Alternate Test Procedure #N09-0020 

In-Situ Inc. Method 1003-8-2009 Biochemical 

Oxygen Demand (BOD) Measurement by Optical 

Probe 
TABLE OF CONTENTS 

DISSOLVED OXYGEN and BOD .............................................................................................3

1. SCOPE AND APPLICATION ............................................................................3

2. SUMMARY OF METHODS ..............................................................................3

3. DEFINITIONS .....................................................................................................3

4. INTERFERENCES ..............................................................................................4

5. SAFETY ..............................................................................................................4

6. EQUIPMENT AND SUPPLIES ..........................................................................4

7. REAGENTS AND STANDARDS ......................................................................5

8. SAMPLE COLLEC/TION, PRESERVATION AND STORRAGE ...................9

9. QUALITY CONTROL ........................................................................................9

10. DISSOLVED OXYGEN METER CALIBRATION ...........................................13

11. DISSOLVED OXYGEN and BOD PROCEEDURES ........................................15

12. BOD CALCULATIONS .....................................................................................17

13. METHOD PERFORMANCE ..............................................................................18

14. POLLUTION PREVENTION .............................................................................19

15. WASTE MANAGEMENT ..................................................................................19

16. REFERENCES ....................................................................................................19

17. TABLES ..............................................................................................................20

APPENDIXES .............................................................................................................................i

Dissolved Oxygen in Saturated Water .............................................................................ii

Density of Water (g/mL) vs. Temperature (°C) ...............................................................vii

BOD Dilution Tables .......................................................................................................ix

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DISSOLVED OXYGEN and BOD 

1. SCOPE AND APPLICATION 

1.1. The Dissolved Oxygen determination is an analytical test in which standardized 

lab procedures are used to determine the oxygen concentration of water, wastewater, 

effluent and polluted water. These tests will measure the oxygen by means of an optical 

electrode compared to a membrane electrode. 

1.2. Biochemical Oxygen Demand (BOD) The BOD determination is an empirical test 

in which standardized laboratory procedures are used to determine the relative oxygen 

consumption of wastewaters, effluents, and polluted waters. 

2. SUMMARY OF METHODS 

2.1. During the last two decades, a new form of electrode was developed based on the 

luminescence emission of a photo active chemical compound and the quenching of that 

emission by oxygen 1 This quenching photophysics mechanism is described by the 

Stern-Volmer equation for dissolved oxygen in a solution 2

: 

[ ] 0

2

0 

SV

2

1

I and I are luminesence in the presence and absence of Oxygen

K is the Stern-Volmer constant for Oxygen quenching

[O ] is the dissolved Oxygen concentration

SV

I K O

I

= +

This equation has been expanded for the fixed photomer on an oxygen optical probe to 

take into account the 2-dimensional photophysics3: 

0 [ 2 2 ] [ ]

1 2

0 

SV

2

x

1 1

I and I are luminesence in the presence and absence of Oxygen

K is the Stern-Volmer constant for Oxygen quenching

[O ] is the dissolved Oxygen concentration

f = Fraction of eac

I KO KO SV SV

If f

+ +

= +

h solid state photomer

The determination of oxygen concentration by luminescence quenching has a linear 

response over a broad range of oxygen concentrations and has excellent accuracy and 

reproducibility 4

. 

2.2. The BOD test is applied in measuring waste loadings to treatment plants and in 

evaluating the BOD-removal efficiency of such treatment systems. The test measures the 

molecular oxygen utilized during a specified incubation period for the biochemical 

degradation of organic material (carbonaceous demand) and the oxygen used to oxidize 

inorganic material such as sulfides and ferrous iron. It also may measure the amount of 

oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand) unless their 

oxidation is prevented by an inhibitor. The seeding and dilution procedures provide an 

estimate of the BOD at pH 6.5 to 7.5.5

3. DEFINITIONS 

3.1. BOD: Biochemical Oxygen Demand 

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3.2. DI Water: Deionized water 

3.3. Duplicate = Repetitive individual analyses of a sample to measure precision. 

3.4. G/GA: A solution of Glucose and Glutamic Acid. 

3.5. RPD, Relative Percent Difference: Measure of precision and/or matrix effects of 

Duplicates. The RPD must be less than or equal to (≤) 20% 

4. INTERFERENCES 

4.1. Chemical compounds that compete or interfere can bias the optical electrode or the 

membrane electrode. 

4.2. Optical electrodes respond to O2 partial pressure, which is a function of dissolved 

inorganic salts. Conversion factors may be calculated from DO concentration verses 

salinity. 

4.3. Chemicals which can pass through or interact with the electrode cap or membrane may 

interfere with either the membrane electrode or optical electrode. 

4.4. High concentrations of oil and grease may plug the membrane or occlude the cap. 

5. SAFETY 

5.1. This method does not address all safety issues associated with its use. The laboratory is 

responsible for maintaining a safe work environment and a current awareness file of 

OSHA regulations regarding the safe handling of any chemicals specified in this method. 

A reference file of material safety data sheets (MSDSs) should be available to all 

personnel involved in these analyses 

6. EQUIPMENT AND SUPPLIES 

6.1.BOD Incubation bottles, ~300mL (capacity to be determined experimentally) with 

ground glass stoppers. 

6.2. Analytical balance that has capacity to measure full BOD bottle to the nearest 0.1 gram. 

6.3.Barometer (or internal barometer in the DO meter) 

6.4.Stirring plate with stir bars. 

6.5.Burette (Class A) with accuracy to the nearest 0.01 mL. 

6.6.BOD air incubator. Thermostatically controlled at 20 ±1°C. Exclude all light to prevent 

possibility of photosynthetic production of DO (Meets requirements of Standard 

Methods 5210 B5

) 

6.7.Dissolved oxygen meter and an In-Situ RDO® probe or suitable luminescence optical 

electrode. Note: other manufacturer’s luminescence optical electrode may not perform 

in the non-stirred mode.
7. REAGENTS AND STANDARDS 

7.1.DEIONIZED WATER (DI) 

7.1.1. Water from a suitable system that produces water that is low in ions and dissolved 

solids. Suitable for analytical analyses. 

7.2.BOD GRADE WATER 

7.2.1. Water from a suitable source (Distilled, Deionized or RO) that is free of all 

chemical interference that would deplete dissolved oxygen or interfere with any 

dissolved oxygen chemical reaction. 

7.3. DISSOLVED OXYGEN DILUTION WATER 

7.3.1. After filling dilution water storage jug with BOD grade water, sparge with air. 

The dilution water must sit for approximately 24 hours to allow the dissolved 

oxygen to come to equilibrium with laboratory temperature and pressure. 

7.4. BOD DILUTION WATER 

7.4.1. After filling dilution water storage jug with Dissolved Oxygen Dilution Water, 

add 1 mL of each of the nutrient solutions listed in 7.6 through 7.9 (add them in 

alphabetical order) per liter of dilution water. Mix well and use that day. 

