ApplicationBrochure 2 BOD 30247570

Selected Applications
Electrochemistry
Application
Brochure
2
SevenExcellence
Seven2Go
SevenGo Duo pro
Biochemical Oxygen Demand

From Theory to Practice
Editorial
Water and air, the two essential fluids on which all life depends,
have become global garbage cans. – Jacques Yves Cousteau
This quote, uttered by the famous French oceanographer, stands at the beginning of a worldwide development
having led to a better consciousness of the exceeding importance of clean water. Since then, the strong increases
of human population and industrial production as well as the cumulative incidence of droughts and desertification
have rendered the availability of clean water one of the most urgent matters across the globe. In many places,
strict regulations have been enacted as to how surface water, drinking water, and wastewater should be moni-
tored and analyzed.
One important analysis to assess water quality is determining the BOD (biochemical oxygen demand).
BOD is an indicator of the amount of organic matter present in freshwater. High BOD levels may indicate that wa-
ter is contaminated with coliform bacteria, other pathogens and organic compounds and therefore unfit for human
consumption.
mg/10 2 1979-90 1991-99

14
12
10
0
Africa North America Latin America Europe Asia and the
and the Caribbean Pacific
Mean BOD (mg/L O2) in surface waters by selected region, 1979-90 and 1991-99 (GEO Data Portal,
compiled from UNEP/GEMS-Water 2004)
Measuring BOD in accordance with regulations implies to accurately follow defined prescriptions, to respect fixed
limits as well as to perform different types of check value analysis and to use the right equipment approved for
this purpose. Especially the numerous calculations, necessary to implement dilution and correction factors, are
prone to many errors.
With this in mind, Mettler-Toledo has developed the SevenExcellence S900/S600 DO/BOD bench meters, offering
predefined and adaptable methods that cope with all those challenges to the point of customizable print-out and
data acquisition. The application brochure at hand is therefore dedicated to all laboratories aiming for a highly
professional and compliant water quality assessment.
With this brochure, SevenExcellence and the widely approved DO sensors InLab® OptiOx or InLab® 605, you will
be capable of setting up your own BOD determination process within the shortest time. Using the expertise con-
tained herein you will benefit from the advantages of a felicitous marriage of computing and electrochemical
measurement combined in one instrument.

We trust that this will bring water quality research and monitoring a big step forward.
Hans Früh, Product Specialist SBU pH Lab

Electrochemistry Application Brochure 2
3
Content
Content
1. Introduction..........................................................................................................................................7
1.1 History.....................................................................................................................................7
1.2 Usage of BOD...........................................................................................................................7
2. BOD Background...................................................................................................................................8
2.1 What is the biochemical oxygen demand?...................................................................................8
2.2 BOD Sequence..........................................................................................................................8
2.3 Different types of oxygen demand values................................................................................... 11
2.4 Water quality and usual BOD values.......................................................................................... 12
3. Procedures to Determine Dissolved Oxygen.........................................................................................13
3.1 DO determination.................................................................................................................... 13
3.1.1 Titrimetric analysis.......................................................................................................... 13
3.1.2 Direct measurement........................................................................................................ 13
3.2 Manometric procedure............................................................................................................. 15
3.3 Comparison of different measurement procedures....................................................................... 15
4. Standards and Norms about BOD Determination................................................................................... 17
4.1 Variation between procedures................................................................................................... 17
4.2 ISO-5815 (International).......................................................................................................... 17
4.2.1 Blank tests..................................................................................................................... 17
4.2.2 Bottle size..................................................................................................................... 17
4.2.3 Nitrification inhibition...................................................................................................... 17
4.2.4 Seeding Water............................................................................................................... 17
4.2.5 Salt solution.................................................................................................................. 17
4.2.6 Dilution water................................................................................................................ 17
4.2.7 Standard solution........................................................................................................... 17
4.2.8 pH adjustment............................................................................................................... 17
4.2.9 Incubator...................................................................................................................... 18
4.2.10 Cleaning...................................................................................................................... 18
4.2.11 DO Measurement.......................................................................................................... 18
4.3 EN/DIN 1899 (European Union)................................................................................................ 18
4.4 Title 40 of the Code of Federal Regulations 40 CFR 136-03 (USA)................................................ 18
4.5 APHA 5210 A-C (USA).............................................................................................................. 18
4.6 EPA Method 405.1 (USA).......................................................................................................... 18
4.7 OECD Water Quality Guidelines................................................................................................. 18
4 Biochemical Oxygen Demand

5. Mettler-Toledo Systems for BOD..........................................................................................................19
5.1 Meters for dissolved oxygen measurement................................................................................. 19
5.2 DO Sensor.............................................................................................................................. 19
5.3 Further Materials...................................................................................................................... 19
5.4 Method types BOD and BCV..................................................................................................... 21
5.5 Method functions and parameters.............................................................................................22
5.5.1 BOD method..................................................................................................................22
5.5.2 BCV: BOD check values..................................................................................................32
5.6 Method start............................................................................................................................34
5.6.1 Concept........................................................................................................................34
5.6.2 Start BCV method...........................................................................................................34
5.6.3 Start BOD method..........................................................................................................36
6. Mettler-Toldeo M020 and M021..........................................................................................................38
6.1 Solution..................................................................................................................................38
6.1.1 Dilution water.................................................................................................................38
6.1.2 Seeded blank stock solution............................................................................................38
6.1.3 Glucose-glutamic acid solution (GGA):.............................................................................38
6.2 Blanks and standard preparation...............................................................................................38
6.2.1 Blank............................................................................................................................38
6.2.2 Seeded blank................................................................................................................39
6.2.3 Glucose-glutamic acid check (GGA).................................................................................39
6.3 Sample preparation.................................................................................................................39
6.4 Determination of initial DO........................................................................................................40
6.4.1 Blank............................................................................................................................40
6.4.2 Seeded blank................................................................................................................40
6.4.3 Standard....................................................................................................................... 41
6.4.4 Sample......................................................................................................................... 41
6.5 Incubation.............................................................................................................................. 41
6.6 Determination of final DO......................................................................................................... 41
6.7 Calculation............................................................................................................................. 41
7. Hints and Tips.....................................................................................................................................48
8. Bibliography.......................................................................................................................................50

5
List of Abbreviations
Introduction
APHA American Pharmacy Association

ATU Allylthiourea
BCV BOD check values
BOD Biochemical oxygen demand
BODn BOD after n days (usually 5/7/20 or ∞)
CBOD Carbonaceous biochemical oxygen demand
COD Chemical oxygen demand
DIN German Institute for Standardization (Deutsches Institut für Normung)
DO Dissolved oxygen
EN European norm
EPA Environmental Protection Agency (USA)
GGA Glucose-Glutamic acid standard solution
ISO International Organization for Standardization
M020 Mettler-Toledo method 020
M021 Mettler-Toledo method 021
MT Mettler-Toledo
OECD Organization for Economic Co-operation and Development
TBOD Total biochemical oxygen demand
uBOD Ultimate biochemical oxygen demand

1. Introduction
1.1 History 1.2 Usage of BOD

During industrial expansion in the Nowadays, the biochemical oxygen
19th century, many major cities demand (BOD) is a widely used
installed sewer systems to cope parameter to determine the qual-
with the rising amount of domestic ity of freshwater or the efficiency of
and industrial wastewater. These sewage treatment plants. Sewage
systems mainly transported the plants in Europe have to determine
untreated wastewater to nearby riv- the BOD5 before introducing treated
ers and lakes, which in turn led to water into a fresh water body.
rapid organic pollution of these. Eu- This has to be checked daily or
trophication was the consequence. weekly depending on local regula-
Due to lack of oxygen, the bodies tions. Fresh water bodies like lakes
of water were no longer able to or rivers are checked at regular
sustain high levels of aquatic life. intervals. It is one of many nec-
The development of procedures to essary parameters that have to
treat wastewater became a neces- be determined in order to use the
sity. A parameter to determine the water as drinking water.
efficiency of these procedures was
also crucial.
The BOD5 was selected by the

Royal Commissions on Sewage
Disposal in 1908 for the sole pur-
pose of determining organic pollu-
tion in river water. They chose five
days as the test period, because it
was reputed to be the maximum
time river water needs to travel from
source to its estuary in the United
Kingdoms. This procedure was ap-
plied in the UK up to the 1970s,
albeit in a slightly revised version.
With increasing globalization and
international standardization, ISO
Standards 8515-1 and 8515-2 have
become the most widespread pro-
cedures for BOD.

7
2. BOD Background
Theory
2.1 What is the biochemical mally seeded with microorganisms This process renders the body of
oxygen demand? that consume oxygen during the water eutrophic. The BOD is one of
The basic idea behind the principle decomposition of organic com- the most important parameters for
is simple. When organic matter pounds. Nutrients are also added. monitoring these processes and it
(leaves, grass, manure, sewage In parallel to the sample, the BODs helps the authorities to assess the
and so on) reaches a body of water of check values such as the stan- right measures for stabilizing or im-
the aerobic bacteria and fungi living dard, the seed (seeded blank) and proving the oxygen content of water
there start to degrade this organic the dilution water (blank) have to bodies.
matter by oxidation. In this process, be determined. All check values are
called respiration, the organisms prepared in the same way as the 2.2 BOD Sequence
consume the oxygen that is present sample and incubated under the A typical process for simple BOD
in their surroundings. same conditions. determination consists of these five
steps:
As water can only hold a limited The oxidation of inorganic materi- Sample taking
amount of dissolved oxygen (DO), als such as sulfides and ferrous Sample preparation
other aerobic species such as zoo- KG:ion as well as the oxidation First/initial measurement
plankton, crustaceans and fish be- of nitrogenous compounds are Incubation
come extinct if the oxygen is not considered interferences. In most Second/final measurement
replenished in due time. There are cases the BOD is determined as
two ways to replenish the DO: diffu- carbonaceous BOD (CBOD). This is Remark:
sion from the atmosphere and pho- achieved by adding an inhibitor that Special attention has to be paid to
tosynthesis. prevents the oxidation of nitrog- thorough cleaning of all equipment
enous compounds. With this setup, used for BOD determination. Don’t
This fact is used to determine the the amount of consumed oxygen use phosphate detergents, and
BOD in water: The amount of the relates directly to the amount of or- rinse everything with tap water and
dissolved oxygen is measured ganic matter that was present in the deionized water after cleaning.
at two points in time in the same water.
sample. Most commonly, these two The steps are described in more de-
points are day zero and day five. As an example: tails in the following.
If nutrients are released into a body
In the time between, the sample of water, the growth of aquatic Taking the sample:
is incubated in a sealed bottle, in plants is promoted. As the plants The sample should be taken shortly
complete darkness and at a con- grow, they also die. The organic before the measurement. If the ini-
stant temperature of 20 ± 2 °C, detritus of these plants stimulates tial measurement of the sample
thus avoiding any replenishment the growth of aerobic microorgan- cannot be performed within 2 hours
of oxygen either by diffusion or by isms. As the microorganisms start after collection, the sample has to
photosynthesis. To allow for proper to degrade the plant matter, they be stored in a refrigerator at a tem-
biodegradation, the sample is nor- consume huge amounts of oxygen. perature below 4 °C.

