Determination of Pyridine and Pyridine-Base Compounds In
River Water and Industrial Wastes
R. C. KRONER, 31. B. ETTINGER, AND W. ALLAN MOORE Environmental Health Center, Public Health Service, Cincinnati, Ohio Trace quantities of pyridine and structurally related pyridine bases may be present in surface waters as a result of discharges from coking operations and petroleum processing. As an aid to the study of the persistence and taste- and odor-producing properties of these compounds, a sensitive, quantitative method of measurement was required. The method requires an initial distillation to remove turbidity and possible interfering compounds and may also be used for concentrating the sample. After dilution to the proper concentration, the sample is buffered
P
YRIDINE and related pyridine bases are known to be pres-
ent in the wastes from by-product coking operations and some oil refining processes. This study of the determination of pyridine and some of its derivatives was designed to provide means for the determination of the small amounts of such materials in certain wastes or for the detection of trace amounts of such materials in surface wat,ers. For the intended use, a reaction capable of detecting as little as 0.01 p.p.m. of pyridine or pyridine base in a 100-ml. sample would be satisfactory. The Koenig ( 7 )reaction has been used with many modifications for the detection of some of the compounds containing the pyridine ring structure. The reaction consists essentially of the addition of a cyanogen halide (usually cyanogen bromide) t’o the pyridine base and subsequent reaction of the compound formed with a suitable aromatic amine. The reaction has been discussed by hIigrdichian ( I O ) , Karrer (S), and Waisman and Elvehjem (13). Lfigrdichian ( 1 0 ) represents the reaction by the following equations:
CN
R BR H
Various investigators have proposed the use of a number of different aromatic amines together with cj-anogen bromide to detect pyridine-type compounds. Tallantyre ( 1I ) used aniline as the coupling agent but obtained a sensitivity of only 1 part in 350,000. Tapia Freses et al. ( 1 2 ) linked pyridine with acetophenone but did not study the reaction extensively. Blekseev (1) employed benzidine in the determination of pyridine in aqueous ammonia solutions and further proposed the use of isoamyl alcohol for extracting the resultant dye. Other investigators have proposed %naphthylamine (S), p-methylaminophenol (Z), and p aininoacetophenone ( 4 , 6 , 1 4 ) in the Koenig reaction. Various investigators have used the acetate ion for increasing the sensitivity of the reaction. McCormack and Smith (8)used potassium acetate as an intensifier in the determination of nicotine (using the Koenig reaction) and obtained a maximum color intensity at a concentration of 0.24 gram of potassium acetate per 100 ml. This was in accord with hlarkwood’s (9) value of 0.2 gram of potassium acetate per 100 ml. On the other hand, von Euler ( 3 )and Bandier and Hald ( 2 ) found that the acetate ion interfered in the reaction, while Kodicek (6) reported that the acet a t e ion had no effect on the color formation. Waisman and Elve-
with sodium acetate. Benzidine hydrochloride and cyanogen bromide are added and the reaction is carried out in a layer of butyl alcohol. The color reaction is complete in about 90 minutes. Factors affecting the reaction, such as time, temperature, concentration of reagent, and pH, are discussed. The reaction is sensitive to 0.005 p.p.m. of pyridine and can be used in the range from 0.005 to 1.0 p.p.m. Other pyridine derivatives that react quantitatively under the same conditions are 1-, 2-, and 3-picoline, 2,4- and 2,6-lutidine, and nicotinic acid.
