association of linear alkylbenzenesulfonates with dissolved humic substances and its effect on...

Association of LinearAlkylbenzenesulfonates withDissolved Humic Substances andIts Effect on BioavailabilityS A M U E L J . T R A I N A , †

D R E W C . M C A V O Y , * , ‡ A N DD O N A L D J . V E R S T E E G ‡

The Ohio State University, 2021 Coffey Road,Columbus, Ohio 43210, and The Procter & Gamble Company,5299 Spring Grove Avenue, Cincinnati, Ohio 45217

The association of C10-, C12-, and C14-linear alkylben-zenesulfonates (LAS) with natural and spec-imen-grade dissolved humic substances (DHS) wasmeasured with fluorescence quenching and withultracentrifugation techniques. Good agreement wasobtained with both of the analytical methods, sug-gesting that fluorescence quenching could be used tomeasure aqueous-phase partition coefficients. LAS-DHS partition coefficients increased with increasinglength of the alkyl chain. Partition coefficients forthe sorption of LAS to alkylammonium surfactant-coated,phyllosilicate clays also increased with increasinglength of the alkyl chain in the LAS molecules. Takentogether, these data indicate the significance ofnonpolar forces in LAS-organic matter interactions.Toxicity studies examined the effects of DHS on thebioavailability to the fathead minnow, Pimephalespromelas. Changes in the uptake and toxicity ofLAS resulting from the addition of DHS were used tocalculate aqueous-phase LAS-DHS partition coef-ficients. Good agreement was found between thepartition coefficients calculated from the responseof the test organism and those obtained with fluores-cence and ultracentrifugation measurements. Thetoxicity studies suggest that the association of LAS withDHS can play a significant role in reducing thebiologically available fraction of LAS in surface waters.

IntroductionAssociation of anthropogenic organic solutes with naturaldissolved humic substances (DHS) plays a significant rolein the environmental fate and effects of xenobiotic solutesreleased to aqueous environments. The partitioning ofrelatively hydrophobic, nonpolar organic solutes with

natural DHS such as humic and fulvic acids can causeincreases in apparent water solubilities, decreases insorption to solid-phase particulates, and reductions inbiological uptake and toxicities (1-3). These effects areattributed to the partitioning of nonpolar, anthropogenicorganic solutes into “low polarity” regions of humic andfulvic acid polymers (1).

Linear alkylbenzenesulfonates (LAS) are anionic sur-factants that have been used extensively by the detergentindustry. Typically, surfactants contain both polar andnonpolar regions. This dual nature confers their surface-active properties. McAvoy et al. (4) indicate that ap-proximately 317 000 t yr-1 LAS is produced in the UnitedStates, and approximately 16 000 t yr-1 is discharged tosurface waters in municipal sewage effluent. Typical surfacewater concentrations immediately below wastewater treat-ment plant outfalls are less than 50 µg L-1 (4). Despite theextensive use of LAS, there are little systematic data on thesorption of LAS to soils and sediments or its associationwith DHS.

Hand and Williams (5) examined structure-activityrelationships for the sorption of various homologues andmixtures of C10-C14-LAS by riverine sediments. Sorptionincreased with increasing chain length, which led theauthors to conclude that nonpolar sorption mechanismswere responsible for LAS retention (5). Brownawell et al.(6) observed nonlinear sorption of C10-, C12-, and C14-LASto selected soils and sediments. Sorption was found toincrease with increasing chain length, increases in ionicstrength or the valence of the cations in the supportingelectrolyte solution, and decreases in pH. Brownawell et al.(6) concluded that the sorption of LAS was due to nonpolar,electrostatic, and specific chemical interactions. In par-ticular, they hypothesized the existence of specific chemicalreactions between sorption sites, Ca2+, and LAS molecules.

Clearly there is some question as to the relativesignificance of polar versus nonpolar chemical reactionson the sorption of LAS. Additionally, no information isavailable on the association of LAS with DHS. Suchassociations could effect the fate, transport, and toxicity ofLAS in aquatic environments. The purpose of the presentstudy was to determine the association of LAS with DHSand to assess its effect on aquatic organisms. We usedfluorescence quenching to examine the effects of surfactantchain length and solution composition ([Na+], [Ca2+], andsynthetic river water) on the association of LAS with DHS.A batch ultracentrifugation technique was also used as anindependent measure of the association constants for LASand Ca-saturated humic acids. The sorption of LAS ontoclay surfaces modified with cationic monoalkyltrimethyl-ammonium surfactants was determined to assess thesignificance of nonpolar interactions on the sorption ofLAS by organic colloids. Finally, the effects of dissolvedorganic substances on the uptake and acute toxicity of LASto fathead minnows was examined.

Experimental SectionMaterials. Water-soluble organic carbon (Carlisle-WSOC)was obtained from the 0-0.1-m depth of a Carlisle muck(Euic, mesic Typic Medisaprist, northwest Ohio) as de-scribed by Traina et al. (7). Humic acid was extracted with

* Corresponding author telephone: 513-627-5570; Fax: 513-627-8198; e-mail address: [email protected].

† The Ohio State University.‡ The Procter & Gamble Company.

Environ. Sci. Technol. 1996, 30, 1300-1309

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0.1 mol L-1 NaOH under a N2 atmosphere as described bySchnitzer (8). The acid-soluble fraction of this extract wasdiscarded. The acid-insoluble fraction (Carlisle-HA) wassuspended in a solution of 0.5% HCl and 0.26% HF in a250-mL polycarbonate centrifuge bottle, capped, shaken24 h on a mechanical shaker, and centrifuged at 7000g. Thesupernatant was discarded, and the HF-HCl treatment wasrepeated three more times on the residue. The residuewas washed with HPLC-grade H2O, then dissolved with 1mol L-1 NaOH under a N2 atmosphere, and finally neutral-ized to pH 7 with HCl. The dissolved Carlisle-HA was thentransferred into 3500 molecular weight cutoff dialysis tubing(Spectrum Medical Industries, Los Angeles, CA) and dialyzedagainst HPLC-grade water until a negative Ag test for Cl-

was obtained. The salt-free solution was then dialyzed withNa-saturated Chelex 100 cation-exchange resin (Bio-Rad,Richmond, CA) to reduce the polyvalent cation content.

