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Determining the Lethal Concentration of Lysol

Cleaning Solution Using Brine Shrimp Assay


Bernadeth B. Asuncion
Jatrice Bianca S. Dela Paz
Camille A. Quiza
Michelle Ann B. Mariacos
Rica Louise F. Prescilla
Students, Bachelor of Science in Pharmacy
School of Natural Sciences
Saint Louis University
Baguio City, Philippines

ABSTRACT
Most poisoning cases are associated with excessive exposure to toxic chemicals such as household
materials. A bioassay using Artemia salina (brine shrimp) was adapted to measure the toxicity of Lysol.
Appropriate amount of Lysol (0.001%, 0.01%, 0.1% to give concentration of 10 g/ml, 100 g/ml, 1000
g/ml respectively) were transferred into vials containing brine shrimp medium. The procedure determines
the LC50 values in g/ml of active compounds of Lysol in the said medium. Activities of active compounds
are manifested as toxic to brine shrimps. The lethal concentration of Lysol resulting 50% mortality of the
brine shrimp LC50 was determined from 6 and 24 hours. The vials were then inspected after. The number
of surviving larvae were counted. The percentage of mortality was calculated at each concentration then
concentration mortality data were analyzed statistically. The concentration at which 50% of the population
died after 6 hours of exposure to Lysol was 100g/mL. Further, after 24 hours of exposure to Lysol, all
died. Since there is good correlation between LD50 using brine shrimp bioassay and LD50 reported in mice,
it can be said that the lethal concentration of Lysol in rats when taken orally after a six-hour exposure is
approximately 100g/mL.

Keywords: Brine shrimp, Lysol, Phenol, Potassium chloride

BACKGROUND INFORMATION
Poisoning is a significant global public
health problem. According to WHO data, updated
2016, in 2012 an estimated 193,460 people died
worldwide from unintentional poisoning. Of
these deaths, 84% occurred in low- and middleincome countries like the Philippines. In the same
year, unintentional poisoning caused the loss of
over 10.7 million years of healthy life (disability
adjusted life years, DALYs). It said that most of
the poisonings are associated strongly with
excessive exposure to, and inappropriate use of,
toxic chemicals such as the household material,
Lysol. In a report by CBC Marketplace,

Toxicologist Shawn Ellis, President of Building


Health Center Inc. who went to various peoples
homes testing air quality in the rooms where their
cleaning products were kept, Lysol was the
worst offender.
Lysol is a germicidal, VOC (Volatile
Organic Compound) product that is used to
disinfect environmental surfaces in homes and in
public places. It is the most common choice of
people in eliminating germs and odors on things
and surfaces that they come in contact with
everyday such as garbage cans, pet areas, under
sinks, around toilet areas, doorknobs, closets,
mattresses and many more. It claims that it kills

99.9% of germs on surfaces for only 30 seconds


and thereby eliminating odors that they cause.
Basing from its primary label, it mainly targets
specific bacteria (Staph and Strep, Salmonella,
E.coli, TB bacteria), fungi (Athletes Foot
fungus) and viruses (rhinovirus 39, rotavirus,
Respiratory Syncytal Virus, adenovirus type II,
Herpes Symplex Virus types 1 and 2, Influenza
type A2).
However, Lysol, as perfect as it sounds
killing cold and flu viruses which other ordinary
non germicidal sprays cant do, is not completely
labeled with its formulation. Although the label
says that it contains Ethanol 60% as its active
ingredient, a blog from Naturalnews.com
enumerated all the possible contents of the
product showing that Lysol is really a very toxic
product when inhaled in excess. Aside from what
is written in the blog that the said product has
been shown to lower IQ of children
developmentally, the following reported contents
and their toxicity is our main concern in having
this study: Ethyl Alcohol 60%, the active
ingredient, can cause developmental toxicity,
gastrointestinal and liver toxicity, neurotoxicity,
and
skin
toxicity;
Butane can
cause
neurotoxicity; Propane can cause neurotoxicity
as well as respiratory; Ethanolamine is
undetermined; water is inert; Fragrance is
unregulated, could literally be anything; Alkyl
(C14, 50%; C12, 40%; C16, 10%) Dimethyl
Benzyl Ammonium Chloride 1.520%; C16; Alkyl
Dimethyl Benzyl Ammonium; Saccharinate; MEA
Borate; Mipa Borate are all undetermined;
Ammonium Hydroxide can cause asthma; and
lastly, n-alkyl Dimethyl Benzyl Ammonium
Chlorides which can cause immunotoxicity,
respiratory toxicity, skin and organ toxicity, and
asthma. It has been restricted for use in cosmetics
in both Canada and Japan.
Considering the above mentioned data,
we can infer that Lysol is indeed a toxic product,
frequently used but neglected by most users to
which the children are most vulnerable. Hence, it
is worth researching and examining available
tests to prove the products toxicity and inform the
public about it. The researchers utilized the Brine
Shrimp Bioassay to establish results.