hjem ( I S ) suggested that the use of different amines influences the results obtained. The effect of light has been reported to be an important factor in the reaction ( 2 , 4 ) . Waisman and Elvehjem (13)suggested that the stability of color in conjunction with light was an important consideration only when amines of high molecular \$eight are used as a coupling agent. -4s a starting point for this investigation, benzidine was chosen as the aromatic amine to be used. Previous investigations by this laboratory, utilizing the Koenig reaction for the detection of anides, had indicated benzidine to have a better combination of .a,isitivity and stability than a number of the other amines proposed. The use of cyanogen bromide was suggested by its ease of preparation. Preliminary investigation showed that the concentration of benzidine and cyanogen bromide had a definite influence on the amount of color produced, while the order of addition of reagents was of no consequence. The details for the preparation of these solutions are given under “Preparation of Reagents.” Exploratory work indicated that the reaction did not proceed a t p H values below 4.0 and that colors formed faded very rapidly a t p H values in excess of 10.0. A satisfactory amount of color was produced in the p H range of 7.0 to 8.0 using a bicarbonate buffer. However, the behavior of the yellow-orange color which resulted was unpredictable, sometimes fading to pink before the reaction was complete. It was, therefore, decided to investigate the method of Alekseev (1). This procedure employs the same reagents already under consideration but carried out the reactions in a two-phase system. The final reaction, which yields a red coloration, apparently takes place in an isoamyl alcohol phase floating on the aqueous phase. While Alekseev’s technique possessed the desired characteristics, difficulty was encountered in obtaining isoamyl alcohol of the required purity. .4 number of different brands of isoamyl alcohol Fvere secured and used in the reaction but in all cases there was color formation in reagent blanks. Moreover, the isoamyl alcohol resisted all efforts to remove the color-producing impurities. As an alternative, n-butyl alcohol was substituted for the isoamyl alcohol n i t h considerably improved success in the matter of colorless blanks and stability of color. The adoption of n-butyl alcohol as a component of the reaction mixture resulted in stabilizing and sensitizing the reaction. The final reaction takes place in the alcohol layer. If the reaction is carried out in aqueous solution and is subsequently extracted u. ith n-butyl alcohol, less color results. Apparently the butyl alcohol
1877
ANALYTICAL CHEMISTRY
1878 extracts intermediate products and then affords a superior medium for the progress of the reaction. FACTORS AFFECTIIVG THE REACTION
Effect of pH. The change fiom an aqucous system to a twophase alcohol-water system necessitated a icsurvey of the pH r e quirements for maximum color development. At this point, i t was observed that the color in samples buffered with solutions containing acetate ions was more intense than samples of comparable pH but lacking the presence of acetate ion. T o establish the pH range most suitable for the rcaction, a series of samples was prepared containing 0.10 p.p.m. of pyridine and 0.50 gram of sodium acetate per 100 ml., the acetate concentration being based on preliminary experimentation. .ifter addition of benzidine, the pH of the aqueous samples m-as adjusted, using small amounts of dilute hydrochloric acid or dilute sodium hydroxide t o produce a p H range from 3.4 t o 11.3. The reaction was then carried out in a two-phase system using n-butyl alcohol and cyanogen bromide. The samplrs werc n-cll shaken and left to age for 4 hours, after which A portion of the colored alcohol layer \vas carefully pipptted off nnd the absorbancy mcasured in the spectrophotometer.
,
( I O 0 PPB PYRIDINE, 25°C)
0'
4b
50
610
70
80
I
9'0
IO0
lil0
Table I.
Effect of Amount of Reagents on Absorhancy per 1.0 Cm. at 520 Wllimicrons
(100-id.miiiples, reaction cari led O I I I a t room teiniwratiire, readings on butyl alc~i11~11 extiact) Cyanogen Bromide. MI. Benzidine, RII. 2 0 7.5 10.0 ?O.OS 0.340 0.354 0.342 0 342 1.0 0.409 2.0 0.369 0.424 0 314 0.415 4.0 0.410 0 461 0 455 0.265 8.06 0.200 0.333 O.?W '
Abore 10 ml. of cvanomm hroiriide fumes are very evident. b Above 4.0 ml. of benzidine precipitate forms and color fadcs.
a
Sodium acetate in the amount of 0.6 gram per 100 in]. of sample causes optimum intensification of the color formed, and also serves to raise the p H of the reaction niisture to the desired pH range. .4ccordingly, -1.0 nil. of 15% rodium acetate solution (0.6 gram) n-as selected as a suitable quantity of the chemical to provide both color intensification and p H control. Esperiments were also conducted to determine whether the sodium ion or the acetate ion i v m the sensitizing agent in the procedure. It \vas found that no substantial difference in the quantity of color resulted, regardless of the amount' of sodium chloride present betn-een the limits of 0 and 1.0 g r a m rlt the same time, comparable samples containing sodium acetate and sodium chloride together showed color in greater concentration than samples containing only sodium chloride. Apparently, the acrtate radical is the intensifying agent in the reaction
1
pH OF 4OUEOUS S O L N
Figure 1.
