Part 20

Procedure.—Add 0.4 cc. of the methylene blue solution to the sample in the 150 cc. bottle. As methylene blue has a slightly antiseptic property be careful to add exactly 0.4 cc. Add the methylene blue solution preferably below the surface of the liquid after filling the bottle with the sample. If the methylene blue is added first do not allow the liquid to overflow as coloring matter will thus be lost. Incubate the sample at 20° C. for ten days. Four days’ incubation may be considered sufficient for all practical purposes in routine plant-control work. If quick results are desired incubate the sample at 37° C. for five days using suitable stoppers to prevent the loss and reabsorption of dissolved oxygen. The bacterial flora at 37° C. is different from that at 20° C. The lower temperature is more nearly the average temperature of surface waters and therefore the higher temperature should be used only when quick approximate results are essential. Observe the sample at least twice a day during incubation. Give a sample in which the methylene blue becomes decolorized a relative stability corresponding to the time required for reduction (see Table 15). For routine filter control ordinary room or cellar temperature gives fairly satisfactory results. For accurate studies, room temperature incubation is very undesirable, as the fluctuations in temperature which are ordinarily not noticed are responsible for appreciable deviations from the true values of relative stability. If the samples are incubated less than 10 days at 20° C. and are not decolorized place a plus sign after the stability value in order to indicate that the stability might have been higher if more time had been allowed. In applying this test to river waters it often happens that the blue coloring matter is removed either partly or completely through absorption by the clay which many rivers carry in suspension. True relative stabilities cannot be obtained for such waters except by determining the initial available oxygen at the start and the biochemical oxygen demand on incubation at 20° C. for 10 days (pp. 71–73). Germicides, such as hypochlorite of lime, if present in sufficient quantity, vitiate the results. If a sample contains free chlorine, therefore, store it about 2 hours, or until the chlorine is gone, and then add methylene blue.

Table 15 gives the relation between the time in days to decolorize methylene blue at 20° C. (t_{20}) and the relative stability number or ratio of available oxygen to oxygen required for equilibrium, expressed in percentage (S).

Table 15.—RELATIVE STABILITY NUMBERS.

───────────────────────────────────┬─────────────────────────────────── Time required for decolorization at│ Relative stability. 20° C. │ ───────────────────────────────────┼─────────────────────────────────── Days. │ Percentage. 0.5│ 11 1.0│ 21 1.5│ 30 2.0│ 37 2.5│ 44 3.0│ 50 4.0│ 60 5.0│ 68 6.0│ 75 7.0│ 80 8.0│ 84 9.0│ 87 10.0│ 90 11.0│ 92 12.0│ 94 13.0│ 95 14.0│ 96 16.0│ 97 18.0│ 98 20.0│ 99 ───────────────────────────────────┴───────────────────────────────────

The theoretical relation is, S = 100 (1 − 0.794t_{20})

The relation between the time of reduction at 20° C. and that at 37° C. is approximately two to one, but if an observer incubates at 37° C. he should work out his own comparative 37° C. table or factor.

A relative stability of 75 signifies that the sample examined contains a supply of available oxygen equal to 75 per cent of the amount of oxygen which it requires in order to become perfectly stable. The available oxygen is approximately equivalent to the dissolved oxygen plus the available oxygen of nitrate and nitrite. Nitrite in sewage is usually so low as to be negligible.

BIOCHEMICAL OXYGEN DEMAND OF SEWAGE AND EFFLUENTS.

RELATIVE STABILITY METHOD.

The relative stability method may be employed to obtain a measure of the putrescible material in sewages and effluents in terms of oxygen demand.

Procedure for effluents.—Divide the total available oxygen, including the oxygen of nitrite and nitrate, by the relative stability expressed as a decimal.

Procedure for sewages.—Make one or two dilutions with fully aerated distilled water of known dissolved oxygen content. Tap water may be employed if it is free from nitrates. Vary the relative proportions of sewage and water to be employed to give a relative stability of 50 to 75. Unless seals are used bring the water as well as the sewage to the temperature at which the mixtures are to be incubated before preparing the dilutions. During the manipulation avoid aeration. Having made the proper dilutions, determine the relative stability of each.

Calculate the oxygen demand in parts per million by the formula:

Oxygen demand = O(1 − p)/Rp

In this formula, O is the initial dissolved oxygen of the diluting water, p is the proportion of sewage; and R is the relative stability of the mixture. Ordinarily the available oxygen in crude sewages, septic tank effluents, settling tank effluents, and trade wastes can be neglected.

