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QSARs and PARs for biodegradation of PCBs
The Science of the Total Environment, 109/110 (1991) 275-281 Elsevier Science Publishers B.V., Amsterdam
275
QSARs and PARs for biodegradation of PCBs J.R. Parsons*, L.C.M. Commandeur, H.E. van Eyseren and H.A.J. Govers Department of Environmental and Toxicological Chemistry, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, Netherlands
ABSTRACT Relationships between the biodegradation rate constants of a number of polychlorinated biphenyls (PCBs) and hydrophobic and electronic structural parameters are compared. There is no simple relationship with octanol-water partition coefficients, indicating that the biodegradation rates of PCBs are probably not determined by their rates of permeation through the bacterial membranes. Biodegradation rate constants correlated much better with both the electronic and hydrophobic properties of the chlorine substituents, which suggests that the reactivity and possibly enzyme binding of PCBs control their biodegradation rates.
INTRODUCTION
There is increasing interest in QSARs which describe the biodegradation (metabolism by microorganisms) of organic chemicals [1, 2]. One of the most important reasons for this interest is that such relationships may make it possible to predict the rates at which chemicals are degraded by microorganisms in the environment. Another use of such relationships is for the study of mechanisms of biodegradation and the processes which control biodegradation rates. In general, the rate of biodegradation of a chemical will be determined by its rate of uptake by and transport within bacterial cells, by its binding to the active site of an enzyme, or by the rate at which it is transformed. Each of these potentially rate-determining steps may give rise to correlations with characteristic molecular structure parameters [3]. In the absence of specific uptake mechanisms, synthetic organic chemicals are probably taken up by bacteria by passive diffusion through the lipid membrane [4]. If the rate of this process determines the rate of biodegradation, correlations with hydrophobic parameters would be expected. If, on the other hand, the biodegradation rate is determined by the binding of the compound to the active site of an enzyme or by the rate of the transformation reaction, correlations with electronic or steric parameters which influence the reactivity of the compound would be * Author to whom correspondence should be addressed. 0048-9697/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved
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expected, although hydrophobicity may also influence the binding of the compound to the active site of the enzyme. Several relationships have been reported between biodegradation rates and hydrophobic parameters such as octanol-water partition coefficients and reversed-phase HPLC retention [1]. Other relationships have been reported with steric parameters such as the Van der Waals radii of substituents on an aromatic ring, and with electronic parameters such as the Hammett substituent constants or the atomic charge differences across a key bond [1]. For some classes of chemicals, for example substituted phenols, correlations have been reported between biodegradation rate parameters and both hydrophobic and electronic parameters [1]. Most of the relationships reported to date have been for compounds which have relatively high biodegradation rates. Few such relationships are known for such poorly degradable, relatively persistent, chemicals as chlorinated aromatic compounds. We compare here relationships between the biodegradation rate constants of a class of chlorinated aromatic chemicals, the polychlorinated biphenyls (PCBs), and hydrophobic and electronic parameters. METHODS Two sets of biodegradation rate constants were used