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Copolymerization of halogenated phenols and syringic acid
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
Copolymerization
23, 261-272 (1985)
of Halogenated
JEAN-MARCBOLLAG Laboratory
of Soil Microbiology,
The Pennsylvania
Phenols and Syringic Acid
ANDSHU-YEN State
University,
LIU University
Park,
Pennsylvania
16802
Received February 23, 1984; accepted July 5, 1984 Enzyme-catalyzed copolymerization of phenols with one to five chlorines (4chlorophenol; 2.4and 2,bdichlorophenol; 4-bromo-2-chlorophenol; 2,3,6- and 2,4,5-trichlorophenol; 2,3,5,6-tetrachlorophenol; and pentachlorophenol) and syringic acid was studied with an extracellular lactase of the fungus Rhizoctonia praticola. This reaction is of interest since it presents a model for explaining ‘the incorporation of anthropogenic compounds into humic substances. When the laccase was incubated together with the halogenated phenols and syringic acid, two types of hybrid products were found: (1) phenols covalently bound to an orthoquinone product of syringic acid resulting in the formation of quinonoid oligomers, and (2) phenols covalently bound to decarboxylated products of syringic acid resulting in the formation of phenolic oligomers. Mass spectra of hybrid oligomers gave typical chlorine isotopic patterns which coincided with their respective chlorophenol monomer. It was concluded that all hybrid products contained only one halogenated phenol molecule and that no dehalogenation took place. o 1985 Academic press, I~C. INTRODUCTION
beled 2,4-D was associated in the soil in a nonextractable form after 35 days. They concluded that it was not 2,4-D but its degradation products which were incorporated into soil organic matter. They did not find 2,4-dichlorophenol and assumed that-if it is formed-it must be rapidly degraded, but they did not consider the possibility of its binding to humic material. McCall et al. (7) also showed the incorporation of 2035% of some form of the ring carbon of 2,4D and 2,4,5-trichlorophenoxyacetic acid into humic substances of the soil. Evidence of the incorporation of pentachlorophenol (PCP) into soil constituents was recently provided by Weiss et al. (8). They found that the major residues of PCP in soil were bound residues which accounted for 28.6% of the applied radioactivity. Previously, we have shown that syringic acid can be polymerized by a lactase from the fungus Rhizoctonia praticola with the formation of a variety of oligomers ranging from dimers to hexamers (9). In the present study we further demonstrate that the enzyme can copolymerize syringic acid products and various chlorophenols. The occurrence of similar reactions in soil may lead
Chlorophenols are major intermediates of phenoxyalkanoate herbicides and other pesticides. They have also been found in pesticide preparations and industrial wastes. Their fate and behavior in the soil environment are of great concern for their toxic properties, particularly since they can easily form soil-bound residues. In soil, many compounds possessing phenolic characteristics and originating from lignin, other organic material, or through synthesis by microorganisms are essential constituents of humus (1, 2). Therefore, it is conceivable that halogenated and other xenobiotic phenols may also be incorporated into organic matter during the humification process (3, 4). Wolf and Martin (5) showed that 14C-ringlabeled 2,4-D (2,4-dichlorophenoxyacetic acid) and chlorpropham (isopropyl 3-chlorophenylcarbamate) or their transformation products were incorporated into humic-like polymers produced by fungi. Smith and Muir (6) found that approximately 15% of the radioactivity derived from carboxyl-labeled [14C]2,4-D and 30% from i4C-ring-la261
004%3575/85 $3.00 Copyright All rights
D 1985 by Academic Press, Inc. of reproduction in any form reserved.
262 to the incorporation organic matter.
