Environ. Sei. Technol. 1984, 18, 416-422
Development of a General Kinetic Model for Biodegradation and Its Application to Chlorophenols and Related Compounds Sujit Banerjee," Philip H. Howard, Arthur M. Rosenberg, Anne E. Dombrowski, Harlsh Slkka, and Denzil L. Tullis Life and Environmental Sciences Division, Syracuse Research Corporation, Syracuse, New York 13210
rn The kinetics of biodegradation of several compounds including phenol, resorcinol, p-cresol, benzoic acid, and various chloro derivatives of phenol, anisole, and resorcinol have been measured in a pure culture system and in two natural waters. Phenol, benzoic acid, and resorcinol supported growth of the organism in the pure culture study, while the other compounds were degraded through a cometabolic process by phenol-grown organisms. The rates spanned 5 orders of magnitude and decreased with increasing chlorine substitution but were relatively independent of the site of substitution. A relationship between rate and lipophilicity was observed, where the rate increased with decreasing lipophilicity and then leveled off. This finding suggested that the rate was controlled by a combination of the following factors: adsorption of substrate to the surface of the cell membrane, penetration of the lipid layer of the membrane, and diffusion through the hydrophilic pores in the membrane. These factors were incorporated into a model that accounted for our results and correlated data from several studies reported in the literature. Among the processes that govern the fate of a chemical in natural ecosystems, microbial degradation is one of the most important and least understood. Until recently, interest in biodegradation had been limited to the establishment of degradative pathways (I), and degradation rates had been treated only in general and qualitative terms. For example, while it was well-known that chlorine substitution enhanced recalcitrance, the extent of enhancement remained undefined. Interest in the quantitative aspects of biodegradation was sparked by the evolution of environmental models (2-4) where it was necessary to represent biodegradation by appropriate rate constants, and quantitative measurements of biodegradation have begun to appear over the past 5 years (5). Our objective was to measure biodegradation rates for a series of related compounds in the presence of both pure and naturally occurring mixed cultures of organisms and to develop a structure-degradability model for their interpretation. In this paper we present our results and validate the model with literature data. Experimental Procedures General and Analytical. All compounds were obtained from Aldrich Chemical Co. and were used without further purification. High-performance liquid chromatographic (HPLC) analyses were made with a Waters Associates pump and a Schoeffel GM-770 or LDC Spectromonitor I11 variable wavelength UV-vis detector. A 10-pm Lichrosorb RP-2 column was used, and the mobile phase consisted of various portions of 5% aqueous acetic acid and acetonitrile. Kinetics. The microorganism used for the pure culture studies was provided by Dr. Martin Alexander, Department of Agronomy, Cornell University. The bacterium was *Address correspondence to this author at the Safety and Environmental Protection Division, Brookhaven National Laboratory, Upton, NY 11973. 416
Environ. Sci. Technol., Vol. 18, No. 6, 1984
isolated on protocatechuic acid (3,4-dihydroxybenzoicacid) and is a Gram-negative nonmotile coccus measuring 0.5-0.75 pm and occurring singly or in pairs. The bacterium was grown in a sterile salts medium containing 0.5 g of (NH4)$04, 0.2 g of KC1, 0.1 g of NaC1, 0.2 g of MgS04.7H20, 0.05 g of CaC12.2H20and 0.02 g of FeC136H20 per L and was maintained at pH 7.2 in a 10 mM phosphate buffer. For the kinetic experiments, the microorganism was grown overnight with phenol (300 mg/L) as the sole carbon source. The cells were harvested by centrifugation for 15 min at lOOOOg at 4 OC, washed twice with 10 mM phosphate buffer, and resuspended in the buffer. Kinetic runs were initiated by allowing the suspension to warm to ambient temperature and by adding to it an aliquot of an. aqueous stock solution of the compound of interest. For the relatively insoluble compounds such as the tri-, tetra-, and pentachlorophenols, stock solutions were prepared in aqueous ethanol. Initial substrate concentrations ranged between 5 and 150 mg/L, and in all cases they were below toxic levels. Cell densities varied between 3 X lo8 and 2 X lo1' cells/mL. The suspension was sampled periodically, diluted with an equal volume of acetonitrile to remove the possibility of deposition of substrate, passed through a 0.2-pm Millipore filter, and analyzed by HPLC. All kinetic measurements were completed within 48 h. Biomass was quantitated by measuring the absorbance of the resting cell suspension (or an appropriate dilution) at 420 nm and by converting it to cell density through the use of eq 1. absorbance = -8.353-4 + 2.273-10 cell density + (n = 10) (r2 = 0.996) (1) 1.243-12 In (cell density) This equation was derived by making several dilutions from a suspension of the microorganism, counting each sample with a Petroff-Hauser counter, and making corresponding absorbance measurements