water

Article Storage Stability and Disinfection Performance on Escherichia coli of Electrolyzed Seawater

Regina G. Damalerio 1, Aileen H. Orbecido 1, Marigold O. Uba 2, Patricio Elvin L. Cantiller 2 and Arnel B. Beltran 1,*

1 Chemical Engineering Department, De La Salle University, Malate, Manila 1004, Philippines; [email protected] (R.G.D.); [email protected] (A.H.O.) 2 Biology Department, De La Salle University, Malate, Manila 1004, Philippines; [email protected] (M.O.U.); [email protected] (P.E.L.C.) * Correspondence: [email protected]; Tel.: +632-524-4611 (local 218)



Received: 22 March 2019; Accepted: 24 April 2019; Published: 10 May 2019


Abstract: The study investigated the effect of storage conditions on the stability of electrolyzed seawater (ESW)’s physicochemical properties (pH, oxidation-reduction potential (ORP), and free chlorine (FC) concentration), and bactericidal efficiency on the fecal coliform Escherichia coli for 30 days. Preliminary experiments were conducted to determine the optimal current and electrolysis time. Two batches of 2750 mL filtered seawater were electrolyzed using 50 mm 192 mm platinum–titanium × mesh electrodes at a current of 1.5 A for 20 min. One hundred milliliters of electrolyzed solution was transferred into each amber glass and high-density polyethylene (HDPE) bottles. The bottles were stored in a dark area at ambient temperature. The results showed an increase in pH and a decrease in ORP and FC concentration through time. Hypochlorous acid remained as the dominant component since the pH levels of the solutions remained below 7.5. FC decay was investigated using Chick’s Law. It was determined that the decay in HDPE bottles (k = 0.066 day 1) was faster compared to amber − − glass bottles (k = 0.046 day 1). Nonetheless, HDPE bottles could still be used as an alternative − − container for 30 days only due to observed instability beyond 30 days. ESW remained effective since no surviving population of E. coli was observed throughout the experimentation.

Keywords: disinfection performance; Escherichia coli; electrolysis; kinetics; storage stability; seawater

1. Introduction Electrochemical disinfection was introduced as an alternative due to its advantages over utilization of chlorine reagents generated from chlorine gas [1–5]. It is performed by passing current through chloride-containing water, which can be made synthetically (brine solution) or collected from a natural source (seawater) [2,6]. The presence of sodium chloride (NaCl) in the solution is important for the formation of oxidizing agents, such as free chlorine (FC), sodium hypochlorite (NaClO), or hypochlorous acid (HOCl) [5,7]. These compounds are responsible for inactivating pathogenic micro-organisms by oxidizing or attacking both the inner and outer membrane of the cell [8–10]. Other advantages of electrochemical disinfection include on-site production, raw material availability, an environment-friendly process, a less-hazardous reaction, low cytotoxicity, and no reported microbial resistance [10]. Furthermore, it has been utilized in several sectors, such as agricultural and food industries, since the disinfectant can be produced over wide pH ranges [11–15]. Xie et al. [12] investigated the effect of acidic electrolyzed water stored at different temperatures on contaminated raw shrimp, while Kasai et al. [16] investigated the effect of electrolyzed seawater (ESW) in naturally contaminated oysters. Moreover, the disinfectant can be applied in sanitizing materials and equipment.

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Osafune et al. [10] concluded that electrolyzed acidic water is effective as it destroyed the cellular structures of the three bacterial species cultivated from the kendo equipment. Seawater has 3.5% salinity, which predominantly consists of sodium and chloride ions. Furthermore, it also contains bromine, which is converted into an oxidant through electrolysis. These oxidizing compounds are detrimental towards pathogenic micro-organisms [4,14,17,18]. Thus, ESW has been employed and studied as a disinfectant [9,14,16–19]. However, the main problem lies in its storage stability through time [1,2]. The disinfectant is considered stable when it is still effective for inactivating pathogenic microorganisms. Residual oxidants or FC remains present and dominant, despite the changes in the physicochemical properties and storage conditions. No studies related to storage stability of ESW can be found in the literature since it is more often viewed as an alternative to water disinfection and as a disinfectant to be utilized immediately. Therefore, there is a possibility that the production and utilization of the disinfectant will be maximized by investigating its storage stability through time. The study could benefit people living near coastal areas, where raw material is abundant and accessible. Furthermore, this could serve as temporary alternative solution when sanitation is inaccessible (e.g., calamities). The storage stability of ESW is determined by investigating the effects of the storage conditions and changes in the physicochemical properties, such as pH, oxidation-reduction potential (ORP), and FC concentration through time, to its bactericidal efficiency on the fecal coliform Escherichia coli. The rate of FC decay in amber glass and high-density polyethylene (HDPE) bottles is investigated using Chick’s Law to determine the possibility of using HDPE bottles as an alternative storage container to amber glass bottles.

