Oxidation of Trimethoprim by Ferrate (VI): Kinetics, Products, and

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Oxidation of Trimethoprim by Ferrate(VI): Kinetics, Products, and Antibacterial Activity George A. K. Anquandah,† Virender K. Sharma,*,† D. Andrew Knight,† Sudha Rani Batchu,‡ and Piero R. Gardinali‡ † ‡

Chemistry Department, Florida Institute of Technology, 150 West University Boulevard, Melbourne, Florida 32901, United States Chemistry Department, Florida International University, 3000 N.E. 151st St, North Miami, Florida 33181, United States

bS Supporting Information ABSTRACT:

Kinetics, stoichiometry, and products of the oxidation of trimethoprim (TMP), one of the most commonly detected antibacterial agents in surface waters and municipal wastewaters, by ferrate(VI) (Fe(VI)) were determined. The pH dependent second-order rate constants of the reactions of Fe(VI) with TMP were examined using acid
base properties of Fe(VI) and TMP. The kinetics of reactions of diaminopyrimidine (DAP) and trimethoxytoluene (TMT) with Fe(VI) were also determined to understand the reactivity of Fe(VI) with TMP. Oxidation products of the reactions of Fe(VI) with TMP and DAP were identified by liquid chromatography-tandem mass spectrometry (LC
MS/MS). Reaction pathways of oxidation of TMP by Fe(VI) are proposed to demonstrate the cleavage of the TMP molecule to ultimately result in 3,4,5,-trimethoxybenzaldehyde and 2,4-dinitropyrimidine as among the final identified products. The oxidized products mixture exhibited no antibacterial activity against E. coli after complete consumption of TMP. Removal of TMP in the secondary effluent by Fe(VI) was achieved.

’ INTRODUCTION The presence of pharmaceuticals in streamwater, wastewater effluent, and drinking water has led to widespread research in their monitoring, degradation, and possible adverse effects to aquatic environments.1,2 Active pharmaceuticals and their residues have been detected in the range of trace levels to μg L
1 in aqueous environment.3,4 Among the different classes of pharmaceuticals detected in the environment, antibiotics are among the most prevalent.5 Antibiotics are frequently used in human and veterinary medicine as well as in aquaculture and farming for both prevention and treating of microbial infections.6 Antibiotics have been detected in freshwater resources and wastewater effluents.4,7 The presence of antibiotics in the aqueous environment can potentially change ecosystems and lead to antibioticresistant bacteria.8
10 Trimethoprim (5-(3, 4, 5-trimethoxybenzyl)pyrimidine-2,4-diamine, TMP) (Figure S1 of the Supporting Information, SI) has shown little evidence of reversibility of its resistance once established.11 TMP acts as an inhibitor to dihydropteroate synthesase by blocking a step in folate production, which has been attributed to the 2,4-diaminopyrimidine moiety (DAP, Figure S1).12,13 TMP has been frequently detected around the world in surface water and wastewater effluent in the concentration range 30
150 ng L
1 and 20
37 000 ng L
1, respectively.14 r 2011 American Chemical Society

TMP is generally not removed by existing conventional water and sewage treatment techniques.15 When TMP is exposed to the commonly used water treatment method of chlorination, mono and dichlorinated products are produced while the 2,4diaminopyrimidine substructure remains intact.16 Ozone oxidation studies of TMP in river water and wastewater have shown g90% removal with ozone,16,17 however, the removal may not necessarily obviate the antibacterial properties completely since the 2,4-diaminopyrimidine substructure was present in some of the degradates.18 The antibacterial study has however demonstrated that oxidation of TMP by ozone or OH radicals resulted in much lower antibacterial activity than that of the TMP.17 The use of ozone raises the concern of production of carcinogenic bromate ion in the treated water.19 Other techniques such as photolysis and TiO2 photocatalysis have been shown to degrade TMP, but some toxicity of oxidation products on V. fischeri was observed.20 This present work describes for the first time that the oxidant, ferrate(VI) (Fe(VI)) degrades TMP completely with cleavage of the original molecule, oxidation of Received: June 29, 2011 Accepted: October 27, 2011 Revised: October 23, 2011 Published: October 27, 2011 10575

