I 4
VERNON T. STACK, Jr. Development Department, Carbide and Carbon Chemicals Co., South Charleston, W. Va.
Toxicity of Alpha, Beta-Unsaturated Carbonyl Compounds to Microorganisms
,Toxicity of aJ3-unsaturated aldehydes and ketones is related to chemical reaction of the carbonyl or a,@conjugated carbonyl groups with the bacteriological system
b Toxicity of a/P-unsaturated esters and neutralized acids results from a probable condensation reaction between the ethylenic linkage and the bacteriological system through 1,4addition
T H E toxicity of a material to microorganisms is generally related to the system in which it is present. Changes in p H or temperature may influence the observed results. The presence of other materials, organic and inorganic, may also have an effect. Therefore, the toxicity of organic chemicals to microorganisms should be evaluated in a system as similar as possible to the one in which biochemical oxidation is to take place. The evaluation presented here is strictly preliminary. The results may be considered quantitative only for the system described, but a qualitative application of the general conclusions is possible. A system identical to the system for the B.O.D. determination was employed for toxicity studies. The toxic materials were diluted in the standard B.O.D. dilution water. Incubation temperature was 20' C. Procedure for Toxicity Test. A series of dilutions of the material to be tested was made in the standard B. 0.D. dilution water. Each dilution contained the same amount of a reference material for biological, oxidation, but not enough to deplete the dissolved oxygen, and the
same amount of the desired bacteriological seed. The dilutions were incubated a t 20' C. for 3 days, and the residual dissolved oxygen was determined. If the residual dissolved oxygen was greater than a blank containing the reference material and bacteriological seed, toxic conditions were assumed to exist. The toxicity threshold is defined as the concentration of the toxic material which produces the same dissolved oxygen residual as the reference material. Any concentration of the toxic material greater than the concentration a t the toxicity threshold will cause the residual dissolved oxygen to increase. The possible effect of other organic materials on the results, through reaction or synergistic actions, waa, anticipated. T o maintain uniform experimental conditions, an aqueous solution of organic materials was prepared as an oxidation reference (Table I). Results obtained in the presence and absence of this oxidation reference did not suggest any synergistic action for the substrate and the toxic material. The fact that results could vary with the concentration and quality of the seed cultures was also considered. A laboratory culture, developed with microorganisms from domestic sewage, was fed with the oxidation reference described above. The resulting culture was more reliably consistent than domestic sewage with respect to concentration of enzymes and microorganisms. This culture served as the source of unacclimated microorganisms for the toxicity evaluations. Determination of the toxicity of a,@unsaturated carbonyl compounds was complicated by their tendency to condense or polymerize. T o ensure a
minimum content of complexes or polymers in the initial materials, products were obtained that had not been stored for an extended period. The majority of these samples contained a polymerization inhibitor, usually hydroquinone. Toxicity of a,@-Unsaturated Aldehydes and Ketones to Unacclimated Microorganisms
The toxicity data for a,@-unsaturated aldehydes and ketones (Table 11) show observed results in accord with expected rates of reactivity. Acrolein, the most reactive, has the most toxic effect. Methacrolein, which should be slightly
Table 1.
Biological Oxidation Reference" Material
Concn., P.P.M.
Ethanol 200 Methanol 100 Acetic acid 500 Acetaldehyde 50 Sodium acetate 200 Ethyl acetate 100 Butyl acetate 100 Benzene 50 Toluene 50 Phenol 10 Ethylene glycol 10 Acetone 20 Methyl isobutyl ketone 20 Polyethylene Glycol 400 10 Ethylene dichloride 5 Diethanolamine 5 Monoethanolamine 10 Formaldehyde 50 a Materials added t o tap water and employed as reference material for biological oxidation in the toxicity test.
