Environ. Sci. Technol. 1984, 18, 187-192
Reverse-Phase Separation Method for Determining Pollutant Binding to Aldrich Humic Acid and Dissolved Organic Carbon of Natural Waterst Peter F. Landrum,” Sheila R. Nlhart, Brian J. Eadle, and Wayne S. Gardner
Great Lakes Environmental Research Laboratory, National Oceanic and Atmospherlc Administration, Ann Arbor, Michlgan 48104 A reverse-phase separation technique was used to determine the binding of “C-radiolabeled organic pollutants [benzo[a]pyrene, anthracene, biphenyl, p,p’-DDT,
2,4,5,2’,4’,5’-hexachlorobiphenyl,2,5,2‘,5’-tetrachlorobiphenyl, and bis(2-ethylhexyl) phthalate] to humic materials in aqueous solution. The humic-bound pollutant was separated from the “freely dissolved” pollutant by using a Sep-Pak C-18 cartridge; humic-bound pollutant passed through, while the unbound pollutants were retained by the column. The partition coefficient (grams of pollutant bound/gram of organic carbon)/(grams of pollutant freely dissolved/milliliter) did not depend on pollutant concentration but was inversely proportional to the concentration of dissolved organic carbon (DOC) in solution. At low DOC (1-2 mg of Aldrich humic acids L-l), the partition coefficient was approximately equal to the octanol-water partition coefficient and inversely proportional to water solubility. The partition coefficient for natural waters was approximately 1order of magnitude lower than that determined for the Aldrich humics a t similar DOC concentrations. The reverse-phase separation was simple and rapid and gave results similar to dialysis techniques. Introduction The physical state and environmental fate of organic pollutants, including pesticides, polychlorinated biphenyls (PCB), and polycyclic aromatic hydrocarbons (PAH), are complicated by the presence of humic and fulvic acids in the aquatic environment. Humic substances in water can increase the apparent water solubility of nonpolar compounds (I+), bind organic compounds either with covalent bonds, as charge-transfer complexes, by hydrogen bonding, or by van der Waals interaction (7-10),hydrolyze pesticides (11-13), interfere with PAH analysis in water (14, 15),photosensitize the pollutant degradation (16,17), and reduce the bioavailability of PAH to aquatic organisms (18, 19). Development of methods to measure the sorption of organic compounds to dissolved humic and fulvic substances is important for predicting pollutant fate in the aquatic environment. Several approaches, including dialysis (20),triacetate filter separations (21),fluorescent polarization techniques (22), and differential solubilization (23,24), have been described to estimate the binding of organic pollutants to humic substances and other large molecules such as proteins and lipoproteins. Our attempt to use dialysis techniques (20) to measure the binding of benzola]pyrene (BaP) resulted in a continually decreasing partition coefficient over time from 4 to 11 days; no apparent equilibrium was approached, a requirement for the dialysis technique. Fluorescence polarization currently requires a glycerol-water solution and a fluorescent pollutant for binding measurements. Since the triacetate filter sorption and separation has only been demonstrated for PCB, its general applicability is unknown. Finally, differential solubilization is a time-consuming technique, GLERL Contribution No. 349.
lacking sufficient precision (24). Our objective was to examine the partitioning behavior of a wide spectrum of compounds with a variety of natural organic matrices. Because none of the above techniques were suitable within our available resources, a complementary reverse-phase technique was investigated to determine pollutant-humic binding. Because humic-bound BaP and other PAHs can be separated from the “freely dissolved” BaP using XAD-4 resins (14, 15), we hypothesized that, in contrast to free PAH compounds, the humic-bound materials would quantitatively pass through a reverse phase column at pHs sufficiently high (>5) to polarize the humic acid compounds and prevent them from sorbing to the reversephase material. We here describe and demonstrate a reverse-phase technique to separate humic-bound pollutants from freely dissolved pollutants and thereby estimate partition coefficients. We compare this method of estimating partition coefficients with the dialysis technique developed by Carter and Suffet (20) and investigate its applicability to a range of environmental pollutants and humic materials. An attempt is made to define the limits of the method. Experimental Section Each of the radiolabeled compounds, [14C]BaP(29.7 mCi mmol-l; Amersham, batch 36), [14C]anthracene (3.3 mCi mmol-l; California Bionuclear Corp., lot no. 770824), [14C]biphenyl(15.9 mCi mmol-’; Pathfinder Laboratories, lot no. 90916), 2,4,5,2’,4’,5’-he~achloro[’~C] biphenyl (HCB) (14.0 mCi mmol-l; Pathfinder Laboratories, lot no. 6043), 2,5,2/,5’-tetra~hloro[~~C] biphenyl(TCB) (6.3 mCi mmol-l; Pathfinder Laboratories, lot no. 708091, [14C]bis(2-ethylhexyl) phthalate (DEHP) (7.32 mCi mmol-I; Pathfinder Laboratories, lot no. 8003131, and [14C]-p,p’-DDT (13.4 mCi mmol-l; Pathfinder Laboratories, lot no. 811213), was