Environ. Sci. Technol. 2010, 44, 284–289
Nitration Processes of Acetaminophen in Nitrifying Activated Sludge S E R G E C H I R O N , †,* E L E N A G O M E Z , ‡ A N D ´ LE HE ` NE FENET‡ UMR 5569 ‘Hydrosciences Montpellier’ University of Montpellier I, 15 Avenue Ch. Flahault, BP 14491, 34093 Montpellier cedex 5, France and Laboratoire Chimie Provence, Aix-Marseille Universite´s-CNRS (UMR 6264), 3 place Victor Hugo, 13331 Marseille cedex 3, France
Received July 16, 2009. Revised manuscript received November 14, 2009. Accepted November 17, 2009.
This work is an attempt to elucidate the quantitative significance of acetaminophen (APAP) nitration in nitrifying activated sludge and to propose a reaction mechanism for this process. The link between nitrification and nitration of APAP was investigated at different scales. Results from field studies showed the occurrence of 3-nitro-APAP and to a lesser extent 3-chloro-5-nitro-APAP at concentration levels in the 50-300 ng/L range in effluents of a full scale wastewater treatment plant (WWTP) operated with nitrogen removal, whereas 3-hydroxy-APAP was eliminated after the nitrification step. Batch experiments with nitrifying activated sludge confirmed APAP transformation by nitration and suggested that nitrifying bacteria may play a role in this transformation process through the release of reactive nitrogen species. In vitro assays provided evidence that nitration through the production of nitrous acid is a very unlikely pathway. In contrast, nitric oxide (•NO) produced by nitrifying bacteria is probably involved in APAP nitration through the formation of peroxynitrite in presence of superoxide anion. The production of 3-nitro-APAP would only account for a few percents of the total transformation rate of APAP in WWTPs. The production of nitrated derivatives is highly relevant because of the potential ecotoxicological risks of these compounds.
Introduction For most pharmaceutical products (PPs), municipal wastewater treatment plants (WWTPs) represent the final treatment step prior to release into the environment. State of the art biological treatment schemes are not fully efficient in eliminating PPs, and biodegradation has been suggested as the most important PPs removal mechanism in activated sludge (1, 2). PPs removal efficiency increases as solids retention time (SRT) increases (3), and WWTPs operated for nitrogen removal have demonstrated increased removal of organic micropollutants (4). However, biodegradation mechanisms are generally not known and little evidence has been published with regards to PPs metabolic pathways in complex communities like those encountered in activated sludge treatment. The significance of hydroxylation pathway has * Corresponding author phone: +33-4-91-10-85-25; fax +33-491-10-63-77; e-mail:
[email protected]. † University of Marseille. ‡ University of Montpellier. 284
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been clearly demonstrated in case of the nonsteroidal antiinflammatory drugs ibuprofen (5) as well as for the antibiotic trimethoprim (6), while hydrolysis pathways have been described for the lipid regulator bezafibrate (7) and the β-blocker atenolol (8) and oxidation of primary and secondary hydroxyl group for the X-ray contrast medium iopromide (9). Another biotransformation pathway of environmental concern might be nitration. In a recent study, abiotic nitration was regarded to be responsible for the transformation of 17R-ethinylestradiol (EE2) into nitrated EE2 derivatives but was ascribed to an artifact of the batch test conditions (10). However, diclofenac (11) and acetaminophen (APAP) in this work have been found to undergo nitration in WWTPs. In agricultural soils, a nonylphenol isomer (4-(1-ethyl-1,4dimethylpentyl)phenol was biologically converted into its 2-nitro metabolite with a 13% yield (12). Consequently, the nitration mechanisms of xenobiotics under biotransformation processes seem to be lacking. Interestingly, nitric oxide (•NO), a powerful precursor of reactive nitrogen species (RNS) and nitrating agents such as peroxynitrite (ONOO-) and nitrogen dioxide radicals (•NO2) (13) are produced during nitrogen removal in WWTPs by different groups of microorganims including ammonia oxidizing bacteria (AOB) (14). Liquid •NO concentration levels of 0.15-0.30 µg N/L have been recently calculated in nitrifying culture, during the stable ammonium conversion phase (15). In mammalian cells, excess production of •NO results in nitration processes of different phenolic compounds (e.g., tyrosine, tryptophane, and dopamine) responsible for physiological and pathophysiological processes (16). Surprisingly, the potential implication of microbial-generated RNS in the nitration of phenolic compounds both of natural and anthropogenic origins in WWTPs including nitrogen removal has not been investigated yet. This will be the main contribution of this work by achieving field studies and by investigating nitration kinetic as well as reaction mechanism aspects through laboratory-scale experiments and finally by using the drug APAP as a mechanistic probe compound. APAP has been selected in this work because it is readily prone to nitration processes (17) and because it is also the most heavily used over-the-counter analgesic in Europe, exhibiting negligible adsorption to sludge and extensive biological transformation (>90%, ref 18). APAP has therefore been detected in WWTPs effluents at high concentration levels in the 1-10 µg/L range (19). The wide occurrence and peculiar chemistry exhibited by APAP made it a suitable xenobiotic to investigate the significance of nitration processes because potential nitrated APAP derivatives would be present at detectable levels in WWTPs effluents, even though nitration processes are quantitatively minor transformation pathways.
