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TECHNICAL REPORTS

Organic Compounds in the Environment

Effects of Ionic Strength, Temperature, and pH on Degradation of Selected Antibiotics

^a U.S. Geological Survey, Organic Geochemistry Research Lab., 4821 Quail Crest Place, Lawrence, KS 66049
^b Univ. of Missouri-Rolla, Dep. of Civil, Architectural, and Environmental Engineering, 1870 Miner Circle Dr., Rolla, MO 65401
^c U.S. Environmental Protection Agency-Region 7, P.O. Box 17-2141, Kansas City, KS 66117. Joint contribution of the U.S. Environmental Protection Agency (Project XP99795901-0), U.S. Geological Survey's Toxic Substances Hydrology Program, and infrastructural support from the Environmental Research Center at the Univ. of Missouri-Rolla

^* Corresponding author (kloftin{at}usgs.gov).

Received for publication May 7, 2007.

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
Aqueous degradation rates, which include hydrolysis and epimerization, for chlortetracycline (CTC), oxytetracycline (OTC), tetracycline (TET), lincomycin (LNC), sulfachlorpyridazine (SCP), sulfadimethoxine (SDM), sulfathiazole (STZ), trimethoprim (TRM), and tylosin A (TYL) were studied as a function of ionic strength (0.0015, 0.050, or 0.084 mg/L as Na₂HPO₄), temperature (7, 22, and 35°C), and pH (2, 5, 7, 9, and 11). Multiple linear regression revealed that ionic strength did not significantly affect ( = 0.05) degradation rates for all compounds, but temperature and pH affected rates for CTC, OTC, and TET significantly ( = 0.05). Degradation also was observed for TYL at pH 2 and 11. No significant degradation was observed for LNC, SCP, SDM, STZ, TRM, and TYL (pH 5, 7, and 9) under study conditions. Pseudo first-order rate constants, half-lives, and Arrhenius coefficients were calculated where appropriate. In general, hydrolysis rates for CTC, OTC, and TET increased as pH and temperature increased following Arrhenius relationships. Known degradation products were used to confirm that degradation had occurred, but these products were not quantified. Half-lives ranged from less than 6 h up to 9.7 wk for the tetracyclines and for TYL (pH 2 and 11), but no degradation of LIN, the sulfonamides, or TRM was observed during the study period. These results indicate that tetracyclines and TYL at pH 2 and 11 are prone to pH-mediated transformation and hydrolysis in some cases, but not the sulfonamides, LIN nor TRM are inclined to degrade under study conditions. This indicates that with the exception of CTC, OTC, and TET, pH-mediated reactions such as hydrolysis and epimerization are not likely removal mechanisms in surface water, anaerobic swine lagoons, wastewater, and ground water.

Abbreviations: -apoOTC, -apooxytetracycline • β-apoOTC, β-apooxytetracycline • A, collision factor • ACTC, anhydrochlortetracycline • ATET, anhydrotetracycline • CAS, chemical abstract service • CTC, chlortetracycline • E_a, activation energy • isoCTC, isochlortetracycline • LC, liquid chromatography • LNC, lincomycin • MS, mass spectrometry • MW, molecular weight • OTC, oxytetracycline: pKa, acid dissociation constant • psig, pounds per square inch gauge • SCP, sulfachlorpyridazine • SDM, sulfadimethoxine • SIM, selected ion monitoring • STZ, sulfathiazole • TET, tetracycline • TRM, trimethoprim • TYL, tylosin A • 4-epi-ACTC, 4-epi-anhydrochlortetracycline • 4-epi-ATET, 4-epi-anhydrotetracycline • 4-epi-CTC, 4-epi-chlortetracycline • 4-epi-isoCTC, 4-epi-iso-chlortetracycline • 4-epi-OTC, 4-epi-oxytetracycline • 4-epi-TET, 4-epi-tetracycline

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
UNMETABOLIZED antibiotics in the aquatic environment have drawn increased attention over the potential connection between antibiotic-resistant infections and human health. In 1999–2000, the U.S. Geological Survey conducted a national stream and ground water reconnaissance study of pharmaceuticals and other wastewater compounds where at least one antibiotic was detected in approximately 50% of the samples analyzed with concentrations generally less than 0.5 µg/L and a maximum concentration of 1.9 µg/L. This study demonstrated a wide-ranging presence of antibiotics in a variety of surface and ground waters (Kolpin et al., 2002).

