Marine Chemistry, 12 (1983) 119--132 119 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

T H E S T R U C T U R E O F M A R I N E F U L V I C A N D HUMIC A C I D S

GEORGE R. HARVEY, DEBORAH A. BORAN, LARRY A. CHESAL and JOHN M. TOKAR

National Oceanic and Atmospheric Administration, Atlantic Oceanographic and Meteoro- logical Laboratories, Ocean Chemistry and Biology Laboratory, 4301 Rickenbacker Causeway, Miami, FL 33149 (U.S.A.)

ABSTRACT

Harvey, G.R., Boran, D.A., Chesal, L.A. and Tokar, J.M., 1983. The structure of marine fulvic and humic acids. Mar. Chem., 12: 119--132.

Fulvic and humic acids were isolated from near surface seawater samples obtained at five biologically diverse sites in the Gulf of Mexico in quantities sufficient for detailed NMR and chemical studies. A gas lift pump and a 1500-I extraction facility provided half- gram quantities of material. The proton NMR spectra of all fulvic and humic acids studied were remarkably similar and differed mainly in the absence of aromaticity in the fulvics. These observations, coupled with published spectra and chemical data, suggest a class structure and general mechanism of formation for marine humic substances. The pro- posed structures are crosslinked, autoxidized, polyunsaturated fatty acids.

INTRODUCTION

Research into the origin, structure and function of seawater humus (fulvic and humic acids) has been hindered by methodological as well as philo- sophical difficulties. It has been difficult to obtain a quantity of material, from different marine environments, sufficient for detailed analysis because concentrations in seawater are usually only a few hundred micrograms per liter. More fundamentally, concepts and assumptions uncritically carried over from soil science have resulted in many research papers which are irrelevent to seawater humus. This latter problem has been discussed in detail by Pocklington (1977).

We have scaled up the procedure (Mantoura and Riley, 1975; Stuermer and Harvey, 1977a) for isolating seawater fulvic and humic acids and semi- automated both the water collection and extraction. We extracted humus from thousands of liters of seawater collected seasonally at five chemically and biologically diverse sites in the Gulf of Mexico (Fig. 1). We thus obtained material sufficient for both chemical and proton NMR studies. The proton NMR spectra of the marine fulvic and humic acids (MFA and MHA) obtained, permitted a more refined interpretation of proton types than had been possible from the single seawater MFA spectrum published previously (Stuermer and Payne, 1976). While this work was in progress, several proton and carbon NMR spectra of marine sedimentary fulvics and

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humics were published (Hatcher et al., 1980a, b). The sedimentary MFA and MHA, isolated by an entirely different procedure, had proton NMR spectra almost identical with our spectra except for the greater prominence of methyl groups. It is clear that marine humus both in the water column and the sediments bear little relationship to terrestrial counterparts.

Based upon our new spectra and the known chemical and physical properties, we propose that MFA and MHA are derived from either light- catalyzed oxidative cross-linking of two or more polyunsaturated fat ty acids or from polyunsaturated glycerides. The proposed structures for which we have new chemical support account for the chemical, physical and spectral properties of MFA and MHA, explains the acid solubility differences, and accounts for the relationship between dissolved and sedimentary marine humus.

MATERIALS AND METHODS

Collection and extraction o f seawater

Seawater was collected from approximately the depth of maximum pro- ductivity as determined by vertical profiles of 14C-uptake and light trans- mission. Collections were made at depths from 4--25 m with a gas lift pump- ing system. Sections of stainless steel tubing were lowered to the sampling depth and compressed nitrogen introduced into the tube about 10 cm above

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its bot tom. Seawater and gas rose together and filled six, vented, stainless steel drums secured in a portable van. When each drum was half full, 250 ml of concentrated HC1 were added from an in-line reservoir. The drums were then filled. We collected thereby ca. 14401 in 2 h. The system is described in detail by Tokar et al. (1981).

Full drums were immediately pressurized with zero-grade nitrogen and the acidified seawater (pH 2) discharged through two 5 × 50-cm glass columns containing 800 cm 3 of Amberlite XAD-2 resin at a controlled flow rate of 500 ml min -1 per column {Fig. 2). Each column extracted 7201 of seawater in ca. 24 h. Prior to use, the resin was cleaned as described by Stuermer and Harvey (1977a).

