OCEAN ACIDIFICATION AND BISPHENOL-A 1 · 2019-03-14 · Jessica Thummel Research Period 6 25 April...

OCEAN ACIDIFICATION AND BISPHENOL-A 1

Effects of Ocean Acidification and Bisphenol-A on Marine Life

Jessica Thummel

Research Period 6

25 April 2018


Abstract

In the world today, pH levels are decreasing in oceanic bodies as carbon emissions are

increasing. Also, due to plastic contamination within the ocean, the amount of bisphenol-A (an

endocrine disruptor) contamination is increasing. Marine ecosystems are negatively affected as

pH levels acidify and BPA contamination increases. In this experiment, gametes were taken from

male and female Echinoidea. Eggs were then fertilized under a microscope. These fertilized eggs

were then submerged in pH levels of 7.8 (with 10 microgram BPA exposure), 8 (with 5

microgram BPA exposure), and 8.2 (with 1 microgram BPA exposure, to mimic the decrease of

pH of ocean water over time). The group with 1 microgram BPA exposure and a pH of 8.2

showed little embryonic mutations, and made it to the end of the blastula stage. As the pH levels

decreased and the amount of BPA increased, more embryonic mutations were visible in early

stages of cell development. At the maximum pH level and BPA exposure, embryos could not

reach 32-cell stage and ultimately died. This is crucial as humans need to find a different energy

source to avoid ocean acidification, and humans need to recycle in order to avoid further BPA

contamination in the ocean. If this is not fixed, the future generations of marine life will not be

able to survive and reproduce.


Context

Ocean acidification is a consequential phenomena taking place today that creates an

altering effect on specific underwater ecosystems. This phenomena can be thanked by excess

carbon dioxide that is emitted into the atmosphere through burning coal and the usage of oil and

gas (Waters, 2016). As the ocean slowly becomes more acidic due to the absorption of carbon

dioxide, there exists a problem such that “increased acidity makes life more difficult for species

[snails and corals] that absorb carbonate from the water” (Ogden, 2013). Snails and corals rely

on the carbonate from the water in order to form outer skeletons for survival (Ogden, 2013). This

proposes that acidified water levels will have a direct negative impact on the formation of

skeletons and on the survival of underwater species like snails and corals. There would exist

mutations in development of future generations of snails and corals, which would cause an

overall decrease in the survival rate.

Another existing problem that affects these specific underwater ecosystems is the oceanic

contamination of bisphenol-A, otherwise widely known by the acronym BPA. BPA is an

endocrine disruptor that is used in creating plastics commonly found in households all over the

world (Fundukian, 2013). Endocrine disruptors are dangerous because they enter physiological

systems acting like a natural hormone, when in reality they are toxic and cause mutations within

body systems. Within many different studies, researchers have found that BPA effects can have

the same effect on many other different species, not just humans. The main concern for the future

is that BPA contamination of bodies of water is going to cause an increased level of embryonic

mutations, eventually causing populations to die off due to the inability to develop and adapt.


Literature Review

Ocean Acidification

Throughout the past century, the pH (potential of hydrogen) of the ocean has steadily

decreased (became more acidic) since the end of the Pre-Industrial Era. The pH level of the

ocean in the Pre-Industrial Era was about an 8.2. The pH level of the ocean today is about 8.

Although it seems like such a small decrease on the pH scale (the pH scale runs from 1, most

acidic,to 10, basic), this decrease may have negative implications on marine ecosystems. One

major factor contributing to the decrease in pH is carbon emissions. As previously described,

carbon emissions are caused directly by the burning of coal and by using gas and oil (Waters,

2016). The level of the potential of hydrogen depends on the amount of carbon emissions

absorbed by the ocean water. There's an additional aspect of this: carbon dioxide dissolves in

water, which causes a greater amount of carbon dioxide to be absorbed. Not only does the ocean

retrieve carbon dioxide through the atmosphere, but the ocean also absorbs carbon dioxide

through photosynthesis undergone by phytoplankton (Riebeek, 2008). This doubles the amount

of carbon dioxide intake, as there are two sources providing the carbon dioxide: humans and

plant-life.

