Advanced Placement Biology ® 292 AP Biology Lab 12 EDVO-Kit # Dissolved Oxygen & Aquatic Primary Productivity Storage: Store entire experiment at room temperature EXPERIMENT OBJECTIVE The objective of this experiment is to understand: 1) the factors that affect the solubility of dissolved gases in aquatic environments, 2) the effects of light and nutrients on photosynthesis, and 3) the relationship between dissolved oxygen, photosynthesis and respiration. Students should be able to describe a method to measure dissolved oxygen, and define primary productivity and factors which influence it. EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com EVT 005057AM 2 EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity AP Biology Table of Contents Lab # 12 Page Experiment Components 3 Experiment Requirements Background Information 3 4 Experiment Procedures Experiment Overview Part A. The Winkler Method for Dissolved Oxygen 7 8 Part B. Primary Productivity Laboratory Extensions 13 20 Study Questions 27 Instructor's Guidelines Notes to the Instructor Pre-Lab Preparations Study Questions and Answers Material Safety Data Sheets Advanced Placement (AP) Program is a registered trademark of the College Entrance Examination Board. These laboratory materials have been prepared by EDVOTEK, Inc. which bears sole responsibility for their contents. EVT 005057AM 1-800-EDVOTEK • www.edvotek.com 29 30 33 34 EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 3 AP Biology Experiment Components This experiment is designed for 10 lab groups A B C D E F G Sodium iodide Sodium hydroxide Manganese sulfate Starch solution Sodium thiosulfate Nitrogen enrichment solution, concentrate Phosphorous enrichment reagents Pipets Lab # Storage: Store entire experiment at room temperature. Requirements All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals. EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc. • • • • • • • • • • • • • • Plastic window screens Sealable containers or BOD bottles, 50 Grow lights Pond/lake water Goggles Sulfuric acid, concentrated Glass pipets Burettes Flasks Beakers Gloves Filter paper Linear graph paper Aluminum foil EDVOTEK - The Biotechnology Education Company ® 1-800-EDVOTEK • www.edvotek.com FAX: (301) 340-0582 • email: edvotek@aol.com EVT 005057AM 12 4 EDVO-Kit # 292 AP Biology Lab # Background Information 12 Dissolved Oxygen & Aquatic Primary Productivity Dissolved Oxygen & Aquatic Primary Productivity Oxygen plays a major role in the biochemical reactions used by organisms to produce energy for life. During oxidative phosphorylation and electron transport, it is generally the final electron acceptor. The greatest number of ATP’s are produced per mole of glucose when oxygen is available. Its concentration in the environment can limit the maximal rates of metabolism. In air, there is ample concentration of oxygen available for utilization by living organisms. It represents nearly 20% of the total gases in the air. There is approximately 200ml of O2 per liter of air, which is 9 mM O2 /L. The situation in aquatic environments is quite different. In aquatic environments, oxygen is not very soluble and its solubility is directly affected by the concentration of dissolved ions such as salt, the pH, and the temperature. Salinity is the content of dissolved salts in water. It is usually expressed in parts per thousand (ppt). The solubility of O2 is inversely proportional to the concentration of salt and temperature. As the salt concentration and temperature increase, the solubility of O2 decreases. Even at its maximum solubility, which is in fresh water at 0°C, its concentration is only 25% of the amount in air. In practical situations, the concentration of O2 in aquatic environments usually does not exceed 3-4% of the concentration of O2 in air. This is only 6.0ml O2/L or 0.3 mM O2 /L. In salt water, O2 concentration would be less since oxygen is less soluble in solutions of increasing ionic strength. Since the maximum concentration of dissolved O2 in water is dependent upon other components dissolved in water, O2 level is often used in water quality and pollution testing. The higher the level of dissolved oxygen (DO), the better the water quality. Because of the low concentration of O2 in aquatic environments, it has been suggested that O2 is a major limiting nutrient for life. It is interesting to note that the largest aquatic animals, the whales, are air breathing mammals. In addition to the chemical factors that influence dissolved oxygen, biological processes such as photosynthesis and respiration affect the maximum amount of aqueous oxygen concentrations. There are both biological demands and chemical demands placed on the oxygen available in water. These are often called the Biological Oxygen Demand (BOD), and the Chemical Oxygen Demand (COD). Photosynthesis in aquatic environments performed by plants, and small phytoplankton which are one-celled plants, will increase the concentration of DO in the water. Respiration will decrease the concentration of DO in an aquatic environment. Therefore during the day, when photosynthesis is at a maximum, the concentration of DO will increase. During the night, the concentration of DO will decrease. Ecologists will often study an ecosystem by measuring or estimating the “primary productivity”. Energy to support the life requirements of 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 5 Dissolved Oxygen & Aquatic Primary Productivity Dissolved Oxygen & Aquatic Primary Productivity animals, plants, and bacteria generally enters an ecosystem as light energy, which is converted by photosynthetic organisms to chemical energy. Primary productivity is defined as the rate at which plants assimilate the energy of sunlight. This rate directly affects the growth of plants and other chlorophyll containing organisms. This in turn affects the growth of animals that feed on plants and others which are at higher trophic levels. Primary productivity therefore is at the base of the trophic structure ( the organization of feeding relationships) in an ecosystem. AP Biology Lab # 12 The equation for primary productivity, which is the utilization of carbon dioxide and water to produce glucose and oxygen during photosynthesis is; 6 CO2 + 6 H2O → C6 H12 O6 + 6 O2 The accumulation of organic matter through the growth and reproduction of the plant is not the only productivity possible. A significant portion of the sugar produced by photosynthesis is used by the plant for other synthetic and maintenance reactions during respiration. Therefore, the gross primary productivity (GPP) is the sum of the organic material produced plus the respiration rate (Rs) of the plant or GPP = NPP + Rs. Net primary productivity (NPP) equals the gross productivity minus the respiration rate (Rs) or NPP = GPP – Rs. Net productivity is therefore a measurement of growth and reproduction. Gross productivity is a measurement of growth and reproduction plus the respiration rate. In terrestrial environments, plant primary productivity is usually measured in terms of the increase in the amount of plant Biomass. In aquatic environments, we measure the plant primary productivity by gas exchange, since the concentration of dissolved oxygen in water is easily determined. The classical method for determining the productivity of an aquatic environment is the light and dark bottle procedure. In a natural setting, for example, sealed bottles containing samples of pond water would be suspended at different depths beneath the surface of the pond. A clear “light” bottle which allows sunlight to enter, and a “dark” bottle which excludes sunlight, would be suspended at each depth. In the laboratory, one can simulate the attenuation of sunlight, which increases as depth increases, by wrapping the light bottles with screens. We will assume that the respiration is equal at all depths. In the light bottles, the production of oxygen by photosynthesis and the consumption of oxygen occur simultaneously. Therefore the change in oxygen levels in the light bottles is a direct measure of the “net productivity”. In the dark bottles, oxygen is consumed since only respiration 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Background Information From the above equation, the amount of oxygen produced can be directly related to the amount of carbon dioxide consumed. For each mole of O2 produced, one mole of CO2 is consumed. One milliliter of O2 gas contains 1.432 mg of oxygen. Therefore, based on formula weights, one can calculate that for each milliliter of oxygen produced, approximately 0.536 mg of carbon has been consumed. 