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The BOD Test from A – Z (aka A Bug’s-Eye-View of the BOD Test) Perry Brake Lab Testing Consultant (253) 565-5350 Presented at OELA.

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Presentation on theme: "The BOD Test from A – Z (aka A Bug’s-Eye-View of the BOD Test) Perry Brake Lab Testing Consultant (253) 565-5350 Presented at OELA."— Presentation transcript:

1 The BOD Test from A – Z (aka A Bug’s-Eye-View of the BOD Test) Perry Brake Lab Testing Consultant (253) 565-5350 pudljumper1@comcast.net Presented at OELA Workshop, 5/24/12 Powerpoint presentation posted at www.perrybrake.com/BODSolutions.html

2 References Specifically, Method 5210B in 18 th,19 th, 20 th, or On-Line * Editions. 22d Ed. is out, but not yet approved Also...  EPA Method 405.1  USGS Method I-1578  AOAC Method 973-44*

3 BO D N N O2O2 O2O2 O2O2 O2O2 O2O2 O2O2 O2O2 N Bacteria Oxygen Organic Material Nitrogen-containing Organic material Nitrifying Bacteria Nitrogen (ammonia, ammonium, nitrites) What is in that BOD Bottle? Plus water, nutrients, buffers, inert material, sometimes interferents

4 What’s happening inside that BOD Bottle? What’s happening inside that BOD Bottle? C x H y O z C x H y O z CO 2 + H 2 0 Examples: C 6 H 12 O 6 – Glucose C 4 H 7 O 4 N – Glutamic Acid C x H y O z N C x H y O z N CO 2 + H 2 0 + NH 4 + - O2O2 NO3NO3 O2O2O2O2 O2O2O2O2 BOD

5 What’s happening inside that BOD Bottle? What’s happening inside that BOD Bottle? C x H y O z C x H y O z CO 2 + H 2 0 Examples: C 6 H 12 O 6 – Glucose C 4 H 7 O 4 N – Glutamic Acid C x H y O z N C x H y O z N CO 2 + H 2 0 + NH 4 + - O2O2 NO3NO3 O2O2O2O2 O2O2O2O2 Inhibitor CBOD

6 What References Say (and Don’t Say) About...  Sampling  Sample preservation/holding time  Equipment  Reagents  Sample Pretreatment  The Analytical Procedure  Calculations/Data Recording  QA/QC and Performance Monitoring

7 What References Say (and Don’t Say) About...  Sampling  Sample preservation/holding time  Equipment  Reagents  Sample Pretreatment  The Analytical Procedure  Calculations/Data Recording  QA/QC and Performance Monitoring

8 Sampling  Type – Grab or Composite  Volume _____________ * BOD ranges from EPA’s Operation of Wastewater Treatment Plants, vol. II, 3 rd Ed., 1991. SampleBOD Range* Vol Range in 300-mL Btl MinMaxMinMax Influent15040038 1° Effluent601607.520 2° Effluent56020240 Digester100040000.31.2 Industrial10030000.412

9 Sampling  Type – Grab or Composite  Volume _____________ * BOD ranges from EPA’s Operation of Wastewater Treatment Plants, vol. II, 3 rd Ed., 1991. SampleBOD Range* Vol Range in 300-mL Btl MinMaxMinMax Influent15040038 1° Effluent601607.520 2° Effluent56020240 Digester100040000.31.2 Industrial10030000.412

10 Sampling  Type – Grab or Composite  Volume  Maximum Volume/bottle - ~295 mL (sample, seed, special nutrient/buffer “pillow”, top off with H 2 O) SampleBOD Range* Vol Range in 300-mL Btl MinMaxMinMax Influent15040038 1° Effluent601607.520 2° Effluent56020240 Digester100040000.31.2 Industrial10030000.412

