A Bug’s-Eye-View of the BOD Test Perry Brake Lab Testing Consultant (253) 565-5350 Presented to the Oregon Water Environment School,

A Bug’s-Eye-View of the BOD Test Perry Brake Lab Testing Consultant (253) 565-5350 pudljumper1@comcast.net Presented to the Oregon Water Environment School, 3/28/12

References Specifically, Method 5210B in 18 th,19 th, 20 th, or On-Line * Editions. 22d Ed. is out…don’t know if it is on-line. Also...  EPA Method 405.1  USGS Method I-1578  AOAC Method 973-44*

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

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

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

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

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

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

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

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

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

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)

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

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

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 6.5 – 7.5  Dechlorination – w/sodium sulfite, but might outgas naturally

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

Sample Pretreatment  Sample prep  Well mixed sample critical  Pre-dilute for 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 6.5 – 7.5  Dechlorination – w/sodium sulfite, but might outgas naturally

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 (if doing CBOD)  Add to samples, seed control, GGA, but NOT blank  TCMP (18 th, 19 th, 20 th editions ofSM)  Also allylthiourea (ATU) in 21 st Edition

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

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 inhibitor to samples, seed control, GGA, but NOT to blank  TCMP (18 th, 19 th, 20 th editions ofSM)  Also allylthiourea (ATU) in 21 st Edition)

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

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

“Steam Distilled” Water

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)

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

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

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)

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

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

Typical Dilution Series  Late Breaking News!! 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

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

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

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

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”)

Determination of Final DO Same process as for initial DO

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

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

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

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

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

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”  Space for reviewer to initial  Space for comments

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

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”

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  Duplicates (if done) - <50% RPD for effluent  Toxicity – check only if toxicity is probable

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

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

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

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

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

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

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

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

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 for 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.

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) suddenly higher than normal  Sample not being thoroughly mixed

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

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.,

Reference Supplement  BOD Checklist (Word®)  Benchsheet (Excel®)  Bottle  Graduated Cylinder  Control Charting (Excel®)  Standards  Duplicates  SM 21 st 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

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A Bug’s-Eye-View of the BOD Test Perry Brake Lab Testing Consultant (253) 565-5350 Presented to the Oregon Water Environment School,

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A Bug’s-Eye-View of the BOD Test Perry Brake Lab Testing Consultant (253) 565-5350 Presented to the Oregon Water Environment School,

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