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Killing Effect of Ozone on House Dust Mites, the Major Indoor Allergen of Allergic Disease

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This study examined whether ozone could kill house dust mites (HDMs), one of the most common causes of allergic diseases, and if an ozone application might be helpful in the environmental control of allergic patients. The experiments were performed in a small chamber (50 cm3), in which the continuous contact between the gaseous ozone and 40–60 live HDMs could be maintained during the reaction time (temperature=25°C and relative humidity=75%). Within the ozone range of 0.19–10.62% (v/v), the higher concentration dose resulted in a more rapid inactivation of the live HDMs. The CT value of ozone showed a linear relationship with the inactivation efficiency (%) of the live HDMs. From our results, it was found that a CT value 400 mg-min/L was required to obtain an almost 100% removal efficiency of the 40–60 live HDMs.
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Killing Effect of Ozone on House Dust Mites, the Major
Indoor Allergen of Allergic Disease
Jae-Hyuk Han a; Byung Soo Oh b; Sung-Yon Choi a; Byoung-Chul Kwon a; Myung
Hyun Sohn a; Kyu-Earn Kim a; Joon-Wun Kang b
aDepartment of Pediatrics, Yonsei University College of Medicine, Youngdong
Severance Hospital. Seoul. Korea
bDepartment of Environmental Engineering, Yonsei University at Wonju. Wonju.
Korea
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Ozone: Science and Engineering, 28: 191–196
Copyright #2006 International Ozone Association
ISSN: 0191-9512 print / 1547–6545 online
DOI: 10.1080/01919510600689679
Killing Effect of Ozone on House Dust Mites, the Major
Indoor Allergen of Allergic Disease
Jae-Hyuk Han,
1
Byung Soo Oh,
2
Sung-Yon Choi,
1
Byoung-Chul Kwon,
1
Myung Hyun Sohn,
1
Kyu-Earn Kim,
1
and Joon-Wun Kang
2
1
Department of Pediatrics, Yonsei University College of Medicine, Youngdong Severance Hospital, Seoul, Korea
2
Department of Environmental Engineering, Yonsei University at Wonju, Wonju, Korea
This study examined whether ozone could kill house dust
mites (HDMs), one of the most common causes of allergic
diseases, and if an ozone application might be helpful in the
environmental control of allergic patients. The experiments
were performed in a small chamber (50 cm
3
), in which the
continuous contact between the gaseous ozone and 40–60 live
HDMs could be maintained during the reaction time (tempera-
ture = 25C and relative humidity = 75%). Within the ozone
range of 0.19–10.62% (v/v), the higher concentration dose
resulted in a more rapid inactivation of the live HDMs. The
CT value of ozone showed a linear relationship with the inactiva-
tion efficiency (%) of the live HDMs. From our results, it was
found that a CT value 400 mg-min/L was required to obtain an
almost 100% removal efficiency of the 40–60 live HDMs.
Keywords Ozone, House Dust Mites, Allergic Disease,
Inactivation
INTRODUCTION
House dust mites (HDMs) are the most frequent cause
of allergic diseases. The fact that house dust is a cause of
allergic diseases was first proposed by Kern in 1921, and
Voorhorst in Denmark proposed, in 1967, that the antigens
of HDMs living in houses are probably the cause of allergy
and reported the relationship between respiratory allergic
disease and HDMs (Voorhorst et al., 1967; Boner et al.,
1985; Arlian et al., 2001). HDMs are mostly present in
mattresses, carpets, cloth sofas, cloths, beddings and cloth
car seats. In Korea, the numbers of HDMs increase signifi-
cantly in August and are at their least in May; however, even
in May their numbers are sufficient to induce allergic
disease, so that the symptoms of allergic diseases are present
all year round (Hong and Lee, 1992). The symptoms of
respiratory sensitivity and respiratory allergic reaction are
induced by the major antigens of HDMs, Dermatophagoides
farinae (Der f) and Dermatophagoides pteronyssinus (Der p)
present mainly in HDM waste, and which bind to the immu-
noglobulin IgE present on mast cell surface to produce
chemical media, such as histamine.