11 Sampling  Type – Grab or Composite  Volume  Minimum Volume/bottle – none, but dilute entire sample for extremely high BOD samples SampleBOD Range* Vol Range in 300-mL Btl MinMaxMinMax Influent15040038 1° Effluent601607.520 2° Effluent56020240 Digester100040000.31.2 Industrial10030000.412

12 What References Say (and Don’t Say) About...  Sampling  Sample preservation/holding time  Equipment  Reagents  Sample Pretreatment  The Analytical Procedure  Calculations/Data Recording  QA/QC and Performance Monitoring

13 Sample Preservation/Holding Time  Preserve at 4°C* if sample cannot be set up in 2 hrs  Holding time: Analyze as soon as possible...  But not to exceed 48 hours (40 CFR 136)  But not to exceed 24 hours (Standard Methods)  But not to exceed 6 hours (some states)  Holding time begins at end of composite  Waiver - permit managers can waive 48-hour requirement __________ * 21 st Edition of SM (and latest 40 CFR 136) says < 6°C but above freezing (unless study shows no difference if frozen)

14 What References Say (and Don’t Say) About...  Sampling  Sample preservation/holding time  Equipment  Reagents  Sample Pretreatment  The Analytical Procedure  Calculations/Data Recording  QA/QC and Performance Monitoring

15 Equipment  DO measurement  Meter (plus barometer if meter has none)  Winkler setup  LDO meter (EPA approved, but Regions require ATP study except Region 6)  Dilution water container(s) w/siphon or gravity flow; glass best, but Nalgene® OK  Thermometer(s) – 1° increment, traceable to NIST  Incubator/Water Bath – no light, 20±1° C, circulation  BOD Bottles – glass most common; Env’l Express plastic OK; 60-, 75- 250-, 300-mL available...300-mL by far most common

16 What References Say (and Don’t Say) About...  Sampling  Sample preservation/holding time  Equipment  Reagents  Sample Pretreatment  The Analytical Procedure  Calculations/Data Recording  QA/QC and Performance Monitoring

17 Reagents  Buffers/Nutrients  Can prepare from reagent-grade chemicals, or purchase “pillows”  Special “pillow” for 300-mL bottle  Standard* (GGA)  Can prepare from chemicals, or...  Purchase ready made  Hach GGA standard is 300 mg/L each G and GA  North Central Labs std is 150 mg/L each G and GA  KHP can backup, but not replace, GGA ________ Standard – solution of known concentration, or expected value Standard – solution of known concentration, or expected value in the case of the BOD test in the case of the BOD test

18 What References Say (and Don’t Say) About...  Sampling  Sample preservation/holding time  Equipment  Reagents  Sample Pretreatment  The Analytical Procedure  Calculations/Data Recording  QA/QC and Performance Monitoring

19 Sample Pretreatment  Sample prep  Well-mixed sample critical  Pre-dilute high-BOD samples  Temperature  20 ± 1°C before reading initial DO (18 th, 19 th, 20 th )  20 ± 1°C before dilution (21 st edition of SM)  pH – if 8.5, adjust to 7.0 – 7.2  Dechlorination – w/sodium sulfite, but might outgas naturally

20 Sampling  Type – Grab or Composite  Volume  Minimum Volume/bottle – none, but dilute entire sample for extremely high BOD samples SampleBOD Range* Vol Range in 300-mL Btl MinMaxMinMax Influent15040038 1° Effluent601607.520 2° Effluent56020240 Digester100040000.31.2 Industrial10030000.412

21 Sample Pretreatment  Sample prep  Well mixed sample critical  Pre-dilute for high-BOD samples  Sample Temperature  20 ± 1°C before reading initial DO (18 th, 19 th, 20 th )  20 ± 1°C before dilution (21 st edition of SM)  pH – if 8.5, adjust to 6.5 – 7.5  Dechlorination – w/sodium sulfite, but might outgas naturally

22 Sample Pretreatment (cont’d)  Other toxic substances – metals, septage  Toxicity will result in higher BOD for increasing dilution in series of bottles --->  Supersaturation – often a problem in winter; can be avoided by vigorous shaking of sample, allowing to sit for at least one hour  Nitrification inhibition - Add pyridine inhibitor to samples, seed control, GGA, but NOT to blank

