5210 BIOCHEMICAL OXYGEN DEMAND (BOD) 5210 A. Introduction 1. General Discussion Biochemical oxygen demand (BOD) testing is used to deter- ‘mine the relative oxygen requirements of wastewaters, effluents, and polluted waters; ts widest application is in measuring waste loadings to treatment plants and in evaluating the plants’ BOD- ‘removal efficiency. BOD testing measures the molecular oxygen used during a specified incubation period to * biochemically degrade organic material (carbonaceous demand), + oxidize inorganic material (¢.g., sulfides and ferrous iron), andlor + measure the amount of oxygen used to oxidize reduced forms of nitrogen (nitrogenous demand) unless an inhibitor is added to prevent such reduction ‘The seeding and dilution procedures provide an estimate of BOD at pit 6 to 8. ‘The methods below measure oxygen consumed in a 5-d period (52108), oxygen consumed after 60 to 90 d of incubation (52100), and continuous oxygen uptake (5210D). Other BOD riethods published elsewhere may ute shorter of longer incuba luon petiods; (ests to determine oxygen-uptake rates; and/or alternative seeding, dilution, ad incubstion conditions to mimic receiving-water conditions, thereby estimating the environmen- lal effects of wastewaters and effluents ‘The ultimate BOD (UBOD) test measures the oxygen required te totally degrade organic material (ultimate carbonaceous de- rand) and/or to oxidize reduced nitrogen compounds (ultimate nitrogenous demand). UBOD values and appropriate kinetic descriptions are needed in water-quality modeling studies [eg.. UBOD:BOD, ratios for relating stream assimilative capacity to regulatory requirements; definition of river, estuary, or lake deoxygenation kinetics; and instream ultimate carbonaceous BOD (UCBOD) values for model calibration} ‘A number of factors (e.g. soluble versus particulate organics, sottleable and floatable solids, oxidation of reduced iron and TApgoved by Sanda Mtiads Commitee, 2016. Jan Tak Group Sues C You cha). Vile D. Hab Robert V-Menepot, Devon A Movgun Kota 8. Panel Lisa Me Raniez Debea A. Walle sulfur compounds, or lack of mixing) may affect the accuracy and precision of BOD measurements. Presently, there are no effective adjustments or corrections to compensate for these factors. 2. Carbonaceous Versus Nitrogenous BOD Microorganisms can oxidize reduced forms of nitrogen, such as ammonia and organic nitrogen, thus exerting nitrogenous demand. Nitogenous demand historically has been considered an injererence in BOD testing: adding ammonia to dilution ‘water contnibutes an extemal source of nitrogenous demand. The interference from nitrogenous demand can now be prevented by aa inhibitory chemical, but i tise sed, the measured oxygen demand isthe sum of carbonaceous and nitrogenous demands Measurements that include nitrogenous demand generally are not useful for assessing the oxygen demand associated with cxganic material. Nitrogenous demand can be estimated diceety fiom ammonia nitogen, and carbonaceous demand can be esti- ‘mated by subtracting the theoretical equivalent of the nitrite and nitrate progvced in uninhibited test results. However, this method is cumbersome and subject to considerable error. Cher {cal inhibition of nitogenous demand provides a more dizect. reliable measute of carbonaceous demand How much nitrogenous compounds oxidize during the 5 incubation period depends on the concentration and type of ‘microorganisms that can carry oUt this oxidation, Such og isms quite often are present in raw or seed primary sewage in adequate numbers to oxidize enough reduced nitrogen forms to contribute oxygen demand inthe S-d BOD test. Most biological teealment plant elflcnts contain enough nitsfying organisms to cause sittification in BOD tests, Because aiteogenous com- pounds can oxidize in such samples, ntriication inhibition (@s directed in 52108 Se) is recommended for secondary-sfduent samples, samples seeded with secondary ellen, and polluted water samples 3. Reference 1. Yoexo, J.C. 1973, Chemical methods for nitifeation contol J. Water Polat. Control Fed 45:53. $210 B. 5-Day BOD Test 1, General Discussion ‘The BOD test is an indirect measurement of organic matter; it measures the change in DO concentration caused by micro- organisms as they degrade organic matter in a sample held in a stoppered bottle incubsted for $ din the datk at 20°C Analysts measure DO before and after incubation, and com pute BOD using the difference between DO measurements. Because initial DO is determined shorty after dilution, all up: org/1021057S MWW.2482.102 oxygen uplake