Day 35: The Case of the Missing Phosphorus

"The most beautiful thing we can experience is the mysterious. It is the source of all true art and all science. He to whom this emotion is a stranger, who can no longer pause to wonder and stand rapt in awe, is as good as dead: his eyes are closed." - Albert Einstein

As I'm analyzing some of the results of my first experiment, I have come across a mystery: what is happening to the phosphorus? This question, if not solved, may stall the completion of my experiment, the experiment which is the ultimate goal of my summer up here. The experimental questions, which nutrient limits the growth of phytoplankton and/or benthic algae, are mysteries in themselves that I am trying to solve. But along the way, there is this interesting problem with the phosphorus results and it is now the phosphorus that draws my attention. I cannot proceed to the next part of the experiment without solving this question, and yet proceeding to the next part may not be as exciting as trying to solve this question. It is the process of discovering and then trying to solve mysteries that makes science wonderfully compelling and intriguing. Though it is frustrating to discover that something went wrong with the methods, it is so much fun to figure out what. And so I have launched into a mystery, the case of the missing phosphorus, where the clues are chemical concentrations, and the tools, chemistry and, of course, logic.

First, I have to take you back to days 13 through 17 when I was running this experiment. Cue harp music....

• Step 1: Take core
• Step 2: Prepare the microcosm
• Step 3: Acclimate the microcosms in the pool
• Step 4:Spike the microcosms with three different nutrients at three different concentrations, this being Phosphorus, Nitrate, and Ammonium at 10 times the maximum lake concentrations of the nutrients, 20 times, and 50 times. 
• Step 5: Take a sample from the "before" microcosm chambers that will be analyzed for Chl a and other plant pigments from both the water to catch the phytoplankton and the top layer of sediment to catch the benthic algae. 
• Step 6: Incubate the microcosms in the pool and take water samples at specific times (before, 0 hours, 6 hours, 10 hours, 24 hours, 48 hours, and 72 hours) and filter the samples to see what the concentrations are of the nutrients (P, N, PO4, NO3-, and NH4+) 
• Step 7: Take a final sample for the Chl a and other pigments to see if the phytoplankton and/or the benthic algal mat took up the nutrient additions
• Step 8: Send a bunch of centrifuge tubes and scintillation vials to a lab to be analyzed in a mass spectrometer to determine the concentrations of the nutrients in the water

That should bring you up to date. I got the results of the water samples back a few weeks ago, when I started running some of the stats on them. One of the results was really bizarre, the phosphorus. I got back the results for the SRP - soluble reactive phosphorus which is the inorganic phosphorus in the form of PO4, called orthophosphate, and is the form of phosphorus that is immediately available for algae to uptake. The first thing I did with the data was graph it, which you can see below. 

This is a line plot of the concentration of phosphorus , specifically SRP, over time in the containers that I spiked with phosphorus. I spiked 2 containers each of lake water at 10x, 20x, and 50x, and also spiked a two containers filled with DI at the 10x concentration, and had two containers as controls with no spikes. Several things you can notice right away. First, we are dealing with really small concentrations.  Below 0.001 mg/L, we can't even detect phosphorus in the water, so this is really small. This roughly means that there is 1 molecule of PO4 for a billion, or 1,000,000,000, molecules of H2O, or you can think of it as one drop of water in a swimming pool. Second thing to notice is that all of these containers are doing roughly the same thing, when we expected each of them to do different things.

This is a plot of the NH4 concentrations to give a comparison to the SRP plot. This is the type of graph we expected to see. At time 0, the nutrient concentration is at the same concentration as what I spiked it at for all the different concentrations. As time passes, all the spiked containers that had lake water in them showed a decrease in the concentration of the nutrient (the purple, light blue, and yellow lines). The concentration in the distilled water containers (the red line) stays the same showing that the container isn't absorbing any of the nutrients. And all of the spiked solutions differs from the control (the dark blue line) showing that something actually happened.

So back to the SRP. Why is it acting so differently from what we expected it to do? The concentrations are so low that it doesn't seem like I added phosphorus at all. At time 0, I would expect that the concentrations would be close to what I spiked at, for 10x it should be 0.1 mg/L P, at 20x it should be 0.04 mg/L, and at 50x it should be at 0.02 mg/L. But when you look at the graph, everything is around 0.003 mg/L which is the same as the control, so it seems like no phosphorus was added.

What could have gone wrong?