VOL. 49, NO. 5
MAY 1957
91 3
less reactive because of the methyl substitution on the a-carbon, is correspondingly less toxic. The other materials fit into the order, as might be expected from the rates of reactivity in a given reaction. Cinnamaldehyde appears to show too much toxicity, but rhe phenyl group probably accounts for this result. Methyl isopropenyl ketone polymerized appreciably when added to water. This should explain its low toxicity in comparison with methyl vinyl ketone. The decreased toxicity of 4-pentenal is attributed to the absence of the conjugated system. The observed toxicity data suggest that the toxicity mechanism functions because of a chemical reaction or reactions. If this is assumed to be true, it is important to consider the form of the materials in a dilute aqueous solution. In the case of a,@-unsaturatedaldehydes and ketones a hydration reaction results, which proceeds through a 1,I-addition reaction to yield a saturated 3,4-addition product (4). 0
I1
R-CHxCHC-Ri
+ HzO 1
OH
The equilibrium of this reaction is significantly to the right because of the electrophilic reactivity of the conjugated system. There is always a tendency for the carbonyl groups of aldehydes and ketones to be hydrated (7). 0
I1
R---CH-CH&R
4-HzO
&
I
OH O H OH R-CH-CH&--R
I/
AH
This reaction is secondary to the 1,4hydration reaction and applies to the @hydroxy derivative. AS the carbonyl group of the @-hydroxy derivative does not possess a high degree of electrophilic reactivity, the equilibrium of the 1,2hydration reaction is far to the left. The net result is that in a dilute aqueous solution the a,?-conjugated carbonyl system is reduced in concentration. The reactive groups in the aqueQUS solution that are likely to enter into a toxicity reaction are the unhydrated, a,@-conjugated carbonyl groups and the carbonyl groups of the @-hydroxy derivative.
9 14
Table 11.
Toxicity of a$-Unsaturated Aldehydes and Ketones to Microorganisms
iMaterial Acrolein Methacrolein Crotonaldehyde 2,4-Hexadienal Cinnamaldehyde 2-Ethyl-3-propylacrolein 4-Pentenala Methyl vinyl ketone Methyl isopropenyl ketone a
Toxicity Threshold t o Unacclimated Microorganisms P.p.m. miM
1.5 3.5 14 18 13 >300 62
1.5 35
0.027 0.050 0.20 0.19 0.098 >2.38 0.74 0.022 0.42
18
0.32 0.043
100 100
36 50
0.51
100
0.52
100
160
1.21
100
120 1.5 35
1.43 0.022 0.42
3
...
...
.*
30 70 70
Included for comparison
In the following discussion these groups are referred to as the reactive carbonyl groups. The conjugated carbonyl groups may enter into a twofold toxicity reaction. A conjugate addition to the ethylenic linkage may occur through a 1,4-addition reaction. The resulting product would still contain a carbonyl group that could enter into condensation reactions. A theoretical toxicity mechanism can be developed if it is assumed that: The a,?-unsaturated aldehydes and ketones are partially hydrated in a dilute aqueous solution by a reversible 1,4addition reaction to yield a ?-hydroxy derivative. The toxic effect is produced by reaction of the reactive carbonyl groups with the bacteriological system. Based on these assumptions are three sets of reactions which govern the quantity of cu,@-unsaturatedaldehyde or ketone required to produce toxic conditions in a given bacteriological system. Condensation, polymerization, or addition reactions decrease the concentration of the reactive carbonyl groups. Biochemical oxidation reactions decrease the concentration of reactive carbonyl groups. The reactive carbonyl groups react with the bacteriological culture to produce the toxic effect, probably a denaturing of the enzymes present. The reactions of the first type usually occur before an industrial waste enters a biochemical oxidation system. They reduce the over-all concentration of reactive carbonyl groups, but this effect is not necessarily in competition with reactions of the second and third types. Such reactions are always in competition for the reactive carbonyl groups. Where n reactive carbonyl groups are required to stop biochemical oxidation effectively, and the third type of reaction progresses slowly, a portion of the reactive carbonyl groups is destroyed before the bacteriological culture is deactivated. Therefore, the initial concentration of reactive carbonyl groups required to produce toxic conditions must be greater
INDUSTRIAL AND ENGINEERING CHEMISTRY
Toxicity Thrsshold t o Acclimated Microorganisms Days of P.p.m. mM Acclimation