Aldrich humic acid (Aldrich-HA), purchased from AldrichChemical Company (Milwaukee, WI), was solubilized in0.1 mol L-1 NaOH under a N2 atmosphere. The resultingsolution was acidified to pH 1.0 with HCl, and the residuewas purified with HF-HCl, followed by dissolution inNaOH, and dialysis with HPLC-grade H2O and Na-saturatedChelex-100 as described above. Suwanee River humic acid(SRHA), obtained from the International Humic SubstancesSociety, was dissolved in 0.1 mol L-1 NaOH under a N2

atmosphere, and dialyzed with H2O and Na-saturatedChelex-100 as described above. All DHS solutions werestored in an amber glass bottle at 4 °C.

Decylbenzenesulfonate (C10-LAS, 98% purity), dodecyl-benzenesulfonate (C12-LAS, 93% purity), and tetradecyl-benzenesulfonate (C14-LAS, 88% purity) were synthesizedat Procter and Gamble. Uniformly14C-ring-labeled C10-,C12-, and C14-LAS were obtained from New England Nuclearand were 93.8, 96.3, and 92.5% pure, with specific activitiesof 26.6, 68.2, and 34.3 µCi mg-1, respectively.

Specimen Na-montmorillonite (SWy-1) was obtainedfrom the Source Clays Repository of the Clay MineralsSociety. Fifty gram samples of clay were washed with 200mL of 1 mol L-1 NaCl, followed by 200 mL of 0.05 mol L-1

sodium acetate buffer (pH 5) to ensure Na saturation andto remove contaminating carbonates. The clays were thenwashed three times with 0.05 mol L-1 NaCl and thendispersed by washing three times with HPLC-grade H2O.The <1-µm fraction of the dispersed clay was removed bysedimentation and then resuspended in 0.05 mol L-1 NaCl.

Separate aliquots of the Na-saturated clay were reactedwith octyl- (C8-TMAC), decyl- (C10-TMAC), dodecyl- (C12-TMAC), tetradecyl- (C14-TMAC), hexadecyl- (C16-TMAC),or octadecyl- (C18-TMAC) trimethylammonium chloridesolutions (synthesized at Procter and Gamble). Sufficientquantities of the TMAC materials were added at ap-proximately 1.25 times the cation exchange capacity of theclay material. After a reaction time of 16 h, the TMAC-claysuspensions were centrifuged at 10 000g, the supernatantswere discarded, and the TMAC-coated clays were washedtwo times with CH3OH followed by two washes with H2Oto remove surfactant molecules bound to the clay particlesthrough nonpolar forces. The total C content of the TMAC-coated clays was estimated assuming 100% coverage of thecation exchange sites by surfactant molecules. The cationexchange capacity of the clay was determined in apreliminary study to be 82 cmol (+) kg-1.

Fluorescence Quenching Measurements. The associa-tion of LAS with DHS was measured with the fluorescence

quenching method of Guathier et al. (9). Briefly, aliquotsof Carlisle-WSOC, Carlisle-HA, Aldrich-HA, or SRHA wereplaced into amber borosilicate bottles containing eitherNaCl, CaCl2, or concentrated synthetic river water (Table1). The sample bottles were then capped with Teflon-linedstoppers and incubated at 25 °C for 18-24 h, after whichan aliquot of C10-, C12-, or C14-LAS was added, and thesolutions were equilibrated at 25 °C for 2 h. Previousexperiments indicated that the effects of DHS on LASfluorescence were constant after 5 min and did not changefor 24 h (data not shown). The total solution volume was20 mL. The final concentration of DHS ranged from 0 to20 mg of C L-1; the LAS concentration was either 0, 5, or10 mg L-1; and the background electrolyte was either 0.03mol L-1 NaCl, 0.01 mol L-1 CaCl2, or synthetic river water(Table 1). All treatments were prepared in triplicate. After2 h, 3 mL of each sample solution was placed into 1-cmstoppered quartz cuvettes, and the fluorescence of LAS wasmeasured with a Perkin Elmer LS 5B spectrofluorometer(Perkin Elmer, Norwalk, CT). The samples were excited at230 nm, and emission intensities were measured at 288nm. Both excitation and emission slits were set at 10 nm.Ultraviolet absorption measurements were made at 230and 288 nm with a Beckman DU 6 UV-vis spectropho-tometer (Beckman Instruments, Fullerton, CA) to allow forcorrection of the “inner filter effect” (9). No attempts weremade to account for the sorption of LAS to the walls of thereaction bottles or the quartz cuvettes.

A modified Stern-Volmer model was used to calculateLAS-DHS association constants and is given as

where Io is the fluorescence emission intensity in theabsence of DHS, I is the fluorescence emission intensity inthe presence of DHS, [DHS] is the concentration of DHSin mg of C L-1, and Koc is the C-normalized LAS-DHSassociation constant (9). Implicit is the assumption thatquenching of LAS fluorescence is due to the formation ofa ground-state complex with DHS (static quenching). Thevalues of I used in eq 1 were corrected for the backgroundfluorescence measured in LAS-free solutions and for theinner filter effect (9).

The effects of temperature and LAS concentration onthe quenching of LAS fluorescence by DHS were measuredto assess the relative contributions of static and dynamicquenching processes. In the studies of temperature effects,Aldrich-HA (either 0, 10, or 20 mg of C L-1) was reactedwith C12-LAS (5 mg L-1) in 0.03 mol L-1 NaCl for 2 h at 25°C. Quartz cuvettes were then filled completely with

TABLE 1

Synthetic River Water Compositioncomponent concn (µmol L-1) concn (mg L-1)

Cl 685 24.3NO3 169 10.5SO4 391 37.5alkalinity (CaCO3) 1100 110.0Ca 1020 40.8Mg 540 13.1K 31 1.2Na 687 15.8pH 7.8a

a pH expressed as -log of the activity of H+ and was adjusted witheither HCl or NaOH.

