CHEMISTRY DEPARTMENT

COLLEGE OF SCIENCE
School Year 2021-2022

Experiment No. 8
Determination of Biological Oxygen Demand of Effluent by Modified
Winkler Method
Cahanding, M.E., Mapas, J. J.*, Siquijor, J. C.
Group 1

1. Introduction

Efficient use of water has been an increasing trend in the 21st century, and one
solution to this demand is the use of recycled water or effluent from wastewater,
especially in agriculture (Toze, 2006). The Philippine Department of Environment and
Natural Resources (DENR) Administrative Order (AO) no. 2016-08 defined effluent as
water discharged from a known source, flowing out of an industrial plant and into bodies
of water and land. Toze mentioned that recycled water is advantageous to urban and
rural communities as it is a constant and reliable source, reducing the water extracted
from the environment. Despite the treatment applied to effluent, Toze also stated that it
can still pose a threat to marine life and public health concerns, primarily the presence
of pathogens. Reducing harmful microbial life is one goal of wastewater treatment, and
a parameter being considered is the amount of dissolved oxygen (DO) in the water.
According to Hauser (2017, p. 127), DO is one of the most useful water measurements
since the biological species present in a water body depend upon the concentration of
DO. Microorganisms utilize DO to thrive, and the amount being consumed is called the
Biological Oxygen Demand (BOD), making this an index of the degree of organic
pollution for wastewater (US Geological Survey, 2018). To determine the BOD of a
sample, a test called BOD5 is performed which measures how much DO is utilized in
five days, and the result obtained would be interpreted following the government
standards, such as the DENR AO 2016-08. The test lasts for five days because it is the
number of days that the scientific community was accustomed to based on the practice
done in the early 1900s in the United Kingdom (Wilhelm, 2009, p. 116). This experiment

Page 1 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

uses a universally accepted classical analysis called the Winkler method, wherein DO
can be collected through a precipitation reaction and then analyzed through redox
titration. There are also instrumental methods for BOD5, however, these are expensive
and require maintenance (Austin, 1983). Whereas in the Winkler method, Austin
discussed that it is highly precise, cheap, and reagents needed are readily available.
Since this technique requires a fragile burette, this becomes a disadvantage, especially
that DO testing is advisable to be done on-site where the sample was taken. Another
minor disadvantage can be the requirement of a freshly made starch indicator (Austin;
Hauser, p. 128).

For this experiment, a modified Winkler method was used because it requires a
lesser amount of sample which is convenient compared to the conventional procedure
as mentioned by Pal and Das (1988). Syringes were utilized for sample collection and
for mixing the sample with the reagents to liberate the iodine needed for the titration.
The solution was then titrated using Na2S2O3 as the titrant and starch as the indicator.
Afterward, the DO concentrations were acquired by stoichiometry, and the difference
from the DO of Day 1 and Day 5 became the BOD. The study aimed to determine if the
BOD of effluent discharged to class A or class II public water supply is acceptable under
AO 2016-08 and to better appreciate how water is kept clean and safe for everyday
needs.

2. Experimental
2.1. Materials and reagents

The equipment used were 50, 100, 250, and 500 mL volumetric flasks, 250 mL
Erlenmeyer flasks, 25 mL burette, 250 mL beaker, and 50 mL syringes. The reagents
utilized were distilled water, manganese (II) sulfate dihydrate (MnSO4·2H2O) crystals,
sodium hydroxide (NaOH) pellets, potassium iodide (KI) crystals, sodium thiosulfate
pentahydrate (Na2S2O3·5H2O), concentrated sulfuric acid (H2SO4), starch, and

Page 2 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

potassium iodate (KIO3). All of the reagents mentioned underwent further preparation to
create the necessary solutions for the experiment proper.

2.2 Preparation of reagents

2.2.1 MnSO4 solution

A 20.0 g of MnSO4·2H2O crystals were taken and dissolved in about 30 mL

distilled water. The resulting solution was filtered into a 50 mL volumetric flask and then
diluted to mark. Filtration was done only in this solution preparation because it was
needed.

2.2.2 Alkaline KI solution

The combination of 18 g NaOH pellets and 10 g KI was dissolved in 30 mL

distilled water in a small beaker submerged in a water bath. Afterward, the solution was
transferred into a 50 mL volumetric flask and diluted to mark.