than n, and the rates of the latter two reactions types determine this initial concentration. The theoretical toxicity mechanism should apply to both saturated and unsaturated aldehydes and ketones, but the reactivity of the conjugated carbonyl systems should favor the toxicity reaction. The development of toxic conditions with lower concentrations of a,@unsaturated aldehydes rather than with saturated aldehydes is expected. Table I11 shows this to be true for unacclimated microorganisms. Polymerization, condensation, and addition reactions affect the concentration of the carbonyl groups in an aqueous solution of a saturated aldehyde. For example, a dilute aqueous solution of formaldehyde contains very little free formaldehyde; the major portion exists as methylene glycol and polymers of formaldehyde (7). The theoretical considerations suggest that the toxic conditions produced by a,@-unsaturated aldehydes and ketones are biostatic in nature. Continuation of the toxic conditions, once established, i s dependent upon the existence of an excess of reactive carbonyl groups. This is probably true, but the data presented in this paper express only the development of toxic conditions. The ability of a biochemical oxidation system to recover from the toxic effects of a,p-unsaturated aldehydes and ketones was observed in the laboratory. Acclimated cultures which were fed just enough material to establish toxic conditions recovered within a few days. This phenomenon was not evaluated. The data for the toxicity of a,@-unsaturated aldehydes and ketones (Table 11) are assumed to reflect the balance among the three sets of theoretical reactions. The observed toxicity data should be dependent upon the experimental conditions. If the conditions are changed, a different toxicity threshold should be established. To illustrate this, toxicity threshold values were determined for methyl vinyl ketone with increasing amounts of bacteriological
TOXlClTY OF CARBONYL COMPOUNDS IO0
Table 111. Toxicity Thresholds of Unacclimated Microorganisms to Saturated Aliphatic Aldehydes Toxicity Thrashold,
Material Formaldehyde Acetaldehyde Propionaldehyde Eutyraldehyde
P.P.M. a5 500 >500
>500
I
I
I I No BOD. for Acrolein or M e t h y l Vinyl Ketone d u e t o towlclty
I
I
i
n
3 80 5n 5 (3
>
8 60 -I 3 I=
W
K
seed added to the experimental system (Table IV). The increase in the toxicity threshold value with an increase in the quantity of seed agrees with the theoretical reactions and is assumed to reflect the denaturing of the enzyme system. The toxicity threshold value of 4.5 p.p.m. for 1 ml. of seed is greater than the 1.5 p.p.m. shown in Table 11. This is attributed to polymerization of the methyl vinyl ketone after 4-month storage. These data amplify the fact that the toxicity threshold values presented in this paper have only a qualitative importance. The data in Figure 1 show the B.O.D. curves for the a,&unsaturated aldehydes and ketones with unacclimated microorganisms. Biochemical oxidation of these materials develops readily when the concentration of the material is less than the toxicity threshold, The concentrations of acrolein and methyl vinyl ketone were greater than the toxicity threshold, and no B.O.D. developed. The concentration of methacrolein in the B.O.D. determination was borderline to the toxicity threshold, and the B.O.D. development was retarded. The slow rate of B.O.D. development for methyl isopropenyl ketone is assumed to be due to polymerization of this material. The other materials show a reasonable development of B.O.D. The observed decrease in the efficiency of the B.O.D. development appears to be related to reactivity, possibly the rate of hydration of the carbonyl groups.
:r:40 I-
IA
0
+ 2 W 0
20
a
0
The ability of microorganisms to develop a tolerance to the toxic effects of a,&unsaturated aldehydes and ketones was evaluated through acclimation of cultures to the particular material. Procedure for Acclimation of Microorganisms to Toxic Organic Materials. A 6-liter container was supplied with air for aeration. Six liters of river water were placed in the container. Approximately 50 p.p.m. of the organic material being studied was added. A lower concentra-
2
4 6 DAYS OF INCUBATION
8
IO
Figure 1 . B.O.D. curves for a,/?-unsaturated aldehydes and ketones with unacclimated microorganisms
tion was used if toxic conditions were expected. Sufficient diammonium phosphate was added to supply the nitrogen and phosphorus required as growth fattors. T h e pH was adjusted to a value between 6 and 8. Thimixture was aerated continuowly, and the C.O.D. was determined daily. Additional organic material was'added when the C.O.D. values indicated that a significant removal of the organic material had occurred. The concentration of the toxic material was gradually in-
Table Iv* Effect
Of Concentration of Unacclimated Microorganisms on Toxicity of Methyl Vinyl Ketone
Bacteriological Seed, M1.
Toxicity Threshold, P.P.M.
1
4.5
2
5.5 6.5
3
IO0
n
z
e
5n
80
z W
? X -I
Toxicity ob a,/?-Unsaturated Aldehydes and Ketones to Acclimated Microorganisms
4
0
60
e -0 c W K
E 40 lL
0
c z
W 0
a 20
0
0
2
4 DAYS
6
8
IO
OF INCUBATION
Figure 2. B.O.D. curves for a,P-unsaturated aldehydes. and ketones with acclimated microorganisms VOL. 49, NO. 5
MAY 1957
915
creased as acclimation advanced, as determined by toxicity tests with the culture. Growth factors other than nitrogen and phosphorus were added by removing one half of the liquid volume from the container once each week and adding an equal amount of tap water. Discussion. Based on the hypothetical reactions of the toxicity mechanism, acclimation can produce an improved tolerance in two ways :
Table V.