in this study. The first set is based on the initial biodegradation rates of PCBs with two to five chlorine substituents by suspensions of Alcaligenes and Acinetobacter strains reported by Furukawa et al. [5]. These values were divided by the initial concentrations of the PCBs to give pseudo-first-order biodegradation rate constants (k(,, Table 1). The second set consists of pseudo-first-order biodegradation rate constants of tetra-, penta-, and hexachlorobiphenyls in continuous cultures of Alcaligenes strain JB1 (Table 1). These data were determined by continuously exposing a 3-methylbenzoate-grown culture to a mixture of the PCBs, as reported previously [6]. Biodegradation rates under these conditions were similar to those in cultures exposed to individual congeners. Octanol-water partition coefficients (Kow) were taken from Shiu and Mackay [7]. The values of the substituent constants for chlorine substituents were taken from Hansch and Leo [8] (Hammett constants: ao = 1.20, O"m 0.37, trp = 0.23; inductive constant, al = 0.46; hydrophobic constant, n = 0.71). In general, the first reaction in the oxidative biodegradation of PCBs is dioxygenation of the least substituted ring in the ortho and meta positions [9]. Assuming that initial attack was in the meta position of the least substituted ring, we summed the a and al values for the least substituted ring to give Ea and Ea z for each congener. Where more than one value of Ea could be =
277
QSARs AND PARs FOR BIODEGRADATION OF PCBs
TABLE 1 Biodegradation rate constants and structural parameters used in this study PCB
2,3 2,6 3,4 3,5 2,2' 2,4' 3,3' 4,4' 2,3,4 2,4,5 2,4,6 2,2',5 2,Y,5 2,4',5 2,4,4' 2,3',4' 2,3,4,5 2,2',3,3' 2,2',5,5' 2,2',6,6' 2,2',4,4',6 2,2',4,5,5' 2,3',4,4',6 2,3',4,5',6 2,2',3,Y,4,4' 2,2',3,3',6,6'
k~, (h-l) a
Log Kowe
p6 b
Y42 c
0.928 0.082 0.882 0.964 0.280 0.982 0.370 0.504 0.640 0.648 0.920 0.102 0.826 0.608 0.804 0.772 0.380 0.146 0.070 ND
> 1
Eol g
E~
0 0 0 0 0.23 0.23 0.37 1.2 0 0 0 0.23 0.37 1.2 1.2 0.23 0 0.6 1.57 1.43 1.43 1.57 1.57 0.37 1.8 1.8
0 0 0 0 0.46 0.46 0.46 0.46 0 0 0 0.46 0.46 0.46 0.46 0.46 0 0.92 0.92 0.92 0.92 0.92 0.92 0.46 1.38 1.38
1.42 1.42 1.42 1.42 1.42 1.42 1.42 1.42 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.84 2.84 2.84 2.84 3.55 3.55 3.55 3.55 4.26 4.26
JBI d
5.0 5.3 5.4 4.9 5.1 5.3 5.3
ND i
> 1 > 1 0.126 0.964 ND 0.324 0.702 0.920 0.062 0.032 0.842 0.436 0.826 0.312 0.516 0.174 ND 0.028
Errf
5.6 5.5 5.6
11.537 0.499 0.786 1.957 0.603 1.271 1.262 0.920 2.658
5.7 5.8 5.8 5.9 5.6 6.1 5.9 6.4
7.0 6.7
aFirst order biodegradation rate constant, bAcinetobacter strain P6 [4]. CAlcaligenesstrain Y42 [4]. dAlcaligenes strain JB1 (this work), eOctanol-water partition coefficient [6]. rSum of Hammett substituent constants [7]. gSum of inductive substituent constants [7]. hSum of substituents' hydrophobic constants [8]. ~No degradation detected.
c a l c u l a t e d , t h e l o w e s t v a l u e w a s used. T h e i n f l u e n c e o f s u b s t i t u e n t s o n t h e m o s t s u b s t i t u t e d r i n g w a s a s s u m e d t o be negligible. T h e h y d r o p h o b i c c o n s t a n t s (re) f o r c h l o r i n e s u b s t i t u e n t s w e r e s u m m e d f o r b o t h rings t o give Xrc as a p a r a m e t e r f o r t h e h y d r o p h o b i c i t y o f t h e m o l e c u l e . C o r r e l a t i o n a n a l y s i s
was carried out with the Statpak package (North West Analytical, Portland, Oregon).
2 7 8
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+
I
0 []
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-1
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+
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I
,
6
,
i
,
I
7
log K o w []
Acinetobacter P6
+
Alcaligenes JB1
Fig. 1. Relationship between log Kowof PCBs and their pseudo-first-order rate constants for biodegradation by two bacterial strains.
