Inhibitory Effect of Different Concentrations of Dishwashing Liquid (Ultra Axion) on the

Succinate Dehydrogenase Activity of Nile Tilapia (Oreochromis niloticus)

Mida Dorothy Manayon

Group 3
S-4L

May 7, 2015

A scientific paper submitted in partial fulfillment of the requirements in Cell Biology laboratory under
Professor Cheryl Agdaca-Natividad, 2nd semester, A.Y. 2014-2015

Abstract
The effect of different concentrations of Dishwashing Liquid to the
succinate dehydrogenase activity of Nile Tilapia (Oreochromis niloticus)
was determined by measuring the rate of respiration based on the DPIP
absorbance of three different concentrations of Dishwashing Liquid (0%,
10%, and 100%). The set-up without dishwashing liquid had the highest
average respiration rate. The set-up with concentrated dishwashing liquid
had the lowest respiration rate. Therefore, as the concentration of
dishwashing liquid increases, the respiration rate of tilapia (Oreochromis
niloticus) decreases as it inhibits succinate dehydrogenase activity.

Introduction
Detergents are used to wash our belongings at home. These chemicals are usually drained
to rivers and other aquatic environment. Detergents have poisonous effects as it has components
that affect aquatic life. According to Lenntech (2015), detergents destroy the mucus layer that
protects the fish from bacteria and parasites and also, it can damage its gills.
Detergents do not only affect the external properties of fishes but also it has toxic effects
internally (Abel, 2006). Abel added that there are components in the detergent that can influence
the permeability of cells. This is due to its amphipathic properties and its interaction to the cell
and proteins. He stated that detergent will solubilize membrane proteins by partitioning it into
the membrane bilayer then binding to water-soluble proteins.
Some of the components that are present in a detergent that affects organisms are Linear
Alkylbenzenesulfonate (LAS) and Sodium dodecyl sulfate, (SDS).
Linear Alkylbenzenesulfonate is an anionic biodegradable surfactant that affects
respiration activity among the lives present in the aquatic environment. LAS inhibits respiration

by inhibiting the enzymes (citrate synthase) present in the Krebs Cycle (Nomura et al. (1997). A
study conducted by Bao-Quey et al. (1994) shows that LAS also affects cytochrome oxidase. The
inhibition is said to not affect the respiration directly but it will still affect the whole respiration
since it affect the enzyme needed in this pathway.
Sodium dodecyl sulfate is an organic compound that inhibits the succinate dehydrogenase
activity. SDS inhibits the enzyme by solubilizing it. By inhibiting succinate dehydrogenase, the
whole activity of cellular respiration will be affected since this enzyme catalyzes the oxidation of
succinate to fumarate.
The range of the activity of the detergent is reported to be affected by varying
concentration as the components will also increase when the amount of detergent increases.
In this study, the inhibitory effect of different concentrations of dishwashing liquid (Ultra
Axion) on the Succinate Dehydrogenase Activity of Nile Tilapia (Oreochromis niloticus) was
observed. It was hypothesized that an increase in the concentration of dishwashing liquid will
decrease the succinate dehydrogenase activity of tilapia.
This study aims to assess the inhibitory effect of dishwashing liquid to the succinate
dehydrogenase activity of Nile tilapia (Oreochromis niloticus).
Specifically, the study was designed:
1. to determine the effect of different concentration of dishwashing liquid to the
succinate dehydrogenase activity of tilapia; and
2. to enumerate different cellular respiration inhibitors present in the dishwashing liquid.

The study was conducted on the 30th of April 2015, Second Semester of the school year
2015-2016, at the Institute of Biological Sciences Laboratory RoomD-332, University of the
Philippines Los Baos.

