the use of the dilution method for determining the effect of industrial wastes on deoxygenation

The Use of the Dilution Method for Determining the Effect of Industrial Wastes onDeoxygenationAuthor(s): H. HeukelekianSource: Sewage Works Journal, Vol. 19, No. 4 (Jul., 1947), pp. 612-620Published by: Water Environment FederationStable URL: http://www.jstor.org/stable/25030506 .

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THE USE OF THE DILUTION METHOD FOR DE TERMINING THE EFFECT OF INDUSTRIAL

WASTES ON DEOXYGENATION *

By H. Heukelekian

Associate Research Specialist, Rutgers University, New Brunswick, N. J.

Certain industrial wastes give a de

creasing B.O.D. with increasing dilu

tion. This has been referred to as the

"sliding scale/' and invalidates the

B.O.D. determination. In some cases

the difficulty from this source can be overcome by the use of a dilution wa

ter containing nitrogen and phosphor us. In other cases, however, this phe nomenon is due not to a deficiency of

nitrogen and phosphorous in the waste, but to the presence of certain toxic

agents which exert an inhibiting effect on the deoxygenation of the waste at

the higher concentrations. The concentrations employed in the

B.O.D. determination are determined

by the B.O.D. of the waste. If the

waste contains large quantities of oxi

dizable material in addition to some

specific toxic ingredient, higher dilu

tions will have to be employed, which

may or may not overcome the inhibi

tion, depending upon the concentra

tion and the potency of the toxic ma

terial. If the material has a low

toxicity or is present in relatively low

concentration, or if the oxidizable por tion of the waste is relatively high, the dilution employed may completely ob

literate the inhibition of the oxidation, and the B.O.D. will not manifest a de

creasing value with increasing concen

tration. In such a case, although the

B.O.D. values obtained from different

dilutions will agree with one another, there is no guarantee that the same

B.O.D. will be exerted in treatment

processes or in the stream when such

high dilutions are not available.

* Paper of the Journal Series, New Jersey

Agricultural Experiment Station, Eutgers

University, Department of Sanitation.

To investigate the deoxygenation rates of such wastes at lower dilutions, the B.O.D. method may be modified as follows: (a) by using pure oxygen to saturate the dilution water or (6) by pooling and reaerating the diluted

samples of a given concentration of the waste during the incubation period. Even with these modifications, the

range of concentration of the waste cannot be materially increased. The direct method of determining oxygen utilization described elsewhere (1) ob viates all these difficulties and makes the use of any concentration of the

waste possible. When the concentration of the oxi

dizable material is low, and the con centration and potency of the toxic

ingredient are high, then over the rela

tively low range of dilutions employed in the regular B.O.D. technique a de

cidedly lower B.O.D. will be obtained at the higher end of the concentrations.

Although such a determination is of little value in regard to B.O.D., it has a definite diagnostic value in indicat

ing the presence of toxic materials and the dilution necessary to overcome the

toxicity. The purpose of this paper is to draw

attention to the possibilities and limi tations in determining the B.O.D. of industrial wastes by the dilution

method. The effect was studied of the addition of certain inorganic and or

ganic materials, which may be present in industrial wastes, on the deoxygena tion of sewage.

Method

Sewage was used as substrate in

studying the effects of artificial addi 612



Vol. 19, No. 4 EFFECT OF INDUSTEIAL WASTES ON DEOXYGENATION 613

tions of chemicals. Various concentra

tions of the chemical were added to

sewage and various dilutions made for

B.O.D. determination, or a definite

volume of sewage was added to the

B.O.D. bottles together with different

concentrations of the chemical. In the

first method both the concentration of

sewage and that of chemical were

variable. In the second method the

volume of sewage was such as to bring

about appreciable, but not complete,

depletion of the oxygen.

Effect of Chemicals on the Dissolved

Oxygen Determination

It was necessary to test first the effect of the chemicals for a possible interference with the D.O. determina tion. This was accomplished by adding the various chemicals to the dilution

water in different concentrations and

TABLE I.?Effect of Various Chemicals on the D.O. Determination

Chemical Cone, (p.p.m.)

Winkler Method

Blank (p.p.m.)

With Chemical (p.p.m.)