Material Methyl acrylate Butyl acrylate Methyl methacrylate Ethyl crotonate Acrylic acida Methacrylic acid" Crotonic acid5 2-Ethyl-3-propylacrylic acid" Sorbic acid" Fumaric acida
By the development of a more highly catalytic enzyme system for the destruction of the reactive carbonyl groups. By increasing the concentration of the enzymes and microorganisms affected by the toxic reaction of the material. Both changes are important, but the second is probably more responsible for the successes shown for acclimated culture in Table 11. The data for toxicity of methacrolein, methyl vinyl ketone, and methyl isopropenyl ketone show no response to acclimation. Apparently, no improvement in the concentration or the biochemical activity of the bacteriological cultures occurred. This situation is indicated in the B.O.D. curves for a,& unsaturated aldehydes and ketones shown in Figure 2. These data, with the exception of methyl vinyl ketone, were obtained at concentrations below the toxicity threshold. Of particular interest is the fact that acclimation has not changed the lag period in the B.O.D. development for methacrolein and methyl isopropenyl ketone. The balance between the degradation and toxicity reactions is apparently unchanged. Therefore, an improvement in the tolerance of the bacteriological cultures through the two changes above cannot occur. Obviously, the presence of the methyl group on the cr or carbonyl carbon has a significant effect on the biochemical oxidation of an a,P-unsaturated aldehyde or ketone. The presence of these groups or larger alkyl groups is expected to reduce the reactivity of the conjugated system. The rate of the toxicity reaction is decreased, but more significant is the retardation of B.O.D. development. This characteristic would seriously hamper the disposal of these materials by biochemical oxidation methods.
Toxicity of a,P-Unsaturated Acids and Esters to Microorganisms
a
Toxicity Threshold t o Unacclimated Microorganisms P.p.m. mM 60 0.68 150
1.17
>300 >300
>3.0 >2.6 1.4 1.2 2.9 >2.1 >2.7 92.6
100
100 250 >300 >300 >300
Toxicity Threshold to Acclimated Microorganisms Days of P.p.m. mM acclimation 140 275
...
... >300 >300 >300
*..
...
...
1.63
2.1
... ... >4.2 >3.5
>3.5
...
... ...
70 70
.. ..
70 70 90
.. .. ..
Neutralized with sodium hydroxide.
0
/I
+ HgO
RCH=CHC-ORx
0
RCH=CH
e
OH
+ RiOH
Attention, therefore, is drawn to the unhydrated unsaturation and the acidity of carboxyl groups when considering probable toxicity reactions of the esters and acids. The toxicity thresholds for a,p-unsaturated acids and esters are shown in Table V. The acids were neutralized before addition to the biological oxidation system. The solutions of the esters were buffered only by the buffering capacity of the B.O.D. dilution water. The observed toxicity thresholds established by methyl and butyl acrylate might possibly be the result of acidic conditions. The data in Figure 3 show that the acidity of acrylic acid can readily break through the buffering capacity of the standard dilution water for the B.O.D. determination. A p H of 5 or less is generally detrimental to bacteriological cultures, and this effect was observed for
acrylic acid a t a concentration of 25 p.p.m. The application of the effect of acidic conditions to the observed results for methyl and butyl acrylate depends upon the rate of hydrolysis. This was evaluated by preparing a series of dilutions of methyl acrylate in the standard dilution water for the B.O.D. determination. Each dilution contained the same amount of an unacclimated culture (Table VI). Unacclimated microorganisms were added to each dilution, 0.3 ml. per 100 ml. These results demonstrate that the toxicity of methyl acrylate is not the result of acidic conditions. Hydrolysis of the ester to the acid to a slight extent is suggested by the decrease in the pH, but this decrease is not enough to be detrimental to bacteriological activity. These data suggest that the toxic effects observed for a,P-unsaturated esters and acids are the result of chemical reactions. The existence of a condensation reaction involving the carbonyl group of the acid or ester and a reactive group on an enzyme is not considered feasible. A condensation might occur
Toxicity of a,&Unsaturated Acids and Esters to Microorganisms
The hypothetical mechanism of toxicity suggested for unsaturated aldehydes and ketones does not appear to be directly applicable to unsaturated acids and esters. The carboxyl groups of these acids and esters do not normally enter into addition or condensation reactions in a manner analogous to carbonyl groups of aldehydes or ketones, although hydration of the ethylenic linkage will occur. Instead, in an aqueous solution an ester may hydrolyze to the acid (3).
