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Enhanced Inactivation of Escherichia coli and MS2 Coliphage by Cupric Ion in the Presence of Hydroxylamine: Dual Microbicidal Effects Hyung-Eun Kim,† Thuy T. M. Nguyen,‡ Hongshin Lee,† and Changha Lee*,† †

School of Urban and Environmental Engineering, Ulsan National Institute of Science and Technology (UNIST), 50 UNIST-gil, Ulju-gun, Ulsan, 689-798, Republic of Korea ‡ Power Engineering Consulting Joint Stock Company 2 (PECC2), 32 Ngo Thoi Nhiem str., Ward 7, District 3, Ho Chi Minh City, Vietnam S Supporting Information *

ABSTRACT: The inactivation of Escherichia coli and MS2 coliphage by Cu(II) is found to be significantly enhanced in the presence of hydroxylamine (HA). The addition of a small amount of HA (i.e., 5−20 μM) increased the inactivation efficacies of E. coli and MS2 coliphage by 5- to 100-fold, depending on the conditions. Dual effects were anticipated to enhance the biocidal activity of Cu(II) by the addition of HA, viz. (i) the accelerated reduction of Cu(II) into Cu(I) (a stronger biocide) and (ii) the production of reactive oxidants from the reaction of Cu(I) with dissolved oxygen (evidenced by the oxidative transformation of methanol into formaldehyde). Deaeration enhanced the inactivation of E. coli but slightly decreased the inactivation efficacy of MS2 coliphage. The addition of 10 μM hydrogen peroxide (H2O2) greatly enhanced the MS2 inactivation, whereas the same concentration of H2O2 did not significantly affect the inactivation efficacy of E. coli Observations collectively indicate that different biocidal actions lead to the inactivation of E. coli and MS2 coliphage. The toxicity of Cu(I) is dominantly responsible for the E. coli inactivation. However, for the MS2 coliphage inactivation, the oxidative damage induced by reactive oxidants is as important as the effect of Cu(I).

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DNA and RNA viruses).5,10−12 The enhanced biocidal activity of Cu(II) in the presence of H2O2 is suggested to result from the production of reactive oxidants via the Cu(II)-catalyzed decomposition of H2O2.10,13,14 Hydroxyl radical (•OH) or high-valent copper species (i.e., Cu(III)) are believed to be formed, causing oxidative damage to the cells.15−19 On the other hand, hydroxylamine (HA), a commonly used reducing agent in contemporary industrial chemistry, was reported to affect the viral protein and viral ribonucleic acid of animal viruses or E. coli T series bacteriophage.20,21 In addition, Cu(II) in combination with HA hinders the biological activity of pancreatic ribonuclease and animal enzymes such as egg white lysozyme and rabbit muscle aldolase.22 Furthermore, the oxidation of HA by dissolved oxygen produces intermediates such as nitrous oxide (N2O) and H2O2,23,24 which can be further decomposed into reactive oxidants by the catalytic reactions with Cu(II). Therefore, it is anticipated that the addition of HA increases the activity of Cu(II)-based

INTRODUCTION The public health risk from waterborne diseases caused by pathogenic microorganisms has been continuously raising concerns, which promotes research on drinking water disinfection. Among the disinfection technologies suggested, chemical disinfectants are found to be effective for inactivating a broad spectrum of microorganisms and are thereby widely used for various water disinfection applications. Cupric ion (Cu(II)) is one of the chemical disinfectants that has been extensively studied for its antimicrobial activity. It has been reported that Cu(II) is effective in inactivating bacteria,1,2 yeast cells,3 and algae,4 but it has minor effects on inactivation of viruses.5 Previous studies revealed that the biocidal activity of Cu(II) is mainly attributed to the cytotoxicity of cellularly generated cuprous ion (Cu(I)).1,6,7 However, it also has been suggested that Cu(II) can induce oxidative stress by accelerating the production of reactive oxygen species (ROS) during the respiration of bacterial cells, which partially contributes to the lethal action of Cu(II).8,9 In combination with auxiliary compounds such as hydrogen peroxide (H2O2), the biocidal activity of Cu(II) was improved to a significant extent, exhibiting distinctive biocidal activity on a wide spectrum of viruses (i.e., single- and double- stranded © 2015 American Chemical Society

