Evidencing Generation of Persistent Ozonation Products of Antibiotics

Jul 24, 2009 - Oxidation Processes in Water Treatment: Are We on Track? Urs von Gunten. Environmental Science & Technology 2018 52 (9), 5062-5075...
0 downloads 0 Views 909KB Size
Download PDF
Environ. Sci. Technol. 2009, 43, 6808–6815

Evidencing Generation of Persistent Ozonation Products of Antibiotics Roxithromycin and Trimethoprim ´ , M. GODEHARDT, J. RADJENOVIC ´ ,* A. HEIN, M. FARRE ´, M. PETROVIC ´ M. JEKEL, AND D. BARCELO Department of Environmental Chemistry, IDAEA-CSIC, c/Jordi Girona 18-26, 08034 barcelona, Spain, Department of Water Quality Control, Technical University of Berlin, KF 4 Straβe des 17. Juni 135, D-10623, Berlin, Germany, Institucio Catalana de Reserca i Estudis Avanzats (ICREA), Barcelona, Spain, and Institut Catala` de Recerca de l’Aigua (ICRA), Parc Cientı´fic i Tecnolo´gic de la, Universitat de Girona, Pic de Peguera, 15, 17003 Girona, Spain

Received March 31, 2009. Revised manuscript received July 14, 2009. Accepted July 15, 2009.

The mechanism of product formation during ozonation of two widely used antimicrobial agents, macrolide roxithromycin and inhibitor of dihydrofolate reductase (DHFR) trimethoprim was studied in laboratory-scale experiments with two types of matrix: distilled water and secondary wastewater effluent. Thestructuresoftheprimaryandsecondaryreactionintermediates were elucidated by quadrupole-time-of-flight (QqToF) instrument, showing that in spite of their high ozone affinity both roxithromycin and trimethoprim oxidation pathway involve to a great degree the · OH radical chain reactions. In total nine ozonation products were detected, whereas two products of roxithromycin exhibited high refractoriness to ozonation, especially in the case of distilled water. Furthermore, the intact tertiary amine moiety of roxithromycin in these products suggests that the antimicrobial activity of the parent compound will be preserved.

Introduction Considering the extent and amounts of usage of antimicrobial agents and the ever increasing tendency to use new antibiotics, it is no surprise that these pharmaceuticals are one of the most ubiquitous environmental contaminants (1). Development of antibiotic resistance of bacteria was demonstrated in surface and groundwater affected by the sewage treatment plant (STP) effluent (2). In light of these facts, advanced drinking and wastewater treatment options should be considered for “at source” removal of trace-level antibiotics and other pharmaceutical residues. Ozonation can be expected to degrade pharmaceuticals with high efficiency (3-5). The main difficulty when treating real water is its high DOC content (e.g., wastewater) that scavenges nonselective hydroxyl radicals ( · OH) (6), as well as selective O3 (7). Besides the oxidant exposure, the efficiency of ozonation will depend on the compound’s affinity toward O3, which will be conditioned by the molecule’s electronic properties and speciation at the given pH (i.e., anionic species are more reactive toward O3 than their neutral equivalents) (6, 8). In some cases partial oxidation by O3 and · OH radicals * Corresponding author phone: +34934006100; fax: +34932145904, ext 432; e-mail: [email protected]. 6808

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 17, 2009

is sufficient for a significant reduction of pharmacological activity and/or toxicity (9, 10). However, ozonation might afford unwanted degradation products, sometimes with increased biological activity (11). In order to evaluate the safety of applying ozonation in DWTPs and STPs, mechanisms of product formation of two frequently encountered antibiotics, inhibitor of dihydrofolate reductase (DHFR) trimethoprim (TMP) and a macrolide antibiotic roxithromycin (ROX) were investigated. TMP was found to produce a toxic response in rainbow trout (12), while for both TMP and ROX ecotoxicological effects on the algal growth were reported (13). The formation of persistent and structurally similar ozonation products of these antibiotics could reflect in their increased hazardousness. To elucidate the structures of ozonation products, analyses were performed on ultraperformance liquid chromatograph (UPLC) coupled to a quadrupole-time-of-flight mass spectrometer (QqToF-MS). The lab-scale ozonation experiments were performed with distilled water (DW) and sewage effluent (SE), and evolution of products and removal efficiencies were compared. Moreover, Daphnia magna assay was used to estimate the toxicity of the parent compounds and their ozonation products.

