Selected Applications - Mettler Toledo · 8 Biochemical Oxygen Demand Electrochemistry Application Brochure 2 Theory 2.1 What is the biochemical oxygen demand? The basic idea behind

Sele

cted

App

licat

ions

SevenExcellence

Seven2Go

SevenGo Duo pro

Electrochemistry Application

Brochure 2

Biochemical Oxygen DemandFrom Theory to Practice

3Biochemical Oxygen Demand Electrochemistry Application Brochure 2

Editorial

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.

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

Water and air, the two essential fluids on which all life depends, have become global garbage cans. – Jacques Yves Cousteau

mg/10 2 1979-90 1991-9914

12

10

8

6

4

2

0Africa North America EuropeLatin America

and the CaribbeanAsia and the

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)

4 Biochemical Oxygen Demand Electrochemistry Application Brochure 2

Cont

ent 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


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 ....................................................................................................................................488. Bibliography ......................................................................................................................................50


Intro

duct

ion List of Abbreviations

APHA American Pharmacy Association

ATU Allylthiourea

BCV BOD check values

BOD Biochemical oxygen demand

BODn BODafterndays(usually5/7/20or∞)

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


1.1 HistoryDuring industrial expansion in the 19th century, many major cities installed sewer systems to cope with the rising amount of domestic and industrial wastewater. These systems mainly transported the untreated wastewater to nearby riv-ers and lakes, which in turn led to rapid organic pollution of these. Eu-trophication was the consequence. Due to lack of oxygen, the bodies of water were no longer able to sustain high levels of aquatic life. The development of procedures to treat wastewater became a neces-sity. A parameter to determine the 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.

1.2 Usage of BODNowadays, the biochemical oxygen demand (BOD) is a widely used parameter to determine the qual-ity of freshwater or the efficiency of sewage treatment plants. Sewage plants in Europe have to determine the BOD5 before introducing treated water into a fresh water body. This has to be checked daily or weekly depending on local regula-tions. Fresh water bodies like lakes or rivers are checked at regular intervals. It is one of many nec-essary parameters that have to be determined in order to use the water as drinking water.

1. Introduction


Theo

ry

2.1 What is the biochemical oxygen demand?The basic idea behind the principle is simple. When organic matter (leaves, grass, manure, sewage and so on) reaches a body of water the aerobic bacteria and fungi living there start to degrade this organic matter by oxidation. In this process, called respiration, the organisms consume the oxygen that is present in their surroundings.

As water can only hold a limited amount of dissolved oxygen (DO), other aerobic species such as zoo-plankton, crustaceans and fish be-come extinct if the oxygen is not replenished in due time. There are two ways to replenish the DO: diffu-sion from the atmosphere and pho-tosynthesis.

This fact is used to determine the BOD in water: The amount of the dissolved oxygen is measured at two points in time in the same sample. Most commonly, these two points are day zero and day five.

In the time between, the sample is incubated in a sealed bottle, in complete darkness and at a con-stant temperature of 20 ± 2 °C,thus avoiding any replenishment of oxygen either by diffusion or by photosynthesis. To allow for proper biodegradation, the sample is nor-

mally seeded with microorganisms that consume oxygen during the decomposition of organic com-pounds. Nutrients are also added. In parallel to the sample, the BODs of check values such as the stan-dard, the seed (seeded blank) and the dilution water (blank) have to be determined. All check values are prepared in the same way as the sample and incubated under the same conditions.

The oxidation of inorganic materi-als such as sulfides and ferrous KG:ion as well as the oxidation of nitrogenous compounds are considered interferences. In most cases the BOD is determined as carbonaceous BOD (CBOD). This is achieved by adding an inhibitor that prevents the oxidation of nitrog-enous compounds. With this setup, the amount of consumed oxygen relates directly to the amount of or-ganic matter that was present in the water.

As an example: If nutrients are released into a body of water, the growth of aquatic plants is promoted. As the plants grow, they also die. The organic detritus of these plants stimulates the growth of aerobic microorgan-isms. As the microorganisms start to degrade the plant matter, they consume huge amounts of oxygen.

This process renders the body of water eutrophic. The BOD is one of the most important parameters for monitoring these processes and it helps the authorities to assess the right measures for stabilizing or im-proving the oxygen content of water bodies.

2.2 BOD SequenceA typical process for simple BOD determination consists of these five steps:Sample takingSample preparation First/initial measurementIncubationSecond/final measurement

Remark: Special attention has to be paid to thorough cleaning of all equipment used for BOD determination. Don’t use phosphate detergents, and rinse everything with tap water and deionized water after cleaning.

The steps are described in more de-tails in the following.

Taking the sample: The sample should be taken shortly before the measurement. If the ini-tial measurement of the sample cannotbeperformedwithin2 hoursafter collection, the sample has to be stored in a refrigerator at a tem-peraturebelow4 °C.

2. BOD Background


Even chilled samples are consid-eredtohaveexpiredafter24 hoursin storage.

Preparing the sample:• Dilution: If the expected BOD is higherthan6 mg/L(seetable1), dilute the sample with dilu-tion water so that the DO doesn’t fallbelow1 mg/Linthebottleafter the incubation, but also avoiding an O2 depletion of less than2 mg/L.Thedilutionwateris de-ionized and air-saturated water with added nutrients. If the sample is diluted, a blank sample consisting of dilution water only has to be simultane-ously analyzed simultaneously.

• Initial DO (base measure-ment): Oxygen oversatura-tion of the sample at the initial measurement has to be avoided(initialDO≤100%).

• Temperature adjustment: The sample temperature before analysisshouldbe20 ± 2 °C(DIN/EN)and20± 1 °C(APHA), respectively. If not, the sample should be cooled or heated. In the latter case an oxygen oversaturation can be avoided by constant stir-ring or regular shaking of the sample during warm-up.

• pH: The pH of the sample shouldbebetweenpH 6and8 (DIN/EN) and between 6.5

and 7.5 (APHA), respectively. If not, neutralize with sulfuric acid or sodium hydroxide.

• Bottles: Either Karlsruher-bottles or Wheaton-bottles can be used. Both have glass grinding for completely airtight sealing. The bottles are filled about half with sample or diluted sample. And a stirring help is added (e.g. a magnetic stir bar).

• Inoculation: The bottle is seeded with microorganisms. The amount of seed should not be excessive and cause lessthan1 mg/LBODinthe

final calculation. For seed sources check chapter 4.2.4.

• Fill the bottle to the glass grind-ing with sample or dilution water depending on the situation.

First measurement:After the preparation, the first DO measurement is done. Depend-ing on the method chosen (check chapter 3), the procedure varies. The temperature for the measure-mentshouldbe20± 1 °C.

After the measurement the bottles are sealed with a ground-in glass

Expected BODn

mg/L of oxygenDilution factor a Exemples of waters b

3 to 6 between 1, 1 and 2 R

4 to 12 2 R, E

10 to 30 5 R, E

20 to 60 10 E

40 to 120 20 S

100 to 300 50 S, C

200 to 600 100 S, C

400 to 1200 200 I, C

1000 to 3000 500 I

2000 to 6000 1000 I

a Volume of diluted sample/volume of the test portionb R: River water;

E: Biologicaly purified municipal sewage;

S: Clarified municipal sewage or lightly contaminetied industrial effluent;

C: Raw municipal sewage;

I: Heavily contaminated industrial effluent.

Table 1 Dilution factors for BODn (Copyright International Organization for Standardization, 2003)


stopper making sure that no air bubbles are sealed in. Note the DO that was measured. This measure-ment step is also known as initial measurement or base measure-ment.

Incubation:The sample is incubated for n days. n is the number of days that are needed for the chosen BOD method. BOD5 takes five days, BOD7 takes seven days and BODn takes n days. For the entire incuba-tion period, the incubator has to be absolutely dark inside and the ther-mostat mustbe set to 20 ± 1 °C.The sample should be stirred at all time.

Second measurement:Afterndays(± 2 h(DIN/EN);± 4 h(APHA)) the second DO measure-ment is done. Take the same pro-cedure as for the first measurement. Note the DO value. This measure-ment step is also known as the final or follow measurement.

