Science Fair Project: Cleaning Up Oil Spills

[Vlance]

Hi Sybee,
I will be testing the different phytol prototypes on the Daphnia (some that have avocado oil because they also worked) to see which one is less toxic. I will not be putting the motor oil on the Daphnia as it would most likely kill them. On the phytol bottle it says it "contains high purity terpenes. Must dilute prior to use based on the information in the MSDS sheets available at TrueTerpenes.com/safety." I am going to be testing the ph of the water to see when the phytol breaks down. I am going to take the ph of the water without the phytol on and just motor oil and then I will put the phytol on and see what changes. Over the next couple of days I will monitor the ph levels of the water until they get back to normal. The ph of pure phytol is very acidic so it will make the water more acidic so measuring it every day will tell me when the phytol is breaking down because the ph will go back to normal for the water. I also do not have much phytol left so I am doing the biodegrading experiment with the other prototype experiments and I want to make the experiment as accurate to real situations as possible with salt, organisms, etc. Is that ok?

Thank you for preparing me with judge questions,
Vlance

*Also for some reason my reply to your last post went to the 5th page, can you still read it?

[SciB]

Hi Vlance,

I think I have seen all your replies but if you had a question i didn't answer, please post again.

If the product information you got with the phytol said nothing about a solvent then i guess you don't need to be concerned about that.

What effect are you measuring on the Daphnia? You can easily count heartbeats if you have a microscope to use to examine them. You can also count how many eggs the Daphnia have and if any babies are hatched. If Daphnia are exposed to a toxic chemical they might stop producing eggs: https://www.eduplace.com/marketing/exps ... 6017_2.pdf

Lastly, you can simply count the number of Daphnia before and after phytol exposure. Be sure to do a control experiment where you just add water with no phytol to the Daphnia. The measurements of eggs, heartbeats and numbers of Daphnia from the control will be compared to the experiment with phytol to see what effect it has if any.

When you said that your phytol preparation was very acidic that made me wonder why. Phytol is chemically an alcohol, meaning that it has an --OH group attached to the long chain molecule. The --OH group makes phytol slightly water soluble but not acidic. I looked up phytol metabolism and found that it can be converted to another form called phytanic acid which does have the acid group --COOH attached to it. Could your phytol contain phytanic acid? Is it being converted to phytanic acid? If so, then how is the acid part being broken down so that the pH as you observed returns to neutral after some time?

The judges may ask you why you think that changing pH [please note--the correct way to write 'pH' is with a lower case 'p', followed by an upper case 'H'] means that phytol is broken down. Be prepared to show them pictures of chlorophyll, phytol and phytanic acid. Also, think about what could happen to phytol in the ocean exposed to plankton and bacteria. How would these organisms break down the phytol? You will have to do some searching and reading online to get this information, but it is out there. You could also call the company that sold you the phytol and tell them about the acidic pH and ask them if the phytol might contain phytanic acid.

If you get stuck on any technical details just ask us. That's what we are here for.

Good luck!

Sybee

[Vlance]

Hi Sybee,
I will definitely count the heart beats and amount of Daphnia that die. I did not know about counting their eggs, how would I do that? I did my pH test today. I did 2 trials for all 4 prototypes, and 3 pH tests for each prototype in each trial (I'm sorry I made it sound very confusing) and I found that no matter what the ratio of the phytol to avocado oil the pH of the water was changed to about 6.5 (I am using color pH strips so it isn't very accurate so if there was a change between all of them it wasn't visible) from 7 and there was 6 drops of the each prototype on the oil spills for each trial. I was confused about why the avocado oil wasn't affecting the pH and I looked it up and found that cooking oil doesn't have a pH. I tried looking up the phytanic acid but all I found was about it being in human bodies. I think that the organisms are going to help process the phytol and break it down. The things I need help on understanding is about the phytanic acid and how things biodegrade.

-Vlance

[SciB]

Hi Vlance,

Your project is really interesting and exciting. I'm glad you decided to expand it with the Daphnia experiment and the phytol degradation part!

Counting eggs in the Daphnia is easy as long as you have a microscope. It doesn't have to be that strong a magnification either because Daphnia are pretty large. You just have to use a small enough drop of water so that they can't move around too much. But don't let it dry or they will die. The eggs are clearly visible as you can see if you look at the link i sent you. Or you can go to Youtube and watch some videos about Daphnia that show the eggs and the heart beating.