Methodology
Three adult tilapias with approximately the same weight were bought from a local wet
market in Batong Malake and were used to observe the effect of different concentration of
dishwashing liquid in cellular respiration. The tilapias were placed in a container.
The tilapias were sacrificed. Their livers, a convenient source of mitochondria, were
extracted for spectrophotometry. Each liver were weighed as the basis of phosphate buffer used.
After weighing, the livers were immersed in a phosphate buffer and were homogenized using a
pre-cooled mortar and pestle to keep the enzymatic activities.
The liver homogenate was decanted in a cold centrifuge tube and was centrifuged at 3500
x g for 10 minutes. The supernatant was decanted in a cold centrifuge tube and was centrifuged
again at 4000 x g for 15 minutes. The supernatant was decanted in a cold test tube since it
contains the mitochondria and the pellet was discarded since it contains debris such as unbroken
cells, fragments of Endoplasmic Reticulum and other larger cellular components.
One mL of liver homogenate was placed in three different test tubes. The homogenate
will provide the succinate dehydrogenase since it is the only enzyme of Krebs cycle bound to
inner mitochondrial membrane. One mL succinate solution was placed in each tube that will
provide the substrate, Succinate. One mL solution of 0.01% DPIP was added in each

experimental tube. It will act as an indicator since it changes color as it accept electrons, from
colorless to blue. With this, reduction of DPIP can be measured spectrophotometrically. DPIP
was added prior to the reading of the absorbance to avoid fast reduction of DPIP.
To observe the effect of different concentration of dishwashing liquid in cellular
respiration, three different concentrations of dishwashing liquid (Ultra Axion) was used. One mL
of 0% dishwashing liquid was placed in the first tube. One mL of 10% dishwashing liquid was
placed in the second tube. One mL of 100% dishwashing liquid was placed on the last tube.
A specific blank was used on each tube. Distilled water was added in some tubes to
obtain equal volumes to their respective blanks. This is to have an accurate measurement of the
DPIP absorbance by the spectrophotometer.
The absorbance was read at 605 nm, since DPIP absorbs light at this wavelength, at twominute intervals for 10 minutes. Table 1 shows the test tube contents for the different set-ups.
Table 1. Test tube contents for the measurement of respiration by dye (DPIP) reduction.

Volume of contents (mL)

Tube/Blank
No.
1
Blank 1
2
Blank 2
3
Blank 3

Liver
Homogenate
1
1
1
1
1
1

Succinate
Solution
1
1
1
1
1
1

dH20
1
2
0
1
0
1

Dishwashing
Liquid
0
0
1 (10%)
1 (10%)
1 (100%)
1(100%)

DPIP solution
1
0
1
0
1
0

Measurement of Respiration by DPIP Reduction was computed using the formula for the
average rate in each tube:

Where R is the average rate of respiration based on DPIP absorbance changes; Ai is the
initial DPIP absorbance; Af is the final DPIP absorbance; and t is the total observation time.
The partial respiratory rates were also computed using the formula:

1
t

Where is the partial respiration rate at observation i; is the absorbance of DPIP at

observation i; 1 is the absorbance of DPIP at observation i-1; and t is the time interval.

Results and Discussion

The study was conducted to determine the inhibitory effect of different Dishwashing
Liquid concentrations on the succinate dehydrogenase activity of tilapia (Oreochromis niloticus).
Tilapia was used because it has a large liver, a convenient source of mitochondria, since the fish
is relatively large. Thus, high amount of succinate dehydrogenase, the only enzyme of Krebs
cycle bound to inner mitochondrial membrane, can be obtained. The liver was then
homogenized.
As mentioned above, different components were placed on three different experimental
tubes. The homogenized liver provides the enzyme, succinate dehydrogenase. Succinate solution

was also added to provide the substrate, Succinate. DPIP was added that will act as an indicator
since it changes color as it accept electrons. Three different concentrations of Ultra Axion
Dishwashing Liquid (0%, 10%, and 100%) were then added to the tubes.
As seen in Table 2, the three tubes have a decreasing absorbance reading of DPIP. This
means that the DPIP was reduced. But, as seen in Fig 1, the degree of the reduction of DPIP
varies for each tube. Tube 1 has the lowest absorbance and Tube 3 has the highest absorbance.
This is due to the concentration of dishwashing liquid since it is the only component that differ
from each tube.

Table 2. Absorbance reading of DPIP in three different test tubes at 605 nm for the measurement
of respiration every 2 minutes for 10 minutes.

