During long term storage, the ORP of ECAS is reduced substantially from that at the point of production, though the rate of reduction depends upon storage conditions, as shown in Figure 1. Irrespective of storage material, ECAS stored at 20 °C demonstrated a greater reduction in ORP over a shorter time period than that stored at 4 °C. The ORP of ECAS stored in polystyrene dropped at a slightly faster rate than that stored in glass. After 14 days, the ORP of ECAS stored in polystyrene and glass at 4 °C was 675 mV and 1048 mV respectively, compared to 1133 mV and 1159 mV respectively for polystyrene and glass at 4 °C. ECAS stored in glass at 4 °C showed the slowest decline in ORP retaining an ORP of greater than 1000 mV for 57 days compared to that of 35 days for ECAS stored in plastic at 4 °C, 14 days for ECAS stored in glass at 20 °C and 7 days for ECAS stored in polystyrene at 20 °C. It is also clear that, although in the short term, storage material and temperature greatly influence the retention of a high ORP, following long term storage the redox potential of the stored solutions begins to equilibrate.

Figure 2 shows the pH of ECAS under long term storage with the inset graph displaying the same data on a limited pH scale of 1.2 to 2.0. Despite the decline in ORP over the same time period, the pH of ECAS remained stable over 398 days, irrespective of storage conditions.

Figure 3 shows the free chlorine level of stored ECAS over a 398 day period. In all cases, the greatest loss in free chlorine occurred over the first 24 h. As was the case with ORP, the rate of loss is the slowest when stored in glass at 4 °C. The detection limit of the test procedure is 0.01 mg/L. By 277 days, all samples except for those stored in polystyrene at 4 °C had free chlorine levels of <0.01 mg/L. ECAS stored in polystyrene at 4 °C had 0.01 mg/L of measurable free chlorine remaining. The time point at which free chlorine levels within the stored ECAS became not statistically different from zero varied according to storage material and temperature. For polystyrene at 20 °C, this was day 7, for glass at 20 °C day 14, for polystyrene at 4 °C day 21 and for glass at 4 °C day 28.

Figure 4 shows the log₁₀ reduction in viable bacteria when using stored ECAS against P. aeruginosa ATCC 9027. Over the first 4 days of storage, zero counts were recorded on all recovery plates indicating that the population of surviving bacteria within the test was fewer than 10 cfu/mL. Basic bactericidal activity is retained in all storage conditions, though the consistency of the result varies depending on storage material and temperature. When stored in polystyrene at 20 °C (Figure 4a), ECAS failed to produce a log₁₀ reduction of 5 (the definition of basic bactericidal activity under EN 1040:2005) on three occasions, compared to two occasions when stored in glass at 20 °C (Figure 4c). When stored at 4 °C in polystyrene or glass (Figure 4b,d respectively), a 5 log₁₀ reduction was achieved at every time point. There was greater variability in the log₁₀ reduction achieved when stored at 20 °C than at 4 °C and the log₁₀ reduction was consistently higher when stored in glass compared to polystyrene. Over the 398 day storage period, the mean log₁₀ reduction achieved under each storage condition (± SD) was 6.634 ± 1.204 in polystyrene at 20 °C, 6.720 ± 1.135 in glass at 20 °C, 7.196 ± 0.6947 in polystyrene at 4 °C and 7.441 ± 0.3402 in glass at 4 °C.

The physiochemical properties of ECAS depend upon the characteristics of the electrochemical cell from which it is produced and its operating parameters [4]. Electrolysis of NaCl in these electrochemical cells yields a solution from both the anodic and cathodic chamber of the cell. Acidic ECAS are produced from the anode and can yield solutions with an ORP of greater than +1100 mV and a pH of lower than 2.7. Whereas the cathodic solutions are basic with a pH of 11 or more and an ORP lower than −800 mV and weak antimicrobial activity compared to acidic ECAS [40]. A modified variant of acidic ECAS can also be produced by directing some of the anodic output back into the cathode chamber, producing a neutralised anolyte often referred to as neutral electrolyzed water [39]. The antimicrobial properties of acidic ECAS have been widely reported, though one of the primary concerns regarding these solutions is the rapid loss of antimicrobial activity post-production [16]; however, this lack of residual activity is likely to contribute to the environmental compatibility of ECAS. Neutralised acidic ECAS have shown greater antimicrobial activity during shelf life trials than their acidic counterparts [41]. Superior antimicrobial activity during storage can be attributed to the greater stability of neutral ECAS, as these solutions generally have lower ORP values at the point of production than those of acidic ECAS, which is considered to be the primary contributor to their bactericidal activity [42].

