Adsorption and transformation of tetracycline antibiotics with aluminum oxide

Chemosphere 79 (2010) 779–785

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Adsorption and transformation of tetracycline antibiotics with aluminum oxide Wan-Ru Chen 1, Ching-Hua Huang * School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA

a r t i c l e

i n f o

Article history: Received 4 August 2009 Received in revised form 11 March 2010 Accepted 12 March 2010

Keywords: Anhydrotetracycline Isomerization Dehydration Surface acidity Chlorotetracycline Oxytetracycline

a b s t r a c t Tetracycline antibiotics (TCs) including tetracycline (TTC), chlorotetracycline (CTC) and oxytetracycline (OTC) adsorb strongly to aluminum oxide (Al2O3), and the surface interaction promotes structural transformation of TCs. The latter phenomenon was not widely recognized previously. Typically, rapid adsorption of TCs to Al2O3 occurs in the first 3 h ([TC] = 40 lM, [Al2O3] = 1.78 g L 1, pH = 5, and T = 22 °C), followed by continuous first-order decay of the parent compound (kobs = 15 ± 1.0, 18 ± 1.0 and 6.2 ± 0.9  10 3 h 1 for TTC, CTC and OTC, respectively) and product formation. The transformation reaction rate of TCs strongly correlates with adsorption to Al2O3 surfaces. Both adsorption and transformation occur at the highest rate at around neutral pH conditions. Product evaluation indicates that Al2O3 promotes dehydration of TTC to yield anhydrotetracycline (AHTTC), epimerization of TTC, and formation of Al-TTC complexes. Al2O3 promotes predominantly the transformation of CTC to iso-CTC. The surface-bound Al(+III) acts as a Lewis acid site to promote the above transformation of TCs. Formation of AHTTC is of special concern because of its higher cytotoxicity. Results of this study indicate that aluminum oxide will likely affect the fate of TC antibiotics in the aquatic environment via both adsorption and transformation. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Tetracyclines (TCs) are among the antibiotics that are used extensively for disease control and in livestock feed for several decades due to their great therapeutic values (Sarmah et al., 2006; Kuemmerer, 2009a,b). The widespread use of TCs and other antibiotics has led to dissemination of these compounds into the water and soil environments (Lindsey et al., 2001; Kumar et al., 2005; Blackwell et al., 2007; Xu and Li, 2010). Although environmental concentrations of antibiotics are typically below the threshold levels to exhibit medicinal treatment effects on bacterial populations and other at-risk species, chronic exposure to low levels of antibiotics alone or along with other toxicants may still exert pressure on the development of antibiotic resistant bacteria and minimize the effectiveness and therapeutic value of antibiotics (Barrett, 2005; Kim et al., 2007; Yu et al., 2009). Understanding the fate of antibiotic contaminants in the water–soil environment is imperative to better assess their risks and develop mitigation strategies. Recent studies have reported strong interactions of TCs with mineral surfaces (Kraemer et al., 1998; Kim et al., 2005; Sassman and Lee, 2005; Pils and Laird, 2007). The most common members of TCs include tetracycline (TTC), oxytetracycline (OTC) and chlorotetracycline (CTC) (Fig. 1). TCs’ electron-rich ketone, carboxyl, * Corresponding author. Tel.: +1 404 894 7694; fax: +1 404 385 7087. E-mail address: [email protected] (C.-H. Huang). 1 Present address: 520 Plant and Soil Science Building, Michigan State University, East Lansing, MI 48824, USA. 0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.03.020