Received: Revised: Accepted: Published: 14416

September 7, 2015 November 13, 2015 November 17, 2015 November 17, 2015 DOI: 10.1021/acs.est.5b04310 Environ. Sci. Technol. 2015, 49, 14416−14423

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Environmental Science & Technology

Figure 1. Inactivation of (a) E. coli and (b) MS2 coliphage by the Cu(II)/HA system as a function of HA concentration (pH 7.0, [Cu(II)]0 = 5 μM).

cells were resuspended in 20 mL PBS and kept in the refrigerator at 4−5 °C.18 The spread plate method27 was employed to determine the population of E. coli: the cells plated on nutrient agar were incubated for 24 h at 37 °C, and the number of colonies were counted. For the virucidal activity test, MS2 coliphage (ATCC 15597B1) was used with E. coli C3000 (ATCC 15597) as a host. The E. coli host was cultivated in a growth broth containing 1% tryptone, 0.8% NaCl, 0.1% yeast extract, 0.01% glucose, 2 mM CaCl2 and 0.001% thiamine. MS2 coliphage was incubated in the prepared E. coli host for 18−24 h at 37 °C. Subsequently, the cultural solution was centrifuged at 3000g for 15 min, and the supernatant was filtered with a 0.22 μm nylon syringe filter.18 The population of MS2 coliphage was determined by the plaque assay method27 using a medium of top and bottom double agar layers (0.5 and 1.5% agar, respectively). Microbial Inactivation Experiments. All experiments were conducted in 60 mL Pyrex flasks at ambient temperature (24 ± 0.5 °C) under vigorous stirring. Except for the experiments using natural waters (unbuffered), a buffered solution of 1 mM PIPES (pH 7.0) was used. E. coli and MS2 coliphage were initially suspended in the buffered solution at approximate populations of 107 CFU/mL (or PFU/mL). The inactivation was initiated by simultaneously adding aliquots of Cu(II) and HA stock solutions to the microorganism suspension. At predetermined time intervals, 1 mL of sample was withdrawn into a 1.7 mL polypropylene tube containing 25 μL of 2 mM EDTA to instantly quench the reactions of Cu(II).1 After diluting to certain concentrations, 0.1 mL of each sample was assayed on triplicate agar plates followed by counting the number of viable cells or viruses. The inactivation experiments were repeated more than three times, and the average values and the standard deviations were presented. For experiments needing deaeration (anoxic conditions), the reaction solution was purged using ultrapure N2 gas for 30 min prior to the experiment. For some experiments, DMP and EDTA were used as Cu(I)- and Cu(II)-chelating agents, respectively,11 and tert-butanol was used as a •OH scavenger. Natural Water Samples. Samples were collected from surface water (Chonsang and Hoeya dams in Ulsan city) and

disinfection systems by converting Cu(II) into Cu(I) as well as by producing reactive oxidants potentially lethal to the cells. Nevertheless, the microbial inactivation using the combined system of Cu(II) and HA has not been studied. The objectives of this study are to assess the biocidal effects of the combined Cu(II)/HA system on Escherichia coli and MS2 coliphage and to gain insight into the mechanism behind the enhanced biocidal actions. For these purposes, the inactivation efficacies of E. coli and MS2 coliphage by the Cu(II)/HA system are examined at different HA doses, and the effects of dissolved oxygen, copper-chelating agents, and H2O2 are investigated. In addition, the level of intracellular oxidants in E. coli cells was monitored in response to the addition of Cu(II) and HA. Inactivation experiments were also conducted in natural water samples from surface waters and groundwater to assess the influences of natural organic and inorganic substances.

