NANOCOLOR 985822 BOD5 Tube Test Instructions

NANOCOLOR 985822 BOD5 Tube Test

Product Information

Specifications

• Method: Tube test for the determination of the biochemical oxygen demand in 5 days (BOD5)
• Principle: Diluting principle according to the German Standard Method DIN 38409-H51
• Incubation: Carried out in Winkler oxygen flasks
• Oxygen Determination: Carried out similarly to the Winkler Method DIN EN 25813-G21 by photometric evaluation of iodine-color
• Reaction Time: 5 days
• Reaction Temperature: 445 nm
• Reagent Set Contents:
  • 3 empty test tubes
  • 15 mL BOD5 R1
  • 15 mL BOD5 R2
  • 30 mL BOD5 R3
• Interferences: Changes in pH-value, accumulation of special microbial metabolites and compounds, iron(II) salts, sulfur dioxide, sulfur hydrogen, algae, nitrified microorganisms

Product Usage Instructions

Preparation and Handling of Diluting Water
To prepare and handle diluting water, refer to the BOD5-Accessories Set (REF 916 918).

Preparation and Handling of BOD5-Nutrient Solutions and Inoculating Water
To prepare and handle BOD5-Nutrient Solutions and inoculating water, refer to the instructions for reagent sets BOD5-Nutrient Mixture (REF 918 994) or BOD5-Nutrient Mixture PLUS (REF 918 995). Make sure to observe the data specified there.

Determination of BOD5
Requisite accessories: BOD5-Accessories Set (REF 916 918), BOD5-Nutrient Mixture (REF 918 992) or BOD5-Nutrient Mixture

Step 1: Control (oxygen consumption of the diluting water)
Fill in a 1 L laboratory flask (BOD5-Accessories Set, REF 916 918) with 500 mL aerated diluting water and 2.5 mL nutrient solution (1.25 mL R1 + 1.25 mL R2 from reagent set BOD5-Nutrient Mixture, REF 918 994).

Step 2: Sample Dilutions
Prepare the sample dilution based on the expected BOD5 of the sample. Use a 1 L laboratory flask (BOD5-Accessories Set, REF 916 918). If there is no experience regarding the expected BOD5 of a sample, prepare at least two, preferably three, different dilutions of the sample to assure accuracy of the determination. For more reliable results, duplicate determinations are recommended.

Expected BOD5 [mg/L O2]| Sample [mL]| Aerated Diluting Water [mL]| Nutrient Solution* [mL]
---|---|---|---
< 5| 500| 0| 1.25 R1 + 1.25 R2
5| 250| 250| 1.25 R1 + 1.25 R2
10| 100| 400| 1.25 R1 + 1.25 R2
25| 50| 450| 1.25 R1 + 1.25 R2
50| 25| 475| 1.25 R1 + 1.25 R2
100| 10| 490| 1.25 R1 + 1.25 R2
200| 5| 495| 1.25 R1 + 1.25 R2
400| 2| 398| 1.0 R1 + 1.0 R2
500| 1| 399| 1.0 R1 + 1.0 R2
500| 1| 499| 1.25 R1 + 1.25 R2

After preparation of the sample dilution based on the above table, close the laboratory flask and mix to enrich the oxygen content by shaking vigorously for a few seconds.

FAQ (Frequently Asked Questions)

• Q: What is the method used for determining BOD5?
  A: The method used is the tube test for the determination of the biochemical oxygen demand in 5 days (BOD5) by using the diluting principle according to the German Standard Method DIN 38409-H51.

• Q: How is the oxygen determination carried out?
  A: The oxygen determination is carried out similarly to the Winkler Method DIN EN 25813-G21 by photometric evaluation of iodine-color.

• Q: What are the interferences that can affect the BOD5 results?
  A: Changes in pH-value, accumulation of special microbial metabolites and compounds, iron(II) salts, sulfur dioxide, sulfur hydrogen, algae, and nitrified microorganisms can cause a decrease of substrate metabolism and a reduction of the oxygen consumption. Iron(II) salts, sulfur dioxide, and sulfur hydrogen consume oxygen and falsify the BOD5-results, meaning they cause false negative results, also. If algae or nitrified microorganisms are present, increased results could occur.

Method

Tube test for the determination of the biochemical oxygen demand in 5 days (BOD5) by using the diluting principle according to the German Standard Method DIN 38409-H51. The incubation of the samples is carried out in Winkler oxygen flasks. The deter-mination of oxygen dissolved in water at day 0 and after 5 days is carried out similarly to the Winkler Method DIN EN 25813-G21 by photometric evaluation of iodine-color.

