I K D U S T R I AI,
460
A N D E N G I N E E K I N G C H E >I I S T R Y
LITERATURE CITED (1) Eckman, J . Am. Med. Assoc., 78, 635 (1922). (2) Hess and Unger, J . Biot. Chem.. 35, 479 (1918). (3) MacLeod and Booher, J . Home Econ., 22, 588 (1930). (4) Morgan and Field, J. Biol. Chena., 88, 9 (1930).
Vol. 24, No. 4
( 5 ) Morgan, Field, and Sichols, J . d g r . Research, 42, 35 (1931). (6) Osborne and Mendel, J. Bid. Chem., 42, 465 (1920). RECEIVED January 11, 1932. Presented before the Division of Agricultural and Food Chemistry a t the 82nd Meeting of the American Chemical Society, Buffalo, N. Y., Sugust 31 t o September 4 , 1931.
Preparation and Biochemical Oxygen Demand of Pure Sodium Soaps G . E. SYMONS ASD -\.11. BUSWELL, State W a t e r Survey Division, Urbana, Ill.
R
ECEKTLk-, interest has been revived in the development of a microcombustion or strictly chemical means
of determining the oxygen demand of sewages in order to eliminate the time and labor necessary in the determination of biochemical oxygen demand. There are several papers in the literature covering some phase of this problem (1-7, 9, 10, 14, f9-21). The use of such a rapid chemical method for the determination of the oxygen demand depends on the correlation of the biochemical oxygen demand with the stoichiometric oxygen demand of the substances found in sewage. A previous paper (20)from this laboratory reported on the oxygen demand of lactose, starch, cellulose, peptone, urea, and sodium palmitate, and concluded that “the biochemical oxygen demand of carbonaceous substances in 30 days is equivalent to 70 to 85 per cent of the theoretical oxygen demand.. . . . . .and (that) of urea in 20 days is equivalent to the calculated theoretical oxygen demand.” This investigation of soaps was to verify the previous data and to establish, if possible, some definite relation between biochemical and chemical oxygen demand (complete oxidation to carbon dioxide and water) of this type of compound.
of this amount was based on the hydrolysis of the soap to the free fatty acid and the beta oxidation of the acid to carbon dioxide and water. An example of the calculation follows:
Sodium propionate (mol. wt., 96.037) 702 -e- 5C02 5Hz0 NanCO, 192.074 = z ___ _
2CHdJHzCOONa
Use of alcohol, ether, etc., for the recrystallization of the soaps was impossible, owing to formation of gels. The sodium content of the soap (index of purity) was determined by treatment with sulfuric acid, digestion to dryness, ignition, and weighing of the sodium sulfate. The isovalerate and stearate decomposed slightly during drying a t 175’ C., as evidenced by the change in the physical appearance (darkened slightly) and the sodium content. The amount of soap needed for each investigation was that which, when dissolved in 2.3 liters of water, would have a n oxygen demand of 5 mg. of oxygen per liter. The calculation
+
224 5 4.287 mg. sodium propionate per liter to require 5 mg. oxygen for complete oxidation.
The molecular weights used were calculated from the International Atomic Weight Table of 1927. Since these amounts were exceedingly small (48.875 mg., maximum; 4.2879 mg., minimum), weighing error was minimized by weighing ten to twenty times the amount needed and dissolving in 1 or 2 liters of dilution water so that 100 ml. of the solution contained the proper quantity. I n Table I are listed the soaps investigated with the purity and physical constants of each. TABLEI. LIST OF SOAPSINVESTIGATED soap
% Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium Sodium
formate 99.95 acetate 99.95 propionate 1 0 0 . 0 n-butyrate 99.9 isobutyrate 1 0 0 . 0 n-valerate 99.45 isovalerate 9 4 . 5 caproate 99.5 99.0 heptylate 98.2 caprylate 98.0 palmitate
Sodium stearate
90.0
Sodium oleate
94.0
0
MELTINQ POINTC Reptd. Ohsvd.