Effects of Ocean Acidification on Echinoidea

As previously stated, ocean acidification implicates negative effects on marine

organisms. The specific marine organism that is going to be focused on is Echinoidea, otherwise

known as the sea urchin. Because the sea urchin is a coral, it relies on calcium carbonate within

the ocean water to form its outer skeleton. The decrease in pH of the water causes a sharp


declination in the amount of calcium carbonate within the ocean (Waters, 2016). With lacking

levels of calcium carbonate, the urchin will not be able to form its test correctly, leaving them

vulnerable to predators. In the urchin, a test is a rigid structure made up of tightly interlocking

plates (“Introduction to the Echinoidea,” n.d.). These tests can be seen in the form of spines on

the sea urchin. The spines serve the main purpose of protection of the sea urchin. If the amount

of calcium carbonate is declining, then there will be a steady decrease in the amount of sea

urchin populations present in the ocean.

Bisphenol-A Contamination in the Ocean

Bisphenol-A (BPA) is a chemical defined as an endocrine disruptor that is found in high

levels in oceanic water today. Generally speaking, the fact that BPA has made its way to the

ocean is a problem that needs to be addressed. The main reason that BPA is abundant in the

ocean is because humans discard their hard plastic trash in bodies of water, eventually carrying

the trash to the ocean. Also, BPA is abundant because humans use epoxy plastic paint to seal

hulls of ships, causing excess of this paint to run off into the ocean (“Hard Plastics Decompose,”

n.d.). This contamination causes negative implications on marine life, as well as the disruption in

the development of future marine generations.

In 2014, a study was conducted by Cassandra D. Finch, Kingsley Ibhazehiebo, et. al., to

address the significant effects that even small amounts of bisphenol-A had on marine life. In this

specific study, the researchers focused on the neurogenic effects that the endocrine disruptor has

on zebrafish during the embryonic developmental stage. The results of this experiment showed

that even a very small dose of BPA (0.0068 micrograms) caused significant neurodevelopmental

disorders in the newly hatched zebrafish population. The zebrafish sample showed to develop


hyperactivity due to the excessive firing of neurons in the brain of the fish. This hyperactivity

was simply caused by the exposure to bisphenol-A during the developmental stages, overall

causing the zebrafish to acquire negative neurogenic effects.

Figure 3. BPA exposure induces precocious neuronal birth in the hypothalamus

(from Kinch, et. al.)

The image above shows the results from the experiment previously described. The red

shows the BPA group compared to the control group. The group that had been exposed to BPA

to the left shows a significantly higher amount of neuron activity in comparison to its control.

The columns to the right of the middle image show another group of embryonic zebrafish. This

group had a decrease in neuron activity when exposed to higher concentrations of BPA. This

figure ultimately shows that the endocrine disruptor has some type of neurogenic effect on

marine populations.

Another study regarding bisphenol-A implications on marine life was conducted by

Masato Kiyomoto, Ayumi Kikuchi, Tatuya Unuma, and Yukio Yokota in 2006. These scientists

looked at the effects that bisphenol-A had on the development of sea urchins. They studied two

different types of urchins, yet still found that small amounts of BPA caused embryological


morphology. The higher the concentration of BPA, the higher the implications. As BPA

concentration increased, the smaller the amount of urchin embryos that successfully hatched.

Concluding Thoughts

Overall, these studies help support the idea that BPA has negative implications on the

development of marine life, as well as the development and viability of future marine

generations. As BPA contamination of ocean water increases, the less viable marine embryos

will be. Also, if humans continue to burn coal and use gas and oil, the more acidic the ocean will

become. Marine ecosystems will begin to die off, as there is no possible way to fight against the

environmental factors surrounding them.


Methodology

Before experimentation begins, the work area is going to be sterilized in order to prevent

contamination. The tables will be disinfected using disinfecting wipes. After disinfecting the

work area, the pH sensor will be set up. The first step in setting up the pH sensor is calibration.

The pH sensor is connected to Logger Pro, a software developed by Vernier. Logger Pro needs

to be started on a laptop. The program will identify the sensor and load a default data-collection

setup. Calibrate the pH sensor by choosing “Calibrate” from the “Experiment” menu and then

click “Calibrate Now.” Then, rinse the tip of the sensor with distilled water, and place the sensor

in the first pH buffer solution (pH buffer solution 4) until the tip is immersed. Then, type in the

first known calibration value in the edit box and click “Keep.” Repeat the previous two steps

with pH buffer solutions 7 and 10. Next, click “Set Sensor Calibration” and click “set.” Finally,

click “Done” to complete the calibration process.