occurring after this measurement is included in the BOD measurement. For sampling and storage procedures, see 5210B.4a. 2. Apparatus 4, Incubation botles: Use 60-mL glass bottles or larger (G00-mL bottles with a flared mouth and ground-glass stopper are preferred), Clean bottles with a detergent, rinse thoroughly,BIOCHEMICAL OXYGEN DEMAND (6210VS5-Day BOD Test and drain before use. Alternatively, use disposable plastic BOD. botiles that are capable of meeting all method quality-control (QO) checks. bs, Air incubator or water bath thermostatically controlled at 20 © 1°C. Exclude all Light to prevent the possibilty of photo- synthetic production of DO. ©. Oxygen-sensitive membrane electrode, polarographic or galvanic, of oxygen-sensitive optical probe with appropriate meter. 8. Reagents Discard reagents if there is any sign of precipitation or bio- logical growth in the stock bottles. Commercial equivalents of these reagents are acceptable, and different stack concentrations may be used if doses are adjusted proportionally. Use reagent grade or belter for all chemicals and use distilled or equivalent reagent-grade water (see Section 1080) to make all solutions. ‘4. Phosphate buffer solution: Dissolve 8.5 g monopotassium phosphate (KH,PO,), 21.75 g dipotassium phosphate (K HPO.) 33.4 g disodium phosphate (Na,HPO,) - 7H,O, and 1.7 g am- ‘onium chloride (NII,CD in about 500 mL reagent-grade Wate and dilute to 1 L. The pHT should be 7.2 without further adjust- rent, Alternatively, dissolve 42.5 g KH,PO, and 1.7 g NH,CLin about 700 mL. reagent-grade water. Adjust pH to 7.2 with 30% sodium hydroxide (NaOH) and dilute to 1 L, b. Magnesium sulfate (MgSO,) soluion: Dissolve 22.5 g MgSO, * THO in reagent-grade water and dilute :o I L, ©. Caleium chloride (CaCl,) solution: Dissolve 27.5 g CaCls in reagent-grade water and dilute to 1 L, d. Ferric chloride (FeCl) solution: Dissolve 0.25 g FeCl, + 61,0 in reagent-grade water and dilute to 1 L. «Acid and alkali solutions, IN, to neutralize caustic or acidic waste samples. 1) Acid—Slowly and while stcring, add 28 ml. cone sulfurie acid (H,SO,) fo reagent-grade water. Dilute to 1 L. 2) Alkali—Dissolve 40 g NaOH in distilled water. Dilute toll, {f Sodium sulfite (Na,SO,) solution: Dissolve 1.575 g Na,S0, in 1000 mL. reagent-grade water. This solution is unstable prepare daily, 1 Nitrification inhibitor: 1) 2-chloro-6-(ichloromethyl) pyridine (TCMP)—U. TCMP or commercial preparations." 2) Allylthiourea (ATU) solution—Dissolve 2.0 g allylthioures (CaHN,S) in about $00 ml. reagent-grade water and dilute to ‘at 4°C, The solution is stable for 2 weeks when stored °C without freezing +h, Glucose-glutamie acid (GGA) solution: Dry reagent-grade alucose and reagent-grade glutamic acid at 103°C for 1h. Add 150 mg glucose and 150 mg glutamic acid to reagent-grade water and dilute to IL. Prepare fresh immediately before use less solution is maintained in a sterile container. Store all GGA mixtures at 6°C without freezing unless manufacturer recommendations state otherwise, Commercial preparations may be used but concentrations may vary, Discard solutions if evi= pure Loveland: CO, oc equivalent up: org/1021057S MWW.2480.102 dence of contamination is indicated (e.g. growth occurs in the stock botle or GGA test results aze consistently low). i Ammonium chloride solution: Dissolve 1,15 g NCI in about 500 ml. reagent-grade water, adjust pH to 7.2 with NaOH solution, and dilute to L L, Solution contains 0.3 mg Nil. J. Source water for preparing BOD dilution water: Use de- mineralized, distilled, or equivalent reagent-grade wate, tap, oF natural water to make sample dilutions (see 5210B.4¢) 4, Preparatory Procedures 4 Sampling and storage: Samples for BOD analysis may degrade significantly during storage between collection and anal- ysis, resulting in low BOD values. 1) Grab samples—If analysis begins within 2h of collection, cold storage is unnecessary. Otherwise, keep sample at =6°C between collection and analysis, Ideally, begin analysis within 6h of sample collection; if impossible due to distance between sampling site and laboratory, then begin analysis within 24 h of collection. The recommended holding time is 24; however, the USS. Environmental Protection Agency (EPA) allows for a 48-h holding time 2) Composite samples—Limsit compositing period to 4b, and keep samples at =6°C during process. Store for the same time and temperature as grab samples, although in this case, holding lime begins when the compositing period ends. . Sample preparation and pretreatment 1) All samples—Check pH if it is not between 6.0 and 8, adjust sample temperature to 20 = 3°C, then adjust pH to between 6.5 and 7.5 using an H,SO, of NaOH solution strong enough not to dilute sample by >0.5%, Exceptions may be justified with natural waters when BOD will be measused atin ‘stu pH values. Dilution-water pH should not be affected by the lowest sample dilution, Always seed samples that have been plL-adjusted. 