Environ. Sci. Technol. 2004, 38, 476-483
Modeling Tetracycline Antibiotic Sorption to Clays RAQUEL A. FIGUEROA, ALLISON LEONARD, AND ALLISON A. MACKAY* Environmental Engineering Program, University of Connecticut, 261 Glenbrook Road, U-2037, Storrs, Connecticut 06269-2037
Sorption interactions of three high-use tetracycline antibiotics (oxytetracycline, chlortetracycline, tetracycline) with montmorillonite and kaolinite clays were investigated under varied pH and ionic strength conditions. Sorption edges were best described with a model that included cation exchange plus surface complexation of zwitterion forms of these compounds. Zwitterion sorption was accompanied by proton uptake, was more favorable on acidic clay, and was relatively insensitive to ionic strength effects. Calcium salts promoted oxytetracycline sorption at alkaline pHs likely by a surface-bridging mechanism. Substituent effects among the compounds in the tetracycline class had only minor effects on sorption edges and isotherms under the same solution pH and ionic strength conditions. At low ionic strength, greater sorption to montmorillonite than kaolinite was observed at all pHs tested, even after normalizing for cation exchange capacity. These results indicate that soil and sediment sorption models for tetracyclines, and other pharmaceuticals with similar chemistry, must account for solution speciation and the presence of other competitor ions in soil or sediment pore waters.
Introduction Veterinary pharmaceuticals are the subject of growing attention because of their potential to pollute both aquatic and soil environments (1-3). Direct release of these pharmaceuticals to the environment occurs in agricultural practice with the discharge of excess feed pellets and animal wastes in aquaculture and by land application of manure containing unmetabolized drugs from animal husbandry. The presence of veterinary antibiotics and other pharmaceuticals, in soils, sediments and aquatic environments is of concern because these bioactive compounds may promote the development and spread of antibiotic-resistance among bacterial populations (4-7) or induce biological responses in nontarget organisms (see review by Daughton and Ternes (2)). Accurate assessments of organism exposures to pharmaceuticals are hampered currently by a lack of applicable environmental fate models. For example, Tolls (8) demonstrated that the models developed to describe sorption of nonpolar organic compounds that interact chiefly by van der Waals forces (e.g., refs 9-11) fail to describe veterinary antibiotic sorption to soils and sediments. Carbon-normalized sorption coefficients (Koc) for pharmaceuticals are one to several orders of magnitude greater than predicted by typical sorption models, * Corresponding author phone: (860)486-2450; fax: (860)486-2298; e-mail:
[email protected]. 476
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and Koc exhibits no correlation with compound hydrophobicity (8, 12). Because sorption is a key determinant of the bioavailability, reactivity and mobility of compounds in the environment, there is a need to understand interactions of pharmaceuticals with soils, sediments and aquatic particles so that robust sorption predictors can be developed for these compounds. The chemistry of tetracycline antibiotics suggests that cation exchange with soil and sediment clay components will be important sorption mechanisms for these antibiotics. As shown in Figure 1, tetracycline antibiotics are positively charged in strongly acidic solutions and anionic under alkaline conditions. Thus, cationic tetracycline species could neutralize negative charge sites under acidic conditions (high sorption) but would be repulsed from clay surfaces at high pH. In general, oxytetracycline sorption capacities of illite and bentonite were decreased when solution pH was increased from strongly acidic to weakly alkaline conditions (13-15). In addition, sorbed oxytetracycline and chlortetracycline were also released from montmorillonite that was immersed in phosphate or citrate buffer, but no release was observed in distilled water at the same pH (13). Slight decreases in montmorillonite sorption capacity were observed when ionic strength was increased from millimolar to molar levels (14). These observations were qualitatively consistent with a cation exchange sorption mechanism in which the presence of other cations from buffer salts or ionic strength adjustments could alter solid-solution distributions of tetracycline antibiotics by competing for negative charge sites on clays. Spectroscopic studies to probe tetracycline-clay interaction mechanisms confirmed the importance of cation exchange and also noted the contribution of zwitterion species to sorption (18). The