23 Convincing Evidence of Toxicity SourceBottleSampleCorrectedDilutionBOD Vol. (mL)Depletionfactor(mg/L) 1204.41566 2104.030120 353.760222 Influent 425.0150750 510*2.630780 65*2.3601380 72*2.61503900*** 81*1.9**300N/A * After ten-fold dilution of entire sample ** Does not meet criterion of at least 2.0 mg/L DO depletion *** Value to be reported

24 Sample Pretreatment (cont’d)  Other toxic substances – metals, septage  Toxicity will result in higher BOD for increasing dilution in series of bottles  Supersaturation – often a problem in winter; can be avoided by vigorous shaking of sample, allowing to sit for at least one hour  Nitrification inhibition - Add pyridine inhibitor to samples, seed control, GGA, but NOT to blank

25 What References Say (and Don’t Say) About...  Sampling  Sample preservation/holding time  Equipment  Reagents  Sample Pretreatment  The Analytical Procedure  Calculations/Data Recording  QA/QC and Performance Monitoring

26 Analytical Procedure  Preparation of Dilution Water  Estimating BOD  Seeding  Dilution of Sample  Determination of Initial DO  Incubation  Determination of Final DO

27 Analytical Procedure  Preparation of Dilution Water  Estimating BOD  Seeding  Dilution of Sample  Determination of Initial DO  Incubation  Determination of Final DO

28 Preparation of Dilution Water  Source water is critical  Distilled often contains Cl 2, NH 3, organics  DI often contains organics  Bad blanks? Try “steam distilled” water  Dilution water for BOD  Aerate, store at 20°, add buffers/nutrients morning of test, settle for one hour (CBOD dilution water can be stored)  Check pH, do blank  D o not add inhibitor to blank

29 “Steam Distilled” Water

30 Preparation of Dilution Water  Source water is critical  Distilled often contains Cl 2, NH 3, organics  DI often contains organics  Bad blanks? Try “steam distilled” water  Dilution water for BOD  Aerate, store at 20°, add buffers/nutrients morning of test, settle for one hour (CBOD dilution water can be stored)  Check pH, do blank  (do not add inhibitor to blank)

31 Analytical Procedure  Preparation of Dilution Water  Estimating BOD  Seeding  Dilution of Sample  Determination of Initial DO  Incubation  Determination of Final DO

32 Estimating BOD SampleBOD Range* Vol Range in 300-mL Btl MinMaxMinMax Influent15040038 1° Effluent601607.520 2° Effluent56020240 Digester100040000.31.2 Industrial10030000.412   Establish correlation with TSS  Do a COD (BOD usually 60-70% of COD)  In PT studies, BOD/COD = ~0.63, BOD/TOC = ~1.58, BOD/CBOD = ~1.16

33 Analytical Procedure  Preparation of Dilution Water  Estimating BOD  Seeding  Dilution of Sample  Determination of Initial DO  Incubation  Determination of Final DO

34 Seeding  Always seed unless known to be unnecessary  Source of seed (need good source of bacteria)  Domestic WWTP – 1° effluent; 2° effluent; influent (often variable)  Industrial WWTP or Private Lab – Synthetic seed  Polyseed® - no nitrifiers  Biosystems® - has nitrifiers (or does it??)  Seed Check  Seed control – should deplete 0.6 – 1.0 mg/L per mL of seed  Glucose/Glutamic Acid – true test of seed  Goal is: average ~198 mg/L; standard deviation <<30.5 mg/L  KHP – OK as supplement

35 KHP Standard (300 mg/L) Parameter Expected Value Parameter Expected Value BOD249 mg/L COD343 mg/L TOC 141 mg/L pH4.4 pH units Total Solids300 mg/L Volatile Solids169 mg/L Conductivity169 mhos Acidity74 mg/L _________ Source: WA Dept of Ecology Manchester Laboratory (lab attained a standard deviation for BOD of 15 mg/L…very precise work)

