# Lab 12: Dissolved Oxygen and Primary Productivity

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## General Overview

"Major misconception: Many students believe that the fish 'breathe' by separating out the oxygen from water. On a previous AP essay, some even described the bubbles rising from the gills as the leftover hydrogen. This is a good time to ensure that the students understand the role of DO in water life, and its use as an indicator of water quality and expected species."
Israel Solon, Greenhill School, Dallas, Texas. 9/12/99

Question: "Can anyone explain the relationship between expected DO and percent saturation, as a function of temperature?"

Answer 1: "The procedure for heating and cooling is important. If cool water is heated carefully without driving out the oxygen, the percent saturation will go up. For example, 5 mg/l of DO in 20-degree water may represent a 50 percent saturation level. Heating that water up to 30 degrees without losing any oxygen may give you an 80 percent saturation level.

"However, if you start with water at, say, 10 degrees that is fully saturated (about 12 mg/l?—I'm too lazy to look up the numbers), as you heat up that water, oxygen will be driven out of solution and the amount of DO in the water will go down, while the saturation level should stay at 100 percent (perhaps 10 mg/l and 20 degrees). If you overheat the water and cool it right away, the mg/l of DO in the cooled water can only be as high as the saturation level when the water was at its higher temp. So 10-degree water rapidly heated to 20 degrees and then cooled to 10 would only contain 10 mg/l of DO. The water would have to have time or opportunity to re-saturate to read 12 mg/l again.

"So, if the percent saturation is lower at higher temps, it could be that the water was even warmer before it cooled to the measured temperature, and lost some DO.

"Let me give you a quick thought, and an example. First, the thought: If you understand how temperature affects relative humidity, the principle is the same. Now, a chart. Let's take some water at 35 degrees Celsius, and cool it without giving it an opportunity to diffuse any additional oxygen. Once again, the numbers may be off, because I'm too lazy to look them up. This is a 'for example,' so don't memorize the numbers for the quiz.

Temp DO Present Max DO Possible Percent Sat
35 10 mg/l 10 mg/l 100
30 10 mg/l 10.5 mg/l 95
25 10 mg/l 11 mg/l 90
20 10 mg/l 12 mg/l 83
15 10 mg/l 13 mg/l 77

"As you can see, as the water cools, if no further gas exchange occurs, the percent saturation changes. If I were a trout, and needed, say, 12 mg/l of DO to live comfortably, water over 20 degrees Celsius would not do it."
Israel Solon, Greenhill School, Dallas, Texas. 10/8/99 and 10/10/99

Answer 2: "I found the discussion of dissolved oxygen in one of my old limnology books to be quite helpful. It goes on for several pages so I am only paraphrasing a few sections here (George K. Reid and Richard D. Wood, Ecology of Inland Waterways and Estuaries, New York: D. Van Nostrand, 1976).

"The discussion of dissolved oxygen is both interesting and instructive. It further solidifies the argument that AP Biology students would benefit from taking a good high school chemistry course FIRST. In 1957 a well-known limnologist, Hutchinson, surmised that one could 'learn more about the nature of a lake from a series of oxygen determinations than from any other kind of chemical data.'

"The volume of oxygen dissolved in water is dependent on:

1. temperature
2. partial pressure of oxygen in the atmosphere
3. concentration of dissolved salts
4. biological activity

"Now, how will any students be able to properly process this without having had chemistry—solubility, equilibrium, and so on?

"With regard to the nomograph—it is used to determine the percent saturation of a given oxygen concentration at various altitudes. Altitude is important to consider because it allows one to take into account pressure. It shows that solubility of oxygen in water increases with decreased temperatures.