Calculation:The formula for the calculation is as follows: Equation 1 BOD calculation undiluted

BODn= (ρ1 – ρ2)

Equation 2 BOD calculation diluted

BODn= (ρ1 – ρ2) – * (ρ3 – ρ4) * V1–Vsam

V1

V1

Vsam[ ]

ρ1 Dissolved oxygen concentration of the test solution at time zero, in mg/Lρ2 Dissolved oxygen concentration of the same test solution after n days, in mg/L.ρ3 Dissolved oxygen concentration of the blank solution at time zero, in mg/Lρ4 Dissolved oxygen concentration of the blank solution after n days, in mg/LVsam Volumeofsampleusedforthepreparationofthetestsolution,in mLVt Total volume of the test solution, in mL

Anexampleforanundilutedsample:Thebottlehasavolumeof250 mL.TheinitialDOofthesampleis7.5 mg/L.Afterfivedaysavalueof3.6 mg/Lis measured.

BOD5 = (7.5 mg/L–3.6 mg/L) = 3.9 mg/L

The BOD5oftheexampleis3.9 mg/L

An example for a diluted sample: The bottle has a volume of 250 mL. Itisfilledwith100 mLofsampleand150 mLofdilutionwater.TheinitialBODvaluesare7.5 mg/Lforthesampleand7.4 mg/Lfortheblank.Afterfivedaysthesamplecontains5.6 mg/Ldissolvedoxygenandtheblank7.3 mg/L.

BOD5= (7.5 mg/L–5.6 mg/L) – * (7.4 mg/L–7.3 mg/L) * = 4.6 mg/L [ 250 mL] 100 mL250 mL–100 mL

250 mL

The BOD5 of the example is 4.6 mg/L.

Theo

ry


2.3 Different types of oxygen demand valuesBODn: The index n defines the time interval in n days that was used for the incubation of the samples. The standardized test is the BOD5. Under laboratory conditions, the oxygen consumption in a sample is measured after five days. Other fre-quently used time intervals areBOD7 and BOD20.

CBOD: The C stands for carbo-naceous biochemical oxygen de-mand. The CBOD quantifies the amount of oxygen consumed dur-ing incubation in the presence of ni-trification inhibitors. It is a measure for all carbonaceous compounds present.

NBOD: The NBOD quantifies the amount of oxygen needed to oxi-dize the present nitrogenous com-pounds (mainly NH3) to nitrate and nitrite by biological degradation. The oxygen demand of nitrifica-tion bacteria is considered interfer-ence to BOD analysis. As shown in Figure 1, the nitrogenous bacteriadon’t begin to consume oxygen until after 7–10 days. This time pe-riod varies hugely depending on the microorganisms in the seed and the composition of the sample.

UBOD: UBOD stands for ultimate biochemical oxygen demand. The

total amount of oxygen that is re-quired for the microorganisms to degrade all organic matter in the sample is represented by this parameter.

Both CBOD and NBOD are parts of the UBOD. Using this parameter, it is possible to predict the impact of an effluent on a lake or a river.

The chemical oxygen demand (COD) is used to measure the amount of organic compounds that can be chemically oxidized in water. It is commonly used to determine the amount of organic pollutants found in fresh water or wastewater. The COD mg/L indicates the mass of oxygen consumed per liter of solution. The consumption is deter-mined by chemical oxidation.

Usually the BOD5isabout70–80%of the COD. This rough estima-tion can also help to determine the dilution factor without doing a BOD5 preliminary test.

The relationship between BOD and COD allows assumptions to be made about the composition of sewage. The COD is always higher than the BOD measured in the same sample.

BOD5=(50...100)%ofCOD:All present materials are easily biodegradable.

BOD5 < 50% of COD: Either thepresent materials are poorly biode-gradable, or they are toxic for the microorganisms that are used.

Figure 1 BOD course over 30 days (EPA)

Second stage: combinedcarbonaceous - plusnitrogenous - demand curve

First stage: carbonaceous - demandcurve

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30TIME, IN DAYS

A

B

BIO

CHEM

ICAL

OXY

GEN

DEM

AND


BOD5=(12...25)%ofCOD:This value is expected for treated sewage.

2.4 Water quality and usual BOD valuesThe amount of organic matter that resides in open freshwater sources fluctuates during seasons.

In spring and autumn, pollen and degrading leaves respectively add organic content to the water. Unpol-luted waters have a BOD range of up to 5 mg/L. Together with otheranalytical parameters such as fecal coliforms, nitrates, pH, tem-perature changes over the year, total dissolved solids (TDS), total

phosphate and turbidity, a clear picture of the water quality can be achieved. Figure 1 gives an over-view on how the BOD values is used to rate fresh water.

Sewage plants have to determine the BOD5 at regular intervals. Depending on the season, the weather, the industry and the ag-riculture in the catchment area of the plant, the standard values for BOD5 vary over the year. The load of organic matter in the sewage will be lighter depending on the size of a community and the dependency on heavy industry and intensive agriculture. See Table 3 for com-monly expected BOD values.

BOD in ppm Water quality

1–2 Very Good: Low levels of organic waste in the water

3–5 Fair: The water is only moderately clean

6–9Poor: The water is somewhat polluted. The water is expected to show bacteria and other microorganisms that are decomposing the present organic waste

>10Polluted: The water contains high amounts of organic waste. There is a chance that manure is being dumped in this water source

Table 2 Water quality values for BOD (Copyright International Organization for Standardiza-tion, 2003)

Table 3 Normal values for BOD in sewage plants

BOD in ppm Sewage

20 For municipal sewage after three stage treatment

600 For untreated sewage in Europe

200For untreated sewage in the USA (the process uses more diluted raw sewage)

Nor

ms

& P

roce

dure

s


3.1 DO determinationTitration or direct measurement is normally used to determine the amount of dissolved oxygen.

3.1.1 Titrimetric analysisThe Winkler titration is the most commonly used titration method for 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).

This acts as an oxidizing agent and releases free iodine (I2) from the potassium iodide (KI). The iodine is titrated with sodium thiosulfate (Na2S2O3).

This is called an iodometric titra-tion. The amount of sodium thiosul-fate (Na2S2O3) is stoichiometrically equivalent to the dissolved oxygen that was in the sample.

3.1.2 Direct measurement

Direct measurement with an electrochemical sensorElectrodes often used for BOD are«Clark Type» electrodes, named af-ter their inventor Dr. Leland Clark. These electrodes contain an anode

and a cathode which are connected by an electrolyte solution. Since the anode and the cathode are po-larized, it is also called a polaro-graphic oxygen sensor. The tip is covered by a polypropylene mem-brane (other materials exist).

This semi-permeable membrane allows gases to pass but prevents contaminants and reducible ions

from passing through. Sheathed by a glass envelope, the cathode builds the core of a Clark Type elec-trode cell. In order to provide higher signal stability and safeguard against drift, the anode has a larger surface. The sensor measures oxy-gen tension amperometrically.

The silver/silver chloride anode pro-vides electrons for the cathode re-action.

Given a constant polarizing voltage, the electrode produces a current which is directly proportional to the partial pressure of oxygen diffusing to the reactive surface of the elec-trode.

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.

Figure 2 Winkler titration


The silver at the anode becomes oxidized. Reduction of oxygen oc-curs at the surface cathode which is exposed at the tip of the electrode. Oxygen molecules diffuse through the semi-permeable membrane and react with the KCI electrolyte solution. The current produced is a result of the following reduction of oxygen at the cathode.

Ag anode: 4 Ag + 4 Cl- 4 AgCl + 4 e-

Pt cathode: O2 + 4 H+ + 4 e- 2 H2O

Direct measurement with optical sensorOptical DO sensors use fluores-cence quenching to measure dis-solved oxygen. Fluorescence is an optical property of certain mol-ecules. When a molecule absorbs light, its energy increases, and

the molecule is said to be excited. Since the excited molecule is un-stable, it quickly loses the absorbed energy typically as light emission (fluorescence) and/or heat.

Because part of the energy of the absorbed light is always converted in to thermal energy, the emitted light inevitably is of lower energy (longer wavelength) than the ab-sorbed light.