The brine shrimp lethality bioassay is


rapid (24 hours), simple (e.g., no aseptic
techniques are required), easily mastered,
inexpensive, and requires small amounts of test
material (2-20 mg or less). The bioassay has a
good correlation with cytotoxic activity in some
human solid tumors and with pesticidal activity.
This test was proposed by Michael et al. and
modified by others. Since its introduction, this in
vivo lethality test has been successively
employed for providing a frontline screen that
can be backed up by more specific and more
sophisticated bioassays once the active
compounds have been isolated (Apu, Muhit et
al.,2010).
Little is known regarding the toxicity of
Lysol, as such, the aim of this study is to
establish its possible lethal concentration via the
Brine Shrimp Bioassay.

MATERIALS AND METHODS


Sample Material
Chemical Structure
Lysol contained cresols such as phenol.
Now, Lysol contains ethanol and benzalkonium
chloride.

Physical characteristics

Lysol is a liquid solution that varies on


color and odor depending on the flavor or scent.
Highly concentrated liquid solution, cost
effective germicide based on a blend of
quaternaries and detergents. It cleans, disinfects
and deodorizes with a neutral pH in dilution. Use
on floors, countertops, washable walls and
appliance exteriors.
In order to study the toxicity of Lysol,
we performed Brine Shrimp lethality bioassay,
which is based on the ability to kill Laboratory
cultured brine shrimp (Artemia salina nauplii).
The assay is considered a useful tool for
preliminary assessment of toxicity. This method
is attractive because it is very simple,
inexpensive, and low toxin amounts are sufficient
to perform the test in a micro scale.
Preparation of the Sample
The Lysol were diluted using distilled
water (1ml/10 ml).

Appropriate amount of Lysol solution


(0.001%, o.o1%, 0.1% to give concentration of
10g/ml, 100 g/ml, 1000 g/ml respectively),
were transferred to clean vials ready for
experiment.
Ten nauplii were selected and transferred
into each sample vial by means of
pipette/dropper, and the final volume in each
volume was adjusted to 5 ml using artificial
water. A drop dry yeast (3gdry yeast/ 3ml
artificial water) was added as food to each vial.
The vials were maintained under illumination.
Survivors were counted with the aid of
magnifying glass, after 6, 24 hours and the deaths
of each dose level, negative control were
determined using only artificial seawater.
Lethal concentration determination
The lethal concentration of Lysol
resulting 50% mortality of the Brine Shrimp
LC50 were determined from 6, 24 hours and the
dose response were transferred into LC50 (Table
4.1).
Hatching the shrimp

Figure: Flow Chart for alternative Dilution for


Brine Shrimp Bioassay
Toxicity Bioassay
Brine Shrimp lethality Bioassay was
carried out to investigate the toxicity of Lysol.
Laboratory Cultured Brine Shrimp were hatched
using Brine Shrimp eggs in a shallow rectangular
dish and were divided by light and dark divisions
filled with artificial sea water, which was
prepared using Sodium Chloride (38g/100ml)
and distilled water. After 48 hours, the active
phototropic nauplii were collected by pipette.
Sample (Lysol) for the experiment were diluted
by distilled water depending each concentrations.

