Environ. Sci. Techno/. 1995, 29, 657-663
Effect of Various Factors on Dehalogenation of Chlorinated Phenols and Anilines durino Oxidative Coupling JERZY DEC AND JEAN-MARC BOLLAG* Laboratory of Soil Biochemistry, Center for Bioremediation and Detoxification, The Pennsylvania State University, University Park, Pennsylvania 16802
According to previous research, dehalogenation of chlorinated phenols in the presence of horseradish peroxidase or Trametes versicolor laccase is due to the free-radical mechanism of an oxidative coupling reaction. This study demonstrated that short reaction time, limited amount of catalyst, or unfavorable pH conditions may result in an insufficient generation of free radicals and a reduction in dehalogenation of chlorinated phenols. The free-radical mechanism of chloride release was also found t o be valid for peroxidase-mediated polymerization of chlorinated anilines. In the case of a tyrosinase-mediated polymerization of chlorophenols, chloride ions were apparently removed from the formed o-quinones during nucleophilic attack by phenoxide ions. The results obtained strongly support the hypothesis that dehalogenation is strictly connected to oxidative coupling and cannot be enhanced independently by adjustment of the reaction conditions.
0013-936x/95/0929-0657$09.00/0
1995 American Chemical Society
Introduction Polymerizationof chlorinated phenols catalyzedby oxidoreductive enzymes is presently considered a potential means for decontamination of aqueous environments (1, 2). The transformation of the substrates to polymers occurs through an oxidative coupling reaction. First, the chlorinated compounds are oxidized by the enzymes to free radicals or reactive quinones; the subsequent coupling of the oxidation products is completed without further involvement of the catalysts. Several studies demonstrated that enzyme-mediated polymerization reactions are accompanied by the release of chloride ions from the substrates, and it was concluded that dehalogenation may participate in the decontamination effect (3- 7 ) . The release of chloride ions was believed to be due to enzyme activity. However, in a recent study (8), a similar dehalogenation was observed not only in the presence of different oxidoreductases (horseradish peroxidase and Trametes versicolor laccase) but also when the polymerization reactions were mediated by an inorganic catalyst, birnessite. Moreover, release of chloride ions was observed during horseradish peroxidase-mediated binding of chlorophenols to humic acid, another detoxification process involving oxidative coupling. These findings indicated that not the enzyme activity but the oxidative couplingreaction is the immediate cause of dehalogenation (8). To determine the patterns of dehalogenation, the reaction mixtures in the previous study were analyzed by high-performance liquid chromatography for remaining substrates and by coulometrictitration for released chloride ions (8). The experimental data were used for calculation of the dehalogenation number (DN), which represented the number of the transformed substrate molecules per one released chloride ion. The dehalogenation patterns were in agreement with the free-radical mechanism of oxidative coupling. This mechanism involves formation of various oligomer products due to the delocalization of the unpaired electron and the occurrence of the free radical in several resonance forms (9). The experimentalDN values indicated that release of the chloride ion may occur only if the unpaired electron of a free radical involved in coupling is located at a chlorine-substituted aromatic carbon (8).No release takes place if, due to the resonance, the unpaired electron is located at an unsubstituted position or if an aromatic carbon with the attached chlorine atom occupies the meta position where the unpaired electron does not occur. According to this scheme, 10 chloride ions may be released from 20 molecules of 2,4-dichlorophenol (DCP) involved in all possible couplings between the four resonance forms of the DCP free radical (8).As a result of these couplings, 10 different dimers may be formed. The predicted DN value (20/10) for such a model is 2, which is in agreement with DN values experimentallyobtained. The dimerization model, however, does not take into account further stages of polymerization. In the case of chlorinated phenols, the polymerization can proceed at least until formation of pentamers that precipitate from aqueous solutions, thus terminating oxidative coupling (10). For that reason, a mathematical model has been developed which permits calculation of the number of monomer
VOL. 29, NO. 3, 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY 1657
molecules (M) involved in oxidative coupling up to the pentamerization of the substrates and the number of chloride ions (0 released from the M molecules (8). The predicted DN value ( M I 0 for DCP, obtained by application of this model was also 2, i.e., in agreement with the experiment. A similar agreement was achieved for other chlorophenols subjected to polymerization, which strongly supported the hypothesis that dehalogenation is related to the location of the unpaired electron in the free-radical molecule. The purpose of the present studywas to further evaluate the problem of dehalogenation. Specific objectives were (1)to examine the effect ofvaried reaction conditions upon dehalogenation patterns of chlorinated phenols incubated with peroxidase and T. versicolor laccase; (2) to determine whether the postulated free-radical mechanism of chloride release also applies to the oxidative coupling of chlorinated anilines; and (3) to evaluate the dehalogenation patterns for oxidative coupling of chlorinated phenols in the presence of tyrosinase, an enzyme which mediates oxidative coupling through the formation of o-quinones.
