Journal of the American Association for Laboratory Animal Science

Vol 61, No 2
Copyright 2022 March 2022
by the American Association for Laboratory Animal Science Pages 181–187

Stability of Free Available Chlorine Levels in

Dilute Sodium Hypochlorite Solutions over a
6-Week Period
Christopher K Gow,1,* Caren Weinhouse,2 Graham O’Brien Johnson,3 and Kim E Saunders1

Animal care and use programs commonly use chlorine and chlorine-based disinfectants to help prevent facility acquired
infections in animals. The Department of Comparative Medicine (DCM) at Oregon Health and Science University (OHSU)
follows the Centers for Disease Control and Prevention (CDC) disinfection guidelines for preparing and storing these disin-
fectants. DCM prepares bottles of dilute solutions of sodium hypochlorite (that is, commercial bleach) daily. In this study, we
tested whether dilute bleach solutions, as prepared following the DCM protocol, remained stable under real-world practice
conditions for up to 6 wk. We tested 4 groups of spray bottles filled with 0.5% bleach solutions in these experiments. Specifi-
cally, we sprayed 2 groups of bottles daily to mimic use while 2 other groups of bottles were not sprayed. We then measured
free available chlorine (FAC) using 2 methods, spectrophotometry and colorimetric strips. All 4 test groups showed stable
maintenance of FAC concentration for the length of the experiment. Mean FAC loss from baseline levels was not significantly
different in the group of bottles not sprayed daily (6% for group 2 at week 5 compared with 7% for Group 4 at week 6). All
bottles in Groups 1 and 3 measured by colorimetric strips showed concentrations at or near 5000 mg/L at all weekly time
points throughout the experiment. This study shows that 0.5% sodium hypochlorite solutions stored and used in a standard
rodent housing room and sprayed daily will maintain acceptable FAC concentrations for at least 5 to 6 wk, perhaps longer.
In addition, we report that colorimetric strips may be a useful and accessible quality control tool for testing freshly prepared
solutions at regular intervals. We conclude that sodium hypochlorite solutions can be prepared on a weekly, biweekly, or
monthly basis with no loss in disinfection effectiveness.

Abbreviations: DCM, Department of Comparative Medicine; OHSU, Oregon Health and Science University; CDC, Centers for
Disease Control and Prevention; FAC, free available chlorine

DOI: 10.30802/AALAS-JAALAS-21-000080

Introduction and financial costs. Some disinfectants, while effective, may

The prevention of animal infection through the accidental cause animal toxicity, including lung and brain injury from
introduction or propagation of pathogens is a high priority in a fume inhalation or occupational health hazards such as fume
research setting. Infections in research animals can spread quickly inhalation, dermal or ocular irritation or burns, the cost of dis-
and confound studies, and may require diagnostic tests, isolation, infectant and of the labor required to prepare and use it must
or treatment of infected animals. Some pathogens may require also be considered.18 Moreover, the CDC offers guidelines for
colony depopulation to ensure the long-term health of animals disinfectant use in human healthcare facilities, but these may not
in a facility. Laboratory animal medicine also presents unique be appropriate for animal research settings. CDC guidelines on
challenges to infection control as animals with varying degrees disinfectant concentrations and use may be excessively stringent
of immune status are routinely used in research experiments. for carefully controlled laboratory environments, causing unnec-
Immunocompromised animals are more susceptible to infection essary expense to programs and potential harm to animals and
and are more likely to require euthanasia due to poor health. staff. Alternatively, disinfectant storage conditions in research
Infectious microorganisms can be introduced easily through environments may be harsher than healthcare environments,
mishandling or improper disinfection of animal housing or speeding degradation and decreasing effectiveness more quickly
care items, but can be successfully avoided using disinfection and suggesting that more stringent protocols are needed.
protocols from the CDC with Environmental Protection Agency Many care and use programs for research animals follow CDC
(EPA)-registered disinfectants, such as alcohols, chlorine and guidelines and use chlorine-based disinfectants, particularly so-
chlorine compounds, formaldehyde, glutaraldehyde, hydrogen dium hypochlorite (bleach), for routine workstation and animal
peroxide, iodophors, phenolics and quaternary ammonium equipment disinfection. In this study, we evaluated the standard
compounds.18 Disinfectant effectiveness (that is, spectrum of operating procedure (SOP) for sodium hypochlorite disinfection
activity, speed of action) must be balanced against health risks in the DCM at OHSU. Per departmental SOP, we prepared a
0.5% (5000 mg/L) working solution daily for the disinfection of
rodent workstations, transfer forceps, equipment and surfaces
Received: 1 July 2021. Revision requested: 11 Aug 2021. Accepted: 15 Nov 2021.
1Department of Comparative Medicine, 2Oregon Institute of Occupational Health that may contact rodents, their caging or their waste. This SOP
Sciences, 3School of Public Health, Oregon Health & Science University, Portland, OR is based on current CDC guidelines and represents current use of
*Corresponding author. Email: topher.gow@gmail.com the guidelines in a representative animal care and use program.