Note: BOD Dilution Water cannot be stored over a standard working day (12 hours) 

7.5. BOD SEED PREPARATION 

7.5.1. The preferred seed is obtained from a biological treatment system processing the 

waste. In this case, use supernatant from settled domestic wastewater, effluent from 

primary clarifiers, diluted mixed liquor from an aeration basin, undisinfected 

effluent, or receiving water from below the point of discharge. Do not use seed 

from effluents that have been disinfected by chlorine or other means. Commercial 

seed sources may be used. Prepare commercial seed as per manufacturer’s 

directions 

7.6. PHOSPHATE BUFFER SOLUTION 

7.6.1. Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4 • 7H2O, and 1.7 g 

NH4Cl in about 500 mL DI water and dilute to 1 L. The pH should be 7.2 without 

further adjustment. (ACS Grade or better) 

7.6.2. Alternatively, dissolve 42.5 g KH2PO4 and 1.7 g NH4Cl in about 700 mL distilled 

water. Adjust pH to 7.2 with 30% NaOH and dilute to 1 L. (ACS Grade or better) 

7.6.3. Commercial reagent can be purchased. 

7.7. MAGNESIUM SULFATE SOLUTION 

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7.7.1. Dissolve 22.5 g MgSO4 •7H2O in DI water and dilute to 1 L. (ACS Grade or 

better) 

7.7.2. Commercial reagent can be purchased. 

7.8. CALCIUM CHLORIDE SOLUTION 

7.8.1. Dissolve 27.5 g CaCl2 in DI water and dilute to 1 L. (ACS Grade or better) 

7.8.2. Commercial reagent can be purchased. 

7.9. FERRIC CHLORIDE SOLUTION 

7.9.1. Dissolve 0.25 g FeCl3•6H2O in distilled water and dilute to 1 L(ACS Grade or 

better) 

7.9.2. Commercial reagent can be purchased. 

7.10. BOD OPTICAL AND MEMBRANE PROBE ZERO SOLUTION 

7.10.1. Sodium Sulfite Zero Solution Dissolve 10 g of sodium sulfite (Na2SO3) in 

200 mL of DI water. Add two drops of saturated cobalt chloride solution to the 

sodium sulfite solution. (ACS Grade or better) 

7.10.2. Saturated Cobalt Chloride solution; Dissolve 4.5 grams of cobalt chloride 

hexahydrate (CoCl2 • 6 H2O) in 10 mL of DI water. (ACS Grade or better) 

7.10.3. A commercial solution of this mixture can be purchased. 

7.11. GLUCOSE-GLUTAMIC ACID SOLUTION (G/GA) 

7.11.1. Dry reagent-grade glucose and reagent-grade glutamic acid at 103o

C for 1 

hour. Store these in a desiccator until needed. Add 0.1500 g (± 0.0001 g) of 

glucose and 0.1500 g (±0.0001 g) of glutamic acid to a 1 liter volumetric flask and 

dilute to volume with BOD Grade water. This solution is good for seven days if 

kept sterile and refrigerated at 4o

C ± 2oC. Replace if solution becomes cloudy or 

shows signs of bacterial or chemical contamination. (ACS Grade or better) 

7.11.2. A commercial solution can be purchased, but concentrations may vary. 

Final concentration must be the same as solution above. Keep solution sterile and 

refrigerated at 4oC ± 2oC. Replace if solution becomes cloudy or shows signs of 

bacterial or chemical contamination 

7.12. SULFURIC ACID 

7.12.1. Concentrated H2SO4, (ACS Grade or better). 

7.13. SULFURIC ACID (6N) 

7.13.1. Slowly and cautiously add 85mL of concentrated sulfuric acid to 

approximately 400mL of DI water, allow it to cool to room temperature and then 

dilute to 500mL. (ACS Grade or better) 

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7.13.2. Commercial solution can be purchased. 

7.14. SODIUM HYDROXIDE (6N) 

7.14.1. Dissolve 120gm in 500mL of DI water. Allow to equilibrate to room 

temperature, transfer to a 1L volumetric flask and dilute to the mark. Mix well by 

inverting flask. (ACS Grade or better) 

7.14.2. Commercial solution can be purchased. 

7.15. MANGANOUS SULFATE SOLUTION 

7.15.1. Dissolve 480 g MnSO4 • 4H2O, 400 g MnSO4 • 2H2O, or 364 g MnSO4 • 

H2O (ACS Grade or better) in DI water, filter, and dilute to 1 L. The MnSO4 

solution should not give a color with starch when added to an acidified potassium 

iodide (KI) solution. (ACS Grade or better) 

7.15.2. Commercial solution can be purchased. 

7.16. ALKALINE IODIDE-AZIDE SOLUTION 

7.16.1. For saturated DO or less-than-saturated DO samples, Dissolve 500 g NaOH (or 

700 g KOH) and 135 g NaI (or 150 g KI) in DI water and dilute to 1 L. Add 10 g 

NaN3 dissolved in 40 mL DI water. This reagent should not give a color with starch 

solution when diluted and acidified. (ACS Grade or better) 

7.16.2. For supersaturated samples—Dissolve 10 g NaN3 in 500 mL DI water. Add 480 g 

sodium hydroxide (NaOH) and 750 g sodium iodide (NaI), and stir until dissolved. 

There will be a white turbidity due to sodium carbonate (Na2CO3). CAUTION, Do 

not acidify this solution because toxic hydrazoic acid fumes may be produced. (ACS 

Grade or better on chemicals) Commercial solution can be purchased. 

7.16.3. Commercial solution can be purchased. 

7.17. SODIUM THIOSULFATE

7.17.1. (Standard Titrant, 0.025 M) Purchase to meet Standard Methods 4500-O C 

criteria or dissolve 6.205 g sodium thiosulfate-pentahydrate in ~ 500 mL DI water 

in a 1000 mL volumetric flask, add 1.5 mL of 6N NaOH or 0.4 grams solid NaOH, 

dilute to 1000 mL, mix well, and standardize. (ACS Grade or better) 

7.17.2. (Standard Titrant, 0.0375 M), Purchase to meet Standard Methods 4500-O C 

criteria or dissolve 9.304 grams of sodium thiosulfate-pentahydrate in ~ 500 mL DI 

water in a 1000 mL volumetric flask, add 1.5 mL of 6N NaOH or 0.4 grams solid 

NaOH, dilute to 1000 mL, mix well, and standardize. (ACS Grade or better) 

7.17.3. Commercial solution can be purchased. 

7.18. STARCH SOLUTION 

7.18.1. Use either an aqueous solution or soluble starch powder mixtures. To prepare an 

aqueous solution, dissolve 2 g laboratory-grade soluble starch and 0.2 g salicylic 

acid, as a preservative, in 100 mL hot DI water. (ACS Grade or better) 

7.18.2. Commercial solution can be purchased. 

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7.19. POTASSIUM BI-IODATE 

7.19.1. 0.0021 Molar potassium bi-iodate solution, (0.0021M): Dissolve 818.8 mg 

KH(IO3)2 in DI water and dilute to 1000 mL in a volumetric flask and mix well. 

(ACS Grade or better) 

7.19.2. 0.00021 Molar potassium bi-iodate solution, (0.00021M): Transfer 100 mL of 

0.0021 M potassium bi-iodate solution to a 1000 mL volumetric flask, dilute to 

1000 mL with DI water and mix well (ACS Grade or better) 

7.19.3. Commercial solution can be purchased. 

7.20. THIOSULFATE STANDARDIZATION 

7.20.1. Dissolve approximately 2 g KI, free from iodate, in a 500 mL Erlenmeyer 

flask with 100 to 150 mL DI water. 

7.20.2. Add 1 mL 6N H2SO4 or a few drops of conc H2SO4. 

7.20.3. Add 20.00 mL of 0.0021 M bi-iodate solution with a Class A pipette and 

mix well. 

7.20.4. Titrate with an appropriate Class A burette the liberated iodine with 

thiosulfate titrant, adding starch toward end of titration (when a pale straw color is 

reached). Continue to titrate the solution until the blue color just disappears. 

7.20.5. Record the mL of thiosulfate titrant used. Read the burette to an accuracy 

of only one half (1/2) of a volume unit between each demarcation. 

