(3) “Chloronaphthalenes”,Amer. Ind. Hyg. Assoc., Hygienic Guide Ser., Jan.-Feb. 1966. (4) Koeman, J. H.,VanVelzen-Blad,H.C.W.,DeVries, R., Vos, 3. G., J . Reprod. Fertil. Suppl., 19,353-64 (1973). (5) Crump-Weisner,H. J., Feltz, H. R., Yates, M. L., U S . Geol. Suru. J . Res., 1,603-7 (1973). (6) Law, L. M., Goerlitz, D. F., Pestic. Monit. J., 8 (l),33-6 (1974). (7) Compton, B., Brzydlo, P. O., Zweig, G., “Field Evaluation of Methods of Collection and Analysis of Airborne Pesticides”,Syracuse Univ. Res. Corp., Vol2, Field Evaluation and Analysis, EPA Contract No. CPA-70-145,EPA-RA-72-004a,1972. (8) R. G.. Brown. A. R.. Jackson. M. D., Anal. Chem., 49, . ~ Lewis. , 1668-71 (1977). (9) Safe, S., Hutzinger, O., Nature, 232,641-2 (1974). (10) Ruzo, L. O., Zabik, M. J., Schuetz, R. D., Bull. Enuiron. Contam. T o x ~ c o ~8,217-8 ., (1972). (11) Hutzinger, O., Safe, S.,Zitko, V., Enuiron. Health Perspect., 15-22 (Apr. 1972). (12) Zabik. M. J., Schuetz, R. D... J . Anric. Food Chem., . . Ruzo. L. 0.. 22,199-201 (1974).
(13) Lewis, R. G., Hanisch, R. C., MacLeod, K. E., Sovocool, G. W., ibid., 24, 1030-5 (1976). (14) Goerlitz,D. F., Law, L. M., J . Assoc. Off. Anal. Chem., 57,17641
(1974). (15) Pellizzari, E. D., “Analysis of Organic Air Pollutants by Gas Chromatography and Mass Spectrometry”, EPA-600/2-77-100, 1977. (16) Safe, S., in National Conference on Polychlorinated Biphenyls, November 19-21, 1974, Chicago, Ill., F. A. Ayer, Ed., EPA-560/674-004 (PB-253,248),p 94, 1976. (17) Erickson, M. D., Zweidinger, R. A., Michael, L. C., Pellizzari, E. D., “Environmental Monitoring Near Industrial Sites: Polychloronaphthalenes”,EPA-560/6-77-019,1977. (18) Erickson. M. D.. Michael, L. C., Zweidinger. R. A.. Pellizzari. E. D., presented in part at 1977 Annual AOAC-Meeting; Washington, D.C., Oct. 27-30, 1977. Received for review November 7,1977. Accepted February 10,1978. Presented at the Division of Industrial and Engineering Chemistry, 174th Meeting, ACS, Chicago,Ill., August 1977. Research supported by EPA Contract No. 68-01-1978.
Investigation of Fulvic Acid Extracted from Sewage Sludge Using Carbon- 13 and Proton NMR Spectroscopy Garrison Sposito’, Gene D. Schaumberg’, Thomas G. Perkins2, and Kenneth M. Holtzclaw Department of Soil and Environmental Sciences and Department of Chemistry, University of California, Riverside, Calif. 9252 1
Carbon-13 and proton NMR spectra were obtained for two representative fulvic acid samples that were extracted from anaerobically digested sewage sludges. The spectra gave evidence for significant amounts of both aliphatic and aromatic constituents, including products from the degradation of polysaccharides and proteins. The results suggested that sludge-derived fulvic acid is in a less oxidized state than fulvic acid extracted from naturally occurring soil organic matter. Carbon-13 NMR spectroscopy appears to be a useful means of characterizing fulvic acids and distinguishing these complex, water-soluble mixtures according to the origin and state of oxidation of the organic matter from which they are extracted. The fulvic acid fraction of the organic matter that occurs in soil or in natural waters generally is believed to be the class of extractable, water-soluble compounds that is most reactive toward metal cations ( I , 2). This organic fraction is a heterogeneous mixture of molecules that differ widely in chemical composition, molecular weight, and functional group acidity, but that possess in toto a significant capability to form soluble complexes with trace metals (e.g., Al, Cu, Fe, and Zn). In this way, fulvic acid (FA) can play an important role in determining the solubility of trace metals in soil solutions and in other natural waters. The current practice of disposing sewage effluent and sludge both in the ocean and on the land poses an environmental problem whose resolution may well depend on an understanding of the chemistry of FA. Because sewage often contains large amounts of potentially harmful trace metals (e.g., Cd, Cr, Ni, and Pb), its disposal provides an opportunity for an unwanted increase in the concentrations of these metals in seawater or in the soil solution ( 3 , 4 ) . Moreover, since 1 Present address, Department of Chemistry, Sonoma State College, Rohnert Park, Calif. 94928. Present address, Church Laboratory of Chemical Biology, California Institute of Technology, Pasadena, Calif. 91125.
