BOD Incubator

Thermostatically controlled at 20± 1°C. All light must be excluded to prevent photosynthesis during the 5-day incubation period.

Barometers

Electronic Models

The laboratory temperature and barometric pressure must be recorded daily, particularly if the dissolved oxygen (DO) meter is calibrated using the water-saturated air or air-saturated water technique. NIST traceable electronic models are available that will monitor both temperature and barometric pressure are available.

Dial type models

Dial type NIST traceable barometers are satisfactory for monitoring barometric pressure. All barometers (electronic and dial type) should be checked to verify they are functioning properly. The internet or local airport can provide information that maybe used to check laboratory barometers. See the calibration section of this document for information on how to check barometers.

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BOD Bottles

Standard glass, 300 mL BOD bottles

Glass BOD bottles are most commonly used by laboratory since they may be washed and reused. Wash BOD bottles using technique described elsewhere in this document.

Image courtesy Environmental Express

Single use, plastic disposable 300 mL BOD bottles

These bottles are acceptable for use providing they are only used once and all quality control criteria are met.

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How BOD Bottles should be cleaned

Use a good lab-grade non-phosphate detergent and bleach Rinse thoroughly with tap water followed by distilled water Allow to dry before storing. Always cover glassware and store in a clean, dry place.

Alternate Cleaning Method without Bleach

Some folks prefer not to use bleach at all in cleaning their BOD labware, as even traces of bleach can kill "bugs" critical to the BOD test. Use a good laboratory grade non-phosphate detergent Rinse thoroughly with tap water followed dilute hydrochloric acid (HCl) (10% solution; 100 mL hydrochloric acid (HCl) per liter of water). Rinse again w/ tap water followed by distilled water. Allow to dry before storing. Always cover glassware and store in a clean, dry place.

Warning: DO NOT MIX hydrochloric acid (HCl) and bleach: It will produce poisonous chlorine gas!!!!

Dilution Water Containers

• Store dilution water in all glass containers only.
• If a dilution water is siphoned into BOD bottles, only Teflon© or glass should be immersed in the water.
• Use only C-flex© and latex rubber tubing to dispense the dilution water into BOD bottles.
• Bottom draw, aspirator bottles also work well.

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Items needed to saturate BOD dilution water with oxygen

Saturating dilution water with oxygen can be source of contamination in BOD testing. Water can be aerated by lightly covering the opening of the glass storage container with link free tissue, cotton or foam rubber. Avoid using material that will slough-off into the dilution water. If a laboratory uses compressed air to aerate the dilution water, use an in-line filter (as shown) to prevent dust and oil from getting into the water. In-line filters are available from most scientific specialty companies.

As a general rule, NEVER put "fish tank" tubing or air stones directly in contact with dilution water. These items can leach oxygen demanding materials. Air stones can harbor bacteria which--unaccounted for--can cause excess DO depletion and subsequent high bias.

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Tubing types

photo: George Bowman, Wisconsin State Laboratory of Hygiene

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Pipet Types

photo: George Bowman, Wisconsin State Laboratory of Hygiene

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Certified thermometers

A factory (or NIST) certified thermometer must be placed in the incubator to verify the temperature is maintained within the 20± 1°C tolerance each day during the 5-day incubation period (including weekends). These may also be used to demonstrate that the temperature of sample refigerators and autosamplers is maintained at temperatures not to exceed 6°C (and samples cannot be frozen). Total immersion type incubator thermometers or electronic thermometers are satisfactory. NIST traceable minimum/maximum or electronic recording thermometer are adequate for checking the temperature on weekends. Always record the temperature in a logbook or bench record.

A partial immersion thermometer is needed to confirm the samples are in the 20± 3°C range prior to preparing the BOD dilutions

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DO Meter and Probe

Dissolved oxygen probe and meter are critical items needed for the BOD testing. Many brands and styles are available and are satisfactory. The polarographic type is the most common style. Most new newer instruments have a built-in barometers, thermometer and on-board software to simplify the calibration process.

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pH Meter and pH Paper

A pH meter is the preferred way to verifying the sample pH is in 6.5-8.0 range prior to testing for BOD. However, short range pH paper (below) is also satisfactory as well. pH paper may not be used to measure pH for permit purposes.

image from Precision Scientific

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Other Supplies Needed for BOD Testing

• Nutrient buffer solutions – commercially available
• Sulfuric acid [H₂SO₄] solution (1N) for adjusting sample pH – available commercially
• Sodium hydroxide [NaOH] solution (1N) for adjusting sample pH – available commercially
• Glucose-glutamic acid (GGA) solution – available commercially
• Nitrification inhibitor (if carbonaceous BOD is desired or allowed in the facility’s NPDES permit) – available commercially (Hach formula 2533 © or equivalent)
• Standard laboratory glassware
• Bleach for sanitizing BOD bottles, dilution water storage containers and tubing used to dispense dilution water.
• Hydrochloric acid [HCl] solution (10% of concentration) for removing scale from BOD bottles.

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