An excited molecule can also lose energy by colliding with another molecule, notably oxygen. As a collision with oxygen provides a path out of the excited state that does not emit light, oxygen re-duces (or quenches) the intensity of the fluorescence. The greater the concentration of oxygen, the greater is the reduction in fluores-cence intensity.

Quenching also reduces the fluo-rescence lifetime. If no oxygen is present, fluorescence will persist for a certain amount of time after the exciting light is shut off. In the presence of oxygen, the lifetime decreases because quenching pro-vides an additional path out of the excited state, thus enhancing the decay of the latter.

Unlike intensity measurements, life-time measurements do not depend on the intensity of the exciting light, and, thus, are less subject to drift. The fluorescence lifetime can be conveniently measured by modulat-ing the exciting light, which causes the emitted light to be modulated at the same frequency, but shifted in phase. The amount of phase shift is a measure of the fluorescence life-time and the oxygen concentration.

When the optical sensor initiates a reading, blue light is emitted by a blue LED. This excites the lumiphore molecules at the sensor’s tip. When excited, lumiphore molecules emit red light: This red light is detected by a photodiode. Oxygen molecules quench the excited lumiphore mol-ecules and prevent the emission of red light. This process is called «dy-namic luminescence quenching.»

The optical sensor measures a phase shift between the red light

Polarization voltage

Anode

Electrolyte

Insulator

Cathode

MembraneMeasuring solution

Figure 3 Polarographic sensor (Mettler-Toledo)

Nor

ms

& P

roce

dure

s


that is returned and the red refer-ence light. The concentration of dissolved oxygen and the red light that returns are inversely propor-tional. This means that a high DO concentration reduces the amount of red light returned to the sensor.

Optical electronics calculate DO concentration and report results to the instrument. DO determination by luminescence quenching has a linear response over a broad range of concentrations and offers a high degree of accuracy and stability.

3.2 Manometric procedureAs distinguished from other methods, this procedure measures only the oxygen consumption through the carbonaceous oxida-tion process. Ammonia oxidation is inhibited.

The water is kept in a sealed bottle that is fitted with a pressure sensor. A substance that absorbs carbon dioxide (typically lithium hydroxide, sodium hydroxide) is placed above the level of the sample. The sample isstoredat20 °Cinabsolutedarkfor the test period.

During this process oxygen is consumed and the released CO2 is absorbed by the aforememen-tioned carbon dioxide absorbing substance, thus making for a drop

in pressure. This can be measured by the pressure sensor and is trans-mitted to a display that shows the consumed quantity of oxygen.

3.3 Comparison of different measurement proceduresEvery method has its benefits and drawbacks. Winkler titration is a method that works without electric-

ity. The major drawbacks, are that it is a manual titration and depends on a double color change during titration.

Both direct measurement sensors are dependent on a meter and cost considerably more when first purchased. A basic grasp of how to use technical equipment is also

1. Blue LED emits blue light

2. Luminophore molecules excited

3. Excited lumiphore molecules emit red light

4. Is oxygen present?

Yes No

5. Oxygen molecules quench excited

lumiphore molecules

5. Excited lumiphore molecules continue to

emit red light

6. Photodiode detects red light. DO concentration and red light

emission are inversely proportional

7. Electronics caluculate DO concentration

mg/L DO reported

Figure 4 Operation mode of optical sensor (Mettler-Toledo)


preparation time. The handling of NaOH or LiOH can be dangerous if not done correctly.

This method can determine high ranges of BOD and is therefore often used in sewage plants and other wastewater treatment facili-ties. All the equipment is in use dur-ing the entire measurement period and cannot be used for other mea-surements.

necessary. Both deliver the real BOD value and are very quick and reliable measurement methods. These methods are very accurate in low DO range measurements and are generally a more scientific ap-proach to the BOD. All these meth-ods allow the measurement equip-ment to be used for other analysis during incubation time.

The manometric method is very easy to use und has a very fast

Table 4 Comparison of main BOD procedures

Winkler-titrationDirect with polarographic sensor

Direct with optical sensor Manometric

Chemicals (without BOD specific chemi-cals)

MnCl2 or MnSO4, KI, H2SO4, Na2S2O3

Electrolytes None LiOH or NaOH

Usage of sample volumeSeparate samples for base and follow mea-surement

Same sample for base and follow measurement



Amount of work needed? High Low Low Very low

Preparation time Long Long Very short Short

Measurement equipment used during incubation?

No No No Yes

Precision medium high high low

Range 0 to 6 mg/L 0 to 6 mg/L 0 to 50 mg/L 0to700 mg/L

Nor

ms

& P

roce

dure

s

Pressure sensor

Support forNaOH pellets

O2

Sample with bacteria

Magnetic stir bar

Figure 5 Manometric procedure (Jouanneau, et al., 2014)


4.2.5 Salt solutionAll nutrient solutions and other salt solutions must be stored in glass bottlesat0 °C to4 °C in thedarkand discarded when biological growth is visible or after 6 months.

4.2.6 Dilution waterThe dilution water is provided with salt solutions, aerated for at least 1 hour and then allowed to rest for another hour. It should contain 8 mg/Lofdissolvedoxygen.

4.2.7 Standard solutionGlucose-Glutamic Acid (GGA) so-lution is used as standard. GGA should be prepared and used on the same day.

4.2.8 pH adjustmentPrior to seed addition, the samples showing alkalinity (pH > 8.5) or acidity (pH < 6.0 harmful to bac-teria are to be neutralized to pH 6.5 to 7.5 with a solution of Hydrochlo-ric acid (HCl) or sodium hydroxide (NaOH) of such strength that the quantity of reagent does not dilute thesamplebymorethan0.5%.

The pH of dilution water should not be affected by the lowest sample dilution.

4.2.2 Bottle sizeMin.100–125 mL,optimal 250–300 mL

4.2.3 Nitrification inhibitionFor ISO 5815-1 with allylthiourea (ATU) (CBOD)For ISO 5815-2 no nitrification inhibitors are used (TBOD)

4.2.4 Seeding water• Urban wastewater with max CODof300 mg/L

• River or lake water contain-ing urban wastewater

• Settlement effluent from a wastewater treatment plant

• Water taken downstream from the discharge of the water to be analyzed or water contain-ing microorganisms adapted to the water to be analyzed and cultivated in the labora-tory (in the case of industrial effluents containing poorly bio-degradable substances)

• Commercially avail-able seeding material

The seed should not contribute morethan0.6to1 mg/Ltotheoxy-gen consumption in the bottle. If the consumption of oxygen by the seed is higher, the test should be re-peated with less seed. Verify the DO of the seed with the seeded blank in advance.

4.1 Variation between proceduresThe standards and norms for regu-lated BOD measurement are the following:ISO 5815EN DIN 189940 CFR 136-03APHA 5210EPA 405.1OECD Water quality guidelines

The differences between them are minor. Their key requirements are pointed out in the following chap-ters. The usage of optical sensors for DO determination is permitted by all mentioned standards and regulations, but no official approval has been published so far.

4.2 ISO-5815 (International)

4.2.1 Blank testsThe blank tests (seeded blank and blank) are carried out simultane-ously or even better, in advance of the determination of the sample, using the dilution water and the seeded dilution water including 2 mL of allylthiourea solution perliter.

For the procedure without nitrifica-tion inhibition the allylthiourea solu-tion is not used. The blank should haveamaximumDOof0.2 mg/L.0.1 mg/Lispreferred.

4. Standards and Norms Relating to BOD Determination


BOD

with

Sev

enEx

celle

nce

4.2.9 IncubatorIncubator,thermostatsetto20 °C ±2 °C(68 °F)

4.2.10 CleaningThe bottles have to be thoroughly cleaned before use. If the Winkler ti-tration procedure is used, rinsingthe bottle several times with tap water and then with deionized water is enough. For the other procedures a more thorough cleaning is neces-sary.

Cleaning agents containing phos-phates are to be avoided.

4.2.11 DO Measurement• Winkler titration procedure:

ISO 5813:1983• Electrochemical: ISO 5814:2012• Optical: ISO/FDIS

17289 in approval• Manometric DIN ISO 16072

4.3 EN/DIN 1899 (European Union)The DIN EN 1899 states that all parameters are taken from the ISO 5813-8515.Thisnormconsistsof two articles.