Io/I ) 1 + Koc[DHS] (1)

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aliquots of the experimental solutions, stoppered, andplaced into the sample chamber of the spectrofluorometer.The temperature of the cuvette holder was then varied overthe temperature range of 15-85 °C with a circulating waterbath. Initial measurements indicated that the solutionsinside of the cuvettes reached the same temperature asthat of the water bath within 5 min. All fluorescenceintensity measurements were made after 15 min of equili-bration at each temperature setting. The excitation shutterwas kept closed between measurements to minimizephotochemical alteration of the experimental solutions.Fluorescence intensity measurements were made over twoheating and cooling cycles, for a total of 32 measurementsper sample. In the LAS concentration study, Aldrich-HA(0 or 10 mg L-1) was equilibrated with C10-, C12-, or C14-LAS(0.5-10 mg L-1) in 0.03 mol L-1 NaCl for 2 h at 25 °C.Fluorescence intensity measurements were then made at25 °C as described above.

Batch Ultracentrifugation Experiments. A batch ul-tracentrifugation method was developed to provide mea-surements of the LAS-DHS association constants inde-pendent of the fluorescence quenching method. Aliquotsof C12-LAS were added to polyallomer ultracentrifuge tubescontaining Aldrich-HA, Carlisle-HA, or SRHA in a back-ground electrolyte of either NaCl or CaCl2. The total volumewas adjusted to 40 mL with HPLC-grade H2O; the sampleswere capped and then allowed to equilibrate at 25 °C. Theconcentrations of NaCl and CaCl2 were 0.03 and 0.01 molL-1; DHS concentrations were 0, 5, and 10 mg of C L-1 foreach humic acid; and the initial concentrations of C12-LASwere 0, 1, 2, 3, 4, 5, and 6 mg L-1. All treatments wereprepared in triplicate. After 2 h, the samples werecentrifuged at 141 000g for 6 h at 25 °C. The supernatantswere decanted and saved for analysis. The sedimentedmaterial was discarded. The walls of the centrifuge tubeswere then extracted with 10 mL of CH3OH, which was savedfor analysis. The concentration of C12-LAS in the super-natants and in the CH3OH extracts was determined byexciting the solutions in 1-cm quartz cuvettes at 230 nmand recording the emission intensity at 288 nm with a PerkinElmer LS-5B spectrofluorometer. Intensities were con-verted to concentrations by comparison to C12-LAS stan-dards and by the method of standard additions. ARosemount/Dohrmann DC 80 C analyzer was also used tomeasure the total DHS concentration in the supernatants.The presence of DHS in the supernatants was alsodetermined by measuring UV absorbance over the wave-length range of 200-300 nm with a Varian/Cary 3 equippedwith 10-cm quartz cuvettes. Stock 0.03 mol L-1 NaCl and0.01 mol L-1 CaCl2 solutions, centrifuged at 141 000g for 6h at 25 °C without LAS or DHS, were used in the referencecells.

The quantity of C12-LAS associated with DHS (LASDHS)was calculated from the difference in the initial and finalsolution concentrations, following the correction for thequantity of C12-LAS sorbed to the walls of the centrifugetubes:

where LASt is the total concentration of C12-LAS in 40 mLof solution, LASs is the concentration of C12-LAS in thesupernatant, LASw is the concentration of C12-LAS extractedfrom the walls of the centrifuge tube with methanol, andMc is the mass of organic C present as DHS. A linear plot

of LASDHS against LASs yielded values of Koc.Sorption of LAS by TMAC-Coated Clay. Two sets of

LAS sorption experiments were conducted with TMAC-coated clays. The first set was designed to examine theeffects of TMAC and LAS chain lengths on LAS sorption.Aliquots of synthetic river water (25.7 mL, Table 1) wereadded to 4.3 mL of TMAC-coated clay slurry (9.6 g of clayL-1) in polycarbonate centrifuge tubes. The resulting clayconcentration was 1.3 g L-1 for all samples. The sampleswere equilibrated for 18 h on a rotary shaker (time sufficientfor thorough mixing and equilibration), then spiked withC10-, C12-, or C14-LAS (1 mg L-1). The ratio of radiolabeledto unlabeled LAS was 1:10. Each treatment was preparedin triplicate. Following a 3-h equilibration period (previousstudies showed equilibration within 30 min, data notshown), the samples were centrifuged for 40 min at 15 000g,and two 5-mL aliquots of the supernatant were removedand assayed by liquid scintillation counting. The clayparticles were then resuspended, and the mixtures (solidsplus remaining supernatant) were assayed by liquid scin-tillation counting. Following removal of the clay slurry,the centrifuge tube walls were extracted with 10 mL of CH3-OH. Recoveries of LAS were generally >90%. Single-pointmass distribution coefficients (DLAS) were calculated as

where Ms is the mass of solid, LASs is the concentration ofLAS in the supernatant, LASt is the mass of LAS in theremaining 20 mL of solid plus supernatant mixture, andLASs × 20 is the mass of LAS in the remaining 20 mL ofsupernatant present in the centrifuge tubes. Single-pointKoc values were calculated by dividing the values of DLAS bythe fractional weight of C(foc) present on the clays as TMAC-C. Values of foc were obtained as described above.

In the second set of measurements, sorption isothermswere conducted with C10- and C14-LAS to determine thelinear range for the mass sorption coefficient (Kd) withrespect to concentration. The C10-LAS experiment usedC12-TMAC-coated clay and radiolabeled LAS concentrationsof 0.05-1 mg L-1. The C14-LAS experiment used C16-TMAC-coated clay and radiolabeled LAS concentrations of 0.1-1.1 mg L-1. The total sample volume in these experimentswas 15 mL. All other experimental protocol and dataanalysis were as described above.

