# Alternate Test Procedure #N09-0021
In-Situ Inc. Method 1004-8-2009 Carbonaceous Biochemical Oxygen Demand (CBOD) Measurement by Optical Probe2 | Page TABLE OF CONTENTS

DISSOLVED OXYGEN and CBOD ...........................................................................................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 M ETER CALIBRATION ...........................................13 11. DISSOLVED OXYGEN and CBOD PROCEEDURES .....................................15 12. CBOD CALCULATIONS ...................................................................................17 13. METHOD PERFORMANCE ..............................................................................17 14. POLLUTION PREVENTION .............................................................................18 15. WASTE MANAGEMENT ..................................................................................18 16. REFERENCES ....................................................................................................18 17. TABLES ..............................................................................................................20

APPENDIXES .............................................................................................................................i Dissolved Oxygen in Saturated Water .............................................................................ii 18. Density of Water (g/mL) vs. Temperature (°C) ...................................................vii 19. CBOD Dilution Tables ........................................................................................ix

## DISSOLVED OXYGEN and CBOD 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. Carbonaceous Biochemical Oxygen Dema nd (CBOD) The CBOD determination is an empirical test in which standardized laboratory procedures are used to determine the relative oxygen consumption of wastew aters, 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 chem ical compound and the quenching of that emission by oxygen 1 This quenching photophysics mechanism is described by the Stern-Volmer equation for di ssolved 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 fi xed photomer on an oxygen optical probe to take into account the 2-dimensional photophysics 3 : 

## [ ] [ ] 2 2 0

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 SV SV KO KO I If f + + = +

h solid state photomer The determination of oxygen concentration by luminescence quenching has a linear response over a broad range of oxygen concen trations and has excellent accuracy and reproducibility 4

. 2.2. The CBOD tests have the wi dest application in measuring waste loadings to treatment plants and in evaluating the CB OD-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 su ch as sulfides and ferrous iron. It also may measure the amount of oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand) unless th eir oxidation is prevented by an inhibitor. The seeding and dilution procedures provide an estimate of the CBOD at pH 6.5 to 7.5. 5 3. DEFINITIONS 3.1. BOD: Biochemical Oxygen Demand 3 | Page3.2. CBOD: Carbonaceous Bioc hemical Oxygen Demand 3.3. DI Water: Deionized water 3.4. Duplicate = Repetitive indi vidual analyses of a samp le to measure precision. 3.5. G/GA: A solution of Glucose and Glutamic Acid. 3.6. RPD, Relative Percent Difference: Measure of precision and/or ma trix effects of Duplicates. The RPD must be less than or equal to ( ≤ ) 20% 3.7. Standard Methods: Standard Methods for th e Examination of Water and Wastewater, 21 st Edition or Online 4. INTERFERENCES 4.1. Chemical compounds that compete or interfer e can bias the optical electrode or the membrane electrode. 4.2. Optical electrodes respond to O 2 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 ma y 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 e nvironment 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 ha s 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 accur acy to the nearest 0.01 mL. 6.6. BOD air incubator. Thermostatically controlle d at 20 ±1°C. Exclude all light to prevent possibility of photosynthetic production of DO (Meets requirements of Standard Methods 5210 B 5 ) 6.7. Dissolved oxygen meter and with In-Situ RDO ® probe or suitable luminescence optical electrode. Note: other manufacturer’s luminescen ce optical electrode may not perform in the non-stirred mode.
4 | Page7. 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 oxyge n 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 la boratory 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 wo rking 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 liquo r from an aeration basin, undisinfected effluent, or receiving water from below th e 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 co mmercial seed as per manufacturer’s directions 7.6. PHOSPHATE BUFFER SOLUTION 7.6.1. Dissolve 8.5 g KH 2 PO 4 , 21.75 g K 2 HPO 4 , 33.4 g Na 2 HPO 4 • 7H 2 O, and 1.7 g NH 4 Cl 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 KH 2 PO 4 and 1.7 g NH 4 Cl 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 7.7.1. Dissolve 22.5 g MgSO 4 •7H 2 O in DI water and dilute to 1 L. (ACS Grade or better) 5 | Page7.7.2. Commercial reagent can be purchased. 7.8. CALCIUM CHLORIDE SOLUTION 7.8.1. Dissolve 27.5 g CaCl 2 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 FeCl 3 •6H 2 O 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 (Na 2 SO 3 ) in 200 mL of DI water. Add two drops of sa turated cobalt chloride solution to the sodium sulfite solution. (ACS Grade or better) 7.10.2. Saturated Cobalt Chloride solution; Di ssolve 4.5 grams of cobalt chloride hexahydrate (CoCl 2 • 6 H 2 O) 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 103 o C for 1 hour. Store these in a desicca tor 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 4 o C ± 2 o C. 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 purchas ed, but concentrations may vary. Final concentration must be the same as solution above. Keep solution sterile and refrigerated at 4 o C ± 2 o C. Replace if solution becomes cloudy or shows signs of bacterial or chemical contamination 7.12. SULFURIC ACID 7.12.1. Concentrated H 2 SO 4 , (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) 7.13.2. Commercial solution can be purchased.