lack of peak shifts between the IR spectrum of tetracycline sorbed to sodium montmorillonite at pH 1.5 and dissolved in solution at pH 1.5 indicated that the surface sorbed species was the cationic form. The absorbance bands of the sorbed species were little changed when the experiment was repeated at pH 5.0 even though the solution phase speciation is dominated by the zwitterion at pH 5 (Figure 1). The similarities in the sorbed-species spectra at pH 5.0 and 1.5 suggest that zwitterion sorption is accompanied by proton uptake, and the resultant sorbed species is the fully protonated cation. Protonation of the sorbed zwitterion was supported by theoretical calculations of an effective pKa1 for tetracycline that was 2.9 units higher in a sodium montmorillonite suspension than in a homogeneous solution (19). Although these studies were conducted with tetracycline, the structural similarity of tetracycline compounds suggest that cation and zwitterion sorption will be important interactions for all antibiotics in this class. The purpose of this investigation was to quantify sorption parameters for tetracycline antibiotics and clays that can be applied under a range of pH and ionic strength conditions. Previous studies of tetracycline antibiotic sorption to clays did not provide a sufficient range of study conditions from which to derive sorption parameters that could be used for making predictions of sorption under other pH and ionic strength conditions. In this study, pure clay minerals, montmorillonite and kaolinite, were chosen as sorbents so that tetracycline interactions with these solids would be isolated from sorption interactions with other components of whole soils or sediments (e.g., hydrophobic partitioning to organic matter, surface complexation to oxyhydroxides). Clay sorption edges and sorption isotherms were obtained under a range of solution conditions by varying pH, ionic 10.1021/es0342087 CCC: $27.50
2004 American Chemical Society Published on Web 12/03/2003
TABLE 1. Clay Characteristics clay type and treatment property surface area
(m2/g)b
cation exchange capacity at pH 5.5 (mequiv/kg)c exchangeable cations (first wash) (mequiv/kg)c Na Ca Mg Al Fe a
FIGURE 1. Tetracycline chemistry and solution speciation. All antibiotics in the tetracycline class have the common base structure which is shown with environmentally relevant proton exchange sites. R1 and R2 groups are reported for oxytetracycline (OTC), tetracycline (TET), and chlortetracycline (CTC). The speciation diagram was calculated for oxytetracycline but is similar for TET and CTC due to the closeness of pKa values all of these compounds. pKa values were obtained from ref 16 for OTC and TET and from ref 17 for CTC. strength, sorbate concentration, and sorbate structure. Sorption distributions were used to assess contributions of cation and zwitterion species to total sorption of tetracycline antibiotics.
Experimental Methods Materials. Hydrochloride salts of tetracycline, chlortetracycline, and oxytetracycline were used as received (USP grade) from USB Corporation (Cleveland, OH). Sodium and calcium salts and sodium hydroxide were all certified ACS grade from Fisher Scientific (Fair Lawn, NJ). Fisher trace-metal grade hydrochloric acid was also used. China clay kaolin powder was from Central Scientific Company (now Sargent-Welch, Buffalo Grove, IL). A low iron-oxide content montmorillonite clay (20) (K-10, acid-washed) was obtained from SigmaAldrich Corp. (St. Louis, MO). All solutions were prepared in high-purity water (18 MΩ‚cm, Barnstead NANOpure Diamond, Dubuque, IA). Analytical. Antibiotic concentrations were quantified by UV/vis spectroscopy (CARY 1, Varian, Inc.). Tetracycline compound absorbances were measured at 360 nm. This wavelength was an isosbestic point for these antibiotics over the pH ranges tested, and, thus, the molar extinction coefficient was independent of sorbate speciation. Absorbance readings were corrected for light scattering effects of unsettled clay particles by subtracting absorbance readings at 700 nm. Metal concentrations were quantified by inductively coupled plasma spectroscopy, mass spectrometry (PE SCIEX, Elan 6000, Environmental Research Institute, Storrs, CT). Preparation and Characterization of Clays. Sodium- or calcium-saturated forms of the clays were prepared by washing 200 mg (kaolinite) or 800 mg (montmorillonite) of clay twice with 20-30 mL of either 1 M sodium chloride or
From ref 25.
b
montmorillonite
kaolinite
Na-saturated 623 ( 3 (600-800)a untreated 429 ( 26 (800-1500)a untreated
Na-saturated 25.5 ( 1 (7-30)a untreated 44 ( 3.6 (20-150)a untreated
26 ( 16 47 ( 9 61 ( 17 120 ( 10 2.5 ( 0.4
1.4 ( 0.4 1.5 ( 0.5 1.1 ( 0.3 6(4 1 ( 0.7
mean ( std dev (n ) 2). c mean ( std dev (n ) 3).