36 Analytical Procedure  Preparation of Dilution Water  Estimating BOD  Seeding  Dilution of Sample  Determination of Initial DO  Incubation  Determination of Final DO

37 Dilution of Sample  Bottle Metho  Bottle Method  Add sample, seed, dilution water to bottle  Never add seed to empty bottle  Add inhibitor only after sample/dilution water  Graduated Cylinder Method  Add sample, seed, dilution water to grad cylinder  Dilution Series  Do enough dilutions to assure at least one has depletion of >2 mg/L, retention of >1 mg/L  Remember – if more than 200 mL of sample is in bottle, use special buffer/nutrient packets

38 Typical Dilution Series  Blank – 1 bottle  Seed control – 1 or 2 bottles, same dilution  Don’t forget...must deplete at least 2 mg/L   G/GA – 1 bottle, ~6 mL, OK...some labs do 2  Effluent – 1 bottle OK if precision is good*  Seed must contribute 0.6 – 1.0 mg/L depletion  Influent – 3 dilutions minimum recommended  Unknown – 3 dilutions minimum, 5 better _________ * Some states do not allow only one

39 Typical Dilution Series Standard Methods staff published a memo (May 19, 2009) saying they never intended to require more than one dilution for any given BOD sample. Standard Methods staff published a memo (May 19, 2009) saying they never intended to require more than one dilution for any given BOD sample. Check www.perrybrake.com/BODSolutions.html for a copy of the memo Check www.perrybrake.com/BODSolutions.html for a copy of the memowww.perrybrake.com/BODSolutions.html

40 Analytical Procedure  Preparation of Dilution Water  Estimating BOD  Seeding  Dilution of Sample  Determination of Initial DO  Incubation  Determination of Final DO

41 Determination of Initial DO  Winkler great...but time consuming  DO meter  Proper calibration critical  Refer to DO chart to see if reading is reasonable  Do all measurements at 20 ± 1° C  Use actual barometric pressure  If using air calibration, use 100% saturation  LDO meter simplifies procedure

42 DO Saturation vs. Temp/Pressure Pressure Temperature (°C) (inches Hg) 17.018.019.020.021.022.023.0 (inches Hg) 17.018.019.020.021.022.023.0 26.68.618.428.258.077.917.757.59 26.68.618.428.258.077.917.757.59 26.88.678.488.318.137.977.817.65 26.88.678.488.318.137.977.817.65 27.08.738.548.378.198.037.877.71 27.08.738.548.378.198.037.877.71 - ------- - ------- 31.110.19.849.649.459.279.098.92 31.110.19.849.649.459.279.098.92 31.310.19.909.709.519.339.158.97 31.310.19.909.709.519.339.158.97

43 Determination of Initial DO  Winkler great...but time consuming  DO meter  Proper calibration critical  Refer to DO chart to see if reading is reasonable  Keep all measurements at 20 ± 1° C  Use actual barometric pressure  If using air calibration, use 100% saturation  LDO meter simplifies procedure

44 Analytical Procedure  Preparation of Dilution Water  Estimating BOD  Seeding  Dilution of Sample  Determination of Initial DO  Incubation  Determination of Final DO

45 Incubation  Water seal plus cap (unless water bath used)  At 20 ± 1° C (NIST-traceable thermometer  In dark  Circulating air/water  For 5 days, ± 2 hours (21 st Ed. says ± 6 hours)  All bottles done together, same incubator (i.e., in same “batch”)

46 Analytical Procedure  Preparation of Dilution Water  Estimating BOD  Seeding  Dilution of Sample  Determination of Initial DO  Incubation  Determination of Final DO

47 Determination of Final DO Same process as for initial DO

48 What References Say (and Don’t Say) About...  Sampling  Sample preservation/holding time  Equipment  Reagents  Sample Pretreatment  The Analytical Procedure  Calculations/Data Recording  QA/QC and Performance Monitoring