6 EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity AP Biology Dissolved Oxygen & Aquatic Primary Productivity Lab # occurs. Therefore, the change in oxygen levels in the dark bottles is a measure of respiration (Figure 1). Addition of the amount of oxygen consumed by respiration in the dark bottle and the oxygen produced in the light bottle, yields gross productivity. The respiration rate includes that of plants, animals, and bacteria. Therefore, only the gross productivity measurement is totally valid. 12 Figure 1: Gross Productivity Determined by Light/Dark Bottle Method The level of dissolved oxygen (DO) in water is a direct measurement of the quality of the water. This is affected by physical conditions such as temperature, salinity, and pH. In addition, it is dramatically affected by both the Biological Oxygen Demand (BOD), and Chemical Oxygen Demand (COD). Lakes loaded with decaying matter have low levels of oxygen, and a high BOD, since the oxygen is being consumed by bacteria and algae. This exercise demonstrates the methodology used for measuring dissolved oxygen and determining the primary productivity of a natural body of water. ( L )ight Bottle Net Productivity DO2 (ml O2/L) Background Information The Winkler method is used to determine dissolved oxygen. This procedure is an iodometric method. The iodide ion is an effective reducing agent which has been widely used for the quantitative analysis of many oxidants. Generally, sodium thiosulfate is used to titrate the iodine liberated by the chemical reaction. The endpoint is determined by the loss of color during titration. This procedure is explained in detail under Part A, Student Experimental Procedures. ( I )nitial Bottle Gross Productivity Respriation ( D )ark Bottle 0 24 L - I = Net Productivity I - D = Respiration L - D = Gross Productivity Incubation Time (Hours) 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 7 Dissolved Oxygen & Aquatic Primary Productivity AP Experiment Overview Biology EXPERIMENT OBJECTIVES: Students will: Understand the factors that affect the solubility of dissolved gases in aquatic environments 2. Understand the effects of light intensity and nutrients on the rate of primary productivity 3. Understand the relationship between dissolved oxygen and the processes of photosynthesis and respiration and the affect on primary productivity 4. Be able to describe a method to measure dissolved oxygen, and define primary productivity and factors which influence it . WORKING HYPOTHESIS If light intensity and the addition of nutrients affect the rate of photosynthesis in aquatic environments, then primary productivity will also be affected. LABORATORY SAFETY Gloves and safety goggles should be worn routinely as good laboratory practice. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM 12 Experiment Procedure 1. Lab # 8 EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity AP Biology Lab # Experiment Procedure 12 Part A: The Winkler Method for Dissolved Oxygen THE WINKLER METHOD FOR DISSOLVED OXYGEN The Winkler method will be used to determine the concentration of dissolved oxygen in water samples. The samples are first treated with an excess of manganese sulfate, sodium iodide, and sodium hydroxide. The white manganese (II) hydroxide precipitate that forms will rapidly react with the dissolved oxygen in the samples to form manganese (III) hydroxide. When the samples are acidified by the addition of sulfuric acid, the manganese (III) oxidizes iodide to iodine. The concentration of the liberated iodine, I2, is titrated with sodium thiosulfate in the presence of a starch indicator solution. It is important to note that the starch is added after the bulk of the iodine has been reduced. In acidic solutions, the starch would be decomposed by a large excess of iodine. For fine analysis, the starch is added after the titration has begun, near the point when the solution has become a faint, pale yellow color. For purposes of this laboratory, the starch can be added at the beginning of titration to facilitate end point detection. The balanced equations for the series of chemical reactions used in the Winkler method are shown below. 4 Mn (OH)2 (s) + O2 + 2 H2O → 4 Mn (OH)3 (s) When acidified by addition of sulfuric acid; 2 Mn (OH)3 (s) + 2 I- + 6 H+ → I2 + 6 H2O + 2 Mn2+ The liberated iodine is titrated to endpoint with sodium thiosulfate as shown below. 2 (S2 O3)-2 + I2 → S4 O6-2 + 2I- When the titration is nearly complete, the solution will begin to change from a purple/brown color to a colorless solution. As can be seen from the above reactions, four moles of sodium thiosulfate titrant, Na2S2O3, are required per mole of dissolved oxygen, O2. The concentration of the sodium thiosulfate titrant has been adjusted so one milliliter of sodium thiosulfate solution equals one milligram of dissolved oxygen per liter of sample, 1 mg DO/L. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 9 Dissolved Oxygen & Aquatic Primary Productivity AP Part A: The Winkler Method for Dissolved Oxygen Forming the Manganese (III) Hydroxide Precipitate 1. Obtain a water sample from the instructor. Samples should be at either 5°, 20° or 30°C. Record the temperature of the water with a thermometer. Different groups will use water at different temperatures. 2. Place the water sample into your 250-300ml “BOD” (Biological Oxygen Demand) bottle. Any container which can be closed with an airtight seal is appropriate. Allow the sample to overflow the container so that it will be completely filled. • Close the container. Turn the container upside down and use a paper towel to remove any water which is around the outside of the stopper or lid. Create manganese hydrogen precipitate in your water sample: • Open the container and carefully pipet 2ml of manganese sulfate into the container. Make sure the pipet tip is below the water surface in the container. • With a fresh pipet, add 2ml of the NaOH/NaI (alkaline-iodide) solution into the sample in the container. Make sure the pipet tip is below the surface of the water in the container. • Stopper or seal your container. Carefully invert the bottle to allow for complete mixing of the sample, manganese sulfate, and NaOH/NaI. • This precipitate is critical to the determination of dissolved oxygen. If a precipitate fails to form, repeat procedure. • Allow the precipitate to settle for 10-15 minutes or until approximately 50% of the volume in the container is occupied by the precipitate. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Lab # 12 Experiment Procedure 3. • Biology 10 AP Biology EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity Part A: The Winkler Method for Dissolved Oxygen Lab # Setting up the Burette 12 4. While waiting for the manganese hydroxide precipitate to settle, set up your burette. • • 5. Test the proper functioning and seal of the petcock at the bottom of the burette. • Experiment Procedure • • 6. Label a beaker ‘Burette Waste Solution’ and place it under the tip of the burette. Close the petcock and fill the burette with distilled water. Open and close the petcock. When open, the water should flow out. When closed, the water should not leak out of the burette tip or around the petcock assembly. Ask your instructor for help if the burette leaks. Practice slightly opening the petcock so that the water just drips out slowly. Completely drain the burette. Close the petcock. • • • 7. Attach a burette to a ring stand with a clamp. Adjust so it is vertical and the volume gradations are easily visible. Allow enough space under the burette for a 500ml flask. To remove any residual water, fill the burette with 3ml of the sodium thiosulfate solution. Open the petcock and allow the sodium thiosulfate solution to drain from the burette. Again, practice opening the petcock so that the sodium thiosulfate solution just drips out slowly. Completely empty the burette. Close the petcock. After closing the petcock, completely fill the burette with the sodium thiosulfate solution. Addition of the Hazardous Concentrated Sulfuric Acid 8. Go to the station set up by the instructor for pipeting the Concentrated Sulfuric Acid. • • Caution! This should be performed by the instructor. The instructor will be wearing gloves and goggles. Exercise extreme caution!! • • Open your container and place it on the lab bench. Standing away from the container, have the instructor pipet 2 ml of the concentrated sulfuric acid directly into your container. The tip of the pipet should be below the surface of the solution in your container. Stopper the container and mix by carefully inverting the container several times. Mix occasionally. You should observe that the precipitate begins to dissolve and the sample will become yellowish as free iodine, I2, is formed in the container. Remember that the quantity of free I2 released in this step is directly equivalent to the concentration of dissolved oxygen in your sample. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 11 AP Part A: The Winkler Method for Dissolved Oxygen Titration of the Free I2 9. Wait 5 minutes and then remove exactly 200ml of solution from the container with a graduated cylinder and transfer the solution to a 500ml flask. Biology Lab # 12 10. Add 1.0ml of starch solution to the pale yellow solution in the 500ml flask. The solution will immediately become purple. Swirl the flask gently to completely mix. 11. Place the 500ml flask under your burette tip. Adjust the burette if necessary. Record the initial starting volume of the sodium thiosulfate in the burette. 12. Slowly and carefully open the petcock of the burette until the sodium thiosulfate begins to drip out of the burette tip. • • Continually swirl the 500ml flask to insure thorough mixing of the sodium thiosulfate with the sample. Close the petcock when the solution in the flask is a pale yellow color. 13. Open the petcock carefully so that the sodium thiosulfate solution drips out slowly. Continue to gently swirl the flask to insure complete mixing. 14. Be careful when adding the titrant. When the solution starts to lighten in color, it should only take another 2-3 drops of titrant. Try not to miss the endpoint. The endpoint is reached when the solution has gone from purple to completely clear. Immediately close the petcock to stop addition of the sodium thiosulfate solution. 15. At this point, all of the free I2 has been converted to sodium iodide by reaction with the sodium thiosulfate. The solution is colorless, since there is no longer any I2. Record the final position of the meniscus of sodium thiosulfate in the burette. Record the ending volume here. ___________ Determine the total volume of sodium thiosulfate which was used to titrate your sample to the endpoint. Calculate the difference between the starting volume recorded in Step 11 and the ending volume recorded above. Record the total volume of sodium thiosulfate used here in ml. ___________ 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Experiment Procedure Record the starting burette volume here. __________ 12 AP Biology EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity Part A: The Winkler Method for Dissolved Oxygen Lab # 16. For the sensitivity required in this laboratory experiment, the total volume in milliliters of the sodium thiosulfate solution used to titrate the sample to the endpoint is exactly equivalent to the dissolved oxygen concentration in milligrams of dissolved oxygen (DO) per liter of sample solution, mg DO/L. 12 17. Record the temperature of your sample and the amount of dissolved oxygen in the chart. Use the nomogram of oxygen saturation graph on this page to determine the % saturation of DO in the sample. If your temperature lies between these values, estimate the % saturation of DO. Record the value in Table 1. Experiment Procedure TABLE 1: Temperature/DO Data Temperature Your DO mg/L 0 5 %DO Saturation Class Mean DO (from nomogram) mg/L Class Mean DO Saturation (from nomogram) 18. Determine the class average data for the water samples at the three temperatures which were available. Record the values in Table 1. 10 15 20 25 30 Water Temperature °C ion rat 00 40 50 60 70 80 120 140 % u Sat 19. Using linear graph paper or Graph #1 provided on page 18, plot Both the lab group and class means percent saturation as function of temperature. 1 90 30 20 10 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Oxygen (mg per liter) Figure 2: Nomogram of Oxygen Saturation 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 13 AP Biology Part B: Primary Productivity PRIMARY PRODUCTIVITY For Part B., samples of natural water will be used to determine gross productivity, net productivity, and respiration rates. To mimic the light attenuation found as one goes deeper beneath the surface of a body of water, screens will be used to reduce the light intensity. Review the Background section of this laboratory for a complete discussion of Primary Productivity. Lab # 12 Day One 1. Obtain 7 clean “BOD” (Biological Oxygen Demand) containers. Any container which holds approximately 250-300ml and which can be closed with an airtight seal is appropriate. • 2. Completely fill each bottle with water sample from the lake or pond. Allow the sample to overflow the container so that it will be completely filled. Stopper or close the container. Turn the container upside down and use a paper towel to remove any water which is around the outside of the stopper or lid. Label the 7 bottles as follows: #1 - Initial #2 - Dark #3 - 100% #4 - 65% #5 - 25% #6 - 10% #7 - 5% 3. Bottle #1 is the initial starting bottle and serves as a baseline. 4. Wrap Bottle #2 in aluminum foil, for it serves as the dark (no light) control. Place in a dark place. No photosynthesis, only respiration, will occur here. 5. Bottles #3 –#7 will simulate the depth in a body of water that natural light will attenuate This is done by wrapping each bottle with a different number of screens. Bottle #3 will have no screens with 100% attenuation of light, Bottle #4 has 1 screen with 65% attenuation, Bottle #5 has 3 screens with 25% attenuation, Bottle #6 has 5 screens with 10% attenuation, and Bottle #7 has 7 screens with 5% attenuation. Cover the bottoms of the bottles so that no light can enter. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Experiment Procedure • 14 AP Biology EDVO-Kit # 292 Part B: Primary Productivity Lab # 12 Dissolved Oxygen & Aquatic Primary Productivity 6. NOTE: Each group will be assigned one of the following number of screens for Bottles 3, 4, & 5. Record the starting time here. ________________. 7. Experiment Procedure Percent Number of Light Screens 100 65 25 10 2 0 1 3 5 8 Put bottles #3-7, labels down, on their sides exposed to a constant source of light. Your teacher will indicate where they should be placed. Leave these bottles overnight. You will fix the amount of dissolved oxygen in the Bottle labeled ‘#1 Initial’. This serves as the starting level of dissolved oxygen in the lake water sample. You will perform step 3 from the Winkler procedure used in Part A of this laboratory. • Open the container labeled “#1 Initial” and carefully pipet 2 ml of manganese sulfate into the container. Make sure the pipet tip is below the surface of the water in the container. • With a fresh pipet, add 2ml of the NaOH/NaI (alkaline-Iodide) solution into the sample in the container. Make sure the pipet tip is below the surface of the water in the container. • Stopper or seal your container. Firmly hold the stopper or lid of your container. Carefully invert the bottle to allow for complete mixing of the sample, manganese sulfate, and NaOH/ NaI. A precipitate will form. • Let the bottle sit on the laboratory bench overnight. Finish processing it with the rest of the bottles tomorrow. Optional Activity If directed by the instructor, place several drops of the pond water on a microscope slide and cover with a coverslip. Observe the organisms found in the natural water sample. Draw what is observed and try to identify the organisms. