Article pubs.acs.org/est
Phototransformation of Cephalosporin Antibiotics in an Aqueous Environment Results in Higher Toxicity Xiao-Huan Wang† and Angela Yu-Chen Lin*,† †
Graduate Institute of Environmental Engineering, National Taiwan University, 71, Chou-Shan Road, Taipei 106, Taiwan S Supporting Information *
ABSTRACT: Photodegradation may be the most important elimination process for cephalosporin antibiotics in surface water. Cefazolin (CFZ) and cephapirin (CFP) underwent mainly direct photolysis (t1/2 = 0.7, 3.9 h), while cephalexin (CFX) and cephradine (CFD) were mainly transformed by indirect photolysis, which during the process a bicarbonate-enhanced nitrate system contributed most to the loss rate of CFX, CFD, and cefotaxime (CTX) (t1/2 = 4.5, 5.3, and 1.3 h, respectively). Laboratory data suggested that bicarbonate enhanced the phototransformation of CFD and CFX in natural water environments. When used together, NO3−, HCO3−, and DOM closely simulated the photolysis behavior in the Jingmei River and were the strongest determinants in the fate of cephalosporins. TOC and byproducts were investigated and identified. Direct photolysis led to decarboxylation of CFD, CFX, and CFP. Transformation only (no mineralization) of all cephalosporins was observed through direct photolysis; byproducts were found to be even less photolabile and more toxic (via the Microtox test). CFZ exhibited the strongest acute toxicity after just a few hours, which may be largely attributed to its 5methyl-1,3,4-thiadiazole-2-thiol moiety. Many pharmaceuticals were previously known to undergo direct sunlight photolysis and transformation in surface waters; however, the synergistic increase in toxicity caused by this cocktail (via pharmaceutical photobyproducts) cannot be ignored and warrants future research attention.
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INTRODUCTION Numerous pharmaceuticals have been detected in natural waterways, including rivers, lakes, regional discharges, and constructed wetlands.1−5 The presence of antibiotics in aqueous environments is becoming a major worldwide concern, as they may strengthen bacterial resistance toward pharmaceuticals.6−8 The cephalosporins, a class of β-lactam antibiotics, disrupt the synthesis of bacterial cell walls, thereby preventing bacterial growth, and have the second highest usage of all antibiotics in Taiwan. We selected the following five cephalosporin antibiotics as target compounds for an in-depth study: cephalexin (CFX), cephradine (CFD), cefotaxime (CTX), cefazolin (CFZ), and cephapirin (CFP). Figure 1 shows the structure of the five target cephalosporins, which differ primarily in the functional groups R1 and R2. Based on statistical data from Taiwan’s Bureau of National Health Insurance,9 cephalexin (73%) and cephradine (10%) were the two most frequently prescribed cephalosporin antibiotics in 2005. All target compounds have been frequently detected in surface waters, with concentrations ranging from 76 to 1117 ng L−1.3,10 High concentrations have been detected in the influents (up to 1100−64000 ng L−1)3,10−13 and effluents (up to 13-2104 ng L−1) of urban wastewater as well as in hospital effluents (up to 10−42900 ng L−1)3,10 in various studies. Organic compounds undergo various degradation pathways in environmental waters, including biotic (biodegradation, bioaccumulation) and abiotic (hydrolysis, photolysis, sorption, oxidation) degradation. However, one study has found that CFD and other cephalosporins had not undergone measurable biodegradation in China’s Xuanwu Lake after seven days14 and © 2012 American Chemical Society
