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Ozonized water as an alternative to alcohol-based hand disinfection
Hans Johan Breidablik, Dag Einar Lysebo, Lene Johannessen, Åse Skare, John
Roger Andersen, Ole T. Kleiven
PII: S0195-6701(19)30056-8
DOI: https://doi.org/10.1016/j.jhin.2019.01.026
Reference: YJHIN 5657
To appear in: Journal of Hospital Infection
Received Date: 4 December 2018
Accepted Date: 29 January 2019
Please cite this article as: Breidablik HJ, Lysebo DE, Johannessen L, Skare Å, Andersen JR, Kleiven
OT, Ozonized water as an alternative to alcohol-based hand disinfection, Journal of Hospital Infection,
https://doi.org/10.1016/j.jhin.2019.01.026.
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Title
Ozonized water as an alternative to alcohol-based hand disinfection
Authors
Hans Johan Breidablik
Center of Health Research, Førde Hospital Trust, Førde, Norway
Dag Einar Lysebo
Haukeland University Hospital, Bergen, Norway
Lene Johannessen
Thelma Indoor Air & Working Environment AS, Microbiology Department, Trondheim,
Norway
Åse Skare
Førde Hospital Trust, Department for Infection Control, Førde, Norway
John Roger Andersen
Faculty of Western Norway University of Applied Sciences, Førde, Norway
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Ole T. Kleiven
Faculty of Western Norway University of Applied Sciences, Førde, Norway
Corresponding author:
Hans Johan Breidablik
hans.johan.breidablik@helse-forde.no
Center of Health Research, Førde Hospital Trust, Svanehaugvegen 2, 6812 Førde, Norway
Tel. +47 90182853
Fax. +47 57839795
Running title:
Ozonized water in hand disinfection
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Summary
Background: Hand hygiene plays a vital role in the prevention of transmission of
microorganisms. Ozone (O
3
) is a highly reactive gas with a broad spectrum of antimicrobial
effects on bacteria, viruses and protozoa. It can easily be produced locally in small
generators, and dissolved in tap water, and quickly transmits into ordinary O
2
in the
surrounding air.
Aim: To compare ozonized tap water and alcohol rub in decontamination of bacterially-
contaminated hands.
Methods: A cross-over study among 30 nursing students. Hands were artificially
contaminated with Escherichia coli (ATCC 25922) then sanitized with ozonized tap water
(0.8 or 4 ppm) or 3 mL standard alcohol-based rub (Antibac 85%). The transient microbes
from fingers were cultivated and colony-forming units (CFU)/mL were counted. The test
procedure was modified from European Standard EN 1500:2013.
Findings: All contaminated hands before disinfection showed a CFU-count >30,000/mL.
The average bacterial counts in (CFU/mL) on both hands combined were 1017 (SD, 1391)
after using ozonized water, and 2337 (SD, 4664) after alcohol hand disinfection. The median
values were 500 (range, 6700) and 250 (range, 16000) respectively, a non-significant
difference (P = 0.713). Twenty percent of participants reported adverse skin effects
(burning/dryness) from alcohol disinfection compared with no adverse sensations with ozone.
Conclusion: Ozonized tap water is an effective decontaminant of E. coli, and could be an
alternative to traditional alcohol fluid hand disinfectants both in healthcare institutions and
public places. Ozonized water may be particularly valuable for individuals with skin
problems.
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Keywords: Ozone, alcohol rub, hand disinfection, E. coli
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Introduction
Our interest in ozone as an alternative hand disinfectant started several years ago when two
eye surgeons in our hospitals developed hand dermatitis and had to stop performing operative
procedures. Both got permission to use ozone gas in tap water in their preoperative hand
hygiene, and this solved their skin problems. In the same period, there was an outbreak of
Giardia lamblia infection in the city of Bergen, Norway, originating from the domestic water
supply, and many kindergartens were advised to start using ozonized tap water in hand
hygiene for children, as it was known that ozone could eradicate protozoans [1]. Some
kindergartens continued using ozone as hand disinfection for several years after, and the staff
reported a clear impression that both the children and staff had fewer infectious diseases
during this period (Christine Andreassen, personal communication). We are also experiencing
a global threat from multidrug-resistant bacteria, and the need to reduce the use of broad-
spectrum antibiotics [2]. Hand disinfection has thus become even more important as a
preventive procedure.
