Developmental Biology Special Problem: Benzalkonium Chloride and Drosophila melanogaster (Fruit Fly) findings

West Visayas State University
College of Arts and Sciences
La Paz, Iloilo City
1
Mutagenic Effects of Benzalkonium Chloride on Drosophila
melanogaster
In Partial Fulfillment of the Requirements In
Bio 206: Development Biology
By
Christopher Jon I. Gaw Put
Mary Grace C. Dumdum
Joanna Rose B. Navarro
BSBS 3B
January 2013

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Table of Contents
Contents Page Number
Introduction to the Study
Research Paradigm
Statement of the Problem
Hypothesis
Significance of the Study
Delimitation of the Study
Review of Related Literature
Methodology
Research Design
Methodology
Materials
Chemicals
Preliminary Activities
Culture Media Preparation
Collecting of Samples
Larva of Drosophila Melanogaster
Experiment Proper
Preparation of Mutagen
Induction of Mutagen
Separation and Transferring of Fruit Flies

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Observation of Visible Mutations
Data Analysis
Results and Discussions
Conclusions
References
Appendix

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Chapter 1
INTRODUCTION TO THE STUDY
Background of the Study
Without genetic variations, some of the basic mechanisms of
evolutionary change cannot operate. Mutations, one of the
primary sources of genetic variation, are the changes in the
DNA. A single mutation can have a large effect.
The fruit fly Drosophila melanogaster is chosen as the
organism of choice because it is a favored organism for the
study of genetic basis of development. It is used extensively in
genetic breeding experiments. It is an ideal testing organism
because it has a short life cycle, exhibits great variability in
inherited characteristics and can be easily raised to produce
large numbers of offspring (Sandhyarani,2011).
Frequency of mutation in fruit flies has been well
documented. Mutation, as an event that gives rise to heritable
alteration in the genotype, in the fruit flies may be classified
as wildtype or a mutant. Visible mutations are easily identified
as the loss of a function or difference in the color of the
wildtype fruit fly.

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In this experiment, benzalkonium chloride was used as an
inducer of mutation. It is an antiseptic agent, commonly found
in household products used for cleaning. Previous studies have
showed that benzalkonium chloride is a mutagen as it affects
mammalian somatic cells, yeasts, or bacteria (ScienceLab.com)
This study aims to determine if the 10% concentration of
benzalkonium chloride affects the phenotype of Drosophila
melanogaster. If mutation does occur, this study aims to observe
the different mutant phenotypes and compare phenotypes between
generations.
Research Paradigm
Independent Variable Dependent Variable
Figure 1.Shows the relationship between the independent and
dependent variables of the study
Phenotype of
Drosophila
melanogaster
Treatment A
10% concentration of
Benzalkonium chloride

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Statement of the Problem
Generally this study will be conducted to determine the
effect of the 10% concentration of benzalkonium chloride to the
phenotype of Drosophila melanogaster.
Specifically this study shall seek to answer the following
questions:
1. What is the effect of the 10% concentration of Benzalkonium
Chloride on the phenotype of Drosophila melanogaster?
Hypothesis
This hypothesis will be tested on this study.
1. Benzalkonium chloride does not significantly affect the
phenotype of Drosophila melanogaster.
Definition of terms

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Significance of the Study
This study would benefit the following:
General public. This study will affirm whether benzalkonium
chloride is indeed a mutagen. This will give information to
people that it is a mutagen and they will be aware of its
possible effects.
This study can also benefit researchers as this can be a
ground for this study.
Delimitation of the Study
This study is limited to assessing the mutagenic effects of
benzalkonium chloride only.Only one concentration will be used
for this study by solving its weight by volume to get its 10%
concentration. There will be 1 trial for this experiment.
Pineapple will be used to collect the fruit flies, and the
offspring of the collected fruit flies will constitute the
parental generation. Sweet potato will then be used as a medium
during the actual conduct of the experiment.

