Mistakes Were Made

Recently, I've been compiling and analyzing my data, and I noticed my trends were not all clean-cut increasing. In fact, population density actually declined in several of the groups exposed to our "medium" amount of oil, or 3 drops. Below are graphs roughly displaying the ratio of the experimental group's population density average to that of their control groups (CH meaning Chaetoceros and the number being the first or second trial and TH meaning Thalassiosira.) The x-axis is the oil drop amount and the y axis is the ratio to the standard. The data point at (0,1) represents the standard itself.

As you can see, the trends aren't all nice and pretty (and please ignore the typo in the second graph title). So I emailed Dr. Edward J. Buskey, the director of  the Gulf of Mexico Research Initiative DROPPS Consortium (whom I had previously emailed about the mysterious ghost and stretched out cells), asking if he had seen similar patterns in previous experiments from his lab and if not, if he believed my results could be due to some outside force (altitude, light level, etc.). His response was as follows:

"Your exposure levels are much higher than we have used in our experiments – your highest levels of 1660 – 2500 ppm (1.6 to 2.5 ppt) are much higher than those used in any scientific study on the effects of oil on phytoplankton that I am familiar with. I would not expect exposure to such high levels of oil to stimulate their growth."

This I considered somewhat weird. Though this amount of oil was nearly impossible to occur in a real spill, why had no researcher ever wondered what the upper limits of oil a cell could metabolize was? Was science so limited to real-world situations that people stopped wondering "Why not?" when faced with testing hypothetical circumstances? He then followed up with this:

"Counting cells can be very challenging – you need to make sure your culture is very well mixed before you sample it since diatoms will tend to sink to the bottom of a culture; you also need to make sure you count only live cells, and that they are not dead or dying cells. And you must count enough cells to eliminate potential counting errors. Fro example, if you only count 4 cells in a sample, your 95% confidence interval (your chance of making an accurate count) is +/- 100% !!  If you count 100 cells it is +/- 20%.

You need to count a lot of cells in each of your experimental treatments  to avoid chance counting errors."

But I hadn't been shaking every bottle we tested to ensure it was "well-mixed," only a select few which I thought had a large proportion of cells settled at the bottom (as in, I'd shake every bottle in one trial, but not during the next trial. I wasn't just shaking up random bottles in each individual trial). And I hadn't been differentiating between dead or living cells (it's hard to do that when A.) a cell is non motile and B.) you're basing your counts off pictures). AND most of my sample pictures usually had below 100 cells in them. Sheesh. Looks like my error margin is going to be as big as the Marina Trench is deep. 

Signing off till next time, this is Erin Butcher

We're Oil Finished Up

As of last week, my hands-on research has concluded. While I still have not calculated everything out (hey, I've got a show performing this and next weekend, and a talent show to host, gimme a break), I have begun writing my research paper and gathering various other sources and similar research papers to compare my research to. I have also begun looking into the context of the Deepwater Horizon spill, the one my experiments were vaguely modeled off of.

From what I gather watching the movie Deepwater Horizon and reading the wiki article, the spill did have (a lot of) foreshadowing. But before I get into detailing the faulty cement and enormous PSI noted in the well just hours before the explosion, let me first detail how the rig worked.

The Deepwater Horizon was a "10 year-old  semi-submersible, mobile, floating, dynamically positioned drilling rig" according to Wikipedia. What does this mean? The semi-submersible part means that the rig is fixed to the top of a two-part pontoon. One part stays above water and keeps the rig hovering above the waves. The second part is buoyant and sits below the surface of the ocean, keeping both the platoon and the rig afloat and stable. The pontoon also had thrusters attached to it that helped to keep it in position. They worked like the propellers on a ship and could be used to reposition the rig if it floated too far from the well center. This is why it's "mobile" and "dynamically positioned."

These types of rigs are used when the seabed is too far down to effectively anchor the rig to it. This means that the only thing actually anchoring the floating rig to the well below was approximately 5,100 feet (1,600 m) of pipe called a marine drilling riser. But that's just the distance to the seafloor, where the cement head of the well lies. The piping was also buried another 13,260 feet (4,000 m) under the sediment to actually reach the oil and gas, meaning in total, the pipe stretched 18, 360 feet (5,600 m) below sea level. If something goes wrong in the drilling riser, such as a leak or a pressure build-up, there was a kill line and a high pressure choke to control the situation. On April 20th, 2010, those precautions proved not enough.