Isolation of the extracted MFA and MHA

Columns were rinsed with 21 of 0.01 N HC1 to remove salts. The MFA and MHA collected were eluted with 2.41 of 1 N NH4 OH in methanol after an initial 4-h soak in the same solvent. To assure complete recovery we then eluted with 21 of methanol.

Ammonia and methanol eluents were combined and concentrated to dry- ness in a rotary evaporator below 40°C. Crude MFA and MHA were redis- solved in the minimum volume of deionized water containing a few micro- liters of NaOH, acidified to pH 2 with 1.0 N HC1, and refrigerated for 48 h to allow complete precipitation of the MHA, which was separated from the soluble MFA by centrifugation and decantation. Both fractions were washed with CH2CI2, freeze~lried, and weighed. They were stored at --20°C. Exposure to both air and light were avoided throughout the isolation. The MFAs obtained were pale yellow powders while the MHAs were yellow-- brown powders.

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Fig. 2. Abbreviated schematic of the pilot plant used for humus extraction. The standard U.S. 55 gallon drum holds 2401 when full. Six drums were used allowing a capacity of 1440 I. The system is run only with compressed nitrogen.

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~ o ~ n N M R

Approximately 10--20 mg of sample was accurately weighed in a small vial. Unde~ a nitrogen stream, 0.5 ml D2 O (Crescent, 99.9% isotopic purity} was added to the sample followed by 1.0 pl of 40% NaOD in D 2 O (99%) to facilitate dissolution. The solution was transferred, under nitrogen, into a D20-rinsed NMR tube (Wilmad, 17.5 cm, ultra-precision) fitted with a pressure cap. All glassware was stored in a 250°C oven until needed. Each sample was prepared shortly before the NMR analysis.

A Varian EM-360A 60 MHz proton NMR spectrometer, coupled with a Varian V-2048 signal averager, was used for all analyses. Instrumental settings were optimized, e.g., 0.3 milligauss rf power and 0.1 milligauss rf filter, and the same settings used for all samples. Prior to each scan, peak height was maximized on methanol which was present in most samples as an internal standard for chemical shift assignment. When methanol was not used, the chemical shift of HDO was set at 4.6 ppm down-field of tetra- methylsilane.

Before each accumulation run, two single scans at different amplitudes were recorded. The signal averager was set to automatically accumulate 64 5-min scans using any prominent peak in the spectrum as the trigger signal. The accumulated data were summed into a single 5-min sweep.

Esterification

About 20 mg of MFA or MHA was allowed to react in a sealed tube with 4 ml of BC13--methanol commercial reagent for 1 h at 60°C. Only a trace of the MHA did not dissolve in the first 10 min of treatment. Subsequent hydrolysis of the BC13 and extraction with CH2 C12 gave a brown oil readily soluble in CDC13 and other organic solvents.

Oxidation o f MFA

Marine fulvic acid from the fall Mississippi station (100 mg) was dissolved in 200 ml of water containing 500 mg of sodium carbonate in a 500 ml three-neck flask fitted with reflux condenser, thermometer, addition funnel and a magnetic stirbar. A solution of 500 mg of potassium permanganate in 15 ml of water was added dropwise at room temperature until the purple color persisted. The reaction mixture was then heated to boiling and the remaining permanganate solution was added over the course of 1 h. The purple solution was cooled, acidified to pH 2 with concentrated hydro- chloric acid and refluxed for 0.5 h. While the reaction mixture was cooling, excess permanganate was reduced by adding portions of solid oxalic acid until the purple color disappeared.

The aqueous solution was concentrated to ca. 1 ml on a r o t ~ evaporator at 60°C and extracted several times with ethyl ether. The evaporation of the

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mixture to near dryness is essential since dicarboxylic acids, especially odd carbon homologs, are extremely water soluble. The combined ether extract was dried over sodium sulfate, concentrated in a nitrogen stream and esterified with BF a/methanol for 10 min at 100°C. The hydrolyzed reaction mixture was extracted with hexane. The hexane was evaporated to dryness in a nitrogen stream and the esters were redissolved in 100 pl of hexane for analysis.