Experimental Section Experiment Setup. For the chemicals used see Supporting Information (SI) Text S1. Ozonation batch experiments were performed in a 3 L standard stirred glass reactor (D ) 150 mm) equipped with four equally spaced baffles and stainless steel six-blade turbine (d ) 75 mm). The scheme of the labscale ozonation plant is presented in SI Figure S1. Pure O2 was continuously supplied to a high-voltage discharge O3 generator (Sorbios, GSG 1.2) at a flow rate of 35 L/h, with the inlet gas O3 concentration of 20 mg/L. The O3/O2 gas was introduced via a steel aeration ring at the bottom of the reactor below the stirrer and dispersed at rotational speed of 500 rpm. The O3 inlet and outlet gas concentrations were measured continuously by two UV-photometers (BMT 961 TPC and BMT 964, λ ) 253.7 nm). Liquid O3 was measured by an O3 sensor (Orbisphere Laboratories) in a short-term water cycle. Samples for the analysis were taken by a plastic tube at the reactor top. The experimental conditions were identical for all experiments: V ) 2 L, 20 °C, initial pH 7.7-7.8, except in the case of the experiments with ROX in DW matrix that was at initial pH 4.4. This was because the spiking solution of ROX had to be acidified due to its poor solubility in water (0.0189 mg/L at 25 °C) (14). In order to detect possible degradation products by UPLC-QqToF-MS, the initial spiking concentration of ROX and TMP was 50 mg/L. Experiments at lower initial concentrations of pharmaceuticals were not possible, since due to the small reactor volume no sample preconcentration could be performed. The dilution correction was performed for each sample, taking into account the volume remaining in the ozonator. Analytical Determinations. Mineralization was followed by measuring the DOC by direct injection of filtered samples into a highTOCII analyzer (Elementar Analysensysteme, Germany) provided with a NDIR detector and calibrated with standard solutions of potassium phthalate. The LOD for the DOC analysis was 0.5 mg/L. Accurate mass analyses were performed using a Waters/ Micromass QqToF-MicroTM system coupled to Waters ACQUITY UPLC system (Micromass, Manchester, UK). Samples were analyzed on a Waters ACQUITY BEH C18 column (10 × 2.1 mm, 1.7 µm particle size). The chromatographic and MS conditions are summarized in SI Text S2. 10.1021/es900965a CCC: $40.75

 2009 American Chemical Society

Published on Web 07/24/2009

Ecotoxicity Assays. For the ecotoxicity assays conducted with Daphnia magna see SI Text S3.

Results SI Tables S1 and S2 summarize the exact mass measurements of TMP and ROX, respectively, and their ozonation products, together with the recalculated mass errors and double bond equivalents (DBEs) given by the MassLynx V4.1 software. The measurements were performed under optimized conditions of collision energy and cone voltage in ESI (+)-MS2 experiments, in the mass range m/z 50-700 for TMP and m/z 100-1000 for ROX, with accuracy threshold of 5 ppm. Structural Elucidation of ROX Ozonation Products. Isolation and CID fragmentation of the protonated molecular ion of ROX, m/z 837, resulted in the MS2 spectrum illustrated in Figure 1a. The most intense fragment ion observed was m/z 679, formed by the loss of 4-methoxy4,6-dimethyl-tetrahydropyran-2,5-diol (cladinose moiety), whereas further expulsion of water and 3-dimethylamino3,4,6-trideoxyhexose (desosamine moiety) generated the fragment ions m/z 661 and 522, respectively. Possibly the cleavage of C14-O2 bond in the oxime alkylether sidechain of m/z 522 gave the signal at m/z 446. After the loss of desosamine and cladinose sugars, cleavages of NsO1 bond and water generated the fragment ion m/z 398, whereas the signal m/z 380 was formed by the subsequent loss of another molecule of water. The total ion chromatogram (TIC) of ozonated samples revealed 5 new products of ROX. Figure 2 illustrates the hypothesized structures of these products based on their MS2 spectra as described just forward, and the proposed degradation mechanism of ozonation of ROX. One of the ozonation products, P694, eluted in the LC chromatogram 0.4 min prior to ROX (Figure S2). The exact mass of the molecular ion m/z 695, shifted upward for 18 Da relative to the m/z 679 fragment ion of ROX suggested that this degradation product was formed by the hydroxylation of the modified ROX molecule that had lost its cladinose moiety. The MS2 spectrum of m/z 695 illustrated in Figure 1b displayed fragment ions m/z 677 and 619, derived from the subsequent cleavages of water and O2-C15 bond in the oxime side chain. The fragment ion m/z 504 suggested that the hydroxylation took place at the side-chain, since this ion corresponded to the cleavage of the acetate and dimethylamino group, and NsO1 bond. The hydroxylation in P694 was presumed to occur at the C14-atom, which was supported by the key fragment m/z 426. The fragment ion m/z 426 was formed analogously to the fragment ion m/z 446 in the spectrum of ROX, i.e., by the breakage of the C14sO2 bond. However, loss of the sOH group from the first C-atom probably resulted in the H-transfer and cyclization in order to stabilize its positive charge, whereas another loss of water might have been favored by the conjugation stabilization of the resulting structure of m/z 426 fragment ion (see insert in Figure 1b). Also, the presence of fragment ions m/z 398 and 380 suggested that the macrolide moiety of ROX probably stayed intact. Product P822 appeared in the LC chromatogram slightly before ROX, at tR ) 6.35 min. The molecular weight (MW) of P822 that was 14 Da lower compared to ROX suggested that it was formed by the cleavage of one methyl group, possibly from the tertiary amine. The product scan of the molecular ion m/z 823 is presented in Figure 1c. It generated the same fragment ions m/z 522, 446, 398, and 380 as seen for the m/z 837 molecular ion of ROX, whereas the indicator of oxidation at the tertiary amine was the fragment ion at m/z 665, analogous to the fragment ion m/z 679 in the spectrum of ROX, yet with mass lowered for 14 Da.