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MATERIALS AND METHODS Reagents. All chemicals were of reagent grade and were used without further purification. The chemicals used include agar, nutrient broth, tryptone, yeast extract for E. coli and MS2 coliphage cultivation (Becton−Dickinson Co.), copper sulfate (CuSO4), hydroxylamine (NH2OH), H2O2, 1,4-piperazinediethanesulfonic acid (PIPES), ethylenediaminetetraacetic acid (EDTA) and 2,9-dimethyl-1,10-phenanthroline (DMP), methanol, tert-butanol for inactivation experiments (Sigma-Aldrich Co.), and 3′-(p-hydroxyphenyl) fluorescein (HPF) and hydroethidine (HE) for intracellular oxidant measurements (Invitrogen Co.). All solutions were prepared using deionized water (>18 MΩ·cm, Millipore Co., USA). All glassware was washed with deionized water and sterilized by autoclave at 121 °C for 15 min prior to use. Culture and Analysis of Microorganisms. E. coli (ATCC 8739) was chosen as a surrogate microorganism for the bactericidal activity test. E. coli stock was incubated in 30 mL of Difco nutrient broth at 37 °C for 18−24 h. The cells were harvested by centrifugation at 3000g for 15 min, and the retained nutrients were removed by washing three times with phosphate-buffer saline (PBS, pH 7.2). The obtained E. coli 14417

DOI: 10.1021/acs.est.5b04310 Environ. Sci. Technol. 2015, 49, 14416−14423

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Figure 2. (a) Cu(I) generation and (b) HCHO production (from methanol oxidation) by the Cu(II)/HA system (pH 7.0, [Cu(II)]0 = 5 μM, reaction time = 30 min, [Methanol]0 = 200 mM for (b)).

Figure 3. Effects of deaeration, copper-chelating reagent, and H2O2 on inactivation of (a) E. coli and (b) MS2 coliphage by the Cu(II)/HA system (pH 7.0, [Cu(II)]0 = 5 μM, [HA]0 = 10 μM, [EDTA]0 = 2 mM, [DMP]0 = 2 mM, [H2O2]0 = 10 μM).

PIPES buffer after centrifugation (5000 rpm, 5 min). The prepared cells were resuspended in the buffered solution and were treated by different systems using Cu(II) and HA for 30 min. Then, the generated fluorescence intensity was measured using a microplate reader (Infinite M200, Tecan Trading AG, Switzerland). A 485 nm excitation and 535 nm emission filter was used for HPF, and a 535 nm excitation and 590 nm emission filter was used for HE. For extracellular oxidant levels, solutions of HPF or HE without E. coli cells were directly used for the fluorescence measurements. The fluorescence intensity ratio (FIR) of each sample, calculated relative to the control containing only the probe compound, was presented. The detailed method for the sample preparation is described elsewhere.26

groundwater (Jeonbuk Province) sources in South Korea. The waters were filtered through 0.22 μm nylon membrane filters upon arrival and were stored at 4 °C until use. Several water quality parameters of the samples were analyzed (refer to the Supporting Information, Table S1). Measurement of Intra/Extracellular Oxidants. The oxidant levels under intracellular and extracellular (free of E. coli cells) conditions were determined using cell-permeable fluorescent probe compounds; HPF is for detecting •OH and Cu(III), and HE is for O2•−.25 For the measurement of intracellular oxidants, E. coli cells incorporating probe compounds were used. Briefly described, the E. coli suspension (107 CFU/mL) was mixed with 10 μM of probe compounds for 1 h at 100 rpm in the dark and washed 2 times with 1 mM 14418

DOI: 10.1021/acs.est.5b04310 Environ. Sci. Technol. 2015, 49, 14416−14423

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Figure 4. Extracellular (cell-free) and intracellular oxidant generation by different Cu(II)-based systems, (a) measurements of •OH and Cu(III) using HPF and (b) O2•− using HE (pH 7.0, [Cu(II)]0 = 5 μM, [HA]0 = 10 μM, [H2O2]0 = 10 μM and 1 mM, reaction time = 30 min).