2. Materials and Methods

2.1. Materials Glassware, platinum–titanium mesh electrodes, and reagents, such as N,N-dimethyl-p- phenylenediamine (DPD) powder, phosphate-buffered saline, nutrient agar, and broth powder, were purchased from several local suppliers. Purified stock culture of E. coli was acquired from the microbiology laboratory. The seawater sample utilized in the experiment was collected five meters away from the beach shore of Oriental Mindoro, Philippines. It was filtered using a PM10 microfilter before being stored at ambient temperature.

2.2. Electrolysis Setup Fifty by 192 mm titanium–platinum mesh electrodes (mesh size: 10 5 mm; electrode surface × area = 23 cm2) were sonicated for 30 min to remove unwanted particles on the surface [18,19], and submerged vertically with a 0.5 cm gap in a 4 L Pyrex beaker. An alternating current (AC) power supply was utilized as an electrical source (Figure1). Twenty each of 120 mL amber glass bottles and 250 mL HDPE bottles were cleaned and labeled prior to the day of the electrolysis experiment. Water 20192019,, 1111,, 980x FOR PEER REVIEW 33 of of 11

Figure 1. Electrolysis Setup.

2.3. Electrolysis of Seawater Figure 1. Electrolysis Setup

2.3. ElectrolysisTwo batches of Seawater of 2750 mL seawater were electrolyzed at a current of 1.5 A for 20 min. The applied current and electrolysis time were determined based on preliminary electrolysis experiments. It wasTwo carried batches out of by2750 investigating mL seawater the were eff ectselectrolyzed of varying at a current current andof 1.5 electrolysis A for 20 minutes. duration The on theapplied physicochemical current and electrolysis properties oftime ESW. were determined based on preliminary electrolysis experiments. It was carried out by investigating the effects of varying current and electrolysis duration on the 2.4.physicochemical Storage Experiment properties of ESW. About 100 mL of ESW was transferred into each amber glass and HDPE bottle. Bottles were stored 2.4. Storage Experiment in a dark place at ambient temperature. The physicochemical properties and bactericidal efficiency of ESWAbout were evaluated 100 mL of for ESW 30 days. was transferred into each amber glass and HDPE bottle. Bottles were stored in a dark place at ambient temperature. The physicochemical properties and bactericidal 2.5.efficiency Preparation of ESW of thewere Working evaluated Culture for of30 E. days. coli A purified stock culture of E. coli was cultivated every two weeks with nutrient agar and maintained 2.5. Preparation of the Working Culture of E. coli at 4–10 ◦C for the duration of the study. A working bacterial suspension, prepared from the stock culture,A purified was standardized stock culture prior of to E. every coli disinfectionwas cultivated experiment. every two This weeks was performed with nutrient by inoculating agar and freshmaintained colonies at of4–10E. coli°C forin sterilethe duration distilled of waterthe study. until A the working turbidity bacterial was adjusted suspension, to equal prepared McFarland from Standardthe stock culture, #0.5, which was approximatesstandardized 1.5prior 10to 8everycolony disinfection forming units experiment. (CFU) per This mL was [20 performed]. A standard by × plateinoculating count wasfresh also colonies performed of E. coli to confirm in sterile the distilled microbial water density until ofthe the turb standardizedidity was adjusted turbidity to[ 1equal,2]. McFarland Standard #0.5, which approximates 1.5 × 108 colony forming units (CFU) per mL [20]. A 2.6.standard Disinfection plate Experimentscount was also performed to confirm the microbial density of the standardized turbidityTo determine [1,2]. the