Next, products P850 and 852 detected at tR ) 7.35 and 6.7 min, respectively, with MWs shifted 14 and 16 Da upward relative to ROX, respectively, were identified as quinone imine analogues of the hydroxylated drug. The strong abundance m/z 695 and 677 fragment ions in the MS2 spectrum of P852 (Figure 1f) shifted for 16 Da compared to the fragment ions m/z 679 and 661of ROX (Figure 1a) affirmed this hypothesis. Moreover, further collision of the product ions afforded the fragment ions m/z 504, 426, 398, and 380, previously observed for the molecular ion m/z 695, evidencing that P852 was generated by the hydroxylation at the C14-atom. On the other side, low intensity molecular ion m/z 851 could afford only one fragment ion at m/z 693 (Figure 1g). Nevertheless, from the exact mass measurements of the molecular ion and sodium adduct ion of P850 it could be concluded that this product was most probably the quinine imine derivative of P852. Both P850 and P852 exhibited a decreased polarity relative to ROX (i.e., elution from the LC column after the parent compound), possibly due to the hydrogen bond between the sOH group and O1-atom at the side-chain. The ozonation product with the molecular ion m/z 839 eluted very closely to ROX, at tR ) 6.47 min, with the MW only 2 Da higher compared to ROX. However, in the extracted ion chromatogram (XIC) another peak appeared at 8.15 min, thus the products were marked as P838A and P838B, eluting at 6.47 and 8.15 min, respectively. The P838B was less intense, whereas CID fragmentation produced a new fragment ion m/z 681, besides the previously observed fragment ions m/z 398 and 380 (Figure 1e). The fragment ion m/z 681 with mass 16 Da higher relative to the m/z 665 fragment ion in the MS2 spectrum of the molecular ion m/z 823 and 14 Da lower relative to the m/z 695 fragment ion of the molecular ion m/z 853 suggested that P838B was generated by demethylation of the tertiary amine and additional hydroxylation. Thus, the fragment ion m/z 681 corresponded to the cleavage of the cladinose moiety from the molecular ion m/z 839. Although the exact position of the sOH group could not be deduced based on the MS2 fragmentation pattern of P838B, it is probable that, as in the case of P694 and P852, it was located at the C14-atom. On the other side, the fragmentation pattern of P838A displayed practically the same fragment ions as the one recorded for P852 (m/z 695, 504, 426, 398, and 380), as presented in Figure 1d. Since the fragment ion m/z 695 corresponding to the cleavage of cladinose moiety indicated the presence of the intact dimethylamino group, it was assumed that P838A was formed by hydroxylation at C14 and cleavage of one of the methyl groups in cladinose sugar, possibly from the methoxy group. The products P838A and P838B eluted 0.07 and 2.15 min after ROX, which can be explained by intramolecular hydrogen bond between the newly attached sOH group and O1-atom, whereas in the case of P838B, the hydrogen bond between the demethylated tertiary amine and neighboring sOH group possibly further decreased the molecule’s polarity. Structural Elucidation of TMP Ozonation Products. The spectrum of TMP obtained in the ESI(+) CID experiments on a QqToF-MS has been previously described by Eichhorn et al. (15). In short, the MS2 fragmentation of the molecular ion of TMP, m/z 291, proceeded via formation of very intense fragments m/z 275, 261, and 230, with the intact diaminopyrimidine moiety and bridging methylene group. The radical fragment ion m/z 276 was generated by the loss of methyl radical ( · CH3), whereas fragment ions m/z 181 and 123 corresponded to the cleavages of the protonated TMP at either side of the central methylene group, with the positive charge possibly stabilized across the rings (see insert in Figure 3a). Samples from ozonation experiments analyzed in the fullscan mode revealed the appearance of four new products along with the degradation of TMP, denominated as P294, VOL. 43, NO. 17, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