BOLLAG
of xenobiotics
into soil
AND
LIU
column (10 Frn) using a radial compression module (RCM-100, Waters Associates, Inc., Milford, Mass.). Isocratic elution was MATERIALS AND METHODS made with a solvent composition of acetoEnzyme. An extracellular fungal lactase nitrile:methanol:water (3:5:2, v/v) at a flow was isolated from the culture filtrate of R. rate of 1 mllmin. Quantitative analysis was praticola and the number of enzyme units carried out with a Waters’ data module used were established in an assay with the using the external standard method. Triplisubstrate 2,6-dimethoxyphenol as previcate samples were evaluated for each treatously described (10). ment. The calibration curves were linear in Substrate incubation and product re- the range between 0 and 20 Fg. covery. Ten micromoles of a halogenated Product characterization was based on phenol dissolved in 1 ml 0.1 N NaOH, 25 electron impact mass spectrometric analpmol syringic acid in 0.8 ml 70% ethanol, ysis (70 eV) with sample introduction by and 12.5 units of R. praticola lactase were direct insertion probe on an AEI MS-902 or mixed with 0.1 M phosphate buffer (pH 6.8) Kratos MS-50 mass spectrometer at temto make a 25ml reaction mixture. All as- peratures varying from 220” to 300°C. says were incubated in the dark for 3 hr at Proton NMR spectra were taken on a 30°C. Boiled enzyme preparations were Bruker 360 MHz instrument with a Fourier used as controls. transform system using deuteriochloroform Precipitates formed in the reaction mix- as a solvent. tures were collected by filtering through a Chemicals. Syringic acid, 2,6-dichloroGelman GN-6 metricel membrane (pore phenol, 2,3,5,6-tetrachlorophenol, 2,4,5size, 0.45 pm). The precipitates on the trichlorophenol, 2,3,6&chlorophenol, and membrane were washed with hexane to re- pentachlorophenol were purchased from move surface contaminants and then sub- Fluka Chemical Corporation (Hauppauge, jected to mass spectrometric analysis. The N.Y.). 4-Chlorophenol, 2,4-dichlorophenol, filtrate was extracted three times with equal and 2-chloro-4-bromophenol were obtained volumes of hexane to recover unreacted from Aldrich Chemical Company (Milchlorophenols and products. The combined waukee, Wise.). Pentachlorophenol and hexane extract and samples from the 2,3,6-trichlorophenol were treated with achexane washing were evaporated to dry- tive charcoal and recrystallized from benness and redissolved in an aliquot of ace- zene before use. The chemical purity of all tonitrile for chromatographic analysis. reagents was analyzed by TLC or HPLC. Analytical methods. Thin-layer chromaRESULTS tography (TLC) was carried out using silica gel F-254 plates (Brinkman Instruments, When the halogenated phenols 4-chloroInc., Westbury, N.Y.) with layer thickness phenol; 2,4- and 2,6-dichlorophenol; 4of 0.25 mm. Products from the hexane ex- bromo-2-chlorophenol; 2,3,6- and 2,4,5tract were separated in a solvent system of trichlorophenol; 2,3,5,6-tetrachlorophenol; ethyl acetate: hexane (1: 1, v/v). and pentachlorophenol were incubated for High-performance liquid chromato3 hr individually with syringic acid in the graphy (HPLC) was performed with a Model presence of a lactase from R. praticola, the 6000A pump, U6K injector, and a 440 uv color of the reaction mixture changed and detector operating at a wavelength of 254 the formation of a precipitate was obnm (Waters Associates, Inc., Milford, served. The liquid phase and the precipitate were separately analyzed. In subsequent Mass.). All samples were passed through experiments it was established that the Sep-Pak C’* cartridges before separation by an 8 mm x 10 cm reverse-phase C-18 products of the two phases had different
COPOLYMERIZATION
OF HALOGENATED
chemical properties. The supematant of the reaction mixture was extracted with hexane and the analysis of this extract is described under Quinonoid Hybrid Oligomers. The precipitate was removed by membrane filtration and washed thoroughly with hexane; the chemical characteristics of the precipitate are described under Phenolic Hybrid Oligomers. If the enzyme assay with the various substrates was done with a boiled enzyme preparation or without enzyme, no color change or product formation (as determined by TLC and HPLC) could be observed. Quinonoid Hybrid Oligomers