effect of ESW on E. coli, 1 mL of the standardized E. coli suspension was mixed with and exposed to 9 mL ESW for one minute. Then, 1 mL of the latter mixture was added to 9 mL of Phosphate-Bu2.6. Disinfectionffered Expe Salineriments for one minute to render the ESW ineffective. A 10-fold serial dilution was 3 performedTo determine by adding the 1 effect mL from of ESW the previouson E. coli, tube 1 mL to of 9 mLthe standardized of sterile distilled E. coli water suspension up to 10 was− dilution. mixed Usingwith and the exposed spread plate to 9 mL technique, ESW for 0.1 one mL minute. aliquot Then, from each1 mL solutionof the latter was mixture inoculated was on added nutrient to 9 agar mL plates.of Phosphate These- wereBuffer performeded Saline for in duplicates. one minute After to render incubation the ESW at 37 ineffective.◦C for 18–24 A 10 hours-fold [ 1serial,2], the dilution plates werewas performed examined forby growthadding and1 mL the from CFU the/mL previous of E. coli tubewas to determined. 9 mL of sterile distilled water up to 10−3 dilution. Using the spread plate technique, 0.1 mL aliquot from each solution was inoculated on 2.7. Electrolysis Setup nutrient agar plates. These were performed in duplicates. After incubation at 37 °C for 18–24 hours [1,2],The the pHplates and were ORP examined values were for measuredgrowth and using the CFU/mL a pocket of pH E. meter coli was (Lutron, determined. PH-211) and an ORP meter (Lutron, ORP-213; Ag/AgCl reference electrode), and ranged from pH 0.00 to 14.00 and ORP 0.02.7. Electrolysis1000.0 mV, Setup respectively. The pH meter was calibrated using standard buffers at a pH of 4.0, 7.0, ± N N and 10.0.The pH The and, ORP-dimethyl-p-phenylenediamine values were measured using (DPD)a pocket colorimetric pH meter (Lutron, method PH employing-211) and a an UV ORP/Vis Spectrophotometermeter (Lutron, ORP at-213 a wavelength; Ag/AgCl reference of 515 nm electrode was used), toand determine ranged from the FCpH concentration 0.00 to 14.00 [and1]. ORP 0.0 ± 1000.0 mV, respectively. The pH meter was calibrated using standard buffers at a pH of 4.0, 7.0,

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3. Theoretical Framework Due to limited literature data, the system is assumed to contain sodium chloride only. Equations (1–6) below show reactions occurring at the anode and cathode sides during electrolysis. Water and chloride ions at the anode are oxidized to oxygen, hydrogen ions, and gaseous chlorine, while water at the cathode is reduced to hydroxides and hydrogen gas. The evolution of chlorine becomes more favorable than oxygen at the anode side due to the increase in hydrogen ion concentration in the solution [6]. Anode Side + 2H O O + 4H + 4e− Eo = 1. 23 V (1) 2 → 2 2Cl− 2e− + Cl (g) Eo = 1.36 V (2) ↔ 2 Cathode Side 2H O + 2e− 2OH− + H Eo = 0.83 V (3) 2 → 2 − Aqueous chlorine formed from gaseous chlorine partially dissociates to hypochlorous acid and chloride and hydrogen ions at increasing concentrations. A further increase in the concentration of hypochlorous acid would result in its decomposition to hypochlorite and hydrogen ions.

+ 4 Cl + H O HOCl + Cl− + H K = 4.0 10− at 25 ◦C (4) 2(aq) 2 ↔ H × + 8 HOCl OCl− + H K = 3.0 10− at 25 ◦C (5) ↔ H × The distribution of chlorine species can be determined from Equations (4) and (5) by expressing it as equilibrium expressions. Simplifying these expressions would result in two equations as functions of pH and/or chloride concentration. Equation 6 determines the ratio of aqueous chlorine to hypochlorous acid, while Equation (7) determines the ratio of hypochlorous acid to hypochlorite [1,21]. h i Cl2 (aq) pK pH+log Cl− = 10 H− (6) [HOCl]