Toxicity Test. Juvenile (3 months of age) fatheadminnows (Pimephales promelas) weighing approximately150 mg were obtained from Aquatic Research Organisms(Hampton, NH) and adapted to laboratory conditions fora minimum of 2 weeks prior to exposure. Acute (96 h)toxicity of C12-LAS was determined in static renewalexposures according to the methods of Peltier and Weber(10). Fish were exposed to five concentrations of testcompound in the presence or absence of Aldrich-HA (0-50 mg of C L-1). Two replicates were prepared for eachconcentration, and five fish were used per replicate. Testsolutions (0.5 L) were maintained at 22 ( 2 °C and wererenewed daily. Survival was assessed during test solutionrenewal. The concentration of C12-LAS was determineddaily using liquid scintillation counting, and the concen-tration of dissolved organic C was measured on a PHOTO-chem organic C analyzer (Sybron/Servomex) or a Dohr-mann Model 190 organic C analyzer. Water qualityparameters (Table 1) were measured according to standardmethods (11).

LASDHS ) [((LASt - LASs) × 40) - (LASw × 20)]/Mc (2)

DLAS ) [(LASt - (LASs × 20))/Ms]/LASs (3)

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Uptake and Depuration Measurements. The effect oforganic C on the uptake and depuration of C12-LAS wasassessed in short-term uptake (24 h) and depurationexposures after Branson et al. (12). Fathead minnows wereexposed to the test compound (55 µg L-1) in the presenceand absence of Aldrich-HA or Carlisle-WSOC (0-25 mg ofC L-1) in 37.8 L of aquaria. Due to the slow uptake of LAS,static exposures could be conducted without significantlyreducing the concentration of test compound in the aquariaduring the study. Aqueous concentrations of test com-pounds were monitored by liquid scintillation countingduring the exposures. Three fish were removed from theexposure tanks at 0, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h ofexposure. After 24 h of exposure, fish were transferred tofresh flowing water and allowed to depurate in the presenceof the DHS. Three fish were sampled after 8, 24, and 72 hof depuration for each treatment. After sampling, the fishwere frozen (-20 °C) until analyzed. Fish were not fedduring the uptake experiments.

Radiochemical liquid scintillation counting of the testcompounds in fish was conducted by a microwave-assistedsolubilization method (13). Briefly, individual fish werethawed, weighed, and placed into a tared scintillation vial.A 5-mL solubilization reagent comprised of 1% NaOH, 1%H2O2, and 1.5% silicon antifoam emulsion (SAG 470, UnionCarbide) was added, and scintillation vials were looselycapped. Samples were processed in a microwave oven(Hotpoint, Model RE 968002) for 4 min at 188 W and thencooled for 1 min. This process was conducted six times.Samples were then acidified with 2 mL of 2 mol L-1 HCl,and liquid scintillation cocktail was added. Samples werecounted for radioactivity on a Beckman LS 7800 liquidscintillation counter. No effort was made to quantifypossible metabolic byproducts of LAS within the fish.

Data Analysis for Bioavailability Experiments. Uptakeand depuration rates were determined using a two-compartment model described by

where Cf is the toxicant concentration in the fish, t is time,Cw is the toxicant concentration in the water, and ku andkd are the uptake and depuration rate constants, respectively(13). Rate constants were estimated by the BIOFACcomputer program (14). Depuration rate constants areexpected to underestimate the elimination of the parentcompound due to metabolism of the test substances.

The 96-h LC50 (concentration lethal to 50% of sampleindividuals) values associated with 95% confidence intervalswere estimated by trimmed Spearman-Karber (15) or Probitanalysis (16). The trimmed Spearman-Karber analysis wasconducted using a computer program developed by Bur-lington Research, Inc. Estimates for Kb‚oc (a biologicallydetermined organic C sorption coefficient, described below)were performed using SAS version 5 (17). Slopes werecompared using the methods described in Sokal and Rolf(18).

Values for the biologically determined organic carbonsorption coefficient, referred to as Kb‚oc, were calculatedfrom the uptake and toxicity data. The Kb‚oc value is similarto the Kb reported by others (2) and is analogous to theC-normalized association constants (Koc) calculated fromthe fluorescence quenching measurements. Derivation of

the Kb‚oc is described in Versteeg and Shorter (13) and wasdetermined by

The estimation of Kb‚oc values from the uptake rate constantsand the LC50 values assumes that LAS bound to DHS arenot available for uptake or are not toxic.

Results and DiscussionFluorescence Quenching Experiments. In the absence ofDHS, changes in the background electrolyte (NaCl, CaCl2,or synthetic river water) had no effect on the shape or theintensity of the fluorescence spectra of LAS. The additionof SRHA to C12-LAS solutions caused a decrease influorescence emission intensity, but no change was ob-served in the peak position. Thus, DHS quenching of LASfluorescence could be quantified with measurements offluorescence intensities at the emission maximum (288 nm).The emission intensity of DHS alone was always <5% ofthe emission intensity of LAS for all solutions, thusinterference due to DHS fluorescence was neglected in ouranalysis. The maximum value for the inner filter effectcorrection factor was 1.7 when the relative fluorescenceemission intensity (Io/I) was linear, which is well within therecommended acceptable range (9).

Representative examples of the effects of DHS on therelative fluorescence emission intensity (Io/I) of C12-LASare shown in Figure 1a,b. The data for Ca-saturated Aldrich-HA was linear over the entire concentration range of DHS(Figure 1a), whereas some curvature was present in thedata from the Ca-saturated SRHA (Figure 1b), and con-siderable deviation from linearity was apparent in many ofthe Na-saturated DHS solutions (data not shown). Ingeneral, there was greater curvature in the presence of NaClthan in either CaCl2 or the synthetic river water solution.It is possible that the curvature observed in the presentstudy (Figure 1b) was due to a combination of static anddynamic quenching mechanisms. In the former case, aground-state complex is formed between the organicpolymer and fluorescent solute. In the latter case, quench-ing could result from dissolved O2 interactions with thehumic substances (19) or just simply due to an increase inthe probability of collisions between the excited LASmolecules and the DHS polymers. An increase in thefrequency of DHS collisions with the LAS molecules shouldbe more prevalent in a monovalent electrolyte such as NaClthan in CaCl2 since the Na-saturated system should promotemore linear, dispersed polymers of DHS (20). Additionally,enhanced charge repulsion between the LAS molecules andthe dissociated anionic functional groups on the DHSpolymers should decrease ground-state complex formationwith LAS in monovalent electrolytes.