7.20.6. Calculate the Molarity of the thiosulfate. 

( )

(0021.01220 ) )( ml titrantethiosulfat

ml Molarity iodatebipotassium Molarity ethiosulfat × × − =

7.20.7. Balanced Equation 

3 2

2 2

23 2 46

2

3 23

IO 5I 6H 3I 3H O

6S O 3I 3S O 6I

Molar Ratio IO : 6S O

−− +

2

− − −

− −

++ → +

+→ +

7.21. BOD BOTTLE VOLUME DETERMINATION (Titrametric) 

(Note: Volume determination is required for Winkler Titration accuracy. See 9.7 for 

QA/QC requirements) 

7.21.1. Fill a BOD bottle to the top with 0.00021 M potassium bi-iodate solution. 

Make sure no bubbles are present. Cap with stopper and pour off excess from the 

water seal. 

7.21.2. Dissolve approximately 4 g KI, free from iodate, in a 500 mL Erlenmeyer 

flask with 50 to 100 mL DI water. 

7.21.3. Add 2 mL 6N H2SO4 or a few drops of concentrated H2SO4. 
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7.21.4. Add the potassium bi-iodate solution from the BOD bottle and mix well. 

7.21.5. Rinse the BOD bottle with DI water and add to the Erlenmeyer flask. 

7.21.6. Titrate with a Class A burette the liberated iodine with standardized 

thiosulfate titrant, adding starch toward end of titration (when a pale straw color is 

reached). Continue to titrate the solution until the blue color just disappears. 

7.21.7. Record the mL of standardized thiosulfate titrant used. Read the burette to 

an accuracy of only one half (1/2) of a volume unit between each demarcation. 

7.21.8. Make sure cap used for volume determination is mated to the BOD bottle 

used. 

7.21.9. Calculate the BOD bottle volume to the nearest 0.1 mL 

(00021.0 12)

( )() ),(

×−

× = Molarity iodatebipotassium

MolarityM mlethiosulfat ethiosulfat BottleBOD mlvolume

7.22. BOD BOTTLE VOLUME DETERMINATION (Gravimetric) 

7.22.1. Weigh a BOD bottle and cap to the nearest 0.1 grams. Record the weight. 

7.22.2. Fill a BOD bottle to the top with DI water. Make sure no bubbles are 

present. Cap with stopper and pour off excess from the water seal. 

7.22.3. Weigh the full BOD bottle and cap to the nearest 0.1 grams. Record the 

weight. 

7.22.4. Make sure cap used for volume determination is mated to the BOD bottle 

used. 

7.22.5. Calculate the volume of the BOD bottle using the Density of Water 

(Appendix 2) at the temperature of the DI water. Record the volume to the nearest 

0.1 mL. 

[ ( ,) ( ] ( ,) BOD Bottle Final weight g BOD Bottle InitialWeight, ) g BOD Bottle volume ml

Density of Water

− =

8. SAMPLE COLLEC/TION, PRESERVATION AND STORRAGE 

8.1. See Title 40 of the Code of Federal Regulations Part 136.3, Table II for information 

regarding required sample collection containers, preservation techniques and holding 

times. 

9. QUALITY CONTROL 

9.1. INITIAL AND ONGOING DEMONSTRATION OF CAPABILITY FOR 

DISSOLVED OXYGEN 
9.1.1. Before new analysts perform the dissolved oxygen analyses or to confirm their 

continuing capability, verify their performance. Have them determine the dissolved 

oxygen with a calibrated meter of four BOD bottles of air saturated water. Utilizing 

the table in the appendixes, calculate the concentration of dissolved oxygen from 

the barometric pressure, temperature and salinity of the air saturated water. 

Calculate the standard deviation for the combined values for each analysis. This 

value is SPooled. Utilize this value to calculate the t-Test for the two experimental 

means. The value determined should be ≤ 2.31 for 95% confidence. The ongoing 

demonstration of capability will be performed at a minimum on a quarterly basis. 

( )

( )

1

2

8 1

Note: Calculate mean from all 8 Data points

Use all 8 data points to calculate pooled standard deviation

Pooled

X X

S

⎡ ⎤ − = ⎢ ⎥

⎢ ⎥ − ⎣ ⎦

∑

1 2

1

2

1

2

2.31 95%

Pooled

X X

t

S

X Meanof the DO Meter Measurements

X Mean of the DOCalculated Measurements

t at confidence

− =

=

=

≤

9.2. DAILY DATA ANALYSES 

9.2.1. Data analysis to be performed on a daily basis or with each set of samples will 

consist of the dissolved oxygen measurement of a duplicate samples and the RPD 

calculated. Typical RPD values based on multiple laboratory analysis of varied 

sample types are illustrated below. 

( )

( )

C C

C C

x

C

C

I D

I D

I

D

−

⎛

⎝

⎜ ⎞

⎠

⎟ +

2

100 = RPD

= Concentration determined for a sample

 = Concentration determined for the duplicate

 

9.3. LOWER LIMIT OF OPERATIONAL RANGE OF THE DO METER 

9.3.1. Determine the lower limit of the meters operational range weekly or every time 

the meter or RDO probe are serviced. Measure the dissolved oxygen with a 

calibrated meter of four BOD bottles of zero solution. Calculate the standard 

deviation for the values for the analysis. This value is S. Utilize this value to 

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calculate the confidence interval for the mean of the zero solution. The mean and 

confidence level must be below the minimum dissolved oxygen measurement 

required for the analysis. 

( )

( )

1

2

4 1

X X

S

⎡ − ⎤ = ⎢ ⎥ ⎢ − ⎥ ⎣ ⎦

∑

2

2

t S Confidence Interval X

t Student Sided t Distribution

×

= ±

=

9.4. BOD DILUTION WATER BLANKS 

9.4.1. Prepare BOD dilution water blanks to represent each type of test being performed 

within the batch. Run these QC samples with each batch of BOD. 

9.4.2. Unseeded BOD blank: A 300-mL bottle of BOD dilution water. (Minimum 3 per 

set) 

9.4.3. Dilution water blanks must meet the dissolved oxygen levels in Table 1 

Table 1: Dilution Water Blanks 

Blank Type Maximum 5 Day Depletion 

for Passing Blanks 

Unseeded BOD ≤ 0.2 mg/L 

9.5. GLUCOSE AND GLUTAMIC ACID CHECK (G/GA) 

9.5.1. Transfer 6 mL of the glucose - glutamic acid solution to a BOD bottle of known 

concentration. 

9.5.2. Add sufficient seed to achieve acceptable dissolved oxygen depletion. 

9.5.3. Fill BOD bottle with BOD dilution water. (Minimum three G-GA checks) 

9.5.4. Determine the 5 Day BOD 

9.5.5. Passing results will have a BOD of 198 (+ 30.5) mg/L. 

9.5.6. Run these QC samples with each batch of BOD samples. It is important to realize 

that GGA is not intended to be an accuracy check in the test. Its sole purpose is to 

demonstrate that the seed is viable and metabolizing in the proper range of activity 

under the conditions of the test.

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9.6. DUPLICATE SAMPLES 

9.6.1. A duplicate sample will be set for each BOD set of seeded and unseeded samples. 

QA/QC criteria for the Relative Percent Difference and Precision and Bias will be 

developed. 

9.6.2. Duplicate checks will be run on all High and Low DO Calibration Standards each 

day the dissolved oxygen meter is used. QA/QC criteria for the Relative Percent 

Difference and Precision and Bias will be developed. 

9.7. DO SPIKING OF SAMPLE 

9.7.1. No DO spiking of samples will be required as accurate spiking of a gaseous 

analyte into a laboratory sample is not easily achieved in most commercial and 

municipal laboratories. 

9.8. BOD BOTTLE VOLUME 

9.8.1. The use of a BOD bottle for Winkler titration volume or BOD dilution volume 

requires that the bottle volume be known. Sections 7.21 through 7.22 describe how 

to calculate the volume. 