[OCl−] = 10pH + pKa (7) [HOCl] The distribution of aqueous chlorine, hypochlorous acid, and hypochlorite ions is plotted in Figure2. It is observed that aqueous chlorine is present in highly acidic conditions and decreases beyond pH 2.0. The concentration in an ideal system decreases as it approaches pH 4.0 and hypochlorous acid dominates the region beyond pH 4.0. Hypochlorous acid is the most important component for ESW due to its high oxidizing property, which is responsible for inactivating pathogenic micro-organisms [8]. Furthermore, it is more stable than aqueous chlorine due to its less volatile nature [1,22]. Hypochlorous acid decreases gradually due to its weak acidic nature and remains predominant until pH 7.5, as illustrated in Figure2b. Beyond pH 7.5, hypochlorite ions become the predominant component of the solution. WaterWater 2019 2019, 11, 11, x, 980xFOR FOR PEER PEER REVIEW REVIEW 5 5of5 of of11 11

(a(a) ) (b(b) )

FigureFigure 2 2.2. .Distribution DistributionDistribution of of of ( a( (a)a ))aqueous aqueous aqueous chlorine chlorine chlorine;; ;and and and ( b (b) ))hypochlorous hypochlorous acid acid acid in in e lectrolyzed electrolyzedelectrolyzed seawater seawater (ESW((ESW)ESW) )at at different different different pH pH levels levels.levels

4.4. Results Results Results and and and Discussion Discussion Discussion 4.1. Electrolysis Parameter Selection 4.1.4.1. Electrolysis Electrolysis Parameter Parameter Selection Selection The preliminary experiments aided in determining the behavior of the physicochemical properties TheThe preliminary preliminary experiments experiments aided aided in in determining determining the the behavior behavior of of the the physicochemical physicochemical during the electrolysis of the seawater sample. It defined the relationship of the three properties and propertiesproperties during during the the electrolysis electrolysis of of the the seawater seawater sample. sample. It It defined defined the the relationship relationship of of the the three three the effect of increasing applied current to the system through time. Figure3a shows the decrease of pH propertiesproperties and and the the effect effect of of increasing increasing applied applied current current to to th thee system system through through time. time. Fig Figureure 3a 3a shows shows theat increasing decrease of electrolysis pH at increasing time and electrolysis applied current. time and ESW applied becomes current. more acidicESW becomes as the current more increasesacidic as the decrease of pH at increasing electrolysis time+ and applied current. ESW becomes more acidic as due to continuous generation of hydrogen (H ) ions. At the end of the electrolysis,+ the pH of ESW at thethe currentcurrent increasesincreases duedue to to continuous continuous generation generation of of hydrogen hydrogen (H (H+) ) ions. ions. At At the the end end of of the the 0.5 A, 1.0 A, and 1.5 A reached values of 5.76, 3.77, and 3.35, respectively. electrolysis,electrolysis, the the pH pH of of ES ESWW at at 0.5 0.5 A, A, 1.0 1.0 A, A, and and 1.5 1.5 A A reached reached values values of of 5.76, 5.76, 3 3.77.77, and, and 3.35, 3.35, respectively. respectively.

(a(a) ) Figure 3. Cont.

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(b)

(c)

Figure 3.3. PreliminaryPreliminary results results of of ten ten minute minutess of of ESW ESW conducted conducted at at 0.5 0.5 A, 1.0 A, and 1.5 A for the following physicochemical properties properties:: ( (aa)) pH pH;; ( (b)) oxidation oxidation-reduction-reduction potential potential (ORP); (ORP); and ( c) free chlorine (FC) concentration.concentration