After the pH sensor is calibrated, flasks are going to be set up in order to create carbonic

acid. 6 separate 1 liter flasks are going to be used in this process. 3 flasks will be filled with 1

liter of spring water and 35 grams of dissolved sea salt. The other 3 flasks will contain 1 liter of

distilled water only. Then, tubing is going to be set up to connect the flasks containing spring

water and dissolved sea salt to the flasks containing only distilled water (there will be 3 pairs of

flasks). When the tubing is set up, 1 Bonne O carbon dioxide tablet will be added to the flask

with distilled water only, which is within the first pair of flasks. Carbonation of the water will

begin on its own, and carbon dioxide bubbling will begin. Continue this bubbling process until

the flask with spring water and dissolved sea salt (within the first pair of flasks) reaches a pH of

7.8. The calibrated pH meter will be used in order to ensure that the water has an exact pH of 7.8.


1 Bonne O carbon dioxide tablet will be added to the flask with distilled water only, which is

within the second pair of flasks. Carbonation of the water will begin on its own, and carbon

dioxide bubbling will begin. Continue this bubbling process until the flask with spring water and

dissolved sea salt (within the second pair of flasks) reaches a pH of 8. The calibrated pH meter

will be used in order to ensure that the water has an exact pH of 8. Finally, 1 Bonne O carbon

dioxide tablet will be added to the flask with distilled water only, which is within the third pair of

flasks. Carbonation of the water will begin on its own, and carbon dioxide bubbling will begin.

Continue this bubbling process until the flask with spring water and dissolved sea salt (within the

third pair of flasks) reaches a pH of 8.2. The calibrated pH meter will be used in order to ensure

that the water has an exact pH of 8.2. Once all three of these desired pH levels have been reached

within each of the flasks, the tubing will be removed and rubber flask stoppers will be placed

into each of the flasks in order to maintain these pH levels.

While the flasks are prepared with proper pH levels, sea urchin tanks will be set up upon

the arrival of the actual sea urchins. Two 10-gallon tanks are going to need to be filled with

deionized/distilled water. Then, 1.2 liters of salt crystals will be added to both tanks so that the

oceanic environment can be mimicked. In order to make sure that the salinity reaches between

32-38 ppt (parts per thousand), a refractometer will be used. If the salinity is below 32 ppt, more

salt will need to be added. If the salinity is above 38 ppt, more water will be added. When the

salinity reaches between the range of 32-38 ppt, add two aquarium heaters to the tanks and set

the temperature to 22 degrees celsius. After 24 hours, check the temperature of the water again,

and continue to adjust the temperature to maintain a constant temperature of 22 degrees celsius.

Finally, place a sponge filter in each tank, and turn them on.


When the sea urchins arrive, place 7 urchins, in their bags, in each tank (tanks set up in

previous paragraph). The tanks will need 15 minutes to regulate in order to let the temperatures

reach equilibrium (22 degrees Celsius). After 15 minutes, the sea urchins will be released from

their bags.

While the sea urchins are acclimating to the tanks, bisphenol-A (BPA) solutions will be

prepared under a fume hood. Because BPA is solid at room temperature, dimethyl sulfoxide

(DMSO) will be added using a clean pipette to aid in the dissolving of the chemical. When

handling chemicals, designated supervisor will need to be present, and the safety listed on the

MSDS sheets will need to be strictly adhered to. 1 microgram of BPA will be added to the flasks

“pH 7.8”, “pH 8”, and “pH 8.2”. Then, 1 milliliter of DMSO will be added to each of the flasks

to dissolve the BPA. When the BPA is completely dissolved, enough water with the respective

pH levels of 7.8, 8, and 8.2 will be added to their respective flasks in order to make a liter

solution. Then, 5 micrograms of BPA will be added to the flasks “pH 7.8”, “pH 8”, and “pH

8.2”. Then, 5 milliliters of DMSO will be added to each of the flasks to dissolve the BPA. When

the BPA is completely dissolved, enough water with the respective pH levels of 7.8, 8, and 8.2

will be added to their respective flasks in order to make a liter solution. Finally, 10 micrograms

of BPA will be added to the flasks “pH 7.8”, “pH 8”, and “pH 8.2”. Then, 10 milliliters of

DMSO will be added to each of the flasks to dissolve the BPA. When the BPA is completely

dissolved, enough water with the respective pH levels of 7.8, 8, and 8.2 will be added to their

respective flasks in order to make a liter solution.