at 420 nm. The kinetic runs were accompanied with or preceded by control experiments in order to ensure that the decrease in substrate concentration was entirely due to biodegradation. First, controls without the microorganism were run, and it was established that abiotic degradation did not occur. Second, results from zero time samples processed through the centrifugation-filtration stage were identical within experimental error with the expected values, and consequently, the possibility of significant absorption to glass or the Millipore filter was eliminated. The major metabolite from phenol was tentatively identified (by HPLC) as catechol. The metabolites for the other compounds were more polar than the starting substrates, and it is likely that they represented hydroxylated derivatives. The studies with natural waters were carried out on two separate occasions. In the first, water was collected from the Jamesville Reservoir and the Seneca River (Seneca Beach Road) in the vicinity of Syracuse, NY, on May 17, 1982. The water temperature at the time of collection was about 15 OC. The samples were stored refrigerated at 5 OC for 1day, following which the kinetic runs were started. Cell counts were performed on each water type by the plate count method using nutrient agar (BBL). The plates were
0013-936X/84/0918-0416$01.50/0
@ 1984 American Chemical Society
on the Monod equation (eq 3) for microbial growth, which
Table I. HPLC Retention Volumes of Some Chlorophenols
(3)
HPLC retention
compound
phenol 2-chlorophenol 3-chlorophenol 4-chlorophenol 2,3-dichlorophenol 3,4-dichlorophenol 2,4-dichlorophenol 2,4,5-trichlorophenol
volume, mL," 13/7-5% aqueous acetic acidlacetonitrile 6.5 9.4 10.6 10.1 14.1 15.2
logb KO,
(4)
1.46 2.15
(5)
2.50 2.39
13.6
3.06
21.4
3.72
2,3,4,5-tetrachlorophenol 31.3 39.7 5.86 pentachlorophenol OObtained with a Merck RP-2 column; average of two determinations. bFrom ref 9.
incubated for 48 h at 32 "C and counted on a NBS Biotran I1 automated colony counter. The kinetic runs were intiated by transferring 125 mL of the water sample to a 250-mL culture flask and adding 1mL of a stock solution of the substrate prepared in distilled water. The flasks were shaken in the dark at room temperature and sampled daily for the analysis of substrate and weekly for biomass analysis. It was found that the substrates degraded to a greater extent in the Seneca River water, and additional water was obtained from the Seneca River on June 28, 1982. The water temperature was 23 "C, and the kinetic experiments that were started immediately after collection were also performed at about 23 OC. Controls of autoclaved water with added chemical were processed along with the test samples for both sets of experiments to ensure that abiotic processes did not contribute to degradation. Measurement of Partition Coefficients. The partition coefficients (KO,) of some of the chlorophenols were obtained through use of the relationship between HPLC retention volume and KO, (7,8). While this relationship has its shortcomings when universally applied, it should be valid for compounds of similar structure. Retention volumes of compounds of known KO, were measured by using an acidic mobile phase to ensure that dissociation did not occur, and the data in Table I led to eq 2 which was used to calculate the partition coefficients of 2,3- and 3,4-dichlorophenol and 2,3,4,5-tetrachlorophenol. log KO, = 5.43 log (retention volume) - 3.09
(r2= 0.98) (2)
The partition coefficients of 4-chloroanisole, 4-chlororesorcinol, and 3,5-dichloroanisolewere measured in triplicate by the general method followed earlier by us (10). Briefly, this involved dissolution of the substrate into purified water saturated octanol and equilibration of the solution with octanol-saturated water. The phases were separated by centrifugation and analyzed by HPLC. The results obtained were as follows: 4-chlororesorcinol(62.8 f 2.5), 4-chloroanisole (598 f 39), and 3,5-dichloroanisole (6320 f 1000). It is important to note that the value for 4-chlororesorcinol refers to that of the undissociated species since the water layer was strongly acidic. When phosphate buffer of pH 7.0 was substituted for water, the KO, dropped to 3.91. Results and Discussion Kinetic Results from Pure Culture Studies. The interpretation of biodegradation rates has typically relied
is analogous to the Michaelis-Menten equation for enzymatic processes (5,11).The parameters p and refer to the actual and maximum growth rate, S is substrate concentration, and K, is the concentration of substrate at which p = 0.5h. The degradation rate of a compound has been expressed by eq 4 which follows from eq 3. Here, S and B represent substrate and biomass, respectively, and Y is the yield factor. At low substrate concentrations where K, > S, eq 4 reduces to eq 5 which predicts that the rate of decrease of substrate is of the first order with respect to substrate and to biomass. This equation has been successfully applied to the biodegradation of several compounds using both pure and mixed cultures (12-15). At high substrate concentrations where [SI > K,,eq 4 yields eq 6 where the rate is of the first order with respect to biomass and is independent of substrate concentration.