Ozone gas (O
3
) was probably first detected by a Dutch chemist (van Marum), but the
first systematic studies were done by Christian Friedrich Schönbein in around 1840 [3]. He
noted the characteristic smell around an electrifier and named the gas based on the Greek
word “ozein” (scent) [4]. Ozone is produced from electric generators when an electrical
discharge (a spark) splits an oxygen molecule into two oxygen atoms, and then the unstable
ozone molecule is formed according to the reaction O + O
2
→ O
3
. According to the balanced
equation 2O
3
⇋ 3O
2
, where ozone quickly decomposes into O
2
(t½ = 20–30 min), requires
that it must be produced in the location where it is used. When it decomposes, O
3
acts as an
oxidant with release of free radicals. Being an oxidant, ozone has antimicrobial properties
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against bacteria, viruses and protozoa [5], and it was first used for general disinfection of
water. Later, ozone was used in food hygiene, fish farming, air purification, hot tubes, and in
some areas of medicine, especially dentistry [6].
Ozone leads to the destruction of both bacteria and viruses [7, 8], by interfering with
metabolism, most likely by inhibiting enzymes. Some ozone breaks through the bacterial cell
membrane, and this leads to cell death. Ozone destroys viruses by diffusing through the
protein coat into the nucleic acid core, resulting in damage to the viral RNA. At higher
concentrations, ozone destroys the capsid by oxidation [9].
Ozone is toxic to humans at higher concentrations, especially to the lungs. The
Norwegian Labour Inspection Authority accepts an 8-h working day average exposure of 0.1
ppm, and this can be exceeded by 200% for periods of 15 min [10]. This limits the use of
ozone gas as a disinfection agent in surroundings with human activity. However, the gas can
be dissolved in tap water for hand washing, and most of the gas then passes in the water
through the outlet of the sink.
Hand hygiene is one of the most important tools available for reducing the spread of
transient microbial pathogens in healthcare and community settings. In the era of multidrug-
resistant bacteria and frequent outbreaks of viral gastroenteritis, hand disinfection has become
an even more essential tool in reducing the spread of microorganisms both in health
institutions and in general society [11]. The main disinfection methods used are handwashing
with soap and alcohol-based solutions [12]. However, both of these are associated with
adverse skin reactions like dryness and dermatitis among sensitive individuals, and
compliance with hygiene recommendations is also variable among health professionals [13,
14]. Among nurses, 25–55% report adverse skin reactions related to hand hygiene procedures
[15]. Meanwhile, the effect of alcohol disinfection on viruses like norovirus and rotavirus is
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unclear, and it has little effect on spores of the bacterium Clostridium difficile [14]. Ozone
gas in higher concentration has also shown to be an effective alternative for sanitizing rooms
[16, 17].
There have been few studies on the potential of using ozonized water as a simple,
cheap and skin-friendly alternative to standard hand disinfection with alcohol. Appelgrein et
al. found ozone to be inferior in effect to propanolol-based hand rubs [18]. Isosu et al. found
ozonized water combined with benzalkonium chloride and alcohol to be an effective
alternative to traditional surgical washing procedures [19]. In a unpublished study where ATP
was used as an indication of the efficacy of hand disinfection, Liceaga et al. found that ozone
combined with soap removed 97.3% of ATP from hands, and noted the lack of studies
regarding ozone and hand sanitation [20]. In a recent study, Nakamura et al. found
that
a 3
log10 cfu reduction was achieved by washing hands with ozonated water or antimicrobial
soap and water. However, ozonated water was not significantly superior to non-antimicrobial
soap and water [21].
The aim of this study was to compare the effect on Escherichia coli-contaminated
hands of ozonized water with standard alcohol-based hand disinfection using a modified
European Standard procedure (EN 1500:2013) [22].