P U R I T Y ~MELTINQP O I N T b Keptd. Obsvd.
1. Purification of the acids by repeated fractional distillation
or fractional crystallization. 2. Neutralization of the acid with carbonate-free sodium hydroxide solution (0.1 N ) . 3. Extraction of the water solution with petroleum ether t o remove excess acid. 4. Boiling of water solution t o remove esters. 5. Evaporation over steam bath and drying at 175’ C. 6. Determination of melting point and purity (from sodium content).
+
5 =
EXPERIMENTAL PROCEDURE PREPARATIOS OF SOAPS. By definition ( l a ) ,“A soap is the salt of any monobasic aliphatic acid containing six or more carbon atoms.” I n this work, however, the sodium salts of the lower fatty acids are also called “soaps” since they are members of a homologous series and consequently undergo the same type of oxidation as the higher members of the series. The preparation of the pure soaps was carried out in the following steps:
+
c.
c.
c.
...
... ... ...
225 240 225
PZB {;;; 1 220
232
)(;{’!
c.
....
...
... ... .. .
2+4.li+Q‘ 254-256.5 344-345 251-253
212-216 235-239 231-235 219-223
350 350 355 265
Dec. Dec. Dec. Dec.
160-170(?)
270
Dee. 200
235
Dec. 235
]
280 285 275 230
Purity based on sodium content. ~ ~ { ~ $ Observed ~ ~ melting ~ ~ points $ uncorrected. ~ d
F: f:fzt:
]
Though there has been considerable controversy (IS,15-1 7‘) in the past concerning the hydrolysis of soaps in concentrations of normal or greater to give the free fatty acid, there is little doubt that in the concentrations used (maximum 0.0003 N ) , there would be such a, hydrolysis. The beta oxidation is the mechanism generally accepted for aerobic decomposition of the fatty acids. OXYGEN DEMAND.The dilution method of determining oxygen demand was used throughout. The dilution water used was entirely different from any heretofore recommended (9, 18),1 The attempt was made to simulate natural conditions under which wastes discharging into streams would be oxidized. 1 Hatfield and Morkert [Sewape W o r h J . , 2, 521 (1930)l have reoommended a similar water since this work waa completed.
April, 1932
INDUSTRIAL AND ENGISEERING CHEMISTRY
The average mineral content of several rivers of Illinois was determined from data of the Illinois State R a t e r Survey on the analyses of fifteen 10-day composites (11). The following rivers were included in the average: Big Vermilion, Embarass, Fox, Illinois, Kankakee, Kaskaskia, Mississippi, Little Wabash, Sangamon, and Vermilion. Using this as a guide, a n artificial water was made in the following manner: 1. Add the various salts to the water. 2. Pass in carbon dioxide until the carbonates are dissolved. 3. Aerate 6 to 8 weeks or longer (use cotton filter to keep out
dust). 4.
Adjust pH to 7.4 by passing in carbon dioxide before using.
Table I1 shows the average analysis of the river waters and the salts added to the synthetic dilution water. It will be noted that potassium nitrate was used in place of sodium nitrate. Potassium was used in view of the recommendations of Foreman (8). Since the content of ammonia and organic nitrogen in the distilled water approximated that found in river water, none was added.
461
4. Incubate the plate 24 hours at 25" C. 5. Pick five different colonies from the plate and transfer to 3, culture bottle containing 95 ml. of dilution water, 5 ml. of peptone broth, and 10 ml. of soap solution. 6. Incubate with constant aeration for 12 to 18hours at 25" C. 7. Inoculate 2.3 liters of dilution water (containing proper amount of soap to demand 5 mg. of oxygen per liter) with 1 ml. of this culture. 8. A control inoculum is built up in the same manner and inoculated into 2.3 liters of dilution water.