The major allergic diseases include asthma, allergic
rhinitis and atopic dermatitis (Jeon et al., 1999). Basic treat-
ments of allergic diseases include removal of the causes,
such as HDM antigens, or maintenance of the surrounding
environment to reduce antigens: Maintenance of beddings
free of HDMs is the main method of HDMs avoidance
(Crank et al., 2001; Choi et al., 2002).
In order to reduce the number of HDMs, it is impor-
tant to maintain the temperature and relative humidity
below 20C and 45%, respectively, and bedding should to
be washed in hot water with the temperature higher than
55C more than once in 2 weeks, and then thoroughly
dried under sunlight (Arlian and Platts-Mills, 1993).
Bedding and sofas should be covered with an antigen-
impenetrable plastic cover to prevent the habitation of
HDMs, and the use of air purifiers and vacuum cleaners
also helps. Although Acarosan and Natamycin, pesticides
containing primiphos methyl and benzyl benzoate, have
been effective in reducing the number of HDMs in recent
years, it is still impossible to completely deal with HDMs
with the use of these pesticides (Cloosterman et al., 1999;
Vyszenski-Moher et al., 2000; Crank et al., 2001).
Ozone, a strong disinfectant and oxidant, has been
used in a variety of industries for the purpose of deco-
loration, deodorizaton and for producing structural
changes in organic compounds (Langlais et al., 1991).
Recently, the use of ozone has been extended to the
sterilization of drinking water and air, the deodorization
Received 05/04/2005; Accepted 11/02/2005
Address correspondence to Joon-Wun Kang, Department of
Environmental Engineering, Yonsei University at Wonju, 234
Maeji, Wonju, Korea 220-710. E-mail: jwkang@dragon.yonsei.
ac.kr
Effect of Ozone on House Dust Mites June 2006 191
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of a wide variety of facilities such as wastewater disposal
or for purifying various facilities, aquariums and bever-
age factories etc. Ozone, with concentrations ranging
from 0.3 to 0.9 mg/L have been reported to be able to
kill E. coli, Vibrio, Salmonella, Yersinia, Pseudomonas,
Staphylococcus and Listeria as well as various kinds of
virus (Sechi et al., 2001). However, no qualitative and
quantitative studies on the killing effect of ozone toward
HDMs have been conducted (Days et al., 1983). Thus,
this study investigated whether ozone would kill HDMs
in the atmospheric phase, and with the continuous expo-
sure of HDMs to gaseous ozone, the CT value of ozone
was measured to find the quantitative ozone dose that
would kill HDMs.
MATERIALS AND METHODS
Collection of HDMs
The target HDMs (Der f and Der p) were supported by
the Department of Parapsychology, College of Medicine.
HDMs living in beddings, sofas and carpets in ordinary
households were collected from dust, gathered by a vacuum
cleaner. The collected HDMs were separated into different
species, reared in an environment suitable for HDMs
growth, using a medium containing both fish food powder
and dried yeast at 1:1 ratio, and maintained at room tem-
perature (21–22C) and 75% relative humidity (Rei et al.,
1997). After an appropriate number of adult HDMs were
obtained, larvae and eggs were removed in culture media.
As an empirical result, 0.01 g culture media contained
approximately 40–60 live HDMs, but without gender
specificity.
Ozonation of HDMs
A chamber (Volume = 50 mL, Length = 4 cm, width =
2 cm and height = 2 cm) was used as an ozone reactor, into
which gaseous ozone could be diffused continuously. The
reactor was made from a glass case to facilitate external
observation. Ozone was generated from high purity oxygen
using an ozone generator (OZONIA, CFS, Switzerland).