Analyses of Cu(I) and Formaldehyde. The concentration of Cu(I) was determined by the neocuproine method.27 The concentration of formaldehyde (HCHO) was measured by DNPH derivatization followed by HPLC analysis with UV absorbance detection at 350 nm. The details are described elsewhere.30

Figures 1a and 1b) increased by approximately 5−100-fold relative to the conditions with Cu(II) alone (Figure S1 in the Supporting Information); the inactivation rate of MS2 coliphage exhibited a good linearity with the dose of HA. Generation of Cu(I) and Reactive Oxidants. The generation of Cu(I) by the Cu(II)/HA system was monitored at different HA concentrations under control (oxic) and anoxic conditions (Figure 2a). The generation of Cu(I) was enhanced as the concentration of HA increased from 0 to 20 μM under both conditions. However, Cu(I) concentrations were slightly higher overall under anoxic conditions. The production of reactive oxidants by the Cu(II)/HA system was also examined by monitoring the HCHO formation in the presence of excess methanol28 (Figure 2b). Under oxic conditions, the concentration of HCHO increased with increasing concentration of HA. However, the HCHO formation under anoxic conditions was negligible due to an absence of significant production of reactive oxidants. Effects of Dissolved Oxygen, Copper-Chelating Agents, and Hydrogen Peroxide. The effects of dissolved oxygen, copper-chelating agents (EDTA and DMP), and H2O2

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RESULTS Enhanced Inactivation of E. coli and MS2 Coliphage by Cu(II) in Combination with Hydroxylamine. The inactivation of E. coli and MS2 coliphage by the Cu(II)/HA system was examined at different input concentrations of HA (Figures 1a and 1b). In control experiments, HA alone had negligible effects on both E. coli and MS2 coliphage (data not shown). A Cu(II) concentration of 5 μM resulted 1.5 log inactivation of E. coli and less than 0.5 log inactivation of MS2 coliphage in 30 min. The addition of HA significantly enhanced the inactivation efficacies of both E. coli and MS2 coliphage. When the concentration of HA was increased, the inactivation rates (calculated from the slopes of inactivation curves in 14419

DOI: 10.1021/acs.est.5b04310 Environ. Sci. Technol. 2015, 49, 14416−14423

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Figure 5. Inactivation of (a) E. coli and (b) MS2 coliphage by the Cu(II)/HA system in natural waters (pH 7.0 for buffered water, [Cu(II)]0 = 5 μM, [HA]0 = 10 μM).

E. coli (6−65%) than for MS2 coliphage (17−30%). The inactivation rate of E. coli was in the order Buffered water > Surface water (Hoeya) > Surface water (Chonsang) > Groundwater, whereas that of MS2 coliphage was in the order Buffered water > Groundwater > Surface water (Hoeya) > Surface water (Chonsang).

on the inactivation of E. coli and MS2 coliphage by the Cu(II)/ HA system were examined. The time-dependent inactivation curves were obtained under different conditions (refer to Figure S2 in the Supporting Information), and the average inactivation rates were presented (Figures 3a and 3b). The E. coli inactivation was significantly enhanced under the anoxic condition compared to the control (Figure 3a), whereas the MS2 coliphage inactivation was marginally inhibited under the anoxic condition (Figure 3b). The addition of Cu(II)- and Cu(I)-chelating agents (EDTA and DMP) almost blocked the microbial inactivation for both E. coli and MS2 coliphage. The addition of 10 μM H2O2 did not significantly affect the E. coli inactivation by the Cu(II)/HA system (Figure 3a). However, the MS2 coliphage inactivation was greatly enhanced by the addition of H2O2 (Figure 3b); the inactivation rate increased by approximately 20-fold compared to the control. Generation of Extra/Intracellular Oxidants. Oxidant levels induced by Cu(II), Cu(II)/HA, Cu(II)/H2O2, and Cu(II)/HA/H2O2 systems (with 0.01 and 1 mM H2O2) were determined under extracellular (cell-free) and intracellular (inside E. coli cells) conditions for •OH and Cu(III) using HPF (Figure 4a) and for O2•− using HE (Figure 4b). The FIR values for •OH and Cu(III) significantly increased only for Cu(II)/H2O2 and Cu(II)/HA/H2O2 systems with 1 mM H2O2 under both extracellular and intracellular conditions (Figure 4a). There were differences in the O2•− level between the extracellular and intracellular conditions (Figure 4b). Under extracellular conditions, the FIR values increased to some degree for most systems; higher FIR values were observed with higher H2O2 concentrations for the Cu(II)/H2O2 and Cu(II)/ HA/H2O2 systems. In contrast, the FIR values under intracellular conditions decreased overall. Microbial Inactivation in Natural Waters. The inactivation of E. coli and MS2 coliphage by the Cu(II)/HA system was examined in natural waters (Figures 5a and 5b; also refer to Figure S3 in the Supporting Information for the timedependent inactivation curves). The inactivation of E. coli and MS2 coliphage was inhibited in natural waters compared to buffered solution; note that the inhibitory effect was greater for

