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The Effects of DEET on Bioluminescent Dinoflagellates, Pyrocystis fusiformis

 

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ABSTRACT

Puerto Rico is home to three bioluminescent bays, protected inlets that hold millions of marine bioluminescent dinoflagellates. These bays are huge tourist attractions, and swimming in them, when permitted, is popular. Often the visitors have sprayed themselves with insect repellants containing the active ingredient DEET. The purpose of this paper is to examine the effects of DEET on the ability of the dinoflagellates to emit light. Four samples of the bioluminescent dinoflagellate Pyrocystis fusiformis were tested; added to each was a different concentration of DEET solution. Increasing concentrations of DEET did have an adverse effect on the dinoflagellates and the bioluminescence measured.

INTRODUCTION

Of the seven bioluminescent bays in the world, there are three in Puerto Rico: one in Vieques called Mosquito Bay, one in the southwest called La Parguera, and one outside Fajardo called La Laguna Grande. Each of these bays is filled with high concentrations of bioluminescent dinoflagellates, unicellular algae of the kingdom Protista. These microorganisms are autotrophic and produce their own energy through photosynthesis. What is most unique about them is that they use part of this energy to emit a bright flash of light whenever they are disturbed. They will bioluminesce only during the dark part of their circadian rhythm, and only when disturbed. Therefore, in naturally occurring concentrations, the bioluminescence can only be observed at night.

The reason for this behavior is contested among experts, but the generally accepted theory is that the dinoflagellates produce the light as a defense mechanism. This "burglar alarm" theory was confirmed in a 1995 study conducted at the Marine Science Institute at the University of California in Santa Barbara (Fleischer and Case 1995). In this study, species that prey upon the dinoflagellate Pyrocystis fusiformis, such as Palaemonetes pugio (grass shrimp), Holmesimysis sculpta (mysids), and Gambusia affinis (mosquito fish) were placed into a dark tank with two different species of predatory squid,Sepis oficinalis and Euprymna scolopes. In another tank, the same number and species of squid and prey were placed into a dark tank along with a high concentration of the bioluminescent dinoflagellatePyrocystis fusiformis. The study found that there was a positive correlation between the concentration of Pyrocystis fusiformis and the number of prey eaten by the squid. On average, the squid ate 16 times more prey than the control when the concentration of dinoflagellates in the water was 20 cells per milliliter. This was because when the grass shrimp, mosquito fish, and mysids preyed uponPyrocystis fusiformis, the dinoflagellates emitted flashes of light. The squid could then use the light as a beacon, leading it to its prey. This behavior helps the dinoflagellate species as a whole; by giving away the location of the mysids, grass shrimp and mosquito fish, the squid can prey upon them more easily and thereby reduce the number of organisms that eat the dinoflagellates.

In order for the dinoflagellates to gather in high concentrations, a set of very specific conditions must be met. First, the bay must be saltwater, but it also must be protected from the open ocean so as not to disperse the dinoflagellates. Therefore, the bay must be fed by a narrow, winding canal. Second, the channel leading to the bay must be shallow so that only surface waters from the ocean, which are rich in the dinoflagellates, will enter the bay. Third, the dinoflagellates must stay at a constant temperature of about 18° to 20°C. Finally, the bay must be surrounded by red mangroves. The leaves of the red mangrove trees fall into the water and decompose, releasing vitamin B12 which is needed for the growth of the dinoflagellates. The mangroves also serve to protect the bays from ocean currents and strong winds (Golden Heron Eco Tour 2010).

Because the bays require such exact conditions in order to form, there are very few in the world, and conservationists work tirelessly to preserve these unique phenomena. The single most harmful species to these dinoflagellates are humans. Light pollution reduces the observable amount of light that the dinoflagellates produce. Global warming, caused at least in part by humans, may heat the bays beyond the tolerance level of the dinoflagellates. Currently dinoflagellates have been able to adjust to slightly higher temperatures, but at the rate that the Earth is heating, the dinoflagellates will not be able to keep up.

Most harmful to the dinoflagellates are chemical pollutants. Heavy metals from nearby cities, as well as industrial waste and sewage runoff, have been found to be disastrous for the dinoflagellates (Lapota et al., 1993). In fact, the dinoflagellates are so sensitive to chemical pollutants that Pyrocystis fusiformis has been used to create a bioassay test called QwikLite that can be used to test water for toxicity (Lapota et al., 2007).

