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

References Specifically, Method 5210B in 18th,19th, 20th, 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*

What is in that BOD Bottle? BOD Bacteria Oxygen Organic Material Nitrogen-containing Organic material Nitrifying Bacteria Nitrogen (ammonia, ammonium, nitrites) BOD O2 BOD O2 O2 O2 BOD N O2 N BOD O2 O2 N Plus water, nutrients, buffers, inert material, sometimes interferents

What’s happening inside that BOD Bottle? CxHyOz O2 CO2 + H20 O2 CO2 + H20 + NH4+ CxHyOzN O2 BOD - NO3 Examples: C6H12O6 – Glucose C4H7O4N – Glutamic Acid

What’s happening inside that BOD Bottle? CxHyOz O2 CO2 + H20 O2 CO2 + H20 + NH4+ CxHyOzN O2 Inhibitor CBOD - NO3 Examples: C6H12O6 – Glucose C4H7O4N – Glutamic Acid

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

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, 3rd Ed., 1991. Sample BOD Range* Vol Range in 300-mL Btl   Min Max Influent 150 400 3 8 1° Effluent 60 160 7.5 20 2° Effluent 5 240 Digester 1000 4000 0.3 1.2 Industrial 100 3000 0.4 12

Sampling Type – Grab or Composite Volume _____________ * BOD ranges from EPA’s Operation of Wastewater Treatment Plants, vol. II, 3rd Ed., 1991. Sample BOD Range* Vol Range in 300-mL Btl   Min Max Influent 150 400 3 8 1° Effluent 60 160 7.5 20 2° Effluent 5 240 Digester 1000 4000 0.3 1.2 Industrial 100 3000 0.4 12

Sampling Type – Grab or Composite Volume Maximum Volume/bottle - ~295 mL (sample, seed, special nutrient/buffer “pillow”, top off with H2O) Sample BOD Range* Vol Range in 300-mL Btl   Min Max Influent 150 400 3 8 1° Effluent 60 160 7.5 20 2° Effluent 5 240 Digester 1000 4000 0.3 1.2 Industrial 100 3000 0.4 12

Sampling Type – Grab or Composite Volume Minimum Volume/bottle – none, but dilute entire sample for extremely high BOD samples Sample BOD Range* Vol Range in 300-mL Btl   Min Max Influent 150 400 3 8 1° Effluent 60 160 7.5 20 2° Effluent 5 240 Digester 1000 4000 0.3 1.2 Industrial 100 3000 0.4 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

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 __________ * 21st Edition of SM (and latest 40 CFR 136) says < 6°C but above freezing (unless study shows no difference if frozen)

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

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

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

Reagents Buffers/Nutrients Standard* (GGA) 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 in the case of the BOD test

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

Sample Pretreatment Sample prep Temperature Well-mixed sample critical Pre-dilute high-BOD samples Temperature 20 ± 1°C before reading initial DO (18th, 19th, 20th) 20 ± 1°C before dilution (21st edition of SM) pH – if <6.0 or >8.5, adjust to 7.0 – 7.2 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 Sample BOD Range* Vol Range in 300-mL Btl   Min Max Influent 150 400 3 8 1° Effluent 60 160 7.5 20 2° Effluent 5 240 Digester 1000 4000 0.3 1.2 Industrial 100 3000 0.4 12

Sample Pretreatment Sample prep Sample Temperature Well mixed sample critical Pre-dilute for high-BOD samples Sample Temperature 20 ± 1°C before reading initial DO (18th, 19th, 20th) 20 ± 1°C before dilution (21st edition of SM) pH – if <6.0 or >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 - Add pyridine inhibitor to samples, seed control, GGA, but NOT to blank

Convincing Evidence of Toxicity   Source Bottle Sample Corrected Dilution BOD Vol. (mL) Depletion factor (mg/L) 1 20 4.4 15 66 2 10 4.0 30 120 3 5 3.7 60 222 Influent 4 5.0 150 750 10* 2.6 780 6 5* 2.3 1380 7 2* 3900*** 8 1* 1.9** 300 N/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 pyridine inhibitor to samples, seed control, GGA, but NOT to blank

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

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

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 Cl2, NH3, 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

“Steam Distilled” Water

Preparation of Dilution Water Source water is critical Distilled often contains Cl2, NH3, 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)

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

Estimating BOD 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 Sample BOD Range* Vol Range in 300-mL Btl   Min Max Influent 150 400 3 8 1° Effluent 60 160 7.5 20 2° Effluent 5 240 Digester 1000 4000 0.3 1.2 Industrial 100 3000 0.4 12

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

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

Parameter Expected Value KHP Standard (300 mg/L) Parameter Expected Value BOD 249 mg/L COD 343 mg/L TOC 141 mg/L pH 4.4 pH units Total Solids 300 mg/L Volatile Solids 169 mg/L Conductivity 169 mhos Acidity 74 mg/L _________ Source: WA Dept of Ecology Manchester Laboratory (lab attained a standard deviation for BOD of 15 mg/L…very precise work)

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

Dilution of Sample Bottle Method Graduated Cylinder 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 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

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

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) 17.0 18.0 19.0 20.0 21.0 22.0 23.0 26.6 8.61 8.42 8.25 8.07 7.91 7.75 7.59 26.8 8.67 8.48 8.31 8.13 7.97 7.81 7.65 27.0 8.73 8.54 8.37 8.19 8.03 7.87 7.71 - - - - - - - - 31.1 10.1 9.84 9.64 9.45 9.27 9.09 8.92 31.3 10.1 9.90 9.70 9.51 9.33 9.15 8.97

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

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

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 (21st Ed. says ± 6 hours) All bottles done together, same incubator (i.e., in same “batch”)

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

Determination of Final DO Same process as for initial DO

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

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” Final BOD result 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 21st Edition of SM allows reporting “0”

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

QA/QC and Performance Monitoring Minimum DO Depletion/Retention Blanks Most zero, a few <0.2, almost none >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

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 < 0.6 mg/L and sometimes >1.0 mg/L per mL of seed in seed control bottles Seed too variable ______ * Applies only to natural seeds; 21st 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) BOD 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 Indicator Possible Cause Possible Solution Reference Blank Usually Source water Incubate several Pg 37 ¶ 10b(5) exceeds is 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) negative water 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 <30 in procedure variations, elimi- nate them etc., etc.,

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

Questions ?

Slides on calculations follow

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

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 / Volsample e.g., for 5-mL sample, DF = 300/5 = 60 Grad Cylinder Method DF = 1000/ Volsample e.g., for 20-mL sample, DF = 1000/20 = 50

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

BOD – Not Seeded BOD = DF(DO1 – DO5) Example: DF = 60 for influent sample DO1 = 8.7 DO5 = 1.7 BOD = 60(8.7 – 1.7) = 420 mg/L

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

BOD - Seeded Must subtract depletion caused by seed BOD = DF[(DO1 – DO5) - f(B1 – B2)] where... “f” = is seed volsample/seed vol seed control for bottle method “f” = is % seedsample/%seedseed control for grad cyl method And B1 and B2 are DO for seed control on Days 1 and 5