1. AP LAB #12 DISSOLVED OXYGEN Introduction to Water Quality &
Ms. Gaynor
AP Biology

2. Why is Water SO Important?
It is a renewable resource that is becoming polluted & destroyed
Recycles through “water (hydrologic) cycle”
WATER = SURVIVAL
~ 80% of body = water
Only 1% of water on Earth = fresh
Used for domestic, industrial, commercial, & recreational use

3. Why Test Water Quality?
To monitor human and ecological impact and its affect on aquatic life/ aquatic habitats
Monitoring means “to collect data” on water to access affects of pollutants
Water quality can disrupt water chemistry  harmful to aquatic food chain

4. AP LAB #12: Dissolved Oxygen
Dissolved Oxygen (DO) = the amount of oxygen gas dissolved in water
95% more O2 in air than in cold water
[DO]
used to determine whether the biological activities requiring O2 are occurring
ALSO an indicator of pollution
O2 important in metabolic processes  indicator of water quality

5. Why is DO important?
Oxygen must be in water in a free state (O2) before organisms can use it
This depends on physical and chemical properties of water.
Sewage and detritus (dead organic matter) depletes DO b/c it requires a lot of O2 as it decomposes

6. How does the O2 get into the water?
primary productivity (photosynthesis)
Atmosphere (air)
Winds
Mixing (waves, currents etc…) 6

7. 6 MAIN Factors that affect [DO]
1. Air pressure above water affects [DO]
Less O2 present at higher elevations (b/c air is less dense)
water at higher elevations contain less O2

8. 6 MAIN Factors that affect [DO] (con’t)
2. Photosynthesis (adds O2) & cell aerobic respiration (uses/takes away O2)
Photosynthesis
in bright light  aquatic producers make more O2
Cellular Respiration (metabolic) activity
aquatic organisms use more O2

9. 6 MAIN Factors that affect [DO] (con’t)
3. As salinity and temperature of water increases  [DO] decreases
Salinity = amount of salts dissolved in water
More Salts= less [DO]
Less Salts= more [DO]
Temperature
Warmer H2O = low [DO]
Cold H2O = higher [DO]

11. 6 MAIN Factors that affect [DO] (con’t)
4. Decomposition activity
As organic (DEAD) material decays, microorganisms that decompose material consume oxygen (O2)
5. Mixing and turbulence
Wave action, waterfalls, and rapids all aerate (add “air” to) water and increase the O2 concentration.

12. 6 MAIN Factors that affect [DO] (con’t)
6. Wind
Windless nights = low(er) [DO]
High winds = High [DO]

13. Where is the greatest [DO]? lowest [DO]?

14. Think: “Ewww” a green lake Think: “Ohhh” a nice clear blue lake
DO and Trophic States
2 classifications
Eutrophic = “well nourished”
high nutrient [ ]  lots of plant life as well as respiration (O2 fluctuates)
GREENER in color (from algae)
Oligotrophic = low nutrient [ ]  low plant growth (always rich in O2 )
CLEAR in color, very “blue”
Think: “Ewww” a green lake
Think: “Ohhh” a nice clear blue lake

15. EUTROPHIC
OLIGOTROPHIC

16. Lab #12: Dissolved Oxygen
Concepts
dissolved O2 (DO)
primary productivity (aka-photosynthesis)
measured in 3 ways:
amount of CO2 used
rate of glucose/ sugar (biomass) formation
rate of O2 production
net productivity vs. gross productivity
cellular respiration 16

17. Reminder… PHOTOSYNTHESIS Cellular Respiration
6CO2 + 6H2O  C6H12O6 + 6O2
carbon water glucose oxygen gas
dioxide (sugar)
Cellular Respiration
C6H12O6 + 6O2  6CO2 + 6H2O + ATP
glucose oxygen gas carbon water energy
(sugar) dioxide

18. Biological Influences &
Dissolved Oxygen
As photosynthesis increases, oxygen levels increase:
CO2 + H2O Biomass + O2
As respiration increases due to decay or organism’s need for ATP increases, oxygen levels decrease:
Biomass + O CO2 + H2O

19. Productivity in a Aquatic Environment
Primary productivity = rate at which organic (carbon based) materials are stored through photosynthesis
Can be measured in three ways:
1. The amt of carbon dioxide used
2. The rate of sugar formation
3. The rate of oxygen production

20. Productivity
gross primary productivity (GPP)= TOTAL Photosynthesis
ALL the sugar (biomass) made during photosynthesis
Net productivity (NNP) = sugar (biomass) that remains after photosynthesis minus sugar being used up by cellular respiration
P– CR = NP
net productivity = gross productivity – respiration

22. Light & Dark Bottle Method
Light bottle represent daytime
Dark bottle can represent night time
BOTTLE #1
(5 light bottles
1 light control bottle)
**exposed to light
**Acts as initial bottle as well
BOTTLE #2
(1 dark bottle)
**NOT exposed to light

23. LIGHT BOTTLES 100%, 65%, 25%, 10%, and 2% 23

24. What is going on in the Light Bottles (2%, 10%, 25%, 65%, 100%)?

25. What is going on in the Dark Bottle (foil wrapped)?

26. You need to isolate variables using the Light & Dark Bottle Method
Light Bottle - Dark Bottle = Gross PP
+O2 & -O O2 only Total + O2
Net PP = Light Bottle – Initial Bottle
+O2 & -O2 starting [DO]
Cellular Respiration = Initial – Dark
You need to isolate variables using the Light & Dark Bottle Method