Even chilled samples are consid- Expected BODn
Dilution factor a Exemples of waters b
mg/L of oxygen
ered to have expired after 24 hours
3 to 6 between 1, 1 and 2 R
in storage.
4 to 12 2 R, E
10 to 30 5 R, E
Preparing the sample:
20 to 60 10 E
• Dilution: If the expected BOD is
40 to 120 20 S
higher than 6 mg/L (see table
100 to 300 50 S, C
1), dilute the sample with dilu-
200 to 600 100 S, C
tion water so that the DO doesn’t
400 to 1200 200 I, C
fall below 1 mg/L in the bottle
1000 to 3000 500 I
after the incubation, but also
2000 to 6000 1000 I
avoiding an O2 depletion of less
than 2 mg/L. The dilution water a
Volume of diluted sample/volume of the test portion
is de-ionized and air-saturated b
R: River water;
water with added nutrients. If E: Biologicaly purified municipal sewage;
the sample is diluted, a blank S: Clarified municipal sewage or lightly contaminetied industrial effluent;
sample consisting of dilution C: Raw municipal sewage;
water only has to be simultane- I: Heavily contaminated industrial effluent.
ously analyzed simultaneously.
• Initial DO (base measure- Table 1 Dilution factors for BODn (Copyright International Organization for Standardization,
2003)
ment): Oxygen oversatura-
tion of the sample at the
initial measurement has to be and 7.5 (APHA), respectively. final calculation. For seed
avoided (initial DO ≤ 100%). If not, neutralize with sulfuric sources check chapter 4.2.4.
• Temperature adjustment: acid or sodium hydroxide. • Fill the bottle to the glass grind-
The sample temperature before • Bottles: Either Karlsruher-bottles ing with sample or dilution water
analysis should be 20 ± 2 °C or Wheaton-bottles can be depending on the situation.
(DIN/EN) and 20 ± 1 °C used. Both have glass grinding
(APHA), respectively. If not, for completely airtight sealing. First measurement:
the sample should be cooled The bottles are filled about half After the preparation, the first DO
or heated. In the latter case with sample or diluted sample. measurement is done. Depend-
an oxygen oversaturation can And a stirring help is added ing on the method chosen (check
be avoided by constant stir- (e.g. a magnetic stir bar). chapter 3), the procedure varies.
ring or regular shaking of the • Inoculation: The bottle is The temperature for the measure-
sample during warm-up. seeded with microorganisms. ment should be 20 ± 1 °C.
• pH: The pH of the sample The amount of seed should
should be between pH 6 and not be excessive and cause After the measurement the bottles
8 (DIN/EN) and between 6.5 less than 1 mg/L BOD in the are sealed with a ground-in glass

9
Theory
stopper making sure that no air Calculation:

bubbles are sealed in. Note the DO The formula for the calculation is as follows:
that was measured. This measure- Equation 1 BOD calculation undiluted
ment step is also known as initial
measurement or base measure- BODn= (ρ1 – ρ2)
ment.
Equation 2 BOD calculation diluted
Incubation:
The sample is incubated for n BODn= [(ρ1 – ρ2) – V1–Vsam * (ρ3 – ρ4)] * V1
V1 Vsam
days. n is the number of days that
are needed for the chosen BOD ρ1 Dissolved oxygen concentration of the test solution at time
method. BOD5 takes five days, zero, in mg/L
BOD7 takes seven days and BODn ρ2 Dissolved oxygen concentration of the same test solution
takes n days. For the entire incuba- after n days, in mg/L.
tion period, the incubator has to be ρ3 Dissolved oxygen concentration of the blank solution at time
absolutely dark inside and the ther- zero, in mg/L
mostat mustbe set to 20 ± 1 °C. ρ4 Dissolved oxygen concentration of the blank solution after n
The sample should be stirred at all days, in mg/L
time. Vsam Volume of sample used for the preparation of the test solution, in mL
Vt Total volume of the test solution, in mL
Second measurement:
After n days (± 2 h (DIN/EN); ± 4 h An example for an undiluted sample: The bottle has a volume of 250 mL.
(APHA)) the second DO measure- The initial DO of the sample is 7.5 mg/L. After five days a value of 3.6 mg/L
ment is done. Take the same pro- is measured.
cedure as for the first measurement.
Note the DO value. This measure- BOD5 = (7.5 mg/L–3.6 mg/L) = 3.9 mg/L
ment step is also known as the final
or follow measurement. The BOD5 of the example is 3.9 mg/L
An example for a diluted sample: The bottle has a volume of 250 mL.
It is filled with 100 mL of sample and 150 mL of dilution water. The initial
BOD values are 7.5 mg/L for the sample and 7.4 mg/L for the blank. After
five days the sample contains 5.6 mg/L dissolved oxygen and the blank
7.3 mg/L.
BOD5= [(7.5 mg/L–5.6 mg/L) – 250 mL–100 mL * (7.4 mg/L–7.3 mg/L)] * 250 mL = 4.6 mg/L
250 mL 100 mL
The BOD5 of the example is 4.6 mg/L.

2.3 Different types of oxygen
demand values
B
BODn: The index n defines the time
BIOCHEMICAL OXYGEN DEMAND

interval in n days that was used Second stage: combined
carbonaceous - plus
for the incubation of the samples. nitrogenous - demand curve
The standardized test is the BOD5.

A
Under laboratory conditions, the
First stage: carbonaceous - demand
oxygen consumption in a sample is curve
measured after five days. Other fre-

quently used time intervals are
BOD7 and BOD20.
CBOD: The C stands for carbo- 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

naceous biochemical oxygen de- TIME, IN DAYS
mand. The CBOD quantifies the Figure 1 BOD course over 30 days (EPA)
amount of oxygen consumed dur-
ing incubation in the presence of ni- total amount of oxygen that is re- Usually the BOD5 is about 70–80%
trification inhibitors. It is a measure quired for the microorganisms of the COD. This rough estima-
for all carbonaceous compounds to degrade all organic matter in tion can also help to determine the
present. the sample is represented by this dilution factor without doing a BOD5
parameter. preliminary test.
NBOD: The NBOD quantifies the
amount of oxygen needed to oxi- Both CBOD and NBOD are parts of The relationship between BOD and
dize the present nitrogenous com- the UBOD. Using this parameter, it COD allows assumptions to be
pounds (mainly NH3) to nitrate and is possible to predict the impact of made about the composition of
nitrite by biological degradation. an effluent on a lake or a river. sewage. The COD is always higher
The oxygen demand of nitrifica- than the BOD measured in the
tion bacteria is considered interfer- The chemical oxygen demand same sample.
ence to BOD analysis. As shown in (COD) is used to measure the
Figure 1, the nitrogenous bacteria amount of organic compounds that BOD5 = (50...100)% of COD:
don’t begin to consume oxygen can be chemically oxidized in water. All present materials are easily
until after 7–10 days. This time pe- It is commonly used to determine biodegradable.
riod varies hugely depending on the the amount of organic pollutants
microorganisms in the seed and the found in fresh water or wastewater. BOD5 < 50% of COD: Either the
composition of the sample. The COD mg/L indicates the mass present materials are poorly biode-
of oxygen consumed per liter of gradable, or they are toxic for the
UBOD: UBOD stands for ultimate solution. The consumption is deter- microorganisms that are used.
biochemical oxygen demand. The mined by chemical oxidation.

11
Norms & Procedures
BOD5 = (12...25) % of COD: In spring and autumn, pollen and phosphate and turbidity, a clear
This value is expected for treated degrading leaves respectively add picture of the water quality can be
sewage. organic content to the water. Unpol- achieved. Figure 1 gives an over-
luted waters have a BOD range of view on how the BOD values is
2.4 Water quality and usual BOD up to 5 mg/L. Together with other used to rate fresh water.
values analytical parameters such as
The amount of organic matter fecal coliforms, nitrates, pH, tem- Sewage plants have to determine
that resides in open freshwater perature changes over the year, the BOD5 at regular intervals.
sources fluctuates during seasons. total dissolved solids (TDS), total Depending on the season, the
weather, the industry and the ag-
BOD in ppm Water quality riculture in the catchment area of
1–2 Very Good: Low levels of organic waste in the water the plant, the standard values for
3–5 Fair: The water is only moderately clean BOD5 vary over the year. The load
Poor: The water is somewhat polluted. The water is expected to of organic matter in the sewage will
6–9 show bacteria and other microorganisms that are decomposing
be lighter depending on the size of
the present organic waste
Polluted: The water contains high amounts of organic waste.
a community and the dependency
>10 There is a chance that manure is being dumped in this water on heavy industry and intensive
source agriculture. See Table 3 for com-
Table 2 Water quality values for BOD (Copyright International Organization for Standardiza- monly expected BOD values.
tion, 2003)
BOD in ppm Sewage

20 For municipal sewage after three stage treatment
600 For untreated sewage in Europe
For untreated sewage in the USA (the process uses
200
more diluted raw sewage)
Table 3 Normal values for BOD in sewage plants

3. Procedures to Determine Dissolved Oxygen
A wide variety of procedures exist to determine the dissolved oxygen. The DO measure-
ment is part of the BOD analysis and is also regulated by ISO and EPA standards and
regulations. The analytical procedures can be divided mainly into two types. One type of
procedure determines the amount of DO at the start and the end of a defined incubation
interval, whereas the other type continuously monitors the pressure decline over time via
manometric measurement.
3.1 DO determination 3.1.2 Direct measurement from passing through. Sheathed

Titration or direct measurement by a glass envelope, the cathode
is normally used to determine the Direct measurement with builds the core of a Clark Type elec-
amount of dissolved oxygen. an electrochemical sensor trode cell. In order to provide higher
Electrodes often used for BOD are signal stability and safeguard
3.1.1 Titrimetric analysis «Clark Type» electrodes, named af- against drift, the anode has a larger
The Winkler titration is the most ter their inventor Dr. Leland Clark. surface. The sensor measures oxy-
commonly used titration method for These electrodes contain an anode gen tension amperometrically.
oxygen determination.
The sample is treated with manga-

nese sulfate (MnSO4) or manganese
chloride (MnCl2) and a solution con-
taining potassium hydroxide (KOH)
and potassium iodide (KI). Manga-
nese hydroxide (Mn(OH)2) reacts
with the dissolved oxygen and forms
a brown, clearly visible precipitate:
(MnO(OH)2).
When acidified with sulfuric acid

(H2SO4) the hydrated manganese
oxide is dissolved back into solu-
tion forming manganese disulfate
(Mn(SO4)2). Figure 2 Winkler titration
This acts as an oxidizing agent and and a cathode which are connected The silver/silver chloride anode pro-
releases free iodine (I2) from the by an electrolyte solution. Since vides electrons for the cathode re-
potassium iodide (KI). The iodine the anode and the cathode are po- action.
is titrated with sodium thiosulfate larized, it is also called a polaro-
(Na2S2O3). graphic oxygen sensor. The tip is Given a constant polarizing voltage,
covered by a polypropylene mem- the electrode produces a current
This is called an iodometric titra- brane (other materials exist). which is directly proportional to the
tion. The amount of sodium thiosul- partial pressure of oxygen diffusing
fate (Na2S2O3) is stoichiometrically This semi-permeable membrane to the reactive surface of the elec-
equivalent to the dissolved oxygen allows gases to pass but prevents trode.
that was in the sample. contaminants and reducible ions

