIR spectra of humic substances from a groundwater obtained at Fuhrberg, Germany, in protonated form: (A) fulvic acids; and (B) humic acids.
The strong absorbance at 1717cm ' is representative of the protonated carboxylic acids and ketonic C = O groups, and the peak at 1216cm -' is the C-O stretching vibration and OH bending deformations of the carboxylic acid groups. Both of these peaks disappeared when the humic substances were treated with K + , which is indicative of salt formation in simple carboxylic acids (MacCarthy and Rice, 1985). In keeping with the low nitrogen contents of these compounds, there was no evidence of secondary amide linkages associated with proteins or peptides, which have been recognized in estuarine humic matter (Filip and Alberts, 1988; Alberts et al., 1991; Filip et al., 1991). The two ultrafilter fractions of the bulk humic substances that represented 60% and 20% of the recovered organic carbon (50,000 -100,000 and 2,00010,000 D, respectively) had FTIR spectra very similar to each other (Fig. 2, B and D). The main difference between these two spectra and that of the starting material is the loss of the two peaks at 1220 and 1717 cm 1, which are attributable to C = O and C-O functionalities in simple carboxylic acids that disappear upon salt formation. Thus, it appears that despite being isolated by precipitation from pH 2 solution and dialysed against deionized water, the humic substances in these two size fractions are still present in a salt form rather than having been reprotonated. The cause of this phenomenon is not known, but may involve steric hindrance of the carboxylic groups during the dialysis procedure. The distinct absorption band at 1385 cm-1 may be attributable to oxy-functional groups in phenolics and/or methyl and methylene groups of aliphatic chains. The 1618-cm -~, C = C ring stretching peak indicates aromatic groups to be present in both fractions.
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~T
t.u (.3 Z < 133 frO o9 t'n <
4000
3500
3000
2500
2000
WAVENUMBER
1500
1000
500
cm -1
Fig. 2. FTIR spectra of bulk humic substances and some molecular size fractionations obtained from groundwater in Germany: (A) protonated bulk humic substances which had not been fractionated over ultrafilters; (B) 50,000-100,000-D (NMW) fraction; (C) 10,000-50,000-D (NMW) fraction; (D) 2,00010,000 D (NMW) fraction; and (E) < 2,000-D (NMW) fraction.
The spectra of the two remaining size fractions of the bulk humic substances show slightly altered, but still strong regions of H-bonding (30003400cm -1) and the presence of the aliphatic C - H stretching (28502970cm-1). The aromatic C = C peaks at 1430 and 1618cm -~ remain weak but visible. The spectrum of the carbon < 2000-D size fraction is highly degraded (Fig. 2, E) and poorly resolved. If it is assumed that the unrecoverable carbon passed through the smallest ultrafilter, this fraction accounts for ,-- 35% of the initial organic carbon in solution. However, only about one-third of that was actually recovered by isolation on the XAD-8 ~ resin column. Therefore, this spectrum is representative of only a very small portion of the total material in this fraction. N M R spectra The ~3C N M R spectra of both the fulvic and humic acids had strong peaks at 130 and 175 ppm (Fig. 3). The former may indicate the presence of C = O in carboxyl groups or secondary amide linkages in peptides and proteins (Piotrowicz et al., 1984; Alberts et al., 1991; Filip et al., 1991); however, the overall low nitrogen content of both the fulvic and humic acids and lack of
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B
i
i
J 200
--
i
i 150
,
I 100
i
i 50
i
i
i
i
0
PPM
Fig. 3. 13C NM R spectra of fulvic and humic acids from a groundwater obtained at Fuhrberg, Germany: (A) fulvic acids; and (B) humic acids.
appropriate peaks in the FTIR spectra (Fig. 1) make the latter less likely. The peak at 130 ppm is usually assigned to alkyl-substituted aryl carbon, e.g. C-l, C-2, C-6 in p-hydroxyl phenols (Benner et al., 1990). The presence of oxygensubstituted aryl carbon, indicative of tannin or lignin structures, is evidenced by the peaks at 116, 145 and 154 ppm. Both the fulvic and humic acids have peaks with these chemical shifts. Peaks at 74 ppm, which are usually assigned to carbohydrate structures, also occur in both spectra. The most noticeable differences in the spectra are the presence of peaks at 17 and 58 ppm in the spectrum of the humic acids (Fig. 3). The sharp peak at 17 ppm is assigned to methyl groups (Wilson et al., 1987) usually of a long chain or paraffinic nature. The latter peak may be attributed to methoxyl substitution on aromatic rings as in lignin. This assignment would be consistent with the presence of peaks at 116, 145 and 154 ppm; however, the peak at 58 ppm is much stronger, particularly in the humic acid, than those at 148 or 153 ppm, indicating additional contribution to the 58-ppm peak by either ether or amino groups (Alberts et al., 1991; Filip et al., 1991). The FTIR spectra of these fulvic and humic acids are relatively simple (Fig. 1), containing primarily carboxylic, and substituted aliphatic and aromatic
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TABLE 2 Amount of carbon as a percentage of the total carbon present in the respective chemical-shift range of the 13C NMR spectra
Fulvic acids (%) Humic acids (%)
220-160 ppm (carbonyl)
160-110 ppm (aromatic)
110-60 ppm (O-alkyl)
60-0 ppm (alkyl)
22.6 19.5
33.3 35.4
8.0 7.9
36.0 42.6
groups. The ~3C N M R spectra confirm these observations. Integration of the peak areas of the two spectra, which were obtained under the same acquisition parameters and are comparable, indicates < 10% of the carbon exists in O-alkyl structures, while at least 70% of the carbon is present in either aromatic or alkyl groups (Table 2). Furthermore, the fulvic and humic acids have very similar distributions of aromatic and aliphatic groups and carbon is equally divided among the two types of groups. Again, the spectral evidence supports the conclusion that the higher E4/E6 ratio of the fulvic acids is due to the smaller molecular size of these molecules rather than decreased conjugation.