Rldeal-Stewart Modification

Blank (p.p.m.)

With Chemical (p.p.m.)

Ferric chloride.

Alum.

Ethylene dichloride...

Gasolene.

Gasolene.

Kerosene.

Kerosene.

Ether.

Methyl alcohol.

Ethyl alcohol.

Butyl alcohol,..

Amyl alcohol.

Chloroform.

Carbon tetrachloride..

Toluene.

Xylene. Benzene.

Formaldehyde.

Formaldehyde. Acetone.

Acetone.

Sodium arsenite.

Sodium arsenite.

Sodium arsenite.

Sodium arsenite.

Potassium cyanide Potassium cyanide Potassium cyanide.... Sodium arsenite.

Potassium dichromate.

Potassium chromate ..

Potassium chromate .'.

Potassium chromate ..

Mercuric bichloride. . .

Cobalt chloride.

Copper sulfate.

Phenol.

Phenol.. Phenol.

Strychnine. Brucine.

200 200

1,000 1,000

100 1,000

100 500

1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000 1,000

100 1,000

100 50 33 17 8

50 33 17

100 100 100 25 10

100 100 100 100 50 10

100 100

8.35

8.35

8.30

8.3

8.3

8.6

8.6

8.4

8.4

8.4

8.4

8.4

8.4

8.4

8.5

8.5

8.4

8.1

8.1

8.1

8.1

8.2

8.2

8.2

8.2

8.2

8.2

8.2

8.5

8.5

8.5

8.5

8.5

8.4

8.4

8.4

8.35

8.35

8.35

8.5

8.5

8.35

8.35

8.20

5.8

7.9

7.8

8.6

8.4

8.4.

8.4

8.5

8.4

8.4

8.4

8.4

8.4

8.4

7.4

8.1

7.5

8.3

2.6

4.7

6.5

7.6

8.0

8.5

8.5

8.5

18.0

15.4

10.5

9.2

8.3

8.4

8.5

6.95

7.45

8.05

7.5

8.2

8.2

8.2

8.2

8.2

8.2

8.2

8.2

8.5

8.0

8.1

8.2

8.3

8.1

8.2

8.4

18.0



614 SEWAGE WORKS JOURNAL July, 1947

determining immediately the dissolved

oxygen. In the majority of cases, only the Winkler method was used. When there was an interference the Rideal Stewart modification was also used in order to eliminate the interference if

possible. The results were compared with the D.O. of the dilution water without the chemical. In Table I, the results of these determinations are as

sembled. Serious interference was en

countered with gasolene, phenol, so

dium arsenite, potassium dichromate, potassium chromate, and strychnine.

The magnitude of the interference varied with the concentration of the chemical.

The interference from sodium arsen

ite could be overcome by the Rideal Stewart modification, provided suffi cient permangenate was added. In

several instances difficulty was encoun

tered in the titration stage with sodi um thiosulfate, due to the recurrence

of the blue color after starch addition. On the basis of the above results

those chemicals that showed little in terference or in which the interference could be overcome by suitable modul ation were selected for the study of the effect on deoxygenation. Phenol and gasolene, which showed an initial

depression of the dissolved oxygen

level, were studied by using the initial blank containing the various concen

trations of the chemicals for compari son with the results after incubation.

Only some of the suggestive results are

incorporated in this paper for the pur pose of illustration.

Inorganic Poisons

The effects of a number of inorganic poisons were studied. Various concen

trations from 100 to 0 p.p.m. were

added to B.O.D. bottles, each contain

ing 20 c.c. of sewage and diluted with sodium bicarbonate water. Dissolved

oxygen determinations were made ini

tially and at the end of 5 days. The results are plotted in Figure 1

as parts per million 02 utilized in 5

? 3 ?J N

?M O

er o:

0 2 4 3 10 20 30 40 50 60 70 80 90 K>0

R P.M.CHEMICAL ADDED

FIGURE 1.?The effect of various concentrations of chemicals on the 5-day oxygen utilization of sewage as determined by the dilution method.