Effect of pH on Color Formation
-c C
>
01
3 0 gms
Sodturn Acetote
0
z
$01-
a:
The data, presented graphically in Figure 1, indicate that litt,le or no color develops a t a p H of 3.4, and the amount of color produced by the reaction is fairly constant from pH 6.2 to 11.3. K a t shown graphically is a slight tendency of the reaction color to fade more rapidly at p H levels in excess of 9.0, Furthermore, the presence of hydrochloric acid in the benzidine solution may cause lowering of the pII upon addition of this reagent. Because of the buffering capacity of sodium acetate, a rough pH control between 6.8 to 8.0 is easily made with indicating p H paper. Effect of Sodium Acetate. The effect of sodium acctate on color formation was determined by adding sodium acetate in various amounts to similar samples of pyridine. Benzidine was then added and the pH adjusted to 6.8 to 8.0 with dilute sodium hydroxide, if too little sodium acetate was used to yield a solution with the desired pH. The reaction was completed with cyanogen bromide in the two-phase water-butyl alcohol system. The data from three similar experiments indicate that: The amount of color formed by pyridine in the complete absence of sodium acetate is variabl(, and unpredictable. The quantity of color formed is grrater in the prescnrc of sodium acetate than in its absenre at comparablr hydrogen ion concentrations. Maximum color formation talws pl:we in the range between 0.2 and 0.8 gram of sodium acetate in 100 nil. of aqueous sample (Figure 2). The color intensity begins to devrease a t about 0.8 gram of sodium acetate per 100 ml. of aqueous sample, and gradually decreases until a complete inhibition of color formation is reitvhpd a t a concentration of about 3.0 gram per 100 ml. of sa,mpl(i (Figure 2).
In the case of this procedure, color may be intensified or decreased by sodium acetate, depending upon its concentration.
s:m Figure 2. Effect of Sodium Acetate on Color Formation 100 p.p.b. of pyridine a t pH 7.1
Effect of Benzidine and Cyanogen Bromide Concentration.
X systematic study of the benzidine and cyanogen bromide requirements was next undertaken Pyridine samples (100 nil.) at a concentration of 0.1 p.p.m. were prepared, the amount of benzidine Tvas held constant, and the quantity of cyanogen bromide was vaiied. The experiment was repeated several times using a larger amount of benzidine in each series. I n all cases, the previously determined figures for pI1 and sodium acetate concentration were used and the reaction carried out in the butyl alcohol phase. The spectrophotometric readings, given in Table I, show that maximum color development occurs with addition of 4.0 1111. of 2% benzidine solution. However, in this range of concentration a precipitate of benzidine occasionally forms in the sample, which hinders the separation of the alcohol-water layers and apparently retards or deteriorates the formation of color. \Vhen higher concentrations of cj-anogen bromide are used, an increacle in the intensity of CQlOr formed is apparent I-se of a minimum quantity of cyanogen bromide, however, is desiiable in view of the hazardous nature of the solution. Experience shows that quantities of reagent that furnish linear results, provide sufficient sensitivity, and are practical for routine analysis are 2.0
V O L U M E 24, NO. 12, D E C E M B E R 1 9 5 2
3
o
.
4
1879
4
i
-
SERIES AT 20.C
V
0
2
Figiire 3 .