amino, and hydroxyl groups contribute to their strong tendency to complex with metals (Lambs et al., 1988; Chen and Huang, 2009). In particular, several studies have demonstrated the strong adsorption of TCs with aluminum oxide surfaces and clays (Gu and Karthikeyan, 2005; Pils and Laird, 2007). Aluminum is abundant in the Earth’s crust and commonly mixed with other metal oxides in the soils. The geographical features (e.g., in the humid tropics with frequent heavy rains and high temperatures) or human activity will enhance the weathering process and result in high aluminum oxide content in the soils (Fageria and Baligar, 2008). Adsorption to aluminum oxides may play an important role in controlling the mobility and spread of trace organic contaminants such as TC antibiotics. However, in addition to adsorbing organic compounds, the Al(III) atom of aluminum oxides has been reported as an acid center and may catalyze the oxidation (Karthikeyan et al., 1999), dimerization (Sohn et al., 2006), dehydration, and isomerization (Pines and Haag, 1960) of organic compounds. Alumina has been one of the most important catalyst materials in industrial processes because of its intrinsic acidity and catalytic activity (Pines and Haag, 1960). Despite the well recognized strong adsorption of TCs to aluminum oxides, the potential impact of aluminum oxide surfaces on the transformation of TCs was not explored. The previous study by Gu and Karthikeyan (2005) adopted two different approaches to measure TTC adsorption on aluminum hydrous oxides and reported that more than 20% of TTC was subjected to other reactions besides adsorption; however, the other reactions were not characterized.

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Fig. 1. Structures of tetracyclines (TCs) and their derivatives.

TC antibiotics are known to be unstable under certain conditions. During long-term storage, abiotic transformation may occur via isomerization, dehydration, substitution, and oxygenation (Walton et al., 1970; Liang et al., 1998). Under alkali conditions, the iso-derivatives of TCs form rapidly via a nucleophilic attack of the C6 hydroxyl group at the C11 carbonyl carbon and the bond cleavage at C11–C11a (Fig. 1). CTC is particularly prone to undergo this isomerization (Waller et al., 1952). In strong acidic media, TCs can undergo reversible epimerization at position C4 to form the corresponding 4-epi-TCs, and may degrade to form anhydro-tetracyclines (AHTCs) by losing a H2O at position C6 (Fig. 1). Studies have shown that some weaker acids such as phosphoric acid and citric acid can accelerate the above acid-catalyzed epimerization and anhydro-TCs formation because they can act as a molecular proton conductor (Walton et al., 1970; Sokoloski et al., 1977; Yuen and Sokoloski, 1977). On the other hand, TTC is relatively stable for long periods in 0.03 N HCl and its epimerization and acid-catalyzed dehydration are insignificant (McCormick et al., 1957). Based on the susceptibility of TCs to the above reactions, we hypothesized that aluminum oxide surfaces may influence transformation of TCs in addition to adsorption. As will be shown later, our study found that aluminum oxide (Al2O3(s)) surfaces promote structural transformation of TCs along with rapid adsorption. The main focus of this study was to elucidate the adsorption and transformation reaction kinetics of three TCs (TTC, CTC and OTC) with Al2O3(s) under various conditions (pH = 5–9, [TTC]0 = 21.3–109.4 lM, and [Al2O3(s)]0 = 0.9–3.3 g L 1) and identify the transformation products. Overall, the results of this study will improve the ability to properly assess the risks of TC residues in the environment.

2. Materials and methods 2.1. Chemicals TTC, epi-TTC, OTC and CTC were obtained from Sigma at 90–98% of purity and used without further purification. Standards of AHTCs

were synthesized according to the method by Clive (1968), in which TTC, CTC and OTC stock solutions were each dosed with 0.1 M H2SO4 and heated to 100 °C to generate the corresponding AHTCs. Unless otherwise specified, all other reagents used (e.g., buffers, ionic strength salt, acids, solvents, etc.) were obtained from Fisher Scientific, Acros or Aldrich at >97% of purity. All solutions were prepared using reagent water from a Millipore Milli-Q Ultrapure Gradient A10 purification system. Stocks of TCs were prepared in methanol at 1.6 mM, protected from light, stored in a 15 °C freezer, and used within a month of preparation. Acetic acid (pH 4–5.5), 4-morpholinepropanesulfonic acid (MOPS) (pH 6–8), or 2-(cyclohexylamino)ethanesulfonic acid (CHES) (pH 9–10) buffers at 10 mM were used to maintain the solution pH. NaCl salt (0.01 M) was used to control ionic strength. The aluminum oxide used in this study was Al2O3(s) (type C, c-Al2O3 powder) from Degussa at >99.6% of purity. The reported surface area and site density of Al2O3(s) were 90.1 m2 g 1 and 3.81 sites nm 2, respectively (Vasudevan and Stone, 1998).

