Oca, Hazel Kaye G.

January 24, 2020

BS Chem – 4A ENVICHEMISTRY

Experiment No. 1
WATER ANALYSIS (pH, DO, and BOD)

OBJECTIVES
 To be able to determine the pH of the water sample.
 To be able to determine the amount of dissolve oxygen in water sample by using a selected procedure
that is Modified Winkler Method

THEORIES AND PRINCIPLES

Dissolved Oxygen
The Winkler Method is a technique used to measure dissolved oxygen in freshwater systems. Dissolved
oxygen is used as an indicator of the health of a water body, where higher dissolved oxygen concentrations are
correlated with high productivity and little pollution. This test is performed on-site, as delays between sample
collection and testing may result in an alteration in oxygen content.
The Winkler Method uses titration to determine dissolved oxygen in the water sample. A sample bottle is
filled completely with water (no air is left). The dissolved oxygen in the sample is then fixed by adding a series
of reagents that form an acid compound that is then titrated with a neutralizing compound that results in a color
change. The point of color change is called the endpoint, which coincides with the dissolved oxygen concentration
in the sample. Dissolved oxygen analysis is best done in the field, as the sample will be less altered by atmospheric
equilibration.

PROCEDURE
Modified Winkler Method (Determination of Dissolved Oxygen)

I. Sample Collection III. Standardization of Sodium thiosulfate

1. Rinse the container (3 times) with
sample
2. Submerge & fill the bottle to the brim
3. Cover bottle with a stopper
4. Collect 3 bottles of the water sample
5. Store it in ice box/refrigerator (4°C)
6. Measure temp. of water sample on site
II. pH (electrode) measurement
1. Calibrate the pH meter
2. Take a 50 mL sample & measure the pH
levels of the water sample.
1. Dissolve approximately 2g KI, free from
iodate in an Erlenmeyer flask w/ 100-
150 mL distilled water
2. Add 1 mL 6N H2SO4, then 20 ml std
biiodate solution.
3. Dilute to 200 mL and titrate w. the
Na2SO3
4. Add starch when a pale straw color is
reached and complete the titration until
the blue color first disappears
5. If the Na2SO3 is the proper strength, 20
mL will be used in the titration
III. Standardization of Sodium thiosulfate
6. Calculate the molarity of S2O3 soln as:
7. Take the average of 4 trials
IV. Procedure for Modified Winkler method
1. Collect the sample to be tested in a 300 mL
BOD bottle (avoid adding air to the liquid
being collected). Fill bottle completely
and add stopper.
2. Remove bottle stopper and add 1 mL of
the MnSO4 solution at the surface of the
liquid.
3. Add 1 mL of the alkaline potassium iodide
sodium azide solution at the surface of the
liquid.
4. Replace the stopper, avoid trapping air
bubbles and shake well by inverting the
bottle several times. Repeat shaking after
floc has settled halfway. Shake the bottle
again and allow the floc to settle a second
time.
5. Add 1 mL of conc’d H2SO4 by allowing
the acid to run down the neck of the bottle
above the surface of the liquid.
6. Restopper, rinse the top of the bottle to
remove any acid and shake well until the
precipitate has dissolved.
7. Titrate a volume of treated sample which
corresponds to 200 mL of the original
sample. This corrects for the loss of some
sample during the addition of reagents.
8. Pour 201 mL of sample from the BOD
bottle into an Erlenmeyer flask.
9. If the solution is reddish brown in color,
titrate with 0.0250 N Na2S2O3 until the
solution is a pale yellow (straw) color. Add
a small quantity of starch indicator and
proceed to step 11.
10. If the solution has no reddish brown color,
or is only slightly colored, add a small
quantity (approx’ly 1 mL) of starch
indicator. If no blue color develops, there
is zero DO. If a blue color develops,
proceed to step 11.
11. Continue to titrate with 0.0250 N
Na2S2O3 to the first disappearance of the
blue color. Record the total volume of
sodium thiosulfate used.
1.
5-day Biological Oxygen Demand Test
I. Preparation of dilution water
1. Aerate a bottle of distilled water long enough
to allow the water to become saturated with
dissolved oxygen (approximately 8 mg/L at
room temperature).
II. Procedure
1. For the BOD analysis prepare 3 dilutions of
50%, 75%, and 100% water sample in the
dilution water. You need 16 BOD bottles for
each water sample.
2. Add 0.25 mL each of phosphate buffer,
MgSO4, CaCl2, and FeCl3 into the water
sample. Label each bottle properly. Gently
swirl the BOD bottle.
3. Add the dilution water into the incubation
bottle to fill it up to the brim.
4. Cover the bottles with the pointed stopper
then remove the excess sample by inverting
the bottle while holding the cover in place
with your index finger. Invert the bottle a few
more times to ensure homogeneity of the
sample.
5. Seal the cover with some clay. Place the
bottles for the BOD5 test inside the incubator
set at 20 C. Incubate the water sample for 5
days.
6. Measure the initial DO for the other 2 bottles
using the Modified Winkler Method.
7. The initial DO of the sample per dilution is
the average DO level of the 2 trials.
8. After five days, measure the final DO of the
BOD5 bottles using the Modified Winkler
Method.
9. The final DO of the sample per dilution is the
average DO level of the 2 trials.
10. Calculate the BOD of the water sample
11. For dilution water check, the DO uptake in 5
days at 20 C should not be more than 0.1
mg/L.
12. If more than one sample dilution meets the
criteria of a residual DO of at least 1 mg/L
and a DO depletion of at least 2 mg/L and
there is no evidence of toxicity at higher
sample concentrations, average the results in
the acceptable range.
  Chemical reactions involved:
Standardization of thiosulfate solution
2 Na2S2O3·5H2O + I2 ↔ Na2S4O6 + 2 NaI + 10 H2O
2 S2O3 2- + I2 ↔ S4O6 2- + 2 I-
The Winkler Method for DO Determination
Mn2+ + 2 OH- ↔ Mn(OH)2 (white precipitate)
Mn2+ + 2 OH– + 0.5 O2 → MnO2 (brown hydrated precipitate) + H2O
Mn(OH)2 + 0.5 O2 → MnO2 + H2O
MnO2 + 2 I– + 4 H+ → Mn2+ + I2 + 2 H2O
I2 + I– → I3–
The Azide Modification with the Winkler Method for DO Determination
2 NO2– + 2 I– + 4 H+ → I2 + N2O2 + 2 H2O
N2O2 + 0.5 O2 + H2O → 2 NO2– + 2 H+
NaN3 + H+ → HN3 + Na+
HN3 + NO2– + H+ → N2 + N2O + H2O

