Sorption of Three Tetracyclines by Several Soils - ACS Publications

Aug 30, 2005 - STEPHEN A. SASSMAN AND. LINDA S. LEE*. Department of Agronomy, Purdue University,. West Lafayette, Indiana 47907. Tetracyclines ...
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Environ. Sci. Technol. 2005, 39, 7452-7459

Sorption of Three Tetracyclines by Several Soils: Assessing the Role of pH and Cation Exchange

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STEPHEN A. SASSMAN AND LINDA S. LEE* Department of Agronomy, Purdue University, West Lafayette, Indiana 47907

Tetracyclines (TCs) are widely used in veterinary medicine for treatment and prevention of disease and are present in animal waste products. Detection of TCs in soil, sediments, and water, and the growing concern of their potentially adverse effect on natural ecosystems have resulted in a need to understand their behavior in aqueous soil systems. TCs have multiple ionizable functional groups such that at environmentally relevant pH values, they may exist as a cation (+ 0 0), zwitterion (+ - 0), or a net negatively charged ion (+ - -), which complicates predicting their sorption, availability, and transport. We investigated the sorption of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) by several soils varying in pH, clay amount and type, cation exchange capacity (CEC), anion exchange capacity (AEC), and soil organic carbon in 0.01 N CaCl2, 0.001 N CaCl2, and 0.01 N KCl. All three TCs are highly sorbed, especially in acidic and high clay soils. When normalized to CEC, sorption tends to decrease with increasing pH. A sorption model in which species-specific sorption coefficients normalized to pH-dependent CEC +-0 (K+00 , and K+-) and weighted by the pHd , Kd d dependent fraction of each species fit the data well across all soils except for a soil rich in gibbsite and high values were more than an order in AEC. Resulting K+00 d and K+-of magnitude larger than values for either K+-0 d d +00 values such that Kd alone described most of the sorption observed as a function of pH for eight soils that varied in their mineralogy and pH (pH ranged from 4 to 8).

Introduction An estimated 16 million kilograms of antimicrobial chemicals are used annually in the United States. Approximately 70% of these are antibiotics used nontherapeutically in livestock production (1). The purpose of administering antimicrobials at sub-therapeutic levels is to promote growth in animals. These growth promoters are administered by addition to water or feed. TCs are the most widely used feed additives (1-3). TCs are poorly absorbed in the digestive tract with 50-80% excreted unmetabolized (4). Animal waste products are typically kept in an incubation pond for an average of 180 days and are then disposed of on arable land (5). TCs are quite persistent in soils and can accumulate with repeated manure application (6). Currently, the primary concern over the use of antibiotics in concentrated animal feeding operations and land application of manure is the * Corresponding author phone: 765-494-8612; fax: 765-496-2926; e-mail: [email protected]. 7452