Contents of reagent set:

• 3 empty test tubes
• 15 mL BOD5 R1
• 15 mL BOD5 R2
• 30 mL BOD5 R3

Hazard warning

Reagent R1 contains manganese(II) chloride 25–83 %, reagent R2 contains sodium hydroxide solution 20–55 %, reagent R3 contains sulfuric acid 51–80 %. H314 Causes severe skin burns and eye damage. P260, P280, P301+330+331, P303+361+353, P304+340, P305+351+338 Do not breathe vapors. Wear protective gloves / eye protection.

• IF SWALLOWED: rinse mouth. Do NOT induce vomiting.
• IF ON SKIN (or hair): Remove / Take off immediately all contaminated clothing. Rinse skin with water / shower.
• IF INHALED: Remove to fresh air and keep at rest in a position comfortable for breathing.
• IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. For further information ask for a safety data sheet.

Interferences:
Changes in pH-value, accumulation of special microbial metabolites and compounds, which are toxic to microorganisms (e. g. mycotoxines, free chlorine, heavy metals) can cause a decrease of substrate metabolism and a reduction of the oxygen consumption. Iron(II) salts, sulfur dioxide and sulfur hydrogen consume oxygen and falsify the BOD5-results, meaning they cause false negative results, also. If algae or nitrified microorganisms are present, increased results could occur.

Sample preparation

At the beginning, the sample is adjusted to room temperature. Then the pH- value is checked. The pH-value of the sample should be between pH 6 and 8, and has to be adjusted, if necessary. If, in this case, a precipitate has been developed, the sample should be homogenized very well or filtrated (membrane filtration kit, REF 916 511). In case of samples containing algae, filtration may also be necessary in order to avoid exaggerated results. Remove free and / or bounded chlorine by addition of sodium sulfite.
Remark: Store the sample in a tightly closed bottle full to the brim at a temperature of 0–4 °C immediately after taking the sample until carrying out the analysis. Start the BOD5 determination as soon as possible or within 24 hours of taking the sample. Samples may also be frozen to keep longer. Homogenise frozen samples after thawing and always use inoculated BOD5-Nutrient Solution (see BOD5 Nutrient Mixture, REF 918 994, or BOD5-Nutrient Mixture PLUS, REF 918 995).

Diluting water, BOD5-Nutrient Solutions and inoculating water:
Details on the preparation and handling of diluting water are given in the BOD5-Accessories Set (REF 916 918). For the use and application of BOD5-Nutrient Solutions and inoculating water, refer to the instructions for reagent sets BOD5-Nutrient Mixture (REF 918 994) or BOD5-Nutrient Mixture PLUS (REF 918 995). Make sure to observe the data specified there.

Determination of BOD5

Requisite accessories: BOD5-Accessories Set (REF 916 918), BOD5-Nutrient Mixture (REF 918 992) or BOD5-Nutrient Mixture PLUS (REF 918 995), graduated cylinders (volume 100 mL and 500 mL), piston pipettes with tips, equipment for incubation with thermostat for 20 ± 1 °C (e. g. water bath or incubator) or as an alternative a dark room with a room temperature of about 20 °C.

Step 1: Control (oxygen consumption of the diluting water)

Step 2: Sample dilutions
Depending on the expected BOD5 of a sample, the most suitable dilution in accordance to the following table must be prepared in a 1 L-laboratory flask (BOD5-Accessories Set, REF 916 918). If there is no experience regarding the expected BOD5 of a sample, at least two, preferably three, different dilutions of this sample should be prepared to assure accuracy of the determination. For more reliable results, we recommend duplicate determinations.

Expected BOD 5 [mg/L O2]| Dilution| Examples for typical waters| Sample [mL]| Aerated diluting water [mL]| *Nutrient Solution [mL] R1                  R2**
---|---|---|---|---|---
< 5| –| R| 500| 0| 1.25| 1.25
4–12| 1 + 1| R, B| 250| 250| 1.25| 1.25
10–30| 1 + 4| R, B| 100| 400| 1.25| 1.25
20–60| 1 + 9| B| 50| 450| 1.25| 1.25
40–120| 1 + 19| C| 25| 475| 1.25| 1.25
100–300| 1 + 49| C, M| 10| 490| 1.25| 1.25
200–600| 1 + 99| C, M| 5| 495| 1.25| 1.25
400–1200| 1 + 199| M, I| 2| 398| 1.0| 1.0
800–2400| 1 + 399| I| 1| 399| 1.0| 1.0
1000–3000| 1 + 499| I| 1| 499| 1.25| 1.25

• BSB5-Nutrient Mixture (REF 918 994) or BOD5-Nutrient Mixture PLUS (REF 918 995)

  • R: River water
  • M: Raw municipal sewage
  • B: Biologically suitable biomass from a sewage plant
  • I: Heavily polluted industrial waste water
  • C: Clarified biomass from a sewage plant or mildly polluted industrial waste water

After preparation of the sample dilution based on the above table, close the laboratory flask and mix to enrich the oxygen content by shaking vigorously for a few seconds.
Open

• 1 Winkler oxygen flask and
• 1 test tube, wash both with some milliliters of the sample dilution and fill to the brim without letting air bubbles in.