The soap solution referred to is that which, when diluted from 100 ml. to 2.3 liters, has a n oxygen demand of 5 mg. per liter. The culture (1 ml.) used for inoculation contained enough peptone to have a n oxygen demand of 0.25 mg. per liter. Fire colonies were picked in order to obtain a mixture of soap-tolerant organisms. The results are calculated as follows:
1. Plot data for oxygen demand of soap plus inoculum, draw curve, and extrapolate to 10 weeks. 2. Plot data for oxygen demand of inoculum (i. e., control), draw curve, and extrapolate to 10 weeks. 3. Subtract curve 2 from curve 1 and obtain curve 3, repreoxygen demand of the soap. TABLE 11. AVERAGERIVER-WATER AND SYNTHETIC DILUTIOX- senting 4. Calculate percentage of theoretical oxygen demand from WATERANALYSIS values on curve 3. Av. RIVERSYNTHETIC DILUTION5. From the percentages thus obtained and the purity of the SALT WLTER ANALYSIS WATERa h A L Y G I S soaps, calculate the percentage of the theoretical oxygen demand P. P. m. P. P. m. (i. e., percentage of complete oxidation) of the pure soaps. KN03 5 NaNOa 4:3 .. NaCl NazS01 MgSO4
MgCOa CaCOa
Si02
Total
10.0 31.8 19.3 71.0 117.7 18.2
-
272.3
ESPERI~IEKTAL RESULTS
10 30 20
70 120
--. . 255
Dissolved oxygen was determined according to Standard Methods (19) except that 202.9 ml. were used for titration ( 2 2 ) . Samples were incubated at 20" C. by immersing the 260-ml. bottles in water. Oxygen demand was determined a t weekly intervals up to 9 weeks. Duplicate determinations were discontinued when it was found that in 135 determinations the deviation averaged 0.04 p. p. m. To conserve incubator space, reagents, and expense of saiiiple bottle., 125-nil. bottles were used in some series. Reagents were added in half the amounts recommended (19). Titration was n itli 0.0125 N sodium thiosulfate, which is direct reading. series of twenty determinations showed agreement to within 0.035 p. p. m. of the results obtained in the larger bottles. Obviously in a study of this kind, conditions of the experiment should be as near as possible those which are encountered in practice. It has been shown (21)that sewage should not be used for inoculation, owing to the fact that the course of the oxidation in the control did not follow that of the substances under investigation. Use of pure cultures was out of the question because pure cultures are not encountered in practice, and, furthermore, information is not available as to what strains will aerobically oxidize all of the soaps used. Further, since soaps are known to exert a bacteriostatic action, it was apparent that the inoculum would have t o be "built up." This led to the development of a special technic for obtaining a mixture of soap-tolerant organisms for inoculating purposes. The method was as follows: 0
1. Add 5 ml. of sewage, 5 ml. of peptone broth (5 grams per liter), and 10 ml. of soap solution to 90 ml. of dilution water (pH 7.4) in a culture bottle, arranged so that filtered air can be
constantly aspirated through the solution. The concentration of soap is then about twice that in the samples for oxygen-demand determinations. 2. Incubate with constant aeration for 24 hours a t 25' C. 3. Streak an agar d a t e (15 cc. of nutrient agar and 1 ml. of soap solution) from th'ls cultbe. I
Sanitary chemical analyses of the six different bottles of dilution water used showed a slight decrease in residue anti alkalinity (from the theoretical) during aeration and standing. The average analysis is as follows: P. p . m.
P. p .
711.
