Timothy Walton

Managing Director
International Eco Trade
T: +1 (954) 309-8251
E-mail: timothy.walton@intellocorp.com

Web: www.intellocorp.com
05 August 2014
acsc

Evaluation of the potential of the disinfection product Ecolox-pro to decontaminate

environmental surfaces contaminated with norovirus

Background
International ECO Trade (IET), (http://intlecotrade.com/ ) has asked the Section of Diagnostic
Technology, Division of Food Microbiology, National Food Institute, Technical University of Denmark
(NFI), to evaluate their disinfection product, Ecolox-pro for its likely potential to act as a disinfectant
of noroviruses (NoVs) on contaminated environmental surfaces.
The evaluation is based on a literature study focusing on issues considered relevant to support
assessment of the efficacy of Ecolox-pro that generates electrolyzed oxidizing water (EOW) to act as
a NoV disinfectant on environmental contaminated surfaces. In addition, the evaluation provides
recommendation on whether the Ecolox-pro should be considered to further undergo laboratory
validation tests to document the efficacy to inactivate NoV on contaminated environmental surfaces.
NFI acts as independent consultants and the evaluation and report is done as commissioned research
payed by WEC.

REG-no. DK 30 06 09 46

The device
Ecolox-pro is a portable, handhold system containing NaCl, a spraying bottle and a power source
transformer (9V 3A) with power cord. The system requires the availability of tap water and power
supply, and allows on-site generation and application of EOW containing 80 ppm hypochlorous acid
(HOCl) (as the active inactivating ingredient). The EOW can be dispensed as spray directly from the
bottle in which it is produced. The WEC has informed that the Ecolox-pro has shown broad-spectrum
antibacterial activity and should be able to decontaminate small surfaces, utensils and equipment for a
range of bacterial pathogens. However, the product has not yet been tested for its efficacy to
inactivate NoV on contaminated surfaces.
Evaluation of Ecolox-pro and recommendation for further tests
NFI has conducted the requested literature study (see summary in appendix 1 and 2 as well as
reference list in Appendix 4) on EOW used as a suspension and spray to disinfect NoV on
contaminated surfaces. To support the conclusions from the literature study, we moreover conducted

Technical University of Denmark

National Food Institute

Mrkhj Bygade 19
2860 Sborg
Denmark

Tel 45 35 88 70 00
Dir. 45 +45 35887356
Fax 45 35 88 70 01

acsc@food.dtu.dk
www.food.dtu.dk

a few simple laboratory tests (see appendix 3) to determine the pH and concentration of generated
free available chlorine (FAC) in the EOW produced using the Ecolox-pro system. Below, we have
listed the main findings and conclusion from these studies as well as our recommendation:
Main findings
In general, EOW produced from electrolyzed brine solution, applied either as liquid or fog, has
been shown in literature to provide an inactivating effect of NoVs (which depending on
circumstances and contact times can reduce NoV and its model viruses (such as murine norovirus
(MNV) with >3 log10 infective viruses). EOW is thus likely to be effective in disinfecting surfaces in
settings to reduce NoV exposures.
The inactivating effect of EOW is more efficient for NoV in suspensions compared to NoV on
contaminated surfaces according to literature. As examples, >3 log10 reduction can be achieved
using EOW as a liquid (38 ppm FAC and pH 5-6) within 30 sec in a suspension or within 10 min
on a surface. When using EOW (~180 ppm FAC) as a fog (where the fogging process has
reduced FAC to >50 ppm), the same reduction of NoV on a surface can be reached within 60 min
contact time.
According to the literature the inactivating effect of EOW on NoV relates to several factors. Among
these are mainly, but not exclusively, the pH and concentration of FAC produced along with the
generation of HOCl as well as the contact time between the virus and disinfectant and the amount
of accompanied organic material present in or at the contaminated matrix.
Laboratory tests conducted at NFI to check pH and FAC of the EOW generated using the Ecoloxpro system by clicking once (as instructed in the manual) or twice at the power source
transformer, showed the following values which could be maintained for at least 4 hours after
production:
o One click: ~100 ppm FAC and pH 7 was measured in the EOW inside the bottle (similar to the
80 ppm HOCl as informed by WEC) while ~200 ppm FAC and pH ~5.5 was measured in the
EOW after being dispensed as a spray.
o Two clicks: ~200 ppm FAC and pH 7 was measured in the EOW inside the bottle while ~400
ppm FAC and pH ~5.5 was measured in the EOW after being dispensed as a spray.
Conclusion
NFI concludes that the pH and FAC concentration of the Ecolox-pro are at such levels to support
that the EOW generated by Ecolox-pro will reduce NoV infectivity on contaminated surfaces.
Supporting data is however needed, to estimate the extent of inactivation at a given contact time
and the more precise efficacy using Ecolox-pro to disinfect NoV on contaminated environmental
surfaces.
Such data should be generated through laboratory tests applying the Ecolox-pro system in
different defined standard test applications using NoV and the close surrogate virus, MNV, in the
presence of organic material.
Recommendation
We recommend the Ecolox-pro to be tested for its potential to inactivate NoV as liquid and spray
at different defined applications (suspension and surface tests) and contact times using quality