Materials and Methods Additional details for the following sections are provided in the Supporting Information (SI). Reagents and Chemicals. Acetaminophen (99%), 4-amino2-chlorophenol(96%),4-amino-2-nitrophenol(97%),4-amino3-nitrophenol (98%), and cotinine-d3 (methyl-d3) were from Sigma-Aldrich (Saint Quentin-Fallavier, France); (2,4-dichlorophenoxy)acetic acid d3 (2,4-D d3) from CIL Cluzeau Info Labo (Courbevoie, France). Synthesis of 3-Nitro-4-hydroxyacetanilide (3-nitroAPAP), 2-Nitro-4-hydroxyacetanilide (2-Nitro-APAP) 3-chloro-5-nitro-4-hydroxyacetanilide (3-Chloro-5-nitro-APAP), and 3,5-Dinitro-4-hydroxyacetanilide (3,5-Dinitro-APAP). The nitration step was conducted by treating 250 mg APAP or 4-amino-2-chlorophenol or 4-amino-2-nitrophenol with 10.1021/es902129c
2010 American Chemical Society
Published on Web 12/08/2009
a five molar excess of sodium nitrite in aqueous acetic acid at pH 4 during 48 h. The N-acetylation step was achieved by treating nitrated compounds with acetic anhydride aqueous solutions at 60 °C and at reflux for 5-10 min. We obtained 3,4-dihydroxyacetalinide (3-OH-APAP) by treating APAP with a 10 molar excess of an equimolar acidic solution (pH 3) of peroxymonosulfate and cobalt(II) which allowed a nearly specific hydroxylation in the ortho position of the phenolic group of APAP. After synthesis, chemicals were further purified by preparative HPLC. Their identity and purity were confirmed by electrospray (ESI) LC-MS/MS. The chemical structures and M/MS spectra of investigated compounds are reported in SI Figure SI1. The MS/MS spectra were all characterized by at least two major fragment ions which are suitable for their unambiguous identification at trace level in environmental samples. The two isomers, 3-nitro-APAP and 2-nitro-APAP, can be potentially generated together but were clearly discriminated by LC-MS analysis due to different fragmentation patterns in the MS/MS mode. The product ion (-) ESI mass spectrum of 3-nitro-APAP using [M-H]- ion at m/z 195 as the precursor ion yields two main fragment ions at m/z 150 and 165 corresponding to [MsNO2-] and [MsHsNO]-, respectively. The MS/MS spectrum of 2-nitroAPAP is depicted in SI Figure SI2 and is characterized by two additional fragment ions at m/z 148 and m/z 123. These latter ions may arise from further losses of OH and COCH3 from m/z 165. Field Sampling. The WWTP selected for this study is located in Aix-en-Provence (175 000 inhabitants serviced, South-East of France). For the secondary treatment processes, this WWTP includes a two-stage secondary biological degradation, with both stages being slurry systems. Stage-1 is a conventional activated sludge with relatively short SRT (0.3 day) for chemical oxygen demand (COD) removal. Stage-2 is a separate activated sludge process with longer SRT (15-20 days) and is optimized for effective nitrification. Samples were collected during the months of October, November, and December 2008. We collected 24 h composite samples during one week of each month using automated samplers in clean baked amber glass bottles from influent and stage-2 secondary effluent. Water samples were filtered using GF/C filters and then frozen at -20 °C until analysis. The filters were discarded. Batch Biodegradation Experiments by Nitrifying Activated Sludge. Five L Erlenmeyer flasks were partially filled with biomass collected from the stage-2 of the Aix-enProvence WWTP with initial mixed liquor suspended solids (MLSS) in the reactors being 2.5 g/L. Each flask was wrapped with aluminum foil. Prior to the spiking with APAP and 3-chloro-APAP dissolved in water at a concentration level of 100 µg/L, the collected biomass was aerated for one day to reduce dissolved organic matter and supplemented with 10 mg/L NH4sN to ensure the growth of nitrifying bacteria. The reactors were daily monitored by measurement of pH and dissolved oxygen and the pH was maintained at 7-7.5 by titration with 0.5 M NaHCO3. Flasks were incubated at a