I should have asked you what the pH of the water was after you added the phytol. Using pH paper is not very accurate and 0.5 pH units from 7.0 may not be enough of a difference to be reliable. The avocado oil may cause problems too because it is not water soluble. You cannot easily measure pH of a non-water solution because what you are measuring is the hydrogen ions and these are present in aqueous solutions.

How are you doing the pH measurement? Where are you putting the pH paper? Remember what I said--phytol is NOT very soluble in water so the pH paper may not give an accurate reading. How long after you put the phytol on the water do you read the pH?

Here's a good product information site from Santa Cruz Biotech: [PDF]Phytol - Santa Cruz Biotechnology
datasheets.scbt.com/sc-250719.pdf

Remember that phytol can be toxic so use it with gloves and safety glasses and dispose of any waste carefully.

You are right that phytanic acid is usually formed by biological action on phytol. I thought of this chemical when you said that your water was acidic after you added the phytol. If the chemical product that you received from the company contains some phytanic acid then that might explain the acidic reaction. Otherwise, there would be no conversion of phytol to phytanic acid without some organism being present--bacteria in sea water, for example.

Be careful what you conclude from your pH measurements. Phytol may be degraded to phytanic acid or something else in the ocean, but probably not in a bowl of water. If you consistently measure a large pH change when you add phytol, then maybe your phytol already contains phytanic acid. I don't know how else to explain it.

I hope this all makes sense to you. Science is always a process of self-education. What we learn in school is basic concepts. Afterwards it is up to us to master all the little details and exceptions--but that's what makes science so much fun and exciting!

Keep asking questions and we'll keep trying to help you understand.

Sybee

[Vlance]

Hi Sybee,
Before I added the phytol (ocean water with oil on it) the pH was 7. I also checked the pH before I put the oil on and it was also 7. After I put the phytol on the ocean water the pH appeared to be around 6.5. This was consistent after 3 pH tests for two trials of pH testing for all prototypes. Today I did another round of pH testing and I found that all of the tests came out as 7 (the pH tests were definitely a different color than yesterday) showing that the phytol had broken down or there was a very small trace left. I will do another test tomorrow to see if the results still come out as 7. I took the pH of the water about 5 minutes after I put it on because I wanted to know right off the bat what it was. I am putting the pH paper in the middle of the container of ocean water with oil and the prototype solution where I know there is definitely ocean water, oil, and the solution. When handling the phytol I wear a smock, gloves, goggles, and a neck warmer that I use to cover my mouth and nose so I don't inhale it. I think that there could possibly be phytanic acid in the phytol. I think it also depends where the people are getting the phytol. If they get it out of some marine algae their are definitely going to be some organisms in it and from the website you sent me a link to it said that it had a purity of about 96%. This made me think that there is something else in there and I think it could be some organisms that are possibly turning the phytol into phytanic acid.

I also wanted to ask you how I do the tests on the Daphnia? How many tests do I do and what part of my different solutions do I do on them and how much water should they be in? I also was wondering if I could watch the Daphnia while they were in a petri dish because I am always accidentally squishing them under the slides. Tell me if some of my questions don't make sense.

-Vlance

[SciB]

Hi VLance,

I wish everyone wrote as clearly as you do. It would make it so much easier to answer questions!

Ok, Now about your pH measurement...
I can understand why you want to put the pH paper in the center of the bowl, but i am concerned that the oil may alter the way the dye in the pH paper works. The fact that you get consistent readings from one measurement to another is very good. That's what a scientist likes to see. Try measuring the pH around the outside of the slick where the phytol is.

I wish I could watch you doing the experiment because there's always some little detail that i forget to ask you. Tell me again step-by-step how you do it:

1. How much water is in a bowl
2. What is the diameter of the water surface
3. How much oil do you add (you pipet the oil in the center of the water, right?)
4. What is the maximum diameter of the oil slick on the surface of the water?
5. Do you wait a few minutes after adding the oil so that it has a chance to make a slick?
6. How much phytol do you add and where do you pipet it?
7. How much smaller is the oil slick after the phytol has worked on it?
8. How long do you allow the phytol to work on the slick before you measure its new diameter?
9. Do you measure the temperature of the water? You should because the temp affects the viscosity of the oil and how quickly the phytol coagulates it.

OK. I think that's all the questions I have for you.

Now, about Daphnia. Don't use a cover slip or you will squash them on a regular slide. If you have to use a cover slip you need a well slide. A well slide has a depression cut into the glass that allows room for larger organism like Daphnia to swim around freely and not get mashed by a cover slip. But you really don't need to use a cover slip. Use your pipetter to catch one and then put it on a regular slide. Don't cover it but try to make the drop as small as possible so the Daphnia can't swim around so much.