-d[SI -
pm
PI dt - -Y Equation 4 applies to idealized situations where the substrate supports growth of the organism. In our studies, only phenol, benzoic acid, and resorcinol supported growth, and the other compounds degraded by essentially a cometabolic process. Although phenol was absent during the degradation of these compounds, the organism was initially grown on phenol, and the enzymes responsible for degradation were therefore induced with phenol. By definition, a cometabolic process is one where the compound undergoing degradation does not support growth (161, and therefore, pm and Y in eq 4 must both equal zero. This, of course, makes the equation unusable. Corresponding equations describing the kinetics and bioenergetics of cometabolism have not been developed, and in their absence it is difficult to compare degradation rates of growth substrates with those that are cometabolized. We have circumvented the problem by redefining cometabolism as a process where utilization of the compound is immeasurably small but nonzero. This allows brnand Y to be defined as small, but also nonzero and enables eq 4 to be extended to cometabolic processes. The dependence of rate on biomass and on initial substrate concentration was thoroughly evaluated with phenol and to a limited extent with the other compounds. The rate-biomass profile for phenol is illustrated in Figure 1, and a linear relationship was obtained in accordance with eq 4. In contrast, the rate was effectively independent of phenol concentration as shown in Figure 2. The two runs in Figure 2 made at low substrate concentrations were somewhat slower than the others. However, since only a few data points were taken for these runs, it is possible that the lag phase could not be fully distinguished from the region where degradation occurred. This would depress the true rate, as observed. In all, 34 runs were made for phenol, and the kinetics (corrected for variations in biomass) showed no dependence on initial phenol concentration (r2= 0.07). Most of the remaining compounds were more toxic than phenol (6), and the independence of rate on substrate concentration could not be verified over quite Environ. Sci. Technol., Vol. 18,
No. 6, 1984 417
T a b l e 11. S u m m a r y o f K i n e t i c D a t a f r o m P u r e C u l t u r e Studies
no.