The liquid phase of the enzyme assays was extracted with hexane. The red color of the hexane fraction indicated the presence of quinonoid products which could be separated from the chlorophenols by TLC (Table 1). The colored spots were extracted with ethyl acetate and subsequently analyzed by mass spectroscopy. Evaluation of the spectra indicated the formation of dimerit and trimeric quinonoid hybrid products. The mass spectra revealed that all products gave identical chlorine patterns as compared to the starting chlorophenol. These results indicated that only one molecule of chlorophenol is incorporated into the hybrid quinonoid dimers and trimers, as shown in the mass spectra of syringic acid and 2,ddichlorophenol, 4-bromo-2-chloro-
phenol, or 2,4,Strichlorophenol (Fig. 1) and in Table 2. The increase in M + 2 shown in the mass spectra was due to the reduction of the quinone molecule to a phenolic compound in the ionization chamber. We could not obtain quinonoid hybrid oligomers from higher chlorinated phenols such as 2,3,5,6-tetrachlorophenol and pentachlorophenol, and syringic acid. However, it is possible that the amount of quinones produced was insufficient for detection with our technique. Phenolic Hybrid Oligomers
Phenolic oligomers which appeared as precipitates in the reaction mixtures were collected by membrane filtration. Mass spectral analysis of the precipitates obtained from the combination of syringic acid and various chlorophenols indicated the presence of dimeric to pentameric hybrid oligomers (Table 2; Figs. 2, 3, and 4). The chlorine isotopic cluster pattern shown on the mass spectra enabled us to determine the number of chlorine atoms present in each hybrid oligomer and thus the type of chlorophenol molecule incorporated. Hybrid oligomers originating from various chlorophenols gave typical chlorine cluster patterns identical to their respective chlorophenol monomers, indicating that only one molecule of chlorophenol was present in the various oligomers and that there was no loss of chlorine. In our earlier studies we established that
TABLE Thin-Layer
Chromatographic
Syringic acid combined with
0 Solvent system: hexane-ethyl
1
Separation of Quinonoid Hybrid Various Chlorophenols
Chlorophenol Monomer (R,)
4-Chlorophenol 2,4-Dichlorophenol 2,6-Dichlorophenol 4-Bromo-2-chlorophenol 2,4,5-Trichlorophenol
0.700 0.86 0.88 0.84 0.93 acetate (1: 1, v/v).
263
PHENOLS
Products
qf Syringic
Acid
Quinonoid product Dimer (I$)
Trimer W,1
0.44 0.62 0.67 0.56 0.77
0.62 0.67 0.56 -
and
a
Y c
p z-
v, z
> I-
0
50
FIG. 1. Mass
50
49
50
50
spectra
69
100
I,
of quinonoid
84
100
loo
200
hybrid
products
150
132 I, Ill,++& ,,,,,.
150
150
of syringic
200
250
acid
200
350
and 2,&dichlorophenol,
m/z
250
+5-v-d~~r-yr.
300
344
Dimrr
250
300
L
ACID
350
ACID
300
ACID
+
+
450
+
4-bromo-2-chlorophenol,
SYRINGIC
400
SYRlNGlC
SYRINGIC
2,4,5-
-450
550
or 2,4,5-trichloropheaol.
400
400
CHLOROPHENOL
TRICHLOROPHENOL
500
4- BROMO-E-
350
2-G - DlCHLOROPHENOL
of Hybrid
Oligomers
of Syringic Acid
C,,H,Wl,
-
-
-
332
-
478 C,,H,,O,ClBr -
342 Ct3Hs0,ClBr 332
CnH704Cl3
C21H1606C~2
434
C,,W',Cl,
298
C2lH,606C12
434
C,,H,O,Clb 298
G3%04C12
Trimer -
Dimer 264”
Quinonoid oligomers (hexane extract)
after Incubation
TABLE
2
C,,W'4C15
G&,oWl, 416
382
C,,H,,O,Cl,
348
C,,H,,O,Cl,
358 C,,H,,O,BrCl 348
CMH,204C12
314
C,,H,204Cl2
‘%HnWl 314
Dimer 280
and Chlorophenols
of a Lactase
from
Rhizoctonia
C22H,707C1,
568
C22H,807C14
534
C22H,,O,Cl3
500
C22H,,07C1,
510 C22H2007BrCI 500
C22H2007CI2
466
C22H2007C12
C22H2,07C1 466
Trimer 432
720 C,OH~SO,OC~S
C,oH26O,oCl4
686
C30H270,0C13
652
C,&,,O,,Cl,
812
C38H34013C14
804 W-W&h 804 GdMWl, 838
652 C30H27010C13
770 W-W,~C4 -
C,BH,~O,,C~,
C3&370,3Cl 770
Pentamer 136
praticola
618 C,o%@,oCh. -
C,oH2tP,oCl2
C,o%O,oCl 618
Tetramer 584
Phenolic oligomers (precipitate)
in the Presence
a m/z value of molecular ion. b Molecular composition based on number of chlorine atoms and m/z value of a given compound.