13
Norms & Procedures
Polarization voltage Quenching also reduces the fluo-

rescence lifetime. If no oxygen is
present, fluorescence will persist
Anode
for a certain amount of time after
Electrolyte the exciting light is shut off. In the
presence of oxygen, the lifetime
Insulator
decreases because quenching pro-
Cathode
vides an additional path out of the
excited state, thus enhancing the
decay of the latter.
Measuring solution Membrane
Unlike intensity measurements, life-
Figure 3 Polarographic sensor (Mettler-Toledo) time measurements do not depend
on the intensity of the exciting light,
The silver at the anode becomes the molecule is said to be excited. and, thus, are less subject to drift.
oxidized. Reduction of oxygen oc- Since the excited molecule is un- The fluorescence lifetime can be
curs at the surface cathode which is stable, it quickly loses the absorbed conveniently measured by modulat-
exposed at the tip of the electrode. energy typically as light emission ing the exciting light, which causes
Oxygen molecules diffuse through (fluorescence) and/or heat. the emitted light to be modulated at
the semi-permeable membrane the same frequency, but shifted in
and react with the KCI electrolyte Because part of the energy of the phase. The amount of phase shift is
solution. The current produced is a absorbed light is always converted a measure of the fluorescence life-
result of the following reduction of in to thermal energy, the emitted time and the oxygen concentration.
oxygen at the cathode. light inevitably is of lower energy
(longer wavelength) than the ab- When the optical sensor initiates a
Ag anode: sorbed light. reading, blue light is emitted by a
4 Ag + 4 Cl-  4 AgCl + 4 e- blue LED. This excites the lumiphore
Pt cathode: An excited molecule can also lose molecules at the sensor’s tip. When
O2 + 4 H+ + 4 e-  2 H2O energy by colliding with another excited, lumiphore molecules emit
molecule, notably oxygen. As a red light: This red light is detected
Direct measurement collision with oxygen provides a by a photodiode. Oxygen molecules
with optical sensor path out of the excited state that quench the excited lumiphore mol-
Optical DO sensors use fluores- does not emit light, oxygen re- ecules and prevent the emission of
cence quenching to measure dis- duces (or quenches) the intensity red light. This process is called «dy-
solved oxygen. Fluorescence is of the fluorescence. The greater namic luminescence quenching.»
an optical property of certain mol- the concentration of oxygen, the
ecules. When a molecule absorbs greater is the reduction in fluores- The optical sensor measures a
light, its energy increases, and cence intensity. phase shift between the red light

that is returned and the red refer-
1. Blue LED emits blue light
ence light. The concentration of
dissolved oxygen and the red light
2. Luminophore molecules excited
that returns are inversely propor-
tional. This means that a high DO
concentration reduces the amount 3. Excited lumiphore molecules emit red light
of red light returned to the sensor.
4. Is oxygen present?
Optical electronics calculate DO Yes No
concentration and report results to
the instrument. DO determination
by luminescence quenching has a
linear response over a broad range 5. Oxygen molecules 5. Excited lumiphore
quench excited molecules continue to
of concentrations and offers a high lumiphore molecules emit red light
degree of accuracy and stability.
3.2 Manometric procedure

As distinguished from other
methods, this procedure measures
only the oxygen consumption
6. Photodiode detects red light.
through the carbonaceous oxida- DO concentration and red light
tion process. Ammonia oxidation is emission are inversely proportional
inhibited. 7. Electronics caluculate DO concentration
The water is kept in a sealed bottle

that is fitted with a pressure sensor. mg/L DO reported
A substance that absorbs carbon Figure 4 Operation mode of optical sensor (Mettler-Toledo)
dioxide (typically lithium hydroxide,
sodium hydroxide) is placed above in pressure. This can be measured ity. The major drawbacks, are that it
the level of the sample. The sample by the pressure sensor and is trans- is a manual titration and depends
is stored at 20 °C in absolute dark mitted to a display that shows the on a double color change during
for the test period. consumed quantity of oxygen. titration.
During this process oxygen is 3.3 Comparison of different Both direct measurement sensors
consumed and the released CO2 measurement procedures are dependent on a meter and
is absorbed by the aforememen- Every method has its benefits and cost considerably more when first
tioned carbon dioxide absorbing drawbacks. Winkler titration is a purchased. A basic grasp of how
substance, thus making for a drop method that works without electric- to use technical equipment is also

15
Norms & Procedures
necessary. Both deliver the real preparation time. The handling of

BOD value and are very quick and NaOH or LiOH can be dangerous if
Pressure sensor
reliable measurement methods. not done correctly.
These methods are very accurate in
low DO range measurements and This method can determine high
Support for
NaOH pellets are generally a more scientific ap- ranges of BOD and is therefore
proach to the BOD. All these meth- often used in sewage plants and
O2
ods allow the measurement equip- other wastewater treatment facili-
Sample with bacteria ment to be used for other analysis ties. All the equipment is in use dur-
during incubation time. ing the entire measurement period
Magnetic stir bar
and cannot be used for other mea-
Figure 5 Manometric procedure The manometric method is very surements.
(Jouanneau, et al., 2014) easy to use und has a very fast
Direct with polarographic

Winkler-titration Direct with optical sensor Manometric
sensor
Chemicals
MnCl2 or MnSO4, KI,
(without BOD specific chemi- Electrolytes None LiOH or NaOH
H2SO4, Na2S2O3
cals)
Separate samples for Same sample for
Same sample for base Same sample for base
Usage of sample volume base and follow mea- base and follow
and follow measurement and follow measurement
surement measurement
Amount of work needed? High Low Low Very low
Preparation time Long Long Very short Short
Measurement equipment used
No No No Yes
during incubation?
Precision medium high high low
Range 0 to 6 mg/L 0 to 6 mg/L 0 to 50 mg/L 0 to 700 mg/L
Table 4 Comparison of main BOD procedures

4. Standards and Norms Relating to BOD Determination
4.1 Variation between procedures 4.2.2 Bottle size 4.2.5 Salt solution
The standards and norms for regu- Min. 100–125 mL, All nutrient solutions and other salt
lated BOD measurement are the optimal 250–300 mL solutions must be stored in glass
following: bottles at 0 °C to 4 °C in the dark
ISO 5815 4.2.3 Nitrification inhibition and discarded when biological
EN DIN 1899 For ISO 5815-1 with allylthiourea growth is visible or after 6 months.
40 CFR 136-03 (ATU) (CBOD)
APHA 5210 For ISO 5815-2 no nitrification 4.2.6 Dilution water
EPA 405.1 inhibitors are used (TBOD) The dilution water is provided with
OECD Water quality guidelines salt solutions, aerated for at least
4.2.4 Seeding water 1 hour and then allowed to rest
The differences between them are • Urban wastewater with max for another hour. It should contain
minor. Their key requirements are COD of 300 mg/L 8 mg/L of dissolved oxygen.
pointed out in the following chap- • River or lake water contain-
ters. The usage of optical sensors ing urban wastewater 4.2.7 Standard solution
for DO determination is permitted • Settlement effluent from a Glucose-Glutamic Acid (GGA) so-
by all mentioned standards and wastewater treatment plant lution is used as standard. GGA
regulations, but no official approval • Water taken downstream from should be prepared and used on
has been published so far. the discharge of the water to the same day.
be analyzed or water contain-
4.2 ISO-5815 (International) ing microorganisms adapted 4.2.8 pH adjustment
to the water to be analyzed Prior to seed addition, the samples
4.2.1 Blank tests and cultivated in the labora- showing alkalinity (pH > 8.5) or
The blank tests (seeded blank and tory (in the case of industrial acidity (pH < 6.0 harmful to bac-
blank) are carried out simultane- effluents containing poorly teria are to be neutralized to pH 6.5
ously or even better, in advance of bio-degradable substances) to 7.5 with a solution of Hydrochlo-
the determination of the sample, • Commercially avail- ric acid (HCl) or sodium hydroxide
using the dilution water and the able seeding material (NaOH) of such strength that the
seeded dilution water including quantity of reagent does not dilute
2 mL of allylthiourea solution per The seed should not contribute the sample by more than 0.5%.
liter. more than 0.6 to 1 mg/L to the oxy-
gen consumption in the bottle. If the The pH of dilution water should not
For the procedure without nitrifica- consumption of oxygen by the seed be affected by the lowest sample
tion inhibition the allylthiourea solu- is higher, the test should be re- dilution.
tion is not used. The blank should peated with less seed. Verify the DO
have a maximum DO of 0.2 mg/L. of the seed with the seeded blank in
0.1 mg/L is preferred. advance.

17
BOD with SevenExcellence
4.2.9 Incubator Part one describes the procedure 4.5 APHA 5210 A-C (USA)
Incubator, thermostat set to 20 °C ± for dilution and nitrification inhibi- The APHA 5210 A-C is identical
2 °C (68 °F) tion, part two describes how it is to the ISO-5815. The APHA
done without these two alterations. 5210 A-C and EPA Method 405.1
4.2.10 Cleaning There are slight modifications but are recommended by title 40
The bottles have to be thoroughly none of the aforementioned points of the Code of Federal Regulations
cleaned before use. If the Winkler ti- are concerned. It mainly differs in 40 CFR 136-03.
tration procedure is used, rinsingthe how the samples are taken.
bottle several times with tap water 4.6 EPA Method 405.1 (USA)
and then with deionized water is 4.4 Title 40 of the Code The APHA procedure is identical
enough. For the other procedures a of Federal Regulations to the EPA Method 405.1.
more thorough cleaning is neces- 40 CFR 136-03 (USA) Both are recommended by title 40
sary. In the federal regulations title 40, of the Code of Federal Regulations
it is mentioned that the standard 40 CFR 136-03.
Cleaning agents containing phos- procedure for BOD is regulated in
phates are to be avoided. the EPA 405.1 and the standard 4.7 OECD Water Quality
procedure 5210 A (the same proce- Guidelines
4.2.11 DO Measurement dure used as in APHA 5210 A-C). The OECD states that all parameters
• Winkler titration procedure: Both procedures are identical. The for the procedure were taken from
ISO 5813:1983 40 CFR 136-03 contains four ap- the DIN EN 1899-1 and 2.
• Electrochemical: ISO 5814:2012 proved methods to determine DO:
• Optical: ISO/FDIS • Winkler titration procedure:
17289 in approval EPA 360.2
• Manometric DIN ISO 16072 • Electrochemical: EPA 360.1
• Optical: EPA approved
4.3 EN/DIN 1899 • Manometric APHA 5210 B
(European Union)
The DIN EN 1899 states that all
parameters are taken from the
ISO 5813-8515. This norm consists
of two articles.

