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Experiment 31
Dissolved Oxygen
Levels in Natural
Waters
Jo A. Beran

The dissolved oxygen levels in natural waters are dependent on temperature

and water flow.

• To develop a proper technique for obtaining a natural water sample Objectives

• To determine the dissolved oxygen concentration of a natural water sample
• To learn the chemical reactions involved in fixing and analyzing a water sample for
dissolved oxygen using the Winkler method

The following techniques are used in the Experimental Procedure: Techniques

Streams, rivers, lakes, and oceans play vital roles in our quality of life. They not only Introduction
are a source of food supplies with the likes of shrimp and salmon but also provide
recreational opportunities in the forms of boating and swimming. Additionally, the
larger bodies of water such as lakes and oceans affect seasonal weather patterns, pro-
ducing changes in rainfall and snowfall and generating conditions for hurricanes and
typhoons.
The aesthetic appearance of smaller bodies of water such as rivers and lakes indi-
cates an immediate perception of the quality of the water. Color, surface growth, and
odor are early indicators of the quality of the water and the nature of its marine life. As
the public water supplies of most larger cities rely on the presence of surface water, water
chemists must be keenly aware of the makeup of that water. “How must the water be
treated to provide safe and clean water to the consumers?”

Courtesy of Thermo Fisher Scientific

A number of water-quality parameters are of primary interest in analyzing a “nat-
ural” water sample: pH, dissolved oxygen, alkalinity, and hardness are but a few. A
quick test, pH, is generally determined with a previously calibrated pH meter; dissolved
oxygen concentrations can be completed with a dissolved oxygen meter (Figure 31.1)
although its availability is less likely than that of a pH meter. Alkalinity and hardness
levels are determined using the titrimetric technique (see Experiments 20 and 21).
The concentration of dissolved oxygen in a water sample is an important indicator of
water quality. Waters with high oxygen concentrations indicate aerobic conditions: clean,
clear, and unpolluted. Low oxygen concentrations indicate anaerobic conditions: high tur-
bidity, foul odors, extensive plant growth on the surface. Dissolved oxygen levels that Figure 31.1 Dissolved oxygen
drop to less than 5 ppm can stress the existing aquatic life. meters can be used for
The solubilities of oxygen in fresh water (saturated solution) at various tempera- determining O2(aq) levels in
tures are listed in Table 31.1. water samples

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Table 31.1 Solubility of Oxygen in Freshwater at Various Temperatures

Winkler Method of The Winkler method of analysis for dissolved oxygen, developed by Lajos Winkler in
Analysis 1888, is the standard experimental procedure for determining the dissolved oxygen
concentration in water and for the calibration of dissolved oxygen meters.
The Winker test is performed in two parts: (1) the water sample is gathered in the
field, where the dissolved oxygen is “fixed” with two reagents, and (2) the sample is
titrated for final analysis in the laboratory within a 48-hour period.
Field Sampling. The natural water sample is carefully collected on-site such that no
air bubbles remain trapped in the flask after collection. The oxygen is fixed by an immedi-
ate oxidation of manganese(II) sulfate to manganese (III) hydroxide in a basic solution:
O2(aq) + 4 MnSO4(aq) + 8 NaOH(aq) + 2 H2O(l)
—› 4 Mn(OH)3(s) + 4 Na2SO4(aq) (31.1)
1
The oxygen is fixed as the manganese(III) hydroxide, an orange-brown color
precipitate—the more precipitate, the greater is the dissolved oxygen concentration.
While on-site, a basic solution of KI-NaN3 is also added to the sample.2 The
manganese(III) hydroxide oxidizes the iodide ion to the triiodide ion, I3⫺, while the man-
ganese(III) reduces to the manganese(II) ion:
2 Mn(OH)3(s) + 3 I–(aq) + 6 H+(aq) —› I3 –(aq) + 6 H2O(l) + 2 Mn2+(aq) (31.2)
The resulting solution now has a slight yellow-brown color due to the presence of I3⫺
([I2•I]⫺).
Laboratory Analysis. The remainder of the dissolved oxygen analysis is completed
in the laboratory (but within 48 hours). The sample is acidified with sulfuric acid to dis-
solve any precipitate. A titration of the sample with a standardized sodium thiosulfate
solution in the presence of a starch indicator determines the amount of I3⫺ generated in
the reactions conducted on-site and provides a direct determination of the dissolved
oxygen concentration in the water sample:
I3 –(aq) + 2 S2O3 2–(aq) —› 3 I–(aq) + S4O6 2–(aq) (31.3)
⫺
The starch indicator forms a deep-blue complex with I3 but is colorless in the pres-
ence of I⫺:
I3⫺•starch (deep blue) l 3 I⫺ + starch (colorless) (31.4)
From equations 31.1–31.3, 1 mole O2 reacts to produce 4 moles of Mn(OH)3, of which
2 moles of Mn(OH)3 react to produce 1 mole of I3–. The I3–, which is the result of the
fixing of the dissolved oxygen, reacts with 2 moles of S2O32– in the titration.
mol S2O32– 1 mol I3–
mol O2 = volume (L) S2O32–
L S2O32– 2 mol S2O32–
2 mol Mn (OH)2 1 mol O2
(31.5)
1 mol I3— 4 mol Mn(OH)3