assayed for radiopurity prior to use. All compounds used for the studies were determined to be greater than 98.8% pure by thin-layer chromatography or high-performance liquid chromatography in combination with liquid scintillation counting. Each of the compounds, [14C]BaP(0.5, 2.0, and 5.0 pg L-l, total solution concentration), [14C] anthracene, [14C]biphenyl,[14C]HCB,[14C]TCB,[14C]DEHP(10, 30, and 50 pg L-l), and [14C]-p,p’-DDT(1pg L-l), was added individually to respective Aldrich humic acid solutions containing 0, 2, 8, or 16 p g of dissolved organic carbon (DOC) mL-’ (pH 5.8-6.8) or a corresponding natural water prefiltered with glass-fiber filters having a nominal pore size in air of 0.3 pm (Gelman Science, Inc.). The natural waters were collected from the Huron River near Ann Arbor, MI, the Grand River near its mouth a t Grand Haven, MI, Lake Erie approximately 1mi off the mouth of the Raisin River a t Monroe, Mi, and Lake Michigan 3 mi offshore a point approximately 3 mi south of Grand Haven. The Aldrich humic acid was prepurified by acid precipitation (twice) and dialysis to remove salt (25). Pollutants were added to 100 mL of humic acid solution
Not subject to U.S. Copyrlght. Published 1984 by the American Chemical Society
Environ. Sci. Technol., Vol. 18, No. 3, 1984
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or natural water and allowed to equilibrate at room temperature for a t least 18 h. Separation of humic-bound BaP from free BaP was initially attempted by using reverse-phase high-pressure liquid chromatography. The components were separated on an 8-cm column packed with Vydac 201SC using a Waters 6000A pump with distilled water as a mobile phase. The flow rate ranged from 0.5 to 3 mL min-l. All separations for partition coefficient determinations were made with Sep-Paks. Two 2-mL aliquots were taken for total 14Cactivity. A 10-mL glass syringe (prewashed once with 10 mL of acetone, twice with 10 mL of deionized water, and once with 5 mL of test solution) was used to pass each solution through a C-18 Sep-Pak cartridge (Waters Associates), and three 2-mL aliquots were taken at a flow rate of approximately 12 mL min-'. One Sep-Pak cartridge was used for each set of four Aldrich humic acid concentrations or each natural water sample for each compound studied. 14Cactivity was determined by liquid scintillation counting with a Beckman LS 150 counter. The samples were counted in 3a70B scintillation cocktail (Research Products International). After the background was subtracted, the samples were corrected for quench by the external standards ratio method. Dissolved organic carbon (DOC) was determined with an Oceanography International corporation Carbon Analyzer (PIR 2000) (26). DOCS were determined prior to the addition of the pollutant to avoid measuring carbon from the carrier, acetone or methanol. For comparative dialysis experiments, solutions containing humic acids and the compound under study were placed in dialysis bags made from membranes with either M, 1000,2000, or 3500 cutoffs, after the method of Carter and Suffet (20). The smallest pore size that would allow apparent equilibrium was used to help ensure that no humics would pass through the pores, particularly when natural waters were studied. The bags containing 5 mL of the humics and pollutant solution were placed in distilled water (100 mL). The concentration of compounds was determined after 4 days both inside and outside the bag. Initial experiments were terminated on a time sequence basis of 2,4,5, and ll days to determine the time required to attain equilibrium. The partition coefficient for dialysis was determined by assuming that the concentration of the compound outside the bag equaled the freely dissolved concentration inside the bag. The partition coefficient was calculated as grams of pollutant per gram of DOC divided by grams of pollutant per milliliter. With this experimental configuration compounds not at equilibrium will have very high partition coefficients that will decline with time as equilibrium is approached. Since humic materials are poorly sorbed to reverse-phase materials at pH values >5 (27) and humic-bound BaP and other PAH can be separated from freely dissolved BaP with XAD-4 resins (14,15),we assumed that humic-bound compounds would pass through the Sep-Pak with the humic acids. Measurements of DOC before and after separation confirmed that Aldrich humic acids quqtitively pass through Sep-Pak cartridges (98 f 3%, n = 8, X f SD) over the range of humic concentrations studied. The partition coefficient was calculated as the ratio of the grams of pollutant per gram of DOC (determined from the '4c activity passing through the Sep-Pak and measured DOC) divided by the grams of pollutant per milliliter trapped (freely dissolved). The amount trapped was determined initially from the 14C activity eluted with the methanol but later from the difference of the I4C activity total per milliliter minus the 14C activity per milliliter 188
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Flow (ml . min-1) Flgure 1. Increase in humlc-bound benzo[a Ipyrene with increased flow through an 8-cm reverse-phase column.