had a low octanol−water partitioning coefficient (logKow = −1.40 to −0.13);15−17 thus they could not be expected to be eliminated through sorption or biotransformation in an aqueous environment. Although cephalosporin antibiotics can be strongly hydrolyzed (t1/2 = 1−10 d at pH >8 or 286 nm (the shortest wavelength received on the Earth’s surface)41 (Figures 1a and 1b). As expected from the UV−vis spectrum, all target compounds underwent direct photolysis but at varying rates; for CFP, direct photolysis rates increased with decreasing pH. This can be explained by its red shift in absorption spectra with decreasing pH. The direct photodegradation rate constants of the target compounds were measured in DI water under simulated sunlight and are listed in Table 1. Pseudo-first-order kinetics was applied with strong correlation coefficients (R2 > 0.99), yielding half-lives of CFZ, CFP, CTX, CFD, and CFX of 0.7, 3.9, 6.9, 31.9, and 33.7 h, respectively (pH 7.0, in DI water). The quantum yields (Φ) are also reported in Table 1 and range from 0.001 to 0.091. 3.2. Photolysis in Natural Waters. Besides direct photolysis, indirect photolysis involving different photosensitizers can also be observed in the natural water matrices. The Jingmei River (pH = 7.8, [NO3−-N] = 1.2−1.8 mg L−1, [DOC] = 3.6−6.0 mg L−1, alkalinity = 80−103.8 mg CaCO3 L−1) served as the sample environment for our study of photodegradation processes of cephalosporins in surface water environments. Compared to photolysis rates observed in DI water, rates observed in Jingmei River water for two cephalosporin antibiotics were significantly enhanced; CFX and CFD were photodegraded at 4.2- and 3.8-fold faster rates (Table 1), respectively. This finding indicates that indirect photolysis may be the primary degradation process for these two compounds. The water parameters in the matrix of natural water are likely to produce transient excited species upon sunlight irradiation and lead to photooxidation of CFX and CFD. However, the photolysis rates observed for CFZ and CFP in the Jingmei River were slower than in DI water, implying that the
electrospray ionization (ESI) interface, we detected byproducts in the degradation mixture (MS/MS in full-scan mode) and obtained their mass spectra (Figure S1, Supporting Information). Chromatographic separation of these byproducts was performed using an Agilent 1200 module (Agilent, Palo Alto, CA, USA) equipped with a ZORBAX Eclipse XDB-C18 column (Agilent, Palo Alto, CA, USA, 150 × 4.6 mm, 5 μm). The signal areas of byproducts were quantified with LC-MS/MS, and the parameters of each byproduct are shown in Text S2, Supporting Information. We used an ACD MS fragmenter (Advanced Chemical Development, Toronto, ON, Canada) to generate a tree fragmentation for each cephalosporin structure, using mass spectrometry fragmentation rules; in addition, the selected ESI ionization mode and the number of fragmentation steps were used to identify photolysis byproducts. Tree fragmentation of each parent compound (the five target cephalosporins) was used to identify the product ions from the mass spectrum of their byproducts, and the product ions were then combined to predict the structure of the byproducts. 2.5. Toxicity Measurements. Microtox acute toxicity testing was performed with Vibrio f ischeri using a Model 500 Analyzer (Microbics Corp., Carlsbad, CA, USA). Acute toxicity testing on bacteria was conducted using the Microtox system, which measures the decrease in light output of the luminescent marine bacterium Vibrio fischeri. Toxicants influencing the enzymatic activity of Vibrio f ischeri reduce bioluminescence,40 which was measured at 5 and 15 min and compared to control. Both samples from dark-control and irradiation were controlled at pH 7 before toxicity testing. Measurement of toxicity was performed within 42 h after irradiation. The EC50 values used in this study are expressed as percentage (% v/v) of the initial sample, where EC50 is the concentration that causes 50% reduction in Vibrio bioluminescence. 12419
dx.doi.org/10.1021/es301929e | Environ. Sci. Technol. 2012, 46, 12417−12426
Environmental Science & Technology
Article
Figure 2. Synthetic water with the same nitrate, FA and bicarbonate concentrations as in (a) Jingmei River-1 sample (pH = 7.8, [NO3−-N] = 1.2 mg L−1, [DOC] = 6.0 mg L−1, alkalinity = 103.8 mg CaCO3 L‑1) and (b) Jingmei River-2 sample (pH = 7.8, [NO3−-N] = 1.8 mg L−1, [DOC] = 3.6 mg L−1, alkalinity = 80 mg CaCO3 L−1) were used to simulate the photodegradation of CFD. Decarbonated-Jingmei River-2 indicates the photolysis behavior in the Jingmei River-2 water after HCO3− was removed. (c) Indirect photolysis of CFD in the presence of 4 mg-C L−1 FA or 5 or 10 mg-N L−1 of nitrate, with varying concentrations of bicarbonate at pH 7.0.