2) Samples containing residual chlorine compound: —If possible, avoid samples containing residual chlorine by sampling ahead of chulorination processes. If residual chlorine is present, dechlrinate sample. Sometimes chlorine will dissipate from sample within Ito 2 h of standing in the light: this often occurs during transport and handling. Ifthe chlorine residual does uot dissipate in a tea sonably short time, destroy it by adding Na,S0, solution, Deter- mine required volume of Na,SO, solution on @ 100- to 1000-ml ‘portion of neutralized sample by adding 10 ml. 1 + 1 acetic acid ot 1 | SO H,SO, and 10 ml. potassium iodide (KI) solution (10 g/100 rmiL) per 1000 mL sample, and then titrating with Na,S0, solution to the starch-iodine endpoint for residual. Add to netalized sample the proportional volume of Na,SO, solution determined by the above test, mix, and check sample for residual chlorine after 10 to 20 min, (Noms: Excess Na,SO, exerts an oxygen demand and reacts slowly with certain organic chloramine compounds that may be present in chlorinated samples.) Do not test chlorinated/dechlori- nated samples without seeding. 3) Samples containing other toxic substances—Certain indus- tial wastes (eg, plating wastes) contain toxic metals. Such samples often require special study and treatment. 4) Samples supersaturated with DO (Table 4500-0:1)—Sam- ples with DO concentrations above saturation at 20°C may be collected in cold waters or in water where photosynthesis occurs, To prevent oxygen loss when incubating such samples, reduceBIOCHEMICAL OXYGEN DEMAND (6210V5-Day BOD Test DO to saturation by bringing sample to about 20 = 3°C in partially filled bottle while agitating by vigorous shaking or aerating with clean, filtered compressed ai, 5) Samples containing hydrogen peroxide—Hydrogen perox- ide remaining in samples from some industrial bleaching pro- cesses (€g. those used at paper mills and textile plants) can cause supersaturated oxygen levels in samples collected for BOD testing, Mix such samples vigorously in open containers long enough to allow hydrogen peroxide to dissipale before setting up BOD tests. Check adequacy of peroxide removal by observing DO concentrations over time during mixing or by using peroxide-specific test strips, Mixing times can vary from 1 to 2h, depending on the amount of hydrogen peroxide present. The peroxide reaction can he considered complete when DO no Tonger increases during 2 30-min period without mixing, © Selection and storage of source water for BOD sample dilution: Obtain water from suitable source (ie. distilled, tap, of reagent-grade water). Make sure water is free of heavy metal, specifically copper (<0.05 mg/L) and toxic substances [e.g.. ciorine (0.10 mg/L}} that can interfere with BOD measure ments, Protect source-water quality by using clean glassware, tubing, and bottles, Deionized (DD water often contains enough forganics and microorganisms to cause the dilution-water QC check to fail (5210B.6c). DI water is not recommended unless dilution-water blanks consistently meet QC limits, Source water may be stored before use as long as the prepared dilution water (5210B.Sa) meets QC criteria in the dilution-water blank (5210B.6e). Such storage may improve the quality of some source waters but may allow biological growth to deteriorate others. Storing prepared dilution water (5210B.5h) for >24h aller adding nutrients, minerals, and buffer is not recommended unless dilution-water blanks consistently meet QC limits. Dis- card stored source water if dilution-water blank shows 0.2 mg/L DO depletion in $ 4 (5210B.6c), 4. Preparation of seed suspension: Each BOD bottle must contain a microorganism population that can oxidize biodegrad- able organic matter in the sample. Domestic wastewater, un- chlorinated or other undisinfected effluents from biological ‘wastewater treatment plants, and surface waters receiving waste- water discharges usually contain satisfactory microbial popula- ions. Some samples (e.g., some untreated industrial wastes, disinfected wastes, high-temperature wastes, wastes with pH values <6 of 8, or wastes stored >6 h alter collection) do not contain a sulficient mierobial population. Seed