1 M calcium chloride and then rinsing with at least two washes of high-purity water. Sodium and calcium were chosen as representative mono- and divalent cations, respectively, and have been used in previous sorption studies (18, 14). An experiment was also conducted using montmorillonite K-10 without any saturating treatment. This was designated as the “untreated” montmorillonite although it was already acidwashed as received from Aldrich (21). As such, the interlayer cations of the untreated montmorillonite consist of both protons and aluminum cations (22). Clay samples were characterized by measuring the specific surface area and the cation exchange capacity. Surface area measurements of the cation-saturated clays were made using the ethylene glycol monoethyl ether (EGME) method (23). The EGME measurements include the total of both external and interlayer areas of the clay sample. Experimental values were in good agreement with previously reported values for montmorillonite and kaolinite (Table 1). Cation exchange capacity (CEC) was determined by compulsive exchange with barium (24) using untreated forms of the clay with the final wash adjusted to either pH 5.5 or 8.5. No significant difference in cation exchange capacity at these two pHs was observed for either clay type although the CEC for K-10 montmorillonite was lower than typically observed for montmorillonites (Table 1). The first wash was collected for analysis of the surfaceexchangeable cations on the untreated clay (Table 1). Sorption Edge Experiments. Ionic strength and pH effects on antibiotic sorption to the clays were assessed using sorption edge experiments. Duplicate batch polypropylene tubes were set up by prewetting sorbents in high-purity water for at least 24 h. A mass of 16 mg of montmorillonite or 200 mg of kaolinite was added to a total of 40 (montmorillonite) or 42 (kaolinite) milliliters of solution containing 10 mM sodium bicarbonate buffer. A constant mass of an antibiotic compound was spiked to each tube from a concentrated stock solution that was prepared fresh daily. The equivalent initial concentration of oxytetracycline in each tube was 0.083 mM. Tube pHs were adjusted between 4 and 9 (0.5 unit increments) with the addition of small volumes of hydrochloric acid or sodium hydroxide. No significant dissolution of clay was seen over this pH range as evidenced by dissolved aluminum and iron measurements. Clay-free controls were prepared at pH 4 to monitor tetracycline compound losses. Sample tubes were wrapped in foil to inhibit photodegradation and allowed to mix end-over-end for a period of 24 h. At the end of this equilibration time, tubes were centrifuged for 60 min at 12 500g’s to separate the clay solids. An aliquot of the supernatant was removed for UV/vis analysis. This experimental protocol was repeated for solutions with total ionic strengths of 110 mM and 510 mM. For these two cases, VOL. 38, NO. 2, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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100 mM and 500 mM, respectively, of sodium chloride was added to the 10 mM bicarbonate buffer before pH adjustment. Sorption coefficients were calculated from mass balances on the equilibration flasks. Mass balances of 100 ( 8% (n ) 12) were achieved by a desorption step (discussed below), and, thus, sorbed antibiotic concentrations were calculated by difference
Cs )
(C0 - Cw)Vw Ms
(1)
where Cs (mmol/kg) is the equilibrium sorbed antibiotic concentration, Co and Cw (mmol/L) are the initial and equilibrium aqueous antibiotic concentrations, respectively, Vw (L) is the volume of solution, and Ms (kg) is the mass of the clay. Cs values were used to calculate sorption coefficients as defined by
KD )
Cs Cw(CEC)
(2)
where KD (L/equiv) is the overall sorption coefficient, Cw (mmol/L) is the equilibrium aqueous antibiotic concentration, and CEC (equiv/kg) is the cation exchange capacity of the clay. Sorption coefficients were normalized to the sorbent cation exchange capacity because interactions of the tetracycline antibiotics with specific charge sites on the clay surface were anticipated. Uptake of sorbates by clays occurs by an adsorptive process, and, thus, mass-normalized KD’s (e.g., Lw/kgclay) are not appropriate. Sorption coefficients can be readily converted to surface area-normalized coefficients using the clay surface area data given in Table 1. Sorption Isotherms. Sorption isotherms were obtained to assess antibiotic distributions between solid and aqueous phases as a function of sorbate concentration. Batch experiments were conducted using an experimental protocol similar to that described for the sorption edge experiments. In this case, the solution pH was maintained constant at pH 5.5, while the mass of an added antibiotic was varied. Duplicate tubes were prepared at each of the 5 antibiotic concentrations from initial concentrations of 0.008 mM to 0.23 mM. As per the sorption edge experiments, the solid-to-water ratio was 4 × 10-4 kg/L (montmorillonite) or 4.76 × 10-3 kg/L (kaolinite), and ionic strengths of 10, 110 and 510 mM were tested by sodium chloride addition, if necessary, to 10 mM sodium bicarbonate buffer.