Vol. 19, No. 4 EFFECT OF INDUSTRIAL WASTES ON DEOXYGENATION 615

days against the various concentrations of the chemicals. The results are to be compared with the oxygen utilized

by the sewage without the chemicals. Sodium arsenate up to 100 p.p.m. had

only slight effect on oxygen utilization. Potassium cyanide, on the other hand, depressed the oxygen utilization

sharply even with extremely low con

centration; for instance, 5 p.p.m. pro

duced 38 per cent inhibition, and 50

p.p.m. resulted in 80 per cent in hibition. Oxygen utilization was neg ligible with 100 p.p.m. of potassium cyanide. Cobalt chloride gave results similar to potassium cyanide except that the inhibition was not quite so

pronounced. Copper sulfate caused a very sharp depressing effect even at low concentrations, amounting to 80

per cent reduction in oxygen utiliza tion with 10 p.p.m. These results were

obtained despite the precipitation of

copper by the bicarbonate in the dilu tion water. The effect of mercuric bichloride was the most striking. Oxy gen utilization with 1 p.p.m. was re duced by 90 per cent and with 3 p.p.m. it was completely inhibited.

The effect of sodium arsenite was

studied by adding 500 p.p.m. to sew

age and introducing various volumes of the treated sewage into B.O.D. bot tles and diluting it with seeded mineral dilution water. D.O. values at the be

ginning and after 5 days as determined

by the Rideal-Stewart modification are

given in Table II. The concentrations of the arsenite refer to the ultimate concentration after dilution in the

TABLE IL?Effect of Sodium Arsenite on the Oxygen Utilization Rate of Sewage*

Cone, (p.p.m.)

50

33

17

D.O. Initial (p.p.m.)

8.0

8.1

8.2

8.3

8.3

D.O. After 5 Days (p.p.m.)

2.4

4.2

5.7

6.9

7.35

B.O.D. (p.p.m.)

56

58

75

84

114

* Incubation period 5 days.

B.O.D. bottles. The B.O.D. values are calculated from the respective deple tions and concentrations of sewage. A 50 per cent lower B.O.D. value was obtained with 50 p.p.m. of sodium arsenite than with 4.0 p.p.m. The

B.O.D. values of the sewage increased with decreasing concentrations of the chemical.

Phenol

Phenol concentrations above 2 p.p.m. completely depleted the dissolved oxy gen in 5 days, and even with, daily poolings and reaerations of the samples

more than 5 p.p.m. phenol depleted the dissolved oxygen in 24 hours, so that the effect of higher concentrations of phenol could not be studied by this

method. Results obtained by the di rect method of oxygen utilization have been reported elsewhere (1). It was found by the latter method that phenol even up to 1,000 p.p.m. concentration had an ultimate oxygen demand, al

though the rate of utilization was re tarded initially.

Gasolene, Kerosene, Alcohols

Gasolene, kerosene, ethyl, methyl, butyl and amyl alcohols above 0.1 c.c.

per liter in concentration depleted com

pletely the dissolved oxygen in 5 days. The results obtained with 0.05 c.c. per liter or less are given in Table III.

Equal quantities of sewage were dis tributed into a number of bottles. Va rious quantities of the above organic

materials were introduced and diluted with sodium bicarbonate dilution wa ter. The immiscible materials were emulsified with a hand emulsifier prior to addition. The depletions are ex

pressed, first, on the basis of mixture of sewage and chemical; then by sub

tracting the demand exerted by the

sewage, the depletions and B.O.D.'s of the chemical are derived. The assump tion was made that the oxygen demand exerted by the sewage in the presence of the chemical was the same as by the

sewage alone. That this assumption



616 SEWAGE WORKS JOUENAL July, 1947

TABLE III.?Effect of Certain Organic Materials on Oxygen Demand of Sewage*

Concen tration

(c.c./liter)

Chemical and

Sewage Deple

tion (p.p.m.)

Chemical Alone Deple

tion (p.p.m.)

0.05

0.025

0.020

0.015

0.010

0.005

0.0025

0

6.5

4.3

3.8

3.2

2.9

2.6

2.4

2.2

4.3

2.1

1.6

1.0

0.7

0.4

0.2

0.015

0.010

0.005

0.0025 0.001

0

8.1 + 7.0

5.0

3.6

2.5

2.2

4.8

2.8

1,4 0.3

0.010

0.005

0.0025

0.001

0

8.2+ 7.5

5.1

3.5

2.4

5.1

2.7

1.1

0.010

0.005

0.0025

0.001

0

8.2+ 6.0

4.4

3.0

2.4

3.6

2.0

0.6

0.005

0.0025

0.001

0

8.2+ 6.4

3.7

2.4

4.0

1.3

0.005

0.0025 0.001

0

8.2+ 5.1

3.1

2.1

3.0

1.0

* Incubation period 5 days.

was justified is shown by the good agreement obtained for the B.O.D. values of the chemicals with different concentrations.