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Environmental Science & Technology amino groups of the pyrimidine moiety, and finally the elimination of antibacterial activity of TMP. In recent years, studies on the oxidation of pharmaceuticals by Fe(VI) have been forthcoming.21
26 Kinetics of the reactions have been studied, but the knowledge of stoichiometry and products of the oxidations are very limited. Significantly, antibacterial activity of the reaction products, after elimination of the parent drug molecule is missing in the literature. The objectives of the present paper are to: (i) determine species-specific rate constants for the reactions of Fe(VI) and TMP, and DAP (Figure S1 of the SI) by studying the kinetics of oxidation as a function of pH (ii) determine stoichiometry and identify oxidized products of the reaction between Fe(VI) and TMP in order to propose reaction pathways, and (iii) determine the antibacterial activity of the reaction mixtures against Escherichia coli.

’ EXPERIMENTAL SECTION Reagents. Trimethoprim (TMP), diaminopyrimidine (DAP), 3,4,5-trimethoxytoluene (TMT), Luria
Bertani broth, sodium acetate, sodium borate, and sodium hydrogen phosphate were all obtained from Sigma-Aldrich or Fisher with purity higher than 97%. Potassium ferrate solid of ∼98% purity used in the experiments was synthesized by the wet method.27 Fe(VI) solutions were prepared by addition of solid Fe(VI) to 1  10
3 M Na2B4O7.10H2O/5  10
3 M Na2HPO4 at pH 9.0. Concentrations of Fe(VI) in the solution were determined spectroscopically at a wavelength of 510 nm using an Agilent 8453 UV
visible spectrophotometer. A molar absorption coefficient, ε510 nm = 1150 M
1 cm
1 was used to determine Fe(VI) concentration at pH 9.0.27 All solutions were prepared using doubly distilled water that had been passed through an 18 MΩ Milli-Q (Millipore) water purification system. Stock solutions of TMP were prepared by dissolving the solid compound in an appropriate buffer solution and lowering the pH to ∼2.0 in order to facilitate the dissolution. The pH of the dissolved TMP was then adjusted to the desired pH. Diaminopyrimidine stock solutions were prepared in 0.01 M Na2HPO4 buffer solution. Solution of trimethoxytoluene was prepared in 12.5 M methanol
0.01 M Na2HPO4 buffer mixture. Methanol aided in dissolution of trimethoxytoluene to achieve the concentration of 2.50  10
2 M. Kinetic Studies. A stopped-flow spectrophotometer (SX. Eighteen MV, Applied Photophysics, and U.K.) with a photomultiplier (PM) detector was used for the kinetic studies in which the substrate was in excess. Time spectra were collected in the wavelength range from 350 to 750 nm. Kinetic traces were collected at a wavelength of 510 nm to determine the pseudofirst-order rate constants.28 Data collected from the stopped flow were analyzed using the nonlinear least-squares algorithm of SX-18MV global software. Rate constants obtained represent the average of six replicate runs. The UV
visible spectrophotometer was used to study kinetic reactions at pH above 8 where reactions were found to have slow rates. Stoichiometry and Product Studies. The stoichiometric molar ratio of TMP and Fe(VI) was determined by mixing equal solution volumes of 10 mL and the reaction mixtures were maintained at pH 9.0. The TMP was prepared in 1.0  10
4 M Na2HPO4, while the Fe(VI) in 1 10
3 M Na2B4O7.10H2O/ 5  10
3 M Na2HPO4. The concentration of the TMP was kept at 1.0  10
4 M and the concentrations of Fe(VI) varied from 1.0  10
4 - 6.0  10
4 M. This resulted in a [Fe(VI):[TMP] ratio in the reaction mixture of 1: 1 to 6: 1. The Fe(VI)