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 15 AP Part B: Primary Productivity Biology Lab # Day Two 1. Fix the dissolved oxygen in Bottles #2, 3, 4 , 5, 6 , 7. This is the beginning of the Winkler procedure. 12 Record the time you begin here. __________ • • • 2. Repeat procedure in step 1 above with Bottles #3, 4, 5, 6, 7. 3. While precipitate is settling in Bottles #2, 3, 4, 5, 6 and 7, review the Winkler procedure used in Part A. Obtain Bottle #1. 4. You should clean and set up the burettes as outlined in Part A, steps 4-7. 5. Begin processing all of the Bottles, #1 - #7, using the Winkler procedure for the determination of dissolved oxygen from Part A. Part A Step 3, the fixing of the dissolved oxygen, has already been completed for all of the bottles. Now continue with Part A Step 8. Continue through steps 16. Record DO values in the appropriate table Table 2 or 3. TABLE 2: Respiration Individual Data Class Mean DO, Initial DO, Dark Bottle Respiration Rate (Initial - Dark) 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Experiment Procedure Open Bottle #2 and carefully pipet 2ml of manganese sulfate into the bottle. Make sure pipet tip is below surface of the water in the container. With a fresh pipet, add 2ml of the NaOH /NaI (alkaline-iodide) solution to the sample in the bottle. Make sure pipet tip is below surface of water in the container. Stopper or seal the bottle. Firmly hold the stopper or lid and carefully invert the bottle to allow for complete mixing of the sample, Manganese sulfate, and NaOH/NaI. A precipitate will form. 16 AP Biology EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity Part B: Primary Productivity Lab # 12 TABLE 3: Productivity of Screen-Wrappen Samples Experiment Procedure # of Screens % Light 0 100 1 65 3 25 5 10 7 5 DO INDIVIDUAL DATA Gross Productivity 6. Net Productivity CLASS MEAN PRODUCTIVITY DO Gross Productivity Net Productivity In reporting the data for productivity, the concentration of dissolved oxygen which has been determined, will be converted from mg/L to ml/L using the following conversion factor: 1 mg DO/L = 0.698ml DO/L. Therefore, [0.698ml D0/L] X (#mg D0/L) = #ml D0/L 1mg D0/L 7. Calculate and record the gross and net productivity for the natural water samples using the following equations. Productivity is a rate term, therefore, divide by the number of hours the experiment ran to arrive at a value of ml DO/L per hour. • Gross Productivity = (Light Bottle (#3, #4, #5, #6, or #7) ml DO/L - Dark Bottle (#2) ml DO/L)/hours • Net Productivity = (Light Bottle (#3, #4, #5, #6. Pr #7) ml DO/L Initial Bottle (#1) ml DO/L) /hours • Respiration rate = (Initial Bottle (#1) ml DO/L - Dark Bottle (#2) ml DO/L) /hours 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 17 AP Biology Part B: Primary Productivity 8. Record the values for the individual experiment conditions here. • Respiration rate = __________ml DO/L/hour. • Bottle 3 Lab # 12 Gross Productivity rate = ___________ ml DO/L/hour Net Productivity rate = ____________ ml DO/L/hour • Bottle 4 Gross Productivity rate = ___________ ml DO/L/hour Net Productivity rate = ____________ ml DO/L/hour Bottle 5 Gross Productivity rate = ___________ ml DO/L/hour Net Productivity rate = ____________ ml DO/L/hour • Bottle 6 Gross Productivity rate = ___________ ml DO/L/hour Net Productivity rate = ____________ ml DO/L/hour • Bottle 7 Gross Productivity rate = ___________ ml DO/L/hour Net Productivity rate = ____________ ml DO/L/hour 9. Record the individual and class average values for respiration in Table 2 and for light of different intensities in the Table 3. The values should be reported in ml DO/L/hour. 10. Record the average class value for the Respiration rate here. __________. 11. Using linear graph paper or Graph #2 provided on page 19, plot the average gross and net productivity on the Y-axis in ml DO/L/hr versus the percent of Light Intensity on the X-axis. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Experiment Procedure • 18 AP Biology EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity Analysis of Results - Graph #1 1. 12 Graph both the lab group and class means percent saturation as function of temperature. 2. Title the Graph_________________________________________________ 3. Determine the independent variable (horizontal (X) axis). Label the graph. 4. Determine the dependent variable (vertical (Y) axis). Label the graph. 5. What hypothesis is being tested in this experiment? Experiment Procedure Lab # 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 19 AP Analysis of Results - Graph #2 1. Graph the average gross and net productivity values for samples as a function of light intensity (%). 2. Title the Graph_________________________________________________ 3. Determine the independent variable (horizontal (X) axis). Label the graph. 4. Determine the dependent variable (vertical (Y) axis). Label the graph. 5. What hypothesis is being tested in this experiment? Biology Lab # 12 Experiment Procedure 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM 20 AP Biology Lab # Experiment Procedure 12 EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity Laboratory Extensions I. EFFECT OF ADDITION OF NITROGEN AND PHOSPHORUS ON PRODUCTIVITY The availability of inorganic nutrients such as nitrogen and phosphorous may also affect primary productivity in aquatic environments. Nutrients are removed during primary productivity. When a nutrient is no longer present in sufficient quantity, a slowing of productivity will result. The addition of specific nutrients may result in an increase in productivity provided all the other necessary nutrients are present in adequate amounts. As in Part B., samples of natural water will be used to determine gross productivity, net productivity, and respiration rates. This experiment will determine the effect of the nutrients nitrogen and phosphorus on primary productivity. Review the Background section of this laboratory for a complete discussion of Primary Productivity. Day One 1. 2. Obtain 5 clean “BOD” (Biological Oxygen Demand) containers. Any container which holds approximately 250-300ml and which can be closed with an airtight seal is appropriate. • Completely fill each bottle with water sample from the lake or pond. Allow the sample to overflow the container so that it will be completely filled. • Stopper or close the container. Turn the container upside down and use a paper towel to remove any water which is around the outside of the stopper or lid. Label 5 bottles as follows: #1 Initial #2 Dark #3 Light + Nitrogen Addition #4 Light + Phosphorous Addition #5 Light Alone 3. Bottle #1 is the initial starting bottle and serves as a baseline. 4. Wrap Bottle #2 in aluminum foil, for it serves as the dark (no light) control. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 21 AP Laboratory Extensions Biology Lab # Bottle #3 is supplemented with a source of nitrogen and exposed to light. With a pipet, add 1.0ml of nitrogen enrichment solution to Bottle #3. The instructor may have set up a pipeting station for this step. 6. Bottle #4 is supplemented with a source of phosphorous and exposed to light. With a pipet, add 1.0ml of phosphorous enrichment solution to bottle 4. The instructor may have set up a pipeting station for this step. 7. Bottle #5 is a control which receives no nutrient supplementation, but is exposed to light. 