Section 1 : DISSOLVED OXYGEN (DO) INTRODUCTION
The DO determination measures the amount of dissolved (or free) oxygen present in water or wastewater. Aerobic bacteria and aquatic life such as fish must have DO to survive. Aerobic wastewater treatment processes use aerobic and facultative bacteria to break down the organic compounds found in wastewater into more stable products that will not harm the receiving waters. Wastewater treatment facilities such as lagoons or ponds, trickling filters and activated sludge plants depend on these aerobic bacteria to treat sewage.
The same type of aerobic wastewater treatment process occurs naturally in streams and ponds if organic matter is present, turning these bodies of water into “aerobic wastewater treatment plants.” If sufficient oxygen is not naturally supplied through wind and turbulence to replace the depleted oxygen, the body of water will develop a low DO and become anaerobic (or septic). The results of septic water bodies include fish kills and anaerobic odors.
If the amount of free or DO present in the wastewater process becomes too low, the aerobic bacteria that normally treat the sewage will die. The process will not operate efficiently and septic conditions will occur. The DO test is used to monitor the process to ensure that there is enough dissolved oxygen present to keep the process from becoming septic.
Aerobic: A condition in which “free” or DO is present in an aquatic environment.
Anaerobic: A condition in which “free” or DO is not present in an aquatic environment.
Indicator solution: A solution used to determine the endpoint (usually a color change) when titrating.
Titrate: To titrate a sample, a chemical solution of known strength is added on a drop by drop basis until a color change, precipitate or pH change in the sample is observed.
Remember, always refer to your NPDES Permit and 40 CFR Part 136 for approved methods.
The 18th Edition of “Standard Methods for the Examination of Water and Wastewater” includes two methods for the determination of DO in wastewater, including the Winkler method (azide modification) and the electrometric method using membrane electrodes and a DO meter.
The Winkler method is based on the ability of the DO in a sample to oxidize chemicals added to the sample. The azide modification of the Winkler method was developed to correct for the presence of nitrites in a sample. These compounds, found in almost all wastewater samples, interfere with the other modifications of the Winkler procedure.
The membrane electrode procedure utilizes a meter and electrode, and is based on the rate at which oxygen molecules diffuse (or pass through) a membrane covering a set of electrodes. The oxygen molecules react with an internal filling solution to develop a small electrical charge between the electrodes which can be read on a meter. The readings on the meter correspond directly to the amount of DO present in the sample.
Section 4: SAFETY AND HYGIENE
When testing for dissolved oxygen, the concerns for safety involve wastewater hazards and exposure to chemicals.
Precautions to follow include:
1. Cover all abrasions and wear good quality latex gloves when in direct contact with raw wastewater.
2. Wash hands frequently, and always wash hands prior to eating or smoking.
3. Clean up all spills of wastewater or buffers immediately.
4. Wear a protective smock, apron or lab coat, and surgical or rubber gloves when working in the laboratory to protect clothes and skin.
5. Read all labels carefully and know what to do in case of a spill.
6. Always use a pipette bulb. NEVER pipette anything by mouth.
7. Always pour acids or bases into water, never add water to the acid or base. Mixing concentrated acids or bases with water can create a significant amount of heat.
When using the electrometric method, analyze immediately. The preferred method is in-stream analysis in the field.
Proper sampling is necessary if laboratory testing is to be accurate. The sample must be representative and handled carefully. No matter how accurate the actual testing is, if the sample is not representative, the results of the test will not reflect actual conditions and can lead to poor plant performance.
The dissolved oxygen determination requires a grab sample. The sample must be analyzed as quickly as possible. Select a sampling location where mixing is thorough and the wastewater quality is uniform. The higher the amount of solids in the sample, the more important a well‑mixed sample becomes. If testing is not done on an hourly basis, choose times when the sample best represents the waste characteristics so that changes can be spotted quickly.
Since variations will occur from plant to plant and with the time of day, it is necessary to develop a testing program which gives the most accurate representation of the characteristics of the flow.
When using the electrometric method, analyze immediately. The preferred method is in-stream analysis in the field.