Part one describes the procedure for dilution and nitrification inhibi-tion, part two describes how it is done without these two alterations. There are slight modifications but none of the aforementioned points are concerned. It mainly differs in how the samples are taken.

4.4 Title 40 of the Code of Federal Regulations 40 CFR 136-03 (USA)In the federal regulations title 40, it is mentioned that the standard procedure for BOD is regulated in the EPA 405.1 and the standard procedure 5210 A (the same proce-dure used as in APHA 5210 A-C). Both procedures are identical. The 40 CFR 136-03 contains four ap-proved methods to determine DO:• Winkler titration procedure:

EPA 360.2• Electrochemical: EPA 360.1• Optical: EPA approved• Manometric APHA 5210 B

4.5 APHA 5210 A-C (USA)The APHA 5210 A-C is identical to the ISO-5815. The APHA 5210 A-C and EPA Method 405.1are recommended by title 40 of the Code of Federal Regulations 40 CFR 136-03.

4.6 EPA Method 405.1 (USA)The APHA procedure is identical to the EPA Method 405.1. Both are recommended by title 40 of the Code of Federal Regulations 40 CFR136-03.

4.7 OECD Water Quality GuidelinesThe OECD states that all parameters for the procedure were taken from the DIN EN 1899-1 and 2.


5.1 Meters for dissolved oxygen measurementThe following meters of the MT portfolio allow DO measurements:• Seven2Go™ (S4), SevenGo™

pro (SG6) and SevenGo ™ Duo pro (SG68) with polaro-graphic sensor InLab® 605

• Seven2Go™ (S9), SevenGo™ pro (SG9) and SevenGo™ Duo pro (SG98) with opti-cal sensor InLab® OptiOx

• SevenExcellence™ with DO/BOD module (e.g. S900) and polaro-graphic sensor InLab® 605 or optical sensor InLab® OptiOx

The method concept of SevenExcel-lence™ does not only allow simple DO measurements but also enables the final BOD to be calculated de-pending on a base and a «follow» measurement. The base is the ini-tial DO measurement of the sample. The «follow» is a follow up mea-surement that has been done taking the base reading into account.

Given these two correlating points, the SevenExcellence™ is able to calculate the BOD taking salinity, temperature, atmospheric pressure as well as the BOD values of the blank, the seeded blank and the standard into consideration. The SevenExcellence™ meter comes with two pre-installed method templates that can be used with

the DO/BOD module to create and adapt the BOD methods to userspe-cific parameters.

The InLab® OptiOx can be equipped with a BOD adapter. This adapter helps to measure DO without spill-ing too much of the content of the bottle, allowing the bottle to be used for an additional Winkler titra-tion. It may also enable the bottle to be resealed in order to continue the measurement for BOD7 or longer BOD intervals (only works in com-bination with a Karlsruher bottle).

The METTLER TOLDEO customer magazineUserCom 17 featuresanarticle(p. 26–27)abouttheproperuse of the InLab® OptiOx together with the BOD adapter and the SG9 instrument.

5.2 DO sensorThe upcoming implementation of optical sensors into the ISO and EPA standards gives way, allow-ing an up-to-date sensor system to take its place among the standard-ized and marketed DO sensors.

Operators will no longer be lim-ited to usage of polarographic and galvanic sensors. Both of them are invasive methods that consume the present oxygen to determine the DO concentration. Moreover, optical sensors are quicker to prepare, as

well as simpler and faster to cali-brate and more stable after calibra-tion.

5.3 Further MaterialsThe determination of BOD needs a variety of accessories. Some of these are essential, others help by simplifying the process.

List of accessories:

Seed capsule: Seed capsules or any other seeds have to be ac-quired in order to perform a BOD analysis. As seed water you can use:• Seed capsules pur-

chased from supplier• Urban wastewater with max.CODof300 mg/L

• River or lake water contain-ing urban wastewater

• Settlement effluent from a wastewater treatment plant

• Water taken downstream from the discharge of the water to be analyzed or water contain-ing microorganisms adapted to the water to be analyzed and cultivated in the labora-tory (in the case of industrial effluents containing poorly bio-degradable substances)

Bottles: The Karlsruher bottle and the Wheaton bottle are commonly used for BOD. The Karlsruher bottle

5. Mettler-Toledo Systems for BOD


works very well with the InLab®

sensors. Both BOD bottle have a ground in glass stopper with a long neck for easy gripping.

The designated filling capacity of a bottle is reached when it is filled with a sample and the ground-in glass stopper is plugged in and no air bubbles are visible. The Karls-ruher bottle comes with a small funnel that holds the overfilled liquid back, in case it has to be opened and resealed again.

Disposable plastic bottles can be used as well. They are thinner, lighter and are always clean and ready for use.

They are usually made of recyclable materials. All bottles have to be washed thoroughly before use.

Incubator: The incubation is best done in an incubator that is dark in-sideandcanberegulatedto20 °C.During incubation the samples have to be stirred, using a multi-stirrer.

OptiOx BOD adapter: The BOD adapter is ideal for use in combina-tion with the Karlsruher bottle. This way the measurement can be car-ried out with a minimum of spilling and the same bottle can be used to measure continuously.

Magnetic stirrer uMix: SevenEx-cellence™ supports the magnetic stirrer uMix. For the BOD analysis it is recommended to stir the sample during the measurement. Magnetic stirring bars are required in addition to the stirrer.

Compact printer: SevenExcellence™ supports the printers USB-P25 and RS-P25. This allows the re-port to be printed directly after the analysis without any possibility for tampering.

LabX direct pH: The LabX direct pH software allows SevenExcellence™ to send data via USB directly to a computer and to convert it automat-ically into an MS Excel, MS Word or a simple Text format.

pH/mV module: For pH measure-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

Figure 6 Karlsruher bottle, Wheaton bottle and plastic bottle

BOD

with

Sev

enEx

celle

nce


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 BCVSevenExcellence™ 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.


5.5 Method functions and parameters5.5.1 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

Method IDStandard name is A8xxx. The last three digits depend onhow many user created methods are saved on theSevenExcellence™. The name can consist of 12 letters.

max. 12 alphanumeric characters

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

ProtectedProtects the method against deletion or modification byother users than the author (logged in user) or administrator.

Yes or no

SOP Standard operation procedure. Yes or no

SOP text Displayed if check box SOP is active. Defines the SOP text. max. 50 alphanumeric characters

BOD

with

Sev

enEx

celle

nce


Configuration


Measurement type Is based on the chosen method type. Dissolved oxygen

Sensor name

A created profile for a sensor can be chosen.If no sensor is chosen, the method will automatically take the sensor that is currently registered in the module. Changing from Polarographic-Sensor to OptiOx opens a warning that the resolution of the measurement is adjusted. See user manual of the used sensor for more details.

List

Salinity correctionIf chosen a new submenu opens up. It allows the method to use the built in salinity correction function. All values of salinity are in ppt. To determine the salinity a separate conductivity sensor and module is necessary.

Yes or no

• 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

Seed AddedTo bottle or 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 factor to be entered. Ratio of seed solution to dilution water.

1.0–999.9

Blank correctionIf this box is checked, and before starting the follow measurement the correlating BCV is chosen, the method will automatically subtract the blank from the measured BOD value.

Yes or no

Bottle volumeThe volume of the bottles you use. Changing the bottle volume will open a warning, saying that all entered bottle settings are changed as well in pro-portion to the new volume.

10…1000 mL

Temperature capture How the temperature should be taken.Internal, or external 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.

• ManualUnder «Sample (BOD)» you will be able to enter a fixed barometric pressure.


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 follow

This is chosen if the same bottles are used for base and follow. There will be different submenus depending on the choice taken here.

Yes or no

Number of bottles(If «same bottles used for base and follow» is selected) Number of bottles used for the analysis. It allows for a maximum of 10 bottles to be analysed simultaneously.

1…10

Number of bottles (base)Number of bottles for the base measurement. It allows for a maximum of 10 bottles to be analysed simultaneously.

1…10

Number of bottles (follow)Number of bottles for the follow measurement. It allows for a maximum of 10 bottles to be analysed simultaneously.