8. Put Bottle #2 in a dark place and Bottles #3, #4, and #5 so that they are exposed to a constant source of light. Your teacher will indicate where they should be placed. Leave these bottles overnight. Record the starting time here. ________________ 9. You will fix the amount of dissolved oxygen in the bottle labeled ‘#1 Initial’. This serves as the starting level of dissolved oxygen in the lake water sample. You will perform step 3 from the Winkler procedure used in Part A of this laboratory. • Open the container labeled “#1 Initial” and carefully pipet 2ml of manganese sulfate into the container. Make sure the pipet tip is below the surface of the water in the container. • With a fresh pipet, add 2ml of the NaOH/NaI (alkaline-Iodide) solution into the sample in the container. Make sure the pipet tip is below the surface of the water in the container. • Stopper or seal your container. Firmly hold the stopper or lid of your container. Carefully invert the bottle to allow for complete mixing of the sample, manganese sulfate, and NaOH/NaI. A precipitate will form. • Let the bottle sit on the laboratory bench overnight. Finish processing it with the rest of the bottles tomorrow. Optional Activity If directed by the instructor, place several drops of the pond water on a microscope slide and cover with a coverslip. Observe the organisms found in the natural water sample. Draw what is observed and try to identify the organisms. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM 12 Experiment Procedure 5. 22 EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity AP Biology Lab # 12 Laboratory Extensions Day Two 1. Fix the dissolved oxygen in Bottles #2, #3, #4 and# 5. This is the beginning of the Winkler procedure. Record the time you begin here. __________ • • Experiment Procedure • Open Bottle #2 and carefully pipet 2ml of manganese sulfate into the container. Make sure pipet tip is below surface of the water in the container. With a fresh pipet, add 2ml of the NaOH /NaI (alkaline-iodide) solution into the sample in the bottle. Make sure pipet tip is below surface of water in the container. Stopper or seal the bottle. Firmly hold the stopper or lid and carefully invert the bottle to allow for complete mixing of the sample, Manganese sulfate, and NaOH/NaI. A precipitate will form. 2. Repeat procedure in step 1 above with Bottles #3, #4, and #5. 3. While precipitate is settling in Bottles #2, #3, #4, and #5, review the Winkler procedure used in Part A. Obtain Bottle #1. 4. You should clean and set up the burettes as outlined in Part A, steps 4-7. 5. Begin processing all of the Bottles #1, #2, #3, #4, and #5 using the Winkler procedure for the determination of dissolved oxygen from Part A. Part A Step 3 , the fixing of the dissolved oxygen, has already been completed for all of the bottles. Now continue with Part A Step 8. 6. In reporting the data for productivity, the concentration of dissolved oxygen which has been determined, will be converted from mg/L to ml/L using the following conversion factor: 1 mg DO/L = 0.698ml DO/L. Therefore, [0.698ml D0/L] 1mg D0/L 7. X (#mg D0/L) = #ml D0/L Calculate and record the gross and net productivity for the natural water samples using the following equations. Productivity is a rate term, therefore, divide by the number of hours the experiment ran to arrive at a value of ml DO/L per hour. • • • Gross Productivity = (Light Bottle (#3, #4, or #5) ml DO/L Dark Bottle (#2) ml DO/L)/hours Net Productivity = (Light Bottle (#3, #4, or # 5) ml DO/L - Initial Bottle (#1) ml DO/L) /hours Respiration rate = (Initial Bottle (#1) ml DO/L - Dark Bottle (#2) ml DO/L) /hours 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 23 AP Biology Laboratory Extensions 8. Record the values for the individual experiment conditions here. • Respiration rate = __________ml DO/L/hour. • Nitrogen enriched Bottle 3 Lab # 12 Gross Productivity rate = ___________ ml DO/L/hour Net Productivity rate = ____________ ml DO/L/hour • Phosphorous enriched bottle 4 Gross Productivity rate = ___________ ml DO/L/hour • Un-enriched Bottle 5 Gross Productivity rate = ___________ ml DO/L/hour Net Productivity rate = ____________ ml DO/L/hour 9. Record the class average values for Respiration Rate in Table 4 and for nitrogen and phosphorous enriched samples in Table 5. The values should be reported in ml DO/L/hour. 10. Record the average class value for the Respiration rate here. __________. 11. Using linear graph paper or Graph #3 on page 25, plot the average gross and net productivity on the Y-axis in ml DO/L/hr versus nitrogen enriched, phosphorous enriched, and un-enriched samples on the Xaxis. TABLE 4: Respiration Individual Data Class Mean DO, Initial DO, Dark Bottle Respiration Rate (Initial - Dark) 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Experiment Procedure Net Productivity rate = ____________ ml DO/L/hour 24 EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity AP Biology Laboratory Extensions Lab # Experiment Procedure 12 TABLE 5: Productivity of Pond Samples - Nitrogen or Phosphorous Added Sample Treatment % Light Nitrogen Added 100 Phosphorous Added 100 Unenriched 100 Little Winter Pond % Light Depth in Intensity Meters 100 0 65 25 0.3 1.0 10 2 3.0 5.0 INDIVIDUAL DATA DO Gross Productivity Intensity Meters 100 0 65 25 3.5 9 10 2 15 25 DO Gross Productivity Net Productivity From the results in Part B of this laboratory, compare the productivity of two separate ponds located in Massachusetts. This exercise is a simulation. In Little Winter Pond, water is murky and filled with algae and weeds. Light does not penetrate far beneath the surface. In Big Winter Pond, a clear pond high in the mountains, light is not attenuated much until you get to a very deep spot near the middle of the lake. The Little and Big Winter Pond have the values as shown in the Tables at left. The values for these two hypothetical ponds will be used for plotting the data. Big Winter Pond Depth in Net Productivity II. PRODUCTIVITY SIMULATION FROM 2 PONDS 1. % Light CLASS MEAN PRODUCTIVITY To simulate the productivity in the two ponds, convert your gross productivity data for the un-enriched sample (5) in ml DO/L/hour to ml DO/L/day. Simply multiply by 24 to get the value per day. # ml DO/L hour 2. X 24 hour day = # ml DO/L day Next, convert the gross productivity values in ml DO/L/day to carbon productivity in mg C/m3/day. For each ml of oxygen produced, 0.536 mg of carbon has been consumed. Therefore, simply multiply the ml DO/L/day values by 0.536 to get the carbon productivity in mg C/m3/day. Also convert the respiration rate to carbon values, again in mg C/m3/day. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 25 AP Analysis of Results - Graph #3 1. Graph the average gross and net productivity values for nitrogen enriched, phosphorous enriched and un-enriched samples. 2. Title the Graph_________________________________________________ 3. Determine the independent variable (horizontal (X) axis). Label the graph. 4. Determine the dependent variable (vertical (Y) axis). Label the graph. 5. What hypothesis is being tested in this experiment? Biology Lab # 12 Experiment Procedure 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM 26 AP Biology EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity Optional Analysis of Results - Graph #4 1. 12 Graph the converted carbon assimilation values versus depth related to light intensity for Little and Big Winter ponds. 2. Title the Graph_________________________________________________ 3. Determine the independent variable (horizontal (X) axis). Label the graph. 4. Determine the dependent variable (vertical (Y) axis). Label the graph. 5. What hypothesis is being tested in this experiment? Experiment Procedure Lab # 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 27 AP Biology Study Questions How does temperature affect the solubility of oxygen in water? 2. How does salinity affect the solubility of oxygen in water? 3. Would you expect boiled water to contain a large amount of dissolved oxygen ? 4. How many moles of sodium thiosulfate are required to completely titrate 6 moles of oxygen. 5. Why is the starch usually added after the titration has begun? 6. From Graph #2 (page 19), were any of the samples from Part B limited by the available light? 7. Were any samples limited by the available nutrients? 8. Explain why the DO concentration of water samples taken from a lake in early morning would be lower than the DO concentration of water samples taken in late afternoon. 