controlled standard procedures including artificial contamination of NoV GI and GII and MNV (as a
NoV surrogate) accompanied by organic material on non-porous material like stainless steel.
The proposed data will not only allow an estimation of the efficacy and required contact time to
inactivate NoV on contaminated surfaces, but also allow estimation on whether Ecolox-pro may
have the potential to act as a virucidal disinfectant, which requires >4 log inactivation of NoVs.

The NFI would be very happy to further discuss the potential efficacy of Ecolox-pro to act as a NoV
disinfectant with WEC.

Best regards,

Anna Charlotte Schultz

Senior Scientist
Division of Food Microbiology

APPENDIX 1
Table 1. Summary of selected studies providing data on inactivation of norovirus and model viruses using the most common chlorine disinfectants.
Process

Free chlorine concentration

Organics

Matrix

Virusa inactivationb (log10)

Reference

Surface

HAV <2 (v)

(Bidawid et al., 2000)

Stainless steal

HAV <1

(Mbithi et al., 1990)

Suspension

HAV >3, <2 (v)

(Abad et al., 1994;Sobsey,

and contact time

ppm (min)
Rinsing with (lots of) water

chloramine-T

3000, 5000 (1)

Chlorination

0.5 (1)

Stool

1989)
Sodium chlorite

30000 (10)

Surface

HAV >3 (v)

(Abad et al., 1997)

Sodium hypochlorite (NaClO)

1250 (10)

Surface

HAV <3 (v)

(D'Souza and Su,

2010;Kawana et al., 1997)

5000 (0.5, 1)

Formica

MNV >2.52, 2.73 (v)

(Mbithi et al., 1990)

2600 (0.5, 1, 3)

10% FBS

Suspension

MNV >4 (v)

(Belliot et al., 2008)

3000, 5000 (1)

Stool

Stainless steel

HAV <1, >4 (v, variable)

(Mbithi et al., 1990)

500, 2500, 5000 (0.5) pH 8.6-10

10% humic

Stainless steel

MNV = 0.1, 1.3, 2.0 (v), 0.2. 1.9, 2.8 (g)

(Julian et al., 2014)

acid (80g/l)

PVC plastic

MNV = 0.2, 1.9, 2.8 (v), 0.5. 1.2, 2.4 (g)

10% stool

Stainless steel

MNV = 0.9, 3 (v)

(Park and Sobsey, 2011)

NoV = 1.48, 3.65, 4.14 (gc)

(Kingsley et al., 2014)

500 (10), 5000 (3.2)

Chlorine
Chlorine dioxide (ClO2)

33, 173, 189 (1)

240 (1)

10% stool
filtrate

Suspension

500, 2500, 5000 (0.5) pH 8.6-10

150, 500 (1) pH 7

NoV ~0.33 (g )
NoV = 0.8, 1.5, 2.8 (gc)

240 (10, 30, 60)

Hypochlorous acid (HOCl) solution

10% humic

Stainless steel

MNV = 0.7, 2.5, 3.0 (v), 0.4, 1.5, 3.4 (g)

acid (80g/l)

PVC plastic

MNV = 0.5, 3.7, 3.8 (v), 0.3. 2.0, 3.5 (g)

Plastic

FCV >5 (99.999%) (v)

(Chander et al., 2012)

Suspension

NoV >3 (>99.9%) (g)

(Park et al., 2007)

18.8, 38, 188 (0.33) pH 5.5-6.2

18.8 (10), 38 (10), 188 (1)

1% stool

Ceramic and stainless steel

NoV >3 (>99.9%) (g)