28. Lab #12: Experimental Design (setup)
This lab has 2 PARTS and will take 2 days!

29. Lab #12: Experimental Design (setup) PART A
Prepare 3 water sampling bottles filled with pond water
Use 3 pond water samples from at different temperatures
refrigerator (cold)
incubator (warm)
room temperature
DO NOT FORGET TO TAKE THE TEMPERATURE AT EACH CONDITION; RECORD ON WORKSHEETS!
Determine the [DO] of each sample using the Winkler Titration Method.
Record your values in your data table
Estimate your % saturation using a nomogram
Record class results

30. Nomograph
To measure how much O2 the water can hold (saturation), you need to be able to read a nomograph.

31. Lab #12: Experimental Design (setup) PART B
Fill 7 water sampling bottles with pond water (BOD bottles for “biological oxygen demand”)
Add piece of plant to each bottle. Make sure that they are the same! Count leaves, measure, etc. CONTROL THE VARIABLES!
Label bottles-I, D, 100%, 65%, 25%, 10%, and 2%
Determine the DO for the initial bottle using Winkler Titration method.
THIS IS YOUR BASE LINE (CONTROL GROUP); the amount of DO ALL your samples start with!
Cover D (dark) bottle with aluminum foil
Cover the remaining bottles with screens (see procedure for details)
Place bottles under the light source.
Wait 24 hours, then measure DO levels of remaining samples using Winkler Titration method.

32. Lab 12: Dissolved Oxygen 32

33. Hints for Setting Up Lab #12
Rinse sampling bottle 3x with sample water
Submerge bottle in water; allow to fill.
Tap bottle to release air bubbles
You do NOT want extra atmospheric O2
While bottle is submerged, replace cap
If there are air bubbles in the bottle, empty and repeat
Preserve sample immediately. Test within 2 hours.

34. More Hints for Setting Up Lab #12
Do NOT leave water samples uncapped! Hello O2 will get in!!!
Use rubber bands to secure the screens
NOTE: We are using HORNWORT plant not Anacharis
Please FIX on your
worksheets!

35. Winkler Titration Method (PART 1) How Do You FIX Samples Correctly?
To “fix” your samples, follow for each sample bottle: (“fix” = trap DO in sample)
Uncap bottle
Add 8 drops of MgSO4 (manganous sulfate) to bottle (can cause cancer/stain)
Add 8 drops of alkaline potassium iodide azide to bottle (can cause cancer/stain)
Cap bottles and mix. A precipitate will form! Allow precipitate to settle to shoulder of bottle.
Add a spoonful (1 g) of sulfamic acid powder to bottle (STRONG ACID)
Cap and mix. Precipitate should dissolve!

36. Winkler Titration Method (PART 2) How Do You Determine the amount of DO in the samples Correctly?
To determine the [DO] of your samples, follow for each sample bottle, including the initial bottle:
Uncap bottle
Carefully fill the titration tube/cup to the 20 mL line.
Fill the titration syringe to the “0” (zero) line with sodium thiosulfate.
Add 1 drop AT A TIME to sample and swirl after each drop. Do this until you get a FAINT yellow color.
Remove titration syringe
Add 8 drops of starch indicator solution
Swirl the sample. The sample should now be BLUE! No blue = no measurable [DO] or too much sodium thiosulfate
Add sodium thiosulfate 1 drop at a time and SWIRL until BLUE color disappears. If you finish the syringe and it is still blue, fill syringe again and continue.
Read titration syringe scale for result in mg DO/liter
Fill in data table; multiple DO in mg/L by to convert to mL/L

37. Chemical Reactions To Preserve DO: Done in the field
FIX
To Preserve DO: Done in the field
O2 + 2 Mn2+ + 2H2O Mn(IV)O2 + 4H+ (pH >10)
Allow precipitate to settle (reaction goes to completion)
2Mn(IV)O2 + 4H+ + 2I Mn2+ + I2 (yellow) + 2H2O (low pH)
DO is preserved (“fixed”)
To Test Sample: This step can be done in the lab.
Na2S2O3 + 4I2 + 5H2O I- + 2SO H+ + Na+ (the titration) Starch + I2 blue (to improve endpoint determination)
SAMPLE

38. Amt of Thiosulfate Used = Amt of DO in water
Titrate w/ Thiosulfate
20 mL
STARTING POINT MIDDLE POINT
Very DARK yellow PALE yellow
Add 8 drops of STARCH
Turns BLUE
PART 1
PART 2

39. ADD ALL THIOSULFATE USED FROM STARTING POINT TO END POINT!
CONTINUE to
Titrate w/ SAME
20 mL Thiosulfate
END POINT Turns CLEAR!!!
ADD ALL THIOSULFATE USED FROM STARTING POINT TO END POINT!
This is your amount of thiosulfate used = amount of DO in water
Each # = 1 mL = 1 ppm = 1 mg/L
1 mg/L x = 1 mL/L

41. Dissolved Oxygen
Can be measured in absolutes (how much O2 is actually in the water) or by % saturation
% saturation - measure of DO compared to how much DO COULD be in the water
Water at a higher temperature CAN NOT hold as much oxygen as cold water (use nonmaogram)

42. Lab 12: Dissolved Oxygen Conclusions to Think About…
 temperature =  dissolved O2
 light =  photosynthesis =  O2 production
O2 is consumed during cellular respiration
 respiration =  dissolved O2 (consumption of O2) 42