Agricultural and human inputs are largely responsible for the environmental presence of antibiotics where multiple classes are in use. This study focused on the commonly used veterinary antibiotics chlortetracycline (CTC), oxytetraycline (OTC), and tetracycline (TET) which are in the tetracycline class of antibiotics, lincomycin (LNC) which is a lincosamide, sulfachlorpyridazine (SCP), sulfadimethoxine (SDM), and sulfathiazole (STZ) which are all sulfonamide antibiotics, trimethoprim (TRM) which is an unclassified antibiotic, and tylosin A (TYL) which is a macrolide antibiotic. These compounds possess different physical and chemical properties as well as various modes of antimicrobial action (Loftin et al., 2005; Qiang and Adams, 2004).

The tetracycline class of antibiotics is a combination of naturally derived and synthetically modified anthracycline compounds. CTC, OTC, and TET (Fig. 1a-c ) have been the most extensively used compounds in this class and were selected for further study. Known tetracycline class degradates are also shown in Fig. 1a-c. Hydrolysis of OTC and TET has been investigated previously by Vej-Hansen and Bundgaard (1978) and Vej-Hansen et al. (1978); however, temperatures in these studies were much higher than those typically encountered in the environment. In addition, data from these studies are not readily amenable to estimation of rate constants at lower temperatures because Arrhenius coefficients are not available.

View larger version (24K): [in this window] [in a new window]   Fig. 1a. Properties and structures of chlortetracycline and common degradates.

View larger version (28K): [in this window] [in a new window]   Fig. 1b. Properties and structures of oxytetracycline and common degradates.

View larger version (28K): [in this window] [in a new window]   Fig. 1c. Properties and structures of tetracycline and common degradates.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Properties and structure of tylosin A.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Properties and structures of other selected veterinary antibiotics studied.

	Materials and Methods

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
Reagents
The following antibiotics were secured from Sigma Chemical (St. Louis, MO): CTC, OTC, TET, TYL, SCP, SDM, STZ, and TRM. LNC was obtained from MP Biomedicals, LLC (formerly ICN Biochemical, Irvine, CA). All tetracycline-class degradates, which included anhydrochlortetracycline (ACTC), 4-epi-anhydrochlortetracycline (4-epi-ACTC), 4-epi-chlortetracycline (4-epi-CTC), isochlortetracycline (isoCTC), 4-epi-oxytetracycline (4-epi-OTC), -apooxytetracycline (-apoOTC), β-apooxytetracycline (β-apoOTC), 4-epi-anhydrotetracycline (4-epi-ATET), anhydrotetracycline (ATET), and 4-epi-tetracycline (4-epi-TET), were obtained from Acros Organics (Geel, Belgium). ¹³C₆–sulfamethazine was obtained from Cambridge Isotopes (Cambridge, MA) and used as an internal standard. No attempt was made to further purify the antibiotics used in this study. Distilled/deionized water was used for all aqueous solutions. All other chemicals were either high-performance liquid chromatography (HPLC) grade or better or American Chemical Society (ACS) certified, including methanol, acetonitrile, formic acid (99%), sodium monohydrogen phosphate, sodium chloride, hydrochloric acid, sodium hydroxide, and glacial acetic acid (Fisher Scientific, Pittsburgh, PA).

Experimental Methods
Antibiotic Standard Mix and Tetracycline-Class Degradate Standard Mixes
Individual antibiotic stock solutions of each compound (CTC, OTC, LNC, SCP, SDM, STZ, TET, TRM, and TYL) were prepared by dissolving each compound in methanol to give a 1.0 g/L stock solution. Three degradate mixes were made to confirm identification of degradate formation containing 4-epi-ACTC, 4-epi-ATET, ATET, and -apoOTC in degradate mix 1; ACTC, ATET, β-apoOTC, and isoCTC in degradate mix 2; and 4-epi-CTC, 4-epi-TET, and 4-epi-OTC in degradate mix 3. Each compound was at 1.0 g/L in methanol and solutions were stored at –20°C until diluted for use.