percentage of chloride released (% C1) and the number of chlorine atoms on the benzene ring. Mathematical Modehg of Dehalogenation. To calculate the theoretical number of substrate molecules (M) involved in pentamerization of the substrates and the theoretical number of chloride ions (0released from the Mmolecules, we used equations developed in the previous study (8):
Materials and Methods
where n is the oligomerization order (n= 1for dimerization, etc.), N is the number of dimerization reactions, R is the number of free-radical resonance forms, and d is the number of chlorines lost during dimerization. Equation 1 is valid for all chlorophenols and chloroanilines; eq 2 can be used for substrates with free radicals occurring in four resonance forms, and eq 3 can be used for substrateswith free radicals occurring in three resonance forms (e.g.,4-chlorophenol and 4-chloroaniline). Dividing the M by the C, the predicted DN values were obtained.
Chemicals. The compounds 2- and 4-chlorophenol (2and 4-CP); 2,4-dichlorophenol (DCP);2-, 3-, and 4-chloroaniline (2-, 3-, and 4-CA); and 2,4-dichloroaniline (DCA) were obtained from Aldrich Chemical Co. (Milwaukee,WI); 3-chlorophenol (3-CP)and 2,4,5-trichlorophenol(TCP)were purchased from Fluka AG (Buchs, Switzerland). Enzymes. Extracellular laccase of T. versicolor was isolated from growth media as described by Leonowicz et al. (11). Oneunit oflaccaseactivityis definedastheamount of enzyme that causes a change in absorbance at 468 nm of l.O/min in 3.4 mL of 1 mM solution of 2,6-dimethoxyphenol in citrate-phosphate buffer (pH 3.8). Absorbance was measured with a Model 2000 spectrophotometer (Bausch and Lomb, Rochester, NY). Horseradish peroxidase, with an RZ (Reinheitszahl) of 1.2 and an activity of 95 unitslmg of solid, and mushroom tyrosinase, with an activity of 2100 units/mg of solid, were purchased from Sigma Chemical Co. (St. Louis, MO).One unit of peroxidase activity is defined as the amount of enzyme that forms 1.0 mg of purpurogallin from pyrogallol in 20 s at pH 6.0 and 20 "C. One unit of tyrosinase activity is defined as the amount of enzyme that causes an increase in absorbance at 280 nm of 0.001/min at pH 6.5 and 25 "C in a 3-mL reaction mixture containing L-tyrosine. Dehalogenation of Chlorinated Phenols and Anilines. Triplicate samples of substrates were incubated with shaking, using specified amounts of enzyme in 5 mL of universal buffer (0.2 M acetic acid, 0.2 M boric acid, 0.2 M phosphoric acid, and 1N NaOH), at a specified pH ranging from 2 to 12. Incubation with boiled enzymes served as control samples. After a 2-h incubation with horseradish peroxidase or a 24-h incubation with tyrosinase or T. versicolor laccase, reaction mixtures were centrifuged and the supernatants analyzed for remaining substrates and released chloride ions as described previously (8).For timecourse experiments w t h the laccase,reactions were stopped after specified incubation times by the addition of 1.5 mL of 1 N NaOH. Specific reaction conditions (substrate concentration, enzyme activity, pH, incubation time, and temperature) are detailed in the figure and table legends. To calculate the experimental DN values, the percentage of substrate transformed (% Tr) was divided by the 658