1
There is uncertainty among chemists as to the oxidation number of manganese in the precipitate—
MnO(OH)2, the hydrated form of MnO2, often represents the form of the precipitate.
2
Sodium azide, NaN3, is added to eliminate interference in the dissolved oxygen analysis caused
by the presence of nitrite ion, NO2–, common in wastewater samples.

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From the data collected and analyzed, the moles of O2 converted to milligrams
divided by the volume of the water sample (in liters) that is titrated results in the dis-
solved oxygen concentration expressed in mg/L or ppm (parts per million) O2:
mg O2
⫽ ppm O2 (31.6)
L sample

A sodium thiosulfate solution is standardized for the experiment with potassium Standard Solution of
iodate, KIO3, a primary standard. In the presence of iodide ion, KIO3 generates a quan- Sodium Thiosulfate
tified concentration of triiodide ion, I3–.
IO3 –(aq) + 8 I–(aq) + 6 H+(aq) —› 3 I3 –(aq) + 3 H2O(l) (31.7) See Experiment 29 for further
explanation and Experimental
This solution is then titrated to the starch endpoint with the prepared sodium thiosul- Procedure.
fate solution.
I3 –(aq) + 2 S2O3 2–(aq) —› 3 I–(aq) + S4O6 2–(aq) (31.8)
For the analysis of the dissolved oxygen concentration in a water sample, the standard
Na2S2O3 solution should have a molar concentration of 0.025 M or less.

Procedure Overview. Three water samples are collected from a source that is Experimental
selected either by the student chemist or the instructor. The samples are immediately
“fixed” with the addition of a basic solution of manganese(II) sulfate and a basic solu- Procedure
tion of KI-NaN3. The samples are stored in the dark on ice and analyzed in the labora-
tory within ideally 6 hours of sampling. The dissolved oxygen concentrations are
reported in units of parts per million (ppm) O2.
Ask your instructor if a standard solution of Na2S2O3 is available. If so, proceed to
Part B of the Experimental Procedure.
Be aware of the number of significant figures when recording data.

Create and design your own Report Sheet for this part of the experiment. A. A Standard 0.025 M
1. Preparation and standardization of 0.1 M Na2S2O3 solution. Refer to Experi- Na 2 S 2 O 3 Solution
ment 29, Parts A and B of the Experimental Procedure for the preparation and
standardization of a 0.1 M Na2S2O3 solution. Prepare only 100 mL of the Na2S2O3
of the solution described in Experiment 29, Part B.1 and standardize the solution
using KIO3 as the primary standard solution (Part B.3–4). Calculate the average
concentration of the Na2S2O3 solution for three trials.
2. Preparation of a standard 0.025 M Na2S2O3 solution. Using a pipet and 100-mL
volumetric flask, prepare a 0.025 M Na2S2O3 solution from the standardized 0.1 M
Na2S2O3. See Prelaboratory Assignment, question 4a.

Disposal: Dispose of the test solutions as directed by your instructor.

1. Prepare the flask for sampling. Thoroughly clean and rinse at least three 250-mL B. Collection of Water
Erlenmeyer flasks and rubber stoppers to fit. Allow to air dry. Sample
2. Collect the water sample. Gently lay the flask along the horizontal surface of the
water. See Figure 31.2, page 352. Slowly and gradually turn the flask upright as
the flask fills being careful not to allow any air bubbles to form in the flask.
Fill the flask to overflowing.
3. “Fix” the dissolved oxygen. Below the surface of the water sample, pipet ~1 mL
of the basic 2.1 M MnSO4 solution into the sample (some overflowing will occur).
Similarly pipet ~1 mL of the basic KI-NaN3 solution. A precipitate should form
(equation 31.1).
4. Secure the sample.
a. Carefully stopper the sample to ensure that no air bubbles become entrapped
beneath the stopper in the water sample. Again, some overflowing will occur.

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Figure 31.2 Allow a gentle flow of water into the flask.

Slowly turn the flask upright as it fills to overflowing

b. Invert and roll the flask to thoroughly mix the reagents. Once the precipitate
settles, repeat the mixing process.
c. Label the sample number for each of the flasks. Store the sample in the dark
and, preferably, in a cool or cold location or on ice.
5. Temperature. Read and record the temperature of the water at the sample site.
Also, write a brief description of the sample site.
6. Analysis should begin within 6 hours of sampling.