The solution was analyzed on a Tracor 550 GC equipped with a 10-m glass capillary column coated with SE-52. Six of the most prominent peaks in the chromatogram had the same retention time and co-eluted with succinic (C4), glutaric (Cs), adipic (C6), pimelic (C7), suberic (Ca), and azaleic (C9) acids. These identities were confirmed by GC--MS on a Varian 3700/112S data system equipped with a 60-m SE-30 capillary. All the diacids had major fragments at m/z 55, 59 and M-31. The spectra matched those of authentic standards. Selected ion-monitoring did not reveal the presence of any diacid heavier than the nine-carbon azaleic.

RESULTS AND DISCUSSION

General

The stations sampled in this study are shown in Fig. 1 and their organic parameters given in Table I. The Gulf Loop site is considered to be highly oligotrophic; it is low in both DOC and humus. Satellite imagery and XBT data confirmed that we were sampling Caribbean water that had intruded into the Gulf of Mexico. The Mississippi station, in contrast, is highly pro-

TABLE I

Summary of organic concentrations in the Gulf of Mexico

Station Day/ Season Depth DOC Humic Fulvic Night (Fall/Spring) (m) (rag 1 -I ) (/~g 1-1 ) (pg 1-1 )

Mississippi D F 3 1.32 114 550 Outflow N F 3 1.50 126 1270

N Sp 4 1.10 176 904

Gulf Loop D F 20 -- 26 724 Intrusion N F 20 0.96 3 190

D Sp 20 0.50 14 234 D Sp 55 0.54 9 58

Yucatan N Sp 10 0.63 29 223

Campeche N Sp 5 0.98 105 754

Cape San D Sp 4 3.19 336 577 Bias D Sp 55 2.20 10 139

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ductive; it is high in both DOC and humus. In contrast to the purely oceanic Gulf Loop station, there is considerable input of fresh water. Surface waters at the Mississippi station usually had salinities of 24--260/00 . The stations at Cape San Blas, Bay of Campeche, and the Yucatan shelf are intermediate in properties between the above extremes. MFAs are present at higher concen- trations than MHAs at all sites although their ratio is variable (see Table I). The primary productivity determined at each station by the ~4C-uptake method was positively correlated with both the total DOC and marine humus (P.B. Ortner, private communication).

Proton NMR spectra of isolated MFAs and MHAs are shown in Figs. 3 and 4. A quantitative summary of the relative amounts of proton types is given in Table II. Several structural characteristics are revealed in these data. First, both MFA and MHA are highly aliphatic. Although the relative proportions of methyl protons (shoulder at 0.8 ppm), methylene (1.2--1.8 ppm), and methylene adjacent to functional groups (1.9--2.5 ppm) vary, they compose greater than 72% of the total carbon-bound hydrogen in all cases. The variable-sized peak centered at about 3.5 ppm is due to protons on etherated or hydrolated carbon, i.e., H--~--OH. That peak has been attributed to

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Fig. 3. Proton NMR spectra of MFA and MHA from two contrasting seasons and environ- ments. The Gulf Loop samples were collected in early December of 1979. The Cape San Bias samples were taken in May of 1980. The sharp spike at about 3.4 ppm in three of the spectra is due to methanol.

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Mississippi Outflow YucatanSta (11) 3 Sto ( [ } 2 Fulvic Acid IOrn HDO-

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Fig. 4. Proton NMR spectra of MFA and MHA from the Mississippi Outflow station in December, 1979 and two contrasting samples from spring 1980 at the Yucatan and Bay of Campeche stations.

sugar residue within the humus structure (Stuermer and Payne, 1976; Hatcher et al., 1980b). That is not necessarily the case because it is impossible to distinguish any polyhydroxylated compounds from sugars in this region when both are mixtures and not pure compounds.

The aromatic proton resonance region (ca. 7.0 ppm) is especially revealing about origin and structure. MFAs have few or no aromatic protons. In con- trast, 3--9% of MHA carbon-bound protons are aromatic with a chemical shift centered at 7.0 ppm. Relative to benzene, which absorbs at 7.2 ppm, electron~ionating substituents such as alkyl or hydroxyl groups on aromatic rings shield aromatic protons, i.e., the absorption peak shifts up-field toward 6 ppm, while electron-withdrawing groups such as nitro, carboxyl, quinone, or halogen deshield causing absorption to shift down-field as far as 9.0 ppm (Silverstein et al., 1974). The latter case is seen in the proton NMR spectra of terrestrial humic acids (Ruggiero et al., 1979). In contrast, the spectra shown here (Figs. 3 and 4) show alkyl and/or hydroxyl substitution on the aromatic rings. The recent work of Hatcher et al. (1980a) documents the extent of this substitution. They determined the percent aromaticity of three sedimentary humics isolated from the northwest Atlantic shelf by both