Go to Youtube and watch some videos about Daphnia and you will see exactly how to count their heartbeats. Their bodies are clear so the organs and the eggs are easy to see. Here's a good video from Carolina Biologicals, the ones who sell Daphnia magna: https://www.youtube.com/watch?v=HhOUwlOdxkA

Your next question is the tough one--how much phytol do you use to test for toxicity and how long do you expose them to it. My answer is that I don't know. What I would have to do if I were doing this experiment would be to put at least three Daphnias (so you can average your results) in a small container of water and add a measured amount of phytol. If you measure the volume of the water in the small container you can calculate how much phytol to add so that the ratio of phytol to water is the same as you used in the oil slick experiment. Now, how long do you let the Daphnia swim in the water with the phytol? That is something you have to determine experimentally. If the chemical alters their heart rate then you may see it right away, but the effects on egg production or survival could take several days exposure. Measure the heart rate of all three Daphnias before you add the phytol, then pipet it on the surface, wait 15 minutes and check their heart rate again.

I'm sure you'll have more questions, so write them down as you think of them and then post them and I will answer them one by one. Oh, and don't forget to answer MY questions so I can see exactly what you did in the oil slick experiment.

Good luck! When is the due date of your project?

Sybee

[Vlance]

Hi Sybee,
Ok, tomorrow I will try testing the pH of the outer rim and see if it is the same as the center. Now I will answer your questions:
1. There is about a 1/2 cup of water in the 12cm in diameter plastic container.
2. I think the diameter of the water surface is about 12cm.
3. I take a pipet and put one drop of oil in the center of the container, and it spreads.
4. The maximum diameter of the oil spill (all of them come out to the same diameter) is 10cm. There is a very small amount of room between the edge of the container and the plastic container but there is enough room to put the phytol solutions on.
5. I wait about a minute or so before I add the solution because the oil is no longer spreading.
6. I add about 6 drops (no sure how much a pipet drop is) and put 1 in the 4 corners of the container and 2 towards the middle of the oil slick.
7. The solutions make the diameter of the oil slick segments (it breaks the oil into segments) after an hour, maximum 3cm and minimum, make some segment's diameter I can't measure (too small). I measure the maximum and minimum diameter of the oil segments to get a range because there are many segments and they all range in various sizes.
8. I leave the solutions on the oil slicks and measure them after 5 minutes, 30 minutes, and 1 hour.
9. I don't actually measure the temperature of the water but all of the water is in the same container in the same place before it is divided into containers, and then all the containers are in the same room and temperature areas after the water is divided up. I am very sure that all the water is the same temperature. I also have done my experiments outside in about the same temperature each time and from my many tests the oil hasn't seemed to have changed.

Thank you for the information on what to do with the Daphnia. I want to make sure that I have all the details you told me right. So I will get a slide and put about 3 Daphnia on it and find the volume of the water in my containers that I have done my experiments in and scale it down to know how much phytol goes on them. I think that I will check their heart rate the same times I measured the diameter of oil segments 5, 30, and 60 minutes. I think that because I am not supposed to inhale the phytol solutions I will put the slide (without a top on) in a petri dish to look at them safely. That way I can keep them and look at them over many days. I also am not sure what to do about feeding them. If I observe their egg count over many days will they starve to death?

Also my project is due either April 11th or after April vacation (I have to clarify with my teacher) so that is why I am finishing everything up this week.

Thank you for all your help,
Vlance

[SciB]

Thanks for the quick clarification. Now i understand how you did the experiment and can make better suggestions.

My first one would have been--don't put phytol in the center of the oil slick, but around the edges. You don't want to split the oil up into a lot of smaller areas but make it contract into as small an area as possible so it can be cleaned up more efficiently. You probably have used up almost all of the phytol, but if you can do a couple more tests, try just adding phytol around the slick and measuring the diameter before and after.

The water temperature is one of your independent variables and you need to know what it is so you can report it. Get an inexpensive thermometer. Digital ones are the easiest to use. You can get them at a store that sells aquarium supplies or buy online.

It would be very useful to know the volume of oil that you used. Does your school lab have a small graduated cylinder that you could use to measure one milliliter (ml)? What I would like for you to do is use the pipet to drip oil into the cylinder and count the drops until you reach 1 ml. Then, by dividing the number of drops into 1 ml you will know the size of your drop and how much oil is on the surface of the water. For example, if it takes 10 drops to equal 1 ml, then each drop is 0.1 ml. If you want to be really scientific you can express the volume in microliters. Each milliliter (one thousandth of a liter) contains 1000 microliters (one millionth of a liter), so 0.1 ml = 100 microliters (100 ul). The 'u' is supposed to be the Greek letter, 'mu', but i don't think this text editor can do Greek letters. Mu means 'micro' in scientific notation, just like 'm' stands for 'milli'.