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March 2022

The DCM uses bleach because of its reasonably broad spectrum (Figure 1). Two of the 4 groups were sprayed daily to mimic
of antimicrobial activity, low toxicity and levels of toxic residues, daily use; the remaining 2 groups were not sprayed (Figure 1).
lack of sensitivity to water hardness, quick action, and low cost. We then used 2 testing methods to evaluate FAC available chlo-
However, some small but notable differences between CDC rec- rine concentrations at weekly time points: spectrophotometry
ommendations and current practice argue for a more stringent and colorimetric chlorine strips.
protocol. For example, the CDC Disinfection Guidelines state The plastic spray bottles we used in these experiments were
that, “hypochlorite solutions in tap water at a pH >8 stored at 32-ounce, white, opaque, high-density polyethylene bottles
room temperature (23°C) in closed, opaque, plastic containers (Wesco Supply, Long Beach, CA) with 9 to 3/4” Adjust-O-Spray
can lose up to 40-50% of their free available chlorine (FAC) over triggers (Wesco Supply, Long Beach, CA). To closely mimic
one month.”18 OHSU DCM staff store dilute sodium hypochlo- practice conditions in rodent housing rooms, we stored bottles
rite solutions in opaque spray bottles in animal care rooms or at in a vacant rodent housing room and maintained these rooms
animal care stations in spaces with fluctuating temperature and on a 12:12-h light: dark cycle, at 19.4 to 22.8 °C and 30% to 70%
light exposure. In addition, tap water pH is not routinely meas- relative humidity (as recorded by a thermo-humidity meter) for
ured when DCM staff prepare dilute solutions. However, current the duration of the study. Because light exposure has been previ-
OHSU DCM protocols could be overly stringent, particularly with ously shown to contribute to sodium hypochlorite degradation,
regard to the requirement to prepare solutions fresh daily, which we ensured maximum light exposure to simulate degradation
increases risk to DCM staff and costs for reagent and labor. The under the most extreme practice conditions.2,5,8,9,11,16,17 To ac-
potential harms of sodium hypochlorite use are highest for the complish this, we positioned bottles in a grid in the middle of
technical staff who prepare working dilutions from purchased a stainless-steel table, approximately 1 m high, directly below a
commercial bleach (5000 mg/L); these risks include potential lighting banister fitted with a compact fluorescent bulb (Figure 2).
for ocular irritation and chemical burns, metal corrosion at high We adjusted the exact positioning under the light banister to
concentrations (>500 ug/L), potential release of chlorine gas expose all bottles to the same average light intensity (lux), as
when mixed with ammonia or acid, and low relative stability confirmed by measuring the center of the bottle grouping with
over very long time periods.18 Therefore, we sought to determine a LX1330B lux meter (Sinometer, ShenZen, China).
whether the current CDC guidelines are appropriate for use in a Sodium hypochlorite solutions. In initial experiments, each
laboratory animal setting. bottle was filled with a 0.5% (5000 mg/L) sodium hypochlorite
In this study, we tested whether dilute bleach solutions, solution using the current DCM protocol. The solutions were
as prepared using the DCM protocol, remained stable under made by diluting 83 mL of 6% Pure Bright Germicidal Ultra
real-world practice conditions for up to 6 wk. The overall goal Bleach (KIK International LLC, Concord, Ontario, Canada) with