One hundred percent saturation does not refer to all the gas that can be held in solution, but instead to the amount that occurs at EQUILIBRIUM under certain stated conditions. Under certain conditions the observed saturation may be over 100 percent. Dissolved salts decrease the oxygen concentration."
Bob Goodman, Hunter College High School, New York, New York. 10/11/99

Answer 3: "If equilibration time is allowed, there is no relationship, as all water will end up at 100 percent saturation. Therefore, the graph that needs to be done is temperature versus DO in mg/l, not percent saturation. Neither the student book nor the teacher's guide is clear on this."
Israel Solon, Greenhill School, Dallas, Texas. 10/12/99

Tip: "Describing net and gross productivity in terms of net and gross pay in their paychecks also helps them understand."
Tricia Glidewell, Marist School, Atlanta, Georgia. 10/14/99

## Equipment and Supply Modifications

Question: "What does it take to make the dissolved oxygen lab successful?"

Answer 1: "1. Very green water. If necessary, grow it in a fish tank. 2. Lots of light without too much heat. Those under-counter fluorescents or the setup used for Fast Plants should do it. 3. Lots of group data—my students seem to always find new ways to add error to procedures, so we always have to 'ignore' some data—something we can only do if we have enough trials to 'overwhelm' the obvious aberrant trial. 4. Luck—this IS biology, and living organisms are living organisms, no matter how small. "Here in Texas, the best green water comes from the stock tank. Works like a charm. A Chlorella culture is the best bet for 'indoor' green-water making."
Israel Solon, Greenhill School, Dallas, Texas. 4/20/99 and 4/21/99

Answer 2: "I have gotten reliable results only with commercially prepared Chlorella cultures (I've maintained mine for two years so far). My very green aquarium water never works... I think there is too much eutrophication in it and bacteria are slurping up all that O2. I do let my Chlorella get nice and green under a fluorescent Gro-Lite and it must be relatively free of bacteria compared to my pea soup, which I grow in an aquarium. The data in this lab have seemed pretty erratic to me over the years that I have done it, but the new manual is certainly an improvement over the old procedure."
Charlotte Freeman, Girls Preparatory School, Chattanooga, Tennessee. 4/20/99

Answer 3: "Instructions: About three days before lab, dilute the culture in 4 liters (or 1 gallon) of spring water and add the Algae Grow media. Do not use distilled water; dechlorinated tap water will work. Keep the culture under continuous moderate light (fluorescent is ideal) for 72 hours or more at 25 degrees Celsius. By the day of the lab, a green mass of algae 1-2 inches deep should be present in the bottom of the container. If you will need the culture early in the week, you may grow the algae for three days the week before the experiment, then remove it from continuous lighting (put it in a window) over the weekend and use it the following week. If you cannot grow the culture under continuous light, start it a week before the lab and let it grow in a window.

"Recommendations: The AlgaGro Concentrate needs to be diluted. Use a clear container if possible. Keep light source 18-24 inches away from container. Monitor temperature (22-25 degrees Celsius optimum). Do not use DISTILLED (DI) water. Do not use 'DRINKING WATER.' Although these may appear similar to the gallon jugs of spring water found in grocery stores, both will inhibit chlorella growth [distilled water = lack of minerals and nutrients due to removal by ionic exchange; drinking water = chlorine kills Chlorella]. The 'algae people' I talked to here at Carolina seem to be in agreement that the water being used is the probable culprit. Chlorella is an extremely good 'grower.' Your use of a grow light for 12-hour intervals is probably not significant. Please monitor the water temperature and assure good water is being used."
Pat Ryan, Carolina Biological Supply Company, Burlington, North Carolina. 5/2/01

Answer 4: "I just performed this lab and it was successful. I think you are adding too much water. I used two tubes of Chlorella in the AlgaGro media from Carolina and only half a gallon of spring water. I kept it under fluorescent lighting and the results were good." Jean Becker, Herricks High, New Hyde Park, New York. 5/2/01

Answer 5: "Transfer the inoculate you received to a smaller volume of sterile spring water and AlgaGro. I start my cultures in 250-500 ml flasks and let them grow under continuous light. When the culture is dense I then transfer the larger volume to a 4-liter flask of media. It may take 4-5 days to achieve the density you need in the 4-liter flask. For faster results try pond water or substitute Elodea for the Chlorella."
—Gail Lima, The Winsor School, Boston, Massachusetts. 5/2/01

