5210 BIOCHEMICAL OXYGEN DEMAND (BOD
Contents
1. or e Minimum DO is less than 1 0 mg L 7a3 8 Precision and Bias There is no measurement for establishing bias of the BOD procedure The glucose glutamic acid check prescribed in 6b 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 BIOCHEMICAL OXYGEN DEMAND BOD 5210 Ultimate BOD Test 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 1 1 mixture of glucose and glutamic acid in the total concen tration 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 concentration mg L 0 280 mg L S 0 100 added concentration 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 within2. New York N Y 6 NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT INc 1982 A Study of the Selection Calibration and Verification of Mathematical Water Quality Models Tech Bull No 367 New York N Y 7 NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT INC 1987 User s Manual for Parameter Estimation for First Order Ultimate BOD Decay BODFO Tech Bull No 529 New York N Y 8 Chu W S amp E W STRECKER 1972 Parameter Identification In Water System Model Dep Civil Engineering Univ Washington Seattle 5210 D Respirometric Method 1 General Discussion a Principle Respirometric methods provide direct measure ment of the oxygen consumed by microorganisms from an air or oxygen enriched environment in a closed vessel under condi tions of constant temperature and agitation b Uses Respirometry measures oxygen uptake more or less continuously over time Respirometric methods are useful for assessing biodegradation of specific chemicals treatability of organic industrial wastes the effect of known amounts of toxic compounds on the oxygen uptake reaction of a test wastewater or organic chemical the concentration at which a pollutant or a wastewater measurably inhibits biological degradation the ef fect of various treatments such as disinfection nutrient addition and pH adjustment on oxidation rates the oxygen requirement for essentially complete oxidation of biologi
3. as a seed source inhibition of nitrification is recommended Do not use seed from effluents that have been disinfected by chlo rine or other means Commercial seed sources may be used but are more likely to be unadapted to the wastewater constituents AGGREGATE ORGANIC CONSTITUENTS 5000 Do not filter seed sources filtering removes the seed microor ganisms When acclimated seed sources are not available develop an acclimated seed in the laboratory by continuously aerating a sample of settled domestic wastewater and adding small daily increments of sample from the waste in question Use a soil suspension activated sludge or a commercial seed preparation to obtain the initial microbial population Determine the exis tence 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 acclimation 5 Testing Procedure a Preparation of dilution water Transfer desired working volume of source water 4c to a suitably sized bottle glass is preferred Check to ensure that the dissolved oxygen concentra tion is at least 7 5 mg L before using water for BOD tests If not add DO by shaking bottle or by aerating with organic free filtered air Alternatively store the water in cotton plugged bot tles long enough for the DO concentration to approach satura tion Add 1 mL each of phosphate buffer MgSO Ca
4. dilution S 0 P lt 1 0 is 2 mg L multiplied by the dilution factor as established by the requirement for a minimum DO depletion of 2 mg L e The lower limit for seeded samples that require dilution S gt 0 P lt 1 0 is approximately 1 mg L as established by the minimum depletion of 2 0 mg L minus the maximum seed cor rection which should be less than about 1 mg L e 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 e The lower detection limit for seeded samples that require no dilution S gt 0 P 1 0 is 0 mg L as established by the difference between the sample DO depletion and the seed cor rection 9 References 1 Young J C 1973 Chemical methods for nitrification control J Water Pollut Control Fed 45 637 2 U S ENVIRONMENTAL PROTECTION AGENCY OFFICE OF RESEARCH AND DEVELOPMENT 1986 Method by Method Statistics from Water Pol lution WP Laboratory Performance Evaluation Studies Quality Assurance Branch Environmental Monitoring and Support Lab Cincinnati Ohio 10 Bibliography THERIAULT E J P D McNamee amp C T BUTTERFIELD 1931 Selection of dilution water for use in oxygen demand tests Pub Health Rep 46 1084 Lea W L amp M S NicHots 1937 Influence of phosphorus and nitrogen on biochemical oxygen demand Sewage Works J 9 34 Rucuuort C C 1941 Report on the cooperative stud
5. 6 2 5 0 25 5 3 5 8 0 4 6 0 0 0 4 6 7 6 2 6 4 0 23 6 2 10 3 2 8 2 9 4 0 10 0 46 8 9 15 4 3 13 3 0 58 12 7 18 2 7 8 1 14 9 0 15 0 69 14 2 20 6 6 16 4 0 80 15 6 25 5 4 17 6 0 20 0 92 16 7 30 2 6 8 2 20 4 0 92 19 5 40 5 3 23 3 0 20 0 92 22 4 50 3 1 8 0 25 5 0 92 24 6 60 4 5 29 0 0 92 28 1 70 3 3 8 1 30 2 0 92 29 3 90 5 4 32 9 0 20 0 92 32 0 Two readings indicate concentrations before and after reaeration None was used Column 1 blank correction none needed in the example Column 4 X 4 57 linear interpolation between values Column 3 Column 5 X dilution factor Ultimate CBOD 34 5 mg L CBOD decay rate 0 03 d calculated with first order equation from 5210C 4 When the weekly DO consumption drops below 1 to 2 of the total accumulative consumption calculate the ultimate BOD using a nonlinear regression method b Wastewater treatment plant samples Use high quality re agent water see Section 1080 for dilution water Add no nitri fication inhibitors if decay rates are desired If seed and nutrients are necessary add the same amounts of each to the dilution water blank Use minimal sample dilution As a rule of thumb the ultimate BOD of the diluted sample should be in the range of 20 to 30 mg L Dilution to this level probably will require two or three sample reaerations during the incubation period to avoid havin
6. atm pressure for 20 min Formula 2533 Hach Chemical Co Loveland CO or equivalent NoTE Some commercial formulations are not pure TCMP Check with supplier to verify compound purity and adjust dosages accordingly 5 12 o Yeast extract solution Add 15 mg laboratory or pharma ceutical grade brewer s yeast extract to 100 mL water Make this solution fresh immediately before each test in which it is used p Nutrient solution Add 2 5 mL phosphate buffer solution 3b 0 65 mL ammonium chloride solution 3c 1 0 mL calcium chloride solution 3d 0 22 mL magnesium sulfate solution 3e 0 1 mL ferric chloride solution 3f 1 mL trace element solution 3n and 1 mL yeast extract solution 30 to about 900 mL water Dilute to 1 L This nutrient solution and those of s n and o above are specifically formulated for use with the OECD method Note A 10 1 concentrated nutrient solution can be made and diluted accordingly 4 Procedure a Instrument operation Follow respirometer manufacturer s instructions for assembly testing calibration and operation of the instrument Nore The manufacturer s stated maximum and minimum limits of measurement are not always the same as the instrument output limits Make sure that test conditions are within the limits of measurement b Sample volume Sample volume or concentration of organic chemicals to be added to test vessels is a function of expected oxygen uptake characterist
7. 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 s 3a e directly to diluted sample at a rate of 1 mL L 0 30 mL 300 mL bottle or use commercially prepared solutions designed to dose the ap propriate bottle size d Addition of seed suspension If seeding is used add seed suspensions to the dilution vessels or to individual BOD bottles before final dilution as described in 5c Do not add seed directly to wastewater samples if they contain materials that are toxic before dilution Generally 1 to 3 mL of settled raw waste water or primary effluent or 1 to 2 mL of a 1 10 dilution of mixed liquor 300 mL bottle will provide a suitable amount of micro organisms Do not filter seed suspension before use Agitate the 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 bottle The DO uptake attributable to the seed added to each bottle generally 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 GGA check results of 198 30 5 mg L For example if 1 mL of seed suspension is required to achieve 198 30 5 mg L BOD in the glucose glutamic acid check then use 1 mL in