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TABLE II

Relative intensities of proton NMR signals according to station, seasoned and depth (The headings Ali, HCO and Aro list percentages of aliphatic, oxygenated carbon and aromatic protons respectively)

Relative intensities of proton NMR signals

Sampling Sample All HCO Aro data

Gulf Loop Fall 20m Humic 89 7 4 intrusion Fulvic {Day) 93 7 0

(Night) 93 4 < 2

Spring 20m Humic 94 3 3 55m Humic 88 5 7

Fulvic 94 4 < 2

Mississippi Fall 4m Humic 93 3 4 outflow Fulvic 93 7 0

Spring 4m Humic 82 10 8

Cape San Blas Spring 4m Humic 80 11 9 Fulvic 72 24 < 2

Campeche Spring 5m Humic 94 2 4

Yucatan Spring 10m Fulvic 93 7 0

proton and carbon-13 NMR. The carbon-13 NMR spectra revealed 2--4 × more aromatic carbon than the proton spectra revealed aromatic protons, requiring that the aromatic rings in marine sedimentary humics at least are tri-, tetra- and penta-substituted.

We propose that less abundant MHAs, with their associated aromaticity but otherwise identical proton distribution, are derived from MFAs. For example, the MHA/MFA ratio is greater in the deep {old) water of the Atlantic than in surface waters (Stuermer, 1975). The lower abundance of aromatic compounds in plankton than in sedimentary organic matter has led others to propose that the aromaticity must form from biogenic precursors released into seawater by plankton (Vasilevskaya et al., 1977, as discussed by Skopintsev, 1981).

Since our studies began, we have noticed from our own work and from reports in the literature that marine humus (MFA and MHA) behaves and exhibits consistent properties as if it were a single chemical reagent. Although concentrations change with time, acid--base titration (Stuermer, 1975; G.R. Harvey, unpublished results), infrared and ultraviolet spectra (Stuermer, 1975; Kerr and Quinn, 1975; G,R. Harvey, unpublished results) and NMR spectra (Stuermer and Payne, 1976; Hatcher et al., 1980a, b; this

127

work) strongly indicate that the substances defined as MFA and MHA are very similar. These observations suggest a common source material and a common mechanism of formation.

The proposed pathway to MFA and MHA

To reasonably account for the origin and structure of marine humus, one must identify a class of biogenic organics produced in all marine environ- ments which in seawater will form water-soluble, oxygenated, aliphatic acids, which can be transformed into alkyl substituted aromatic rings.

A related problem has been studied in classical organic chemistry, i.e., in the curing of drying oils. Polyunsaturated vegetable oils, e.g., tung and linseed, composed of C16 to C26 polyunsaturated fatty acids (PUFAs), undergo free radical oxidative cross-linking in air, especially in sunlight and in the presence of transition metals (Kirschenbauer, 1960). The oxidative cross-linking includes hydroxyl, carbon--carbon, ether and peroxide bonds with both saturated and aromatic rings forming in the process. Because of the high concentration of oils used in the above~lescribed process (usually for surface finishing of wood), polymers are formed.

In the sea, biota are an abundant source of PUFAs usually as glycerides (Ackman et al., 1968; Williams, 1968). Similar cross-linking should occur in PUFAs released into oxygenated, sunlit seawater, except that polymeriz- ation should not occur due to the very low concentrations present, i.e. about 10 -9 gem -3 vs. the 0.8gem -3 of PUFAs in neat drying oil.

PUFAs could cross-link while adsorbed on particles, but much more likely is that the two- or three-PUFA chains of unsaturated glycerides, which are already in close proximity, oxidatively cross-link. Subsequent hydrolysis of the glycerol ester bonds would produce a di- or tri-carboxylic, cross-linked, hydroxylated acid having the physical, chemical and spectroscopic properties of marine humus. This proposed pathway is illustrated by the three structures shown in Fig. 5.