2.2. Kinetic experiments All glassware was soaked in 5 M HNO3 for at least 12 h, thoroughly rinsed with reagent water and dried prior to use. Batch kinetic studies with 20–110 lM of parent TC and 0.8–3.5 g L 1 of Al2O3 were conducted in 60 mL screw-cap amber glass bottles with Teflon septa. A suspension containing Al2O3 particles, pH buffer, and NaCl was initially prepared and constantly stirred by a stir bar on a submersible stirrer in a 22 °C water bath. Reaction was initiated by adding a known amount of the TC stock to the suspension. Sample aliquots were periodically taken from the reactor and centrifuged for 20 min to remove oxide particles. The supernatant was acidified by adding 5 lL of concentrated HCl to improve the analytical results of TCs and analyzed by HPLC. The TCs were stable in the presence of HCl (an example shown in Fig. S1, Supplementary material), in agreement with the report by McCormick et al. (1957). To desorb TCs from the Al2O3 surfaces, 10 lL of concentrated HCl was added to 1 mL of sample aliquot from the

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reactor. The acidified sample was shaken for three minutes and centrifuged for 20 min and then the supernatant was analyzed by HPLC. All samples were stored in amber vials at <5 °C and analyzed by HPLC within 2 d. For each testing parameter, at least two replicate experiments were conducted. Control experiments without Al oxide showed no significant degradation (<5%) of any of the tested compounds during the reaction periods (typically 24 h). 2.3. Analysis of TCs TCs were analyzed by an Agilent 1100 high performance liquid chromatography (HPLC) system with a Zorbax RX-C18 reversephase column (4.6  250 mm, 5 lm) at an injection volume of 100 lL and a flow rate of 1 mL min 1. TCs were detected at 275 and 365 nm by a diode-array UV/Vis detector. The mobile phase A consisted of 0.01 M oxalic acid and 10 lM EDTA, while mobile phase B was pure acetonitrile. The mobile phase gradient was as follows: 0–3 min 96% A and 4% B, 3–10 min a linear gradient to 73.7% A and 26.3% B, 10–19 min 73.7% A and 26.3% B, 19–25 min linear gradient to 96% A and 4% B, and then the final composition was maintained for the remainder of the 30-min run time. 2.4. Product identification After more than 75% of the parent TC had disappeared, reaction suspensions were filtered and analyzed by an Agilent 1100 HPLC/ DAD/MSD system equipped with a single-quadrupole mass spectrometer with an electrospray ionization source. The separation was performed by a Zorbax SB-C18 column (2.1  150 mm, 5 lm) at a flow rate of 0.2 mL min 1 with two mobile phases as described above. MS analysis was conducted using positive electrospray ionization at both low and high fragmentation voltages (110 and 250 eV) with a mass scan range of m/z 50–1000. The drying gas was at 10 L min 1 at 350 °C, the nebulizer pressure 172 kPa, and the capillary voltage 4000 V. 3. Results and discussion 3.1. Adsorption and transformation of TCs in the presence of Al2O3(s) In the absence of Al2O3, TTC, OTC and CTC were stable under the employed experimental conditions and less than 5% of the parent compounds were lost after 24 h. In contrast, significant loss of TCs from the aqueous phase occurred in the presence of Al2O3 (Fig. 2a). The dotted and solid lines in Fig. 2a represent samples with and without acid (HCl) desorption (see Section 2.2), respectively. The TC concentration measured without acid desorption

b

1.6

Adsorption (%)