8. depletion may be reported as the BOD even if it is less than 2 0 mg L 3 When all dilutions result in a residual DO lt 1 0 select the bottle having the lowest DO concentration greatest dilution and report BOD mg L gt as P In the above calculations do not make corrections for DO uptake by the dilution water blank during incubation This cor rection is unnecessary if dilution water meets the blank criteria stipulated in 6c 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 b Reporting Average the test results for all qualified bottles within each dilution series Report the result as BOD if nitrifi cation is not inhibited Report results as CBOD if nitrification is inhibited Samples showing large differences between the com puted BOD for different dilutions for example greater than 30 may indicate the presence of a toxic substance or analytical problems When the effect becomes repetitive investigate to identify the cause Identify results in the test reports when any of the following quality control parameters is not met e Dilution water blank exceeds 0 20 mg L 6c e Glucose glutamic acid check falls outside acceptable limits I 6b e Test replicates show more than 30 difference between high and low values e Seed control samples do not meet the above criteria in all dilutions 6d
9. of ammo nium chloride 3c magnesium sulfate 3e calcium chloride 3d ferric chloride 3f and trace element solution 37 to approximately 800 mL water Add 10 mg nitrification inhibitor TCMP L Add sufficient seed from a suitable source as de scribed in J 4h to give a lag time less than 6 h usually 25 mL supernatant from settled primary effluent L test solution is suf ficient Dilute to 1 L Adjust temperature to 20 1 C Prepare a seed blank by diluting 500 mL or more of the seed solution to 800 mL with distilled water Add the same amount of buffer nutrients and TCMP as in the test solution and dilute to 1 L Adjust temperature to 20 1 C Place test solution and seed blank solution in separate reaction vessels of respirometer and incubate for 5 d at 20 C Run at least three replicates of each The seed corrected oxygen uptake after 5 d incubation should be 260 30 mg L If the value of the check is outside this range repeat the test using a fresh seed culture and seek the cause of the problem 7 Precision and Bias a Precision No standard is available to check the accuracy of respirometric oxygen uptake measurements To obtain labora tory precision data use a glucose glutamic acid mixture 6 above having a known theoretical maximum oxygen uptake value Tests with this and similar organic compound mixtures have shown that the standard deviation expressed as the coef 5 13 ficient of variation C is appr
10. 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 interlabo ratory tests is recommended as a control limit for individual laboratories Alternatively each laboratory may establish its control limits by performing a minimum of 25 glucose glutamic acid checks 6b 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 glu tamic acid checks Compare calculated control limits to the single laboratory tests presented above and to interlaboratory results If the glucose glutamic acid test results are outside the range of 198 30 5 re evaluate the control limits and investi gate 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 or identify such tests clearly in all data records and reports 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 corrected for seed and multiplied by the dilution factor Detection limits are established by the minimum DO depletion and minimum DO residuals as follows 5 7 e The lower detection limit for unseeded samples that require
11. water from the reservoir as necessary Cover dam with clean aluminum foil to retard evap oration If a 2 L BOD bottle is used fill reservoir with sample and cover with a polyethylene cap before incubation Place a clean magnetic stirring bar in each bottle to mix contents before making DO measurement or taking a subsample Do not remove the magnets until the test is complete Alternatively use a series of 300 mL BOD bottles as described in 5210B if larger bottles are not available or incubation space is limited Some analysts have reported satisfactory results with 2 chloro 6 trichloro methyl pyridine Nitrification Inhibitor Formula 2533 Hach Co Loveland CO or equivalent Wheaton 2 L BOD bottle No 227580 1000 North Tenth St Millville NJ or equivalent AGGREGATE ORGANIC CONSTITUENTS 5000 b Reservoir bottle 4 L or larger glass bottle Close with screw plastic cap or non rubber plug c Incubator or water bath thermostatically controlled at 20 1 C Exclude all light to prevent the possibility of photosynthetic production of DO d Oxygen sensitive membrane electrode See Section 4500 0 G 2 3 Procedure a River water samples Preferably fill large BOD bottle gt 2 L or alternatively 6 or more 300 mL BOD bottles with sample at 20 C Add no nutrients seed or nitrification inhibitor if in bottle decay rates will be used to estimate in stream rates Do not dilute sample unless it is known by pretesti
12. 5 2 AGGREGATE ORGANIC CONSTITUENTS 5000 5210 BIOCHEMICAL OXYGEN DEMAND BOD 5210 A 1 General Discussion The biochemical oxygen demand BOD determination is an empirical test in which standardized laboratory procedures are used to determine the relative oxygen requirements of wastewa ters effluents and polluted waters The test has its widest ap plication in measuring waste loadings to treatment plants and in evaluating the BOD removal efficiency of such treatment sys tems The test measures the molecular oxygen utilized during a specified incubation period for the biochemical degradation of organic material carbonaceous demand and the oxygen used to oxidize inorganic material such as sulfides and ferrous iron It also may measure the amount of oxygen used to oxidize reduced forms of nitrogen nitrogenous demand unless their oxidation is prevented by an inhibitor The seeding and dilution procedures provide an estimate of the BOD at pH 6 5 to 7 5 Measurements of oxygen consumed in a 5 d test period 5 d BOD or BOD 5210B oxygen consumed after 60 to 90 d of incubation ultimate BOD or UBOD 5210C and continuous oxygen uptake respirometric method 5210D are described here Many other variations of oxygen demand measurements exist including using shorter and longer incubation periods and tests to determine rates of oxygen uptake Alternative seeding dilution and incubation conditions can be chosen to mimic receiving
13. 5e is recommended for samples of secondary effluent for samples seeded with second ary effluent and for samples of polluted waters 3 Reference 1 Young J C 1973 Chemical methods for nitrification control J Water Pollut Control Fed 45 637 5210 B 5 Day BOD Test 1 General Discussion The method consists of filling with diluted and seeded sample to overflowing an airtight bottle of specified size and incubating it at the specified temperature for 5 d Dissolved oxygen is measured initially and after incubation and the BOD is com puted from the difference between initial and final DO Because the initial DO is determined shortly after the dilution is made all BIOCHEMICAL OXYGEN DEMAND BOD 5210 5 Day BOD Test oxygen uptake occurring after this measurement is included in the BOD measurement For sampling and storage procedures see 4a below 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 b Air incubator or water bath thermostatically controlled at 20 1 C Exclude all light to prevent possibility of photosyn thetic 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 Commer cial equivalents of these reagents are