9.8.2. The laboratory can determine the precision and bias of a large number of BOD 

bottles by selecting 10 bottles at random, determine their volume accurately, use a 

Known Value of 300 mL, and calculate the precision and bias utilizing the 

equations: 

1

( )

n

Known Value Experimental Value

i

X X

Bias

n

=

−

=

∑

2

1

( )

Precision

1

n

Known Value Experimental Value

i

X X

n

=

−

= −

∑

9.8.3. All DO and BOD values calculated with the default Known Value of 300 mL will 

have the Bias and Precision reported in the final value. 

9.8.4. The laboratory will repeat the precision and bias analysis at a minimum on a 

quarterly basis and keep control records of the precision and bias. 

9.9. BOD WORKING RANGE AND DETECTION LIMIT 

9.9.1. The working range is equal to the difference between the maximum initial DO (7 

to 9 mg/L) and minimum DO residual of 1 mg/L corrected for seed, and multiplied 

by the dilution factor. Detection limits are established by the minimum DO 

depletion and minimum DO residuals as follows (See formula at 12.2 for formula 

variable definitions): 

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9.9.2. The lower limit for seeded samples that require dilution (S > 0; P < 1.0) is 

approximately 1 mg/L as established by the minimum depletion of 2.0 mg/L minus 

the maximum seed correction, which should be less than about 1 mg/L.

9.9.3. The lower limit for seeded samples that require dilution (S > 0; P < 1.0) is 

approximately 1 mg/L as established by the minimum depletion of 2.0 mg/L minus 

the maximum seed correction, which should be less than about 1 mg/L.

9.9.4. The lower limit for unseeded samples that require no dilution (S = 0; P = 1.0) is 

equal to the detection limit of the DO measurement method (~0.1 mg/L).

9.9.5. The lower detection limit for seeded samples that require no dilution (S > 0; P = 

1.0) is 0 mg/L, as established by the difference between the sample DO depletion 

and the seed correction.

10. DISSOLVED OXYGEN METER CALIBRATION 

10.1. WINKLER TITRATION 

10.1.1. Prepare dissolved oxygen dilution water by sparging with air a volume of 

BOD grade water in a carboy with a venting cap. Allow saturated dilution water to 

vent to the atmosphere for approximately 24 hours at laboratory temperature. 

Transfer the air saturated water to the BOD bottle with the minimum of agitation. 

10.1.2. Fill a BOD bottle of known volume to the top with air saturated dilution 

water. Make sure no bubbles are present. Cap with stopper used to calibrate the 

BOD bottle. 

10.1.3. Remove the BOD bottle stopper, add 1mL of the manganous sulfate 

solution to the BOD bottle by inserting the pipette tip below the surface of the 

water or by transferring the solution by touching the tip of the pipette to the side of 

the BOD bottle above the surface of the water. Care must be taken to transfer the 

solution quickly with minimum water agitation. Cap with stopper and pour off 

excess from the water seal. Mix well by inversion. 

10.1.4. Remove the BOD bottle stopper, add 1mL of the alkaline iodide-azide 

solution to the BOD bottle by inserting the pipette tip below the surface of the 

water or by transferring the solution by touching the tip of the pipette to the side of 

the BOD bottle above the surface of the water. Care must be taken to transfer the 

solution quickly with minimum water agitation. Cap with stopper and pour off 

excess from the water seal. Mix well by inversion. 

10.1.5. Allow a golden brown flock to form and precipitate to the bottom third of 

the bottle. 

10.1.6. Carefully remove the stopper and immediately add 1mL of concentrated 

H2SO4 solution to the BOD bottle by inserting the pipette tip below the surface of 

the water. Care must be taken to transfer the solution quickly with minimum water 

agitation. Cap with stopper and pour off excess from the water seal. Mix well by 

inversion. The solution will be a golden brown color 

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10.1.7. Transfer by Class A volumetric pipette a measured amount of the contents 

of the BOD bottle into a 500mL wide mouth flask quickly, but without introducing 

air bubbles. 

10.1.8. Titrate the solution using standardized thiosulfate titrant in a Class A 

burette until it is a pale yellow. 

10.1.9. Add approximately 3 drops of the starch solution to the pale yellow 

solution. The solution should turn dark blue. 

10.1.10. Continue to titrate the solution until the blue color just disappears. 

10.1.11. Record the mL of standardized thiosulfate titrant used. Read the burette to 

an accuracy of only one half (1/2) of a volume unit between each demarcation. 

10.1.12. Calculate the concentration of dissolved oxygen: 

( )

( ) ( ) 32 ( / 2 ( , )1 4

( ,)

1. ml (thiosulfate) = ml of titrant used

2. Molarity (thiosulfate) = 

ml thiosulfate Molarity thiosulfate Dissolved oxygen mg l

volume initial sample ml ml ml sample volume initial sample ml

× × =

⎛ ⎞ − × ×⎜ ⎟ ⎝ ⎠

Molarity of standardized thiosulfate 

3. ml (sample) = ml of Winkler sample titrated

4. volume (initial sample) = volume of BOD bottle 

)

10.1.13. Record this dissolved oxygen value to the nearest 0.01 mg/L. 

10.2. BAROMETRIC DETERMINATION: AIR SATURATED WATER 

10.2.1. Determine the barometric pressure for your laboratory in atmospheres or 

mm Hg (Torr). 

10.2.2. Convert laboratory barometric pressure to atmospheres. 

10.2.3. Determine the temperature for your laboratory in centigrade and Kelvin. 

10.2.4. Determine the DO in air saturated water from the tables and formulas in 

Appendix 1. 

10.3. BAROMETRIC DETERMINATION: WATER SATURATED AIR

10.3.1. Determine the barometric pressure for your laboratory in atmospheres or 

mm Hg (Torr). 

10.3.2. Convert laboratory barometric pressure to atmospheres. 

10.3.3. Determine the temperature for your laboratory in centigrade and Kelvin. 

10.3.4. Determine the DO in water saturated water from the tables and formulas in 

Appendix 1. 

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10.4. CALIBRATION OF DISSOLVED OXYGEN METER 

10.4.1. Operate the meter and electrode as per instrument manufacturer’s 

instructions. Sample may be stirred on not. Allow the meter to come to equilibrium 

prior to accepting dissolved oxygen value. 

10.4.2. Calibrate the meter as per instrument manufacturer’s instructions with 

both the following standards: 

10.4.2.1. High Dissolved Oxygen Standard consisting of one of the following 

10.4.2.1.1. Winkler titration. 

10.4.2.1.2. Water Saturated Air. 

10.4.2.1.3. Air Saturated Water. 

10.4.2.2. Low Dissolved Oxygen Standard as required by instrument response or 

Lower Reporting Limit. 

10.4.2.2.1. Sodium Sulfite with CoCl2-6 H2O 

11. DISSOLVED OXYGEN and BOD PROCEEDURES 

11.1. DISSOLVED OXYGEN DETERMINATION 

11.1.1. Calibrate the DO meter as per manufacturer’s directions and Section 10 

above. 

11.1.2. Fill a BOD bottle to the top with sample prepared as per 11.2-11.3 leaving 

no air gaps or bubbles or place dissolved oxygen RDO probe in the bottle it was 

collected in or place dissolved oxygen probe in sample stream. 

11.1.3. Allow the dissolved oxygen meter to come to equilibrium with the sample. 

Stirring, agitation, or flow may accelerate the dissolved oxygen meter equilibrium. 

11.1.4. Record the dissolved oxygen concentration. 

11.2. SAMPLE PREPARATION AND DETERMINATION FOR BOD 

11.2.1. Bring the sample to ambient room temperature. 

Note: Transfer the sample to a cylindrical container if it is not already in one. 

Non-cylindrical containers do not allow for even mixing by magnetic stirrers.

11.2.2. Add a magnetic stir bar to the sample and mix the sample on a magnetic 

stirrer. 

Note: It is important the sample is allowed to stir until all of the solids are 

suspended. Failure to do this will cause a bias in the sampling.