2.2.3 0.125 M Na2S2O3 stock solution

Approximately 7.8 g of Na2S2O3 was weighed in a 250 mL beaker and dissolved

with 100 mL pre-boiled distilled water. Then, the solution was transferred into a 250 mL
volumetric flask and the washings from the beaker were added to the latter. The
contents of the volumetric flask were diluted to mark.

2.2.4 0.5 M H2SO4 solution

A volume of 6.94 mL of concentrated H2SO4 was diluted in a 500 mL volumetric

flask and then, diluted to mark.

2.2.5 1% starch solution

A 0.50 g of soluble starch was moistened with a small amount of distilled water
until the formation of a smooth paste. Then, 100 mL of boiling water was poured slowly

Page 3 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

and the solution was mixed thoroughly until the starch dissolved. The resulting solution
was cooled to room temperature.

2.2.6 0.0125 M Na2S2O3 standard solution

Approximately 0.0125 M Na2S2O3 was prepared into a 500 mL volumetric flask.

2.3. Standardization of Na2S2O3 solution

A mass of 0.15 g KIO3 was weighed to the nearest 0.1 mg and dissolved in 50
mL pre-boiled distilled water. The solution was then transferred into a 100 mL volumetric
flask, diluted to mark, and mixed thoroughly. Afterward, three 10.00 mL aliquots of the
prepared KIO3 solution were delivered into three Erlenmeyer flasks. The following
reagents were added one at a time into each Erlenmeyer flask specifically, 20 mL
distilled water, 1 g KI crystals, and 10 mL 0.5 H2SO4. This mixture was swirled to mix
and immediately titrated with the standard S2O32- solution while constantly swirling.
When the solution became pale yellow, 1 mL of starch solution was added and the
titration continued until the blue color disappeared. These steps were repeated for the
second and third aliquots.

IO3- + 5 I- + 6 H+ → 3 I2 + 3 H2O

I2 + 2 S2O32- → 2 I- + S4O62-

Scheme 1. Reaction mechanism for the standardization of Na2S2O3 titrant

After all of the mentioned procedures were done, the molarities of the Na2S2O3
titrant from the triplicates were calculated based on the stoichiometric relationships
illustrated in Scheme 1. Afterward, the following statistical data were computed
specifically the mean molarity, standard deviation (SD), and percent relative SD
(%RSD).

Page 4 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

2.4. Analysis of unknown sample

A total of four 50 mL syringes that are fresh from the pack were labeled
individually as days 1A, 1B, 5A, and 5B, respectively. Each syringe drew the water
sample from 6 feet below the surface of the water body and care was observed to not
allow air bubbles to be trapped inside the syringe. The tips were dried with a tissue, and
then, 20 mL of the sample was expelled from each syringe or until the plunger is exactly
at the 30 mL mark. Lastly, the tip of the syringes labeled Day 5A and 5B was sealed
with paraffin film and stored in the dark for five days.

The processing of the samples started with Day 1A and 1B by drawing MnSO4
solution until the 35 mL mark. The mixture was mixed cautiously by tipping the syringe
repeatedly. Afterward, the alkaline iodide solution was drawn up to the 40 mL mark and
again, mixed in a manner similar to before. The H2SO4 was then drawn up to the 50 mL
mark and mixed well until all brownish-yellow particles dissolved. It was ensured from
these procedures that no air bubbles were trapped in the syringes during the extraction
of reagents. Furthermore, the content of the syringes was expelled into separate 250
mL Erlenmeyer flasks. The syringes were rinsed with distilled water and the washings
were added to those flasks along with about 5 mL of the freshly prepared starch
solution. Titration was then performed with the standardized Na2S2O3 and the DO was
calculated in ppm. The computations were also done to obtain the mean DO
concentration, SD, and %RSD. These procedures were then repeated for the Day 5A
and 5B syringes after the five-day incubation period. Lastly, the differences of the mean
DO concentrations of the Day 1 group and Day 5 group were calculated to finally
determine the BOD of the water sample.

Page 5 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

3. Results and Discussion

The experiment yielded data that was used for statistical analysis and the
determination of the BOD5 value of the water sample. All of the key results and
implications are discussed in the following paragraphs.