In this experiment, the effects of N,N-diethyl-meta-toluamide (DEET) were tested on Pyrocystis fusiformis. DEET is a common active ingredient found in insect repellants and is effective in repelling ticks and mosquitoes (EPA 2010). Because mosquitoes are abundant near the bioluminescent bays in Puerto Rico, visitors often spray themselves with insect repellant. According to the website of a tour service at Mosquito Bay in Vieques, people "who have sprayed themselves with DEET adversely affect the bioluminescent bay" (Golden Heron Eco Tour 2010). This claim is made without reference to any evidence. My paper strives to answer the question, If DEET from a person's skin enters the bay, how will it affect the bioluminescent dinoflagellates?

HYPOTHESIS

I predicted that the DEET would adversely affect the luminance of the dinoflagellates. The dinoflagellates subjected to a 0.01% DEET solution would produce 10% less light than the control over the 10-day test period. The ones in a 0.10% solution would produce 20% less light than the control, and the ones in a 1.00% solution would produce 40% less light.

MATERIALS

1. Four 250-ml Erlenmeyer flasks
2. Four 50-ml flasks of Pyrocystis fusiformis and F/2 medium
3. One Vernier light sensor and Lab Pro
4. One fluorescent 13-watt bulb and small desk lamp
5. One 24-hour light timer
6. Two large black garbage bags and duct tape
7. Four 10-ml solutions of different concentrations of DEET (composed of DEET and distilled water)
8. Two pipettes

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Eye and hand slits in screen to allow for manipulation of the flasks while maintaining a dark testing area.


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Testing area with Vernier light sensor (left) secured to a fixed position so as to increase consistency of data.


METHOD

Eye and hand slits in screen to allow for manipulation of the flasks while maintaining a dark testing area.
Testing area with Vernier light sensor (left) secured to a fixed position so as to increase consistency of data.

1. Prepare the dinoflagellates by pouring 50 ml of the F/2 medium and dinoflagellate mixture into each of the four Erlenmeyer flasks.

2. Create an experimentation area of near total darkness by using the garbage bags and duct tape. I used the garbage bags to cover the open end of a shelf and cut three holes into the bag, one for each hand and the other as an eye slit. I then taped another bag over that to create a curtain. Inside the experimentation area there was never more than 0.4 lumens per square meter even when outside lights were on.

3. Place the 13-watt lamp and four flasks into the experimentation area. The bulb should be approximately 14 cm from the dinoflagellates so that the dinoflagellates will receive ~7,000 lux. This is the ideal amount of light for the dinoflagellates to produce bioluminescence (Widder 2010).

4. Set the timer to turn the lamp on for 12 hours and then off for 12 hours. I set the timer to turn the lamp on at 4 a.m. and off at 4 p.m. so that I could test at 8 p.m. All readings were taken at 8 p.m. because the dinoflagellates are most luminescent four hours after the end of the light half of the cycle (Haddock et al., 2010).

5. Let the dinoflagellates become accustomed to the cycle by letting them sit undisturbed for two days.

6. Prepare the 10-ml DEET solutions using the pipettes. For the 0% solution, drop 200 drops of distilled water into a separate container; it will be referred to as the control. To create the other three solutions, drop 198 drops of distilled water and 2 drops of DEET into a separate container, producing a 1.00% solution. Next, drop 20 drops of the 1.00% solution and 180 drops of distilled water into another container to create a 0.10% solution. Then drop 20 drops of the 0.10% solution and 180 drops of distilled water into another separate container to create the 0.01% solution. Using the above method, create another 0.10% solution and a 1.00% solution so as to have four different 10-ml solutions of DEET. In this experiment, the concentrations of DEET were chosen arbitrarily because no previous studies have tested DEET on any type of dinoflagellate. In order to create a wider scope, the concentrations increased in a logarithmic fashion; that is to say, the first concentration was 0.01% DEET, the second (0.10% DEET) was ten times greater than the first, and the third (1.00% DEET) was ten times greater than the second. The error for all concentrations of the solutions is ±0.05%.

7. Label the flasks 0%, 0.01%, 0.10%, and 1.00%.

8. Pour the DEET solutions into the appropriate flasks and swirl gently to disperse the solutions evenly. Leave the flasks in one cycle of 12 hours of light and then 12 hours of darkness before proceeding to Step 9.

9. Take illuminance (lux) readings from each culture using the Vernier light sensor and Lab Pro and by swirling the flask directly against the light sensor.

10. Record data.

11. Repeat Steps 9 and 10 every day at the same time to ensure consistency of results.

Pyrocystis fusiformis was used in this experiment because it is one of the brightest of the marine dinoflagellates. The species that can be found in the bays of Puerto Rico is called Pyrodinium bahamense. F/2 medium is an enriched seawater solution that has been found to be ideal for growing dinoflagellates (Haddock et al., 2010). It is used in this experiment as growth medium for the dinoflagellates.