11.2.3. If pH of sample is <6.5 or >7.5 neutralize the sample to approximately a 

pH of 7.0 using either sulfuric acid or sodium hydroxide. 

15 | Page
11.2.4. Aliquots of the neutralized sample are transferred to 300 mL BOD bottles 

using the procedures listed below. These BOD samples must be at concentrations 

that will deplete by at least 2 mg/L DO and have at least 1 mg/L DO left after five 

days of incubation. Therefore make enough dilutions (minimum of 3) of the 

prepared sample to bracket the predicted BOD. See Appendix for dilution charts.

11.2.5. Use a magnetic stirrer to stir all samples, including dilutions, while an 

aliquot is being taken. These samples and their dilutions must be in cylindrical 

containers to insure an even distribution of solids while being stirred. 

11.2.6. Sampling aliquots of the stirred sample or dilution will be taken with a 

wide tipped pipette. 

11.2.7. The minimum aliquot volume transferred to a BOD bottle will be 3 mL. If 

a smaller volume is needed to meet the DO depletion requirements, then you must 

make dilutions to the sample. 

11.2.8. Only pipette the aliquot volume required for a single BOD bottle. Do not 

transfer multiple aliquot volumes in your pipette draw. This will lead to solids 

settling out and sample bias. 

11.2.9. Dilutions of a sample will be made by transferring a measured volume of 

the stirred sample by wide tipped pipette (minimum of a 10 mL pipette) to a 

volumetric flask and diluting to volume with BOD Dilution water. This sample is 

then transferred to a cylindrical container and sampled as outlined above. 

11.2.10. If the sample is being prepared as a seeded sample, add enough prepared 

seed to the sample to achieve acceptable dissolved oxygen depletion. 

11.2.11. Add BOD Dilution water to each BOD sample bottle so as to completely 

fill the bottle with no air spaces or bubbles when the stopper is placed in the bottle. 

11.2.12. If the DO measurement is to be taken with a non-stirred RDO probe, cap 

the BOD bottle and invert sample multiple times to mix well. 

11.2.13. Place the dissolved oxygen RDO probe in the bottle, make sure no bubbles 

are introduced to the sample or air gaps in the bottle and allow the dissolved 

oxygen meter to come to equilibrium. Sample may be stirred or not. Allow the 

meter to come to equilibrium prior to accepting dissolved oxygen value. 

11.2.14. Record the DO of the sample, stopper the bottle making sure no bubbles 

are introduced to the sample or air gaps in the bottle, add DI water to the water seal 

if needed, cap with a water snap lid to reduce evaporation, and incubate for 5 days 

at 20°C ± 1°C. Exclude light to avoid growth of algae in the bottles during 

incubation. 

16 | Page
11.2.15. Upon completion of the 5-day incubation± 6 hours, record the DO’s of the 

depleted samples with a calibrated DO meter. Sample may be stirred or not. Allow 

the meter to come to equilibrium prior to accepting dissolved oxygen value. 

11.2.16. Calculate the BODs from the formula below. 

11.2.17. Only bottles, including seed controls, giving a minimum DO depletion of 

2.0 mg/L and a residual DO of at least 1.0 mg/L after 5 d of incubation are 

considered to produce valid data, because at least 2.0 mg oxygen uptake per L is 

required to give a meaningful measure of oxygen uptake and at least 1.0 mg/L must 

remain throughout the test to ensure that insufficient DO does not affect the rate of 

oxidation of waste constituents. 

11.3. SEED BOD UPTAKE 

11.3.1. Seed BOD Uptake: Typically a 10, 20, and 30 mL sample of seed added to 

3 separate BOD bottles and diluted with BOD dilution water. Run these QC 

samples with each batch of seeded BOD. 

11.3.2. Calculate the DO uptake per mL of seed added to each bottle using either 

the slope method or the ratio method. 

11.3.3. For the slope method, plot DO depletion in milligrams per liter versus 

mLs of seed for all seed control bottles having a 2.0 mg/L depletion and 1.0 

minimum residual DO. The plot should present a straight line for which the slope 

indicates DO depletion per mL of seed. The DO-axis intercept is oxygen depletion 

caused by the dilution water and should be less than 0.20 mg/L 

11.3.4. For the ratio method, divide the DO depletion by the volume of seed in 

mLs for each seed control bottle having a 2.0 mg/L depletion and greater than 1.0 

mg/L minimum residual DO and average the results. 

12. BOD CALCULATIONS 

12.1. SEED UPTAKE: RATIO METHOD CALCULATION 

For each Seed Uptake bottle having 2.0 mg / L minimum Dissolved Oxygen depletion and 

at least 1.0 mg / L residual Dissolved Oxygen , calculate Seed Uptake as follows :

Dissolved Oxygen SeedUptakeS

SeedUptake

Δ

= 0

0

n

n

ample

ml Seed

∑

∑

17 | Page
12.2. BOD5 CALCULATION 

( ) 1 2 ( ) 5

For each test bottle having 2.0 mg / L minimum Dissolved Oxygen depletion and 

at least 1.0 mg / L residual Dissolved Oxygen , calculate BOD as follows :

D1 Dissolved Oxygen of diluted sa

, /

D D SVS

BOD mg l P

=

− − =

mple immediately after preparation, mg / L,

D2 Dissolved Oxygen ofdiluted sample after 5 d incubation at 20 C, mg / L,

S seed uptake, DO / mL seed suspension added per bottle 

S 0 if samples are not se

= °

= Δ

( ) = eded ,

Vs volume of seed in the respective test bottle, mL, and

P decimal volumetric fraction of sample used; 1/ P dilution factor

=

= =

13. METHOD PERFORMANCE 

13.1. BLANKS 

13.1.1. Dissolved oxygen data analysis performed on a daily basis or with each set 

of samples will consist of the measurement of duplicate samples and the RPD 

calculated. Blank RPDs for BOD of the RDO to membrane electrode from a 

multiple laboratory study are in Section 17.1. 

13.1.2. Bias and precision for RDO blank measurement compared to the 

membrane electrode for multiple laboratory study are in Section 17.1. 

13.1.3. Lower of operational range of the RDO DO Meter 

13.1.4. The lower limit of operational range of the DO Meter must be below the 

lowest dissolved oxygen value required. Typical values for RDO confidence 

intervals based on multiple laboratory analysis are illustrated Section 17.2. 

13.2. GLUCOSE/GLUTAMIC ACID 

13.2.1. The mixture of glucose and glutamic acid was developed in order to 

provide a standard with a stable metabolic rate that could be used in the 5 day BOD 

test under a wide variety of applications. F Test results showing method 

comparability of BOD for the RDO to the membrane electrode are in Section 17.3.

13.2.2. GGA recoveries of BOD for the RDO and membrane from a multiple 

laboratory study are in Section 17.3. 

13.2.3. GGA DO RPDs for the BOD of the RDO to membrane electrode from a 

multiple laboratory study are in Section 17.3.

13.2.4. Bias and precision for the RDO GGA BOD values are compared to the 

membrane electrode from a multiple laboratory study in Section 17.3 

18 | Page
13.3. OTHER WATER AND WASTEWATER BOD MATRIXES 

13.3.1. Typical RPD values of the RDO to membrane electrode based on multiple 

laboratory analysis of varied sample types are illustrated in Section 17.4 The 

matrixes include wastewater influent, wastewater effluent, surface water, and 

industrial waste from Significant Industries and Categorical Industries.

14. POLLUTION PREVENTION 

14.1. There are no standards or reagents used in this method at the concentrations 

required that pose a threat to the environment. Refer to Waste Management for correct 

disposal of all chemicals.