Buffered Systems
Hydrolysis was investigated by varying ionic strength, pH, and temperature. All conditions were replicated in duplicate and reactions were conducted in amber glass bottles (250 mL) with temperature controlled at 7, 22, and 35°C in the absence of light. A 4-mmol L^–1 aqueous solution of sodium monohydrogen phosphate was prepared from autoclaved, deionized, distilled water for all hydrolysis reactions, ionic strength was adjusted with ACS-grade sodium chloride to either 0.0015, 0.050, or 0.084 mg/L as Na₂HPO₄, followed by pH adjustment with aqueous hydrochloric acid or with sodium hydroxide solutions to a pH of 2, 5, 7, 9, or 11 to give a total of 90 reactors where the antibiotics were amended as a mixture in each reactor yielding an initial reactor concentration of 2 mg/L for each antibiotic. Ionic strength was calculated based on contributions of Na₂HPO₄ and NaCl, while contributions of Na⁺ and Cl^– from NaOH and HCl used to adjust pH were ignored because the largest contribution was 0.00035% of the total Na⁺ or Cl^– concentration at pH 2 and therefore should have a negligible influence on ionic strength. Samples were collected daily for 3 wk after the initial addition of antibiotics and were stored frozen at –20°C until analysis. Time, temperature, and pH were recorded during the duration of the study at each sampling.

Liquid Chromatography Mass Spectrometry (LCMS) Analysis
Antibiotics (20 µL injection) were measured by reverse-phase liquid chromatography (LC) with an Agilent 1100 series LC/MS (Model 1946D, Wilmington, DE) in electrospray positive mode using a Phenomenex Luna 3-µm C18(2) 3.0 x 150-mm column preceded by a Security C18 guard cartridge (Phenomenex, Torrance, CA) at a flow rate of 0.360 mL/min and a column oven temperature of 30°C. A gradient separation was used where mobile-phase A (aqueous 0.3% formic acid solution) and mobile-phase B (100% methanol) were varied over 25 min with a 5-min post-column equilibration. The gradient started at 5% mobile-phase B initially and increased to 100% by 23 min. The MS fragmentor voltage was optimized at 190 V. The quantifying (QI) and confirming ions (CI) are listed in Fig. 1a-c, 2, and 3.

The electrospray source settings were optimized with the gas temperature adjusted to 350°C, drying gas flow rate at 12.0 L/min, and nebulizer pressure at 35 psig. Data were imported into Target Chromatographic Analysis Software (ThermoQuest Thru-Put software, Orlando, FL) version T4.12 with the T4.13 patch for quantitation. ¹³C₆–sulfamethazine (MS ions– QI: 285.0, CI: 186.0) was used as an internal standard. Six-point calibration curves were developed by diluting the previously frozen 2.0 mg/L antibiotic reactor mix at a given ionic strength and pH. Three different antibiotic degradation mixes were used for confirmation of known tetracycline-class degradation products by standard addition in selected samples.

Determination of Pseudo First-Order Rate Constants, Half-Lives, and Arrhenius Parameters
It is common for hydrolysis reactions to be second order overall and pseudo first-order with respect to the compound of interest. Initially, plots of ln (C_AB/C_{AB 0}) versus time were developed for each set of conditions (pH, temperature, and ionic strength). Pseudo first-order rate constants were determined by using the slope of a linear fit of ln (C_AB/C_{AB 0}) versus time for each set of conditions (Eq. [1]). Confidence intervals (CI) were determined at the = 0.05 level (95% CI).

[1]

where C_{AB 0} = antibiotic concentration at t = 0; C_AB = antibiotic concentration at some time (t); k' = pseudo first-order rate constant (1/time); and t = time.