log [TCs] (µM)

a

corresponded to the unreacted parent compound in the aqueous phase, while the concentration measured with acid desorption corresponded to the unreacted parent compound in the aqueous phase plus the desorbable portion of the parent compound from the Al2O3 surfaces. Fig. 2a shows that the decrease of TC concentration in the presence of Al2O3 was most rapid during the first 30 min, followed by relatively fast loss within 3 h. After 3 h, the loss of TC from solution slowed down appreciably, yet the log(TC concentration) decreased linearly with time. The initially rapid then slower and continuous disappearance of TCs and detection of transformation products (see discussion later) indicate that both adsorption and transformation of TCs occurred with Al2O3. We hypothesized that the initial rapid loss of TCs was predominantly due to adsorption to Al2O3 surfaces whereas the later slower loss was related to transformation. The rate of TC transformation was approximated by applying linear regression to the linear section of the log([TC])-versus-time plot (Fig. 2a) and the slope yielded an observed first-order rate constant kobs in h 1. Note that the transformation kinetics were difficult to measure during the initial reaction period because adsorption of TCs to Al2O3 occurred quickly within the initial 3 h, the rate of TC transformation was much slower than adsorption, and complete desorption of TCs from the Al2O3 surfaces was not possible (see the discussion below). The above approach reasonably distinguished transformation kinetics from adsorption kinetics based on available data. However, possibility exists that the actual surface reaction kinetics could be more complex than what were quantified here. The data collected with and without acid desorption showed similar characteristics, i.e., the log([TC])-versus-time curves had similar shapes (Fig. 2a) and linear regressions of both trendlines yielded comparable kobs values (Table 1). The adsorption of TCs to Al2O3 surfaces was estimated by the differences between the initial TC concentration and the intercepts of the two regression lines at t = 0, respectively. We call the adsorption determined from the data without acid desorption as the ‘‘total adsorption (AT)”, and the adsorption determined by the data with acid desorption the ‘‘irreversible adsorption (AI)”. Note the irreversibility is with respect to HCl extraction. The AT was in the range of 43–57% and AI in 15–21% of the total initial TC under the employed experimental conditions (Fig. 2b and Table 1). Apparently, HCl addition could desorb about only half of the adsorbed TCs from the Al2O3 surface. In general, TTC and OTC showed comparable adsorption while CTC had greater adsorption. CTC also showed the fastest transformation rate (18 ± 1.0  10 3 h 1), followed by TTC (15 ± 1.1  10 3 h 1), and OTC (6.2 ± 0.9  10 3 h 1). Overall, the trends in adsorption and transformation rate among CTC, TTC and OTC obtained in this study are consistent with

1.4

1.2 TTC TTC (desorbed by HCl) CTC CTC (desorbed by HCl) OTC OTC (desorbed by HCl)

1.0

0.8 0

5

10

15

Time (h)

20

25

100 80

AI AT

60 40 20 0 TTC

CTC

OTC

TC Species

Fig. 2. Kinetics of removal of TCs by Al2O3 at pH 5.0. (a) Semi-logarithmic plot is linear after the first 3 h. (b) Adsorption percentage of TCs determined by two different quenching methods. AI and AT refer to irreversible adsorption and total adsorption, respectively. (Initial total [TCs]0 = 40 lM, [Al2O3(s)] = 1.78 g L 1).

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Table 1 Reaction rate constants and adsorption of TCs with Al2O3. Compound

TTC CTC OTC

Reaction rate kobs (10

3

h

1

)

R2

Adsorption (%)