49 Calculations/Data Recording  Dilution Factor  BOD – Seeded  BOD – Not Seeded  Benchsheet  Reporting Results

50 Benchsheet Must include:  Date/time sample taken, set up, final reading  ID of sampler and analyst  ID of sample  Sample pH  Sample temp when initial DO reading taken  Bottle numbers  Volume of seed in each bottle or graduated cylinder  Volume of sample in each bottle or graduated cylinder  Initial and final DO for each bottle  Space for calculation of “f”, “DF”  Final BOD result  Space for reviewer to initial  Space for comments

51 Calculations/Data Recording  Dilution Factor  BOD – Seeded  BOD – Not Seeded  Benchsheet  Reporting Results

52 Reporting Results  Report average of dilutions that deplete >2, retain >1 mg/L, unless signs of toxicity  If toxicity indicated, report highest result that met depletion/retention criteria  If no bottle depleted 2 or more mg/L, report as directed by regulatory agency  21 st Edition of SM allows reporting “0”

53 What References Say (and Don’t Say) About...  Sampling  Sample preservation/holding time  Equipment  Reagents  Sample Pretreatment  The Analytical Procedure  Calculations/Data Recording  QA/QC and Performance Monitoring

54 QA/QC and Performance Monitoring  Minimum DO Depletion/Retention  Blanks Most zero, a few 0.2  Check Standard (G/GA) Average ~198 mg/L (175 – 235 good goal) Standard deviation <15 mg/L  Matrix/Matrix Spike Duplicate (New in 22d edition) No guidance for limits in Standard Methods  Duplicates (if done) - <50%RPD for effluent  Toxicity – check only if toxicity is probable

55 Performance Monitoring Summary  Bias  Total Precision  Within-batch precision  Between-batch precision  Detection Limit - No “MDL” per se  Working Limits  Minimum  Maximum

56 Performance Monitoring Summary  Bias  Total Precision  Within-batch precision  Between-batch precision  Detection Limit - No “MDL” per se  Working Limits  Minimum  Maximum Control charting is good way to monitor bias and precision

57 Control Chart for Repeated Analysis of a Standard (e.g., GGA)

58 Another Reference  Sampling  Sample Prep/Holding Times  Equipment  Reagents  Sample Pretreatment  Procedure  Calculations/Data Recording  QA/QC  Method Performance  Appendices  Prep of Solutions  Sources of Reagents  Trouble Shooting  Glossary

59 Troubleshooting Sequence  Isolate Problem(s)  Blanks  G/GA (bias or total-precision problem)  Duplicates (within-batch precision problem  Seed Strength  Environmental Samples  Identify/Rank Probable Causes  Try Possible Fixes  One Problem at a time  One fix at a time

60 Problem Causes/Fixes - Blanks  Sometimes exceeds 0.2 mg/L  Supersaturated dilution water  Labware contaminated  Usually exceeds 0.2 mg/L Source water problem  Quite often negative  Temperature control problem  Photosynthesis  Sometimes negative, sometimes positive  DO measurement (e.g., atmospheric pressure not used)  High blanks for CBOD, OK for BOD  Inhibitor being added to blank

61 Problem Causes/Fixes – G/GA  Standard Deviation approaching 30.5 mg/L  Variable seed  Meter calibration problems  Inattention to detail  Standard Deviation >30.5 mg/L  MANY sources of imprecision! BIG problem!  Average >> 198 mg/L (precision OK)  Seed too strong  Average << 198 mg/L (precision OK)  Seed too weak

62 Problem Causes/Fixes – Duplicates*  Relative percent differences (RPD**) exceed 50% for samples in 5-20 mg/L range (e.g., effluents)  Poorly mixed seed  Contaminated bottles  Faulty DO meter/probe  Countless other things that change from bottle to bottle within a batch _________ * Duplicates – two samples done exactly the same way ** RPD – Difference divided by average of two results

63 Problem Causes/Fixes Seed Strength*  DO depletion < 0.6 mg/L per mL of seed in seed control bottle *  Seed too weak  DO depletion > 1.0 mg/L per mL of seed in seed control bottle *  Seed too strong  DO depletion sometimes 1.0 mg/L per mL of seed in seed control bottles  Seed too variable ______ * Applies only to natural seeds; 21 st edition of SM omits 0.6 – 1.0 mg/L seed depletion requirement.