Time
0
2
4
6
8
10

1
0.031
0.024
0.020
0.017
0.014
0.012

Absorbance
Tubes
2
0.015
0.010
0.009
0.009
0.008
0.005

3
0.014
0.016
0.008
0.008
0.006
0.005

0.07
0.06

Absorbance

0.05
0.04
Tube 3
0.03

Tube 2
Tube 1

0.02
0.01
0
0

10

Time (min)

Fig 1. Absorbance reading of DPIP in 3 different test tubes at 605 nm for the measurement of
respiration every 2 minutes for 10 minutes.

According to Abel (2006), not only detergents can damage gills but also detergents can
have a toxic effect to the internal of fish. Abel added that this is due to the influence of detergent
to the permeability of cells due to its amphipathic properties and its interaction to the proteins.
Detergent will solubilize membrane proteins by partitioning into the membrane bilayer and will
bind to water-soluble proteins. The range of the activity of the detergent is reported to be
affected by varying concentration, temperature, and additives.
As seen in Table 3, all the tubes have an increasing amount of partial respiratory rate. The
trend of the partial respiratory rate of each tube is accelerating (See Fig 2). Thus, respiration is
present in each tube.

Table 3. Partial respiratory rate of the 3 different test tubes for 10 minutes.

Time (min)

Partial Rates
Tubes
1
-0.0350
-0.0020
-0.0015
-0.0015
-0.0010

2
4
6
8
10

2
-0.0025
-0.0005
0
-0.0005
-0.0015

3
0.0010
-0.0040
0
-0.0010
-0.0050

0
2

-0.005
-0.01

Axis Title

-0.015
Tube 3
-0.02

Tube 2
Tube 1

-0.025
-0.03
-0.035
-0.04

Time (min)

Fig 2. Partial respiratory rate of the 3 different test tubes for 10 minutes.

Though respiration is still active in the tubes, the tubes average rate differs from each
other. As seen in Table 4, Tube 1 has the highest average respiration rate, with -0.0019, and Tube
3 has the lowest with -0.0009. Tube 2 has an average rate of -0.0010.

Table 4. Average rate of the 3 different test tubes based on the DPIP absorbance.

Average Rate

Tubes
2
-0.0010

1
-0.0019

3
-0.0009

Average Rate
Tube 3

Tube 2

Tube 1

-0.002

-0.0018 -0.0016 -0.0014 -0.0012

-0.001

-0.0008 -0.0006 -0.0004 -0.0002

Fig 3. Average rate of the 3 different test tubes based on the DPIP absorbance.

According to Lal et al. (1984), detergents affect respiration of aquatic life due to the
properties of its components. The Dishwashing Liquid used has the following components (as
seen on the product package): Linear Alkylbenzenesulfonate (LAS), magnesium dodecylbenzene
sulfonate,

sodium

dodecyl

sulfate

(SDS),

Lauramidopropylamine

oxide,

Myristamidopropylamine oxide, and PCMX.

Linear Alkylbenzenesulfonate, LAS, is a biodegradable surfactant that affects respiratory
function on aquatic life. According to Nomura et al. (1997), LAS inhibits respiration by
inhibiting the enzymes like citrate synthase, present in the Krebs Cycle. Based on the study
Bao-Quey et al. (1994) conducted, LAS also affects cytochrome oxidase. This inhibition will
affect the respiration rate of the fish since it will inhibit the enzymes present in this metabolic
pathway.
Another component of the detergent is the Sodium dodecyl sulfate, SDS, which is an
anionic denaturing detergent. This component disrupts membranes and denatures proteins by
breaking protein-protein interaction (Life Technologies, 2015). With this, proteins needed in
cellular respiration might be affected. Also, a study conducted by Bishop et al. (1967) shows that
SDS has an effect to the succinate dehydrogenase activity. SDS will solubilize this enzyme and
will affect the organisms respiration since the enzyme is responsible for catalyzing the oxidation
of succinate to fumarate.
These explain the drastic drop observed on the average respiration rate between
the tube without detergent and the tube with a concentrated detergent (See Fig 3). A higher
concentration of the detergent means a higher concentration of the components that affect the
respiration of the fish.

