the bod test from a – z (aka a bug’s-eye-view of the bod test) perry brake lab testing...

The BOD Test from A – ZThe BOD Test from A – Z(aka (aka A Bug’s-Eye-View A Bug’s-Eye-View

of the BOD Testof the BOD Test))Perry BrakeLab Testing Consultant(253) [email protected]

Presented at OELA Workshop, 5/24/12

Powerpoint presentation posted at www.perrybrake.com/BODSolutions.html

ReferencesReferences

Specifically, Method 5210Bin 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*

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

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

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

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 special nutrient/buffer “pillow”, top off with Hnutrient/buffer “pillow”, top off with H22O)O)

SampleBOD


Min Max Min Max


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


Min Max Min Max


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 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)

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

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 concentration, or expected value Standard – solution of known concentration, or expected value in the case of the BOD testin the case of the BOD test

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 7.0 – 7.2 – if <6.0 or >8.5, adjust to 7.0 – 7.2 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


Min Max Min Max


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

Sample TemperatureSample Temperature 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 Toxicity will result in higher BOD for

increasing dilution in series of bottles increasing 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 - Nitrification inhibition - Add pyridine Add pyridine inhibitor to samples, seed control, GGA, but inhibitor to samples, seed control, GGA, but NOTNOT to blank to blank

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 Toxicity will result in higher BOD for

increasing dilution in series of bottlesincreasing 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 - Nitrification inhibition - Add pyridine Add pyridine inhibitor to samples, seed control, GGA, but inhibitor to samples, seed control, GGA, but NOTNOT to blank 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

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 o notnot add inhibitor to blank 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 (do notnot add inhibitor to blank) add inhibitor to blank)

Estimating BODEstimating BOD

Sample BOD Range*Vol Range in 300-mL

Btl

Min Max Min Max


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

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)

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

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

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

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”)

Determination of Final Determination of Final DODO

Same process as Same process as

for initial DOfor initial DO

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” Final BOD resultFinal BOD result Space for reviewer to initialSpace for reviewer to initial Space for commentsSpace for comments


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”

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

Matrix/Matrix Spike Duplicate Matrix/Matrix Spike Duplicate (New in 22d edition)(New in 22d edition)No guidance for limits in No guidance for limits in Standard MethodsStandard Methods

Duplicates (if done) - <50%RPD for effluentDuplicates (if done) - <50%RPD for effluent Toxicity – check only if toxicity is probableToxicity – 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 seed in seed control bottle 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 seed in seed

control bottle 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 per mL of seed in seed sometimes >1.0 mg/L per mL of seed in seed control bottlescontrol 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 requirement.omits 0.6 – 1.0 mg/L seed depletion 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) BOD suddenly higher than Influent (and TSS) BOD 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 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 SupplementReference 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

QuestionsQuestions

?

Slides on calculations follow


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

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

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

the bod test from a – z (aka a bug’s-eye-view of the bod test) perry brake lab testing...

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