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Chapter 2
REVIEW OF RELATED LITERATURE
Benzalkonium chloride is considered a quaternary ammonium
compound (QACs) which are cationic surfactants that are widely
used as disinfectants (BfR, 2012). In household products, they
are primarily found in fabric softeners, detergents and hair
products (Ferk, 2007). According to ScienceLab.com, benzalkonium
chloride (BAC) is mutagenic for mammalian somatic cells,
bacteria or yeasts. It contains a material which may cause
damage to the kidneys, liver, heart, gastrointestinal tract,
cardiovascular system, and central nervous system. Benzalkonium
chloride (BAC)may affect genetic material and cause adverse
reproductive effects based on laboratory experiments done on
animals.
Benzalkonium chloride may be harmful when ingested
accidentally. Animal experiments have indicated that ingestion
of less than 150 gram may be fatal or may produce serious damage
to the health of an individual. It can also produce chemical
burns to the eye and skin following direct contact. Skin contact

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is harmful as it may result to systemic effects following
absorption.
Benzalkonium chloride is an antiseptic agent which must not
be applied to areas which have not been fully rinsed as it is
inactivated by organic compounds. Application of BAC may include
disinfecting instruments and preserving drugs in low
concentration form (chemicalland).
In a study done by Ferk and his team, he studied the
genotoxic effects of benzalkonium chloride (BAC) in mammalian
and plant cells at environmentally relevant concentrations. BAC
was tested in four genotoxicity tests in the
Salmonella/microsome assay with strains TA 98, TA 100 and TA
102, in the single cell gel electrophoresis (SCGE) assay with
primary rat hepatocytes and in micronucleus (MN) assays with
peripheral human lymphocytes and with root tip cells of
Viciafaba. Results show that with BAC, a moderate but
significant effect was found at an exposure of 1.0mg/l. Findings
show that this chemical includes moderate but significant
genotoxic effects in eukaryotic cells at concentrations which
are found in wastewaters and indicate that their release into
the environment may cause genetic damage in exposed organisms.
The results show that BAC that was tested cause DNA migration

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and MN formation in mammalian and plant-derived cells but no
gene mutation in bacteria.
A disinfectant containing benzalkonium chloride has been
identified as the most probable cause of birth defects and
fertility problems in caged mice. In the cosmetic safety
database, there is moderate to strong evidence that it is an
immune, skin, and respiratory toxicant. One or more in vitro
tests on mammalian cells exposed to BAC show positive mutation
effects.
According to an article by ET CBC News, scientists at the
University of Ireland in Galway found that when they added the
disinfectant benzalkonium chloride to common bacteria called
Pseudomonas aeruginosa, increasing the amount of germ-fighting
solution over time, the bacteria learned to survive. The
researchers discovered that over time the bacteria became more
efficient at ridding their cells of both the disinfectant and
antibiotic. They also found that a mutation in their DNA made
them resistant to antibiotics.

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CHAPTER III
Research Design and Methodology
Research Design
An experimental method was used in the study to assess the
mutagenic effects of 10% benzalkonium chloride to Drosophila
melanogaster. It consists of a 10% benzalkonium chloride
concentration.
Methodology
Materials
The materials that were utilized are overripe pineapple,
sweet potato, wide-mouth bottles, cheesecloth, rubber bands,
tray, medicine dropper, gas range, pan, fork/spoon, peeler,
water, inoculating loops, cotton, ether/chloroform, dissecting
and compound microscopes, camera, high precision balance and
wild-type fruit flies.
Chemical(s)
The chemical that was used to anaesthetize the Drosophila
melanogaster was chloroform.

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Preliminary Activities
Culture Media Preparation
Overripe pineapple (Ananascomosus (l.) merr.) was used as
the culture medium for this experiment. The medium was crushed
until it is soft and placed in a sterile bottle to ensure a “no-
mold” culture media. This was also be used for the collection of
fruit flies prior to the experiment.
Collecting of Samples
The bottle with the cultured medium was placed beside a
trash bin and was left there for several hours until the bottle
was filled with fruit flies. The cheesecloth was then sterilized
and was used to cover the sanitized bottle which was secured by
the rubber bands. The collected fruit flies were left to breed
until visible larva could be seen.
Larva of Drosophila melanogaster
The larva of the collected fruit flies was used as the
parental generation when it reached its adult stage after six
(6) days.

