Laboratory
20
Effects of Pollution
(LM pages 267–273)
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 PREPARATIONS1
20.1 Effect of Pollutants on Ecosystems (LM pages 268-270)
_____
hay infusion cultures (culture media sets, Carolina 13-1206)
_____
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 13-2450)
_____
Gammarus culture (Carolina 14-2355, amphipods, or Nasco Science LM00205, fairy
shrimp)
_____
containers for spring water
_____
1% sulfuric acid (H2SO4) (Carolina 89-3300, -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.
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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.
20.2 Cultural Eutrophication (LM pages 271-272)
_____
Daphnia culture (Carolina 14-2314)
_____
spectrophotometer
_____
cuvettes for spectrophotometer
_____
Chlorella (common pond algae) (Carolina 15-2069)
_____
test-tube racks
_____
petroleum jelly
_____
petri dishes, small
_____
microscopes, binocular dissecting
_____
lens paper
_____
indigo carmine (Carolina 86-8550)
_____
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 0.635 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 268-270)
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.
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Hay Infusion (LM pages 268-269)
Experimental Procedure: Hay Infusion (LM page 269)
Table 20.1 Hay Infusion Cultures*
Types of Culture
Diversity of Life
(List Organisms)
Relative Quantity of 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.
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20.2 Cultural Eutrophication (LM page 271-272)
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
Percent of Lake Filtered
10
24%
50
120%
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
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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.