a bug’s-eye-view of the bod test perry brake lab testing consultant (253) 565-5350...
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A Bug’s-Eye-A Bug’s-Eye-View View
of the BOD Testof the BOD TestPerry BrakeLab Testing Consultant(253) 565-5350pudljumper1@comcast.net
Presented to the Oregon Water Environment School, 3/28/12
ReferencesReferences
Specifically, Method 5210Bin 18th,19th, 20th, 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*
BOBODD
BOD
BOD
BOD
N
N
O2
O2
O2
O2
O2
O2
O2
N
Bacteria
Oxygen
Organic Material
Nitrogen-containingOrganic material
Nitrifying Bacteria
Nitrogen (ammonia, ammonium, nitrites)
Whatis inthatBODBottle?
Plus water, nutrients, buffers, inert material, sometimes interferents
What’s happening inside What’s happening inside that BOD Bottle?that BOD Bottle?
CCxxHHyyOOzz CO2 + H20
Examples: C6H12O6 – Glucose C4H7O4N – Glutamic Acid
CCxxHHyyOOzzNN CO2 + H20 + NH4+
-
O2
NO3
OO22
OO22
What’s happening inside What’s happening inside that BOD Bottle?that BOD Bottle?
CCxxHHyyOOzz CO2 + H20
Examples: C6H12O6 – Glucose C4H7O4N – Glutamic Acid
CCxxHHyyOOzzNN CO2 + H20 + NH4+
-
O2
NO3
OO22
OO22
BOD
What’s happening inside What’s happening inside that BOD Bottle?that BOD Bottle?
CCxxHHyyOOzz CO2 + H20
Examples: C6H12O6 – Glucose C4H7O4N – Glutamic Acid
CCxxHHyyOOzzNN CO2 + H20 + NH4+
-O2
NO3
OO22
OO22
Inhibitor
What’s happening inside What’s happening inside that BOD Bottle?that BOD Bottle?
CCxxHHyyOOzz CO2 + H20
Examples: C6H12O6 – Glucose C4H7O4N – Glutamic Acid
CCxxHHyyOOzzNN CO2 + H20 + NH4+
-O2
NO3
OO22
OO22
Inhibitor
What’s happening inside What’s happening inside that BOD Bottle?that BOD Bottle?
CCxxHHyyOOzz CO2 + H20
Examples: C6H12O6 – Glucose C4H7O4N – Glutamic Acid
CCxxHHyyOOzzNN CO2 + H20 + NH4+
-O2
NO3
OO22
OO22
Inhibitor
CBOD
What References Say (and What References Say (and Don’t Say) About...Don’t Say) About...
SamplingSampling Sample preservation/holding timeSample preservation/holding time EquipmentEquipment ReagentsReagents Sample PretreatmentSample Pretreatment The Analytical ProcedureThe Analytical Procedure Calculations/Data RecordingCalculations/Data Recording QA/QC and Performance MonitoringQA/QC and Performance Monitoring
What References Say (and What References Say (and Don’t Say) About...Don’t Say) About...
SamplingSampling Sample preservation/holding timeSample preservation/holding time EquipmentEquipment ReagentsReagents Sample PretreatmentSample Pretreatment The Analytical ProcedureThe Analytical Procedure Calculations/Data RecordingCalculations/Data Recording QA/QC and Performance MonitoringQA/QC and Performance Monitoring
SamplingSampling Type – Grab or CompositeType – Grab or Composite VolumeVolume
__________________________
* * BOD ranges fromBOD ranges from EPA’s EPA’s Operation of Wastewater Operation of Wastewater Treatment Plants, Treatment Plants, vol. II, 3vol. II, 3rdrd Ed., 1991. Ed., 1991.
SampleBOD
Range* Vol Range in 300-mL Btl
Min Max Min Max
Influent 150 400 3 8
1° Effluent 60 160 7.5 20
2° Effluent 5 60 20 240
Digester 1000 4000 0.3 1.2
Industrial 100 3000 0.4 12
SamplingSampling Type – Grab or CompositeType – Grab or Composite VolumeVolume
__________________________
* * BOD ranges fromBOD ranges from EPA’s EPA’s Operation of Wastewater Operation of Wastewater Treatment Plants, Treatment Plants, vol. II, 3vol. II, 3rdrd Ed., 1991. Ed., 1991.