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development and dissemination of antibiotic-resistant pathogens in the environment (7, 8). However, antibiotics and their transformation products are found to be persistent and/ or mobile in the environment; therefore, may also pose a threat to biota and disrupt indigenous microbial populations (9-11). TCs are amphoteric molecules having multiple ionizable functional groups (Figure 1) that exist predominantly as zwitterions (Figure 2) at pH values typical of the natural environment. These zwitterions tend to aggregate in aqueous and aqueous mixed solvent solutions with increasing aggregation in the presence of divalent cations (12-14). At alkaline pH values (pH > 7) where the hydroxyl groups (pKa2) become increasingly more negative and the C-4 nitrogen (pKa3) begins to deprotonate, TCs can complex with metals as has been observed spectroscopically for several metals including 1:1 and 2:1 metal-TC complexes with Ca and Mg, respectively (12-15). Both aggregation and the formation of metal-TC complexes can lead to enhanced aqueous solubility (12), changes in molecular conformation (12-15), and decreased gastrointestinal absorption (15). In a pH range of 2-6, TCs undergo a reversible epimerization at position C-4 to form 4-epi-TCs, which is enhanced by the presence of anions such as formate, acetate, citrate, oxalate, and phosphate (16). The 4-epi-TCs have been reported to exhibit greater water solubility and decreased antibacterial activity. Under acidic conditions, dehydration at position C-5a and at position C-6 occurs forming the anhydroTCs and epianhydroTCs. These products retain antibacterial activity and are reported to be more toxic to bacteria than the parent compounds (4). Under alkaline conditions, the hydroxyl group at C-6 of the TCs is cleaved forming the iso-TCs. Chlortetracycline is known to be reversibly transformed to ketochlortetracycline depending on pH and ionic strength (4). Sorption studies for TCs are limited and have focused primarily on isolated clays (17-19), organoclays (18), or humic materials (20). From these studies, cation exchange comes forth as the primary sorption mechanism responsible for sorption of TCs especially under acidic conditions, although several other secondary mechanisms have been proposed. In all studies, TC sorption decreased with increasing pH and increasing ionic strength, and sorption by clays appeared to be enhanced in the presence of Ca versus Na. Infrared spectroscopic and X-ray diffraction analyses of TC in aqueous Ca-montmorillonite and Na-montmorillonite systems (17) showed that TC sorbed through complexation with Ca in the interlayer at pH values between 1.5 and 8.7. At pH ) 11, Ca-TC complexes were observed, but appeared to be external to the clay inner-layer, and TC sorption was reduced. TCs may also associate with exposed Al ions on clay edges (18) in which sorption may also decrease with increasing pH. In the current study, we investigated the sorption of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) by several soils varying in pH, clay amount and type, cation and anion exchange capacities, and soil organic carbon from 0.01 N CaCl2, 0.001 N CaCl2, and 0.01 N KCl. We hypothesized that (1) cation exchange would dominate the sorption process; (2) differences in sorption between soils could be approximately described by soil CEC and pH; and (3) calcium complexation with the + - - species may enhance sorption at higher pH values. We also evaluated a simple empirical model for estimating sorption across soils using a weighted fraction of the species present as a function of pH and assuming species-specific sorption to cation exchange sites. 10.1021/es0480217 CCC: $30.25

 2005 American Chemical Society Published on Web 08/30/2005

FIGURE 1. Molecular structure and ionization constants of the TCs. Rica (24). EPA-14 is a soil from an eroded hillside in southeast Ohio (25). Eustis-26 is a subsurface soil from Florida. A1A2 and A3A2 are subsurface soils from Korea (26). Soils were air-dried, gently crushed to pass through a 2-mm sieve, thoroughly mixed, and stored in closed containers at room temperature prior to use. Soils were characterized in previous studies unless otherwise noted in Table 1. Soil cation exchange capacity (CEC) will increase with increasing pH due to the acid functional groups in organic matter and some clay edges. Therefore, CEC was determined using a KCl saturation method as described by Zelazny et al. (27) that allows for adjusting pH. CEC values were determined at the soil’s natural pH as well a few determinations above and below the natural soil pH to allow for proper estimation of CEC at the pH values measured at equilibrium in the sorption studies. FIGURE 2. Speciation of tetracycline as a function of pH.

Materials and Methods Chemicals. All chemicals were analytical-reagent grade or higher purity and solvents were HPLC grade. Tetracycline hydrate (99%), oxytetracycline dihydrate (g98%), and chlortetracycline hydrochloride (g97%) were purchased from Aldrich (Milwaukee, WI). Calcium chloride dihydrate, citric acid monohydrate, ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA), oxalic acid dihydrate, sodium acetate trihydrate, acetonitrile, and methanol were purchased from Mallinckrodt Baker (Paris, KY). Water was purified using a Nanopure system (Barnstead, Dubuque, IA). Soils. Eight soils representing a range in texture, pH, organic carbon (OC), and CEC were selected (Table 1). Drummer-1, Raub, and Toronto-4 soils are surface soils from Indiana (21-23). 7CB is a surface soil from Northern Costa