The gaseous ozone was diffused into the chamber, contain-
ing the 40–60 live HDMs, at a flow rate of 50 mL/min fixed
using a flow meter. The steady state ozone concentrations
in the chamber were obtained after at least 3 minutes and
varied between 0.19–10.62% (v/v). The reactor was fixed on
the stand of a microscope (Reichert, Germany) with 200X
magnification, for counting of the numbers of live and
moving HDMs during ozonation. This set-up enabled the
numbers of the live and moving HDMs to be counted in
real time during the runs. The temperature and relatively
humidity inside the reactor were 25C and 75%, respec-
tively. To determine whether the HDMs were alive was
decided by observing their movement for a few seconds.
Even if only a little movement was seen, the HDMs were
regarded as living, as they constantly move when alive. The
inlet and outlet concentrations of the gaseous ozone were
measured using 2% potassium iodide solution in a KI trap
connected to one end of the reactor. Figure 1 shows the
schematic diagram of lab-scale ozone reactor.
For the first experiment, the HDMs in the reactor were
examined to see if they were killed during continuous expo-
sure to ozone maintained at a concentration of 0.48% (v/v)
and the time required recorded. Con-currently, control
HDMs were investigated in a closed state without gas
injection and with exposure to oxygen (99.999%) instead
of ozone. For the second experiment, to determine the
effectiveness of various ozone concentrations and exposure
times, the HDMs were exposed to 0.19, 0.29, 0.63 and
1.18% of constantly maintained ozone, and the number
of live HDMs recorded at variable exposure time of 0, 10,
20, 30, 40, 60, 80, 100 min. For the third experiment, no
ozone gas was introduced into the reactor after a single
injection of 0.10% ozone. The ozone concentration in the
reactor changed naturally with time, and the correlation
between the CT value (product of ozone concentration and
exposure time) and HDMs inactivation efficiency was
investigated. For the fourth experiment, since the ozone
gas remaining in the reactor after killing HDMs could be
harmful to people, the time required for the ozone concen-
tration to reduce to a level not harmful to people (0.10 ppm)
was also determined. Thus, after opening the reactor lid
under a hood, the ozone was naturally aerated and its
concentration was recorded with time.
RESULTS AND DISCUSSION
HDMs Inactivation with Ozone
In order to observe the variations in live HDMs due to
continuous exposure to gaseous ozone, the oxygen spar-
ing (99.999%) was used as a blank test. This is because
tested ozone gas concentrations were 0.19–1.18% (v/v) in
the main oxygen gas stream (99.2–98.2% (v/v)). We also
performed a blank test in a closed state. In this case, only
ambient air was initially contained in the reactor. In
FIGURE 1. Schematic diagram of lab-scale ozone reactor.
192 J.-H. Han et al. June 2006
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Figure 2, the inactivation efficiencies of HDMs under
each experimental condition were compared as a function
of reaction time. No reduction in the number of live
HDMs in the closed state was observed during the reac-
tion time. Sparging of oxygen to the reactor containing
live HDMs was also negligible in inactivating the live
HDMs. An increase in active HDMs was particularly
observed during oxygen exposure.
In the case of ozone sparging, the live HDMs were
reduced relatively rapidly with increasing time, with
no HDMs alive 45 min after the introduction of
0.48% (v/v) ozone (test #1). In test #2, only 3
HDMs were remaining. A similar inactivation rate
was observed for two identical trials (tests #1 and
#2 in Figure 2) of the ozone sparging experiments.
This result clearly indicates that continuous exposure
to gaseous ozone is significantly fatal to the live
HDMs. In addition, the dead mites were observed
one hour later to reaffirm that they were actually
dead. As a result, no mites initially regarded as
dead were reevaluated as live. It was also found that
the remaining 3 HDMs in test #2 were observed to be
dead. This phenomenon indicates that once HDMs
were exposed to ozone, they were damaged, and
then killed. In order to illuminate the reason for
this result, further detailed experiments are now in
progress.
