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BOD Test (BOD5)


1. General Discussion



    a. Principle: The method consists of filling with sample, to overflowing, an airtight bottle of the specified size and incubating it at the specified temperature for 5 d. DO (dissolved oxygen) is measured initially and after incubation, and the BOD is computed from the difference between initial and final DO. Because the initial DO is determined shortly after the dilution is made, all oxygen uptake occurring after this measurement is included in the BOD measurement.

    b. Sampling and storage: Samples for BOD analysis may degrade significantly during storage between collection and analysis, resulting in low BOD values. Minimize reduction of BOD by analyzing sample promptly or by cooling it to near-freezing temperature during storage. However, even at low temperature, keep holding time to a minimum. Warm chilled samples to 20 3oC before analysis.

    1) Grab samples If analysis is begun within 2 h of collection, cold storage is unnecessary. If analysis is not started within 2 h of sample collection, keep sample at or below 4oC from the time of collection. Begin analysis within 6 h of collection; when this is not possible because the sampling site is distant from the laboratory, store at or below 4oC and report length and temperature of storage with the results. In no case start analysis more than 24 h after grab sample collection. When samples are to be used for regulatory purposes make every effort to deliver samples for analysis within 6 h of collection.

    2) Composite samples Keep samples at or below 4oC during compositing. Limit compositing period to 24 h. Use the same criteria as for storage of grab samples, starting the measurement of holding time from end of compositing period. State storage time and conditions as part of the results.



2. Apparatus



    a. Incubation bottles: Use glass bottles having 60 mL or greater capacity (300-mL bottles having a ground-glass stopper and a flared mouth are preferred). Clean bottles with a detergent, rinse thoroughly, and drain before use. As a precaution against drawing air into the dilution bottle during incubation, use a water seal. Obtain satisfactory water seals by inverting bottles in a water bath or by adding water to the flared mouth of special BOD bottles. Place a paper or plastic cup or foil cap over flared mouth of bottle to reduce evaporation of the water seal during incubation.

    b. Air incubator or water bath, thermostatically controlled at 20 1oC. Exclude all light to prevent possibility of photosynthetic production of DO.



3. Reagents



    Prepare reagents in advance but discard if there is any sign of precipitation or biological growth in the stock bottles. Commercial equivalents of these reagents are acceptable and different stock concentrations may be used if doses are adjusted proportionally.

    a. Phosphate buffer solution: Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO47H2O, and 1.7 g NH4Cl in about 500 mL distilled water and dilute to 1 L. The pH should be 7.2 without further adjustment. Alternatively, dissolve 42.5 g KH2PO4 or 54.3 g K2HPO4 in about 700 mL distilled water. Adjust pH to 7.2 with 30% NaOH and dilute to 1 L.

    b. Magnesium sulfate solution: Dissolve 22.5 g MgSO47H2O in distilled water and dilute to 1 L.

    c. Calcium chloride solution: Dissolve 27.5 g CaCl2 in distilled water and dilute to 1 L.

    d. Ferric chloride solution: Dissolve 0.25 g FeCl36H2O in distilled water and dilute to 1 L.

    e. Acid and alkali solutions, 1N, for neutralization of caustic or acid waste samples.

    1) Acid Slowly while stirring, add 28 mL conc sulfuric acid to distilled water. Dilute to 1 L.

    2) Alkali Dissolve 40 g sodium hydroxide in distilled water. Dilute to 1 L.

    f. Sodium sulfite solution: Dissolve 1.575 g Na2SO3 in 1000 mL distilled water. This solution is not stable; prepare daily.

    g. Nitrification inhibitor, 2-chloro-6-(trichloromethyl) pyridine.*

   h. Glucose-glutamic acid solution: Dry reagent-grade glucose and reagent-grade glutamic acid at 103oC for 1 h. Add 150 mg glucose and 150 mg glutamic acid to distilled water and dilute to 1 L. Prepare fresh immediately before use.

    i. Ammonium chloride solution: Dissolve 1.15 g NH4Cl in about 500 mL distilled water, adjust pH to 7.2 with NaOH solution, and dilute to 1 L. Solution contains 0.3 mg N/mL.

    j. Dilution water: Use demineralized, distilled, tap, or natural water for making sample dilutions.