Materials and methods
The test procedure was modified from European Standard EN 1500:2013, which specifies a
method for simulating practical conditions for establishing whether a product for hygienic
hand sanitation reduces the release of transient microbes after use on the artificially-
contaminated hands of volunteers. For testing one product at a time, a crossover design was
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used. The 30 test persons were all nursing students (26 women/4 men) at the end of their
second year of study (average age 23 years). None of the students had visible signs of
dermatitis on their hands during the study days. On day one, the 30 students were divided
randomly into two equal groups of 15, one using ozonized water 0.8 ppm and the other 4.0
ppm. Six days later the same students used the reference disinfectant, ethanol supplemented
with propan-2-ol (Antibac, 85% with glycerol, www.antibac.no). The test organism was non-
pathogenic E. coli (ATCC® 25922), as recommended in the standard procedure. This
bacterial strain is often used as a reference strain in microbial research.
E. coli was cultured for 18–24 h at 36 °C on Tryptic Soy Agar plates (Thermo Fisher
Scientific, USA). A single colony was then inoculated into 10 mL Tryptic Soy Broth (TSB),
and cultivated for 18–24 h. The 10-mL bacterial solution was then used to inoculate 1 L TSB
before further cultivation for 18–24 h. The final concentration of cultured E. coli was
estimated as >10
9
colony-forming units (CFU)/mL. The 1 L solution was divided into two
500-mL glass containers.
The test subjects prepared their hands by washing for 1 min with soft soap and
lukewarm tap water to remove natural transients. The hands were then dried with paper
towels, and immersed up to the mid-metacarpals for 5 s, one hand in each of the glass vessels
containing bacterial solution. Hands were air-dried in a horizontal position with the fingers
spread and rotating for 3 min. Immediately after drying, the fingertips were rubbed on the
base of a Petri dish containing 10 mL of TSB using separate Petri dishes for each hand.
Without further delay, the test subject then performed the hygienic hand rub procedure, using
ozonized water or the reference hand rub (3 mL of 85% Antibac) according to information
provided by the manufacturer. The hygienic hand rubbing time for both ozone water and
Antibac was 30 s. After cleaning, the fingertips were dried and rubbed on the base of new
Petri dishes containing 10 mL of TSB. Immediately after performing the tests, 1 mL of pre-
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or postvalue sampling fluid was transferred into Eppendorf tubes (Thermo Fisher Scientific),
and brought to the laboratory in a cooling bag. Within 24 h, 10 µL of the pre-or postvalue
samples were spread on two parallel MacConkey agar plates (Mac3, Thermo Fisher
Scientific) and incubated for 18–24 h at 36 ± 1 °C before counting the number of CFU. The
pre-test value was the number of CFU sampled from the left or right hand prior to hand
sanitization treatment, and the post-test value was the number of CFU after treatment.
Calculation of CFU/mL was performed by multiplying the arithmetic mean of the plate CFU
counts for each hand by the dilution factor of 100. For both reference and test procedures, the
counts from the right or left hand of each subject were averaged separately to obtain pre-
values and post-values. CFU counts >300 per 10 µL were not counted and were noted as
>30,000 CFU/mL.
Ozonized water was produced by two separate generators, a Water Ozonator
CYS300C from Cleanzone delivering 0.8 ppm in tap water, and a BioSure CSS from Ozone
Scandinavia delivering 4.0 ppm in tap water. For measuring the ozone gas concentration in
surrounding air, three different sensors were used (EcoZone Monitor, EZ-1X, Eco Sensors,
Inc. type A-21ZX and Murco Portable Ozone Detectorand), and we also measured the level
of ozone in the tap water. The tap water was regular domestic water of approximately 20°C
from the local water reservoir in Førde, Norway. Water here is filtered through a layer of
marble sand, and then disinfected with chlorine and UV light. It has a pH of 7.8–8.3 [23]. The
tap water-flow were different. For the ozone 0.8 ppm generator the flow was about 8 L/min,
while the 4.0 ppm the BioSure CSS delivered only 2 L/min.
Ethics
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The students were informed in a lesson about the study and invited to participate. The
resulting participants gave their written consent. The study was approved by the Regional
Committee for Medical Research Ethics and the Norwegian Data Inspectorate (reference
number: 2017/943).
Statistical analysis
Categorical data is presented with numbers and percentages. The raw data (CFU/mL) after
using ozonized water and alcohol disinfection are displayed within figures. CFU/mL values
from both hands combined after using ozone water and alcohol disinfection are presented as
the average values along with standard deviations (SD), and also as the medians with ranges.
