2) Dilutions prepared directly in BOD bottles - Using a wide-tip volumetric pipet, add the desired sample volume to individual BOD bottles of known capacity. Add appropriate amounts of seed material either to the individual BOD bottles or to the dilution water. Fill bottles with enough dilution water, seeded if necessary, so that insertion of stopper will displace all air, leaving no bubbles. For dilutions greater than 1:100 make a primary dilution in a graduated cylinder before making final dilution in the bottle. When using titrimetric iodometric methods for DO measurement, prepare two bottles at each dilution. Determine initial DO on one bottle. Stopper second bottle tightly, water-seal, and incubate for 5 d at 20°C. If the membrane electrode method is used for DO measurement, prepare only one BOD bottle for each dilution. Determine initial DO on this bottle and replace any displaced contents with dilution water to fill the bottle. Stopper tightly, water-seal, and incubate for 5 d at 20°C. Rinse DO electrode between determinations to prevent cross-contamination of samples.
Use the azide modification of the iodometric method (Section 4500-O.C) or the membrane electrode method (Section 4500-O.G) to determine initial DO on all sample dilutions, dilution water blanks, and where appropriate, seed controls.
If the membrane electrode method is used, the azide modification of the iodometric method (Method 4500-O.C) is recommended for calibrating the DO probe.
g. Determination of initial DO: If the sample contains materials that react rapidly with DO, determine initial DO immediately after filling BOD bottle with diluted sample. If rapid initial DO uptake is insignificant, the time period between preparing dilution and measuring initial DO is not critical but should not exceed 30 min.
h. Dilution water blank: Use a dilution water blank as a rough check on quality of unseeded dilution water and cleanliness of incubation bottles. Together with each batch of samples incubate a bottle of unseeded dilution water. Determine