10

Aquatic Humic Substances

Prof. Dr. Fritz H. FrimmelEngler-Bunte-Institut, Universität Karlsruhe, Engler-Bunte-Ring 1, 76131 Karlsruhe,Germany, E-mail: Fritz.Frimmel@ciw.uni-karlsruhe.de

1 Introduction and Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . 302

2 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

3 Hydrophobic and Hydrophilic Fractions . . . . . . . . . . . . . . . . . . . . . . 304

4 Chemical Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3064.1 Elemental Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3074.2 Molecular Size Distribution and Charge . . . . . . . . . . . . . . . . . . . . . . 3094.3 Spectral Absorbance and Fluorescence . . . . . . . . . . . . . . . . . . . . . . . 3114.4 Infrared Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3124.5 Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3154.6 NMR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3164.7 Soft Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

5 Interaction of Humic Matter with Other Water Constituents . . . . . . . . . . . 317

6 Fate of Humics in Technical Application of Water . . . . . . . . . . . . . . . . . 319

7 Viewing the Function in Aquatic Ecosystems . . . . . . . . . . . . . . . . . . . . 319

8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

AOX adsorbable organically bound halogensAPC anion particle chargeAPCI atmospheric pressure chemical ionizationCP cross-polarizationCPMAS cross-polarization magic angle spinningDBP disinfection by-productDOC dissolved organic carbonDOM dissolved organic matter

301

ESI electrospray ionizationFA fulvic acidsFAB fast atom bombardmentFD field desorptionFFF field flow fractionationsFI field ionizationFT Fourier transformHA humic acidshAA hydrolyzable amino acidhCH hydrolyzable carbohydrateHS humic substancesIR infraredLC/DOC liquid chromatography with detection of organic carbonMALDI matrix-assisted laser desorption/ionizationMAS magic angle spinningMS mass spectroscopyMX Ames mutagenic compoundsNHS nonhumic substancesNMR nuclear magnetic resonanceNOM natural organic matterPAH polycyclic aromatic hydrocarbonPY-GC/MS pyrolysis with gas chromatography/mass spectroscopyROS refractory organic substancesSOS synthetic organic substancesTOC total organic carbon

1

Introduction and Historical Outline

Traditionally, the brown organic matter ofsoils has been connected with the expressionof humic substances. Reflecting the refrac-tory yellow-brown organic substances of bio-logical origin, systematic studies on theisolation of humic substances from soil dateback to the end of the 18th century, whenAchard (1786) first extracted peat with alkali.The name `humus' for the brownish-coloredorganic matter in soil was most likelyintroduced by Saussure (1804), while theacidic character of the main components wasrecognized by Döbereiner (1822) and as-signed as `Humussäuren' (humus acids).Berzelius (1839) and others finally used

the term `humic acid' for the alkali-soluble,acid-insoluble fraction of soil organic matter.

It is clear that understanding the functionofhumicmatter insoils is stronglydependenton an understanding of the role of moistureand water. However, this aspect has beenneglected in many contributions. In particu-lar,humicsubstances inaquaticsystemswerenot recognized for a long time, mainly due totheir low concentrations, the lack of suitableanalytical tools, and (presumably) the miss-ing relevance of the problem in daily life. In1938, the `Gelbstoff' was recognized as color-giving matter in sea water (Kalle, 1938) andthe first differentiations were made betweencoastal run-off humics and open-ocean hu-micsubstances (Stuermerand Harvey,1974).The fluorescence of the humic compounds

10 Aquatic Humic Substances302

was measured as one of their specific proper-ties inaquatic systems(GhassemiandChrist-man,1968).The discovery ofRook(1974) thathumic matter is a precursor of chloroformformed by chlorination of natural waters ledto an avalanche of investigations on theoxidative reactivity of this material. Severalof the topics concerning aquatic humicsubstances and their behavior in coagulation,sorption on activated carbon, ion-exchangeand membrane processes, and reactions withozone and chlorine have been detailed in bothbooks and journals (Suffet and MacCarthy,1989; Allard et al. , 1991; Odegaard, 1999).

