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., B i o c h e m J . , 34, 712 (1940). J . prakt. C h e m . , 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. C h e m . 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 that 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
ANALYTICAL CHEMISTRY
1882 B.O.D. test reported by hrieger and Moore (24). Jenkins and Hewitt (If) indicate that 10 p.p.m. of chromium as chromate may reduce nitrification by 50% in a trickling filter while altering the amount of carbonaceous oxidation only slightly. When 100 p.p.m. of chromium was used, both nitrification and carbonaceous oxidation were effected but the effluent was still passahle. I n a study of the B.O.D. test it was indicated that chromates may be reduced when added to full-strength seiyage and left during a 5day incubation period. These same authors (IS)have indicated that the addition of 10 p.p,m, of chromate to sewage in an activated sludge pilot plant eliminatrd nitrification without greatly impairing carbonaceous oxidation. In further work they (12) reported similar results in a stud\- of the effect of chromate in the B.O.D. test.
Ioc
80
e
2 60
+ z w
0
II:
p" 4 0
duction of chromates in sewage over several days, but they ohtained no information on the redox potential under which this occurred or the mechanism of its reduction. The review of the literature indicated so much confusion in assigning a value for the toxic level of chromium that it was considered necessary to choose exprrimcntal techniques that could use the same organisms under various environmental conditions. The clawical dilution B.0 D test (2) has a very limited concentration range, but it would serve as the technique for measuring toxicity a t low organic matter concentration under aerohic conditions. The Sierp or inanomctric B . 0 D. test as recently developed (7, 8) offers the choice of a very wide range of organic matter concentration as well as a choice of oxygen concentrations. The use of nitrates as the source of oxygen added to full-strength sewage would permit the study of the rate of oxygen uptake by a sewage sample under anaerobic conditions. Allen ( I ) found that highly oxygenated substances Twrr especially toxic to methylene blue reduction in the relative stability test. Because chromates carry a high content of oxygen the relative stability test should provide the means of studying the relative toxicity of chromic versus chromate ion, and indicate the fate of the oxygen in the chromate ion. The recording apparatus for studying relative stability of sewage developed in this lahoratory (10) was usrd in this study. Thus, these three B.O.D. tests hare permitted the study of the toxicity of chromic and chromate ions under aerobic and anaerobic conditions, under various organic matter concentrations, and under varying oxygen tensions. METHODS OF STUDY
20 I
0
I
4
8
12
1 16
20
I? P.M.CHROMIUMt 6
Figure 1.
Interference or Toxicity of Various Concentrations of Chromic Ion on B.O.D. 7.alues
Coburn (6) indicates that trivalent chioniium is very toxic in sludge digestion tanks in spite of its inso1uk)ilitya t sludge d i g s tiori pH values. He states that hexavalent chromium is so soluble that it mill not appear in the digestion tank, going off, instead. in the sewage. However, he intimates that it would be toxic in the digester if present. On the other hand, Barnes and Braidech (4)found that 5000 p.p.m. of trivalent chromium and 10,000 p,p.m. of hexavalent chromium could be tolerated in the digestion tanks, although the organisms produced only 41 and 68%, r e speetively, of the normal amount of gas. Southgate (18) agrees with Barnes and Braidech (4) that' trivalent chromium has very little effect on digestion. However, it has been reported ( 1 6 ) that the presence of 1 p.p.m. of hexavalent chroinium seriously interferes with digestion. I n unpublished work on sludge digestion, the senior author found hexavalent chromium highly toxic and trivalent chromium of low toxicity when gas production was used as the measure of toxicity. However, it is not known whether the chromate ion can act as a hydrogen acceptor in the anaerobic oxidation of organic matter in the same way as nitrate acts in the anacrobic digestion of paper pulp (19). Methane-producing bacteria must operate in a medium which has a very low redox potential (51, while nitrates are normally reduced a t redox potentials higher than the Eo of methylene blue on the basis of the relative stability test. It has been pointed out (11-13) that chromates retard nitrate ion formation, but there is no information concerning the possibility that chromates may retard nitrate reduction by niicroorganisms. Jenkins and I l r v i t t (11 ) have pointed out $light re-