RESULTS AND DISCUSSION The data in Table 1 show that, in general, biodegradation rates of PCBs decrease with increasing number of chlorine substituents and that there are significant differences in the relative rates of biodegradation by the three strains. The much higher values of k~, for strain JB 1 compared with those for the other strains is probably at least partly a result of differences in experimental conditions. As is shown in Fig. 1, there is no simple relationship between log Kow of the PCBs and their biodegradation rate constants, such has been reported for, e.g., chlorophenols [10] and alkyl esters of p-aminobenzoic acid [11]. (For clarity, only data for strains P6 and JB 1 are shown in this figure.) In general, the rates of diffusion of organic chemicals through biological membranes increase with increasing hydrophobicity until, for highly hydrophobic chemicals, the rates of diffusion in the unstirred water layers surrounding the membrane become rate limiting [12]. This indicates that the biodegradation rates of these compounds are not determined by their rates of permeation through the cell membranes. The biodegradation rates therefore apparently depend on factors influencing reactivity or binding to enzymes. Correlations of biodegradation rates of aromatic compounds with Hammett substituent parameters have been reported for substituted benzoic acids [13] and mono- and disubstituted phenols and anilines [14]. Recently, Peijnenburg et al. described structure-activity relationships for abiotic and
QSARs A N D PARs FOR B t O D E G R A D A T I O N O F PCBs
279
microbial dehalogenation of haloaromatic compounds in sediments [15]. In these relationships, they used as structural parameters a , a t , the substituents' Taft steric parameter E s and the carbon-halogen bond strength. The first reaction in the biodegradation of PCBs is dioxygenation to give 2,3-dihydroxylated [9], or in some cases 3,4-dihydroxylated [16], metabolites. Dioxygenations are reactions with molecular oxygen in which both oxygen atoms are incorporated into the hydroxylated metabolites. Although the mechanism of dioxygenation has not been fully characterized, it is generally considered to involve initial attack by oxygen to give a peroxy intermediate [13, 17]. For the purposes of this study, we assumed initial attack at an unchlorinated m e t a position of the least chlorinated ring, which could lead to both 2,3- and 3,4-dihydroxylated products. Hammett constants for o r t h o substituents are poorly characterized, but the value of 1.2 given by Hansch and Leo [8] was determined for 2-chlorotoluene from the t3C-H coupling constant [18] and would seem applicable to PCBs. No correlations were found for the biodegradation rate constants reported for strains Y42 and P6. For example, for the relationship of log k~ for strain P6 with Z a the value of r 2 was 0.06. Similarly, no correlations were found between log k(, and go- and log Kow or Ea and En (r 2 = 0.06 and 0.12, respectively). The biodegradation rate constants listed in Table 1 for strain JB1 were determined under identical conditions in continuously growing cultures exposed to a mixture of PCBs. These rate constants can therefore be reliably compared. The data for 2,3',4,5',6-pentachlorobiphenyl were not used in this analysis as this PCB has only chlorinated m e t a positions on the least chlorinated ring. For the remaining eight PCBs there was a poor correlation between log k~, and Zo- with r 2 = 0.52. However, a much better correlation was obtained using the parameters Z a and Zrr: log k~, = 0.26 (_+0.44) (r 2 = 0.86,
F = 14.8,
1.44 (_+0.26) Ea + 0.58 (_+0.17) En
(1)
s.e.r. = 0.20)
The experimental values of log k~, are plotted against those predicted from Eqn (1) in Fig. 2. Including the three parameters Eo-, Za~ and En in the relationship gave a slightly higher r 2 value of 0.87, but an F value of 9.2. For the six PCBs of this series for which values of log Kow were available, the correlation of log k(~ with Z a and log Kow ( r 2 = 0.71) was worse than that with Ea and 2 n (r 2 = 0.86). Similar calculations for initial attack at the o r t h o positions and those using other values for a o gave much poorer correlations (data not given). These results suggest that the rates at which the tetra-, penta- and hexachlorobiphenyls are degraded by strain JB1 are to a large extent determined
280
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2
t _Q 2d ID1 0
o
[]
-1 0
1
2
log kb (calc) ( h - l )
Fig. 2. Comparison of experimental log k~, values and those calculated from Y~trand Y.n [Eqn (1)].