Table 1. Standardized molarity and dispersion data of the Na2S2O3 titrant

Molarity (M) 0.124
SD (M) 0.000623
%RSD 0.00502%

The standardized molarity of the Na2S2O3 titrant is reliable, precise, and

repeatable as evidenced by a very low %RSD presented in Table 1. This indicates that
the method of preparing and standardizing the titrant was acceptable. Not only that, a
reliable titrant concentration can increase the accuracy of the BOD5 value, making the
analysis credible.

Table 2. BOD5 value from the comparison of DO concentration between days 1 and 5
DAY 1
O2 concentration (ppm) 352.3
SD (ppm) 2.527
%RSD 0.007174
DAY 5
O2 concentration (ppm) 343.2
SD (ppm) 3.863
%RSD 1.126%
BOD5 (mg/L) 9.04
Effluent Standard
PASSED
(Class A, < 20 mg/L)

Page 6 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

As presented by Table 2, the effluent analyzed passed Section 7.2 of the DENR
AO no. 2016-08. The BOD was valid because it was supported by the low %RSD of the
DO concentration on both days 1 and 5. Having such %RSD values prove that the
analysis produced a reliable and precise outcome. Lastly, there was a trend in the data
wherein the DO concentration decreased by the day as evidenced by the higher initial
concentration on the first day while lower final concentration on the fifth day. This
observation can be expected because of the microorganisms that consume the oxygen
in the water sample. All in all, the test revealed that the effluent discharged into class A
water may contain few microbial life and is authorized to undergo conventional
treatment. In terms of its potability, the treatment process for this effluent must comply
with the latest Philippine National Standards for Drinking Water (PNSDW) which is the
Department of Health (DOH) AO no. 2017-0010.

The reason behind the valid results of the analysis was the reliability of the
Winkler method. This technique ensures that discrepancies between the DO
concentrations of each trial will be minimal since oxygen saturation is affected by
temperature and atmospheric pressure (Hauser, p. 127). Through this, the data showed
why there was little variation between the amount of DO of each replicate as revealed
by the %RSD. Moreover, the problem with the said ambient conditions was the reason
behind the immediate analysis of the Day 1 samples and sealing of Day 5 samples with
paraffin film. An additional step was performed by storing the Day 5 samples in the dark
to combat substantial changes in DO content. Wilhelm (p. 116) explained that
incubation in the dark ensures that possible photosynthetic organisms in the samples do
not produce additional oxygen. Regarding the minimal increase of %RSD for the Day 5
group, the rise in this quantity may be due to the prolonged storage of the samples
because oxygen content is erratic as elaborated before.

Page 7 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

O2 + 4 Mn(OH)2 + 2 H2O → 4 Mn(OH)3

2 Mn(OH)3 + 2 I- + 6 H+ → 2 Mn2+ + I2 + 6 H2O
I2 + 2 S2O32- → 2 I- + S4O6-
Scheme 2. Reaction mechanism showing the stoichiometric relationship of O2 and I2.

Unfortunately, there is a problem when it comes to BOD5 wherein the amount of

DO from days 1 and 5 cannot be identified directly. This is why determining the DO
concentration in the water sample consists of a series of reactions as shown in Scheme
2 since direct titration of oxygen is unsuitable due to the effects of ambient conditions
mentioned. To carry out the analysis, Scheme 2 illustrated the reactions required to
determine the DO in the sample. A precipitation reaction was done in order to fix or trap
the current amount of DO by drawing Mn(OH)2 and alkaline KI into the
sample-containing syringe, producing a brown Mn(OH)3 precipitate. Afterward, the
H2SO4 was added, wherein the addition of this reagent made the mixture suitable for
titration by dissolving the precipitate and to also liberate iodine from the alkaline KI.
From the processes presented, Hauser (p. 128) and Rice et al. (2017) discussed that
the iodine formation starts from the oxidation of Mn2+ ions by DO, forming a brown
precipitate under alkaline conditions. This oxidized manganese then oxidizes the iodide
once the H2SO4 is added, yielding the desired iodine molecules. This iodine is needed
because it will be used for the titration procedure as it is stoichiometrically equivalent to
oxygen (Hauser, p. 128; Rice et al., 2017). Once the iodine was obtained, the starch
indicator and Na2S2O3 titrant were added, and this is where the modification to the
Winkler method may have made the analysis quick and efficient. Normally, the titrant is
dispensed first into the iodine-containing solution until the pale straw color, where the
starch will be administered. This is because having a high iodine concentration will
make the blue-black color gradually disappear. Converting the iodine back into iodide
ions, as demonstrated in the third elementary step in Scheme 2, will increase indicator
sensitivity and cause rapid color disappearance (Seely, 2020). Fortunately, the modified
Winkler method allows the starch to be added before the titrant as this technique added
a lesser volume of the alkaline KI than the conventional method. A small volume of the