6809

FIGURE 1. Spectra obtained in ESI(+)-MS2 experiments at QqToF instrument (cone voltages 35-40 V, collision energies 20-30 eV) for roxithromycin (ROX) and its ozonation products: (a) ROX, (b) P694, (c) P822, (d) P838A, (e) P838B, (f) P852, and (g) P850. P322, P324, and P338, whereas their proposed structures and degradation mechanism are illustrated in Figure 4. On the basis of the order of LC elution of these products, only P338 was deduced to be less polar than the parent compound, whereas P294, P322, and P324 had an increased polarity. 6810

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 17, 2009

Interestingly, ozonation product P324 was determined to be identical to the product of degradation of TMP by nitrifying activated sludge (i.e., M324) (15). The MS2 fragmentation pattern of P324 coincided with the one reported for M324 by Eichhorn et al. (15), except for the absence of two fragment

FIGURE 2. Degradation mechanism of ozonation of ROX in DW and SE matrix. ions m/z 307 and 297, corresponding to the losses of water and CO. The key fragment ion for placing the sOH group at the C5-atom was m/z 143 (cleavage of C5sC7 bond and formation of cyclohexendione, with the stabilized positive charge over tautomeric keto-enol and imine-enamine structures), which excluded the hydroxylation at the C7-atom (see insert in Figure 3d). At Figure 3b is presented the ESI(+)-MS2 spectrum of protonated P294 with the molecular ion m/z 295. The hypothesized structure of the molecular ion m/z 295 (i.e., hydroxylation at C4-atom and cleavage of one methyl group at the trimethoxybenzene portion) was supported by its fragment and radical fragment ions formed. The cleavage of one amino group, and amino group and · CH3 radical generated fragment ions m/z 278 and 263, respectively. On one side, formation of the resonantly stabilized carbonyl group at the hydroxylated C4-atom and posterior loss of CO, · CH3 radical -CH2 group produced a relatively intense signal at m/z 236. On the other side, expulsion of the amino group from the diaminopyrimidine structure led to an increase in its degree of unsaturation and consequent loss of CO, which after cleavages of · OCH3 radical and -CH2 group resulted in the fragment ion m/z 205. In spite of the assumed modification of trimethoxybenzene ring, the abundant fragment ion m/z 181 appeared. Nevertheless, mesomeric resonance stabilization across the ring was still possible. Moreover, the homolytic cleavage of one of the · OCH3 radical in m/z 181 probably induced the formation of another double bond (i.e., carbonyl group) and another resonantly stabilized radical fragment ion m/z 148. In the product ion scan of the molecular ion m/z 323, only three signals were recorded at m/z 291, 259, and 231 (Figure 3c). The loss of · NH2 radical and O2 generated the

radical fragment ion m/z 291. Also, cleavage of · OCH3 radical, O2 and amino group rendered the radical fragment ion m/z 259, which after expulsion of CO gave another radical fragment ion m/z 231. These fragments together with the exact mass measurements of the molecular ion and its sodium adduct evidenced that P322 was most probably formed by the introduction of carbonyl groups at C4 and C7-atom. As far as P338 is concerned, its MW that was 48 Da higher relative to TMP suggested that it corresponded to a trihydroxylated product (Figure 3e). Loss of the methoxy group generated the fragment ion m/z 307 with the positive charge delocalized over the benzene ring, whereas expulsion of another methoxy group resulted in further unsaturation of the molecule and the intense, resonantly stabilized fragment ion m/z 275. Further loss of O2 and · NH2 radical from the m/z 275 possibly afforded the radical fragment ion m/z 243, whereas the radical fragment ion m/z 215 was derived from the loss of another methoxy group. From the abovementioned reasoning of product ions of the molecular ion m/z 339, the exact sites of hydroxylation could not be revealed, although it most probably occurred at the C4-atom, similar to the products P294. Ozonation of TMP and ROX in DW and SE. In Figures 5 and 6 are illustrated qualitative profiles of ROX and TMP, respectively, and their ozonation products, obtained by normalization of the peak areas the initial values of peak areas of TMP and ROX (i.e., at t ) 0), and plotted versus specific O3 consumption (Zspec) calculated for the initial DOC content (DOC0) (see SI Text S4, for calculating Zspec). The employment of Zspec made it possible to describe and compare the ozonation of pharmaceuticals in matrices having different DOC0 dimensionless with respect to the duration of oxidation (16). Also, quantified concentrations of ROX and TMP VOL. 43, NO. 17, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