These amounts of nitrogen together with what was added in the inoculum (very small) may not have been great enough to meet the metabolic demands of the bacteria. During the courbe of oxidation, the nitrate that had been added to the synthetic water was not reduced. The oxygen demand of the blanks averaged less than 1 p. p. m. in 10 weeks. During the preparation of the cultures, the heterogeneity of the organisms was greatly reduced. Nost of the inoculating cultures showed a predominance of micrococci and short rody the latter were shownto be Aerobacter aerogenes and Eschzrichia coli. After inoculation, it was found that the samples coiltained 10,000 to 12,000 bacteria per milliliter. I n eleven series, forty-five separate investigations were made. Of these, thirty-five produced results. However, only twenty-two of these thirty-five investigations appeared to show normal oxidation as evidenced by the velocity constants. The data (expressed in per cent of the theoretical oxygen demand) on the runs which showed normal oxidation are presented in Table 111. Included for comparison are the 5-, lo-, and 20-day data as read from the curves. Also, the velocity constants are listed. The calculation of the velocity constants was made according to the Phelps equation: K
where K
=
t =
L = X =
1
-
t
L L-x
log -
velocity constant time in days ultimate oxygen demand (70 days) oxygen demand in t days
I n deriving the value of L it was assumed that the oxvgendemand value obtained after 70 days of incubation couid be
INDUSTRIAL AND ENGINEERING CHEMISTRY
462
I
taken as the ultim a t e o x y g e n demand. Warrant for this assumption is found on comparing the oxygen-demand values obtained after 56 and 63 days with t h e 70-day value. As an example of the method of comp u t a t i o n , use will be made of the results obtained in the second run with sodium heptylate ( T a b l e 111). For this experiment the value of L was 87.0 in terms of the percentage of the theor e t i c a l oxygen req u i r e m e n t which h a d b e e n satisfied when the biochemical reaction ceased. I n the same experiment, the o x y g e n d e m a n d a f t e r 28 days was 78.1, likewise expressed as a p e r c e n t a g e . For that particular exp e r i m e n t the velocity constant obtained from t h e Phelps equation was
=
0 0353
The \ elocity con* t a n t s corresponding to other periods w-ere computed iii t h e s a m e manner, and the results obt a i n e d w e r e then averaged t o give the constants recorded at the b o t t o m of Table 111. In most cascs (there were a few exceptions), the velocity constants c o r r e s p o n d in g to the o b s e r v a t i o n s made at 7 days of in c u b a t i o n TI e r c relatively high. It also appeared that huge errors might be introduced in the value of the velocity constant when the observed oxygen de-
Vol. 24, No. 4
mand approached the value of L. These end values, therefore, were generally omitted in deriving the average value of the velocity constant. The average velocity constant of all the runs was about 0.0324. This indicates that the reaction is about one-third as fast as ordinary oxygen-demand rates with K values of 0.1 a t 20" C. Those runs which did not fit the equation showed, in some cases, excessive lag phases and variable rates. I n most instances, however, they reached (in 10 weeks) a percentage of the theoretical demand comparable to the demands obtained in normal runs. (A few runs required 20 weeks.) The 20-day demand appears to have been between 80 and 90 per cent of the ultimate demand, except in a few cases where K was low. I n interpreting this statement, it must be remembered that the ultimate demand was never equal to 100 per cent of the theoretical oxygen demand. The fact that theoretically complete oxidation was not obtained may have been due to lack of sufficient nitrogen, lack of symbiotic conditions, or bacteriostatic action of the soaps. (Note the decrease in demand from formate to butyrate.) Lack of agreement between different series might be ascribed to the same reasons, since experimental errors, as already shown, would account for the difference of 0.04 p. p. m. between duplicate determinations, corresponding to an error of only 0.8 per cent with depletions of 5 p. p. m. Table IV shows the average ultimate (10-week ) demand obtained from the data in Table I11 and also the average demand determined from all the data, regardless of the course of oxidation. As all the data presented in Table 111 fit the Phelps equation given above and hence are typical orderly time reactions, no curves have been presented. TABLEIV. AMOUNT OF COMPLETE OXIDATIONOF SOAPS SOAP
Av. ULTIMATE Av. MAX. ( 1 0 - w ~ ~ ~D)E M A N D OBTAINED IN DEMAND-DIFFERENTSERIESb
OXYQEN
%
%
Sodium formate 80.0C 78.4 Sodium acetate 77.7 75.5 88. O C Sodium propionate 69.2 Sodium &butyrate 60.3C 88.3 Sodium isobutyrate 64.4C 75.8 Sodium valerate 62.O C 82.5 88.SC 88.8 Sodium isovalerate 88.o c 84.0 Sodium caproate 80.0 80.0 Sodium heptylate 81.OC ' 81.0 Sodium caprylate 74.4 Sodium palmitate 76.8 68.3 Sodium stearate 68.3 55.0 Sodium oleate 55.0 0 Average of data on Table 111, normal oxidation rates. b Average of all data without regard to type of curve. C One run only.