4. Procedure



    a. Preparation of dilution water: Place desired volume of water (3j) in a suitable bottle and add 1 mL each of phosphate buffer, MgSO4, CaCl2, and FeCl3 solutions/L of water. Seed dilution water, if desired, as described in (4d). Test dilution water as described in (4h)  so that water of assured quality always is on hand.

    Before use, bring dilution water temperature to 20 + 3C. Saturate with DO by shaking in a partially filled bottle or by aerating with organic-free filtered air. Alternatively, store in cotton-plugged bottles long enough for water to become saturated with DO. Protect water quality by using clean glassware, tubing, and bottles.

    b. Dilution water storage: Source water (3j) may be stored before use as long as the prepared dilution water meets quality control criteria in the dilution water blank (4h). Such storage may improve the quality of some source waters but may allow biological growth to cause deterioration in others. Preferably do not store prepared dilution water for more than 24 h after adding nutrients, minerals, and buffer unless dilution water blanks consistently meet quality control limits. Discard stored source water if dilution water blank shows more than 0.2 mg/L DO depletion in 5 d.

    c. Glucose-glutamic acid check: Because the BOD test is a bioassay its results can be influenced greatly by the presence of toxicants or by use of a poor seeding material. Distilled waters frequently are contaminated with copper; some sewage seeds are relatively inactive. Low results always are obtained with such seeds and waters. Periodically check dilution water quality, seed effectiveness, and analytical technique by making BOD measurements on a mixture of 150 mg glucose/L and 150 mg glutamic acid/L as a "standard" check solution. Glucose has an exceptionally high and variable oxidation rate but when it is used with glutamic acid, the oxidation rate is stabilized and is similar to that obtained with many municipal wastes. Alternatively, if a particular wastewater contains an identifiable major constituent that contributes to the BOD, use this compound in place of the glucose-glutamic acid.

    Determine the 5-d 20C BOD of a 2% dilution of the glucose-glutamic acid standard check solution using the techniques outlined in (4d-j). Adjust concentrations of commercial mixtures to give 3 mg/L glucose and 3 mg/L glutamic acid in each GGA test bottle. Evaluate data as described in (6), Precision and Bias.


    d. Seeding:


    1) Seed source It is necessary to have present a population of microorganisms capable of oxidizing the biodegradable organic matter in the sample. Domestic wastewater, unchlorinated or otherwise undisinfected effluents from biological waste treatment plants and surface waters receiving wastewater discharges contain satisfactory microbial populations. Some samples do not contain a sufficient microbial population (for example some untreated industrial wastes, disinfected wastes, high-temperature wastes, or wastes with extreme pH values). For such wastes seed the dilution water or sample by adding a population of microorganisms. The preferred seed is effluent or mixed liquor from a biological treatment system processing the waste. Where such seed is not available, use supernatant from domestic wastewater after settling at room temperature for at least 1 h but no longer than 36 h. When effluent or mixed liquor from a biological treatment process is used, inhibition of nitrification is recommended.

    Some samples may contain materials not degraded at normal rates by the microorganisms in settled domestic wastewater. Seed such samples with an adapted microbial population obtained from the undisinfected effluent or mixed liquor of a biological process treating the waste. In the absence of such a facility, obtain seed from the receiving water below (preferably 3 to 8 km) the point of discharge. When such seed sources also are not available, develop an adapted seed in the laboratory by continuously aerating a sample of settled domestic wastewater and adding small daily increments of waste. Optionally use a soil suspension or activated sludge, or a commercial seed preparation to obtain the initial microbial population. Determine the existence of a satisfactory population by testing the performance of the seed in BOD tests on the sample. BOD values that increase with time of adaptation to a steady high value indicate successful seed adaptation.

    2) Seed control Determine BOD of the seeding material as for any other sample. This is the seed control. From the value of the seed control and a knowledge of the seeding material dilution (in the dilution water) determine seed DO uptake. Ideally, make dilutions of seed such that the largest quantity results in at least 50% DO depletion. A plot of DO depletion, in milligrams per litre, versus millilitres of seed for all bottles having a 2 mg/L depletion and a 1.0-mg/L minimum residual DO should present a straight line for which the slope indicates DO depletion per millilitre of seed. The DO-axis intercept is oxygen depletion caused by the dilution water and should be less than 0.1 mg/L (4h)Alternatively, divide DO depletion by volume of seed in millilitres for each seed control bottle having a 2-mg/L depletion and a 1.0-mg/L residual DO. Average the results for all bottles meeting minimum depletion and residual DO criteria. The DO uptake attributable to the seed added to each bottle should be between 0.6 and 1.0 mg/L, but the amount of seed added should be adjusted from this range to that required to provide glucose-glutamic acid check results in the range of 198 30.5 mg/L. To determine DO uptake for a test bottle, subtract DO uptake attributable to the seed from total DO update (see 5).