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sewage itself has a FA fraction, the trace metals added to the coastal or to the soil environment may be soluble species already, without further interaction between them and the components of naturally occurring FA. It is clear, therefore, that the properties of sludge-derived FA must be known in order to predict accurately the fates of the added trace metals. The FA fraction of sewage sludge has only recently been the object of detailed investigation (5-9). Since this organic material is quite heterogeneous, physical methods, such as IR and NMR spectroscopy, may prove to be more useful in its initial characterization than wet-chemical techniques. Infrared spectroscopy has been shown to be effective in elucidating the functional group character of sewage effluent and sludge, especially in relation to the known functional groups in soilderived FA ( 5 , 8 ) .Carbon-13 and proton NMR spectra of FA extracted from soil and from seawater have been published very recently by Stuermer and Payne (IO). These investigators found that ‘3C-NMR spectroscopy is an effective tool for establishing the gross chemical structure of FA and for comparing FA samples of different origin. In this paper we report 13C-and ‘H-NMR spectra for two FA samples extracted from anaerobically digested sewage sludges. The two fulvic acids studied are of different chemical composition but are representative of FA extracted from anaerobically digested sludges produced in California. The principal objectives of our investigation were to determine the general classes of organic constituents in sludge-derived FA and to evaluate the potential of ‘3C-NMR spectroscopy as a method of distinguishing sludge FA from that originating in soil organic matter and seawater.
Experimental The fulvic acid fractions of two anaerobically digested sewage sludges were extracted and purified according to the method of Holtzclaw et al. (7).The sludge materials had been collected from the surface matter in drying lagoons located near the cities of Ontario and Rialto in southern California. Representative data on the trace metal content in these
1978 American Chemical Society
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sludges, as well as on the yields and ash contents (54%) of their fulvic acid fractions, have been published (7,8, 11).(In the papers cited, Ontario sludge is given the code designation "A" while Rialto sludge is designated "Bl''.) Ontario sludge tends to have a high content of the metals Al, Cd, Cu, Fe, Ni, and Zn, whereas Rialto sludge is quite low in these metals, insofar as sewage sludges from California are concerned (12).
Samples of lyophilized FA weighing 1.000 g each were dissolved in 10 mL of 5 N NaOH under N2 to provide solutions for analysis by 13C-NMR spectroscopy. The concentration of NaOH employed was found necessary to ensure that the FA solutions contained no colloidal matter. These solutions were maintained carefully under an N2 atmosphere and stored in a refrigerator prior to the NMR analysis. Although the possibility of amide and ester hydrolysis reactions in the FA solutions at 22 "C during the NMR analysis cannot be discounted, these reactions, if they occurred, would not affect the interpretation of the I3C-NMR spectra to be given. A sample of 0.5127 g of Rialto FA for proton NMR analysis was dried overnight at 50 "C, further dried to constant weight over P205 under vacuum, then dissolved in 100 g D2O (Aldrich Chemical Co., 99.7 mol % DzO). The transfer of the dried FA sample from the P205desiccator to the flask containing DzO was carried out in a glove bag under an N2 atmosphere. This FA solution was maintained in the N2 atmosphere at room temperature prior to 'H-NMR analysis. The 13C-NMR spectra were obtained on a Bruker WH 90-D-18 multinuclear NMR spectrometer operated in the Fourier transform mode, with a D20 lock and full proton decoupling. Ten-millimeter NMR sample tubes were employed to contain both the fulvic acid solutions and the solution of dioxane in DzO that served as the external standard. The 'H-NMR spectra were obtained on the Bruker instrument using a 5-mm NMR sample tube, with TMS* (Na salt of 2,2 dimethyl-2-silapentane-5-sulfonicacid) included in the sample as an internal standard. All spectra were obtained at 22 f 3 "C. Chemical shifts are reported in ppm downfield from TMS or TMS*.