64 Problem Causes/Fixes – Environmental Samples  No dilutions leave at least 1.0 mg/L DO residual  Samples not dilute enough  No dilutions deplete at least 2.0 mg/L DO  Samples too dilute  Significant increase in BOD for more dilute bottles in dilution series  Matrix is interfering (toxicity)  Influent (and TSS) BOD suddenly higher than normal  Sample not being thoroughly mixed

65 Reference  Sampling  Sample Prep/Holding Times  Equipment  Reagents  Sample Pretreatment  Procedure  Calculations/Data Recording  QA/QC  Method Performance  Appendices  Prep of Solutions  Sources of Reagents  Trouble Shooting  Glossary

66 Trouble Shooting Guide Appendix C Sample IndicatorPossible Cause Possible Solution Reference Blank Usually Source water Incubate several Pg 37 ¶ 10b(5) exceedsis unsuitable blanks using Pg 20, ¶ 8a(3)(b) 0.2 mg/L alternate waters, choose best Sometimes Bulk dilution Aerate water day Pg 36 ¶ 10b(2) negativewater contami- before test, add Pg 20 ¶ 8a(3)(b) inated nutrients/buffer Pg 16 ¶ 7f etc. GGA Standard Devia- Random variations Find sources of Pg 38 ¶ 10c tion > 15 but <30in procedure variations, elimi- nate them etc., etc.,

67 Reference Supplement  BOD Checklist (Word®)  Benchsheet (Excel®)  Bottle  Graduated Cylinder  Control Charting (Excel®)  Standards  Duplicates  SM 21 st and 22d Ed. Changes  Toxicity in BOD Testing  The “Perfect” Seed  Statistics used in BOD Testing  Bonus documents  EPA letters  NELAP Guidance Perry Brake Revised May 2011

68 Questions ?

69 Slides on calculations follow

70 Calculations/Data Recording  Dilution Factor  BOD – Not Seeded  BOD – Seeded  Benchsheet  Reporting Results

71 Calculations/Data Recording  Dilution Factor  BOD – Not Seeded  BOD – Seeded  Benchsheet  Reporting Results

72 Dilution Factor  Ratio of final volume to volume of sample  Bottle Method  DF = 300 / Vol sample e.g., for 5-mL sample, DF = 300/5 = 60  Grad Cylinder Method  DF = 1000/ Vol sample e.g., for 20-mL sample, DF = 1000/20 = 50 e.g., for 20-mL sample, DF = 1000/20 = 50

73 Calculations/Data Recording  Dilution Factor  BOD – Not Seeded  BOD – Seeded  Benchsheet  Reporting Results

74 BOD – Not Seeded  BOD = DF(DO 1 – DO 5 )  Example: DF = 60 for influent sample DO 1 = 8.7 DO 1 = 8.7 DO 5 = 1.7 DO 5 = 1.7 BOD = 60(8.7 – 1.7) = 420 mg/L BOD = 60(8.7 – 1.7) = 420 mg/L

75 Calculations/Data Recording  Dilution Factor  BOD – Not Seeded  BOD – Seeded  Benchsheet  Reporting Results

76 BOD - Seeded  Must subtract depletion caused by seed  BOD = DF[(DO 1 – DO 5 ) - f(B1 – B2)] where... “f” = is seed vol sample /seed vol seed control for bottle method “f” = is % seed sample /%seed seed control for grad cyl method And B1 and B2 are DO for seed control on Days 1 and 5


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