6 | Page7 | Page 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 MnSO 4 • 4H2O, 400 g MnSO 4 • 2H 2 O, or 364 g MnSO 4 • H 2 O (ACS Grade or better) in DI water, filter, and dilute to 1 L. The MnSO4 solution should not give a color with star ch when added to an acidified potassium iodide (KI) solution. (A CS 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-satura ted 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 NaN 3 dissolved in 40 mL DI water. This reag ent should not give a color with starch solution when diluted and acidifi ed. (ACS Grade or better) 7.16.2. For supersaturated samples—Dissolve 10 g NaN 3 in 500 mL DI water. Add 480 g sodium hydroxide (NaOH) and 750 g sodium i odide (NaI), and s tir until dissolved. There will be a white turbidity due to sodium carbonate (Na 2 CO 3 ). CAUTION , Do not acidify this solution because toxic hydrazoic acid fumes may be produced. (ACS Grade or better on chemicals) Comme rcial 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 thiosu lfate-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 st andardize. (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, a dd 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 solubl e 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.8 | Page 7.19. POTASSIUM BI-IODATE 7.19.1. 0.0021 Molar potassium bi-iodate solution, ( 0.0021 M): Dissolve 818.8 mg KH(IO 3 ) 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, ( 0.00021 M): 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 6 N H2SO4 or a few drops of conc H 2 SO 4 . 7.20.3. Add 20.00 mL of 0.0021 M bi-iodate soluti on with a Class A pipette and mix well. 7.20.4. Titrate with an appropriate Class A burett e the liberated iodine with thiosulfate titrant, adding starch toward end of titra tion (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 thiosulfat e titrant used. Read the bure tte to an accuracy of only one half (1/2) of a volume un it between each demarcation. 7.20.6. Calculate the Molarity of the thiosulfate.

) ( ) (0021.01220 )( titrantethiosulfat ml iodatebipotassium Molarity ml ethiosulfat Molarity − × ×

=

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 DETERM INATION (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 st opper 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 6 N H2SO4 or a few drops of concentrated H 2 SO 4 . 7.21.4. Add the potassium bi-iodate solution from the BOD bottle and mix well.9 | Page 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 liberate d iodine with standardized thiosulfate titrant, adding starch toward end of titra tion (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 thiosulfat e 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

12) (00021.0 )() ( ),( ×− ×

= iodatebipotassium Molarity ethiosulfat mlethiosulfat MolarityM mlvolume BottleBOD

7.22. BOD BOTTLE VOLUME DETERM INATION (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 w ith 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. R ecord the volume to the nearest 0.1 mL. 

## [ ] ( ,) ( ( ,) BOD Bottle Final weight g BOD Bottle InitialWei , ) g ht 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 containe rs, preservation techniques and holding times. 9. QUALITY CONTROL 9.1. INNITIAL 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 perform ance. 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 salinit y of the air saturated water. Calculate the standard deviation for the combined values for each analysis. This value is S Pooled . Utilize this value to calculate the t-Te st 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 measurem ent 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 probe are serviced. Measur e 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 calculate the confidence interval for the mean of the zero solution. The mean and confidence level must be below the minimum dissolved oxygen meas urement required for the analysis. 