5. Mettler-Toledo Systems for BOD
5.1 Meters for dissolved oxygen the DO/BOD module to create and well as simpler and faster to cali-
measurement adapt the BOD methods to userspe- brate and more stable after calibra-
The following meters of the MT cific parameters. tion.
portfolio allow DO measurements:
• Seven2Go™ (S4), SevenGo™ The InLab® OptiOx can be equipped 5.3 Further Materials
pro (SG6) and SevenGo ™ with a BOD adapter. This adapter The determination of BOD needs
Duo pro (SG68) with polaro- helps to measure DO without spill- a variety of accessories. Some of
graphic sensor InLab® 605 ing too much of the content of the these are essential, others help by
• Seven2Go™ (S9), SevenGo™ bottle, allowing the bottle to be simplifying the process.
pro (SG9) and SevenGo™ used for an additional Winkler titra-
Duo pro (SG98) with opti- tion. It may also enable the bottle to List of accessories:
cal sensor InLab® OptiOx be resealed in order to continue the
• SevenExcellence™ with DO/BOD measurement for BOD7 or longer Seed capsule: Seed capsules or
module (e.g. S900) and polaro- BOD intervals (only works in com- any other seeds have to be ac-
graphic sensor InLab® 605 or bination with a Karlsruher bottle). quired in order to perform a BOD
optical sensor InLab® OptiOx analysis.
The METTLER TOLDEO customer As seed water you can use:
The method concept of SevenExcel- magazine UserCom 17 features an • Seed capsules pur-
lence™ does not only allow simple article (p. 26–27) about the proper chased from supplier
DO measurements but also enables use of the InLab® OptiOx together • Urban wastewater with
the final BOD to be calculated de- with the BOD adapter and the SG9 max. COD of 300 mg/L
pending on a base and a «follow» instrument. • River or lake water contain-
measurement. The base is the ini- ing urban wastewater
tial DO measurement of the sample. 5.2 DO sensor • Settlement effluent from a
The «follow» is a follow up mea- The upcoming implementation of wastewater treatment plant
surement that has been done taking optical sensors into the ISO and • Water taken downstream from
the base reading into account. EPA standards gives way, allow- the discharge of the water to
ing an up-to-date sensor system to be analyzed or water contain-
Given these two correlating points, take its place among the standard- ing microorganisms adapted
the SevenExcellence™ is able to ized and marketed DO sensors. to the water to be analyzed
calculate the BOD taking salinity, and cultivated in the labora-
temperature, atmospheric pressure Operators will no longer be lim- tory (in the case of industrial
as well as the BOD values of the ited to usage of polarographic and effluents containing poorly
blank, the seeded blank and the galvanic sensors. Both of them are bio-degradable substances)
standard into consideration. The invasive methods that consume the
SevenExcellence™ meter comes present oxygen to determine the DO Bottles: The Karlsruher bottle and
with two pre-installed method concentration. Moreover, optical the Wheaton bottle are commonly
templates that can be used with sensors are quicker to prepare, as used for BOD. The Karlsruher bottle

19
works very well with the InLab® Disposable plastic bottles can Magnetic stirrer uMix: SevenEx-
sensors. Both BOD bottle have a be used as well. They are thinner, cellence™ supports the magnetic
ground in glass stopper with a long lighter and are always clean and stirrer uMix. For the BOD analysis it
neck for easy gripping. ready for use. is recommended to stir the sample
during the measurement. Magnetic
The designated filling capacity of They are usually made of recyclable stirring bars are required in addition
a bottle is reached when it is filled materials. All bottles have to be to the stirrer.
with a sample and the ground-in washed thoroughly before use.
glass stopper is plugged in and no Compact printer: SevenExcellence™
air bubbles are visible. The Karls- Incubator: The incubation is best supports the printers USB-P25
ruher bottle comes with a small done in an incubator that is dark in- and RS-P25. This allows the re-
funnel that holds the overfilled side and can be regulated to 20 °C. port to be printed directly after the
liquid back, in case it has to be During incubation the samples analysis without any possibility for
opened and resealed again. have to be stirred, using a multi- tampering.
stirrer.
LabX direct pH: The LabX direct pH
OptiOx BOD adapter: The BOD software allows SevenExcellence™
adapter is ideal for use in combina- to send data via USB directly to a
tion with the Karlsruher bottle. This computer and to convert it automat-
way the measurement can be car- ically into an MS Excel, MS Word or
ried out with a minimum of spilling a simple Text format.
and the same bottle can be used to
measure continuously. pH/mV module: For pH measure-
Figure 6 Karlsruher bottle,
Wheaton bottle and plastic bottle ments, SevenExcellence™ can
additionally be equipped with a
pH/mV module. As such, money
and space are saved for an addi-
tional meter. Coming with the fa-
miliar Mettler-Toldeo interface, it is
a viable solution to check the
sample’s pH value prior to the BOD
analysis. A recommended pH elec-
trode is InLab® Expert Pro-ISM.
Conductivity module: For conduc-

tivity or salinity measurements the
SevenExcellence™ can be equipped
Figure 7 Incubator with multi stirrer

with a conductivity module. It al-
lows the determination of salinity in
the sample, dilution water or seed
solution prior to the BOD analysis.
InLab® 741 is the recommended
conductivity sensor.
5.4 Method types BOD and BCV

SevenExcellence™ methods of type
BOD and BCV are used to determine
a simple BODn value based on a
base and a follow measurement.
For more enhanced analysis which
require check values like blank or
standard BOD determination, the
BCV (BOD check values) method
type is used in combination with a
BOD method. In this chapter the pa-
rameters of BOD and BCV methods
are explained.
Calculation of BOD in consider-

ation of salinity, pressure and tem-
perature as well as water blanks,
seeded blanks and standards allow
for a complete analysis with the
click of a few buttons.
In the following pages it will often

be referred to base and follow. Base
is the specification of the first mea-
surement on day 0. Follow is the
follow-up measurement in consid-
eration of a base value. Together
they allow the calculation of the
BODn of the examined sample.

21
5.5 Method functions and parameters
5.5.1 BOD method
When creating or modifying a Mettler-Toledo method, several parameters can be chosen and edited. If the
parameter is light blue it is a fixed value that cannot be changed, if it is dark blue it can be edited. Standard
method functions when choosing the T0007 template are:
Title
Parameter Description Value
Method type Displays the chosen method type. Cannot be changed. BOD
Standard name is A8xxx. The last three digits depend on
Method ID how many user created methods are saved on the max. 12 alphanumeric characters
SevenExcellence™. The name can consist of 12 letters.
Title Title of Method. It can consist of 30 letters. max. 30 alphanumeric characters
Author Automatically displays the name of the logged in user. Info
Created on Automatically displays the date of creation. Info
Modified on Automatically displays the date of modification. Info
Modified by Automatically displays the name of the logged in user. Info
Protects the method against deletion or modification by
Protected Yes or no
other users than the author (logged in user) or administrator.
SOP Standard operation procedure. Yes or no
SOP text Displayed if check box SOP is active. Defines the SOP text. max. 50 alphanumeric characters

Configuration
Measurement type Is based on the chosen method type. Dissolved oxygen
A created profile for a sensor can be chosen.
If no sensor is chosen, the method will automatically take the sensor that is
Sensor name currently registered in the module. Changing from Polarographic-Sensor to List
OptiOx opens a warning that the resolution of the measurement is adjusted.
See user manual of the used sensor for more details.
If chosen a new submenu opens up. It allows the method to use the built in
Salinity correction salinity correction function. All values of salinity are in ppt. To determine the Yes or no
salinity a separate conductivity sensor and module is necessary.
• Salinity of seed solution The salinity in ppt for the chosen media. 0.0…70.0 ppt
• Salinity of dilution water The salinity in ppt for the chosen media. 0.0…70.0 ppt
• Salinity of undiluted sample The salinity in ppt for the chosen media. 0.0…70.0 ppt
To bottle or
Seed Added
To dilution water
• To bottle If the seed is added directly to the bottle. In the bottle submenu it will be
necessary to specify the amount of seed to be added.
• To dilution water If the seed is added to the dilution water it is necessary to note the factor of
dilution here. In the bottle submenu it will be calculated trough the factor
and the entered volume of sample.
• Seed dilution factor Only shows up if «to dilution water» is selected. It allows the seed dilution
1.0–999.9
factor to be entered. Ratio of seed solution to dilution water.
If this box is checked, and before starting the follow measurement the
Blank correction correlating BCV is chosen, the method will automatically subtract the blank Yes or no
from the measured BOD value.
The volume of the bottles you use. Changing the bottle volume will open a
Bottle volume warning, saying that all entered bottle settings are changed as well in pro- 10…1000 mL
portion to the new volume.
Internal, or external
Temperature capture How the temperature should be taken.
or manual
• Internal The built-in temperature sensor is used.
• External A secondary temperature sensor is added to the sample. It can be selected
from the submenu, that opens up if this parameter is chosen.
• Manual Under «Sample (BOD)» you will be able to enter a fixed temperature.
Barometric pressure capture How the pressure should be measured. Automatic or manual
• Automatic The built-in pressure or manual sensor is used.
Under «Sample (BOD)» you will be able to enter a fixed barometric
• Manual
pressure.

23
Sample (BOD)
Sample ID ID of the sample. max. 30 characters
Comment Comment on the sample. max. 30 characters
Sample type sample (blank, seeded blank or standard in BCV method respectively). Sample
Same bottles used for base and This is chosen if the same bottles are used for base and follow. There will
Yes or no
follow be different submenus depending on the choice taken here.
(If «same bottles used for base and follow» is selected) Number of bottles
Number of bottles used for the analysis. It allows for a maximum of 10 bottles to be analysed 1…10
simultaneously.
Number of bottles for the base measurement. It allows for a
Number of bottles (base) 1…10
maximum of 10 bottles to be analysed simultaneously.
Number of bottles for the follow measurement. It allows for a
Number of bottles (follow) 1…10
maximum of 10 bottles to be analysed simultaneously.
(If «manual» was selected before in «Configuration») This allows the user to
Temperature 0…60 °C
set a manually temperature.
(If «manual» was selected before in «Configuration») This allows the user to 500.0…
Barometric pressure
set a manually pressure. 1100.0 mbar
Bottles: Shows a list with detailed information about all bottles of the analysis. Tapping any line allows editing
the bottle’s settings. If the option "different bottles for base and follow" has been chosen before, the base and
follow tap have to be edited separately. When open, the bottle’s menu shows a list of bottle-IDs that are by default
set to 1-10. If tapped, the bottle-IDs can be edited separately.
Sample (BOD): Bottles

Shows the correlating sample ID that was entered
Sample ID
under «Sample (BOD)».
Bottle ID Name of that specific bottle. max. 12 letters
Comment Comment on that specific bottle. max. 30 letters
The volume of sample added to the bottle previously max was writtenn with a
Sample volume 0.1...1000 mL
dot at the end.
If «to dilution water» was selected it is calculated depending on the maximum
volume and the volume taken by the sample.
Seed volume 0.1...1000 mL
If «to bottle» was selected the volume of seed solution in mL
that was added to the bottle can be entered here
Volume of dilution water added to the bottle in mL. Calculated depending on the
Dilution volume Info
maximum volume, the volume taken by the sample and the added seed