6811

FIGURE 3. Spectra obtained in ESI(+)-MS2 experiments at QqToF instrument (cone voltage 30 V, collision energies 15-25 eV) for trimethoprim (TMP) and its ozonation products: (a) TMP, (b) P294, (c) P322, (d) P324, and (e) P338. together with the DOC concentration are illustrated in SI Figures S5 and S6, respectively. In the case of both TMP and ROX, the ozonation products formed were the same in DW and SE. However, in the case of ROX unexpected persistence of P852 and P694 was observed during the ozonation in DW, even at the end of the experiment after 23 min. Moreover, the continuously increasing liquid O3 concentration as well as calculated decreasing Zspec indicated that practically no further consumption of O3 was occurring. More importantly, the peak areas of P852 and P694 reached 100 and 60% relative to the initial peak area of ROX (Ao) after 0.65-0.70 g O3/g DOC0 consumed. The other three identified products P822, P838A, and P850 were encountered in smaller quantities, whereas P838B was detected in traces and only in 6812

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 17, 2009

a few samples. The products P838A, P838B, and P850 were resulting from the secondary oxidation reactions. On the contrary to DW matrix, SE enhanced the ozonation of ROX and its degradation products. As it can be seen from the Figure 5b, product P694 reached it maximum of 20% of Ao after 0.65 g O3/g DOC0, being completely degraded until the end of the experiment (i.e., 18 min). Although the product P852 again reached 100% of Ao as in the case of DW, ozonation in SE was more efficient with P852 being slowly oxidized to only 12% of Ao after 1.24 g O3/g DOC0 consumed. The DOC measured in the experiments with DW was practically constant during the entire experiment, which can be assigned to the formed persistent primary ozonation products and possibly other degradates (SI Figure S4).

FIGURE 4. Degradation mechanism of ozonation of TMP in DW and SE matrix.

FIGURE 5. Peak areas of ROX and its ozonation products normalized to the initial value of peak area of ROX (t ) 0) presented vs Zspec calculated for the DOCo in the ozonation experiment with (a) distilled water (DW) and (b) sewage effluent (SE). [, ROX; 1, P852; 9, P694; 1, P822; b, P838A, ... P850. The degradation of TMP was slightly faster in the experiment with SE than with DW, since it occurred after 0.89 and 1.62 g O3/g DOC0 consumed, respectively. The most abundant products in both cases were P294 and P324, whereas P322 and P338 were apparently formed only in traces (see Figure 6). Their complete degradation was achieved after 1.48 and 1.81 g O3/g DOC0 in the SE and DW, respectively. As far as mineralization is concerned, the removal of only ∼10% of DOC0 in DW matrix was achieved, indicating that in spite of the disappearance of the primary intermediates other unidentified ozonation products might still be present in the finished water (SI Figure S5). Toxicity Determined in Daphnia magna Tests. Ecotoxicity results showed no acute toxic effects for ROX and TMP in the tested concentration range. Only slight acute toxicity was observed for TMP, with EC50 of 7.9 mg/L, which is much

FIGURE 6. Peak areas of TMP and its ozonation products normalized to the initial value of peak area of TMP (t ) 0) presented vs specific O3 consumption (Zspec) calculated for the DOCo in the ozonation experiment with (a) DW and (b) SE. [, TMP; 1P324; 9, P294, ... P338. higher than its usual environmental concentrations. However, limited growth rate of D. magna occurred in presence of ROX after 5 days of exposure, in comparison to other substances or the blanks. The growth of Daphnias exposed to 10 mg/L of ROX was the 72% of the growth of Daphnias in a blank solution (as determined in unpaired t test, P < 0.0001). For the test solutions containing final ozonation products of TMP and ROX no acute toxicity was noted and no statistically significant growth inhibition. Nevertheless, dilutions that had to be performed due to the limited amount of sample led to lowered concentrations of these products VOL. 43, NO. 17, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

6813

(e.g., in the case of ozonation products of ROX, P852, and P694, their expected overall concentrations were approximately 4 and 2 mg/L in 1:3 and 1:6 dilutions). Chronic toxicity of these compounds should be further investigated, especially considering the fact that the tertiary amine moiety responsible for the molecule’s activity remained unchanged. Furthermore, to determine quantitatively the antibiotic activity of the identified degradates antibiotic potency tests should be performed. They would allow a direct comparison of bacterial growth inhibition by the original drugs and their ozonation products.