CFU/mL scores from each hand are presented as average values. The Wilcoxon signed-rank
test was used to test for differences in CFU/mL values after using ozone water or alcohol
disinfection. The Mann-Whitney U-test was used to test if CFU/mL was different using
ozone water with 4,0 ppm versus 0,8 ppm. A P-value < 0.05 was considered significant. The
analysis was conducted using SPSS software version 24, and figures were prepared using
GraphPad Prism version 8.
Results
All 30 participants completed the study. Eight of the students (27%) reported varying degrees
of previous skin problems connected to handwashing and disinfection.
In connection to the experiment, 20% of participants reported some adverse skin
effects (burning/dryness) from using the alcohol disinfection method, but continued the
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procedure as planned. Their post-CFU counts showed no special pattern. None reported
adverse sensations using ozonized water. Half of the participants reported that they felt their
hands became smoother/softer after ozone use, and after the experiment the majority (77%)
said they would prefer ozonized water disinfection if the two methods were equal in
antimicrobial effect.
Table I summarizes the bacteriological results. From all 60 hands (30 participants,
each with left and right hand) the pre-tests gave a CFU count >30 000 /mL. The average post-
test value for both hands combined in CFU/mL after using ozonized water was 1017 (SD,
1391), and for regular alcohol hand disinfection was 2337 (SD, 4664). The median post-test
value (in CFU/mL) after using ozonized water was 500 (range, 6700), and for regular alcohol
hand disinfection was 250 (range, 16 000), P=0.713. Where disinfection used the higher
ozone concentration (4.0 ppm), values were not statistically different in CFU/mL compared
to those for the lower concentration (0.8 ppm) (P=0.142).
More hands (n = 26) showed zero post-value CFU-counts from fingers using alcohol
versus ozone (n = 10) (Figure 1). There was greater individual variation in the CFU-counts
for alcohol (0–12,500) compared with ozone (0–4800), especially for the left hand. Skin
symptoms from alcohol disinfection earlier or during the study was not correlated with higher
CFU-counts in post-tests.
The measured level of ozone gas in the air around the face-level of the students never
exceeded 0.01 ppm (one-tenth of the exposure limit of 0.1 ppm for 8 h). However, when
asked afterwards, 77% of the students reported they had noticed the characteristic smell of
ozone gas, which also represent an extra security factor in connection with ozone use.
Discussion
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This study indicated that ozonized water on average eradicates E. coli from artificially-
contaminated fingers as effectively as 85% alcohol. The variation in results between
individuals, and between the two hands of the same individual, was higher for alcohol
disinfection than disinfection using ozonized water. The concentration of ozone gas in the
surrounding air was low, and there were no adverse skin reactions with the ozone method.
The ozone method was preferred by most of the nursing students. The ozone hand
disinfection method is simple, cheap and leaves no residual waste, and when dissolved in
water ozone also seems to be safe.
Our conclusion is that ozonized water could be an alternative to traditional fluid hand
disinfectants with alcohol, for example in institutions. Appelgrein et al. also found an effect
of ozone, but this was inferior to alcohol [19]. In their study with 4.0 ppm ozone, they used a
lower concentration of alcohol (60%), but the amount was double (3+3 ml) and the exposure
time was longer (3 min.), and also on one of the hands they used a delayed 3 hour method. It
is therefore not comparable in all the details to our study.
The seemingly better effect of 0.8 ppm ozone compared to 4.0 ppm was surprising,
but both the different technical design of the generators and especially the different water
flow could be possible explanations for this observation. Under ideal circumstances we
should have used the same generator producing different concentrations, but CYS 300C can
only deliver 0.8 ppm at maximum output.
Other studies have shown that the presence of organic carbon in water can severely reduce
ozone effectiveness when used as a method to eradicate the bacterial load [24].
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In the present study, we found differences in effect between the left hand and right
hand in CFU-counts post-disinfection with alcohol, but less difference with ozone. Fierera et
al. found significant heterogeneity in bacterial community composition between left and right
hands from the same individual depending on handedness, gender and time since last hand
washing [25]. We do not have data on the handedness of our participants. However, the
spreading of a sparse amount of alcohol disinfectant (3 ml) to all parts of both hands can be
more difficult, even under ideal circumstances, than the simpler procedure of just holding and
rubbing both hands under a greater volume of running tap water (8 L/min) containing ozone.