of this project was to provide practical guidance in use and 917 mL of tap water (1:12 dilution) using 250 mL and 1000 mL
storage of bleach solutions in research animal settings and to polypropylene graduated cylinders. Initial spectrophotometry
provide programs with technical information on testing options results indicated that this protocol did not reliably produce a
available for determining the stability of sodium hypochlorite 5000 mg/L solution (data not shown). Therefore, in subsequent
used under specific storage and use conditions. experiments, we adjusted the dilution to achieve a starting con-
Prior studies have shown that the decomposition rate of so- centration of 5000 mg/L, as confirmed by spectrophotometry.
dium hypochlorite is mainly dependent on pH, concentration, We empirically determined that the starting concentration of the
temperature, and ambient light exposure.5,8-11,15-17 The ideal purchased bleach product was 4.5%, rather than the expected
storage conditions to maximize sodium hypochlorite solution 6%. Thus, the final protocol used 111 mL of bleach product and
stability would include maintaining a solution at a pH of 9 to 889 mL of tap water (1:9 dilution) to achieve a final concentra-
11, at temperatures below 30 °C, and in an opaque bottle with tion of 5000 mg/L.
little to no ambient light exposure.4-6 One predictive model of Mimicking daily use of bottles. We sprayed bottles in Groups
FAC loss showed that a 1.25% commercially available sodium 1 and 2 (Figure 1) to mimic the effects of volume depletion and
hypochlorite solution, stabilized to a pH of 11.9, degrades 10% introduction of room air into spray bottles as a result of daily
after 660 d at 25 °C.13 However, this model does not consider
real-world variables. We added to the existing literature by test-
ing the role of air introduction into bleach spray bottles due to
daily use (via using spray bottles to mimic daily practice) and
by testing for the loss of FAC over a 6-wk period when stored
and used in an active laboratory animal care facility. We used 2
methods to determine FAC concentrations—spectrophotometry,
a highly quantitative approach that may not be available to some
programs, and colorimetric strips, which are widely available
and easy to use but semiquantitative. We hypothesized that we
would find be no significant difference in FAC concentrations
over a 6-wk period, and that a difference in FAC concentrations
would not develop between bottles sprayed daily and those that
are not. If true, these results would indicate that dilute bleach
solution remains stable for up to 6 wk under active use condi-
tions, suggesting that less frequent solution preparation would Figure 1. Experimental design by group, bottle identification, testing
be acceptable, representing a saving of both cost and effort. method, and storage conditions. The study design included 2 groupings
of 6 bottles each: the first group (group 1 and 2) was sprayed daily to
mimic use and the second group (group 3 and 4) was not sprayed, to test
Materials and Methods the role of air introduction in sodium hypochlorite degradation. Three
bottles in each group (group 2 and 4) were monitored for free available
Experimental design. We tested 4 groups of 3 spray bottles chlorine (FAC) using spectrophotometry and three by colorimetric strips
each (total of 12 bottles) of sodium hypochlorite solution (group 1 and 3).
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 Guidance on storage and testing of a dilute bleach solution