Discussion Ozonation Mechanism of ROX. While the SE matrix was buffered to pH 7.8, the initial pH in the experiments with DW was pH 4.4 due to the preparation of ROX solution (see Experimental Section). Considering the pKa of ROX (i.e., pKa ) 8.8), it will be mainly present in the form of protonated tertiary amine (CsNdOsR group) in both DW and SE, which makes this group inactive for O3 attacks (10). Dodd et al. (17) estimated that at pH 7.7 the oxidation of ROX will proceed mainly through O3 attack at the deprotonated dimethylamino group, whereas only 20% of the · OH chain reactions contribution can be expected. The · OH radicals are generated in reactions of O3 with functional moieties of the organic matter and/or from its autocatalytic decomposition (5). Among the identified ozonation products, the formation of only two of them, P822 and P838B, involved the demethylation of the tertiary amine. Moreover, the attached sOH group at the C14-atom of the side-chain in the P838 was most probably resulting from the · OH radical attack. Likewise, the generation of the most abundant product P852 could be elaborated by the · OH radical attack at C14-atom, whereas the following cleavage of the cladinose sugar in P852 along the radical chain pathway led to the formation of another very abundant product P694. Also, formation of the ketoderivative P850 is in accordance with previously published studies (18). The products P852, P694, and P822 were formed as primary intermediates concomitantly with the disappearance of ROX, whereas other two products P838A, P838B, and P850 were possibly resulting from the secondary oxidation reactions. Besides the reported intermediates, there were no other lower MW ozonation products detected in ESI (+) and ESI (-) mode on a QqToF-MS. Furthermore, although byproduct such as aldehydes, ketones, and acids were probably formed at the end of the ozonation reactions, they could not be detected by QqToF-MS due to the interferences of matrix components and poor sensitivity of QqToF analyzer for such low MW compounds. Macrolide antibiotics are thought to bind to rRNA and block the tunnel that channels the nascent peptides away from the peptidyl transferase center inside the ribosome, thus impeding the ribosomal activity, whereas the main role in this binding is assigned to the dimethylamino group of the desosamine sugar (19). In the study of Lange et al. (20) the decrease in growth suppression effect of clarithromycin of Pseudomonas putida was assigned to its transformation into N-oxide as the major product of ozonation. As can be seen from the elucidated ozonation mechanism of ROX, the two most abundant products P694 and P852 have the dimethylamino group preserved. Therefore, the persistency of these products in the ozonation experiments with DW even at very high O3 doses is a matter of concern, and should be included in future studies when monitoring ROX in ozonation treatment. On the other side, their degradability when ozonating SE was enhanced, although after 1.24 g O3/g DOCo consumed ∼12% of P852 was still remaining in the reactor. Ozonation Mechanism of TMP. The speciation of TMP is characterized by two pKa constants: pKa1 ) 3.2 (21) and 6814