such samples by adding a population of suitable microorganisms; the preferred seed comes ftom a sample-telated biological wastewater treat- rent system of receiving water. In this case, use supernatant from seltled domestic wastewater, effluent from primary elarili- ers, diluted mixed liquor from an aeration basin, undisinfected effluent, or receiving water from below the discharge point. If using effluent or mixed liquor from a biological treatment pro- cess as a seed source, nittfication inhibition is recommended, Do not use seed from effluents that have been disinfected by clorine or other means. Commercial seed sources may be used according to manufacturer's preparation instructions but are more likely o be unadapted to the wastewater constituents. Do not filter seed sources; filering removes the seed microorganisms. adapted seed sources are unavailable, develop an acclimated seed in the laboratory by continuously aerating a sample of settled domestic wastewater and adding small daily increments pe: org/1021057S MWW.2480.102 of sample from the waste in question, Use a soil suspension, activated sludge, or a commercial seed prepazation to obtain the initial microbial population, Determine the existence of a satis- factory population by testing the seed’s performance in BOD tests on the sample. BOD values that increase during adaptation to-a steady high value indicate successful seed acclimation. 5. Testing Procedure 4 Preparation of diluion water: Transfer desired working volume of source water (S210B.4c) to a suitably sized botle (las is preferred). Check to ensure that the DO concentration is at least 7.5 mg/L before using water for BOD tests. If not, add DO by shaking bottle or aerating it with organi-free Giltered ait Alternatively, store the water in cotton-plugged bottles long enough for the DO concentration to approach saturation, Add 1 mL each of phosphate buffer, MgS0., CaCl, and FeCl, solution/L. to prepared source water (5210B 4c), Mix thoroughly and bring temperature to 20 = 3°C. Prepare dilution water immediately before use, unless dilution-water blanks (5210B.60) show that the water is acceptable after longer storage times. If dilution-water blanks show a DO depletion >0.2 mg/L. then improve purification or use water from another source. Do not add oxidizing agents or expose dilution water to ultraviolet light, to try to bring the dilution blank into range. b, Sample temperature adjusiment: Bring sample teraperature to 20 * 3°C before making dilutions. € Preparation of dilutions: Using dilution water prepared as in a above, make at least three dilutions of prepared sample estimated to produce, atthe end of the test, atleast one dilution that would result in a residual DO of =10 mg/l. and a DO uptake of =2.0 mg/L after a 5-d incubation. Two dilutions are allowed if experience with a particular sample source produces at least one bottle with acceptable minimum DO depletions and residual limits (5210B.6a). Individual laboratories should eval- uate the need for more than tree dilutions when historical sample data are unavailable. A more rapid analysis, such as COD. (Section 5220), may be correlated approximately with BOD and serve as a guide in selecting dilutions. In the absence of prior knowledge, use the following percentages of wastewater when preparing dilutions: 0.01 to 1.0% for stzong industial wastes, 1 to 5% for raw and settled wastewater, 5 to 25% for biologically treated effluent, and 25 to 100% for polluted river waters. The ‘number of botiles to be prepared for each dilution depends on DO technigue and number of replicates desired. Prepare dilu- UUons in volumetric containers (Class A glass or equivalent) and then transfer to BOD botles, or else prepare directly in BOD bottles. Either dilution method can be used to transfer sample to respective BOD bottles. 1) Dilutions prepazed in volumetric containers—Using a wide- tipped pipet or graduated cylinder, add desired amount of pre- pared sample to individual volumetric eylinders or flasks. Mix sample well immediately before pipetting to avoid solids loss via sotiing. For dilutions greater than 1:300, make a primary dilu- tion before making final dilution in volumetric cylinders of flasks, Fill eylinders or flasks atleast two-thirds full with dilution water and sample without entraining air. Add appropriate amounts of seed suspension (J d below) and nitrification inkib- itor ( € below), Dilute to final level with dilution water ¢ a above). Mix well but avoid entraining air. Siphon mixed dilutionBIOCHEMICAL OXYGEN DEMAND (6210V5-Day BOD Test