Comparison of HDMs Inactivation Rate at Various
Ozone Doses
In Figure 3, the efficiency for inactivating live HDMs
was investigated with diverse ozone doses (0.19–1.18%)
in the reactor by continuous ozone injection. Under all
experimental conditions, the number of HDMs regarded
as live initially increased as some of the HDMs initially
regarded as dead as they had not moved earlier, started to
move on the introduction of ozone. When the ozone
concentration was maintained at 0.19%, the number of
live HDMs gradually decreased with increasing expo-
sure time, but never to fewer than 23. At a 0.29%
ozone dose, 2 HDMs were alive after 100 min of
exposure. No HDMs were moving after 60 and 30 min
with exposure to 0.63 and 1.18% ozone gas, respec-
tively. From the results, the inactivation rate of HDMs
was found to increase with increasing ozone concentra-
tion in the reactor. In this experiment, it was also
observed that the inactivation of HDMs was affected
by their size distribution (0.28 mm ·0.19 mm–3.4 mm
·0.23 mm). Particularly, at an ozone dose of 0.19%,
all the live HDMs remaining after an 80 min ozone
contact time were relatively bigger (above 3.0 mm ·
2.1 mm) than those that had died. This result provides
significantly important information of the bigger
HDMs having a higher resistance to gaseous ozone.
However, continuous exposure to the gaseous ozone
over 0.29% was able to inactivate even the HDMs
with a strong resistance.
As an objective comparison, Figure 4 shows the inacti-
vation efficiency (%) of the live HDMs as a function of the
ozone contact time. The calculation of the inactivation
efficiency (%) was based on the initial time when the
number of live HDMs was most prevalent (peak number
in Figure 3). At an ozone dose of 0.19%, the inactivation
efficiency was 11% after 40 min, 43% after 60 min, 67%
after 80 min and 68% after 100 min. At 0.29%, the inacti-
vation efficiency was 44% after 40 min, 59% after 60 min,
81% after 80 min and 96% after 100 min. At 0.63%, the
inactivation efficiency was 91% after 40 min and 100%
after 100 min. At 1.18%, the inactivation efficiency was
complete after 30 min of exposure. This result indicates
that the live HDMs can be dramatically inactivated within
a short reaction time by exposure to excess ozone.
Time (min)
0 20 40 60 80 100 120
Number of live HDMs
0
10
20
30
40
50
60
70
Blank test (closed state)
Blank test (oxygen)
Ozone (test #1)
Ozone (test #2)
FIGURE 2. Blank tests and the effect of ozone on inactivating
HDMs (Initial number of HDMs = 41–50; ozone dose = 0.48% (v/v);
oxygen = 99.99%).
Time (min)
0 20 40 60 80 100 120
Number of live HDMs
0
20
40
60
80
100
0.19 % (v/v)
0.29 % (v/v)
0.63 % (v/v)
1.18 % (v/v)
FIGURE 3. Inactivation of HDMs at variable ozone doses (Initial
number of HDMs = 40–68).
Effect of Ozone on House Dust Mites June 2006 193
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Relationship Between Inactivation of HDMs and
Ozone CT Value
In this experiment, the gaseous ozone injection was
stopped after the desired ozone concentration had been
reached (19 mg O
3
/L of O
2
), and the residual ozone in
the reactor then measured at various HDMs counting
points (0, 5, 10, 20, 40 min). Figure 5a shows the com-
parison of the residual ozone concentration and the
inactivation efficiencies (%) of HDMs as a function of
the reaction time. The residual ozone concentration gra-
dually decre-ased with increasing contact time. In Figure
5b, the percentage of HDMs inactivated was plotted as a
function of the ozone CT value, the product of the ozone
concentration (mg/L) and exposure time (min) (Federal
Register, 1989), which showed a linear relationship.