Sorption Studies. A batch-equilibration method was used to measure sorption of TCs by soil from an aqueous solution of 0.01 N CaCl2, 0.001 N CaCl2, or 0.01 N KCl. Sorption isotherms were measured using five initial concentrations ranging from 0.4 to 65 µmol/L (0.2-29 mg/L). This concentration range was chosen because incubation ponds receiving wastes from concentrated swine operations have been reported to contain concentrations of this magnitude (6, 7, 28, 29). A soil mass to solution volume ratio of 0.6 g to 35 mL was chosen so that the solution concentrations were above the limits of quantitation for the analytes. Soils were transferred into preweighed 40-mL glass centrifuge tubes equipped with Teflon-lined closures, a solution containing one of the TCs was added to the tube, and the tube was capped. Tubes were wrapped with aluminum foil to minimize photodegradation and placed on a rotary end-over-end mixer (30 rpm) at room temperature (22 ( 2 °C). Control samples with no soil containing analyte solution and with soil

TABLE 1. Selected Soil Propertiesa soil

pHb

OCc (%)

clayd (%)

CECe (cmolc/kg)

AEC (cmolc/kg)

ZPNCg

clay typeh

Drummer-1 (21) Raub (23) Toronto-4 (21) EPA-14 (25) Eustis-26 7CB (24) A1A2 (26) A3A2 (26)

7.49 6.03 4.18 3.80 5.35 5.30 5.52 4.87

2.91 1.35 1.34 0.48 0.47 7.5 1.38 0.70

21 24 21 64 2 7 41 82

26.5 14.6 11.3 18.6 0.76 18.9 1.27 3.41

ndi nd 0.03f nd nd nd 0.42f 0.38f

ndi nd I,K,V S>V>I>K S > I, K K > S, V, I K nd Gb > Go K > He

a Soil properties reported from other studies are cited after the soil ID. b pH of 0.6 g of soil mixed with 35 mL of 0.01 N CaCl in the absence 2 of TCs. c Organic carbon content estimated by loss on ignition. d Particle size determination by pipet method or hydrometer methods. e Cation exchange capacity using 1 M KCl at the soil pH listed (27). f Anion exchange capacity at the natural soil pH using 1 M KCl (20, 27). g Zero point of net charge. h Determined by XRD analysis of less than 2 µM fraction: S ) smectite, I ) illite, K ) kaolinite, V ) vermiculite, Gb ) gibbsite (AlOOH), Go ) goethite (FeOOH), He ) hematite (Fe2O3). i Not determined, which for ZPNC, values are likely less than 1.

