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AP
Biology Labs |
 Advanced Placement Biology |
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A P Biology Labs |
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Advanced Placement Biology Lab #12
STUDENT INSTRUCTIONS Objectives Before doing this laboratory exercise, you should understand: • the biological importance of carbon and oxygen cycling
in ecosystems After completing the laboratory exercise, you should be able to: 1. measure primary productivity based on changes in dissolved oxygen
in a controlled experiment
Oxygen is critical for the life processes of nearly all organisms. In the aquatic environment, oxygen must be in solution in a free state before it is available for use by organisms. The dissolved oxygen (DO) concentration in a body of water is often used as a benchmark indicator of water quality. Desirable fish species such as trout and perch require a minimum of 8 ppm dissolved oxygen to survive. Less desirable fish such as carp can survive at dissolved oxygen levels as low as 2 ppm. Below 2 ppm only invertebrates such as sludge worms and mosquito larvae can survive. Oxygen enters water via diffusion from the air and photosynthesis
by aquatic plants. Physical factors such as the temperature and
salinity of the water and the partial pressure of oxygen in the
air influence the rate at which oxygen enters the water. In the
absence of mixing by winds, currents, tides or other flows, the
only way that oxygen is distributed through water is by diffusion.
At 20° C, oxygen diffuses 300,000 times more slowly in water
than in air. In contrast to the relatively uniform distribution
of oxygen in air, spatial distribution of oxygen in water can be
highly variable. Oxygen can be consumed at lower regions of an aquatic
environment faster than it can be replaced from the surface, resulting
in gradients of oxygen concentration and / or anaerobic conditions
in some parts of a body of water. Water Pollution and Dissolved Oxygen Water pollutants can decrease dissolved oxygen concentration, usually by stimulating microbial growth and thereby increasing the demand for oxygen for microbial respiration. Many organic pollutants, such as sewage, can be directly metabolized by microbes in oxygen-requiring processes. Therefore a large influx of sewage stimulates growth of microbes that metabolize it, with a subsequent decrease in dissolved oxygen. Decomposition of dead organisms is also carried out by microbes through oxygen-requiring processes. Any pollution event that kills large numbers of organisms (such as spills of herbicides or pesticides) results in proliferation of decomposers and the use of oxygen for decomposition. Although the effect is not direct, fertilizer pollution can diminish dissolved oxygen in the same way. The pollution first stimulates over-proliferation of aquatic plants, which may first produce additional oxygen, but eventually the plants will die and require decomposition. Fish kills as the result of fertilizer pollution are often the result of oxygen starvation that occurs as large masses of dead plant material are decomposed. Exercise 12-A In this exercise, you will determine the effect of temperature on dissolved oxygen. You will start with a single water sample, divide it into three portions, and let each portion equilibrate at a different temperature. Then you will measure dissolved oxygen. Water samples are not usually saturated; the amount of oxygen dissolved in a water sample is often only part of what the water could hold. You will use a graphical device called a nomograph to determine what percent of saturation is represented by the levels of dissolved oxygen you measure in the different temperature samples. Primary Productivity The fertility of any body of natural water depends on the productivity of the green plants within it. The primary productivity of an ecosystem is defined as the rate at which sunlight is stored by plants in the form of organic materials (carbon-containing compounds). Aquatic green plants use carbon from the carbon dioxide dissolved in the water for carbohydrate synthesis according to the basic equation for photosynthesis: light
Plants respire constantly, but photosynthesize only when light is available. The balance of the two processes, respiration and photosynthesis, determines whether the plant is a net consumer or producer of oxygen and indicates the net productivity. Exercise 12-B In this laboratory exercise, you will be measuring oxygen production by the alga Chiarella under different light conditions, using the light and dark bottle method. In this method, the dissolved oxygen concentrations of samples of ocean, lake, or river water or of laboratory algae cultures are measured and compared after incubation in light and darkness. You will expose samples to a range of light intensities, from full light to 2% of full light, and measure the changes in dissolved oxygen concentration after overnight incubation. Total oxygen production in a sample bottle is the sum of any increase in total oxygen plus the amount of oxygen consumed by respiration during the incubation period. In the bottles kept in darkness, the change in dissolved oxygen (DO) concentration from the initial concentration is a measure of respiration, since photosynthesis cannot occur in the absence of light. In the bottles exposed to light, both photosynthesis and respiration occur; therefore, the change in DO concentration in these samples is a measure of net productivity. The difference between the final DO concentrations in the light bottle and the dark bottle is the total oxygen productivity and therefore an estimate of gross productivity.