An increase increase in in the the acidity acidity of the of solution the solution correlates correlates to an increasing to an increasing amount of oxidizingamount of compounds, oxidizing suchcompounds as aqueous, such chlorine as aqueous and chlorine hypochlorous and hypochlorous acid, in the solution. acid, in the Thus, solution. high values Thus,of high ORP values and FC of concentrationORP and FC concentration are expected are [1,2 expected,12,13,18]. [1, However,2,12,13,18 stability]. However, in ORP stability was observedin ORP was from observed six minutes from onwardssix minutes at all onwards currents, at while all currents stability, while in FC concentration stability in FC was concentration achieved from was six achieved minutes onwardsfrom six atminutes currents onwards of 0.5 Aat andcurrents 1.0 A of (Figure 0.5 A 3andb–c). 1.0 The A (Fig stabilityure 3b of–c). the The FC stability concentration of the couldFC concentration mean that thecould maximum mean that concentration the maximum of hypochlorousconcentration acidof hypochlorous was attained acid at a givenwas attained current. at Based a given on Figurecurrent.2, electrolyzedBased on Fig solutionsure 2, electrolyzed generated solutions at currents generated of 0.5 A andat currents 1.0 A fall of in0.5 the A regionand 1.0 where A fall hypochlorous in the region acidwhere is thehypochlorous predominant acid component. is the predominant There would component. be minimal There changes would in thebe chlorineminimal specieschanges due in tothe a lowchlorine concentration species ofdue aqueous to a chlorinelow concentration and slow dissociation of aqueous of hypochlorous chlorine and acid slow to hypochlorite dissociation ions. of Thehypochlorous electrolyzed acid solutions to hypochlorite at 1.5 A predominantly ions. The electrolyzed contain aqueous solutions chlorine. at 1.5 A Due predominantly to its volatile contain nature, someaqueous of thechlorine. aqueous Due chlorine to its volatile could nature, have dissociated some of the to aqueous hypochlorous chlorine acid could to achieve have dissociate equilibrium,d to ashypochlorous stated in Equation acid to (6).achieve equilibrium, as stated in Equation (6). From the the preliminary preliminary results, results, the currentthe current 1.5 A 1.5was A utilized was utilized to determine to determine the maximum the electrolysismaximum timeelectrolysis to be used time for to thebe used actual for experimentation. the actual experimentation. Two runs of Two electrolysis runs of were electrolysis conducted were and conducted samples wereand samples collected were every collected five minutes every for five evaluation minutes of for physicochemical evaluation of properties.physicochemical It is shown properties. in Figure It 4is thatshown stability in Fig wasure 4 achieved that stability at an electrolysiswas achieved time at ofan 20 electrolysis min (pH = time4.54, of ORP 20 minutes= 988 mV, (pH FC == 4.54,48.97 ORP mg/ L= Cl9882). mV, ESW FC generated = 48.97 mg/L at 20 Cl min2). ESW was determinedgenerated at to 20 contain minutes a maximumwas determined of 86.47% to contain hypochlorous a maximum acid. of 86.47% hypochlorous acid.

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(a) (b)

Figure Figure4. Behavior 4. Behavior of (a) pH of (anda) pH ORP and; and ORP; (b) and FC (concentrationsb) FC concentrations at 1.5 A at for 1.5 30 A minutes for 30 min of ofelectrolysis electrolysis.

TheThe power requiredrequired forfor ESW ESW generated generated at at a currenta current of 1.5of 1.5 A for A 20for min 20 minutes was determined was determined using the usingformula the [ 4formula]: [4]: P = V I (8) 푃 = 푉 × ×퐼 (8) where: where: 푃 P: Power requirement,: wattsPower requirement, watts 푉: Observed voltage, volts V: Observed voltage, volts 퐼: Current applied, ampere I: Current applied, ampere The energy consumption can be calculated by multiplying the calculated power and electrolysis time [4]:The energy consumption can be calculated by multiplying the calculated power and electrolysis time [4]: 퐸 = 푃 × 푡 = 푉 × 퐼 × 푡 (9) E = P t = V I t (9) where: × × × where: E: Energy consumption, watt-hour 푃: Power requirement, watts E: Energy consumption, watt-hour 푉: Observed voltage, volts P: Power requirement,퐼: wattsCurrent applied, ampere V: Observed voltage,t: voltsElectrolysis time, hours I: Current applied, ampere Thet: Electrolysis calculated time, power hours required for generating one liter of ESW is 2.62 watts. If the system is upscaled to produce 1 kiloliter of the disinfectant, the power requirement is about 2620 watts (2.62 kW). On theThe other calculated hand, the power energy required consumption for generating of 1 kiloliter one of liter electrolyzed of ESW is seawater 2.62 watts. is about If the 873 system watt- hours.is upscaled to produce 1 kiloliter of the disinfectant, the power requirement is about 2620 watts (2.62 kW). On the other hand, the energy consumption of 1 kiloliter of electrolyzed seawater is about 4.2.873 Storage watt-hours. Effect on Physicochemical Properties 4.2. Storage Effect on Physicochemical Properties 4.2.1. Evaluation for 30 Days 4.2.1.Fig Evaluationure 5 illustrates for 30 the Days behavior of pH and ORP of stored ESW in amber glass and HDPE bottles throughoutFigure 5 the illustrates 30-day the storage. behavior Initially, of pH the and ESW ORP in of storedamber ESW bottles in amber (pH = glass 4.21, and ORP HDPE = 1010 bottles mV) containthroughouted 83.7 the1% 30-day hypochlorous storage. acid Initially,, while the the ESW ESW in in amber HDPE bottles bottles (pH (pH= =4.21, 5.74, ORP ORP= = 1010920 mV) mV) containcontaineded 96.86% 83.71% hypochlorous hypochlorous acid. acid, It while can be the observed ESW in that HDPE there bottles is a gradual (pH = 5.74,increase ORP in= pH920 and mV) a decreasecontained in 96.86% ORP throughout hypochlorous the storage acid. It period. can be observed Slow changes that therein pH is and a gradual ORP coul increased be attributed in pH and to a thedecrease nature in of ORP the throughoutacid and storage the storage conditions period. that Slow minimized changes decomposition in pH and ORP of couldhypochlorous be attributed acid throughto the nature time.of the acid and storage conditions that minimized decomposition of hypochlorous acid through time.