compound
1 2 3 4 5 6 7 8 9 10
phenol 2-chlorophenol 3-chlorophenol 4-chlorophenol 2,4-dichlorophenol 3,4-dichlorophenol 2,3-dichlorophenol 2,4,5-trichlorophenol 2,3,4$-tetrachlorophenol aniso1e 4-chloroanisole 3,5-dichloroanisole resorcinol 4-chlororesorcinol benzoic acid p-cresol 2,6-dichlorophenol 2,2'-biphenol pentachlorophenol
11
12 13 14 15 16 17 18 19
rate, mol.cell-'.h-'
(k% SD)
1.083-16 (77) 3.493-17 (38) 4.323-17 (47) 5.443-17 (32) 3.763-19 (47) 6.843-19 (38) 2.663-18 (19) 1.433-20 (77) 7.143-22 (41) 2.863-17 (8) 2.293-18 (36) 1.453-20 (62) 7.903-18 (9) 11.2E-18 (31) 6.843-17 (18) 1.723-17 (76) NW NW NW
na
krel
log K o w b
log Dc
34 10 5 5 11 5 12 8 4 4 6 9 4 2 4 5
150 000 49 000 61 000 76 000 530 960 3 700 20 1 40 000 3 200 20 11000 16 000 96 000 24 000
1.46 2.15 2.50 2.39 3.06 3.33d 3.15d 3.72 5.03d 2.11 2.78" 3.80" 0.80 1.80e 1.87 1.94
1.46 2.13 2.49 2.39 2.98 3.31 3.03 3.121 3.4d
0.80 0.5ge -1.13 1.96
5.86
3.4d
"Number of kinetic runs. bFrom ref 9 unless otherwise noted. CCalculatedwith pK, values from ref 17 by the method in ref 18 unless otherwise noted. Calculated from eq 2. e Measured in this study. f Calculated with pK, values from ref 19. NO detectable degradation. 140
T
220
1
Hours
Figure 1. Dependence of the rate of phenol disappearance on biomass concentration. The symbols refer to the following biomass in cells per milllllter. (0)3.05E8, (A)6.09E8, (V)1.22E9, (0)1.83E9, (A)2.44E9, and (0) 3.05E9.
as wide a range. Nevertheless for 2-chloro-, 3,4-dichloro-, and 2,4,5-trichlorophenol, the kinetics were found to be invariant over at least a fivefold change in substrate concentration. Hence, all the rate data were processed with eq 6, and the results are summarized in Table 11. Two of the compounds studied, biphenol and 2,6-dichlorophenol, resisted degradation under all conditions. The recalcitrance of the latter compound can be understood if the degradation pathway is considered. The first step in phenol metabolism is hydroxylation to catechol (20),and this route is unavailable to 2,6-dichlorophenol. A suitable alternate pathway is also evidently unavailable, as might be expected for a cometabolic process. While changes in regiochemistry have been documented in cometabolic degradation (21))they are quite rare. The unreactivity of biphenol would seem to suggest that the enzyme system responsible for the degradation of monocyclic compounds does not act on larger bicyclic derivatives. 418
Environ. Sci. Technol., Vol. 18, No. 6, 1984
b
1
i
3
4
5
6
HOURS
Figure 2. Dependence of rate of phenol degradation on inltial phenol concentration. The biomass was 3.73E10 cells per mL.
Correlation of Data from Pure Culture Studies. The relationship between our kinetic data and the octanol-water partition coefficient (Kow)is illustrated in Figure 3. The correlation shows that the rate increases with decreasing lipophilicity and then appears to level off for compounds with log Kow k3 + k4, and eq 10 results. For hydrophobic compounds, k l / k z
0
I
I
I
1
I
3
2
4
I 5
I
6
log KO,
Figure 3. Variation of log kmlwith log Kow. The numbers refer to the compounds in Table 11.
adsorption-desorption of the substrate from solution to the cell wall, k3 governs the rate /i3(hydrophobic)
~
S
+
B
S-B xp
\
\
k4(hydrophillc)
of penetration of the compound through the lipid layer, and k4 is the rate constant for diffusion through the hydrophilic pores, It is assumed that active transport does not occur and that k3 and k4 are rate limiting; Le., the enzymatic process after diffusion through the cell membrane is rapid, and the adsorption-desorption equlibrium on the cell wall governed by k l and k z is also established rapidly. If the steady-state approximation is applied to the intermediate S-B, then eq 7 which is analogous to the
Michaelis-Menten equation can be derived. Here, [B], is total biomass ([B] [S-B]). Now, at high substrate concentrations where most of the organism is associated with the substrate, k l [ S ] > k z k3 h4 and eq 7 simplifies to eq 8. For hydrophobic compounds, k4 will be negligibly
+
+ +
small, and it is likely that k3 will be inversely related to
KO,. On the other hand, for hydrophilic compounds, k4 will be dominant, and the rate will bear no relationship to KO,. Thus, at high substrate concentrations, curve B in Figure 4 will result. At low substrate levels where k 2 + k3 + k4 > k l [SI, eq 7 reduces to eq 9. Now, since the adsorption-desorption
(9)