Although DO tests should be performed as quickly as possible, samples which are to be tested using the modified Winkler procedure may be preserved by either of the following methods:
1. If the samples must wait for a time period of up to two hours before testing and have no iodine demand, they may be stored by adding manganous sulfate solution, alkaline‑iodide‑azide solution, sulfuric acid solution and shaking well (making sure that no air bubbles are trapped or formed).
(Protect the samples stored at this point from strong sunlight and titrate as soon as possible.)
2. Those samples which have an iodine demand may be preserved for 4 to 8 hours by adding 0.7 mL concentrated sulfuric acid (H2SO4) and 1.0 mL sodium azide solution (2.0 g NaN3/100 mL distilled water) to the DO bottle. This will stop the biological activity and maintain the dissolved oxygen levels if stored at the same temperature as the sample or if water‑sealed and kept at a temperature of 10 to 20°C. As soon as possible, complete the procedure using 2.0 mL manganese sulfate solution, 3.0 mL Alkaline‑iodide‑azide solution and 2.0 mL concentrated sulfuric acid.
SAMPLE CONTAINERS
For the Winkler analysis, samples should be collected in narrow‑mouth glass‑stoppered BOD bottles with 300 mL capacity with tapered, ground‑glass, pointed stoppers and flared mouths.
Samples must be taken so that the bottle will be completely filled with liquid that has not been in contact with air and no air is trapped under the stopper. An APHA type sampler, or equivalent should be used to collect samples for dissolved oxygen determination. For depths greater than 2 meters use a Kemmerer‑type sampler, or equivalent.
Sample containers must be clean, dry and free of contaminants. The best method to prepare sample containers is by acid cleaning with dichromate cleaning acid, which can either be bought already prepared, or mixed at the plant. CAUTION: DICHROMATE IS EXTREMELY HAZARDOUS AND CAN CAUSE SEVERE BURNS. IN CASE OF CONTACT OR SPILL, RINSE THOROUGHLY WITH LARGE QUANTITIES OF TAP WATER. Wear rubber gloves and safety glasses whenever using cleaning acid. After cleaning with acid, rinse with tap water, followed by distilled water. If possible, allow the containers to dry upside‑down on a rack.
If cleaning acid is not available, use a laboratory grade detergent that is low in phosphorus and nitrates. Wash with a good cleaning brush, and rinse thoroughly with tap water, followed by distilled water. Allow the bottles to dry in an upside‑down position.
All sample containers should be cleaned immediately after the test has been completed to prevent accumulation of residues in the containers which can affect the test.
Section 6: EFFECTS OF TEMPERATURE ON DO
The solubility of oxygen in water is greater at cooler temperatures. When using the membrane electrode method to measure DO, the meter must be temperature calibrated to produce accurate results.
Section 7: INTERPRETATIONS
Some common ranges for DO are listed below:
Sample Common Range (mg/L)
Influent Usually 0; >1 is very good
Primary Effluent Usually 0; >1 is good
Secondary Effluent >5 is good
50% to 95% saturation;
6 to 8 is excellent
Oxidation Ponds 1 to 25
Activated Sludge;
Aeration tank outlet 1 to 3
Quiz 4.1
1. What is dissolved oxygen (DO)?
2. What are the sampling requirements for a DO sample that is to be tested in the laboratory?
3. What are the approved methods for determination of DO?
4. Can a DO sample be preserved? If so, how?
Section 8: MODIFIED WINKLER METHOD
The sample is treated with manganous sulfate, alkaline‑iodide‑azide reagent and finally sulfuric acid. The first two chemicals combine with dissolved oxygen to form a compound which, when acid is added, releases free iodine (from the potassium iodide). Because the amount of iodine released is equal to the amount of oxygen present, the sample can be titrated with either sodium thiosulfate or phenylarsine oxide (PAO) to determine the amount of dissolved oxygen present. Under specific conditions, the amount of sodium thiosulfate or PAO used is equivalent to the amount of dissolved oxygen present in the sample.
EQUIPMENT AND REAGENTS
See Appendix B for the procedures for preparation of the reagents used in this method.