Centrifugation

Acid added before centrifugation

Irreversible

Total

Centrifugation

Acid added before centrifugation

15 ± 1.1 18 ± 1.0 6.2 ± 0.9

10.9 ± 0.3 17.2 ± 0.9 6.4 ± 0.1

18.8 ± 0.9 22 ± 2.3 15.8 ± 0.2

43 ± 1.9 57 ± 1.4 44 ± 1.6

0.991 0.993 0.961

0.998 0.994 0.999

the literature. Pils and Laird (2007) reported stronger adsorption of CTC than TTC to soil clays. The authors attributed this to enhanced polarity of CTC’s functional groups due to the additional chlorine atom at C7 position and thus stronger polar–polar interactions with clays’ surface sites. Aga et al. (2005) investigated the persistence of TCs and related products in manure-amended soils and found that only OTC (out of CTC, TTC and OTC) remained at measurable levels in subsurface soils after several months of incubation, suggesting that OTC is most persistent. In previous studies, the reactivity order of CTC > TTC > OTC with MnO2 have been observed (Rubert and Pedersen, 2006, Zhang et al., 2008). Dissolution of Al2O3(s) under the employed experimental conditions (pH = 5, [TTC]0 = 40 lM, [Al2O3(s)]0 = 1.78 g L 1) was measured and found to yield up to 0.45 mM of dissolved Al (Table S1, Supplementary material). The presence of TTC, however, did not increase Al2O3 dissolution, contrary to the result by Gu and Karthikeyan (2005). The lower impact of TTC on Al oxide dissolution is likely due to lower TTC concentration and different oxide phases (Al2O3 versus aluminum hydrous oxide) employed in this study. 3.2. Effect of reaction conditions To further understand the involved transformation, reaction kinetics were evaluated for the influence of TTC concentration, Al2O3 oxide concentration, solution pH, and the presence of oxygen. A series of experiments with varying Al2O3 loadings but a fixed TTC loading, or with varying TTC loadings but a fixed Al2O3 loading, were conducted at pH 5 to assess the effect of each reactant on the reaction kinetics. Loading refers to the initial reactant concentration employed. When TTC loading was increased from 20 to 106 lM at a fixed 1.78 g L 1 of Al2O3 loading, TTC transformation rate constant kobs decreased with increasing TTC loading (Fig. 3a). In a similar manner, adsorption (AT and AI) of TTC to Al2O3 surfaces also decreased with increasing TTC concentration (Fig. 3b), suggesting limited available surface sites under the employed Al2O3 loading. When Al2O3 loading was increased from 0.9 to 3.3 g L 1, TTC transformation rate constant kobs and adsorption (AT and AI) of TTC to Al2O3 surfaces increased almost linearly with increasing Al2O3 loading (Fig. 4a and b). As expected, more

b

0.04

100

AI AT

Adsorption (%)

-1

Rate constant kobs (h )

a

surface sites were available at a higher Al2O3 loading and thus led to greater adsorption of TTC. Based on the data in Figs. 3b and 4b, the Al2O3 surfaces adsorb TTC on the average of 10 lmol per gram (Table S2, Supplementary material). Dividing 10 lmol g 1 by the reported specific surface area of 90.1 m2 g 1 for Al2O3 (Vasudevan and Stone, 1998) yields site density of 0.07 sites nm 2. This site density appears to be lower than those (e.g., 3.81 sites nm 2) reported for other adsorbates. The effect of pH was examined at pH 5–9 with fixed TTC and Al2O3 loadings. The kobs was greatest at near pH 7 and decreased at pH lower or higher pH values (Fig. 5a). Similarly, the adsorption (AT and AI) of TTC to Al2O3 also showed a maximum near pH 7 and decreased when the pH was either decreased or increased from 7. The observed pH dependence can be rationalized by evaluating the charges of TTC and Al2O3 surfaces. The reported zero-point-ofcharge (pHzpc) of Al2O3 was in the range of 7.5–8.7 (Stumm, 1992). Within the experimental pH range of 5–9, the Al2O3 surface was positively charged when pH was below its pHzpc, while negatively charged when pH is above the pHzpc. As pH increases from 5 to 9, the dominant species of TTC changed from being neutral or zwitterionic (H2L+/ at pKa1 < pH < pKa2 = 7.78) to negatively charged (HL at pH > pKa2 = 7.78). Electrostatic repulsion between similar charges of TTC and Al2O3 surfaces was greater at either lower pH (positive–positive repulsion) or higher pH (negative–negative repulsion), thus creating a maximum electrostatic attraction at the intermediate pH range. Similar bell-shaped pH dependence with highest adsorption around pH 7 was also observed by Gu and Karthikeyan (2005). The surface complexes of zwitterionic species of TTC was previously suggested by Figueroa et al. (2004) as the major contributor to the adsorption mechanism of TTC to montmorillonite and kaolinite clays. Overall, the strong correlation between TTC’s transformation rate constant and adsorption to Al2O3 surfaces (Figs. 3–5) strongly indicates that interactions of TTC molecules with Al2O3 surface sites is crucial for TTC transformation. Previous studies have reported that Al oxide surfaces can catalyze oxidation of 1-naphthol by oxygen, in which the Al(OH)3 surfaces promote the reaction by binding to the oxidation product of 1-naphthol and facilitating its removal from the aqueous phase