are useful for nonregulatory reporting and for spot checking.

Unfortunately, reading the color changes is subjective and is
influenced by the light source and individual ability to judge
subtle differences in color.14 More accurate methods include
spectrophotometry and iodometric titration. Spectrophotom-
etry uses a photometer to accurately measure colorimetric
changes that correspond with FAC concentrations. However,
spectrophotometry is technically more challenging and requires
equipment that ranges in price from a few hundred dollars to
several thousand dollars.14
We measured the FAC concentrations in samples from Groups
2 and 4 using a DR2000 spectrophotometer (Hach, Loveland,
CO) set to method 80 in accordance with USEPA DPD Method
8021.3 This spectrophotometer has a resolution of 2 significant
digits. Because we expected concentrations of FAC chlorine
to exceed the upper range of the instrument (2.00 mg/L), the
samples were diluted by transferring 4 µL of sample to a 10 mL
glass sample cell (Hach, Loveland, CO) filled with deionized
water. The diluted sample was then vortexed before proceeding
with the remaining instructions for method 8021. A 1:2500 dilu-
tion factor was used to provide the highest resolution between
expected readout values while minimizing error introduced by
dilution. All samples were analyzed in triplicate.
FAC concentrations were estimated in samples from Groups
1 and 3 by using Bartovation extra high-level chlorine test strips
(Queens, NY). These test strips detect FAC at the following
concentrations: 0, 1000, 2500, 5000, 7500, 10000. One unblinded
individual tested the samples following the manufacturer’s
protocol. An individual test strip was briefly submerged in
the solution sample for one second, removed, and left on the
Figure 2. Set-up of the table and bottles within the empty rodent hous- benchtop for 30 s. The test strip color was evaluated within the
ing room. Twelve bottles (3 rows by 4 columns) were positioned at next 10 s. All samples were analyzed in triplicate.
the center of a stainless-steel table, approximately 1 m tall, directly
below the lighting banister. The exact positioning was adjusted until Statistical analysis. Statistical analysis was performed using
all bottles were exposed to the same average light intensity (lux), as Prism 9.1.2 (GraphPad Software, San Diego, CA). Group data
confirmed by a light meter. were the mean ± 1 SD and individual data as the mean ± 1 SEM.
Multiple unpaired t tests were used to compare group data for
pH and FAC. Statistical significance was defined as a P value
use. On days 0 to 42 of the experiment, we gently swirled each
of less than 0.05 for all analyses.
Group 1 and Group 2 bottle to thoroughly mix the solution
and adjusted the nozzle until it produced a fine mist before
continuing to spray for a total of 50 times per weekday based Results
on our estimation of a standard workweek and the number of Environmental parameters. Environmental parameters meas-
sprays required to saturate the working surface of an animal ured in the experimental space (laboratory animal housing
transfer station. room) included room temperature, relative humidity, and light
Sample collection for pH and FAC analysis. We sampled intensity (Figure 3A-C). Room temperature ranged from 18.9 to
solutions weekly for the 6 wk duration of the experiment. We 21.1 °C, with a mean temperature of 21 °C (SD = 0.8) (Figure 3A).
gently swirled bottles before twisting off the trigger spray. We Relative humidity (as measured with a monitor placed at
transferred a 10 mL sample by a plastic serological pipette into bottle height) ranged from 17% to 32%, with a mean of 20%
a labeled 25 mL glass sample cell (Hach, Loveland, CO). We (SD = 4) (Figure 3B). Light intensity (as measured with a lux
replaced the trigger spray and rinsed the serological pipette with meter centered over experimental bottles) ranged from 345 to
tap water before proceeding to the next bottle. We collected sam- 359 lx, with a mean of 352 lx (SD = 3) (Figure 3C). All environ-
ple cells into an enclosed cardboard box at room temperature mental parameter measurements were within ranges commonly
until all samples were ready for analysis. We then measured the accepted as normal in animal housing units, with the exception
pH for all samples using an Orion 720A Plus digital pH meter of relative humidity, which was lower than usual in our experi-
(Fisher Scientific, Waltham, MA). The pH meter has a default ment. However, relative humidity is commonly measured in
resolution of 3 significant digits and an accuracy of ±0.002 pH. animal spaces at the level of building ducts, rather than at the
We calibrated the unit at the beginning of each time point us- table height level measured here.
ing 3 standard solutions (pH 4, pH 7, pH 10). We adjusted the pH. pH values were generally stable in all bottles over the
samples to a pH between 6 and 7 using 1N sulfuric acid before course of the experiment, despite slight decreases with time
proceeding to FAC analysis. (Figure 4). Bottles in Groups 1 and 2 (Sprayed Daily) showed
Several methods can be used to measure and monitor FAC. a mean baseline pH of 11.5 (SD = 0.05), which fell to 11.0
The selection of specific methods depends on ease, precision and (SD = 0.06) by week 5 (Figure 4). pH values were not recorded for
accuracy, available resources, and equipment. Semiquantitative bottles in Groups 1 and 2 beyond week 5 because daily spraying
methods include colorimetric strips that convert changes in completely depleted the bottles’ contents. Bottles in Groups 3
FAC concentration to a visual color comparator. These strips and 4 (not sprayed daily) showed a mean baseline pH of 11.5
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Journal of the American Association for Laboratory Animal Science
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Figure 4. pH of solutions that were sprayed (Group 1 and 2) or not

sprayed daily (Group 3 and 4) over time (mean ± 1 SD [error bars]). The
pH values were generally stable throughout the experiment despite a
slight decrease with time. Groups 1 and 2 (sprayed daily) had a baseline
pH of 11.5 that decreased to 11.0 by week 5. Groups 3 and 4 (not sprayed
daily) had a baseline pH of 11.5 that decreased to 11.2 by week 6. There
was a statistically significant difference (P < 0.05) at weeks 4 and 5. Group
1 and 2 bottles (sprayed daily) had pH values of 11.2 and 11.0 compared
with Group 3 and 4 bottles (not sprayed daily) that had pH values of 11.3
and 11.2 on weeks 4 and 5, respectively. Note that daily spraying com-
pletely depleted the bottle contents of Group 1 and 2 by week 5.