14. 8 p 12 4 mg L 2 12 6 3 12 6 CV 44 4 11 6 w mean CV coefficient of variation 5 10 Bias was assessed by determining the BOD of a known concentration of glucose 150 mg L and glutamic acid 150 mg L This solution has a UBOD of 321 mg L to 308 mg L depending on extent of nitrification The results of the study conducted in triplicate were Estimated Theoretical UBOD BOD Percent mg L mg L Difference 276 308 321 10 14 310 308 321 1 3 303 308 321 2 6 By statistical model 6 References 1 Martone C H 1976 Studies Related to the Determination of Bio degradability and Long Term BOD MLS thesis Dep Civil Engi neering Tufts Univ Medford Mass 2 NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT INc 1986 A Review of the Separation of Carbona AGGREGATE ORGANIC CONSTITUENTS 5000 ceous and Nitrogenous BOD in Long Term BOD Measurements Tech Bull No 461 New York N Y 3 NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT Inc 1982 A Review of Ultimate BOD and Its Kinetic Formulation for Pulp and Paper Mill Effluents Tech Bull No 382 New York N Y 4 BARNWELL T 1980 Least Squares Estimates of BOD Parameters J Environ Eng Div Proc Amer Soc Civil Eng 107 EE6 1197 5 NATIONAL COUNCIL OF THE PAPER INDUSTRY FOR AIR AND STREAM IMPROVEMENT INc 1982 A Proposal to Examine the Effect of Mixing on Long Term BOD Test NE82 01
15. Cl and FeCl solution L to prepared source water J 4c Mix thoroughly and bring temperature to 20 3 C Prepare dilution water immediately before use unless dilution water blanks J 6c show that the water is acceptable after longer storage times If the dilution water blanks show a DO depletion greater than 0 20 mg L obtain a satisfactory water by improving purification or use water from another source Do not add oxidizing agents or expose dilution water to ultraviolet light in attempts to bring the dilution blank into range b Sample temperature adjustment Bring samples to 20 3 C before making dilutions c Preparation of dilutions Using the dilution water prepared as in f 5a make at least three dilutions of prepared sample estimated to produce a residual DO of at least 1 0 mg L and a DO uptake of at least 2 0 mg L after a 5 d incubation Five dilutions are recommended if experience with a particular sample does not produce at least three bottles having acceptable minimum DO depletions and residual limits 6a 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 strong industrial wastes 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 b
16. acceptable and different stock concentrations may be used if doses are adjusted propor tionally Use reagent grade or better for all chemicals and use distilled or equivalent water preferably sterilized for making all solutions a Phosphate buffer solution Dissolve 8 5 g KH POu 21 75 g K HPOg 33 4 g Na HPO 7H 0 and 1 7 g NH Cl 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 KH PO and 1 7 g NH Cl 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 MgSO 7H O in distilled water and dilute to 1 L c Calcium chloride solution Dissolve 27 5 g CaCl in dis tilled water and dilute to 1 L d Ferric chloride solution Dissolve 0 25 g FeCl 6H O in distilled water and dilute to 1 L e Acid and alkali solutions 1N for neutralization of caustic or acidic waste samples 1 Acid Slowly and 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 Na SO in 1000 mL distilled water This solution is not stable prepare daily g Nitrification inhibitor 1 2 chloro 6 trichloromethyl pyridine Use pure TCMP or commercial preparations 2 Allylthiourea ATU solution Dissolve 2 0 g allylthio urea C4HgN S in about 500 mL wa
17. al DO The plot should present a straight line for which the slope indicates DO depletion per milliliter of seed The DO axis intercept is oxygen depletion caused by the dilution water and should be less than 0 20 mg L see 6c For the ratio method divide the DO depletion by the volume of seed in milliliters for each seed control bottle having a 2 0 mg L deple tion and greater than 1 0 mg L minimum residual DO and average the results Seed dilutions showing widely varying de pletions per milliliter of seed 30 suggest the presence of toxic substances or large particulates in the seed suspension In this case check or change the seed source 7 Data Analysis and Reporting a Calculations 1 For each test bottle having 2 0 mg L minimum DO deple tion and at least 1 0 mg L residual DO calculate BOD as follows AGGREGATE ORGANIC CONSTITUENTS 5000 Di D3 OV P BOD mg L where D DO of diluted sample immediately after preparation mg L D DO of diluted sample after 5 d incubation at 20 C mg L S oxygen uptake of seed A DO mL seed suspension added per bottle J 6d S 0 if samples are not seeded V volume of seed in the respective test bottle mL and P decimal volumetric fraction of sample used 1 P dilution factor 2 If DO depletion is less than 2 0 mg L and sample concen tration is 100 no dilution except for seed nutrient mineral and buffer solutions actual seed corrected DO
18. cally oxidizable matter the need for using adapted seed in other biochemical oxygen uptake measurements such as the dilution BOD test and stability of sludges Respirometric data typically will be used comparatively that is in a direct comparison between oxygen uptakes from two test samples or from a test sample and a control Because of inherent differences among uses among seed cultures among applica tions of results and among instruments a single procedure for respirometric tests applicable to all cases cannot be defined Therefore only basic recommendations and guidelines for over all test setup and procedure are given Follow manufacturer s instructions for operating details for specific commercial instru ments c Types of respirometers Four principal types of commercial respirometers are available Manometric respirometers relate oxygen uptake to the change in pressure caused by oxygen consumption while maintaining a constant volume Volumetric respirometers measure oxygen uptake in incremental changes in gas volume while maintaining a constant pressure at the time of reading Electrolytic respirometers monitor the amount of oxy gen produced by electrolysis of water to maintain a constant oxygen pressure within the reaction vessel Direct input respi rometers deliver oxygen to the sample from a pure oxygen supply through metering on demand as detected by minute pressure differences Most respirometers have been instrumente
19. ck for residual chlorine Re seed the sample see 4h below 4 Samples containing toxic substances Certain industrial wastes contain toxic metals or organic compounds These often require special study and treatment 5 Initial oxygen concentration If samples contain dissolved oxygen concentrations above or below the desired concentration agitate or aerate with clean and filtered compressed air for about 1 h immediately before testing Minimum and maximum actual DO concentrations will vary with test objectives In some cases AGGREGATE ORGANIC CONSTITUENTS 5000 pure oxygen may be added to respirometer vessels to increase oxygen levels above ambient 6 Temperature adjustment Bring samples and dilution water to desired test temperature 1 C before making dilutions or transferring to test vessels e Sample dilution Use distilled water or water from other appropriate sources free of organic matter In some cases re ceiving stream water may be used for dilution Add desired sample volume to test vessels using a wide tip volumetric pipet or other suitable volumetric glassware Add dilution water to bring sample to about 80 of desired final volume Add appro priate amounts of nutrients minerals buffer nitrification inhib itor if desired and seed culture as described in Js 4f and h below Dilute sample to desired final volume The number of test vessels to prepare for each dilution depends on test objectives and number of