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concentration was monitored until no residual Fe(VI) was detectable. After completion of the reaction, solutions were filtered using 0.45 μm nylon filters into HPLC vials. The concentrations of the TMP in the resulting reaction mixtures were quantified by the use of a Waters Alliance 2695 HPLC with an Alltima C18 column (250  10 mm, 5 μm) at a wavelength of 271 nm. A binary mobile phase consisting of 70% Solvent A (0.1% HCOOH and acetonitrile at 1:9 (v/v)) and 30% B (0.1% formic acid in water) at a flow rate of 0.4 mL min
1 were used in an isocratic elution mode. The mixed solutions, after completion of the reaction between Fe(VI) and TMP, were also subjected to product analysis. The possible inorganic products from the reaction of TMP and Fe(VI) analyzed were NO2
, NO3
, and NH3. NO3
or NO2
was analyzed by using the Waters Alliance 2695 HPLC with a Waters ion chromatography (IC)-PAC Anion column (4.6  75 mm). Borate gluconate eluant at a flow rate of 1.0 mL min
1 was used and the injected volume of the sample was 100 μL. Possible ammonia evolution was tested using an Orion model 95
12 ammonia electrode in combination with an Orion benchtop pH/ISE meter model 720A. The organic products formed from the reactions of Fe(VI) with TMP, and Fe(VI) with DAP were studied by LC-MS/MS. The LCQ advantage max ion trap mass spectrometer (IT-MS) was operated under electrospray ionization (ESI) attached to quaternary surveyor LC system (ThermoFinnigan, San Jose, CA). The separations were achieved on a Luna C-18 column (150 cm x 4.6 mm x 5 μm - Phenomenex, Torrance, CA). A binary gradient mobile phase at a flow rate of 0.5 mL/min was used. The mobile phase consisted of methanol (A) and 0.1% formic acid with water (B). Phase A was maintained at 10% for the first 2 min, then the percentage was increased to 80% during the next 3 min, then back to 10% in the next 3 min and was maintained at the same level for the last 3 min. The ESI-IT-MS/ MS was operated in the positive ion mode with the capillary temperature at 315 °C and a spray voltage of 4500 V. Data were acquired in the full scan mode (m/z 50
400) for identifying intermediates and MS/MS information on the identified products was obtained in the data dependent scan mode with a collision induced dissociation energy (CID) of 35 eV. Antibacterial Activity Study. Experiments were performed with E. coli 01K1H7 wild type strain at 1  106 CFU/mL. The initial concentration of TMP in the reaction mixtures was TMP 1.0  10
4 M while the concentration of Fe(VI) concentrations varied from 1.0
8.0  10
4 M. All reactions were performed at pH 9.0. After completion of reaction, an aliquot of the reaction mixture was taken and subjected to HPLC analysis. The completion of the reaction was determined when no Fe(VI) remained in the reaction mixture. The residual TMP in each of the reaction samples was determined by the HPLC techniques. The broth microdilution method29 was used to test the biological activity of the mixed solution without filtering (Text S1 of the SI). Control experiments were also performed whereby Fe(VI) which has been reduced to Fe(III) were mixed without TMP. A detailed description of this study is given in Text S1 of the SI. All experiments were conducted in triplicate. The corresponding absorbance readings from the plate reader were converted to growth inhibition percentage by using eq 1. Ið%Þ ¼ fðAmax
AÞ=Amax g  100%

ð1Þ

The Amax represents the maximum absorbance reading; implying there is 0% growth inhibition and the inhibition growth I, thus 10576

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varies from 0 to 100%. Nonlinear regression analysis was used to quantitatively evaluate the antibacterial activity. The relationship between sample dilution and the corresponding growth inhibition I, (%) was fitted using the four parameters sigmodal regression given as eq 2. ð2Þ þ ðY
Y Þ=1 þ ðe
ðX -XoÞ=b Þ I% ¼ Y min

max

min

where Ymax and Ymin represent the maximum and minimum values of the growth inhibition respectively, b is a slope (dimensionless), and Xo is the TMP concentration that provoked a response halfway between the baseline and maximum (EC50). SigmaPlot 2001 software was used to fit the experimental data using eq 2. Removal study. Secondary effluent samples were collected from the Melbourne Reclamation Facility (Florida, USA). The secondary effluent sample had water characteristics as: pH = 7.25, TSS = 0.9 mg/L, nitrogen as N = 11.30 mg/L, phosphorus as P = 1.63 mg/L, BOD = 1.7 mg/L and DOC= 13.9 mg/L. The sample was spiked with 1.7  10
6 M TMP and various concentrations of Fe(VI) were added to determine the removal of TMP from the effluent. The concentrations of TMP remaining in the mixed solutions, after completion of the reactions, were determined using the LC/MS as described above.