# ( ) ( ) 1 2 4 1 X X S ⎡ ⎤

−
= ⎢ ⎥

− ⎢ ⎥ ⎣ ⎦



## ∑

2 2 t S Confidence Interval X
t Student Sided t Distribution × = ±

=

10 | Page9.4. CBOD DILUTION WATER BLANKS 9.4.1. Prepare CBOD dilution water blanks to represent each type of test being performed within the batch. Run these QC samples with each batch of CBOD. 9.4.1.1. Unseeded CBOD blank: A 300 mL bottle of BOD dilution water with approximately 0.1 g Nitrification In hibitor. (Minimum 3 per set) 9.4.2. 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 CBOD ≤ 0.2 mg/L

9.5. GLUCOSE AND GLUTAMIC ACID CHECK (G/GA) 9.5.1. Transfer 6 mL of the glucose - glutam ic acid solution to a BOD bottle of known concentration. 9.5.2. Add sufficient seed to achieve ac ceptable dissolved oxygen depletion. 9.5.3. Fill BOD bottle with BOD dilution water and approximately 0.1 g Nitrification Inhibitor. (Minimum three G-GA checks) 9.5.4. Determine the 5 Day CBOD 9.5.5. Passing results will have a CBOD of 198 ( + 30.5) mg/L. 9.5.6. Run these QC samples with each batch of CBOD samples. It is important to realize that GGA is not inte nded 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. 9.6. DUPLICATE SAMPLES 9.6.1. A duplicate sample will be set for each CBOD set of seeded and unseeded samples. QA/QC criteria for the Relative Pe rcent Difference and Precision and Bias will be developed. 9.6.2. Duplicate checks will be run on all Hi gh 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. 11 | Page9.8. BOD BOTTLE VOLUME 9.8.1. The use of a BOD bottle for Winkler titration volume or CBOD 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 CBOD 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 K nown 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 CBOD 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 a nd bias analysis at a minimum on a quarterly basis and keep control r ecords of the precission and bias. 9.9. CBOD WORKING RANGE AND DETECTION LIMIT 9.9.1. The working range is equal to the diff erence 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 re siduals as follows (See formula at 12.2 for formula variable definitions): 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 th e 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 th e 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 di fference between the sample DO depletion and the seed correction.

12 | Page10. 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 w ith a venting cap. Allow sa turated dilution water to vent to the atmosphere for approximate ly 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 th e 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, ad d 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 pi pette 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 agit ation. Cap with stopper and pour off excess from the water seal . Mix well by inversion. 10.1.4. Remove the BOD bottle stopper, add 1m L 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 pi pette 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 agit ation. 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 a nd precipitate to the bottom third of the bottle. 10.1.6. Carefully remove the stopper and i mmediately add 1mL of concentrated H 2 SO 4 solution to the BOD bottle by inserti ng 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 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 st arch solution to the pale yellow solution. The solution sh ould 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 thiosulf ate titrant used. Read the burette to an accuracy of only one half (1/2) of a volume unit betw een each demarcation. 10.1.12. Calculate the concentration of dissolved oxygen:

13 | Page14 | Page 

## ( ) ( ) ( ) 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 va lue to the nearest 0.01 mg/L. 10.2. BAROMETRIC DETERMINATION: AIR SATURATED WATER 10.2.1. Determine the barometric pressure fo r 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 la boratory in centigrade and Kelvin. 10.2.4. Determine the DO in air saturated wate r from the tables and formulas in Appendix 1. 10.3. BAROMETRIC DETERMINATION: WATER SATURATED AIR 10.3.1. Determine the barometric pressure fo r 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 la boratory in centigrade and Kelvin. 10.3.4. Determine the DO in water saturated water from the tables and formulas in Appendix 1. 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 CoCl 2 -6 H 2 O11. DISSOLVED OXYGEN AND CBOD PROCEEDURES 11.1. DISSOLVED OXYGEN DETERMINATION 11.1.1. Calibrate the DO meter as per manufactur er’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 oxyge n RDO probe in the bo ttle 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 accelerat e the dissolved oxygen meter equilibrium. 11.1.4. Record the dissolved oxygen concentration. 11.2. SAMPLE PREPARATION AND DETERMINATION FOR CBOD 11.2.1. Bring the sample to ambien t 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 ac id or sodium hydroxide. 11.2.4. Aliquots of the neutralized sample are transferred to 300 mL BOD bottles using the procedures listed below. These CBOD sa mples 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 diluti ons (minimum of 3) of the prepared sample to bracket the predicted CB OD. See Appendix for dilution charts