Copper binding capacities (CuBC 's) CuBC's of the fulvic and humic acids isolated from the Fuhrberg groundwater were the same (Table 1) and are in general agreement with values for CuBC's of humic substances from a number of sources, including groundwater (Stevenson, 1982; Thurman, 1985b). In the environment, metal complexation by humic substances is governed by many competitive reactions of metals for binding sites. To investigate this process, a series of competition experiments were conducted to determine the effectiveness of major potential competing ions to bind and block CuBC sites on the organic molecules. The ions chosen for the competition studies were Ca 2÷ and Mg 2÷ as the major divalent cations found in most surface and groundwaters, and A13÷ and Fe 3÷ to represent trivalent cations found in these waters and believed to bind more strongly to natural organic matter than most divalent metals (Alberts and Giesy, 1983; Pott et al., 1985). Ca 2+ and Mg 2÷ had no apparent effect on the overall CuBC values of either fulvic or humic acids from groundwater (Table 1), as the CuBC of both remained the same as when no competing ions other than Na ÷ were present. A13÷ and Fe 3÷ had a greater effect on blocking CuBC sites than did the divalent alkaline earths. However, only 16-20% of the sites on the humic acids and 10-14% on the fulvic acids were blocked by the trivalent ions. Considering that the amount of competing ion added to the solutions was
FULVIC AND HUMIC ACIDS ISOLATED FROM GROUNDWATER
4000
3500
3000
2500
2000
1500
1000
327
500
WAVENUMBER cm -1
Fig. 4. F T I R spectra of g r o u n d w a t e r humic acids complexed with: (A) H + ; (B) Ca 2+ ; (C) Mg 2+ ; (D) A13+ ; (E) Fe 3+ ; (F) Pb z+ ; and (G) Cu 2+ .
calculated to block 90-100% of the available sites, there seems to be little effect of these cations on the CuBC's of these materials, unlike unfractionated natural organic matter in some surface waters, where up to 90% of the CuBC may be blocked by the addition of A13+ and Fe 3+ (Alberts and Giesy, 1983; Giesy and Alberts, 1984). Examination of the F T I R spectra of the groundwater humic acids from the competition experiments and those which were complexed with Cu 2+ , Pb 2+ or H + (Fig. 4) shows that the major changes apparent in all spectra of metal-humic acid complexes are the decreased intensity of the peaks at 1231 and 1717 c m - 1 and the increased intensity of peaks at 1385 and 1617 c m - ~ due to deprotonation of simple carboxylic acids and asymmetric and symmetric stretching of cations coordinated to - C O 0 - groups, respectively. These changes are very similar to those observed in the F T I R spectra of Laurentian soil fulvic acids when complexed with Cu 2+ (Wang et al., 1990). In addition to these changes, peaks appeared at 458, 617, 780, 960 and 1092cm -~ in the
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J.J. ALBERTS ET AL.
spectra of all the metal-humic acid complexes, except that with Cu 2+ . These peaks are usually associated with Si-O bonds and often observed when humic acids have high ash contents, which are suspected to be clay minerals (Filip and Alberts, 1988). However, in this case, the samples were of low ash content at the start of the experiment (Table 1) and were not exposed to clay minerals during the study. Therefore, it is possible that the increased absorbances at these wave numbers may indicate humic acid-O-metal-On X binding similar to that expected for Si-O bonding to humic acids. The lack of the peaks in the humic acid-Cu 2+ spectra may indicate that Cu 2÷ is binding to the organic molecules through a different mechanism. An alternative explanation is binding through humic acid-O functional groups which is multidentate as opposed to unidentate, as in the case of other metals. Both possibilities would in part explain the lack of apparent competition of metals for Cu 2+ binding sites in these humic acids. CONCLUSIONS Bulk humic substances isolated from a groundwater aquifer in Fuhrberg, Germany, had a bimodal size distribution, with ,~40% of the total carbon being in the 50,000-100,000-D (NMW) size fraction and ,,~ 50% of the total carbon in the < 10,000-D (NMW) size fraction. Fulvic and humic acids isolated from these bulk humic substances were relatively low in nitrogen and had low H/C and N/C atomic ratios. They were very similar to each other in elemental composition, FTIR and ~3C N M R spectra, and copper binding capacities. They were relatively simple mixtures of molecules dominated by carboxylic acids, and aromatic and aliphatic groups, the latter two being in approximately equal proportions. The copper binding capacities of both fulvic and humic acids were similar to values obtained for humic substances from a number of sources. Studies with Ca 2+, Mg 2+, A13+ and Fe 3+ indicate that copper binding was not significantly affected by competition reactions from any of these elements, and that carboxyl groups may be the main site of complexation. ACKNOWLEDGEMENTS The authors wish to thank Dr. F6rster of Bruker Analytische Messtechnik for obtaining the N M R spectra. One of us (J.J.A.) gratefully acknowledges the support from the Alexander yon Humboldt Foundation, Bonn, through a Senior U.S. Scientist Award. This work is a joint contribution of the University of Georgia's Marine Institute (No. 707), the Institut ffir Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, F.R.G., and the Institut Okologische Chemie des Forschungzentrum fiir Umwelt and Gesundheit, F.R.G.