SODIUM NITRATE METHOD.

For the determination of the biochemical oxygen demand the sodium nitrate method may be used. The method is based on the biochemical consumption of oxygen from sodium nitrate by a sewage or polluted water during an incubation period of ten days at 20° C. A reasonable excess of sodium nitrate does not give a higher oxygen demand, as do higher dilutions with aerated water. The oxygen absorbed from the air in applying the method to sewages is negligible.

Reagent.—Sodium nitrate solution. Dissolve 26.56 grams of pure sodium nitrate in 1 liter of distilled water. One cc. of this solution in 250 cc. of sewage represents 50 parts per million of available oxygen. The strength of the sodium nitrate solution may be varied to suit conditions.

Procedure for sewages.—Ordinarily disregard the initial available oxygen as it is very small compared with the total biochemical oxygen demand. Add measured amounts of the sodium nitrate solution to the sewage in bottles holding approximately 250 cc. which have been completely filled and stoppered. Incubate for 10 days at 20° C. A seal is not required during incubation. The appearance of a black sediment and the development of a putrid odor during incubation indicates that too little sodium nitrate has been added. Methylene blue solution in proper proportion may be added at the start to serve as an indicator during the incubation. Domestic sewage usually varies in its oxygen demand from 100 to 300 parts per million, approximately 30 per cent of which is used up at 20° C. in the first 24 hours. At the end of the incubation period determine the residual nitrite and nitrate. Determine the nitrate by the aluminium reduction method and direct Nesslerization. To convert the nitrogen into oxygen equivalents, multiply the nitrite nitrogen by 1.7 and the nitrate nitrogen by 2.9. The difference between the available oxygen added as sodium nitrate and that found as nitrite and nitrate at the end of the incubation period is the biochemical oxygen demand.

Procedure for industrial wastes.—Employ the same procedure using larger quantities of the sodium nitrate solution. Make the reaction alkaline to methyl orange and acid to phenolphthalein. Adjust an acid reaction with sodium bicarbonate and a caustic alkaline reaction with weak hydrochloric acid. If the liquid is devoid of sewage bacteria seed it with sewage after adjusting the reaction.

Procedure for polluted river waters.—Determine the initial available oxygen. Unless the river water is badly polluted add 10 parts per million of sodium nitrate oxygen. Collect carefully, avoiding aeration, three samples in 250 cc. bottles. To one sample add a definite quantity of sodium nitrate solution and incubate. Incubate the other two samples for the determination of the residual free oxygen, nitrite, and nitrate. If there is free oxygen left, the bottle containing the sodium nitrate solution may be discarded. If there is no free oxygen determine residual nitrite and nitrate as directed under the procedure for sewage (p. 72) and calculate the oxygen demand.

ANALYSIS OF SEWAGE SLUDGE AND MUD DEPOSITS.

COLLECTION OF SAMPLE.

Collect a representative sample of the material. In general more than one sample should be taken from a spot and a large number of samples should be collected rather than a few large samples. If the surface layer is darker and a lower layer consists of pure clay sample only the surface layer. Samples may be analyzed either separately or as composites of careful mixtures. After the sample has settled a few minutes roughly drain or siphon the excess water. Allow sewage sludge to stand for one hour before draining it free from excess water unless it is essential to determine the moisture content of the sample originally collected. If sludge cannot be analyzed within twenty-four hours it is better not to use air-tight bottles and to add small quantities of chloroform and keep in the ice box to retard decomposition. At the time of collection carefully examine mud from the bottom of surface water for evidence of sewage pollution and macroscopic and microscopic animal and plant organisms. Record the predominant species. Note the physical appearance of the material, particularly its color, odor, and consistency. Express all analytical results in percentage on a dry basis.

REACTION.

Determine the reaction by diluting a definite quantity of the wet sludge and titrating by the methods given under alkalinity and acidity (pp. 35–39 and 39–41).

SPECIFIC GRAVITY.

Weigh to the nearest tenth of a gram a wide-mouthed flask of 100 to 300 cc. capacity, according to the quantity of material available. Then completely fill the flask with distilled water to the brim and weigh it again. Empty the flask and fill it completely with fresh sewage sludge or mud. If the material is of such consistency that it flows readily fill the flask to the brim and weigh. The specific gravity is equal to the weight of the sludge or mud divided by the weight of an equal volume of distilled water.