Brine Shrimp eggs were hatched in a


shallow rectangular dish filled with artificial
water which was prepared by dissolving 0.38g
rock salt in 100mL of distilled water. A plastic
divider with several 2mm holes was clamped in
the dish to make two unequal compartments. The
eggs were sprinkled into the larger compartment
which was darkened, while the smaller
compartment was illuminated. After a week, the
phototropic nauplii were collected from the
illuminated side, having separated by the divider
from their shells.
Counting nauplii and analysis of data
After 6 hours, the vials were inspected
using a magnifying glass and the number of
surviving larvae were counted. The percentage of
mortality was calculated at each concentration.
The concentration mortality data were analyzed
statistically. The effectiveness or the
concentration-mortality relationship of the
sample is expressed as a median lethal

Sam
ple
Lysol

concentration (LC50) value which represents the


concentration of the chemical that produces death
in half of the subjects after a certain exposure
period. Since it is desirable to correct the
mortality in the experimental treatments by the
mortality that occurs in the control treatment,
LC50 was determined using Abbots Formula
(Abbot, 1925). It is a mathematical formula used
to correct mortality in the untreated check. The
adjusted value is permissible when mortality in
control does not exceed 20% (Sreeshma&Nair,
2014).

Figure 4.1 shows the lethality (%) of


brine shrimps against concentration of Lysol.
Upon observation of the brine shrimps after 6
hours of their exposure to Lysol, the
concentration at which 50% of the population of
brine shrimps was identified as 100g/mL.

RESULTS AND DISCUSSION

REFERENCES

Vial 1
(10g/mL)
6hrs
12.5%

24hrs
100%

Mortality (%)
Vial 2
(100g/mL)
6hrs
50%

24hrs
100%

Vial 3
(1,000g/mL)
6hrs
100%

24hrs
100%

Table 4.1 Mortality of Artemia salina (%)


Table 4.1 shows the corrected percentage
mortality as computed by Abbots Formula. The
concentration at which 50% of the population of
the brine shrimps died (LC50) after 6hours of
exposure to Lysol is 100g/mL. Further, after 24
hours of exposure to Lysol, all the brine shrimps
died. However there are several factors to
consider in brine shrimp assays, such as the
solvent (Chao, 2014), nutrition and environment,
in general, that may have contributed to
inaccuracy of results.

CONCLUSION
Since there is a good correlation between
LD50 using brine shrimp bioassay and LD50
reported in mice (Logarto et al, 2001), it can be
said that the lethal concentration of Lysol in rats
when taken orally after a six-hour exposure is
approximately 100g/mL. However, the results
collected may not be applicable to humans.

Abbot WS. (1925). A method of computing the


effectiveness of an insecticide. J Econ
Entomol 1925; 18: 265-267
Logarto et. al. (2001). Comparative study of the
assay of Artemia salina L. and the
estimate of the medium lethal dose
(LD50 value) in mice, to determine oral
acute toxicity of plant extracts.
Sreeshma, L.S. & Nair, B.R. (2014). Brine
Shrimp Lethality Assay in Two Species
of Biophytum Dc. (Oxalidaceae).
International Journal of Pharmacy and
Pharmaceutical Sciences. Vol 6, Issue 4.
582-586
Wu, C. (2014). An important player in brine
shrimp lethality bioassay: The solvent.
Journal of Advanced Pharmaceutical
technology and research. 2014 Jan-Mar.
Barendse, S. (2014, November 26). Is Lysol Toxic
To Humans? Retrieved May 17, 2016,
from
Natural
News
Network:
http://blogs.naturalnews.com/lysoltoxic-humans/