After preparing the BPA solutions, gametes will be extracted from the sea urchins. In

order to accomplish this, a syringe containing 1.5 milliliters of .5 molar solution of potassium


chloride will be prepared. This solution will be used in order to extract the gametes. Take an

urchin from the tank, then carefully insert the needle of the syringe through the soft tissue

surrounding the urchin’s mouth. Once the needle is inserted, the solution will be injected into the

central cavity. After the solution is injected, swirl the urchin gently so that the solution mixes

inside. When the solution is mixed, invert the urchin on a ring clamp over a petri dish with

seawater attaining the pH of 8.1 (this is the average pH level of the ocean). Within 10 minutes,

sperm will release from the gonopores in the aboral side. These previous steps in the paragraph

will need to be repeated until both gametes have been collected and are in labeled petri dishes.

Following collecting the gametes, the embryos will be fertilized. In order to accomplish this,

pipettes will be used to extract .1 milliliter of the eggs and .1 milliliters of sperm from the petri

dishes. The eggs and the sperm will be deposited onto a concave slide, into gamete solution, each

via their own micropipette.

When each embryo is fertilized, separate concave slides will be set up with the

bisphenol-A solutions. For the control group (the group with no BPA), there will be 3 subgroups

of 3 concave slides. The first subgroup of concave slides will have embryos immersed in the

water with a respective pH of 7.8. The second group of concave slides will have embryos

immersed in water with a respective pH of 8. The final group will have embryos immersed in

water with a respective pH of 8.2. Each group of slides will then be placed under a digital

microscope with a magnification of 40X. When this step is completed, a timer will be set up

timing the embryonic development for 48 hours. After 48 hours have been reached, view the

footage to see how long it took or if any mutations took place for the embryo to reach the 32 cell

stage. Then, record the time it took for the embryo to reach the 32 cell stage. For the B group, 1


microgram of the bisphenol-A solution will be placed on three subgroups containing 3 concave

slides. The first subgroup of concave slides will have embryos immersed in the water with a

respective pH of 7.8. The second group of concave slides will have embryos immersed in water

with a respective pH of 8. The final group will have embryos immersed in water with a

respective pH of 8.2. Each group of slides will then be placed under a digital microscope with a

magnification of 40X. When this step is completed, a timer will be set up timing the embryonic

development for 48 hours. After 48 hours have been reached, view the footage to see how long it

took or if any mutations took place for the embryo to reach the 32 cell stage. Then, record the

time it took for the embryo to reach the 32 cell stage. For the C group, 5 micrograms of the

bisphenol-A solution will be placed on three subgroups containing 3 concave slides. The first

subgroup of concave slides will have embryos immersed in the water with a respective pH of 7.8.

The second group of concave slides will have embryos immersed in water with a respective pH

of 8. The final group will have embryos immersed in water with a respective pH of 8.2. Each

group of slides will then be placed under a digital microscope with a magnification of 40X.

When this step is completed, a timer will be set up timing the embryonic development for 48

hours. After 48 hours have been reached, view the footage to see how long it took or if any

mutations took place for the embryo to reach the 32 cell stage. Then, record the time it took for

the embryo to reach the 32 cell stage. For the D group, 10 micrograms of the bisphenol-A

solution will be placed on three subgroups containing 3 concave slides. The first subgroup of

concave slides will have embryos immersed in the water with a respective pH of 7.8. The second

group of concave slides will have embryos immersed in water with a respective pH of 8. The

final group will have embryos immersed in water with a respective pH of 8.2. Each group of


slides will then be placed under a digital microscope with a magnification of 40X. When this

step is completed, a timer will be set up timing the embryonic development for 48 hours. After

48 hours have been reached, view the footage to see how long it took or if any mutations took

place for the embryo to reach the 32 cell stage. Then, record the time it took for the embryo to

reach the 32 cell stage.