Summary and Conclusion

To determine the inhibitory effect of different concentrations of detergent on the
succinate dehydrogenase activity of tilapia, three tilapia livers were subjected to three different
concentrations of Dishwashing Liquid. The respiration for each set-up was determined by
measuring the average rate of respiration based on DPIP absorbance.
Results showed that there is a reduction of DPIP in all the tubes. Thus, respiration is
observed since free electrons from this metabolic pathway will be donated to DPIP.
As seen in Table 3, the partial respiratory rate for all the tubes was increasing. Tube 1 has
a partial respiration rate from -0.0350 to -0.0010; Tube 2 has a partial respiration rate from 0.0025 to -0.0015; and Tube 3 has a partial respiration rate from 0.0010 to -0.0050. This shows
that the rate of respiration varies on each tube. This is due to the different concentration of
dishwashing liquid since it is the component that only differ in each tube.
As seen in Table 4, the Tube 1 has the highest average respiration rate (-0.0019) among
the tubes while Tube 3 has the lowest (-0.0009). This is due to the components present in the
dishwashing liquid that inhibits the respiration of the fish.
Such components are Linear Alkylbenzenesulfonate (LAS) and Sodium dodecyl
sulfate, (SDS). LAS inhibits respiration by inhibiting the enzymes like citrate synthase, present
in the Krebs Cycle. SDS inhibits the succinate dehydrogenase activity by solubilizing the
enzyme. This affected the fish respiration since the enzyme is responsible for catalyzing the
oxidation of succinate to fumarate.

With these, it can be concluded that respiration is inhibited due to the inhibitory effect of
detergent not only to the succinate dehydrogenase activity but also to the other enzymes present
in the metabolic pathway.
Thus, as the concentration of dishwashing liquid increases, the rate of respiration among
tilapia decreases.

Computations
Average Respiration Rate
Tube 1:

1 =

0.012 0.031
= 0.0019
10

Tube 2:

2 =

0.005 0.015
= 0.0010
10

Tube 3:

3 =

0.005 0.014
= 0.0009
10

Partial Respiration Rate

Tube 1:

1 =

0.024 0.031
= 0.0350
2

2 =

0.020 0.024
= 0.0020
2

4 =

0.008 0.009
= 0.0005
2

3 =

0.017 0.020
= 0.0015
2

5 =

0.005 0.008
= 0.0015
2

4 =

0.014 0.017
= 0.0015
2

0.012 0.014
=
= 0.0010
2

Tube 2:

Tube 3:

1 =

0.0016 0.0014
= 0.0010
2

2 =

0.008 0.016
= 0.0040
2

1 =

0.010 0.015
= 0.0025
2

3 =

0.008 0.008
= 0
2

2 =

0.009 0.010
= 0.0005
2

4 =

0.006 0.008
= 0.0010
2

3 =

0.009 0.009
= 0
2

5 =

0.005 0.006
= 0.0050
2

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invertebrates. Volume 6. pp 279298
Bao-Quey, H. & Darhi, W. 1994. Effects of Linear Alkylbenzene Sulfonate (LAS) on the
Respiratory Functions of Tigerperch (Terapon jarbua). Institute of Fisheries, National
Taiwan Ocean University. pp 205-210
Bishop, D. G. 1967. The Solubilization of the Cytoplasmic Membrane of Bacillus subt ilis by
Sodium Dodecyl Sulphate. Department of Chemistry and Department of Bacteriology. pp
454-459
Lal, H. et al. 1984. Effect of synthetic detergents on some of the behavioral patterns of fish
fingerlings (Cirrhina mrigala) and its relation to ecotoxicology. Volume 32. Bulletin
of Environmental Contamination and Toxicology. pp 109-115
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https://lifetechnologies.com/ph/en/home/life-science/protein-biology/protein-biologylearning-center/protein-biology-resource-library/pierce-protein-methods/detergents-celllysis-protein-extraction.html
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