According to the different approaches forobtaining detailed information on substan-ces of a humic nature, a number of specificexpressions have been used to address theirexistence, from different viewpoints (Table1). Many of these are not well defined, andreflect only one of the main aspects of theauthor's work in which they are introduced.Others, such as humic acid (HA) and fulvicacid (FA) are defined operationally followinga specific protocol for their isolation.

Specialattentionhasbeenpaid to theroleofhumic substances (HS) in natural aquaticsystems. The influence of natural organicacids on pH and buffering of Swedish surfacewater (Köhler, 1999), limnology of humicwaters (Keskitalo and Eloranta, 1999), for-

mation of aggregates of dissolved organicmatter (DOM) and xenobiotics and theirdegradation induced by light (Kulovaara,1995) and a special issue on natural organicmatter in water (Egeberg et al. , 1999), havebeen published.

2

Occurrence

HS are ubiquitous, and as old as life on ourplanet. The matter must be seen as left-oversof biological material that have lost theirreadily bioconsumable components, and bythis have formed a polydisperse and hetero-geneous mixture of relatively bio-unreactivematter. The interaction of HS with biota leadsto a poorly defined state in between degrada-tion and biosynthesis (Meybeck, 1982).

In aqueous samples, the concentrations oforganic compounds are mostly expressed astotal organic carbon (TOC) or dissolvedorganic carbon (DOC). Differentiation be-tween both parameters is normally carriedout by 0.45 mm membrane filtration. DOCconcentrationsofaquatic systemsrange from<1 mg Lÿ1 in most groundwaters to severaltens of mg Lÿ1 in the brown water of swamps.Rivers typically show DOC concentrations ofa few mg Lÿ1. The contribution of HS to theDOC as defined by specific isolation proce-dures can be found in the range between 40%to 70%. Some typical values for most abun-dant aquatic systems are given in Table 2.

Humic matter in aquatic systems can beattributed to two main sources:

1) Terrestrial origin from plants and soil(allochtonous substances).

2) Material resulting from biological activ-ities within the water body itself (au-thochtonous substances).

In addition, water from waste water treat-ment plants can contribute significantly to

2 Occurrence 303

Tab. 1 Common expressions for materials of thehumic type

BOM Biogenic organic matter

DOC Dissolved organic carbonFA Fulvic acidsHA Humic acidsHS Humic substancesNOM Natural organic matterPOC Particulate organic carbonROS Refractory organic substancesSOC Soil organic compounds (carbon)TOC Total organic carbon

the load of refractory organic substances(ROS) in aquatic systems. The structure andcharacter of the materials typical of thedifferent sources of HS are partly conservedin the ROS. Breakdown products from ligninand polysaccharides, for example, are geo-genic. Microbial activity can be traced bypeptide-derived structures, and civilizationleaves its foot prints in form of anthropogenicproducts and traces from technical activities.The different resulting probes and theirrelative amounts are also strongly influencedby their biochemical reactivity. It is interest-ing to note that despite the wide variety ofsources and reaction pathways, there is quitea uniformity in the gross properties of theresulting humic matter. However, identifica-tion of the specific matrix-dependent influ-ences needs powerful methods for the deter-mination of sensitive parameters.

3

Hydrophobic and Hydrophilic Fractions

Humic substances are ambiguous in theircharacter, and structural regions dominatedby hydrocarbon bonds lead to lipophilicinteractions. On the other hand, polar groupswith hetero elements such as oxygen, nitro-

gen, and sulfur contribute to the hydrophiliccharacter. Many of these functional groupsare subject to acid±base equilibria. Therefore,their contribution to the hydrophilic charac-ter of HS is strongly pH-dependent (Perdueand Parrish, 1987; De Wit et al. , 1993a).