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 3, 1995
n=4
M =z ( n
+ l)NRn-'
n=l
n=4
C = CRn-'d
+ 2(n - l)NR"-' +
n=l
1/6(n - l ) ( n - 2 ) ( n - 3)(8NR - 2 N p ) (2) n=4
C= CRn-'d
+ ( n - l)NR"-' +
n=l
1/6(n - l ) ( n - 2 ) ( n- 3)(3NR - NP) (3)
Results The rate of dehalogenation was found to depend on the followingfactors: the pH ofthe reaction mixture, the activity of the applied enzyme, the incubation time, the chemical structure of the substrate, and the type of enzyme. pH Dependence and Stoichiometry of Dehalogenation. The effect of pH was examined using DCP incubated with horseradish peroxidase or a laccase of T. versicolor in the pH range from 2 to 12. In the case of horseradish peroxidase, the percentage of DCP transformed was extensive over a broad pH range (from pH 3 to pH 10) (Figure 1). However, the maximum removal of DCP (97.8% at pH 6) did not coincide with the maximum release of chloride ions (36.6%at pH 10). The number oftransformed DCP molecules per one released chloride ion (DN)gradually changed from 2.2 at pH 3 to 0.5 at pH 12. The pH range for transformation of DCP by T. versicolor laccase was relatively narrow with a maximum of DCP transformation (69.8%)occurring at pH 6 (Figure 2). This coincided with the greatest chloride release (17.6%at pH 6). The DN values changed rapidly, from 6.3 at pH 2 to 2.0 at optimal pH 6 and to 4.6 at pH 8. Effect of Enzyme Activity. Horseradish peroxidasemediated transformation and dehalogenation of DCP depended on both enzyme activityand H202concentration (Figure 3). With increasing amounts of these two components, the removal of DCP increased from 20.8% for 1 unit/mL of enzyme and 1 mM H202 to 98.9% for 9 units/ mL of enzyme and 9 mM H202. A slight decrease in the removal of DCP at 18 mM H202 (97.7%)was probably due to inhibition by excess H202. The rate of dehalogenation increased in accordance with DCP removal, from 4.1% of
-
100 r
i
40
ru
80
1
30
35 30
25
Be
20
p
m v)
15
z -
10 8 5 0
2
0
4
0
6
10
12
14
PH
--W-
CHLORIDE IONS
2.4-DICHLOROPHENOL
-
2.4-DICHLOROPHENOL CHLORIDE IONS
-D-
-0-
pH 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 I l O I l l ( 1 2 DN I n.d. I 2.2 I 2.2 I 1.9 1 1.7 I 1.6 1 1.7 I 1.6 I 1.2 0.7 1 0.5
FIGURE 1. Effect of pH on the transformation and dehalogenation of 2,4-dichIorophenol(9.0 mM) by horseradish peroxidase (9.0 units/ mL 18.0 mM H202). Incubations were performed for 2 h at 25 "C. DN is the experimental dehalogenation number. For pH 2, DN was not determined (n.d.1. The SD for percent of DCP remaining ranged from 0.1 to 3.9, and for percent of chloride released ranged from 0.1 to 2.6.