Half-life (t_1/2), by definition, represents the amount of time necessary for a compound to degrade to one-half of its initial concentration. With substitution and rearrangement, Eq. [1] gives

[2]

Half-lives were calculated for each rate constant, and then all individual half-lives were averaged together for a given condition.

Temperature effects on degradation rates were assessed by data reduction with the linearized form of the Arrhenius equation (Eq. [3])

[3]

where A = preexponential factor or collision factor; E_a = experimental activation energy (kJ/K/mol); R = universal gas constant = 8.3141 kJ/K/mol; and T = absolute temperature (°K).

Statistical Analysis
Linear regressions were conducted using SPSS version 10.0.5 (SPSS, Inc., Chicago, IL) to assess relationships between k', ionic strength (I), pH (P), and temperature (T). First- (I, P, T), second- (IP, IT, PT), and third-order interactions (IPT) also were investigated between the three measured independent variables. Correlations were considered statistically significant at = 0.05. Confidence intervals at 95% ( = 0.05) were calculated for duplicate degradation rates.

	Results and Discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
Effect of Ionic Strength on Pseudo First-Order Rate Constants
The pseudo first-order rate constant (k') with respect to antibiotic concentration (degradation rates) was found to lack correlation ( = 0.05) as a function of I for those compounds exhibiting degradation in this study, which included CTC, OTC, TET, and TYL (pH 2 and 11) based on linear regression analysis. From these results, I was eliminated as a variable for calculation of degradation rates, and variation in the data set was treated as dependent on pH and temperature only. This had the result of increasing the number of replicates for a given set of conditions from n = 2 to n = 6. The remainder of the compounds (LIN, SCP, SDM, STZ, and TRM) did not exhibit any degradation during the timeframe of this study and, therefore, were not subject to statistical analysis.

Pseudo First-Order Rate Constants and Half-Lives
Average k' with 95% confidence intervals ( = 0.05) in parentheses and average half-lives (t_{1/2 avg}) are summarized as a function of pH and temperature in Tables 1 and 2 for CTC, OTC, TET, and TYL (pH 2 and 11). In general, degradation occurred according to the following trend CTC OTC > TET (Table 1 and Fig. 4 ). Literature values for OTC degradation seem slightly longer from this study when compared with Pouliquen et al. (2007) who reported half-lives of 59, 51, and 51 d at 8°C buffered around neutral pH in deionized water, freshwater, and seawater, respectively. In the study reported herein, half-lives were 23 (pH 5), 26 (pH 7), and 43 d (pH 9) at 7°C (Table 1). Hydrolysis of TYL was observed at all study temperatures for pH 2 and at 22 and 35°C for pH 11 with half-lives less than 6 h for all three temperatures for pH 2, and half-lives of 40 and 21 h for pH 11.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Comparison of rates of degradation of chlortetracycline (CTC) and oxytetracycline (OTC) normalized against the rate of degradation of tetracycline (TET) as a function of pH at 22°C.

Hydrolysis of LIN, SCP, SDM, STZ, and TRM was not expected because these compounds did not possess structural features (Fig. 3) that can be readily hydrolyzed at the temperatures relevant to environmental conditions. Amide (LIN) and sulfonamide (SCP, SDM, and STZ) type linkages typically are recalcitrant to hydrolysis at lower temperatures and require higher concentrations of strong acids or bases than may be encountered in the environment. Furthermore, the sulfonamide functionality is usually more difficult to hydrolyze than the amide functionality due to steric and electronic effects (Morrison and Boyd, 1983). Specifically, hydrolysis studies of clindamycin, which is in the same antibiotic class as lincomycin having a chlorine atom in place of a hydroxyl group, have exhibited less than 10% degradation over 2 yr from pH values ranging between 1 and 6.5 at 25°C. Lincomycin was one of the degradation products observed and found to be the predominant product over time at pH values greater than 5, indicating that lincomycin like clindamycin is stable as well (Oesterling, 1970). Additionally, Manzo and Martínez de Bertorello (1978) showed that two sulfaisoxazole derivatives underwent hydrolysis of the sulfamide bond or the isoxazole ring at sulfuric acid concentrations (35–80% by weight) at temperatures of 79°C or greater. Extrapolation of this data suggests that at 35°C the faster degrading derivative would have a half-life of approximately 8 d in 70% sulfuric acid (Manzo and Martínez de Bertorello, 1978). Two other studies subjected honey samples to acidic hydrolysis conditions to release the conjugated sugars from the sulfonamides present (10% trichloroacetic acid, 63°C, for 60 min– Verzengnassi et al. (2002), and 2 mol L^–1 HCl, 45 min, room temperature– Maudens et al., 2004) with no mention of degradation of the sulfonamides. Trimethoprim does not possess any hydrolyzeable groups or substituted amino groups that could epimerize.

