It is late June and Tony has just shown up at your door to do the mid-season sample pick-up. While chatting about the lake, you start digging through your freezer looking for the nutrient bottles. You know that there are five of them in here somewhere. Under the bag of mixed vegetables? Hiding out behind the chocolate ice cream? You finally find them under the bag of tater-tots. Tony takes them and puts them in a cooler with all of the other nutrient bottles he has collected this trip. Once he gets back to the University, he stores all of those bottles in one of four freezers that we have just for this purpose.

When it is time to analyze the samples, they are placed into a sink with warm water to speed up the thawing process. Because the bottles are placed into water, it is very important to be sure that any information written on them is in permanent marker, and that any paper labels are covered with tape (paper labels don’t hold up so well when wet!). Once the bottles are defrosted, we will set up two tubes for Total Nitrogen (TN) analysis and two tubes for Total Phosphorus (TP).

Because we have so many little plastic bottles of frozen water, it is important that all of the required information is on each bottle. The machine you see is the spectrophotometer. The cuvette is in the foreground.

After all of the tubes have been set-up, they go through a digestion step. A chemical is added to the tubes and they are then placed into an autoclave (scientific word meaning pressure cooker) where they cook (I mean autoclave!) at 250 degrees F for an hour. This process takes all of the nitrogen or phosphorus that is in the tube and turns it into a measurable form. In undigested lake water the nutrients are present in many different forms that include: dissolved in the water, attached to soil material, as well as incorporated into algal cells and zooplankton bodies. Without the digestion step we would greatly underestimate the amount of nutrients that are in the lake water.

Once the tubes are cooked and cooled, they are ready for analysis. At this point the process for TN and TP differs. TN samples are acidified and then analyzed. TP analysis differs in that we add a color reagent to the tubes. This reagent reacts with the phosphorus in the sample to create a blue color. The more phosphorus in the sample, the darker the blue color in the tube. The actual analysis takes place on a machine known as a spectrophotometer. The simplified version of how it works is some sample is placed into a cuvette (see picture) and this is placed inside the machine. Light of a precise wavelength is passed through the sample and the machine determines how much of that light is absorbed by the sample. For TN the machine actually does readings at three different wavelengths. TP samples are analyzed at a single wavelength.

It is very important that the bottles be filled to the shoulder with water, because we sometimes have to go back and redo analyses (don’t overfill them though, or they will burst in the freezer).

Along with tubes that contain lake water, each run of TN and TP contains tubes that have known amounts of nitrogen or phosphorus (referred to as standards). We use information generated by analyzing the standards to help us determine the amount of nutrients in the lake samples.

After all the analyses are done we end up with a number associated with each standard and each sample. We then use the standards to create what is known as a regression. Figure 1 shows an example of the phosphorus regression. The known standards are scaled on the bottom of graph and the absorbency values on the left side. The points within the graph show the absorbency values for our standards (0, 25, 50, 100 and 150 ug/L) and the solid line indicates the relationship between phosphorus concentration and absorbency value. We take this information and can use it to turn the absorbency value of an unknown sample (for example, 250) into a phosphorus concentration (80 ug/L). Nitrogen works the pretty much the same way.

Once TN and TP values have been determined for your samples, we quality check them to make sure that there is good agreement (remember, we run two of each). If the numbers aren’t within 5% of each other, we start the process over with the water that is left in the nutrient bottle. The data are then entered into a computer file where we can use them to calculate statistics and create graphs.

Total Nitrogen and Total Phosphorus play a large role in determining water quality in our lakes. These nutrients determine how much algae a lake can support, which in turn impacts our use and enjoyment of the lake. The data generated from these samples also helps the state meet Clean Water Standards...not bad for a sample that was hanging out with tater-tots.

Dan Obrecht