Pentachlorophenol
2,3,5,6-Tetrachlorophenol
2,4,5-Trichlorophenol
2,3,6-Trichlorophenol
4-Bromo-2chlorophenol
2,6-Dichlorophenol
2,4-Dichlorophenol
Reaction of syringic acid with 4Chlorophenol
Formation
s z
8
F s m” ?
%
2
% z g ij 5
BOLLAG
266 SYRINGIC
AND LIU
ACID
+
4 - CHLOROPHENOL
28o DIMER
TETRAMER 584
TRIMER
100
200
300
400
m/z 100
s
1
SYRINGIC
ACID
+
4 - BROMO
- 2 - CHLOROPHENOL
I
I
TRIMER 510
100
200
l,...,l.,~"~'~~l 400
300
i' 500
600
m/z FIG. 2. Mass spectra of phenolic hybrid products of syringic acid and I-chlorophenol 2-chlorophenol.
oligomers of syringic acid are coupled by aryl-oxygen-aryl (C-O) linkages (9). The binding of PCP to syringic acid products had to be by C-O linkage exclusively at the position of l-0-4’, since this is the only way to achieve a molecular ion which will fit a
or I-bromo-
given molecular composition containing five chlorine atoms. It can be concluded that PCP-syringic acid hybrid oligomers consist of one molecule of PCP bound to one to several molecules of decarboxylated syringic acid prod-
COPOLYMERIZATION SYRINGIC
400
OF ACID
HALOGENATED ‘2.6
+
500
267
PHENOLS
DICHLOROPHENOL
600
700
800
m ./z
SYRINGIC
ACID
+
2.4.5
-TRICHLOROPHENOL DIMER
FIG. 3. Mass trichlorophenol.
spectra
of
phenolic
hybrid
products
ucts. The possible structural configurations of PCP-syringic acid hybrid oligomers are presented in Fig. 5. The molecular compositions of the hybrid oligomers from other chlorophenols and syringic acid (listed in Table 2) can be derived when the chlorine content and molecular ion are taken into consideration. According to the molecular compositions, each hybrid oli-
of syringic
acid and 2,6-dichlorophenol
or 2,4-S-
gomer consists of one chlorophenol and one to several molecules of syringic acid derivatives. Therefore, it appears reasonable to assume that the hydroxyl group of the chlorophenols is the reactive site. Consequently, the binding of chlorophenols other than PCP to syringic acid products should take place via C-O linkage as in the case of the PCP-syringic acid complex. A
268
BOLLAG SYRINGIC
350
400
ACID
+
AND
LIU
2.3.5.6 - TETRACHLOROPHENOL
450
560
m/z
SYRINGIC
350
400
ACID
450
+
PENTACHLOROPHENOL
500
550
600
650
700
m/z
FIG. 4. Mass pentachlorophenol.
spectra
of phenoiic
hybrid
products
model is proposed in Fig. 6 in which the structural configurations of phenolic and quinonoid oligomers from a reaction of syringic acid and 2,4,5-TCP is presented. It is most likely that hybrid oligomers higher than hexamers were formed but that they could not be detected since the mass spectrometer was only programmed to a mass range of 800.
of syringic
acid and 2,3,5,6-tetrachlorophenol
or
Zdentzjkation of 3-Methoxy-5-(2’,4’,5’trichlorophenoxy)benzoquinone(l,2) (MTPQ), a Hybrid Product of 2,4,5TCP and Syringic Acid The quinone dimer from the combination of 243TCP and syringic acid was isolated by TLC and further purified by HPLC for chemical characterization. High resolution mass spectrometric analysis of the quinone
COPOLYMERIZATION
OF
HALOGENATED
269
PHENOLS
m/z 416
PENTACnLOROPHENOL
FIG.
SYRINGIC
5. Proposed
structures
ACID
of hybrid
qf
products
hybrid dimer yielded an elemental composition of C,,H,O,Cl, (calculated, 333.9567; found, 333.9575). The increase in M + 2 could be explained by the reduction of the quinone molecule to a phenolic compound PUINONOID
syringic
m/z 332 OCH3
2.4,5-TRICHLOROPHENOL
SYRINOIC
ACID
m/r
m/z 196
FIG.