temperature-controlled of 25 °C and mixed with magnetic stirrers at 150 rpm. Shaking ensured a sufficient supply of oxygen to keep the dissolved oxygen concentration higher than 3 mg/L. The chemical-treated reactors were further subdivided into batch-1 (no allylthiourea), batch-2 with allylthiourea (final concentration of 5 mg/L) to inhibit nitrification (20), and into batch-3 with HgSO4 (final concentration of 0.4 g/L) for full inactivation of sludge. At a predetermined sampling time, 20 mL aliquots were withdrawn from each reactor. Samples were centrifuged at 3500 rpm for 10 min and stored at -20 °C after adding 100 µL formaldehyde for preservation until analysis. Concentrations of APAP, 3-chloro-APAP and targeted transformation products were analyzed by online SPE (10 mL) LC-MS/MS (see
SI). The reported results are averages of experiments performed in triplicate. NH4sN, NO2sN, and NO3sN concentrations were measured using an ion chromatographic analyzer to track the nitrification activity in each batch reactor. The effect of sorption on the removal of both APAP and its transformation products was assessed by the use of the batch-3 experiments. No chemical removal could be attributed to sorption processes under our experimental conditions (see SI). All results are reported as an average of three experiments. Variability in repeated experiments was under 12%. Peroxynitrite Reactions. Peroxynitrite anion (ONOO-) was prepared from isoamylnitrite in presence of H2O2 and quantified spectrophotometrically (ε302 ) 1670 ( 50 M-1cm-1) as previously described (21). Excess H2O2 was removed by treatment with MnO2. In spite of this, the peroxynitrite preparation could contain residues of H2O2. In presence of H2O2, APAP underwent oxidative coupling transformation (see SI Figure SI3). Consequently, a little part of APAP transformation might be ascribed to H2O2 itself and not solely to peroxynitrite. To avoid the rapid decomposition of ONOO-, stock solutions of peroxynitrite were prepared in KOH 0.1 M (final concentration of 15-20 mM). Peroxynitrite was allowed to react with 70 µM of APAP or 3-chloro-APAP in 2 mL of 0.1 M phosphate buffer, pH 7.0-7.5, that also contained (0.2-20 mM) bicarbonate, (0.2-20 mM) chloride ion, and (5-50 mg/ L) dissolved organic matter (DOM), to be closer to real nitrifying activated sludge samples. The reactions were performed by adding aliquots (100-150 µL) of stock peroxynitrite solution to the APAP/3-chloro-APAP solutions with vigorous stirring for a few seconds and at room temperature. Solutions were filtered before direct analysis by LC/MS. Horseradish Peroxidase Assays. Reaction mixtures (1 mL) contained 100 mM phosphate buffer (pH 7.4), 70 µM APAP, 80 nM of horseradish peroxidase, 200 µM H2O2 and 100 µM nitrite ions. Mixtures were equilibrated at 25 °C and reactions were initiated by the addition of H2O2. Deionized distilled water was used to prepare stock solutions of H2O2 and horseradish peroxidase. Concentrations were estimated by absorbance spectroscopy using the extinction coefficient of 43.6 M-1cm-1 at 240 nm for H2O2 and 89.5 × 103 M-1cm-1 at 403 nm for horseradish peroxidase. Reactions were terminated by the addition of 1 mL ice-cold methanol:water (90:10, v/v) containing 2 mM ascorbic acid (22). Water Sample Analysis. WWTP influent (20 mL) or effluent (50 mL) samples as well as samples from laboratory batch experiments (10 mL) were analyzed by online solid phase extraction (SPE) coupled to LC-MS/MS as previously described (23). Limits of detection (LODs) were 85, 58, 35, 32, 48, 21 ng/L in WWTP influent samples and 9.2, 4.5, 3.2, 2.8, 4.1, 1.6 ng/L in WWTP effluent samples for 3-OH-APAP, APAP, 3-nitro-APAP, 3-chloro-APAP, 3-chloro-5-nitro-APAP and 3,5-dinitro-APAP, respectively. For matrix effect correction in ESI ionization mode, two deuterated internal standards were added to samples, one for positive ionization mode (cotinine d3) and the another one for negative ionization mode (2,4-D d3), before extraction.