0.03

0.02

0.01

[Al2O3]0 = 1.78 g L-1, pH 5 0.00

80 60 40 20 0

0

20

40

60

80

[TTC]0 (µM)

100

120

20

40

60

80

100

[TTC]0 (µM)

Fig. 3. Effect of TTC loading on TC transformation kinetics. (a) Reaction rate constant kobs. (b) Adsorption.

120

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b

0.020

100

Adsorption (%)

-1

Rate constant kobs (h )

a

0.015

0.010

[TTC] 0= 40 µM, pH 5

0.005

60 40 20

0.000 0

1

2

3

AI AT

80

0

4

0

1

2

3

4

-1

-1

[Al2O3]0 (g L )

[Al2O3]0 (g L )

Fig. 4. Effect of Al2O3 loading on TTC transformation kinetics. (a) Reaction rate constant kobs. (b) Adsorption.

0.06

b

[Al2O3]0 = 1.78 g L-1, [TTC] 0= 40 µM

Adsorption (%)

Rate constant kobs (h-1)

a

0.04

0.02

0.00 5

6

7

8

9

pH

100

AI AT

80 60 40 20 0 5

6

7

8

9

pH

Fig. 5. Effect of pH on TTC transformation kinetics. (a) Reaction rate constant kobs. (b) Adsorption.

(Karthikeyan et al., 1999). To evaluate whether oxygen was involved in the transformation of TCs observed in this study, experiments were conducted in the absence of oxygen with constant nitrogen purging. Another setup with constant oxygen purging was conducted in parallel. Experiments show that changing oxygen content had little impact on TCs’ adsorption, transformation rate, or product formation in the presence of Al2O3. Thus, the catalyzed transformation of TCs by Al2O3 is not oxidation; instead, dehydration and isomerization catalyzed by Al2O3’s acidic surfaces are more likely. 3.3. Transformation product analysis Reaction mixtures of TTC with Al2O3 at pH 5 were stopped by centrifugation after 23 h (about 25% of the parent TTC was transformed). The supernatant was analyzed by HPLC–UV to yield five peaks (Fig. S2, Supplementary material): epi-TTC (8.88 min), TTC (11.18 min) and three transformation products with longer retention times (16.74, 18.47 and 19.31 min). The UV spectra of the five peaks are shown in Fig. S3, Supplementary material. The supernatant was also analyzed by LC/MS to yield five peaks with molecular ions of m/z 445a, 445b, 559a, 559b and 427 (Table S3 and Fig. S4, Supplementary material). The two peaks with the same m/z 445 are epi-TTC and TTC, and exhibited the same fragmentation pattern with two major fragments of M 17 and M-35, corresponding to [M+H NH3]+ and [M+H NH3 H2O]+ ions, respectively. It has been shown that the NH3 loss likely involves the C2 amino group and the H2O loss involves the C6 hydroxyl group (Dalmazio et al., 2007). The two product peaks at 16.74 and 18.47 min showed nearly identical LC/MS (Products I and II in Table S3) and UV spectra