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REFERENCES Aiken, G.R., McKnight, D., Wershaw, R.L. and MacCarthy, P. (Editors), 1985. Humic Substances in Soil, Sediment and Water. Wiley, New York, NY, 692 pp. Alberts, J.J. and Filip, Z., 1989. Sources and characteristics of fulvic and humic acids from a salt marsh estuary. Sci. Total Environ., 81/82: 353-361. Alberts, J.J. and Giesy, J.P., 1983. Conditional stability constants of trace metals and naturally occurring humic materials: Their application in equilibrium models and verification with field data. In: R.F. Christman and E.T. Gjessing (Editors), Aquatic and Terrestrial Humic Materials. Ann Arbor Science Publishers, Ann Arbor, MI, pp. 333-348. Alberts, J.J., Halverson, J.E. and Orlandini, K.A., 1986. The distribution of plutonium, americium and curium isotopes in pond and stream sediments of the Savannah River Plant, South Carolina, USA. J. Environ. Radioactivity, 3: 249-271. Alberts, J.J., Filip, Z., Price, M.T., Williams, D.C. and Williams, M.C., 1988. Elemental composition, stable carbon isotope ratios and spectrophotometric properties of humic substances occurring in a salt marsh estuary. Org. Geochem., 12: 455-467. Alberts, J.J., Filip, Z. and Leversee, G.J., 1989a. Interaction of estuarine organic matter with copper and benzo(a)pyrene. Mar. Chem., 28: 77-87. Alberts, J.J., Wahlgren, M.A., Orlandini, K.A. and Durbahn, C.A., 1989b. The distribution of 239"24°pu, 238pu, 24JAm and 137Cs among chemically-defined components of sediments, settling particulates and net plankton of Lake Michigan. J. Environ. Radioactivity, 9: 89-103. Alberts, J.J., Hatcher, P.G., Price, M.T. and Filip, Z., 1991. Carbon-13 nuclear magnetic resonance analysis, lignin content and carbohydrate composition of humic substances from salt marsh estuaries. In: B. Allard, H. Bor6n and A. Grimvall (Editors), Humic Substances in the Aquatic and Terrestrial Environment. Lecture Notes Earth Sci., Vol. 33, Springer, Berlin, pp. 195-203. Benner, R., Hatcher, P.G. and Hedges, J.I., 1990. Early diagenesis of mangrove leaves in a tropical estuary: Bulk chemical characterization using solid-state ~3C N M R and elemental analyses. Geochim. Cosmochim. Acta, 54: 2003-2013. Christman, R.H. and Gjessing, E.T. (Editors), 1983. Aquatic and Terrestrial Humic Materials. Ann Arbor Science Publishers, Ann Arbor, MI, 538 pp. Filip, Z. and Alberts, J.J., 1988. The release of humic substances from Spartina altern(/tora (Loisel.) into sea water as influenced by salt marsh indigenous microorganisms. Sci. Total Environ., 73: 143-157. Filip, Z. and Smed-Hildmann, R., 1992. Does fossil plant material release humic substances into groundwater? Sci. Total Environ., 117/118:313-324. Filip, Z., Alberts, J.J., Cheshire, M.V., Goodman, B.A. and Bacon, J.R., 1988. Comparison of salt marsh humic acid with humic-like substances from the indigenous plant species Spartina altern(/tora (Loisel.), Sci. Total Environ., 71: 157-172. Filip, Z., Newman, R.H. and Alberts, J.J., 1991. Carbon-13 nuclear magnetic resonance characterization of humic substances associated with salt marsh environments. Sci. Total Environ., 101: 195-203. Giesy, J.P. and Alberts, J.J., 1984. Trace metal speciation: The interaction of metals with organic constituents of surface waters. In: R.J. Ward (Editor), Effects of Trace Elements on Aquatic Ecosystems. Electric Power Res. Inst., Palo Alto, CA, EA-3329, pp. 11-1-11-31. Giesy, Jr., J.P., Briese, L.A. and Leversee, G.P., 1978. Metal binding capacity of selected Maine surface water. Environ. Geol., 2: 257-268. Giesy, J.P., Geiger, R.A., Kevern, N.R. and Alberts, J.J,, 1986. UO22+-humate interactions in soft, acid, humate-rich waters. J. Environ. Radioactivity, 4: 39-64.