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containing CaCl2 solution without analyte were processed with the sorption samples. After equilibration, tubes were centrifuged (630g for 20 min) and supernatant (≈33 mL) was decanted into wide-mouth amber jars. One drop of 6 M HCl was added resulting in pH values from 2 to 3 to minimize degradation, epimerization, and sorption to silanol groups that may be present in glassware. Residual solution remaining in the soil was determined gravimetrically and solute concentration in the residual solution was assumed to be the same as that measured in the supernatant. For estimating isotherm pH without introducing artifacts in the analysis of TCs, separate tubes were prepared at the lowest and highest applied concentration for each TC-soil isotherm and pH was measured after equilibration without centrifugation; TCs can adsorb to glass electrodes. Soil Extraction for TCs. Optimizing soil extraction efficiency was done using CTC as the probe compound and Toronto-4 as the model soil. Several aqueous-cosolvent solutions were tested that varied in the type and fraction of an organic cosolvent (ethanol, methanol, ethyl acetate, or dioxane alone or in binary mixtures), type and concentration of complexing agent (formic, citric, or oxalic acid, EDTA, or H3PO4), and type and concentration of salt (NaCl, CaCl2, NH4Cl). Soil (0.6 g) was pretreated with 25 mg/L chlortetracycline in 0.01 N CaCl2 in a manner analogous to the equilibration step previously described for the sorption studies. Following removal of the supernatant, soils were extracted with 25-30 mL of a variety of extracting solutions. From this screening test, extraction using two 30-mL portions of 0.5 M sodium chloride/0.25 M oxalic acid/ethyl alcohol (25/25/50 v/v/v) was selected for use in the sorption studies. The first and second extractions were conducted for 4 and 20 h, respectively, and combined supernatants were analyzed by HPLC. All sorption data were modeled with the Freundlich sorption model (Cs ) KfCwN) where Cs (mmol/kg) and Cw (mmol/L) are the equilibrium sorbed and solution phase concentrations, respectively, and Kf (mmol1-N LN kg-1) and N (unitless) are the Freundlich model coefficients representing sorption magnitude and nonlinearity, respectively. Isotherms were fitted with the Freundlich sorption model using SAS v. 8.2 as described in the equations above with forced zero intercepts and equal weighting across the data ranges. If the resulting fit included N ) 1 within the 95% confidence interval, the linear sorption model, Cs ) KdCw where Kd (L/kg) is the linear sorption coefficient, was used to fit the data. HPLC Analysis. Samples were analyzed on a Shimadzu high-performance liquid chromatography (HPLC) system equipped with a UV detector (SPD-10Avp; λ ) 370 nm), a fluorescence detector (RF-10AXL; Ex ) 390 nm, Em ) 512 nm), a 4.0 × 20 mm C-18 guard column, and a Supelcosil LC-18 reverse-phase column (4.6 × 150 mm, 5-µm particles; Supleco, Bellefonte, PA). The injection volumes were 200 µL, 25 µL, and 10 µL for analysis of the aqueous phase, no-soil control samples, and soil extraction samples, respectively. Mobile phase A was an aqueous solution containing 50 mM sodium acetate, 25 mM calcium chloride, and 12.5 mM EDTA adjusted to pH 6.5 using 2 M sodium hydroxide. Mobile phase B was methanol. The volume fraction of solvent B was 25, 35, or 45% for the analysis of OTC, TC, CTC, respectively. The mobile phase flow rate was 1.2 mL/min. External calibration curves were used to estimate sample concentrations from the integrated peak areas. Calibration curves were linear from 0 to 1 mg/L for the 200 µL injections and from 0 to 30 mg/L for the 10 and 25 µL injections. All samples were analyzed within 24 h; loss of parent compounds from acidified controls became apparent by 72 h. The limit of quantitation with fluorescence detection was 0.1 ng per injection. Note that TCs can undergo reversible epimerization in which the 4-epi-TCs tend to have decreased antibacterial activity and 7454

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TABLE 2. Extraction of Chlortetracycline from Toronto #4 Soil at a Soil Mass to Solution Ratio of 0.6 g:25 mL and a 20 h Extraction Time

extraction solution

extraction efficiency (%)

1 M formic acid 1 M citric acid 0.67 M oxalic acid 0.67 M oxalic acid/ethanol (50:50) 0.2 M NaCl/0.5 M oxalic acid/ethanol (25:25:50) 1 M NaCl/0.5 M oxalic acid/ethanol (25:25:50) 1 M NaCl/1 M oxalic acid/ethanol (25:25:50)

0.3 3.5 41.3 49.3 58.2 75.9 79.1

higher water solubility, thus potentially slightly different sorption affinities. HPLC conditions were not optimized to separate the epimers for independent quantification. Epimerization is reversible such that epimer ratios present in the aqueous soil slurries at equilibrium may be altered with extraction and analysis techniques. To properly assess the latter would have been a project in itself, which we chose not to undertake.