by electronic effects of the chlorine substituents, but that the hydrophobic effects of the substituents are also important. Surprisingly, the increasing Ere with higher chlorination has a positive influence on the biodegradation rate. The hydrophobicity of these compounds lies in the range where diffusion through the unstirred water layers surrounding the membrane are expected to be rate limiting for uptake by the cells (see above) [12]. Therefore, the favourable effect of increased hydrophobicity of the higher chlorinated congeners may be related to stronger binding to the active site of the enzyme. Clearly, the value for r2 of 0.86 for Eqn (1) as well as the differences in (relative) biodegradation rate constants for different strains, mean that other factors also have an important influence on biodegradation rates. These factors may be related to such biological factors as enzyme specificity. Nevertheless, despite their shortcomings, studies such as this do yield insights into the processes which determine biodegradation rates and may eventually make prediction of such rates possible. REFERENCES 1 J.R. Parsons and H.A.J. Govers, Quantitative structure-activity relationships for biodegradation. Ecotoxicol. Environ. Saf., 19 (1990) 212-227. 2 P. Kuenemann, P. Vasseur and J. Devillers, Structure-biodegradability relationships, in W. Karcher and J. Devillers (Eds), Practical Applications of Quantitative StructureActivity Relationships (QSAR) in Environmental Chemistry and Toxicology, Kluwer, Dordrecht, 1990, pp. 1-12. 3 J.C. Dearden, Physico-chemical descriptors, in W. Karcher and J. Devillers (Eds),
QSARs AND PARs FOR BIODEGRADATION OF PCBs
4 5
6 7
8 9
10
1! 12
13
14 15
16
17 18
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Practical Applications of Quantitative Structure-Activity Relationships (QSAR) in Environmental Chemistry and Toxicology, Kluwer, Dordrecht, 1990, pp. 25-59. J.N. Bateman, B. Speer, L. Feduik and R.A. Hartline, Naphthalene association and uptake in Pseudomonas putida, J. Bacteriol., 16 (1986) 155-161. K. Furukawa, K. Tonomura and A. Kamibayshi, Effect of chlorine substitution on the biodegradability of polychlorinated biphenyls. Appl. Environ. Microbiol., 35 (1978) 223-227. J.R. Parsons and D.T.H.M. Sijm, biodegradation kinetics of polychlorinated biphenyls in continuous cultures of a Pseudomonas strain. Chemosphere, ! 7 (1988) 1755-1766. W.Y. Shiu and D. Mackay, A critical review of aqueous solubilities, vapor pressures, Henry's law constants, and octanol-water partition coefficients of the polychlorinated biphenyls. J. Phys. Chem. Ref. Data, 15 (1986) 911-929. C. Hansch and A.L. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley, New York, 1979. S.W. Hooper, C.A. Pettigrew and G.S. Sayler, Ecological fate, effects and prospects for the elimination of environmental polychlorinated biphenyls (PCBs). Environ. Toxicol. Chem., 9 (1990) 655-667. S. Banerjee, P.H. Howard, A.M. Rosenberg, A.E. Dombrowski, H. Sikka and D.L. Tullis, Development of a general kinetic model for biodegradation and its application to chlorophenols and related compounds. Environ. Sci. Technol., 18 (1984) 416-422. J.R. Parsons, A. Opperhuizen and O. Hutzinger, Influence of membrane permeation of biodegradation kinetics of hydrophobic compounds. Chemosphere, 16 (1987) 1361-1370. G.L. Flynn and S.H. Yalkowsky, Correlation and prediction of mass transport across membranes. I: Influence of alkyl chain length on flux-determining properties of barrier and diffusant. J. Pharm. Sci., 61 (1972) 838-852. W. Reineke and H.-J. Knackmuss, Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on 1,2-dioxygenation of benzoic acid. Biochim. Biophys. Acta. 542 (1978) 412-423. P. Pitter, Correlation of microbial degradation rates with the chemical structure. Acta Hydrochim. Hydrobiol., 13 (1985) 453-460. W.J.G.M. Peijnenburg, M.J. 't Hart, H.A. den Hollander, D. van de Meent, J.H. Verboom and N.L. Wolfe, The Development of a Structure-Activity Relationship for the Reduction of Halogenated Aromatic Hydrocarbons in Anaerobic Water-Sediment Systems, Rep. No. 718907002, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands, 1990. R. Unterman, D.L. Bedard, M.J. Brennan, L.H. Bopp, F.J. Mondello, R.E. Brooks, D.P. Mobley, J.B. McDermott, C.C. Schartz and D.K. Dietrich, Biological approaches for polychlorinated biphenyl degradation. Basic Life Sci., 45 (1988) 253-269. J.M. Wood, Chlorinated hydrocarbons: oxidation in the biosphere. Environ. Sci. Technol., 16 (1982) 291A-297A. R.E. Hess, C.D. Schaeffer Jr. and C.H. Yoder, 13C-H coupling constants as a probe of ortho-substituent effects. J. Org. Chem., 26 (1971) 2201-2203.
275
QSARs and PARs for biodegradation of PCBs J.R. Parsons*, L.C.M. Commandeur, H.E. van Eyseren and H.A.J. Govers Department of Environmental and Toxicological Chemistry, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, Netherlands
ABSTRACT Relationships between the biodegradation rate constants of a number of polychlorinated biphenyls (PCBs) and hydrophobic and electronic structural parameters are compared. There is no simple relationship with octanol-water partition coefficients, indicating that the biodegradation rates of PCBs are probably not determined by their rates of permeation through the bacterial membranes. Biodegradation rate constants correlated much better with both the electronic and hydrophobic properties of the chlorine substituents, which suggests that the reactivity and possibly enzyme binding of PCBs control their biodegradation rates.