info a suitable number of BOD boitles, taking care not to let solids settle in cylinder or ask during wansfer. When a cylinder or flask contains >67% of sample after dilution, nutrients may be limited in the diluted sample and subsequently reduce bio- Togical activity. In such samples, add the nutrient, mineral, and bulfer solutions (52108 3a~d) diecty to diluted sample at arate of 1 mL/L (0.30 mL/300-mL bottle), or use commercially pre~ ‘pared solutions designed to dose the appropriate container size 2) Dilutions prepared directly in BOD bottles—Using a wide- Lip volumetsic pipet or graduated cylinder, add desired sample volume to individual BOD bortles. Mix sample well insmediately before pipetting to avoid solids loss via settling. For dilutions greater than 1:500, make a primary dilution before making final Uilution in the bot. Fill each BOD bottle approximately two- thirds full with dilution water and/or sample without entaining air, Add appropriate amounts of seed suspension (.d below) and nitrification inhibitor ({e below) to individual BOD bottles. Fill remainder of BOD boltle with dilution water. When a botle contains 67% of sample after dilution, nutrients may be limited in the diluted sample and subsequently reduce biological activ ity. In such samples, add the nutrient, mineral, and buffer solu tions (5210B.3a-) directly to diluted sample at arate of J mL (0.30 mL/300-mL. bottle), or use commercially prepared solu UUons designed to dose the appropriate bottle size. Addition of seed suspension: If seeding is used, add seed suspensions (o dilution vessels or individual BOD bottles before final dilution, as described in above. Do not add seed directly to wastewater samples before dilution if they contain toxic material. Generally, 1 to 3 mi. of settled raw wastewater or primary event or 1 to 2 mL of @ 1:10 dilution of mixed liquor/300-ma. bottle will provide enough microorganisms. Do not filler seed suspension before use. Agitate seed suspension during transfer to ensure that the same quantity of microorganisms is added to each BOD bottle ‘Always record the exact volume of seed suspension added to each boille, The DO uptake attibatable to added seed generally should be between 0.5 and 1.0 mg/L, but adjust sed amount as needed to provide GGA check results of 198 ~ 30:5 mg/L. For example, if | mL seed suspension is required to achieve 198 * 30.5 mg/l. BOD in the GGA check, then use 1 ml. in each BOD bottle receiving the test wastewater. «Addition of nitrification inhibitor: Sanaples that may requize nitvification inhibition’ include, but are not limited to, biologi cally tated effluents, samples seeded with biologically treated effluents, and river waters. Note the use of nitification inbibition and the related chemical used when reporting results. (Nove: TCMP is the preferred nitrification inhibitor but requires ban- dling and transfer in a solid form. ATU is not always effective in inhibiting nitrification within the S-d incubation period, and concentration’ >2 mg/L may increase carbonaceous BOD (CBOD) measurements and/or adversely affect the azide modi fication of the iodametric method ) Seed all samples to which nitvfictton inhibitor has been added 1) Nitification inhibition using TCMP—Add 10 mg TCMP/L to luted sample, 3 mg TCMP to each 300-mL bottle, of proportional amounts to ther sized bottles ater intial sample dition bu before final filling of botles with dilution water. Do not add TCMP to BOD bottles before they are atleast two-thirds filled with diluted sample. (Nor: TCMP dissolves slowly and can float on top of sample if not mixed well) Some commercial TCMP formulations are not 100% TCMP: adjust dosage appropriately pe: org/1021057S MWW.2489.102 2) Nitificaton inhibition using ATU—Add I mL. ATU solu- lion [5210B.3g2))/L diluced sample or 0.3 mL/300-mL test bot- te, Do not add ATU to BOD bottles until they are at least two-thirds filled with diluted sample. f Sealing bottles: Completely ll each botle by adding exiough dilution water so insestion of stopper leaves no bubbles in the bottle, Mix sample by tuming bottle manually several times unless immediately using a DO probe with a stirer to measure initial DO concentration. As a precaution agains! draw- ing air into the dilution bottle during incubation, use a water seal, Obain satisfactory water seals by inverting bottles in @ 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 water seal duting incubation, £8. Determination of intial DO: Use the azide modification of the iodometric method (Section 4500-0.C), membrane-clectrode method (Section 