Clownfish Behavior and Health Effects of Increased Ocean Acidity, Salinity and Temperature

Clownfish and ClimateChange:
The Effects of Increased Salinity, Temperature, and Ocean Acidity on the
Behavior and Health of the Common Clownfish (Amphiprion ocellaris)

By April Dawson

High School Senior

Boulder High School Boulder, Colorado

Research has only recently been conducted on fish and the effect that
ocean acidification has on them. Clownfish and damsels are often used
as representative species because they are easy to keep in captivity. As
was described in my introductory video, the results of the initial
experiments with clownfish showed that the carbon dioxide had very
few physical effects on the fish but had severe behavioral effects. What
was not discussed in my video was the cause of these changes. GABAa
is a well known inhibitory neurotransmitter. When GABAa bonds to a
receptor it allows Cl- ions into the neuron which prevents it from firing.
When carbon dioxide was added to seawater the GABAa inhibitor still
able to bind to its receptor, but the distribution of ions inside the fish
was altered. When the receptor site opens Cl- ions do not enter the
cell but escape it instead. So when GABAa binds to a receptor in a
clownfish in acidic water the neuron is excited rather than inhibited as
it would be in an unaffected clownfish. We know that GABAa is being
affected because the introduction of gabazine, a GABAa antagonist,
results in normal fish behavior despite high carbon dioxide. Most
aquatic animals use GABAa as an inhibitor so this effect is likely to be
seen with fish and animals other than clownfish and damsels.

This diagram shows the GABA neurotransmitter bonding with its receptor site. The result
is Cl- entering the cell. A cell with more carbon dioxide will experience Cl- exiting the cell
rather than entering it.

My experiment will differ from past experiments in that I will combine increased ocean
acidification with increased temperature and salinity. All of these changes are expected to occur
in the near future.

The Idea is to see if these other two variables affect the behavioral changes seen in carbon
Dioxide exposed fish. I will also be interested to see if they result in more changes in the fish’s
state of health than carbon dioxide alone did.

Rather than only doing a control Vs. experimental group analysis I will also do a before Vs. after
analysis. This will provide more accurate results as to what changes occurred. To keep track of
which fish is which I took pictures of each fish from both its right and left side. I printed these
pictures in groups according to the Specific fish and then the experimental group that that fish
was a part of. Because of this design I was not able to keep the fish in groups of 6 as I had
originally planned because identifying individuals accurately would be nearly impossible. Instead
I planned to keep them in pairs and run the experiment 3 times with different fish each time.
This design has the added benefit of showing repeatability to my results.

• Experimental Conditions:
I am considering “Normal” to be the average measured value of
the great barrier reef, one of the most common places to find
clownfish.

• Normal Seawater: pH 8.2
• Past experiments tested: mainlypH 8.2, 7.8, 7.6
• My Experiment: pH 7.8 (expected by 2100)

• Normal Salinity: 35 ppt (parts per trillion)
• My experiment: 42 ppt

• Normal Temperature: 25 C
• My Experiment: 28 C

To understand what variable any resulting variations from the original
experiments are caused by I will need to test every combination of my
3 variables. To do this I will need 7 experimental groups and one
control group.

The experimental combinations are as follows with the variable(s)
written after a number being the one(s) to be altered:

1.) CO2
2.) Salinity
3.)Temperature
4.) CO2 + NA
5.) CO2 + Temp
6.) Temp + NA
7.) CO2 + Temp + NA

Hypothesis
• I expect that the same results seen in past tests with
ocean acidification will occur in all 4 tanks that I add
carbon dioxide to. So I expect the results of the tests to
be as follows:

Health tests
Weight: No change
Respiration: No change
Eating: No change
Sensory Tests
Lateralization: The fish will no longer show this trait
Net: The fish will swim towards the net rather than away from it
Food: The fish will swim away from the food rather than towards it
Tap test: The fish will always jump at the tap instead of stopping after a
few taps

• I expect to see weight loss, a drop in eating, and
quicker respiration in fish exposed to higher
temperatures. I also expect that the tanks that have
higher temperature combined with higher CO2 will see
more dramatic changes in these health results than will
the tank with high temperature only.
• I don’t expect to see any change in either behavior or
health with the increase in salinity when it is alone or
combined with the other variables. The reason I am
testing it is because I want to see if the NA+ Ions affect
the GABAa receptor/Cl- process at all. I am curious but
I don’t expect it to cause any changes.