Measure (BOD)
Parameter Description
Sensor name Originates from «Configuration». Info
DO unit DO unit to be shown in the report. mg/L or ppm
BOD unit BOD unit to be shown int the report. Not editable. mg/L
Defines how many digits are displayed for the DO measurement. By default set
DO resolution 1, 2, 3
to 2.
Defines how many digits are displayed for the BOD
BOD resolution 1, 2, 3
measurement. By default set to 2.
Automatic or
Endpoint type Allows a choice of 3 different options for the endpoint. manual or
timed
• Automatic Measurement ends, when chosen criteria are met.
• (if «Automatic» is chosen) There are several criteria the user can choose
from.
• Strict: Value varies less than 0.03 mg/L during the last 20 seconds.
• Standard: Value varies less than 0.08 mg/L during the last 20 seconds.
• Fast: Value varies less than 0.08 mg/L during the last 10 seconds.
• User-defined:
• dE: Defines the measured value interval. As soon as the change in
the measured value over the time period dt is less than dE, the
• Endpoint criteria
measured value will be acquired. This occurs within the defined
time interval.
• dt: Defines the time component, in [sec] for dE/dt. dt>tmin and
tmax>dt.
• tmin: Earliest possible time for the measured value acquisition,
in [sec].
• tmax: Latest possible time for the measured value acquisition,
in [sec].
Measurement ends, when user taps on «Take manual endpoint» during
• Manual
measurement.
• Timed Measurement ends when the defined measurement time has passed.
• Endpoint time (if «Timed» is chosen) Time until the analysis is finished. 1…1'000'000 s
Stir If a stirrer is connected, this option allows its use during a measurement. Yes or no
• Stirring speed If selected, this option allows regulation of stirring speed. 10…100%

25
Report Parameters for printing the report can be set here
If enabled, measurement data is printed to the device that is defined in
Print Yes or no
the SevenExcellence settings
Print format Two options on what shall be printed Summary or User defined
Covers all important data concerning date, time, user and all
• Summary
parameters according to the settings of the measurement type
User defined
If selected, options, which are freely selectable, appear
Values and Calculations V alues and calculations of this particular method can be exported or
Yes or No
printed.
• Data Data of this particular method can be exported or printed. Yes or No
• Info Information of this particular method can be exported or printed. Yes or No
Additional method functions that can be added:
Instruction Allows the user to add a pop up window containing instructions

Instruction The instruction can be edited here. max. 120 characters
What user interaction is needed for the message to vanish and the analysis to
Continue after Confirmation or Time span
continue
• Time span The instruction will vanish after a pre-set time
• Time Time that will pass in seconds 1...1'000'000
The instruction will vanish when confirmed by tapping the «OK» button on
• Confirmation
the screen
The condition that causes the instruction. If not checked the instruction will
Condition Yes or no
appear in all measurements with this method
The formula for the condition. For more information about the Mettler-Toledo max. 120 aplhanumeric
• Formula
formula syntax check the SevenExcellence™ user guide characters

If additional waiting or stirring is necessary for the procedure this
Wait/Stir
parameter can be added
Wait time Time to wait before the next step is initiated, in seconds 1...1'000'000 s
If the sample has to be stirred in this wait time.
Stir Yes or no
If checked it allows the stirring speed to be adjusted.
• Stirring speed The stirring speed in %. 10…100%
Instruction Allows the user to add a pop up window containing instructions. Yes or no
• Text The instruction can be edited here. max. 120 characters
The condition that causes the wait/stir step. If not checked, the wait/stir
Condition Yes or no
step will happen in all measurements with this method
The formula for the condition. For more information about the Mettler- Max. 120 alphanumeric
• Formula
Toledo formula syntax check the SevenExcellence™ user guide characters
This parameter checks the properties of the sensor prior to the measurement.
Sensor check
Slope and calibration date can be checked
Check slope The slope of the sensor is checked
• Min. slope The minimal value of the sensor’s slope 10…200%
• Max. slope The maximal value of the sensor’s slope 10…200%
Check calibration date The last calibration date is checked
• Monitoring period Days or hours Days or hours
• Max. elapsed period The length of time that can pass between calibrations. 1 to 100
If enabled, the method is interrupted if one of the parameter is outside of the set
Interrupt outside
limits. If disabled, the analysis continues but the final result has the status OK*, Yes or no
limits
signifying that there was an anomaly during measurement.

27
In this method function, the user can enter a calculation based
on the results of his measurement. It is possible to set result limits and
Calculation decide to interrupt the measurement when exceeding the
limits, see chapter «Formula Syntax» of the SevenExcellence
operating manual.
Name Name for the calculation max. 30 characters
Unit The unit will be used after the calculation
Formula Formula that allows the results of your analysis to be evaluated
Decimal places How many decimal places the new result wil have 0…6
Result limits User set limits
–1'000'000 to
• Lower limit Set a lower limit
1'000'000
–1'000'000…
• Upper limit Set an upper limit
1'000'000
• Interrupt outside limits If enabled, the method is interrupted if one of the parameter is outside of the
set limits. If disabled, the analysis continues but the final result has the sta-
tus OK*, signifying that there was an anomaly during measurement.

Analysis (BOD):
Analysis (base)
The user can set a temperature limit with a minimum and maximum value. The
Temperature limits Yes or no
standard procedure for BOD states that it should be between 19 and 21 °C.
Set a maximum temperature. Unit is chosen in the user settings of the
Max. temperature
SevenExcellence™.
Set a minimum temperature. Unit is chosen in the user settings
Min. temperature
of the SevenExcellence™.
Action when outside Choose what action is taken, when your measurement is outside of the set
limits limit.
• Save and report: The value is saved as a result and a note (OK*) is
added to the report that it is outside of the set range.
• Repeat: The measurement is repeated until terminated by the user or
the set limits are matched.
• Skip bottle: The results from the bottle are skipped and ignored. The
analysis continues with the next bottle.
• Interrupt: The whole analysis is interrupted and stopped.
If a measurement result is outside the set limits an instruction window pops up
Show instruction which has to be confirmed by tapping OK. This can be disabled by unselecting Yes or no
the checkbox.
If the sample is oversaturated with oxygen due to dissolved chemicals, adjust-
Max DO limit Yes or no
ments can be made here.
Max. DO The maximal saturation of oxygen in %. 90…200%
limits limit.
the checkbox.
If a minimum DO is required to meet certain requirements adjustments can be
Min DO limit Yes or no
made here.
Min. DO Minimum value of DO in mg/L the sample should have. 0.1…7 mg/L
limits limit.
• Repeat: The measurement is repeated until terminated by the user or Selections
If a measurement result is outside the set limits, an instruction window pops up
the checkbox.

29
Analysis (BOD):
Analysis (follow)
The time tolerance limit states how much time can pass between the two
Time tolerance limit Yes or no
measurements
Tolerable positive and negative deviation from wanted incubation time (i.e.: 5
Time tolerance 0.1…12 ours
days ± 2 hours).
limits limit.
• Warn and continue: The value is saved as a result and a note Selections
(OK*) is added to the report that it is outside of the set range.
the checkbox
If a minimum DO is required to meet certain requirements, adjustments can be
Min. DO limit Yes or no
made here
Min. DO Minimum value of DO in the bottle the sample must not fall below. 0.1…15 mg/L
limits limit.
•Interrupt: The whole analysis is interrupted and stopped.
the checkbox

Analysis (BOD):
Analysis results
Min. BOD limit of bottle Settings for minimum BOD value that is required for the bottle at hand.
Min BOD The minimum amount of BOD required for a valid analysis result. 0.1…15 mg/L
Limit applied to If the set minimum BOD should apply to the corrected or
uncorrected BOD value.
• Corrected BOD: After deduction of blank, seeded blank and salinity
Selections
correction.
• Uncorrected BOD: Before deduction of blank, seeded blank and
salinity correction.
Action when outside limits Choose what action is taken, when your measurement is outside of the set
limit.
• Save and report: The value is saved as a result and
a note (OK*) is added to the report that it is outside of the set range.
• Skip bottle: The results from the bottle are skipped and ignored.
The analysis continues with the next bottle.
If a measurement result is outside the set limits an instruction
Show instruction window pops up which has to be confirmed by tapping OK. This can be dis- Yes or no
abled by unselecting the checkbox
For certain standards, the BOD caused by the seed is not
Seed correction limits
permitted to be too high
Min. correction factor The minimum correction factor caused by the seed’s own BOD
Max correction factor The maximal correction factor caused by the seed’s own BOD
hoose what action is taken, when your measurement
C
is outside of the set limit.
• Save and report: The value is saved as a result and
a note (OK*) is added to the report that it is outside
of the set range.
Action when outside limits Selections
• Repeat: The measurement is repeated until terminated
by the user or the set limits are matched.
Show instruction window pops up which has to be confirmed by tapping OK. Yes or no
This can be disabled by unselecting the checkbox.
This function checks whether the final BOD result complies with the corpora-
BOD limits of sample
tions proprietary limits of water quality.
0.01 to
Max. BOD The maximum BOD for the sample.
1’000’000 mg/L
0.01…
Min. BOD The minimum BOD for the sample.
1’000’000 mg/L
Choose what action is taken, when your measurement
is outside of the set limit.
Action when outside limits Selections
Show instruction window pops up which has to be confirmed by tapping OK. Yes or no
This can be disabled by unselecting the checkbox.

31
M020 is a pre-installed method on SevenExcellence™ for BOD. It works with 3 bottles of 300 mL whereof 10 mL
are sample and 290 mL dilution water. In this method no seed is added. This generally applies to sample origi-
nating from sewage plants. To create an own method out of M020, its method ID can be changed in order to get
a modifiable copy.
5.5.2 BCV: BOD check values

Several national and international BOD standards require determining additional results to check that the whole
setup is working properly. The following values can be determined in the BCV-type methods:
Blank value: the BOD value is determined in dilution water without any sample or seed (biological organisms).
Normally a limit for maximum BOD value of e.g. 0.2 mg/L is given. A blank correction of the samples BOD is
generally not recommended.
Seeded blank value: the BOD value is determined in dilution water with seed, but without sample. The BOD con-
tribution of the seed added to the sample must not be higher than 1 mg/L. That’s why the BOD of the seed has
to be determined prior to the BOD analysis of the sample. The result of the seeded blank analysis is then required
for both, to determine the seed volume that has to be added to the sample and to tcorrect the BOD of the sample.
Standard: the BOD is measured in a sample with well-known BOD value, typically glucose and glutamic acid
(GGA). It is used to make sure that the seed is producing sufficient but not excessive oxidative potential and to
check for potential toxic compounds present in the blank or seed.
For a BCV determination, a user-defined combination can be created, based on the three mentioned values and
depending on individual requirements. The results of the BCV can be implemented later in any subsequent BOD
determination, by just linking the corresponding “check value ID” in the start analysis screen, when performing a
BOD follow measurement.
The standard parameters for creating a new BCV method using the T0006 template are the same as with the BOD
method (T0007). The difference is in being able to choose blank, seeded blank and standard. These three op-
tions are found in method function Configuration. All three can be chosen at the same time.