CALCULATIONS
 Molarity of thiosulfate solution
𝟔 𝒎𝒎𝒐𝒍 𝑺𝟐 𝑶𝟑 𝟐− 𝟏
𝑴𝑺𝟐 𝑶𝟑 𝟐− = 𝟎. 𝟎𝟎𝟒𝟏𝟔𝟕 𝑴 𝑰𝑶𝟑 − 𝒙 𝟐𝟎. 𝟎𝟎 𝒎𝑳 𝒙 𝒙
𝟏 𝒎𝒎𝒐𝒍 𝑰𝑶𝟑 − 𝑽𝒐𝒍 𝑺𝟐 𝑶𝟑 𝟐− (𝒊𝒏 𝒎𝑳)
 Dissolved oxygen in the sample
𝒎𝒈 𝟏 𝒎𝒎𝒐𝒍 𝑶𝟐 𝟑𝟐 𝒎𝒈 𝑶𝟐 𝟏
𝑫𝑶 ( ⁄𝑳) = 𝑴𝑺𝟐𝑶𝟑 𝟐− 𝒙 𝑽𝑺𝟐𝑶𝟑 𝟐− (𝒊𝒏 𝒎𝑳) 𝒙 𝟐−
𝒙 𝒙
𝟒 𝒎𝒎𝒐𝒍 𝑺𝟐 𝑶𝟑 𝟏 𝒎𝒎𝒐𝒍 𝑶 𝟐 𝟎. 𝟐𝟎𝟎 𝑳 𝒔𝒂𝒎𝒑𝒍𝒆

 BOD of the water sample

𝒎𝒈 (𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝑫𝑶 − 𝒇𝒊𝒏𝒂𝒍 𝑫𝑶)
𝑩𝑶𝑫 ( ⁄𝑳) =
𝑷
P = decimal volumetric fraction of sample used.
DATA
Standardization of thiosulfate solution
Trial Initial vol. Final vol. Vol. used Wt. KI (g) Molarity 𝑺𝟐 𝑶𝟑 𝟐−
No. (mL) (mL) (mL) (mmol/mL)
1 2.39 21.5 19.11 2.1628 0.026166
2 0.1 18.30 18.20 2.1200 0.027474
3 21.5 39.6 18.10 2.05 0.027626
4 17.9 35.7 17.8 2.05 0.028092
 Average molarity of 𝑺𝟐 𝑶𝟑 : 0.0273395 ≈ 0.02734 mmol/mL
𝟐−
pH (electrode) measurement
Raw sample = 9.41 pH