(SD = 0.05) that fell to 11.2 (SD = 0.04) by week 6 (Figure 4).
Unpaired t tests comparing sprayed daily (combined data from
Groups 1 and 2) and not sprayed daily (combined data from
Groups 3 and 4) conditions showed a statistically significant
difference (P < 0.05) at weeks 4 and 5, although effect sizes
were small and unlikely to reflect functionally significant dif-
ferences in disinfectant chemistry. Specifically, mean pH values
for sprayed daily bottles were 11.2 and 11.0 for weeks 4 and 5,
respectively. Mean pH values for unsprayed daily bottles were
11.3 and 11.2 for weeks 4 and 5, respectively.
Free available chlorine. Spectrophotometry measurements of
FAC for 2 groups of bottles (Group 2 [sprayed daily] and Group
4 [not sprayed daily]) revealed that initial FAC levels were near
the target starting concentration of 5000 mg/L; measured FAC
concentrations were 4880 mg/L (SD = 23) in Group 2 and 4950
mg/L (SD = 12) in Group 4 (Figure 5C). FAC concentration fell
slightly with time for all bottles, reaching a final concentration
of 4600 mg/L (SD = 21) for Group 2 (at week 5) and 4630 mg/L
(SD = 31) for Group 4 (at week 6) (Figure 5A-B). As noted above,
Sprayed Daily bottles were empty by the end of week 5, due
to daily spraying. Mean FAC loss from baseline levels was not
significantly different between the 2 groups (6% for Group 2 at
week 5 compared with 7% for Group 4 at week 6).
Colorimetric chlorine strips were used to evaluate FAC levels
for the remaining 2 groups of bottles: Group 1 (sprayed daily)
and Group 3 (not sprayed daily). All bottles in both groups
showed concentrations at or near 5000 mg/L at all weekly time
Figure 3. Environmental parameters recorded as (A) room tempera- points throughout the experiment (5 wk for Group 1 and 6 wk
ture (°C), (B) relative humidity (%), and (C) light intensity (lux). The for Group 2).
room temperature and relative humidity were measured using a ther-
mo-humidity meter placed at the level of the bottles. (A) Room tem-
perature remained relatively stable throughout the experiment except Discussion
for a 7-d period where the temperature dropped below the lower limit This study evaluated an existing CDC guideline-based SOP
(19.4 °C) to 18.9 °C. (B) Relative humidity varied between 17% and
32% throughout the experiment. (C) Light intensity was measured
for sodium hypochlorite solution preparation for laboratory
daily with a light meter and was very stable throughout the experi- animal use. The data showed that FAC levels remained stable
ment with an average of 352 lx. for up to 6 wk. In addition, spray bottles in active use were de-

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 Guidance on storage and testing of a dilute bleach solution