Maybe the phosphorus is precipitating out of the solution by calcium? This means that the calcium will bind to the phosphorus, so the phosphorus isn't available in a form that algae can use, and it is also not detected in the water because it is no longer soluble. Ok, so let's look at the results again. If this was happening, this would only happen in the containers that had calcium in it, the containers with lake water in it. So let's look at the containers that didn't have lake water in them, but had deionized water in them. Do you remember that post I had about making DI water? DI water doesn't have any salts or ions in it, therefore, it wouldn't have calcium in it for the phosphorus to bind to. Therefore, the containers with DI water in them shouldn't show any precipitation. But when we look at these containers, the concentrations do the exact same things as the other containers. So maybe it's not the precipitation.

Maybe I messed up and added something other than phosphorus to the containers? Mistakes happen, and this is a possibility that I can't really test for. I do know that I have four containers that look similar, but have different labels, so it is possible that they were switched. I know we didn't add nitrate or ammonium instead of phosphorus because these containers were also analyzed for nitrate and ammonium and these concentrations were no different from the control. The third container was the sulfuric acid, which in such dilute concentrations wouldn't really harm anything and is undetectable by all the measurements we've made. I just doubt that I messed up like that. With LeeAnn's help, we were so cautious, double checking the containers, the volumes to add to each container... I distinctly remember asking LeeAnn if she had the right container because phosphorus was the first one we spiked. I really don't think this was the problem.

Maybe the solution I added doesn't really contain phosphorus? Maybe it was mixed incorrectly from the start? Again, mistakes happen. So I ran a quick and dirty experiment using a 10 year old Hach kit (prepackaged easy to use chemistry set) which uses ascorbic acid to determine if phosphorus is present. If you are interested in the actual chemical reaction taking place read the parenthesized sentence, if not, just know that if there is phosphorus present, the liquid turns blue. (Principle: Ammonium molybdate and potassium antimonyl tartrate react in acid medium with orthophosphate to form a heteropoly acid—phosphomolybdic acid—that is reduced to intensely colored molybdenum blue by ascorbic acid.) And guess what, it turned blue! 

The results of the ascorbic acid test: There is phosphorus in the solution I added. The first tube contains 5 ml of the stock solution at 14 mg/L of P. The second tube is a dilution at 5 mg/L of P, then 2 mg/L of P and 1 mg/L of P so we can see the slight gradient between the samples. The 14 mg/L is the darkest blue because it has the most phosphorus present, and it gets progressively lighter as there is less phosphorus in the tubes.

Maybe the container is absorbing the phosphorus? This means that the phosphorus is sticking to the container walls instead of staying in the water and being available for the algae to take it up. If the container was not absorbing nutrients, we would see a straight line in the DI containers (as we can see in the NH4 results). If the container does absorb nutrients, the DI container concentrations would decrease over time, but in SRP, they don't. In SRP, the concentration is similar to all the other containers. But just incase, I ran another experiment to confirm that the containers aren't absorbing the nutrients, again using the Hach kit. 

Here is the set up. I had one container for each concentration, 10x, 20x, and 50x, and I have two types of containers, the container I ran the incubation in, and the container I used to transport water samples from the pool to the lab to filter. Then I filled one group of containers with lake water (left) and distilled water (right). Then I spiked the containers with phosphorus using the same methods I used in the field - spiking with the same concentrations as in the field and taking a 5 ml water sample seconds after the spike, after stirring the water with the sampling syringe. I then put the samples into centrifuge tubes and added the contents of the Hack kit. 

This is my control: Pure samples so I know what it would look like if there is no phosphorus present. On the left is DI water and on the right is lake water from Left Lake.

This is the water form the 1 liter incubation containers at the 50x, 20x and 10x spikes in the DI water. Again, these are very small concentrations, 0.1mg/L, 0.04mg/L, and 0.02mg/L respectfully. But even at these concentrations, all of the tubes were tinged blue, with the darkest blue with the highest concentrations. (These pictures really didn't capture this very well, unfortunately.)

This is the water form the 1 liter incubation containers at the 50x, 20x and 10x spikes in the lake water. Again, tinges of blue in all of them, so phosphorus is present. This also seems to mean that the incubation chambers are not taking up phosphorus below the detection limit (where it won't turn blue).

This is the water from the 450 ml transportation containers at the 50x, 20x and 10x spikes in the DI water. There's blue!

This is the water from the 450 ml transportation containers at the 50x, 20x and 10x spikes in the lake water. Also all blue, though it seems a lot less than the DI concentrations. Is the lake water doing something then?

And the case of the missing phosphorus is still active. Where can it have gone?! Maybe something went wrong in the analysis of the water samples back at the lab? Maybe the algae took it up really quickly - though the DI control still doesn't make sense. 

The mystery continues...