INTRODUCTION
There is increasing interest in QSARs which describe the biodegradation (metabolism by microorganisms) of organic chemicals [1, 2]. One of the most important reasons for this interest is that such relationships may make it possible to predict the rates at which chemicals are degraded by microorganisms in the environment. Another use of such relationships is for the study of mechanisms of biodegradation and the processes which control biodegradation rates. In general, the rate of biodegradation of a chemical will be determined by its rate of uptake by and transport within bacterial cells, by its binding to the active site of an enzyme, or by the rate at which it is transformed. Each of these potentially rate-determining steps may give rise to correlations with characteristic molecular structure parameters [3]. In the absence of specific uptake mechanisms, synthetic organic chemicals are probably taken up by bacteria by passive diffusion through the lipid membrane [4]. If the rate of this process determines the rate of biodegradation, correlations with hydrophobic parameters would be expected. If, on the other hand, the biodegradation rate is determined by the binding of the compound to the active site of an enzyme or by the rate of the transformation reaction, correlations with electronic or steric parameters which influence the reactivity of the compound would be * Author to whom correspondence should be addressed. 0048-9697/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved
276
J . R PARSONS ET AL.
expected, although hydrophobicity may also influence the binding of the compound to the active site of the enzyme. Several relationships have been reported between biodegradation rates and hydrophobic parameters such as octanol-water partition coefficients and reversed-phase HPLC retention [1]. Other relationships have been reported with steric parameters such as the Van der Waals radii of substituents on an aromatic ring, and with electronic parameters such as the Hammett substituent constants or the atomic charge differences across a key bond [1]. For some classes of chemicals, for example substituted phenols, correlations have been reported between biodegradation rate parameters and both hydrophobic and electronic parameters [1]. Most of the relationships reported to date have been for compounds which have relatively high biodegradation rates. Few such relationships are known for such poorly degradable, relatively persistent, chemicals as chlorinated aromatic compounds. We compare here relationships between the biodegradation rate constants of a class of chlorinated aromatic chemicals, the polychlorinated biphenyls (PCBs), and hydrophobic and electronic parameters. METHODS Two sets of biodegradation rate constants were used in this study. The first set is based on the initial biodegradation rates of PCBs with two to five chlorine substituents by suspensions of Alcaligenes and Acinetobacter strains reported by Furukawa et al. [5]. These values were divided by the initial concentrations of the PCBs to give pseudo-first-order biodegradation rate constants (k(,, Table 1). The second set consists of pseudo-first-order biodegradation rate constants of tetra-, penta-, and hexachlorobiphenyls in continuous cultures of Alcaligenes strain JB1 (Table 1). These data were determined by continuously exposing a 3-methylbenzoate-grown culture to a mixture of the PCBs, as reported previously [6]. Biodegradation rates under these conditions were similar to those in cultures exposed to individual congeners. Octanol-water partition coefficients (Kow) were taken from Shiu and Mackay [7]. The values of the substituent constants for chlorine substituents were taken from Hansch and Leo [8] (Hammett constants: ao = 1.20, O"m 0.37, trp = 0.23; inductive constant, al = 0.46; hydrophobic constant, n = 0.71). In general, the first reaction in the oxidative biodegradation of PCBs is dioxygenation of the least substituted ring in the ortho and meta positions [9]. Assuming that initial attack was in the meta position of the least substituted ring, we summed the a and al values for the least substituted ring to give Ea and Ea z for each congener. Where more than one value of Ea could be =
277
QSARs AND PARs FOR BIODEGRADATION OF PCBs