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K611e, W., 1981. Erfahrungen bei der Aufbereitung eines reduzierten huminstoff-hattigen Grundwassers in Wasserwerk Fuhrberg der Stadtwerke Hannover AG. In: Neue Technologien in der Trinkwasserversorgung. Dtsch. Ver. G. Wasserwirtsch., Hannover, Schriftenr. Wasser, No. 102, pp. 339-349. MacCarthy, P. and Rice, J.A., 1985. Spectroscopic methods (other than NMR) for determining functionality in humic substances. In: G.R. Aiken, D.M. McKnight, R.I. Wershaw and P. MacCarthy (Editors), Humic Substances in Soil, Sediment, and Water. Wiley, New York, NY, pp. 527-559. McCarthy, J.F., 1989. Bioavailability and toxicity of metals and hydrophobic organic contaminants. In: I.H. Suffet and P. MacCarthy (Editors), Aquatic Humic Substances. Am. Chem. Soc., Washington, DC, Adv. Chem. Ser., 219: 263-277. Mfiller-Wegener, U., 1988. Interactions of humic substances with biota. In: F.H. Frimmel and R.F. Christman (Editors), Humic Substances and Their Role in the Environment. Wiley, New York, NY, pp. 179-192. Piotrowicz, S.R., Harvey, G.R., Boran, D.A., Weisel, C.P. and Springer-Young, M., 1984. Cadmium, copper, and zinc interactions with marine humus as a function of ligand structure. Mar. Chem., 14: 333-346. Pott, D.B., Alberts, J.J. and Elzerman, A.W., 1985. The influence ofpH on the binding capacity and conditional stability constants of aluminum and naturally-occurring organic matter. Chem Geol., 48: 293-304. Rashid, M.A., 1985. Geochemistry of Marine Humic Compounds, Springer, New York, NY, 300 pp. Stevenson, F.J., 1982. Humus Chemistry. Wiley, New York, NY, 443 pp. Stewart, A.J., 1984. Interactions between dissolved humic materials and organic toxicants. In: K.E. Cowser (Editor), Synthetic Fossil Fuel Technologies: Results of Health and Environmental Studies. Butterworth, Boston, MA, pp. 505-521. Suffet, I.H. and MacCarthy, P., 1989. Aquatic Humic Substances. Am. Chem. Soc., Washington, DC, Adv. Chem. Ser. 219, 864 pp. Thurman, E.M., 1985a. Organic Geochemistry of Natural Waters. Martinus Nijhoff/Dr. W. Junk Publishers, Dordrecht, 497 pp. Thurman, E.M., 1985b. Humic substances in groundwater. In: G.R. Aiken, D.M. McKnight, R.I. Wershaw and P. MacCarthy (Editors), Humic Substances in Soil, Sediment, and Water. Wiley, New York, NY, pp. 87-103. Thurman, E.M. and Malcolm, R.L., 1981. Preparative isolation of aquatic humic substances. Environ. Sci. Technol., 15: 463-466. Wang, Z.-D., Pant, B.C. and Langford, C.H., 1990. Spectroscopic and structural characterization of a Laurentian fulvic acid: notes on the origin of the color. Anal. Chim. Acta, 232: 43-49. Weber, J.H., 1988. Binding and transport of metals by humic materials. In: F.H. Frimmel and R.F. Christman (Editors), Humic Substances and Their Role in the Environment. Wiley, Chichester, pp. 165-178. Wilson, M.A., Vassallo, A.M., Perdue, E.M. and Reuter, J.H., 1987. Compositional and solid-state nuclear magnetic resonance study of humic and fulvic acid fractions of soil organic matter. Anal. Chem., 59:551-558.
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Fulvic and humic acids isolated from groundwater: Compositional characteristics and cation binding J.J. A l b e r t s a, Z. F i l i p b a n d N. H e r t k o r n c
"University of Georgia Mar&e Institute, Sapelo Island, GA 31327, USA blnstitut 3~r Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Aussenstelle Langen, Paul-Erhlieh-Strasse 29, D-6070 Langen, Germany Clnstitutj~r Okologisehe Chemie/GSF, Schulstrasse 10, D-8050 Attaching, Germany (Received August 22, 1991; revised and accepted June 3, 1992)
ABSTRACT Alberts, J.J., Filip, Z. and Hertkorn, N., 1992. Fulvic and humic acids isolated from groundwater: Compositional characteristics and cation binding. J. Contam. Hydrol., 11: 317-330. Samples of humic substances were obtained from a waterworks at Fuhrberg, Germany. The material had a bimodal molecular size distribution with ~40°/o of the total carbon in the 50,000-100,000-D (nominal molecular weight, NMW, in daltons) size fraction and ~ 50% of the carbon in the < 10,000-D (NMW) size fraction. The fulvic and humic acids isolated from the bulk humic substances were low in nitrogen content and had low H/C atomic ratios. Furthermore, the fulvic and humic acids had very similar elemental, spectral and copper binding characteristics. Over 70% of the carbon in both the fulvic and humic acids was present in aromatic or aliphatic groups, with 13C N M R analyses indicating approximately even distribution among the two types. Competitive elemental binding studies indicated that Ca 2÷ , Mg 2÷ , AI 3÷ and Fe 3+ do not effectively compete for copper binding sites on these compounds. In humic acids, these cations are predominantly bond by carboxylic groups. INTRODUCTION
Humic substances of the fulvic and humic acid type comprise a large portion of the ubiquitous organic matter reservoir in natural waters (Thurman, 1985a). They are known to complex many heavy metals, radionuclides and anthropogenic organic compounds (Alberts et al., 1986, 1989a, b; Giesy et al., 1986; Weber, 1988; Suffet and MacCarthy, 1989). Furthermore, this complexation by humic substances can enhance or retard uptake and toxicity of these compounds by organisms (Stewart, 1984; Mfiller-Wegener, 1988; McCarthy, 1989). While considerable study of humic substances has Correspondence to: J.J. Alberts, University of Georgia Marine Institute, Sapelo Island, GA 31327, USA. 0169-7722/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
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been conducted with materials isolated from soils, sediments, surface waters and estuaries (Stevenson, 1982; Christman and Gjessing, 1983; Aiken et al., 1985; Rashid, 1985; Alberts et al., 1988, 1989a; Filip and Alberts, 1988; Filip et al., 1988, 1991; Alberts and Filip, 1989) very little is known of the characteristics of these materials isolated from groundwaters (Thurman, 1985a, b), which are often the source of drinking water to large populations. In this study, we have characterized humic substances from a groundwater aquifer in Fuhrberg, Lower Saxony, Germany. This groundwater aquifer is important to the drinking water supply for the city of Hannover, Germany. The bulk humic substances from these groundwaters were examined with respect to their molecular size spectrum, while the humic and fulvic acids isolated from those humic substances were characterized with respect to their FTIR (Fourier transform infrared) and nuclear magnetic resonance (NMR) spectral characteristics and copper binding capacities (CuBC's). METHODS
Site description and &olation of humic substances The humic substances characterized in this investigation originated from the waterworks at Fuhrberg, Germany, where a local groundwater aquifer yields a brownish-colored water. This porous aquifer is composed of homogeneous alluvial sand, located at a depth of 30-70 m. The untreated groundwater has a pH of 6.5 and organic carbon content of 10.0 g m -3 C. Inorganic elemental concentrations (mol m -3) of the groundwater are: Na + , 1.09; K + , 0 . 0 8 ; C a 2+ , 1.80; Mg 2+, 0.37, total Fe, 280; total Mn, 20; N H 4 +, 60; C l - , 1.30; NO2-, 0.1; NO3-, 0.03; and SO42-, 1.46 (K611e, 1981). The groundwater is decolorized with ion-exchange resins, which yield a dark-brown concentrate. This concentrate was collected, filtered through paper filters, freeze-dried and used as bulk humic substances and the source of humic and fulvic acids examined in this study. Bulk humic substances were used for the molecular size fractionation experiment. However, fulvic and humic acids, which had been isolated from the bulk humic substances, were used for all subsequent analyses. These fulvic and humic acid fractions were isolated by dissolving the bulk humic substances in base (pH 12, NaOH) then acidifying to pH 1.0 (HC1). The fulvic and humic acids were separated by centrifugation, with subsequent purification of the fulvic acid fraction with XAD-8 ® macroreticular resin (Robin & Haas ~) and the humic acid fraction by dialysis (Thurman and Malcolm, 1981; Alberts et al., 1988). Both fractions were then freeze-dried.