Results and Discussion Field Observations. Table 1 summarizes the concentrations of APAP in µg/L and its targeted transformation products (3-OH-APAP, 3-chloro-APAP, 3-nitro-APAP, 3-chloro-5-nitroAPAP, and 3,5-dinitro-APAP) for triplicate composite samples collected before and after the nitrification stage in a WWTP. A major impurity in APAP drug substance, 3-chloro-APAP, (24) that can be also formed upon reaction of APAP with hypochlorite (25) was selected as a representative compound to investigate the chlorinating transformation pathway of APAP. We included 3-OH-APAP, formed upon hydroxyl radical reactivity (26) and upon monooxygenase activity (27), VOL. 44, NO. 1, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Summary of the Average Concentrations and Standard Deviations of APAP and Its Targeted Transformation Products in µg/L for 24 h Composite Samples Collected before and after the Nitrification Step in a WWTPa sampling month
APAP
3-OH-APAP
3-chloro-APAP 3-nitro-APAP 3-chloro-5-nitro-APAP 3,5-dinitro-APAP
stage-2 influent October 2008 3.45 ( 0.21 0.96 ( 0.11 November 2008 5.35 ( 0.32 1.44 ( 0.17 December 2008 6.75 ( 0.41 1.86 ( 0.22
0.24 ( 0.02 0.85 ( 0.04 0.76 ( 0.04
nd nd nd
nd nd nd
nd nd nd
stage-2 effluent October 2008 0.19 ( 0.02 nd November 2008 0.35 ( 0.03 nd December 2008 0.64 ( 0.05 nd
nd nd nd
0.18 ( 0.02 0.26 ( 0.03 0.32 ( 0.03
0.028 ( 1 × 10-3 0.105 ( 5 × 10-3 0.085 ( 3 × 10-3
nd nd nd
a nd, not determined (below LODs of the analytical method). LODs: 85, 58, 35, 32, 48, 21 ng/L in stage-2 influent samples and 9.2, 4.5, 3.2, 2.8, 4.1, 1.6 ng/L in stage-2 effluent samples for 3-OH-APAP, APAP, 3-nitro-APAP, 3-chloro-APAP, 3-chloro-5-nitro-APAP and 3,5-dinitro-APAP, respectively.