will be directly related to KO,, whereas k3 will vary inversely with KO,,and the kinetics could display a positive or negative dependence on KO,depending on the relative sensitivities of the two relationships. For hydrophilic substrates, the rates will be independent of KO, as discussed above. Hence, at low substrate concentrations, either curve A or curve B in Figure 4 could result. Our experimental data in Figure 3 follows curve B in Figure 4 as expected from the model, and we conclude that for the more lipophilic compounds, lipid penetration is rate determining. The scatter observed for the relatively hydrophilic compounds is expected, since in this region there is no relationship between rate and KO,,and the effect of other rate-controlling factors such as steric or electronic contributions becomes apparent. It is also clear why substitution of a chlorine atom in phenol or resorcinol leads to no major differences in rate, but the same substitution has a sizable effect on anisole. The KO,values for phenol, resorcinol, and their monochloro derivatives are in the region where rate is independent of KO,;i.e., k4 is rate determining. Conversely, the chloroanisole by virtue of its higher KO,is in the region where k3 begins to be important, and chlorine substitution in anisole leads to a rate decrease. An intriguing aspect of the data in Figure 3 is that compounds such as benzoic acid and the tetrachlorophenol, which will be ionized in the medium, are apparently governed by the same correlation that applies to the undissociated and neutral compounds in the series. Scherrer and Howard (28) have discussed the effect of dissociation on partitioning and have suggested that the distribution coefficient D be used in place of KO,for acidic or basic compounds. This coefficient is the concentration ratio of a compound in octanol to that of all related species, whether neutral or ionic, in water. Values for D are included in Table 11, and they differ appreciably from KO, for the tri- and tetrachlorophenols, 4-ch1ororesorcino1, and benzoic acid. However, these differences do not distort the correlation for reasons which will be apparent from the following discussion. Consider first, the effect of dissociation on eq 8 which is appropriate for high substrate concentrations and where the observed rate constant equals k3 kq. For hydrophilic
+
Environ. Sci. Technol., Vol. 18, No. 6, 1984
419
Table 111. Summary of Rate Data in Natural Waters
no. 1 4 5 8 9 15 19 1 4 5 8 9 15 19 1 15
compound Seneca River (first collection) phenol 4-chlorophenol 2,4-dichlorophenol 2,4,5-trichlorophenol 2,3,4,5-tetrachlorophenol benzoic acid pentachlorophenol Seneca River (second collection) phenol 4-chlorophenol 2,4-dichlorophenol 2,4,5-trichlorophenol 2,3,4,5-tetrachlorophenol benzoic acid pentachlorophenol Jamesville Reservoir phenol benzoic acid
initial concn, mg/L
1% time, h
rate, M*h-'
cell density, cells/mL
rate, rnol.h-'.cell-'
19 20 21 20 7 15 7
20 170 200
3.23-6 6.53-6 2.33-6
1E3 2E4 1E5
3E-12 4E-13 2E-14
400 0
1.4E-7 4.23-6
3E5 1E3
5E-16 4E-12
15 14 20 19 7 18 7
23 200 60 640
1.8E-6 1.3E-6 l.lE-6 9.33-8
2E3 4E4 6E6 2E4
9E-13 3E-14 2E-16 5E-15
0 360
1.5E-6 l.lE-7
1E5 4E3
2E-14 3E-14
15 15
140 20
4.93-7 9.83-7
2E5 1E5
3E-15 1E-14
compounds such as benzoic acid and 4-ch1ororesorcino1,
k4 will be the dominant rate constant, and replacement of KO,with D will move these values along the flat portion of curve B in Figure 4,and the general shape of the curve will therefore be unaffected. For lipophilic compounds such as the tri- and tetrachlorophenols, ionization will lower the lipid-water distribution of the substrates. Nevertheless, these compounds will still be preferentially partitioned into the lipid phase, and transport through the hydrophilic pores will still be negligible. The rate-controlling step will continue to be diffusion through the lipid phase, and since the chlorophenols will be undissociated in the lipid, the rate constant k3 which correlates with KO, rather than with D will dominate. In other words, while dissociation will alter the partitioning somewhat, the change will be insufficient to affect the rate-limiting step which is related to KO,. Thus, the position of these compounds in Figure 3 will remain virtually unchanged. For compounds of lower KO,,dissociation may cause the rate-determining step to be changed, and in this case the appropriate point on curve B in Figure 4 will move to the hydrophilic region of the curve. The situation is expected to be slightly more complex at low substrate concentrations where the measured rate