’ RESULTS AND DISCUSSION Kinetics. Initially, the oxidation of TMP by Fe(VI) was studied at pH 7.3 and 25 °C by performing spectral measurements during the reaction (Figure S2 of the SI). Fe(VI) decayed without the apparent formation of Fe(VI)-TMP intermediate in the time scale of the studied reaction. The kinetics of the reaction was followed at 510 nm at different concentrations of TMP under pseudo-first-order conditions. The inset of Figure S3 shows the decrease in absorbance of Fe(VI) with time, which could be fitted nicely to a single exponential decay. This suggests that the reaction is first-order with respect to the concentration of Fe(VI). The pseudo-firstorder rate constants (k0 ) were obtained at different concentrations of TMP and a plot of k0 versus [TMP] showed a linear relationship (Figure S3). A log
log plot of the data from Figure S3 gave a slope of 1.00 ( 0.03; indicating the reaction is also first-order with respect to the concentration of TMP. The second-order rate constants (k), for the reaction of Fe(VI) with TMP were then determined at different pH (Figure 1). Generally, the values of k increased with decrease in pH, similar to results from most studies with Fe(VI).22,24,30,31 Similar kinetic studies at different pH were also performed for the reaction of Fe(VI) with DAP and the results are shown in Figure 1. The trend was similar to those observed for reaction of Fe(VI) with TMP. The variation in the values of k with pH in Figure 1 can be explained by considering reactions between acid
base species of Fe(VI) (H3FeO4+ h H+ + H2FeO4, pKa1 = 1.9; H2FeO4 h H+ + HFeO4
, pKa2 = 3.5; HFeO4
h H+ + FeO42‑, pKa2 = 7.2332) and trimethoprim (H2TMP2+ h H+ + HTMP+, pK0 a1 = 3.2;33 HTMP+ h H+ + TMP, pK0 a2 = 7.234). The pH dependence of k for the reaction of Fe(VI) and trimethoprim could be modeled by eq 3. k½FeðVIÞtot ½TMPtot ¼

∑

i ¼ 1, 2, 3, 4, j ¼ 1, 2, 3

Figure 1. Second-order rate constants for the oxidation of TMP, DAP and TMP as a function of pH at 25 °C. (Experimental conditions: [TMP]initial and [DAP]initial = 1  10
3 - 5  10
3 M and [Fe(VI)]initial ≈ 10
4 M; Solid lines were drawn using kinetic model given in eq 3).

where [Fe(VI)]tot = [H3FeO4+] + [H2FeO4] + [HFeO4
]+ [FeO42‑]; [TMP]tot = [H2TMP2+] + [HTMP+] + [TMP]; αi and βj represent the respective species distribution coefficients for Fe(VI) and TMP, i and j represent each of the species of Fe(VI) and TMP respectively and kij is the species-specific second-order rate constant for the reaction between the Fe(VI) species i and the TMP species j. While there are twelve possible reactions from the mathematical expression in eq 3, only three reactions (eqs 4-6) were needed to fit the experimental values of k (solid line in Figure 1). H2 FeO4 þ HTMPþ f FeðOHÞ3 þ ProductðsÞk22 ¼ ð1:6 ( 0:2Þ  103 M
1 s
1

ð4Þ

HFeO4
þ HTMPþ f FeðOHÞ3 þ ProductsðsÞk32 ¼ ð8:0 ( 0:5Þ  101 M
1 s
1

ð5Þ

FeO4 2- þ HTMP f FeðOHÞ3 þ ProductsðsÞk42 ¼ ð2:5 ( 2:1Þ  101 M
1 s
1

ð6Þ

The rate constants for reactions 4
6 demonstrate that the diprotonated (or monoprotonated) species of Fe(VI) react faster with TMP species than the monoprotonated (or unprotonated) species of Fe(VI). This is consistent with the general trend predicted using theoretical calculations.35 The species-specific rate constants for the oxidation of 2,4-diamino pyrimidine (HDAP+ h H+ + DAP, pK0 a2 = 7.434) were also obtained using eq 3 and are shown in eqs 7-9. H2 FeO4 þ HDAPþ f FeðOHÞ3 þ ProductðsÞ k22 ¼ ð2:1 ( 0:2Þ  102 M
1 s
1

ð7Þ

HFeO4
þ HDAPþ f FeðOHÞ3 þ ProductsðsÞ k32 ¼ ð2:4 ( 0:1Þ  101 M
1 s
1

kij αiβj ½FeðVIÞtot ½TMPtot

ð8Þ

FeO4 2
þ HDAP f FeðOHÞ3 þ ProductsðsÞ k42 ¼ ð1:4 ( 0:4Þ  101 M
1 s
1

ð3Þ 10577

ð9Þ

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Figure 2. Stoichiometry of the reaction between Fe(VI) and TMP at pH 9.0. ([Fe(VI)]R = [Fe(VI)] reacted with TMP).