SampleBOD
Range* Vol Range in 300-mL Btl
Min Max Min Max
Influent 150 400 3 8
1° Effluent 60 160 7.5 20
2° Effluent 5 60 20 240
Digester 1000 4000 0.3 1.2
Industrial 100 3000 0.4 12
SamplingSampling Type – Grab or CompositeType – Grab or Composite VolumeVolume
Maximum Volume/bottle - ~295 mL (sample, seed, Maximum Volume/bottle - ~295 mL (sample, seed, special nutrient/buffer “pillow”, top off with Hspecial nutrient/buffer “pillow”, top off with H22O)O)
SampleBOD
Range* Vol Range in 300-mL Btl
Min Max Min Max
Influent 150 400 3 8
1° Effluent 60 160 7.5 20
2° Effluent 5 60 20 240
Digester 1000 4000 0.3 1.2
Industrial 100 3000 0.4 12
SamplingSampling TypeType – Grab or Composite– Grab or Composite VolumeVolume
Minimum Volume/bottleMinimum Volume/bottle – none, but dilute – none, but dilute entire sample for extremely high BOD entire sample for extremely high BOD samplessamples
SampleBOD
Range* Vol Range in 300-mL Btl
Min Max Min Max
Influent 150 400 3 8
1° Effluent 60 160 7.5 20
2° Effluent 5 60 20 240
Digester 1000 4000 0.3 1.2
Industrial 100 3000 0.4 12
What References Say (and What References Say (and Don’t Say) About...Don’t Say) About...
SamplingSampling Sample preservation/holding timeSample preservation/holding time EquipmentEquipment ReagentsReagents Sample PretreatmentSample Pretreatment The Analytical ProcedureThe Analytical Procedure Calculations/Data RecordingCalculations/Data Recording QA/QC and Performance MonitoringQA/QC and Performance Monitoring
Sample Sample Preservation/Holding TimePreservation/Holding Time
Preserve at 4°CPreserve at 4°C** if sample cannot be set up if sample cannot be set up in 2 hrsin 2 hrs
Holding timeHolding time: Analyze as soon as possible...: Analyze as soon as possible... But not to exceed But not to exceed 4848 hours (40 CFR 136) hours (40 CFR 136) But not to exceed But not to exceed 2424 hours ( hours (Standard Methods)Standard Methods) But not to exceed But not to exceed 66 hours (some states) hours (some states)
Holding time beginsHolding time begins at end of composite at end of composite WaiverWaiver - permit managers can waive 48- - permit managers can waive 48-
hour requirementhour requirement____________________
* * 2121stst Edition of SM (and latest 40 CFR 136) says Edition of SM (and latest 40 CFR 136) says << 6°C but 6°C but above freezing (unless study shows no difference if frozen)above freezing (unless study shows no difference if frozen)
What References Say (and What References Say (and Don’t Say) About...Don’t Say) About...
SamplingSampling Sample preservation/holding timeSample preservation/holding time EquipmentEquipment ReagentsReagents Sample PretreatmentSample Pretreatment The Analytical ProcedureThe Analytical Procedure Calculations/Data RecordingCalculations/Data Recording QA/QC and Performance MonitoringQA/QC and Performance Monitoring
EquipmentEquipment DO measurementDO measurement
Meter (plus barometer if meter has none)Meter (plus barometer if meter has none) Winkler setupWinkler setup LDO meter (EPA approved, but Regions require ATP LDO meter (EPA approved, but Regions require ATP
study except Region 6)study except Region 6) Dilution water container(s)Dilution water container(s) w/siphon or gravity w/siphon or gravity
flow; glass best, but Nalgene® OKflow; glass best, but Nalgene® OK Thermometer(s)Thermometer(s) – – 1° increment, traceable to NIST1° increment, traceable to NIST Incubator/Water BathIncubator/Water Bath – – no light, 20±1° C, no light, 20±1° C,
circulationcirculation BOD BottlesBOD Bottles – – glass most common; Env’l Express glass most common; Env’l Express
plastic OK; 60-, 75- 250-, 300-mL available...300-mL by far plastic OK; 60-, 75- 250-, 300-mL available...300-mL by far most commonmost common
What References Say (and What References Say (and Don’t Say) About...Don’t Say) About...
SamplingSampling Sample preservation/holding timeSample preservation/holding time EquipmentEquipment ReagentsReagents Sample PretreatmentSample Pretreatment The Analytical ProcedureThe Analytical Procedure Calculations/Data RecordingCalculations/Data Recording QA/QC and Performance MonitoringQA/QC and Performance Monitoring
ReagentsReagents Buffers/NutrientsBuffers/Nutrients
Can prepare from reagent-grade chemicals, or Can prepare from reagent-grade chemicals, or purchase “pillows”purchase “pillows”
Special “pillow” for 300-mL bottleSpecial “pillow” for 300-mL bottle StandardStandard** (GGA) (GGA)
Can prepare from chemicals, or...Can prepare from chemicals, or... Purchase ready madePurchase ready made
Hach GGA standard is 300 mg/L each G and GAHach GGA standard is 300 mg/L each G and GA North Central Labs std is 150 mg/L each G and GANorth Central Labs std is 150 mg/L each G and GA
KHP can backup, but not replace, GGAKHP can backup, but not replace, GGA________________** Standard – solution of known concentrationStandard – solution of known concentration
What References Say (and What References Say (and Don’t Say) About...Don’t Say) About...