• Keep in mind that research has found some fish
to be more tolerant of acidification than others.
This means that most fish may be affected at a
certain pH but others will show no changes.
While this makes analyzing results a little more
difficult, it does offer the possibility of selection
in the fish’s natural habitat. It was not until a pH
of 7.6 was reached that all fish were affected. At
this point many fish had lost smell completely
rather than following the opposite scent trail.

Health
Weight Test
• Weight: The weight of each
fish will be measured. I
expect that fish that are
stressed for extended periods
of time may experience
weight loss while healthy fish
will most likely increase in
weight. Fish will be fed an
excess of food at each
feeding to insure that food
availability does not affect
weight gain/loss.
• I have not found previous
research that monitored fish
weight so I have nothing to
compare this to.

Sensory
Turkey Baster Tests
• Turkey Baster Test, part 1: • Turkey Baster Test, Part 2: a
Before experimentation, the second turkey baster with a
fish were trained to expect black tip will be introduced
food when a common to the tank for the first time
turkey baster is lowered after variable exposure.
into the tank. As a result, Instead of food, this baster
the fish will bite the turkey will shoot cold water
baster whenever it enters towards the fish if they bite
the water. If the fish still it. If, within a few days, the
exhibit this behavior after fish learn to bite the normal
treatment then their long baster and avoid the baster
term memory, or the fish with the black tip, then they
equivalent associations, are will have shown the ability
in tact. to form new associations.

Sensory
T-Tests
• T-Test Part 1: All fish have what is called lateralization. This means
that when faced with a decision as to turn left or right, an
individual fish will choose either left or right the majority of the
time and this behavior will remain constant for the fish over time.
This could be compared to the human being left or right handed.
However one side is not more common as it is with humans. The
preference of each fish will be recorded using a "T" shaped
tank before the experiment and then the fish will be tested again
during the experiment to see if the treated fish will retain their
lateralization or not.

Sensory
T-Tests
• T-Test Part 2: A net, T-Test Part 3: After not
representing a predator, being fed for 24 hours
will be placed at one end
of the "T" shaped tank. food in a tea strainer (so
This will be repeated 6 that the fish can't eat
times at random ends of it) will be placed at
the tank. Fish that wish to random ends of the Tee.
avoid being caught will
swim opposite the net If the fish turns towards
even if it defies their the food despite its
lateralization preference. lateralization preference,
After treatment some fish then it retains both the
may loose the ability to
accurately avoid the net. instinct to get the food
and the olfactory sense
that allows it to smell the
food.

Sensory
Unused Tests-T Test Part 3
• T-Test Part 3: After not being fed for 24 hours food in
a tea strainer (so that the fish can't eat it) will be
placed at random ends of the Tee. If the fish turns
towards the food despite its lateralization preference,
then it retains both the instinct to get the food and
the olfactory sense that allows it to smell the food.
• This test worked for the practice fish that I tested it
on before testing any of my experimental fish.
However I had to replace the water in the T tank each
time I ran the fish through so the process took over
an hour. With 16 fish to test this was simply not
practical.

Health
Unused Tests-Respiration Test
• Respiration test: Fish breathe by pumping water through their
gills, a process that is clearly visible to the naked eye. Respiration
will be visually monitored for one minute to produce a breaths
per minute rate. In general, a stressed fish will breathe either very
quickly or very slowly while a healthy fish will lie somewhere in
between.