A fine example for studying hydrophobic/hydrophilic interactions of aquatic HS is theisolation procedure for different fractionsusing nonionic macroporous sorbents com-posed of styrene-divinylbenzene (e.g., XAD-2, XAD-4) or acrylic esters (e.g., XAD-7, XAD-8). Figure 1 shows a commonly used protocolfor obtaining standardized material.

It must be borne in mind that the productsobtained are operationally defined. As aconsequence, the yield of the different frac-tions will vary according to the reactionconditions applied, and the adsorbents used.This pitfall can be partly compensated by theadvantage of producing huge amounts ofstandard materials which can be used for awhole suite of characterization and structuralidentification procedures. In any case, it ismost important to provide a clear and detaileddescription of the isolation procedure ap-plied. Freeze-drying of the isolated fractionsis often used to stabilize the final samples andprevent significant chemical and biologicalreactions during storage.

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Tab. 2 Typical ranges for dissolved organic carbon (DOC) concentrations and humic contribution in selectedaquatic systems

Source DOC (mg L±1) HS (mg L±1) Reference

Sea water 0.2 ± 2.0 0.06 ± 0.6 Thurman, 1985Groundwater 0.1 ± 2.0 0.03 ± 0.6 Thurman, 1985River 1 ± 10 0.5 ± 4.0 Thurman, 1985Donau 1.7 1.0 Frimmel and Geywitz, 1983Rhein 2.2 1.3 Abbt-Braun et al., 1991Lake 1 ± 50 0.5 ± 40 Thurman, 1985Bodensee 1.2 0.6 Abbt-Braun et al., 1991Starnberger See 3.2 1.5 Frimmel and Geywitz, 1983Waste watereffluent (Neureut) 8.6 1.7 Frimmel and Abbt-Braun, 1999

The operational definition of the fractionsobtained is further reflected in their hydro-phobic and hydrophilic character. It is widelyaccepted to attribute the low pH-values of

nonadsorbable organic material (NHS�nonhumic substances) to `neutral hydrophil-ic substances'. The fulvic acid fraction iscalled the `ionic hydrophilic' matter, and the

3 Hydrophobic and Hydrophilic Fractions 305

Fig. 1 Isolation scheme for solid (s) anddissolved (l) fractions of aquatic humicsubstances.

humic acids have `ionic hydrophobic' char-acter. The fraction of `neutral hydrophobic'material is retained on the adsorbent (resin),and can be isolated by extraction with organicsolvents. A total material balance based onorganic carbon is suited to quantify therelative yields of the fractions, and by thisthe efficiency of the isolation procedureapplied.

In addition to the adsorption/desorptionon resins, several other methods have beenapplied for the isolation and fractionation ofaquatic organic matter, leading to differentoperationally defined cuts. Some of themethods and their specific advantages anddisadvantages are shown in Table 3 (Leenh-eer, 1985).

4

Chemical Structure

Characterization of the composition of aquat-ic humic material ideally should be per-formed in the matrix of the aquatic sampleconcerned. However, the analytical methods

appliedmayaskforaspecificpre-treatmentofthe sample. Often, higher concentrations oreven solid samples are required. This leads tothe fundamental question of whether theresults obtained can be transferred directly tothe original sample and the structure of itsdissolved or dispersed matter. Artifact for-mation and steric changes must be consid-ered and quantified wherever possible. Todeal with this complex situation it is advisableto apply as many independent methods aspossible to gather structural information onHS from different points of view. Figure 2provides an overview of the most commonmethods for characterization of HS (Frim-mel, 1990).

