Time Estimate for Entire Lab: 2.0 hours. Special Requirements. Seventh Edition Changes This was Laboratory 19 in the previous edition.

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1 Laboratory 20 Effects of Pollution (LM pages ) Time Estimate for Entire Lab: 2.0 hours Special Requirements 1. Living material (order in advance for timely delivery): hay infusion cultures; Gammarus; Chlorella (common pond algae); and Daphnia 2. Growth required: One week to prepare your own hay infusion (optional); seed germination must be started at least 4 days prior to lab. 3. Fresh material (obtain locally, close to time of use): hay or grass (optional); sterile spring water Seventh Edition Changes This was Laboratory 19 in the previous edition. MATERIALS AND PREPARATIONS Effect of Pollutants on Ecosystems (LM pages ) hay infusion cultures (culture media sets, Carolina ) jar, wide-mouth, with screw-cap, 1-gallon size eyedropper slides and coverslips microscopes, compound light lens paper sterile spring water (local purchase or Carolina ) Gammarus culture (Carolina , amphipods, or Nasco Science LM00205, fairy shrimp) containers for spring water 1% sulfuric acid (H 2 SO 4 ) (Carolina , -3301) water, boiled and cooled to 31 C petri dishes filter paper for germinating seeds bean seeds ph meter beakers, 50 and 200 ml hot plate graduated cylinder thermometer, Celsius Hay infusion cultures. Culture media sets are suitable and can be purchased. Instructions are included in each set. Use a 1-gallon, wide-mouth jar covered loosely with screw-cap, and maintain the culture in a welllighted place, but not in direct sunlight. To prepare the various types of cultures needed, follow these directions: 1. Control culture: Prepare the culture according to the directions provided by the supply house. 2. Enriched medium: Do not dilute the nutrient solution as much as recommended by the supply house. 1 Note: Materials and Preparations instructions are grouped by exercise. Some materials may be used in more than one exercise.

2 84 3. Lack of oxygen: Prepare the same as the control culture, but use minimal oxygen tightly cover the jar with a screw-cap or plastic wrap, and do not aerate. 4. ph 4: Adjust one culture to ph 4 using 1% sulfuric acid. Seed germination. Line two petri dishes with two layers of filter-paper disks. In dish 1 (the control), dampen the paper with spring water. In dish 2 (the acidic solution), dampen the paper with an acidic solution (ph 4), which simulates acid rain. Arrange four to six bean seeds in each petri dish, and cover with two filterpaper disks. Replace the lids on the petri dishes, and label the dishes appropriately. The seeds should germinate in at least four days. Gammarus. Large quantities of spring water (100 ml per group) will need to be prepared for the Gammarus culture. Use plain spring water in the control culture. To simulate thermal pollution, use plain spring water that has been boiled and cooled to 31 C. For acid pollution, use spring water adjusted to ph 4 with 1% sulfuric acid. Expect some Gammarus in each laboratory to die. Therefore, purchase half again as many as you determine you will need. Other species may also be used. 1% sulfuric acid solution. Add 1 ml of sulfuric acid to 100 ml of water Cultural Eutrophication (LM pages ) Daphnia culture (Carolina ) spectrophotometer cuvettes for spectrophotometer Chlorella (common pond algae) (Carolina ) test-tube racks petroleum jelly petri dishes, small microscopes, binocular dissecting lens paper indigo carmine (Carolina ) water, distilled probe or dissecting needle Spectrophotometer and tubes (cuvettes). The most common instrument for teaching use is the Bausch and Lomb Spectronic 20. For this laboratory, the wave-length indicator should be set at 635 nm (or mm). The instrument must be calibrated for zero and infinite absorbance. Calibrate for infinite absorbance by using the left-hand knob to line up the needle with the left-most marker (an infinity symbol). Then insert a clean cuvette of distilled water and adjust for 100% transmittance using the right-hand knob. The culture should be just visibly green. The Daphnia should be starved in clean, aged water (stale tap water that has been allowed to stand for a few days to allow chlorine to escape) to for an hour or so prior to the lab. This clears their gut, since feces production would foul the experimental vessel and reduce the apparent feeding rate. 0.1% carmine solution. Dissolve 0.1 g of indigo carmine in 100 ml of distilled water. EXERCISE QUESTIONS 20.1 Effect of Pollutants on Ecosystems (LM pages ) Hypothesize how acid rain and thermal pollution might affect (1) the producers, and therefore the ecosystem; and (2) the first-order consumers, and therefore the ecosystem. 1. Acid rain has a low ph that is harmful to plants. The increase in temperature caused by thermal pollution may reduce members of some species of producers as well. If plants (producers) are destroyed, the ecosystem is destroyed. 2. Thermal pollution removes oxygen from the water, oxygen that is necessary for consumers. If first-order consumers die, all the rest die.