1…10

Temperature(If «manual» was selected before in «Configuration») This allows the user to set a manually temperature.

0…60°C

Barometric pressure(If «manual» was selected before in «Configuration») This allows the user to set a manually pressure.

500.0… 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


Sample IDShows the correlating sample ID that was entered under «Sample (BOD)».

Bottle ID Name of that specific bottle. max. 12 letters

Comment Comment on that specific bottle. max. 30 letters

Sample volumeThe volume of sample added to the bottle previously max was writtenn with a dot at the end.

0.1...1000 mL

Seed volume

If «to dilution water» was selected it is calculated depending on the maximum volume and the volume taken by the sample. If «to bottle» was selected the volume of seed solution in mL that was added to the bottle can be entered here

0.1...1000 mL

Dilution volumeVolume of dilution water added to the bottle in mL. Calculated depending on the maximum volume, the volume taken by the sample and the added seed

Info

BOD

with

Sev

enEx

celle

nce


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

DO resolutionDefines how many digits are displayed for the DO measurement. By default set to 2.

1, 2, 3

BOD resolutionDefines how many digits are displayed for the BODmeasurement. By default set to 2.

1, 2, 3

Endpoint type Allows a choice of 3 different options for the endpoint.Automatic ormanual ortimed

• Automatic Measurement ends, when chosen criteria are met.

• Endpoint criteria

• (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 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].

• ManualMeasurement ends, when user taps on «Take manual endpoint» during 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%


Report Parameters for printing the report can be set here


PrintIf enabled, measurement data is printed to the device that is defined in the SevenExcellence settings

Yes or no

Print format Two options on what shall be printed Summary or User defined

• SummaryCovers all important data concerning date, time, user and all parameters according to the settings of the measurement type

User definedIf selected, options, which are freely selectable, appear

Values and Calculations Values and calculations of this particular method can be exported or printed.

Yes or No

• 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

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


Instruction The instruction can be edited here. max. 120 characters

Continue afterWhat user interaction is needed for the message to vanish and the analysis to continue

Confirmation or Time span

• Time span The instruction will vanish after a pre-set time

• Time Time that will pass in seconds 1...1'000'000

• ConfirmationThe instruction will vanish when confirmed by tapping the «OK» button on the screen

ConditionThe condition that causes the instruction. If not checked the instruction will appear in all measurements with this method

Yes or no

• FormulaThe formula for the condition. For more information about the Mettler-Toledo formula syntax check the SevenExcellence™ user guide

max. 120 aplhanumeric characters

Additional method functions that can be added:

BOD

with

Sev

enEx

celle

nce


Sensor checkThis parameter checks the properties of the sensor prior to the measurement. 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

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 status OK*, signifying that there was an anomaly during measurement.

Yes or no

Wait/StirIf additional waiting or stirring is necessary for the procedure this parameter can be added


Wait time Time to wait before the next step is initiated, in seconds 1...1'000'000 s

StirIf the sample has to be stirred in this wait time. If checked it allows the stirring speed to be adjusted.

Yes or no

• Stirring speed Thestirringspeedin%. 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

ConditionThe condition that causes the wait/stir step. If not checked, the wait/stir step will happen in all measurements with this method

Yes or no

• FormulaThe formula for the condition. For more information about the Mettler-Toledo formula syntax check the SevenExcellence™ user guide

Max. 120 alphanumeric characters


Calculation

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 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

• Lower limit Set a lower limit–1'000'000 to1'000'000

• Upper limit Set an upper limit–1'000'000… 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.

BOD

with

Sev

enEx

celle

nce


Analysis (BOD): Analysis (base)


Temperature limitsThe user can set a temperature limit with a minimum and maximum value. The standardprocedureforBODstatesthatitshouldbebetween19and21°C.

Yes or no

Max. temperatureSet a maximum temperature. Unit is chosen in the user settings of the SevenExcellence™.

Min. temperatureSet a minimum temperature. Unit is chosen in the user settings of the SevenExcellence™.

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. • 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.

Show instructionIf a measurement result is outside the set limits an instruction window pops up which has to be confirmed by tapping OK. This can be disabled by unselecting the checkbox.

Yes or no

Max DO limitIf the sample is oversaturated with oxygen due to dissolved chemicals, adjust-ments can be made here.

Yes or no

Max. DO Themaximalsaturationofoxygenin%. 90…200%







Yes or no

Min DO limitIf a minimum DO is required to meet certain requirements adjustments can be made here.

Yes or no

Min. DO Minimum value of DO in mg/L the sample should have. 0.1…7 mg/L






Selections

Show instructionIf a measurement result is outside the set limits, an instruction window pops up which has to be confirmed by tapping OK. This can be disabled by unselecting the checkbox.

Yes or no


Analysis (BOD): Analysis (follow)


Time tolerance limitThe time tolerance limit states how much time can pass between the two measurements

Yes or no

Time toleranceTolerable positive and negative deviation from wanted incubation time (i.e.: 5 days ± 2 hours).

0.1…12 ours


Choose what action is taken, when your measurement is outside of the set limit. • Warn and continue: The value is saved as a result and a note

(OK*) is added to the report that it is outside of the set range. • Interrupt: The whole analysis is interrupted and stopped.

Selections

Show instructionIf a measurement result is outside the set limits an instruction window pops up which has to be confirmed by tapping OK. This can be disabled by unselecting the checkbox

Yes or no

Min. DO limitIf a minimum DO is required to meet certain requirements, adjustments can be made here

Yes or no

Min. DO Minimum value of DO in the bottle the sample must not fall below. 0.1…15 mg/L





analysis continues with the next bottle. •Interrupt: The whole analysis is interrupted and stopped.

Show instructionIf a measurement result is outside the set limits an instruction window pops up which has to be confirmed by tapping OK. This can be disabled by unselecting the checkbox

Yes or no

BOD

with

Sev

enEx

celle

nce


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

correction. • Uncorrected BOD: Before deduction of blank, seeded blank and

salinity correction.

Selections

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. • 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 analysiscontinueswiththenextbottle. • Interrupt: The whole analysis is interrupted and stopped.

Show instructionIf a measurement result is outside the set limits an instruction window pops up which has to be confirmed by tapping OK. This can be dis-abled by unselecting the checkbox

Yes or no

Seed correction limitsFor certain standards, the BOD caused by the seed is not 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


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.

• 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.

Selections


Yes or no

BOD limits of sampleThis function checks whether the final BOD result complies with the corpora-tions proprietary limits of water quality.

Max. BOD The maximum BOD for the sample.0.01 to1’000’000 mg/L

Min. BOD The minimum BOD for the sample.0.01… 1’000’000 mg/L



added to the report that it is outside of the set range.

Selections


Yes or no


M020 is a pre-installed method on SevenExcellence™forBOD.Itworkswith3bottlesof300 mLwhereof10 mLare 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 valuesSeveral 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.

BOD

with

Sev

enEx

celle

nce


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™forBCV.Itworkswitheachthreebottlesof300 mLtode-termineblank,seededblankandstandard.Theblankworkswith300 mLdilutionwater.Theseededblankworkswith10mLseedand290 mLdilutionwater.Thestandardworkswith6 mLsample,2 mLseedand292 mLdilu-tion water.

To create an individual method out of M021, the method ID can be changed in order to get a modifiable copy.


5.6 Method start5.6.1 ConceptFor a simple BOD analysis without check values, the procedure is as follows:

Determine base value with BOD methodIncubate for n daysDetermine 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 method2. Determine base value with BOD method3. Incubate for n days4. Determine follow value with BCV method5. Determine follow value with BOD method

5.6.2 Start BCV methodThe 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

BOD

with

Sev

enEx

celle

nce


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


5.6.3 Start BOD methodThe 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

BOD

with

Sev

enEx

celle

nce


Figure 15 Start analysis screen M020 follow measurement


Appl

icat

ions

BO

D &

BCV 6. METTLER TOLEDO M020 and M021

6.1 Solutions pH 6.2 Buffer• Dissolve 12.2 g Na2HPO4 in about200 mLofdistilledwater.AdjustpHto7.2with~15 mL30%KOHanddiluteto250 mL.