Figure 8 Configuration screen M021
Blank: Adds method functions «Blank (BOD)» and «Measure (Blank)» to the method and allows the insertion of
«Analysis (Blank)». The parameters of each method function are the same as in method functions of a BOD type
method.
Seeded Blank: Adds method functions «Seeded blank (BOD)» and «Measure (Seeded blank)» to the method and
allows the insertion of «Analysis (Seeded blank)». The parameters of each method function are the same as in
method functions of a BOD type method.
Standard: Adds method functions «Standard (BOD)» and «Measure (Standard)» to the method and allows the
insertion of «Analysis (Standard)». The parameters of each method function are the same as in method functions
of a BOD type method.
M021 is a pre-installed method on SevenExcellence™ for BCV. It works with each three bottles of 300 mL to de-
termine blank, seeded blank and standard. The blank works with 300 mL dilution water. The seeded blank works
with 10 mL seed and 290 mL dilution water. The standard works with 6 mL sample, 2 mL seed and 292 mL dilu-
tion water.
To create an individual method out of M021, the method ID can be changed in order to get a modifiable copy.

33
5.6 Method start
5.6.1 Concept
For a simple BOD analysis without check values, the procedure is as follows:
Determine base value with BOD method

Incubate for n days
Determine follow value with BOD method
If results for blank BOD, seeded blank BOD and/or standard BOD have to be considered, procedure is as follows:
1. Determine base value with BCV method
2. Determine base value with BOD method
3. Incubate for n days
4. Determine follow value with BCV method
5. Determine follow value with BOD method
5.6.2 Start BCV method

The start screen for the method shows the type of the analysis, method ID and its type. For the measurement
of the initial DO concentration, choose «Base» as method step. Enter a check value ID and if necessary write a
comment. By clicking «Start» the analysis begins. Follow the instructions on the screen. Repeat for additional
check value IDs. After the measurement, place the bottles in the incubator for 5 (or more) days.
Figure 12 Start analysis screen M021 base measurement

To measure DO concentration after incubation time (follow measurement), start the same method again. When
choosing the method step «Follow», the check value ID field changes from text to a list icon. By tapping on it, one
can choose the appropriate check value ID from the list. Again, if necessary, write a comment, then click «Start».
Follow the instructions on the screen.
Figure 13 Start analysis screen M021 follow measurement

35
5.6.3 Start BOD method
The start screen for the method shows the type of the analysis, method ID and type. For the measurement of the
initial DO concentration, choose «Base» as method step. Enter a sample ID and, if necessary, write a comment.
By clicking «Start», the analysis begins. Follow the instructions on the screen. Repeat for additional sample IDs.
After the measurement, place the bottles in the incubator for 5 (or more) days.
To measure DO concentration after incubation time (follow measurement), start the same method again. When
choosing the method step «Follow», the sample ID field changes from text to a list icon. By tapping on it one can
choose the appropriate sample ID from the list. If desired, the results of a BCV method can now be linked to the
current BOD analysis. This is done in the next field by selecting an appropriate “Associated check value ID” from a
list showing all positively validated checkvalue determinations available on the meter. The results of the selected
check values will then be taken into account for final BOD calculations of the sample. Again if necessary write a
comment then click «Start». Follow the instructions on the screen.
Figure 14 Start analysis screen M020 base measurement

Figure 15 Start analysis screen M020 follow measurement

37
6. METTLER TOLEDO M020 and M021
Applications BOD & BCV
6.1 Solutions Alternatively Seeded blank stock solution

pH 6.2 Buffer • Allylthiourea (C4H8N2S) (ATU): Dilute the content of one PolySeed®
• Dissolve 12.2 g Na2HPO4 in 200 mg diluted to 200 mL capsule in 500 mL of dilution wa-
about 200 mL of distilled water. with deionized water. ter and discard the capsule. Aerate
Adjust pH to 7.2 with ~15 mL • Dechlorination the solution for 1 h and decant the
30% KOH and dilute to 250 mL. • Sodium sulfite solution: supernatant. For best results the
• Dissolve 1.70 g NH4Cl in about Dissolve 0.394 g Na2SO3 in PolySeed® solution should be used
500 mL distilled water, adjust 250 mL distilled water. This solu- within 6 h of preparation. Check the
pH to 7.2 with KOH solution, and tion is not stable, prepare daily. salinity of the solution and enter it
dilute to 1 L. Solution contains in the method function «configura-
0.3 mg N/mL. Neutralization agents: tion» of the BOD method.
• Acid: Add 7 mL conc. sul-
Alternatively furic acid to distilled wa- Standard stock solution
• Dissolve 8.5 g KH2PO4, 21.75 g ter. Dilute to 250 mL. • Glucose-glutamic acid-standard
K2HPO4, 33.4 g Na2HPO4×7H2O, • Alkali: Dissolve 10 g sodium solution (GGA), 2*100 mg/L
and 1.7 g NH4Cl in about hydroxide in distilled water. (100 mg glucose, 100 mg glu-
500 mL distilled water and Dilute to 250 mL. tamic) or dilute 6.667 mL of a
dilute to 1 L. The pH should be Voluette™ ampoule, 2*300 mg/L,
7.2 without further adjustment. Dilution water (300 mg glucose, 300 mg glu-
Use demineralized or distilled water. tamic acid, with 13.333 mL of
Nutrients • Add 1 mL of the following solu- dilution water). Check salinity of
• Magnesium sulfate solution: tions: pH 7.2 Buffer, MgSO4, the solution and enter it in the
Dissolve 5.63 g MgSO4×7H2O in CaCl2, and FeCl3. Fill up to ap- method function configuration of
distilled water and prox. 950 mL using demineral- the BOD method (this only has
dilute to 250 mL. ized or distilled water. Check to be done once for the whole
• Calcium chloride solution: and adjust the pH using either package).
Dissolve 6.88 g CaCl2 in distilled the acid or the alkali solution,
water and dilute to 250 mL. so that the pH lies between 6.5 Alternatively
• Ferric chloride solution: Dissolve and 7.5. Fill up to 1000 mL with • Dry reagent-grade glucose
0.0625 g FeCl3×6H2O in distilled demineralized or distilled water. and reagent-grade glutamic
water and dilute to 250 mL. Check the salinity (unit ppt) of acid at 103 °C for 1 h. Add
the solution and enter it in the 150 mg glucose and 150 mg
Nitrification inhibitor method function «configura- glutamic acid to distilled
• 2-chloro-6-(trichloromethyl) tion» of the BOD method. water and dilute to 1 L.
pyridine (TCMP) 200 mg diluted
to 200 mL with deionized water.

6.2 Blanks and standard 6.2.3 Glucose-glutamic acid 6.3 Sample preparation
preparation check (GGA) 1) Check the pH of all samples
6.2.1 Blank As the BOD test is a bioassay, re- before testing, unless previous
Together with each batch of sam- sults can be influenced greatly by experiences have proven that the
ples, incubate three bottles of un- the presence of toxicants or by the pH is within the acceptable range.
seeded dilution water. The BOD of use of a weak seeding material. Samples exhibiting an alkalinity
the dilution water obtained this way Distilled waters are frequently con- > 8.5 pH or an acidity <6.0 pH are
provides you with the blank value. taminated with copper, and some to be neutralized to a pH between
The DO uptake of the blank should sewage seeds might exhibit poor 6.5 and 7.5 using a solution of HCl
not be more than 0.2 mg/L and biological activity, both leading to or sulfuric acid. Or use sodium hy-
preferably not more than 0.1 mg/L. low BOD results. droxide (NaOH) of such strength,
that the quantity of reagent does
Discard all dilution water having a The quality of the dilution water, the not dilute the sample by more than
DO uptake greater than 0.2 mg/L seed and your analytical procedure 0.5%. Furthermore, the pH of the
and prepare a fresh batch. Repeat is checked by determining the BOD dilution water should not be af-
all BOD determinations using the of a «standard» check solution such fected by the lowest sample dilu-
fresh dilution water. To avoid vain as GGA, on a regular basis. tion. Don’t seed the samples until
BOD analysis by contaminated dilu- the pH is adjusted properly.
tion water or vessels, perform blank Glucose has an exceptionally high
measurements separately, prior to and variable oxidation rate but 2) In some samples chlorine will
each BOD analysis. when it is used with glutamic acid, dissipate within 1 to 2 hours of
the oxidation rate is diminished, standing in the light. This often oc-
6.2.2 Seeded blank so that it is similar to that obtained curs during sample transport and
This is the seed control to determine with municipal waste water. The handling. For samples in which
the seed DO uptake in dilution wa- best option is, to determine the chlorine residuals do not dissipate
ter. Ideally, make dilutions of seed 5 day 20 °C BOD of a 2% dilu- in a reasonably short time, remove
so that the largest quantity results tion of the glucose-glutamic acid the chlorine by adding Na2SO3 solu-
in at least 50% DO depletion. standard check solution simultane- tion (see chapter 7). All chlorinated
ously and in exactly the same way samples have to be decholorinated
A plot of DO depletion, in milligrams as you determine the BOD of your prior to seed addition.
per liter, versus milliliters of seed for sample.
all bottles having more than 2 mg/L 3) Industrial wastewaters often
oxygen depletion and a minimum Adjust concentrations of commer- contain other toxic substances like
residual DO of 1.0 mg/L, should cial mixtures to give 3 mg/L glu- toxic metals. Such samples often
present a straight line for which the cose and 3 mg/L glutamic acid in require special treatment. If pos-
slope indicates DO depletion per each GGA test bottle. sible dilute strong enough.
milliliter of seed.

39
Bottle (300 mL) Seed volume [mL] Dilution water volume [mL] (trichloro methyl) pyridine (TCMP)
1 0.00 300.0 solution or 1 mL of 1 mg/L allylthio-
2 0.00 300.0 urea (ATU) solution to each 300 mL
3 0.00 300.0 bottle before capping, or add suf-
Table 5 Dilution blank ficient amounts of TCMP or ATU to
the dilution water to make a final
Bottle (300 mL) Seed volume [mL] Dilution water volume [mL] concentration of 10 mg/L.
1 10.0 290.0
2 10.0 290.0 Samples that may require nitrifi-
3 10.0 290.0 cation inhibition are biologically
Table 6 Seeded blank treated effluents, samples seeded
with biologically treated effluents
Dilution water volume and river waters. Note the use of
Bottle (300 mL) Standard volume [mL] Seed volume [mL]
[mL] nitrogen inhibition in the reporting
1 6 2 292.0 results.
2 6 2 292.0
3 6 2 292.0 7) If algae are present, consider a
Table 7 Oxygen standard previous filtration of the samples to
avoid unusually low BOD results.
Dilution water volume A filter pore size of 1.6 μm is ap-
Bottle (300 mL) Sample volume [mL] Seed volume [mL]
[mL] propriate. Filtering can change BOD
1 10.0 0.0 300 results radically. If filtration is car-
2 10.0 0.0 300 ried out, the filter pore size shall be
3 10.0 0.0 300 recorded in the test report.
Table 8 Municipal sewage sample
8) If samples contain high amounts
4) Fresh samples of cold water ing them for at least 1 hour while of BOD, dilute according to table 1.
or samples, where photosynthe- agitating by vigorous shaking or by
sis occurs, may be oversaturated stirring and aerating with clean, fil- 6.4 Determination of initial DO
with oxygen at the measuring tem- tered compressed air. When doing BOD determinations
perature of 20 °C, thus leading to of the samples (Method M020,
undetected loss of oxygen during 5) Bring samples to 20 ± 1 °C be- «BOD») and BOD of the check
incubation. To prevent this, reduce fore making dilutions. values (Method M021, «BCV») si-
the DO to saturation by heating the multaneously, always perform the
samples to a temperature of 20 °C 6) If nitrification inhibition is de- BCV measurement prior to the BOD
in partially filled bottles and keep- sired, add 1 mL of 2-chloro-6- measurement.