Results and Discussions The only antibiotic loss from solution in this study was sorptive uptake by the clay sorbents. A minimum of three clay-free controls was included in each experiment to account for sorptive losses to polypropylene or losses due to chemical transformations. Compound recoveries from controls were 100 ( 7% (n ) 36) for oxytetracycline and 99 ( 4% (n ) 7) for tetracycline. Chlortetracycline exhibited losses of up to 11% over the 24 h equilibration time. The instability of chlortetracycline under alkaline conditions has been reported in prior studies (15). Complete compound mass recovery was also observed in the clay-containing tubes that showed a decrease in aqueous antibiotic concentrations by the end of the equilibration period. To verify the mass balance in these tubes, the solution phase was adjusted to pH 9 to convert all tetracycline species to anionic forms that have a repulsive interaction with the clay surfaces. Mass recoveries were 100 ( 8% (n ) 12). In a comparison test, some clay solids were extracted with methanol to close the mass balance. Less than 5% of the sorbed mass could be extracted by this common solid-phase extraction procedure, indicating 478
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FIGURE 2. Sorption of oxytetracycline to sodium-saturated montmorillonite (b) and kaolinite (O) clays at a total ionic strength of 10 mM. The solid line (s) denotes the best fit of eq 3 to the montmorillonite data, and the dashed line (- -) denotes a model fit that considers only cation species interactions with the clay. that this extraction method is not appropriate for antibiotic sorbate-clay sorbent pairs. We note that the use of pH buffer implicitly affected the magnitude of sorption coefficients because all measurements were made in the presence of competing sodium ions. Strictly, the reported sorption coefficients and capacities will only describe sorption distributions in other systems with similar concentrations of sodium. Tetracycline antibiotic sorption to clays with sodium concentrations that differ from those tested here can be estimated with knowledge of the selectivity coefficient in the presence of Na+ (see “Mechanism of Oxytetracycline Sorption to Clay”). In the case of calciumcontaining systems, equilibrium thermodynamic calculations indicated that calcite (and siderite) solubility was not exceeded over pH range tested. Sorption Edge Experiments. Oxytetracycline sorption to montmorillonite and kaolinite showed a pH dependence that was consistent with the sorbate and clay chemistries (Figure 2). Clay surfaces are negatively charged over the entire pH range investigated because of isomorphic substitution in clay layers and variable charge edge sites (22), but the oppositely charged cationic oxytetracycline species is only dominant at pH values below 3.6 (Figure 1). Although oxytetracycline showed less sorption as the pH was increased, the sorption coefficients did not exhibit the characteristic “edge” that would be expected if only the cationic oxytetracycline species interacted with the clay surface. Rather, the sorption coefficients exhibited a gradual decrease as the solution pH was raised from 6 to 8. This decrease appeared to mimic the zwitterion abundance between pH 6 and 8 (Figure 1), indicating that the zwitterionic form of oxytetracycline was also an important sorbate at typical environmental pHs. The sorption edge data for oxytetracycline were fit with an empirical model to assess individual sorption coefficients for both the cationic and zwitterionic forms of this antibiotic. Given the near-complete mass recoveries when the solution pH was raised to 9, the model assumed that anionic species exhibited no interactions with the clay surfaces. Thus, the overall sorption coefficient at any pH was the sum of contributions from sorption of cationic species and zwitterionic species (26)
KD )
CsOTC+ + CsOTC0 Cw
OTC+
+ Cw
OTC0
1 (CEC ))K
D
OTC+
R+ + KDOTC0R0 (3)
where KD (L/equiv) is the total sorption coefficient, KDOTC+ and KDOTC0 (L/equiv) are the cation and zwitterion sorption
TABLE 2. Sorption Coefficients for Oxytetracycline and Tetracycline Species Oxytetracycline sorbent and solution conditions KdOTC+ (L/equiv) Na-montmorillonite 10 mM 110 mM 510 mM untreated montmorillonite 10 mM Na-kaolinite 10 mM 110 mM 510 mM
FIGURE 3. Sorption edges for oxytetracycline and Na-montmorillonite at three different ionic strengths: 10 mM (b), 110 mM (O), and 510 mM (2).
r2
70800 ( 3600a 10700 ( 1100 5600 ( 800
3500 ( 100 1500 ( 100 1700 ( 100
0.98
59800 ( 3200
8500 ( 300
0.96
8700 ( 1900 8750 ( 1200 1390 ( 980
1640 ( 110 1300 ( 90 1070 ( 90
0.80
KdTC0
r2
3000 ( 170
0.94
Tetracycline sorbent and solution conditions KdTC+ Na-montmorillonite 10 mM a
coefficients, respectively, and R+ and R0 are the mass fractions of cation and zwitterion in solution, respectively. The General Linear Model Procedure using the Separate Slopes Model (SAS Institute Inc., Cary, NC) was used to fit eq 3 to the sodium montmorillonite sorption edge. Best fits were obtained with KDOTC+ of 70 800 ( 3600 L/equiv and KDOTC0 of 3500 ( 100 L/equiv. For comparison, a fit assuming only cation interactions is also shown in Figure 2. This “cationonly” fit was obtained by setting KdOTC0 equal to 0 in eq 3 to yield KDOTC+ ) 138 000 L/equiv and clearly does not describe our observations. Although eq 3 is empirical and does not indicate the mechanism(s) of oxytetracycline interactions with montmorillonite, fits of eq 3 to sorption edges do give insight into the relative contributions of oxytetracycline species to the overall compound sorption. The oxytetracycline cation interaction with the clay surface was about 20 times stronger than for the zwitterion, according to the fit shown in Figure 2. Consequently, the cation was still a large contributor to overall oxytetracycline sorption even when the dominant solution phase species was the zwitterion. For example, at pH 5.5, R0 ) 0.978, while R+ is only 0.0115. Comparison of the two terms in eq 3 after substituting these R values indicated that 19% of the overall sorption could be ascribed to the cation and the remaining 81% was the zwitterion contribution, despite its greater abundance in the solution phase. The zwitterion contribution did not account for greater than 99% of the oxytetracycline sorbed until the solution pH was increased to pH 7. Effect of Ionic Strength. Ionic strength effects on oxytetracycline sorption were probed by conducting sorption edge experiments at three different salt concentrations. Sodium montmorillonite sorption coefficients showed similar decreasing trends with increasing pH at all ionic strengths; however, less overall sorption was observed with increasing salt concentration (Figure 3). To assess how salt concentrations changed cation sorption and zwitterion sorption to the clay, these sorption edges were also fit with eq 3 (Table 2). For the 110 mM and 510 mM fits, the oxytetracycline solution speciation (i.e., R values) was corrected for ionic strength effects using the Davies equation (27). These fits indicated that the oxytetracycline cation always exhibited a stronger interaction with the sodium montmorillonite than did the zwitterion species; however, oxytetracycline cation sorption was more sensitive to salt concentration than was zwitterion sorption (Table 2). KDOTC+ decreased by a factor of almost 13 when the ionic strength was increased from 10 mM to 510 mM, while KDOTC0 only decreased by a factor of about 2. Thus, the interactions of individual species must be considered
KdOTC0 (L/equiv)
54900 ( 4900
( one standard deviation; n ) 22 samples.