+
20
100
1 unit/mL 3 units/mL 9 unitsimL
--*-
--e-
--*-
H202. mM I 1 I 2 I 4.5 I 9 I la HRP, unIlsimLI 9 I 3 I 1 I 9 I 3 I 1 I 9 I 3 I 1 I 9 I 3 I 1 I 9 I 3 I 1 12.71 2.41 2.512.51 2 . 2 1 z.olz.1 I 2.2 I 2.01 I . S I i . a i i . e l 1 .a I I .711.6 DN
FIGURE 3. Effect of enzyme activity and concentration of HZOZon the transformation and dehalogenation of 2.4-dichlorophenol (9.0 mM) by horseradish peroxidase (HRP). DN is the experimantal dehalogenation number. Incubations were performed at pH 5 for 2 h at 25 "C. The SO for percent of DCP remaining ranged from 0.1 to 5.0, and for percent of chloride released ranged from 0.1 to 1.2. 100
r
8
z
'zs
00
15
d
60 10
9 I
40
9
20
5
0
80
5 a
x
60
P
-
40
v)
0
N
f
=
8
20
0 0
2
4
6
8
10
12
14
0.1
+ --m-
2,CDICHLOROPHENOL CHLORIDE IONS
10
1
PH
chloride ions released with 1unit/mL of enzyme and 1mM H202 to 27.9%with 9 units/mL of enzyme and 18 mM H202 (Figure 3). The DN values gradually changed with increasing dosages of horseradish peroxidase and H202 (Figure3), from 1.6 for 1 unit/mL and 18 mM H202 to 2.7 for 9 units/mL and 1 mM H202. The increase in H202 concentration resulted in the enhancement of dehalogenation (reduced
1000
Units/mL U
2,4-DICHLOROPHENOL CHLORIDE IONS
--)-
EA. unilsimL 10.151 0.3 1 0 . 6 DN 110.41 5.4 I 4.3
FIGURE 2. Effect of pH on the transformation and dehalogenation of 2,4-dichlorophenol (9.0 mM) by the laccase of T. versicolor (5 units/ml). DN is the experimental dehalogenation number. For pH 9-12, DN was not determined (n.d.). Incubations were performed for 24 h at 25 "C. The SD for percent of DCP remaining ranged from 0.4 to 3.0, and for percent of chloride released ranged from 0.1 to 0.4.
100
I I
1.2 I 2.5 I 5 I 10 I 20 I 40 I 80 1 1 6 0 3.2 I 2.9 I 2.6 I 2.2 I 2.1 I 2.1 I 2.0 I 2.0
FIGURE 4. Effect of enzyme activity (EA) on the transformation and dehalogenation of 2,4-dichlorophenol (9.0 mM) by the laccase of T. versicolor. DN is the experimental dehalogenation number. Incubations were performed at pH 6 and 25 "C for 24 h. The SD for percent of DCP remaining ranged from 0.1 to 3.0, and for percent of chloride released ranged from 0.1 to 0.8.
DNvalues),whereas the increase in enzyme activity caused an insignificant reduction of dehalogenation. Incubation with increasing activities of the Trametes laccase caused an increase in DCP removal (from 8.3% for 0.15 unit/mL to 99.7% for 160 units/mL) that coincided with an increase in the release of chloride ions (from 0.4% for 0.15 unit/mL to 25.1% for 160 units/mL) (Figure 4). The DN values gradually decreased with increasing enzyme activity, from 10.4for 0.15 unit/mL of enzyme to 2.0 for 80 VOL. 29, NO. 3,1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY 1669
loo
r
TABLE 2
.-.-
0
10
5
Percentage Transformation (YO Tr) and Dehalogenation (YOCI) of Chlorinated Anilines by Horseradish Peroxidasea and Respective Dehalogenation Numbers (DN)
20
15
Time DN
I I
1 min 6.8
--
-
I
1 hr
I
2.3
I2
hrs 2.2
I
I
4 hrs 2.1
I 8 hrs I 1 2.1 I
16 hrs 1 24 hrs 2.1 I 2.0
FlQURE 5. Effect of incubation time on the transformation and dehalogenation of 2.4-dichlorophenol(9.0 mM) by the laccase of T. versicolor (20 units/"). DN is the experimental dehalogenetion number. Incubations were performed at pH 6 and 25 "C for the indicated times. The S D for percent of DCP remaining ranged from 0.1 to 3.9, and for percent of chloride released ranged from 0.1 to 1.4. TABLE 1
Percentage Transformation (% Tr) and Dehalogenation (% CI) of Chlorapkenols by Tyrosinasea and Horseradish Peroxitlase" and Respective Dehalogenation Numbers (DN) chlorophenols 2-chlorophenol 3-chlorophenol 4-chlorophenol 2,4-dichlorophenol 2,4,5-trichlorophenoI
O/O
2.3 0.2 6.9 24.0 12.5
6.1 50.0 2.9 2.0 2.2
"Thesubstrates(9.0 mM)were incubated with tyrosinase(1200 units/ mL) for 24 h at 25 "C and pH 6. The SD for % Tr ranged from 0.3 to 2.4 and for % CI from 0.1 to 2.3. The substrates (9.0 mM) were incubated with horseradish peroxidase ( 4 units/mL 9.0 mM HzOz) for 2 h at 25 "C and pH 5. The SD for % Tr ranged from 0.4 to 4.2 and for % CI from 0.01 to 3.2.