According to this result, a CT value of 400 mg-min/L
was required for an almost 100% removal efficiency for
about 60 live HDMs. The following empirical equation
was obtained:
y¼0:2677ðR2¼0:9422;p<0:05Þ
y : inactivity efficiency ð%Þ
x:Cðozone concentration;mgO3=LofO
2Þ
Tðexposure time;minÞ
From this formula, the exposure time needed to kill
a specified number of HDMs with certain ozone con-
centration could be predicted. For example, the CT
value needed to kill 35% of the HDMs would be 130,
indicating that about 10 min of exposure time would
be needed for an ozone concentration of 13 mg/L.
Obviously, a method of detection as to whether the
HDMs are really killed is highly desirable. The
mechanism involved in the killing HDMs due to
ozone remains unclear; however, ozone might damage
genes within the cell nucleus due to its strong oxidative
ability. However, further studies are needed to deter-
mine an accurate mechanism in order to reduce the
antigenicity of waste products secreted by HDMs.
Effect of various ambient factors such as temperatures
and relative humidities on HDM inaction are also
subjectedtobeexploredinfuturestudies.
Ventilation of Residual Gaseous Ozone
When HDMs are killed by spraying of ozone gas,
the residual ozone could worsen any respiratory symp-
toms (Phillips et al., 1999). According to the ozone
alarm system in Korea, a low level is indicated when
the ozone concentration in air per hour is higher than
0.12 ppm (v/v), a medium level when higher than
0.3 ppm, and a high level when higher than 0.5 ppm.
When a low level ozone alarm is given, people are
advised to avoid outdoor exercise, and respiratory
patients, the elderly and children are advised to restrain
Time (min)
0 20406080100120
Inactivation efficiency (%)
0
20
40
60
80
100
120
0.19 % (v/v)
0.29 % (v/v)
0.63 % (v/v)
1.18 % (v/v)
FIGURE 4. Percentage of HDMs inactivated with ozone at var-
ious ozone doses (Initial number of HDMs = 51–80).
0
2
4
6
8
10
12
14
16
18
20
0 1020304050
Time (min)
Ozone Conc. (mg/L)
0
20
40
60
80
100
120
Inactivation efficiency (%)
Residual ozone
Inactivation efficiency (%)
(a)
y= 0.2667x
R2= 0.9422
Ozone CT value
0 100 200 300 400 500
Inactivation efficiency (%)
0
20
40
60
80
100
120
(b)
FIGURE 5. Changes in ozone concentration and HDMs inactiva-
tion efficiency with time following ozone exposure (Initial number of
HDMs = 62).
194 J.-H. Han et al. June 2006
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from outdoor activity. Although the ozone concentrations
under 0.12 ppm might not affect respiratory disease, con-
centrations higher than 0.14 ppm slightly lower the lung
functions (Berrington and Pedler, 1998). Furthermore, the
symptoms of asthma can worsen if asthmatic patients are
exposed to ozone concentrations exceeding 0.16 ppm for
more than 6.7 hours, and the concentration regarded as
immediately dangerous to life or health (IDLH) is 5 ppm
(Krishna et al., 1995; Peden et al., 1997; Kehrl et al., 1999;
Gottschalk et al., 2000). Therefore, it is of great importance
to ventilate residual ozone following ozone introduction.
In this experiment, the residual ozone concentration
after HDMs inactivation with gaseous ozone was
assumed to be 16 mg/L (0.80% or 8000 ppm), the
trends of the ozone concentration remaining in the
reactor when keeping the reactor closed or open for
natural ventilation were investigated. As shown in
Figure 6, when the reactor was kept closed, the amount
of ozone consumed was measured at below 2 mg/L
(0.09% or 900 ppm) for 45 min. When aerated with
natural air, it took 45 min for the ozone concentration
to decrease from 16 to 0.8 mg/L (0.05% or 500 ppm).
This result indicates that the residual ozone concen-
tration (0.8 mg/L) in the reactor at the termination of
the experiments is still sufficient to damage signifi-
cantly humans. Therefore, a compulsory ventilation
(or destruction) system will be required for removing
ozone. The development of methods to process com-
pletely and safely and quickly aerate ozone could
greatly aid in managing the environment of allergic
patients.