4
6
8 TIME
IN
10 12 HOURS
14
16
IS
I
Effect of Time and Temperature on Color Formation
100 p.p.b. of p>ridinr, pN i.1, reactions carried out in darkness
nil. of the benzidine solution and 5.0 nil. of cyanogen bromide. As no substantial difference is caused l)y the order in which the reagents are added, the cyanogen hromide should be added last to minimize fumes of the chemical from open vessels. Effect of Time and Temperature. Investigation of the effect of time and temperature on the production of color with t,he reagents selected was nest undertaken. Three series of samples were livepared containing 0.1 p.p.m. of pyridine. Series 1 was placed in a constant, temperature rooiii a t 20" C., series 2 was treated riinilarly a t 37" and series 3 was held a t room temper:itui,e of 25' C . -It meusured time intervals samples were removed and the absorbancy was determined. The data, shown in Figure 3, indicate that, with an increase in temperature the reaction reaches a maximum color development in a shorter period of t h e , although the maximum color formed is approsiinately the same regardless of temperature. i\laximum color development takes place in about 4 hours a t 37" C., while a temperature of 20" C. requires 10 hours, but in all cases the masiilium color persists unr11:inged for several hours. At :I room temperature of 25" C. between 6 and 8 hours are req u i i d for complete color development. After 4 hours the reaction is approsinlately 9Oy, complete and further color development is very OW. Hence, good results can be obtained in routine anal!-Pis if the optical densit>-of the developed color is read after 4 hours. A t any stage of the reaction the amount of color app:rrently is pi'oportional to the coilcentration of pyridine. This permits deterniiiiatioiis to be made with a fair degree of accuracy based on readings of the samples after a relatively short storage period, provided that mechanical operatioiis do not introduce a substantial time differential in the observations. Because of the effect. of time and temperature on color tievelopnient., standard solutions and port'ions of samples to be analyzed should be set up simultaneously and under the same conditions. From the standpoint of accuracy and sensitivity, it is preferable to compare the colors after complete color formation and stabilization have taken place. Effect of Light. Duriiig studies on the effcct of time and temperature on the development of color, it was concurrently determined that (Figure 4):
good linear properties. \\-lien the reaction was carried out i n tiiffused light. the formation of the brown color or fading was less pronounced. To secure niaximum accuracy for the pyridine measui'cmelit, samples should be stored in the dark during the required reaction time. Reactions of Other Pyridine-Base Compounds. This reaction is not specific for pyridine alone. Other pyridine-base compounds will also react under the conditions stipulated. Various investigators differ regarding the specificity of the reaction for different pi-ridine bases, probably because of the variety of conditions and reagents used in the application of the Koenig reaction. Table I1 lists a few of the compounds that have been examined. Because of the difficulty of purchasing or piqiaring chemically pure compounds of t,he pyridine series, no really decipive study of the different pyridine-type compounds that will enter into the react,iona has been made. Specificity of Reaction for Pyridine Bases. The specificity of the procedure was given a limited investigation bg- testing the reactivity of represent,ative compounds having various functional organic groups. The groups included were phenols, amines, aldehydes, ketones, and one nitrile. COLOR DEVELOPED IN TOTAL DARKNESS HCOCOR
DEVELOPED IN DIRECT SUNLIGHT
r,,
Saniples exposed to strong sunlight a t room temperature reached a maximum of color formation in a shorter period of time than samples stored in the dark. The maximum color formed in the light-exposed samples was less th:m the maximum of similar samples not exposed to strong light. Samples developed in strong sunlight exhibit a tendency to fade iiiore rapidly than samples stored in the dark.
In addition, in direct sunlight, without pyridine or pi-ridinebase compounds present, the reagents alone will develop a light brown color unlike the red color resulting in the presence of pyridine. In several cases, where linear series of pyridine samples were prepared, discrepant results were noted on light-exposed samples, whereas similar series reacted in total darkness exhibited
0
2
6
4
Figiire 4.
8 IO T I M E I N HPI!RC
---
12
14
16
18
Effect of Light on Color Formation
100 p.p.b. of pJridine, pII 7.1, 2.5' C.
The data (Table 111) indicat,e that a number of the phenols are apparently reactive in concentrations of about 500 p.p.m., and that 1-naphthol ip reactive at a concentration of about 100 p.p.ni. I n bot,h cases, reactivity may be due to trace iiiipurit,ies in the original compounds. However, hecause of the relatively large quantities required to produce color Formation and because of the conditions prescribed under "Preparation of Samples," no interference in the pyridine-base measurement should be encountered from phenols. 1-Naphthylainine and furfural show a greater tendency to react with the pyridine-base reagents than the previously nientioned phenol$. \l'hile the screening procedure proposed will not i'e.........
Table 11. Compound Pyridine Quinoline Quinaldine 1-Picoline 2-Picoline 3-Picoline Picolinic acid 1-ilminopyridine Nicotinic acid 2.4-Lutidine
Reactivity of Various Pyridiue Bases
Reactioii
f
-
=; +-, - 0
Approximate Sensitivity, P.P.JT. 000.5
.... ...
1.0
0.05
0.1
.... ....
10
Color
W a r e Length a t J Iaxi 111i i ni .ibsorlntion, AIp
Red
.j20
Orange-red Red Red
510
........ ........