+
and 160 units/mL of enzyme. Effect of Incubation Time. Both DCP transformation and release of chloride ions gradually increased with time in the presence of T. versicolor laccase (Figure 5). After 1 min, DCP removal and chloride release were 17.8% and 1.3%,respectively; after 24 h, they were 90.7% and 23.1%, respectively. As indicated by DN values, after 1 min of incubation, about three times fewer molecules lost a chlorine atom (DN 6.8)than after a 1-hor longer incubation (DN 2.3 for 1 h and 2.0 for 24 h). SubstrateSpecificity. When various chlorophenols were incubated with horseradish peroxidase, the greatest substrate removal and dehalogenation were observed for DCP (94.0%Tr and 24.0% Cl), followed by TCP, 4-CP, and 2-CP (Table 1). The lowest rates (10% Tr and 0.2% C1) were observed for 3-CP, a phenol with chlorine in the metu position. The pattern was reversed with tyrosinase, with the greatest rate of removal and dehalogenation observed 000
DN
2-chloroaniline 3-chloroaniline 4-c h I o roa niIine 2,4-dichloroaniline
59.7 53.7 92.8 44.4
0
ndb 24.4 3.8 5.2
2.2 24.3 4.3
for monochlorophenols, including 3-CP (98.2% Tr and 50.3% Cl). During transformation of chloroanilines, the pattern of substrate removal and dehalogenation for the various chlorine atom positions differed from that determined for chlorophenols (Table 2). The greatest rate of chloroaniline removal was observed fOr4-CA (92.8%),and the lowest was for the double-halogenated DCA (44.4%). No dehalogenation was observed for 2-CA, although a relatively large removal (59.7%)of this substrate was determined. The DN values for 4-CA and DCA were greater (more than two times in the case of DCA) than those for the respective chlorophenols incubated with horseradish peroxidase (Tables 1 and 2). The DN value for 3424, however, was 2-fold less than that obtained for 3-CP.
Discussion
tyrosinase horseradish peroxidase Tr YO CI DN YO Tr YO CI DN
100.0 26.0 3.8 14.0 98.2 50.3 2.0 10.0 100.0 59.9 1.7 20.0 14.3 0.8 8.9 94.0 21.0 0.2 35.0 83.5
Yo CI
+
2,4-DICHLOROPHENOL CHLORIDE IONS
1
% Tr
a The substrates (1.6 mM) were incubated at 25 "C with horseradish peroxidase (4 units/mL 3.5 mM H20J at pH 5 for 2 h. The SD for % Tr rangedfrom 0.6 to 3.8and for % CI from 0.4 t o 1.4. Not determined.