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DISCUSSION Generation of Biocides by the Cu(II)/HA System. Dual biocidal effects (i.e., effects of Cu(I) and reactive oxidants) are anticipated in the Cu(II)/HA system. First, HA as a reducing agent (abiotically) converts Cu(II) into Cu(I) (reaction 1).23 Cu(I) is more cytotoxic and is directly responsible for the biocidal actions of copper compounds. Previous studies have suggested that the bactericidal action of Cu(II) results from the cytotoxicity of Cu(I) that is reductively generated by intracellular cell components.1,2 2NH 2OH + 4Cu(II) → N2O + H 2O + 4Cu(I) + 4H+ (1)

The enhanced generation of Cu(I) by HA was observed under both oxic and anoxic conditions (Figure 2a). Under oxic conditions, concentrations of Cu(I) were lower because dissolved oxygen can oxidize HA as well as Cu(I) (refer to the reactions below). Second, the Cu(II)/HA system produces reactive oxidants (•OH or Cu(III)) capable of causing potential oxidative damage to microorganisms by the Fenton-like reaction of the Cu(I) and H2O2 generated in situ. The Cu(I) generated by reaction 1 undergoes a series of single-electron transfer reactions in the presence of dissolved oxygen to produce reactive oxidants (reactions 2−4).10,17,29

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Cu(I) + O2 → Cu(II) + O2•−

(2)

Cu(I) + O2•− + 2H+ → Cu(II) + H 2O2

(3)

Cu(I) + H 2O2 → Cu(II) + •OH + OH− or Cu(III) + 2OH−

(4)

DOI: 10.1021/acs.est.5b04310 Environ. Sci. Technol. 2015, 49, 14416−14423

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Environmental Science & Technology The reaction of HA with dissolved oxygen can also serve as a source of H2O2 for the Fenton-like reaction. In the presence of oxygen, HA is known to be oxidized to N2 via a two-step process (Reactions 5 and 6)24 in which H2O2 is produced as an intermediate. 2NH 2OH + O2 → N2 + H 2O2 + 2H 2O

(5)

2NH 2OH + H 2O2 → N2 + 4H 2O

(6)

Cu(II) + H 2O2 → Cu(I) + O2•− + 2H+

(7)

Cu(III) + H 2O2 → Cu(II) + O2•− + 2H+

(8)

These reactions are responsible for the increase in the extracellular O2•− level; reaction 2 is responsible for the Cu(II)/HA system, and reactions 7 and 8 are mainly for the Cu(II)/H2O2 and Cu(II)/HA/H2O2 systems. However, in the intracellular region, the presence of Cu(II) (which may exist in complexed forms with biomolecules of cell components) appears to favor the consumption of O2•− regardless of the presence of HA and H2O2. The reactions of cell-bound Cu(II) or Cu(III) with O2•− may be responsible for the O2•− level drop (reactions 9 and 10), but further study is needed to clarify the mechanism.