6.4.1 Blank will automatically take the different step» base to follow in the «Start
For the M021 method (BCV), fill concentrations into account. analysis»-screen, and that you se-
three bottles to the glass grinding lect the corresponding «check value
with dilution water. Measure the ini- 6.4.4 Sample ID» / «sample ID» from the list un-
tial (base) DO and seal the bottles. For the M020 method (BOD) fill derneath.
Incubate and stir for 5 days. three bottles partly with dilution wa-
ter. Add 10 mL of sample to each In method M020 (BOD) the «Start
6.4.2 Seeded blank bottle. Fill the bottles to the glass analysis»- screen offers the addi-
For the M021 method (BCV), fill three grinding with dilution water. Mea- tional option to assign the correct
bottles partly with dilution water. sure the initial (base) DO and seal «Associated check value ID», thus
Add 10 mL of seeded stock solu- the bottles. Incubate and stir for 5 allowing the meter to take the re-
tion. Fill the bottles to the glass days. sults from check value determina-
grinding with dilution water. Mea- tion, performed with M020 (BCV) in
sure the initial (base) DO and seal parallel, into account for final calcu-
the bottles. Incubate and stir for Remark: After sealing of the bottles lation and validation of the samples
5 days. for incubation, abslolutely no air BOD analysis.
must remain in the bottles.
6.4.3 Standard 6.7 Calculation
For M021 method (BCV) 6 mL of a 6.5 Incubation If the M020, M021 or any methods
mixture of 150 mg glucose/L and Incubate samples, seed controls, basing on them is used for the BOD
150 mg glutamic acid/L is added to dilution water blanks, and glucose- measurement, no further calcula-
each of the three bottles to give a glutamic acid checks at 20 °C ± tions are necessary.
final GGA concentration of 3 mg/L. 1 °C and in total darkness for 5 SevenExcellence™ will deliver any
After the addition of 2 mL of seed days. Stir during the entire incuba- results according to the data (vol-
the initial DO (base) is measured tion period. umes, dilution factors, salinity) en-
and the bottles are sealed and in- tered in the method.
cubated in the same way as the 6.6 Determination of final DO
samples. After 5 days of incubation, deter-
mine the final DO (follow measure-
The BCV method in SevenExcellence ment) firstly in the blank, seeded
can easily be adapted by the user blank and standard bottles using
so as to employ different concentra- the M021 method and secondly in
tions of GGA solution. This is done the samples using method M020
by modifying the volume of stan- (BOD).
dard in the bottle submenu of the
method function «Standard (BOD)». At the start of either method, make
In the statistical report, the meter sure that you change from «Method

41
Application M020
Biochemical Oxygen Demand (BOD) Sample
The BOD5 is determined according to APHA (USEPA) 5210B method. The method can easily be adapted to
individual requirements regarding BOD content, incubation time, bottle volume, seed addition, and numbers
of multiple determinations.
Preliminary procedures
Sample Municipal sewage
(sample preparation, conditioning, calibration, etc.)
Sample size 10 mL Dilution water preparation:

- Add 1 mL each of phosphate/NH4Cl buffer, MgSO4, FeCl3, CaCl2
and optionally Na2SO3 solution to 1 L of distilled water. Always
Sensor InLab® OptiOx (51344621)
prepare freshly before use.
Sample preparation:
SevenExcellence™
Instruments - Homogenize sample before use.
with DO/BOD module
- Check the pH of the sample and adjust it so that it lies between
6.4 and 7.4
- uPlace™ electrode arm - Add a stirrer bar to each bottle.
- 3 BOD bottles (300 mL)
- Rainin pipette 1 mL,10 mL - Fill the bottles as described in chapter 6.4.4.
- Volumetric flask - Fill the bottles up with dilution water.
Accessories - Homogenizer - Take measures to prevent O2 oversaturation of samples prior to
- Laboratory balance «base» measurement by heating and agitating.
- Incubator with Multistirrer and
- Calibrate the DO sensor prior to each measuring series
3 stirrer bars
(base and follow).
- Wash bottle & waste beaker
Operation procedure
0.3 NaH2PO4 / 0.03 NH4Cl
Buffers / Standards buffer pH 7.2 (pH adjusted
- Insert the InLab® OptiOx into the bottle by holding the bottle at a
with KOH 30%)
45° angle and immerse it so that the blue ring reaches the neck of
the bottle (~7 cm).
Nutrients:
- 0.09 M MgSO4 - For BOD5 Incubate bottles for 5 days ± 3 h at 20 ± 1 °C and in
- 0.9 mM FeCl3 complete darkness, stirring constantly.
- 0.19 M CaCl2 - Take the bottles out of the incubator and prepare the sensor as
pH adjustment: described above
Reagents - 1N H2SO4
- Place the sensor in the bottle
- 1N NaOH
- 30% KOH - Start stirring and start the method by selecting method step
Dechlorination Follow
(only if required): - Follow the instructions on the meter
- 0.4 M Na2SO3
Remarks
Waste disposal
A gently stirring is normally needed for a stable measurement. For
Hans Früh, Yves Moser owners of a uMix magnetic sitrrer the method can be adapted ac-
Author, Version MSG pH Lab cordingly. To adapt the method to other parameters, save it under a
Version 1.0 new name and change the volumes accordingly under the method
function «Sample».

Comments
• Make sure that no air bubbles are present, either on the sensor’s membrane or in the vortex
• If oversaturated with O2 stir, the sample for a few minutes
• Store all solutions in a temperature controlled environement at 20 °C for at least 60 minutes prior to use
• For statistical reasons use at least three bottles per sample or check value
• Literature: EN1899-1 Part 1; ISO 5815:1989 (modified); USEPA Standard Methods for the Examination
of Water and Wastewater; 5210 5-day Biochemical Oxygen Demand (BOD, Section 5210B); G.C. Delzer and
S.W. McKenzie, ‘Five day biochemical oxygen demand’, U.S. Geological Survey TWRI Book 9, chapter A7.2;
U.S. Environmental Protection Agency Biochemical oxygen demand, 40 CFR Part 136, July 1, 1996.
Results
Sample DO base [mg/L] DO follow [mg/L] O2 depletion [mg/L] Concentration [mg/L] Mean [mg/L] Rel. std. dev. [%]
municipal 174.4 4.4
sewage
Bottle 1 8.00 2.11 5.89 176.7
Bottle 2 7.80 1.78 6.02 180.6
Bottle 3 8.00 2.47 5.53 165.9
Method
Title DO resolution 2
Method type BOD BOD resolution 2
Method ID M020 Endpoint type Automatic
Title BOD BOD sample Endpoint criteria Standard
Author METTLER TOLEDO Stir no
Created on 2013-07-24 16:53:55 Analysis (BOD)
Modified on 2013-07-24 16:57:00 Analysis (base)
Modified by METTLER TOLEDO Temperature limits no
Protect yes Max. DO limit yes
SOP no Max. DO 100%
Configuration Action when outside limits Repeat
Measurement type Dissolved Oxygen Min. DO limit no
Sensor name Show instruction no
Salinity correction no Analysis (follow)
Seed added To bottle Time tolerance limit yes
Blank correction no Time tolerance 3 Hours
Bottle volume 300 mL Action when outside limits Save and report
Temperature capture Internal Show instruction yes
Barometric pressure capture Automatic Min. DO limit yes
Sample (BOD) Min. DO 1 mg/L
Sample ID Sample01 Action when outside limits Save and report
Comment - Show instruction yes
Sample type Sample Analysis results
Same bottles used for base Min. BOD limit of bottle no

yes
and follow Seed correction limits no
Number of bottles 3 BOD limits of sample no
Measure (BOD) Report
Sensor name Print no
DO unit mg/L Signature:
BOD unit mg/L

43
Application M021
BOD Check Values (BCV)
The check values for BOD5 are determined according to APHA (USEPA) 5210B method. This method allows
for the determination of blank, seeded blank and standard solutions. The results can then be implemented
in a corresponding BOD5 determination.
Dilution water, Seed and

Sample Preliminary procedures
oxygen standard
(sample preparation, conditioning, calibration, etc.)
Blank 300 mL
Sample size Seeded Blank 10 mL -D
ilution water preparation:
Standard 6 mL - Add 1 mL each of phosphate/NH4Cl buffer, MgSO4, FeCl3, CaCl2
and optionally Na2SO3 solution to 1 L of distilled water. Always
Sensor InLab® OptiOx (51344621) prepare freshly before use.
SevenExcellence™ with Sample preparation:
Instruments pH/mV or conductivity or - Homogenize seed before use.
DO/BOD module - Check the pH of the seed and adjust it so that it lies between 6.4
- uPlace™ electrode arm and 7.4.
- 9 BOD bottles (250 mL) - Add a stirrer bar to each bottle.
- Rainin pipette 1 mL,10 mL - Fill the bottles as described in chapter 6.4.
Accessories - Incubator with Multistirrer
- Take measures to prevent O2 oversaturation prior to «base»
and 9 stirrer bars
measurement.
- Wash bottle
- Waste beaker - Calibrate the DO sensor prior to each measuring series.
BOD standard solution for

Operation procedure
dilution method (300 mg/L
glucose, 300 mg/L glutamic
Buffers / Standards
acid) 0.3 NaH2PO4 / 0.03 - Always measure the BCV prior to the BOD.
NH4Cl buffer pH 7.2 (pH - Immerse the InLab® OptiOx in the bottle so that the blue ring
adjusted with KOH 30%) reaches the neck of the bottle (7 cm).
- Polyseed bacteria Nutrients - Make sure that no air bubbles are present, either on the sensor's
- 0.09 M MgSO4 membrane or in the vortex underneath.
- 0.9 mM FeCl3 - All measurements are performed at 20 °C ± 1.0 °C using the
- 0.19 M CaCl2 same conditions regarding the stirring speed (4) and stirrer type.
pH adjustment:
- Start the method.
Reagents - 1N H2SO4
- 1N NaOH - For BOD5 Incubate bottles for 5 days ± 3 h at 20 ± 1 °C and in
Dechlorination (only if complete darkness stirring constanty.
required):
- 0.4 M Na2SO3 Remarks
Nitrification inhibitor
Waste disposal A gently stirring is normally needed for a stable measurement. For
owners of a uMix magnetic sitrrer the method can be adapted ac-
Hans Früh, Yves Moser
cordingly. To adapt the method to other parameters, save it under a
Author, Version MSG pH Lab
new name and change the volumes accordingly under the method
Version 1.0
function «Sample».