Calculation of LAS-humic acid Koc values with eq 1explicitly assumes that all quenching is due to static ground-state complex formation. Before this equation could beused in the present study, it was necessary to evaluate thepotential for the dynamic quenching of LAS fluorescenceby DHS. This was done by examining the effects oftemperature and surfactant concentration on the quenchingof LAS by Aldrich-HA. If collisional processes are respon-sible for the nonlinear quenching of LAS by DHS, then theyshould become more prevalent with increases in solutiontemperature. At a DHS concentration of 10 mg of C L-1,

dCf/dt ) kuCw - kdCf (4)

LC50oc/LC50f or Ku/Kuoc ) Kb‚oc[DHS] + 1 (5)


there was little or no effect of temperature on the quenchingof C12-LAS by Na-saturated Aldrich-HA, suggesting thatstatic ground-state complex formation was the dominantquenching mechanism at low DHS concentrations (Figure2). However, the value of Io/I did increase with increasingtemperature at a DHS concentration of 20 mg of C L-1. Itis likely that the greater concentration of DHS increasedthe frequency of collisions between “free” excited C12-LASmolecules and Aldrich-HA polymers resulting in greaterdynamic quenching.

If dynamic quenching contributed significantly to humicacid-induced reductions in LAS fluorescence, then it is likelythat increases in LAS concentration should have increasedthe probability of collisions between LAS and humic acidmolecules, increasing the value of Io/I. There was no effectof surfactant concentration on the quenching of LASfluorescence by Na-saturated Aldrich-HA at 10 mg of C L-1

(Figure 3). Thus, it appears that static and not dynamic

quenching was dominant at lower DHS concentrations (<10mg of C L-1).

In the present study, we calculated values of log Koc byrestricting the use of eq 1 to the linear region of thequenching plots (generally DHS concentrations of 0-10mg L-1). The contribution of nonpolar forces to LAS-DHSassociations is evidenced by increases in log Koc withincreasing chain length (Table 2). Similar effects of chainlength have been reported for LAS sorption by soils andsediments (5, 6). Hand and Williams (5) measured log Kd

values of 0.75-2.3, 1.7-3.2, and 2.25-4.1 for C10-, C12-, andC14-LAS sorption by soils and sediments, respectively. Con-version to log Koc yields values of 2.80-3.82, 3.75-4.94, and4.30-5.62 for C10-, C12-, and C14-LAS, respectively. Thevalues listed in Table 2 are either close to (in the case ofC10-LAS) or within the range observed by Hand and Williams(5).

FIGURE 1. Fluorescence quenching titration of C12-LAS with (a)Aldrich humic acid and (b) Suwanee River humic acid in 0.01 molL-1 CaCl2. The excitation and emission wavelengths were 230 and288 nm, respectively. Vertical error bars denote 1 SD.

FIGURE 2. Effect of temperature and DHS concentration on thequenching of C12-LAS fluorescence by Aldrich humic acid in 0.03mol L-1 NaCl. Humic acid concentration expressed as mg of C L-1.Vertical error bars denote 1 SD.

FIGURE 3. Effect of surfactant concentration on the quenching ofC10-, C12-, and C14-LAS fluorescence by Aldrich humic acid (10 mgof C L-1) in 0.03 mol NaCl L-1. Vertical error bars denote 1 SD.


A slight dependence of Koc on the valence of thecounterions present in the supporting electrolyte wasobserved (Table 2). Variations in pH did not seem to affectthis dependency. In all cases, the values of log Koc

determined in the presence of Na+ were less than thosemeasured in the presence of Ca2+ or the synthetic riverwater solution. These results are consistent with thesuggestion that specific complexes can form between LAS,Ca2+, and a sorbent with anionic functional groups likeDHS (6). It is also likely that Na+ was less effective thanCa2+ in shielding the anionic charges present on the DHSmolecules. This could have resulted in greater chargerepulsion between the sulfonate groups on the LAS andanionic sites on the humic substances, thus lowering thevalues of log Koc.

Gauthier et al. (21) and Chiou et al. (22, 1) have reportedsignificant effects of DHS composition on the associationof nonpolar organic solutes with dissolved humic and fulvicacids. In general, the extent of partitioning increased withincreases in the concentration of nonpolar structures inthe humic and fulvic acids. In particular, the partitioncoefficients for p,p′-DDT and 2,4,5,2′,5′-PCB were more than10 times greater for Aldrich-HA than for SRHA (22). Aldrich-HA is considerably less polar than SRHA, containing 65.31%versus 54.22% C and 25.05% versus 39.00% O (22, 23), andthus it is expected to react more strongly with low polaritysolutes. Chiou et al. (1) also observed the partitioningcoefficients of organic solutes to be 5-7 times greater forsoil-derived humic acids than for aquatic humic acids. Theybelieved that this effect was due to the differences in DHSmolecular size and polarity. In the present study, therewas little or no effect of DHS type on the values of log Koc

(Table 2). For the same electrolyte solutions, the Koc valuesfor Aldrich-HA and SRHA differed by <0.15 log unit, andin the presence of CaCl2 the association constants weregreater for SRHA than for Aldrich-HA (Table 2). It is possiblethat differences in the nonpolar composition of the DHSwere masked by specific-ion interactions between LAS, theelectrolyte ions, and oxygen-containing functional groupsin the DHS.

Ultracentrifugation Experiments. Ultracentrifugation(141 000g for 6 h) of Aldrich-HA, Carlisle-HA, and SRHA in

0.03 mol L-1 NaCl only reduced the DHS concentrationsin the supernatant by <5% (as determined by the DohrmannC-analyzer and with UV absorbance measurements). Littleor no pellets were present on the bottom of the centrifugetubes. Increasing the centrifugation time to 24 h had nosignificant effect on the removal of Na-saturated humicacid from solution. In contrast, ultracentrifugation (141000gfor 6 h) of each of the humic acids in 0.01 mol L-1 CaCl2

caused the deposition of dark-colored pellets on the bottomof the centrifuge tubes and removed all detectable humicacid from solution. In the absence of C12-LAS, no DHScould be detected with the CR-80 C-analyzer, and the UVspectra from 200-300 nm showed no deviations frombaseline. Apparently the CaCl2 caused sufficient coilingand flocculation of the humic acid polymers to allowsedimentation at 141 000g. Since this was not the case inthe NaCl solution, no measurements of C12-LAS associationwith Na-saturated humic acid were made in the ultracen-trifugation experiments.