when assessing ionic strength effects on the overall sorption of oxytetracycline to montmorillonite. Mechanisms of Oxytetracycline Sorption to Clay. The model fits that are reported in Table 2 were used to calculate system-independent interaction parameters for oxytetracycline and sodium-montmorillonite. Since the KDOTC+ and KDOTC0 fits were obtained from an empirical model (eq 3), they apply only to the ionic strength conditions tested. To extend these results to other solution conditions, the mechanism by which the OTC cation and zwitterion species interact with the montmorillonite surface must be considered. The strong dependence of KDOTC+ on ionic strength suggests that cation exchange is the most important mechanism for this species. Cation exchange has been proposed for the related compound tetracycline, based upon sorbed and dissolved species IR spectra obtained at pH 1.5 where the cation accounts for 99% of all dissolved compound mass (Figure 1) (18). Oxytetracycline cation sorption coefficients (KDOTC+) were used to calculate a selectivity coefficient for this cation in the presence of the competing sodium ion. The experimental conditions of our sorption studies were best described as a minor constitutentsoxytetracycline (10 mM). For this case, the selectivity coefficient, Ks,OTC:Na (equiv/equiv), is related to the oxytetracycline cation sorption coefficient as follows (28, 29):
Ks,OTC:Na ) KDOTC+ [Na+]
(4)
If eq 4 is log-transformed, the relationship between the salt concentration and cation sorption coefficient should have a slope of -1 and an intercept equal to the log of the selectivity coefficient:
log KDOTC+ ) - log [Na+] + log Ks,OTC:Na
(5)
A linear regression of eq 5 using data from Table 2 gave a slope of -0.66 and an intercept corresponding to Ks,OTC:Na ) 3000 equiv/equiv (r2 ) 0.98). The change in KDOTC+ with an increase in salt concentration from 10 to 110 mM was about an order of magnitude as predicted by eq 4; however, KDOTC+ decreased by a factor of 2 when the salt concentration increased by a factor of about 5 from 110 to 510 mM. This trend contributed to the lower than expected slope for the fit of eq 5 and may be an artifact of oxytetracycline exclusion from montmorillonite interlayers at high ionic strengths. Norrish (30) observed the interlayer spacing of sodium VOL. 38, NO. 2, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Increase in solution pH over the duration of oxytetracycline sorption edge experiments with Na-montmorillonite (b) and untreated montmorillonite (O). The concentration of oxytetracycline sorbed by the surface complex mechanism was calculated using the best fit KDOTC0 values from Table 2 with the equilibrium aqueous phase concentrations of oxytetracycline in the zwitterion form at the final pH of each tube. All sorption edges were measured at an ionic strength of 10 mM sodium bicarbonate. montmorillonite to decrease sharply from 40 to 20 Å (OTC dimensions of 6 Å (18)) at ionic strengths of 0.3 M. Consequently, the selectivity coefficient is only reported to one significant figure. The low sensitivity of KDOTC0 to ionic strength relative to KDOTC+ suggests that a surface complexation mechanism is important for the oxytetracycline zwitterion species. Surface protonation has been proposed to explain sorption of amino acids and the structurally related compound, tetracycline, under solution conditions at which zwitterion species of these compounds dominate (18, 19, 31, 32). Surface protonation of oxytetracycline can be represented by the following reaction
OTC0 + ≡ - H+ ) ≡ - HOTC+
(6)
where OTC0 is the dissolved oxytetracycline zwitterion, and ≡-HOTC+ denotes the fully protonated sorbed oxytetracycline species. ≡-H+ denotes a surface-layer proton which could originate from water coordinated to the clay surface or to cations in the interlayer or from a proton balancing charge in the interlayer (31). This surface complexation mechanism was supported by several lines of evidence in our experiments. First, most tubes in the sorption edge experiments exhibited an increase in pH over the time course of the experiment that was proportional to the amount of oxytetracycline zwitterion sorbed (Figure 4). Second, the extent of zwitterion sorption was affected by the clay treatment. Greater overall oxytetracycline sorption was observed for untreated montmorillonite clay than for the sodium form (Figure 5). When the sorption edges were fit with eq 3, KDOTC+ for the untreated clay was not significantly different than for sodium montmorillonite (t-test, 95% C.I.); however, the zwitterion sorption coefficient, KDOTC0, was 2.4 times greater on the untreated clay (Table 2). Finally, the net increase in pH over the experiment duration was smaller for untreated montmorillonite (Figure 4). Considered as a whole, these observations suggest that the solution phase served as a source of free protons when the proton distribution in the interlayer was perturbed by reaction with oxytetracycline. In the case of the acidic untreated montmorillonite, less uptake of free protons occurred because more surface-bound protons are available from hydronium ions or easily-hydrolyzed aluminum cations in the inner layer (22, 33). Thus, the enhanced uptake of oxytetracycline zwitterions on the more acidic clay 480