. 11.2.5. Use a magnetic stirrer to s tir all samples, including dilu tions, while an aliquot is being taken. These samples and their dilutions must be in cylindrical containers to insure an even dist ribution of solids wh ile 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 as set by Standard Methods. 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 fo r a single BOD bottle. Do not transfer multiple aliquot volumes in your pipette dr aw. 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 (minim um 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 contai ner and sampled as outlined above. 15 | Page11.2.10. Add approximately 0.1 g of Nitr ification Inhib itor (2-chloro-6- (trichloro-methyl) pyridine) to each 300mL bottle before adding BOD dilution water. 11.2.11. If the sample is being prepared as a seeded sample, add enough prepared seed to the sample to achieve acceptable dissolved oxygen depletion. 11.2.12. Add BOD Dilution water to each CBOD sample bottle so as to completely fill the bot tle with no air spaces or bubbles when the stopper is placed in the bottle. 11.2.13. If DO measurement is to be taken with a non-stirred RDO probe, cap the BOD bottle and invert sample multiple times to mix well. 11.2.14. Place the dissolved oxygen probe 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 prio r to accepting dissolved oxygen value. 11.2.15. Record the DO of the sample, stopper the bottle, add DI water to the water seal if needed, cap the water seal, and incubate for 5 days at 20°C ± 1°C. Exclude light to avoid growth of al gae in the bottles during incubation. 11.2.16. Upon completion of the 5-day incuba tion± 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. 11.2.17. Calculate the BODs from the formula below. 11.2.18. Only bottles, including seed controls, giving a minimum DO depletion of 2.0 mg/L and a residual DO of at l east 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 CBOD Uptake: Typically a 10, 20, and 30 mL sample of seed added to 3 separate BOD bottles with approximately 0.1 g Nitrification Inhibitor and diluted with BOD dilution water. R un these QC samples with each batch of seeded CBOD. 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 stra ight 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

16 | Page11.3.4. For the ratio method, divide the DO deplet ion by the volume of seed in mLs for each seed control bottle having a 2.0 mg/L depletion and great er than 1.0 mg/L minimum residual DO and average the results. 12. CBOD 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 

## ∑ ∑

12.2. CBOD 5 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 CBOD as follows :

D1 Dissolved Oxygen of diluted 

### , / S 

### D D SV CBOD mg l P

= 

### − − = sample immediately after preparation, mg / L, D2 Dissolved Oxygen of diluted 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 = ° = Δ

= 

## ( ) seeded , 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 CBOD of th e RDO to membrane electrode from a multiple laboratory study are in Section 17.1. 13.1.2. Bias and precision for R DO blank measurement compared to the membrane electrode for multiple laboratory study are in Section 17.1. 13.1.3. Lower of operational rang e 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.

17 | Page13.2. GLUCOSE/GLUTAMIC ACID 13.2.1. The mixture of glucose and glutamic aci d was developed in order to provide a standard with a stable metabolic rate th at could be used in the 5 day CBOD test under a wide variety of applications. F Te st results showing method comparability of the CBOD for the RDO to the memb rane electrode are in Section 17.3. 13.2.2. GGA recoveries of CBOD for the RDO and membrane from a multiple laboratory study are in Section 17.3. 13.2.3. GGA DO RPDs for CBOD for the RDO to me mbrane electrode from a multiple laboratory study are in Section 17.3. 13.2.4. Bias and precision for the RDO GGA CBOD values are compared to the membrane electrode from a multiple laboratory study in Section 17.3 13.3. OTHER WATER AND WASTEWATER MATRIXES 13.3.1. Typical RPD values of the RDO to membrane electrode based on multiple laboratory analysis of varied sample t ypes are illustrated in Section 17.4 The matrixes include wastewater influent, wastewater effluent , surface water, and industrial waste from Significant In dustries and Categorical Industries. 14. POLLUTION PREVENTION 14.1. There are no standards or reagents used in this me thod at the concentrations required that pose a threat to the environmen t. Refer to Waste Management for correct disposal of all chemicals. 15. WASTE MANAGEMENT 15.1. It is the laboratory’s responsibility to co mply with all federal, state, and local regulations governing waste management, par ticularly the hazardous waste identification rules and land disposal restri ctions, and to protect air, water, and land by minimizing and control all releases from fume hoods and be nch operations. Compliance with all sewage discharge permits and regulat ions is also required. 16. REFERENCES 1. Mingoarranz, F. J.; More no-Bondi, M. C.; Garcia-Fresn adillo, 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.