........ ......
Tpllow
. . 520 553
... ...
No inaxiniuiii in visible rnnge 480
OrangeSeiiow Oiange480 yellow a Reaction questionable, coloi niay be due t o Iiniiurities. b Substantial amounts of color d e \ elop in both nlcohol and n a t e r ~ , h a s e .
2,B-Lutidine
C"
+"
1 0
1 0
ANALYTICAL CHEMlSTRY
1880 Table 111. Compound Phenol m-Cresol o-Cresol Pentachlorophenol 2,4,6-TrichlorophenoI Thymol 1-Naphthol Aniline 1-Naphthylamine Tyrosine Benzaldehvde Furfural Formaldehyde Acetophenone Scetone Acetonitrile
Specificity of Reaction Reaction
+ +
Approxiinate Sensitivity, P.P.RI. 5 5
Color Pink Yellow
5 10 ...
...
100
...
10
,..
...
1.0
...
...
... ...
......... .........
Orange-pink
.........
Pink
.........
.........
Red-pink
......... ......... .........
.........
move these materials prior to the final reaction, no interference is expected in the normal area of application of the procedure. Aromatic amines may interfere with the procedure by competition with benzidine in the reaction. I n the presence of substantial amounts of benzidine the amount of such side reaction would be expected to be small.
the solution dropwise until the reddish brown color of excess bromine is apparent. Remove the excess bromine by adding a solution of 10% potassium cyanide dropwise until the bromine color is discharged. All of the free bromine must be removed from the solution; otherwise an intense side reaction takes place between the rengents themselves, making any analytical determination impossible. To prevent the presence of free bromine, the following procedure is recommended: Make up a 0.1% solution of indigo carmine (indigo disulfonate) and place 2 drops of the solution on a spot plate. Add 1.0 ml. of the cyanogen bromide to the indicator. If bromine is present in excess, the dye will be decolorized immediately to a pale yellow. When the bromine is removed by a small excess of potassium cyanide, the addition of 1.0 ml. of cyanogen bromide to 2 drops of the indicator will not change the original blue color. The solution is then ready for use and is stable for 2 or 3 weeks. Prepare the cyanogen bromide under a well ventilated hood and avoid inhalation of the fumes during preparation and use. Store in a glass-stoppered bottle. SODIUM ACETATE. rl 15% solution of the C.P. chemical in distilled water is used. n-BmYL ALCOHOL, C.P. Run a blank determination on every batch of untested butyl alcohol. If a positive blank is obtained, the n-butyl alcohol may be purified by washing. Using a sepnratory funnel, wash each liter of contaminated alcohol nith three successive portions of 5% hydrochloric acid. Follow nith three washings using successive 100-ml. portions of distilled water. Mallinckrodt’s n-butyl alcohol has shown consistent absence of interfering materials.
DETERMINATIOh OF PYRIDINE AND RELATED BASES
Recommended Procedure for Preparation of Sample. So far, the procedure described has been applied only to polluted aerobic river waters. For this work, simple distillation, after addition of sufficient sodium hydroxide to produce a p H in excess of 10, has sufficed. Pyridine in trace quantities distills over readily. About 98% of the pyridine distills over in the first 20% of the distillate and complete recovery is obtained in the first 40% of the distillate. The rate of distillation of other pyridine bases has not been investigated. However, quantitative recovery of %picoline and 3-picoline has resulted from the distillatory purification procedure described in detail below. The properties which will permit separation of pyridine bases from more complicated n aste solutions are obvious. Most of the pyridine-base compounds are volatile from alkaline solutions and their salts are not volatile from highly acid solutions. These materials may be recovered from wastes or river waters by simple distillation or steam distillation from neutral or alkaline aqueous solutions Moreover, many interfering organic materials may be extracted by organic solvents from aqueous solutions containing substantial amounts of free mineral acids without extracting the pyridine bases. A universally applicable procedure for preliminary preparation of samples for analysis cannot be given a t this time. The following procedure has been applied satisfactorily to prepare samples of polluted river waters for determination of pyridine and 2- and 3picoline.