Effects of pH on Degradation Rates
In general, CTC degrades more quickly than OTC, and OTC degrades more quickly than TET for pH 7 through 11. At pH 5, OTC degrades more quickly than CTC or TET, and degradation rates for CTC and TET are roughly equivalent. Degradation rates for CTC and TET are approximately equal at pH 2 with OTC being slightly more stable (Table 1). Figure 4 is a visual representation of this phenomenon where the degradation rate of CTC and OTC are normalized against the degradation rate of TET as a function of pH at 22°C. It is interesting to note that the activation energy increases proportional to pH to approximately pH 7 and then either decreases slightly or plateaus (Table 2). It is suspected that this results from a change in the predominant reaction pathway from acidic pH values versus neutral and basic pH values.

There are a variety of complex physicochemical interactions taking place in the tetracycline antibiotics that make it difficult to determine from this study which forces are responsible for variations in reactivity. Structurally, tetracycline can be considered the base structure for this group of compounds with CTC having a chlorine atom attached at the 10 position of the D ring and OTC having a hydroxyl group attached at the 6 position of the B ring (Fig. 1a-c). It is suspected that reactivity is affected for these three antibiotics by the presence of the substitutions of Cl (R₁) and OH (R₂) for CTC and OTC, respectively, versus hydrogen in both positions for TET. These substitutions may directly change the acidity or basicity of the reactive site on the molecule, induce steric hindrance, or enhance or detract from the cross-conjugated systems present in the tetracycline class of antibiotics. It is also possible that the changes in pH cause changes in structural conformation that leads to steric hindrance at the reactive centers (Morrison and Boyd, 1983; Mayr and Ofial, 2005). Therefore, this hindrance would be responsible for different reaction pathways becoming more energetically favorable as a function of pH. Additionally, with three acid dissociation constants (pKa), the tetracyclines have two to three reactive species with various degrees of ionization present depending on pH and temperature (Qiang and Adams, 2004).

In a more general sense, it is known that different degradation pathways dominate under acidic versus basic conditions for the tetracyclines (Halling-Sørensen et al., 2002), and therefore, it is expected that reaction rates and activation energies would be affected because different transition states would occur. Similar tetracycline-class degradates were observed in this study as a function of pH and have been reported previously in the literature (Halling-Sørensen et al., 2002, 2003; Loke et al., 2003). The epimerization of the dimethylamino group, which is a pH-mediated reaction, attached to position 4 of the A ring was the initial degradation product for CTC and TET and to a lesser extent for OTC. Further degradation of both the parent compounds (CTC and TET) and their epimers under acidic conditions leads to dehydration of the anhydro- or 4-epi-anhydro- constituent (Khan et al., 1990; Halling-Sørensen et al., 2002). However, OTC has not been observed to form stable anhydro structures, but instead has been reported to form - and β- apoOTC's (Khan et al., 1992; Halling-Sørensen et al., 2003; Loke et al., 2003). Alkaline conditions degrade the CTC, OTC, and TET and their epimers to the corresponding iso- or 4-epi-iso- compound (Khan et al., 1990, 1992; Halling-Sørensen et al., 2002, 2003; Loke et al., 2003).