18 | Page4. Titze, J.; Walter, H.; Jacob, F.; Friess, A.; Parlar, H., Evaluation of a new optical sensor for measuring dissolved oxygen by comp arison 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 | Page20 | Page 17. TABLES 17.1. DO BLANK PERFORMANCE.

RPD RDO to Membrane: CBOD Blanks RPD NSRDO Initial RPD SRDO Initial RPD NSRDO Final RPD SRDO Final Average 1.34 0.73 1.54 0.73 Standard Deviation 1.18 0.62 1.57 0.93

Bias and Precision CBOD Blank NSRDO SRDO Membrane Overall Bias (mg/L) -0.10 -0.10 -0.09 Overall Precision (mg/L) 0.12 0.12 0.11

17.2. LOWER OF OPERATIONAL RA NGE OF THE RDO DO METER

RDO Stirred and Non-Stirred Lo wer 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 0.0417.3. GLUCOSE/GLUTAMIC ACID PERFORMANCE

Glucose-Glutamic Acid CBOD

Laboratory # NonStirred RDO

Mean (mg/L) Standard Deviation (Pooled) %RSD

1 202 5.48 2.71% 2 172 † † 3 218 10.34 4.75% 4 167 19.42 11.64% 5 182 15.97 8.78% 6 231 25.08 10.86% 7 221 8.25 3.74% 8 172 19.19 11.16% Overall 191 16.09 8.43%

Table I: Glucose-Glutamic Acid CBOD

Laboratory # Stirred RDO

Mean (mg/L) Standard Deviation (Pooled) %RSD

1 205 5.45 2.66% 2 177 † † 3 205 43.23 21.07% 4 200 16.86 8.43% 5 201 17.38 8.64% 6 244 25.50 10.45% 7 ‡ ‡ ‡ 8 206 18.05 8.76% Overall 207 19.60 9.23%

†: Results are too few to calculate a Standard Deviation ‡: Laboratory did not submit SRDO data.

21 | PageTable J: Glucose-Glutamic Acid CBOD

Laboratory # Membrane

Mean (mg/L) Standard Deviation (Pooled) %RSD

1 203 5.12 2.53% 2 182 † † 3 210 6.53 3.11% 4 203 10.43 5.14% 5 205 12.58 6.14% 6 244 21.35 8.74% 7 222 5.35 2.41% 8 212 22.35 10.54% Overall 210 15.21 7.24%

†: Results are too few to calculate a Standard Deviation ‡: Laboratory did not submit SRDO data.