Deepwater Horizon was also an exploratory rig at the time, meaning it's main purpose was solely to drill the well, and then cap it off so drilling vessels in the future could come, reopen it, and take what they needed.

On April 20th, 2010, the day the spill took place, a team of  engineers sent from the company British Petroleum (BP) were set to test the cement just recently placed at the very bottom of the newly-completed well. The cement was supposed to act as a stopper for the oil and gas, preventing it from uncontrollably flowing into the marine drilling riser while they finished drilling and capped it off, as well as a stabilizer for the marine drilling riser, to keep it upright and prevent it from moving around. The team left the rig before they could even start the test, likely because they were set to head back to BP HQ to report that the well was "safe" and ready for active oil pumping. Why the big rush? You see, Deepwater Horizon  was 43 days behind BP's set schedule. Every extra day the rig took cost the company an extra half a million dollars because the company was actually loaning the rig from another company called Transocean. Needless to say, they wanted the drilling done ASAP.

Because the cement test was never run, (therefore, because they didn't know if the cement had been laid properly so it wouldn't leak) it was decided between the remaining BP employees and the rig crew to conduct what is commonly called a "negative pressure test." This means they were going to temporarily flush out the drilling fluid (called "mud") in the kill line, choke line, and drilling riser with the lighter-density seawater and see how much pressure the seawater was going to exert on the mud in doing so. This simulates the well becoming "active," as when the oil actually starts flowing, it will act like the seawater and push up on the mud until it comes out of the drilling riser and into an oil transport vessel. If all is well in the drilling riser, then there should be no pressure detected. The mud should have to be removed manually in order to allow the water to flow up the pipe.

So in conclusion, the PSI reading on the negative pressure test should have been 0. Instead, it came out to 1,260-1,400 (sources differ as far as I've seen, but the point is it was really high). This means that the seawater was pushing up so hard on the mud, safety valves could barely hold back all the fluid from rushing up the pipe all at once. But for some reason, (a reason which none of the surviving rig crew or BP employees can agree on), the rig was never evacuated. Instead, the pressure test was considered a "success." 

The crew and employees then decided to leave some of the seawater in the riser instead of replacing it with mud again. This is a tactic that supposedly was used to preemptively help tankers encourage the oil to flow back up when they reopened the well in the future. But experienced drillers shake their heads at this, saying it was an unsafe move that would only be used save them time and money. Leaving lighter-density seawater in the riser was inviting any sort of small leak to grow bigger due to a lack of sufficient pressure within the well. 

Now it's around 9 pm. The rig sits humming atop the waves as some of the crew call their families. The work's completed, they tell them, they're going to be home soon. No one is paying serious attention to the sensors. Suddenly, there's a rise in pressure in the drilling risers. Oil and gas are leaking through the leaks in the cement at the bottom of the well into the riser. The countdown to the explosion has begun.

9:49 pm. A crew member by the name of Andrea Fleytas is looking over the positioning system of the rig on the bridge of Deepwater. She feels the floor beneath her jolt. The monitor next to her displaying the layout of the rig begins blaring magenta over nearly all of the rig sections. She panics. Magenta is the worst color to see on a drilling rig. It signals the most dangerous amount of combustible gas possible has flooded in from the well.

At approximately 9:56, the rig is abruptly consumed in fire. Boom. 11 crew members never found and presumed dead. Boom. 17 physically injured by fire and debris. Boom. 4.9 million barrels of oil doomed to bubble into the Pacific.

This explosion was classified as an accident, but there were so many preventable causes. BP would end up paying over $54 billion dollars to settle federal and state claims as of 2015, an amount hardly necessary if this truly was an "accident."

Truthfully, I don't believe there's any amount British Petroleum could have payed to make up not only for the loss of 11 valuable lives, but also the immeasurable damage they caused to the marine environment during and after the spill. Although the days immediately before the spill have been painted with blurry perspectives as to who was more responsible for ignoring the warning signs, the BP employees or the rig crew, what is undeniably true is that everyone felt the company's pressure to hurry up as the time ticked on and their profits declined. Corners were cut and safety regulations were ignored all so the company could meet its monthly quota. No amount of money can ever forgive that.

Signing off till next time, this is Erin Butcher.

Oil Unaffected

After a couple weeks break from posting, it's time I finally get back into documenting my findings. To begin, I'd like to skip over the phytoplankton and get to the more exciting second part of the experimentation Mr. Soderblom and I conducted a few weeks ago. As I blogged about before, we exposed groups of Artemia to the phytoplankton which had been feeding off the oil for a solid week. Well, when I returned the next week to observe them, I discovered that despite the contaminated groups' fecal matter appearing darker than the control groups, there were no obvious signs of toxic overload or death. Actually, none of the Artemia from any group died or appeared sickly. This is surprising for two reasons. 