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FIGURE 5. Sorption edges for oxytetracycline and montmorillonite with different treatments: sodium-saturated (b), calcium-saturated (4), and untreated clay (O). All sorption edges were measured at an ionic strength of 10 mM sodium bicarbonate. surface is consistent with the surface protonation mechanism of eq 6 and the prior microscopic observation that the structurally related compound tetracycline is sorbed in cationic form at pH when the zwitterion species dominates in solution (18). Various modeling approaches have been used to derive system-independent or intrinsic sorption parameters for surface complexation reactions such as eq 6 (e.g., refs 34 and 35). We attempted to derive an intrinsic sorption coefficient for the oxytetracycline zwitterion using FITEQL (36). Input data points of the zwitterion sorption coefficient (KDOTC0R0) as a function of pH were computed using best fit KDOTC0 values from Table 2 with the appropriate R0. Poor convergence of the model fits occurred, perhaps because the KDOTC0 values used to calculate data points contained uncertainties from apportioning total oxytetracycline sorption to cation and zwitterion contributions (e.g., eq 3 fits to Figures 3 and 5). Despite the lack of an intrinsic parameter for zwitterion sorption, application of KDOTC0 values reported in Table 2 to predict oxytetracycline sorption at intermediate ionic strengths would give estimates within the generally accepted bounds of 2 to 3 times factor of variation for sorption coefficients (26) because of the low sensitivity of zwitterion sorption to ionic strength. Note that hydrophobic interactions of the net neutral oxytetracycline zwitterion with montmorillonite can be neglected. We measured the octanol-water partition coefficient for the oxytetracycline zwitterion to be 0.11 at pH 5.5 (unpublished results). Negligible clay sorption of oxytetracycline was estimated from hydrophobic mineral sorption models (26). In addition, a methanol wash of the clay recovered less than 5% of sorbed oxytetracycline mass, indicating that physical adsorption by van der Waals interactions is not a significant interaction mechanism for oxytetracycline. Cation Effects. The type of cations initially present on the clay surface will affect oxytetracycline sorption by competing for surface sites. A small test for cation competition was performed by desorbing oxytetracycline from sodium montmorillonite in the presence of either sodium or calcium at pH 5.5 and 110 mM ionic strength. The apparent KDs were 7000 ( 480 and 5700 ( 600 (all n ) 3), respectively, for Na+ and Ca2+. The mean apparent KD for calcium appeared to be lower suggesting that less sorption occurred in the presence of Ca2+. At low pHs, when oxytetracycline sorption occurs by cation exchange, lower sorption coefficients were observed for calcium-saturated montmorillonite than for the sodiumsaturated form (Figure 5). Generally, the replacing power of a cation increases with its charge (22), and thus if calcium
FIGURE 6. Effect of antibiotic structure on the sorption of antibiotics to Na-montmorillonite: oxytetracycline (b), tetracycline (4), and chlortetracycline (O). All sorption edges were measured with an ionic strength of 10 mM sodium bicarbonate. is preferentially held by the clay, then it could displace oxytetracycline at pH 5.5 and lower pHs more effectively than would sodium cations, giving rise to lower apparent KDs. Furthermore, clay surface charge neutralization by calcium is enhanced relative to sodium by its more tightly bound waters of hydration (ref 37 as cited by ref 38). In contrast, calcium enhanced the sorption of oxytetracycline to montmorillonite under alkaline conditions. Sorption coefficients for calcium-saturated montmorillonite were higher than for sodium-saturated clay at pH greater than 7 (Figure 5) even though the use of sodium buffer displaced about 30% of calcium from Ca-saturated montmorillonite. Studies with structurally related compounds suggest that the sorption enhancement observed for Ca-montmorillonite occurred through cation bridging of calcium to oxytetracycline anions (18). Accounting for the distribution of calcium and antibiotic between clay and solution phases in our experiment gave a ratio of 1 mole of oxytetracycline sorbed per 2 mol of sorbed calcium. Calcium is known to complex tetracycline antibiotics in solution with complexation constants for the divalent anions of oxytetracycline, tetracycline and chlortetracycline being an order of magnitude greater than for the monovalent anions (17, 39). Thus, if cation bridging were the mechanism of oxytetracycline sorption to calcium montmorillonite, even greater sorption coefficients would be expected at pH above 9 when divalent anionic species dominate solution speciation (Figure 1). Sorption edges were not conducted under a wide enough range of pH conditions to assess the contributions of the divalent oxytetracycline species or to assess surface complexation constants for oxytetracycline sorption by calcium bridging. Effect of Tetracycline