Results and Discussion Equilibration Time. CTC sorption was measured as a function of time for a low OC sandy soil and a high OC silty clay loam to determine an equilibration time that would be near equilibrium with minimal transformation or irreversible binding. CTC concentrations were analyzed in the aqueous phase at 2, 4, 8, 24, and 48 h and sorbed concentrations weremeasured by extraction in sacrificed samples at 4 and 23 h. Typical of sorption phenomena, over 50% of the sorption was attained in the first 4 h followed by a slow increase in sorption over time. Soil-water distribution coefficients at a given time (Kd,t) calculated using Cs estimated by loss of compound from the aqueous phase relative to applied aqueous concentrations (by difference) and by extraction were similar at 4 h. Kd,t values measured by extraction at 23 h were within 8% of the values estimated by difference at 7 h. Kd,t values estimated by difference after 48 h were within 10% of those estimated at 20 h; therefore, a contact time of 20 h was chosen for assessing reversible sorption processes near equilibrium. This contact time also resulted in a minimal difference between sorption isotherms constructed using Cs estimated by difference and measured directly by extraction. Extraction Efficiency and Mass Balance. Selection of a solvent for extraction was based on tests with CTC. A summary of a small subset of the extracting solutions that were screened for their ability to extract CTC from Toronto-4 soil is provided in Table 2. On the basis of extraction efficiency, extraction using two 30-mL portions of 1 M sodium chloride/ 0.5 M oxalic acid/ethyl alcohol (25/25/50 v/v/v) was selected, which resulted in a solution pH of below 2. The solution was gently heated with stirring to dissolve sodium oxalate before adding it to soils. Parent compound stability in the extracting solutions (no soil present) showed that only 8%, 4%, and 7 (Figure 2). Cation exchange is thermodynamically more favorable than hydrophobic partitioning-type processes (30); therefore, cation exchange may dominate even when only a small fraction of the aqueous-phase species exists as a cation (31). Cation exchange has been shown to dominate for other organic bases well above solution pH ) pKa + 2 (32). Greater sorption by the lower pH soils where TCs exist as a cation (+00) suggests cation exchange as a significant sorption mechanism. Also, although the net charge of a zwitterion is neutral (+-0) and the + - - species has a net negative charge, the negative and positive charges are spatially separated and may act independently similar to what has been shown for reactivity of soil cation and anion

FIGURE 3. Representative sorption isotherms for tetracycline, oxytetracycline, and chlortetracycline on several soils at 23 ( 3 °C with an equilibration time of 20 h. Applied concentrations: 0.3956 uM in 0.01 N CaCl2. Sorbed concentrations were calculated by the difference between the mass applied and measured in the aqueous phase after equilibration. exchange sites (33). Therefore, cation exchange may have a significant contribution to the overall sorption of TCs at pH values well above 5.5. Linear sorption coefficients for the TCs were normalized to cation exchange (K/d, L/eq) and plotted as a function of the soil pH measured in equilibrium with each TC (Figure 4) similar to what has been done for pH-adjusted clays (19). For soils exhibiting nonlinear behavior, K/d values were estimated at equilibrium aqueous phase concentrations (Ceq,w) of 5 × 10-5 mM using the Freundlich model fits; this Ceq,w was selected because it was within or very close to the Ceq,w range common to all TC-soil isotherms. Log K/d values decrease with increasing pH. The trend that emerges clearly demonstrates that CEC is a primary contributor to sorption of TCs by whole soils. CTC sorption for the gibbsite-rich variable-charge soil A1A2 appears greater than the trend from the other soils (Figure 4B) (note sorption isotherms for OTC and TC on this soil were not measured). The A1A2 soil has a zero point of net charge (ZPNC) of 5.1 (Table 1); therefore, at pH ) 5.5 (CTC sorption equilibrium pH), a significant number of both cation and anion exchange sites are available, which will favor sorption of the zwitterion. The other oxidic soil A3A2 has significant AEC; however, both the soil ZPC (1.7, Table 1) and AEC relative to CEC at the isotherm pH of VOL. 39, NO. 19, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Sorption Model Coefficients for Sorption of Tetracycline and Oxytetracycline from 0.01 N CaCl2 at 23 ( 3 °C after a 20 h Equilibration with Applied Concentrations of 0.4-65 µM (R2 Values Reflect the Goodness of the Model Fit) TC soil