100-
p>. 7 5 4
m
%
m $50X
I
+ 2
-
0
c
2
25-
I
300
400 500 WAVELENOTW I N YlLLlMlCRONS
600
Figure 5 . Absorbancy Characteristics of Color Produced from Pyridine at pH 7.1
Apparatus. Becknian Model B spectrophotometer equipped with 1.0-cm. cells. Procedure. Pipet off an aliquot portion of the purified sample of such size as to contain between 0.001 and 0.02 mg. of pyridine Take a 500-ml. portion of the original sample containing a mixbases as “pyridine.” ture of pyridine bases and add sodium hydroxide until the p H is Dilute the sample portion to 100-ml. with distilled water, using above 10. Transfer the material to an all-glass distilling apa glass-stoppered bottle to contain the solution. paratus (Pyrex Catalog h-0. 3360) or to a Kjeldahl distilling apPrepare a series of standards, ranging from 0.01 to 0.2 p.p.m. paratus. Add several boiling chips to prevent bumping and a of pyridine, and a blank of 100-ml. volume and treat in parallel pea-sized portion of silicone grease to prevent excessive foaming with the sample. For routine work one standard a t 0.1 p.p.m. when the solution boils. Distill over 450 ml. Stop the distillais sufficient. tion, add 50 ml. of distilled water, and continue the distillation until 500 ml. of purified sample have been collected. Add 4.0 ml. of 15% sodium acetate and 2.0 ml. of the benzidine Preparation of Reagents. STANDARD PYRIDINE SOLUTION. reagent. Shake well. Check the p H of the solution 15-ith indicating p H paper and adDissolve 1.0 gram of C.P.pyridine in distilled water and make up just to 6.8 to 8.0 if necessary. to 1 liter. Dilute 5.0 ml. of the stock solution to 500 ml. with Add 25 ml. of n-butyl alcohol and 5.0 ml. of cyanogen brodistilled water. This solution contains 10 p.p.m. of pyridine. mide. Shake well and store the samples in the dark for 3.5 to BENZIDIKEHYDROCHLORIDE SOLUTION,C.P. Dissolve 2.0 4.0 hours a t normal room temperature. grams of C.P. benzidine hydrochloride in 100 ml. of distilled water Pipet off very carefully about 5.0 ml. of the colored butyl by stirring and add 2 or 3 drops of concentrated hydrochloric acid alcohol layer. A4void inclusion of any water droplets during to complete solution of the benzidine crystals. Filter if necessary. the pipetting procedure; otherwise the sample will not be clear CYANOGEN BROMIDESOLUTIOK.Dissolve 1.0 gram of poand will be unfit for spectrophotometric reading. Place the tassium cyanide in 100 ml. of distilled water and add bromine to
V O L U M E 24, NO. 12, D E C E M B E R 1 9 5 2 pipetted sample in a 1.0-cm. cell and read the absorbancy a t 520 Ink (Figure 5 ) against the similarly prepared blank extract. t Compute the pyridine content of the sample on a proportional basis, using the standards to determine the relation between color and pyridine concentration. Evaluation of Results. If the sample analyzed contains one known pyridine base, analysis for the pyridine-base content yields a well defined quantity, but when the sample contains a niisture of pyridine bases of unknown proportions the measurement is less definite in terms of total concentration. When the reaction is carried out as specified, the amount of color produced per unit weight of pyridine base is a maximum in the case of pyridine. Also, those pyridine-base compounds which are known to react in this procedure possess maximum absorbancies close to that of pyridine (Table 11). Hence, when a mixture of pyridine bases is examined and the specific constituents are not definitely known, the results should be reported as pyridine. When so reported, the value is indicative of the minimum amount of pyridine base material which could be present. SUMMARY
Because of the presence of pyridine-base compounds in byproduct coke and oil refinery wastes, a sensitive method for their detection is desirable. Pyridine, picolines, lutidines, and nicotinic acid give colored reaction products by the Koenig reaction suitable for their spectrophotometric determination. Phenol, 0- and nz-cresol, 1naphthol, 1-naphthylamine, and furfural interfere. Factois affecting the determination of pyridine, such as pH, temperature, solvent, time, amounts of reagents, and effect of sunlight, were studied. The maximum color formation took place in the pH range of
1881