SamplingSampling Sample preservation/holding timeSample preservation/holding time EquipmentEquipment ReagentsReagents Sample PretreatmentSample Pretreatment The Analytical ProcedureThe Analytical Procedure Calculations/Data RecordingCalculations/Data Recording QA/QC and Performance MonitoringQA/QC and Performance Monitoring
Sample PretreatmentSample Pretreatment
Sample prepSample prep Well-mixed sample criticalWell-mixed sample critical Pre-dilute high-BOD samplesPre-dilute high-BOD samples
TemperatureTemperature 20 ± 1°C before reading initial DO (1820 ± 1°C before reading initial DO (18thth, 19, 19thth, ,
2020thth)) 20 ± 1°C before dilution (2120 ± 1°C before dilution (21stst edition of edition of SMSM))
pHpH – if <6.0 or >8.5, adjust to 6.5 – 7.5 – if <6.0 or >8.5, adjust to 6.5 – 7.5 DechlorinationDechlorination – w/sodium sulfite, but – w/sodium sulfite, but
might outgas naturallymight outgas naturally
SamplingSampling TypeType – Grab or Composite– Grab or Composite VolumeVolume
Minimum Volume/bottleMinimum Volume/bottle – none, but dilute entire – none, but dilute entire sample for extremely high BOD samplessample for extremely high BOD samples
SampleBOD
Range* Vol Range in 300-mL Btl
Min Max Min Max
Influent 150 400 3 8
1° Effluent 60 160 7.5 20
2° Effluent 5 60 20 240
Digester 1000 4000 0.3 1.2
Industrial 100 3000 0.4 12
Sample PretreatmentSample Pretreatment
Sample prepSample prep Well mixed sample criticalWell mixed sample critical Pre-dilute for high-BOD samplesPre-dilute for high-BOD samples
TemperatureTemperature 20 ± 1°C before reading initial DO (1820 ± 1°C before reading initial DO (18thth, 19, 19thth, ,
2020thth)) 20 ± 1°C 20 ± 1°C before dilutionbefore dilution (21 (21stst edition of edition of SMSM))
pHpH – if <6.0 or >8.5, adjust to 6.5 – 7.5 – if <6.0 or >8.5, adjust to 6.5 – 7.5 DechlorinationDechlorination – w/sodium – w/sodium sulfitesulfite, but , but
might outgas naturallymight outgas naturally
Sample Pretreatment Sample Pretreatment (cont’d)(cont’d)
Other toxic substancesOther toxic substances – metals, septage – metals, septage Toxicity will result in higher BOD for increasing Toxicity will result in higher BOD for increasing
dilution in series of bottles dilution in series of bottles SupersaturationSupersaturation – often a problem in winter; – often a problem in winter;
can be avoided by vigorous shaking of can be avoided by vigorous shaking of sample, allowing to sit for at least one hoursample, allowing to sit for at least one hour
Nitrification inhibition (if doing CBOD)Nitrification inhibition (if doing CBOD) Add to samples, seed control, GGA, but Add to samples, seed control, GGA, but NOTNOT blank blank TCMP (18TCMP (18thth, 19, 19thth, 20, 20thth editions of editions ofSMSM)) Also allylthiourea (ATU) in 21Also allylthiourea (ATU) in 21stst Edition Edition
Convincing Evidence of Convincing Evidence of ToxicityToxicity
Source Bottle Sample Corrected Dilution BOD
Vol. (mL) Depletion factor (mg/L)
(mg/L)
1 20 4.4 15 66
2 10 4.0 30 120
3 5 3.7 60 222
Influent 4 2 5.0 150 750
5 10* 2.6 30 780
6 5* 2.3 60 1380
7 2* 2.6 150 3900***
8 1* 1.9** 300300 N/AN/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 Sample Pretreatment (cont’d)(cont’d)
Other toxic substancesOther toxic substances – metals, septage – metals, septage Toxicity will result in higher BOD for increasing Toxicity will result in higher BOD for increasing
dilution in series of bottlesdilution in series of bottles SupersaturationSupersaturation – often a problem in – often a problem in
winter; can be avoided by vigorous winter; can be avoided by vigorous shaking of sample, allowing to sit for at shaking of sample, allowing to sit for at least one hourleast one hour
Nitrification inhibitionNitrification inhibition Add inhibitor to samples, seed control, GGA, Add inhibitor to samples, seed control, GGA,
but but NOTNOT to blank to blank TCMP (18TCMP (18thth, 19, 19thth, 20, 20thth editions of editions ofSMSM)) Also allylthiourea (ATU) in 21Also allylthiourea (ATU) in 21stst Edition) Edition)
What References Say (and What References Say (and Don’t Say) About...Don’t Say) About...