4500-0.G), or optical-probe method (Section 4500-0.H) to determine inital DO on all sample dilutions, dilution-water blanks, and, where appropriate, seed conteols. Replace any displaced contents with enough diluted sample or dition water to fill the bottle, stopper all bottles tightly, and water seal before beginning incubation. After preparing dilution, ‘measure initial DO within 30 min. If using the membrane- electrode method or optical probe method, calibrate DO probe daily by following the manufacturer's calibration procedure ‘Make frequent calibration checks daily to ensure accurate DO readings and, ideally, perform a Winkler titration as needed to verify calibration. If using the azide modification of the titi- ‘metric iodomettic method, prepare aa extra bot for initisl DO determination for each sample dilution ‘Sample incubation: Incubate at 20 * 1°C the stoppered and sealed BOD bottles containing desired dilutions ([¢ above), seed controls (5210B.6d), dilution-water blanks (S210B.60), and GGA checks (5210B.68), Exclude ight to avoid algae growth in bottles during incubation. i. Determination of final DO: After S d+ 6 b of incubation, determine DO in all sample dilutions, blanks, and checks as in 5210B.6g, using the azide modification ofthe titrimesric method (Section 4500-0.C), membrane-clectrode method (Section 4500.0.G), or optical-probe method (Section 4500-0.H). 6. Quality Control Checks ‘The QC practices considered to be an integral part of each ‘method are summarized in Table S020. ‘4, Minimum residual DO and minimum DO depletion: Only hotles (including seed controls) whose DO depletion is 2,0 mg/L and residual DO is 1.0 mg/L alter 5 d of incubation are considered to produce valid data, because =2.0 mg oxygen Uuptake/L is requized to give a meaningful measure of oxygen ‘uptake and =1,0 mgil. must remain to ensure that waste con- stituents’ oxidation rates were not limited by insufficient DO. However, there ate exceptions—for reporting purposes only— ‘when ‘esting undiluted samples and all botes” DO depletion is 0 mg/L and residual DO is <1.0 mg/L (see 521087) , Glucose-gluiamic acid check: The GGA check is the pri- mary basis for establishing the BOD test's accuracy and preci sion, as well as the principal measure of seed quality and set-up procedure. Together with each batch of BOD or CBOD samples, thheck seed effectiveness and analytical technique by using pro-BIOCHEMICAL OXYGEN DEMAND (6210VS5-Day BOD Test cedures in S210B.5 to make BOD measurements on an equal weight mixture of glucose and glutamic acid as follows: Add sufficient amounts of standard glucose and glutamic acid solu- tions (5210B. 3) to give 3.0 mg glucose/l. and 3.0 mg glutamic acidiL in each of three test bottles 20 mL GGA solution/l. seeded dilution water, of 6.0 mL/300-mL bottle). Commercial solutions may contain other GGA concentrations; adjust doses accordingly. Add nitrification inhibitor if seed is obtained from fa source that is nitrifying, and also to all CBOD GGA checks. Eyaluate data as described in 5210B.8, The resulting average BOD/CBOD for the three bottles, after comtecting for dilution and seeding, must fall into the contol-limit range established in S5210B.8a, If the average value falls outside this range, evaluate the cause and make appropriate corrections. Consistently high values can indicate too much seed suspension, contaminated dilution water, or mitification; consistently low values can indi cate poor seed quality or quantity or else the presence of a toxic rmaterial. If low values persist, prepare a new GGA mixture and check the dilution-water and seed sources, «. Dilution-water-quality check: Wita each batch of dilution water, incubate two or more bottles of dilution water containing ruisient, mineral, and buller solutions but no seed oF niltification inhibitor. Dilution water checks must be analyzed with each batch of samples; the dilution-water blank serves as a check onthe quality fof unseeded dilution water and cleanliness of incubation bots. Determine intial and final DO for each bole (5210B.Se and), and average result, The average DO uptake in 5 d must not be 2022 mg/L and preferably =0.1 mg/L before making seed comrec- lions. I average diution-water blank is 0.2 mgIL, record the data and clearly identity such samples in data records. . Seed control: Determine the seed suspension's BOD as for any other sample. This isthe seed control, Ideally, make three dilutions of seed so the smallest quantity depletes =2.0 mg/L. DO and the largest quantity leaves =1.0 mg/l. DO residual after 5 dof incubation, Determine