here. - Perry Brake
Download
Report
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