Environ. Sci. Technol. 2005, 39, 2660-2667
Interaction of Tetracycline with Aluminum and Iron Hydrous Oxides CHENG GU Environmental Chemistry and Technology Program, University of Wisconsin-Madison, Madison, Wisconsin 53706 K. G. KARTHIKEYAN* Department of Biological Systems Engineering, Environmental Chemistry and Technology Program, 460 Henry Mall, University of Wisconsin, Madison, Wisconsin 53706
The effect of solution chemistry (pH, sorbate-to-sorbent ratio, ionic strength (I)) and reaction time on the sorption of tetracycline to the hydrous oxides of Al (HAO) and Fe (HFO) was examined. Sorption to HAO increased with increasing pH up to pH 7 (no such trend for HFO) above which it decreased at higher pH values for both the hydrous oxides. Experimental results indicate that ligand-promoted dissolution is occurring during tetracycline sorption to these hydrous oxides. Ligand-promoted dissolution was more significant for HAO than HFO attributable to the difference in labile surface sites between these two sorbents. The ability of tetracycline to form strong complexes with Al and Fe will increase the solubility of these minerals. Sorption of tetracycline was quite rapid and equilibrium was achieved after 8 h. However, soluble metal (Me: Al or Fe) concentrations attained equilibrium only after 24 h. Ligandpromoted dissolution appears to be a two-step process; initially, 1:1 Me-tetracycline soluble complexes are formed and as the reaction progresses 2:1 complexes existed. Increasing I (from 0.01 to 0.5 M) decreased the sorption extent only at higher sorbate-to-sorbent ratios suggesting the dominance of inner-sphere type complexes at low equilibrium tetracycline concentrations. Spectroscopic evidence indicates that tricarbonylamide and carbonyl functional groups of tetracycline could be responsible for sorption to mineral surfaces. Our research findings will increase understanding of the environmental occurrence, fate, and transport characteristics of antibiotics, which are considered as emerging organic contaminants.
Introduction Antibiotics are used extensively as human infection medicine, veterinary medicine, and husbandry growth promoters. In the United States (U.S.) alone, annual antibiotic production exceeded 50 million lbs by the late 1990s (1). Currently, over 70% of all the antibiotics manufactured are for agricultural use (2). Most of the antibiotics are poorly absorbed by human and animals after intake, with about 25-75% of added compounds leaving the organisms unaltered via feces or urine (3). Because of the dispersion of manure and sewage sludge in fields as fertilizers, antibiotics have the potential to reach soil and aquatic environments. In the first national recon* Corresponding author phone: (608)262-9367; fax: (608)262-1228; e-mail:
[email protected]. 2660
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naissance survey on emerging organic contaminants conducted by the U.S. Geological Survey, antibiotics were detected in half of the sites from a network of 139 streams across 30 states (4). A total of six antibiotic compounds were detected including two sulfonamides, one tetracycline, fluoroquinolone, macrolide, and trimethoprim in our Wisconsin (WI) statewide survey of wastewater treatment plants. Compared to other well-known xenobiotics such as pesticides, PAHs, and PCBs, there is very little information available on the fate and transformation of antibiotics in soil/water environments. A striking difference between the antibiotics and the above xenobiotics is in their hydrophobicity (log Kow of well-known xenobiotics ) 3-7) and aqueous solubility (10-5 to 0.1 mM for these xenobiotics). The high polarity (e.g., log Kow ) -1.97 to -0.47* for tetracycline, *pHdependent (5)) and aqueous solubility (0.52-117* mM for tetracycline, also *pH-dependent (6)) of antibiotics can enhance their environmental mobility. Sorption processes, in particular, are important, since association of antibiotics with mineral particles and organic matter will determine their transportability in surface runoff, leachability through soils, and mobility in aquifers. Bioassay studies also revealed that the antibiotics would lose their antibacterial activity when they are adsorbed to soils (7, 8). Tetracyclines constitute one of the most important antibiotic families, ranking second in production and usage worldwide (9). Tetracyclines have been detected in soils (5, 10, 11), surface waters (12), groundwater samples collected near waste and wastewater lagoons (13), and hog lagoon samples (14). In our statewide survey of wastewater treatment plants in WI, the compound tetracycline was the most frequently detected antibiotic (among 25 antibiotics), being present in 80% of the wastewater influent and effluent samples. As shown in Figure 1a, tetracycline possesses tricarbonylamide (C-1:C-2:C-3), phenolic diketone (C-10:C11:C-12), and dimethylamine (C-4) groups that confer a marked pH dependent behavior on solubility and lipophilicity. Tetracycline has three pKa’s (3.3, 7.68, and 9.69) causing it to exist as a cationic, zwitterionic, and anionic species under acidic, moderately acidic to neutral, and alkaline conditions (Figure 1b), respectively. The ionization behavior can be expected to significantly influence tetracycline sorption to soil components. Hydrous oxides of aluminum (Al; HAO) and iron (Fe; HFO) are important mineral components of environmental particles. In highly weathered soils, HAO/HFO can account for as much as 50% of the total soil mass (15). Poorly crystalline or amorphous Al and Fe oxides are short-range-ordered minerals and exist as a discrete phase or in association with clay minerals and organic matter. Although they may not be found in large quantities in soils, these minerals are considered as major “sinks” for many inorganic and organic contaminants because of their high surface area and reactivity (16-18). Although there are a few studies on the interaction of tetracycline with clay minerals (e.g., 5, 10, 19, 20), investigations related to tetracycline-hydrous oxide systems are rare. It is fairly well-known that sorption of chelating compounds, such as ethylenediaminetetraacetic acid (EDTA) and salicylic acid, possessing carboxylic and phenolic functional groups capable of forming stable complexes with metals (Me), will significantly increase the solubility of oxide minerals through ligand-promoted dissolution (21-25). Although there is a lack of agreement on the coordination sites, many studies have shown that tetracycline forms strong complexes with various Me cations in solution (26-32). Since tetracycline 10.1021/es048603o CCC: $30.25
2005 American Chemical Society Published on Web 03/12/2005
FIGURE 1. (a) Structure and (b) pH-dependent speciation of tetracycline (TC). possesses similar functional groups and can form strong Me complexes, ligand-promoted dissolution can be expected to occur during its interaction with Al and Fe hydrous oxides. The major goal of this study, therefore, was to understand the role of hydrous oxide minerals (HAO and HFO) in influencing the environmental fate and transformation of tetracycline. Specific focus was on the effect of important solution chemistry variables (pH, ionic strength (I), sorbateto-sorbent ratio) and reaction time on the sorption of tetracycline to HAO and HFO. Spectroscopic methods were used to elucidate the mechanisms of interaction.