to assess the significance of the hydroxylating pathway, whereas 2- or 3-nitro-APAP and 3-chloro-5-nitro-APAP were probes for the nitrating pathway. Field data first revealed APAP removal rates in the 90-95% range in agreement to previous findings (19), providing evidence of the WWTP efficiency. The 3-OH-APAP concentration level was about one-third of that of APAP, probably making hydroxylation the most predominant biodegradation pathway of APAP in WWTP. The unexpected high level of occurrence of 3-chloroAPAP in the 7-15% range of the APAP concentration is not currently understood. There was a trend for full elimination of 3-chloro-APAP and 3-OH-APAP during stage-2 treatment. APAP and 3-chloro-APAP underwent nitration processes and were transformed into 3-nitro-APAP and 3-chloro-5-nitroAPAP with an average nitration yield of 4.9 and 10%, respectively. Nitration was a minor transformation pathway of APAP. However, the nitrated derivatives appeared to be more stable than the chlorinated or hydroxylated ones in nitrifying activated sludge and were released into the environment. The following section is an attempt to check if nitrifying bacteria are responsible for nitration processes. Transformation of Trace Concentrations of APAP and 3-chloro-APAP in Batch Experiments. A laboratory study was conducted to investigate the role of nitrifying bacteria in the formation of nitrated derivatives of APAP and 3-chloroAPAP. A series of two batch tests involving enriched nitrifying community were conducted at initial NH4sN concentrations of 10 mg/L to ensure cell growth and at initial APAP and 3-chloro-APAP concentrations of 100 µg/L, close to environmental concentrations. In such systems, a mixed community including both autotrophic nitrifying bacteria and fast-growing heterotrophic bacteria was probably developed. In batch-1, no inhibition of nitrification was achieved while in batch-2, oxygenase activity including ammonia monooxygenase activity was selectively inhibited by allylthiourea. The ammonium was typically completely oxidized after 144 h experiment. Nitrite accumulated up to 2.4 mg/L NO2sN after 60 h and was completely oxidized into nitrate after 172 h, suggesting good performances of nitrifying activities in batch-1 experiments. Nitrite oxidation rate was lower than the ammonium oxidation rate which explains nitrite accumulation in the reactors. In contrast, the changes in ammonium and nitrate concentrations were negligible in batch-2 experiments. The operational performance of the experimental system was very stable which allowed to obtain meaningful results. The removal of APAP and 3-chloro-APAP was slightly slower in batch-2 (90%) with allylthiourea than in batch-1 (95%) tests. Figure 1a (batch-1) and Figure 1b (batch-2) show the results from two representative experiments with time variation in APAP and 3-chloro-APAP concentrations. The calculated degradation half-lives (t1/2) of APAP using apparent first order rate kinetic k (t1/2 ) Ln2/ k) reached 1.77 ( 0.14 and 2.43 ( 0.19 days in batch-1 and batch-2 experiments, respectively. A similar increase in half286
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FIGURE 1. APAP (×), 3-chloro-APAP (0) depletion, and 3-OH-APAP (g), 3-nitro-APAP (4), 3-chloro-5-nitro-APAP (O) formation in batch test with nitrifying activated sludge (a) without nitrification inhibition (Batch-1) and (b) with nitrification inhibition (Batch-2). The second axis shows 3-OH-APAP, 3-nitro-APAP, and 3-chloro-5-nitro-APAP. lives from 1.74 ( 0.12 (batch-1) to 2.79 ( 0.17 days (batch-2) was also recorded for 3-chloro-APAP. More interestingly, 3-OH-APAP, 3-nitro-APAP and 3-chloro-5-nitro-APAP were identified based on their retention time and their MS/MS fragmentation patterns in all experiments (SI Figure SI4). However, a great difference was observed in the formation profiles of these three metabolites between the two experiments. In batch 1 and at 72 h reaction time, nitrated metabolite concentrations reached up to 4.1-4.6% of the initial concentrations of APAP and 3-chloro-APAP while in batch-2, this percentage of formation was only 0.2-0.25%. The higher nitration rates of APAP and 3-chloro-APAP in batch-1 experiment provides clear evidence that oxygenase activity and probably AOB might play a key role in the bionitration of APAP and 3-chloro-APAP. Additional experiments were carried out by adding 10 mg/L tyrosine in the batch-1 tests. Such a high concentration level was allowed due to the low toxicity of tyrosine. Tyrosine was used as a potential scavenger of nitrogen dioxide radicals (•NO2) since tyrosyl radical (TyrO•) reacts with (•NO2) at near diffusionlimited rate (k ) 1.7 × 109 M-1s-1 (28)). In these experiments, 3-nitro-APAP and 3-chloro-5-nitro-APAP were not produced while their parent compounds were degraded at the same