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 17, 2009

pKa2 ) 7.1 (22). The active fraction for the O3 attack in TMP molecule at circumneutral pH is the diaminopyrimidine moiety, whereas in the more acidic environment and prevalence of mono- and diprotonated TMP, the O3 attack would occur at the C9- and C13-atom of the trimethoxytolyl moiety (17). Also, according to the available data (23), the CdN double bond is virtually intact, and the addition of O3 at nitrogen-containing aromatic compounds only takes place at CdC bonds. The most abundant product was P294, which most probably resulted from the consecutive · OH attacks at the methoxy group and hydroxylation at the C4-atom of the diaminopyrimidine ring, possibly by O3 attack. On the other side, introductions of sOH and carbonyl groups at C4- and C7-atoms of TMP molecule, respectively, rendered the product P322, detected in small amounts. The substitution of the sOH group by sCdO group at the C4-atom can be explained by the mesomeric resonant stabilization and migration of the double bond from the electron-acceptor CsN bond to the electron-donor sOH group. Another relatively abundant product was P324, with the carbonyl group at C4- and sOH group at C5-atom of the diaminopyrimidine ring, previously reported as microbial metabolite of TMP from batch experiments with nitrifying activated sludge (15). Although the exact mass measurements unambiguously identified P338 as a trihydroxylated derivative of TMP, it eluted 0.25 min later at the LC column. The only plausible explanation for this phenomenon is the existence of intramolecular hydrogen bonds between the attached sOH groups, and/or the sOH group at the C7 and sNH2 group at the C6-atom. Moreover, all of the above-mentioned ozonation products were results of primary oxidation reactions and were further degraded until the end of the experiments with both DW and SE. Similar to the ozonation of ROX, there were no lower MW products detected in the experiments with TMP. Although direct oxidation reactions can be held responsible for the formation of P324, the most abundant product detected, P294, as well as P322 and P338 can rather be assigned to a combined pathways of nonselective · OH radicals and selective O3 attacks. This clearly indicates that, as in the case of ROX, the specificity of TMP toward O3 was compromised by the high reaction rates of · OH radicals present in the solution. The antibacterial activity of TMP is derived from the 2,4-diamino-5-methylpyrimidine moiety, which blocks the bacterial folate synthesis by occupying available dihydrofolate reductase enzymes (24). The substitution of this fraction of the TMP molecule could imply the reduction of its antibacterial activity, however the ecotoxicological effect of these products should be further studied. Practical Implications. Although the affinity toward O3 has been reported to be very high for both TMP and ROX (∼80% contribution of direct oxidation by O3), with secondorder apparent rate constants >3 × 105 M-1s-1 at pH 7.7 (17), the · OH radicals had a significant role in the product formation. When applied in DWTPs and STPs, ozonation can be expected to be efficient for the treatment of removal of ROX and TMP. However, identified ozonation products of ROX are of concern due to their persistency and preserved antimicrobial activity (i.e., dimethylamino group). The observed growth inhibition of D. magna by ROX (72% at 10 mg/L) suggests its potential chronic toxicity that could be passed on to degradates. The identified ozonation products of TMP did not show persistency to ozonation, although their biological activity should be further studied. At more realistic concentration of pharmaceuticals better transformation during ozonation can be expected, since the O3 concentration will exceed the pharmaceutical concentration by a larger factor. Nevertheless, future monitoring studies of full-scale

ozonation treatment plants should include these products besides the original compounds, especially in the case of water with lower DOC content (i.e., groundwater, surface water).

Acknowledgments J.R. gratefully acknowledges the JAE Program (Junta para la Ampliacio´n de los Estudios), cofinanced by CSIC (Consejo Superior de Investigaciones Cientı´ficas) and European Social Funds. This work was financially supported by the European Union through projects INNOVA MED (INCO-CT- 2006517728) and RECLAIM WATER (Contract No. 018309) and the Department of Water Quality Control (Technical University of Berlin).

Supporting Information Available The explanation of Zspec, experimental conditions of QqToFMS analysis, chemicals used, ecotoxicity assays, scheme of the lab scale ozonation plant, TICs representing ozonation products, DOC concentration profiles vs time and tables summarizing the exact mass measurements for TMP and ROX. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Hirsch, R.; Ternes, T.; Haberer, K.; Kratz, K.-L. Occurrence of antibiotics in the aquatic environment. Sci. Total Environ. 1999, 225 (1-2), 109–118. (2) Harwood, V. J.; Whitlock, J.; Withington, V. Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis: Use in predicting the source of fecal contamination in subtropical waters. Appl. Environ. Microbiol. 2000, 66 (9), 3698–3704. (3) Vieno, N. M.; Ha¨rkki, H.; Tuhkanen, T.; Kronberg, L. Occurrence of pharmaceuticals in river water and their elimination in a pilot-scale drinking water treatment plant. Environ. Sci. Technol. 2007, 41 (14), 5077–5084. (4) Ternes, T. A.; Stu ¨ ber, J.; Herrmann, N.; McDowell, D.; Ried, A.; Kampmann, M.; Teiser, B. Ozonation: a tool for removal of pharmaceuticals, contrast media and musk fragrances from wastewater. Water Res. 2003, 37 (8), 1976–1982. (5) Huber, M. M.; Go¨bel, A.; Joss, A.; Hermann, N.; Lof¨fler, D.; McArdell, C. S.; Ried, A.; Siegrist, H.; Ternes, T. A.; Von Gunten, U. Oxidation of pharmaceuticals during ozonation of municipal wastewater effluents: A pilot study. Environ. Sci. Technol. 2005, 39 (11), 4290–4299. (6) Von Gunten, U. Ozonation of drinking water: Part I. Oxidation kinetics and product formation. Water Res. 2003, 37 (7), 1443– 1467. (7) Buffle, M.-O.; Schumacher, J.; Salhi, E.; Jekel, M.; von Gunten, U. Measurement of the initial phase of ozone decomposition in water and wastewater by means of a continuous quenchflow system: Application to disinfection and pharmaceutical oxidation. Water Res. 2006, 40 (9), 1884–1894. (8) Hoigne´, J.; Bader, H. Rate constants of reactions of ozone with organic and inorganic compounds in watersII: Dissociating organic compounds. Water Res. 1983, 17 (2), 185–194.