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Figure 5. The pH and ORP distribution of ESW stored in both bottles throughout the 30-day Figure 5. The pH and ORP distribution of ESW stored in both bottles throughout the 30-day storage period. storage period.

FigureAnAn averageaverage 5. The pHpH and ofof 6.536.53 ORP was was distribution measured measured of forESW for 30-day 30stored-day oldin old both solutions solutions bottlesstored throughout stored in in both boththe bottles.30 bottles.-day There There was was a storage period. 2.39%a 2.39% increase increase of of hypochlorous hypochlorous acid acid in in amber amber glass glass bottles bottles and and anan 11.51%11.51% decreasedecrease inin HDPE bottles. TheThe increaseincrease of of the the hypochlorous hypochlorous acid acid in in amber amber glass glass bottles bottles may may be be attributed attributed to to partial partial dissociation dissociation of An average pH of 6.53 was measured for 30-day old solutions stored in both bottles. There was remainingof remaining aqueous aqueous chlorine. chlorine. On On the the other other hand, hand, the the decrease decrease of ORP of ORP from from 845.50 845.50 to 833 mV mV, to 833 for bothmV, a 2.39% increase of hypochlorous acid in amber glass bottles and an 11.51% decrease in HDPE bottles. bottles,for both could bottles be, could due to be the due increasing to the increasing decomposition decompo of hypochloroussition of hypochlorous acid [1]. acid [1]. The increase of the hypochlorous acid in amber glass bottles may be attributed to partial dissociation TheThe averageaverage initial initial FC FC concentrations concentrations of ESWof ESW are are 51.85 51.85 mg/ Lmg/L for amber for amber glass glass bottles bottles and 51.23 and mg51.23/L of remaining aqueous chlorine. On the other hand, the decrease of ORP from 845.50 mV to 833 mV, formg/L HDPE for bottles HDPE (Figure bottles 6 (Fi). Unlikegure 6). in Unlike pH and in ORP, pH FC and in ORP, both bottlesFC in bothexperienced bottles an experienced exponential an for both bottles, could be due to the increasing decomposition of hypochlorous acid [1]. declineexponential through decline time. through By day time. 30, approximately By day 30, approximately 22.72% and 8.80% 22.72% of theand initial 8.80% FC of concentration the initial FC The average initial FC concentrations of ESW are 51.85 mg/L for amber glass bottles and 51.23 remainedconcentration in amber remained glass in and amber HDPE glass bottles, and respectively.HDPE bottles, respectively. mg/L for HDPE bottles (Figure 6). Unlike in pH and ORP, FC in both bottles experienced an exponential decline through time. By day 30, approximately 22.72% and 8.80% of the initial FC concentration remained in amber glass and HDPE bottles, respectively.