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Preparation of culture medium with mutagen concentration:
In preparing the 10 grams, 10% benzalkonium chloride
concentration, the researchers weighed 1 mL of benzalkonium
chloride via graduated cylinder which was followed by the
addition of 10 grams mashed sweet potato. The mutagen and the
culture medium were mixed thoroughly and were placed in a wide-
mouth bottle.
Culturing fruit flies and induction of mutagen:
From the earlier collection of wild type fruit flies from
the overripe pineapple, it was cultured using the sweet potato
medium with benzalkonium chloride culture media. The fruit flies
were allowed to mate and their offspring/s were perceived from
larva to adult. Before the offspring develop into adult, the
collected fruit flies were released so that only the parental
generation will remain inside the bottle. The parents were then
allowed to mate until they produced offspring which were our
(F1) generation. When the offspring developed into adult, the
females were separated from the males to prevent them from
mating. Their phenotypes were observed and were also recorded.
They were combined later to mate and their offspring (F2)

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generation will again have their phenotypes be observed and
recorded.
Separation of Male and Female Virgins Drosophila melanogaster
and Pairing and Transferring of Male and Virgin Female
Drosophila melanogaster to a vial:
The Drosophila melanogaster that was anesthetized were
transferred in a microscope slide for clear viewing of the
structures of male and female under a microscope. After viewing,
the male and female virgin flies were then paired and
transferred to a vial that contained the cultured medium.
Observation of Visible Mutations
The adult Drosophila melanogaster of the F1 generation and
F2 generation were used in observing the visible mutations under
the aid of a microscope. These mutations were identified based
on the differences in appearance of a wild type fruit fly and a
mutant one.

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Data collection and Analysis
All of the recorded data, the observations in the phenotype
were collected for analysis. All the results will be presented
in tabular form and graphs.

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Chapter 4
Results and Discussions
This study was conducted in order to assess the mutagenic
effects of benzalkonium chloride on Drosophila melanogaster and
to observe and compare subsequent mutations between generations.
Parental Generation: 8 virgin females
5 males
Table 1: Fruitflies in the First Filial Generation (F1
Generation)
Fruitfly Number
of
Males:
Male
Frequency:
Percentage: Number
of
Females:
Female
Frequency:
Percentage:
Wildtype 4 0.2 20% 7 0.35 35%
Mutant 0 0 0 9 0.45 45%
Total
number:
4 16

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Graph 1: Fruitflies in the First Filial Generation (F1
Generation)
Our F1 generation showed us that out of the 20 fruit flies,
there were 11 wild type fruit flies and 9 mutant female fruit
flies. Out of the 9 mutant female fruit flies, there were 2
fruit flies that showed 2 kinds of mutation.
The following graph and table will show you the incidence
of mutation for the F1 generation in terms of its body color and
wing shape.
Wildtype Male
20%
Mutant Female
45%
Wildtype Female
35%
F1 Generation

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Table 2: Incidence of Mutation in the First Filial Generation in
terms of body color(F1 Generation)
Graph 2: Incidence of Mutation in the First Filial Generation in
terms of body color(F1 Generation)
Tan colored body
(Male)
20%
Tan colored body
(Female)
35%
Yellow colored
body (Female)
35%
Ebony colored
body (Female)
10%
Incidence of Mutations in the body of Fruit
Flies (F1 Generation)
Body Number
of
Males:
Male
Frequency:
Percentage: Number
of
Females:
Female
Frequency:
Percentage:
Tan 4 0.2 20% 7 0.35 35%
Yellow 0 0 0 7 0.35 35%
Ebony 0 0 0 2 0.1 10%

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The results of our F1 generation showed us that in terms of
body color, it was found that only females expressed either of
two mutant forms in this generation. Out of the total 20 fruit
flies from the F1 generation, 4 males and 7 females expressed
their tan coloration (wild type fruit fly). While there were, 9
females who showed either yellow or ebony coloration. Out of the
9 mutant fruit flies, 7 expressed the color yellow in their body
and 2 expressed the ebony color.
Table 3: Incidence of Mutation in the First Filial Generation
(F1 Generation)
Parts Number
of
Males:
Male
Frequency:
Percentage: Number
of
Females:
Female
Frequency:
Percentage:
Short
winged
0 0 0 1 0.05 5%
Blistered
wing
0 0 0 1 0.05 5%

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The results in our F1 generation showed us that in terms of
wing type and head type, it was found that out of the 9 mutant
female fruit flies, 2 of which showed two kinds of mutation in
terms of its body color and wing type. 1 fruit fly expressed a
short type kind of wing and another 1 expressed a blistered kind
of wing type.
Table 4: Fruit Flies in the Second Filial Generation (F2
Generation)
Fruitfly Number
of
Males:
Male
Frequency:
Percentage: Number
of
Females:
Female
Frequency:
Percentage:
Wildtype 4 0.09 9% 13 0.32 32%
Mutant 8 0.195 19% 16 0.390 39%
Total
number:
12 29