Hypochlorous acid (HOCl) fog

190 in liquid state,

filtrate

Ceramic

NoV >3 (>99.9%), Average >6 (g)

(10 min using a flow of 0.4L/min)

>50 in fogged state (60)

(Julian et al., 2014)

MNV >3.5 (v), >3 (>99.9%), Average >5 (g)

Viruses used to assemble this table are the human pathogens, hepatitis A virus (HAV) and noroviruses (NoV) and the animal models, feline calicivirus (FCV) and
b
murine norovirus (MNV). The level of inactivation is provided according to the method applied for counting remaining viruses after treatment demonstrated in either
viability (v, for infectious viruses) counted by 50% tissue culture infectious dose or plaque assays or in genomic titre (g) measured by reverse transcriptasepolymerase
c
chain reaction RT-PCR which may be combined with virus binding to porcine gastric mucin binding magnetic bead (PGM-MB), prior isolation of genome. Bold:
Conditions in contact times and free available chlorine of specific processes which have resulted in >4 log reduction.

APPENDIX 2
Summary of literature
8
NoVs are shed in amounts up to 10 particles per gram primarily in an organic matrix such as feces or
vomit from infected people (Atmar et al., 2008). NoV is resistant to most commonly used surface
disinfectants. Among several disinfectants that have been evaluated for efficacy to control NoV on
surfaces, chlorine containing products have shown most efficient (Kingsley et al., 2014) and free
chlorine as hypochlorite (sodium hypochlorite, NaClO, or bleach) is recommended to decontaminate
contaminated surfaces to control NoV (Park and Sobsey, 2011). EOW are chlorine-based
disinfectants that can be generated using battery power to electrolyze brine (NaCl) solutions resulting
in the formation of HOCl (Martinez-Huitle and Brillas, 2008). For the decontamination of NoVs, we
found no studies describing the effect of EOW produced from electrolyzed brine solution used as a
spray, but we found several studies applying EOW as a liquid and one study using EOW as a fog (see
table 1, Appendix 1). In general, The inactivating effect of EOW on NoV relates mainly but not
exclusively to the pH and concentration of FAC produced along with the generation of HOCl as well as
the contact time between the virus and disinfectant and the amount of accompanied organic material
present on the contaminated surface (Julian et al., 2014).

REG-no. DK 30 06 09 46

The efficacy of EOW to inactivate the uncultivable NoVs and cultivable model surrogate viruses in
aqueous suspensions and on environmental surfaces has been tested by measuring reduction in
infectivity and/or nucleic acids applying culture-dependent and independent assays in a number of
different studies. The data obtained from a selection of these studies including chlorine based
disinfections are summarized in table 1. Interpretations and comparisons of these data are very
difficult, because of the lack of standardization in initial viral load, virus exposure, simultaneous
presence of organic material, assay methods and the lack of comparative studies simultaneously
including e.g. NoV and specific candidate surrogate viruses. Virucidal efficacy is defined as >4 log10
reduction in the number of viable viruses (an inactivation of 99.99%), thus this reduction in virus titre is
necessary to demonstrate virucidal activity (Anon, 2011). However, this level is seldom accomplished
for NoV and its model surrogate viruses by commonly used cleaning and disinfection methods (Barker
et al., 2004;Sattar et al., 1994) including most of the disinfectants listed in table 1.
A particular study by (Park et al., 2007) are very relevant for the evaluation of the Ecolox-pro system,
because it describes the inactivating effect of EOW used in suspension and as a fog to inactivate NoV
and its best regarded surrogate, MNV (Lopman et al., 2012), in liquid suspension and on surfaces. In
this study, a reduction of >3 log10 in NoV genome titre could be achieved using EOW in suspension
tests at concentrations of 18.8 -188 ppm AFC and 20 sec contact time, while the same reduction on
contaminated surfaces of stainless steel required 10 min contact with 18.8 - 38 ppm AFC or 1 min
contact with 188 ppm AFC. When using EOW as a fog to decontaminate NoV and MNV on ceramic
tiles distributed within a confined space, reductions of >3 log10 in genome titers of both viruses and
>3.5 log10 in variable MNV could be obtained using 60 min contact time and effective FAC
concentration of >50 ppm (Park et al., 2007).