CONCLUSIONS 1. All of the sodium salts of the fatty acids studied are capable of being oxidized biochemically. 2. When proper bacterial conditions are obtained, the oxidation fits the curve of a n orderly time reaction, and the 20day biochemical oxygen demand has a definite relation to the ultimate (10-week) demand. The velocity constants approximate an average value of 0.0324. 3. Theoretically complete oxidation was not obtained even in 10 weeks, although, practically, oxidation had ceased st 8 weeks. The average percentage of complete oxidation varied considerably for the different soaps (55-89 per cent). 4. The biochemical oxygen demand of the sodium salts of the fatty acids apparently does not bear any definite relation to the theoretical oxygen demand for complete oxidation to carbon dioxide and water.
ACKNOWLEDGMENT The authors are indebted to E. J. Theriault, of the United States Public Health Service, for helpful criticisms and suggestions concerning calculations of the velocity constants.
Plpril, 1932
INDUSTRIAL AND ENGINEERING CHEMISTRY
LITERATURE CITED Adeney, Fifth Rept. Royal Commission on Sewage Disposal, App VI, 13-23 (1908). Adeney and Dawson, Sci. Proc. Roy. Dublin SOC.,18, 199-202 (1926). Cooper and Nicholas, J. SOC.Chem. Ind., 47, 320-2T (1928). Cooper and Read, Ibid., 46, 154-6T (1927). Cooper and Read, Ibid., 46, 156-7T (1927). Cooper and Read, Ihid., 46, 413-81‘ (1927). Elder, IND.ENG.CHEM.,21, 560 (1929). Foreman, N. J. State Dept. Health, Public Health News, 13, 132-6 (1928). Greenfield, Elder, and McMurray, IND.ENG. CHEM., 18, 1276-9 (1926). Horowita-Wlassowa, Goldberg, and Goldberg, Brch. Hyg., 98 2 3 3 4 0 (1927). Illinois State Water Survey. Bull. 5 (1907). International Critical Tables. Vol. V. D. 446. McGraw-Hill, 1926. Krafft and Wiglow. Be?., 29, 1329 (i896)
463
(14) Levine, Sopperland, and Burke, Iowa State College, Eng. Expt. Sta., Bull. 68 (1923). (15) Lewkowitsch, “Oils, Fats and Waxes,” 5th ed., vol. 1, p. 133, Macmillan, 1913. (16) McBain, Trans. Faraday SOC.,9, 99 (1913). (17) MoBain, J. SOC.Chem. Ind., 37, 249-521‘ (1918). (18) Mohlman, Edwards, and Swope, 1x0. ENG. CHEM.,20, 242-6 (1928). (19) Standard Methods for m’ater Analysis, 6th ed., A. P. H. A.. 1925. (20) Symons and Buswell, IND. ENQ.CHEM.,Anal. Ed., 1, 161 (1929;. (21) Symons and Buswell, Ibid., 1,214 (1929). (22) Theriault, U.S. Pub. Health Bull. 151 (1925). RECEIVED Auguat 10, 1931. Presented before the Division of Water, Sewage, and Sanitation at the 81st Meeting of the American Chemical Society, Indianapolis. Ind., March 30 to April 3, 1931. This paper ia a n abatrsct of a thesis submitted in partial fulfilment of the requirement8 for the degree of master of science in chemistry in the Graduate School of the University of Illinois.