The results indicate that gasolene at all the concentrations employed in creased the oxygen demand of the sew

age. The B.O.D. of the gasolene varied from 66 to 86 mg. per c.c. with an

average of 78. There is no indication of decreasing B.O.D. values with in

creasing concentrations of gasoline, which means that within this range gasolene did not exert an inhibiting action.

Kerosene, when added at a concen

tration of 0.015 c.c. per liter, com

pletely depleted the dissolved oxygen content of the mixture, indicating that it exerts a higher oxygen demand than

gasolene. The B.O.D. values of kero

sene varied from 480 to 560 mg. per c.c, with an average of 533 mg. per c.c.

The oxygen demand of kerosene is

nearly 7 times greater than gasolene, but it falls in the same category in that it has no deleterious effect on oxygen utilization within this narrow range of concentrations.

Similarly, ethyl alcohol has an even

higher oxygen demand, and no retard

ing effect on oxygen utilization within the narrow range of concentrations in

which dissolved oxygen could be main tained. The oxygen demand of methyl alcohol is somewhat lower than that of ethyl alcohol, while that of butyl alcohol is higher. Amyl alcohol has an

average oxygen demand of 1,100 mg.

per c.c. These materials, therefore, fall

into a group varying in their oxygen demand but having no effect on oxida tion rates within the narrow range of

concentrations which could be employed in the dilution method of B.O.D. de

terminations. Their effect at higher concentrations cannot be determined

by this method.

Other Organic Substances

The effects of another group of chem

icals, namely, carbon tetrachloride, to

luene, xylene, benzene, chloroform,

ethylene chloride, acetone, formalde

hyde, and ether, are given in Table IV

and differ greatly from these grouped



Vol. 19, No. 4 EFFECT OF INDUSTEIAL WASTES ON DEOXYGENATION 617

TABLE IV.?Effect of Various Chemicals on Oxygen Depletions (P.P.M. in 5 Days)

Cone, (c.c./liter)

Carbon Tetra chloride

Toluene Xylene Benzene Chloro form Ether Acetone Formalde

hyde Ethylene Chloride

1.0

0.5

0.25

0.1

0.05

0.025

0.01

0.005

0.0025

0.001

0

0.5

0.7

0.7

1.3

1.6

1.9

1.9

1.9

1.9

2.0

2.1

0.6

0.7

8.4+

8.4+

8.4+

8.4+

8.4+

2.1

0.6

0.6

8.4+

8,4+ 8.4+

8.4+

8.4+

2.1

1.4

8.4+

8.4+

8.4+

8.4+

8.4+

8.4+

2.1

8.4+ 6.1

4.4

2.9

2.6

2.7

2.1

2.2

2.1

2.1

2.1

8.4+

8.4+

8.4+ 5.1

3.6

2.9

2.4

2.3

2.2

2.5

2.1

8.2+

8.2+

5.9

6.1

5.5

5.2

2.1

5.2

3.9

2.9

2.3

2.1

4.0

3.1

2.5

2.2

2.3

2.0

2.2

in Table III. The concentrations em

ployed are much higher than in the

former group and the effects are much

less pronounced. With various concen

trations of carbon tetrachloride the

oxygen utilization was lower than by

sewage alone, indicating that this ma

terial exerts no oxygen demand of its

own but instead inhibits the oxidation

of sewage. Toluene, xylene, and ben

zene fall into a group in which the

addition of 1 c.c. per liter showed some oxygen utilization which was

lower than the control, but with con

centrations as low as 0.0025 c.c per liter all the oxygen was depleted, show

ing an oxygen demand greater than

the dissolved oxygen content of the di

lution water. Chloroform depleted

completely the dissolved oxygen" when

added in concentration of 1 c.c. per

liter, and ether likewise depleted the

oxygen at 0.25 c.c. per liter concentra

tion.- With lower concentrations the

oxygen depletions decreased but were

not lower than the control, indicating that little inhibiting action was ex

erted. Acetone also increased the oxy

gen demand of sewage. Formaldehyde, in the low concentrations employed, in

creased the oxygen utilization.