• This test was not completed because placing the fish in a confined
area caused their respiration rate to increase so rapidly that any
differences between healthy fish and unhealthy fish would have
been impossible to determine

Health
Unused Tests-Eating Test
• Eating Test: After not being fed for 24 hours, each fish will be offered as
much food as it will eat in one sitting. The mass of the food consumed
will be closely measured by zeroing a scale with food and recording the
absolute value of the mass of the food removed from the scale. Healthy
fish will eat larger amounts than will unhealthy fish. The mass of food
eaten will be compared to body mass of the individual fish rather than
being directly compared to the amount of food eaten by other fish that
may vary in size.

• This test was not used because when the fish were place in a confined
space where their eating could be observed they refused to take food.
This test worked quite successfully a few times for a fish that was alone
in a tank but whenever 2 fish were separated they would become too
stressed to accept food. I attempted leaving the fish alone for several
minutes before offering food but they still would not try to eat it.

Sensory
Unused Tests-Tap Test
• Each fish will be placed in a container with an automatic spinning
weight that will hit the outside of the container every 10 seconds.
Fish will jump when the weight hits the side of the tank. Over time
they will remember that nothing bad happens after the weight hits
the tank and stop reacting. A fish with no working short term
memory will not be able to make this association and jump each
time the weight hits the side of the container.

• This worked for a number of fish but many either never jumped at
the sound or jumped every time during the pretest. This offered
nothing to compare to after experimental conditions were initiated.

Results: Part 1
• Due to the time constraints of high school, applying for college, and
other extracurricular activities I was only able to test one round of fish
before having to present my findings. Thus my experiment will not be
scientifically valid until I am able to run at least 6 fish per group
(providing the experiment being run more than once) and show
consistent results. As of right now I have placed 2 fish from each of the
8 sets of variables in experimental conditions and run them through
the health and sensory tests both before experimentation and after 1
week of variable exposure.

• I also had a parasite get into my salt only tank. This killed one of the fish
and the other had to be moved to a quarantine tank for medication.
This salt only tank was unable to be tested

• I will present the data that I have collected so far but keep in mind
that it is not yet able to be accepted as scientific fact.

Weight Test-Results
• Total: Average weight change was a loss of .76%
• Tanks with CO2-lost .6% of their initial body weight on average, 21% more weight retained than the total
average. The CO2 only tank gained 1.125% of their body weight,

• Tanks with heat lost 1.27% of body weight, 67% more loss than the total average.

• Tanks with salt lost 1.1% of their body weight on average, 44.7% more than the total average. To calculate this I
doubled the values for the CO2 + salt tank since CO2 appears to have no effect on the fish’s weight

• Control tank lost 3.65% on average. However one fish picked on the other constantly and the fish that was picked
on was the one that lost an excessive amount of weight. This is a good example as to why this needs to be
repeated as many times as possible to assure accurate results.

• Note: while most of the averages showed a drop in weight, many fish gained weight as well. There were also a
few tanks that showed one fish gaining weight or remaining the same with the other fish loosing weight. This is
likely because of the clown fish’s aggression in groups. One fish will become dominant (the eventual female) and
pick on the other (male). This hierarchy can result in loss of weight from the stress of being picked on or because
the dominant fish is more aggressive during feeding. To lean more about this visit the background on clownfish
page.

• If this data were from a larger pool I would conclude that heat is detrimental to health and caused the fish to
loose weight. I would not make a conclusion about salt without a salt only tank.

Weight Data
Data
Control Heat CO2 CO2 + Heat

Fish #1 Fish #1 Fish #1 Fish #1
Before: 2.51 Before: 1.74 Before:1.71g Before: 2.25g
After:2.5 After: 1.78 After:1.72g After: 2.23g

Before: 2.45g Before: 2.38 Before:2.39g Before: 1.77g
After: 2.37g After:2.38 After:2.43g After:1.71g