The data generated from this study suggest that that both temperature and storage material significantly influence the rate at which ORP and chlorine decline in acidic ECAS, with temperature having a greater influence on long term storage than the storage material. Fabrizio and Cutter [30] also determined that ECAS were more stable when stored at 4 °C than when stored at 25 °C over three days, with ORP remaining stable over this time period and chlorine levels increasing in the stored solution. The ECAS used in their study had a pH of 2.6, an ORP of 1150 mV and a free chlorine concentration of 50 ppm (at point of production) and they found that freshly produced and “aged” EO water (stored for 24 h at 4 °C) both demonstrated potent bactericidal activity. As with our study, pH remained constant throughout short term storage. A study by Len et al.[36] showed that ECAS (EO water) stored at 25 °C lost 40% of its “active chlorine” after 58 days storage under closed conditions. However, in our study, by this time point, in excess of 99.9% of free chlorine had been lost under all storage conditions compared to levels recorded at the point of production. Len et al. stored their ECAS in 1.7 L volumes in glass vessels compared to the 30 mL samples stored in our study. The ECAS used in the current study had an ORP of 1180 mV, a pH of 1.37 and a free chlorine concentration of 570 mg/L at the point of production compared to that of 1020–1120 mV, pH 2.5–2.6 and 53–56 mg/L chlorine used by Len et al. They suggested that the primary mechanism of chlorine loss under closed conditions (in the absence of evaporation) was self-decomposition of chlorine species in solution. The ECAS samples used in the study by Len et al. were pH adjusted up to 9 though they found that more rapid chlorine loss was observed at lower pH values. This could account for the rapid chlorine loss observed in the current study. This study found that pH remained stable throughout storage. Len et al. observed only slight reductions in ORP, as did Hsu and Kao [37] who also observed a stable pH during storage, but more rapid loss of chlorine in those ECAS with the lowest pH and highest chlorine levels at the point of production.

Cui et al.[39] studied neutral and acidic ECAS stored for 30 days, also finding that pH remained stable during the storage period. The acidic ECAS used in their study had a pH of 3.01, ORP of 1079.1 and chlorine concentration of 25 mg/L and they found that in addition to pH, ORP remained stable over the storage period under closed conditions, whilst chlorine concentrations decreased by 80% and 96% in dark and light conditions respectively and to undetectable levels in open conditions after 30 days. Evaluating bactericidal activity against Salmonella Enteritidis and Escherichia coli O157:H7 demonstrated that acidic ECAS stored for 30 days was no less effective than at the point of production. Although the authors recognised that acidic ECAS have potent bactericidal activity due to lower pH and higher ORP than neutral ECAS, they concluded that available chlorine may be the primary factor responsible for bactericidal activity. This is in contrast to the work of Venkitanarayanan et al.[43] who suggested that ORP, pH and chlorine act in combination. Our study also demonstrated bactericidal activity in the absence of detectable chlorine, supporting the assertion that the bactericidal activity of these solutions is not wholly dependent on chlorine concentration. Nisola et al.[35] stored neutral and acidic ECAS for 30 days at 25 °C and tested them against E. coli O157:H7 and Salmonella Typhi. The acidic ECAS was produced with a pH of 2.0, ORP of 1271 mV and chlorine concentration of 173 mg/L. At the point of production acidic ECAS was the superior biocide, the authors suggesting that the low pH sensitised bacterial membranes to chlorine penetration. Under closed conditions a slight decrease in ORP was observed with a concomitant increase in pH and chlorine, which stabilised after 10 days. Following 30 days storage, 1.1 and 1.0 log₁₀ reductions in S. typhi and E. coli were observed respectively compared to >8 log₁₀ reductions at the point of production. Conversely, in the current study a >7 log₁₀ reduction in viable P. aeruginosa was measured after 398 days storage in all conditions with the exception of polystyrene at 20 °C. The ECAS evaluated in the current study failed to achieve a >5 log₁₀ reduction in viable bacteria on five occasions, all of which occurred when stored at 20 °C though on four of those occasions solutions stored under the same conditions achieved a >5 log₁₀ reduction at a later date. ORP and free chlorine were found to decline more slowly at 4 °C than at 20 °C and also more slowly when stored in glass vessels than polystyrene vessels. Failure to achieve a >5 log₁₀ reduction on these independent test dates may have been due to headspace being left in the vessels, although every effort was made to exclude this at the point of production. The presence of headspace would have allowed more rapid volatilisation of Cl₂ from solution compared to the suggested self-decomposition in the absence of airspace [36]. The ECAS used in the current study are capable of meeting or exceeding a modified EN 1040:2005 up to 277 days post production under all storage conditions tested and up to 398 days in three of the four conditions tested. This activity is most reliably achieved with samples stored at 4 °C rather than 20 °C, and in glass rather than polystyrene vessels and whilst storage in glass vessels would be impractical on a day-to-day basis, this research has also demonstrated retention of basic bactericidal activity when stored in plastic for a period of up to 13 months. Under the storage conditions tested, temperature had more of an influence on the physiochemical parameters and antimicrobial efficacy of ECAS than storage material. Previous work has shown that acidic ECAS are potent bactericidal and sporicidal agents [5]. Whilst high-level efficacy may be dependent on a combination of ORP, pH and chlorine concentration [43], the physiochemical parameters of solutions stored under differing conditions equilibrate after long term storage, suggesting that for long-term storage applications, storage temperature and material may be less important than for short-term storage applications.