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Molecular size fractionation
Fifty mg of bulk humic substances were dissolved in 300mL of KH2PO 4 buffer solution (0.1 M KHzPO4-0.1 M NaOH, v/v; pH = 7.4). Ten mL of solution were taken for dissolved organic carbon (DOC) analyses. The remaining solution was placed in an Amicon ® Model 404, Stirred Ultrafiltration Cell containing a X M - I O 0 ® ultrafilter. The solution volume was reduced to 100mL under nitrogen pressure (10 psi or ~ 68.95 kPa) and the solution volume of the retentate was again made to 300mL with KHzPO4 buffer solution. The ultrafiltrate, containing materials of < 100,000 D (nominal molecular weight, NMW, in daltons), were collected in 100-mL aliquots. The process was repeated until the ultrafiltrate was colorless. The retentate was collected, 25 m L of solution were taken for DOC analyses, and the remainder was acidified to pH 2 (6 N HC1) and allowed to stand overnight. The precipitate was collected by centrifugation and dialysed (1000 D) against three changes of deionized water before freeze-drying. The ultrafiltrates were combined and sequential ultrafiltration was continued as above using XM50 ~" (20 psi or --~ 137.90kPa N2) and Y M - I O ® (35 psi or ~ 2 4 1 . 3 2 k P a N2) ultrafilters to obtain molecular size fractions of 50,000-100,000 and 10,00050,000 D (NMW), respectively. The ultrafiltrate volume from the Y M - I O ® ultrafilter was so large, almost 3 L, that the organic matter was concentrated on an XAD-8 ® resin column (Thurman and Malcolm, 1981). The organic matter isolated in this manner was again dissolved in KHzPO 4 buffer and ultrafiltered, as above, over a YM-2 ~ (molecular size 2000 D NMW, 75 psi or ~ 5 1 7 . 1 1 K P a N2). The ultrafiltrate which passed the YM-2 ® filter was acidified and humic substances ( < 2000 D NMW) remaining in solution were isolated on XAD-8 ® resin. Volumes and DOC analyses of all ultrafiltrates, retentates and starting solutions were determined to allow carbon mass-balance calculations to be made for all fractions. Copper binding capacities (CuBC's)
Aliquots of humic or fulvic acids (,-~ 5 mg) were dissolved in ~ 50 mL of deionized water with the addition of 1-2 mL of 0.2 N NaOH. The solutions were made to volume with deionized water and aliquots of these solutions were pipetted into beakers. For copper binding studies, the methods of Giesy et al. (1978) were used. Briefly, 5 M NaNO3 was used as an ionic strength adjuster (2 mL/100 mL analyte) and sufficient Cu 2+ ion was added to produce a free Cu 2+ concentration between 3.2-10 -7 and 1.6.10 -4 M. The pH of the solutions were adjusted to 5.1 + 0.1 by dropwise addition of dilute HC1 to minimize hydroxyl-copper complex formation. All volume changes were
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recorded. Samples were covered and allowed to stand overnight ( ~ 18 h) at room temperature (19.6_+2.7°C). The pH was again measured before determining the free Cu 2+ ion concentration (procedure described below). Analyses were conducted in triplicate and standard curves were prepared immediately prior to estimating the Cu 2+ ion concentrations. Experiments to determine the effect of competitive ions on CuBC's were conducted as above, except that the competing ion was added with the ionic strength adjustor. The solutions containing the humic or fulvic acids and competing ions were allowed to equilibrate overnight before the Cu 2+ ion solution was added and the pH was again adjusted to 5.1. The solutions were once again equilibrated overnight before free Cu 2+ concentrations were determined. In all competitive ion studies, humic and fulvic acid samples not containing competing ions were analyzed in parallel with the competition studies so that differences due to daily electrode variations were eliminated. Following free Cu 2+ ion determinations, the humic acid samples were dialysed against deionized water for three days, freeze-dried and retained for F T I R spectral analyses.