Three methods or procedures r e r e used in the study of the toxicity of tri- and hexavalent chromium. Two of these were carried out under aerobic conditions and the other was conducted in the absence of free osygcn. The first of the aerobic tests was the classical dilution B.O.D., which was modified by the addition of equal amounts of chromium ion into two bottles, one containing only dilution water and the other containing dilution n-ater and sewage. The dissolved osygcn content (total iodine titration) of both bottles was determined at the end of the standard &day, 20" C. incubation period, and the difference was taken as thc B.O.D. of the sample. The ot,her aerobic test was carried out in the Sierp apparatus, wing the procedure as outlined hj- Falk and Rudolfs (7) anti as niodified by Gelman and Heukeleliian ( 8 ) . Various concentrations of chromium were introduced directly into a full-strength saniple of sewage, and the amount of oxygen required hy the sample was measured directly. This method proved particularly satisfactory, in measuring both the rate of oxygc 7 utilization and the total amount of oxygen used a t any given time. The pH of the sample was taken a t the end of incubation, as the authors believe that a pH of 8 provides an excellent indication of adequate carbon dioxide absorption, while an extremely high pH indicates contamination of the sample by some of the carbon dioxide , absorbent. Where the Heukelekian modification is to be u ~ c d it is strongly recommended that a pH value be taken of all samples incubated. The procedure used in the anaerobic or nitrate test was that described by Ingols (IO). This is essentially a modified relative stability test in xhich the amount of combined oxygen available to anaerobic bacteria is constant in all samples a t the beginning of the run. A limited amount of nitrate mas added to the full strength of sewage in order to get an end point in the control within 16 to 24 hours. h constant amount of available oxygen was provided by the use of a given amount of sodium nitratc with the trivalent chromium; then, with the hexavalent chromium, the total oxygen from the nitrate plus chromate was generally kept constant. Thus, four bottles were set up for one run with the nitrate and chromate solution placed in the bottles hrforc the
V O L U M E 2 4 , NO. 1 2 , D E C E M B E R 1 9 5 2
1883
sewage was added. The nitrate and chromate solutions were made up so that each contained the same amount of coinbined o\ygcn per milliliter. The control contained nitrate while the other three contained the same total volume (generally 4.0 nil.) of nitrate plus chromate solutions before the sewage was added. Fiecluently, the dye was added to thesewage before it was added to thc individual bottles The time required for the reduction of the iiitiate or chromate was taken as the point a t which the nicth~leneblue started to lose color as discussed in the original article (IO). The relative toxicities of different Concentrations of rhroinium were indicated by the increase in time taken for different samples to decolorize. Even in the control, the test with cei tniri sewage samples did not give B.O.D. values which were numerically comparable with the dilution B.O.D. values. However, in R study of reproducibility, replicates have hem shown to give excellent agrecment. Thus, the relative B.O.D. values and the relative toxicity from the chromium ions are considered valid The sewage samplrs used in the study were entirely domestic and 7wre taken from a local sewage treatment plant a t the same time each week in order to obtain as much uniformity of character as possible. The mean B.O.D. of the settled waste was about 200 p.p.m , and values varied very little froin this figure during the timr rovered by the studies reported here,
100
80
---- DILUTION
The data in Figure 2 show that there v a s no toxicity a t the oxygen concentrations resulting from saturation with pure osygen, while the hexavalent chromium was very toxic under anaerohic conditions. At first it was thought that a concentration of 4 p.p.m. of chromium (as chromate) r a s much more toxic than is reported in Figure 2 with nitrate reduction, until it mas notcd that there was no chromate left in the sample after incubatiou'at these concentrations. This mould confirm the observation of Jenkins and Hewitt (11) concerning the reduction of chromate when it had been added to undiluted sewage and left for scvrral days. Thus, when the chromate oxygen was added to the arailahle nitrate oxygen, the B.O.D.value showed a much lower apparent toxic effert. When higher amounts of chromate were used, the time of reduction was increased but the chromate waa eventually reduced. When a saiiqdv was sct up in duplicate, 20 p.p.ni. of chromium n-as completely reduced, yet the methylene blue in a replicate was not redurrd in anothpr 20 days. A test of a portion of the sample used to determine rhromates indicated that the organisms remaining were capable of oxidizing the organic matter present under aerobic conditions. A sample of sterile sei\ age gave no change in the chromate concentration over a period of 20 days. Because the dilution and manometric techniques vary both concentration and oxygen tension under aerobic conditione. the manometric technique was varied to use air on one series of tests in comparison with oxygen. The results, as shown in Table I, indicated that under aerobic conditions different oxygen tension had no effect on toxicity.
Table I. Effect of Oxygen Tension upon Toxicity of Both Oxidized and Reduced Forms of Chromium in Manometric B.0.1). Procedure
0:
: b
60
I-
Concn.,
z
P.P.hl.