346
6. Proposed
mh
structures
of hybrid
pentachlorophenol
OLIOOYER
Cl
PHENOLIC
and
in the ionization chamber. NMR analysis further verified the structural configuration of the molecule (Table 3). The results clearly indicated that 2,4,Strichlorophenol was covalently bound to a monomethoxy
Cl&,+-$,
m/r
acid
196
OLIQOYER
500
products
m/t 652
of
syringic
acid
and
2.4,5-trichlorophenol.
BOLLAG
270
AND LIU
TABLE Proton
NMR
Data
for
a Quinonoid
Hybrid product
3
Hybrid Dimer, 3-Methoxy-5-(2’,4’,5’-trichlorophenoxy)benzoquinone(l,2), of 2,4,5-Trichlorophenol and Syringic Acid
Chemical shift (w-d
Splitting integral
7.64 7.33 6.05 5.18 3.89
orthoquinone derived from syringic acid through a C-O linkage. Consequently, the product was identified as 3-methoxy-5(2’,4’,5’ - trichlorophenoxy) benzoquinone(12).
Quantitative Analysis of the Formation of MTPQ The decrease in 2,4,5-TCP and the formation of the hybrid quinone dimer (MTPQ) were monitored at different time intervals for a period of 3.5 hr. As shown in Fig. 7, the concentration of the hybrid quinone dimer gradually increased during the 2.5-hr reaction period while the concentration of 2,4,5-TCP decreased. Approximately 42% of the metabolized 2,4,5-TCP was recovered as the hybrid quinone dimer after 2 hr of incubation.
Singlet Singlet Doublet Doublet Singlet
Proton assignments
Coupling (H-4
Ha Hb Hc Hd Methoxy
(1H) (1H) (1H) (1H) (3H)
2.5 2.5
the hybrid oligomers (e.g., Fig. 4), suggesting that these products are not involved in the copolymerization reaction with chlorophenols. On the other hand, the products with m/z 290, 306, and their corresponding trimers and tetramers (9) are no longer detected in the present mass spectra, indicating their reaction with chlorphenols. Previously, we reported that mono- and disubstituted chlorinated phenols such as 2,4-dichlorophenol or 4-bromo-2-chloro-
2,4,5-TRICHLOROPHENOL
DISCUSSION
Syringic acid is a representative of the numerous humus monomers. It is easily oxidized when exposed to phenoloxidases (9, 11). In our laboratory, a number of investigations have been undertaken on the laccase-catalyzed polymerization and copolymerization of syringic acid (9, 12, 13). In the present study if was found that decarboxylated syringic acid products and monomethoxy-orthoquinone are able to complex with differently substituted halogenated phenols. Syringic acid dimers with ml z 350 and 364, and their corresponding trimers and tetramers with m/z 502, 516 and m/z 654, 668, respectively (9), usually appeared in the mass spectrum together with
;t
.I
c
HYBRID
1 .----.-.-. ,*----
.A*’
0 0
PRODUCT
120
60
180
MINUTES
FIG. 7. Disappearance of 2,4,5-trichlorophenol and formation of a quinonoid hybrid dimer [3-methoxy-5(2’,4’,5’-trichlorophenoxy)benzoquinone(l,2)] during incubation of 2,4,5-trichlorophenol and syringic acid in the presence of a lactase of R. praticola.