Results and Findings

The results of this experiment show that under rather normal conditions of the ocean (pH

8.2, 1 microgram BPA exposure), the embryonic development of the Echinoidea showed rather

normal development in the blastula stage. Under future conditions (pH 8, 5 microgram BPA

exposure & pH 7.8, 10 microgram BPA exposure), embryonic development showed major

mutations, and only some embryos with the pH 8, 5 microgram BPA exposure made it to the end

of the blastula stage. With a pH of 8 and 5 microgram BPA exposure, the first mitotic division

began approximately 50 minutes post-fertilization (compared to the 80 minute norm). With a pH

of 7.8 and 10 microgram BPA exposure, the first mitotic division began approximately 20

minutes post-fertilization. This early start of mitotic division is important to note because cells

need to take the full 80 minutes in order to duplicate and communicate properly. If this does not

happen, then the embryos will ultimately rupture. After analysis of footage, a Pearson Chi

Square Test was run in order to determine the probability of association of mutations with the

environmental conditions. Pictures from trials, data tables, graphs, and Pearson Chi Square Test

results are located in the Appendix.


Discussion/Limitations

Within this project, several assumptions were made: the pH of the ocean will continually

absorb carbon dioxide, the average amount of BPA contamination and the salinity of the water

remains constant. In this experiment, the water on the concave slides had a constant salinity of 34

parts per thousand. It is noteworthy that the salinity of ocean water may actually fluctuate

between 34-38 ppt due to the complex composition of salts. This fluctuation was not noted

within the experiment. Also, the main assumption is that the pH of ocean water will continually

decrease in the future. The ocean is proven to absorb carbon dioxide endlessly; however, it might

not decrease as much as the projected levels in this experiment. Finally, the average amount of

BPA contamination was most definitely assumed. There is no definitive way to test the total

exposure of BPA in the ocean today, since it is such a vast body of water.

Conclusion

The results of this experiment support the proposed hypothesis. Under normal conditions (pH

8.2, 1 microgram BPA exposure), the embryos showed normal development with little evidence

of abnormal growth. Today, there is potential of sea urchin embryos surviving. However, as pH

decreases and BPA contamination increases, the time until the first mitotic division decreases,

meaning that cells are not replicating genetic code correctly. At the maximum pH level and

maximum amount of BPA contamination (compared to in the literature review), cells began

rupturing and ultimately could not survive. This ultimately poses the idea that future generations

of Echinoidea may not be able to even begin the early life stages in embryological development.

The implications of this research show that further research will need to be conducted in order to


deduct a solid conclusion. Possible ways this could be expanded is by testing an even smaller

amount of BPA on Echinoidea embryos. Another way to expand on this is to test each variable

independently: trials with only acidifying pH levels and trials with only increasing BPA values.

This is to ensure which variable is causing the big change in the embryological development.


References

Fundukian, L. J. (2013). The Gale encyclopedia of public health. Detroit: Gale, Cengage

Learning.

Hard plastics decompose in oceans, releasing endocrine disruptor BPA. (n.d.). Retrieved

December 21, 2017, from

https://www.acs.org/content/acs/en/pressroom/newsreleases/2010/march/hard-plastics-de

compose-in-oceans-releasing-erine-disruptor-bpa.html

Introduction to the Echinoidea. (n.d.). Retrieved December 21, 2017, from

http://www.ucmp.berkeley.edu/echinodermata/echinoidea.htm

Kinch, C. D., Ibhazehiebo, K., Jeong, J., Habibi, H. R., & Kurrasch, D. M. (2015). Low-dose

exposure to bisphenol A and replacement bisphenol S induces precocious hypothalamic

neurogenesis in embryonic zebrafish. Proceedings of the National Academy of Sciences,

112(5), 1475-1480. doi:10.1073/pnas.1417731112

Kiyomoto, M., Kikuchi, A., Unuma, T., & Yokota, Y. (2005). Effects of ethynylestradiol and

bisphenol A on the development of sea urchin embryos and juveniles. Marine Biology,

