Winkler test for dissolved oxygen

The Winkler test is used to determine the concentration of dissolved oxygen in water
samples. Dissolved Oxygen, abbreviated D.O., is widely used in water quality studies and
routine operation of water reclamation facilities. An excess of manganese(II) salt, iodide
(I-) and hydroxide (HO-) ions are added to a water sample causing a white precipitate of
Mn(OH)2 to form. This precipitate is then oxidized by the dissolved oxygen in the water
sample into a brown manganese precipitate. In the next step, a strong acid (either
hydrochloric acid or sulfuric acid) is added to acidify the solution. The brown precipitate
then convert the iodide ion (I-) to Iodine. The amount of dissolved oxygen is directly
proportional to the titration of Iodine with a thiosulfate solution [1].

Sample method
In the first step, Manganese(II) sulfate (at 48% of the total volume) is added to an
environmental water sample. Next, Potassium iodide (15% in potassium hydroxide 70%)
is added to create a pinkish-brown precipitate. In the alkaline solution, dissolved oxygen
will oxidize manganese(II) ions to the tetravalent state.

2 Mn(OH)2(s) + O2(aq) → 2 MnO(OH)2(s)

MnO(OH)2 appears as a brown precipitate. There is some confusion about whether the
oxidised manganese is tetravalent or trivalent. Some sources claim that Mn(OH)3 is the
brown precipitate, but hydrated MnO2 may also give the brown colour.

4 Mn(OH)2(s) + O2(aq) + 2 H2O → 4 Mn(OH)3(s)

The second part of the Winkler test reduces acidifies the solution. The precipitate will
dissolve back into solution. The acid facilitates the conversion by the brown, Manganese-
containing precipitate of the Iodide ion into elemental Iodine.

The Mn(SO4)2 formed by the acid converts the iodide ions into iodine, itself being
reduced back to manganese(II) ions in an acidic medium.

Mn(SO4)2 + 2 I-(aq) → Mn2+(aq) + I2(aq) + 2 SO42-(aq)

Thiosulfate solution is used, with a starch indicator, to titrate the iodine.

2 S2O32-(aq) + I2 → S4O62-(aq) + 2 I-(aq)

Analysis
From the above stoichiometric equations, we can find that:
 1 mole of O2 → 2 moles of MnO(OH)2 → 2 mole of I2 → 4 mole of S2O32-

Therefore, after determining the number of moles of iodine produced, we can work out
the number of moles of oxygen molecules present in the original water sample. The
oxygen content is usually presented as mg dm-3.

Limitations
The success of this method is critically dependent upon the manner in which the sample
is manipulated. At all stages, steps must be taken to ensure that oxygen is neither
introduced to nor lost from the sample. Furthermore, the water sample must be free of
any solutes that will oxidize or reduce iodine.

Instrumental methods for measurement of dissolved oxygen have widely supplanted the
routine use of the Winkler test, although the test is still used to check instrument
calibration.

BOD5
To determine five-day biological oxygen demand (BOD5), several dilutions of a sample
are analyzed for dissolved oxygen before and after a five-day incubation period at 20
degrees Celsius (68 degrees Fahrenheit) in the dark. In some cases, bacteria are used to
provide a source of oxygen to the sample; these bacteria are known as "seed". The
difference in DO and the dilution factor are used to calculated BOD5. The resulting
number (usually reported in parts per million or milligrams per liter) is useful in
determining the relative organic strength of sewage or other polluted waters.

The BOD5 test is an example of analysis that determines classes of materials in a sample.

The BOD5 test

BOD measures the rate of oxygen uptake by micro-organisms in a sample of water at a
temperature of 20°C and over an elapsed period of five days in the dark.

There are two recognized methods for the measurement of BOD.

Dilution method

To ensure that all other conditions are equal, a very small amount of micro-organism seed
is added to each sample being tested. This seed is typically generated by diluting
activated sludge with de-ionized water. The BOD test is carried out by diluting the
sample with oxygen saturated de-ionized water, inoculating it with a fixed aliquot of
seed, measuring the dissolved oxygen (DO) and then sealing the sample to prevent
further oxygen dissolving in. The sample is kept at 20 °C in the dark to prevent
photosynthesis (and thereby the addition of oxygen) for five days, and the dissolved
oxygen is measured again. The difference between the final DO and initial DO is the
BOD. The apparent BOD for the control is subtracted from the control result to provide
the corrected value.

The loss of dissolved oxygen in the sample, once corrections have been made for the
degree of dilution, is called the BOD5. For measurement of carbonaceous BOD (cBOD),
a nitrification inhibitor is added after the dilution water has been added to the sample.
The inhibitor hinders the oxidation of nitrogen.

BOD can be calculated by:

• Undiluted: Initial DO - Final DO = BOD

• Diluted: ((Initial DO - Final DO)- BOD of Seed) x Dilution Factor

BOD is similar in function to chemical oxygen demand (COD), in that both measure the
amount of organic compounds in water. However, COD is less specific, since it measures
everything that can be chemically oxidised, rather than just levels of biologically active
organic matter.

Manometric method

This method is limited to the measurement of the oxygen consumption due only to
carbonaceous oxidation. Ammonia oxidation is inhibited.

The sample is kept in a sealed container fitted with a pressure sensor. A substance that
absorbs carbon dioxide (typically lithium hydroxide) is added in the container above the
sample level. The sample is stored in conditions identical to the dilution method. Oxygen
is consumed and, as ammonia oxidation is inhibited, carbon dioxide is released. The total
amount of gas, and thus the pressure, decreases because carbon dioxide is absorbed. From
the drop of pressure, the sensor electronics computes and displays the consumed quantity
of oxygen.