If you can also measure the volume of a drop of the phytol, that would be really helpful. You can't assume that the drop size for phytol will be the same as for oil because they have different viscosities and surface tension. Use your 1 ml vessel and count drops again. Make sure the vessel is clean so you can reuse the phytol. Calculate the volume of one drop of phytol in ul.

Now when you do your report you can say that a specific volume of phytol was able to cause a specific contraction of an oil slick produced by a specific volume of oil in a certain length of time. These 'specifics' are very important to scientists who might want to reproduce your experiments. When you write a scientific paper, you have to include a Methods section and that is where you put all the details of chemical types, suppliers, volumes, temperatures, times, etc.

Daphnia eat algae mostly, so I have kept them in an aquarium under lights. Carolina Biologicals sells Daphnia magna cultures and their food: https://www.carolina.com/daphnia/daphni ... hnia+magna
I would expose them to phytol in the same containers that you did the oil slick experiments. That way you eliminate one variable that could affect your results.

You can also get the well slides ('concavity' slides) from Carolina: https://www.carolina.com/browse/product ... ubmit=true

You will need to do the heart beat measurements individually, so get the slides that have three wells so you can put one daphnia in each well. Be sure you watch the video on how to do this. You might want to get some extra disposable pipets if you don't have enough. I would not leave the daphnia in slides. They will be stressed and maybe die. Keep them in your container with the phytol and just take them out to check them. That way there won't be any phytol on the slide, although you can put the slide into a Petri dish if the microscope objective lens can still work through that distance. You want to use a low magnification, like 40X, but the lens still has to be a certain distance from the slide and if the cover of the Petri dish is in the way then you won't be able to use it.

I think I have answered all your questions, but if not, I'll be here for your next post.

Sybee

[Vlance]

Hi Sybee,
I do not have enough phytol to do another test of my solutions. I need the solutions for the Daphnia now. I think that I can justify what I did with putting the phytol in the middle though. If the container represented the ocean that oil slick was gigantic and it would not have come together just by putting it on the outside. When I first did one of my tests I put a couple drops around the outside and it wasn't able to reach everything, but by putting some in the middle all of the oil contracted. It didn't go into one giant clump but the segments became much smaller so that they can be isolated and treated very fast.

I can definitely use a graduated cylinder to find the volume of the what amount of solution and oil I am putting on the water.

I actually bought the Daphnia from Carolina Biologicals. Will the culture come with their food?

I am confused by what you are saying about putting the Daphnia into the containers I did the slick in. I won't be able to put them under the microscope in the containers. Do you mean while I wait I put them in plastic containers? I will put the slides the Daphnia are on in a petri dish to look under a microscope to look at and then I will put the slide in the plastic container. Do that work?

I also want to ask if in a previous post I was on the right track for why there might be phytanic acid in the phytol.

I took the pH again today and found no difference between the outer edge of the water in the container and in the center.

-Vlance

[SciB]

Hi VLance,

Sorry, I didn't mean to leave out the details. What I was thinking, and you may have a better idea, was to put three Daphnia in spring water in one of your cups to do the treatment. What I forgot to say was that in order to check the heartbeat you need to suck them up with a pipet and put each one into one of the wells of a slide as it shows in the video. If you don't have time to get the well slides, just pipet a single drop onto a regular slide but don't put on a cover slip.

Check the heart rate several times before you add the phytol then wash the Daphnia from the slide back into the cup and add phytol. After a certain length of time (15 minutes?) suck up the Daphnia in the pipet, put them on a slide and check the heart beats again. When you are done, return them to the cup carefully.

I don't know if this is the best way to do the experiment, but it seemed like the closest to the conditions that you used for the oil slick, so that's why I suggested it. The idea is to keep the independent variables as consistent as possible.

As far as I know, Carolina Biologicals does not send Daphnia food with the culture that you purchase. They sell it separately. Here is their guide to caring for Daphnia: https://www.carolina.com/teacher-resour ... tr10492.tr
If you don't want to take the time to order their Daphnia food you can just sprinkle some baker's yeast in the water as they suggest--just don't use too much (read the Guide on feeding).