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Graph3: Fruit Flies in the Second Filial Generation (F2
Generation)
Our F2 generation showed us that out of the 41 fruit flies,
there were 17 wild type fruit flies and 24 mutant fruit flies.
Out of the 24 mutant fruit flies, there were 5 fruit flies that
showed 2 kinds of mutation.
The following graph and table will show you the incidence
of mutation for the F2 generation in terms of its body color and
wing shape.
Wildtype Male
10%
Mutant Male
19%
Wildtype Female
32%
Mutant Female
39%
F2 Generation

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Table 5: Incidence of Mutation in the Second Filial Generation
(F2 Generation)
Graph 4: Incidence of Mutation in the Second Filial Generation
(F2 Generation)
Ebony colored
body (Female)
10%
Tan colored body
(Female)
32%
Ebony colored
body (Male)
19%
Yellow colored
body (Female)
29%
Tan colored body
(Male)
10%
F2 Generation
Body Number
of
Males:
Male
Frequency:
Percentage: Number
of
Females:
Female
Frequency:
Percentage:
Tan 4 0.09 9% 13 0.317 31.7%
Yellow 0 0 0 12 0.29 29%
Ebony 8 0.195 19.5% 4 0.09 9%

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The results of our F2 generation showed us that in terms of
body color, it was found that out of the total 41 fruit flies,
24 mutant fruit flies expressed either yellow or ebony body
coloration. Out of 24 mutant fruit flies, 8 males and 4 females
expressed the ebony coloration. While there were 12 females that
expressed the yellow body coloration.
Discussions:
Abnormal body coloration occurred most likely due to
benzalkonium chloride affecting genes (the yellow genes),
located on the X chromosome, which blocks the synthesis of brown
pigment resulting to fruit flies with yellow bodies. Also,
according to Bruce Alberts (1985), the yellow gene (y) is
involved in pattern-specific melanin pigmentation of the cuticle
of the adult fly and of larval mouth parts of Drosophila
melanogaster and has mainly neural functions. This means that
BAC is strong enough to alter such gene for the phenotypic
observation of the mutant fruit flies that exhibited yellow body
coloration. Other abnormal body coloration is due to a defect in
their ebony gene which is responsible for the building up of the
tan colored pigments in the normal fruit fly. Ebony was found to
be exclusively localized to neuropile and epithelial glial cells

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(Richardt, 2002). The spatial localization of Ebony protein
within the adult nervous system, including the visual system and
protocerebrum, was studied in order to examine the abundance of
the protein at different times of day.Since the ebony gene is
defective, this resulted to the black pigments accumulating all
over the body thus we have obtained an ebony colored body fruit
fly. In addition, according to Wittkopp, True, and Caroll (2002)
Ebony is required to suppress some melanin formation, and is
expressed in cells that will produce both melanizedand non-
melanized cuticle. Ectopic expression of Ebony inhibits melanin
formation, but increasing Yellow expression can overcome this
effect.
Another mutation that we have observed is with the wing
type of the fruit flies. The short winged fruit flies have a
defect in their vestigial gene on its second chromosome. Flies
with short wing type have a recessive mutation (The
Exploratorium, 2013). Short wing gene is located in chromosome I
between 63.5 and 64.This gene proved to have a very pleiotropic
manifestation, on changes in the wings and the eyes being the
most common ones. The alterations in the wings are manifold; the
wings may be stretched out in an abnormal direction, not
backwards as is normal, but held out from the sides of the body
at an angle of 90 degrees. The wings may exhibit incisions of

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very different size and number; if only one incision is present
this is usually found in the medial margin of the wing, but the
medial, lateral and posterior margin may all have incisions
(Eker, 2002).
Blistered wings were also observed on the mutant ebony
Drosophila melanogaster. According to Martin, Zusman, and Li
(1999), a new laminin α chain is assigned to the locus of wing
blister which is essential for embryonic viability and is
involved in processes requiring cell migration and cell
adhesion.Blistered gene in accordance to Fristrom, Gotwalset.
al. (1994) both developmental and genetic analyses suggest that
it is required during metamorphosis for the initiation of
intervein development and the concomitant inhibition of vein
development.
A single wingless and legless Drosophila melanogaster was
perceived on the second filial generation. Wingless trait
according to Bishop, Huggler, et. al. (2010) or (mutation H) is
an autosomal recessive gene. Meaning, two copies of an abnormal
gene must be present in order for the disease or trait to
develop.A mutation in a gene on one of the first 22 nonsex
chromosomes leads to wingless trait.