passing through the Sep-Pak. Determination of the freely dissolved fraction by difference was faster and easier than eluting with methanol and did not have the potential drawback of errors due to incomplete elution of the compounds by methanol. Accountability (amount of humicbound 14Cactivity passing through the column plus the amount eluted from the Sep-Pak with methanol divided by the total 14C activity from the original solution) was nearly quantitative, 92 f 8% (Xf 1 SD). In contrast, water containing no humic material yielded low accountability (67 f 12%,8 f SD, n = 3) because methanol did not quantitively remove bound contaminant from the Sep-Pack. The grams of pollutant were determined from the 14Cactivity and the specific activity of the compound tested. The dialysis method was compared to the reverse-phase method using Student's t test. Interaction of pollutant and humic concentrations on the partition coefficients was determined from multiple linear regression; the slopes of the lines were compared to zero.
Results and Discussion Factors Affecting Reverse-Phase Separation. Initial separation of humic-bound and dissolved BaP by reverse-phase high-pressure liquid chromatography indicated that the amount of humic-associatedBaP varied with flow rate but approached an asymptotic value (Figure 1). Slow flow rates and a relatively long column appeared to allow sufficient contact time to alter the binding equilibrium; some BaP apparently desorbed from the humic material and reduced the calculated partition coefficients. The much shorter (1 cm long) (2-18 Sep-Pak columns had sufficient capacity to absorb the freely dissolved compound but allowed fast flow rates (N12 mL mi&) using a syringe. The minimal contact time between the column and humic-pollutant complex minimized the potential for pollutant-humic acid complex dissociation as a result of column interactions. In the absence of humic acid, no breakthrough was found for BaP, anthracene, biphenyl, or DDT with the volumes (150-200 mL) required for one set of humic acid solutions a t each pollutant concentration and flow rates of 12 mL min-l. However, some breakthrough of TCB, HCB, and DEHP occurred in the absence of humic acids even a t low flow rates. To compensate for this breakthrough, we used an empirical correction factor when the amount of breakthrough in the absence of humics was small, The correction factor was determined by the following equation: N
BF = CH(BNH/CNH) where BF = relative amount of breakthrough (ng mL-l), CH = concentration of pollutant in the presence of humics
Table I. Comparison of Partition Coefficients for Aldrich Humic Acids Determined Using Dialysis and Reverse-Phase Separation Methods
compound
pollutant concn, ng mL-'
partition coefficients ( X 10-4)
-
reverse phasea
dialysis'
p,p'-DDT
1.14 i 0.1 13.0 i 1.0 28.0 t 6.0 ( n =6) ( n =6 ) ( n =4 ) anthracene 55.96 i 0.5 0.9 i 0.1 2.9 i 0.3 ( n = 9) ( n =6 ) ( n =4) biphenyl 14.3 i 1.5 0 . 1 c 0.01 NDb ( n = 3) ( n =3) 2,5,2', 5'19.6 f 0.3 3.5 i 0.1 2.6 .I 0.2 tetrachlorobiphenyl ( n = 3) ( n= 3) ( n = 2)
Time (days) Figure 2. Decline in humic-benzo[a ] pyrene partition coefficient determined using the dialysis technique with increased equilibration time.
' xt SE. All experiments were performed at 9.3-9.5 mg L-' humic acid determined as dissolved organic carbon. ND = no partitioning detectable.
formation on their use with different pollutants. Paired experiments with both methods determined the respective partition coefficients of several of the compounds for both Aldrich humics and DOC in natural water. If the binding of biphenyl with Huron River DOC is omitted, the reverse-phase partition coefficients correlate well with those from dialysis for both Aldrich humic acid [log reversephase partition coefficient = 0.54 + 0.84 log dialysis partition coefficient r2 = 0.70 (Table I)] and dissolved organic carbon in natural waters [log reverse-phase partition coefficient = 1.67 0.51 log dialysis partition coefficient r2 = 0.68 (Table II)]. If both methods measure the same binding sites and if the experiments are not biased, the regression line should pass through the origin and have a slope of unity. Comparison of the two methods showed significantly higher partition coefficients for dialysis with natural waters (p