W V
.4ir
Chroiniuin Valence +3 +6 l l a n o m e t r i c B.O.D. Values, P.P.M. Oxygen Air Oxygen
-_
E 40 a 20
A-
Thct rcwdtsJ nr shown in Talrlt' 11. iiidicared that a givrn of chroniium producrtl an approxiiiia tely constant amount of I3.O.D. redurtion. IYhrn this amount of B.O.D. was related to the total B.O.D., the percentage of thc reduction was lees at the higher B.O.D. values. aillorlilt
I
0
4
6
16
12
P P M CHROMIUM
20
+3
Figure 2. Interference or Toxicit) of \ ariocis Concentrations of Chromate Ion on B.O.D. Values
.\t times the srwage was supplcnit~iitc~l with vaTious h o \ m organic subst,ancee for the dual purpose of giving :i wider range of v:ilues with which t o work in the aerobic tests and for supplying an adequate amount or specific typr of Food in the aiiaerohic piwdure. RESULlS
1Iany preliminary runs 1Yit.h the yariouP B.0.1). tcchiiiqucs up in order to determine the range of toxicities that could he vzpected for the different techniques with the two chromium vnieiic4es. The results shown in Figures 1 and 2 were all obtained from one sample of sewage, but they are typical. The data for the trivalent chromium in Figure 1 show that in thc nianometric oxygen technique even 20 p.p.m. of chromium had very little effcc-t. This mas also true for the rates of osygen utilization and the M a y B.O.D. values, except for an initial heightened lag in soiiie samples where the control also showed some lag. The data also showed that the values from the dilution B.O.D. technique n-ere lo\~\-orthan those n-here nitrates served as the sourc(2 of
DISCUSSION
.4n understanding of the rncchanism of the toxic reaction would make it much easier to dctermiur a reasonable toxic level for any substance. Thus, this discussion is devoted largely to attempting to develop a concept of the mechanism of chromium tosicity from data in the literature and those given in this paper. It is now known that mercury, a heavy metal, forms a tight lrond with the sulfhydryl radical of casential enzymes and is thcrei'orcl not only toxic \ r u t I- of the chromic ion. The authors’ experimental results xere negative. As a third consideration, it would appear that the trivalent chromium could interfere with the bacteria itself in some nonspecific fashion. Heukelekian (9) indicates that many of the light metals show that chromium and the other metals are more toxic at a lower organic matter concentration, as is shown by the authors’ data and by the individual reports in the literature (6, I , $ ) . The authors interpret these data to mean that these medium sized particles (betmen sodium and mercury) exert their toxic influence by some nonspecific mechanical interference with metaholism. This would mean that a search for a specific toxic levcl Tyould be very tedious, a t best. Khile the toxic effect of the chromium iri the chromate ion is slightly lower than that from the same chromic ion concentration undcr aerobic conditions, the data show that the t’oxiclevcl of the chromate is also dependent upon the food concentration. This would indicate that the mechanism of the chromate ion toxicity under aerohic conditions may be the same as that for the chromic ion. Homver, the very high relative toxicity of the chromate iou under anaerobic conditions indicates that there may be a more specific mechanism under anaerobic conditions than has bren suggested as the mechanism for t,he chromic ion or for the chromate ion under aerobic conditions. On the basis that chromates are apparently toxic to both the oxidation of ammonia and reduction of nitrates, an explanation considered the possibility that the chromates were interfering with the enzyme systems responsible for nitrification because of a similarity on the spatial configuration of the chromate and nitrate ions. However, nitrate ion atoms are in a single plane, while chromate ion atoms form a t,etrahedron. The sulfate ion is also a tetrahedron and would he even more likely to cause interference than the chromate ion because of its size. Since the sulfate ion fails to cause interference in nitrate metabolism, it does not seem likely that spatial configuration is the cause of chromate toxicity. When it is maintained at p H 7.0 and 20” C., the chromate ion apparently has a redox potential similar to that of the nitrate ion. Thus, it is concluded that it interferes with the nitrate ion metaholism because of this similarity in redox potent,ials. This hypothesis implies that chromates pass into the cell and do not react with any particular cellular substance as does the mercuric ion. It further assumes that the chromates are reduced under those conditions and b y those organisms which reduce nitrates. The last assumption immediately raises the question of the possible production of chromate ions by the enzymatic oxidation of chromic ions. The occurrence of such oxidative transformation has not been observed except in possibly trace amounts over a period of 6 weeks in three series of experiments. However, chromium in its trivalent form is not a free metal ion but is highly hydrated in the center of a Werner complrs, shile the nitrogen in