149(1), 57-63. doi:10.1007/s00227-005-0208-x

Ogden, L. E. (2013). Marine life on acid. BioScience, 63(5), 322-328.

doi:10.1525/bio.2013.63.5.3

Riebeek, H. (2008, June 30). The ocean's carbon balance : Feature articles. Retrieved December

21, 2017, from https://earthobservatory.nasa.gov/Features/OceanCarbon/

Waters, H. (2016, November 29). Ocean acidification. Retrieved December 21, 2017, from

http://ocean.si.edu/ocean-acidification

https://www.acs.org/content/acs/en/pressroom/newsreleases/2010/march/hard-plastics-decompose-in-oceans-releasing-erine-disruptor-bpa.html

https://www.acs.org/content/acs/en/pressroom/newsreleases/2010/march/hard-plastics-decompose-in-oceans-releasing-erine-disruptor-bpa.html

http://www.ucmp.berkeley.edu/echinodermata/echinoidea.htm

https://earthobservatory.nasa.gov/Features/OceanCarbon/

http://ocean.si.edu/ocean-acidification


Appendix

Pre- and Post-experimental photos from pH 8.2 with 1 microgram of BPA exposure


Pre- and Post-experimental photos from pH 8 with 5 micrograms of BPA exposure


Pre- and Post-experimental photos from pH 7.8 with 10 micrograms of BPA exposure


Data Tables

After 24 Hours

pH- 8.2, 1 μg BPA Slide 1 Slide 2 Slide 3

Time to reach 32 cell stage

20 hours 17 hours 24 hours

Mutations/Observations

Cells started division normally within 80

minutes; cells showing normal

development; most reached end of

blastula stage; altered development not applicable for this

slide

Cells started division a little earlier than

normal; cells showing normal development, however some have abnormal cell sizes; altered development

showed at 16-cell stage (10 hours in)

Cells started division within 80 minutes as

normal; showing normal development;

reached end of blastula stage; altered

development not applicable for this

slide

After 24 Hours

p-H- 8, 5 μg BPA Slide 1 Slide 2 Slide 3


12 hours 10 hours 9 hours


First mitotic division was early; started

approx. 50 minutes; cells already showing

signs of abnormal development; altered development began at

6-cell stage (approximately 4

hours in)

First mitotic division showing normal

signs, still early; at 8-cell stage the cells

began to show abnormal

development; altered development began at

6-cell stage

First mitotic division started sooner than

expected; cells immediately showed

abnormal development and

different sized cells; altered development began at 6-cell stage


After 24 Hours

pH-7.8, 10 μg BPA Slide 1 Slide 2 Slide 3


N/A*

N/A* N/A*


Cells began mitotic division within an hour (early); all

cells started bubbling and

eventually ruptured completely; altered development began

at first mitotic division (<1 hour)

A lot of the cells did not even start cell division; the ones that did ended up rupturing; altered

development began at 2-cell stage (<1

hour)

Cell division is altered; cell division is by 2; some cells

are significantly bigger than others; altered

development began at first mitotic division (<1 hour)

*None reached 32 cell stage

pH 8.2, 1 Microgram BPA Exposure

Cell Time to Reach 32-Cell Stage

1 19

2 24

3 18

pH 8, 5 Microgram BPA Exposure

Cell Time to Reach 32-Cell Stage

1 11

2 8

3 12

4 9

5 8


pH 7.8, 10 Microgram BPA Exposure

Cell Time to Reach 32-Cell Stage*

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

*None reached 32-cell stage


Data Analysis

hrs to 32 Cell

Abnormalities Observed

Pearson Chi-Squared Probability

hrs to 32 Cell

pH 8.2, 1 μg BPA 20.33 1 pH 8.2, 1 μg BPA 0.504488

pH 8, 5 μg BPA 9.60 4 pH 8, 5 μg BPA 0.000082

pH 7.8, 10 μg BPA 0 8

pH 7.8, 10 μg BPA 0

OCEAN ACIDIFICATION AND BISPHENOL-A 1 · 2019-03-14 · Jessica Thummel Research Period 6 25 April...

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Transcript of OCEAN ACIDIFICATION AND BISPHENOL-A 1 · 2019-03-14 · Jessica Thummel Research Period 6 25 April...