DO uplake per milliliter of seed by dividing the DO depletion by the volume of seed in milliliters for each seed control bottle with a 2.0 mg/L depletion and >1.0 mg/L. ‘minimum residual DO, and averaging the results, Seed dilations showing widely varying depletions per milliliter of seed (30%) suggest the presence of toxic substances of large paztculates in the seed suspension; check or change the seed source 7. Data Analysis and Reporting . Calculations: 1) For each test bole with at least 2.0 mg/L. DO depletion and at least 1.0 mg/L residual DO— before seed covrection, calculate BOD as follows = Dd - Ns Bo, mgt, = P= P9= Sts where: D, = DO of diluted sample immediatly after preparation, mel D, ~ DO of silted sample afer 5 d incubation a 20°C, mg/L, 8 = oxygen uptake of seed [4 DO. seed suspension added et bottle (52108. 64) ($ ~ Of samples ae unseeded), = volume of seed in especive test bottle, ml, and pe: org/1021057S MWW.2489.102 P= decimal volumetic fraction of sample uted; UP ~ dilution factor 2) If DO depletion is <2.0 mg/L and sample concentration is 100% (no dilution except for seed, nutrient, mineral, and buffer solutions), actual seed-corrected DO depletion may be reported as the BOD even if itis <20 mel. 3) When all dilutions result in a residual DO <1.0, select the bottle with the highest DO concentration (usually the greatest dilution) and report: = dy ~ Vs $ BOD,,mglL > 4) If all dilutions result in DO depletion <2.0 mg/L. and the sample was diluted, select the bottle with the largest volume of sample (the least dilution) and caleulate the report as if the dilution had depleted 2.0 mg/L: ©, BOD, my/L « Vs In the above calculations, do not make corrections for DO uptake by the dilution-water blank dusing incubation, », Reporting: Average test results forall qualified botles in each dilution series. Report the result as BOD, if nittfication is not inhibited; report it as CBOD, if nitilicaion is inhibited. Samples With large differences between the computed BOD for different dilutions (eg, the highest value is >30%% larger than the lowest value) may indicate a toxic substance or analytical problems, When the effect becomes repetitive, investigate to identify the cause ‘Toxicity should be claimed only aller thorough investigation using tespirometric (5210D) or equivalent methods. lentfy results inthe test reports when any of the following QC conditions occur + dilution-water blank average is >0.2 mg. (S210B 6c), + GGA check falls outside acceptable limits (5210B.65), + test replicates show >30% difference between highest and lowest values, ‘+ none of the seed control samples mest the sbove criteria (5210B.64), oF + all itis result in a residual DO <1. mg_L [5210B.768)] 8, Precision and Bias ‘There is no measurement for establishing the BOD test's bias ‘The GGA check prescribed in S210B.6b is intended to be a reference point for evaluating dilution-water quality, seed effec tiveness, and analytical technique. Single-laboratory tests using & 300-mg/L mixed GGA solution provided the following results snber of month: 1“ ‘amber of triplicates: at Average monthly recovery 204 met ‘Average monthly stndard deviation 104 mgit, In a series of interlaboratory studies," 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 Ld mixture of GGA ranging from 3.3 to 231 mg/L total concentration, The regression equations for mean value, X, and standard deviation, S, from these studies were:BIOCHEMICAL OXYGEN DEMAND (6210V/Utimate BOD Test X = 0.558 (added concenttation, mg/L) + 0.280 mg/l, 'S = 0,100 (added concentration, mg/L.) 0.547 mg/L @ Control limits: Applying the above equations to the 300-mg/L GGA primary standard yields an average 5- BOD of 198 mg/L with a standard deviation of 30.5 mg/L. Because many factors affect BOD tests in maulti-laboratory studies, resulting in extremely variable test results, one standard deviation (as deter- ‘mined by interlaboratory tests) is recommended as a control limit for individual laboratories. Alternatively, each laboratory may establish its own contro! limits by performing at least 25 GGA. checks (5210B.6b) aver several weeks or months and calculating the mean and standard deviation, Use the mean =3 standard Udeviations as the contol limit for future GGA checks. Compare calculated control limits to the single-laboratory tests presented above and to interlaboratory results. If any GGA test results are ‘ouside the acceptable control-imit range, identify them clearly in all data records, investigate source of the problem, and make appropriate corrections. ‘When nitrification