• Dissolve 1.70 g NH4Cl in about 500 mLdistilledwater,adjustpH to 7.2 with KOH solution, and diluteto1 L.Solutioncontains0.3 mgN/mL.

Alternatively• Dissolve 8.5 g KH2PO4,21.75 g

K2HPO4, 33.4 g Na2HPO4×7H2O, and 1.7 g NH4Cl in about 500 mLdistilledwaterand diluteto1 L.ThepHshouldbe7.2 without further adjustment.

Nutrients• Magnesium sulfate solution:

Dissolve 5.63 g MgSO4×7H2O in distilled water and diluteto250 mL.

• Calcium chloride solution: Dissolve6.88 gCaCl2 in distilled wateranddiluteto250 mL.

• Ferric chloride solution: Dissolve 0.0625 g FeCl3×6H2O in distilled wateranddiluteto250 mL.

Nitrification inhibitor• 2-chloro-6-(trichloromethyl) pyridine(TCMP)200 mgdiluted to200 mLwithdeionizedwater.

Alternatively• Allylthiourea (C4H8N2S) (ATU): 200mgdilutedto200 mLwith deionized water.

• Dechlorination• Sodium sulfite solution:

Dissolve 0.394 g Na2SO3 in 250 mLdistilledwater.Thissolu-tion is not stable, prepare daily.

Neutralization agents:• Acid:Add7 mLconc.sul-

furic acid to distilled wa-ter.Diluteto250 mL.

• Alkali:Dissolve10 gsodium hydroxide in distilled water. Diluteto250 mL.

Dilution waterUse demineralized or distilled water.• Add1 mLofthefollowingsolu-

tions: pH 7.2 Buffer, MgSO4, CaCl2, and FeCl3. Fill up to ap-prox.950 mLusingdemineral-ized or distilled water. Check and adjust the pH using either the acid or the alkali solution, so that the pH lies between 6.5 and7.5.Fillupto1000 mLwithdemineralized or distilled water. Check the salinity (unit ppt) of the solution and enter it in the method function «configura-tion» of the BOD method.

Seeded blank stock solutionDilute the content of one PolySeed® capsule in500 mLofdilutionwa-ter and discard the capsule. Aerate the solution for 1 h and decant the supernatant. For best results the PolySeed® solution should be used within 6 h of preparation. Check the salinity of the solution and enter it in the method function «configura-tion» of the BOD method.

Standard stock solution• Glucose-glutamic acid-standard solution(GGA),2*100 mg/L(100 mgglucose,100mgglu-tamic)ordilute6.667 mLofaVoluette™ampoule,2*300 mg/L,(300 mgglucose,300 mgglu-tamicacid,with13.333 mLofdilution water). Check salinity of the solution and enter it in the method function configuration of the BOD method (this only has to be done once for the whole package).

Alternatively• Dry reagent-grade glucose

and reagent-grade glutamic acidat103 °Cfor1h.Add150 mgglucoseand150 mgglutamic acid to distilled wateranddiluteto1 L.


6.2 Blanks and standard preparation6.2.1 BlankTogether with each batch of sam-ples, incubate three bottles of un-seeded dilution water. The BOD of the dilution water obtained this way provides you with the blank value. The DO uptake of the blank should not be more than 0.2 mg/L andpreferablynotmorethan0.1 mg/L.

Discard all dilution water having a DO uptake greater than 0.2 mg/Land prepare a fresh batch. Repeat all BOD determinations using the fresh dilution water. To avoid vain BOD analysis by contaminated dilu-tion water or vessels, perform blank measurements separately, prior to each BOD analysis.

6.2.2 Seeded blankThis is the seed control to determine the seed DO uptake in dilution wa-ter. Ideally, make dilutions of seed so that the largest quantity results inatleast50%DOdepletion.

A plot of DO depletion, in milligrams per liter, versus milliliters of seed for allbottleshavingmorethan2 mg/Loxygen depletion and a minimum residual DO of 1.0 mg/L, shouldpresent a straight line for which the slope indicates DO depletion per milliliter of seed.

6.2.3 Glucose-glutamic acid check (GGA)As the BOD test is a bioassay, re-sults can be influenced greatly by the presence of toxicants or by the use of a weak seeding material. Distilled waters are frequently con-taminated with copper, and some sewage seeds might exhibit poor biological activity, both leading to low BOD results.

The quality of the dilution water, the seed and your analytical procedure is checked by determining the BOD of a «standard» check solution such as GGA, on a regular basis.

Glucose has an exceptionally high and variable oxidation rate but when it is used with glutamic acid, the oxidation rate is diminished, so that it is similar to that obtained with municipal waste water. The best option is, to determine the 5 day 20 °C BOD of a 2% dilu-tion of the glucose-glutamic acid standard check solution simultane-ously and in exactly the same way as you determine the BOD of your sample.

Adjust concentrations of commer-cial mixtures to give 3 mg/L glu-coseand3 mg/Lglutamicacid ineach GGA test bottle.

6.3 Sample preparation1) Check the pH of all samples before testing, unless previous experiences have proven that the pH is within the acceptable range. Samples exhibiting an alkalinity > 8.5 pHoranacidity<6.0pHareto be neutralized to a pH between 6.5 and 7.5 using a solution of HCl or sulfuric acid. Or use sodium hy-droxide (NaOH) of such strength, that the quantity of reagent does not dilute the sample by more than 0.5%. Furthermore, the pH of thedilution water should not be af-fected by the lowest sample dilu-tion. Don’t seed the samples until the pH is adjusted properly.

2) In some samples chlorine will dissipate within 1 to 2 hours of standing in the light. This often oc-curs during sample transport and handling. For samples in which chlorine residuals do not dissipate in a reasonably short time, remove the chlorine by adding Na2SO3 solu-tion (see chapter 7). All chlorinated samples have to be decholorinated prior to seed addition.

3) Industrial wastewaters often contain other toxic substances like toxic metals. Such samples often require special treatment. If pos-sible dilute strong enough.


4) Fresh samples of cold water or samples, where photosynthe-sis occurs, may be oversaturated with oxygen at the measuring tem-perature of 20 °C, thus leading toundetected loss of oxygen during incubation. To prevent this, reduce the DO to saturation by heating the samplestoatemperatureof20 °Cin partially filled bottles and keep-

ing them for at least 1 hour while agitating by vigorous shaking or by stirring and aerating with clean, fil-tered compressed air.

5)Bringsamplesto20±1 °Cbe-fore making dilutions.

6) If nitrification inhibition is de-sired, add 1 mL of 2-chloro-6-

(trichloro methyl) pyridine (TCMP) solutionor1 mLof1 mg/Lallylthio-urea(ATU)solutiontoeach300 mLbottle before capping, or add suf-ficient amounts of TCMP or ATU to the dilution water to make a final concentrationof10 mg/L.

Samples that may require nitrifi-cation inhibition are biologically treated effluents, samples seeded with biologically treated effluents and river waters. Note the use of nitrogen inhibition in the reporting results.

7) If algae are present, consider a previous filtration of the samples to avoid unusually low BOD results. A filter pore size of 1.6 μm is ap-propriate. Filtering can change BOD results radically. If filtration is car-ried out, the filter pore size shall be recorded in the test report.

8) If samples contain high amounts of BOD, dilute according to table 1.

6.4 Determination of initial DOWhen doing BOD determinations of the samples (Method M020, «BOD») and BOD of the check values (Method M021, «BCV») si-multaneously, always perform the BCV measurement prior to the BOD measurement.

Table 5 Dilution blank

Table 6 Seeded blank

Bottle (300 mL) Seed volume [mL] Dilution water volume [mL]

1 0.00 300.0

2 0.00 300.0

3 0.00 300.0

Bottle (300 mL) Seed volume [mL] Dilution water volume [mL]

1 10.0 290.0

2 10.0 290.0

3 10.0 290.0

Table 7 Oxygen standard

Bottle (300 mL) Standard volume [mL] Seed volume [mL]Dilution water volume [mL]

1 6 2 292.0

2 6 2 292.0

3 6 2 292.0

Bottle(300 mL) Sample volume [mL] Seed volume [mL]Dilution water volume [mL]

1 10.0 0.0 300

2 10.0 0.0 300

3 10.0 0.0 300

Table 8 Municipal sewage sample

Appl

icat

ions

BO

D &

BCV


6.4.1 BlankFor the M021 method (BCV), fill three bottles to the glass grinding with dilution water. Measure the ini-tial (base) DO and seal the bottles. Incubate and stir for 5 days.