The inferior results for ozone 4.0 ppm when delivered in only 2 L/min of water could also
point in this direction.
Adverse skin reactions connected to hand hygiene are prevalent among health professionals
[12]. Among the participants in this study, 20% of the participant reported adverse skin
effects (burning/dryness) from using the alcohol disinfection method whereas none reported
these problems when using ozonized water. We find this to be an interesting observation. The
standard method for comparing hand disinfectants (EN 1500:2013) has been criticized for not
being in line with clinical practice, and for not testing microorganisms other than E. coli [26].
To be more in line with the present advised practice in healthcare institutions, we modified
the Standard by using only 3 mL/30 s for alcohol hand disinfection, and used a higher
concentration of alcohol (Antibac 85%) than the 60% in the Standard.
This study was performed in optimal circumstances with well-informed and
professional healthcare students knowing the background and correct technique for hand
disinfection. The hand disinfection with alcohol was administrated and observed by a nurse
specializing in hygiene. The effect of hand wash/hand disinfection on contaminated hands
can therefore be expected to be poorer in general public health circumstances, and especially
among children. The broad-spectrum antimicrobial effect of ozone, including naked viruses
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and protozoa, could represent an important potential in public health efforts aimed at
communicable diseases. We have not found any publication regarding the potential for
developing bacterial resistance to ozone. This study confirms that ozonized water possesses
antimicrobial properties in line with alcohol, and that it could have a place in effective hand
hygiene protocols, especially targeted at individuals with skin problems. For some, this
becomes a major problem resulting in sick leave and, eventually, working disability. This was
the situation for one of the authors of this study (DEL) before he obtained permission to use
ozone in his preoperative hand disinfection procedure.
There are few studies about the effect of ozone as an antimicrobial agent in human
medicine. We need further studies comparing regular hand washing with soap vs. ozonized
water, and on the use of ozonized water as primary prevention of prevalent infections in
schools and kindergartens. Research should also focus on the possibility of using ozone as an
alternative to soap washing and alcohol disinfection for persons with contact dermatitis.
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Acknowledgment
We thank the nurse students for their participation and assistance in performing this study,
and two bioengineers at the Microbiological Department of Førde Hospital, Torill Aarø and
Torunn Ytreland, for their excellent assistance.
The study had a small internal financial grant from Helse Førde Medical Trust, and no
external grants.
Potential conflicts of interest: One of the authors (DEL) has a small ownership in a firm that
manufactures ozone generators. For the other authors: Nothing to declare.
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Table I. Data for Escherichia coli cultivation CFU/mL (colony-forming units/mL). Samples
were taken from left and right hands treated with a standard solution of
non-pathogenic E.
coli (ATCC® 25922), then disinfected with ozonized tap water (ozone) or alcohol rub
(alcohol; Antibac, 85% v/v). All pre-tests had CFU > 30 000/mL.
Hands Ozone
(0.8 and 4.0
ppm combined)
Ozone
(0.8 ppm) Ozone
(4.0 ppm) Alcohol
(85%)
Both hands,
mean (SD) 1017 (1391) 600 (643) 1433 (1795) 2337 (4664)
Both hands,
median (range) 500 (6700) 300 (2500) 700 (6600) 250 (16000)
Right hand,
mean (SD) 550 (942) 240 (232) 860 (1256) 797 (2147)
Right hand,
median (range) 300 (4800) 200 (800) 400 (4800) 100 (10500)
Left hand, mean
(SD) 467 (574) 360 (526) 573 (617) 1540 (3294)
Left hand,
median (range) 250 (2000) 200 (2000) 300 (1800) 100 (12500)
N = 30 in all analyses. SD: Standard deviation.
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Figure Legends
Figure 1a: Individual distribution of CFU-counts/mL after disinfection of hands with 3 mL of
85% alcohol for 30 s, and fig. 1b: Individual distribution of CFU-counts/mL after disinfection
of hands with ozonized water for 30 s.
Illustration 1. Equipment (generators) used for ozonizion of tap water: BioSure CSS and
CYS300C
Model
BioSure CSS
Ozone concentration
in water
2
–
8 ppm
Voltage
100V
–
240V,
50/60Hz
Energy Usage
60 watts
Size (in.)
17.1 x 12.9 x 6.9
Weight
7.5 kg
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