20. clusion of ammonia in the dilution water contributes an exter nal source of nitrogenous demand The interference from nitrog enous demand can now be prevented by an inhibitory chemical If an inhibiting chemical is not used the oxygen demand mea sured is the sum of carbonaceous and nitrogenous demands Measurements that include nitrogenous demand generally are not useful for assessing the oxygen demand associated with organic material Nitrogenous demand can be estimated directly from am monia nitrogen Section 4500 NH and carbonaceous demand can be estimated by subtracting the theoretical equivalent of the nitrite and nitrate produced in uninhibited test results However this method is cumbersome and is subject to considerable error Chem ical inhibition of nitrogenous demand provides a more direct and more reliable measure of carbonaceous demand The extent of oxidation of nitrogenous compounds during the 5 d incubation period depends on the concentration and type of microorganisms capable of carrying out this oxidation Such organisms usually are not present in raw or settled primary sewage in sufficient numbers to oxidize sufficient quantities of reduced nitrogen forms in the 5 d BOD test Many biological treatment plant effluents contain sufficient numbers of nitrifying organisms to cause nitrification in BOD tests Because oxidation of nitrogenous compounds can occur in such samples inhibition of nitrification as directed in 5210B
21. d to permit data collection and processing by computer Reaction vessel contents are mixed by using a magnetic or mechanical stirring device or by bubbling the gaseous phase within the reaction vessel through the liquid phase All respirometers re move carbon dioxide produced during biological growth by suspending a concentrated adsorbent granular or solution within the closed reaction chamber or by recirculating the gas phase through an external scrubber d Interferences Evolution of gases other than CO may introduce errors in pressure or volume measurements this is uncommon in the presence of dissolved oxygen Incomplete CO absorption will introduce errors if appropriate amounts and con centrations of alkaline absorbent are not used Temperature fluctuations or inadequate mixing will introduce error Fluctua tions in barometric pressure can cause errors with some respi rometers Become familiar with the limits of the instrument to be used e Minimum detectable concentration Most commercial re spirometers can detect oxygen demand in increments as small as 0 1 mg but test precision depends on the total amount of oxygen consumed at the time of reading the precision of pressure or volume measurement and the effect of temperature and baro metric pressure changes Upper limits of oxygen uptake rate are BIOCHEMICAL OXYGEN DEMAND BOD 5210 Respirometric Method determined by the ability to transfer oxygen into the solution from th
22. dilute to 1 L 1 mL 10 mg Mg f Ferric chloride solution 0 018N Dissolve 4 84 g FeCl 6H O in water and dilute to 1 L 1 mL 1 0 mg Fe g Potassium hydroxide solution 6N Dissolve 336 g KOH in about 700 mL water and dilute to 1 L Caution Add KOH to water slowly and use constant mixing to prevent excessive heat buildup Alternately use commercial solutions containing 30 to 50 KOH by weight h Acid solutions 1N Add 28 mL conc H SO or 83 mL conc HCI to about 700 mL water Dilute to 1 L i Alkali solution 1N Add 40 g NaOH to 700 mL water Dilute to 1 L j Nitrification inhibitor Reagent grade 2 chloro 6 trichlo romethyl pyridine TCMP or equivalent k Glucose glutamic acid solution Dry reagent grade glucose and reagent grade glutamic acid at 103 C for 1 h Add 15 0 g glucose and 15 0 g glutamic acid to distilled water and dilute to 1 L Neutralize to pH 7 0 using 6N potassium hydroxide 3g This solution may be stored for up to 1 week at 4 C l Electrolyte solution for electrolytic respirometers Use manufacturer s recommended solution m Sodium sulfite solution 0 025N Dissolve 1 575 g Na SO in about 800 mL water Dilute to 1 L This solution is not stable prepare daily or as needed n Trace element solution Dissolve 40 mg MnSO 4H O 57 mg H BO3 43 mg ZnSO 7H O 35 mg NH4 Mo7Oo4 and 100 mg Fe chelate FeCl EDTA in about 800 mL water Dilute to 1 L Sterilize at 120 C and 200 kPa 2
23. e gas phase which typically is related to mixing intensity Transfer limits typically range from less than 10 mg O L h for low intensity mixing to above 100 mg O L h for high intensity mixing f Relationship to dilution BOD Variations in waste compo sition substrate concentration mixing and oxygen concentra tions from one wastewater source to another generally preclude use of a general relationship between oxygen uptake by respi rometers and the 5 d 20 C BOD see 5210B above Reason ably accurate correlations may be possible for a specific waste water The incubation period for respirometric measurements need not be 5 d because equally valid correlations can be made between the 5 d dilution BOD and respirometric oxygen uptake at any time after 2 d The point of common dilution and respirometric BOD seems to occur at about 2 to 3 d incubation for municipal wastewaters Correlations between respirometric measurements and 5 d BOD for industrial wastes and specific chemicals are less certain Respirometric measurements also can provide an indication of the ultimate biochemical oxygen de mand UBOD see Section 5210C In many cases it is reason able to consider that the 28 to 30 d oxygen uptake is essentially equal to the UBOD More commonly respirometers are used as a diagnostic tool The continuous readout of oxygen consumption in respirometric measurements indicates lag toxicity or any abnormalities in the biodegradati
24. each BOD bottle receiving the test wastewater e Addition of nitrification inhibitor Samples that may require nitrification inhibition include but are not limited to biologi cally treated effluents samples seeded with biologically treated effluents and river waters Note the use of nitrogen inhibition and the chemical used when reporting results NOTE TCMP is the preferred nitrification inhibitor but requires handling and transfer in a solid form Allylthiourea is not always effective in inhibiting nitrification within the 5 d incubation period and con centrations above 2 mg L may cause increases in carbonaceous BOD measurements ATU concentrations above 2 mg L also can adversely affect the azide modification of the iodometric meth od Seed all samples to which nitrification inhibitor has been added The amount of seed should be consistent with that re quired to achieve GGA test results in the range of 198 30 5 mg L 6b 1 Nitrification inhibition using 2 chloro 6 trichloromethy pyridine TCMP Add 10 mg TCMP L to diluted sample or 3 mg TCMP to each 300 mL bottle or sample dilution vessel or proportional amounts to other sized bottles after initial sample dilution but before final filling of the bottles with dilution water Do not add TCMP to BOD bottles before they are at least two thirds filled with diluted sample Nore TCMP dissolves slowly and can float on top of the sample if not mixed well 5 5 Some commercia