Overview The Winkler method is commonly used to measure DO. In this method, a series of solutions is added to the water sample which reacts with the dissolved oxygen in the sample to release free iodine. The quantity of free iodine released is proportional to the amount of free dissolved oxygen in the original sample. The amount of free iodine in the sample can be determined by adding a starch indicator solution to the sample, which turns blue in the presence of free iodine, then titrating with sodium thiosulfate until a colorless endpoint is reached. The amount of sodium thiosulfate needed to titrate the iodine is directly proportional to the concentration of dissolved oxygen in the original sample. Procedure 1. Label 3 BOD bottles, one 4° C, one 25° C, and one 30° C. 2. Fill the BOD bottles. The most important aspects to this process are 1) not to trap any air in the bottle and 2) to avoid introducing any turbulence, since turbulence will mix air into the samples and increase the dissolved oxygen levels. There are several different ways to fill the bottles: 1) submerge the bottle in the sample, let it fill, then cap it while it is still submerged; 2) pour the sample very gently from a beaker, creating as little turbulence as possible; 3) use the 60 ml syringe (take special care since it is easy to introduce air by pushing the sample out fast); or 4) use the 60 ml syringe with a piece of tubing attached. The last method works well if the sample container has a narrow mouth or is very deep. If you use a beaker or syringe, tip the BOD bottle as you fill it and let the sample run gently down the side. If you are using tubing, place the end of the tubing at the bottom of an upright BOD bottle and introduce sample gently. Fill the bottle until it overflows significantly. This is to ensure there is no air trapped in the bottle to give elevated oxygen readings. Cap bottles tightly after filling. 3. After filling the three bottles, fix the oxygen in each by the following procedure: a) Uncap each bottle. b) Add 8 drops of manganous sulfate solution to each bottle. c) Add 8 drops of alkaline potassium iodide azide to each bottle. d) Cap bottles and mix. A precipitate will form. Allow the precipitate to settle to the shoulder of the bottle before proceeding. e) Use spoon to add 1 gram (1 spoon) of sulfamic acid powder to each bottle. f) Cap and mix until reagent and precipitate dissolve. The samples are now fixed. This is an optional stopping point. Samples can be stored at room temperature until convenient to continue. a) Uncap the 4° C bottle and fill titration tube to 20 mL line. Care should be taken to be as precise as possible. Variations in filling from group to group and from bottle to bottle will result in inconsistent data. b) Fill titrator to the 0 line with sodium thiosulfate. Read the volume across the concave edge of the plunger. c) Add one drop at a time to sample, swirling between each additional drop until the sample is a faint yellow color. d) Remove the titration syringe and cap without disturbing the syringe. Add 8 drops of starch indicator solution. e) Replace the lid of the titration tube and swirl the sample. The solution should turn blue. If the solution does not turn blue, there is not a measurable amount of oxygen present, or too much sodium thiosulfate was added in step c. Pour out the sample, refill the titration tube from the BOD bottle, and start the titration again (step b). f) Continue the titration with the sodium thiosulfate already in the syringe. Add one drop at a time, swirling the sample after the addition of each drop, until the blue color disappears. If the blue color does not disappear after the addition of the whole syringe of sodium thiosulfate, refill and continue. When the titration is complete, add 10 ppm from the first syringe to the amount added from the second syringe. g) Read the syringe at the bottom of the plunger. The number represents ppm DO which is equal to mg oxygen per liter of water. Record data in Table 12.1 5. Repeat steps a-e above for the 25° C sample and the 30° C sample. 6. Using the nomograph of oxygen saturation, estimate the percent saturation of dissolved oxygen in your samples and record this value in the table below. Line up the edge of a ruler with the temperature of the water on the top scale and the dissolved oxygen on the bottom scale, then read the percent saturation from the middle scale. Record the data in Table 12.1.