1. Manganous sulfate solution *
2. Alkaline potassium iodide‑sodium azide solution */+
3. Sulfuric acid (H2SO4), concentrated +
4. Starch indicator solution
5. Sodium thiosulfate (Na2S2O3 5H2O), 0.025 N *
6. Phenylarsine oxide (PAO), 0.025 N *
7. Potassium bi‑iodate (KH(IO3)2), 0.025 N
8. Distilled or deionized water
* These reagents are poisonous and should be handled with extreme caution.
+ These reagents are corrosive and should be handled with extreme caution.
EQUIPMENT
1. Burette, graduated to 0.1 mL
2. Burette stand
3. 300 mL glass stoppered BOD bottles
4. 500 mL wide‑mouthed Erlenmeyer flasks
5. Pipettes with elongated tips and minimum volume of 1.0 mL (+/‑ 0.1 mL)
6. Pipette bulb
7. 250 mL graduated cylinders
8. Distilled water rinse bottle
LABORATORY PROCEDURE
1. Collect the sample to be tested in a 300 mL BOD bottle taking special care to avoid adding air to the liquid being collected. Fill bottle completely and add stopper.
2. Remove bottle stopper and add 1 mL of the manganous sulfate solution at the surface of the liquid.
3. Add 1 mL of the alkaline‑potassium iodide‑sodium azide solution at the surface of the liquid.
4. Replace the stopper, avoid trapping air bubbles and shake well by inverting the bottle several times. Repeat shaking after floc has settled halfway. Allow floc to settle a second time.
5. Add 1 mL of concentrated sulfuric acid by allowing the acid to run down the neck of the bottle above the surface of the liquid.
6. Restopper, rinse the top of the bottle to remove any acid and shake well until the precipitate has dissolved.
7. Titrate a volume of treated sample which corresponds to 200 mL of the original sample. This corrects for the loss of some sample during the addition of reagents. This volume calculated using the formula:
mL of sample to titrate = 200 x [300/(300‑2)] = 201 mL
8. Pour 201 mL of sample from the BOD bottle into an Erlenmeyer flask.
NOTE: Since variations occur in the actual volume of each BOD bottle, do not pour 99 mL of sample out of the BOD bottle and assume that 201 mL will be left.
9. If the solution is reddish‑brown in color, titrate with 0.0250 N sodium thiosulfate or 0.0250 N PAO until the solution is a pale yellow (straw) color. Record the amount of titrant used. Add a small quantity of starch indicator and proceed to step 11.
10. If the solution has no reddish‑brown color, or is only slightly colored, add a small quantity (approximately 1 mL) of starch indicator. If no blue color develops, there is zero dissolved oxygen. If a blue color develops, proceed to step 11.
11. Titrate with 0.0250 N sodium thiosulfate or 0.0250 N PAO to the first disappearance of the blue color. Record the total number of mL of sodium thiosulfate or PAO used.
CALCULATIONS
Calculate the concentration of DO in the sample using the following formula:
mg/L DO = (mL titrant x normality of titrant x 8000)/equivalent volume of sample titrated
Example:
If 10.0 mL of titrant are used, then:
mg/L DO = (10.0 x .025 x 8000)/200 = 10.0
If the normality of the titrant is exactly 0.025 N and an equivalent volume of 200 mL of sample is titrated, the amount of oxygen dissolved in the original sample (in mg/l) will be equal to the number of mL of sodium thiosulfate or PAO used in the titration.
INTERFERENCES
In the determination of DO by the Winkler method, there are various materials which cause interferences, including iron salts, organic matter, excessive suspended matter, sulfide, sulfur dioxide, residual chlorine, chromium, cyanide, and certain oxidizing and reducing agents.
If the sample contains 5 mg/L or more of ferric iron salt, add potassium fluoride as the first reagent in the azide modification. Alternately, eliminate ferric iron (Fe III) interference by using 90% phosphoric acid (H3PO4) instead of sulfuric acid for acidification. (This procedure has not been tested for Fe III concentrations above 20 mg/L).
PRECISION AND ACCURACY
DO in a water sample can be determined with a standard deviation of +/‑ 0.020 mg/L in distilled water and about +/‑ 0.060 mg/L in wastewater and secondary effluents. In the presence of an interference, even with the proper modifications, the standard deviation may be as high as +/‑ 0.10 mg/L.