Discussion of proposed pathway

Because the relationship of MFA to MHA shown in Fig. 5 is by necessity highly simplified, it requires five explanatory comments:

(1) The triglyceride shown is composed of an 18:2, 18:3, and a 16:1 fatty acid array and is not meant to be an exclusive solution or a unique ease. Hundreds of possible polyunsaturated triglycerides probably exist in marine organisms, but their precise structures are not important to the present dis- cussion.

(2) The conversion of the triglyceride through allylic radical intermediates to the cross-linked product is well-documented classical organic chemistry. Six-membered earbocyclic rings are generally thermodynamically favored in such condensations, but five-membered carbocyclic, furan and pyran rings

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:H 3

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Fig. 5. Proposed pathway to marine humic substances by oxidative crosslinking of poly- unsaturated lipids catalyzed by ultraviolet light and transition metals.

(not shown) may also be present. It is not even necessary to invoke Diels- Alder-type reactions, which are difficult on unactivated olefins. Also not shown in the proposed humus structures are the likely hydroperoxide and peracid functionalities which are known products of oxidative cross-linking (Schauenstein, 1967). As a competing reaction, oxidative cleavage at the double bonds must be occurring to produce free, C6--C9 aldehydes (Gsch- wend et al., 1980; Sauer, 1981) and other small hydrophylic compounds. Basically, our proposal is that the primary conversion of the unsaturated triglycerides is to a flexible, oxygen and carbon cross-linked structure, water- soluble but basically hydrophobic.

(3) Those MFA structures which have formed six-membered carbocyclic rings can go on to form aromaticity as shown for the MHA in the figure. The entire process is a dynamic cont inuum which is perturbed or interrupted only by sampling.

The greater degree of cross-linking and the aromaticity in the MHA, i.e., decreased entropy, is probably responsible for its insolubility in acid solution (Fuoss and Strauss, 1948). The MHA certainly continues to be acted upon by the reagents shown until totally insoluble structures are formed or mixing takes it out of the reactive euphotic zone.

(4) Since the proposed pathway is a light-assisted free-radical process,

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incorporation of oxygen- or nitrogen-containing molecules is likely. No nitrogen is shown in the proposed structures, though elemental analysis shows MFA and MHA to contain about 3% N. We believe the incorporated nitrogen functions to be incidental to the basic structures shown. Incorpo- ration of amino acids or sugars is not excluded, but are regarded as appendages to the main structures.

(5) The more extensive oxidative cross-linking that would be expected in the older sedimentary humics would cause the methyl protons in the NMR spectrum to appear more prominent as more methylene protons were shifted downfield. Methyl protons are the least reactive in free radical or carbonium ion reactions. Branching or short methylene chains are thus not necessary to explain the small differences between dissolved and sedimentary humics. Finally, we must emphasize that the structures shown are by no means exact solutions but graphically represent families of logically similar structures.

Chemical evidence from this study

The correlation of the proposed MFA and MHA structures with two chemical reactions was tested. First, the structures predict that the carboxyl groups are quite free and unhindered and should easily esterify. Both MFA and MHA are completely esterified by BCla --methanol in ~ 10 min at 60°C. The esters are soluble in common organic solvents. Proton NMR and thin film infrared spectra confirmed the conversions to methyl esters.

The crosslinking reactions shown in Fig. 5 start at C-9 of the carbon chains, then continue in both directions with further oxidation. Oxidation of an MFA should produce a series of dicarboxylic acids up to the C9 azaleic acid. We oxidized a MFA isolated from the Mississippi station and indeed found only C4--C9 diacids (see Methods section).

Neutron activation analysis of a MHA revealed 7000 ppm of C1, 680 ppm of Br and 530 ppm of I had been incorporated. These halide concentration factors are in the reverse order of their abundance in seawater but in the correct order of the ease of oxidation of the halide anions. We interpret this observation as evidence that the MHA was formed by oxidative processes. The same halide concentration factors are seen in sedimentary organic matter (Harvey, 1980).

Supporting evidence from the literature

A number of published experiments and observations lend support to our hypothesis:

(1) When emulsions of PUFAs are irradiated with UV light in air, hydro- xylation and oxidative cross-linking occur to produce water-soluble products (Baker and Wilson, 1966a). The reported IR- and UV spectra are the same as those of MFA and are consistent with our proposed structures. Air, light and water are all required for the reactions to occur. Interestingly, the water-

130

soluble products of this reaction were investigated because of their potent anti-tumor activity (Baker and Wilson, 1966b).