inhibitors are used, GGA test results out- side the contral-limit range often indicate that incorrect amounts of seed were used. Adjust the amount of seed added to the GGA, test so results fall within range (5210B.66). , Working range and reporting limit: The working range is equal tothe difference between the maximum initial DO (7 to 9 mg/L.) and minimum DO residual of 1 mg/L corrected for seed ‘and muliplied by the dilution factor, including any intermediate dilutions performed (5210B.5e) Reporting limits ate established by the minimum DO deple- tion and minimum DO residuals as follows: + The lower reporting limit for unseeded samples that require no dllution—except for nutrient, mineral, and buffer solu- tions (S = 0; P = 10}—is equal to the DO measurement method's detection limit (0.1 mgil.) + The lower reporting limit for seeded samples that require no dilution—except for seed, nutrient, mineral, and buffer so- lutions (S > 0; P = 1.0}—is the difference between sample DO depletion and seed correction. 9, References 1. You, LC. 1973, Chemical methods for nitifeation conto. J. Water Polat. Control Fed. 45:68. 2. US. ExwnosnesTat Pronacrio AGENCY, OFFICE OF RESEARCH AD Davmorami. 1986, Method-by-Method Statistics from Water Pol- lation (WP) Laboratory Performance Evaluation Studies. Quality ‘Assurance Beaneh, Environmental Monitoring and Support Tab, Cincinnati, Ohio, 10. Bibliography ‘Tennar, EJ, PD. McNawst & CT, Romana, 1931 Selection of| diluion water for use in oxygen demand tests, Pub. Health Rep 41084, Las, WL. & MS, Nieiots. 1937, Infuence of phosphorus and nitrogen on biochemical oxygen demand. Sewage Works J. 9:3. ‘Recimorr, C.C. 1941. Report on the cooperative study of dilution Waters made for the Standard Methods Committee of the Federation of Sewage Works Associations. Sewage Works J. 15:68. Momaux, FW., E Heawre, GR Baers & HK Rass 1950. [Experience with modified methods for BOD. Sewage Ind. Wastes 231 oust. LC. GN, MeDenworr & D, esas, 1981. Alterations inthe HOD procedire forthe 15th edtion of Standard Methods for the [Examination of Waler and Wastewater. J. Water Poll. Control Ped. 581253 5210 C. Ultimate BOD Test 1. General Discussion ‘The ultimate BOD testis an extension ofthe Sd dilution BOD test 52108) bot with a numberof specific test equirements and diferences in aplication. Be familia withthe 5210B procedure before conducting tests for UBOD. «@. Principle: The method consists of placing a single sample dilution in ful, aiztght botles and incubating under specified conditions for an extended period, depending on wastewater, efilueat, river, or estuary quality DO is measured (with probes) initially and intermitently during the test. From the DO versus time series, UBOD is calculated by an appro- priate slatistical technique. For more accuracy, run (eats i teplicate Bottle size and incubation time are lexble to accommodate individual sample characteristics and laboratory limitations. Ia- cubation temperature, however, is 20°C. Most effluents and some naturally occurring surface waters contain materials whose oxygen demands exceed the DO available in air-saturated water, in such cases, either dilute sample or monitor DO frequently to ensure that low DO or anaerebic conditions do not occur. Re- aerate sample when DO concentrations approach 2 mg/L pe: org/1021057S MWW.2882.102 Because bacterial growth requites nutrients (e.g. nitrogen, phosphorus, and trace metals), the necessary amounts may be added to dilution water, along with a buifer to keep pH in the bacterial-growth range and enough seed for an adequate bactezial population. (No specific nutrient or buffer formulations are in- cluded here because of the wide range of water and wastewater characteristics and Varied applications of UBOD data) That said, if the result will be used to estimate the oxidation rate of naturally occurring surface waters, adding nutvents and seed probably will accelerate the decay rate and prodiuce misleading results, If only UBOD is desited, adding supplemental nutrients that accelerate decay and shorten test duration may be advanta geous, When using nutrients, aso add them to the dilution-water lank How much nitrogenous compounds will oxidize during the prescribed incubation period depends on how many relevant oxidizing microorganisms are present. These organisms may be too scarce in wastewaters to oxidize significant quantities of reduced nitzogen, but abundant in naturally occurring surface waters, Results may be erratic when a nitrification inhibitor is ‘used,? so do NOT use one unless prior experimental evidence on