rate than in batch-1 experiments (SI Figure SI5). Consequently, the following section is an attempt to check if the microbial-generated RNS are able to account for the nitration of APAP and 3-chloro-APAP in batch-1 experiments. Reactivity of APAP and 3-Chloro-APAP with Different Reactive Nitrogen Species. Nitration Rate in Presence of Nitrite Ion. Due to the rather high production of nitrite (2 mg/L NO2sN) in batch-1 experiment, the potential contribution of abiotic nitration processes to the formation of 3-nitro-APAP and 3-chloro-5-nitro-APAP was first investigated by conducting experiments with nitrite (0.1 mM) at different pH. Nitration rate was clearly pH dependent and was only relevant at pH e 6 (see Table 2). Nitration is probably correlated with concentrations in free nitrous acid (pKa (HNO2/NO2-) ) 3.4) which suggests that HNO2 is likely involved in the process. However, only 2-nitro-APAP was detected (see SI Figure SI6). In presence of HNO2, APAP is probably oxidized into N-acetyl-p-benzoquinone imine, which undergoes aromatic nitration through the Michaeltype addition of nitrite ion to generate 2-nitro-APAP (29). The lack of dinitro-APAP is probably due to the electronicwithdrawing character of the nitro group impairing 2-nitroAPAP nitration. 2-nitro-APAP was neither determined in batch-1 experiments nor in field studies. Consequently, nitration is very unlikely to arise from NO2sN produced by AOB. Nitration Rate in Presence of Horseradish Peroxidase (HRP). An alternative pathway for the production of nitrated phenolic compounds which is receiving increasing attention is that dependent on heme proteins (e.g., peroxidases (30)). In this latter case, the key substrate in the generation of RNS is the nitrite ion (NO2-), while nitration occurs through the formation of •NO2 radical in presence of H2O2 (30). In presence of HRP, a representative peroxidase, APAP was transformed into 3-nitro-APAP and 3,5-dinitro-APAP (see SI Figure SI7). The nitration reaction was assumed to be in strong competition with the classical peroxidase coupling reaction to dimeric or trimeric compounds, resulting in the low nitration yields (5.5%). Surprisingly, 3,5-dinitro-APAP prevailed over 3-nitro-APAP (Table 2). This result is not consistent with previous findings involving myeloperoxidase, where only 3-nitro-APAP was determined (17), but the reasons for this discrepancy were not further investigated. Since dinitro-APAP was not detectable in batch experiments and in field studies with a LOD of 1.6 ng/L, the peroxidasemediated nitration pathway could be ruled out. Nitration Rate in Presence of Peroxynitrite. Nitric oxide (•NO) is produced during the nitrification step in WWTPs (15). Reactivity of •NO and reaction mechanisms conducting to nitrating species have been discussed extensively in biochemistry (28) and are summarized in the following equations (1-6). 10 -1 -1 •NO + O•2 f ONOO k ) 1.9 × 10 M s -
+
ONOO + H h ONOOH pKa ) 6.8
(1) (2)
ONOO- + H+ f •NO2 + •OHk ) 5.3 × 109M-1s-1 (3) 4 -1 -1 ONOO- + CO2 f •NO2 + CO•3 k ) 1 × 10 M s
(4) 8 -1 -1 2•O•2 + CO3 f O2 + CO3 k ) 4 × 10 M s
•NO +
CO•3
-
+ OH f
NO2
+
HCO3k
9
(5) -1 -1
) 3.5 × 10 M s
(6) Briefly, •NO is a free radical with selective reactivity. •NO reacts at a near diffusion-limited rate with superoxide anion O2•- to yield peroxynitrite anion (ONOO-), a powerful oxidizing and nitrating specie (eq 1). During nitrification, AOB oxidize NH3 to NO2- in two steps (31). The first one is the oxidation of NH3 to hydroxylamine (NH2OH) with a simultaneous release of •NO and N2O as side-products (32). The second step consists in the oxidation of NH2OH to NO2-, yielding two pairs of electrons. One pair of the electrons is used in the first step of the ammonia oxidation, while the second pair allows for the reduction of oxygen to O2•- under oxic conditions or the reduction of NO2- to •NO and N2O under more anoxic conditions (31). Consequently, •NO and O2•- might be