One, the phytoplankton we fed them was not their preferred diet. Mr. Soderblom usually feeds these brine shrimp with Nannochloropsis, a smaller, non-motile species of algae that are more spherical in shape than the motile Tetraselmis. But, given that both are green algae, I guess it's not really much a surprise that the shrimp were still able to thrive off the substitute species. It'd basically be like switching out someone's Gatorade for Sprite. Sure, it's a soda and not a sport's drink, but it's not as severe of a change as if the drink had been switched to gasoline instead.

The second and more relevant reason is that this means that even if the petroleum levels reach an insanely high concentration in their food source, it will not cause these critters to keel over and die. It also possibly alludes to this trend continuing up the food web, with the initial exposure not really severely affecting any of the consumers too drastically. 

Something to keep in mind, though, is that this is a single generation of shrimp we're observing. It is incredibly possible that if the Artemia were allowed to reproduce, they'd develop mutations or become sterile due to the prolonged exposure to the toxic hydrocarbons within the oil. Or, on the flip side, it's also plausible to believe that because the phytoplankton (usually) increased in cell density with rising oil levels, the shrimp population would similarly increase and start a chain reaction of increased population among all levels of the oceanic food chain. (This DOES NOT mean I endorse dumping oil into the ocean in an attempt to increase marine life quantity because as seen with all the sad Dawn commercials, while oil slicks may not harm algae, they will kill birds, otters, and cute, tiny ducklings.)

Moving on from that, we also did something cool with our microscopes. We both attempted immersion viewing. Immersion viewing involves placing a drop of a viscous, clear substance with the consistency of honey on top of the slide cover. Once you adjust the 10X and 40X objectives to be in focus and have selected a cell to focus on, you move the stage down and switch your objectives to be in between the 40X and 100X. You place a drop (just one) of the substance on your slide and then fully switch to the 100X objective. Very slowly, you move the stage up till the lens of the objective becomes just submerged in the substance. Now comes the hard part. You have to refocus the objective onto the cell you originally focused on, and let me tell you, it is much harder than it seems. I was never once able to do it without Mr. Soderblom's help. But once you do have it focused, you can view the inside of the cell. Below are pictures we captured of the Nannochloropsis and Tetraselmis, respectively. On the Tetraselmis, you can just barely see one of it's three flagella that help to propel it through its environment.

Anyways, that's all the exciting news I've got. For the next trial, we're allowing the Artemia to continue growing with the contaminated algae to see how more prolonged exposure will affect them. This will probably be one of our last experiments as it is time I move on to compare my research to other previous studies. Mr. Soderblom has already started me off with a study conducted in the 1800's.

I've also taken out a book detailing the causes behind the Deepwater Horizon disaster and I plan to watch Deepwater Horizon starring Mark Wahlberg. Though the reasoning behind the actual explosion is unnecessary for my research, I would like to answer a few of my own questions about it. What's the possibility of a spill that size happening again? What events lead up to it? Were they preventable? And if it was, can I do anything, such as lower my oil and gas consumption, to prevent myself from funding companies like British Petroleum who cut safety corners like a grandmother with supermarket coupons on a quiet, Sunday afternoon? 

Signing off till next time, this is Erin Butcher.

Feed Me, Artemia

Welp, our experiments with the Tetraselmis have come to a conclusion. I haven't computed the data yet, and will update this post as soon as I do, but based solely off of visual observation, it seems as though these cultures reacted to the levels of oil in the same manner that the Thalassiosira did (with the middle amount of oil yielding the smallest cell density). While neither Mr. Soderblom nor I have come up with any solid conclusions as to why this happens, I will be emailing the GoMRI (Gulf of Mexico Research Initiative) to see if they perchance have noticed similar patterns in their research and hopefully have some theories by the time next week rolls around.

But the reason I'm so anxious to post despite having no data yet is that this week, Mr. Soderblom and I came to the conclusion that it would be fun to see how zooplankton react to the contaminated phytoplankton, which is something we originally thought I might not have time for! (Yay!) This section of experimentation will lend an insight into how the oil gets metabolized as it works its way up the oceanic food chain and help me to better contextualize the reactions of the phytoplankton cultures.