The external supply of H2O2 to the Cu(II)/HA system (i.e., the Cu(II)/HA/H2O2 system) accelerates the production of reactive oxidants. Indeed, the HCHO formation (from methanol oxidation) was enhanced 2.6-fold by the addition of H2O2 (5 μM Cu(II)/10 μM HA vs 5 μM Cu(II)/10 μM HA/ 10 μM H2O2; data not shown). According to recent studies,18,31,32 Cu(III) (rather than • OH) is the dominant oxidant produced by the copper-based Fenton-like reaction (reaction 4) under neutral pH conditions. Our previous study on the Cu(II)/H2O2 system suggested that Cu(III) has strong biocidal activity that is more selective toward a virus surrogate, MS2 coliphage.18 Both biocidal effects described above are mediated by the redox reactions of the Cu(II)/Cu(I) couple. EDTA and DMP hinder these redox reactions by forming stable complexes with Cu(II) and Cu(I), consequently eliminating the biocidal activity of the Cu(II)/HA system (Figures 3a and 3b). Inactivation of E. coli. The enhanced inactivation of E. coli by the Cu(II)/HA system is mainly attributed to the biocidal effect of Cu(I) generated by the reduction of Cu(II). This claim is supported by the increased inactivation rate of E. coli under anoxic conditions (Figure 3a). Compared to oxic condition, the anoxic condition increased the inactivation rate of E. coli by 55% (Figure 3a), which was consistent with the observation that the Cu(I) concentration increased by 58% (from 1.2 to 1.9 μM) under the same conditions (10 μM HA addition in Figure 2a). It appears that the generation of reactive oxidants by the Cu(II)/HA system does not significantly affect the E. coli inactivation, which is supported by multiple observations in this study. First, the HCHO formation (i.e., the indicator for the generation of reactive oxidants) was negligible under the anoxic condition (Figure 2b), whereas the E. coli inactivation was enhanced. Second, the intracellular oxidant levels in E. coli cells did not increase with the Cu(II)/HA treatment. The FIR for • OH and Cu(III) only increased in Cu(II)/H2O2 and Cu(II)/ HA/H2O2 systems with a relatively high dose of H2O2 (1 mM) (Figure 4a), in which the reactive oxidants may play a role in the E. coli inactivation.18 In addition, the E. coli cells treated with the Cu(II)/HA system exhibited different morphologies from those treated with the Cu(II)/H2O2 system with high doses of reagents (refer to Figure S4a−S4d in the Supporting Information). The Cu(II)/HA treatment did not significantly disrupt the integrity of cell membranes (Figure S4c). Moreover, an interesting observation is that the intracellular O2•− level decreased for all of the treatment systems (i.e., the FIR values were less than unity), whereas the extracellular O2•− level in the bulk solution generally increased (Figure 4b) (found to cause no lethal damage to the cells1). Based on the chemistry of Cu(II) with HA and H2O2, there are several routes for the formation of O2•−, which include the oxygen reduction by Cu(I) (reaction 2) and the oxidation of H2O2 by Cu(II) (reaction 7; particularly important in the Cu(II)/H2O2 system) or Cu(III) (reaction 8).

≡ Cu(II) + O2•− → Cu(I) + O2 ≡ Cu(III) + O2•− → Cu(II) + O2

(9) (10)

