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Safety: wear an apron, goggles, and gloves; keep chemicals off skin
Day 1
1.) Label a cup with 500mL spring water "control" and another cup with 400mL spring water and 100mL Chlorella "algae"
2.) Cover both with plastic wrap and poke 4 holes in each
3.) Let sit for 72 hours under a light bank
Day 4
1.) Label 2 DO bottles "control" and "algae"
2.) Fill each bottle from the corresponding cup by submerging it at a 45 degree angle; fill slowly and cap underwater *limit turbulence*
3.) Winkler Method: Oxygen Fixation
a.) Add 8 drops of manganese (II) sulfate and 8 drops of alkaline potassium iodide azide to each bottle; cap and mix by inverting
b.) Allow precipitate to settle below the shoulder of the bottle and add a 1g spoonful of sulfamic acid powder *SAFETY NOTE: be careful with the sulfamic acid powder*
c.) Cap and mix until reagent and precipitate dissolve
4.) Add 400mL of spring water, 100mL of Chlorella, 30 Daphnia, and 10 Hydra to each of the 4 cups
5.) Label cups for constant uninhibited and window/natural, cover one cup with one layer of screening, and cover the fourth cup with three layers of screen fabric
6.) Cover each cup in plastic wrap with 4 holes and place under corresponding light source
Day 7
1.) Repeat steps 1-3c from Day 4 for the 4 variable cups
2.) Winkler Method: Titration
a.) Fill 6 titration sampling vials to the 20mL line; one for each fixed solution in the DO bottles *label vials*
b.) Fill each titration syringe (one for each DO bottle) with 1.0mL sodium thiosulfate
c.) Insert syringe into the vials' lid, add 1 drop at a time, swirling between each drop until the solution is post-it yellow
d.) Remove the syringe and cap together
e.) Add 8 drops of starch indicator solution, replace lid with syringe and swirl (should turn dark blue)
f.) Continue to add sodium thiosulfate until solution is clear
*for c and f record how many drops are added*
Note: each 0.1mL of sodium thiosulfate = 1ppm DO or 1mg DO/L water
3.) Clean lab station and materials

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Daphnia

Arthropod

Diet: algae, bacteria, protozoa

Hydra

Diet: small aquatic invertebrates (including Daphnia)

Tentacles

In a mutualistic relationship with certain types of algae (algae are protected from predators, photosynthetic products are a food source for the Hydra)

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Constant light with no inhibition will produce the highest DO levels because photosynthesis requires light energy; constant light with inhibition and natural light will produce lower DO levels because there will be less light energy for photosynthesis.

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In this lab we sought to determine how species interact using Chlorella, Daphnia, and Hydra and measuring dissolved oxygen. We tested two controls, one with only water and one with only algae. We also decided to run four variations on light: natural light, three with constant light (one with no inhibition, 1 layer of inhibition screening, and one with 3 layers of inhibition screening). We used the Winkler Method to test the water in all 5 cups using oxygen fixation and titrations to find out how much dissolved oxygen was in the water. Based on the levels of dissolved oxygen we were able to explain the relationship between species and how light affected those relationships.

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Independent Variable- amount/intensity of light

Dependent Variable- DO levels
Constants- amount/number of cultures, type of water

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The only cup not under constant light is the cup labeled "natural light." Constant light cups include control (water) and algae only, however, that is not specified in their names like it is with the other three cups under constant light.

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Hydra alive in constant light (no inhibition

Most Hydra dead, most/all Daphnia dead

Visible Chlorella in all

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Titration Totals
Note: y-axis is mL of sodium thiosulfate; titration 1- red bar should read 1.0; titration 2- orange bar should read 0.40 (this graph program takes away "extra" zeros)

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Dissolved Oxygen Totals
Note: blue bar should read 12.0; olive green bar should read 9.0

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Constant light (no inhibition) ended up with the highest amount of DO with 15.4ppm. Constant light (1 layer) was second with 12.0ppm. Constant light (3 layers) was third with 9.0ppm. Tied for fourth was algae only and natural light with 8.7ppm. Last was the control (water) with 8.2ppm.

Patterns:
All five cups with algae were higher than the one cup with only water.
Natural light is lower than every cup under constant light (excluding water) other than the algae only cup which it is tied with.
All values are within 1.0ppm of each other except constant light (no inhibition) and constant light (1 layer).

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Claim:
Constant light (no inhibition) had the highest DO level because of the mutualistic relationship between algae and hydra as well as the amount of light.
Evidence:
Constant light (no inhibition) has the highest DO level whereas algae only in constant light is tied for the second lowest DO level. Constant light with 1 and 3 layers are both lower than constant light (no inhibition). Hydra and Chlorella are in a mutualistic relationship (see background).
Reasoning:
Algae only and constant light (no inhibition) are 5.7ppm apart, the latter being the highest DO level. These two conditions both have algae and are both under constant light that isn't inhibited.; therefore, the only difference between the two is that constant light (no inhibition) has Daphnia and Hydra. Additionally, Hydra is in a mutualistic relationship with Chlorella where the Hydra gets rid of Chlorella's predators and the Chlorella is photosynthetic, producing the products of photosynthesis which are a food source for Hydra. Therefore, by killing/eating the Daphnia, the Hydra protect the Chlorella which are then able to undergo more photosynthesis and produce more oxygen to raise the DO level.

Because the cup with constant light (no inhibition) has a higher DO level than both constant lights with screening that inhibits light. We can assume that because the only difference between the cups is the light inhibiting screening that the cup without inhibition has the highest DO level because it gets the most light. This is because light energy is needed for the Chlorella to undergo photosynthesis which produces oxygen, raising the DO level

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Our hypothesis is technically correct, however, we were able to realize that out hypothesis wan't complete. We didn't consider how the controls would compare to our variations. We were also correct on that photosynthesis was the cause of higher DO levels, but we did not consider the implications of the other organisms, specifically the relationship between Hydra and Chlorella.

It surprised us that the algae only cup didn't have higher DO levels. I believe that this may be because the algae we put in the other cups had sat longer in the water, allowing the water they were in to become more oxygen rich and allowing the algae to reproduce and increase population density.

Our results showed us that the relationship between Hydra and Chlorella was more important than we had originally thought (see CER). It also confirmed that we thought with regards to light. We believed that less light would create less DO, and that is what happened in our three constant light variant cups.

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Run the experiment again with the constant light cups except removing the Daphnia. The mutualistic relationship would be irrelevant without a Chlorella predator and Hydra wouldn't have a food source. I'd predict this would raise DO levels even more because the Chlorella wouldn't be prey and the Hydra would die off, no longer to consume any oxygen at all for cellular respiration.

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Google Sites: Mr. Colonna's Website
Carolina Species Interaction for AP Biology handout
Species Interaction Kit for AP Biology inquiry activity handout