Environ. Sci. Technol. 1996, 30, 1300-1309
Association of Linear Alkylbenzenesulfonates with Dissolved Humic Substances and Its Effect on Bioavailability SAMUEL J. TRAINA,† D R E W C . M C A V O Y , * ,‡ A N D DONALD J. VERSTEEG‡ 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 alkylbenzenesulfonates (LAS) with natural and specimen-grade dissolved humic substances (DHS) was measured with fluorescence quenching and with ultracentrifugation techniques. Good agreement was obtained with both of the analytical methods, suggesting that fluorescence quenching could be used to measure aqueous-phase partition coefficients. LASDHS partition coefficients increased with increasing length of the alkyl chain. Partition coefficients for the sorption of LAS to alkylammonium surfactant-coated, phyllosilicate clays also increased with increasing length of the alkyl chain in the LAS molecules. Taken together, these data indicate the significance of nonpolar forces in LAS-organic matter interactions. Toxicity studies examined the effects of DHS on the bioavailability to the fathead minnow, Pimephales promelas. Changes in the uptake and toxicity of LAS resulting from the addition of DHS were used to calculate aqueous-phase LAS-DHS partition coefficients. Good agreement was found between the partition coefficients calculated from the response of the test organism and those obtained with fluorescence and ultracentrifugation measurements. The toxicity studies suggest that the association of LAS with DHS can play a significant role in reducing the biologically available fraction of LAS in surface waters.
Introduction Association of anthropogenic organic solutes with natural dissolved humic substances (DHS) plays a significant role in the environmental fate and effects of xenobiotic solutes released to aqueous environments. The partitioning of relatively hydrophobic, nonpolar organic solutes with * Corresponding author telephone: 513-627-5570; Fax: 513-6278198; e-mail address:
[email protected]. † The Ohio State University. ‡ The Procter & Gamble Company.
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 30, NO. 4, 1996
natural DHS such as humic and fulvic acids can cause increases in apparent water solubilities, decreases in sorption to solid-phase particulates, and reductions in biological uptake and toxicities (1-3). These effects are attributed to the partitioning of nonpolar, anthropogenic organic solutes into “low polarity” regions of humic and fulvic acid polymers (1). Linear alkylbenzenesulfonates (LAS) are anionic surfactants that have been used extensively by the detergent industry. Typically, surfactants contain both polar and nonpolar regions. This dual nature confers their surfaceactive properties. McAvoy et al. (4) indicate that approximately 317 000 t yr-1 LAS is produced in the United States, and approximately 16 000 t yr-1 is discharged to surface waters in municipal sewage effluent. Typical surface water concentrations immediately below wastewater treatment plant outfalls are less than 50 µg L-1 (4). Despite the extensive use of LAS, there are little systematic data on the sorption of LAS to soils and sediments or its association with DHS. Hand and Williams (5) examined structure-activity relationships for the sorption of various homologues and mixtures of C10-C14-LAS by riverine sediments. Sorption increased with increasing chain length, which led the authors to conclude that nonpolar sorption mechanisms were responsible for LAS retention (5). Brownawell et al. (6) observed nonlinear sorption of C10-, C12-, and C14-LAS to selected soils and sediments. Sorption was found to increase with increasing chain length, increases in ionic strength or the valence of the cations in the supporting electrolyte 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 particular, they hypothesized the existence of specific chemical reactions between sorption sites, Ca2+, and LAS molecules. Clearly there is some question as to the relative significance of polar versus nonpolar chemical reactions on the sorption of LAS. Additionally, no information is available on the association of LAS with DHS. Such associations could effect the fate, transport, and toxicity of LAS in aquatic environments. The purpose of the present study was to determine the association of LAS with DHS and to assess its effect on aquatic organisms. We used fluorescence quenching to examine the effects of surfactant chain length and solution composition ([Na+], [Ca2+], and synthetic river water) on the association of LAS with DHS. A batch ultracentrifugation technique was also used as an independent measure of the association constants for LAS and Ca-saturated humic acids. The sorption of LAS onto clay surfaces modified with cationic monoalkyltrimethylammonium surfactants was determined to assess the significance of nonpolar interactions on the sorption of LAS by organic colloids. Finally, the effects of dissolved organic substances on the uptake and acute toxicity of LAS to fathead minnows was examined.