Materials and Methods Materials. Tetracycline hydrochloride was obtained from Sigma-Aldrich Chemical (St. Louis, MO) and was used without any purification. Important physicochemical properties of tetracycline are (5, 6) MW: 444.43; aqueous solubility: 0.52117 mM; log Kow: -1.97 to -0.47; and pKa: 3.3, 7.7, and 9.7. The tetracycline solution was thoroughly mixed with radiolabeled 7-3H-tetracycline (specific activity ) 5 Ci mmol-1, American Radiolabeled Chemicals Inc.) to provide a stock with 95% at pH between 4.2 and 9.3 (Figure SI-1 in Supporting Information) indicating its stability against degradation. However, it is important to confirm whether epimerization is occurring in the presence of hydrous oxides. Using analytical standards,
FIGURE 4. (a) UV spectra of tetracycline (TC, 0.05 mM) in the presence and absence of Al as a function of pH (molar ratio of tetracycline:Al ) 1:10); (b) UV spectra of supernatant remaining after interaction of tetracycline with HAO as a function of pH ([TC]initial ) 0.1 mM; ionic strength ) 0.01 M NaCl; equilibration time ) 24 h). The UV spectra for tetracycline-Al complexes formed in solution ([TC] ) 0.05 mM; molar ratio of tetracycline:Al ) 1:10 at pH 5.02) and for tetracycline ([TC] ) 0.05 mM; pH ) 4.51) are also included for comparison. we determined the RT in the HPLC chromatogram for 4-epitetracycline and tetracycline to be 8.91 and 9.46 min, respectively. The HPLC chromatograms of the supernatant remaining after reaction with HAO and HFO (Figures SI-2 and SI-3 in Supporting Information, respectively) clearly showed that the major peak present is due to free tetracycline and that contributions from 4-epitetracycline are insignificant. Also, the tetracycline peak height in the supernatant was inversely related, not to the extent of sorption (as determined by LSC), but to the sum of sorption amount and the magnitude of difference between HPLC and LSC measurements. As shown in Figure SI-4 (Supporting Information), the UV spectrum of 4-epitetracycline is similar to that of tetracycline (especially between 300 and 400 nm), and those for the supernatant from reaction with HAO resembled the UV spectra of Al-tetracycline complexes (Figure 4b). Therefore, epimerization of tetracycline cannot account for the observed differences between HPLC and LSC measurements. A concomitant increase in soluble Al and Fe concentration was observed as the initial tetracycline level was increased (Figure 3a and 3b), which also correlated well with the difference in sorption amounts determined using HPLC and LSC. Therefore, we formulated a hypothesis that ligand-promoted dissolution of HAO and HFO is occurring during tetracycline sorption, and the difference between independent measurements is due to the formation of tetracycline-Me (Me: Al or Fe) complexes. Chromatographic separation and direct quantification of Al/Fe-tetracycline complexes proved to be extremely difficult. We hypothesize that the Al-tetracycline complex is multivalent with a high polarity causing difficulties in chromatographic separation/quantification. The tetracycline-promoted dissolution was more pronounced for HAO than HFO. The solubility of HAO increased significantly in the presence of tetracycline with an increase of up to 2 orders of magnitude noticeable at 10-3 M. Also, increasing levels of tetracycline produced a proportional increase in HAO dissolution. In comparison, the effect on HFO was limited (note: difference in y-axis scale for Figure 3a and 3b). Tetracycline forms strong complexes with Me such as Al and Fe as supported by the formation constants (log K) of 12.5 and 13.4, respectively (39). These values compare favorably with other well-known chelating agents (log K for Fe complexation with nitrilotriacetic acid, citric acid, and EDTA is 15.9, 11.4, and 25, respectively (40); log K for Al-EDTA complexation is 19.07 (41)), except EDTA which has higher log K values. Figure 4a shows the UV spectra of tetracycline at different pH in the presence and absence of Al. In the absence of Al,