A similar behavior has been observed in the Cu(II)/H2O2 system;18 the intracellular O2•− level decreased when increasing the dose of Cu(II). Inactivation of MS2 Coliphage. In contrast with the case of E. coli, reactive oxidants (mainly Cu(III)) appear to play a role in the MS2 coliphage inactivation by the Cu(II)/HA system. A slightly higher inactivation rate of MS2 coliphage under the oxic condition compared to anoxic condition supports the involvement of reactive oxidants (Figure 3b). In particular, the addition of 10 μM H2O2 greatly enhanced the MS2 coliphage inactivation (Figure 3b), implying that MS2 coliphage is highly susceptible to the oxidative damage induced by reactive oxidants, which is consistent with previous observations of MS2 coliphage inactivation by the Cu(II)/ H2O2 system.18 The addition of 200 mM tert-butanol (a •OH scavenger) did not affect the MS2 coliphage inactivation efficacy in the Cu(II)/HA system (data not shown), confirming that Cu(III) is the responsible oxidant rather than •OH.18,31,32 Despite the role of reactive oxidants, the direct cytotoxicity of Cu(I) cannot be neglected for the MS2 coliphage inactivation by the Cu(II)/HA system. Under the anoxic condition, where reactive oxidants are barely produced (Figure 2b), substantial inactivation of MS2 coliphage was observed (Figure 3b), indicating that the cytotoxicity of Cu(I) is also important. To sum up, the MS2 coliphage inactivation by the Cu(II)/HA system results from the simultaneous biocidal actions of Cu(I) and reactive oxidants. Environmental Implications. The Cu(II)/HA system has potential applications as a disinfection technology for many water uses such as wastewater, reused water, industrial and agricultural water, and possibly drinking water; note that significant inactivation of bacteria and viruses was achieved by relatively low concentrations of reagents (usually micromolar levels of Cu(II), HA, and H2O2). The concentration of Cu(II) used in this study (5 μM) is even lower than the WHO drinking water guideline value (2 mg/L = 31.4 μM33) and the drinking water standards in the United States (1.3 mg/L = 20.5 μM34) and in Korea (1 mg/L = 15.7 μM35). The Cu(II)/HA system exhibited substantial activity in natural water (Figures 5a and b) even if the disinfection efficacy was somewhat decreased by manifold factors (e.g., copper-chelation, oxidant scavenging, and undesired consumption of HA by natural organic and inorganic substances). In particular, the employment of H2O2 in the Cu(II)/HA system (i.e., the Cu(II)/HA/H2O2 system) 14421