    Techniques for adding seeding material to dilution water are described for two sample dilution methods (4f).

    e. Sample pretreatment: Check pH of all samples before testing unless previous experience indicates that pH is within the acceptable range.

    1) Samples containing caustic alkalinity (pH > 8.5) or acidity (pH < 6.0) - Neutralize samples to pH 6.5 to 7.5 with a solution of sulphuric acid (H2SO4) or sodium hydroxide (NaOH) of such strength that the quantity of reagent does not dilute the sample by more than 0.5%. The pH of dilution water should not be affected by the lowest sample dilution. Always seed samples that have been pH adjusted.

    2) Samples containing residual chlorine compounds - If possible, avoid samples containing residual chlorine by sampling ahead of chlorination processes. If the sample has been chlorinated but no detectable chlorine residual is present, seed the dilution water. If residual chlorine is present, dechlorinate sample and seed the dilution water (4f). Do not test chlorinated/dechlorinated samples without seeding the dilution water. In some samples chlorine will dissipate within 1 to 2 h of standing in the light. This often occurs during sample transport and handling. For samples in which chlorine residual does not dissipate in a reasonably short time, destroy chlorine residual by adding Na2SO3 solution. Determine required volume of Na2SO3 solution on a 100 to 1000 mL portion of neutralized sample by adding 10 mL of 1 + 1 acetic acid or 1 + 50 H2SO4, 10 mL potassium iodide (KI) solution (10 g/100 mL) per 1000 mL portion, and titrating with Na2SO3 solution to the starch-iodine end point for residual. Add to neutralized sample the relative volume of Na2SO3 solution determined by the above test, mix, and after 10 to 20 min check sample for residual chlorine. (NOTE: Excess Na2SO3 exerts an oxygen demand and reacts slowly with certain organic chloramine compounds that may be present in chlorinated samples.)

    3) Samples containing other toxic substances - Certain industrial wastes, for example, plating wastes, contain toxic metals. Such samples often require special study and treatment.

    4) Samples supersaturated with DO - Samples containing more than 9 mg DO/ L at 20C may be encountered in cold waters or in water where photosynthesis occurs. To prevent loss of oxygen during incubation of such samples, reduce DO to saturation at 20C by bringing sample to about 20C in partially filled bottle while agitating by vigorous shaking or by aerating with clean, filtered compressed air.

    5)  Sample temperature adjustment - Bring samples to 20 1C before making dilutions.

   6) Nitrification inhibition - If nitrification inhibition is desired add 3 mg 2-chloro-6-(trichloro methyl) pyridine (TCMP) to each 300-mL bottle before capping or add sufficient amounts to the dilution water to make a final concentration of 10 mg/L. (NOTE: Pure TCMP may dissolve slowly and can float on top of the sample. Some commercial formulations dissolve more readily but are not 100% TCMP; adjust dosage accordingly.) Samples that may require nitrification inhibition include, but are not limited to, biologically treated effluents, samples seeded with biologically treated effluents, and river waters. Note the use of nitrogen inhibition in reporting results.

    f. Dilution technique: Make several dilutions of sample that will result in a residual DO of at least 1 mg/L and a DO uptake of at least 2 mg/L after a 5 d incubation. Five dilutions are recommended unless experience with a particular sample shows that use of a smaller number of dilutions produces at least two bottles giving acceptable minimum DO depletion and residual limits. A more rapid analysis, such as COD, may be correlated approximately with BOD and serve as a guide in selecting dilutions. In the absence of prior knowledge, use the following dilutions: 0.0 to 1.0% for strong industrial wastes, 1 to 5% for raw and settled wastewater, 5 to 25% for biologically treated effluent, and 25 to100% for polluted river waters.