Still greater errors may occur in waters having high organic suspended solids or heavy pollution. Avoid errors due to carelessness in collecting samples, waiting too long to complete testing, or improper testing techniques.
Quiz 4.2
1. What reagents are required for DO determinations using the modified Winkley titration method?
2. What equipment, apparatus or instrumentation is required for DO determinations using the modified Winkler titration method?
3. What interferences affect DO determinations by the modified Winkler titration procedure?
4. If 200 mL of a prepared DO sample requires 8.6 mL of 0.0275 N sodium thiosulfate titrant, what is the DO in mg/L?
Section 9: THE ELECTROMETRIC METER METHOD
There are two types of DO probes available, including a laboratory probe and a field probe. Both types operate on the same principle. The laboratory probe is designed to be used in the laboratory only, while the field probe may be used in the field or the laboratory. If the field probe is used in the laboratory, a magnetic stirrer is required.
An advantage of the field probe is that it eliminates the need to collect, transport and then perform analysis on a sample. The field probe allows the dissolved oxygen to be determined on‑site, without special sampling equipment.
Oxygen‑sensitive membrane probes contain two solid metal electrodes immersed in an electrolyte solution which allows the passage of an electrical current. The electrode pair is covered with a semi-permeable membrane which allows the passage of DO molecules. When the oxygen molecules pass through the membrane, they cause the electrical current already present to change. This change is reflected by the movement of the needle on the meter.
The more DO that is present in the sample, the more the current will change and the higher the reading on the meter will be. The meter is designed so that the amount of DO can be read directly.
EQUIPMENT AND REAGENTS
Reagent chemicals are only required for the membrane procedure if the modified Winkler method is used to calibrate the DO meter.
1. DO meter
2. DO probe, laboratory or field
3. Probe membrane kit
4. Equipment needed for Winkler method to calibrate DO meter
Special care is required in handling lab equipment. DO probes and meters cannot take rough handling. Do not drop or hit probes or meters against hard objects as this can break probes and knock meters out of alignment. Avoid hitting membranes against bottle lips, as they puncture and tear easily and will have to be replaced frequently.
LABORATORY PROCEDURE
Since there are many models and manufacturers of DO meters and probes, this manual will not describe the operating procedures for all meters. For best results, follow the manufacturer’s instruction for operation of meters and probes.
Condensation may collect on the membrane. This will interfere with the readings. Shake off excess water and gently blot dry with a laboratory tissue. The membrane must be stretched tightly across the probe and will not work correctly if air bubbles are present in the probe underneath the membrane. Be sure that the O‑ring holding the membrane to the probe fits securely.
Allow the instrument to warm up for at least 15 minutes before taking the first reading of the day. Leave the instrument on between measurements. This will eliminate the need for warm‑up periods between tests.
Calibrate at least once per day. Distilled or deionized water, and not effluent, should be used for the probe calibration. Always stir the sample while taking a reading.
METER CALIBRATION
To be certain that the DO meter reading provides the correct DO content of the sample, the probe must be calibrated. There are three acceptable methods for the calibration of the meter: modified Winkler, saturated air, and saturated water. It is recommended that the Winkler method be used periodically if one of the other methods is used on a regular basis.
A. Calibration With Modified Winkler Method
1. Take a sample (distilled, deionized or tap water) that does not contain substances which interfere with either the probe reading or the modified Winkler procedure.
2. Carefully divide this reference sample into 4 clean BOD bottles.
3. Prepare 3 of the bottles for the Winkler titration procedure.
4. Titrate all of the prepared portions and average the results.
5. Place the probe in the fourth BOD bottle of the sample for 3 to 5 minutes while agitating or stirring.
6. Calibrate the probe reading to agree with the averaged results of the modified Winkler method.
NOTE: Any error in the modified Winkler method will affect the calibration of the probe.
B. Calibration With Saturated Water
1. Aerate sample for 10‑15 minutes.
2. Measure temperature, then look up the calibration value on the DO concentration vs temperature chart. Correct as necessary for altitude or salinity of samples.
3. Put probe in sample, wait 3 to 5 minutes, and calibrate.
NOTE: This method depends on 100% saturation of DO in the sample. Since 100% saturation is difficult to achieve, an error may be introduced.