Results and Discussion Before embarking on a discussion of the 13C and l H NMR spectra of the sludge-derived fulvic acids, it is useful to have as a point of reference a summary of the known chemical properties of these organic acid mixtures. Table I gives the elemental composition of Ontario and Rialto FA (8) along with that of the FA samples extracted from soil and from seawater by Stuermer and Payne (10). Note that all four FA samples have the characteristic properties of relatively low N content (1-6%), relatively high 0 content, and % C I 50 ( I ) . The H/C ratios given in Table I suggest that sludge-derived FA will have a fewer number of sites of unsaturation and a greater content of polyhydroxy1 compounds than either soil or seawater FA, while the C/N ratios suggest that both sludge and seawater FA will be much richer in protein decomposition products than soil FA. The sludge-derived FA samples have the additional distinguishing property of a large S content. Sulfur was not determined in the soil and seawater FA samples studied by Stuermer and Payne (IO),but its content in them may be expected to range between 0.1 and 2.0% ( I ) . Infrared spectral analysis of the sludge-derived FA samples shows that S03H groups are present (8).These functional groups, whose existence is confirmed in potentiometric titration studies (9), may account for a significant portion of the total S. The ultimate source of these functional groups probably is the organic sulfonate compounds that are found in anionic surfactants. Thus, these groups would not be expected to occur in significant quantities in FA extracted from naturally occurring organic matter. Insofar as other acidic functional groups are 932
Environmental Science & Technology
concerned, however, Ontario and Rialto FA are similar to other fulvic acids in comprising COOH groups, phenolic OH, and N-containing functional groups ( 8 , 9 ) . The 13C-NMR spectra of Ontario and Rialto FA are presented in Figures 1and 2, respectively. These spectra, which are quite similar despite differences in the chemical composition of the two fulvic acids, show the complex absorption patterns and broad resonances that are expected for complicated molecular systems (13,14),but their resolution is better than that in any l3C spectrum of fulvic acid reported previously. The interpretation of the spectra is given in the following paragraphs and is based on standard references (14-16). A lengthy discussion of the problems of interpretation for mixtures of macromolecules has been given by Stuermer and Payne (10). The resonances between 10 and 60 ppm are characteristic of aliphatic carbon atoms. These are fairly prominent in the two sewage sludge FA samples, more so than what is observed
Table 1. Elemental Composition of Fuivic Acids Extracted from Sewage Sludge, Soil Organic Matter, and Seawater (Water and Ash-Free Basis) %
FA sample
c
Ontariosludge(8) 30. Rialto sludge(8) 45. Podzol soil (IO) 47. Sargasso Sea 50.
yo n
nic
yo N
5.2 7.3 4.5 6.8
2.1 1.9 1.1 1.6
1.8 3.1
s
CIN
19.2 17.0
0.5
110.
6.4
9.0
%
12.
o
51. 40. 48. 37.
4.5 ND ND
(70)
I 6D 7I O4XPOINE
3hTARIO FllLVIC ACIC
IJC
hiNR SPECTRUM
h
L L C L I I D I S nwD " i T 0 r i C i
2M
2 3
2m
90
L4ULI,C
:13glr.