pleted of diluted solution by 5 wk, suggesting that bottles can

be refilled with fresh solution when empty without any loss of
disinfection effectiveness in practice. This finding showed that
laboratory animal programs can reduce labor and reagent costs
associated with daily sodium hypochlorite solution preparation
without compromising animal or technician safety. Our data
also showed that actual concentrations of commercial bleach
concentrates may be significantly lower than what is reported
on consumer labels. This was also noted in another study that
reported variability in advertised and measured concentrations
of commercial bleach samples from different countries.12 These
findings strongly suggest that laboratory animal programs
should empirically test the starting concentrations of purchased
commercial bleach brands to develop dilution protocols that
yield the desired 5000 mg/L disinfection concentration for use
in animal spaces.
Prior studies show that extrinsic factors, including light,
temperature, and air in bottle headspace, are important pre-
dictors of sodium hypochlorite solution stability.1,2,5,6,8-13,15-17
We found that these factors remain reasonably stable in OHSU
DCM animal housing spaces. We acknowledge that natural
fluctuations in environmental parameters can be expected in
any representative animal care and use program. Temperature
was largely stable, apart from a 7-d period during which room
temperature dropped to 18.9 °C, a 0.5 °C dip from the room
minimum, possibly due to the absence of major thermal output
(active racks with live animals) in the study room. Relative
humidity levels, while not previously reported to affect FAC
degradation, were out of the intended range for the majority
of our experiment. We tracked temperature and relative hu-
midity by using a thermo-humidity meter at the level of the
bottles. These measures are highly dependent on placement
of the meter within the room. Furthermore, relative humid-
ity in animal rooms is commonly measured at the level of the
building ducts. Light intensity, reported as lux, was within the
range prescribed by the Guide for the Care and Use of Laboratory
Animals, which recommend that empty rooms not exceed 400 lx
approximately 1 m from the ground.7 Conditions in this experi-
ment represented the most extreme light conditions likely in
an animal housing room. Prior studies found that light quality
and quantity influence bleach stability.2,5,8,9,11,16,17 Most, if not
all, rodent housing rooms are completely devoid of natural
light, so the compact fluorescent light tested in this experiment
is most relevant to practice. Compact fluorescent bulbs emit a
small amount of UVA, UVB, and infrared radiation, which we
hypothesized might speed FAC degradation.19 However, we
found minimal decreases in FAC over the course of the study,
such that environmental parameters reported here did not ap-
preciably affect FAC concentrations. These FAC results may not
Figure 5. Degradation curves of dilute sodium hypochlorite solu- be generalizable to laboratory animal spaces with less tightly
tions that were (A) Sprayed daily or (B) Not Sprayed Daily (mean ± controlled environmental parameters.
1 SEM [error bars]). (C) Group degradation curves (mean ± 1 SD
[error bars]). Spectrophotometry measurements were taken for Group
Prior studies also found that intrinsic factors, including pH
2 and Group 4 bottles. (A) This depicts the individual degradation and baseline FAC concentrations, contribute to bleach stability.
curves for bottles 4, 5, and 6 (Group 2) over 5 wk. All time points Commercial bleach is commonly manufactured to a final pH
were performed in triplicate. (B) This depicts the individual degra- greater than 11 because sodium hypochlorite solutions are more
dation curves for bottles 10, 11, and 12 (Group 4) over 6 wk. All time stable at higher pH.5,16 Dilution of sodium hypochlorite with
points were performed in triplicate. (C) Initial FAC levels for Group
2 were 4880 mg/L (SD = 23) and 4950 mg/L (SD = 12) for Group 4. water lowers pH, which may speed solution degradation. Deg-
FAC concentration decreased slightly with time for all bottles, to a fi- radation increases at pH levels between 11 and 7 and increases
nal concentration of 4600 mg/L (SD = 21) for Group 2 (at week 5) and precipitously at pH < 7.1,5 Here, we found that the current OHSU
4630 mg/L (SD = 31) for Group 4 (at week 6). Mean FAC loss from DCM protocol yields a dilute sodium hypochlorite solution of
baseline levels was slightly higher in the not sprayed daily (Group pH approximately 11, and that this pH was relatively stable
4) bottles (6% for Group 2 at week 5 compared with 7% for Group 4
at week 6), although this difference was not statistically significant. throughout the experiment, indicating that common practice
Note that daily spraying completely depleted the bottle contents of and conditions will produce and maintain solutions of reason-
Group 2 by week 5. able stability. Initial FAC concentrations can also affect solution

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Journal of the American Association for Laboratory Animal Science
March 2022