25
HOURS
+
chloroanilines
ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 3, 1995
Dehalogenation of Chlorophenols underVarious Reaction Conditions. Reaction conditions significantly affected dehalogenation of chlorophenols changing experimental DN values. It appears that the increase in DN values (reduced dehalogenation) resulted mainly from insufficient generation of free radicals. There are three probable factors responsible for the deficit of free radicals: (1) short incubation times; (2) limited amounts of catalysts; and (3) nonoptimal pH values of the reaction mixture. According to Dordick et al. (I,?), at early reaction times, predominantly monomer radicals are initiated with little polymerization. Thus, as long as only a few monomer radicals are present, coupling of the monomers occurs infrequently. Consequently, the release of chloride ions should also be limited, and the experimental DN values should be elevated, as observed for the 1-min incubation of DCP (DN 6.8) with the laccase of T. versicolor (Figure 5). As this reaction proceeded, the coupling and dehalogenation became more frequent, and the experimental DN values dropped to approximately 2, Le., to the level of the predicted DN value (2.0). As mentioned previously, the deficit of free radicals may also be due to insufficient quantities of enzyme. Experimental DN values for DCP incubated with low activities of the Trumetes laccase ranged from 10.4 for 0.15 unitlmL to 2.6 for 5 units/mL (Figure 4). At higher enzyme levels, greater than 10 units/mL, DN values approached the predicted DN value of 2. Coupling rates for radicals with chlorine atoms next to the unpaired electron were probably lower than those for other radicals due to steric hindrance caused by the chlorine atom. Apparently, the difference in coupling rates is much more marked (elevated DN values)
when there is a deficit of free radicals due to low enzyme activity. The lack of free radicals may also result from an insufficient amount of electron acceptors. The reduction in the concentration of H202 (the electron acceptor) during incubation of DCP with horseradish peroxidase resulted in an increase in the DN values from 1.6 for 18 mM H202 to 2.7 for 1 mM H202 (Figure 3). During incubation of DCP with the Trametes laccase, the generation of free radicals was seriously affected by the third of the discussed factors, Le., the pH value of the reaction mixture. The experimental DN value was equal to 2 at pH 6 and 7 only; it increased rapidly at both higher and lower pH values (Figure 2). Apparently, this increase was due to diminished efficiency of free radical production by the Trumetes laccase under unfavorable pH conditions. Horseradish peroxidase was found to be much less sensitive to low pH. This enzyme was able to generate sufficient free radicals at pH 3 to maintain the DN value (2.2) at almost the predicted level (Figure 1). An increase in pH, however, resulted in DN values that were much less than 2 (Figure 1). The decrease in the DN value to 1.5may still be explained by a preference for 0-para radical coupling (even with C1 release) over 0-ortho or orthoortho radical coupling. However, the cause for the decline in the DN value to 0.5 has not yet been determined. DN values below 1.5 indicate a significant enhancement of dehalogenation, i.e., a prevalence of coupling routes involving radicals with a C1 atom next to the lone electron. This supposition, however, contradicts the effect of steric hindrance caused by Cl atoms in favor of coupling between the radicals without C1. In addition, the eliminationof steric hindrance is not enough to account for lowering the DN value to 0.5, indicating that both C1 atoms should be removed from the DCP molecule. Such lowering of the DN value should require an extensive release of chloride ions from oligomer radicals instead of only the observed small release (8). In the case of DCP, however, even release of chloride ions from oligomers should be limited because chlorine atoms in DCP oligomers primarily assume the meta position. Thus, the cause of the enhanced dehalogenation at high pH may be due to other phenomena. It is possible that the decrease in DN values below 1.5 is related to the fact that, at high pH, chlorophenols occur as phenolate anions. The decrease may also result from further oxidation of free radicals to phenoxonium ions. Another possible explanation is that the aromatic ring may be cleaved with a release of chloride ions due to oxidation of chlorophenols in alkaline solution. Dehalogenation of Chloroanilines. The predicted DN values for chlorinated anilines obtained by application of the