The main advantages of this method compared to the dilution method are:

• simplicity: no dilution of sample required, no seeding, no blank sample

• direct reading of BOD value
• continuous display of BOD value at the current incubation time.

Furthermore, as the BOD measurement can be monitored continuously, a graph of its

evolution can be plotted. Interpolation of several graphs on a similar water may build an
experience of its usual evolution, and allow an estimation of the five days BOD after as
early as the first two days of incubation.[2]

Test Limitations
The test method involves variables limiting reproducibility. Tests normally show
observations varying plus or minus ten to twenty percent around the mean.[3]:82

[Toxicity

Some wastes contain chemicals capable of suppressing microbiological growth or

activity. Potential sources include industrial wastes, antibiotics in pharmaceutical or
medical wastes, sanitizers in food processing or commercial cleaning facilities,
chlorination disinfection used following conventional sewage treatment, and odor-control
formulations used in sanitary waste holding tanks in passenger vehicles or portable
toilets. Suppression of the microbial community oxidizing the waste will lower the test
result.[3]:85

Appropriate Microbial Population

The test relies upon a microbial ecosystem with enzymes capable of oxidizing the
available organic material. Some waste waters, such as those from biological secondary
sewage treatment, will already contain a large population of microorganisms acclimated
to the water being tested. An appreciable portion of the waste may be utilized during the
holding period prior to commencement of the test procedure. On the other hand, organic
wastes from industrial sources may require specialized enzymes. Microbial populations
from standard seed sources may take some time to produce those enzymes. A specialized
seed culture may be appropriate to reflect conditions of an evolved ecosystem in the
receiving waters.[3]:85-87

History of the use of BOD

The Royal Commission on River Pollution, which was established in 1865 and the
formation of the Royal Commission on Sewage Disposal in 1898 led to the selection in
1908 of BOD5 as the definitive test for organic pollution of rivers. Five days was chosen
as an appropriate test period because this is supposedly the longest time that river water
takes to travel from source to estuary in the U.K. In 1912, the commission also set a
standard of 20 ppm BOD5 as the maximum concentration permitted in sewage works
discharging to rivers, provided that there was at least an 8:1 dilution available at dry
weather flow. This was contained in the famous 20:30 (BOD:Suspended Solids) + full
nitrification standard which was used as a yardstick in the U.K. up to the 1970s for
sewage works effluent quality. The value of BOD is less than COD.[citation needed]

The United States includes BOD effluent limitations in its secondary treatment
regulations. Secondary sewage treatment is generally expected to remove 85 percent of
the BOD measured in sewage and produce effluent BOD concentrations with a 30-day
average of less than 30 mg/L and a 7-day average of less than 45 mg/L. The regulations
also describe "treatment equivalent to secondary treatment" as removing 65 percent of the
BOD and producing effluent BOD concentrations with a 30-day average less than 45
mg/L and a 7-day average less than 65 mg/L.[4]

Typical BOD values

Most pristine rivers will have a 5-day carbonaceous BOD below 1 mg/L. Moderately
polluted rivers may have a BOD value in the range of 2 to 8 mg/L. Municipal sewage that
is efficiently treated by a three-stage process would have a value of about 20 mg/L or
less. Untreated sewage varies, but averages around 600 mg/L in Europe and as low as
200 mg/L in the U.S., or where there is severe groundwater or surface water infiltration.
(The generally lower values in the U.S. derive from the much greater water use per capita
than in other parts of the world.)[1]

Instructions
Things You'll Need: pH test kit or chlorine test kit
Step

Test the chlorine level in the water with a pH kit or other water testing kits.
Testing usually involves dipping a paper tab into the water and comparing
the colors revealed against a chart. High chlorine levels or other pH
imbalances should be treated.

1. Scope

1.1 These test methods cover the determination of ammonia nitrogen, exclusive of
organic nitrogen, in water. Two test methods are included as follows:

Sections
Test Method A—Direct Nesslerization 7 to 15
Test Method B—Ion Selective Electrode 16 to 24

1.2 Test Method A is used for the routine determination of ammonia in steam
condensates and demineralizer effluents.

1.3 Test Method B is applicable to the determination of ammonia nitrogen in the range
from 0.5 to 1000 mg NH3N/L directly in reagent and effluent waters. Higher
concentrations can be determined following dilution. The reported lower range is based
on multiple-operator precision. Lower limits have been obtained by two of the twelve
laboratories participating in the round robin.

1.4 Both test methods A and B are applicable to surface and industrial waters and
wastewaters following distillation. The test method for distillation given in Appendix X1
has been used in the past to meet requirements for predistillation of samples being
analyzed for ammonia.

1.5 The values stated in SI units are to be regarded as standard. No other units of
measurement are included in this standard.

1.6 This standard does not purport to address all of the safety concerns, if any, associated
with its use. It is the responsibility of the user of this standard to establish appropriate
safety and health practices and determine the applicability of regulatory limitations prior
to use.

1.7 The distillation method now appears as Appendix X1 and is provided as

nonmandatory information only. The automated colorimetric phenate method has been
discontinued.

7.1 This test method is suitable for the rapid routine determination of ammonia nitrogen
in steam condensates and demineralized water. See Appendix X1 for the distillation test
method.

16.1 This test method is applicable to the measurement of ammonia in reagent and
effluent water.