degradation. Solutions with relatively higher initial FAC in the CDC guidelines (Table 1). This provides room for error.
concentrations generally degrade more quickly than solutions If a laboratory animal program has concerns about a specific
with lower starting FAC.2,8,17 We found that the OHSU DCM microorganism that requires a high concentration of sodium
protocol (adjusted for empirically measured commercial bleach hypochlorite, they may opt to use a higher concentration, or,
concentration) yields dilute bleach solutions that vary minimally alternatively, they may choose a different disinfection protocol
in starting FAC concentrations and that this minimal variance that is more powerful (that is, vaporized hydrogen peroxide,
has negligible effects on solution degradation rate. chlorine dioxide).
Measurement of FAC by spectrophotometry may not be feasi- This experiment has several limitations. First, our results may
ble for all laboratory animal programs. Therefore, we tested the be limited by our small sample size. We tested only 3 bottles
effectiveness of inexpensive and readily available colorimetric per group, which is significantly fewer than the number of
strips for evaluation of FAC concentrations. Colorimetric strips bottles in use in OHSU DCM. However, our sample size was
have lower resolution than spectrophotometry and can therefore consistent with other studies evaluating bleach stability, and we
not detect small fluctuations in FAC concentration. However, in tested each bottle in triplicate to increase precision.10,11 Second,
this study, we found that fluctuations in FAC in dilute sodium the colorimetric strips we used have poor resolution at high
hypochlorite solutions over 5 to 6 wk were not detectable on concentrations, and we were unable to find alternative strips
colorimetric strips; all bottles showed concentrations at 5000 with better resolution. Manufacturers produce most colorimet-
mg/L, as measured by the strips. However, the FAC fluctuations ric strips for FAC level estimation to determine the safety of
that occurred in this study were minor, and this experiment drinking water treated with low levels of chlorine; they produce
spanned the full length of the likely “lifespan” of a bottle of few colorimetric strips for use in high concentration solutions.
solution in regular use, suggesting that colorimetric strips are In this experiment, all bottles tested with colorimetric strips
a useful tool for a quick determination of adequate disinfection were at the 5000 mg/L mark during the entire length of the
capacity of a given dilute solution. These strips may be a useful experiment. However, these strips would not record less than a
quality control tool for detecting the FAC of freshly prepared 50% loss in FAC; the next “color bar” records 2500 mg/L FAC.
solutions, manufacturing changes in starting bleach concentra- Therefore, these strips would only detect large errors or changes
tions, and errors in dilution. These events are not unlikely, as in manufacturing protocol. The colorimetric strips were also
evidenced by the lower-than-expected starting concentration read by one unblinded individual. Results could have been
in the commercial bleach product used in this experiment. As strengthened by blinding the reader to the bottle conditions.
mentioned in the materials and methods section, we altered the However, this study was performed during the COVID-19
dilution equation from our SOP after determining that the FAC pandemic under modified operations at OHSU. Modified
of the stock bleach bottle was lower than that stated on the bottle. operations and campus research requirements dictated that
Typically, the consumer does not know the lag in time between only one individual at a time could work in a small, enclosed
creation of the sodium hypochlorite solution and its distribu- space. Third, we only tested 1 starting concentration of a dilute
tion and use; increased distribution lag times may contribute sodium hypochlorite solution (0.5% or 5000 mg/L) generated
to lower stock bleach concentrations. The CDC guidelines for from a single commercial brand of bleach. Care should be taken
disinfection and sterilization in healthcare settings and the in- when extrapolating to other bleach brands without empirical
structions given by the bleach manufacturer recommend testing testing of FAC concentrations because starting concentration
the solution using a quantitative method to fine tune dilutions can influence bleach degradation. Fourth, we did not test for
to the specific concentration required.12,18 However, the impor- efficacy of solution disinfection through methods recognized
tance of using a 5000 mg/L sodium hypochlorite concentration by the Association of Official Analytical Chemists against com-
may depend on disinfection use. For example, OHSU DCM’s mon microorganisms; instead, we relied on published sodium
working solution (0.5%) is a much higher concentration than hypochlorite concentrations previously reported to be effective.
that required to kill a large majority of the microorganisms listed Last, we recognize that daily use in this study was an estimated
average and that factors such as variability in how much people
spray to clean surfaces and in how rooms are used contributes
Table 1. Microbicidal activity of chlorine and chlorine compounds. to how quickly bottles are emptied. Consideration for average
The table, according to the CDC guidelines on disinfection, lists the room usage across facilities and animal care technician room
FAC concentration and contact time required to render the listed mi- task schedules will determine the exact rate at which sodium
croorganisms inactive.
hypochlorite solutions are replaced; our data indicate that
FAC Concentration Required Contact bottles can be prepared weekly, every other week, or possibly
Microorganism for Disinfection (ppm) Time monthly. The findings of our study also support the need for a
Mycoplasma 25 15 s resource that shares detailed “true use” protocols for disinfect-
Vegetative bacteria <5 30 s ants to promote uniformity in protection.
Mycobacterium tuberculosis 1000 <10 min The goal of this project was to provide practical guidance for
Bacillus atrophaeus spores 100 5 min the DCM on bleach stability. Here we show that in real-world
Mycotic agents 100 <1 h conditions, 0.5% sodium hypochlorite solutions stored and used
in a standard rodent housing room in opaque plastic bottles
Clostridium difficile 5000 ≤10 min
sprayed daily will maintain acceptable FAC concentrations for
25 different viruses 200 10 min
5 to 6 wk, perhaps longer. Our results should directly inform
Candida 500 30 s practice changes that reduce technician time in preparing so-
Staphyloccous aureus 100 <10 min dium hypochlorite bottles daily, reduce waste and help conserve
Salmonella cholerasuis 100 <10 min resources, and reduce overall facility costs.
Pseudomona aeruginosa 100 <10 min
FAC, Free Available Chlorine
ppm, parts per million

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 Guidance on storage and testing of a dilute bleach solution

Acknowledgments acidic hypochlorous acid solution with microbicidal activity.

