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Cardinal Laboratories, LLC

Standard Operating Procedure

Procedure No.: 4010 Revision Date:


Subject: Biological Oxygen Demand, 5 Day (Including

(SM 5210 B, EPA Method 405.1 )

Revision #/Date: Approved By: Date


Revised 01/2008

Biological Oxygen Demand, 5 Day

(Including Carbonaceous)
EPA Method: 5210 B

Scope and Application

This method determines the BOD of a water sample.


This method is used to determine BOD5 & CBOD5 in surface waters,

treated effluents and untreated effluents. The test measures the
oxygen utilized during a specified period for the biochemical
degradation of organic material (carbonaceous). It also may measure
oxidation of sulfides and ferrous iron as well as a reduction of nitrogen
unless an inhibitor is used.


General Requirements (See procedure 0010)

Corrosives (See procedure 0020)

Sample Preservation-Preparation:

Samples must be unpreserved. Collect in plastic or glass bottles. If

the analysis is performed more than 2 hours after collection, refrigerate
to 4 C. Maximum holding time is 48 hours.


Oxidation of reduced forms of nitrogen exerts nitrogenous demand.

This can be prevented by addition of nitrification inhibitor. If inhibitor
is not added the measured oxygen demand is the sum of carbonaceous
and nitrogenous demand. Poor quality dilution water will appear as
sample BOD, resulting in a positive bias that is amplified by the dilution

Revised 01/2008

- 300 ml incubation bottles:

Wash with 1% bleach solution and rinse three times with ultra
pure water.
Do not acid clean. Invert to drain.

- Air incubator:
Thermostatically controlled to 20 C 1 C. Sealed to prevent
light entering.

- Stir plate
- Stir bar
- 25ml buret
- Volumetric pipettes, various
- Graduated cylinders, various
- Dissolved oxygen meter:
Orion meter using membrane electrode to take direct oxygen
- Tubing and glass siphon (from dilution water to BOD bottles)


- Sulfuric Acid Solution (H 2SO4) 1.0N:

Slowly add 28ml concentrated sulfuric acid to ultra pure water
dilute to 1L.

- Sodium Hydroxide solution (NaOH) 1.0N:

Dissolve 40g of NaOH in ultra pure water and dilute to 1L.

- Nitrification inhibitor: commercially available.

Hach product #2533-34

- Manganous Sulfate (MnSO4) solution: commercially available.

- Alkali-Iodide-Azide solution:
Dissolve 500g NaOH (or 700g KOH) and 135g NaI (or 150g KI)
ultra pure water and dilute to l liter. Add 10g NaN 3 dissolved
40 ml of ultra pure water.
CAUTION: this solution is highly corrosive.

- Sodium Thiosulfate (Na2S2O3 5H2O) .0250N: commercially


- Starch indicator: commercially available.

- Polyseed-TM: commercially available.

Revised 01/2008
- Sodium Sulfite solution: Dissolve 1.575g Na2SO3 in 1-L ultrapure
water. Prepare daily.

- Sulfuric acid (H2SO4) concentrated: ACS grade or better.

- BOD Standard: commercially available.

- BOD Nutrient buffer pillows: Commercially available.

- Potassium Iodide - (KI)

- Chlorine powder pillows: commercially available.


1.0 Preparation of dilution water

1.1 Add two BOD nutrient buffer pillows to 19L of ultra pure water:
(Cat. # 14863-98 to 19L / Cat. #14862-98 to 6 L). Aerate for
15-30 minutes using lab grade compressed air.

1.2 Before use bring dilution water temperature to 20C.

2.0 Standardization of dilution water

2.1 Pour off three incubation bottles with the freshly prepared
dilution water.
Cap and set one aside.

2.2 To the remaining two bottles, add 1 ml MnSO 4 solution, cap

and invert
to mix.

2.3 Carefully add 1 ml alkali-iodide-azide to each bottle, cap and

invert to mix.

2.4 Carefully add 1 ml concentrated H 2SO4 to each bottle and

cap. Invert
several times of mix well.

2.5 Pour off 100ml from the incubation bottle, discard.

Revised 01/2008
2.6 Titrate remaining 200ml with 0.0250N sodium thiosulfate to a
light straw
color. Add 1-2 ml of starch indicator and continue titration to
a clear

2.7 The DO of the dilution water should be between 7.5 - 9.0

(1 ml sodium thiosulfate = 1 mg DO/ liter).

3.0 Calibration of DO meter

3.1 Put DO probe into bottle previously capped and set aside
(step 2.1)

3.2 Turn knob from off position to C. Let reading stabilize about

3.3 Turn to MG/L CAL and enter the DO of the dilution water. Use
three buttons on the display set to scroll to the correct

3.4 Turn knob to MG/L. After the meter has stabilized, it will give
two beeps
and a reading. It is then ready to measure DO results for

4.0 Sample pretreatment: The following parameters must be checked before

setting up

4.1 pH should be adjusted to between 6.5 - 7.5 using a minimum of 1N

acid or 1N Sodium Hydroxide.