Structure. Sorption edges were determined for several compounds in the tetracycline class to assess effects of chemical structure on compound interactions with Na-montmorillonite. All compounds in this class of antibiotics share the same base structure and functional groups as tetracycline and thus are expected to show similarities in sorption mechanisms with clays, as has been qualitatively indicated in previous studies (13-15). The compounds tested here were tetracycline, oxytetracycline (differs from the base structure by the addition of a hydroxyl group), and chlortetracycline (addition of a chloro group) (Figure 1). Substituents on the base tetracycline structure had little effect on the sorption of these antibiotics to montmorillonite. The sorption edges for oxytetracycline, chlortetracycline and the base compound tetracycline were very similar (Figure 6). Equation 3 was fit to the sorption edge data to account for slight differences in compound pKas by deriving species-
specific sorption coefficients (Table 2). There was no significant difference between the cation sorption coefficients for tetracycline and oxytetracycline or the zwitterion sorption coefficients for these two compounds at the 95% confidence interval. The closeness between the overall KDs for these two compounds and chlortetracycline suggested that speciesspecific sorption coefficients for chlortetracycline would be of similar magnitude to the values reported for oxytetracycline and tetracycline. Sorption coefficients are not reported for chlortetracycline above pH 7 because of compound instabilities in alkaline solution (15). Thus, although substituent structures affect tetracycline stability, little difference in sorption under the same solution conditions is observed from compound to compound in the tetracycline class of antibiotics. Sorption Isotherms. Isotherms for individual tetracycline species could only be obtained for the zwitterion. The previous calculations using the oxytetracycline sorption edge parameters indicated that isotherms measured at pH 7 would contain zwitterions as the only sorptive species in solution. To achieve similar conditions for the cation, the solution pH must be less than 2.5. Isotherms were not measured under this acidic condition because data interpretation would be confounded by dissolution of the clay. Rather, an intermediate pH of 5.5, typical of groundwater or pure rainwater was chosen, recognizing that these were “mixed” isotherms with both solution-phase cation and zwitterion mechanisms contributing to oxytetracycline sorption. The sorption isotherms were nonlinear under all conditions tested (Figure 7). Sorption isotherms were fit with a Langmuir isotherm that assumed only one type of surface site
Cs )
QbCw 1 + bCw
(7)
where Q (mmol/kg) is the maximum adsorption capacity and b (L/mmol) is a measure of the energy of adsorption. The oxytetracycline sorption capacity on montmorillonite was not significantly different at all ionic strengths tested (Table 3). Assuming an oxytetracycline molecule occupies a surface area of 190 Å2 (18), a sorption capacity of 72 mmol/ kg corresponds to 14% clay coverage on an area basis or 20% of the available cation exchange sites. Slightly greater sorption capacities were observed for tetracycline and chlortetracycline than oxytetracycline; however, it is not clear whether these differences could be attributed to lower polarity of tetracycline and chlortetracycline or uncertainty in the fits (e.g., note wide variation in “b” values, Table 3). The low sensitivity of oxytetracycline sorption capacities to ionic strength at pH 5.5 (Table 3) is consistent with sorption edge observations showing low sensitivity of KdOTC0 values to ionic strength (Table 2). At pH 5.5, about 80% of the total oxytetracycline sorption occurred through the zwitterion complexation mechanism, and fits to the sorption edges had KDOTC0 values that varied by only a factor of 2 over the ionic strengths tested. For an initial concentration of 0.083 mM oxytetracycline, KD was estimated to be 950 L/kg at 10 mM ionic strength according to eq 7 with Q ) 72 mmol/kg and b ) 51 L/mmol, compared to 1800 L/kg with eq 3 using KDOTC+ ) 70 800 L/equiv and KDOTC0 ) 3500 L/equiv at pH 5.5. Thus, results from sorption isotherms were consistent with observations from the sorption edges. Clay Type Effects. The differences in oxytetracycline sorption to different clay types were probed by comparing sorption to montmorillonite and kaolinite clays. The sorption edges for these two clays indicated a more favorable interaction of the fully protonated oxytetracycline molecule with montmorillonite than kaolinite: Greater oxytetracycline sorption to montmorillonite than to kaolinite was observed VOL. 38, NO. 2, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 7. Sorption isotherms for (a) oxytetracycline and Na-montmorillonite with 10 mM ionic strength; (b) oxytetracycline and Namontmorillonite with 110 mM ionic strength; (c) tetracycline and Na-montmorillonite with 10 mM ionic strength; and (d) oxytetracycline and Na-kaolinite with 110 mM ionic strength. Solid lines are best fits of a Langmuir isotherm (eq 7) (parameters reported in Table 3).