l8C
73
I63
I50
160
ma "On Y x c ~ m ~ --I~ ~ w
SllnrmXIL
:
830
IM
,
0
IKC
90
E3
70
M
h . L # w m C:
!€
40
X
rn
IC
00
PPM FROM T U S
Flgure 1. 22.638
MHz I3C-NMR spectrum of Ontario FA Result of 37 900 scans using 90" pulse and total pulse delay and acquisition time of 1.36 s
PPM FROM T M S
Figure 2. 22.638 MHz I3C-NMR spectrum of Rialto FA Result of 45 444 scans using 90' pulse and total pulse delay and acquisition time of 1.36 s
for soil FA (10).This fact may reflect the input of both animal and industrial wastes into sewage sludge; soil organic matter which derives from the decomposition of plants usually has only a small percentage of aliphatic compounds in its FA fraction ( I ) . Resonances in the range 50-110 ppm generally represent carbon atoms in polyhydroxyl compounds as well as those in certain amino acids (16, 1 7 ) . These resonances are the most pronounced in the spectra of the two sludge FA samples. The particularly sharp resonances in the range 60-65 ppm may correspond, e.g., to CHzOH in polysaccharides or, possibly, to amino acids and other protein decomposition products. The broad resonances at 72 and 101 ppm likely are due to polysaccharides in some phase of degradation. Fulvic acid extracted from soil does not exhibit resonances as prominent as those in Figures 1 and 2 in the polyhydroxyl range (10).In fact, the resonances are no more pronounced than those for aliphatic constituents. This difference between the sludge and soil FA spectra probably stems from the anaerobic conditions under which the sludges were produced. Under anaerobic conditions, the decomposition of polysaccharides and proteins should be arrested somewhat. I t is worthwhile to note in passing that the IR spectra of Ontario and Rialto FA show deep absorptions near 1045 cm-l that may be assigned to C-0 in polysaccharides (8). The resonances in the range 110-160 ppm indicate aromatic, heteroaromatic, and olefinic compounds. These resonances show prominently around 125 ppm, which suggests the significant presence of substituted benzene rings. The same conclusion applies to soil FA (10). The resonances between 160 and 185 ppm may be assigned to carbon atoms in carboxyl and in amide groups. These resonances are not particularly strong in the 13C spectra of the two sludge FA samples, in contrast with the spectrum of soil FA, where, for example, the carbonyl C resonance is the most prominent feature (10).This result is in accordance with the IR spectra of sludge and soil FA samples, which show that sludge FA tends to exhibit a relatively weaker absorption than soil FA at 1720 cm-l, indicative of C=O in COOH (8).The difference in the spectra once again may be explained on the basis of the anaerobic conditions that produced the sludge samples and that would inhibit oxidation and COOH production. The same explanation would apply to the weak resonance near 193 ppm, characteristic of C=O in aldehydes and ketones, that appears in the I3C-NMR spectrum for Ontario FA. The ‘H-NMR spectrum of Rialto FA is presented in Figure 3. The broad resonances centered at 0.90 and 1.30 ppm indicate protons on aliphatic carbon atoms (CH3-R and R’CH2-R, respectively), while the sharp resonances near 2.0 ppm indicate protons on carbons adjacent to carbonyl groups, to aromatic molecules, and to alkenes (18).The broad, pronounced resonance near 3.8 ppm may be assigned primarily to protons in polysaccharide decomposition products (13,191. This resonance, which is the most prominent feature in the ‘H-NMR spectrum of Rialto FA (excepting the HzO-HDO resonance), corroborates the assignment of the strong resonances at 72 and 101 ppm in the l3C-NMR spectrum. The complicated series of resonances in the range 7.3-8.2 ppm in Figure 3 indicates that a complex variety of aromatic protons exists in sludge-derived FA. Conclusions
The l3C-NMR spectrum of sludge-derived FA shows the presence of significant numbers of carbon atoms in both aliphatic and aromatic constituents. The spectrum gives a characteristic picture of sludge FA, in that resonances due to polysaccharide and protein decomposition products figure prominently while those due to carbonyl groups are weak,
Figure 3. 90.020 MHz ’H-NMR spectrum of Rialto FA dissolved in D20 Result of 6 022 scans using 90” pulse. Resonance marked “Acetone” due to solvent impurity in sample tube. Features marked with asterisk are spinning sidebands due to intense H2O-HDO and TMS’ resonances
relative to FA extracted from more oxidized material such as soil organic matter. This picture of sludge FA is quite consistent with expectations based on the data in Table I. The H/C and C/N ratios, for example, both indicated that polysaccharide and proteinaceous materials would be significant in sludge FA. On the other hand, the presence of S in sludge FA is not uniquely discernible in the I3C-NMR spectra, possibly because a major part of the S simply may be found in S 0 3 H groups substituted onto aromatic rings. This substitution would contribute resonances in the region around 144 PPm. Carbon-13 NMR spectroscopy appears to be an effective means for investigating the organic structures to be found in complex, heterogeneous mixtures such as FA. It seems quite possible that detailed structural information about sludgederived FA could be obtained by separating this material into fractions using column chromatography and then studying the fractions with 13C- and ‘H-NMR spectroscopy. This possibility currently is under investigation. It is worthwhile to point out that the differences between sludge and soil fulvic acids shown in their 13C-NMR spectra imply that the water soluble organic compounds in sewage sludges would be microbiologically unstable in the soil environment. Thus, it would be expected that the initial speciation of organic molecules and of trace metals in the soil solution after the addition of sludge may change significantly with time as the compounds more susceptible to microbial attack (e.g., surfactants and polysaccharides) are degraded and the functional group character of the material changes.