Hydrolysis was observed at pH 2 and 11 for TYL, but not at 5, 7, or 9. No tylosin was measured in the first sample at pH 2 indicating complete degradation in less than 12 h. Paesen et al. (1995) observed that tylosin A hydrolysis (60°C) occurred much more readily as acidity or basicity increased with t_1/2 on the order of 11 to 20 d at pHs of 6 to 8, 0.25 h at pH 2, and 7.40 h at pH 11, which seems consistent with the results presented here considering the temperature differential in each study.

Potential Hydrolysis in Surface Water, Anaerobic Swine Lagoons, Wastewater, and Ground Water
Surface water, anaerobic swine lagoons, wastewater, and ground water affected by surface recharge typically have temperatures that range from 0 to 35°C and perhaps higher in tropical locations where pH values commonly range from 6 to 8.5 (Freeze and Cherry, 1979; Hem, 1986; Do et al., 2003; Loftin et al., 2005; Loftin, 2006). On the basis of hydrolysis results from this study, the slowest hydrolysis rates tend to occur around neutral pH, and therefore, it is unlikely that hydrolysis of LNC, sulfonamides, TYL, or TRM would be an important degradation mechanism. CTC, OTC, and TET, however, may be degraded under these same conditions, although complexation with certain buffers has been reported to have a catalytic effect on epimerization rates of OTC and TET (Vej-Hansen and Bundgaard, 1978; Vej-Hansen et al., 1978). Conversely, Lambs et al. (1988) has shown that complexation with calcium and magnesium cations inhibit epimerization of several tetracyclines.

Loke et al. (2000) indicated that TYL degrades mostly to tylosin B with minor amounts of tylosin D in swine manure amended microcosms (pH 6 to 8); however, it is not clear whether this occurred by an abiotic or biotic mechanism or both. Previous research by Loftin (2006) showed mixed results regarding abiotic degradation of LIN, OTC, STZ, and TYL in swine manure microcosms from two different anaerobic swine lagoons. Degradation was observed in both lagoons for TYL with t_1/2 values on the order of 15 d; no degradation was observed in either lagoon for STZ. However, abiotic degradation was observed in only one of the study lagoons for LIN and OTC with t_1/2 values on the order of 100 d and 15 d, respectively, with comparable pH (7.4 and 7.9) and temperature (10.0 and 6.0°C) values between both study lagoons. These results suggest that other abiotic mechanisms besides hydrolysis in addition to biotic mechanisms are involved in degradation of these antibiotics when compared with this study's hydrolysis rates (i.e., oxidation/reduction, sorption, catalysis).

	Conclusions

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
In general, half-lives of CTC, OTC, TET, and TYL (pH 2 and 11) are on the order of hours to 9.7 wk, whereas LNC, SCP, SDM, STZ, and TYL (pH 5, 7, and 9) are much more stable in the range of aqueous solutions addressed in this study. Degradation generally followed the trend CTC OTC > TET. Epimerization was the dominant initial transformation process for CTC and TET; however, further degradation was indicated by the continued disappearance of the parent compound and a relatively constant ratio between the parent compound and its respective epimer representing equilibrium. If the epimer had been the stopping point of the degradation pathway, then the concentration of the parent compound would have remained constant once equilibrium was reached. The lifetime of the epimer is important because it can readily convert back to the more biologically active parent form and, therefore, warrants additional study to determine the lifetime of the respective epimers. Other intermediate degradation products of CTC also were observed with time and included anhydrochlortetracycline, isochlortetracycline, and their epimers. Similar products also were observed for tetracycline. On the basis of the results from this study, hydrolysis of LNC, sulfonamides, TYL, and TRM is not expected in surface water, ground water, or anaerobic lagoons. In contrast, the tetracyclines tend to degrade quite rapidly under conditions similar to those encountered in natural water and lagoons.

	ACKNOWLEDGMENTS

 
The authors would like to express their appreciation for the help of two undergraduate assistants, John Akin and Tito Martinez, from the Univ. of Kansas (Lawrence), and Dr. Jacque Gibbons, Kansas State Univ. (Manhattan), for valuable discussions on the statistical treatment of this data set. Any use of trade, product, or firm names in this paper is for descriptive purposes only and does not imply endorsement by the U.S. Government.

	NOTES

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	REFERENCES

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