SamplingSampling Sample preservation/holding timeSample preservation/holding time EquipmentEquipment ReagentsReagents Sample PretreatmentSample Pretreatment The Analytical ProcedureThe Analytical Procedure Calculations/Data RecordingCalculations/Data Recording QA/QC and Performance MonitoringQA/QC and Performance Monitoring
Analytical ProcedureAnalytical Procedure
Preparation of Dilution WaterPreparation of Dilution Water Estimating BODEstimating BOD SeedingSeeding Dilution of SampleDilution of Sample Determination of Initial DODetermination of Initial DO IncubationIncubation Determination of Final DODetermination of Final DO
Analytical ProcedureAnalytical Procedure
Preparation of Dilution WaterPreparation of Dilution Water Estimating BODEstimating BOD SeedingSeeding Dilution of SampleDilution of Sample Determination of Initial DODetermination of Initial DO IncubationIncubation Determination of Final DODetermination of Final DO
Preparation of Dilution Preparation of Dilution WaterWater
Source water is criticalSource water is critical Distilled often contains ClDistilled often contains Cl22, NH, NH33, organics, organics DI often contains organicsDI often contains organics Bad blanks? Try “Bad blanks? Try “steam distilledsteam distilled” water” water
Dilution water for BODDilution water for BOD Aerate, store at 20°, add buffers/nutrients Aerate, store at 20°, add buffers/nutrients
morning of test, settle for one hour (CBOD morning of test, settle for one hour (CBOD dilution water can be stored)dilution water can be stored)
Check pH, do blankCheck pH, do blank DDo not add inhibitor to blanko not add inhibitor to blank
““Steam Distilled” WaterSteam Distilled” Water
Preparation of Dilution Preparation of Dilution WaterWater
Source water is criticalSource water is critical Distilled often contains ClDistilled often contains Cl22, NH, NH33, organics, organics DI often contains organicsDI often contains organics Bad blanks? Try “steam distilled” waterBad blanks? Try “steam distilled” water
Dilution water for Dilution water for BODBOD Aerate, store at 20°, add buffers/nutrients Aerate, store at 20°, add buffers/nutrients
morning of test, settle for one hour (morning of test, settle for one hour (CBODCBOD dilution water can be stored)dilution water can be stored)
Check pH, do blankCheck pH, do blank (do not add inhibitor to blank)(do not add inhibitor to blank)
Analytical ProcedureAnalytical Procedure
Preparation of Dilution WaterPreparation of Dilution Water Estimating BODEstimating BOD SeedingSeeding Dilution of SampleDilution of Sample Determination of Initial DODetermination of Initial DO IncubationIncubation Determination of Final DODetermination of Final DO
Estimating BODEstimating BOD
Sample BOD Range*Vol Range in 300-mL
Btl
Min Max Min Max
Influent 150 400 3 8
1° Effluent 60 160 7.5 20
2° Effluent 5 60 20 240
Digester 1000 4000 0.3 1.2
Industrial 100 3000 0.4 12
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
Analytical ProcedureAnalytical Procedure
Preparation of Dilution WaterPreparation of Dilution Water Estimating BODEstimating BOD SeedingSeeding Dilution of SampleDilution of Sample Determination of Initial DODetermination of Initial DO IncubationIncubation Determination of Final DODetermination of Final DO
SeedingSeeding Always seed unless known to be unnecessaryAlways seed unless known to be unnecessary Source of seed (need good source of bacteria)Source of seed (need good source of bacteria)
Domestic WWTP – 1° effluent; 2° effluent; influent (often Domestic WWTP – 1° effluent; 2° effluent; influent (often variable)variable)
Industrial WWTP or Private Lab – Synthetic seedIndustrial WWTP or Private Lab – Synthetic seed Polyseed® - no nitrifiersPolyseed® - no nitrifiers Biosystems® - has nitrifiers (or does it??)Biosystems® - has nitrifiers (or does it??)