The experiment was conducted over a 10-day period. During this period, the dinoflagellates received an equal amount of F/2 medium and an equal amount of light from the lamp. In order for the dinoflagellates to receive equal amounts of light, the flasks were rotated every night after the testing was conducted. The data points below represent the maximum illuminance that was given off during the time that each flask was being swirled. In order to take these readings, the computer application LoggerPro 3 was used. The light sensor received the data from the dinoflagellates, and LoggerPro 3 was used to create an illuminance versus time graph. The greatest illuminance was used in each reading, no matter what time during the test it was observed. Day 1 refers to the time 24 hours after the DEET solutions were added. Day 2 is 24 hours after that.

RESULTS

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The control sets the baseline for this data set. Over the 10-day test period, the control produced a mean of 3.88 lux per day. After the first day, the dinoflagellates subjected to the 0.01% DEET solution produced more bioluminescence than the dinoflagellates subjected to the 0.10% DEET solution. By Day 2, the dinoflagellates in the 0.01% solution had rebounded to nearly the baseline. By Day 3, the dinoflagellates in the 0.10% solution had also rebounded to approximately the baseline. The dinoflagellates in the 1.00% solution produced no bioluminescence at all for the entire 10-day test period. The graph below represents the above data as a line graph. Each concentration of DEET solution is represented by a color and a shape, as shown on the right side of the graph.

ANALYSIS

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Illuminance of Four Different Cultures of Pyrocystis fusiformis in Various Concentrations of DEET Solution Over a 10-Day Period


Illuminance of Four Different Cultures of Pyrocystis fusiformis in Various Concentrations of DEET Solution Over a 10-Day Period

The hypothesis was not supported. After Day 1, the dinoflagellates in the control, the 0.10% DEET solution and 0.01% DEET solution showed a positive increase in illuminance over time, and the dinoflagellates in the 1.00% DEET solution produced no bioluminescence at all. It is most likely that 10 ml of a 1.00% DEET solution is sufficient to completely eradicate 50 ml of dinoflagellates. Another possible explanation is that the DEET inhibited the dinoflagellates from producing bioluminescence, but this is highly unlikely because even after ten days, the dinoflagellates showed no response to a physical stimulus.

The most significant portion of the data is the Day 1 result. Both the dinoflagellates subjected to the 0.01% DEET solution and the dinoflagellates subjected to the 0.10% DEET solution produced significantly less light than the control. After Day 1, there was no significant difference between the light that the control produced and the light that the dinoflagellates subjected to the 0.10% DEET and the 0.01% DEET solutions produced. Therefore, it can be postulated that the dinoflagellates have the ability to rebound from the stress caused by DEET as long as they have not passed the tolerance threshold. The tolerance threshold is the "point at which a stimulus of increasing strength is first perceived or produces its specific response" (Webster's Dictionary 1989). In the case of this experiment, the dinoflagellates have a threshold of tolerance for DEET that, if passed, will apparently kill the dinoflagellates. The 1.00% DEET solution was over that threshold, and the 0.10% DEET solution was under the threshold. Further testing would be necessary to determine the exact point at which the dinoflagellates are harmed so much by the DEET that they cannot rebound.

Both the dinoflagellates subjected to the 0.10% DEET solution and the dinoflagellates subjected to the 0.01% DEET solution rebounded by the time the next reading was taken on the second day of the experiment, 48 hours after the DEET solutions had been added to the dinoflagellates. It is possible that both cultures rebounded at the same rate, but it is much more likely that the dinoflagellates in the 0.01% DEET solution rebounded earlier than the dinoflagellates in the 0.10% DEET solution. However, it is not possible to determine the exact amount of time it would take each culture to rebound without further testing.

In a 1993 study led by David Lapota, a marine biologist and bioluminescent dinoflagellate expert, similar results were reached using Pyrocystis lunula, a close relative of Pyrocystis fusiformis, as the test subject (Lapota et al., 1993). The study investigated the effects of tributyltin chloride (TBTCl), copper sulfate (Cu2SO4) and zinc sulfate (ZnSO4) on Pyrocystis lunula. The team found that the amount of light given off by the dinoflagellates was inversely proportional to the toxicity of the pollutant. They also found that the dinoflagellates rebound from toxins as long as they do not pass the tolerance threshold.

Experimental Limitations
This experiment was conducted outside of a laboratory and on a small scale, and there are many improvements that could be made.

1. The sample size for the experiment could be larger. Only three different test subjects and one control were involved. This was due to the high cost of the dinoflagellates. A subsequent test would involve at least three separate controls and three different tests each of the various concentrations of DEET solution.

2. More concentrations of DEET should be used; this experiment tested only three. In order to more accurately pinpoint the threshold of tolerance of the dinoflagellates, many tests would need to be conducted.