Answer 6: " I called the instructor at Clemson University of the AP Biology Institute and she suggested to put it in open containers (large beakers of equal size). I put it under a fluorescent light table and had growth within two days. The mistake is the jug. She thinks that the new spring water jugs have UV protectant in the plastic." Amy Litz, Summerville High School, Summerville, South Carolina. 5/7/01

Answer 7: "Try a sunny window, or use algae from a local pond. If that fails, you can use any aquatic plant (Elodea) or filamentous algae." Scott Stein, Springside School, Philadelphia, Pennsylvania. 5/2/01

Answer 8: "Use duckweed—it works well in this lab, in my experience."
Bruce Faitsch, Guilford High School, Guilford, Connecticut. 5/4/01

## Pre-Lab Preparation

Question: "How do you keep Chlorella cultures going?"

Answer: "I order a Chlorella culture from Carolina Biological and their prepared AlgaGro medium. I put the medium in 1000 ml Erlenmeyer flasks and divide the culture between the two. I cover the tops with Saran Wrap or parafilm. I have reordered the medium once and am about to do it again. I will microwave two additional well-washed Erlenmeyer flasks that have a little water in the bottom. (This gets them sterile enough to keep other microbes from overtaking the cultures.) I will pour out the hot water, pour the new media solution in... and inoculate both with Chlorella from the old flasks. I keep them under an 18-inch Gro-Lite that is mounted below a cabinet so it is fairly close to the actual culture. They will grow all summer and be nice and dark green by fall and I will do that lab early in the fall. There are cheaper ways to do it by using pond water, etc., or even making up your own recipe, but I have found the most success with the sterile solutions from Carolina and consider the extra cost worth my time trying to sterilize my own pond water, etc. This is the end of the third year on these cultures and I may start fresh as they are not looking as nice and green as last year...but they are still alive. They will tend to settle at the bottom, but just before lab I put a gentle magnetic stirrer treatment to them and they re-suspend."
Charlotte Freeman, Girls Preparatory School, Chattanooga, Tennessee. 5/10/99

## Procedure Modifications

Tip: "Have any of you used the Chemetrics oxygen determination for this lab? It's fast and easy; and if you keep the lighting even, it's pretty reliable. Using the disposable ampoules, we can do several temperatures and several salinities in a 45-minute class, with each team doing repeats, then pooling class data. The phone number of Chemetrics company is (800) 356-3072. They are very accommodating and usually get your order out the same day they receive the PO. For those of you lucky enough to be able to do this over the phone or by fax, it's very convenient. You have to buy the kits, including the set of sealed ampoules for standards, but after that, you need only order refills for the test ampoules. I highly recommend doing it this way, for its convenience, speed, and lack of mess. We've even taken the kits to the Long Island Sound on ecology field trips."
Barb Beitch, Hamden Hall Country Day School, Hamden, Connecticut. 9/13/99 and 9/14/99

Tip: "I used both Chemetrics and Hach kits last year for Lab 12. My problem with the Chemetrics kit was that the kit did not go up to a high enough DO, and so the 100 percent light bottles did not have a DO as high as they should have when we measured them with the Chemetrics. We realized we needed to use the Hach kits for those bottles. The kits worked fine for the light-limited bottles (I had nice green water!). The Hach Company, in Loveland, Colorado, is in the business of producing kits for testing of every conceivable contaminant in water. Their kits are widely used by professionals and many schools. They have a very informative catalog, and have always been very helpful to me as a teacher."
Anne Soos, Stuart Country Day School, Princeton, New Jersey. 9/14/99 and 9/16/99

Tip: "I, too, use the Chemetrics DO test ampoules, but became frustrated with my students trying to use the color standards. They make another size of the DO vials that can be read with their DO test meter (called the 'SAM Single Analyte Photometer;' catalog #I-2002) and it is great for Lab 12. The meter and 30 ampoules runs \$195, but you only need to spend that much one time since you only need one meter (each reading takes about 2 seconds!). I have found that one-time expense to be well worth it. Once you have the meter, you just order the ampoules each year (they cost \$19 for 30 ampoules; catalog #R-7513).... just call them and they will answer any questions and send out a catalog; or go to their Web site at www.chemetrics.com."
Robert Dennison, Jersey Village High School, Houston, Texas. 9/16/99