25. esidual DO in all dilutions is less than 1 0 mg L see 7 When using membrane electrodes for measuring DO make frequent calibration checks to ensure accurate DO readings see 4500 O C b Glucose glutamic acid check The glucose glutamic acid check is the primary basis for establishing accuracy and preci sion of the BOD test and is the principal measure of seed quality and set up procedure Together with each batch of samples 5 6 check seed effectiveness and analytical technique by using pro cedures in 5 to make BOD measurements on an equal weight mixture of glucose and glutamic acid as follows Add sufficient amounts of standard glucose glutamic acid solution 3h to give 3 0 mg glucose L and 3 0 mg glutamic acid L in each of three test bottles 20 mL GGA solution L seeded dilution water or 6 0 mL 300 mL bottle Commercial solutions may contain other glucose glutamic acid concentrations adjust doses accord ingly Add nitrification inhibitor if seed is obtained from a source that is nitrifying Evaluate data as described in 8 Precision and Bias The resulting average BOD for the three bottles after correction for dilution and seeding must fall into the range of 198 30 5 mg L If the average value falls outside this range evaluate the cause and make appropriate corrections Consis tently high values can indicate the use of too much seed suspen sion contaminated dilution water or the occurrence of nitrifica tion consi
26. f reduced nitrogen This situation may be reversed in naturally occurring surface waters Erratic results may be obtained when a nitrification inhibitor is used therefore the specified method precludes use of a nitrogen inhibitor unless prior experimental evidence on the particular sample suggests that it is acceptable Monitor NO N and NO N to compute the oxygen equivalency of the nitrification reaction When these values are subtracted from the DO vs time series the carbonaceous BOD time series can be constructed b Sampling and storage See Section 5210B 4a 2 Apparatus a Incubation bottles Glass bottles with ground glass stop pers 2 L or larger capacity Glass serum bottles of 4 to 10 L capacity are available Alternatively use nonground glass bottles with nonbiodegradable plastic caps as a plug insert Do not reuse the plugs because discoloration occurs with continued use Re place plugs every 7 to 14 d Do not use rubber stoppers that may exert an oxygen demand Clean bottles with a detergent and wash with dilute HCl 3N to remove surface films and precip itated inorganic salts rinse thoroughly with DI water before use Cover top of bottles with paper after rinsing to prevent dust from collecting To prevent drawing air into the sample bottle during incubation use a water seal If the bottle does not have a flared mouth construct a water seal by making a watertight dam around the stopper or plug and fill with
27. from the bottle for these analyses Refill bottle as necessary from the reservoir bottle Preserve NO N NO N subsample with H SO to pH lt 2 and refrigerate If the purpose of the UBOD test is to assess the UBOD and not to provide data for rate calculations measure nitrate nitrogen concentration only at Day 0 and on the last day of the test kinetic rate estimates are not useful when the nitri fication reaction is not followed Calculate oxygen consumption during each time interval and make appropriate corrections for nitrogenous oxygen demand Correct by using 3 43 X the NH3 N to NO N conversion plus 1 14 X the NO N to NO N conversion to reflect the stoi chiometry of the oxidation of NH to NO or NO When using a dilution water blank subtract DO uptake of the blank from the total DO consumed High quality reagent water without nutrients typically will consume a maximum of 1 mg DO L in a 30 to 90 d period If DO uptake of the dilution water is greater than 0 5 mg L for a 20 d period or 1 mg L for a 90 d period report the magnitude of the correction and try to obtain higher quality dilution water for use with subsequent UBOD tests BIOCHEMICAL OXYGEN DEMAND BOD 5210 Ultimate BOD Test 5 9 TABLE 5210 I UBOD RESULTS FoR WASTEWATER SAMPLE a 2 3 4 5 6 Accumulated Average Average DO Consumed Average DO Blank DO by Samplet NO N NBOD CBOD Day mg L mg L mg L mg L mg L meg L 0 8 1 0 0 0 0 0 3 5
28. g dissolved oxygen concentrations fall below 2 mg L Use 2 L or larger BOD bottles alternatively multiple 300 mL BOD bottles for each dilution Add desired volume of sample to each bottle and fill with dilution water Fill a BOD bottle with dilution water to serve as a dilution water blank Treat blank the same as all samples Follow procedure given in 3a and incubate for at least as long as UBOD test 4 Calculations An example of results obtained for a wastewater sample undiluted without seed and nutrients is given in Table 5210 I UBOD can be estimated by using a first order model described as follows BOD UBOD 1 e where BOD oxygen uptake measured at time t mg L and k first order oxygen uptake rate The data in Table 5210 I were analyzed with a nonlinear regression technique applied to the above first order model However a first order kinetic model may not always be the best choice Significantly better statistical fits usually are obtained with alternative kinetic models including sum of two first order and logistic function models 5 Precision and Bias The precision of the ultimate BOD test was assessed with a series of replicate tests in a single laboratory Interlaboratory studies have not been conducted UBOD Reference Replicate No mg L Precision Summary 2 1 154 p 151 mg L 2 154 3 145 CV 3 5 5 1 10 3 2 11 1 3 9 6 p 10 0 mg L 4 9 9 5 9 8 CV 5 8 6 9 6 6 1 12
29. h to cause deterioration in others Storage of prepared dilution water 5g for more than 24 h after adding nutrients minerals and buffer is not recommended unless dilution water blanks consistently meet quality control limits Discard stored source water if the dilution water blank shows more than 0 20 mg L DO depletion in 5 d see 6c d Preparation of seed suspension It is necessary to have present in each BOD bottle a population of microorganisms capable of oxidizing the biodegradable organic matter in the sample Domestic wastewater unchlorinated or otherwise undis infected effluents from biological wastewater treatment plants and surface waters receiving wastewater discharges usually con tain satisfactory microbial populations Some samples for ex ample some untreated industrial wastes disinfected wastes high temperature wastes wastes having pH values less than 6 or greater than 8 or wastes stored more than 6 h after collection do not contain a sufficient microbial population Seed such samples by adding a population of suitable microorganisms The pre ferred seed is obtained from a biological treatment system pro cessing the waste In this case use supernatant from settled domestic wastewater effluent from primary clarifiers diluted mixed liquor from an aeration basin undisinfected effluent or receiving water from below the point of discharge When efflu ent or mixed liquor from a biological treatment process is used
30. hin 30 min If the membrane electrode method is used take care to eliminate drift in calibration between initial and final DO readings If the azide modification of the titrimetric iodometric method is used pre pare an extra bottle for initial DO determination for each sample dilution h Sample incubation Incubate at 20 C 1 C the stoppered and sealed BOD bottles containing desired dilutions 5a seed controls 6d dilution water blanks J 6c and glucose glu tamic acid checks 6b Exclude light to avoid growth of algae in the bottles during incubation i Determination of final DO After 5 d 6 h of incubation determine DO in all sample dilutions and in all blanks and checks as in 6b d using the azide modification of the titri metric method or the membrane electrode method 6 Quality Control Checks a Minimum residual DO and minimum DO depletion Only bottles including seed controls giving a minimum DO depletion of 2 0 mg L and a residual DO of at least 1 0 mg L after 5 d of incubation are considered to produce valid data because at least 2 0 mg oxygen uptake L is required to give a meaningful mea sure 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 Exceptions occur for reporting purposes only when the depletions for tests using undiluted samples in all bottles fall below 2 0 mg L and when the r