6.2 to 11.3 when the reaction was allowed to proceed in the dark. Sodium acetate was used not only to act as a buffer but also to sensitize the reaction. An increase in temperature caused maximum color formation in a shorter period of time. However, it was found that consistent results could be obtained a t room temperature with a reaction time of 4 hours. The separation of pyridine and related compounds from interfering materials was effected by distillation from an alkaline solution. The method was found to be sensitive to 5 parts per billion of pyridine. LITERATURE ClTED
(1) dlekscev, R. I., Zavodskaya Lab., 8, 807-9 (1939). (2) Bandier. E.. and Hald. J.. Biochem. J.. 33. 264 (1939). (3) Euler, H. ron, Schlenk,’F., Heiwinkel, H,, and Hogherg, B., Z.physiol. CAem., 256,208 (1938). (4) Harris, L. J., and Raymond, W. D., Biochem. J., 33, 2037 (1939). (5) Karrer, P., “Organic Chemistry,” 3rd ed., p. 786, New York, Elsevier Publishing Co., 1947. (6) Kodicek, E., Biochem J . , 34, 712 (1940). J . prakt. Chem., 69,105 (1904). 17) Koenig, W., (8) hIcCormack, K.E., and Smith, H., IND.ENG.CHEX,,ANAL. ED.,18,508 (1946). (9) Markwood, L. N., J . Assoc. Ofic.Agr. Chemists, 22, 427 (1939). (10) Rligrdichian, V.,“Chemistry of Organic Cyanogen Compounds,” AMERICASCHEMICAL SOCIETYMonograph Series, Xo. 105, p. 110, iYew York, Reinhold Publishing Corp., 1947 (11) Tallantyre, S.B., J . Soc. Chem. I n d . , 49, 466 (1930). (12) Tapia Fteses, A , , Sanchez Moreno Tira, C., and Canaleg, Cockburn, J., A e t a s trabnjos congr. peruano qiaim., 2, I, 337 (1943). (13) TTaisman, H. .I.,and Elvehjem, C. A.. IND.ENG.C m x , ANAL. ED.,13,221 (1941). (14) Wollish, E. G., Kuhnis, G. P.. and Price, R. T.. Ibid., 21, 1412 (1949).
RECEIVED for review May 28, 1962. Bocepted September 24.
1952,
Study of Chromium Toxicity by Several Oxygen Demand Tests R . S. INGOLS AND E. S. KIRKPATRICK State Engineering Experiment Station, Georgia Institute of Technology, Atlanta, Ga. The toxicity of chromium in its various forms has been studied, using three techniques for determining B.O.D. The data indicate that the toxic level of chromium is controlled by several variable factors, including the presence or absence of oxygen, the valence of the chromium, the type of organism (autotrophic us. heterotrophic), and the amount of organic matter present (rate of metabolism). A possible mechanism for the chromate ion toxicity under anaerobic conditions is discussed, and i t is suggested that the mechanism for chromic ion toxicity is simply one of inert, sniall particle interference.
T
HE presence of chromium compounds i n many industrial effluents discharging to sewage treatment plants and rivers makes it important to understand the factors involved in the toxicity of such compounds toward either the microorganisms in the sewage treatment plant or the aquatic plants and animals of the river. The Research Committee (17) of the Federation of Sen age and Industrial Wastes Associations has recently reviewed the literature on the toxicity of chromium as part of a larger study on the toxicity of industrial wastes. According to this review, chromium has been reported to have about 40% toxicity a t 3 concentration of only I p.p.m. under some experimental conditions (14), while under other conditions (4)a concentration of 5000 p.p.m. or more waa required for approximately the same toyicity. The data reported in the present paper indicate that
bacteria can tolerate high concentrations of chromium where large amounts of organic matter are present, as in sludge digestion. This map be one factor responsible for the large divergence in reported toxic levels. Some authors (4, 15, 20) indicate t h a t the valence of the chromium is not important in defining the toxicity limits, while others (5, 6, I S , 14, 16, 18) claim that there is a difference in the toxicity of the tri- and hexavalent chromium. Krieger and Moore (14)report that both trivalent and hexavalent chromium are toxic a t 1 or 2 p.p.m. in the dilution B.O.D. test, but that the trivalent form is generally more toxic. Dawson and Jenkins (6) indicate that trivalent chromium is more toxic to activated sludge than the hexavalent chromium. However, the concentration for obvious toxicity to activated sludge is approximately 10 times that for a definite toxic reaction in the dilution