About the phytanic acid contamination. I would call the company that makes the phytol and tell them that you measured a slightly acid pH in the water to which you added phytol and ask them if they thought this could be due to phytanic acid. You can also ask them where they extract the phytol from and whether they think some of it could be degraded to phytanic acid. I don't see any other explanation for the acidic reaction, do you? The only way you will know the answer to this question is to talk to technical support at the company directly [NO email] and ask them. Scientists have to talk to tech support people frequently because their products don't always work the way they say. The companies welcome feedback from researchers because sometimes there is a serious problem with a batch of chemical and the company has to recall it. This is rare but not impossible.

Hope this helps.

Sybee

[Vlance]

Hi Sybee,
I did my tests on the Daphnia. I used 3 Daphnia for each prototype and found that when there was more phytol or pure phytol it decreased the Daphnia heart rate after 30 minutes and an hour after the solutions were put on. I checked the Daphnia heart rate at the same times that I checked the oil segments in my other tests, 5, 30, and 60 minutes. I was wondering if 3 Daphnia was enough to prove my testing. All the Daphnia either had the same or very close to the same heart rates for each check in so I think that 3 Daphnia is good but I wanted to get your opinion.

-Vlance

[SciB]

Congratulations! Your results sound good. Three runs per experiment is usually sufficient. Do you know how to calculate standard deviation and standard error of the mean for statistical analysis of your data? If not, I would recommend a quick visit to Google to learn how to do that. There are websites that will calculate these statistical values for you. All you have to do is put in your three readings and the app will average them and give you the standard deviation. This statistical measurement allows you to be more confident in saying whether two sets of readings are significantly different or not.

In your case, this is very important because you tested phytol on the Daphnia and you want to be able to use statistical support to state that there is or is not an effect of phytol on their heart rate, eggs or survival.

If you need help with this let me know. It's not hard, but if you haven't had statistics in a math course yet then it will be unfamiliar and maybe confusing.

Sybee

[Vlance]

Hi Sybee,
I was wondering when averaging the heart rate of all of the Daphnia do I round to the nearest whole number or do I keep the decimals?

Average Heart Rate of Daphnia for all Prototypes
Prototype Average Heart Rate (beats per minute)
Control 149
Prototype 1 (1:3) 149
Prototype 2 (2:2) 149
Prototype 3 (3:1) 145
Prototype 4 (4:0) 137

This is what my average table looks like now (the tables lines did not paste with the numbers and the table posted weirdly. The Control Daphnia average heart rate was 149.)

Vlance

[SciB]

That's great, Vlance! Your Daphnia heart rates for controls are right where they should be. Did you notice any eggs when you were examining them?

Just report the heart rate to three significant figures (or two if it falls below 100 bpm). When you average the counts, round up if it is above 0.5 or leave as is if it's 0.5 or below. Omit the decimal in your report because you cannot have a fraction of a heart beat.

I'm confused by your use of the word 'prototype'. To me a prototype is the first or original model of something, like the old flip-top phones were the prototypes of today's smart phones. Can you explain what you mean?

Didn't you say that your project was due Apr. 11, 2018? Are you still working on it? Let me know if you need help.

Sybee

[Vlance]

Hi Sybee,
As you can see the pure phytol dropped the heart rate of the Daphnia to low levels out of the range established by the control group, so therefore I am recommending Prototype 1 to be used because although it isn't as fast at contracting the oil as others, it has less phytol and did not hurt the Daphnia. I was actually unable to count the eggs as not any of them had visible eggs in them.

My science teacher told me to call all the different solutions I create prototypes. So when I refer to Prototype 1 I am talking about the first solution I created that is 1 milliliter phytol and 3 milliliters avocado oil mixed together. When I refer to Prototype 2, it is the second prototype I created that is 2 milliliters phytol and 2 milliliters avocado oil mixed together. Prototype 3 is the third prototype I created that is 3 milliliters phytol and 1 milliliter avocado oil mixed together. Lastly, Prototype 4 is just pure phytol.

I actually was incorrect about the date my project was due. My lab report was due today, and I will be presenting to my class on April 23rd. Then the Regional Science Fair is on May 4th.

I was hoping that you could give me some insight on how to present to the judges. Last year I went to the Regional Science Fair and I used cilantro to clean lead out of the water (it worked) but I didn't have enough testing because the professional testing cost so much (only 1 round of tests). I used note cards to know what I was going to say, but I don't know if judges like this or not. I also had a problem where one of my judges made me skip my speech and go to the conclusion because they felt it was too long. How should I handle a situation like that? If it happened again do I give in or try to continue my speech?

Vlance