the UV spectra of tetracycline were similar in the pH range of 1.93∼6.32, with the characteristic absorption peak at a wavelength of 360 nm (used in HPLC analysis as well). On the other hand, the UV spectra of Al-tetracycline complexes were pH dependent. At pH 1.72, significantly lower than the pKa1 of tetracycline (pH ) 3.3), the UV spectrum was unaffected by the presence of Al. However, with increasing pH, a significant shift in the absorption peak (to a wavelength of 390 nm) occurred (Figure 4a). It, therefore, appears that the tricarbonylamide group (pKa1) in ring A might be involved in complexation reaction with Al. As shown in Figure 4b, the UV spectra of supernatant after interaction with HAO matches very well with that of Al-tetracycline complex and, importantly, are different from that of tetracycline and 4-epitetracycline (Figure SI-4 in Supporting Information contains UV spectra for 4-epitetracycline at various pH values). With Fe absorbing significant UV radiation between 300 and 400 nm, it was not possible to distinguish the band for Fetetracycline complexes. Effect of Sorbate-to-Sorbent Ratio and Ionic Strength. Isotherms for tetracycline sorption onto HAO and HFO (Figure 5) indicate that I effect on tetracycline sorption is dependent on surface coverage. An increase in I (from 0.01 to 0.5 M NaCl) lowered the extent of sorption only at surface coverages exceeding 0.073 and 0.036 mol kg-1 for HAO and HFO, respectively, and the corresponding equilibrium tetracycline concentrations are 0.347 and 0.182 mM. The isotherms were generated at a pH of 5.3 when the zwitterionic form is the predominant tetracycline species. Therefore, increased competition from both Na+ and Cl- with increasing I can be expected to lower sorption levels. However, a lack of competitive effect at low surface coverages is suggestive of initial complex formation via the inner-sphere (I. S.) mechanism (17, 42). At higher surface coverages, the isotherm data indicates the likelihood of the presence of both I. S. and weak outer-sphere (O.S.) type complexes. The isotherm data were all adequately described using the Freundlich isotherm equation, and the parameters are listed in Table 1. The n values were always less than 1 (Table 1), suggesting that the sorption sites on hydrous oxide minerals are not homogeneous (34). The sorption capacity (Kf) decreased with increasing I attributable to the effect of competing ions on tetracycline sorbed as O.S. complexes. The isotherms resembled the L-type curves that are normally observed when the adsorbate has a high affinity for the sorbent at low surface coverage and a decreasing affinity with increasing surface coverage (34). The isotherm for HAO at 0.01 M I displayed a significant increase in sorption capacity VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Isotherms for tetracycline sorption to (a) HAO and (b) HFO at different ionic strength (I) values (pH ) 5.3 ( 0.05; equilibration time ) 24 h). Ce is the equilibrium tetracycline concentration and qe is the amount of tetracycline sorbed onto the hydrous oxides. Error bars ((1 standard deviation) if not shown are within the symbols.
FIGURE 6. Surface charge characteristics of HAO and HFO as a function of increasing tetracycline surface coverage (pH ) 5.3 ( 0.05; ionic strength ) 0.01 M NaCl; equilibration time ) 24 h). qe is the amount of tetracycline sorbed onto the hydrous oxides. Error bars ((1 standard deviation) if not shown are within the symbols.
TABLE 1. Freundlich Isotherma Parameters for Tetracycline Sorption to HAO and HFO sorbent
Kf
n
r2
Hydrous Al Oxide (HAO), pH ) 5.3 ( 0.05 ionic strength ) 0.01 M 150 0.95 ionic strength ) 0.1 M 118 0.93 ionic strength ) 0.5 M 83.0 0.91
0.99 0.99 0.99
Hydrous Fe Oxide (HFO), pH ) 5.3 ( 0.05 ionic strength ) 0.01 M 59.1 0.85 ionic strength ) 0.1 M 35.1 0.81 ionic strength ) 0.5 M 14.6 0.75
0.99 0.99 0.98
a Freundlich isotherm: q ) K × C n; where q is the amount of e f e e tetracycline sorbed onto the hydrous oxides in mol kg-1; Ce is the equilibrium tetracycline concentration in M; and Kf and n are dimensionless Freundlich isotherm constants.
at high surface coverage. As proposed by Molis et al. (21) and Poirier and Cases (43), during sorption at high tetracycline concentrations the adsorbed molecules would orient themselves parallel to each other to have the lowest Gibbs free energy. The adsorbed layer can accommodate additional tetracycline molecules via hydrophobic interactions. A progressive decrease in the surface charge of both the hydrous oxides (experiments at pH 5.3 ( 0.05, below the pHZPC) with increasing tetracycline sorption levels (Figure 6) can be considered to provide evidence for I.S. complex formation (17, 21). A sharp decrease in zeta potential at low 2664
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sorption densities (