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Chapter 5
Conclusions:
Based on the gathered results, the following conclusions
were made:
The results of the experiment showed that the Benzalkonium
Chloride induces mutation to the F1 generation and F2 Generation
of Drosophila melanogaster.
The observed mutations in this study were: the yellow and
ebony colored body of the fruit fly, and the short winged and
blistered wing type.
References:
Alberts B. (1985). Molecular analysis of the yellow gene (y)
region of Drosophila melanogaster.Retrieved January 14,
2013 from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC391346/pdf/pnas
00361-0222.pdf

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Bundesinstitut fur Risikobewertung. (2012). Health assessment of
benzalkonium chloride residues in food. Retrieved on
December 29, 2012 from:
http://www.bfr.bund.de/cm/349/health-assessment-of-
benzalkonium-chloride-residues-in-food.pdf
Chemical land (2012).General Description of antiseptic agent.
Retrieved on December 29, 2012 from:
http://chemicalland21.com/lifescience/phar/benzalkonium%20c
hloride.htm
Chemwatch (2008).Benzalkonium chloride. Retrieved on December
29, 2012 from: http://datasheets.scbt.com/sc-257126.pdf
Eker R. (2002).The Short wing gene in Drosophila melanogaster
and the effect of temperature on its
manifestation.Retrieved January 14, 2013 from
http://www.ias.ac.in/jarch/jgenet/30/357.pdf
ET CBC News. (2009). Disinfectant use could cause superbugs.
Retrieved on December 30, 2012 from:
http://www.cbc.ca/news/health/story/2009/12/28/disinfectant
s-superbugs.html
Ferk F. (2007). Benzalkonium chloride and dimethyldioctadecyl-
ammonium bromide (DDAB), two common quaternanry ammonium
compounds, cause genotoxic effects in mammalian and plant

La Paz, Iloilo City
28
cells at environmentally relevant concentrations. Retrieved
on December 29, 2012 from:
http://mutage.oxfordjournals.org/content/22/6/363.full.pdf
Fristrom D., Gotwals P., et. al. (1994).Blistered: a gene
required for vein/intervein formation in wings of
Drosophila. Retrieved on January 14, 2013 from
http://dev.biologists.org/content/120/9/2661.full.pdf
Huggler A., Patel G. (2010). Analysis of the Mode of Inheritance
of the Mutant White Eye Trait and Wingless Trait in live
Drosophila melanogaster and the Mapping of Sepia Eye Color
Mutation to Identify the Particular Chromosome on which the
Mutation is Located in Drosophila using Virtual FlyLab.
Retrieved on January 14, 2013 from http://by423-01-fall-
2010.wikispaces.com/BY423-01+Group+1+Report
Martin D., Zusman S., and Li X. (1999).Wing blister, A New
Drosophila Laminin α Chain Required for Cell Adhesion and
Migration during Embryonic and Imaginal Development.
Retrieved January 14, 2013 from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2148222/
Richardt, A., Rybak, J., et al. (2002). Ebony protein in the
Drosophila nervous system: optic neuropile expression in

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glial cells. Retrieved January 13, 2012 from
http://www.sdbonline.org/fly/sturtevant/ebony4.htm
ScienceLab.com (2012).Benzalkonium chloride MSDS. Retrieved on
December 30, 2012 from:
http://www/sciencelab.com/msds.php?msdsId=9923038
The Exploratorium © (2013). Mutant fruit flies. Retrieved
December 30, 2012 from
http://www.exploratorium.edu/exhibits/mutant_flies/mutant_f
lies.html
WittkoppPJ., True JR., Carroll SB. (2002). Reciprocal functions
of the Drosophila Yellow and Ebony proteins in the
development and evolution of pigment patterns.Retrieved
January 14, 2013 from
http://www.ncbi.nlm.nih.gov/pubmed/11934851

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Appendi

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Normal Wild-type Fruit Flies
Yellow-bodied Fruit flies

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Ebony Fruit flies with blistered wings
Ebony Fruit flies with proportioned body

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Hunchbacked Ebony Fruit flies
Short-winged Fruit fly Wingless and legless Fruit Fly

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Fruit flies with weird antennae
Fruit flies with acute abdomen
Solving for the 10% Benzalkonium Chloride Concentration:
Mass/ Volume = 10g x 0.1 = 1 mL of Benzalkonium Chloride mixed
with 9 mL of distilled water

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