(Fig. S2), suggesting that they may be isomers. These two products may be Al2-epi-TTC and Al2-TTC complexes, respectively, because they (i) had mass increase by 114 compared to TTC (m/z 559 versus m/z 445) and (ii) exhibited nearly the same UV spectra as the proposed 2:1 Al:TTC complex by Gu and Karthikeyan (2005) (Fig. S5, Supplementary material). However, due to low ionization efficiency, the current LC/MS spectra are insufficient to determine the structures of these two complexes. Since this study employed comparable Al oxide-to-TTC ratio and solution pH as Gu and Karthikeyan (2005) and found dissolved Al at >10 times of the initial TTC concentration, the 2:1 metal to TTC complexation stoichiometry is reasonable. The product peak at 19.31 min had similar HPLC retention time as the synthesized anhydrotetracycline (AHTTC) (Fig. S6, Supplementary material). The longer retention time of AHTTC than that of TTC on the reverse-phase (C18) liquid chromatography is expected since removal of the C6 hydroxyl group makes the compound less polar (Gratacos-Cubarsi et al., 2007). This product also showed similar UV spectrum as AHTTC (Figs. S3 and S7, Supplementary material) with a bathochromic shift from 365 nm to 420 nm compared to TTC (Gratacos-Cubarsi et al., 2007). Furthermore, this product’s LC/MS spectrum showed a molecular ion of m/z 427 (i.e., M-18) and only the [M+H NH3]+ but not the [M+H NH3 H2O]+ fragment, confirming the structure is produced by the loss of a water from the parent TTC. In contrast, Gu and Karthikeyan (2005) did not report AHTTC formation, probably because their HPLC monitoring time (12 min) was not long enough. Separate experiments were conducted with 40 lM TTC and 0.017 M Al3+ in 0.03 M HCl at room temperature. About 35% of TTC was transformed after 72 h to yield significant amounts of AHTTC and epi-AHTTC. In the absence of Al3+, TTC was stable in 0.03 M HCl

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without any transformation. These results further confirmed the role of Al(III) in promoting dehydration of TTC to yield AHTTC. The generation of products over time was also monitored (Fig. 6). Due to the lack of authentic standards and different ionization efficiency among TTC and its products, the product concentration was estimated by HPLC–UV absorbance (275 nm) based on the crude assumption that the products’ UV absorbance coefficients are comparable to TTC’s at 275 nm (Fig. S2, Supplementary material). As shown in Fig. 6, formation of aluminum complexes of TTC and epi-TTC accompanied the disappearance of TTC over time. AHTCC was generated as TTC was decreased in the first 2 h but decreased slightly over time. Interestingly, epi-TTC concentration decreased noticeably during the first 2 h but remained constant afterwards. The initial sharp decrease of epi-TTC is likely due to rapid adsorption to Al2O3 surfaces. Owing to similar structures, epiTTC should have comparable tendency as TTC to convert to its anhydro-form in the presence of Al2O3; this was confirmed in a separate experiment in which continuous decrease of epi-TTC occurred when only epi-TTC was reacted with Al2O3. The observation that the overall epi-TTC concentration did not decrease over time in Fig. 6 suggests that epi-TTC was generated as a transformation product of TTC, counterbalancing transformation of epi-TTC in the presence of Al2O3. Experimental data also showed increasing epimer-to-TTC ratio (in free or complex forms) over the reaction time course with Al2O3 (Table S4, Supplementary material), further indicating the impact of Al2O3 to promote TTC epimerization. As mentioned earlier, aluminum oxides are well known catalysts for organic compound transformation because of the intrinsic acidity associated with Al(+III) (Pines and Haag, 1960). The reactions catalyzed by alumina such as dehydration of alcohols and skeletal isomerization are also typically acid-catalyzed (Pines and Haag, 1960; Hashimoto et al., 1986; Llorens et al., 1998). The results presented above strongly indicate that TTC adsorbs strongly to Al2O3 surfaces, forms Al-complexes from dissolution of Al2O3, and undergoes acid-promoted transformation including dehydration and epimerization. The surface-bound Al(+III) acts as a Lewis acid site to catalyze dehydration of TTC to AHTTC, and serves as a molecular proton conductor like phosphate and citric acid (Sokoloski et al., 1977; Yuen and Sokoloski, 1977) to facilitate epimerization of TTC. The reaction products of CTC (m/z 479) and OTC (m/z 461) were also analyzed by HPLC–UV and LC/MS. For CTC, formation of two M + 114 Al-complexes (Products III and IV in Table S3) was also observed but AHCTC was not detected (Fig. S1, Supplementary material). The most abundant product was iso-CTC, which was identified based on matched retention time with a standard, the lack of UV absorptivity at 365 nm, the m/z ratio of 479, and the formation of a [M+H NH3 CO]+ but not a [M+H NH3 H2O]+ frag-