15. WASTE MANAGEMENT 

15.1. It is the laboratory’s responsibility to comply with all federal, state, and local 

regulations governing waste management, particularly the hazardous waste identification 

rules and land disposal restrictions, and to protect air, water, and land by minimizing and 

control all releases from fume hoods and bench operations. Compliance with all sewage 

discharge permits and regulations is also required.

16. REFERENCES 

1. Mingoarranz, F. J.; Moreno-Bondi, M. C.; Garcia-Fresnadillo, D.; de Dios, C.; 

Orellana, G., Oxygen-sensitive layers for optical fiber devices. Mikrochim. Acta 1995,

121, (1-4), 107-18. 

2. Garcia-Fresnadillo, D.; Marazuela, M. D.; Moreno-Bondi, M. C.; Orellana, G., 

Luminescent Nafion Membranes Dyed with Ruthenium(II) Complexes as Sensing 

Materials for Dissolved Oxygen. Langmuir 1999, 15, (19), 6451-6459. 

3. Borisov, S. M.; Klimant, I., Ultrabright Oxygen Optodes Based on 

Cyclometalated Iridium(III) Coumarin Complexes. Anal. Chem. (Washington, DC, U. S.) 

2007, 79, (19), 7501-7509. 

4. Titze, J.; Walter, H.; Jacob, F.; Friess, A.; Parlar, H., Evaluation of a new optical 

sensor for measuring dissolved oxygen by comparison with standard analytical methods. 

Monatsschr. Brauwiss. 2008, (Mar./Apr.), 66-80. 

5. 5210 Biochemical Oxygen Demand. In Standard Methods for the Examination of 

Water and Wastewater, 21 ed.; Eaton, A., Ed. APHA: 2005; pp 5-2 : 5-7. 

19 | Page
20 | Page

17. TABLES 

17.1. DO BLANK PERFORMANCE 

RPD RDO to Membrane: BOD Blanks

RPD NSRDO 

Initial

RPD SRDO 

Initial

RPD NSRDO 

Final

RPD SRDO 

Final

Average 1.25 1.00 1.43 0.99

Standard 

Deviation 1.51 1.58 1.71 1.74

Bias and Precision 

BOD Blank 

 NSRDO SRDO Membrane 

Overall Bias (mg/L) -0.10 -0.10 -0.10 

Overall 

Precision (mg/L) 0.12 0.12 0.12 

17.2. LOWER OF OPERATIONAL RANGE OF THE RDO DO METER 

RDO Stirred and Non-Stirred Lower Limit of Determination 

Zero Solution Reading 

Laboratory # Mean 

(mg/L) 

Pooled Standard 

Deviation 

(mg/L) 

Calculated 

Confidence Interval 

(mg/L) 

1 0.053 0.025 0.03 

2 0.072 0.053 0.07 

3 0.043 0.006 0.01 

4 0.022 0.007 0.01 

5 0.047 0.021 0.04 

6 0.162 0.008 0.01 

7 0.067 0.047 0.10 

Overall 0.071 0.037 0.04 
17.3. GLUCOSE/GLUTAMIC ACID PERFORMANCE 

Glucose-Glutamic Acid BOD 

Laboratory # 

Non Stirred RDO 

Mean 

(mg/L) 

Standard 

Deviation (Pooled) 

%RSD 

1 198 10.03 5.06% 

2 197 7.63 3.86% 

3 211 19.70 9.32% 

4 203 9.25 4.57% 

5 173 10.92 6.31% 

6 188 21.39 11.35% 

7 203 20.89 10.28% 

8 194 14.76 7.61% 

9 207 23.64 11.40% 

Overall 197 17.07 8.66% 

Glucose-Glutamic Acid BOD 

Laboratory # 

Stirred RDO 

Mean 

(mg/L) 

Standard 

Deviation (Pooled) 

 %RSD 

1 203 7.09 3.49% 

2 199 7.18 3.61% 

3 198 28.36 14.30% 

4 179 6.96 3.88% 

5 204 9.32 4.56% 

6 255 24.10 9.46% 

7 231 28.57 12.39% 

8 205 23.53 11.47% 

Overall 212 24.39 11.50% 

21 | Page
Glucose-Glutamic Acid BOD 

Laboratory # 

Membrane 

Mean 

(mg/L) 

Standard 

Deviation (Pooled) 

%RSD 

1 201 6.21 3.09% 

2 230 9.18 3.99% 

3 214 24.66 11.55% 

4 205 14.82 7.23% 

5 179 6.70 3.74% 

6 208 12.92 6.23% 

7 257 23.24 9.03% 

9 233 27.29 11.71% 

10 206 24.09 11.70% 

Overall 214 19.97 9.35% 

22 | Page
RDO Compared to Membrane Glucose-Glutamic Acid BOD F Tests 

Laboratory # # of 

Data Sets 

Degrees of 

Freedom 

F Test 

NSRDO 

Upper F Level 

95% 

F Test 

SRDO 

1 15 14 2.61 2.48 1.30 2 3 2 1.45 19.00 0.61 3 5 4 1.57 6.39 3.02 4 8 7 2.57 3.79 3.66 5 4 3 0.38 9.28 1.08 6 12 11 2.74 2.82 1.92 7 6 5 1.24 5.05 1.08 8 10 9 3.42 3.18 1.10 9 8 7 1.04 3.79 0.95 

Overall 71 62 1.37 1.52 1.49 

Precision and Bias 

Glucose-Glutamic Acid BOD 

NSRDO SRDO 

Overall Bias (mg/L) -0.93 11.32 15

Overall Precision (mg/L) 17.91 39.90 

RPD RDO to Membrane BOD GGA 

RPD NSRDO Initial RPD SRDO Initial RPD NSRDO Final RPD SRDO Final RPD NSRDO Differen

Average 2.55 2.31 15.36 6.08 8.72 

Standard Deviation 3.03 2.46 15.34 6.35 8.56 
17.4. OTHER WATER AND WASTEWATER BOD MATRIXES 

RPD RDO to Membrane BOD Seed 

RPD NSRDO Initial RPD SRDO Initial RPD NSRDO Final RPD SRDO Final RPD NSRDO Differen

Average 2.87 2.34 10.55 6.04 11.86 

Standard Deviation 2.39 2.56 9.50 11.96 16.64 

RPD RDO to Membrane BOD Influent 

RPD NSRDO Initial RPD SRDO Initial RPD NSRDO Final RPD SRDO Final RPD NSRDO Differen

Average 2.94 2.45 13.56 5.18 27.49 

Standard Deviation 3.04 2.90 14.53 13.02 65.79 

RPD RDO to Membrane BOD Effluent 

RPD NSRDO Initial RPD SRDO Initial RPD NSRDO Final RPD SRDO Final RPD NSRDO Differen

Average 1.25 1.00 1.43 0.99 18.01 

Standard Deviation 2.85 2.60 10.27 6.02 27.45 

RPD RDO to Membrane BOD Industrial 

RPD NSRDO Initial RPD SRDO Initial RPD NSRDO Final RPD SRDO Final RPD NSRDO Differen

Average 2.61 2.18 8.85 4.12 18.13 

Standard Deviation 3.67 3.70 11.01 8.61 28.20 
RPD RDO to Membrane BOD Surface Water 

RPD NSRDO Initial RPD SRDO Initial RPD NSRDO Final RPD SRDO Final RPD NSRDO Differen

Average 1.25 1.00 1.43 0.99 18.01 

Standard Deviation 1.72 1.35 23.91 2.30 28.82 

Table X: BOD Tap Water 

RPD NSRDO 

Initial RPD SRDO Initial RPD NSRDO Final RPD SRDO 

Average 2.58 2.02 3.60 2.17 

Standard Deviation 2.35 1.55 2.49 2.07 
APPENDIXES 

i | Page
ii | Page

Dissolved Oxygen in Saturated Water
 SOLUBILITY OF OXYGEN IN WATER EXPOSED TO WATER-SATURATED AIR 