31. ial as for any other sample This is the seed control Typically the seed volume in the seed control should be 10 times the volume used in seeded samples i Incubation Incubate samples at 20 C or other suitable temperature 1 0 C Take care that the stirring device does not increase the temperature of the sample 5 Calculations To convert instrument readings to oxygen uptake refer to manufacturer s procedures Correct oxygen uptake for seed and dilution by the following equation C A B S4 Sg 1000 N where C corrected oxygen uptake of sample mg L A measured oxygen uptake in seeded sample mg BIOCHEMICAL OXYGEN DEMAND BOD 5210 Respirometric Method B measured oxygen uptake in seed control mg S volume of seed in Sample A mL Sg volume of seed in Sample B mL and N volume of undiluted sample in Sample A mL 6 Quality Control Periodically use the following procedure to check distilled water quality instrument quality instrument function and ana lytical technique by making oxygen uptake measurements using a mixture of glucose and glutamic acid as a standard check solution Adjust water for sample formulation to test temperature and saturate with DO by aerating with clean organic free filtered air Protect water quality by using clean glassware tubing and bottles Prepare a test solution by adding 10 mL glucose glutamic acid solution 3k 6 mL phosphate buffer 3b 2 mL each
32. ics and oxygen transfer capability of the instrument Small volumes or low concentrations may be required for high strength wastes Large volumes may be re quired for low strength wastes to improve accuracy c Data recording interval Set instrument to give data read ings at suitable intervals Intervals of 15 min to 6 h typically are used d Sample preparation 1 Homogenization If sample contains large settleable or floatable solids homogenize it with a blender and transfer rep resentative test portions while all solids are in suspension If there is a concern for changing sample characteristics skip this step 2 pH adjustment Neutralize samples to pH 7 0 with H SO or NaOH of such strength s 3h and i that reagent quantity does not dilute the sample more than 0 5 3 Dechlorination Avoid analyzing samples containing re sidual chlorine by collecting the samples ahead of chlorination processes If residual chlorine is present aerate as described in 5 below or let stand in light for 1 to 2 h If a chlorine residual persists add Na SO solution Determine required volume of NaSO solution by adding 10 mL 1 1 acetic acid or 1 50 H SO and 10 mL potassium iodide solution 10 g 100 mL to a portion of the sample Titrate with 0 025N Na SO solution to the starch iodine end point see Section 4500 C1 B Add to the neutralized sample a proportional volume of Na SO solution determined above mix and after 10 to 20 min che
33. ide Hydrogen perox ide remaining in samples from some industrial bleaching pro cesses such as 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 for sufficient time to allow the hydrogen peroxide to dissipate before setting up BOD tests Check adequacy of peroxide re moval by observing dissolved oxygen concentrations over time during mixing or by using peroxide specific test strips Mixing times can vary from 1 to 2 h depending on the amount of hydrogen peroxide present The peroxide reaction can be con sidered complete when the DO no longer increases during a 30 min period without mixing c Selection and storage of source water for BOD sample dilution Obtain water from suitable source distilled tap or receiving water Make sure the water is free of heavy metals specifically copper and toxic substances such as chlorine that can interfere with BOD measurements Protect source water quality by using clean glassware tubing and bottles Deionized water often contains sufficient amounts of organics and micro organisms to cause failure of the dilution water quality control check 6c Source water may be stored before use as long as the prepared dilution water 5a meets quality control criteria in the dilution water blank 6c Such storage may improve the quality of some source waters but may allow biological growt
34. l TCMP formulations are not 100 TCMP adjust dosage appropriately 2 Nitrification inhibition using allylthiourea ATU Add 1 mL ATU solution J 3g L diluted sample or 0 3 mL 300mL test bottle or sample dilution vessel Do not add ATU to BOD bottles until they are at least two thirds filled with diluted sample f Sealing of bottles Complete filling of each bottle by adding enough dilution water that insertion of the stopper leaves no bubbles in the bottle Mix the sample by turning the bottle manually several times unless a DO probe having a stirrer is used immediately to measure initial DO concentration As a precau tion against drawing air into the dilution bottle during incuba tion use a water seal Obtain satisfactory water seals by invert ing 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 g Determination of initial DO 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 appropri ate seed controls Replace any displaced contents with sufficient diluted sample or dilution water to fill the bottle stopper all bottles tightly and water seal before beginning incubation After preparing dilution measure initial DO wit
35. ng or by experi ence to have a high ultimate BOD gt 20 mg L Measure DO in each bottle stopper and make an airtight seal Incubate at 20 C in the dark Measure DO in each bottle at intervals of at least 2 to 5 d over a period of 30 to 60 d minimum of 6 to 8 readings or longer under special circumstances To avoid oxygen depletion in sam ples containing NH3 N measure DO more frequently until ni trification has taken place If DO falls to about 2 mg L reaerate as directed below Replace sample lost by the cap and DO probe displacement by adding to 2 mL sample from the reservoir bottle When DO approaches 2 mg L reaerate Pour a small amount of sample into a clean vessel and reaerate the remainder directly in the bottle by vigorous shaking or bubbling with purified air medical grade Refill bottle from the storage reservoir and measure DO This concentration becomes the initial DO for the next measurement If using 300 mL BOD bottles pour all of the sample from the several bottles used into a clean vessel reaerate and refill the small bottles Analyze for nitrate plus nitrite nitrogen NO N NO N see Sections 4500 NO and 4500 NO on Days 0 5 10 15 20 and 30 Alternatively determine NO N and NO N each time DO is determined thereby producing corresponding BOD and nitrogen determinations If the ultimate demand occurs at a time greater than 30 d make additional analyses at 30 d inter vals Remove 10 to 20 mL
36. on reaction The change in the normal shape of an oxygen uptake curve in the first few hours may help to identify the effect of toxic or unusual wastes entering a treatment plant in time to make operating corrections g Relationship to other test methods and protocols This method supports most of the protocols and guidelines established by the European Organization for Economic Co operation and Development OECD that require measurement of oxygen uptake h Sampling and storage 1 Grab samples If analysis is begun within 2 h of sample collection cold storage is unnecessary Otherwise keep sample at or below 4 C from the time of collection Begin analysis within 6 h of collection when this is not possible store at or below 4 C and report length and temperature of storage Never start analysis more than 24 h after grab sample collection 2 Composite samples Keep samples at or below 4 C 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 the end of the compositing period State storage time and conditions with results 2 Apparatus a Respirometer system Use commercial apparatus and check manufacturer s instructions for specific system requirements reaction vessel type and volume and instrument operating char acteristics b Incubator or water bath Use a constant temperature room incubator chamber or water bath to con