Carbon tetrachloride, therefore, does

not have a B.O.D., since the oxygen

demand exerted in its presence is

lower than the oxygen demand of sew

age. The B.O.D. of toluene, xylene and

benzene cannot be calculated from the

results at hand since in the concentra

tions employed these substances de

pleted the dissolved oxygen completely. The B.O.D. of the rest of the materials can be calculated by assuming that the

oxygen demand of the sewage was not

affected by the presence of the chemi

cals.

The results given in Table V indi cate that chloroform and ether have a

small oxygen demand. Within the

range of concentrations used, the B.O.D. 's do not exhibit decreasing values and, therefore, have not ex

erted an inhibiting influence. The oxy gen demand of these materials is lower than those given in Table III.

Acetone exhibits definitely increas

ing B.O.D. values with lower concen

trations. Formaldehyde, at the concen

trations used, had an appreciable and

TABLE V.?Calculated 5-Day B.O.D.

Values of Materials

Cone. (c.c./ liter)

1.0

0.5

0.25

0.1

0.05

0.025

0.01

0.005

0.0025

0.001

Chloro form (mg./ c.c.)

8.0

9.2

8.0

10.0

Ether (mg./ c.c.)

30

30

32

30

Acetone (mg./c.c.)

38

80

136 310

Formal dehyde (mg./c.c.)

310 360 320

Ethylene dichloride (mg./c.c.)

1.8

1.8

2.0




constant B.O.D. Ethylene chloride had an extremely small B.O.D. value.

Discussion

The results indicate the limitations and possibilities in the use of the di lution method of B.O.D. for assaying the toxic properties of industrial

wastes. With inorganic inhibiting agents, which have no oxygen demand of their own, it is a simple matter to use a suitable well-seeded organic sub

strate, to add various concentrations of the toxic chemical, to dilute with

well oxygenated mineral dilution wa

ter, and to determine the oxygen utili zation over suitable periods of time.

Domestic sewage serves as a good

substrate for such a study. It is only necessary that the volume used should

bring about sufficient depletion of oxy gen. If too much sewage is used, the

oxygen will be completely depleted, with the result that the effect of the chemical agent added cannot be accu

rately estimated. If the volume of

sewage is too small, then the effect of chemicals on oxygen depletions cannot be evaluated with precision on account of the narrow range of depletions.

The chemical should be added after the sewage is diluted, in order to pre vent a temporary high concentration with its inhibiting influence. Further

more, the chemical itself should not

interfere with the D.O. determination. The effects of various concentrations

of the chemical on oxygen utilization rates over a period of at least twenty days could be studied, but this would be very laborious. However, if a fixed

volume of sewage were used to cover

the entire period of study, it would cause too little depletions and result in

less accuracy for the shorter periods. Changing the volume of sewage accord

ing to the length of incubation would

introduce objectionable variables.

The results presented with such in

organic poisons for a 5-day period of

incubation are suggestive of the possi

bilities of the use of this method for evaluation of the toxic concentrations. The possibility of adaptation by se

lection and cultivation of a specialized flora tolerating the initial toxic concen trations cannot be readily studied.

Organic wastes with an oxygen de mand at low concentrations and an in

hibiting effect at higher concentrations offer additional difficulties, and pre clude the possibility of studying such effects over a wide range of concentra tions. By pooling and reaerating the

B.O.D. bottles during the incubation

period, before complete depletion, the

range of concentrations may be some

what extended. Caution should be ex

ercised when dealing with solvents by this technique, as losses would occur

during reaeration, causing an error.

Dilution water saturated with oxygen gas may also serve this purpose.