Experimental Section Materials. 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 described by Traina et al. (7). Humic acid was extracted with
0013-936X/96/0930-1300$12.00/0
1996 American Chemical Society
0.1 mol L-1 NaOH under a N2 atmosphere as described by Schnitzer (8). The acid-soluble fraction of this extract was discarded. The acid-insoluble fraction (Carlisle-HA) was suspended in a solution of 0.5% HCl and 0.26% HF in a 250-mL polycarbonate centrifuge bottle, capped, shaken 24 h on a mechanical shaker, and centrifuged at 7000g. The supernatant was discarded, and the HF-HCl treatment was repeated three more times on the residue. The residue was washed with HPLC-grade H2O, then dissolved with 1 mol L-1 NaOH under a N2 atmosphere, and finally neutralized to pH 7 with HCl. The dissolved Carlisle-HA was then transferred into 3500 molecular weight cutoff dialysis tubing (Spectrum Medical Industries, Los Angeles, CA) and dialyzed against HPLC-grade water until a negative Ag test for Clwas obtained. The salt-free solution was then dialyzed with Na-saturated Chelex 100 cation-exchange resin (Bio-Rad, Richmond, CA) to reduce the polyvalent cation content. Aldrich humic acid (Aldrich-HA), purchased from Aldrich Chemical Company (Milwaukee, WI), was solubilized in 0.1 mol L-1 NaOH under a N2 atmosphere. The resulting solution was acidified to pH 1.0 with HCl, and the residue was purified with HF-HCl, followed by dissolution in NaOH, and dialysis with HPLC-grade H2O and Na-saturated Chelex-100 as described above. Suwanee River humic acid (SRHA), obtained from the International Humic Substances Society, was dissolved in 0.1 mol L-1 NaOH under a N2 atmosphere, and dialyzed with H2O and Na-saturated Chelex-100 as described above. All DHS solutions were stored in an amber glass bottle at 4 °C. Decylbenzenesulfonate (C10-LAS, 98% purity), dodecylbenzenesulfonate (C12-LAS, 93% purity), and tetradecylbenzenesulfonate (C14-LAS, 88% purity) were synthesized at Procter and Gamble. Uniformly14C-ring-labeled C10-, C12-, and C14-LAS were obtained from New England Nuclear and were 93.8, 96.3, and 92.5% pure, with specific activities of 26.6, 68.2, and 34.3 µCi mg-1, respectively. Specimen Na-montmorillonite (SWy-1) was obtained from the Source Clays Repository of the Clay Minerals Society. Fifty gram samples of clay were washed with 200 mL 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 and to remove contaminating carbonates. The clays were then washed three times with 0.05 mol L-1 NaCl and then dispersed by washing three times with HPLC-grade H2O. The 90%. Single-point mass distribution coefficients (DLAS) were calculated as
DLAS ) [(LASt - (LASs × 20))/Ms]/LASs
(3)
where Ms is the mass of solid, LASs is the concentration of LAS in the supernatant, LASt is the mass of LAS in the remaining 20 mL of solid plus supernatant mixture, and LASs × 20 is the mass of LAS in the remaining 20 mL of supernatant present in the centrifuge tubes. Single-point Koc values were calculated by dividing the values of DLAS by the fractional weight of C(foc) present on the clays as TMACC. Values of foc were obtained as described above. In the second set of measurements, sorption isotherms were conducted with C10- and C14-LAS to determine the linear range for the mass sorption coefficient (Kd) with respect to concentration. The C10-LAS experiment used C12-TMAC-coated clay and radiolabeled LAS concentrations of 0.05-1 mg L-1. The C14-LAS experiment used C16-TMACcoated clay and radiolabeled LAS concentrations of 0.11.1 mg L-1. The total sample volume in these experiments was 15 mL. All other experimental protocol and data analysis were as described above. Toxicity Test. Juvenile (3 months of age) fathead minnows (Pimephales promelas) weighing approximately 150 mg were obtained from Aquatic Research Organisms (Hampton, NH) and adapted to laboratory conditions for a minimum of 2 weeks prior to exposure. Acute (96 h) toxicity of C12-LAS was determined in static renewal exposures according to the methods of Peltier and Weber (10). Fish were exposed to five concentrations of test compound in the presence or absence of Aldrich-HA (050 mg of C L-1). Two replicates were prepared for each concentration, and five fish were used per replicate. Test solutions (0.5 L) were maintained at 22 ( 2 °C and were renewed daily. Survival was assessed during test solution renewal. The concentration of C12-LAS was determined daily using liquid scintillation counting, and the concentration of dissolved organic C was measured on a PHOTOchem organic C analyzer (Sybron/Servomex) or a Dohrmann Model 190 organic C analyzer. Water quality parameters (Table 1) were measured according to standard methods (11).