CO2 + Salt Salt +Heat Salt + Heat + CO2 Heat
Fish #1
Before: 1.54g Fish #1 Fish #1 Omitted
After:1.54g Before: 2.31g before: 1.43
After:2.26g after: 1.40
Fish #2
Before: 1.65g Fish #2 Fish #2
After: 1.64g Before:1.56g Before: 1.98
After:1.5g After: 1.98

T-Test-Results
• Control: 15% average change
• 28.6% total change

• All Heat tanks average change: 27.5%, heat only: 35%
• All CO2 tanks average change: 18.75%, CO2 only: 20%
• All Salt tanks average change: 15%

• Notable: CO2 + Heat resulted in both fish having drastically
altered lateralization with one fish’s lateralization being
completely reversed. However, the CO2 + Heat + salt tank and
the CO2 + salt showed little change. As is expected with such
little data there appears to be some inconsistency.

T-Test-Percent of Time Turned Right
Data

30%
Before: 70% 10% Before: 70% 60% Before: 70% Change Before: 60% 30%
Change Change After: 40% Change
After: 80% After: 10% After: 90%

20% 10% Before: 20% 10% 50%
Before: 50% Change Before: 60% Change Change
Before: 30% Change
After: 30% After:70% After: 10% After: 80%

Fish #1
Before: 80% 20% Fish #1 20% Fish #1 10% Omitted
After: 60% Change Before: 30% Change before: 70% Change
After: 10% after: 60%
Fish #2
Before: 80% No Fish #2 Fish #2
After: 80% Change Before: 50% 40% Before: 90% No
After:10% Change After: 90% Change

T-Test

I could find no consistency in the T-test turn
results so I can not make any conclusions until
I repeat my experiment.

T-Test-Percent of Time Turned Away From Net
Data

-33%
Before: 66% -16% Before: 100% 0% Before: 100% Change Before: 66% -0%
Change Change After: 66% Change
After: 50% After: 100% After: 66%

-17% 0% Before: 50% +33% -67%
Before: 83% Change Before: 83% Change Change
Before: 100% Change
After: 66% After: 83% After: 83% After: 33%

Fish #1
Before: 66% -16% Fish #1 -33% Fish #1 -17% Omitted
After: 50% Change Before: 100% Change before: 83% Change
After: 66% after: 66%
Fish #2
Before: 100% -17% Fish #2 Fish #2
After: 83% Change Before: 100% 0% Before: 50% +33%
After: 100% Change After: 83% Change

Turkey baster tests #1 and #2
• All fish were attracted to the original turkey baster
after testing had started. I tested them 4 days into
experimental conditions and again 7 days into
experimentation with the same results both times.
• All fish were afraid to come up to the turkey baster
with the black tip when it was put into the water
initially. I tested them at 5 days and again at 7. At the 7
day test I added food to the baster and while the fish
came closer to it because they saw the food they were
still wary of it.
• No variation in results was seen for any fish

Conclusion
• Although very little can be determined from the data collected so far I found it more
important that I learned a lot about the process of scientific research.

• Improvements that could have been made:

• More tests could be run on the fish. So many tests that I planned to run were not run,
therefore; very few sets of data were left to analyze changes in the fish.
• I would use bigger tanks so that I could keep more than 2 fish in them. Even though I used 30
gallon sumps the 10 gallon main tanks I used did not allow for more than 2 fish without
deaths from fighting. Perhaps a good solution to this would be to use tank dividers.
• Higher quality heaters and pH controllers would have offered more accurate experimental
conditions. The funding for these expensive items was not there.
• I would like to run experimental conditions longer next time and test the fish several times
during this period to find out exactly how long after conditions are initiated that the fish
begin to be affected.

Thank you…

• http://www.newscientist.com/article/dn21355-
carbon-dioxide-encourages-risky-behaviour-in-
clownfish.html?DCMP=OTC-rss&nsref=deep-sea
• http://wottsupwiththat.com/2012/01/18/co2-
increases-to-make-drunken-clownfish/
• http://www.newscientist.com/article/dn21355-
carbon-dioxide-encourages-risky-behaviour-in-
clownfish.html

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