Transcript here. - Perry Brake

The BOD Test from A – Z
(aka A Bug’s-Eye-View
of the BOD Test)
Perry Brake
Lab Testing Consultant
(253) 565-5350
[email protected]
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?
Bacteria
Oxygen
BOD
BO O
D
2
O2
O2
O2
BOD
N
O2
O2
Nitrogen-containing
Organic material
Nitrifying Bacteria
N
BOD
Organic Material
N
O2
Nitrogen (ammonia,
ammonium, nitrites)
Plus water, nutrients, buffers, inert material, sometimes interferents
What’s happening inside
that BOD Bottle?

O2
CxHyOz
CxHyOzN
O2
CO2 + H20
CO2 + H20 + NH4
+
BOD
Examples:
C6H12O6 – Glucose
O2
NO3 -
C4H7O4N – Glutamic Acid
What’s happening inside
that BOD Bottle?

O2
CxHyOz
CxHyOzN
O2
Inhibitor
Examples:
C6H12O6 – Glucose
CO2 + H20
CO2 + H20 + NH4
+
CBOD
O2
NO3
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
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
_____________
*
BOD ranges from EPA’s Operation of Wastewater Treatment
Plants, vol. II, 3rd Ed., 1991.
Sampling


Type – Grab or Composite
Volume
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
_____________
*
BOD ranges from EPA’s Operation of Wastewater Treatment
Plants, vol. II, 3rd Ed., 1991.
Sampling



Type – Grab or Composite
Volume
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
Maximum Volume/bottle - ~295 mL (sample, seed,
special nutrient/buffer “pillow”, top off with H2O)
Sampling



Type – Grab or Composite
Volume
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
Minimum Volume/bottle – none, but dilute
entire sample for extremely high BOD samples
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



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
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
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
Minimum Volume/bottle – none, but dilute entire sample
for extremely high BOD samples
Sample Pretreatment

Sample prep



Sample Temperature




Well mixed sample critical
Pre-dilute for high-BOD samples
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)
(mg/L)
Influent
1
20
4.4
15
66
2
10
4.0
30
120
3
5
3.7
60
222
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**
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



Dilution water for BOD


DI often contains organics
Bad blanks? Try “steam distilled” water
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



Dilution water for BOD


DI often contains organics
Bad blanks? Try “steam distilled” water
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

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 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
KHP Standard (300 mg/L)
Parameter
BOD
COD
TOC
pH
Total Solids
Volatile Solids
Conductivity
Acidity
_________
Expected Value
249 mg/L
343 mg/L
141 mg/L
4.4 pH units
300 mg/L
169 mg/L
169 mhos
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
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



G/GA – 1 bottle, ~6 mL, OK...some labs do 2
Effluent – 1 bottle OK if precision is good*



Don’t forget...must deplete at least 2 mg/L
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
26.8
27.0
31.1
31.3
8.61
8.67
8.73
10.1
10.1
8.42
8.48
8.54
9.84
9.90
8.25
8.31
8.37
9.64
9.70
8.07
8.13
8.19
9.45
9.51
7.91
7.97
8.03
9.27
9.33
7.75
7.81
7.87
9.09
9.15
7.59
7.65
7.71
8.92
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
Control charting
is good way to
monitor bias
and precision
Limit - No “MDL” per se
 Working Limits


Minimum
Maximum
Control Chart for Repeated
Analysis of a Standard (e.g., GGA)
Repeated Analysis of a Standard
UAL
UWL
Mean
230.00
LWL
LAL
Daily
220.00
Concentration
210.00
200.00
190.00
180.00
170.00
160.00
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
Te st Numbe r
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




Usually exceeds 0.2 mg/L Source water problem
Quite often negative



Temperature control problem
Photosynthesis
Sometimes negative, sometimes positive


Supersaturated dilution water
Labware contaminated
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
 Seed
too weak
Average << 198 mg/L (precision OK)
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

Seed too strong
DO depletion > 1.0 mg/L per mL of seed in seed
control bottle *
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

No dilutions deplete at least 2.0 mg/L DO
 Samples

too dilute
Significant increase in BOD for more dilute
bottles in dilution series


not dilute enough
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
Blank
Usually
exceeds
0.2 mg/L
Source water
is unsuitable
Incubate several
blanks using
alternate waters,
choose best
Pg 37 ¶ 10b(5)
Pg 20, ¶ 8a(3)(b)
Sometimes
negative
Bulk dilution
water contami-
Pg 36 ¶ 10b(2)
inated
Aerate water day
before test, add
nutrients/buffer
Random variations
in procedure
Find sources of
variations, eliminate them
Pg 38 ¶ 10c
etc.
GGA
Standard Deviation > 15 but <30
etc., etc.,
Reference
Pg 20 ¶ 8a(3)(b)
Pg 16 ¶ 7f
Reference Supplement
Perry Brake
Revised May 2011
 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
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