Kfa or Kda,b

Drummer-1 Raub Toronto-4 EPA-14 Eustis-26

3102 ( 287 ( 41 104 601 ( 3844 312 447 ( 17 966 12.8 ( 0.9

OTC

Na

84c

0.74 ( 0.02 0.49 ( 0.02

R2

Kfa or Kda,b

0.980 0.997 0.962 0.937 0.995

1229 ( 28 92 ( 18 83 878 ( 2871 269 097 ( 13 271 13.5 ( 2.0

R2

N

0.988 0.991 0.967 0.941 0.982

0.59 ( 0.03 0.50 ( 0.03

Kf (mmol1-N LN kg-1), Kd (L/kg), and N (unitless). b If the Freundlich model fit resulted in an N estimate that included unity in its 95% confidence interval, then the linear isotherm model with a zero intercept was fit to the isotherm data. c ( standard deviation. a

TABLE 4. Sorption Model Coefficients for Sorption of Chlortetracycline from Three Electrolyte Solutions at 23 ( 3 °C after a 20 h Equilibration with Applied Concentrations of 0.4-65 µM (R2 Values Reflect the Goodness of the Model Fit) 0.01 N CaCl2 soil

Kfa or Kda,b

Drummer-1 5706 ( 98c Raub 304 ( 93 Toronto-4 90758 ( 3709 EPA-14 164973 ( 7343 Eustis-26 22.0 ( 3.4 7CB 732 ( 224 A1A2 204 ( 46 A3A2 423 ( 116

Na

0.01 N KCl

R2

Kfa or Kda,b

Na

0.992 6234 ( 235 0.68 ( 0.04 0.987 356 ( 145 0.958 39580 ( 2253 0.950 227 657 ( 6733 0.57 ( 0.03 0.986 17.1 ( 4.3 0.84 ( 0.04 ndd 0.60 ( 03 0.993 192 ( 38 0.65 ( 0.03 0.992 969 ( 192

0.001 N CaCl2

R2

Kfa or Kda,b

0.959 10494 ( 207 0.66 ( 0.05 0.968 778 ( 363 0.947 17662 ( 570 0.975 352 911 ( 10 491 0.53 ( 0.05 0.987 17.2 ( 5.1 0.988 ndd 0.57 ( 0.02 0.994 107 ( 28 0.72 ( 0.02 0.997 697 ( 236

Na

R2

0.991 0.73 ( 0.06 0.988 0.974 0.986 0.49 ( 0.06 0.981 0.51 ( 0.03 0.996 0.68 ( 0.04 0.99

a K (mmol1-N LN kg-1), K (L/kg), and N (unitless). b If the Freundlich model fit resulted in an N estimate that included unity in its 95% confidence f d interval, then the linear isotherm model with a zero intercept was fit to the isotherm data. c (standard error. d Not determined.

FIGURE 5. Sorption of CTC by pH-altered soils from a CTC concentration in 0.01 N KCl (K) or 0.01 N CaCl2 (Ca) yielding equilibrium CTC concentrations of Cw ) 0.01 ( 0.005 µM across soils shown as a function of pH for (A) log(Kd, L/kg) and (B) sorption normalized to CEC, log(Kd, L/eq).

FIGURE 4. Log of the CEC-normalized K/d values estimated at Cw ) 0.05 µM versus pH for (A) TC, OTC, and CTC in 0.01 N CaCl2 and (B) CTC in 0.01 N CaCl2, 0.001 N CaCl2, and 0.01 N KCl solutions. Circle about data groups reflects sorption by the same soil. 4.87 is much lower, thus reducing the effective AEC compared to the A1A2 soil (34). Recent work by Jones et al. (35) for sorption of OTC at a buffered pH of 5.5 for 30 soils showed no apparent correlation to CEC. However, the CEC reported 7456