Instrumental analyses F T I R spectra of all isolates and starting materials were collected from 2% KBr disks using a Bruker ® Model 48 F T I R Spectrophotometer. Ultraviolet/ visible spectra were collected with a Carlo-Erba ® Recording Spectrophotometer (Model Spectrocomp 601). Solid-state 13C N M R spectra were obtained by the methods of cross-polarization and magic angle spinning (CP-MAS) on a Bruker ® Model MSL 200 spectrometer, operated at 50.3 MHz. Typically, 12,000 scans were acquired with a pulse width of 5/~s and an acquisition time of 25.6 ms; a 4.5-/ts proton preparation pulse, followed by a cross-polarization contact time of 1 ms and a pulse delay of 4 s were used. The samples were spun at 6 kHz. Free Cu 2+ ion concentrations were determined with an Orion :R~ Model 942900 copper-selective ion electrode and an Orion '~R;Model 900200 Double Junction Reference Electrode. Elemental analyses (C, H, N and O by difference) were conducted with a Carlo-Erba ~: C H N Elemental Analyzer (Model EA 1108). DOC analyses were performed with an Astro ® Model 1850 DOC Analyzer. RESULTS AND DISCUSSION
Molecular size distribution The ultrafilter fractionation of the bulk humic substances resulted in 58% recovery of the organic carbon in the original sample. This was significantly
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lower than recoveries for humic acids isolated from estuarine sources, which normally had carbon recoveries of 96-100% (J.J. Alberts, unpublished data, 1990). Since there was little evidence of loss of humic acid carbon on the ultrafilters from the estuarine samples, it must be inferred that the losses of the groundwater humic substances were due to small molecules, which were isolated on the initial ion-exchange resin, but were subsequently lost during the isolation of the < 2000-D (NMW) fraction on the XAD-8 ® resin, perhaps by nonreversible adsorption to the XAD resin. The carbon which could be accounted for was bimodal in distribution, with ~ 6 0 % in the 50,000-100,000-D (NMW) size fraction. Another 20% of the carbon was in the 2,000-10,000-D (NMW) fraction. Less than 1% of the carbon was in the > 100,000-D (NMW) fraction and 7% was in the 10,00050,000-D (NMW) fraction. Ten percent of the carbon was in the < 2000-D fraction (NMW), but this number is an underestimate if the unrecovered carbon was small molecular size material. The latter would agree with studies which show that groundwater humic substances are of relatively small molecular size (Thurman, 1985b).
Elemental analyses The fulvic and humic acids isolated from the groundwater had very similar elemental compositions (Table 1). Both were low in nitrogen content. The fulvic acids had nitrogen contents similar to fulvic acids recovered from some groundwater samples from the U.S.A., but the humic acids were significantly lower in nitrogen content as compared to groundwaters in the U.S.A. (Thurman, 1985b). Accordingly, the atomic ratios of N/C in the humic acids reported here are significantly less than those of the U.S. groundwaters, while the fulvic acids have similar values. The atomic ratios of H/C of the humic acids presented here are similar to the U.S. groundwater material, but the fulvic acids from the Fuhrberg groundwater are low relative to groundwater fulvic acids as reported by T h u r m a n (1985b). The low H/C ratios in the fulvic and humic acids from Fuhrberg groundwater indicates considerable unsaturated character in both. The UV/visible spectra of both the fulvic and humic acids were featureless and decreased monotonically with increasing wave number. The ratio of light absorbance at 465 nm to that at 665 nm (E4/E6, Table 1) indicates that the fulvic acids are either composed of smaller molecules than the humic acids, or that they have a lesser degree of conjugation. The former conclusion would agree with the ultrafiltration results reported above. The E4/E6 ratio for the humic acids is high relative to soil humic acids (Stevenson, 1982), but the E4/E6 for both the fulvic and humic acids agree with values reported for groundwaters by T h u r m a n (1985b).
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TABLE I Elemental composition *j , E4/E 6 ratios and copper binding capacities (CuBC's) of groundwater humic and fulvic acids Sample
Ash (wt%)
C (wt%)
H (wt%)
N (wt%)
Fulvic acids Humic acids
1.51 2.26
54.74_+ 1.31 54.43 + 0.15
4.23 + 0.11 4.61 + 0.13
1.02 + 0.08 t.69 _+0.10
Fulvic acids Humic acids
E4/E 6
H/C (*2)
N/C 1,2,
l 1.7 7.6
0.93 + 0.02 1.01 +_0.03
0.016 _+0.001 0.026 +_0.002
Competing ions *~
Fulvic acids Humic acids
CuBC .3
AI 3+
Fe 3+
Ca 2+
Mg 2-
0.311 + 0.005 0.316_+ 0.0
90 84
86 80
100 100
100 100
*~All values corrected for ash content of humic and fulvic acids. *2Atomic ratios. *3Copper binding capacity as/~g-at. Cu 2+/mg humic or fulvic acids. *4Reported as percent of original CuBC remaining after equilibration with the competing ions.
While the groundwater fulvic and humic acids characterized here have many similarities to those from groundwaters in the U.S.A., there are some significant differences. These differences may be the result of fossil plant material inputs into the groundwater humic substances of this region of Germany (Filip and Smed-Hildmann, 1992). F T I R spectra The F T I R spectra of the fulvic and humic acids from this groundwater are relatively uncomplex and dominated by absorbances due to aromatic, aliphatic and carboxylic groups (Fig. 1). The spectra are very similar to each other and dominated by a large and complex band of peaks at the 3000-3400cm -~ region, which can be attributed to hydrogen bonding and O - H stretching. The presence of aliphatic groups is evidenced by the weak doublet between 2850 and 2970 cm -~ . The peak at 1618 cm-J and broad band around 1400 cm -~ , represents the C = C stretching of substituted aromatic rings. The absorption in these regions relative to the aliphatic doublet at 28502970 cm-1 appears somewhat stronger in the humic acids than in the fulvic acids, thus indicating that the humic acids may have greater aromatic character than the fulvic acids. If this is true, then the higher E4/E 6 ratio for the fulvic acids is likely to be due to the presence of smaller molecules.