TABLE 1 Biodegradation rate constants and structural parameters used in this study PCB
2,3 2,6 3,4 3,5 2,2' 2,4' 3,3' 4,4' 2,3,4 2,4,5 2,4,6 2,2',5 2,Y,5 2,4',5 2,4,4' 2,3',4' 2,3,4,5 2,2',3,3' 2,2',5,5' 2,2',6,6' 2,2',4,4',6 2,2',4,5,5' 2,3',4,4',6 2,3',4,5',6 2,2',3,Y,4,4' 2,2',3,3',6,6'
k~, (h-l) a
Log Kowe
p6 b
Y42 c
0.928 0.082 0.882 0.964 0.280 0.982 0.370 0.504 0.640 0.648 0.920 0.102 0.826 0.608 0.804 0.772 0.380 0.146 0.070 ND
> 1
Eol g
E~
0 0 0 0 0.23 0.23 0.37 1.2 0 0 0 0.23 0.37 1.2 1.2 0.23 0 0.6 1.57 1.43 1.43 1.57 1.57 0.37 1.8 1.8
0 0 0 0 0.46 0.46 0.46 0.46 0 0 0 0.46 0.46 0.46 0.46 0.46 0 0.92 0.92 0.92 0.92 0.92 0.92 0.46 1.38 1.38
1.42 1.42 1.42 1.42 1.42 1.42 1.42 1.42 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.13 2.84 2.84 2.84 2.84 3.55 3.55 3.55 3.55 4.26 4.26
JBI d
5.0 5.3 5.4 4.9 5.1 5.3 5.3
ND i
> 1 > 1 0.126 0.964 ND 0.324 0.702 0.920 0.062 0.032 0.842 0.436 0.826 0.312 0.516 0.174 ND 0.028
Errf
5.6 5.5 5.6
11.537 0.499 0.786 1.957 0.603 1.271 1.262 0.920 2.658
5.7 5.8 5.8 5.9 5.6 6.1 5.9 6.4
7.0 6.7
aFirst order biodegradation rate constant, bAcinetobacter strain P6 [4]. CAlcaligenesstrain Y42 [4]. dAlcaligenes strain JB1 (this work), eOctanol-water partition coefficient [6]. rSum of Hammett substituent constants [7]. gSum of inductive substituent constants [7]. hSum of substituents' hydrophobic constants [8]. ~No degradation detected.
c a l c u l a t e d , t h e l o w e s t v a l u e w a s used. T h e i n f l u e n c e o f s u b s t i t u e n t s o n t h e m o s t s u b s t i t u t e d r i n g w a s a s s u m e d t o be negligible. T h e h y d r o p h o b i c c o n s t a n t s (re) f o r c h l o r i n e s u b s t i t u e n t s w e r e s u m m e d f o r b o t h rings t o give Xrc as a p a r a m e t e r f o r t h e h y d r o p h o b i c i t y o f t h e m o l e c u l e . C o r r e l a t i o n a n a l y s i s
was carried out with the Statpak package (North West Analytical, Portland, Oregon).
2 7 8
JR.
PARSONS
ET
AL.
+
I
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[]
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-1
[] []
0
-2
i
4
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i
i
L
i
5
r
i
+
+
[]
L
I
,
6
,
i
,
I
7
log K o w []
Acinetobacter P6
+
Alcaligenes JB1
Fig. 1. Relationship between log Kowof PCBs and their pseudo-first-order rate constants for biodegradation by two bacterial strains.
RESULTS AND DISCUSSION The data in Table 1 show that, in general, biodegradation rates of PCBs decrease with increasing number of chlorine substituents and that there are significant differences in the relative rates of biodegradation by the three strains. The much higher values of k~, for strain JB 1 compared with those for the other strains is probably at least partly a result of differences in experimental conditions. As is shown in Fig. 1, there is no simple relationship between log Kow of the PCBs and their biodegradation rate constants, such has been reported for, e.g., chlorophenols [10] and alkyl esters of p-aminobenzoic acid [11]. (For clarity, only data for strains P6 and JB 1 are shown in this figure.) In general, the rates of diffusion of organic chemicals through biological membranes increase with increasing hydrophobicity until, for highly hydrophobic chemicals, the rates of diffusion in the unstirred water layers surrounding the membrane become rate limiting [12]. This indicates that the biodegradation rates of these compounds are not determined by their rates of permeation through the cell membranes. The biodegradation rates therefore apparently depend on factors influencing reactivity or binding to enzymes. Correlations of biodegradation rates of aromatic compounds with Hammett substituent parameters have been reported for substituted benzoic acids [13] and mono- and disubstituted phenols and anilines [14]. Recently, Peijnenburg et al. described structure-activity relationships for abiotic and
QSARs A N D PARs FOR B t O D E G R A D A T I O N O F PCBs
279