Over the narrow range of concentra

tions that could be employed by this

method, some interesting observations were made. There were only a few

organic materials which did not exert an oxygen demand. Even materials

which are commonly employed for in

hibition of microbial activity had some

oxygen demand. This emphasizes the

concept that toxicity is relative and is a function of concentration. A ma

terial may be toxic at higher concen

trations and oxidizable at lower con

centrations. The method lends itself

only to the lower and oxidizable con

centrations and is not applicable to the

higher and toxic range. The only substance in this study

which did not exert any oxygen de

mand was carbon tetrachloride. The

ability of microorganisms to utilize hy drocarbons has been studied by many

investigators. Zobell (2) has reviewed

the available literature.

On the basis of their effects on oxy

gen utilization within the range of

concentration employed the organic ma

terials tested may be grouped under

the following categories:



Vol. 19, No. 4 EFFECT OF INDUSTRIAL WASTES ON DEOXYGENATION 619

(1) Those with a high oxygen de

mand, but no apparent toxic effect; examples: kerosene, ethyl alcohol,

methyl alcohol, butyl alcohol, and amyl alcohol. The lack of toxicity is sug gested by the absence of the decreasing B.O.D. values with increasing concen

trations.

(2) Those having an oxygen demand at low concentrations and a toxic effect at higher concentrations; examples: acetone, toluene, xylene, benzene.

(3) Those without any oxygen de mand but with a toxic effect ; example :

carbon tetrachloride.

(4) Those having a slight oxygen demand and low toxicity; examples: chloroform, ether, ethylene chloride.

(5) Those with low oxygen demand, but no toxicity; example: gasolene.

It should be emphasized that the

grouping of the chemicals, in these cate

gories applies only within the range of concentrations which the method

permitted, and may be subject to

change with another method in which

higher concentrations can be studied. The possibility of increasing the

B.O.D. of sewage by using preservatives such as chloroform and formaldehyde is indicated.

Mixed organic wastes of unknown character have been studied by this

method, but no reliable results over a

wide enough range of concentrations could be obtained. Despite attempts to reaerate the oxygen at times became

completely depleted, thus invalidating the results.

The many difficulties in obtaining reliable results on B.O.D. values of some industrial wastes are recognized. The difficulties can be made use of in

detecting the presence of inhibiting agents, determining the dilution neces

sary to overcome the inhibition, and in

preparing the material for suitable bio

logical treatment. When such dilution is reached and used in the B.O.D. de

termination, the results should be

fairly constant. The range which pro

duces decreasing B.O.D. values would indicate the inhibiting zone, and the lower concentrations with constant

B.O.D. values would indicate the true

oxygen demand. This is because, with out such dilution, biological treatment is not practical, and in a majority of cases a dilution high enough to permit biological action is obtained in the re

ceiving stream. When the wastes con

sist of inorganic materials alone, then the B.O.D. determination is needless, but the B.O.D. technique can be used to assay the effect of such wastes when

they become mixed with oxidizable or

ganic materials.

Summary and Conclusions

The effect of inorganic substances on the oxygen utilization can be studied

readily by introducing various concen trations of the compounds into a suit able volume of sewage after dilution.

Toxicity is indicated by decreasing oxy gen utilization with increasing concen trations. The study of the effect of

organic substances is subject to the limitation imposed by the low concen trations that can be employed by the dilution method, since most of the or

ganic compounds have an oxygen de mand. Even materials used as preser

vatives are oxidized at low enough concentrations.

The B.O.D. of these organic ma terials can be determined by subtract

ing the oxygen demand of the sewage from the total utilized. However, the B.O.D. thus determined applies only to the concentrations employed. If it

were possible to use higher concentra

tions, a different value might be ob tained. Many organic substances are

oxidizable at low concentrations and toxic at high concentrations; that is, they have an oxygen demand at low concentrations and not only remain un oxidized at higher concentrations, but also retard the oxidation of an other

wise oxidizable substrate, such as sew

age. Thus within only a narrow range can these effects be studied by the di




lution method. The same difficulties are encountered when an attempt is

made to appraise the B.O.D. of mixed unknown wastes containing both an

oxidizable organic material and an in

hibiting agent. By the direct method, the oxygen utilization over any range of concentrations can be studied.

References

1. Heukelekian, H., to be published. 2. Zobell, C. E., "Action of Microorganisms

on Hydrocarbons. ' ' Bad. Eeviews, 10,

1 (1946).



the use of the dilution method for determining the effect of industrial wastes on deoxygenation

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