The higher reactivity of the protonated species than the deprotonated species has been attributed to the larger spin density on the oxo ligands of protonated species.35 Relatively, the k32 and k42 differ by ∼3 fold in the case of TMP, but are similar for DAP. This indicates the involvement of the methylene group of TMP in the oxidation by Fe(VI). However, no such possibility exists in the oxidation of DAP by Fe(VI). The reactivity of Fe(VI) with TMT was also performed in the pH range from 5.0
9.0. The rates were similar with and without 1.25  10
2 M TMT present in the reaction mixture. This suggests no significant reaction between the Fe(VI) and TMT occurred. Stoichiometry and Products. Two sets of experiments were conducted to determine the stoichiometry of the reaction between Fe(VI) and TMP at pH 9.0. In the first set, solutions of different concentrations of Fe(VI) were mixed with solutions that had a fixed initial concentration of TMP. An increase in the concentration of the Fe(VI) resulted in a decrease in the concentration of the TMP, however, Fe(VI) could also react simultaneously with buffered water (Table S1 of the SI). Hence, Fe(VI) and buffered solutions were mixed and the decay of Fe(VI) was monitored in the second set of experiments. The amount of Fe(VI) reacted with only TMP was obtained from this set of experiments. The plot [TMP] vs the [Fe(VI)]reacted showed a linear relationship with the slope (Δ[TMP]/Δ[Fe(VI)]) being
0.20 ( 0.02 (Figure 2). This indicates that the stoichiometric ratio is ∼5:1 ([Fe(VI)]:[TMP]) and the stoichiometry may be written as follows: 5FeðVIÞ þ TMP f 5FeðIIIÞ þ productðsÞ

ð10Þ

Next, a study on the products of reaction 10 was performed. The presence of amine groups in the TMP (see Figure S1 of the SI) suggests the possibility of the formation of inorganic products such as ammonia, nitrite, and nitrate. A fixed concentration of TMP of 2.0  10
4 M was reacted with different concentrations of Fe(VI). Ammonia analyses were conducted using an ammonia electrode in all the reaction mixtures. There was no significant evolution of NH3 as the concentration of Fe(VI) was increased in the reaction mixture, suggesting that there was no NH3 released in the oxidation process. The same reaction mixture samples were also subjected to analysis for NO2
and NO3
. No detectable levels of either of the ions were observed. This suggests that the amine groups of TMP were not converted to inorganic oxy-nitrogen compounds or ions.

Figure 3. Integrated peak areas in the reaction of Fe(VI) reaction with TMP and DAP at pH 9.0 and 25 °C. ([TMP] = 1.0  10
4 M and [DAP] = 1.0  10
4 M, filled symbols
reaction between Fe(VI) and TMP; unfilled symbols - reaction between Fe(VI) and DAP; DNP*
DAP found in oxidation of DAP by Fe(VI)).