    Prepare dilutions either in graduated cylinders or volumetric glassware, and then transfer to BOD bottles or prepare directly in BOD bottles. Either dilution method can be combined with any DO measurement technique. The number of bottles to be prepared for each dilution depends on the DO technique and the number of replicates desired.

    When using graduated cylinders or volumetric flasks to prepare dilutions, and when seeding is necessary, add seed either directly to dilution water or to individual cylinders or flasks before dilution. Seeding of individual cylinders or flasks avoids a declining ratio of seed to sample as increasing dilutions are made. When dilutions are prepared directly in BOD bottles and when seeding is necessary, add seed directly to dilution water or directly to the BOD bottles. When a bottle contains more than 67% of the sample after dilution, nutrients may be limited in the diluted sample and subsequently reduce biological activity. In such samples, add the nutrient, mineral, and buffer solutions (3a through e) directly to individual BOD bottles at a rate of 1 mL/L (0.33 mL/ 300-mL bottle) or use commercially prepared solutions designed to dose the appropriate bottle size.

    1) Dilutions prepared in graduated cylinders or volumetric flasks - If the azide modification of the titrimetric iodometric method is used, carefully siphon dilution water, seeded if necessary, into a 1 to 2L capacity flask or cylinder. Fill half full without entraining air. Add desired quantity of carefully mixed sample and dilute to appropriate level with dilution water. Mix well with a plunger-type mixing rod; avoid entraining air. Siphon mixed dilution into two BOD bottles. Determine initial DO on one of these bottles. Stopper the second bottle tightly, water-seal, and incubate for 5 d at 20C. If the membrane electrode method is used for DO measurement, siphon dilution mixture into one BOD bottle. Determine initial DO on this bottle and replace any displaced contents with sample dilution to fill the bottle. Stopper tightly, water-seal, and incubate for 5 d at 20C.

    2) Dilutions prepared directly in BOD bottles - Using a wide-tip volumetric pipette, add the desired sample volume to individual BOD bottles of known capacity. Add appropriate amounts of seed material either to the individual BOD bottles or to the dilution water. Fill bottles with enough dilution water, seeded if necessary, so that insertion of stopper will displace all air, leaving no bubbles. For dilutions greater than 1:100 make a primary dilution in a graduated cylinder before making final dilution in the bottle. When using titrimetric iodometric methods for DO measurement, prepare two bottles at each dilution. Determine initial DO on one bottle. Stopper second bottle tightly, water-seal, and incubate for 5 d at 20C. If the membrane electrode method is used for DO measurement, prepare only one BOD bottle for each dilution. Determine initial DO on this bottle and replace any displaced contents with dilution water to fill the bottle. Stopper tightly, water-seal, and incubate for 5 d at 20C. Rinse DO electrode between determinations to prevent cross-contamination of samples.

    Use the azide modification of the iodometric method (Section 4500-O.C) or the membrane electrode method (Section 4500-O.G) to determine initial DO on all sample dilutions, dilution water blanks, and where appropriate, seed controls.

    If the membrane electrode method is used, the azide modification of the iodometric method (Method 4500-O.C) is recommended for calibrating the DO probe.

    g. Determination of initial DO: If the sample contains materials that react rapidly with DO, determine initial DO immediately after filling BOD bottle with diluted sample. If rapid initial DO uptake is insignificant, the time period between preparing dilution and measuring initial DO is not critical but should not exceed 30 min.

    h. Dilution water blank: Use a dilution water blank as a rough check on quality of unseeded dilution water and cleanliness of incubation bottles. Together with each batch of samples incubate a bottle of unseeded dilution water. Determine initial and final DO as in 4g and j. The DO uptake should not be more than 0.2 mg/L and preferably not more than 0.1 mg/L Discard all dilution water having a DO uptake greater than 0.2 mg/L and either eliminate source of contamination or select an alternate dilution water source..

    i. Incubation: Incubate at 20oC 1oC BOD bottles containing desired dilutions, seed controls, dilution water blanks, and glucose-glutamic acid checks. Water-seal bottles as described in 4f.

    j. Determination of final DO: After 5 d incubation determine DO in sample dilutions, blanks, and checks as in 4g.