6.4.2 Seeded blankFor the M021 method (BCV), fill three bottles partly with dilution water. Add 10 mL of seeded stock solu-tion. Fill the bottles to the glass grinding with dilution water. Mea-sure the initial (base) DO and seal the bottles. Incubate and stir for 5 days.

6.4.3 StandardFor M021 method (BCV) 6 mL of a mixture of 150 mg glucose/L and 150 mgglutamicacid/Lisaddedtoeach of the three bottles to give a final GGA concentration of 3 mg/L. After the addition of 2 mL of seedthe initial DO (base) is measured and the bottles are sealed and in-cubated in the same way as the samples.

The BCV method in SevenExcellence can easily be adapted by the user so as to employ different concentra-tions of GGA solution. This is done by modifying the volume of stan-dard in the bottle submenu of the method function «Standard (BOD)». In the statistical report, the meter

will automatically take the different concentrations into account.

6.4.4 SampleFor the M020 method (BOD) fill three bottles partly with dilution wa-ter. Add 10 mL of sample to eachbottle. Fill the bottles to the glass grinding with dilution water. Mea-sure the initial (base) DO and seal the bottles. Incubate and stir for 5 days.

Remark: After sealing of the bottles for incubation, abslolutely no air must remain in the bottles.

6.5 IncubationIncubate samples, seed controls, dilution water blanks, and glucose-glutamic acid checks at 20 °C ±1 °C and in total darkness for 5days. Stir during the entire incuba-tion period.

6.6 Determination of final DOAfter 5 days of incubation, deter-mine the final DO (follow measure-ment) firstly in the blank, seeded blank and standard bottles using the M021 method and secondly in the samples using method M020 (BOD).

At the start of either method, make sure that you change from «Method

step» base to follow in the «Start analysis»-screen, and that you se-lect the corresponding «check value ID» / «sample ID» from the list un-derneath.

In method M020 (BOD) the «Start analysis»- screen offers the addi-tional option to assign the correct «Associated check value ID», thus allowing the meter to take the re-sults from check value determina-tion, performed with M020 (BCV) in parallel, into account for final calcu-lation and validation of the samples BOD analysis.

6.7 CalculationIf the M020, M021 or any methods basing on them is used for the BOD measurement, no further calcula-tions are necessary. SevenExcellence™ will deliver any results according to the data (vol-umes, dilution factors, salinity) en-tered in the method.


Application M020Biochemical 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.

Sample Municipal sewage

Sample size 10 mL

Sensor InLab® OptiOx (51344621)

InstrumentsSevenExcellence™ with DO/BOD module

Accessories

- uPlace™ electrode arm- 3 BOD bottles (300 mL)- Rainin pipette 1 mL,10 mL- Volumetric flask- Homogenizer- Laboratory balance- Incubator with Multistirrer and

3 stirrer bars - Wash bottle & waste beaker

Buffers / Standards0.3 NaH2PO4 / 0.03 NH4Cl buffer pH 7.2 (pH adjusted withKOH30%)

Reagents

Nutrients:- 0.09 M MgSO4 - 0.9 mM FeCl3 - 0.19 M CaCl2pH adjustment:- 1N H2SO4

- 1N NaOH-30%KOHDechlorination (only if required):- 0.4 M Na2SO3

Waste disposal

Author, VersionHans Früh, Yves MoserMSG pH Lab Version 1.0

Preliminary procedures (sample preparation, conditioning, calibration, etc.)

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 prepare freshly before use.

Sample preparation:

- Homogenize sample before use.

- Check the pH of the sample and adjust it so that it lies between 6.4 and 7.4

- Add a stirrer bar to each bottle.

- Fill the bottles as described in chapter 6.4.4.

- Fill the bottles up with dilution water.

- Take measures to prevent O2 oversaturation of samples prior to «base» measurement by heating and agitating.

- Calibrate the DO sensor prior to each measuring series (base and follow).

Operation procedure

- Insert the InLab® OptiOx into the bottle by holding the bottle at a 45°angleandimmerseitsothattheblueringreachestheneckofthe bottle (~7 cm).

- For BOD5Incubatebottlesfor5days±3hat20±1°Candin complete darkness, stirring constantly.

- Take the bottles out of the incubator and prepare the sensor as described above

- Place the sensor in the bottle

- Start stirring and start the method by selecting method step Follow

- Follow the instructions on the meter

Remarks

A gently stirring is normally needed for a stable measurement. For owners of a uMix magnetic sitrrer the method can be adapted ac-cordingly. To adapt the method to other parameters, save it under a new name and change the volumes accordingly under the method function «Sample».

Appl

icat

ions

BO

D &

BCV


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•Storeallsolutionsinatemperaturecontrolledenvironementat20 °Cforatleast60minutespriortouse• 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. EnvironmentalProtectionAgencyBiochemicaloxygendemand,40CFRPart136,July1,1996.

MethodTitle

Method type BOD

Method ID M020

Title BOD BOD sample

Author METTLER TOLEDO

Created on 2013-07-24 16:53:55

Modified on 2013-07-24 16:57:00

Modified by METTLER TOLEDO

Protect yes

SOP no

Configuration

Measurement type Dissolved Oxygen

Sensor name

Salinity correction no

Seed added To bottle

Blank correction no

Bottle volume 300 mL

Temperature capture Internal

Barometric pressure capture Automatic

Sample (BOD)

Sample ID Sample01

Comment -

Sample type Sample


yes

Number of bottles 3

Measure (BOD)

Sensor name

DO unit mg/L

BOD unit mg/L

DO resolution 2

BOD resolution 2

Endpoint type Automatic

Endpoint criteria Standard

Stir no

Analysis (BOD)Analysis (base)

Temperature limits no

Max. DO limit yes

Max. DO 100%

Action when outside limits Repeat

Min. DO limit no

Show instruction no

Analysis (follow)

Time tolerance limit yes

Time tolerance 3 Hours

Action when outside limits Save and report

Show instruction yes

Min. DO limit yes

Min. DO 1 mg/L



Analysis results

Min. BOD limit of bottle no

Seed correction limits no

BOD limits of sample no

Report

Print no

Signature:

ResultsSample DO base [mg/L] DO follow [mg/L] O2 depletion [mg/L] Concentration [mg/L] Mean [mg/L] Rel. std. dev. [%]municipal sewage

174.4 4.4

Bottle 1 8.00 2.11 5.89 176.7Bottle 2 7.80 1.78 6.02 180.6Bottle 3 8.00 2.47 5.53 165.9


Application M021BOD 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.

SampleDilution water, Seed and oxygen standard

Sample sizeBlank 300 mLSeededBlank 10 mLStandard 6 mL

Sensor InLab® OptiOx (51344621)

InstrumentsSevenExcellence™ with pH/mV or conductivity or DO/BOD module

Accessories

- uPlace™ electrode arm- 9 BOD bottles (250 mL)-Raininpipette1 mL,10 mL- Incubator with Multistirrer

and 9 stirrer bars- Wash bottle- Waste beaker

Buffers / Standards

BOD standard solution for dilution method (300 mg/L glucose, 300 mg/L glutamic acid) 0.3 NaH2PO4 / 0.03 NH4Cl buffer pH 7.2 (pH adjustedwithKOH30%)

Reagents

- Polyseed bacteria Nutrients- 0.09 M MgSO4

- 0.9 mM FeCl3- 0.19 M CaCl2 pH adjustment:- 1N H2SO4

- 1N NaOHDechlorination (only if required):- 0.4 M Na2SO3

Nitrification inhibitor

Waste disposal

Author, VersionHans Früh, Yves MoserMSG pH Lab Version 1.0

Preliminary procedures (sample preparation, conditioning, calibration, etc.)

- 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 prepare freshly before use.

Sample preparation:

- Homogenize seed before use.

- Check the pH of the seed and adjust it so that it lies between 6.4 and 7.4.