ECAS was produced from a saturated sodium chloride solution and softened mains water in an electrochemical cell (Bridge Systems Ltd.: Fife, UK) at a rate of 200 mL/min from the anodic chamber and 400 mL/min from the cathodic chamber. ECAS was produced at an ORP of 1183.1 mV, a pH of 1.37 and a free chlorine concentration of 570 mg/L. ECAS was collected in two sets of containers; 30 mL thick-walled glass Universal bottles with rubber-sealed aluminium screw caps, and 30 mL polystyrene Universal bottles with high density polyethylene screw caps. The containers were filled sufficient to exclude air, sealed and stored under dark conditions at either 4 °C or 20 °C. At the every time point, three samples of ECAS stored under each of the four conditions were analysed for ORP, pH, free and total chlorine and tested for bactericidal activity.

The redox potential and pH of ECAS were measured immediately after collection (Sartorius PT-10P portable meter with PYP12 pH electrode and CEPTRL/87 ORP electrode, calibrated according to manufacturer’s instructions; Sartorius: Epsom, UK). At each time point, three ECAS samples stored in glass and plastic at 4 °C and 20 °C were tested similarly. Furthermore, free and total available chlorine were assayed using a chlorine DPD test (Palintest: Gateshead, UK) following appropriate dilution in sterile deionized water.

At each time point, three ECAS samples stored in glass and plastic at 4 °C and 20 °C were subjected to a modified version of EN 1040:2005, Staphylococcus aureus ATCC 6538 was removed from the regimen as previous research had shown it to be more susceptible to the bactericidal activity of ECAS than P. aeruginosa ATCC 9027 (data not shown). A dilution-neutralization assay was undertaken in accordance with EN 1040:2005. A stock culture of P. aeruginosa ATCC 9027 was streaked onto Tryptone Soya Agar (TSA; Oxoid: Basingstoke, UK) and incubated at 37 °C for 18–24 h. A single, isolated colony was subcultured onto TSA for 18–24 h. Colonies were suspended in diluent (1 g/L Tryptone, 8.5 g/L NaCl) to a density of 1.5–5 × 10⁸ cfu/mL (test suspension). 1 mL of test suspension and 1 mL water were added to a Universal bottle and incubated at 20 °C for 2 min ± 1 s. 8 mL of stored ECAS (equilibrated to 20 °C) was added for a contact time of 5 min ± 10 s followed by immediate removal to neutralizer (Letheen Broth, BD: Oxford, UK) for 5 min ± 10 s. Duplicate 1 mL volumes were transferred directly from the neutralizer and from 10-fold dilutions made into diluent, to a sterile petri dish. Twenty microliters melted TSA cooled to 45 °C was added to each petri dish. Plates were incubated at 37 °C and counted at 24 and 48 h with the higher number used for biocide evaluation.

An analysis of variance (ANOVA) was performed (GraphPad Prism 5.03, GraphPad Software Inc.: La Jolla, CA, USA), followed by Dunnett’s multiple comparison test against a chlorine level of 0.0 mg/L with a P value of <0.05 being regarded as significant in order to determine at what time point the free chlorine levels within the stored ECAS became not statistically different from zero.