Close the Winkler oxygen flask, without letting air bubbles in, by slowly pressing in the obliquely cut glass stopper and incubate in a water bath or in an incubator for 5 days at 20 ± 1 °C in the dark. Close the test tube without letting air bubbles in and immediately start the measurement of dissolved oxygen according to step 3.

Proceed in the same way for all other samples or sample dilutions.
Remark: The added laboratory flask in the BOD5-Accessories Set can be used for all preparations of any water samples to be tested (control, sample dilutions). Before using, the flask must be washed thoroughly by using tap water, after every preparation and before every new preparation, respectively.

Step 3: Measurement of dissolved oxygen

• Preliminary remarks:
  The added test tubes in the reagent set NANOCOLOR® BOD5 can be used for all measurements of dissolved oxygen. Before using for a new determination of dissolved oxygen the test tube is directly be emptied down the drain and thoroughly washed with tap water. Additional empty test tubes (REF 916 80) can be ordered at MACHEREY-NAGEL.

• Measurement of dissolved oxygen on day 0: The measurement of dissolved oxygen in test tubes filled at the beginning of the test (day 0) has to be started immediately.

• Measurement of dissolved oxygen on day 5: The measurement of the concentration of dissolved oxygen in the incubated Winkler flasks after 5 days of incubation starts with the filling of one empty test tube (for double determinations two empty test tubes) to the brim, with the water sample to be tested (control and sample dilutions). After the filling, the test tubes are carefully closed without letting air bubbles in, and the determination of dissolved oxygen is carried out as in the following chapter “Procedure” described.

Procedure:

Measurement:
For NANOCOLOR® photometers and PF-11 / PF-12 see manual, test 8-22.

Photometers of other manufacturers:
For other photometers check whether measurement of round glass tubes is possible. Verify factor for each type of instrument by measuring standard solutions.

Step 4: Evaluation
Important: The BOD5 value is only calculated for samples in which the residual oxygen concentration after 5 days incubation still amounts to at least 2 mg/L O2 and where the oxygen consumption lies between 2 and 6 mg/L O2.

Analytical Quality Control:
NANOCONTROL BOD5 (REF 925 82)

Table for evaluation

Table for evaluation for BOD5 – Test 8-22

• Sample: ____
• Date: __
• COD [mg/L O2]: _____
• pH-value: __
• Total Phosphate [mg/L P]: __
• Ammonia-N [mg/L NH4-N]: _____
• Nitrite-N [mg/L NO2-N]: ____
• Nitrate-N [mg/L NO3-N]: ___

Results of the determination of BOD5:

Results of the determination of BOD 5:

Date of test beginning (day 0):  Date of test end (day 5):
Control:| O C0 [mg/L O 2]| O C5 [mg/L O 2]| O C [mg/L O2] **= (O C0 – OC5)**|
 |  |

Sample dilutions:| Dilution D| O S0 [mg/L O 2]| O S5 [mg/L O 2]| O S [mg/L O2] **= (O S0 – OS5)| BOD 5 [mg/L O2] **= [D x (O S – OC) + OC]
 |  |  |  |
 |  |  |  |
 |  |  |  |
 |  |  |  |
 |  |  |  |
 |  |  |  |

 | BOD 5 [mg/L O2]: **__**
OC = Oxygen consumption of the control after an incubation period of 5 days OS = Oxygen consumption of the sample dilution after an incubation period of 5 days OC0 = Oxygen concentration in the control at the beginning of the test (day 0) OS0 = Oxygen concentration in the sample dilution at the beginning of the test (day 0) OC5 = Oxygen concentration in the control at the end of the test (day 5) OS5 = Oxygen concentration in the sample dilution at the end of the test (day 5)

D  = Reciprocal value of the sample dilution (e. g. sample dilution 1+199 –> V = 200)

MACHEREY-NAGEL GmbH & Co. KG · Neumann-Neander-Str. 6–8 · 52355 Düren · Germany
Tel.: +49 24 21 969-0
Fax: +49 24 21 969-199
info@mn-net.com
www.mn-net.com

References

• MACHEREY-NAGEL Webshop | MACHEREY-NAGEL

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