Zinc in. Water Supplies EDWARD BARTOW AND OTIS MELVIN WEIGLE,State t-niversity of Iowa, Iowa City, Iowa disappearance of all but one of The limits of permissible zinc in drinking the m a n y s p r i n g s a t B a x t e r n a t u r a l w a t e r s . Zinc water have been questioned, and this ineestiSprings, Kans. sometimes occurs in tap gation was undertaken to secure data concerning Water coming in contact with water, having bpen d i s s o l v e d these limits. Methods of analysis were checked, zinc in g a l v a n i z e d iron pipes from the zinc coating of galand a method was adapted to test samples in the a n d cisterns f r e q u e n t l y disvanized iron pipes and storage solves considerable zinc. Weintanks. field. The more accurate standard method of the land (24) found 5 p. p. m. of Underground waters occurA. 0. A. C . was used in the laboratory. zinc in the water supply of the ring in zinc-mining regions freA survey was made of natural waters of the chemical l a b o r a t o r y of t h e quently c o n t a i n considerable Missouri-Kansas-Oklahoma zinc district. Waters University of Tubingen. This zinc. The alternate exposure containing as high as 50 p . p . m. of zinc were contamination was traced to the of the zinc ore to air and moisgalvanized pipes leading into the ture favors its s o l u t i o n . The found. Animals and humans were drinking buildings. same principle a p p l i e s t o the waters known to contain zinc. A water which w a s d r a w n leaching effect of the s u r f a c e Water containing varying amounts of zinc from a well through a galvanized water on the e x t e n s i v e piles sulfate was fed to rats. The rats drank 50 of waste rock m a t e r i a l from iron pipe, several hundred yards p . p . m. of zinc as zinc sulfate over a period of which most of the zinc ore has in l e n g t h , w a s r e p o r t e d by been removed. Schwarz (18) to c o n t a i n 32.4 several weeks with apparently no harmful results. Parker and Bailey (16)report p. p. m. of zinc oxide. Many Zinc in many drinking waters comes f r o m w a t e r s f r o m zinc m i n e s and families using this water comgalvanized iron pipes. If a pure zinc were used concentration mills with a zinc plained of intestinal troubles. in the galvanizing, there should be less zinc content as high as 1862 p. p. m. Abbott ( 1 ) reports that the dissolved by the water. Mine waters analyzed b y water of Devil’s Lake, N. D., W a r i n g (23) s h o w as high as which seemed more toxic to fish It seems advisable that, until further informa6500 p. p. m. of z i n c . S u c h than its composition indicated, tion is obtained, 5 p . p . m. or less of zinc should waters have a taste so astringent showed after careful analysis 15 remain the standard for drinking water. that they are not likely to be p. p. m. zinc. used bv men or animals. Jackson (10) s a.w : Two- springs in Newton County, Mo., on the road from I have on numerous occasions examined water for zinc which Joplin to Seneca, about a quarter of a mile north of Shoal Creek, known as East Spring and West Spring, were notable has had very marked physiological action with 5 or 6 p. p. m. of zinc present. Notably in Falmouth Foreside, a summer place for their large content of zinc (4)-120.5 and 132.4 p. p. m., near Portland, Me., where they put in new water-supply mains respectively. These analyses were made by Hillebrand (5) of galvanized piping and started to drink it early in the spring in 1891. He says those waters were supposed to have a without flushing, the entire town was badly affected by it. I peculiar composition, owing to the strong metallic astringent ordered the pipes thoroughly flushed two or three times, using well water as drinking supply in the meantime, after which no taste left in the mouth after swallowing. further trouble occurred. One of the authors (Weigle) spent considerable time in The same difficulty arose with the new annex to our Natural April, 1931, in an unsuccessful effort to locate these springs. Science building where many of the fish and smaller animals were I t seems probable that the extensive underground excava- killed by the water which was used, and where a number of the instructors and professors were made very ill. This water tions in the zinc-ore bearing vicinity have intercepted the contained about 8 p. p. m. of zinc. In this case also, after flushing flow of these waters. The same explanation is given for the thoroughly several times, no further trouble occurred.
I S C o c c u r s in a fen.
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,