ment in MS spectrum (Zurhelle et al., 2000). Because CTC has a stronger tendency to form an iso-derivative than TTC and OTC (Waller et al., 1952), it is likely that Al2O3 surfaces promoted such isomerization of CTC rather than dehydration. Reacting at the slowest rate with Al2O3, OTC yielded only one transformation product (Product V in Table S3) with the same m/z 461 as OTC, and AHOTC was not detected (Fig. S1). Product V may be iso-derivative of OTC since its UV spectrum shows much lower UV absorptivity at 365 nm. While preparing the standards of AHTCs, it required a higher concentration of H2SO4 and a longer reaction time for OTC than TTC and CTC to yield the same amount of anhydro derivative, indicating the formation of AHOTC is less favorable (Clive, 1968). The C6–OH and C5–OH groups of OTC are positioned favorably for hydrogen bond formation, and such interactions may help stabilize the C6–OH of OTC and lower the tendency to form AHOTC. 3.4. Environmental relevance While earlier studies have reported strong adsorption of TC antibiotics to aluminum oxide, this study has identified the capacity of Al oxide surfaces to catalyze structural transformation of TCs – a phenomenon that was not readily recognized previously. The transformation reaction rate is strongly influenced by surface complex formation between TC molecule and Al2O3 (i.e., adsorption) and solution pH. Since adsorption was the greatest and transformation reaction rate the fastest at around neutral pH conditions, both processes involving Al oxide are likely to occur in the aquatic environment and affect the fate and transport of TC antibiotics. Among the TCs examined, the reactivity towards Al2O3 followed the order of CTC > TTC > OTC. Owing to its Lewis acidity, the surface-bound Al(+III) facilitates the acid-catalyzed dehydration and isomerization of TCs. Reaction patterns and product formation vary with the structure of TCs. The formation of AHTTC from TTC promoted by Al2O3 merits particular concern because anhydro-derivatives of TCs in general have high toxicity (Klimova and Ermolova, 1976). Formation of AHCTC and AHOTC was not observed in this study within 72 h, most likely because the surface-promoted formation of iso-CTC was the dominant pathway for CTC, and the kinetics of AHOTC formation from OTC is comparatively slower than in the other two TCs. Further studies are needed to evaluate the persistence and fate of AHTTC, and determine the fate and potential impact of TC–aluminum complexes. Acknowledgement This material is based upon work supported by the National Science Foundation under Grant 0229172. Appendix A. Supplementary material

TCs concentration (µM)

40

epi-TTC TTC Al2-TTC complex (m/z 559)

35 30

Al2-epi-TTC complex (m/z 559)

25

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chemosphere.2010.03.020.

AHTTC (m/z 427)

References

20 15 4 2 0 0

5

10

15

20

Time (h) Fig. 6. Product evolution of TTC in reaction with Al2O3. Measurement was conducted by HPLC UV–Vis absorbance at 275 nm.

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