37. ottles to be prepared for each dilution depends on the DO technique and the number of replicates desired Prepare dilutions in volumetric containers Class A glass or equivalent and then transfer to BOD bottles or prepare directly in BOD bottles Either dilution method can be combined with any DO measurement technique 1 Dilutions prepared in volumetric containers Using a wide tipped pipet add desired amount of prepared sample to individual volumetric cylinders or flasks Mix the sample well immediately before pipetting to avoid loss of solids by settling For dilutions greater than 1 100 make a primary dilution before making final dilution in the bottle Fill cylinders or flasks at least two thirds full of dilution water without entraining air Add BIOCHEMICAL OXYGEN DEMAND BOD 5210 5 Day BOD Test appropriate amounts of seed suspension 5d and nitrification inhibitor J 5e Dilute to final level with dilution water J 5a Mix well but avoid entraining air Siphon mixed dilution into a suitable number of BOD bottles taking care not to let solids settle in the cylinder or flask during transfer 2 Dilutions prepared directly in BOD bottles Using a wide tip volumetric pipet add the desired sample volume to individual BOD bottles Fill each BOD bottle approximately two thirds full with dilution water Add appropriate amounts of seed suspension T 5d and nitrification inhibitor J 5e to the individual BOD bottles When a bottle
38. ow 4 C and report length and temper ature 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 sam ples for analysis within 6 h of collection 2 Composite samples Keep samples at or below 4 C 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 b Sample preparation and pretreatment 1 All samples Check pH if it is not between 6 0 and 8 0 adjust sample temperature to 20 3 C then adjust pH to 7 0 to 7 2 using a solution of sulfuric acid H SO or sodium hydrox ide NaOH of such strength that the quantity of reagent does not dilute the sample by more than 0 5 Exceptions may be justi fied with natural waters when the BOD is to be measured at in situ pH values 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 pos sible avoid samples containing residual chlorine by sampling ahead of chlorination processes If residual chlorine is present dechlorinate sample In some samples chlorine will dissipate within 1 to 2 h of standing in the light This dissipation often occurs during sample trans
39. oximately 5 for samples having total oxygen uptakes of 50 to 100 mg L and 3 for more concentrated samples Individual instruments have different readability limits that can affect precision The minimum re sponse or sensitivity of most commercial respirometers ranges from 0 05 to 1 mg oxygen Check manufacturer s specifications for sensitivity of the instrument at hand b Control limits To establish laboratory control limits per form a minimum of 25 glucose glutamic acid checks over a period of several weeks or months and calculate mean and standard deviation If measured oxygen uptake in 5 d at 20 C is outside the 260 30 mg L range re evaluate procedure to identify source of error For other samples use the mean 3 standard deviations as the control limit c Working range and detection limits The working range and detection limits are established by the limits of each commercial instrument Refer to manufacturer s specifications 8 References 1 Youna J C amp E R BAUMANN 1976 The electrolytic respirometer Factors affecting oxygen uptake measurements Water Res 10 1031 2 Youn J C amp E R BAUMANN 1976 The electrolytic respirometer Use in water pollution control plant laboratories Water Res 10 1141 3 ORGANIZATION FOR ECONOMIC CO OPERATION AND DEVELOPMENT 1981 Method 5 2 Annex V Part C in OECD Guidelines for Testing of Chemicals OECD Paris France 4 Youna J C 1973 Chemical methods for ni
40. port and handling For samples in which chlorine residual does not dissipate in a reasonably short time destroy chlorine residual by adding Na SO solution De termine required volume of Na SO solution on a 100 to 1000 mL portion of neutralized sample by adding 10 mL 1 1 acetic acid or 1 50 H SO 10 mL potassium iodide KI solution 10 g 100 mL per 1000 mL sample and titrating with NaSO solution to the starch iodine end point for residual Add to neutralized sample the proportional volume of NaSO solu tion determined by the above test mix and after 10 to 20 min check sample for residual chlorine NOTE Excess NaSO exerts an oxygen demand and reacts slowly with certain organic chlo ramine compounds that may be present in chlorinated samples Do not test chlorinated dechlorinated samples without seeding 3 Samples containing other toxic substances Certain indus trial wastes for example plating wastes contain toxic metals Such samples often require special study and treatment 5 4 4 Samples supersaturated with DO Samples containing DO concentration above saturation at 20 C 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 by bringing sample to about 20 3 C in partially filled bottle while agitating by vigorous shaking or by aerating with clean filtered compressed air 5 Samples containing hydrogen perox
41. replicates desired f Nutrients minerals and buffer Add sufficient ammonia nitrogen to provide a COD N P ratio of 100 5 1 or a TOC N P ratio of 30 5 1 Add 2 mL each of calcium magnesium ferric chloride and trace mineral solutions to each liter of diluted sample unless sufficient amounts of these minerals are present in the original sample Phosphorus requirements will be met by the phosphate buffer if it is used 1 mL 50 mg L COD or ultimate BOD of diluted sample usually is sufficient to maintain pH between 6 8 and 7 2 Be cautious in adding phosphate buffer to samples containing metal salts because metal phosphates may precipitate and show less toxic or beneficial effect than when phosphate is not present For OECD compatible tests substitute the nutrient mineral and buffer amounts listed in 3p for the above nutrient mineral buffer quantities g Nitrification inhibition If nitrification inhibition is desired add 10 mg 2 chloro 6 trichloromethyl pyridine TCMP L sample in the test vessel Samples that may nitrify readily in clude biologically treated effluents samples seeded with biolog ically treated effluents and river waters h Seeding See 5210B 4d for seed preparation Use sufficient amounts of seed culture to prevent major lags in the oxygen uptake reaction but not so much that the oxygen uptake of the seed exceeds about 10 of the oxygen uptake of the seeded sample Determine the oxygen uptake of the seeding mater
42. stently low values can indicate poor seed quality or quantity or the presence of a toxic material If low values persist prepare a new mixture of glucose and glutamic acid and check the sources of dilution water and source of seed c Dilution water quality check With each batch of samples incubate one or more bottles of dilution water that contains nutrient mineral and buffer solutions but no seed or nitrification inhibitor This dilution water blank serves as a check on quality of unseeded dilution water and cleanliness of incubation bottles Determine initial and final DO as in Js 5e and g The DO uptake in 5 d must not be more than 0 20 mg L and preferably not more than 0 10 mg L before making seed corrections If the dilution water blank exceeds 0 20 mg L discard all data for tests using this dilution water or clearly identify such samples in data records d Seed control Determine BOD of the seed suspension as for any other sample This is the seed control Ideally make three dilutions of seed such that the smallest quantity gives at least 2 0 mg L DO depletion and the largest quantity results in at least 1 0 mg L DO residual after 5 d of incubation Determine the DO uptake per milliliter of seed added to each bottle using either the slope method or the ratio method For the slope method plot DO depletion in milligrams per liter versus milliliters of seed for all seed control bottles having a 2 0 mg L depletion and 1 0 mini mum residu