The species of zooplankton we have used are called Artemia, or in layman terms, Brine shrimp. They're pretty big, about five could fit on my nail (approximately 8-12 mm depending on age and sex), and very creepy. Their nearly translucent body allows me to see both of their compound eyes at all times, no matter the direction they're facing. Their bodies are covered with a thin exoskeleton and have a total of 19 segments, 11 of which house pairs of appendages that move a way that reminds me of millipedes, if millipedes could swim. The Artemia species is believed to have existed for about 5.5 million years. They're used for toxicology assays, aquaculture, and have even been launched into lower orbit to see how radiation impacts life (they were taken on a total of 7 missions guys). Below are pictures starring them, the first being from our own experiment, and the rest from Google.

As you can see in the first picture, we put about twenty in each tube. Then, half of the solution they're swimming in is their own water from their previous holding tank. The other half is from the phytoplankton cultures. We've filled ten test tubes, four controls, and two of each level of oil (so two tubes containing the culture with 1 drop of oil, etc.). I believe the zooplankton will react similarly to the phytoplankton with accelerated growth rates, but we'll just have to see.

Signing off till next time, this is Erin Butcher.

Teenage Mutant Phytoplankton

Alright, alright well this week was certainly interesting. As of last Friday, our last diatom cultures, the Thalassosira, reached maturity after being exposed to the oil. (I don't think I mentioned it in my last post, but we decided to use our batch of Thalassiosira to compare to the Chaetoceros since they're both diatoms and we had the culture ready to be used.) Looking at these bottles, I can tell the difference between each of the cultures is going to be much more minute.

After measuring and calculating out the cell density, I found the average density of the standard (aka, the control) cultures was 1.73x10^6 cells/mL.  The culture that had been exposed to one drop (or 166 ppm of oil) increased in cell density to 4.11x10^6 cells/mL. After what we saw with the Chaetoceros cultures, this increase not alarming in the slightest. What was surprising though was that our culture containing three drops (about 500 ppm) decreased significantly in cell density to 9.77x10^5 cells/mL. This trend wasn't consistent, as the ten drop (1660 ppm) culture showed another increase from the control at 2.44x10^6 cells/mL. Likely, the inconsistency can be contributed to either a foreign contaminate or a lack of proper aeration.

But this off-trend pattern of cell density wasn't the only alarming trait of these cultures. At 1660 ppm, we noticed some of the cells had oblonged themselves and attached to other cells. We also noticed strange "ghost" cells forming in the oil drops. Below is first a picture of a normal cell group within the 1660 ppm culture, and then an example of the stretched cells and the translucent cells at the 40X objective.

A researcher at the Dauphin Lab had some interesting theories about why the cells did this. The translucent cells could be formed from dissolved organic carbon (DOC) produced by the Thalassiosira. "In a culture, DOC congeals through random interactions such that the "sticky" DOC particles aggregate into larger particles collectively called "Transparent Exopolymeric Substances" (aka TEP).  TEP is reactive and will cause aggregations, especially with crude oil droplets," according to Mr. Jeffery Krause. He also said that depending on how we aerated the culture, the phytoplankton could have reacted with the oil. He didn't specify how, as he's only been at the lab since 2012 and didn't study the Deepwater spill personally, but he did refer me to a "Gulf of Mexico Initiative (funding agency) research consortium" that solely researches with those types of interactions.

Overall, I'd say this part of the experiment did not reveal anything about my hypothesis, but it did help to deepen my understanding of phytoplankton on a cellular level. 

Today, I'll be going back to Mr. Soderblom's to record data on the green phytoplankton we began experimenting with last Friday and have now probably reached maturity.

Signing off till next time, this is Erin Butcher.

They Kept on Growing

Our Chaetoceros cultures have reached maturity in one week. Just looking at the bottles, I can tell right away that the culture with the most contaminate, 2500 ppm or 10 drops of oil to be exact, did anything but die. The color is slightly darker than the 250 and 750 ppm cultures, indicting that instead of complete annihilation, the oil has caused an abundance of growth. 

Very carefully, we took samples of each of the contaminated bottles and placed one drop on individual slides. We captured pictures of the tiny cells, now darker in appearance because of their oil-heavy meals, and I used these pictures to calculate cell density. (For this, we calculated the area in a single field of view or FOV on a microscope at 10X, and then found out how many FOVs would be in one slide cover and multiplied the amount of cells in the FOV we captured in our picture by the ratio of a FOV to the slide cover.)

Below are pictures of the 2500 ppm culture at 10X and 40X respectively.