TABLE 3. Langmuir Fits of Tetracycline Sorption Isotherms for Clays b (L/mmol)
r2
51 ( 22 64 ( 27 38 ( 14
0.77 0.78 0.71
3(3 8(2 2(2
0.76 0.95 0.88
Tetracycline Na-montmorillonite - pH 5.5 10 mM 112 ( 18 110 mM 111 ( 28
34 ( 19 11 ( 5
0.73 0.82
Chlortetracycline Na-montmorillonite - pH 5.5 10 mM 167 ( 28 110 mM 56 ( 9
14 ( 6 183 ( 215
0.88 0.68
sorbent and solution conditions
Q (mmol/kg)
Oxytetracycline Na-montmorillonite - pH 5.5 10 mM 72 ( 7a 110 mM 68 ( 6 510 mM 58 ( 6 Na-kaolinite - pH 5.5 10 mM 30 ( 27 110 mM 13 ( 2 510 mM 20 ( 14
a
( standard error (n ) 12).
for all pH values, even after sorption coefficients were normalized to the number of sorption sites (CEC) (Figure 2). Whether differences in sorption between the two clay types could be attributed to the oxytetracycline cation or zwitterion interaction mechanisms was less evident. The “knee” between pH 6 to 8 in the sorption edges for kaolinite were less pronounced than in the case of montmorillonite; however, a 2-species model (i.e., eq 3) was still required to fit the total oxytetracycline sorption as a function of pH (Table 2). In the case of kaolinite, fitting errors were from 20 to 70% for KDOTC+ and KDOTC0. Consequently, only the 10 mM case gave KDOTC+ and KDOTC0 that were significantly greater for montmorillonite than for kaolinite (t-test, 95% C.I.) (Table 2). The kaolinite sorption capacities on a mass basis were 2 to 3 times lower than for montmorillonite and corresponded to coverage of 482
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45% of the clay surface area (Table 3). The differences in magnitude of oxytetracycline sorption interactions with these two clays indicate that clay type must also be considered when estimating tetracycline antibiotic interactions with soil clay components. Environmental Significance. The strong interaction of tetracycline antibiotics with clays indicates that environmental fate models must account for sorption to clay mineral components of soils and sediments to predict accurately the mobility of these, and other ionogenic pharmaceutical compounds. The sorption edge modeling approach undertaken in this work enables estimation of both cation and zwitterion species contributions to overall pharmaceutical sorption to clays, and hence effects of pH and ionic strength can be incorporated into sorption models for these compounds. The modeling results demonstrated the importance of tetracycline zwitterions as sorptive species, although the net charge of these compounds is zero. In addition to tetracyclines, other pharmaceutical classes, such as fluoroquinolone and sulfonamide antibiotics, also have speciation dominated by zwitterion forms at environmentally relevant pHs. Thus, the general trend of cation plus zwitterion species interactions with soil and sediment clay components is expected to be true for these pharmaceutical classes as well as for tetracycline antibiotics. Note that the KDs estimated for pure clay minerals may not be directly applicable for estimating sorption to clay components in real soils and sediments because clays in environmental solids may be coated with organic matter or oxide particles, thus blocking pharmaceutical sorption sites. Furthermore, the presence of competing solutes in soil and sediment pore waters will also affect the mobility and bioavailability of tetracycline and other ionogenic pharmaceutical compounds. The results of this research indicate that pharmaceutical sorption interactions with clays are controlled by the ionic functional groups of the base compound structure within a pharmaceutical class, with little influence of other nonionic substituents on the base structure. The soil KDs for fluoro-
quinolone antibiotics reported by Nowara et al. (40) also showed little difference among the compounds tested, despite differences in substituents on the 4-fluorquinolone carboxylic acid base structure. Thus, identification of the key sorption interactions of the base structure within a pharmaceutical class, using approaches similar to this study, may yield reasonable models for predicting sorption of other compounds in the class, provided that nonpolar substituent effects are reasonably small (i.e. ∆ log Kow ) 3 log units).
Acknowledgments Funding for this research was provided by the NSF (Grant BES-0225696) and by the University of Connecticut through the Research Foundation, the Department of Civil and Environmental Engineering, and the Environmental Engineering Program. We thank two anonymous reviewers for their thoughtful comments and discussion of this manuscript.
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Received for review March 10, 2003. Revised manuscript received September 30, 2003. Accepted October 17, 2003. ES0342087
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