9. Explain why the DO concentration of water samples taken from a swiftly flowing stream would be higher than the DO concentration of water samples taken from a lake. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Lab # 12 Experiment Procedure 1. 28 AP Biology EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity Notes: Lab # Experiment Procedure 12 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity AP Notes to the Instructor Biology OVERVIEW OF LABORATORY INVESTIGATIONS The "hands-on" laboratory experience is a very important component of the science courses. Laboratory experiment activities allow students to identify assumptions, use critical and logical thinking, and consider alternative explanations, as well as help apply themes and concepts to biological processes. www.edvotek.com ORGANIZING AND IMPLEMENTING THE EXPERIMENT Visit our web site for information about EDVOTEK's complete line of experiments for biotechnology and biology education. Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. Technical Service Department E O DV -TE C H S E RV I C E Mon - Fri 9:00 am to 6:00 pm ET If you do not find the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. www. edvotek.com 1-800-EDVOTEK (1-800-338-6835) FAX: (301) 340-0582 web: www.edvotek.com ET email: edvotek@aol.com pm Mo -6 Please have the following information: n - Fri 9 am In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at 1-800-EDVOTEK (1-800-3386835). • The experiment number and title • Kit Lot number on box or tube • The literature version number (in lower right corner) • Approximate purchase date 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Lab # 12 Instructor's Guide EDVOTEK experiments have been designed to provide students the opportunity to learn very important concepts and techniques used by scientists in laboratories conducting biotechnology research. Some of the experimental procedures may have been modified or adapted to minimize equipment requirements and to emphasize safety in the classroom, but do not compromise the educational experience for the student. The experiments have been tested repeatedly to maximize a successful transition from the laboratory to the classroom setting. Furthermore, the experiments allow teachers and students the flexibility to further modify and adapt procedures for laboratory extensions or alternative inquiry-based investigations. Online Ordering now available 29 30 AP Biology Lab # EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity Pre-Lab Preparations • Part A requires 2 liters each of fresh tap water at 5°, 20°, and 30°C. The water should be held covered at these temperatures the day before the laboratory. • A natural source of water such as lake, pond, seawater, or an algal culture of Chlorella will work for Part B. Depending on the size of the bottles or flasks to be used, collect 15 liters of natural source water. • The solutions should be prepared 1 or 2 days before the laboratory. The sodium thiosulfate should be prepared the day before and stored in the refrigerator. Working stocks are made on the day of the laboratory. 12 Instructor's Guide Part A. The Winkler Method for Dissolved Oxygen 1. Preparation of the sodium thiosulfate solution: • Boil 4 liters of distilled water for 10-15 minutes (to be completed before lab session). • On the day of the first laboratory session, add all of the sodium thiosulfate (component E) to boiled and cooled distilled water to make 2 liters final volume. Boiling depletes the oxygen in the water so that it will not interfere with the analysis. • Store carefully sealed in the refrigerator. 2. The thiosulfate solution will not be standardized since fine “analytical” results are not needed. When the thiosulfate is properly measured on a balance, titration is not necessary for classroom results. 3. Just prior to the titration step in Part A, Student Experimental Procedures: 4. • Prepare the sodium thiosulfate working solution for the class. • Add 250ml of the working stock solution to 750ml of boiled and cooled distilled water. On the day of the lab, set up measuring/pipeting stations for: • • • • • • Tap water (250-300ml) Manganese Sulfate (2ml) NaOH/NaI (2ml) Sodium thiosulfate (50ml) Concentrated sulfuric acid (2ml) Starch solution (1ml) 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity 31 AP Biology Pre-Lab Preparations Lab # 5. 6. Prepare the manganese sulfate solution: • Add all of the manganese sulfate (component C) to a total volume of 250ml of distilled water. • Filter with #1 filter paper and store covered at room temperature. Wearing gloves and goggles, prepare the NaI/NaOH solution: Add all of the NaOH pellets (component B) to a large beaker. • Add distilled water to a volume of 150 - 175ml. Caution: The solution will get hot. • Add all of the NaI (component A) and adjust to a final volume of 250ml with distilled water. Mix. • Store covered at room temperature. 2. 3. 4. On the day of the lab, prepare a working nitrogen enrichment solution for the class: • Add 10ml of the stock solution (component F) to 500ml of distilled water. • Store covered in the refrigerator. Students will use 1ml of the working solution to provide nitrogen enrichment for their sample. Prepare the phosphorous enrichment stock solution: • Add all the contents of component G to 100ml distilled water. Mix. • Store covered in the refrigerator. On the day of the lab, prepare a working phosphorous enrichment solution for the class: • Add 10ml of stock solution to 500ml of distilled water. • Students will use 1ml of the working solution to provide phosphorous enrichment for their sample. Part B will require 50 BOD type bottles (250-300ml). You may substitute 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Instructor's Guide • Part B. Primary Productivity 1. 12 32 AP Biology EDVO-Kit # 292 Dissolved Oxygen & Aquatic Primary Productivity Pre-Lab Preparations Lab # 12 250ml Erlenmeyer flasks. Small juice bottles with caps can also be used successfully. 5. Based on Table 1, assign each group a different light percentage (corresponding number of screens). The screens will be used to cover Bottles 3, 4, and 5. 6. On the day of the lab, set up measuring/pipeting stations for: • • • • • Instructor's Guide 7. Pond water (250-300ml) Nitrogen enrichment solution (1ml) Phosphorous enrichment solution (1ml) Manganese sulfate (2ml) NaOH/NaI (2ml) Set up microscopes if the optional activity in Student Experimental Procedures B. #11 is to be done by students. 1-800-EDVOTEK • www.edvotek.com Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/ laboratory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1996,1997, 1998, 1999, 2000, 2001, 2005,2007 EDVOTEK, Inc., all rights reserved. EVT 005057AM Please refer to the kit insert for the Answers to Study Questions Section V - Reactivity Data Material Safety Data Sheet IDENTITY (As Used on Label and List) Emergency Telephone Number Manufacturer's Name EDVOTEK, Inc. Incompatibility (301) 251-5990 (301) 251-5990 Date Prepared 14676 Rothgeb Drive Rockville, MD 20850 09-18-2002 ACGIH TLV Sulfur oxides Conditions to Avoid May Occur X Will Not Occur Section VI - Health Hazard Data Inhalation? Ingestion? Skin? Yes Yes Yes Causes eye and skin irritation. Material is irritating to mucous membranes and upper respiratory tract. Health Hazards (Acute and Chronic) IARC Monographs? NTP? OSHA Regulation? No data Signs and Symptoms of Exposure A stolid mask-like appearance of face, sleepiness d weakness skin and eye irritation, irritating to mucous membranes and upper respiratory Section II - Hazardous Ingredients/Identify Information OSHA PEL Hazardous Polymerization Carcinogenicity: Signature of Preparer (optional) Hazardous Components [Specific Chemical Identity; Common Name(s)] Strong acids Hazardous Decomposition or Byproducts Route(s) of Entry: Telephone Number for information Address (Number, Street, City, State, Zip Code) X Stable Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Manganese Sulfate Monohydride Section I Conditions to Avoid Unstable Stability May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements. ® Medical Conditions Generally Aggravated by Exposure