ANALYTICAL CHEMlSTRY ammonia i> water-free and the ammonia is nonionized. The hydrogen of the ammonia is involved in chemosynthetic action, uhilc the chromic ion has no hydrogen. The question has been raised as to whether the chromates are reduced \Tithin the bacterial cell or are reduced by the chemical reaction of a barttxrial by-product outside the cell. This is obviouslv very difficult to answer, but a large number of experiments have bern carried out to gain a better understanding of the situation. The free sulfhydryl radical of cysteine can reduce chromate a t pH 8.0 Thus, chromates flowing in a stream over sludge beds may be reduced even in the presence of dissolved oxygen. This condition has been found in a small stream receiving an industrial waste containing both organic matter and chromates. It is realized that the mechanism whereby chromates are reduced under anaerobic conditions may be the result of the production of by-product sulfhydryl radicals from organic sulfur-bearing compounds. I n order to evaluate the necessity of organic sulfhydryl radical production, methionine (with a thio-ether group) was added to a sample of sewage and its stimulation t o chromate reduction was compared with the stimulation from a similar amount of dextrose. The dextrose and methionine required the same time foi reducing the chromate and methylene blue, indicating that deytrose u a s as good a hydrogen donor as a sulfhydrylbearing organic compound. It has been observed that the dye is decolorized after the chromate is reduced. I n order for the bacteria to produce organic sulfhydryl radicals such as mercaptans (thiols), it is understood by the authors that the bacteria would have to be operating in a medium mith a redox potential less than the Eo of methylene blue. If the methylene blue color persisted, one would assume that the bacteria ere operating in a medium of higher potential than is normal for sulfhydryl production. llethiouine, cystine, sodium nitrite, and sodium sulfide are all possible bv-products oi anaerobic metabolism, but they do not cause direct reduction of chromate a t pH 8 0; this does not facilitate reduction of chromate under the same anaerobic condition, possibly because the esccss sulfide ip too tosic for the bacteria. Because of the similarity in the redox potential of nitrate formation and carbonaceous oxidation, it may he that the chromate also interferes somewhat in carbonaceous oxidation by the similarity of the chromate potential to the carbonaceous oxidations. The general similarity of the toxicity ofchromateandchromic ions under aerobic conditions does not lend much weight to this idea. LITERATURE CITED
(1) Allen, L. A., A p p l . Bacteriol., 2, 26 (1949). (2) Am. Public Health Assoc., New York, “Standard Methods for the Analyses of Water and Sewage,” 9th ed., 1946. (3) Barker, H. A., A x h . Mikrobiol., 7, 420 (1936). (4) Barnes, G. E., and Braidech, M. &I., E n g . Sews Record, 129,
496 (19421. (5) Coburn, S.E., Sewage W o r k s J . , 21, 522 (1949). (6) Dawson, P. S. S., and Jenkins, S. H., Sewage a n d I n d . W a s t e s , 22, 490 (1950). (7) Falk, L. L., and Rudolfs, W., Sewage W o r k s J., 19 1000 (1947). (8) Gelman, I., and Heukelekian, H., Sewage and I n d . W a s t e s , 23,
1267 (1961). (9) Heukelekian, H., private communication. (10) Ingols, R. S.,Sewage W o r k s J., 21, 984 (1949). (11) Jenkins, S. H.. and Hewitt, C. H., J . SOC.Claern. I n d . , 59, 41 (1940). (12) Jenkins, S. H., and Hewitt, C. H., ,Vature, 147, 239 (1941). (13) Jenkins, S. H., and Hewitt, C. H., Suraeyor, 101, 211 (1942). (14) Krieger, H. S., and Moore, W. A., “Determination of the Toxicity of Trivalent and Hexavalent Chromium on Oxygen Utilization of Sewage,” U. S. Pub. Health Service, January 1949. (15) Laboon, J. F., Sewage W o r k s J., 21, 197 (1949). (16) Public W o r k s , 73, No. 11, 32 (1942). (17) Rudolfs, W.,e t al., Sewage a n d I n d . W a s t e s , 22, 1157 (1950). (18) Southgate, B. A , , “Treatment and Disposal of Industrial Tastes,” London, England, H. M. Stationery Office, 1948. (19) Straub, C. P., Sewage W o r k s J., 16, 30 (1944). (20) Wise, \V. S . , Ibid., 17, 338 (1945). R E C E I V Efor D revien. 3Iay 14, 19.52, Accepted N o r e m b e r 3 , 1952