4.2 Samples should be checked for total residual chlorine using Hach
chlorine powder pillows. If chlorine is present, it should be removed
by addition of sodium sulfite.

4.2.1 To 100 mls of sample, add 1ml of 6N H 2SO4, 1g potassium

iodide (KI) crystals. Stir to mix.

4.2.2 Titrate to a pale yellow with sodium sulfite solution, add 1

ml of starch indicator and titrate until sample just becomes

Revised 01/2008
4.2.3 Add to neutralized sample a proportionate amount of
sodium sulfite. Mix well.

4.2.4 After 10-20 minutes, check for residual chlorine. Avoid

excess sodium sulfite, it exerts an oxygen demand
and may positively bias results.

4.3 Samples must be brought to room temperature before aliquoting.

4.4 Dissolved Oxygen level on sample should be taken directly from

the sample
bottle. If D.O. is greater than 9.0, reduce by purging with organic
free compressed air at 20 C.

4.5. If sample is analyzed for CBOD, add 2 aliquots of the nitrification

inhibitor prior to initial DO reading.

5.0 Dilution Technique

5.1 Several dilutions should be made for each sample. Choose

dilution volumes based on historical data or follow these general

1. Strong Industrial Wastes:

Prepare an initial dilution of 10X and prepare bottles in
the range of 3mls to 30mls using the diluted

2. Raw & Settled Wastewater:

Prepare bottles with 3 to 15mls of sample.

3. Biologically Treated Effluents:

Prepare bottles using 15 to 75mls of sample.

4. River Water:
Prepare bottles using 75 to 300mls of sample.

5.1.1 For samples where content of organisms is unknown, seed

the samples to ensure biological degradation. Use 6 ml
per incubation bottle.

5.1.2 To make polyseed-TM: Empty one capsule of polyseed-TM

into 200ml of unaerated dilution water. ( This is dilution
water with the nutrients added but not aerated.)
Aerate gently for 1 hour. Filter thru #4 Whatman filter
paper. Make daily.

5.1.3 Standard should be prepared with 3 dilutions of 3.0 mls

each and requires seed.

Revised 01/2008
5.2 Fill with dilution water by using the siphon apparatus under the
level of the sample. Rinse after each bottle. Measure initial DO
with oxygen probe and replace displaced water with dilution
water. This measurement should be made as quickly as
possible after dilution.

5.3 Cap tightly making sure no air bubbles are entrapped in the
incubation bottle, cover with plastic cap and place in the
incubator. A dilution water blank should be run with each sample set. If
samples have been seeded, a seeded dilution water blank
should be run. If CBODs are analyzed, a dilution water blank with
nitrification inhibitor should be run.

5.4 Incubate for 5 days at 20 C 1 C.

5.5 Measure final DO.

5.5.1 Calibrate meter per steps 2.0-3.5.

6.0 Calculations:

6.1 In order to have a valid dilution for calculation:

6.1.1 The difference between the final and initial D.O. must be
>2.0 and

6.1.2 The final D. O. should not be <1.0.

6.1.3 Report results that are <10 to 2 significant figures.

Results that are >10 are reported to 3 significant


SEED VALUE:mg/L Depletion = Initial DO - Final DO


Revised 01/2008
BOD mg/L = (Initial DO-Final DO) X 300
mls sample


BOD mg/L = (Initial DO - Final DO - Seed Value) X 300

mls sample


If all sample depletions are <2.0 the result is BDL. If the largest
sample volume used was not 300 mls. YOU MUST ADJUST THE


(2.0) X 300
mls sample* * This is the largest volume of sample used.


If all Final DOs are <1.0 the result will be reported as >.
To calculate this, use the initial DO of the dilution with the *smallest
sample volume.

Calculation: (Initial DO - 1.0) X 300

mls sample*


True value is 200 mg/L

Calculation: BOD mg/L = (Initial DO - Final DO - Seed Value)


Waste Disposal

Samples are disposed of in the sink with running water.

Quality Control

* Each analytical batch analyzed for BOD or CBOD must be

accompanied by a method blank. Blanks are taken through
the incubation steps.

Revised 01/2008
The unseeded blanks should not exceed a BOD of 0.2mg/L.

The DO of seeded blanks should fall within the range of 0.6-


* All volumes and D.O. readings, must be entered in a notebook

form for data review.

* The current acceptable limits for QC parameters are 70-130%.


Standard Methods for the Examination of Water and Wastewater 18th

edition 1992
Method 5210B, pg 5 - 1 thru 5 - 6.

Methods for Chemical Analysis of Water and Wastes

EPA 600/4-79-020, Method 405.1 - pp.405.1-1 thru 405.1-2

National Environmental Methods Index. April 1994. Method 5210B. 7

April 2004.

Revised 01/2008