Uptake and Depuration Measurements. The effect of organic C on the uptake and depuration of C12-LAS was assessed in short-term uptake (24 h) and depuration exposures after Branson et al. (12). Fathead minnows were exposed to the test compound (55 µg L-1) in the presence and absence of Aldrich-HA or Carlisle-WSOC (0-25 mg of C L-1) in 37.8 L of aquaria. Due to the slow uptake of LAS, static exposures could be conducted without significantly reducing the concentration of test compound in the aquaria during the study. Aqueous concentrations of test compounds were monitored by liquid scintillation counting during the exposures. Three fish were removed from the exposure tanks at 0, 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h of exposure. After 24 h of exposure, fish were transferred to fresh flowing water and allowed to depurate in the presence of the DHS. Three fish were sampled after 8, 24, and 72 h of depuration for each treatment. After sampling, the fish were frozen (-20 °C) until analyzed. Fish were not fed during the uptake experiments. Radiochemical liquid scintillation counting of the test compounds in fish was conducted by a microwave-assisted solubilization method (13). Briefly, individual fish were thawed, 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, Union Carbide) was added, and scintillation vials were loosely capped. Samples were processed in a microwave oven (Hotpoint, Model RE 968002) for 4 min at 188 W and then cooled 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 were counted for radioactivity on a Beckman LS 7800 liquid scintillation counter. No effort was made to quantify possible metabolic byproducts of LAS within the fish. Data Analysis for Bioavailability Experiments. Uptake and depuration rates were determined using a twocompartment model described by
dCf/dt ) kuCw - kdCf
(4)
where Cf is the toxicant concentration in the fish, t is time, Cw is the toxicant concentration in the water, and ku and kd are the uptake and depuration rate constants, respectively (13). Rate constants were estimated by the BIOFAC computer program (14). Depuration rate constants are expected to underestimate the elimination of the parent compound due to metabolism of the test substances. The 96-h LC50 (concentration lethal to 50% of sample individuals) values associated with 95% confidence intervals were estimated by trimmed Spearman-Karber (15) or Probit analysis (16). The trimmed Spearman-Karber analysis was conducted using a computer program developed by Burlington Research, Inc. Estimates for Kb‚oc (a biologically determined organic C sorption coefficient, described below) were performed using SAS version 5 (17). Slopes were compared using the methods described in Sokal and Rolf (18). Values for the biologically determined organic carbon sorption coefficient, referred to as Kb‚oc, were calculated from the uptake and toxicity data. The Kb‚oc value is similar to the Kb reported by others (2) and is analogous to the C-normalized association constants (Koc) calculated from the fluorescence quenching measurements. Derivation of
the Kb‚oc is described in Versteeg and Shorter (13) and was determined by
LC50oc/LC50f or Ku/Kuoc ) Kb‚oc[DHS] + 1
(5)
The estimation of Kb‚oc values from the uptake rate constants and the LC50 values assumes that LAS bound to DHS are not available for uptake or are not toxic.
Results and Discussion Fluorescence Quenching Experiments. In the absence of DHS, changes in the background electrolyte (NaCl, CaCl2, or synthetic river water) had no effect on the shape or the intensity of the fluorescence spectra of LAS. The addition of SRHA to C12-LAS solutions caused a decrease in fluorescence emission intensity, but no change was observed in the peak position. Thus, DHS quenching of LAS fluorescence could be quantified with measurements of fluorescence intensities at the emission maximum (288 nm). The emission intensity of DHS alone was always