Seed CheckSeed Check Seed control – should deplete 0.6 – 1.0 mg/L Seed control – should deplete 0.6 – 1.0 mg/L per mL of seedper mL of seed Glucose/Glutamic Acid – true test of seedGlucose/Glutamic Acid – true test of seed
Goal is: Goal is: averageaverage ~198 mg/L; ~198 mg/L; standard deviationstandard deviation <<30.5 mg/L <<30.5 mg/L KHP – OK as supplement KHP – OK as supplement
KHP Standard (300 mg/L)KHP Standard (300 mg/L)
ParameterParameter Expected Expected ValueValue
BODBOD 249 mg/L249 mg/LCODCOD 343 mg/L343 mg/LTOCTOC 141 mg/L141 mg/LpHpH 4.4 pH units4.4 pH unitsTotal SolidsTotal Solids 300 mg/L300 mg/LVolatile SolidsVolatile Solids 169 mg/L169 mg/LConductivityConductivity 169 mhos169 mhosAcidityAcidity 74 mg/L74 mg/L
__________________Source: WA Dept of Ecology Manchester Laboratory Source: WA Dept of Ecology Manchester Laboratory
(lab attained a standard deviation for BOD of 15 (lab attained a standard deviation for BOD of 15 mg/L…very precise work)mg/L…very precise work)
Analytical ProcedureAnalytical Procedure
Preparation of Dilution WaterPreparation of Dilution Water Estimating BODEstimating BOD SeedingSeeding Dilution of SampleDilution of Sample Determination of Initial DODetermination of Initial DO IncubationIncubation Determination of Final DODetermination of Final DO
Dilution of SampleDilution of Sample
Bottle MethoBottle Method Add sample, seed, dilution water to bottleAdd sample, seed, dilution water to bottle Never add seed to empty bottleNever add seed to empty bottle Add inhibitor only after sample/dilution waterAdd inhibitor only after sample/dilution water
Graduated Cylinder MethodGraduated Cylinder Method Add sample, seed, dilution water to grad cylinderAdd sample, seed, dilution water to grad cylinder
Dilution SeriesDilution Series Do enough dilutions to assure at least one has Do enough dilutions to assure at least one has
depletion of depletion of >>2 mg/L, retention of 2 mg/L, retention of >>1 mg/L1 mg/L Remember – if more than 200 mL of sample is in Remember – if more than 200 mL of sample is in
bottle, use special buffer/nutrient packetsbottle, use special buffer/nutrient packets
Typical Dilution SeriesTypical Dilution Series
Blank Blank – 1 bottle– 1 bottle Seed controlSeed control – 1 or 2 bottles, – 1 or 2 bottles, samesame dilution dilution
Don’t forget...must deplete at least 2 mg/LDon’t forget...must deplete at least 2 mg/L G/GA – 1 bottle, ~6 mL, OK...some labs do 2 EffluentEffluent – 1 bottle OK if precision is good – 1 bottle OK if precision is good**
Seed must contribute 0.6 – 1.0 mg/L depletionSeed must contribute 0.6 – 1.0 mg/L depletion InfluentInfluent – 3 dilutions minimum – 3 dilutions minimum
recommendedrecommended UnknownUnknown – 3 dilutions minimum, 5 better – 3 dilutions minimum, 5 better__________________** Some states do not allow only one Some states do not allow only one
Typical Dilution SeriesTypical Dilution Series
Late Breaking News!!Late Breaking News!!
Standard Methods Standard Methods staff published a memo (May staff published a memo (May 19, 2009) saying they never intended to require 19, 2009) saying they never intended to require more than one dilution for any given BOD sample.more than one dilution for any given BOD sample.