C. Calibration With Saturated Air
1. Place probe in moist air calibration chamber (such as a BOD bottle filled halfway with distilled water). Do not turn on probe stirrer.
2. Wait 5 minutes.
3. Measure the air temperature, look up the calibration value, correct as necessary for altitude or salinity of samples and calibrate.
CALCULATIONS
There are no calculations for this method. The DO concentration is read directly from the meter.
INTERFERENCES
There are very few substances which will interfere with the DO method when utilizing the electrometric meter. Salinity caused by dissolved inorganic salts (such as sea water, estuaries, and industrial or manufacturing processes) can influence the probe’s readings. Reactive compounds and gases (like hydrogen sulfide and other sulfur compounds) can interfere with the reading by reducing probe sensitivity. Chlorine residual can create a positive interference. Read the manufacturer’s instructions for the care and handling of the DO electrode and meter for specific remedies, if any, for these interferences.
PRECISION AND ACCURACY
Using most commercially available membrane electrode systems, an operator can obtain an accuracy of +/‑ 0.1 mg/L DO and a precision of +/‑ 0.05 mg/L DO.
Quiz 4.3
1. What equipment, apparatus or instrumentation is required for the DO determinations by the membrane electrode method?
2. What are the accepted methods of calibration of the DO meter and electrode?
3. What interferences can affect DO determinations by the membrane electrode procedure?
A Quality Assurance/Quality Control program is required by the NPDES permit. Quality Assurance (QA) is a set of operating principles that must be followed during sample collection and analysis. Lab bench sheets must be maintained that document when the sample was collected, how it was preserved, and what results were obtained.
Quality Control (QC) includes any testing which is done to prove that the results are reliable. One of every ten samples analyzed should be a Quality Control check. This may include duplicate samples, spike samples, reagent blank analyses and known QC samples obtained from outside sources.
Duplicate sample analysis involves analyzing the same sample twice and comparing the results. The closer the results, the more accurate the analysis. Results should not differ by more than 10%. Spike sample analysis involves adding known amounts of analyte to a sample and calculating the percent recovery. These are discussed further in Chapter 10.
In DO testing, a QC program includes checking DO results from different locations in tanks and streams to determine the most representative location for testing. Duplicate samples should be analyzed when utilizing the Winkler Method to test for variability. Use the Winkler Method to check the calibration of the DO meter when using the membrane electrode method. Sample bench sheets are included in Appendix C.
1. What is dissolved oxygen (DO)?
2. What are the sampling requirements for a DO sample that is to be tested in the laboratory?
Samples for DO measurement in the laboratory must be collect in a 250 - 300 mL BOD bottle.
3. What are the approved methods for determination of DO?
Winkler (azide modification) and membrane electrode methods.
4. Can a DO sample be preserved? If so, how?
Yes, if the Winkler method is to be used for the DO determination. Add the reagents of the Winkler procedure and store for up to two hours, protected from strong sunlight, or add sulfuric acid and store at 10 to 20°C for up to 8 hours.
1. What reagents are required for D.O. determinations using the modified Winkler titration method?
Manganous sulfate solution, alkaline-potassium iodide-sodium azide solution, concentrated sulfuric acid, phenylarsine oxide or sodium thiosulfate and starch indicator.
2. What equipment, apparatus or instrumentation is required for DO determinations using the modified Winkler titration method?
300 mL BOD bottle, 500 mL Erlenmeyer flask, 250 mL graduated cylinder, 25 or 50 mL burette and stand, 1 to 10 mL measuring pipettes, and pipette bulb.
3. What interferences affect DO determinations by the modified Winkler titration procedure?
Iron salts, organic matter, excess suspended solids, sulfide or sulfur dioxide, residual chlorine, chromium or cyanide, and certain oxidizing and reducing compounds.
1. What equipment, apparatus or instrumentation is required for the DO determinations by the membrane electrode method?
Dissolved oxygen meter and electrode (laboratory or field models), BOD bottle, Modified Winkler procedure reagents and equipment.