Tip: "I use the kits from Chemetrics (800-356-3072): dissolved oxygen kit #K-7512 and refill boxes of ampoules #12-7512. Use uniform direct lighting from above. Be consistent. Fast and easy!"
Barbara Beitch, Hamden Hall Country Day School, Hamden, Connecticut. 11/1/99

Tip: "After trying the Winkler Titration once, I switched to snap tests. I got them from Wards. They aren't cheap (unless you compare them to a probe) but if you have the kids set up the productivity lab in the lab, and then do not have access to the lab room 24 hours later, snap tests are the way to go! All you do is snap off the skinny end of the ampoule right in the sample; the culture is drawn up into it and turns a lovely shade of blue depending on the DO. Apparently many people use these as I saw them mentioned often when I was reading the photosynthesis question last year at Clemson!"
Mary Jane Davis, Red Bank Catholic High School, Red Bank, New Jersey. 11/1/99

Tip: " I'm considering completing the DO lab during the photosynthesis unit next year rather than in the ecology unit for a more direct link in the students' minds. I always make sure that the kids know the reasons for gross productivity and net productivity in that lab, and how to calculate it. I like to use specific amounts of duckweed in the DO bottles so you can get better control of the amount of producer biomass. This eliminates an unneeded variable and lends itself to a clearer understanding of the dynamics involved. Also, I suggest using a light meter to get a uniform magnitude of light. Distance from a light source alone involves too many variables; age of the bulb, reflector efficiency, etc."
Bruce Faitsch, Guilford High School, Guilford, Connecticut. 6/5/01

## Trouble Shooting and Cleanup

Question: "This is my first year teaching AP; we just ran the dissolved oxygen lab and my results are wacky—the net productivity values are negative! I'm assuming that this means that there was more productivity in the initial bottle than in the light bottles—how could this be? Shouldn't the results decrease with number of screens added, with the dark bottle having the lowest value? Please help!!"

Answer: "Negative net productivity tells you that your water was very low in producers—so low, that it could not replace the oxygen being used by the consumers. You probably have mucho bacteria and very little phytoplankton. If this is the case, the number of screens won't make a difference, as there wasn't enough photosynthesis going on in full light to be inhibited by lack of light.

"I have always had the best luck using water from a pond that already has a stable ecosystem in it. Any small or medium-size pond with fish, maybe some ducks, green stuff growing on the bottom, etc., should work fine. I have also used slow-moving streams. Avoid such water immediately after heavy rains—the sediment kills off the algae and the bacterial load will be high. In my travels around Texas, I have been about 80 percent successful in just asking 'any good yucky water around?' Another idea would be to set up a goldfish tank and let it run under light until it is good and green. Three days may not have been enough for the Chlorella culture. Did you add nutrients? I don't have the instructions, as I tend to avoid anything that requires advance planning.

"All is not lost. If you get the same results, your students need to explain them to you. You can then provide them with the data from the teacher's guide so they can come up with the 'correct' answers. Their understanding of the 'what,' 'why,' and 'how' of this lab is more important than the technique. On the other hand, I tell my AP students I am grading their technique—it keeps them on their toes. I even remember to actually do it once or twice a year :). The objectives of this lab do not require perfect results as long as the students:

• Understand what DO is
• Can explain net and gross productivity and explain negative net productivity
• Can set up an experiment to measure productivity and photosynthesis
• Can describe the relationships among photosynthesis, respiration, and DO

"This can be a frustrating lab. For a light source, I have had good luck with the inexpensive fluorescents designed to go under kitchen cabinets. I prop them about 5 to 10 cm above the lab table, with something official, like a couple of books or the boxes from the DO test kits, and lay the bottles flat on the table. The light is nice and close, but is not hot enough to provide me with cooked algae. I have also used the setups for Fast Plants as a light source.