AT ATMOSPHERIC PRESSURE (101.3 KPA)1

Oxygen Solubility mg/L Oxygen Solubility mg/L

Temperature o

C Chlorinity: 0 5.0 10.0 15.0 20.0 25.0 Temperature oC Chlorinity: 0 5.0 10.0 15.0 20.0 25.0 

0.0 14.621 13.728 12.888 12.097 11.355 10.657 26.0 8.113 7.711 7.327 6.962 6.615 6.285 

1.0 14.216 13.356 12.545 11.783 11.066 10.392 27.0 7.968 7.575 7.201 6.845 6.506 6.184 

2.0 13.829 13.000 12.218 11.483 10.790 10.139 28.0 7.827 7.444 7.079 6.731 6.400 6.085 

3.0 13.460 12.660 11.906 11.195 10.526 9.897 29.0 7.691 7.317 6.961 6.621 6.297 5.990 

4.0 13.107 12.335 11.607 10.920 10.273 9.664 30.0 7.559 7.194 6.845 6.513 6.197 5.896 

5.0 12.770 12.024 11.320 10.656 10.031 9.441 31.0 7.430 7.073 6.733 6.409 6.100 5.806 

6.0 12.447 11.727 11.046 10.404 9.799 9.228 32.0 7.305 6.957 6.624 6.307 6.005 5.717 

7.0 12.139 11.442 10.783 10.162 9.576 9.023 33.0 7.183 6.843 6.518 6.208 5.912 5.631 

8.0 11.843 11.169 10.531 9.930 9.362 8.826 34.0 7.065 6.732 6.415 6.111 5.822 5.546 

9.0 11.559 10.907 10.290 9.707 9.156 8.636 35.0 6.950 6.624 6.314 6.017 5.734 5.464 

10.0 11.288 10.656 10.058 9.493 8.959 8.454 36.0 6.837 6.519 6.215 5.925 5.648 5.384 

11.0 11.027 10.415 9.835 9.287 8.769 8.279 37.0 6.727 6.416 6.119 5.835 5.564 5.305 

12.0 10.777 10.183 9.621 9.089 8.586 8.111 38.0 6.620 6.316 6.025 5.747 5.481 5.228 

13.0 10.537 9.961 9.416 8.899 8.411 7.949 39.0 6.515 6.217 5.932 5.660 5.400 5.152 

14.0 10.306 9.747 9.218 8.716 8.242 7.792 40.0 6.412 6.121 5.842 5.576 5.321 5.078 

15.0 10.084 9.541 9.027 8.540 8.079 7.642 41.0 6.312 6.026 5.753 5.493 5.243 5.005 

16.0 9.870 9.344 8.844 8.370 7.922 7.496 42.0 6.213 5.934 5.667 5.411 5.167 4.933 

17.0 9.665 9.153 8.667 8.207 7.770 7.356 43.0 6.116 5.843 5.581 5.331 5.091 4.862 

18.0 9.467 8.969 8.497 8.049 7.624 7.221 44.0 6.021 5.753 5.497 5.252 5.017 4.793 

19.0 9.276 8.792 8.333 7.896 7.483 7.090 45.0 5.927 5.665 5.414 5.174 4.944 4.724 

20.0 9.092 8.621 8.174 7.749 7.346 6.964 46.0 5.835 5.578 5.333 5.097 4.872 4.656 

21.0 8.915 8.456 8.021 7.607 7.214 6.842 47.0 5.744 5.493 5.252 5.021 4.801 4.589 

22.0 8.743 8.297 7.873 7.470 7.087 6.723 48.0 5.654 5.408 5.172 4.947 4.730 4.523 

23.0 8.578 8.143 7.730 7.337 6.963 6.609 49.0 5.565 5.324 5.094 4.872 4.660 4.457 

24.0 8.418 7.994 7.591 7.208 6.844 6.498 50.0 5.477 5.242 5.016 4.799 4.591 4.392 

25.0 8.263 7.850 7.457 7.083 6.728 6.390 

NOTE: 

1. The table provides three decimal places to aid interpolation. When computing 

saturation values to be used with measured values, such as in computing DO deficit in a 

receiving water, precision of measured values will control choice of decimal places to be 

used. 

2. Equations are available to compute DO concentration in fresh water1–3 and in 

seawater1 at equilibrium with water-saturated air. Figures and tables also are 

available.3

Calculate the equilibrium oxygen concentration, C*, from equation: 

ln C* = -139.34411 + (1.575701 x 105

/T) - (6.642308 x 107/T 2) + (1.243800 x 1010/T 3) - 

(8.621949 x 1011/T 4

) - Chl [(3.1929) x 10 2) - (1.9428 _ 101/T) + (3.8673 x 103/T 2)] 

where: 

iii | Page
C* = equilibrium oxygen concentration at 101.325 kPa, mg/L, 

T _ temperature (°K) = °C + 273.150, (°C is between 0.0 and 40.0 in the equation; the 

table is accurate up to 50.0), and 

Chl = Chlorinity (see definition in Note 4, below). 

Example 1: At 20°C and 0.000 Chl, ln C* = 2.207442 and C* = 9.092 mg/L; 

Example 2: At 20°C and 15.000 ChL, 

ln C* = (2.207442) – [15.000 x (0.010 657)] 

= 2.0476 and C* = 7.749 mg/L. 

When salinity is used, replace the chlorinity term (-Chl[. . .]) by: 

- S(1.7674 x 10-2) - (1.0754 x 101

/T) + (2.1407 x 103/T 2) 

where: 

S = salinity (see definition in Note 4, below). 

3. For nonstandard conditions of pressure: (Excel worksheet is available) 

( )( )

( )( ) ⎥

⎥

⎦

⎤

⎢

⎢

⎣

⎡

−−

−− = θ

θ

11

11

*

WV

WV

P

P

P P

P

PCC

where: 

Cp = equilibrium oxygen concentration at nonstandard pressure, mg/L, 

C* = equilibrium oxygen concentration at standard pressure of 1 atm, mg/L. 

P = nonstandard pressure, atm, 

PWV = partial pressure of water vapor, atm, computed from: ln PWV = 11.8571 x 

(3840.70/T) - (216961/T 2

), 

T = temperature, °K, 

θ = 0.000975 - (1.426 x 10-5t) + (6.436 x 10-8t

2

), and 

t = temperature, °C. 

iv | Page
N.B.: Although not explicit in the above, the quantity in brackets in the equation for Cp 

has dimensions of atm1 per Reference 4, so that P multiplied by this quantity is 

dimensionless. 

Also, the equation for ln PWV is strictly valid for fresh water only, but for practical 

purposes no error is made by neglecting the effect of salinity. 

An equation for Pwv that includes the salinity factor may be found in Reference 1. 

Example 3: At 20°C, 0.000 Chl, and 0.700 atm, 

Cp = C* P (0.990092) = 6.30 mg/L. 

4. Definitions: 

Salinity: Although salinity has been defined traditionally as the total solids in water after 

all carbonates have been converted to oxides, all bromide and iodide have been replaced 

by chloride, and all organic matter has been oxidized (see Section 2520), the new scale 

used to define salinity is based on the electrical conductivity of seawater relative to a 

specified solution of KCl in water.5

 The scale is dimensionless and the traditional 

dimension of parts per thousand (i.e., g/kg of solution) no longer applies. 

Chlorinity: Chlorinity is defined in relation to salinity as follows: 

Salinity = 1.806 55 x chlorinity 

Although chlorinity is not equivalent to chloride concentration, the factor for converting a 

chloride concentration in seawater to include bromide, for example, is only 1.0045 (based 

on the relative molecular weights and amounts of the two ions). Therefore, for practical 

purposes, chloride concentration (in g/kg of solution) is nearly equal to chlorinity in 

seawater. For wastewater, it is necessary to know the ions responsible for the solution’s 

electrical conductivity to correct for their effect on oxygen solubility and use of the 

tabular value. If this is not done, the equation is inappropriate unless the relative 

composition of the wastewater is similar to that of seawater. 