The amount of C12-LAS sorbed to the centrifuge tubewalls (as determined by methanol extraction) in solutionscontaining 0.01 mol L-1 CaCl2 and no DHS was <12%. Thetotal recovery of C12-LAS was >95% in these samples. Theamount of LAS sorbed to the centrifuge walls decreased to<10% in the presence of DHS at 5 and 10 mg of C L-1.Replicate samples containing LAS without DHS were storedin the centrifuge tubes for 6 h without centrifugation. Thesolutions were then decanted, and the tube walls wereextracted with methanol as described above. These samplesshowed the same loss of LAS to the walls of the centrifugetubes as was measured in the samples subjected tocentrifugation (data not shown). Thus, centrifugation at141 000g for 6 h did not result in the sedimentation of LASmolecules in the absence of humic acid. The determinationof C12-LAS concentrations in the supernatants by fluores-cence intensity measurements and by the Dohrmann CR-80 C-analyzer always differed by <5%.

The association of C12-LAS with Ca-saturated Aldrich-HA, Carlisle-HA, and SRHA increased linearly with increasesin surfactant concentration (Figure 4). Values of log Koc

determined by the ultracentrifugation method in 5 and 10mg of DHS L-1 were identical at the 5% level and agreedwell with the values measured with fluorescence quenching,suggesting that the latter analytical method provided a goodestimate of the LAS-DHS association constants.

Sorption of LAS by TMAC-Coated Clay. Increases inthe length of the alkyl chains on LAS generally resulted ingreater LAS sorption by TMAC clays (Figure 5). Since theion exchange sites on the clays were satisfied by theadsorbed quaternary ammonium surfactant ions, thesorption of LAS by these surfactant-coated clays must haveresulted from nonpolar interactions between the LAS andadsorbed TMAC molecules. Increases in the TMAC chainlength, from C8 to C12, resulted in large increases in LASsorption. Further increases in TMAC chain length had littleor no effect on LAS sorption. The enhanced sorption ofLAS by clays coated with C12-TMAC (and larger TMACmolecules) may be due to an increased expansion of theinterlamellar spacing of the clay particles. The C8-TMAC-coated SWy-1 clay has a vertical lattice spacing of 1.36 nm.This spacing increases to 1.45 nm for C12-TMAC, to 1.55nm for C14-TMAC, to 1.58 nm for C16-TMAC, and to 1.77nm for C18-TMAC-coated clays (24). The greater inter-lamellar spacing (C12 > C8) may have facilitated greatersorption of LAS by intercalated TMAC molecules. With the

TABLE 2

DHS-LAS Association Constants Measured withFluorescence Quenchinga

log Koc (L kg-1)

DHS electrolytesolution

pH C10-LAS C12-LAS C14-LAS

Aldrich-HA NaCl 6.6 3.97 4.73 5.31CaCl2 6.1 4.08 4.87 5.69CaCl2 6.1 4.86b

SRW 7.5 4.92Carlisle-WSOC NaCl 6.3 4.65

CaCl2 6.1 4.81SRW 7.7 4.83

Carlisle-HA NaCl 5.8 3.92 4.81 5.36CaCl2 6.3 4.11 4.91 5.63SRW 7.3 4.88

Suwanee NaCl 6.6 4.58River-HA CaCl2 5.9 4.93

CaCl2 5.9 4.92b

SRW 7.3 4.88a Log Koc values determined in 0.03 mol L-1 NaCl, 0.01 mol L-1 CaCl2,

or synthetic river water (SRW, see Table 1). b Log Koc values determinedwith 10 mg of C12-LAS L-1; 5 mg L-1 LAS used in all other measurements.


exception of the clays coated with C8-TMAC, the log Koc

values in Figure 5 are all larger than those measured for theassociation of LAS with DHS (Table 2). This response isconsistent with coverage of the polar sites on the claysurfaces by adsorbed TMAC molecules and the subsequentpartitioning of LAS into quaternary ammonium surfactantcoatings, which should be less polar than DHS.

Sorption isotherms for C10-LAS on C12-TMAC-coated clayand C14-LAS on C16-TMAC-coated clay are shown in Figure6. Both isotherms were linear over the range of added LASfrom 0 to 1 mg L-1, indicating that LAS sorption by organicsorbents is likely through nonspecific partitioning processes.The linear regions in Figure 6 yielded log Kd values of 4.38and 4.98, corresponding to log Koc values of 5.36 and 5.73for C10- and C14-LAS, respectively. These log Koc values aresimilar to the ones obtained from single-point measure-ments of Kd (Figure 5) and again indicate a greater affinityof LAS for the more lipid-like adsorbed TMAC moleculesthan for DHS. Clearly, the presence of polar functional

FIGURE 4. Association of C12-LAS with DHS as (a) Aldrich humicacid, (b) Carlisle humic acid, and (c) Suwanee River humic acid in0.01 mol L-1 CaCl2. All measurements made after separation of DHSby ultracentrifugation. Vertical bars denote 1 SD.

FIGURE 5. Effect of alkylammonium surfactant chain length on thesorption of LAS by surfactant-coated montmorillonite in the syntheticwater solution. Vertical error bars denote 1 SD.

FIGURE 6. Sorption of C10- and C14-LAS on C12- and C16-TMAC coatedmontmorillonite in the synthetic water solution. Vertical error barsdenote 1 SD.


groups on DHS (for example, carboxyls, hydroxyls, etc.)reduces the extent of “hydrophobic” interactions with LAS.