"The bottom line, though, is that you are dealing with living organisms, which, under carefully controlled laboratory conditions, tend to do whatever they please."
&mdashIsrael Solon, Greenhill School, Dallas, Texas. 9/16/99 and 9/17/99

Tip: "While I was recently going over the dissolved oxygen data from Lab 12, I realized that the nomograph in edition D (page 140) of the AP Lab Manual is very poor and inaccurate. I compared it to a chart of 100 percent oxygen solubility at different temperatures, and in all cases for the chart values at 100 percent, the nomograph reads between 70 and 75 percent. I subsequently pulled out the nomograph from edition C, and it is correct. So, I have three recommendations:

1. Copy the nomograph from the previous edition for your kids to use; OR
2. Give them a chart of temperature and 100 percent solubility of DO, and let them calculate their own percent solubilities; OR
3. Do both (which is what I will do). I like my kids to see and use a nomograph, but I also like them to realize they are approximations, and are not really very precise.

"On another note for this lab, I believe there is a mistake in question #5 on page 139. The question has the students graph percent saturation as a function of temperature. This section of the lab is looking at the relationship between DO and temperature. The percent saturation could be 100 percent in all cases, and a straight line would result for the graph, not the inverse relationship I want them to see. SO, I tell my kids to graph their average dissolved oxygen (NOT percent saturation) results as a function of temperature. This way I reinforce the inverse relationship between temperature and gas solubility. I then talk about the percent saturation with my kids and go over some environmental factors that could change the percent saturation values like cooling of water without wave action (lower), or a lot of algae near the surface actively photosynthesizing could raise the percent saturation over 100 percent."
Franklin M. Bell, St. Mary's Hall, San Antonio, Texas. 3/6/00

Tip: "The nomograph in edition D of the lab manual gives incorrect percent saturation values. They are usually off by 20-40 percent. The nomograph in edition C (green cover) is the one you want to use. Also, I would recommend that you change the graphing question on page 139 (#5) so that your students graph class mean DO versus temperature. I see the purpose of this graph to show the inverse relationship between temperature and dissolved gases. If you graph percent saturation, you may or may not see this relationship, especially if all of your samples are 100 percent (or close to 100) saturated. Instead of an inverse relationship, the graph will probably show a straight horizontal line."
Franklin Bell, St. Mary's Hall, San Antonio, Texas. 10/5/00

Tip: "I am unfamiliar with the lab manual... [but] the nomograph is modified from C.H. Mortimer, published in Limnology by Robert G. Wetzel (Saunders Publishing Co., Harcourt Brace Jovanovich, 2nd ed., 1983). Requests for permission to reproduce from Wetzel should be directed to: Permissions, Harcourt Brace Jovanovich, Inc., Orlando, FL 32887."
Harry Padden, Washington Township High School, Sewell, New Jersey. 10/7/00

## Conducting Lab Using Probes and Computer/Calculator

Tip: "My AP Biology class just finished the Aquatic Productivity Lab using a long-term computer interfacing setup. We used pH probes to follow the carbonic acid (CO2) levels over time (90 hours) in four miniature aquatic ecosystems. The samples were taken from a small pond and included some visible algae and other 'critters.' The bottles were placed under a light stand with a timer set for 12 hours light/12 hours dark.

"Our dissolved oxygen readings came out as follows:

Start-12 ppm

After 90 hours (alternating 12 hours light/12 hours dark):

Control-17.6 ppm
1 Screen-5.6 ppm
2 Screens-4.0 ppm
4 Screens-1.6 ppm

"A bottle kept in continuous darkness had 1.4 ppm. This experiment is one I highly recommend for computer interfacing. The changes over time (90 hours) in the various systems with the changing day/night cycles are something that the 'regular' method of doing this lab misses entirely."
Jeff Smith, Indiana Academy, Muncie, Indiana. 4/26/99