- Add a stirrer bar to each bottle.

- Fill the bottles as described in chapter 6.4.

- Take measures to prevent O2 oversaturation prior to «base» measurement.

- Calibrate the DO sensor prior to each measuring series.

Operation procedure

- Always measure the BCV prior to the BOD.

- Immerse the InLab® OptiOx in the bottle so that the blue ring reaches the neck of the bottle (7 cm).

- Make sure that no air bubbles are present, either on the sensor's membrane or in the vortex underneath.

-Allmeasurementsareperformedat20 °C±1.0 °Cusingthesame conditions regarding the stirring speed (4) and stirrer type.

- Start the method.

- For BOD5Incubatebottlesfor5days±3hat20±1°Candincomplete darkness stirring constanty.

Remarks

A gently stirring is normally needed for a stable measurement. For owners of a uMix magnetic sitrrer the method can be adapted ac-cordingly. To adapt the method to other parameters, save it under a new name and change the volumes accordingly under the method function «Sample».

Appl

icat

ions

BO

D &

BCV


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•Storeallsolutionsinatemperaturecontrolledenvironementat20 °Cforatleast60minutespriortouse• For statistical reasons use at least three bottles per sample or check value

Results

Sample DO base [mg/L] DO follow [mg/L] O2 depletion [mg/L]Concentration [mg/L]

Mean [mg/L]Rel. std. dev. [%]

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

MethodTitle

Method type BOD Check Values

Method ID M021

Title BOD check values (BCV)

Author METTLER TOLEDO

Created on 2013-07-24 17:22:32

Modified on 2013-07-24 17:25:37

Modified by METTLER TOLEDO

Protect yes

SOP no

Configuration

Measurement type Dissolved Oxygen

Sensor name

Check value ID CV001

Blank yes

Seeded blank yes

Standard yes

Salinity correction no

Seed added To bottle

Blank correction no

Bottle volume 300 mL

Temperature capture Internal

Barometric pressure capture Automatic

Blank (BOD)

Blank ID CV001

Comment -

Sample type Blank


yes

Number of bottles 3

Measure (Blank)

Sensor name

DO unit mg/L

BOD unit mg/L

DO resolution 2

BOD resolution 2



Stir no

Analysis (Blank)Analysis (base)


Max. DO limit yes

Max. DO 100%


Min. DO limit no

Show instruction no

Analysis (follow)





Analysis results

Max. BOD limit of bottle yes

Max. BOD 0.2 mg/L




Seeded blank (BOD)

Seeded blank ID CV001

Comment -

Sample type Seeded blank


yes

Number of bottles 3

Measure (Seeded blank)

Sensor name

DO unit mg/L

BOD unit mg/L

DO resolution 2

BOD resolution 2



Stir no

Analysis (Seeded blank)Analysis (base)


Max. DO limit yes

Max. DO 100%



Min. DO limit no

Analysis (follow)





Min. DO limit yes

Min. DO 1 mg/L


Analysis results


O2 depletion limits no

Show instruction no

Standard (BOD)

Standard ID CV001

Comment -

Sample type Standard


yes

Number of bottles 3

Measure (Standard)

Sensor name

DO unit mg/L

BOD unit mg/L

DO resolution 2

BOD resolution 2



Stir no

Analysis (Standard)Analysis (base)


Max. DO limit yes

Max. DO 100%



Min. DO limit no

Show instruction no

Analysis (follow)





Min. DO limit yes

Min. DO 1 mg/L



Analysis results


Seed correction limits no

BOD limits of standard yes

Max. BOD 237 mg/L

Min. BOD 163 mg/L



Report

Print no

Signature:

Appl

icat

ions

BO

D &

BCV



Hin

ts a

nd T

ips 7. Hints and Tips

Ifsamplesarenottestedwithin2hoursaftersamplecollection,storethembelow4 °C(39 °F)toinhibitbacterialgrowth.

Bringthetemperatureofallsamplesto±20 °C(68 °F)beforeyoustarttheanyDOmeasurement.

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.Fill100mLoftheundilutedsampleintoa250 mLErlenmeyerflask.Add10 mLofsulfuricacid(0.02 N)and10 mLofpotassiumiodidesolution(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 used in titration x mL to dechlorinate100mL 0.025 N sodium thiosulfate =

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%aroundthenominalvolume(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.



Bibl

iogr

aphy 8. Bibliography

APhA. (1999). 5210 Biochemical oxygen demand A-C. USA: Ameri-can Public Health Association.

Copyright International Organiza-tion for Standardization. (2003). ISO 5815-1.Copyright InternationalOrganization for Standardization.

Delzer, G., & McKenzie, S. (2003). FIVE-DAY BIOCHEMICAL OXYGEN DEMAND. In USGS TWRI Book 9 A7 (p. Chapter 7). USA: USGS TWRI.

DIN Deutsches Institut für Normung e.V., Berlin. (1998). DIN EN 1899-1. Berlin: DIN Deutsches Institut für Normung e.V., Berlin.

DIN Deutsches Institut für Normung e.V., Berlin. (1998). DIN EN 1899-2. Berlin: DIN Deutsches Institut für Normung e.V., Berlin.

EPA. (2003). P40 CFR Ch. I ART 136 – GUIDELINES ESTABLISHING TEST PROCEDURES FOR THE ANAL-YSIS OF POLLUTANTS. USA: Envi-ronmental Protection Agency.

EPA. (2007, January). Standard Operating Procedure for Dissolved Oxygen Micro Method, Winkler Titration. USA: EPA. EPA. (n.d.). Method 405.1. USA: EPA.

Gossett-Johnson, S. (2012). Bio-chemical Oxygen Demand and Carbonaceous Biochemical Oxygen Demand. USA: www.WaterWorldCE.com.

Harvey, D. (n.d.). Chapter 11: Elec-trochemical Methods. In D. Harvey, Analytical Chemistry 2.0.

In-Situ Inc. (2012). Rugged Dis-solved Oxygen (RDO) Sensors Use the Latest Advancements in Optical Technology. USA: In-Situ Inc.

International Organization for Stan-dardization. (2003). ISO 5815-2. International Organization for Stan-dardization.

Jouanneau, S., Recoules, L., Du-rand, M., Boukabache, A., Picot, V., Primault, Y., et al. (2014). Methods for assessing biochemical oxygen demand (BOD): A rebiew. Water Research 49, pp. 62-82.

Mettler-Toledo. (n.d.). Seven- Excellence™ Version 2.0 Workbook. Sales Introduction.

MITTAL, S., & RATRA, R. (2000). TOXIC EFFECT OF METAL IONS ON BIOCHEMICAL OXYGEN DEMAND. In Wat. Res. 34 (pp. 147-152). Great Britain: Elsevier.

OECD. (n.d.). DATA SHEETS FOR SURFACE WATER QUALITY STAN-DARDS. OECD.

Penn, M. R., Pauer, J. J., & Mi-helcic, J. R. (2012). Environmental Exological Chemistry Voll. II. USA: EOLSS.

Stütz, A. (2012). Vereinfachte BSB-Analyse mit OptiOx™. UserCom 17, pp. 26-27.

For more information

GEPTM

1Evaluation

2Selection

3Installation

5Routine

Operation

4Qualification

Quality certificate. Development, production and testing according to ISO 9001.

Environmental management system according to ISO 14001.

“European conformity”. The CE conformity mark provides you with the assurance that our products comply with the EU directives.

www.mt.com//pH

Mettler-Toledo AG, AnalyticalCH-8603 Schwerzenbach, SwitzerlandTel. +41 22 567 53 22Fax +41 22 567 53 23

Subject to technical changes© 04/2015 Mettler-Toledo AG, 30247570Marketing pH Lab / MarCom Analytical

Various factors can affect your pH, redox, conductivity, dissolved oxy-gen or ion measurements. Take 5 minutes to localize your risks and get the neccessary support:

Discover the Safest Path to the Topwith Good Electrochemistry Practice™

www.mt.com/GEP

Documents

Selected Applications - Mettler Toledo · 8 Biochemical Oxygen Demand Electrochemistry Application Brochure 2 Theory 2.1 What is the biochemical oxygen demand? The basic idea behind