shortened from 33.6 to 11.9, 31.9 to 14.6, and 6.9 to 4.3 h, respectively; however, CFP and CFZ were not particularly affected. Since •OH is a strong oxidizing agent and has high reactivity with most organic substrates, its importance to target compounds depends on its steady-state concentrations ([•OH]ss) in natural waters. The bimolecular rate constants measured for the reactions of CFX (7.1 × 109 M−1 S−1), CFD (1.1 × 1010 M−1 S−1), and CTX (8.1 × 109 M−1 S−1) with •OH were all approximately 1010 M−1 s−1 (the measurement method was described in texts S1 and S3, Supporting Information). While these rate constants are near the diffusion-controlled limit, the [•OH]ss in natural waters is an important factor in this reaction. For most natural surface waters, which have [•OH]ss =10−1610−18 M,43,51 the calculated half-lives of CFX, CFD, and CTX are rather long and likely mean that •OH reaction is a relatively minor elimination pathway compared to the effect of other photoreactants. 3.2.3. Bicarbonate − Enhanced Nitrate System. Bicarbonate, which is responsible for alkalinity, is the most common inorganic anion present in natural surface water. Because of high molar concentrations of HCO3− in surface water (1−3 mM28,52), •OH may collide with HCO3− easily to produce carbonate radicals (•OH + HCO3− /CO32‑→ CO3•− + H2O, k = 1.5 × 107 M−1 S−1).53 In contrast to •OH, carbonate radical is a more selective oxidant. The steady-state concentrations of carbonate radicals (10−13−10−14 M)30 are expected to be significantly higher than those of •OH in natural surface waters. As shown in Table 1, the indirect photolysis rates of CFX, CFD, and CTX in the presence of HCO3− and NO3− were 7.5-,
matrix of natural water competed with the light source and hindered light transmission, resulting in a reduced photolysis rate. We concluded that direct photolysis is most likely the main photolytic pathway for CFZ and CFP in natural surface water systems. In the case of CTX, both direct and indirect photolysis appear to be equally important. To better understand the effect of specific water parameters/photosensitizers and their combining/competing effect on photolysis rates of cephalosporin drugs, sets of water spiked with DOM, nitrate, and bicarbonate were tested in the laboratory, and results are described below. 3.2.1. Effect of DOM. Suwannee River Fulvic Acid standard (FA) was used to study the effect of DOM on the photolysis rates of cephalosporins. DOM progresses through an excited triplet state (e.g., 3DOM*) to form excited species (e.g., singlet oxygen (1O2), peroxyl radicals (ROO•), and aqueous electrons (eaq−)) that photooxidize organic contaminants. On the other hand, DOM can also react with and serve as the sink for these excited species. Our experimental results showed photodegradation rates of CFX, CFD, and CTX in FA-spiked water that were slightly higher than degradation rates observed in DI water (Table 1), implying that DOM-derived photoreactants could react and consume the target compounds. 3.2.2. Effect of Nitrate. Nitrate under solar light is the major precursor for •OH;29,42−44 it is commonly present in surface waters and ranges in concentration from 0.1−5 mg-N L−1;45−48 with higher concentrations (9−15 mg-N L−1) observed in sites with intense agricultural activity.49,50 Table 1 shows that the photodegradation rates of CFX, CFD, and CTX were enhanced by the presence of 2.7 mg L−1 NO3− and that their half-lives 12420
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results suggest that after exposure of a typical surface water (HCO3− = 1−3 mM28,52) to a simulated sunlight with intensity = 765 W/m2 (equivalent to midday, midsummer sun in Southern California),55 the amount of HCO3− present should always be sufficient and will mostly likely not create observable differences on cephalosporin photolysis rates. 3.3. Photolysis Byproducts. Approximately 2.0−8.5% CFD was found to be dehydrogenated (oxidized) to CFX in DOM, nitrate, nitrate + bicarbonate solutions, and in the Jingmei River water (Figure 3). However, direct photolysis of CFD did not