2. What are the accepted methods of calibration of the DO meter and electrode?
3. What interferences can affect DO determinations by the membrane electrode procedure?
Standard Methods for the Examination of Water and Wastewater, APHA-AWWA-WEF, 18th Edition, 1992, Method 4500-O.
Methods for Chemical Analysis of Water and Wastes, U.S. EPA 600/4-79-020, March 1979, Method 405.1.
NOTES:
1. Manganous sulfate solution
a. Dissolve 480g MnSO4 4H2O, or 400g MnSO4 2H2O, or 364g MnSO4 H2O in distilled water, filter and dilute to 1 liter. This solution should not produce a blue color with starch indicator when added to an acidified potassium iodide (KI) solution.
2. Alkaline‑iodide‑sodium azide solution
a. Dissolve 500g NaOH (or 700g KOH) and 135g NaI (or 150g KI) in 600 to 700 mL of distilled water.
CAUTION: This mixture produces a great deal of heat and caustic fumes. Take extreme care in mixing and handling. Use a borosilicate glass (Pyrex, Kimax, etc.) beaker and avoid breathing fumes or bodily contact with the solution.
b. After the solution has cooled, dilute to 1 liter while slowly and carefully stirring. (If possible, the use of a magnetic stirrer is highly recommended.)
c. Dissolve 10 g sodium azide (NaN3) in 40 mL distilled water.
CAUTION: This solution is poisonous.
d. Add the NaN3 solution, with constant stirring, to the cooled alkaline iodide solution. Allow final mixture to cool to room temperature before using.
NOTE: Sodium azide will decompose over time and is no good after 3 months. Note that sodium and potassium salts can be used interchangeably. This reagent should not produce a blue color with starch indicator when diluted and acidified.
3. Sulfuric acid (H2SO4), concentrated
Use concentrated reagent‑grade acid (H2SO4). Handle Carefully: This chemical will burn hands and clothing. Rinse affected parts with large quantities of tap water to prevent injury and remove contaminated clothing, as residual may still damage skin.
CAUTION: When working with alkaline azide and sulfuric acid, keep a nearby faucet running for frequent hand rinsing.
4. Starch indicator solution
Use either an aqueous solution or soluble starch powder mixture. Prepare an aqueous solution by dissolving 2 g of laboratory grade soluble starch powder and 0.2 g of salicylic acid (as a preservative) in 100 mL of hot distilled water.
5. Sodium thiosulfate standard solution, 0.0250 N
a. Dissolve 6.205 g Na2S2O3 5H2O in distilled water.
b. Add 1.5 mL 6 N NaOH or 0.4 g solid NaOH and dilute to 1 liter.
c. Standardize with bi‑iodate solution.
a. Dissolve approximately 2 g KI, free from iodate, in an Erlenmeyer flask with 100 to 150 mL distilled water.
b. Add 1 mL of 6N H2SO4 or a few drops of concentrated H2SO4, then 20.0 mL standard bi‑iodate solution.
c. Dilute to 200 mL and titrate with the sodium thiosulfate titrant solution. Add starch when a pale straw color is reached and complete the titration until the blue color first disappears. If the sodium thiosulfate is the proper strength, 20.0 mL will be used in the titration. If not, adjust the solution to the proper normality.
6. Standard potassium bi‑iodate solution, 0.0250 N
Dissolve 812.4 mg of potassium bi‑iodate (KH(IO3)2) in distilled water and dilute to 1 liter.
7. Standard phenylarsine oxide, 0.0250 N
Commercially prepared 0.0250 N PAO is available from several scientific supply companies. As an alternative 0.0250 N PAO may be prepared by diluting a higher normality solution.
CAUTION: PAO is very poisonous. Care must be taken in preparation and handling of this reagent.
8. Copper sulfate‑Sulfamic acid solution
a. Dissolve 32 g technical grade sulfamic acid (NH2SO2OH), without heat, in 475 mL distilled water.
b. Dissolve 50 g technical grade copper sulfate (CuSO4 5H2O) in 500 mL distiller water.
c. Mix the copper sulfate and sulfamic acid solutions together and add 25 mL glacial acetic acid (CH3COOH).
Source: https://www.dep.state.pa.us/dep/deputate/waterops/redesign/NPDES%20Lab/Chapter%204.doc
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