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and used in the correlation was measured at the soils’ natural pH values, which ranged from 3.6 to 7.5, and not the CEC that was operational at buffered isotherm pH of 5.5. The effect of pH on TC sorption was confirmed by measuring CTC sorption in triplicate from a single CTC concentration in 0.01 N KCl by four soils (Drummer-1, Raub, Toronto-4, and EPA-14) for which the pH was altered by addition of HCl or KOH. The applied CTC concentration was varied to achieve similar aqueous CTC concentrations at equilibrium of Cw ) 0.01 ( 0.005 µM across soils. In a similar manner, sorption by pH-altered Drummer soil was also measured from 0.01 N CaCl2. Sorption clearly decreased with increasing pH (Figure 5A) except for Drummer soil. Drummer soil is high in OC, thus high in pH-dependent CEC; CEC

TABLE 5. Estimates of Species-Specific Sorption Coefficients Assuming Only Sorption through Cation Exchange Estimated at Equilibrium Concentrations of 0.05 µM chemical

electrolyte

na

b K+00 d

b K+-0 d

R2 b

CTC CTC CTC TC OTC

0.01 N CaCl2 0.001 N CaCl2 0.01 N KCl 0.01 N CaCl2 0.01 N CaCl2

5 5 5 4 4

3.72E+06 1.31E+07 5.10E+06 7.35E+06 6.43E+06

1.05E+05 0.00E+00 7.15E+04 1.47E+05 1.46E+05

0.82 0.86 0.97 0.97 0.98

K+d

c

0.00E+00 5.84E+04 1.30E+04 0.00E+00 0.00E+00

R2 c

d K+00 d

R2 d

0.87 0.88 0.89 0.98 0.98

4.17E+06 1.31E+07 4.78E+06 8.04E+06 7.14E+06

0.84 0.87 0.89 0.96 0.95

a Number of soils included in the fit. b K +00, K + -0, and the goodness of fit (R2) when K +00 and K +-0 optimized for best fit. c K + - - and R2 when d d d d d Kd+00, and Kd+-0 were fixed at previously determined values and only Kd+- - optimized for best fit. d Kd+00 and R2 when only Kd+00 is used to optimize for best fit to the data.

FIGURE 6. Fits of K/d ) Kd+ 0 0f+00 + Kd+ - 0f+ - 0 + Kd+ - -f+ - - (dash-dot line) and K/d ) Kd+ 0 0f+00 (solid line) to the CEC-normalized sorption coefficient (L/eq) at Cw ) 5 × 10-5 mM versus pH for each solute-electrolyte system where Kd+ 0 0, Kd+ - 0, and Kd+ - - are species-specific CEC-normalized sorption coefficients (L/eq), and f+00, f+ - 0, and f+ - - are the species fraction for +00, + - 0, and + - species, respectively. decreased from 22.59 cmolc/kg at a pH ) 7.98 to 8.75 cmolc/ kg at a pH of 5.59. When sorption is normalized to CEC, the resulting sorption coefficients (K/d, L/eq) decrease with increasing pH for all soils (Figure 5B). Electrolyte Effect. Trends in CTC sorption with soil isotherm pH values were similar regardless of the electrolyte composition (Figure 4B); however, the magnitude of sorption was impacted by changes in electrolyte composition. If cation exchange is the controlling process, decreasing the competing inorganic cation concentration (e.g., 0.01 to 0.001 N CaCl2) or reducing the selectivity of the inorganic cation (e.g., substituting K+ for Ca2+) is expected to yield the following trends at a given pH: sorption from 0.001 N CaCl2 > 0.01 N KCl g 0.01 N CaCl2. Ca2+ can also complex with CTC, which can further suppress sorption if complexes prefer water; Ca2+ has been shown to enhance the aqueous solubility of CTC (12). Complexation with Ca2+ may alternatively enhance sorption by serving as a bridge to the soil surface as has been observed for sorption of TC by pure clays relative to a Na+ solution (17-19). Assessing the electrolyte effects in this study

is further complicated by the shifts in soil-solution pH, and subsequently CEC, which occur upon electrolyte addition. However, when the differential shifts in pH and CEC are considered, the trend in sorption hypothesized assuming cation exchange as the controlling process is followed (Figure 4B). Empirical Modeling Assessment. Assuming cation exchange as the primary sorption mechanism and that each ionic species has its own sorption magnitude, similar to what has been done for other ionizable compounds on soils (36) and for OTC and TC on pure clays (19), the following weighted sum model was employed:

K*d ) Kd+00f+00 + Kd+ -0f+ -0 + Kd+ - - f+- where Kd+00, Kd+ -0, and Kd+ - - are the individual CECnormalized sorption coefficients (L/eq) at Cw ) 5 × 10-5 mM and f+00, f+-0, and f+ - - are the species fraction for +00, + - 0, and + - - species, respectively. This model inherently assumes sorption to only negatively charged sites; therefore, VOL. 39, NO. 19, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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differences in the resulting TC species-specific K/d values can be considered to reflect the influence of the negatively charged functional groups on the ability of TCs’ cationic functional group to interact with soil cation exchange sites. Of the species-specific sorption coefficients, Kd+00 was expected to be the greatest. To obtain Kd+00 and Kd+ - 0 values, the model was fit to all sorption data measured at pH e 6.2, where >95% was either +00 or + -0, thus less than 5% of the species was + - -. These two coefficients were then fixed and the model fit to all the data including data at pH > 6.2 to obtain an estimate of Kd+ - -. For comparison, model fits to the data using only Kd+00 were run as well. All coefficients were constrained to be g 0. Sorption data for the gibbsiterich A1A2 soil, which has a significant fraction of active anion exchange sites, were omitted from the modeling attempt. Model fits are summarized in Table 5. The expected trend of Kd+00 - - > Kd+-0 > Kd+ - - resulted for all systems except the CTC-0.001 N CaCl2. Also consistently for all soluteelectrolyte combinations, Kd+00 was orders of magnitude larger than either of the other two coefficients as expected. Similar Kd+00 values and goodness of fit resulted when fitting was done with only Kd+-0 (Kd+00 and Kd+ - - set to zero). FTIR data for OTC zwitterions sorbed to organo-treated clays in Na-dominated systems at pH ) 5 indicated the presence of hydrophobic interactions, which are not accounted for in this simple model (37). Given the small data set and the wide range in properties represented by the soils, further speculation is not warranted. However, the modeling exercise supports the hypothesis that cation exchange is the primary process driving sorption for the soil and electrolytes used in this study. Environmental Significance. Sorption and transport of tetracyclines (TCs) is complex given that they can exist as cations, zwitterions, and anions at environmentally relevant pH values, and undergo epimerization. Although there are several processes that may impact sorption of TCs, batch sorption data and modeling results in the current work clearly demonstrated that TC sorption is dependent on pH and cation exchange capacity (CEC). Similar to the findings of Figueroa et al. (19) for pure clays, all three TCs appeared to behave similarly regardless of their different structural substituents. However, these same studies as well as others with pure clays indicate that CEC alone is not sufficient to account for differences observed between different clay types. In the current study with natural soils, a single CECnormalized sorption coefficient for the cation (Kd+00) described most of the sorption observed from a given CaCl2 or KCl electrolyte solution as a function of pH for eight soils that varied in their mineralogy and pH (pH ranged from 4 to 8). We hypothesize that the apparent lack of correlation to CEC in a recent OTC sorption study with 30 soils buffered at a single pH valued of 5.5 (35) was because the CEC values used were measured at the natural soil pH and not at the isotherm pH of 5.5.

Acknowledgments This work was funded in part by the U.S. Environmental Protection Agency National Risk Management Research Laboratory (Cincinnati, OH) under Cooperative Agreement 82811901-0 and the School of Agriculture, Purdue University.

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Received for review December 14, 2004. Revised manuscript received May 7, 2005. Accepted July 15, 2005. ES0480217

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