3 85 Hay Infusion (LM pages ) Experimental Procedure: Hay Infusion (LM page 269) Table 20.1 Hay Infusion Cultures* Types of Culture Diversity of Life Relative Quantity of Organisms (List Organisms) (High, Medium, or Low) Control culture Will vary Medium Enriched medium Will vary High Lack of oxygen Will vary Low ph 4 Will vary Low *Results will depend partially on the particular hay infusion culture obtained. Seed Germination (LM page 269) Experimental Procedure: Seed Germination (LM page 269) Table 20.2 Seed Germination ph Observations Control solution Acidic solution Generally, seeds germinating in the presence of water with a low ph will imbibe this water, thus resulting in the loss of a variety of enzymatic activities. Optimal soil conditions for the germination of most seeds include a neutral or slightly acidic ph. Gammarus (LM page 270) Experimental Procedure: Gammarus (LM page 270) Control Culture (LM page 270) 4. Where do they spend their time in the container? near the bottom 5. How do they spend their time? They swim around in an apparently random fashion. Occasionally, they lie still on the bottom of the container. 6. What percentage of time is spent moving? 90 to 95% 7. Do they use all of their legs in swimming? No, they move by flexing the appendages on the side of their body, one side at a time. 8. Which legs are used in jumping and climbing? the thoracic appendages 9. Do Gammarus avoid each other? yes 10. What do Gammarus do when they bump into each other? They pass each other quickly, going off in a new direction. Thermal Pollution (LM page 270) 3. Observe and record any effects on the animals behavior. The Gammarus immediately sink to the bottom. They spend most of their time lying motionless. Occasionally, they swim for a few seconds at a time. Acid Pollution (LM page 270) 1. What was the ph of the control culture? Determine from previous data (number 2 under Control Culture ). 3. Observe and record any effect on the organisms behavior. At ph 4, the Gammarus swim more actively than previously. At the beginning, they spend all of their time at the surface. Later, they swim to the bottom. Almost no time is spent lying still on the bottom.

4 Cultural Eutrophication (LM page ) Experimental Procedure: Predation by Daphnia (LM page 271) 4. a. Absorbance before feeding. Measurements will vary. b. Absorbance after feeding. Measurements will vary. 5. Absorbance is lower after allowing Daphnia to feed. Explain. The Daphnia have eaten the algae, lowering the absorbance. Observational Data (LM page 271) 5. Does the carmine travel completely through the gut in 30 minutes? yes 6. If food retention time in the gut is less than 30 minutes, how would this bias the results of your measure of absorbance in this Experimental Procedure? The absorbance would not only reflect the algae actually left after the Daphnia had fed for 30 minutes but also the suspended fecal particles from the crustaceans. The absorbance would be too high. Experimental Procedure: Case Study in Cultural Eutrophication (LM page 272) Table 20.3 Daphnia Filtering Number of Daphnia/Liter 10 24% % Percent of Lake Filtered Table 20.4 Cultural Eutrophication Number of Condominiums Phosphorus Added Increase in Algal Population 10 1 kg 30% 20 2 kg 60% 30 3 kg 90% 40 4 kg 120% 50 5 kg 150% 3. How many condominiums would you allow the developer to build? no more than forty

5 87 LABORATORY REVIEW 20 (LM page 273) 1. Any effect on seeds would typify an effect on what type of population in an ecosystem? producers 2. Any effect on Gammarus would typify an effect on what type of population in an ecosystem? consumers 3. Stable organic chemicals are subject to what process as they move through food chains? biological magnification 4. What type of pollution results when water from rivers and ponds is used to cool industrial processes? thermal 5. In your experiment, did you add acid or base to adjust the hay infusion culture to ph 4? acid 6. What condition does acid rain result in that can be harmful to organisms? low ph 7. What trophic level is most subject to the effects of biological magnification? top carnivores 8. Cultural eutrophication results when there is an excess of what type of substances? nutrients 9. Overenrichment causes which types of populations to increase in size beyond the ordinary? producers and decomposers 10. In your experiment, what two factors caused the producer population (Chlorella) to increase in size? increased nutrients, decreased predation 11. What are free-floating algae called? phytoplankton Thought Questions 12. Use biological magnification to show that pollution affects all living things, including humans. Pesticides concentrate as they move from producer through consumer populations. Human beings are a type of consumer, therefore pesticides will concentrate in their bodies. 13. When pollutants enter the environment, they have far-ranging effects. Give an example from this laboratory. Acid rain precipitates far away from the source of pollutants that brought about acids in the atmosphere.

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