Literature Cited (1) Schnitzer, M., Khan, S.U., “Humic Substances in the Environ-
ment”, Marcel Dekker, New York, N.Y., 1972. (2) Reuter, J. H., Perdue, E. M., Geochim. Cosmochim. Acta, 41,325 (1977). ( 3 ) Morel, F.M.M., Westall, J. C., O’Melia, C. R., Morgan, J. J., Enuiron. Sci. Technol., 9,756 (1975). (4) Page, A. L., “Fate and Effects of Trace Elements in Sewage Sludge When Applied to Agricultural Lands”, USEPA, Rep. No. EPA670/2-74-005, Cincinnati, Ohio, 1974. ( 5 ) Manka, J., Rebhun, M., Mandelbaum, A., Bortinger, A,, Enuiron. Sci. Technol.. 8. 1017 (1974). (6) Chian, E.S.K.,‘DeWalle, F;.B., ibid., 11,158 (1977). (7) Holtzclaw, K. M., Sposito, G., Bradford, G. R., Soil Sci. SOC. Am. J.. 40. 254 (1976). ( 8 ) Sposito, G., Holtzclaw, K. M., Baham, J., ibid., p 691. (9) Sposito, G., Holtzclaw, K. M., ibid., 41,330 (1977). (10) Stuermer, D. H., Payne, J . R., Geochim. Cosmochcm. Acta, 40, 1- 1 - na - - (1976) (11) Holtzclaw, K. M., Keech, D. K., Page, A. L., Sposito, G., Ganje, T. J., Ball, N. B., J. Enuiron. Qual., 7, 124 (1978). \ - - . - I .
(12) Lund, L. J., University of California, Riverside, Calif., private communication. 1976. ~.. (13) James, T. L., “Nuclear Magnetic Resonance in Biochemistry”, Academic Press. New York. N.Y.. 1975. . (14) Stothers, J. B., “Carbon-13 NMR Spectroscopy”, Academic Press, New York, N.Y., 1972. ~
I
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(15) Levy, G. C., Nelson, G. L., "Carbon-13 Nuclear Magnetic Resonance for Organic Chemists", Wiley-Interscience,New York, N.Y., 1972.
(16) Johnson, L. F., Jankowski, W. C., "Carbon-13 NMR Spectra", Wiley, New York, N.Y., 1972. (17) Horsley, W., Sternlicht,H., Cohen,J. S.,J . A m , Chem. SOC.,92, 680 (1970). (18) Kemp, W., "Organic Spectroscopy", Wiley, New Yo&, N.Y., 1975. (19) Sadtler Res. Labs., Sadtler Stand. Spectrum No. 6245 (D-glucose), 1969.
Received for reuieu; August 29, 1977. Accepted February 21, 1978. f k ~ e a r c hsupported in part by the Kearney Foundation of Soil Science and in part with federal funds from the Environmental Protection Agency under grant number R804516010. The contents of this paper do not necessarily reflect the views and policies of EPA, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. T h e Bruker W H 90-D-18 NMR spectrometer was supported by Bio-medical Sciences Grant No. 5 SO5 RR07010-09from the National Institute of Health and by NSF @ant " 0 . MPS75-06138 t o the Department of Chemistry, Uniuersity of California, Riuerside.