3. The concentrations of DEET in each test could be more accurate. In an ideal situation, the exact amount of dinoflagellates, F/2 medium and DEET would be known. In order for this to happen, the experiment would require a source of stock culture and F/2 medium (in my experiment both came together) as well as a method of extracting miniscule amounts of DEET instead of relying upon diluting the DEET with distilled water.

4. In my experiment, I arbitrarily chose concentrations of DEET solution. A better experiment would first test how much DEET was washed off of a person's skin during the time that they were swimming in a bioluminescent bay. This would allow the tests to better test the claim that swimming in the bays with DEET insect repellant on one's body is harmful to the bay.

5. The dinoflagellates would be kept in a sterile environment at a perfectly constant temperature with a perfectly even distribution of light. This could be obtained in a laboratory setting by placing the flasks in a biological incubator, a device that closely regulates the temperature, light intensity, humidity and CO2 in the chamber.

6. In addition to using a control, subsequent experiments should involve baseline readings for every test subject. When the dinoflagellates are first obtained from the supplier, they may produce varying levels of bioluminescence. It would skew the results if one flask of dinoflagellates produced more bioluminescence than another before the experiment began.

CONCLUSION

The results of the experiment can be summarized as follows: the bioluminescent dinoflagellatePyrocystis fusiformis has a tolerance for N,N-diethyl-meta-toluamide, also known as DEET. When levels of DEET are within this range of tolerance, the dinoflagellates have the ability to rebound from their decreased light output levels. When the threshold of tolerance is passed, it appears that the dinoflagellates are destroyed since they show no response at all when stimulated, even after ten days. The reason that DEET affects the dinoflagellates is unknown; in fact, it is still contested among scientists as to how DEET works as an insect repellant (Luukinen et al., 2010). It is most likely that because relatively large amounts of DEET are harmful to humans, DEET is also harmful to the single-celled dinoflagellates. This experiment confirms that a single exposure of DEET is harmful to bioluminescent dinoflagellates. What this experiment does not address is the issue of repetitive exposure.

In a real-life situation, the dinoflagellates would be exposed to DEET from swimmers' bodies on a daily basis. In a subsequent study, various concentrations of DEET would be added to different cultures of dinoflagellates every day. Results of this experiment, as well as experiments proposed in this paper, would aid scientists and conservationists in evaluating the policies regarding swimming in some bioluminescent bays. Results from this experiment could also contribute to the use of bioluminescent dinoflagellates as a bioassay tool. With knowledge of the effects of DEET on Pyrocystis fusiformis, patterns could be derived that help to detect the levels of DEET in marine environments.

BIBLIOGRAPHY

"Bioluminescent Bay in Vieques, Puerto Rico." Golden Heron Eco Tour. 2002. Retrieved from http://www.golden-heron.com/biobay.html

Fleisher, K., and J. Case. "Cephalopod predation facilitated by dinoflagellate luminescence." Biological Bulletin 189 (1995): 263-271.

Haddock, S.H.D., C.M. McDougall, and J.F. Case. "The Bioluminescence Web Page." 2010. Retrieved from http://lifesci.ucsb.edu/~biolum/

"The Insect Repellent DEET." Environmental Protection Agency. 2007. Retrieved from http://www.epa.gov/opp00001/factsheets/chemicals/deet.htm

Lapota, D., A.R. Osorio, C. Liao, and B. Bjorndal. "The use of bioluminescent dinoflagellates as an environmental risk-assessment tool." Marine Pollution Bulletin 54:12 (December 2007): 1857-67. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/17928009

Lapota, D., G.J. Moskowitz, D.E. Rosenberger, and J. Grovhoug. "The use of stimulable bioluminescence from marine dinoflagellates as a means of detecting toxicity in the marine environment." Naval Command Control and Ocean Surveillance Center (NCCOSC), Naval Facilities Engineering Command, 93-12127. 1993.

Luukinen, B., K. Buhl, and D. Stone. "DEET Technical Fact Sheet." National Pesticide Information Center, Oregon State University Extension Services. 2008. Retrieved from http://npic.orst.edu/factsheets/DEETtech.pdf

Santos, S. "Recipe for F/2 medium stock." 2009. Unpublished manuscript, Biology Sciences, Auburn University, Auburn, Alabama. Retrieved from http://www.auburn.edu/~santosr/protocols/SymbioCultureMediaAntibiotics.pdf

"Threshold." Webster's Dictionary of the English Language. New York: New Lexicon, 1989.

Widder, E. "Bioluminescent dinoflagellates." 2007. Retrieved from http://www.mbari.org/earth/mar_tech/EITS/18Growing%20dinos.pdf

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