constant is ( k 1 / k 2 )(k3 + k4). The arguments made above for the effects of dissociation on k3 + k4 remain unchanged, but the additional effect of k l / k 2must now also be considered. This ratio governs the adsorption-desorption equilibria and is therefore related to D rather than to KO,. As before, dissociation of hydrophilic compounds will merely move the points in question along the flat portion of the curve in Figure 4, but for lipophilic compounds, deviations from the curve may occur. Kinetic Results from Natural Water Studies. In order to test the applicability of our model to environmental situations, we measured the rates of degradation of a number of phenols and benzoic acid in two natural water-the Seneca River (SR) and the Jamesville River (JR). The measurements were made on two occasions as described earlier. Rates were calculated from the linear portion of the decay curve and are expressed on a per cell basis by dividing the rate by the biomass at or just before the point at which degradation started. Benzoic acid and phenol were the only compounds which degraded in JR water, whereas organisms in the SR water 420
Environ. Sci. Technol., Vol. 18, No. 6, 1984
proved to be more active. This is not surprising since the SR receives domestic and industrial effluents, while the JR is relatively clean, and the organisms in the SR are probably adapted to phenolic compounds to a greater degree than those from the JR. Examination of the data in Table I11 reveals that the reactivity of the systems studied followed the order SR > JR > pure culture. The greater reactivity of the microorganisms in the SR water over those in the JR water is to be expected if the former is adapted to a greater extent to phenolic compounds. The slower rate for the pure culture system is probably caused by reduced metabolic activity in the resting cell suspension compared to the natural water system. A high degree of variability exists for the two sets of data for the SR water. In the first set of results, trichlorophenol and pentachlorophenol are recalcitrant, and the tetrachlorophenol degrades, while the converse is true for the second set. We attribute these differences to variable adaptation times rather than to a fundamental difference in the degradation mechanism. Spain and co-workers (24, 25) have shown that adaptation in natural water samples can be affected by several factors such as substrate concentration, prior exposure, and the presence of specific microorganisms. The variation in the relative degradation rates particularly for benzoic acid and phenol points out the biological variability that occurs with time even at the same collection site. Similar results have been noted by Pfaender and Bartholomew (26). In this context, the experience of Paris et al. (129,who found little variability in rate with the source of the organism, is surprising, and it is possible that the high degree of consistency that they observed is attributable to the relatively high reactivity of their substrates. The SR data (first collection) appears to be governed by curve B in Figure 4, and the relationship to KO,is illustrated in Figure 5. The one exception, of course, is that of the trichlorophenol which should have degraded according to our model but proved to be recalcitrant in practice, and we attribute this difference to a long adaptation time for the compound. A similar fit of the second set of SR data to our model is less satisfactory, particularly for pentachlorophenol which deviates sharply from the line. A mechanistic change may be responsible for the deviation since both sites ortho t o the phenolic group are substituted, and therefore, catechol formation cannot oc-
11
-
.
1 .
12-
Table v. Biodegradation of Phthalate Esters by Organisms in Activated Sludge"
15
compound
krel
Rt, min
dimethyl phthalate
6.0
0.18
4.2
0.22 0.27 0.35 0.46 0.56 0.68 0.91
diethyl phthalate dipropyl phthalate
01
dibutyl phthalate dipentyl phthalate dihexyl phthalate diheptyl phthalate dioctyl phthalate
04
13-
-
6.8 8.5
5.8 4.4 4.6
1
aData obtained from ref 27.
e!
o4
?i
1
-E
0'
5
015
Table VI. Biodegradation of Phenols"
I
14-
compound p-methoxyphenol
O8
p-acetylphenol phenol p-cyanophenol p-nitrophenol p-cresol p-chlorophenol p-bromophenol "Data obtained from ref 28.
15-
9.
o5 16-
I
I
I
2
I
1
4
Figure 5. Variation of the rate of degradation of substrates in Seneca River water with Kow. ( 0 )First collection;(0)second collection. The numbers refer to the compounds In Table 111.