Results
Concentration Rel. std. dev.
Sample DO base [mg/L] DO follow [mg/L] O2 depletion [mg/L] Mean [mg/L]
[mg/L] [%]
BCV
Blank 1 8.00 7.9 0.10 0.10 0.10 0.041
Blank 2 7.80 7.75 0.05 0.05
Blank 3 8.00 7.85 0.15 0.15
Seeded Blank 1 7.90 7.35 0.55 16.5 16.5 0.49
Seeded Blank 2 7.86 7.29 0.57 17.1
Seeded Blank 3 7.92 7.39 0.53 15.9
Standard 1 7.56 2.86 4.70 235 236 1.70
Standard 2 7.60 2.92 4.68 234
Standard 3 7.55 2.79 4.76 238
Comments
• Make sure that no air bubbles are present, either on the sensor's membrane or in the vortex
• If oversaturated with O2 stir, the sample for a few minutes
• Store all solutions in a temperature controlled environement at 20 °C for at least 60 minutes prior to use
• For statistical reasons use at least three bottles per sample or check value
Method
Title Measure (Blank)
Method type BOD Check Values Sensor name
Method ID M021 DO unit mg/L
Title BOD check values (BCV) BOD unit mg/L
Author METTLER TOLEDO DO resolution 2
Created on 2013-07-24 17:22:32 BOD resolution 2
Modified on 2013-07-24 17:25:37 Endpoint type Automatic
Modified by METTLER TOLEDO Endpoint criteria Standard
Protect yes Stir no
SOP no Analysis (Blank)
Configuration Analysis (base)
Measurement type Dissolved Oxygen Temperature limits no
Sensor name Max. DO limit yes
Check value ID CV001 Max. DO 100%
Blank yes Action when outside limits Save and report
Seeded blank yes Min. DO limit no
Standard yes Show instruction no
Salinity correction no Analysis (follow)
Seed added To bottle Time tolerance limit yes
Blank correction no Time tolerance 3 Hours
Bottle volume 300 mL Action when outside limits Save and report
Temperature capture Internal Show instruction yes
Barometric pressure capture Automatic Analysis results
Blank (BOD) Max. BOD limit of bottle

yes
Blank ID CV001
Max. BOD 0.2 mg/L
Comment -
Action when outside limits Save and report
Sample type Blank
Show instruction yes
Same bottles used for base
yes
and follow
Number of bottles 3

45
Seeded blank (BOD) Analysis (Standard)
Seeded blank ID CV001 Analysis (base)
Comment - Temperature limits no
Sample type Seeded blank Max. DO limit yes
Same bottles used for Max. DO 100%

yes
base and follow Action when outside limits Save and report
Number of bottles 3 Show instruction yes
Measure (Seeded blank) Min. DO limit no
Sensor name Show instruction no
DO unit mg/L Analysis (follow)
BOD unit mg/L Time tolerance limit yes
DO resolution 2 Time tolerance 3 Hours
BOD resolution 2 Action when outside limits Save and report
Endpoint type Automatic Show instruction yes
Endpoint criteria Standard Min. DO limit yes
Stir no Min. DO 1 mg/L
Analysis (Seeded blank) Action when outside limits Save and report
Analysis (base) Show instruction yes
Temperature limits no Analysis results
Max. DO limit yes Min. BOD limit of bottle no
Max. DO 100% Seed correction limits no
Action when outside limits Save and report BOD limits of standard yes
Show instruction yes Max. BOD 237 mg/L
Min. DO limit no Min. BOD 163 mg/L
Analysis (follow) Action when outside limits Save and report
Time tolerance limit yes Show instruction yes
Time tolerance 3 Hours Report
Action when outside limits Save and report Print no
Show instruction yes Signature:
Min. DO limit yes
Min. DO 1 mg/L
Action when outside limits Save and report
Analysis results
Min. BOD limit of bottle no
O2 depletion limits no
Show instruction no
Standard (BOD)
Standard ID CV001
Comment -
Sample type Standard
Same bottles used for base
yes
and follow
Number of bottles 3
Measure (Standard)
Sensor name
DO unit mg/L
BOD unit mg/L
DO resolution 2
BOD resolution 2
Endpoint type Automatic
Endpoint criteria Standard
Stir no

47
7. Hints and Tips
Hints and Tips
If samples are not tested within 2 hours after sample collection, store them below 4 °C (39 °F) to inhibit bacterial
growth.
Bring the temperature of all samples to ± 20 °C (68 °F) before you start the any DO measurement.
Tables exist for BOD-testing at different temperatures. Find the right one and adapt your test parameters to your
needs.
The effect of toxic metal ions can be compensated by adding EDTA or by diluting the sample accordingly.
High chlorine values can be counter-measured by adding sodium thiosulfate. To determine the right amount of
sodium thiosulfate to be added to the sample, proceed as follows:
1. Fill 100 mL of the undiluted sample into a 250 mL Erlenmeyer flask. Add 10 mL of sulfuric acid (0.02 N) and
10 mL of potassium iodide solution (100 g/L).
2. Add 1–3 drops of starch indicator solution. This will turn the sample deep blue.
3. Titrate to colorless with sodium thiosulfate solution (0.025 N). Note the used volume.
4. Calculate the amount you need:
mL 0.025 N sodium thiosulfate = mL used in titration x mL to dechlorinate

100
5. Add the calculated amount of sodium thiosulfate to your samples and mix them. Let the samples rest for
10–20 min.
Most toxic materials, such as phenols, formaldehyde and cyanide, have a negative effect on the growth of the
involved microorganisms. To avoid this, dilute your sample accordingly. As a way to check your chosen dilution,
always use triple determination.
Check the volume of your bottles. To comply with regulations, the bottle’s volume should not scatter more than
1% around the nominal volume (i.e.: 300 ± 3 mL).
If an unknown sample is tested use a COD test to find an approximation (see Chapter Different types of oxygen
demand values 2.3).
If an unknown sample is tested use different dilutions for your analysis and discard the ones that are outside of
the set parameters.

49
8. Bibliography
Bibliography
APhA. (1999). 5210 Biochemical EPA. (2007, January). Standard Mettler-Toledo. (n.d.). Seven-
™
oxygen demand A-C. USA: Ameri- Operating Procedure for Dissolved Excellence Version 2.0 Workbook.
can Public Health Association. Oxygen Micro Method, Winkler Sales Introduction.
Titration. USA: EPA. EPA. (n.d.).
Copyright International Organiza- Method 405.1. USA: EPA. MITTAL, S., & RATRA, R. (2000).
tion for Standardization. (2003). TOXIC EFFECT OF METAL IONS ON
ISO 5815-1. Copyright International Gossett-Johnson, S. (2012). Bio- BIOCHEMICAL OXYGEN DEMAND.
Organization for Standardization. chemical Oxygen Demand and In Wat. Res. 34 (pp. 147-152).
Carbonaceous Biochemical Oxygen Great Britain: Elsevier.
Delzer, G., & McKenzie, S. (2003). Demand.
FIVE-DAY BIOCHEMICAL OXYGEN USA: www.WaterWorldCE.com. OECD. (n.d.). DATA SHEETS FOR
DEMAND. In USGS TWRI Book 9 A7 SURFACE WATER QUALITY STAN-
(p. Chapter 7). USA: USGS TWRI. Harvey, D. (n.d.). Chapter 11: Elec- DARDS. OECD.
trochemical Methods. In D. Harvey,
DIN Deutsches Institut für Normung Analytical Chemistry 2.0. Penn, M. R., Pauer, J. J., & Mi-
e.V., Berlin. (1998). DIN EN 1899-1. helcic, J. R. (2012). Environmental
Berlin: DIN Deutsches Institut für In-Situ Inc. (2012). Rugged Dis- Exological Chemistry Voll. II. USA:
Normung e.V., Berlin. solved Oxygen (RDO) Sensors Use EOLSS.
the Latest Advancements in Optical
DIN Deutsches Institut für Normung Technology. USA: In-Situ Inc. Stütz, A. (2012). Vereinfachte BSB-
e.V., Berlin. (1998). DIN EN 1899- Analyse mit OptiOx™. UserCom 17,
2. Berlin: DIN Deutsches Institut für International Organization for Stan- pp. 26-27.
Normung e.V., Berlin. dardization. (2003). ISO 5815-2.
International Organization for Stan-
EPA. (2003). P40 CFR Ch. I ART dardization.
136 – GUIDELINES ESTABLISHING
TEST PROCEDURES FOR THE ANAL- Jouanneau, S., Recoules, L., Du-
YSIS OF POLLUTANTS. USA: Envi- rand, M., Boukabache, A., Picot, V.,
ronmental Protection Agency. Primault, Y., et al. (2014). Methods
for assessing biochemical oxygen
demand (BOD): A rebiew. Water
Research 49, pp. 62-82.

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Selected Applications

Biochemical Oxygen Demand

mg/10 2 1979-90 1991-99

Biochemical Oxygen Demand

4 Biochemical Oxygen Demand

Biochemical Oxygen Demand

APHA American Pharmacy Association

6 Biochemical Oxygen Demand

1.1 History 1.2 Usage of BOD

The BOD5 was selected by the

Biochemical Oxygen Demand

8 Biochemical Oxygen Demand

Biochemical Oxygen Demand

stopper making sure that no air Calculation:

The BOD5 of the example is 4.6 mg/L.

10 Biochemical Oxygen Demand

BIOCHEMICAL OXYGEN DEMAND

The standardized test is the BOD5.

measured after five days. Other fre-

CBOD: The C stands for carbo- 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Biochemical Oxygen Demand

BOD in ppm Sewage

12 Biochemical Oxygen Demand

3.1 DO determination 3.1.2 Direct measurement from passing through. Sheathed

The sample is treated with manga-

When acidified with sulfuric acid

Biochemical Oxygen Demand

Polarization voltage Quenching also reduces the fluo-

14 Biochemical Oxygen Demand

3.2 Manometric procedure

The water is kept in a sealed bottle

Biochemical Oxygen Demand

necessary. Both deliver the real preparation time. The handling of

Direct with polarographic

16 Biochemical Oxygen Demand

Biochemical Oxygen Demand

18 Biochemical Oxygen Demand

Biochemical Oxygen Demand

Conductivity module: For conduc-

20 Biochemical Oxygen Demand

5.4 Method types BOD and BCV

Calculation of BOD in consider-

In the following pages it will often

Biochemical Oxygen Demand

22 Biochemical Oxygen Demand

Biochemical Oxygen Demand

Sample (BOD): Bottles

24 Biochemical Oxygen Demand

Biochemical Oxygen Demand

Additional method functions that can be added:

Instruction Allows the user to add a pop up window containing instructions

26 Biochemical Oxygen Demand

Biochemical Oxygen Demand

28 Biochemical Oxygen Demand

Biochemical Oxygen Demand

30 Biochemical Oxygen Demand

Biochemical Oxygen Demand

5.5.2 BCV: BOD check values

32 Biochemical Oxygen Demand