Page 8 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

alkaline KI entails less amount of iodine would be produced in this scenario. However,
errors may still arise from performing this experiment. Since the Winkler method is
considered an iodometric method, there are sources of error to watch out for namely,
the loss of iodine due to evaporation and unfresh or improperly prepared starch solution
(Rice et al.; Seely). To remedy these, Seely mentioned that the titration of the
iodine-containing sample must be made in cold solutions to minimize loss through
evaporation or, alternatively, quick titration of the solution right after the oxidation of
iodide to prevent volatilization of iodine. Also, the starch solution must be prepared
properly or added with a preservative, such as mercuric ions, to prolong its shelf life. All
these principles that govern the Winkler method for the BOD5 prove that this technique
ensures the precision and reliability of DO concentrations measurement, which is
important, especially in protecting public health.

4. Conclusion

The effluent dispensed to class A water was analyzed through BOD5, resulting in
a BOD of 9.04 mg/L which passed the 20 mg/L limit of the DENR AO no. 2016-08. This
analysis was considered valid, precise, and repeatable because of the low %RSD
specifically, 0.007174% for Day 1 and 1.126% for Day 2. Another factor that may have
added to the validity of the results is the 0.000623% RSD of the standardized Na2S2O3
titrant which is very low. The favorable dispersions of data for the BOD5 were due to the
technique used for the experiment called the modified Winkler method. This is because
the modification, where lesser volumes of samples and reagents were used, may
minimize the random and systematic errors. Having smaller amounts make it easier to
handle the setups and counteract the effects of ambient conditions. This analysis of
effluent is a good introduction to redox titration. Through this experiment, students can
better understand how redox reactions are used in chemical analyses and appreciate
how such a titrimetric technique can protect public health. The study can be further
explored by employing the modified Winkler method to analyze the effluent dispensed to
class C water which is for agriculture, fishing, and other maritime activities. Determining

Page 9 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

and controlling the BOD of the effluent is vital to not amplify the BOD from the algae
present since high BOD may result in hypoxia which is detrimental to marine life.

Page 10 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

References

Austin, J. (1983). A simplified and inexpensive method for measuring dissolved oxygen

in water. Journal of Biological Education, 17(3), 187-188.

https://doi.org/10.1080/00219266.1983.9654536

Hauser, B. (2017). Drinking Water Chemistry: A Laboratory Manual. Taylor & Francis

Group.

Pal, T., & Das, P. K. (1988). Spectrophotometric determination of dissolved oxygen in

water through the formation of an argentocyanide complex with silver sol.

Analyst, 113(10), 1601-1603. https://doi.org/10.1039/AN9881301601

Rice, E. W., Baird, R., Bridgewater, L., Eaton, A. D., & Water Environment Federation

(Eds.). (2017). Standard Methods for the Examination of Water and Wastewater.

American Public Health Association. https://doi.org/10.2105/SMWW.2882.216

Seely, O. (2020, June 29). Iodometric Determination of Cu in Brass. Chemistry

LibreTexts. Retrieved May 12, 2022, from

https://chem.libretexts.org/Ancillary_Materials/Laboratory_Experiments/Wet_Lab

_Experiments/Analytical_Chemistry_Labs/Iodometric_Determination_of_Cu_in_B

rass

Toze, S. (2006, 24 February). Reuse of effluent water—benefits and risks. Agricultural

Water Management, 80(1-3), 147-159.

https://doi.org/10.1016/j.agwat.2005.07.010

US Geological Survey. (2018, June 5). Biological Oxygen Demand (BOD) and Water.

USGS.gov. Retrieved May 7, 2022, from

Page 11 of 12
 CHEMISTRY DEPARTMENT
COLLEGE OF SCIENCE
School Year 2021-2022

https://www.usgs.gov/special-topics/water-science-school/science/biological-oxyg

en-demand-bod-and-water#overview

Wilhelm, F. M. (2009). Encyclopedia of Inland Waters || Pollution of Aquatic Ecosystems

I. 110-119. https://doi.org/10.1016/B978-012370626-3.00222-2

Page 12 of 12