Check Check www.perrybrake.com/BODSolutions.html for a copy of the memofor a copy of the memo
Analytical ProcedureAnalytical Procedure
Preparation of Dilution WaterPreparation of Dilution Water Estimating BODEstimating BOD SeedingSeeding Dilution of SampleDilution of Sample Determination of Initial DODetermination of Initial DO IncubationIncubation Determination of Final DODetermination of Final DO
Determination of Initial Determination of Initial DODO
WinklerWinkler great...but time consuming great...but time consuming DO meterDO meter
Proper calibration criticalProper calibration critical Refer to DO chart to see if reading is reasonable Refer to DO chart to see if reading is reasonable
Do all measurements at 20 ± 1° CDo all measurements at 20 ± 1° C Use actual barometric pressureUse actual barometric pressure If using air calibration, use 100% saturationIf using air calibration, use 100% saturation
LDO meterLDO meter simplifies procedure simplifies procedure
DO Saturation vs. Temp/PressureDO Saturation vs. Temp/Pressure
PressurePressure Temperature (°C)Temperature (°C)
((inches Hginches Hg) ) 17.017.0 18.018.0 19.019.0 20.020.0 21.021.0 22.022.0 23.023.0
26.626.6 8.618.61 8.428.42 8.258.25 8.078.07 7.917.91 7.757.75 7.597.59
26.826.8 8.678.67 8.488.48 8.318.31 8.138.13 7.977.97 7.817.81 7.657.65
27.027.0 8.738.73 8.548.54 8.378.37 8.198.19 8.038.03 7.877.87 7.717.71
-- -- -- -- -- -- -- --
31.131.1 10.110.1 9.849.84 9.649.64 9.459.45 9.279.27 9.099.09 8.928.92
31.331.3 10.110.1 9.909.90 9.709.70 9.519.51 9.339.33 9.159.15 8.978.97
Determination of Initial Determination of Initial DODO
WinklerWinkler great...but time consuming great...but time consuming DO meterDO meter
Proper calibration criticalProper calibration critical Refer to DO chart to see if reading is reasonable Refer to DO chart to see if reading is reasonable
Keep all measurements at 20 ± 1° CKeep all measurements at 20 ± 1° C Use actual barometric pressureUse actual barometric pressure If using air calibration, use 100% saturationIf using air calibration, use 100% saturation
LDO meterLDO meter simplifies procedure simplifies procedure
Analytical ProcedureAnalytical Procedure
Preparation of Dilution WaterPreparation of Dilution Water Estimating BODEstimating BOD SeedingSeeding Dilution of SampleDilution of Sample Determination of Initial DODetermination of Initial DO IncubationIncubation Determination of Final DODetermination of Final DO
IncubationIncubation
Water seal plus cap Water seal plus cap (unless water bath (unless water bath used)used)
At 20 ± 1° C At 20 ± 1° C (NIST-traceable thermometer(NIST-traceable thermometer
In darkIn dark Circulating air/waterCirculating air/water For 5 days, ± 2 hours For 5 days, ± 2 hours (21(21stst Ed. says ± 6 Ed. says ± 6
hours)hours)
All bottles done together, same All bottles done together, same incubator (i.e., in same “batch”)incubator (i.e., in same “batch”)
Analytical ProcedureAnalytical Procedure
Preparation of Dilution WaterPreparation of Dilution Water Estimating BODEstimating BOD SeedingSeeding Dilution of SampleDilution of Sample Determination of Initial DODetermination of Initial DO IncubationIncubation Determination of Final DODetermination of Final DO
Determination of Final Determination of Final DODO
Same process as Same process as
for initial DOfor initial DO
What References Say (and What References Say (and Don’t Say) About...Don’t Say) About...
SamplingSampling Sample preservation/holding timeSample preservation/holding time EquipmentEquipment ReagentsReagents Sample PretreatmentSample Pretreatment The Analytical ProcedureThe Analytical Procedure Calculations/Data RecordingCalculations/Data Recording QA/QC and Performance MonitoringQA/QC and Performance Monitoring
Calculations/Data Calculations/Data RecordingRecording
Dilution FactorDilution Factor BOD – Not SeededBOD – Not Seeded BOD – SeededBOD – Seeded BenchsheetBenchsheet Reporting ResultsReporting Results
Calculations/Data Calculations/Data RecordingRecording
Dilution FactorDilution Factor BOD – Not SeededBOD – Not Seeded BOD – SeededBOD – Seeded BenchsheetBenchsheet Reporting ResultsReporting Results
Dilution FactorDilution Factor
Ratio of final volume to volume of Ratio of final volume to volume of samplesample
Bottle MethodBottle Method DF = 300 / VolDF = 300 / Volsamplesample
e.g., for 5-mL sample, DF = 300/5 = 60e.g., for 5-mL sample, DF = 300/5 = 60
Grad Cylinder MethodGrad Cylinder Method DF = 1000/ VolDF = 1000/ Volsamplesample e.g., for 20-mL sample, DF = 1000/20 = 50e.g., for 20-mL sample, DF = 1000/20 = 50
Calculations/Data Calculations/Data RecordingRecording
Dilution FactorDilution Factor BOD – Not SeededBOD – Not Seeded BOD – SeededBOD – Seeded BenchsheetBenchsheet Reporting ResultsReporting Results
BOD – Not SeededBOD – Not Seeded
BOD = BOD = DF(DODF(DO11 – DO – DO55)) Example: DF = 60 for influent sampleExample: DF = 60 for influent sample
DODO11 = 8.7 = 8.7
DODO55 = 1.7 = 1.7
BOD = 60(8.7 – 1.7) BOD = 60(8.7 – 1.7) = 420 mg/L= 420 mg/L
Calculations/Data Calculations/Data RecordingRecording
Dilution FactorDilution Factor BOD – Not SeededBOD – Not Seeded BOD – SeededBOD – Seeded BenchsheetBenchsheet Reporting ResultsReporting Results
BOD - SeededBOD - Seeded
Must subtract depletion caused by seedMust subtract depletion caused by seed BOD = DF[(DOBOD = DF[(DO11 – DO – DO55) - f(B1 – B2)]) - f(B1 – B2)] where... where...