mathematical modeling (2.5 for 2-CA; none for 3-CP; 3.0 for 4-CA; and 2.0 for DCA) were the same as those for the respective chlorophenols (8). However, unlike for chlorinated phenols, the experimental DN values for chlorinated anilines (Table 2) differed from the predicted ones. The experimental DNvalues also differed from those determined experimentally for chlorophenols (Table 1). Nevertheless, oxidative coupling and dehalogenation of chloroanilines may still be explained by the same freeradical mechanism that was proposed in our previous studies for chlorinated phenols (8). Figure 6 presents all possible couplings between the three resonance forms of the 4-CA free radical, resulting in the formation of six different dimers and the release of four
chloride ions. The experimental DN value for 4-CA (3.8) was somewhat higher than the predicted one (3.0)and the DN experimentally determined for 4-CP (2.91, indicating a reduced dehalogenation. The possible cause of this reduction may lie in the difference between electronegativities of the oxygen and nitrogen atoms attached to the benzene ring. Nitrogen is less electronegative than oxygen;therefore, the electron density in the vicinity of chlorine atoms in chloroanilines is greater than that in chlorophenols. This could result in an increased repulsion of radicals approaching for coupling, leading to a reduction of chlorine atom removal from4-CA. This reasoning is consistent with the finding of Simmons et al. (13)that N-N radical coupling (resulting in no chloride release) predominated over N-ortho and N-para radical coupling (the ratio was 2.6: 1:l). Of these three pathways, only N-para coupling involves release of chlorine. At the same time, no coupling between ortho and para radicals (which would result in C1 release) was reported, probably due to insufficient yields of these products (13). No chloride ions were detected in the reaction mixture containing2-CA,and the DNvalue could not be determined; at the same time, the substrate removal was as great as 59.7% (Table 2). The lack of chloride ion release was apparently due to the same competition between two radicals with the unpaired electron in either ortho position (one with and one without a chlorine atom), as previously discussed for 2-CP (8). Due to steric hindrance, the rate of coupling for the radical having the chlorine atom next to the unpaired electron must be lower than that for the radical with the unsubstituted carbon. Consequently, dehalogenationmust be reduced. However, unlike for 2-CP (experimental DN, 6.1; predicted DN, 2.5), in the case of 2-CA, the competition resulted in acomplete lack of chloride ion release from the ortho position. Again, the cause may be the difference between electronegativities of nitrogen and oxygen. This difference resulted in the reduction of chloride ion release from the para position in 4-CA. Because the distance from the ortho position to nitrogen is less than that from the para position, the electronegativity effect in 2-CA was greater and resulted in the complete lack of dehalogenation. The electronegativity effect may also explain the reduced dehalogenation of DCA (experimental DN, 5.2; predicted DN, 2.0). This reduction was greater than that for 4-CA (experimental DN, 3.8, predicted DN, 3.0). The probable cause was that only the para-substituted chlorines were released from DCA, because no chloride release from the ortho position would be expected,as was observed for 2-CA. Similar to 3-CP, no release of chloride ions should be expectedfrom 3-CAdue to substitution of the chlorine atom at the meta position, where the unpaired electron does not occur. The observed limited dehalogenation (2.2%)could be explained by the same effect as discussed in our previous study for 3-CP, i.e., release of chloride ions from oligomer radicals (8). However, no similar release from oligomer radicals was observed for 2-CA (Table 2). The probable reason is that, in oligomers, the 2-CAchlorine atoms assume only the ortho and meta position but not the para position. The 3-CA chlorine atoms, on the other hand, frequently assume the para position in oligomers. This indicates that chlorine atoms released from 3-CA (and perhaps also from 3-CP) probably occupied only the oligomer para position. Dehalogenation of Chlorophenols in the Presence of Tyrosinase. Transformation of chlorophenols in the presVOL. 29, NO. 3, 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 1661
6 6- 6- 6 catalyst
CI
CI
CI
CI
1
2
3 Possible couplings:
1-1
1-2
1-3
2-2
2-3
3-3 FIGURE 6. Proposed coupling reactions between free radicals generated during catalytic oxidation of 4-chloroaniline.