C. Sample Analysis 1. Prepare the titrant. Prepare a clean buret. Add 3 to 5 mL of the standard Na2S2O3
solution to the buret, roll the solution to wet the wall of the buret, and dispense
through the buret tip and discard. Use a clean funnel to fill the buret—dispense a
small portion through the buret tip. Read and record the volume of Na2S2O3 solu-
tion in the buret (Technique 16A.2), using all certain digits plus one uncertain digit.
Place a white sheet of paper beneath the receiving flask.
Read and record the buret to the
correct number of significant figures. 2. Prepare sample 1
a. Remove the stopper from the 250-mL Erlenmeyer flask. To the collected water
sample, add ~1 mL of conc H2SO4 (Caution!) and stir or swirl to dissolve any
precipitate. The sample can now be handled in open vessels.
Data Analysis, A
b. Transfer a known, measured but exact volume (~200 mL, ±0.1 mL) to a receiving
flask (either a beaker or Erlenmeyer flask) for the titrimetric analysis (Part C.3).
3. Titrate water sample 1. Slowly dispense the Na2S2O3 titrant into the water sam-
ple. Swirl the flask as titrant is added (Technique 16C.4). When the color of the
analyte fades to a light yellow-brown, add ~1 mL of the starch solution. Continue
slowly adding titrant—when one drop (ideally, half-drop) results in the disappear-
ance of the deep-blue color of the I3–•starch complex, stop the titration and again
(after ~15 seconds) read and record the volume of titrant in the buret.
4. Additional trials. Repeat the analysis for the two remaining samples.
Data Analysis, A 5. Calculations. Calculate the dissolved oxygen concentration for each sample
expressed in ppm O2 (mg O2/L sample).

Disposal: Dispose of the test solutions as directed by your instructor.

The Next Step The biological oxygen demand (BOD) of a water sample is a measure of the organic
material in a water sample that is consumable by aerobic bacteria. The O2(aq) concentra-
tion is measured when a sample is taken and then again five days later, that period being
the incubation period for the aerobic bacteria to consume a portion of the O2(aq) to biode-
grade the organic material. Research the importance and significance of BOD levels in
natural waters and develop an experiment to determine the BOD for a water analysis.

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Experiment 31 Prelaboratory Assignment

Dissolved Oxygen Levels
in Natural Waters
Date __________ Lab Sec. ______ Name ____________________________________________ Desk No. __________
1. For a natural water sample, what range of dissolved oxygen concentrations may you expect? Explain your reasoning.

2. How does the dissolved oxygen concentration in a water sample change (if at all) with
a. ambient temperature changes?

b. atmospheric pressure changes?

c. the volume of the flask collecting the water sample?

d. the amount of organic matter in the water sample?

e. the depth of the body of water (e.g., lake, river, or ocean)?

3. Experimental Procedure, Part A.1. A 100-mL volume of a primary standard 0.0110 M KIO3 solution is prepared. A 25.0-mL
aliquot of this solution is used to standardize a prepared Na2S2O3 solution. A 15.6-mL volume of the Na2S2O3 solution
titrated the KIO3 solution to the starch endpoint. What is the molar concentration of the Na2S2O3 solution?
IO3 –(aq) + 8 I–(aq) + 6 H+(aq) —› 3 I3 –(aq) + 3 H2O(l)
I3 –(aq) + 2 S2O3 2–(aq) —› 3 I–(aq) + S4O6 2–(aq)

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4. Experimental Procedure, Part A.2. What is the procedure for preparing 250 mL of 0.0210 M Na2S2O3 for this
experiment from a 100-mL volume of standard 0.106 M Na2S2O3?

5. a. An analysis of a water sample for dissolved oxygen levels provided the data in the following table. Complete the
analysis for Trial 1 (See Report Sheet.) of the water sample. Record the calculated values with the correct number
of significant figures.

B. Standard 0.025 M Na2S2O3 Solution Calculation Zone

1. Sample volume (mL) ________

200.0 Part B.6
2. Buret reading, initial (mL) ________
3.85
3. Buret reading, final (mL) ________
18.25
4. Volume of Na2S2O3 dispensed (mL) ________ Part B.7
5. Average molar concentration of
Na2S2O3 (mol/L) ________
0.0213
6. Moles of Na2S2O3 dispensed (mol) Part B.8
Show calculation. ________
7. Moles of I3– reduced by S2O32– (mol),
Equation 31.3.
Part B.9
Show calculation. __________
8. Moles of O2 (mol)
Equations 31.2 and 31.1.
Show calculation. ________ Part B.10
9. Mass of O2 (mg)
Show calculation. ________
10. Dissolved oxygen, ppm O2 (mg/L)
Show calculation. ________

5.b. For Trials 2 and 3, the dissolved oxygen levels were 10.9 ppm and 11.1 ppm respectively.
a. What is the average dissolved oxygen level in the water sample?

b. What are the standard deviation and the relative standard deviation (%RSD) of the dissolved oxygen level in the
water sample?