5. Calculation



    For each test bottle meeting the 2.0-mg/L minimum DO depletion and the 1.0-mg/L residual DO, calculate BOD5 as follows:

    When dilution water is not seeded:



                                                    D1 D2

                        BOD5, mg/L =                                


    When dilution water is seeded:



                                        (D1 D2) (B1 B2)f

                 BOD5, mg/L =                                           






    D1 = DO of diluted sample immediately after preparation, mg/L,

    D2 = DO of diluted sample after 5 d incubation at 20oC, mg/L,

    P  = decimal volumetric fraction of sample used,

    B1 = DO of seed control before incubation, mg/L (4d),

    B2 = DO of seed control after incubation mg/L (4d), and

    f  = ratio of seed in diluted sample to seed in seed control = (% seed in diluted sample)/(% seed in seed control).



    If seed material is added directly to sample or to seed control bottles:



    f = (volume of seed in diluted sample)/(volume of seed in seed control)



    Report results as CBOD5 if nitrification is inhibited.

    If more than one sample dilution meets the criteria of a residual DO of at least 1 mg/L and a DO depletion of at least 2 mg/L and there is no evidence of toxicity at higher sample concentrations or the existence of an obvious anomaly, average results in the acceptable range.

    In these calculations, do not make corrections for DO uptake by the dilution water blank during incubation. This correction is unnecessary if dilution water meets the blank criteria stipulated above. If the dilution water does not meet these criteria, proper corrections are difficult ; do not record results or, as a minimum, mark them as not meeting quality control criteria.



6. Precision and Bias



    There is no measurement for establishing bias of the BOD procedure. The glucose-glutamic acid check prescribed in 4c is intended to be a reference point for evaluation of dilution water quality, seed effectiveness, and analytical technique. Single-laboratory tests using a 300-mg/L mixed glucose-glutamic acid solution provided the following results:



                                                                    Number of months: 14

                                                                 Number of triplicates: 421

                                                          Average monthly recovery: 204 mg/L

                                            Average monthly standard deviation: 10.4 mg/L



    In a series of interlaboratory studies,1 each involving 2 to 112 laboratories (and as many analysts and seed sources), 5-d BOD measurements were made on synthetic water samples containing a 1:1 mixture of glucose and glutamic acid in the total concentration range of 3.3 to 231 mg/L. The regression equations for mean value, X, and standard deviation, S, from these studies were:



                                    X = 0.658 (added level, mg/L) + 0.280 mg/L

                                    S = 0.100 (added level, mg/L) + 0.547 mg/L



    For the 300-mg/L mixed primary standard, the average 5-d BOD would be 198 mg/L with a standard deviation of 30.5 mg/L. When nitrification inhibitors are used, GGA test results falling outside the 198 30.5 control limit quite often indicate use of incorrect amounts of seed. Adjust amount of seed added to the GGA test to achieve results falling within this range.

    a. Control limits: Because of many factors affecting BOD tests in multilaboratory studies and the resulting extreme variability in test results, one standard deviation, as determined by interlaboratory tests, is recommended as a control limit for individual laboratories. Alternatively, for each laboratory, establish its control limits by performing a minimum of 25 glucose-glutamic acid checks (4c) over a period of several weeks or months and calculating the mean and standard deviation. Use the mean 3 standard deviations as the control limit for future glucose-glutamic acid checks. Compare calculated control limits to the single-laboratory tests presented above and to interlaboratory results. If control limits are outside the range of 198 30.5, re-evaluate the control limits and investigate source of the problem. If measured BOD for a glucose-glutamic acid check is outside the accepted control limit range, reject tests made with that seed and dilution water.

    b. Working range and detection limit: 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 multiplied by the dilution factor. A lower detection limit of 2 mg/L is established by the requirement for a minimum DO depletion of 2 mg/L.



7. Reference


  1. U.S. ENVIRONMENTAL PROTECTION AGENCY, OFFICE OF RESEARCH AND DEVELOPMENT. 1986. Method by Method Statistics from Water Pollution (WP) Laboratory Performance Evaluation Studies. Quality Assurance Branch, Environmental Monitoring and Support Lab., Cincinnati, Ohio.

8. Bibliography


YOUNG, J.C., G.N. MCDERMOTT & D. JENKINS. 1981. Alterations in the BOD procedure for the 15th edition of Standard Methods for the Examination of Water and Wastewater. J. Water Pollut. Control Fed. 53:1253.


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