ence of tyrosinase resulted in extensive dehalogenation of monochlorophenols (e.g., 60% for 4-CP) and limited dehalogenation of DCP (0.8%)and TCP (0.2%). As Table 1 demonstrates, the pattern of substrate transformation for this enzyme differed from that observed for horseradish peroxidase. The reason is that the tyrosinase-mediated oxidation of chlorophenols leads to the formation of o-quinones (14) not the generation of free radicals, and oxidative coupling is governed by a different mechanism. The experimental DN values (Table 1) indicated that dehalogenation of chlorophenols in the presence of tyrosinase may result from nucleophilic attack of the o-quinone by a phenoxide anion. As shown in Figure 7, which presents the mechanism of oxidative coupling for 2-CP, chlorinated o-diphenols are formed during the first step of the tyrosinase-mediated oxidation of chlorophenols to o-quinones. In neutral and alkaline environments, these substances occur as phenoxide anions and can couple with o-quinones (9). It appears that there are two possible routes of nucleophilic substitution of chlorinated o-quinones by these ionized species: one with and one without chloride release (Figure 7 ) . As a result, one chloride ion may be released from 4 molecules of 2-CP participating in the two-route coupling. The predicted DN value (4.0) obtained by application of the above model is in close agreement with the experimental DN value (3.8),suggesting that coupling rates for the two routes of 2-CP transformation should be nearly equal. 662
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 3, 1995
The tyrosinase-mediated oxidationof 3-CP (Table 1)may result in the formation of two o-quinones: one with the new oxygen between the original oxygen and the chlorine and the other with the new oxygen on the opposite side of the ring. The experimental DN value (2.0) indicated that the second o-quinone was mostly involved in oxidative coupling (the first o-quinone was identical to that originating from 2-CP). It appeared that the prevailing route of subsequent nucleophilic attack of the second o-quinone by phenoxide anions involved the removal of a chlorine atom (probably due to its paru-substitution status in the quinone). Apparently, almost no coupling through the attack at the ortho position took place. Also, the involvement of the first o-quinone in the coupling was apparently significantly limited. The o-quinone formed during tyrosinase-mediated oxidation of 4-CP (Table 1) is identical to the second o-quinone resulting from 3-CP. The experimental DN value for 4-CP (1.7) was lower than that for 3-CP (2.0) because no other oxidation products were formed to sporadically and competitively participate in oxidative coupling, as was the case for 3-CP. The experimental DN values for DCP (8.9) and TCP (35.0) indicated that the nucleophilic attack of o-quinones resulting from the above substrates by the respective phenoxide anions was mainly restricted to the unsubstituted para position. The limited release of chloride ions (Table 1)
W oltho- Hydroxylation
Ionization
-
OH C
I
W
0' OH
O
H
Route 2
CI
FIGURE 7. Proposed reaction pathways during transformation of 2-chlorophenol in the presence of tyrosinase.
may be attributed to sporadic nucleophilic attack on the C1-substituted para position. Conclusions. The results of the present investigations confirm our previous findings (8). There are two major aspects that determine the significance of both studies. First, the mechanism of dehalogenation has been elucidated. Previously, it was believed that enzymes were the primary cause of chloride ion release (3, 6, 7). Our recent study made it clear that dehalogenation is a consequence of an oxidative coupling reaction (@ TheIpresent . research demonstrated that both chlorinated phenols and anilines can be dechlorinated. Depending on the type of catalyst, chloride ions can be released either through the free-radical mechanism or during nucleophilic addition of phenoxide ions to the formed o-quinones. The second important aspect of this research relates to the detoxification potential of the oxidative coupling processes ( I , 2). The discovery that oxidoreductasemediated polymerization of chlorinated phenols is accompanied by chloride release raised expectations that dehalogenation may contribute to the overall detoxification effect (3,6, 7). It has been postulated that polymerization can be avoided and the rate and extent of chloride release increased by manipulation of the reaction conditions (7). However, our previous research indicated that oxidative coupling and dehalogenation may not be separated (8). The only way to enhance dehalogenation is to increase oxidative coupling. As demonstrated in this study, such an enhancement can be achieved through changes in the reaction conditions (varyingpHvalues, amounts of catalyst and/or electron acceptor, or incubation times). Due to the specificity of the oxidative coupling mechanism, however, even under the most favorable conditions only a partial dehalogenation is possible. This, in turn, determines that only a limited increase in the detoxification effect can be expected.
Acknowledgments Primary funding for this research project was provided by the Office of Research and Development, Environmental Protection Agency (EPA Grant R-820921). The EPA does not necessarily endorse any commercial products used in the study, and the conclusions represent the views of the authors and do not necessarily represent the opinions, policies, or recommendations of the EPA.
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Received for review May 10, 1994. Revised manuscript received November 1 I, 1994. Accepted December 8, 1994.@ ES940284L @
Abstract published in Advance ACS Abstracts, January15,1995.
VOL. 29, NO. 3. 1995 /ENVIRONMENTAL SCIENCE &TECHNOLOGY
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