““f” = is seed volf” = is seed volsamplesample/seed vol /seed vol seed controlseed control for for bottlebottle method method
““f” = is % seedf” = is % seedsamplesample/%seed/%seedseed control seed control for for grad cylgrad cyl method method
And B1 and B2 are DO for seed control on Days 1 and 5And B1 and B2 are DO for seed control on Days 1 and 5
Calculations/Data Calculations/Data RecordingRecording
Dilution FactorDilution Factor BOD – SeededBOD – Seeded BOD – Not SeededBOD – Not Seeded BenchsheetBenchsheet Reporting ResultsReporting Results
BenchsheetBenchsheet
Must include:Must include: Date/time sample taken, set up, final readingDate/time sample taken, set up, final reading ID of sampler and analystID of sampler and analyst ID of sampleID of sample Sample pHSample pH Sample temp when initial DO reading takenSample temp when initial DO reading taken Bottle numbersBottle numbers Volume of seed in each bottle or graduated cylinderVolume of seed in each bottle or graduated cylinder Volume of sample in each bottle or graduated cylinderVolume of sample in each bottle or graduated cylinder Initial and final DO for each bottleInitial and final DO for each bottle Space for calculation of “f”, “DF”Space for calculation of “f”, “DF” Space for reviewer to initialSpace for reviewer to initial Space for commentsSpace for comments
Calculations/Data Calculations/Data RecordingRecording
Dilution FactorDilution Factor BOD – SeededBOD – Seeded BOD – Not SeededBOD – Not Seeded BenchsheetBenchsheet Reporting ResultsReporting Results
Reporting ResultsReporting Results
Report Report averageaverage of dilutions that deplete of dilutions that deplete >>2, 2, retain retain >>1 mg/L, unless signs of toxicity1 mg/L, unless signs of toxicity
If toxicity indicated, report If toxicity indicated, report highesthighest result result that met depletion/retention criteriathat met depletion/retention criteria
If no bottle depleted 2 or more mg/L, report If no bottle depleted 2 or more mg/L, report as directedas directed by regulatory agency by regulatory agency
2121stst Edition of Edition of SM SM allows reporting “0”allows reporting “0”
What References Say (and What References Say (and Don’t Say) About...Don’t Say) About...
SamplingSampling Sample preservation/holding timeSample preservation/holding time EquipmentEquipment ReagentsReagents Sample PretreatmentSample Pretreatment The Analytical ProcedureThe Analytical Procedure Calculations/Data RecordingCalculations/Data Recording QA/QC and Performance MonitoringQA/QC and Performance Monitoring
QA/QC and Performance QA/QC and Performance MonitoringMonitoring
Minimum DO Depletion/RetentionMinimum DO Depletion/Retention BlanksBlanks
Most zero, a few <0.2, almost none >0.2Most zero, a few <0.2, almost none >0.2 Check Standard (G/GA)Check Standard (G/GA)
Average ~198 mg/L (175 – 235 good goal)Average ~198 mg/L (175 – 235 good goal)Standard deviation Standard deviation <<15 mg/L15 mg/L
DuplicatesDuplicates (if done) (if done) - <50% RPD for effluent - <50% RPD for effluent ToxicityToxicity – check only if toxicity is probable – check only if toxicity is probable
Performance Monitoring Performance Monitoring SummarySummary
BiasBias Total PrecisionTotal Precision
Within-batch precisionWithin-batch precision Between-batch precisionBetween-batch precision
Detection Limit - Detection Limit - No “MDL” No “MDL” per seper se Working LimitsWorking Limits
MinimumMinimum MaximumMaximum
Performance Monitoring Performance Monitoring SummarySummary
BiasBias Total PrecisionTotal Precision
Within-batch precisionWithin-batch precision Between-batch precisionBetween-batch precision
Detection Limit - Detection Limit - No “MDL” No “MDL” per seper se Working LimitsWorking Limits
MinimumMinimum MaximumMaximum
Control chartingis good way to
monitor biasand precision
Control Chart for RepeatedControl Chart for RepeatedAnalysis of a Standard (e.g., GGA)Analysis of a Standard (e.g., GGA)
Repeated Analysis of a Standard
160.00
170.00
180.00
190.00
200.00
210.00
220.00
230.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Test Number
Co
nce
ntr
ati
on
UAL
UWL
Mean
LWL
LAL
Daily
Another ReferenceAnother 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 Troubleshooting SequenceSequence