concomitantly generated during nitrification, favoring the formation of ONOO-. ONOO-, with a pKa of 6.8, is readily protonated in environmental samples (eq 2). The protonation of ONOO- generates peroxynitrous acid (ONOOH) which is a unstable specie yielding two radicals, the hydroxyl (•OH) radical and the nitrogen dioxide (•NO2) radical (eq 3). Both radicals that are released are responsible for the oxidation and the nitration effects attributed to ONOOH. ONOO- can also react faster with CO2 than with most environmental molecules to form a reactive adduct. Concentrations of CO2 in activated sludge are relatively high due to the high levels of bicarbonate. This suggests that the reaction of peroxynitrite with CO2 might be the predominant pathway for peroxynitrite disappearance in activated sludge treatment. In this case, the nitrating pathway is favored over the hydroxylating one because this adduct undergoes homolysis process generating the carbonate radical (CO3•-) and the •NO2 radical at the expense of •OH and with a 30% yield (eq 4). Oxidizing, hydroxylating, and nitrating properties of peroxynitrite with respect to phenolic compounds have been investigated in depth in biochemistry (33-35). The goal of this work was therefore to identify some relevant environmental parameters (e.g., the bicarbonate, chloride, and DOM content as well as pH) that may influence the different reaction pathways between APAP and peroxynitrite. In presence of bicarbonate (5 mM), chloride (5 mM) and at pH 7.5, the reaction of APAP with peroxynitrite produced hydroxylated derivatives (3-OH-APAP), nitrated derivatives (3-nitro-APAP) and chlorinated derivatives (3-chloro-APAP and 3-chloro-5-nitro-APAP) as reported in SI Figure SI8. This reaction also involved a dimer and a trimer of APAP which
TABLE 2. Formation of Chlorinated and Nitrated Derivatives of APAP in µM for the Reaction of APAP with Different Reactive Nitrogen Speciesa experimental conditions APAP + 0.1 mM NO2-/pH 6 APAP + 0.1 mM NO2-/pH 7.5 APAP + HRP (80 nM) /H2O2 (0.2 mM)/NO2- (0.1 mM)/pH 7.5 APAP + 5 mM ONOO-/pH 7.5 3-chloro-APAP + 5 mM ONOO-/ pH 7.5
3-nitro-APAP 2-nitro-APAP 3-chloro-APAP 3-nitro-5-chloro-APAP 3,5-dinitro-APAP nitration yield (%) nd nd 0.75 ( 0.08
3.11 ( 0.18 nd nd
nd nd nd
nd nd nd
nd nd 3.12 ( 0.22
4.4 ( 0.3 nd 5.5 ( 0.3
5.25 ( 0.29 nd
nd nd
0.12 ( 0.01 nd
0.35 ( 0.04 6.27 ( 0.37
nd nd
7.5 ( 0.5 10.0 ( 0.6
a Incubations contained in all experiments 70 µM APAP or 3-chloro-APAP, 5 mM chloride, 5 mM bicarbonate, and 10 mg/ L DOM at 25°C. Incubation time 24 h in 100 mM phosphate buffer before direct injection analysis by LC/MS. nd: not determined.
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FIGURE 2. Yield of 3-nitro-APAP (O) formation as a function of [HCO3-]. Yield of 3-nitro-APAP (4) and 3-chloro-APAP (0) as a function [Cl-], in both cases for the reaction of peroxynitrite with APAP. [APAP] 0.07 mM. [ONOO-] ) 5 mM in 100 mM phosphate buffer (pH 7.5) at 25 °C. were indicative of a free radical-coupling reaction of two or three phenoxyl radicals of APAP and which supported a radical mechanism for APAP nitration (13). Nitration yields were always below 10% (Table 2). The nitration reaction was first investigated between pH 4 and 9. The variation in the yields of hydroxylation and nitration with pH at 25 °C is reported in SI Figure SI9. Nitration yield was maximum at pH 7-7.5 while hydroxylation yield was the highest at pH 4.5. At pH below 6, part at least of the nitration processes observed in the reactions was probably linked to the formation of nitrous acid either from little nitrite contamination of the stock peroxynitrite solutions or because peroxynitrite itself produced nitrate and nitrite upon its decomposition. In spite of this, nitration appeared to be facilitated at environmental pH. When varying the concentrations in bicarbonate (see Figure 2), the formation rate of 3-nitro-APAP underwent a 6-fold increase between 2 and 20 mM of bicarbonate and then leveled off. The enhancement by bicarbonate of APAP nitration is probably accounted for by an increase of •NO2 production but also by the production of CO3•-. This latter radical being a stronger oxidant with higher selectivity than •NO2 (E° (CO3•-/CO32-) ) 1.59 V versus E° (•NO2 /NO2-) ) 1.04 V), it can more easily oxidize the phenol moiety of APAP in complex matrices to produce the phenoxyl radical (E° (PhO•/PhOH) ) 