Appendix References 

1. BENSON, B.B. & D. KRAUSE, JR. 1984. The concentration and isotopic 

fractionation of oxygen dissolved in freshwater and seawater in equilibrium with the 

atmosphere. Limnol. Oceanogr. 29:620. 

2. BENSON, B.B. & D. KRAUSE, JR. 1980. The concentration and isotopic 

fractionation of gases dissolved in fresh water in equilibrium with the atmosphere: I. 

Oxygen. Limnol. Oceanogr. 25:662. 

v | Page
vi | Page

3. MORTIMER, C.H. 1981. The oxygen content of air-saturated fresh waters over ranges 

of temperature and atmospheric pressure of limnological interest. Int. Assoc. Theoret. 

Appl. Limnol., Communication No. 22, Stuttgart, West Germany. 

4. SULZER, F. & W.M. WESTGARTH. 1962. Continuous D. O. recording in activated 

sludge. Water Sewage Works 109: 376. 

5. UNITED NATIONS EDUCATIONAL, SCIENTIFIC & CULTURAL 

ORGANIZATION. 1981. Background Papers and Supporting Data on the Practical 

Salinity Scale 1978. Tech. Paper Mar. Sci. No. 37. 4500-O D. Permanganate 

Modification 
Density of Water (g/mL) vs. Temperature (°C) 

Whole degrees are listed down the left hand side of the table, while tenths of a degree are listed across the top. So 

to find the density of water at say 5.4 °C, you would first find the whole degree by searching down the left hand 

column until you reach '5'. Then you would slide across that row until you reach the column labeled '0.4'. The 

density of water at 5.4 °C is 0.999957 g/mL. 

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 

0 0.999841 0.999847 0.999854 0.999860 0.999866 0.999872 0.999878 0.999884 0.999889 0.999895

1 0.999900 0.999905 0.999909 0.999914 0.999918 0.999923 0.999927 0.999930 0.999934 0.999938

2 0.999941 0.999944 0.999947 0.999950 0.999953 0.999955 0.999958 0.999960 0.999962 0.999964

3 0.999965 0.999967 0.999968 0.999969 0.999970 0.999971 0.999972 0.999972 0.999973 0.999973

4 0.999973 0.999973 0.999973 0.999972 0.999972 0.999972 0.999970 0.999969 0.999968 0.999966

5 0.999965 0.999963 0.999961 0.999959 0.999957 0.999955 0.999952 0.999950 0.999947 0.999944

6 0.999941 0.999938 0.999935 0.999931 0.999927 0.999924 0.999920 0.999916 0.999911 0.999907

7 0.999902 0.999898 0.999893 0.999888 0.999883 0.999877 0.999872 0.999866 0.999861 0.999855

8 0.999849 0.999843 0.999837 0.999830 0.999824 0.999817 0.999810 0.999803 0.999796 0.999789

9 0.999781 0.999774 0.999766 0.999758 0.999751 0.999742 0.999734 0.999726 0.999717 0.999709

10 0.999700 0.999691 0.999682 0.999673 0.999664 0.999654 0.999645 0.999635 0.999625 0.999615

11 0.999605 0.999595 0.999585 0.999574 0.999564 0.999553 0.999542 0.999531 0.999520 0.999509

12 0.999498 0.999486 0.999475 0.999463 0.999451 0.999439 0.999427 0.999415 0.999402 0.999390

13 0.999377 0.999364 0.999352 0.999339 0.999326 0.999312 0.999299 0.999285 0.999272 0.999258

14 0.999244 0.999230 0.999216 0.999202 0.999188 0.999173 0.999159 0.999144 0.999129 0.999114

15 0.999099 0.999084 0.999069 0.999054 0.999038 0.999023 0.999007 0.998991 0.998975 0.998959

16 0.998943 0.998926 0.998910 0.998893 0.998877 0.998860 0.998843 0.998826 0.998809 0.998792

17 0.998774 0.998757 0.998739 0.998722 0.998704 0.998686 0.998668 0.998650 0.998632 0.998613

18 0.998595 0.998576 0.998558 0.998539 0.998520 0.998501 0.998482 0.998463 0.998444 0.998424

19 0.998405 0.998385 0.998365 0.998345 0.998325 0.998305 0.998285 0.998265 0.998244 0.998224

20 0.998203 0.998183 0.998162 0.998141 0.998120 0.998099 0.998078 0.998056 0.998035 0.998013

21 0.997992 0.997970 0.997948 0.997926 0.997904 0.997882 0.997860 0.997837 0.997815 0.997792

22 0.997770 0.997747 0.997724 0.997701 0.997678 0.997655 0.997632 0.997608 0.997585 0.997561

23 0.997538 0.997514 0.997490 0.997466 0.997442 0.997418 0.997394 0.997369 0.997345 0.997320

24 0.997296 0.997271 0.997246 0.997221 0.997196 0.997171 0.997146 0.997120 0.997095 0.997069

25 0.997044 0.997018 0.996992 0.996967 0.996941 0.996914 0.996888 0.996862 0.996836 0.996809

26 0.996783 0.996756 0.996729 0.996703 0.996676 0.996649 0.996621 0.996594 0.996567 0.996540

27 0.996512 0.996485 0.996457 0.996429 0.996401 0.996373 0.996345 0.996317 0.996289 0.996261

vii | Page
viii | Page

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 

28 0.996232 0.996204 0.996175 0.996147 0.996118 0.996089 0.996060 0.996031 0.996002 0.995973

29 0.995944 0.995914 0.995885 0.995855 0.995826 0.995796 0.995766 0.995736 0.995706 0.995676

30 0.995646 0.995616 0.995586 0.995555 0.995525 0.995494 0.995464 0.995433 0.995402 0.995371
ix | Page

BOD Dilution Tables 

Table 1: Dilution Table for Unseeded Samples 

Anticipated BOD Dilution Factor % Dilution ml of Sample per 

300 ml Bottle 

Dilution BOD 

Range 

5 1 100 300 2-7 

9 2 50 150 4-14 

13 3 33 100 6-60 

16 4 25 75 8-25 

22 5 20 60 10-35 

32 7.5 13 40 15-50 

45 10 10 30 20-70 

65 15 607 20 30-100 

90 20 5.0 15 40-140 

120 25 4.0 12 50-175 

130 30 3.3 10 60-200 

160 37 2.7 8 75-250 

200 42 2.4 7 85-300 

225 50 2.0 6 100-350 

260 60 1.7 5 120-400 

325 75 1.3 4 150-500 

475 100 1.0 3 200-700 

Table 2: Dilution Table for Seeded Samples 

Assume Initial DO is > 8.0 mg/L and the Seed Correction is between 0.5 mg/L and 1.0 mg/L 

Anticipated BOD Dilution Factor % Dilution ml of Sample per 

300 ml Bottle 

Dilution BOD 

Range 

4 1 100 300 2-6 

8 2 50 150 4-12 

12 3 33 100 6-18 

16 4 25 75 8-24 

22 5.5 18.3 55 11-33 

30 7.5 13.3 40 15-45 

40 10 10 30 20-60 

48 12 8.3 25 24-72 

60 15 6.7 20 30-90 

80 20 5 15 40-120 

100 25 4 12 50-150 

120 30 3.3 10 60-180 

150 37.5 2.7 8 75-180 

200 50 2 6 100-225 

240 60 1.7 5 120-360 

300 75 1.3 4 150-450 

400 100 1 3 200-600