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and a disulfide-dithiol interchange between membrane proteins. Cell Biochem. Biophys. 2008, 51 (1), 45−50. (8) Kimura, T.; Nishioka, H. Intracellular generation of superoxide by copper sulphate in Escherichia coli. Mutat. Res., Genet. Toxicol. Environ. Mutagen. 1997, 389 (2−3), 237−242. (9) Stohs, S. J.; Bagchi, D. Oxidative mechanisms in the toxicity of metal ions. Free Radical Biol. Med. 1995, 18 (2), 321−336. (10) Nieto-Juarez, J. I.; Pierzchła, K.; Sienkiewicz, A.; Kohn, T. Inactivation of MS2 coliphage in Fenton and Fenton-like systems: role of transition metals, hydrogen peroxide and sunlight. Environ. Sci. Technol. 2010, 44 (9), 3351−3356. (11) Patikarnmonthon, N.; Nawapan, S.; Buranajitpakorn, S.; Charoenlap, N.; Mongkolsuk, S.; Vattanaviboon, P. Copper ions potentiate organic hydroperoxide and hydrogen peroxide toxicity through different mechanisms in Xanthomonas campestris pv. campestris. FEMS Microbiol. Lett. 2010, 313 (1), 75−80. (12) Yamamoto, N.; Hiatt, C. W.; Haller, W. Mechanism of inactivation of bacteriophages by metals. Biochim. Biophys. Acta, Spec. Sect. Nucleic Acids Relat. Subj. 1964, 91 (2), 257−261. (13) Barbouti, A.; Doulias, P.-T.; Zhu, B.-Z.; Frei, B.; Galaris, D. Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage. Free Radical Biol. Med. 2001, 31 (4), 490−498. (14) Ozawa, T.; Hanaki, A. The first ESR spin-trapping evidence for the formation of hydroxyl radical from the reaction of copper(II) complex with hydrogen peroxide in aqueous solution. J. Chem. Soc., Chem. Commun. 1991, No. 5, 330−332. (15) Hanna, P. M.; Mason, R. P. Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique. Arch. Biochem. Biophys. 1992, 295 (1), 205−213. (16) Johnson, G. R. A.; Nazhat, N. B.; Saadalla-Nazhat, R. A. Reaction of the aquocopper(I) ion with hydrogen peroxide: evidence against hydroxyl free radical formation. J. Chem. Soc., Chem. Commun. 1985, No. 7, 407−408. (17) Johnson, G. R. A.; Nazhat, N. B.; Saadalla-Nazhat, R. A. Reaction of the aquacopper(I) ion with hydrogen peroxide. Evidence for a CuIII(cupryl) intermediate. J. Chem. Soc., Faraday Trans. 1 1988, 84 (2), 501−510. (18) Nguyen, T. T. M.; Park, H.-J.; Kim, J. Y.; Kim, H.-E.; Lee, H.; Yoon, J.; Lee, C. Microbial inactivation by cupric ion in combination with H2O2: Role of reactive oxidants. Environ. Sci. Technol. 2013, 47 (23), 13661−13667. (19) Yamamoto, K.; Kawanishi, S. Hydroxyl free radical is not the main active species in site-specific DNA damage induced by copper (II) ion and hydrogen peroxide. J. Biol. Chem. 1989, 264 (26), 15435− 40. (20) Franklin, R.; Wecker, E. Inactivation of some animal viruses by hydroxylamine and the structure of ribonucleic acid. Nature 1959, 184, 343−345. (21) Vízdalová, M.; Pillich, J. The inactivation of Escherichia coli bacteriophages of T series by hydroxylamine and UV radiation. Folia Microbiol. 1968, 13 (2), 153−155. (22) Lin, Y.-C. Inactivation of pancreatic ribonuclease with hydroxylamine-oxygen-cupric ion. Biochim. Biophys. Acta, Protein Struct. 1972, 263 (3), 680−682. (23) Anderson, J. H. The copper-catalysed oxidation of hydroxylamine. Analyst 1964, 89 (1058), 357−362. (24) Tomat, R.; Rigo, A.; Salmaso, R. Kinetic study on the reaction between O2 and hydroxylamine. J. Electroanal. Chem. Interfacial Electrochem. 1975, 59 (3), 255−260. (25) Gomes, A.; Fernandes, E.; Lima, J. L. F. C. Fluorescence probes used for detection of reactive oxygen species. J. Biochem. Biophys. Methods 2005, 65 (2−3), 45−80. (26) Kim, J. Y.; Park, H.-J.; Lee, C.; Nelson, K. L.; Sedlak, D. L.; Yoon, J. Inactivation of Escherichia coli by nanoparticulate zerovalent iron and ferrous ion. Appl. Environ. Microbiol. 2010, 76 (22), 7668− 7670.

showed excellent virucidal activity (>4 log inactivation of MS2 coliphage in 1 min; refer to Figure S2b in the Supporting Information), implying that the Cu(II)/HA/H2O2 system can be successfully applied for special purposes needing the control of viral strains. A concern regarding the potential toxicity of HA may be raised. However, HA was completely decomposed within minutes, and no residual HA was detected after the reaction (data not shown). For better application of the Cu(II)/HA system, impacts of various substances from different water matrices need to be studied further. The development of heterogeneous systems utilizing immobilized copper compounds will also be beneficial.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b04310. Inactivation rates of E. coli and MS2 coliphage by the Cu(II)/HA system as a function of HA concentration (Figure S1), time-dependent inactivation curves for Figure 3 (Figure S2), time-dependent inactivation curves for Figure 5 (Figure S3), TEM images of untreated and treated E. coli cells by Cu(II), Cu(II)/HA, and Cu(II)/ H2O2 (Figure S4), water quality parameters of natural water samples (Table S1) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +82-52-217-2812; fax: +82-52-217-2809; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIP) (NRF-2015R1A5A7037825) and by grants (14IFIPC088924-01 and 15IFIP-B088091-02) from the Smart Civil Infrastructure Research Program and the Industrial Facilities & Infrastructure Research Program funded by the Ministry of Land, Infrastructure, and Transport of the Korean government.

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