0.99 V) and bicarbonate than •NO2 does. The phenoxyl radical intermediate would then react with •NO2 at near diffusion-limited rate similarly to tyrosine radical (13). The inhibition of 3-nitro-APAP formation at elevated bicarbonate could be accounted for by the depletion of the reactive species O2•- and •NO with CO3•- (eqs 5 and 6). Surprisingly, the DOM content in the 5-50 mg/L range hardly impaired the nitration yield of APAP (SI Figure SI10) probably due to the high selectivity of reaction between APAP and CO3•- and between •NO2 and phenoxyl radicals of APAP. Chlorination was a very limited reaction pathway in presence of chloride ions, accounted for less than 0.2% transformation of the APAP initial concentration. Chlorination appeared to be achieved at the expense of nitration since simultaneously to a decrease in 3-nitro-APAP concentration, an increase in 3-chloro-APAP concentration was observed (see Figure 2). The unexpected production of chlorinated derivatives might arise from the oxidation of chloride by peroxynitric acid (O2NOOH) into hypochlorous acid (HOCl), a potential chlorinating agent for APAP. The 288
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production of O2NOOH probably resulted from the reaction of ONOO- with excess of CO2 in aerated solutions containing H2O2 residues from ONOO- stock solutions (36, 37). Further details on these reactions are provided in the Supporting Information. Finally, the transformation of 3-chloro-APAP upon reaction with peroxynitrite was also investigated. One transformation pathway was nitration leading to 3-chloro5-nitro-APAP (see SI Figure SI11). In this case, the nitration yield (10.0%) was slightly higher than that recorded for APAP nitration (7.5%, see Table 2) under the same experimental conditions. The reaction was probably facilitated by an electron-withdrawing substituent and the lower pKa value of chlorinated phenols with respect to phenol, favoring the more reactive anionic form at neutral pH (38). The reported product formation of 3-nitro-APAP and 3-chloro-5-nitro-APAP and the reported nitration yields show a trend which is consistent with batch experiment and field data and support the notion that nitration of APAP and 3-chloro-APAP in nitrifying activated sludge might be induced by •NO produced by AOB or others nitrifying bacteria rather than by HNO2 due to pH reduction during the nitrification stage of treatment. Environmental Significance. A previous work from our group has shown the relevance of photonitration as a transformation pathway of phenolic compounds in shallow surface waters (39). This work provides evidence that in systems such as nitrifying activated sludge, where biomass is exposed to dynamic changes in nitrite, ammonium, and oxygen concentration, nitration of APAP is also relevant. APAP was selected as a probe in this work but others important phenolic pollutants such as bisphenol A or nonylphenol might also undergo similar nitration processes, potentially accounting for part of their degradation rate in nitifying activated sludge (40, 41). Nitration is probably linked to the generation of •NO during the nitrification stage of sewage water treatment. The emission of •NO is predominantly generated by nitrifying bacteria such as AOB. However, another potential source of •NO in WWTP is denitrifying bacteria (15). Even though nutrient removal strategies have improved the whole effluent quality of municipal WWTPs, there is a potential risk to generate harmful transformation products. The formation and occurrence of nitro-APAP derivatives in urban wastewater cycle is a matter of concern for the protection of aquatic organisms due to their potential persistence and increased hydrophobicity relative to APAP and due to the phytotoxic and genotoxic properties of nitrophenols (42, 43). Additional measurements of nitrated compounds are required in WWTPs to better quantify the risk. However, if the occurrence of stable nitrated metabolites is confirmed in WWTPs, they would have to be addressed in the risk assessment analysis by investigating their toxicological effects.
Supporting Information Available Experimental details and details on the formation of 3-chloroAPAP upon reaction of APAP with peroxynitrite. This material is available free of charge via the Internet at http:// pubs.acs.org.
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