Other Limits Recommended % (Optional) Emergency First Aid Procedures CAS # 10034-96-5 Flush eyes or skin with copious amounts of water for atleast 15 minutes while removing contaminated clothing and shoes. Remove to fresh air. Section VII - Precautions for Safe Handling and Use Section III - Physical/Chemical Characteristics Boiling Point No data Vapor Pressure (mm Hg.) No data Vapor Density (AIR = 1) No data Steps to be Taken in case Material is Released for Spilled Specific Gravity (H 0 = 1) 2 2.950 Melting Point No data Evaporation Rate (Butyl Acetate = 1) No data Sweep up, place in a bag and hold for waste disposal. Avoid raising dust. Ventilate area and wash soil site after material pick up is complete. Waste Disposal Method The material should be dissolved in 1) water 2) acid solution 3) Oxidized to a water-soluble state. Solubility in Water Precautions to be Taken in Handling and Storing Keep tightly closed , wash thoroughly after handling Appearance and Odor Other Precautions White to pale-pink powder None Section IV - Physical/Chemical Characteristics Flash Point (Method Used) LEL Flammable Limits No data UEL Extinguishing Media Noncombustible-use extinquishing media appropriate to surrounding fire condition Special Fire Fighting Procedures Section VIII - Control Measures Respiratory Protection (Specify Type) X Eye Protection Chemical resistant gloves Other Protective Clothing or Equipment Emits toxic fumes under fire conditions Other Mechanical (General) Protective Gloves Unusual Fire and Explosion Hazards Special Yes Local Exhaust Ventilation Wear SCBA and protective clothing to prevent contact with skin and eyes. Safety goggles SCBA, safety gloves and goggles Work/Hygienic Practices Material Safety Data Sheet Section V - Reactivity Data May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200. Standard must be consulted for specific requirements. Stability Unstable IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Sodium Iodide Section I 14676 Rothgeb Drive Emergency Telephone Number 301-251-5990 Telephone Number for information 301-251-5990 Rockville, MD 20850 Date Prepared 09-19-2002 EDVOTEK Address (Number, Street, City, State, and ZIP Code) Conditions to Avoid Stable Incompatiblity (Materials to avoid) Manufacturer's Name NIOSH/MSHA approved respirator X Incompatibles Alkali metals, bromine triflouride, strong oxidizers, perchloric acid, perchloryl Hazardous Decomposition or Byproducts thermal decomposition products may include toxic and corrosive fumes of iodides May Occur Hazardous Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Skin? Yes Yes Ingestion? Yes Health Hazards (Acute and Chronic) May cause irritation. Chronic ingestion may result in hypothyroidism. Signature of Preparer (optional) Carcinogenicity: NTP? IARC Monographs? None OSHA Regulation? No Data Section II - Hazardous Ingredients/Identity Information Hazardous Components (Specific Chemical Identity; Common Name(s)) OSHA PEL CAS # 7681-62-5 No Data ACGIH TLV Other Limits Recommended Signs and Symptoms of Exposure % (Optional) Inhalation: mucous membrane irritation Eye/skin contact: flush w/ water Medical Conditions Generally Aggravated by Exposure No data Contains no hazardous components Emergency and First Aid Procedures Treat symptomatically and supportively. Eye/skin contact: flush w/ water Inhalation: remove to fresh air. Section III - Physical/Chemical Characteristics Boiling Point 1304ϒC 1.91 Vapor Pressure (mm Hg.) Specific Gravity (H20 = 1) Section VII - Precautions for Safe Handling and Use at 25ϒC Melting Point 1 Vapor Density (AIR =1) N.D. = No Data N.D. 3.667 Steps to Be Taken in case Material Is Released or Spilled Sweep up, place in a bag and hold for waste disposal. Avoid raising dust. 661ϒC Evaporation Rate (Butyl Acetate =1) N.D. Ventilate area and wash spill site after pickup. Waste Disposal Method Follow federal, state and local regulations. Solubility in Water 184% at 25ϒC Precautions to be Taken in Handling and Storing Appearance and Odor odorless, white powder Section IV - Fire and Explosion Hazard Data Flash Point (Method Used) Extinguishing Media Flammable Limits Other Precautions LEL Store away from incompatibles Avoid contact UEL Section VIII - Control Measures Use suitable agent for type of surrounding fire Special Fire Fighting Procedures Move container from fire area if possible. Avoid breathing vapors or dust. Unusual Fire and Explosion Hazards None Respiratory Protection (Specify Type) NIOSH/MSHA approved respirator Special Yes No Mechanical (General) No Other None Protective Gloves Eye Protection Safety goggles PVC gloves Other Protective Clothing or Equipment Impervious clothing and equipment to prevent contact Ventilation Work/Hygienic Practices Local Exhaust Avoid contact Section V - Reactivity Data Material Safety Data Sheet IDENTITY (As Used on Label and List) Manufacturer's Name EDVOTEK, Inc. (301) 251-5990 (301) 251-5990 Date Prepared 14676 Rothgeb Drive Rockville, MD 20850 09-19-2002 Sodium Hydroxide CAS # 1310-73-2 2mg/m3 ACGIH TLV Ingestion? Skin? Yes Yes Yes None identified IARC Monographs? NTP? OSHA Regulation? None identified Call physician. Ingestion: Do not induce vomiting. Give water followed by vinegar, juice or egg white Inhalation: Move to fresh air. Skin/eye contact: flush with water Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled 1390°C Specific Gravity (H 0 = 1) 2 2.13 Vapor Pressure (mm Hg.) 20°C Melting Point 318°C NO data Evaporation Rate (Butyl Acetate = 1) NO data Dispose of properly Wear SCBA and protective clothing. Carefully place material into clean, dry container and cover. Waste Disposal Method Follow all federal, state, and local laws. Precautions to be Taken in Handling and Storing Keep container tightly closed. Store in corrosion-proof area. Store in a dry area. Isolate from incompatible materials. 10% appreciable Other Precautions Appearance and Odor White pellets, odorless None Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL UEL NA Extinguishing Media Inhalation? Health Hazards (Acute and Chronic) Emergency First Aid Procedures No data Boiling Point Solubility in Water Section VI - Health Hazard Data Medical Conditions Generally Aggravated by Exposure % (Optional) Section III - Physical/Chemical Characteristics Vapor Density (AIR = 1) X No data No data No data Signs and Symptoms of Exposure Ingestion: Severe burns to mouth, throat, and stomach, nausea & vomiting Inhalation: irritation Skin/eye contact: severe irritation or burns Other Limits Recommended 2mg/m3 Conditions to Avoid May Occur Will Not Occur None Section II - Hazardous Ingredients/Identify Information OSHA PEL Hazardous Polymerization Carcinogenicity: Signature of Preparer (optional) Hazardous Components [Specific Chemical Identity; Common Name(s)] Water, strong acids, metals, combustible materials, organic materials Zinc, aluminous, peroxide, halogenated hydroca Hazardous Decomposition or Byproducts None identified Route(s) of Entry: Telephone Number for information Address (Number, Street, City, State, Zip Code) Moisture Incompatibility Section I Emergency Telephone Number X Stable Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Sodium Hydroxide Conditions to Avoid Unstable Stability May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements. ® NA NA Section VIII - Control Measures Respiratory Protection (Specify Type) NIOSH/MSHA approved respirator Yes Use extinquishing media appropriate for surrounding fire Yes Mechanical (General) Special Fire Fighting Procedures Protective Gloves Wear protective equipment and self-contained breathing apparatus. Floof material with water Contact with moisture or water generate sufficient heat to ignite other materials. React with metals to produce hydrogen gas which can form explosive mixture with air. Material Safety Data Sheet Work/Hygienic Practices Other Eye Protection Neoprene gloves Other Protective Clothing or Equipment Unusual Fire and Explosion Hazards Special Local Exhaust Ventilation No None Safety goggles Uniform, apron Avoid contact Section V - Reactivity Data May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200. Standard