Bioavailability Studies. The effect of LAS associationwith DHS on bioavailability and toxicity to fish wasinvestigated. Exposure of fathead minnows to C12-LAS inthe presence of 8-24 mg of C L-1 Carlisle-WSOC resultedin a reduced uptake of LAS into fish (Figure 7). Although theeffect of the humic substance on the uptake rate constantwas variable, a significant linear relationship (p ) 0.035)between the ratio of the uptake rate constants in the absenceand presence of Carlisle-WSOC (Ku/Ku-ha) and the humicsubstance concentration was observed. The biologicallydetermined C12-LAS-DHS association constant (log Kb‚oc)measured for Carlisle-WSOC was 4.83. This value wasidentical to the measured log Koc value for Carlisle-WSOCby fluorescence quenching in synthetic river water.

Studies into the effect of Aldrich-HA on the uptake ofC12-LAS indicated enhanced C12-LAS uptake in the presenceof a low concentration of Aldrich-HA, 2-6 mg of C L-1,followed by reduced LAS uptake at 12 and 24 mg of C L-1

of Aldrich-HA (Figure 8). To better understand theimportance of the enhanced uptake of LAS into fish at lowAldrich-HA concentrations, we conducted acute toxicitytests with LAS in the presence of 2.6-55 mg of C L-1 Aldrich-HA. The LC50 concentration for C12-LAS was 1.0 mg/L-1 inthe absence of Aldrich-HA. At all concentrations of Aldrich-HA, the LC50 value for C12-LAS increased (less toxic) andthe slope of the regression of toxicity ratio on Aldrich-HAconcentration was significantly greater than zero (p )0.0007) (Figure 9). The log Kb‚oc for C12-LAS and Aldrich-HA determined from these toxicity values was 4.45. Theincrease in the uptake of LAS in the presence of lowconcentrations of Aldrich-HA suggests increased affinityof the LAS humic acid complex for fish tissue. Since thisapparent uptake does not affect toxicity, we speculate theLAS-Aldrich humic acid complex is associated with theexternal surface of the fish, perhaps the mucous layer.Additional studies are needed to better understand theimportance of this uptake; however, at this time it appearsto have little toxicological significance. If, however, weeliminate the 1 and 6 mg L-1 Aldrich-HA values from the

LAS uptake data set (Figure 8), a linear relationship (r )0.96) with a calculated log Kb‚oc of 4.69 is observed. Thisvalue is very similar to those measured by fluorescencequenching for C12-LAS in Aldrich-HA solutions.

Environmental Significance. Knowing the associationconstant of LAS with DHS, one can now estimate thebioavailable fraction of LAS via mass balance and massaction equations. The total mass balance for LAS in asolution containg DHS is given by

where [LASt] is the total concentration of dissolved LAS,[LAS] is the concentration of “free” LAS not associated withDHS, and [LAS-DHS] is the concentration of LAS “bound”with DHS . The mass action equation for the associationof LAS with DHS can be written as

where and [DHS] is the concentration of the unbound DHS.

FIGURE 7. Effect of Carlisle-WSOC on the uptake of C12-LAS by thefathead minnow, Pimephales promelas.

FIGURE 8. Effect of AldrichHA on the uptake of C12-LAS by the fatheadminnow, Pimephales promelas.

FIGURE 9. Effect of AldrichHA on the LC50 of C12-LAS by the fatheadminnow, Pimephales promelas.

[LASt] ) [LAS] + [LAS-DHS] (6)

[LAS] + [DHS] S [LAS-DHS] (7)


The equilibrium distribution of LAS can then be describedby

Combining eqs 6 and 8 and solving for the free fraction, Rdefined as [LAS]/[LASt], provides an indication of thedependence of LAS speciation on the concentration of LASof the form:

In the present study, an average log Koc value of 4.83(mean of the values for C12-LAS in Table 2) and the valueof 4.45 measured in the toxicology assay were used with eq8 to estimate the fractions of free C12-LAS in natural waters.The fraction of bound LAS was taken as 1 - R. The smallerKoc value serves both to bracket the model calculation andto estimate the uncertainty in the free LAS prediction. Fora log Koc of 4.83 and a DHS concentration of 15 mg of C L-1,approximately 50% of the C12-LAS is free and approximately50% is associated with humic acid polymers (Figure 10). Asmaller quantity of LAS is estimated to be associated withthe humic acid polymers for a given DHS concentration ifa log Koc value of 4.45 is used. Clearly these values areestimates that will vary with the nature of the DHS presentin a given environment. It should be noted that the majorityof the DHS used in this study were humic acids. Since theLAS associates with DHS primarily through nonpolarinteractions, it can be anticipated that the quantity of freeLAS will be greater in those waters dominated by fulvicacids. Additionally, the aqueous speciation of LAS willdepend on the specific mixture of LAS homologues presentin a given environment and on competition between DHSand solid-phase sorbents for LAS molecules. Nevertheless,the data in the present study indicate that the aqueousspeciation of LAS in natural waters can be influenced bynatural DHS. This in turn attenuates the toxicity of LAS tofathead minnows, as illustrated in Figure 9, and possiblyto other aquatic organisms not examined in this study.

SummaryThe general agreement between the values of log Koc

measured with fluorescence quenching and ultracentrifu-gation indicates that both techniques can be used toquantify the effects of DHS on the speciation of LAS innatural waters. However, limitations do exist for each

method. Fluorescence quenching measurements must berestricted to low concentrations of DHS (<10 mg of C L-1)to minimize nonlinearity in the quenching plots. Deter-mination of log Koc values with ultracentrifugation requiresthat a sufficient concentration of divalent ions be presentin the supporting electrolyte to promote coagulation,flocculation, and sedimentation of the organic polymers.Nevertheless, when used within these constraints bothmethods produced log Koc values similar to each other.

