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freedom from toxic materials, favorable pH and osmotic pressure conditions, optimal amount of nutrients, and the presence of significant amount of population of mixed organisms of soil origin. Through long years of experience, it has been found that synthetic dilution water prepared from distilled water or demineralized water is best for BOD work, because the presence of such toxic substances as chloramine, chlorine, and copper can be easily controlled. The maintenance of favorable pH can be assured by buffering the dilution water at about pH 7.0 using potassium and sodium phosphates. The potassium and sodium ions, along with the addition of calcium and magnesium ions, can also maintain the proper osmotic pressure, as well as provide the necessary nutrients in terms of these elements. The phosphates, of course, provide the necessary phosphorus nutrient requirement. Ferric chloride, magnesium sulfate, and ammonium chloride supply the requirements for iron, sulfur, and nitrogen, respectively.

A sample submitted for analysis may not contain any organism at all. Such is the case, for example, of an industrial waste, which can be completely sterile. For this situation, the dilution water must be seeded with organisms from an appropriate source. In domestic wastewaters, all the organisms needed are already there; consequently, these wastewaters can serve as good sources of seed organisms. Experience has shown that a seed volume of 2.0 mL per liter of dilution water is all that is needed.

Laboratory calculation of BOD. In the subsequent development, the formulation will be based on the assumption that the dilution method is used. If, in fact, the method used is direct, that is, no dilution, then the dilution factor that appears in the formulation will simply be ignored and equated to 1.

The technique for determining the BOD of a sample is to find the difference in dissolved oxygen (DO) concentration between the final and the initial time after a period of incubation at some controlled temperature. This difference, converted to mass of oxygen per unit volume of sample (such as mg/L) is the BOD.

Let I be the initial DO of the sample, which has been diluted with seeded dilution water, and F be the final DO of the same sample after the incubation period. The difference would then represent a BOD, but since the sample is seeded, a correction must be made for the BOD of the seed. This requires running a blank.

Let I represent the initial DO of a volume Y of the blank composed of only the seeded dilution water; also, let F be the final DO after incubating this blank at the same time and temperature as the sample. If X is the volume of the seeded dilution water mixed with the sample, the DO correction would be (I - F)(X/Y). Letting D be the fractional dilution, the BOD of the sample is simply

In this equation, if the incubation period is five days, the BOD is called the five-day biochemical oxygen demand, BOD5. It is understood that unless it is specified, BOD5 is a BOD measured at the standard temperature of incubation of 20°C. If incubation is done for a long period of time such as 20 to 30 days, it is assumed that all the BOD has been exerted. The BOD under this situation is the ultimate; therefore, it is called ultimate BOD, or BODu.

BODu, in turn, can have two fractions in it: one due to carbon and the other due to nitrogen. As mentioned before, carbon reacts with oxygen; also, nitrogen in the form of ammonia, reacts with oxygen. If the BOD reaction is allowed to go to completion with the ammonia reaction inhibited, the resulting ultimate BOD is called ultimate carbonaceous BOD or CBOD. Because Nitrosomonas and Nitrobacter, the organisms for the ammonia reaction, cannot compete very well with carbonaceous bacteria (the organisms for the carbon reaction), the reaction during the first few days of incubation up to approximately five or six days is mainly carbonaceous. Thus, BOD5 is mainly carbonaceous. If the reaction is uninhibited, the BOD after five or six days of incubation also contains the nitrogenous BOD. BOD is normally reported in units of mg/L.

Experience has demonstrated that a dissolved oxygen concentration of 0.5 mg/L practically does not cause depletion of BOD. Also, it has been learned that a depletion of less than 2.0 mg/L produces erroneous results. Thus, it is important that in BOD work, the concentration of DO in the incubation bottle should not fall below 0.5 mg/L and that the depletion after the incubation period should not be less than 2.0 mg/L.

Example 2.1 Ten milliliters of sample is pipetted directly into a 300-mL incubation bottle. The initial DO of the diluted sample is 9.0 mg/L and its final DO is 2.0 mg/L. The initial DO of the dilution water is also 9.0 mg/L, and the final DO is 8.0 mg/L. The temperature of incubation is 20°C. If the sample is incubated for five days, what is the BOD5 of the sample?

Solution:

(I - F) - (I - F)( X/ Y) _ (9-2) - (9-8)([ 300-10]/300)

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