C.K.G. thanks the OHSU branch of the Oregon State Laboratory Biocontrol Sci 22:223–227. https://doi.org/10.4265/bio.22.223.
Animal Medicine Residency Consortium for funding this project. 10. Johnson BR, Remeikis NA. 1993. Effective shelf–life of prepared
sodium hypochlorite solution. J Endod 19:40–43. https://doi.org/
10.1016/S0099-2399(06)81040-X.
References 11. Kobylarz J. 2017. Efficacy of chlorine sanitizer spray bottles exposed
1.
Camps J, Pommel L, Aubut V, Verhille B, Satoshi F, Lascola B, to various temperatures and ambient light. BCIT Environmental
About I. 2009. Shelf life, dissolving action, and antibacterial activ- Public Health Journal. https://doi.org/10.47339/ephj.2017.74
ity of a neutralized 2.5% sodium hypochlorite solution. Oral Surg 12. Lantagne DS. 2009. Viability of commercially available bleach
Oral Med Oral Pathol Oral Radiol Endod 108:e66–e73. https:// for water treatment in developing countries. Am J Public Health
doi.org/10.1016/j.tripleo.2009.03.034. 99:1975–1978. https://doi.org/10.2105/AJPH.2009.160077.
2.
Clarkson RM, Moule AJ, Podlich HM. 2001. The shelf–life of 13. Lantagne PED, Preston K, Blanton E, Kotlarz N, Gezagehn H,
sodium hypochlorite irrigating solutions. Aust Dent J 46:269–276. van Dusen E, Berens J, Jellison K. 2011. Hypochlorite solution ex-
https://doi.org/10.1111/j.1834-7819.2001.tb00291.x. piration and stability in household water treatment in developing
3.
Eaton AD, Clesceri LS, Greenberg AE, Franson MAH. countries. J Environ Eng 137:131–136. https://doi.org/10.1061/
2017. American Public Health Association, American
(ASCE)EE.1943-7870.0000299.
Water Works Association, and Water Environment Federation.
14. Murray A, Lantagne D. 2015. Accuracy, precision, usability, and
4500–Cl−CHLORIDE. In: Standard methods for the examination
cost of free chlorine residual testing methods. J Water Health
of water and wastewater, 23rd ed. Washington (DC): American
13:79–90. https://doi.org/10.2166/wh.2014.195.
Public Health Association.
15. Nicoletti MA, Siqueira EL, Bombana AC, Oliveira GG. 2009.
4.
Farr JP, Smith WL, Steichen DS. Bleaching agents (survey),
Shelf–life of a 2.5% sodium hypochlorite solution as determined by
p. 271–300. In: Kirk–Othmer encyclopedia of chemical technology.
New York (NY): Chichester Wiley. Arrhenius equation. Braz Dent J 20:27–31. https://doi.org/10.1590/
5.
Frais S, Ng YL, Gulabivala K. 2008. Some factors affecting the S0103-64402009000100004.
concentration of available chlorine in commercial sources of 16. Piskin B, Turkun M. 1995. Stability of various sodium hypochlorite
sodium hypochlorite. Int Endod J 34:206–215. https://doi.org/ solutions. J Endod 21:253–255. https://doi.org/10.1016/S0099-
10.1046/j.1365-2591.2001.00371.x. 2399(06)80991-X.
6.
Gallandat K, Kolus RC, Julian TR, Lantagne DS. 2021. A system- 17. Rutala WA, Cole EC, Thomann CA, Weber DJ. 1998. Stability
atic review of chlorine–based surface disinfection efficacy to inform and bactericidal activity of chlorine solutions. Infect Control Hosp
recommendations for low–resource outbreak settings. Am J Infect Epidemiol 19:323–327. https://doi.org/10.2307/30141372.
Control 49:90–103. https://doi.org/10.1016/j.ajic.2020.05.014. 18. Rutala WA, Weber DJ. 2008. Guideline for disinfection and sterili-
7.
Institute for Laboratory Animal Research. 2011. Guide for the zation in healthcare facilities. Washington (DC): Center for Disease
care and use of laboratory animals: 8th ed. Washington (DC): The Control
National Academies Press. 19. United State Food and Drug Administration. [Internet]. 2017.
8.
Iqbal Q, Lubeck–Schricker M, Wells E, Wolfe MK, Lantagne D. Compact fluorescent lamps (CFLs) – Fact Sheet/FAQ. [Cited 1
2016. Shelf–life of chlorine solutions recommended in ebola virus July 2021]. Available at: https://www.fda.gov/radiation–emit-
disease response. PLoS One 11:e0156136. https://doi.org/10.1371/ ting–products/home–business–and–entertainment–products/
journal.pone.0156136. compact–fluorescent–lamps–cfls–fact–sheetfaq#:~:text=into%20
9.
Ishihara M, Murakami K, Fukuda K, Nakamura S, Kuwabara M, this%20area.–,Do%20CFLs%20emit%20UV%3F,measuring%20
Hattori H, Fujita M, Kiyosawa T, Yokoe H. 2017. Stability of weakly equipment%20must%20be%20used.

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