From the data, I was able to compute that the cell density of the 2500 ppm culture was on average 2,510,000 cells/mL. Compare this to the control cultures, which averaged a cell density of 860,000 cells/mL. That's nearly a 1:3 ratio (actually 1:2.97, but close enough).

The 250 and 750 ppm cultures came out to 1.24x10^6 and 1.31x10^6 cells/mL.

This data disproves my hypothesis of the 2500 ppm of oil killing all the plankton, but it does illustrate something amazing. A concentration of 2500 ppm of crude oil is an impossibility in a real spill. With the constant current of the ocean dispersing the oil over its surface and the lack of any boundaries (besides the shore) containing the oil to one section of the sea, the oil would never be able to remain all in one area long enough to reach a concentration that high. So the fact that the diatoms were able to not only survive but thrive under these unrealistically strenuous conditions shows that these organisms are perfectly capable of coping with spill conditions without the need of any clean up whatsoever. (Whoa.) 

Moving on, after we recorded all the necessary data, we had to discontinue our experiment cultures. The reasoning behind this is that with every day these cultures are allowed to grow, the chance of an uncontrollable contaminate such as a bacteria or virus polluting and overtaking the cultures becomes more probable. This would lead to confounding evidence as we wouldn't be able to determine whether cell death would be due to the oil or another contaminate.

For the next round of experiments, Mr. Soderblom and I decided to move away from diatoms to a green or brown phytoplankton. This is because, while diatoms are the most populous group of plankton in the Louisiana Bay area, they are not the select food source for zooplankton. Diatoms, unlike other species, have a sodium metasilicate shell covering their cell walls. This makes them harder to digest and somewhat  toxic to zooplankton when consumed in large quantities. Green and brown phytoplankton, however, are eaten by almost every species of zooplankton and even constitute the diet of other creatures of the ocean, such as fish, sponges, sea stars, and even whales.

So the two species we have decided to culture for the next round of experimentation are Chlorella vulgaris and Tetraselmis suecica (I'm not exactly sure on the Tetraselmis species, but I'm pretty sure it's that one). Both are between 10 and 12 micrometers in width, making them easy to observe, and the Tetraselmis is motile, making it fun to watch. Below are pictures of the Chlorella and Tetraselmis respectively at 10X and 40X.

We plan on exposing these buggers to oil starting this Friday.

Signing off till next time, this is Erin Butcher.

An Unexpected Turn and Experimentation

Well, as stated in my last post, Mr. Soderblom and I had hoped that the Cyclotella species would be the diatom to survive the best, as it is the easiest to observe, but alas, not everything goes as planned. Unfortunately, all of our Cyclotella cultures turned clear after one week of allowed growth, aerated or not, meaning a majority of the cells died due to unknown causes.

Our Chaetoceros cultures, however, have taken really well. Their bottles are a darkish yellow color, even the cultures that ended up taking 5 days to ship, meaning they are thriving quite well. The aeration seems to have aided in growth rate, so we will be using this method in our experiment.

We officially began experimentation today!! To start, we expanded our best culture (the one that took 5 days to ship, surprisingly) by diluting it with more medium (in a 1:1 ratio) to allow it to grow. In the end, we had 1 liter of the expanded culture. Then, we separated the culture into 5 bottles, each getting around 200mL. 

Two of the bottles we designated as controls. The other three were contaminated with the oil. Now after having sifted through some research, I found that oil concentrations in the ocean water of the Louisiana Bay were about 160-260 ppm near the time the drill was capped off. We decided that in order to get noticeably different results for each bottle, we would have one bottle be within the range of the actual spill, and two with more exaggerated levels. In the end, the bottles held 250, 750, and 2500 ppm of crude oil (or 1, 3, and 10 drops from a pipette). I hypothesize that the 250 ppm won't cause much difference, the 750 ppm will have accelerated the growth rate, and that the 2500 ppm will precipitate, turn clear, and kill all of the plankton. The oil sat on top and looks really sludgy. (I thought it was funny the researchers who gave us the sample labeled it "sweet crude").

We took pictures of Chaetoceros yesterday. They're notably smaller than the Cyclotella at about 8 micrometers by 4 micrometers. They also aren't very neatly-shaped. (The third is from Google and apparently, they enjoy sticking together but it's hard for us to tell if ours are exhibiting that behavior). (At 4X and 40X objective respectively).

We also took pictures of the Thalassiosira and plan on using this species if we have the proper amount of time. (at 4X and 40X)

As you can see, they're small and hard to count, but we'll just have to do our best.

Signing off till next time, this is Erin Butcher.