Determination of DO and BOD in the water sample

A Initial DO Titration
Sample Initial Vol. Final. Vol Vol. used (mL) Dissolve oxygen
dilution % (mL) (mL) 𝑺𝟐 𝑶𝟑 𝟐− Content (mg/L)
1 (0mL) 1.9 9.6 7.7 8.420566
2 (50mL) 9.6 16.1 6.5 7.10827
3 (100mL) 16.1 22.4 6.3 6.889554
4 (150mL) 22.4 28.7 6.3 6.889554
Average residual DO (in the acceptable range): 7.326986 mg/L
B. Final DO Titration
Sample No. Initial Vol. Final. Vol Vol. used (mL) Dissolve oxygen
(mL) (mL) 𝑺𝟐 𝑶𝟑 𝟐− Content (mg/L)
1 (0mL) 0 1.1 1.1 0.22
2 (50mL) 1.1 2.1 1.0 4.00
3 (100mL) 2.1 3.3 1.2 2.40
4 (150mL) 3.3 33.3 30.0 40.00
Average DO depletion (in the acceptable range): 3.2mg/L
C. BOD of the water sample
Sample BOD
dilution %
1 (0mL) 0
2 (50mL) 0.518045
3 (100mL) 1.496518
4 (150mL) 16.555223

DISCUSSION
The relevant chemical reactions occurring throughout the procedure are outlined below:
Mn2+ + 2OH– + 1/2 O2 R oxygen-manganese complex + H2O (1)
oxygen-manganese complex + 4H+ + 2I– R I2 + Mn2+ + 2H2O (2)
I2 + 2Na2S2O3 R Na2S4O6 + 2NaI 3)
The objectives of the experiment is to determine the dissolved oxygen level in the river water sample
ascertain whether they comply with Malaysian Water Standard. There are two sample of solution which are
dissolved oxygen and azide modification by using Winkler method.
Addition of the manganous sulfate and the alkaline-iodide results in the formation of an insoluble oxygen-
manganese complex (1), the precipitate in step 2. The oxygen is stable in this form for several days. Both the
manganous sulfate and the alkaline iodide are added in excess to ensure reaction with all of the oxygen. Treatment
with the sulfuric acid dissolves the complex and liberates free iodine (2), imparting the distinctive yellow-gold
color. The amount of free iodine is proportional to the amount of oxygen dissolved in the original sample. By
titrating a measured portion of the sample against a standardized sodium thiosulfate solution (3), the amount of
free iodine—and the corresponding amount of oxygen—is determined. The starch “indicator” (which forms a
distinctly colored complex with the free iodine) is used to provide an unmistakable visual endpoint for the
titration.
The concentration of dissolved oxygen (DO) is one of the most important indicators of the overall health
of a body of water. Waters with consistently high levels of DO (> 6 mg/L) typically support the most diverse
biological communities. Waters with consistently low DO levels (< 3 mg/L) may be virtually devoid of aquatic
life or may harbor only a few species adapted to such conditions.

CONCLUSION
Dissolved oxygen analysis measured the amount of oxygen dissolved in an aqueous solution. Oxygen gets
into the water by diffusion from the surrounding air by rapid movement(aeration) and also as a waste product of
photosynthesis process. Dissolved oxygen analysis can be used to determine the health or cleanliness of a river
or stream, the amount and type of biomass a freshwater system can support and the amount of decomposition
occurring in the river or stream. In this experiment, there are two sample of solution which is 60 mL of BOD
bottles and 300 mL of BOD bottles. For sample using 300 mL of BOD bottles, the oxygen dissolve is 7.33 mg/L.
The result shows that the water sample content does not comply with Philippine Standard water which the value
of oxygen dissolves still in range 5 until 7 mg/L. Thus, the objective of the experiment is achieved.

REFERNCES
 EnviChem Laboratory Manual - Experiment No.1 “pH, DO and BOD” prepared by r.a.mindo
 https://serc.carleton.edu/microbelife/research_methods/environ_sampling/oxygen.html
 https://www.webpages.uidaho.edu/fish503al/002%20Oxygen/FISH%20503%20Winkler%20titration%2
0lab%20day%20I.pdf