22 | PageRDO Compared to Membrane Glucose-Glutamic Acid CBOD F TestsLaboratory ## ofData SetsDegrees ofFreedomF TestNSRDOUpper F Level95%F TestSRDOUpper F Level95%114131.1442.5771.1352.577221††††3542.5116.38843.8816.3884873.4663.7872.6123.787512111.6122.8181.9092.8186651.3805.0501.4275.0507322.38219.000‡‡815140.7372.4840.6522.484Overall6556or541.1201.5501.2991.560†: Results are too few to calculate a Standard DeviationPrecision and BiasGlucose-Glutamic Acid CBODNSRDOSRDOMembraneOverall Bias (mg/L)-7912Overall Precision (mg/L)272523RPD RDO to Membrane CBOD GGARPD NSRDO InitialRPD SRDO InitialRPD NSRDO FinalRPD SRDO FinalRPDNSRDO DifferenceRPDSRDO DifferenceAverage2.102.0011.904.4710.293.74Standard Deviation1.491.749.654.299.094.3123 |Page17.4.OTHER WATER AND WASTEWATER CBOD MATRIXESRPD RDO to Membrane CBOD InfluentRPD NSRDO InitialRPD SRDO InitialRPD NSRDO FinalRPD SRDO FinalRPDNSRDO DifferenceRPDSRDO DifferenceAverage1.931.979.552.8511.536.91Standard Deviation1.732.999.314.1810.8311.39RPD RDO to Membrane CBOD EffluentRPD NSRDO InitialRPD SRDO InitialRPD NSRDO FinalRPD SRDO FinalRPDNSRDO DifferenceRPDSRDO DifferenceAverage4.253.738.765.5319.6814.02Standard Deviation8.879.8913.5014.4128.9524.31RPD RDO to Membrane CBOD IndustrialsRPD NSRDO InitialRPD SRDO InitialRPD NSRDO FinalRPD SRDO FinalRPDNSRDO DifferenceRPDSRDO DifferenceAverage1.811.587.652.6113.026.64Standard Deviation2.051.9011.692.9216.449.57RPD RDO to MembraneCBOD Surface WaterRPD NSRDO InitialRPD SRDO InitialRPD NSRDO FinalRPD SRDO FinalRPDNSRDO DifferenceRPDSRDO DifferenceAverage2.101.893.772.0716.2910.95Standard Deviation1.662.287.773.8914.9816.5924 |Page25 |PageTable U: CBOD SeedRPD NSRDO InitialRPD SRDO InitialRPD NSRDO FinalRPD SRDO FinalRPDNSRDO DifferenceRPDSRDO DifferenceAverage2.091.5112.067.1411.764.63Standard Deviation1.361.3510.3018.7511.655.65Table Y: CBOD Tap WaterRPD NSRDOInitialRPD SRDOInitialRPD NSRDOFinalRPD SRDOFinalAverage2.421.871.891.58Standard Deviation1.581.441.851.70

### APPENDIXES

i | Pageii | Page DISSOLVED OXYGEN IN SATURATED WATERSOLUBILITY 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

o C 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 water 1–3 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 10 5 / T ) - (6.642308 x 10 7 / T 2 ) + (1.243800 x 10 10 / T 3 ) - (8.621949 x 10 11 / T 4 ) - Chl [(3.1929) x 10 2 ) - (1.9428 _ 10 1 / T ) + (3.8673 x 10 3 / T 2 )]
where:

iii | PageC * = 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 ch lorinity term (-C hl[. . .]) by:

- S (1.7674 x 10 -2 ) - (1.0754 x 10 1 / T ) + (2.1407 x 10 3 / 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,

P WV = partial pressure of water va por, atm, computed from: ln P WV = 11.8571 x (3840.70/ T ) - (216961/ T 2 ),

T = temperature, °K,
θ = 0.000975 - (1.426 x 10 -5 t ) + (6.436 x 10 -8 t 2 ), and

t = temperature, °C.

iv | PageN.B.: Although not explicit in the above, th e quantity in brackets in the equation for Cp has dimensions of atm 1 per Reference 4, so that P multiplied by this quantity is dimensionless. Also, the equation for ln P WV is strictly valid for fresh water only, but for practical purposes no error is made by negl ecting 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 defi ned traditionally as the to tal 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 elec trical conductivity of s eawater 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 no t equivalent to chlori de 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 near ly 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 e quation 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 | Pagevi | Page 3. MORTIMER, C.H. 1981. The oxygen content of air-saturated fresh waters over ranges of temperature and atmospheric pr essure of limnological interest. Int. Assoc. Theoret. Appl. Limnol., Communication No. 22, Stu ttgart, West Germany. 4. SULZER, F. & W.M. WESTGARTH. 1962. C ontinuous D. O. recording in activated sludge. Water Sewage Works 109: 376. 5. UNITED NATIONS EDUCATIONAL, SCIENTIFIC & CULTURAL ORGANIZATION. 1981. Bac kground Papers and Supporting Data on the Practical Salinity Scale 1978. Tech. Paper Mar. Sci. No. 37. 4500-O D. Permanganate Modification18. 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 | Pageviii | 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 0.995371ix | Page 19. CBOD DILUTION TABLES Table 1: Dilution Table for Unseeded Samples Anticipated CBOD Dilution Factor % Dilution ml of Sample per 300 ml Bottle Dilution CBOD 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 CBOD Dilution Factor % Dilution ml of Sample per 300 ml Bottle Dilution CBOD 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