(2) Wheeler (1972) observed the development of UV absorption in the 270--300 nm range, yellow color, and particle formation when he irradiated linolenic acid (18:3) on sterilized seawater. The UV spectrum of the pro- ducts is the same as that of MFA (Stuermer, 1975).

(3) Boon et al. (1980) found only saturated triglycerides in the sediments off Namibia (Walvis Bay, southwest Africa) despite abundant poly- unsaturated glycerides in the plankton of the region. They cautiously suggested biohydrogenation in the sediments to explain the discrepancy, but it is well known that polyunsaturated lipids are very fragile outside the living cell and quickly undergo the types of reactions shown in Fig. 5 (chemical supply houses recommend that PUFAs be shipped in solid carbon dioxide to deter yellowing).

(4) Stuermer and Harvey (1977b) showed that the saturated protons in Sargasso Sea fulvic acid were due to fat ty acid moieties, not hydrocarbons. High-pressure hydrogenation followed by a reductive cleavage then low- pressure hydrogenation produced a series of unbranched hydrocarbons with chain lengths of 14, 16, 18, and 20. Since marine hydrocarbons have pre- dominately odd-numbered chain lengths, these carbon chains must have been originally fat ty acids before they became part of the MFA. Further, these fat ty acids had to be linked by ether or peroxide bonds because the reductive cleavage conditions used could no t have broken a saturated carbon-- carbon bond and left the chain lengths intact.

A curious series of alkyl aromatic hydrocarbons was also produced by the reductive cleavage reactions described above (Stuermer, 1975; Gagosian and Stuermer, 1977). The mass spectra of these compounds indicated every possible isomeric C9--C~2 alkyl subsitution on benzene. This series of com- pounds had never been reported in the literature. Although the chemistry is complex, one can envisage how benzene rings substi tuted with 9--12 carbon alkyl groups could be cleaved out of the proposed molecular structures shown in Fig. 5, especially by the powerful reagent, tr iphenylphosphine dibromide, which was used between the high- and low-pressure reductions.

(5) Finally, the proposed polyfunctional structures easily account for the metal complexing ability we measured. The MFA is a strong complexer of Zn while the MHA interacts strongly with Cu (Piotrowicz et al., 1981). Further work is in progress.

Final c o m m e n t

The chemical concepts proposed here are certainly vulnerable to criticisms of insufficient or incorrect interpretations of data but they are readily testable. The proposed structures do explain more chemical, physical and spectral data on marine humus than previous attempts, including that of Stuermer and Harvey (1977b).

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ACKNOWLEDGMENTS

We t h a n k Drs. S.R. P io t rowicz , P.M. Williams, P. Pa rke r and R. Zika fo r p ro f i t ab le discussions and suggest ions on this work . R. Zika p rov ided o p e n access to the N M R s p e c t r o m e t e r , R. Duce p rov ided the n e u t r o n ac t iva t ion analysis and P.B. Or t ne r furn ished the p r o d u c t i v i t y da ta . Mr. Russel Lang furn ished the mass spec t ra . Technica l assis tance a t sea and in Miami was pro- v ided b y M. T r u d e a u and J. Acuna . The first d raf t s o f this m a n u s c r i p t were grea t ly i m p r o v e d b y the crit ical reviews o f Drs. P.B. Or tner , S.R. P io t rowicz and D.K. A t w o o d . The off icers and crews o f the R.V. ' E n d e a v o r ' (URI ) and O.S.S. ' R e s e a r c h e r ' (NOAA) suppl ied exce l l en t phys ica l s u p p o r t services. This w o r k was s u p p o r t e d b y the Off ice of Marine Pol lu t ion Assess- men t , U.S. Na t iona l Oceanic and A t m o s p h e r i c Admin i s t r a t i on ( N O A A ) a s p ro j ec t ROME (Role o f Organics in the Marine Env i ronmen t ) .

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Baker, N. and Wilson, L., 1966b. Water soluble inhibitors of tumor respiration formed from ultraviolet-induced oxidation of linoleic and linolenic acids. J. Lipid Res., 7: 349--356.

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