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Experiment 31 Report Sheet

Dissolved Oxygen Levels
in Natural Waters
Date __________ Lab Sec. ______ Name ____________________________________________ Desk No. __________

A. A Standard 0.025 M Na2S2O3 Solution

Prepare a self-designed Report Sheet for this part of the experiment. Review the Report Sheet of Experiment 29 for
guidance. Submit this with the completed Report Sheet.

B. Collection of Water Sample

Sampling site: Temperature: ___________°C
Characterize/describe the sampling site.

C. Sample Analysis Sample 1 Sample 2 Sample 3

1. Sample volume (mL) _________________ _________________ _________________

2. Buret reading, initial (mL) _________________ _________________ _________________

3. Buret reading, final (mL) _________________ _________________ _________________

4. Volume Na2S2O3 dispensed (mL) _________________ _________________ _________________

5. Molar concentration of Na2S2O3 (mol/L), Part A _________________________

6. Moles of Na2S2O3 dispensed (mol) _________________ _________________ _________________

7. Moles of I3– reduced by S2O32– (mol) _________________ _________________ _________________

8. Moles of O2 (mol) _________________ _________________ _________________

9. Mass of O2 (mg) _________________ _________________ _________________

10. Dissolved oxygen, ppm O2 (mg/L) _________________ _________________ _________________

Data Analysis, B
11. Average dissolved oxygen, ppm O2 _________________________

12. Standard deviation _________________________ Data Analysis, C

13. Relative standard deviation (%RSD) _________________________ Data Analysis, D

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Write a short summary based on an interpretation of your analytical data.

Laboratory Questions
Circle the questions that have been assigned.
1. Part B. The water chemist waits until returning to the laboratory to fix the water sample for the dissolved oxygen analy-
sis. Will the reported dissolved oxygen concentration be reported as too high, too low, or remain unchanged? Explain.
2. Part B.3. A solution of MnSO4 is added to fix the dissolved oxygen in the collected sample.
a. What is the meaning of the expression, “fix the dissolved oxygen,” and why is it so important for the analysis of
dissolved oxygen in a water sample?
b. Only an approximate volume (~1 mL) of MnSO4 is required for fixing the dissolved oxygen in the sample. Ex-
plain why an exact volume is not critical.
3. Part B.4. No precipitate forms! Assuming the reagents were properly prepared and dispensed into the sample, what
might be predicted about its dissolved oxygen concentration? Explain.
4. Part B.5. A water chemist measured and recorded the air temperature at 27°C when he should have measured the
water temperature, which was only 21°C. As a result of this error, will the dissolved oxygen concentration be re-
ported as being higher or lower than it should be? Explain.
5. Part C.3. The color of the analyte did not fade to form the light yellow-brown color but remained intense even after
the addition of a full buret of the S2O32– titrant, even though a precipitate formed in Part B.4. What can be stated
about the dissolved oxygen concentration of the sample? Explain.
6. Assuming a dissolved oxygen concentration of 7.0 ppm (mg/L) in a 200-mL water sample,
a. how many moles of Mn(OH)3 will be produced with the addition of the MnSO4 solution?
b. how many moles of I3– will be produced when the KI-NaN3 solution is added to the above solution?
c. how many moles of S2O32– will be needed to react with the I3– that is generated?
d. and also assuming the concentration of the S2O32– titrant to be 0.025 M, how many milliliters of titrant will be
predictably used for the analysis.
7. A nonscientist brings a water sample to your laboratory and asks you to determine why there was a fish kill in the
nearby lake. Having recently finished this experiment, what might you tell that person about the legitimacy of a test
for dissolved oxygen? What reasoning would you use to maintain the integrity of your laboratory?
8. a. Fish kills are often found near the discharge point of water from cooling waters at electrical generating power
plants. Explain why this occurrence may occur.
b. Fish kills are often found in streams following heavy rainfall in a watershed dominated by farmland or denuded
forestland. Explain why this occurrence may occur.
9. Explain how the dissolved oxygen concentrations may change starting at the headwaters of a river and ending at the
ocean. Account for the changes.
*10. Salt (ocean) water generally has a lower dissolved oxygen concentration than freshwater at a given temperature.
Explain why this is generally observed.

356 Dissolved Oxygen Levels in Natural Waters