(9) Huber, M. M.; Ternes, T. A.; Von Gunten, U. Removal of estrogenic activity and formation of oxidation products during ozonation of 17R-ethinylestradiol. Environ. Sci. Technol. 2004, 38 (19), 5177–5186. (10) Mcdowell, D. C.; Huber, M. M.; Wagner, M.; Von Gunten, U.; Ternes, T. A. Ozonation of carbamazepine in drinking water: Identification and kinetic study of major oxidation products. Enivron. Sci. Technol. 2005, 39 (20), 8014–8022. (11) Huber, M. M.; Canonica, S.; Park, G.-Y.; Von Gunten, U. Oxidation of pharmaceuticals during ozonation and advanced oxidation processes. Enivron. Sci. Technol. 2003, 37 (5), 1016–1024. (12) Gagne´, F.; Blaise, C.; Andre´, C. Occurrence of pharmaceutical products in a municipal effluent and toxicity to rainbow trout (Oncorhynchus mykiss) hepatocytes. Ecotoxicol. Environ. Saf. 2006, 64 (3), 329–336. (13) Yang, L. H.; Ying, G. G.; Su, H. C.; Stauber, J. L.; Adams, M. S.; Binet, M. T. Growth-inhibiting effects of 12 antibacterial agents and their mixtures on the freshwater microalga Pseudokirchneriella subcapitata. Environ. Toxicol. Chem. 2008, 27 (5), 1201– 1208. (14) Syracuse Research Corporation Physical Properties database, www.syrres.com. (15) Eichhorn, P.; Ferguson, P. L.; Pee´rez, S.; Aga, D. S. Application of ion trap-MS with H/D exchange and QqTOF-MS in the identification of microbial degradates of trimethoprim in nitrifying activated sludge. Anal. Chem. 2005, 77 (13), 4176– 4184. (16) Drewes, J. E.; Jekel, M. Behavior of DOC and AOX using advanced treated wastewater and groundwater recharge. Water Res. 1998, 32 (10), 3125–3133. (17) Dodd, M. C.; Buffle, M.-O.; von Gunten, U. Oxidation of antibacterial molecules by aqueous ozone: moiety-specific reaction kinetics and application to ozone-based wastewater treatment. Environ. Sci. Technol. 2006, 40 (6), 1969–1977. (18) Pe´rez-Estrada, L. A.; Malato, S.; Gernjak, W.; Agu ¨era, A.; Thurman, E. M.; Ferrer, I.; Ferna´ndez-Alba, A. R. Photo-fenton degradation of diclofenac: Identification of main intermediates and degradation pathway. Environ. Sci. Technol. 2005, 39 (21), 8300– 8306. (19) Schlu ¨ nzen, F.; Zarivach, R.; Harms, J.; Bashan, A.; Tocilj, A.; Albrecht, R.; Yonath, A.; Franceschi, F. Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature 2001, 413 (6858), 814–821. (20) Lange, F.; Cornelissen, S.; Kubac, D.; Sein, M. M.; von Sonntag, J.; Hannich, C. B.; Golloch, A.; Heipieper, H. J.; Mod¨er, M.; von Sonntag, C. Degradation of macrolide antibiotics by ozone: A mechanistic case study with clarithromycin. Chemosphere 2006, 65 (1), 17–23. (21) Qiang, Z. M.; Adams, C. Potentiometric determination of acid dissociation constants (pK(a)) for human and veterinary antibiotics. Water Res. 2004, 38 (12), 2874–2890. (22) Roth, B.; Strelitz, J. Z. The protonation of 2,4-diaminopyrimidines. I. Dissociation constants and substituent effects. J. Org. Chem. 1969, 34, 821–836. (23) Bailey, P. S. The reactions of ozone with organic compounds. Chem. Rev. 1958, 58 (5), 925–1010. (24) Walsh, C. Antibiotics: Actions, Origins, Resistance; ASM Press: Washington, DC, 2003.

ES900965A

VOL. 43, NO. 17, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

9

6815