6.0-, and 5.3-fold faster than were direct photolysis rates and were 2.6-, 1.8-, and 3.1-fold faster than when only NO3− was present in the solution. As a result, bicarbonate appears to enhance the photolysis rates in the nitrate system. Vione et al. 31 has shown that bicarbonate was able to induce a considerable formation of the carbonate radicals, and this higher availability of less-reactive species than the •OH would enhance the photolysis of compounds that are sufficiently reactive toward the carbonate radical. Studies have shown that carbonate radical reacts rapidly with compounds bearing easily oxidizable moieties, such as amino acids36 and aromatic anilines bearing electron-donating substituents.33 The carbonate radical may also be a significant reactant in the oxidation of sulfur-containing compounds.53 CFX and CFD have structures similar to amino acids, while CTX has an aromatic aniline’s structure, and all three contain a sulfur atom. This may explain the rapid indirect photolysis rates (t1/2 = 1.3−5.3 h) observed for these three target cephalosporins. Synthetic water with the same nitrate, DOM, and bicarbonate concentrations as in the Jingmei River-1 (pH = 7.8, [NO3−-N] = 1.2 mg L−1, [DOC] = 6.0 mg L−1, alkalinity = 103.8 mg CaCO3 L−1) was used to simulate the photodegradation of target compounds and compare it to results from Jingmei River water1; the data are shown in Table 1. The relative reaction rate constants (krel = ksimulated/kJingmei) were calculated to be krel = 0.8, 1.0, 0.8, 1.0, and 0.8 for CFX, CFD, CTX, CFZ, and CFP, respectively. The photolysis fate of target compounds in the Jingmei River was closely simulated by the synthetic water experiments, indicating that levels of nitrate, DOM, and bicarbonate are the strongest determinants in the fate of cephalosporins in natural surface water environments. Laboratory studies on CFD (as shown in Figure 2a) further suggest that in a natural surface water system such as the Jingmei River, NO3−, HCO3−, and DOM all contribute to indirect photodegradation. Synthetic waters with FA, NO3−, or FA + NO3− underestimated the CFD photolysis in natural environment; however, synthetic waters containing NO3− + HCO3− overestimated the photolysis rate. Only in the FA + NO3− + HCO3− system did photolysis rates (t1/2 = 8.4 ± 0.3 h) match rates of Jingmei River water, indicating the competing effects among these three important water parameters. In a previous study, Jiang et al.14 also noted that DOM and NO3− alone do not explain the much faster photolysis rate observed in lake water for CFD and other cephalosporins. Figure 2(b) again shows the important role of bicarbonate on surface water photochemistry; in the decarbonated Jingmei River-2 water (removing HCO3− from the Jingmei River-2 sample), CFD photolysis rates were greatly reduced and closely matched rates from the system containing NO3− + FA alone (t1/2 = 16.9 h). CFX exhibits very similar behavior (Figure S3b, Supporting Information). As shown in Figure 2(b), bicarbonate significantly enhanced photolysis rates of cephalosporins in natural waters (2.6 times faster for both CFD and CFX in Jingmei River waters as compared to decarbonated river waters). Huang and Mabury30 also demonstrated the important role of bicarbonate in the N,Ndimethylaniline photoreaction in decarbonated Thames River water, concluding that carbonate radicals were the main reagents produced in sunlight surface waters responsible for the removal. In phenol photodegradation experiments, bicarbonate enhanced the rate of nitrate photolysis in solutions containing [HCO3−] > 10 mM in the absence of DOM.54 However, in our system in the presence of 4 mg C L−1 FA, bicarbonate was able to enhance the rate at only 0.1−5 mM [HCO3−] level, and the photolysis rate remained fairly constant (RSD < 8%) in this range at pH 7. Our
Figure 3. CFD (△) was dehydrogenated (via oxidation) to produce CFX (○) under simulated sunlight in DOM (FA), NO3−, HCO3− solutions and Jingmei River-1. ([NO3−-N] = 1.2 mg L−1; [HCO3−] = 2 mM; DOM (FA) = 3 mg C L−1). CFX was not observed in the darkcontrol samples.
result in CFX. Previous studies have demonstrated oxidation of 1,4-cyclohexadiene to benzene in an aqueous solution.56 As Figure 3 shows, CFX was quickly produced and peaked at 14 h, followed by degradation within 42 h of irradiation in an NO3− + HCO3− system. The faster production and degradation of CFX in a NO3− + HCO3− system vs in NO3− or FA systems indicate the importance of bicarbonate in enhancing rates of photodegradation. Besides its higher drug usage (CFX, 73%; CFD, 10%), this finding may help to explain why a higher concentration of CFX than of CFD was detected in surface water. Our investigation found that CFX was typically detected at 0.3 μg L−1 in the Jingmei River and 53−243 ng L−1 in the Sindian River (Taipei region), while CFD was not detected in the Jingmei River (