Technical University of Denmark

National Food Institute

Mrkhj Bygade 19
2860 Sborg
Denmark

Tel 45 35 88 70 00
Dir. 45 +45 35887356
Fax 45 35 88 70 01

acsc@food.dtu.dk
www.food.dtu.dk

These results suggest that HOCl produced from electrolyzed brine solution as a liquid or fog is likely to
be effective in disinfecting surfaces in common settings to reduce NoV exposures and thereby control
virus spread via fomites.

APPENDIX 3
Laboratory tests of the Ecolox-pro system
During preliminary tests carried out at the NFI, concentration of ~100 ppm FAC (pH 7), similar to the
informed ~80 ppm HOCl as informed WEC, was detected when measuring the EOW inside the bottle.
However, up to 200 ppm AFC (pH ~5.5) was found in the EOW when dispensed as a spray and
produced following manufactures instructions which recommend one click at the power bottom. When
clicking twice at the power bottom, the FAC measured in the liquid inside the bottle was ~200 ppm (pH
7) which increased to ~400 ppm (pH ~5.5) after being dispensed as spray. These values maintained
at least up to 4 hours after the production of the EOW.
Likely potential of Ecolox-pro to inactivate NoV on contaminated surfaces.
Although there are substantial differences in size and power between equipments producing EOW
such as the above selected literature study (Park et al., 2007) and the handheld product (Ecolox-pro),
similar FAC concentrations are generated in the two specific EOW products. According to our results it
is even possible to increase the concentration of FAC generated by Ecolox to higher levels. It is
therefore reasonable to believe that the Ecolox-pro may possess a similar reducing effect on NoV and
the surrogate MNV (>3 log10) as the above mentioned example and thus be suitable for use to
decontaminate NoV contaminated environmental surfaces. It should however be underlined that the
effect on NoV inactivation using EOW as spray compared to fog has not been described. Given the
relatively small volume (400 ml) of the EOW produced using the Ecolox-pro and the intension to apply
it in a simple manner such as a limited amount of spray on a defined contaminated area, this may be a
limiting factor in the competition with larger equipments producing and fogging EOW to disinfect larger
areas of contaminated surfaces. However, there are situations where other factors such as the
possibility for acute, easy and on-site access to rapidly generate and apply EOW could have
advantages which favour small handheld systems such as the Ecolox-pro.

REG-no. DK 30 06 09 46

To allow for better and more precise estimation of the effect of Ecolox-pro to inactivate NoV on
contaminated environmental surfaces, data obtained from the specific system are needed. Such data
should cover the applications and contact times required to reduce NoV with 3 and if possible 4 log10
of viable viruses (as required to demonstrate virucidal efficacy) (Anon, 2011). We therefore
recommend WEC to have such data generated by laboratory tests following quality controlled
standard procedures including artificial contamination of NoV and MNV (as a NoV surrogate)
accompanied by organic material on non-porous material like stainless steel (Rabenau et al.,
2014;Lopman et al., 2012).

Technical University of Denmark

National Food Institute

Mrkhj Bygade 19
2860 Sborg
Denmark

Tel 45 35 88 70 00
Dir. 45 +45 35887356
Fax 45 35 88 70 01

acsc@food.dtu.dk
www.food.dtu.dk

APPENDIX 4

Reference List

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fomites. Appl. Environ. Microbiol. 60:3704-3710.
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FEMS Microbiol. Lett. 156:107-111.
3. Anon, 2011. prEN 14476:2011: Chemical disinfectants and antiseptics. Virucidal quantitative
suspension test for chemical disinfectants and antiseptics used in human medicine.Test method
and requirements (phase 2/ step 1). Brussels. Comit Europen de Normalisation; 2011.
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Graham, D. Y., 2008. Norwalk virus shedding after experimental human infection. Emerg. Infect.
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5. Barker, J., Vipond, I. B., and Bloomfield, S. F., 2004. Effects of cleaning and disinfection in
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10. Julian, T. R., Trumble, J. M., and Schwab, K. J., 2014. Evaluating Efficacy of Field-Generated
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Technical University of Denmark

National Food Institute

Mrkhj Bygade 19
2860 Sborg
Denmark

Tel 45 35 88 70 00
Dir. 45 +45 35887356
Fax 45 35 88 70 01

acsc@food.dtu.dk
www.food.dtu.dk

13. Lopman, B., Gastanaduy, P., Park, G. W., Hall, A. J., Parashar, U. D., and Vinje, J., 2012.
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