43. ter and dilute to 1 L Store at 4 C The solution is stable for not more than 2 weeks h Glucose glutamic acid solution Dry reagent grade glucose and reagent grade glutamic acid at 103 C 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 unless solution is maintained in a sterile condition Store all glucose glutamic acid mixtures at 4 C or lower Commercial preparations may be used but concentrations may vary Nitrification Inhibitor Formula 2533 2 TCMP on sodium sulfate Hach Co Loveland CO or equivalent 5 3 i Ammonium chloride solution Dissolve 1 15 g NH Cl 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 Source water for preparing BOD dilution water Use de mineralized distilled tap or natural water for making sample dilutions see 4c 4 Preparatory Procedures a 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 is begun within 2 h of collec tion cold storage is unnecessary If analysis is not started within 2 h of sample collection keep sample at or below 4 C 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 bel
44. ters contain materials with oxygen demands exceeding the DO available in air saturated water Therefore it is necessary either to dilute the sample or to monitor DO frequently to ensure that low DO or anaerobic conditions do not occur When DO concentrations approach 2 mg L the sample should be reaerated 5 8 Because bacterial growth requires nutrients such as nitrogen phosphorus and trace metals the necessary amounts may be added to the dilution water together with buffer to ensure that pH remains in a range suitable for bacterial growth and seed to provide an adequate bacterial population However if the result is being used to estimate the rate of oxidation of naturally occurring surface waters addition of nutrients and seed probably accelerates the decay rate and produces misleading results If only UBOD is desired it may be advantageous to add supple mental nutrients that accelerate decay and reduce the test dura tion When nutrients are used they also should be used in the dilution water blank Because of the wide range of water and wastewater characteristics and varied applications of UBOD data no specific nutrient or buffer formulations are included The extent of oxidation of nitrogenous compounds during the prescribed incubation period depends on the presence of micro organisms capable of carrying out this oxidation Such organ isms may not be present in wastewaters in sufficient numbers to oxidize significant quantities o
45. trification control J Water Poll Control Fed 45 637 9 Bibliography HEUKELEKIAN H 1947 Use of direct method of oxygen utilization in waste treatment studies Sew Works J 19 375 CALDWELL D H amp W F LANGELIER 1948 Manometric measurement of the biochemical oxygen demand of sewage Sew Works J 20 202 GELLMAN I amp H HEUKELEKIAN 1951 Studies of biochemical oxidation by direct methods Sew Ind Wastes 23 1267 Jenkins D 1960 The use of manometric methods in the study of sewage and trade wastes Jn Waste Treatment p 99 Pergamon Press Oxford U K Montcomery H A C 1967 The determination of biochemical oxygen demand by respirometric methods Water Res 1 631 CEDENA F A DROHOBYCZAR M I BEACH amp D Barnes 1988 A novel approach to simplified respirometric oxygen demand determina tions Proc 43rd Ind Waste Conf Purdue Univ Lafayette Ind
46. trol temperature to 1 C Exclude all light to prevent oxygen formation by algae in the sample Use red actinic coated bottles for analysis outside of a darkened incubator 3 Reagents Formulations of reagent solutions are given for 1 L volumes but smaller or larger volumes may be prepared according to need Discard any reagent showing signs of biological growth or chemical precipitation Stock solutions can be sterilized by au toclaving to provide longer shelf life a Distilled water Use only high quality water distilled from a block tin or all glass still see Section 1080 Deionized water may be used but often contains high bacterial counts The water must contain less than 0 01 mg heavy metals L and be free of chlorine chloramines caustic alkalinity organic material or acids Make all reagents with this water When other waters are required for special purpose testing state clearly their source and quality characteristics b Phosphate buffer solution 1 5N Dissolve 207 g sodium dihydrogen phosphate NaH PO H O in water Neutralize to pH 7 2 with 6N KOH 3g below and dilute to 1 L c Ammonium chloride solution 0 71N Dissolve 38 2 g am monium chloride NH Cl in water Neutralize to pH 7 0 with KOH Dilute to 1 0 L 1 mL 10 mg N d Calcium chloride solution 0 25N Dissolve 27 7 g CaCl in water and dilute to 1 L 1 mL 10 mg Ca e Magnesium sulfate solution 0 41N Dissolve 101 g MgSO 7H O in water and
47. water conditions thereby providing an estimate of the environmental effects of wastewaters and effluents The UBOD measures the oxygen required for the total degra dation of organic material ultimate carbonaceous demand and or the oxygen to oxidize reduced nitrogen compounds ul timate nitrogenous demand UBOD values and appropriate ki netic descriptions are needed in water quality modeling studies such as UBOD BOD ratios for relating stream assimilative capacity to regulatory requirements definition of river estuary or lake deoxygenation kinetics and instream ultimate carbona ceous BOD UCBOD values for model calibration 2 Carbonaceous Versus Nitrogenous BOD A number of factors for example soluble versus particulate organics settleable and floatable solids oxidation of reduced Approved by Standard Methods Committee 2001 Joint Task Group James C Young chair George T Bowman Sabry M Kamhawy Terry G Mills Marlene Patillo Ray C Whittemore Introduction iron and sulfur compounds or lack of mixing may affect the accuracy and precision of BOD measurements Presently there is no way to include adjustments or corrections to account for the effect of these factors Oxidation of reduced forms of nitrogen such as ammonia and organic nitrogen can be mediated by microorganisms and exert nitrogenous demand Nitrogenous demand historically has been considered an interference in the determination of BOD and the in
48. y of dilution waters made for the Standard Methods Committee of the Federation of Sewage Works Associations Sewage Works J 13 669 Mouioan F W E Hurwitz G R BARNETT amp H K RAmer 1950 Experience with modified methods for BOD Sewage Ind Wastes 22 31 Young J C G N McDermott amp 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 5210 C Ultimate BOD Test 1 General Discussion The ultimate BOD test is an extension of the 5 d dilution BOD test as described in 5210B but with a number of specific test requirements and differences in application The user should be familar with the 5210B procedure before conducting tests for UBOD a Principle The method consists of placing a single sample dilution in full airtight bottles and incubating under specified conditions for an extended period depending on wastewater effluent river or estuary quality Dissolved oxygen DO is measured with probes initially and intermittently during the test From the DO versus time series UBOD is calculated by an appropriate statistical technique For improved accuracy run tests in triplicate Bottle size and incubation time are flexible to accommodate individual sample characteristics and laboratory limitations In cubation temperature however is 20 C Most effluents and some naturally occurring surface wa
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