Organic products of the oxidation of TMP by Fe(VI) were studied at different molar ratios of added Fe(VI) to TMP (filled symbols, Figure 3A). Mass spectra of the identified organic products of the oxidation of TMP by Fe(VI) after separation by chromatography are shown in Figures S4A
F of the SI. Among the identified intermediates and final products had m/z = 307 (TMP
OH), m/z = 305 (TMP=O), m/z = 197 (TMBA), and m/z = 171 (DNP) (Table S2 of the SI; Figure 3A). TMP
OH was observed at a 0.5:1 molar ratio of Fe(VI): TMP, which decreased with the increase in concentration of Fe(VI) until it degraded completely at a molar ratio of 2.7: 1. The formation of nitropyrimidine DNP, was observed when excess amount of Fe(VI) was reacted with TMP. DAP as a possible intermediate before yielding DNP was not seen. This could be attributed to competing reaction rates of Fe(VI) with TMP and DAP which have similar rate constants at pH 9.0. Formation of DNP as the final product from DAP most likely occurred through several intermediates, which also reacted with Fe(VI). In a separate experiment, the reaction between Fe(VI) and DAP also formed DNP as the product (unfilled symbols, Figure 3). This supports the DAP as the possible intermediate in the reaction of Fe(VI) with TMP. Products and their levels in the reaction mixture indicate that the oxidation of TMP by Fe(VI) involved oxidation and cleavage steps. Proposed reaction pathways for the reaction of Fe(VI) with TMP are presented in Figure 4, which are based on identified organic products. A number of initial reaction sites on TMP are possible including the exocyclic amino groups of the diaminopyridine ring and an activated bridging methylene group. In pathway (1), Fe(VI) preferentially attacks the reactive bridging methylene group of TMP to undergo oxidation to form intermediate, TMP
OH with m/z of 307. The methylene bridge is activated due to the presence of both the electron withdrawing trimethoxybenzene and diaminopyimide rings which can stabilize the resulting intermediate through resonance. Activated methylene oxidation by iron complexes has previously been reported for a number of substrates including cinnamyl alcohol, 10578

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Figure 4. Reaction pathways for oxidation of TMP by Fe(VI).

tetrahydronaphthalene, fluorene, diphenylmethane, and dihydroanthracene.36 The decrease in TMP and the formation of TMP
OH were seen at a Fe(VI) to TMP molar ratio of 0.5:1 (Figure 3). At higher molar ratios of Fe(VI) to TMP, a new product, TMPdO, with m/z 305, was observed (Figure 3). This indicates that TMP
OH underwent further reaction to yield the ketone product, TMPdO (pathway (2)). Both reaction pathways have also been suggested in the reaction of TMP with permanganate.37 Additionally, Fe(VI) has been shown to selectively oxidize secondary alcohols to ketones.38 Significantly, TMP
OH showed a continuous decrease with the increase in the amount of Fe(VI) while TMPdO remained in the solution as a stable product (Figure 3). This is not surprising considering no proton is available on TMP=O for abstraction by Fe(VI). Moreover, diaryl ketons has resonance stabilization, which may not be allowing further oxidation of TMP=O. Therefore, pathway 3 is proposed in which cleavage of TMP
OH takes place, to produce DAP and TMBA of m/z 111 and m/z 197, respectively. The amino product, DAP, showed further reactivity with Fe(VI) (Figure 1), hence its concentration decreased and a new product, DNP with m/z of 171 (Figure S4E) was formed (pathway 4). The results of the reaction between Fe(VI) and DAP, shown in Figure 3 (unfilled symbols), further indicate the decrease of DAP and concomitant formation of DNP (Figure 3). There was no further decrease in DNP, analogous to the reaction of Fe(VI) with TMP (Figure 3). Formation of nitro compounds was also observed in the reaction of Fe(VI) with arylamines.39,40 Antibacterial Activity after Oxidation. The results of E. coli growth experiments in the absence and presence of Fe(VI) treatment of TMP are presented in Figure 5A. Increases in the concentration of Fe(VI) eventually resulted in no growth

inhibition of E. coli at a concentration of Fe(VI) of 8.2  10
4 M, which corresponded to a molar ratio of 8:1 of Fe(VI) added to TMP in the reaction mixture. This implies that oxidation of the TMP by Fe(VI) resulted in elimination of the antibacterial properties of TMP. This is illustrated by the shift of the dose
response curve from the left to the right, where the leftmost curve is the untreated TMP. The antibacterial properties of TMP have been attributed to the 2,4-diaminopyrimidine moiety,12,13 and since this moiety was altered to 2,4- dinitropyrimidine, antibacterial properties were no longer evident. Another possible explanation can be attributed to the lower concentration of the TMP as a result of the reaction, or to any oxidized product(s) that might still have antibacterial properties but less than the minimum inhibition concentration (MIC). A MIC of ∼0.2 x10
6 M and EC50 of ∼8.0  10
6 M were estimated from the dose
response plot. In other studies where E. coli (ATCC 25922) were used with temperatures of 22 °C, 28 and 35 °C and corresponding incubation times of 24 - 28 h, 44 - 48 h, 16 - 20 h respectively, all resulted in a MIC median of 0.21  10
6 M .41 A control experiment in which E. coli growth was monitored in the presence of Fe(III) showed no growth inhibition of the bacteria, similar to when excess of Fe(VI) was used to consume all TMP from the reaction. Correlating the residual concentration of the TMP in respective samples as the Fe(VI) dosage was increased in reaction, a potency equivalent quotient (PEQ) value can be calculated for each sample using eq 11. PEQ ¼ EC50, 0 =EC50, X