Several attempts have been made to com-pose defined chemical structures, but veryoften these activities have led to misinter-pretation. It should be borne in mind thatmost of the models of chemical structure areseen from a specific method-defined view. Agood structural model should well agree withthe state of knowledge and the specificaspects investigated. A basic suggestion fora chemical structure, including the relativeamounts of aromatic and saturated moieties,

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Tab. 3 Isolation methods for aquatic organic matter

Method Advantages Disadvantages

Membrane filtration Defined pore sizeHigh yields

Inorganic impurities (ash content);Membrane fouling

Vacuum distillation Low decomposition at low temperature Coprecipitation of organic matter andinorganic salts

Freeze drying Structure protective SlowNeeds pretreatment for good results

Sorbent extraction Exclusion of salts Limited solubility of humic matterPartly irreversible sorption

Alumina High sorption capacityNo organic bleed

Irreversible sorption and chemical changeof sorptive possible

Activated carbon High sorption capacityChemical impurities

Strong irreversible sorptionBiofouling

Ion exchange resin Specific ionic interaction Resin bleedLimited regeneration

Non ionic resins Broad pH-rangeSimple fractionation

Irreversible sorption possibleResin bleed

was provided by Christman et al. (1989)(Figure 3). In the meantime, more sophisti-cated structures based on force field calcu-lation of lowest energy have been proposed(Sein et al., 1999).

4.1

Elemental Composition

In general, freeze-dried samples are used toanalyze for the elemental composition of

fulvic acids (FA) and humic acids (HA). Thecontent of carbon, hydrogen, oxygen, nitro-gen, and sulfur provides the initial informa-tion on the origin of the sample. The ashcontent reflects the type of isolation proce-dure, and the amount of trace elements (e.g.,phosphorus), metals and chlorine can betypical for the genuine matrixof the system. Itwas shown that the water content of theanalyzedsamplessignificantly influences theresults, and therefore should be determined

4 Chemical Structure 307

Fig. 2 Analytical multiple method approach for characterization of aquatic organic substances (HS).

Fig. 3 Basic suggestion for a chemical structure of humic substances.

carefully, e.g. by the Karl-Fischer method(Huffman and Stuber, 1985).

Despite the different aquatic sources, thereis a somewhat uniform composition of theisolated samples. Basic data for typical aquat-ic systems where samples were taken for theisolation of humic substances are shown inTable 4.

The elemental composition of the isolatedhumic (HA) and fulvic (FA) acid fractions isgiven in Table 5.

Methods have become available for thedirect determination of carbon, nitrogen, andhalogens in the aqueous samples, the lowerlimits of detection being ~10 mg Lÿ1. Based onthe findings that most freeze-dried humicsamples contain ~50% carbon (w/w) (Ta-ble 5), it is easy to estimate the total mass ofHS in the aquatic samples. All other proper-ties of aquatic HS can be compared on thebasis of this carbon mass base, and carbonmass-specific data of different HS have

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Tab. 4 Origin, symbols and basic specification of aquatic samples

Sample Sampling date DOC(mg L±1)

pH(22 8C)

A (254 nm)(m±1)

A (436 nm)(m±1)

Electrical conductivity(mS cm±1; 22 8C)

Brown waterHO10 10.94 29.4 4.1 120.5 9.1 30HO12 07.96 22.9 4.2 98.2 10.0 40HO13 08.96 26.6 3.5 90.5 7.5 ±HO14 07.97 25.7 3.5 117.5 9.6 46HO15 11.97 24.9 3.7 110.9 7.0 48

Soil seepageBS1 11.95 63.2 4.2 197.8 11.6 63

GroundwaterFG1 01.96 8.1 7.5 23.7 1.2 550

Secondary effluentABV2 03.95 8.6 7.9 12.4 0.9 800

Tab. 5 Elemental composition of FA and HA isolated according to the XAD procedure (Abbt-Braun et al. ,1990, 1991)

Sample Mass %C H N O S

Ash %100% - S (C, H, N, O, S)

Brown waterHO10 FA 54.13 3.75 0.85 41.20 0.69 ±0.62HO10 HA 52.45 3.49 1.17 36.40 0.77 5.72

Soil seepageBS1 FA 53.30 3.62 0.70 41.80 0.26 0.32BS1 HA 53.35 3.66 1.06 36.50 0.35 5.08

GroundwaterFG1 FA 55.62 4.84 1.44 36.50 1.62 ±0.02FG1 HA 51.70 3.95 2.87 28.35 ND 13.13

Secondary EffluentABV2 FA 50.58 5.03 2.92 33.20 2.81 5.46ABV2 HA 43.27 5.42 6.86 31.00 1.60 11.85

ND�not determined.

turned out to be a very useful way ofcharacterizing the samples. Some typicaldata for the elemental composition of aque-ous HS are given in Table 5.