Table IV. Biodegradation of Aliphatic Alcohols by Organisms in Activated Sludge" krel
log KO,
1.0 3.0
1.02 1.48
1-hexanol 8.3 13 1-heptanol 1-octanol 38 a Data obtained from ref 23.
2.41 2.80
1-pentanol
log Kow
13.2
1.34 1.35 1.46
20.4 457 1.00 6.61
309 112 105
1.6 1.91 1.92 2.39 2.59
1 6
log KO,
compound 1-butanol
krel
1.99
cur. I t may be recalled that 2,6-dichlorophenol did not conform to the correlation in our pure culture studies for what we believe to be similar reasons. Correlation of Literature Data. The success of our model in correlating our kinetic resulb prompted us to test the generality of the model by applying it to literature data, where a series of homologous compounds had been studied in the same system. Yonezawa and Urushigawa (23) measured the biodegradation of aliphatic alochols by activated sludge, and some of their results are summarized in Table IV. In contrast to our results with phenols, their rates increased with increasing KO,,and thus, k l / k 2 in Figure 4 would appear to be dominant. According to our model this situation will arise at low substrate concentrations where eq 10 applies, and the kinetics show a first-order dependence on substrate. Yonezawa and Urushigawa used concentrations of approximately 50 pg/L and observed first-order kinetics, and our model is therefore entirely consistent with their results. In a separate study, the same authors measured the biodegradation of phthalate esters by activated sludge (27). Their results are listed in Table V where Rt (min), the retention time of a substrate on a reversed-phase column, is proportional to Kow. The R, - log krel relationship re-
sembles curve B in Figure 4,and our model would suggest that permeation of the lipid layer was rate limiting. The degradation of phthalate esters was also studied by Wolfe et al. (149, and their rates varied inversely with KO, in accordance with curve B in Figure 4. These authors, however, obtained a better correlation with the rate of alkaline hydrolysis but did not rationalize their results through a model. Interpretation of their results through our model is straightforward. As a final example, consider the data (Table VI) of Paris et al. (28), who measured the degradation of para-substituted phenols. Their rates were well correlated with the van der Waals radii but showed no correlation whatsoever with KO,. All the substrates, however, were fairly polar, and the rate data can easily be accomodated by our model if k4 in Figure 4 dominates. In other words, penetration of the cell occurs through the hydrophilic pores, and no correlation with KO,is therefore expected.
Conclusions In this work, biodegradation rates were measured for a number of phenols and related compounds. A model for the semiquantitative evaluation of these rates was developed and was found to apply to a number of previously reported studies. The model is based on the premise that penetration of the bacterial cell membrane is rate determining and that enzymes capable of degrading the compounds of interest are available. For lipophilic compounds, this suggests that the rate can be related to the octanolwater partition coefficient. As with most structure-activity relationships, the model assumes a constancy of degradation mechanism, and in its present form its environmental application is likely to be limited. Some exceptions have already been encountered, and while the deviations of these compounds from the model can be rationalized after the fact, they cannot be reliably predicted. The model also does not take into account factors such as toxicity, synergism, and antagonism or the effects of extracellular enzymes. However, the model can be used to estimate rate data by interpolation. For example, if rates for a series Environ. Sci. Technol., Vol. 18, No. 6 , 1984 421
of related compounds are available and can be related by the model, then it should be possible to estimate the rates of other compounds in the series whose properties fall within the range of the series.
Acknowledgments We thank Robert Brink and Robert Boethling for several discussions and Martin Alexander for providing us with the organism. Registry No. Phenol, 108-95-2; 2-chlorophenol, 95-57-8; 3chlorophenol, 108-43-0; 4-chlorophenol, 106-48-9; 2,4-dichlorophenol, 120-83-2;3,4-dichlorophenol, 95-77-2; 2,3-dichlorophenol, 576-24-9;2,4,5-trichlorophenol, 95-95-4; 2,3,4,5-tetrachlorophenol, 4901-51-3; anisole, 100-66-3; 4-chloroanisole, 623-12-1; 3,5-dichloroanisole, 33719-74-3;resorcinol, 108-46-3;4-chlororesorcinol, 95-88-5; benzoic acid, 65-85-0; p-cresol, 106-44-5.
Literature Cited (1) Gibson, D. T. In “The Handbook of Environmental
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