Water Temperature °C Oxygen (mg per liter) Topics for Discussion 1. How does temperature affect solubility of oxygen in water? 2. Design an experiment to determine how salinity affects the solubility
of oxygen in water. What variables would you need to be sure to
control? Overview The productivity per square meter of a water column within an aquatic ecosystem can be measured by the Light-Dark Bottle Winkler method. When using this method, temperature should be held constant so that only one variable is being tested. Each student group will measure an initial sample, a dark sample, a plain light sample, and four samples wrapped with different numbers of plastic screens. The screens filter the light available in the bottle, simulating the effect of increasing depth in a pond. Number of Screens Percent Light 0 100% Procedure 1. Work in groups of up to 5 students depending on class size.
Obtain seven BOD bottles and rinse them well. a) Uncap the bottle. b) Add 8 drops of manganous sulfate solution to the bottle. c) Add 8 drops of alkaline potassium iodide azide to the bottle d) Cap bottle and mix. A precipitate will form. Allow the precipitate to settle to the shoulder of the bottle before proceeding. e) Use spoon to add 1 gram (1 spoon of sulfamic acid powder to the bottle. f) Cap and mix until reagent and precipitate dissolve. The sample is now fixed. NOTE: If your sample contains a heavy algae load, the algae will form a black precipitate which will not go into solution. This will not affect your results. 8. Lay the remaining bottles on their sides under a fluorescent
or grow light, seam side down, and leave overnight. 1. Obtain bottles from light source. 2. Fix the samples by following steps a-f below. a) Uncap each bottle. b) Add 8 drops of manganous sulfate solution to each bottle. c) Add 8 drops of alkaline potassium iodide azide to each bottle. d) Cap bottles and mix. A precipitate will form. Allow the precipitate to settle to the shoulder of the bottle before proceeding. e) Use spoon to add 1 gram (1 spoon) of sulfamic acid powder to each bottle. f) Cap and mix until reagent and precipitate dissolve. 3. Determine the amount of dissolved oxygen in each bottle, including the Initial sample, by the following method: a) Uncap the sample bottle and carefully fill titration tube to the 20 mL line. b) Fill titrator syringe to the 0 line with sodium thiosulfate. c) Add one drop at a time to sample, swirling between each additional drop until the sample is a faint yellow color. d) Remove the titrator syringe and cap from the titration tube. Add 8 drops of starch indicator solution. e) Replace the lid of the titration tube and swirl the sample. The solution should turn blue. If the solution does not turn blue, there is not a measurable amount of oxygen present, or too much sodium thiosulfate was added in step c. Pour out the sample, refill the titration tube from the BOD bottle, and start the titration again (step 3a). f) Add sodium thiosulfate one drop at a time, swirling the sample after the addition of each drop, until the blue color disappears. If the blue color does not disappear after the addition of the whole syringe of sodium thiosulfate, refill and continue. When the titration is complete, add 10 ppm from the first syringe to the amount added from the second syringe. g) Read titrator scale on the syringe for result in mg DO per liter. 4. Enter the amount of dissolved oxygen (DO) for each bottle in
Table 12.2. Multiply the dissolved oxygen concentration in mg/l
by 0.698 to convert it to mLlL, the units most commonly used to
report dissolved oxygen levels. Initial x 0.698 = Dark x 0.698 = Light Bottle x 0.698 = 1 screen x 0.698 = 3 screens x 0.698 = 5 screens x 0.698 = 8 screens x 0.698 = 5. Calculate the respiration rate by the following equation: Respiration = [Initial (mL O2/L) - Dark (mL 02/L)] Respiration rate = 6. Calculate the net productivities of the remaining cultures by the following equation, Enter the data in Table 12.3. Net Productivity = [Light(mL 02/L) – Initial(mL 02L)] 7. Assume the algae in each bottle consumed the same amount of oxygen through respiration as did the algae in the dark bottle, Each culture of algae therefore had a gross oxygen production equal to the net oxygen production plus the amount consumed in respiration: Gross Productivity = [(Light - Initial) + (Initial - Dark)] = (Light Bottle - Dark Bottle) Calculate the gross productivity of each bottle and enter it in
Table 12.3. Use mL 02/L as units for dissolved oxygen and hours
for time. No screen 1 screen 3 screens 5 screens 8 screens Table 12.4 Class Average Gross and Net Productivities Bottle Ave. DO Ave. Net Ave Gross No screen 1 screen 3 screens 5 screens 8 screens 9. Was growth in any of the sample bottles limited by the availability of light? Why do you conclude this? 10. Plot the average gross and net productivities (mL 02/L)/nr as a function of light intensity (%). Which is the independent variable? Which is the dependent variable? |