microbial dehalogenation of haloaromatic compounds in sediments [15]. In these relationships, they used as structural parameters a , a t , the substituents' Taft steric parameter E s and the carbon-halogen bond strength. The first reaction in the biodegradation of PCBs is dioxygenation to give 2,3-dihydroxylated [9], or in some cases 3,4-dihydroxylated [16], metabolites. Dioxygenations are reactions with molecular oxygen in which both oxygen atoms are incorporated into the hydroxylated metabolites. Although the mechanism of dioxygenation has not been fully characterized, it is generally considered to involve initial attack by oxygen to give a peroxy intermediate [13, 17]. For the purposes of this study, we assumed initial attack at an unchlorinated m e t a position of the least chlorinated ring, which could lead to both 2,3- and 3,4-dihydroxylated products. Hammett constants for o r t h o substituents are poorly characterized, but the value of 1.2 given by Hansch and Leo [8] was determined for 2-chlorotoluene from the t3C-H coupling constant [18] and would seem applicable to PCBs. No correlations were found for the biodegradation rate constants reported for strains Y42 and P6. For example, for the relationship of log k~ for strain P6 with Z a the value of r 2 was 0.06. Similarly, no correlations were found between log k(, and go- and log Kow or Ea and En (r 2 = 0.06 and 0.12, respectively). The biodegradation rate constants listed in Table 1 for strain JB1 were determined under identical conditions in continuously growing cultures exposed to a mixture of PCBs. These rate constants can therefore be reliably compared. The data for 2,3',4,5',6-pentachlorobiphenyl were not used in this analysis as this PCB has only chlorinated m e t a positions on the least chlorinated ring. For the remaining eight PCBs there was a poor correlation between log k~, and Zo- with r 2 = 0.52. However, a much better correlation was obtained using the parameters Z a and Zrr: log k~, = 0.26 (_+0.44) (r 2 = 0.86,
F = 14.8,
1.44 (_+0.26) Ea + 0.58 (_+0.17) En
(1)
s.e.r. = 0.20)
The experimental values of log k~, are plotted against those predicted from Eqn (1) in Fig. 2. Including the three parameters Eo-, Za~ and En in the relationship gave a slightly higher r 2 value of 0.87, but an F value of 9.2. For the six PCBs of this series for which values of log Kow were available, the correlation of log k(~ with Z a and log Kow ( r 2 = 0.71) was worse than that with Ea and 2 n (r 2 = 0.86). Similar calculations for initial attack at the o r t h o positions and those using other values for a o gave much poorer correlations (data not given). These results suggest that the rates at which the tetra-, penta- and hexachlorobiphenyls are degraded by strain JB1 are to a large extent determined
280
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2
t _Q 2d ID1 0
o
[]
-1 0
1
2
log kb (calc) ( h - l )
Fig. 2. Comparison of experimental log k~, values and those calculated from Y~trand Y.n [Eqn (1)].
by electronic effects of the chlorine substituents, but that the hydrophobic effects of the substituents are also important. Surprisingly, the increasing Ere with higher chlorination has a positive influence on the biodegradation rate. The hydrophobicity of these compounds lies in the range where diffusion through the unstirred water layers surrounding the membrane are expected to be rate limiting for uptake by the cells (see above) [12]. Therefore, the favourable effect of increased hydrophobicity of the higher chlorinated congeners may be related to stronger binding to the active site of the enzyme. Clearly, the value for r2 of 0.86 for Eqn (1) as well as the differences in (relative) biodegradation rate constants for different strains, mean that other factors also have an important influence on biodegradation rates. These factors may be related to such biological factors as enzyme specificity. Nevertheless, despite their shortcomings, studies such as this do yield insights into the processes which determine biodegradation rates and may eventually make prediction of such rates possible. REFERENCES 1 J.R. Parsons and H.A.J. Govers, Quantitative structure-activity relationships for biodegradation. Ecotoxicol. Environ. Saf., 19 (1990) 212-227. 2 P. Kuenemann, P. Vasseur and J. Devillers, Structure-biodegradability relationships, in W. Karcher and J. Devillers (Eds), Practical Applications of Quantitative StructureActivity Relationships (QSAR) in Environmental Chemistry and Toxicology, Kluwer, Dordrecht, 1990, pp. 1-12. 3 J.C. Dearden, Physico-chemical descriptors, in W. Karcher and J. Devillers (Eds),
QSARs AND PARs FOR BIODEGRADATION OF PCBs
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