ð11Þ

EC50,0 in eq 11 represents the EC50 value calculated from the measured dose
response relationship for the TMP without any 10579

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Figure 6. Residual TMP in secondary effluents of Water Reclamation facility after adding Fe(VI) at pH 9.0 (In mixed solution: [TMP] = 1.7  10
6 M).

Figure 5. (A) Dose
response relationships for initial [TMP] = 1.0  10
4 M with increasing concentration of Fe(VI) at 37 °C and 18 h inoculation, and (B) Potency equivalent quotient plots with the corresponding TMP oxidation by Fe(VI).

oxidizing Fe(VI) added and EC50,x represents the EC50 for the corresponding plots with  M Fe(VI) dosed to the TMP to cause oxidation (Figure 5A). The individual calculated PEQ’s are plotted against the [TMP]/[TMP]o in each sample (Figure 5B). This plot was used to evaluate the quantitative relationship between the decrease of the TMP compound and corresponding changes in its biological activity. The straight line represents in which a loss of one mole of TMP would result in the loss of one PEQ of the TMP antibacterial properties. The experimental data represented in Figure 5B showed a negative deviation. This indicates that not only the oxidation products had no antibacterial properties, but also interfered with the inhibition properties of TMP.17 Furthermore, the unequal negative deviation from the ideal at different dosages of Fe(VI) suggest that the oxidation products formed at various dosages of Fe(VI) caused different interference(s) to the inhibition properties of TMP. A possible role of Fe(III) in the negative deviation (Figure 5B) may not be significant because no proportional deviation with increase in the concentration of Fe(III), produced from Fe(VI), was seen. The mixture of products resulting from the Fe(VI) reactions with TMP had insignificant antibacterial potency in comparison to the TMP. Removal Study. Results of the removal experiments, using secondary effluent water demonstrate that Fe(VI) is effective in oxidizing TMP (Figure 6). The secondary effluent water was relatively clean since it had undergone treatment and did not supposedly have many reactive compounds. At a concentration of 1.7  10
6 M TMP, Fe(VI) removed TMP completely

(Figure 6). However, demand for Fe(VI) was more than predicted by stoichiometry of the reaction. This indicates that the other components present in the effluent competed to cause an increase in the demand of Fe(VI). Additionally, if excess pollutants present in the secondary effluent have higher rate constants for oxidation with Fe(VI) than TMP, the demand for Fe(VI) would increase for complete removal of TMP from the secondary effluent water. The water matrices such as dissolved organic matter (DOM) would also increase the demand for Fe(VI) since they would be in higher concentration compared to the TMP. Nevertheless, Fe(VI) showed a potential to remove TMP and demand of Fe(VI) dose would vary with the concentration of TMP and constituents of water matrices.

’ ASSOCIATED CONTENT

bS

Supporting Information. Supporting Information (Tables S1
S2, Figures S1
S4, and Text S1). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: 321-674-7310; fax: 321-674-8951; e-mail: vsharma@ fit.edu.

’ ACKNOWLEDGMENT The authors wish to thank the Center for Ferrate Excellence. Authors also thank Professors Clayton Baum, Nasri Nesnas, and Mary Sohn for their comments and Christa Simmers for guidance with E. coli experiments. The authors also wish to thank the anonymous reviewers for their comments which improved the manuscript greatly. ’ REFERENCES (1) Miege, C.; Choubert, J. M.; Ribeiro, L.; Eusebe, M.; Coquery, M. Fate of pharmaceuticals and personal care products in wastewater treatment plants—Conception of a database and first results. Environ. Pollut. 2009, 157, 1721–1726. (2) Mohring, S. A. I.; Strzysch, I.; Fernandes, M. R.; Kiffmeyer, T. K.; Tuerk, J.; Hamscher, G. Degradation and elimination of various sulfonamides during anaerobic fermentation: A promising step on the 10580

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