9 16
ACRYLIC
ACID, Pp.m
Figure 3. pH of acrylic acid solutions in standard dilution water for B.O.D. determination
INDUSTRIAL AND ENGINEERING CHEMISTRY
TOXICITY O F CARBONYL COMPOUNDS
1
100
-----
, a
B
eo
Crotonic Acid
z
Sorbic Acid 2-Ethyl-3-Propyl Acrylic Acld
e
5
I
Acr IIC ACld Melhcrylic ~ c t d Fumoric Acid
60
c 4
8 z
’a e
60
Y
L 8 E 40
P 8
U
0 U
c
40
I-2
w
0
I
6
eo
n
eo
0
0
0
2
6
4 6 DAYS OF INCUBATION
0
2 CHZ=CH- !i OCzH6
+ CHa-NHz0
/
J
CHzCHz OCzHs
Table VI.
6
e
IO
12
Figure 5. B.O.D. curves for a,@-unsaturated esters with both acclimated and unacclimated microorganisms
a,p-unsaturated esters do not respond as readily. The data in Figure 5 show generally retarded rates of oxidation for esters, only ethyl crotonate showing a significant improvement with acclimated microorganisms. The ready oxidation of the acids and data for the hydrolysis of methyl acrylate suggest that the retarded B.O.D. development of the esters may be related to a slow hydrolysis of the ester to the acid. Conclusions
The hypothetical mechanism for toxicity of a,@-unsaturatedesters and acids is similar, therefore, to that for a,@-unsaturated aldehydes and ketones, except that only the reactivity of the conjugated carboxyl system is considered. The conjugated carboxyl system of the acid and esters have a slower rate of reactivity than the similar system in aldehydes an! ketones. Therefore, the balance between the toxicity reaction and biochemical degradation should result in less toxicity for acids and esters (Table V). The a,@-unsaturated acids (Figure 4) are readily oxidized biochemically. The
4
DAYS OF INCUBATION
Figure 4. B.O.D. curves for a,@-unsaturatedacids with unacclimated microorganisms
through a conjugate addition similar to the addition of methylamine to ethyl acrylate (2).
2
0
10
8
The order of toxicity for analogous a,@unsaturated compounds is aldehyde, ketone, ester, and acid. The threshold values of toxicity observed in a particular biochemical system are quantitative for that system only. Qualitative application of the data should be true for other systems. The mechanism of toxicity produced by aldehydes and ketones appears to be related to a chemical reaction of the carbonyl group or the a,@-conjugated carbonyl system with the bacteriological system. The a,P-conjugated carbonyl system produces the greater toxic effect.
Hydrolysis of Methyl Acrylate in Standard Dilution Water for B.O.D. Determination
PH
Concn.,
P.P.M.
Initial
1 day
2 days
3 days
250 500 1000 2000
6.88 6.90 6.89 6.91
6.85 6.78 6.72 6.77
6.72 6.68 6.58 6.68
6.62 6.63 6.50 6.65
The degrees of toxicity for a,@-unsaturated aldehydes and ketones appear to be modified by the balance of three reactions:
1. Polymerization, condensation, or addition reactions of the a,@-unsaturated aldehyde or ketone. 2. The biochemical degradation of the carbonyl groups or the a,@-conjugated carbonyl system. 3. The toxicity reactions. The toxicity of an a,@-unsaturated aldehyde or ketone to unacclimated microorganisms is decreased by any substitution of an alkyl group that reduces the rate or degree of reactivity of the a,& conjugated carbonyl system. The toxicity threshold established by an a,P-unsaturated ester is not the result of a hydrolysis to the acid and the development of acidic condition. The toxic effects of a,P-unsaturated esters and neutrahed a,P-unsaturated acids appear to be the result of a reaction which is possibly a condensation of the ethylenic linkage with the bacteriological system through a 1,4-addition reaction. The acclimation of microorganisms to a,@-unsaturatedaldehydes which have an alkyl substitution on the a carbon and to a,@-unsaturated ketones is limited by a retarded B.O.D. development which favors the toxicity reaction. literature Cited
Royals, E. E., “Advanced Organic Chemistry,” p. 626, Prentice-Hall, New York, 1954. Zbid..> AD. 6- 6- 7 . Ibid.,p. 668. Ibid., p. 753.
RECEIVED for review May 25, 1956 I ACCEPTED October 31, 1956 VOL. 49, NO. 5
M A Y 1957
917