In most cases the sorption of LAS to TMAC-coated clays,as measured by log Koc, was greater than those valuesdetermined for the association of LAS with DHS. Thisresponse, along with the linear nature of the sorptionisotherm, indicates that LAS sorption by organic sorbentsis likely through nonspecific partitioning processes. Thus,it seems likely that the association of LAS with DHS isprimarily due to hydrophobic interactions between alkylchains of the surfactant molecules and nonpolar regionsof the humic substances. However, the log Koc values doshow a slight but consistent dependence on the valence ofthe cations in the supporting electrolyte solutions. In allcases, the values of log Koc determined in the presence ofNa+ were less than those measured in the presence of Ca2+

or the synthetic river water. Although this suggests theoccurrence of specific interactions between LAS, Ca2+, andDHS, an electrolyte-induced-change in charge repulsionbetween LAS and DHS cannot be ruled out. In any event,specific ion effects are minor when compared to nonpolarinteractions between LAS and DHS.

Bioassay measurements indicated that DHS can alterthe bioavailability of LAS to aquatic organisms. The extentof this effect will likely depend upon the specific mixtureof LAS homologues and the chemical composition of theDHS present in the natural waters in question. In thepresent study, model calculations show that approximately50% of the total dissolved LAS will be associated with humicacid molecules at DHS concentrations of 15 mg L-1. Clearlythe environmental fate and behavior of LAS will beinfluenced by the presence of dissolved organic carbon inaquatic environments.

AcknowledgmentsWe want to thank Marshall E. Pritchard, Charlotte E. White,and Shirley J. Shorter for their technical assistance. Partialsupport for salaries and research at The Ohio StateUniversity were provided by The State of Ohio, The FederalGovernment, and The Procter & Gamble Company.

Literature Cited(1) Chiou, C. T.; Malcolm, R. L.; Brinton, T. I.; Kile, D. E. Environ.

Sci. Technol. 1986, 20, 502-508.(2) Landrum, P. F.; Nihart, S. R.; Eadie, B. J.; Herche, L. R. Environ.

Toxicol. Chem. 1987, 6, 11-20.(3) Chin, Y. P.; Weber, W. J.; Eadie, B. J. Environ. Sci. Technol. 1990,

24, 837-842.(4) McAvoy, D. C.; Eckhoff, W. S.; Rapaport, R. A. Environ. Toxicol.

Chem. 1993, 12, 977-987.(5) Hand, V. C.; Williams, G. K. Environ. Sci. Technol. 1987, 21, 370-

373.(6) Brownawell, B. J.; Chen, H.; Zhang, W.; Westall, J. C. Adsorption

of Surfactants. In Organic Substances and Sediments in Water:Processes and Analytical; Baker, R. A., Ed.; Lewis Publishers:Chelsea, MI, 1991; pp 127-147.

(7) Traina, S. J.; Spontak, D. A.; Logan, T. J. J. Environ. Qual. 1989,18, 221-227.

(8) Schnitzer, M. Organic Matter Characterization. In Methods ofSoil Analysis: Part 2sChemical and Microbiological Properties;Page, A. L., Miller, R. H., Keeney, D. R., Eds.; American Societyof Agronomy: Madison, WI, 1982; pp 581-594.

FIGURE 10. Model calculation of the effect of DHS on the aqueousspeciation of C12-LAS. The log Koc of 4.83 is the mean of the valuesfor C12-LAS listed in Table 2, and the value of 4.45 is from the toxicityassay.

Koc ) {[LAS-DHS]}/{[LAS][DHS]} (8)

R ) 1/(1 + Koc[DHS]) (9)


(9) Gauthier, T. D.; Shane, E. C.; Guerin, W. F.; Seitz, W. R.; Grant,C. L. Environ Sci. Technol. 1986, 20, 1162-1166.

(10) Peltier, W. H., Weber, C. I., Eds. Methods for Measuring the AcuteToxicity of Effluents to Freshwater and Marine Organisms; EPA600/4-85-013; U.S. Environmental Protection Agency: Cincin-nati, OH, 1985.

(11) APHA. Standard methods for the examination of water andwastewaters; APHA: Washington, DC, 1985.

(12) Branson, D. R.; Blau, G. E.; Alexander, H. C.; Neely, W. B. Trans.Am. Fish. Soc. 1975, 104, 785-792.

(13) Versteeg, D. J.; Shorter, S. J. Environ. Toxicol. Chem. 1992, 11,571-580.

(14) Blau, G. E.; Agin, G. L. A Users Manual for BIOFAC: A ComputerProgram for Characterizing the Rates of Uptake and Clearanceof Chemicals in Aquatic Organisms; The Dow Chemical Com-pany: Midland, MI, 1978.

(15) Hamilton, M. A.; Russo, R. C.; Thurston, R. V. Environ. Sci.Technol. 1977, 11, 714-719; 1978, 12, 417 (correction).

(16) Finney, D. J. Probit Analysis; Cambridge University Press:Cambridge, MA, 1971.

(17) SAS Institute. SAS User’s Guide: Statistics Version 5; SAS: Cary,NC, 1985.

(18) Sokal, R. R.; Rohlf, F. J. Biometry; W. H. Freeman & Company:New York, NY, 1981.

(19) Danielsen, K. M. Partitioning of pyrene to sediment organicmatter and humic substances. M.S. Thesis, The Ohio StateUniversity, 1995; p 72.

(20) Stevenson, F. J. Humus Chemistry. Genesis, Composition, Reac-tions; John Wiley & Sons: New York. 1982; p 443.

(21) Gauthier, T. D.; Seitz, W. R.; Grant, C. L. Environ. Sci. Technol.1987, 21, 243-248.

(22) Chiou, C. T.; Kile, D. E.; Brinton, T. I.; Malcolm, R. L.; Leenheer,J. A. Environ. Sci. Technol. 1987, 21, 1231-1234.

(23) Malcolm, R. L.; MacCarthy, P. Environ. Sci. Technol. 1986, 20,904-910.

(24) Jaynes, W. F.; Bigham, J. M. Clays Clay Miner. 1987, 35, 440-448.

Received for review July 11, 1995. Revised manuscript re-ceived November 29, 1995. Accepted December 1, 1995.X

ES950512R

X Abstract published in Advance ACS Abstracts, February 15, 1996.


association of linear alkylbenzenesulfonates with dissolved humic substances and its effect on...

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