Figure 6. FC distribution of ESW stored in both bottles throughout the 30-day storage period.

TheFigure rate 6. ofFC FC distribution decay of of ESW ESW during stored storagein both bottles can be throughout evaluated the using 30-day Equation storage (10)period [1, 13].

C The rate of FC decay of ESW during storageln t can= bekt evaluated using Equation (10) [1,13]. (10) Figure 6. FC distribution of ESW stored in both Cbottleso − throughout the 30-day storage period 퐶푡 ln = −푘푡 where: (10) The rate of FC decay of ESW during storage퐶표 can be evaluated using Equation (10) [1,13]. where: Ct: FC concentration at any storage period퐶푡 t, mg/L ln = −푘푡 퐶푡: FC concentration at any storage period t, mg/L (10) Co: Initial FC concentration, mg/L 퐶표 퐶표: Initial1 FC concentration, mg/L k: Rate ofwhere: FC decay, day− t : Storage period of퐶푡: ESW, FC day concentration at any storage period t, mg/L 퐶표: Initial FC concentration, mg/L

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푘: Rate of FC decay, day−1 The results are illustrated푡: Storage in Figure period7, and of it ESW is determined, day that stored ESW samples experienced single-stageThe results first-order are illustrated decay due in Fig toure HOCl 7, and decomposition. it is determined The that first-order stored ESW decay samples was also experienced observed insingle other-stage studies first-order [1,13 ,decay22]. due Stored to HOCl samples decomposition. in amber glass The first bottles-order exhibited decay was slower also observed FC decay in (kother= studies0.046 day [1,131,22), compared]. Stored samples to stored in samples amber glass in HDPE bottles bottles exhibited (k = slower0.066 dayFC decay1). Nonetheless, (k = −0.046 − − − − HDPEday−1), compared bottles could to stored be used samples as a storage in HDPE container bottles (k due = −0.066 to the day minimal−1). Nonetheless difference, HDPE between bottles the could two decaybe used constants. as a storage container due to the minimal difference between the two decay constants.

Figure 7. FC decay during the 30-day storage period. Figure 7. FC decay during the 30-day storage period 4.2.2. Extension of 30-Day Storage Evaluation 4.2.2.The Extension storage of experiment 30-Day Storage was extended Evaluation for two weeks to gather more information on the effect of storage conditions on the stability and disinfection performance. The remaining ESW in the 28-day-old The storage experiment was extended for two weeks to gather more information on the effect of bottles was used to evaluate the properties and bactericidal efficiency on days 42 and 49. From Table1, storage conditions on the stability and disinfection performance. The remaining ESW in the 28-day- it can be observed that there are minimal changes in the pH for both bottles. The oxidation-reduction old bottles was used to evaluate the properties and bactericidal efficiency on days 42 and 49. From potential of stored ESW in HDPE bottles had a minimal difference between days 28 and 42 but Table 1, it can be observed that there are minimal changes in the pH for both bottles. The oxidation- decreased suddenly to 632 mV by day 49. Meanwhile, FC in both bottles continued to decline until it reduction potential of stored ESW in HDPE bottles had a minimal difference between days 28 and 42 reached less than 5 mg/L. The reason for the decrease in ORP and FC may be attributed to a decrease of but decreased suddenly to 632 mV by day 49. Meanwhile, FC in both bottles continued to decline hypochlorous acid and escape of chlorine gas through repeated opening of the bottles. until it reached less than 5 mg/L. The reason for the decrease in ORP and FC may be attributed to a decrease of hypochlorous acid and escape of chlorine gas through repeated opening of the bottles. Table 1. pH, ORP, and FC concentration of ESW stored in both bottles at days 0, 28, 42, and 49.