FULVIC AND HUMIC ACIDS ISOLATED FROM GROUNDWATER
LU o z < tn cr
I
hi
t
323
i#
O co 1:13 ,<
4000
3500
3000
2500
2000
WAVENUMBER
1500
1000
500
c m -1
Fig. 1. FTIR spectra of humic substances from a groundwater obtained at Fuhrberg, Germany, in protonated form: (A) fulvic acids; and (B) humic acids.
The strong absorbance at 1717cm ' is representative of the protonated carboxylic acids and ketonic C = O groups, and the peak at 1216cm -' is the C-O stretching vibration and OH bending deformations of the carboxylic acid groups. Both of these peaks disappeared when the humic substances were treated with K + , which is indicative of salt formation in simple carboxylic acids (MacCarthy and Rice, 1985). In keeping with the low nitrogen contents of these compounds, there was no evidence of secondary amide linkages associated with proteins or peptides, which have been recognized in estuarine humic matter (Filip and Alberts, 1988; Alberts et al., 1991; Filip et al., 1991). The two ultrafilter fractions of the bulk humic substances that represented 60% and 20% of the recovered organic carbon (50,000 -100,000 and 2,00010,000 D, respectively) had FTIR spectra very similar to each other (Fig. 2, B and D). The main difference between these two spectra and that of the starting material is the loss of the two peaks at 1220 and 1717 cm 1, which are attributable to C = O and C-O functionalities in simple carboxylic acids that disappear upon salt formation. Thus, it appears that despite being isolated by precipitation from pH 2 solution and dialysed against deionized water, the humic substances in these two size fractions are still present in a salt form rather than having been reprotonated. The cause of this phenomenon is not known, but may involve steric hindrance of the carboxylic groups during the dialysis procedure. The distinct absorption band at 1385 cm-1 may be attributable to oxy-functional groups in phenolics and/or methyl and methylene groups of aliphatic chains. The 1618-cm -~, C = C ring stretching peak indicates aromatic groups to be present in both fractions.
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~T
t.u (.3 Z < 133 frO o9 t'n <
4000
3500
3000
2500
2000
WAVENUMBER
1500
1000
500
cm -1
Fig. 2. FTIR spectra of bulk humic substances and some molecular size fractionations obtained from groundwater in Germany: (A) protonated bulk humic substances which had not been fractionated over ultrafilters; (B) 50,000-100,000-D (NMW) fraction; (C) 10,000-50,000-D (NMW) fraction; (D) 2,00010,000 D (NMW) fraction; and (E) < 2,000-D (NMW) fraction.
The spectra of the two remaining size fractions of the bulk humic substances show slightly altered, but still strong regions of H-bonding (30003400cm -1) and the presence of the aliphatic C - H stretching (28502970cm-1). The aromatic C = C peaks at 1430 and 1618cm -~ remain weak but visible. The spectrum of the carbon < 2000-D size fraction is highly degraded (Fig. 2, E) and poorly resolved. If it is assumed that the unrecoverable carbon passed through the smallest ultrafilter, this fraction accounts for ,-- 35% of the initial organic carbon in solution. However, only about one-third of that was actually recovered by isolation on the XAD-8 ~ resin column. Therefore, this spectrum is representative of only a very small portion of the total material in this fraction. N M R spectra The ~3C N M R spectra of both the fulvic and humic acids had strong peaks at 130 and 175 ppm (Fig. 3). The former may indicate the presence of C = O in carboxyl groups or secondary amide linkages in peptides and proteins (Piotrowicz et al., 1984; Alberts et al., 1991; Filip et al., 1991); however, the overall low nitrogen content of both the fulvic and humic acids and lack of
FULVIC AND HUMIC ACIDS ISOLATED FROM GROUNDWATER
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B
i
i
J 200
--
i
i 150
,
I 100
i
i 50
i
i
i
i
0
PPM
Fig. 3. 13C NM R spectra of fulvic and humic acids from a groundwater obtained at Fuhrberg, Germany: (A) fulvic acids; and (B) humic acids.
appropriate peaks in the FTIR spectra (Fig. 1) make the latter less likely. The peak at 130 ppm is usually assigned to alkyl-substituted aryl carbon, e.g. C-l, C-2, C-6 in p-hydroxyl phenols (Benner et al., 1990). The presence of oxygensubstituted aryl carbon, indicative of tannin or lignin structures, is evidenced by the peaks at 116, 145 and 154 ppm. Both the fulvic and humic acids have peaks with these chemical shifts. Peaks at 74 ppm, which are usually assigned to carbohydrate structures, also occur in both spectra. The most noticeable differences in the spectra are the presence of peaks at 17 and 58 ppm in the spectrum of the humic acids (Fig. 3). The sharp peak at 17 ppm is assigned to methyl groups (Wilson et al., 1987) usually of a long chain or paraffinic nature. The latter peak may be attributed to methoxyl substitution on aromatic rings as in lignin. This assignment would be consistent with the presence of peaks at 116, 145 and 154 ppm; however, the peak at 58 ppm is much stronger, particularly in the humic acid, than those at 148 or 153 ppm, indicating additional contribution to the 58-ppm peak by either ether or amino groups (Alberts et al., 1991; Filip et al., 1991). The FTIR spectra of these fulvic and humic acids are relatively simple (Fig. 1), containing primarily carboxylic, and substituted aliphatic and aromatic
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TABLE 2 Amount of carbon as a percentage of the total carbon present in the respective chemical-shift range of the 13C NMR spectra
Fulvic acids (%) Humic acids (%)
220-160 ppm (carbonyl)
160-110 ppm (aromatic)
110-60 ppm (O-alkyl)
60-0 ppm (alkyl)
22.6 19.5
33.3 35.4
8.0 7.9
36.0 42.6
groups. The ~3C N M R spectra confirm these observations. Integration of the peak areas of the two spectra, which were obtained under the same acquisition parameters and are comparable, indicates < 10% of the carbon exists in O-alkyl structures, while at least 70% of the carbon is present in either aromatic or alkyl groups (Table 2). Furthermore, the fulvic and humic acids have very similar distributions of aromatic and aliphatic groups and carbon is equally divided among the two types of groups. Again, the spectral evidence supports the conclusion that the higher E4/E6 ratio of the fulvic acids is due to the smaller molecular size of these molecules rather than decreased conjugation.