must be consulted for specific requirements. Stability Unstable Conditions to Avoid Stable X incompatibles Incompatiblity (Materials to avoid) Strong acids, strong oxidizing agents, lead, silver and mercury salts, and iodines IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Sodium Thiosulfate Section I Manufacturer's Name 14676 Rothgeb Drive Emergency Telephone Number 301-251-5990 Telephone Number for information 301-251-5990 Rockville, MD 20850 Date Prepared 09-19-2002 EDVOTEK Address (Number, Street, City, State, and ZIP Code) Hazardous Decomposition or Byproducts Sulfur oxides May Occur Hazardous Polymerization Will Not Occur X Incompatibles Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Signature of Preparer (optional) Skin? Yes Health Hazards (Acute and Chronic) Carcinogenicity: Yes Ingestion? Yes Eye/skin irritation NTP? IARC Monographs? OSHA Regulation? No data Section II - Hazardous Ingredients/Identity Information Hazardous Components (Specific Chemical Identity; Common Name(s)) OSHA PEL ACGIH TLV Other Limits Recommended % (Optional) CAS # 10102-17-7 Signs and Symptoms of Exposure Irritation Medical Conditions Generally Aggravated by Exposure No data Does not contain any hazardous components Emergency and First Aid Procedures Sodium thiosulfate pentahydrate Skin/eye contact flush with copious amounts of H20 for at least 15 minutes . Remove to fresh air & remove contaminated clothing Section III - Physical/Chemical Characteristics Boiling Point N.D. Vapor Pressure (mm Hg.) N.D. Vapor Density (AIR =1) N.D. Section VII - Precautions for Safe Handling and Use Specific Gravity (H20 = 1) 1.729 Melting Point Evaporation Rate (Butyl Acetate =1) N.D. Water soluble Waste Disposal Method Cautiously add to a large stirred excess of water, adjust pH to neutral. Flush the aqueous solution down Precautions to be Taken in Handling and Storing White crystals, no odor Section IV - Fire and Explosion Hazard Data Flammable Limits UEL Wear SCBA and protective clothing to prevent contact with skin and eyes Emits toxic fumes under fire conditions. Avoid incompatibles Other Precautions LEL Use media for surrounding fire Special Fire Fighting Procedures Unusual Fire and Explosion Hazards Ventilate area and wash spill site after pickup. drain with plenty of water. Appearance and Odor Extinguishing Media Sweep up, place in a bag and hold for waste disposal. Avoid raising dust. N.D. Solubility in Water Flash Point (Method Used) Steps to Be Taken in case Material Is Released or Spilled Do not breathe dust. Avoid contact. Section VIII - Control Measures Respiratory Protection (Specify Type) NIOSH/MSHA approved respirator No No Special Mechanical (General) Yes Other None Protective Gloves Eye Protection Safety goggles Chemical resistant Other Protective Clothing or Equipment None Ventilation Work/Hygienic Practices Local Exhaust Wash thoroughly after handling Material Safety Data Sheet Section V - Reactivity Data May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200. Standard must be consulted for specific requirements. Stability Unstable Conditions to Avoid Stable X Incompatiblity (Materials to avoid) IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Nitrogen Enrichment Solution Section I Manufacturer's Name Emergency T Telephone Number 301-251-5990 Telephone Number for information T 301-251-5990 EDVOTEK Address (Number, Street, City, State, and ZIP Code) Rockville, MD 20850 Incompatibles Strongg reducingg agents, g , metals,, stron acids,, strongg bases Hazardous Decomposition or Byproducts May Occur Hazardous Polymerization X Incompatibles Section VI - Health Hazard Data Route(s) of Entry: Skin? es Ingestion? g Ys Ye Yes Ye Mayy cause mucous membranes b and upper pp respiratory p y tract irritation. Eye/skin y irritation 0 09-15-02 Signature of Preparer (optional) Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? No Data Hazardous Components (Specific Chemical Identity; Common Name(s)) OSHA PEL ACGIH TLV L LV Other Limits Recommended % (Optional) Signs and Symptoms of Exposure Irritation CAS # 7757-79-1 CAS# 12125-02-9 No Data Contains no hazardous components p Emergency and First Aid Procedures N.D.= Not Determined Boiling Point N.D. Vapor Pressure (mm Hg.) V N.D. N.D. Specific Gravity (H20 = 1) N.D. Melting Point Avoid raisingg dust. A N.D. Evaporation Rate (Butyl (B t lA Acetate t t =1) 1 N.D. W Solubility in Water Observe all federal, state, and local regulations soluble Appearance and Odor Clear liquid, characteristic odor Section IV - Fire and Explosion Hazard Data Flash Point (Method Used) Precautions to be T Taken in Handling and Storing Flammable Limits LEL Other Precautions Extinguishing Media Keep away from incompatibles UEL Avoid contact A Water spray W Special Fire Fighting Procedures Wear W ear SCBA A and pprotective clothing g to pprevent contact with skin and eyes y Respiratory Protection (Specify Type) T Ventilation No Local Exhaust Special es Unusual Fire and Explosion Hazards Emits toxic fumes. Contact with other material mayy cause fire. Protective Gloves Chemical resistant Work/Hygienic Practices No Other None Eye Protection Safety goggles Avoid contact and incompatibles A Section V - Reactivity Data Material Safety Data Sheet IDENTITY (As Used on Label and List) Emergency Telephone Number Manufacturer's Name EDVOTEK, Inc. Incompatibility (301) 251-5990 (301) 251-5990 Date Prepared 14676 Rothgeb Drive Rockville, MD 20850 Incompatibles Alkaloids, antipyrine, choral hydrate, lead acetate, pyrogallol, resorcinol Hazardous Decomposition or Byproducts Hazardous Polymerization phosphorous oxides, sodium oxide Conditions to Avoid May Occur X Will Not Occur None Section VI - Health Hazard Data Route(s) of Entry: Telephone Number for information Address (Number, Street, City, State, Zip Code) X Stable Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Phosphorous Enrichment Reagents Section I Conditions to Avoid Unstable Stability May be used to comply with OSHA's Hazard Communication Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements. ® Inhalation? Ingestion? Skin? Yes Yes Yes Health Hazards (Acute and Chronic) None 09-19-2002 Carcinogenicity: IARC Monographs? NTP? Signature of Preparer (optional) OSHA Regulation? No data Signs and Symptoms of Exposure Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Other Limits Recommended Medical Conditions Generally Aggravated by Exposure % (Optional) Inhalation: remove to fresh air Skin/eye contact: flush with water Ingestion: treat symptomatically and supportively. Seek medical attention Section VII - Precautions for Safe Handling and Use Section III - Physical/Chemical Characteristics Boiling Point Steps to be Taken in case Material is Released for Spilled Specific Gravity (H 0 = 1) 2 decomposes Vapor Pressure (mm Hg.) No data Vapor Density (AIR = 1) No data Solubility in Water No data Melting Point Evaporation Rate (Butyl Acetate = 1) No data Keep container closed and store away from incompatibles Avoid contact Flammable Limits LEL UEL Extinguishing Media Water spray, CO2, Dry chemical powder or appropriate foam Special Fire Fighting Procedures Wear SCBA and protective clothing to prevent contact with skin and eyes Move container from fire area if possible. Do not scatter spilled material with high pressure water Unusual Fire and Explosion Hazards Avoid breathing fumes Observe all federal, state, and local regulations Other Precautions Odorless, white powder No data Waste Disposal Method Precautions to be Taken in Handling and Storing Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Sweep up, place in a bag and hold for waste disposal. Avoid raising dust. Ventilate area and wash spill site after pickup. No data Soluble Appearance and Odor No Data Emergency First Aid Procedures CAS # 7558-79-4 Contains no hazardous components Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Protective Gloves NIOSH/MSHA approved respirator Local Exhaust No Mechanical (General) Yes Work/Hygienic Practices Other Eye Protection YEs Other Protective Clothing or Equipment Special Wear equipment to prevent contact Avoid contact No None Safety goggles