Comparison of Associations of Different Hydrocarbons with Clay Particles in Simulated Seawater Philip A. Meyers" and Terrence G. Oas' Department of Atmospheric and Oceanic Science, The University of Michigan, Ann Arbor, Mich. 48109
Some aspects of the association of hydrocarbons and smectite clay in simulated seawater were investigated using NaCl solutions in laboratory experiments. Both n-eicosane and n-eicosene displayed identical association behaviors with this clay. Association increased linearly with increasing hydrocarbon concentration in water. The amount of n-alkane associated with smectite increased with carbon chain length from C17 to C28, reaching a maximum of 70% removal from water. This may be due to decreased accommodation in water as hydrocarbon chain length becomes larger. The level of association of aromatic hydrocarbons was generally low, and isoalkanes were more effectively removed from water than n-alkanes of the same number of carbons. A process contributing to the removal of petroleum hydrocarbons from natural waters is the association of these compounds with settling particles and eventual burial in sediments. Because crude petroleum and refined petroleum fractions consist of mixtures of hydrocarbons, selective interactions during sorption can lead to different amounts of removal of various components. The result of such selective association would be that certain petroleum fractions would preferentially become incorporated into sediments while others would remain in the overlying water. Furthermore, the reverse of this process, selective desorption, would be likely to enhance any fractionation that occurs during sorption because the more readily desorbed components will also be the more soluble and therefore less easily sorbed ones. This has been indicated both by laboratory experiments ( I ) and by field data ( 2 ) . In view of the probable importance of hydrocarbon-settling particle interactions in transferring petroleum components from water to sediments, we initiated a number of laboratory experiments designed to compare relative amounts of association of different hydrocarbons by a common clay mineral. The results of these studies are reported here and are relevant to predicting fractionation of petroleum components in marine waters. Experimental The laboratory experiments in this study were conducted using a procedure adapted from one developed by Meyers and Present address, Department of Oceanography, Florida State University, Tallahassee, Fla. 32306. 934
Environmental Science & Technology
Quinn ( 3 , 4 )for fatty acid-mineral association experiments. Seawater was simulated with a distilled water solution containing 34 g NaCl per kg of solution. The pH of this solution was not adjusted. The resulting solution pH of 6-7 may have enhanced association by about 10%relative to typical seawater of pH 8, assuming hydrocarbon association decreases with increasing pH in a way like that of fatty acids ( 4 ) .Hydrocarbons dissolved in 50 pL of benzene or ethyl acetate were introduced into 950-mL volumes of NaCl solution in separatory funnels and dispersed by vigorous shaking for at least 30 s. The amount of solvent used in each experiment was kept constant to minimize any effect upon hydrocarbon adsorption. Fifty mg of sodium smectite from the same lot utilized by Meyers and Quinn ( I , 3 , 4 ) for previous experiments were dispersed in 10 mL distilled water to achieve maximum swelling and then added to the hydrocarbon-NaC1 solution mixtures. After making the volume up to 1 L, the mixtures were again vigorously shaken for at least 30 s. A minimum settling period of 2 days at ambient temperatures (around 20 "C) was used in all experiments and was sufficient for nearly all the clay to flocculate and settle. After this period the settled clay was drawn out the bottom of the separatory funnel and dried. Associated hydrocarbons were removed from the dried smectite by covering with 10 mL benzene/methanol, 1/1,held a t 45 "C 18 h followed by two 10-mL petroleum ether extractions. This extraction scheme gave quantitative recovery of n-eicosane. An internal standard of 100 pg n-tetracosane was added to the extraction mixture, and the solvent evaporated to dryness at 25 "C. The residue was immediately redissolved in petroleum ether in preparation for analysis by gas-liquid chromatography. The extracted clay was collected on a preweighed Millipore HA filter, dried at 60 "C for one week, and weighed. Analysis of the extracted hydrocarbons was done on a Hewlett-Packard 5711A flame ionization gas chromatograph equipped with 4 m X 2.1 mm i.d. columns packed with 3% SP-2100 on 80-100 Supelcoport (Supelco, Bellfonte, Pa.) operated isothermally in the range 150-300 "C depending upon the hydrocarbon being investigated. Quantitation was achieved using a Hewlett-Packard 3380 A electronic integrator. Controls were run on all procedures, and the reported results are corrected values. Eight replicate experiments using n-eicosane at a concentration of 126 pg/L yielded a coefficient of variation of f8.0%. This value is assumed to be valid for all other hydrocarbons used in this study.
0013-936X/78/0912-0934$0 t.OO/O @ 1978 American Chemical
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