Isolate Problem(s)Isolate Problem(s) BlanksBlanks G/GA (bias or total-precision problem)G/GA (bias or total-precision problem) Duplicates (within-batch precision problemDuplicates (within-batch precision problem Seed StrengthSeed Strength Environmental SamplesEnvironmental Samples
Identify/Rank Probable CausesIdentify/Rank Probable Causes Try Possible FixesTry Possible Fixes
One Problem at a timeOne Problem at a time One fix at a timeOne fix at a time
Problem Causes/Fixes - Problem Causes/Fixes - BlanksBlanks
Sometimes exceeds 0.2 mg/LSometimes exceeds 0.2 mg/L Supersaturated dilution waterSupersaturated dilution water Labware contaminatedLabware contaminated
Usually exceeds 0.2 mg/L Usually exceeds 0.2 mg/L Source water Source water problemproblem
Quite often negativeQuite often negative Temperature control problem Temperature control problem PhotosynthesisPhotosynthesis
Sometimes negative, sometimes positiveSometimes negative, sometimes positive DO measurement (e.g., atmospheric pressure not DO measurement (e.g., atmospheric pressure not
used)used) High blanks for CBOD, OK for BODHigh blanks for CBOD, OK for BOD
Inhibitor being added to blankInhibitor being added to blank
Problem Causes/Fixes – Problem Causes/Fixes – G/GAG/GA
Standard Deviation approaching 30.5 Standard Deviation approaching 30.5 mg/Lmg/L Variable seedVariable seed Meter calibration problemsMeter calibration problems Inattention to detailInattention to detail
Standard Deviation >30.5 mg/LStandard Deviation >30.5 mg/L MANY sources of imprecision! MANY sources of imprecision! BIGBIG problem! problem!
Average >> 198 mg/L (precision OK)Average >> 198 mg/L (precision OK) Seed too strongSeed too strong
Average << 198 mg/L (precision OK)Average << 198 mg/L (precision OK) Seed too weakSeed too weak
Problem Causes/Fixes – Problem Causes/Fixes – Duplicates*Duplicates*
Relative percent differences (RPDRelative percent differences (RPD****) ) exceed 50% for samples in 5-20 mg/L exceed 50% for samples in 5-20 mg/L range (e.g., effluents)range (e.g., effluents) Poorly mixed seedPoorly mixed seed Contaminated bottlesContaminated bottles Faulty DO meter/probeFaulty DO meter/probe Countless other things that change from Countless other things that change from
bottle to bottle within a batchbottle to bottle within a batch__________________** Duplicates – Duplicates – two samples done exactly the same two samples done exactly the same
wayway**** RPD – Difference divided by average of two results RPD – Difference divided by average of two results
Problem Causes/Problem Causes/FixesFixes Seed Strength*Seed Strength*
DO depletion < 0.6 mg/L DO depletion < 0.6 mg/L per mL of seedper mL of seed in in seed control bottleseed control bottle
Seed too weakSeed too weak DO depletion > 1.0 mg/L DO depletion > 1.0 mg/L per mL of seedper mL of seed in in
seed control bottle seed control bottle Seed too strongSeed too strong
DO depletion sometimes < 0.6 mg/L and DO depletion sometimes < 0.6 mg/L and sometimes >1.0 mg/L for seed control bottlessometimes >1.0 mg/L for seed control bottles
Seed too variableSeed too variable
____________* * Applies only to natural seeds; 21 Applies only to natural seeds; 21stst edition of edition of
SM SM omits 0.6 – 1.0 mg/L seed depletion omits 0.6 – 1.0 mg/L seed depletion requirement.requirement.
Problem Causes/Problem Causes/FixesFixes – – Environmental SamplesEnvironmental Samples
No dilutions leave at least 1.0 mg/L DO No dilutions leave at least 1.0 mg/L DO residualresidual Samples not dilute enoughSamples not dilute enough
No dilutions deplete at least 2.0 mg/L DO No dilutions deplete at least 2.0 mg/L DO Samples too diluteSamples too dilute
Significant increase in BOD for more dilute Significant increase in BOD for more dilute bottles in dilution seriesbottles in dilution series Matrix is interfering (toxicity)Matrix is interfering (toxicity)
Influent (and TSS) suddenly higher than Influent (and TSS) suddenly higher than normalnormal Sample not being thoroughly mixedSample not being thoroughly mixed
ReferenceReference
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 GuideTrouble Shooting Guide
Appendix CAppendix CSample 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 SupplementReference Supplement
BOD Checklist (Word®) Benchsheet (Excel®)
Bottle Graduated Cylinder
Control Charting (Excel®) Standards Duplicates
SM 21st 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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