Table 1. pH, ORP, and FC concentration of ESW stored in both bottles at days 0, 28, 42,Free and Chlorine 49. Container Type Days pH ORP (mV) Concentration (mg/L) Free Chlorine Concentration AmberContainer Glass Type Days 0 pH 4.21ORP (mV) 1010 51.85 (mg/L) 28 6.70 853.50 12.67 Amber Glass 420 4.21 6.61 1010 85551.85 5.17 4928 6.70 6.71 853.50 815.5012.67 2.95 High-Density Polyethylene (HDPE) 042 6.61 5.74 855 9255.17 51.23 2849 6.71 7.24 815.50 7972.95 8.77 42 7.26 788.50 2.93 High-Density Polyethylene (HDPE) 490 5.74 7.30 925 62351.23 0.99 28 7.24 797 8.77 42 7.26 788.50 2.93 4.3. Storage Effect on Disinfection Performance 49 7.30 623 0.99 The storage effect on the bactericidal efficiency of ESW on the fecal coliform E. coli was observed for4.3. 49Storage days. Effect The on freshly Disinfection generated Performance solution achieved complete inactivation of 1.5 108 CFU/mL × of E. coli due to its slightly acidic (pH < 6.00) and highly oxidizing nature (ORP > 900 mV). It was The storage effect on the bactericidal efficiency of ESW on the fecal coliform E. coli was observed hypothesized that the effectiveness of the disinfectant would decline through time due to changes in for 49 days. The freshly generated solution achieved complete inactivation of 1.5 × 108 CFU/mL of E. the physicochemical properties. However, no growth was detected for both bottles. A similar result coli due to its slightly acidic (pH < 6.00) and highly oxidizing nature (ORP > 900 mV). It was was observed in another study where no surviving population was detected for 30 days, using acidic hypothesized that the effectiveness of the disinfectant would decline through time due to changes in

Water 2019, 11, 980 10 of 11 and neutral electrolyzed water on Salmonella typhii and Escherichia coli O157:H7 [2]. The main reason for this result could lie in the amount of hypochlorous acid present. Slow changes were observed throughout storage for ESW both in amber glass and HDPE bottles. From an initial solution containing 83.71% hypochlorous acid in amber glass bottles and 96.86% in HDPE bottles, the hypochlorous acid in 30-day-old solutions became 85.71%. The amount of hypochlorous acid is still high, and this could be enough to inactivate an average initial population of 1.5 108 CFU/mL. The 49-day-old ESW × solutions have neutral pH and a lower oxidation-reduction potential (ORP = 815.5 mV for amber glass and ORP = 632 mV for HDPE bottles). Despite the low and almost negligible FC concentration, hypochlorous acid remains predominant for stored ESW in both bottles, since the pH of the solution is still below 7.5 [1,2,8].

5. Conclusions The changes in the physicochemical properties affect the distribution of chlorine species in ESW stored in both amber glass and HDPE bottles. The increase in pH and decrease in ORP and FC concentration are due to a decreasing amount of hypochlorous acid through time. Equation (10) was used to investigate the FC decay of ESW through time. Therefore, it was observed that the decline was faster in HDPE bottles (k = 0.066 day 1), compared to amber glass bottles (k = 0.046 day 1). Further − − − − investigation revealed that instability in the physicochemical properties occurred in 49-day-old solutions stored in HDPE bottles. However, it did not affect the effectiveness of ESW as a disinfectant for the fecal coliform E. coli. No surviving population of E. coli was observed throughout the experimentation, and this could be due to the predominance of hypochlorous acid in the solution. Based on the results, the use of HDPE bottles for storage is recommended for 30 days only. Further investigation is needed for a full understanding on ESW’s storage stability. The physicochemical properties and bactericidal efficiency may be evaluated by changing the storage conditions, such as storage time, temperature, and exposure to light and atmosphere. The antimicrobial assay can also be modified using different microbial species.

Author Contributions: All authors collaborated on this work. Conceptualization, A.B.B.; and R.G.D.; methodology, A.B.B.; R.G.D.; P.E.L.C.; and M.O.U.; formal analysis, A.H.O.; A.B.B.; M.O.U.; P.E.L.C.; and R.D; investigation, R.G.D.; writing—original draft preparation, R.G.D.; writing—review and editing, A.H.O.; A.B.B.; M.O.U.; P.E.L.C.; and R.G.D.; supervision, P.E.L.C.; and M.O.U. Funding: This research was funded by the University Research Coordination Center of De La Salle University-Manila, grant number 56 N 3TAY 14–3TAY 15. Acknowledgments: The authors would like to acknowledge the technical staff of the Microbiology Laboratory for their kind assistance and support. Conflicts of Interest: The authors declare no conflict of interest.

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