Copper binding capacities (CuBC 's) CuBC's of the fulvic and humic acids isolated from the Fuhrberg groundwater were the same (Table 1) and are in general agreement with values for CuBC's of humic substances from a number of sources, including groundwater (Stevenson, 1982; Thurman, 1985b). In the environment, metal complexation by humic substances is governed by many competitive reactions of metals for binding sites. To investigate this process, a series of competition experiments were conducted to determine the effectiveness of major potential competing ions to bind and block CuBC sites on the organic molecules. The ions chosen for the competition studies were Ca 2÷ and Mg 2÷ as the major divalent cations found in most surface and groundwaters, and A13÷ and Fe 3÷ to represent trivalent cations found in these waters and believed to bind more strongly to natural organic matter than most divalent metals (Alberts and Giesy, 1983; Pott et al., 1985). Ca 2+ and Mg 2÷ had no apparent effect on the overall CuBC values of either fulvic or humic acids from groundwater (Table 1), as the CuBC of both remained the same as when no competing ions other than Na ÷ were present. A13÷ and Fe 3÷ had a greater effect on blocking CuBC sites than did the divalent alkaline earths. However, only 16-20% of the sites on the humic acids and 10-14% on the fulvic acids were blocked by the trivalent ions. Considering that the amount of competing ion added to the solutions was
FULVIC AND HUMIC ACIDS ISOLATED FROM GROUNDWATER
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3500
3000
2500
2000
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1000
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500
WAVENUMBER cm -1
Fig. 4. F T I R spectra of g r o u n d w a t e r humic acids complexed with: (A) H + ; (B) Ca 2+ ; (C) Mg 2+ ; (D) A13+ ; (E) Fe 3+ ; (F) Pb z+ ; and (G) Cu 2+ .
calculated to block 90-100% of the available sites, there seems to be little effect of these cations on the CuBC's of these materials, unlike unfractionated natural organic matter in some surface waters, where up to 90% of the CuBC may be blocked by the addition of A13+ and Fe 3+ (Alberts and Giesy, 1983; Giesy and Alberts, 1984). Examination of the F T I R spectra of the groundwater humic acids from the competition experiments and those which were complexed with Cu 2+ , Pb 2+ or H + (Fig. 4) shows that the major changes apparent in all spectra of metal-humic acid complexes are the decreased intensity of the peaks at 1231 and 1717 c m - 1 and the increased intensity of peaks at 1385 and 1617 c m - ~ due to deprotonation of simple carboxylic acids and asymmetric and symmetric stretching of cations coordinated to - C O 0 - groups, respectively. These changes are very similar to those observed in the F T I R spectra of Laurentian soil fulvic acids when complexed with Cu 2+ (Wang et al., 1990). In addition to these changes, peaks appeared at 458, 617, 780, 960 and 1092cm -~ in the
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spectra of all the metal-humic acid complexes, except that with Cu 2+ . These peaks are usually associated with Si-O bonds and often observed when humic acids have high ash contents, which are suspected to be clay minerals (Filip and Alberts, 1988). However, in this case, the samples were of low ash content at the start of the experiment (Table 1) and were not exposed to clay minerals during the study. Therefore, it is possible that the increased absorbances at these wave numbers may indicate humic acid-O-metal-On X binding similar to that expected for Si-O bonding to humic acids. The lack of the peaks in the humic acid-Cu 2+ spectra may indicate that Cu 2÷ is binding to the organic molecules through a different mechanism. An alternative explanation is binding through humic acid-O functional groups which is multidentate as opposed to unidentate, as in the case of other metals. Both possibilities would in part explain the lack of apparent competition of metals for Cu 2+ binding sites in these humic acids. CONCLUSIONS Bulk humic substances isolated from a groundwater aquifer in Fuhrberg, Germany, had a bimodal size distribution, with ,~40% of the total carbon being in the 50,000-100,000-D (NMW) size fraction and ,,~ 50% of the total carbon in the < 10,000-D (NMW) size fraction. Fulvic and humic acids isolated from these bulk humic substances were relatively low in nitrogen and had low H/C and N/C atomic ratios. They were very similar to each other in elemental composition, FTIR and ~3C N M R spectra, and copper binding capacities. They were relatively simple mixtures of molecules dominated by carboxylic acids, and aromatic and aliphatic groups, the latter two being in approximately equal proportions. The copper binding capacities of both fulvic and humic acids were similar to values obtained for humic substances from a number of sources. Studies with Ca 2+, Mg 2+, A13+ and Fe 3+ indicate that copper binding was not significantly affected by competition reactions from any of these elements, and that carboxyl groups may be the main site of complexation. ACKNOWLEDGEMENTS The authors wish to thank Dr. F6rster of Bruker Analytische Messtechnik for obtaining the N M R spectra. One of us (J.J.A.) gratefully acknowledges the support from the Alexander yon Humboldt Foundation, Bonn, through a Senior U.S. Scientist Award. This work is a joint contribution of the University of Georgia's Marine Institute (No. 707), the Institut ffir Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, F.R.G., and the Institut Okologische Chemie des Forschungzentrum fiir Umwelt and Gesundheit, F.R.G.
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