CONCLUSIONS
This study investigated the effect of ozone on house dust
mites (HDMs) by their continuous exposure to ozone
gas, which confirmed HDMs could be inactivated by
ozone. At an ozone dose of 0.48% (v/v) (temperature =
25C and relative humidity = 75%), no live HDMs (initial
number of HDMs = 41–50) were found after 40 min of
ozone introduction. At diverse ozone doses (0.19–1.18%),
the inactivation rate of HDMs due to ozone exposure
increased with increasing ozone dose. This result indicates
that the live HDMs can be dramatically reduced within a
short reaction time due to exposure to excess ozone.
From the result comparing the residual ozone concentra-
tion to the inactivation efficiency (%) of HDMs as a func-
tion of the reaction time, a CT value of 400 mg-min/L was
required to obtain an almost 100% removal efficiency for
the 40–60 live HDMs (temperature = 25C and relative
humidity = 75%). With the formula obtained from CT
value and inactivation efficiency (%), the exposure time
needed to kill a specified number of HDMs at given ozone
concentrations can be predicted.
When the ozone remaining in the reactor was naturally
ventilated, 16 mg/L of residual ozone decreased to under
1 mg/L, but for a safer environment for humans, a com-
pulsory ventilation (or destruction) system will be
required.
ACKNOWLEDGMENT
This work was supported by grant No.( R01-2002-000-
00313-0) from the Basic Research program of the Korea
Science & Engineering Foundation.
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... This method will produce OH -which can shift the equilibrium reaction or produce OH * radicals to promote degradation of ammonia and urea nitrogen. Due to ozone is known as strong disinfectant and oxidant, it is often used by industry to deodorize, decolouration, and to produce structural changes in organic compounds [7]. Ozone also proven in reducing COD by 27% which is 2.5 folds greater than oxidation of ordinary organic compounds or biological treatment. ...
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... This method will produce OHwhich can shift the equilibrium reaction or produce OH * radicals to promote degradation of ammonia and urea nitrogen. Due to ozone is known as strong disinfectant and oxidant, it is often used by industry to deodorize, decolouration, and to produce structural changes in organic compounds [7]. Ozone also has proven in reducing COD by 27% which is 2.5 folds greater than oxidation of ordinary organic compounds or biological treatment. ...
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In order to produce large amounts of antigens of Dermatophagoides farinae (DF) and D. pteronyssinus (DP), both of which are very important species as the main inhalant allergens causing allergic diseases, mass-rearing techniques of DF and DP mites were studied. A mixture of 50% fish food powder and 50% dried yeast gave the highest production of both DF and DP, showing 37.0-fold and 51.8-fold increase in number after 12 weeks, respectively. When the same amount of culture media were used, the larger surface of the rearing container gave better production rate in both cases of DF and DP, showing 188.2-fold and 200.8-fold increase, respectively in a 154 (cm)^2 surface container (14cm in diameter) compared to a 79(cm)^2 surface container (10cm in diameter) after 12 weeks. Several different temperature and relative humidity conditions were compared for finding the maximum mass production. The highest production of DF mites resulted when 28℃ and 64% RH were provided, showing 815-fold increase in number after 10 weeks, and followed by 28℃ and 52% RH showing 773.3-fold increase after 10 weeks. In the case of DP mass rearing, the maximum production resulted when 25℃ and 75% RH were given, showing 1,391.7-fold increase in number after 10 weeks, and almost the same production resulted under conditions of 28℃ and 64% RH giving 1,385-fold increase in number after 10 weeks. When a 154(cm)^2 surface container was used, the optimum amount of culture media was 50g, and satisfactory result was obtained when the culture was started with 1,500 seed mites. During 20 weeks' observation period, the peak in number was obtained after 10 weeks of the culture in all test groups of DF and DP, and thereafter the number decreased.
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