4.2

Molecular Size Distribution and Charge

Aquatic natural organic matter (NOM) is thedomain of higher molecular weights andcolloidal dimension. To obtain quantitativeinformation on the charge and size distribu-tion, the systems must be determined innatural concentrations. However, due to thehydrophilic character of the humic matterand the polarity of water as solvent, theexperimental methods are limited. Gel chro-matography with different detection systemshas been used successfully for the molecularsize-based characterization of HS (De Nobiliet al. , 1989; Huber and Frimmel, 1991). Asevere pitfall of the method is the lack ofauthentic material for calibration purposes.In case the retention time is combined withother specific structural data, the molecularsize and mass values become more reliable(Perminova et al. , 1998). Figure 4 shows thegel chromatograms with DOC detection ofthe aqueous samples. The experimental set-up for the procedure is given in Table 6. Thefraction eluting at short retention time (tR �20 min) is due to high-molecular weightrefractory material; this is dominant in thebrown water and soil water samples. Thefractions with a retention time which ismaximum at ~23 min are assigned to some-what smaller molecules with higher hydro-philicity. At tR >25 min, eluting material (andespecially the fraction at tR�31 min) is due to

4 Chemical Structure 309

Fig. 4 Gel chromatograms with DOC- and UV-detection of aqueous FA samples (brown waterHO10; soil seepage water BS1; groundwater FG1;waste water ABV2; brown coal waste water SV1).

organic acids which are dominant in thewaste water FAs. This seems to be typical formatter from aerobic digestion processes, aswas shown recently in an investigation of asmall riverine system (Hesse et al. , 1997).Comparison with the chromatograms ob-tained with A(254 nm) detection showsclearly that the refractory high-molecularweight substances show a relatively highUVabsorbance and the low-molecular weightsubstances (especially the acids in the secon-dary effluent FAs) show a decreased specificUV absorbance. The chromatographic frac-tionation may appear useful to identify andassign well-defined structures.

The power of the gel chromatographicapproach lies in the direct applicability ofnatural aquatic samples, the excellent repro-ducibility, and the structural information onthe fractions which can be obtained frommultidimensional detection such as DOCand specific elements, in addition to spectralabsorbance in the UV and visible range, andfluorescence.

Interpretation of the fractions obtained canbedoneaccordingto theunderstandingof theretention times (tR ) for the different types ofcompounds (Huber and Frimmel, 1996). At

short tR appear the poorly UV absorbingpolysugars which are obvious in samplesfrom secondary effluents, and those fromrelatively young and biologically influencedsystems. The following main fraction is dueto refractory, humic-type compounds. Thenarrow fraction is characterized as the `salt'peak, and is caused by mono- and poly-functional fatty acids. Fractions at higherretention times are mostly due to sorptioneffects, and should not be interpreted on thebasis of molecular size.

Agglomeration of HS is strongly influ-enced by the charge density of the macro-molecules. An experimental approach todetermine the anionic particle charge(APC) can be carried out in an electrochem-ical deviceby titrationwithpolydiallyldimeth-yl-ammonium chloride. Typical values foraquatic HS are 1 ± 3 mmol anionic particlecharge per mg DOC (Weis and Frimmel,1989). The anionic functional groups can beoccupied by metal cations, leading to a partialor total neutralization. The different metalcomplexes canbe compared with one anotheronthebasisof this thestability.Asequencefordecreasingstability is shownbelow(WeisandFrimmel, 1990).

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Tab. 6 Experimental set up for LC/DOC measurements