COPOLYMERIZATION
OF HALOGENATED
phenol can be oxidatively coupled to higher oligomers (14), but higher chlorinated phenols such as trichlorophenols, 2,3,5,6tetrachlorophenol, and pentachlorophenol were not transformed by the R. praticola lactase (S.-Y. Liu, unpublished data). Konishi and Inoue (15) found that a lactase from Trametes versicolor can transform pentachlorophenol to quinonoid products. In their experiments, the reactions were carried out with high concentrations of pentachlorophenol (500 ppm) at pH 6.2, 30°C for 2 days. Repeating their experiment under the same conditions, we obtained similar results with a lactase from T. versicolor, but not with the R. praticola laccase (unpublished data). When chlorophenols, syringic acid, and the lactase were incubated together, the primary reaction appeared to be the copolymerization of the two substrates. In our previous experiments with chlorinated phenols as the only substrate, oligomers were clearly determined (16), while in the present study only hybrid products and oligomers of syringic acid were found in the mass spectral analysis. Apparently, the formation of oligomers from chlorophenols alone was suppressed. It is suggested that a sequence of reactions is involved in the copolymerization of chlorophenols with syringic acid products. It appears that oxidation of syringic acid is a prerequisite for this copolymerization. Our previous studies, in which the formation of multiple products of syringic acid has been established, support this view (9). The evidence presented in this paper clearly demonstrates the chemical binding between chlorophenols and syringic acid products. These findings imply the potential incorporation of xenobiotics into soil organic matter during the humitication process. Such products are known as bound residues (4, 17), and are a cause of concern because of their environmental impact. Particularly, phenols are of concern due to their inherent toxicity and ever-increasing prevalence as byproducts in industry. Sev-
PHENOLS
271
era1 studies indicate that xenobiotics bound to humic substances can later be released by the activity of microorganisms (18, 19) or earthworms (20), thus posing a delayed health hazard. ACKNOWLEDGMENTS The preparation of this article was supported in part by a research grant from the Environmental Protection Agency (R-808165) and by the Pennsylvania Agriculturaf Experiment Station (Journal Series No. 6830). The views expressed herein do not necessarily reflect the opinion of the Environmental Protection Agency and no endorsement of that agency should be inferred. REFERENCES 1. K. Haider, J. P. Martin, and Z. Filip, Humus biochemistry, in “Soil Biochemistry” (E. A. Paul and A. D. McLaren, Ed.), Vol4, p. 195, Marcel Dekker, New York, 1975. 2. W. Flaig, H. Beutelspacher, and E. Rietz, Chemical composition and physical properties of humic substances, in “Soil Components” (J. E. Gieseking, Ed.), Vol. 1, p. 1. Springer-Verlag. New York, 1975. 3. R. Bartha, Pesticide residues in humus. ASM News 46, 356 (1980). 4. J.-M. Bollag and M. J. Loll, Incorporation of xenobiotics into soil humus, Experientiu 39. 1221 (1983). 5. D. C. Wolf and J. P. Martin, Decomposition of fungal mycelia and humic-type polymers containing carbon-r4 from ring and sidechain labeled 2.4-D and chlorpropham, Soil Sci. Sot. Amer. J. 40, 700 (1976). 6. A. E. Smith and D. C. G. Muir, Determination of extractable and non-extractable radioactivity from prairie soils treated with carboxyl- and ring-labelled [14C] 2.4-D. Weed RPS. 20, 123 (1980). 7. P. J. McCall, S. A. Vrona, and S. S. Kelley, Fate of uniformly carbon-14 ring labeled 2.4.5trichlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid, J. Agric. Food Chem. 29, loo (1981). 8. U. M. Weiss, I. Scheunert, W. Klein, and E Korte, Fate of pentachlorophenol-r4C in soil under controlled conditions, J. Agric. Food Chem. 30, 1191 (1982). 9. S.-Y. Liu, R. D. Minard, and J.-M. Bollag, Oligomerization of syringic acid, a lignin derivative, by a phenoloxidase. Soil Sci. Sot. Amer. J. 45, 1100 (1981). 10. J.-M. Bollag, R. D. Sjoblad. and S.-Y. Liu. Characterization of an enzyme from Rhizocronia praticola which polymerizes phenolic compounds, Canad. .I. Microbial. 2.5, 229 (1979).
272
BOLLAG
11. T. Ishihara and M. Ishihara, Oxidation of syringic acid by fimgal lactase, Mokuzai Gakkaishi 22, 371 (1976). 12. J.-M. Bollag, S.-Y. Liu, and R. D. Minard, Crosscoupling of phenolic humus constituents and 2,4-dichlorophenol, Soil Sci. Sot. Amer. J. 44, 52 (1980). 13. J.-M. Bollag, R. D. Minard, and S.-Y. Liu, Crosslinkage between anilines and phenolic humus constituents, Environ. Sci. Technol. 17, 72 (1983). 14. J.-M. Bollag, R. D. Sjoblad, and R. D. Minard, Polymerization of phenolic intermediates of pesticides by a fungal enzyme, Experientia 33, 1564 (1977). 15. K. Konishi and Y. Inoue, Detoxification mechanism of pentachlorophenol by the lactase of Coriolus versicolor, Wood Res. 18, 463 (1972).
AND LIU 16. R. D. Minard, S.-Y. Liu, and J.-M. Bollag, Oligomers and quinones from 2,4-dichlorophenol, J. Agric. Food Chem. 29, 250 (1981). 17. D. D. Kaufman, Bound and conjugated pesticide residues, in “Bound and Conjugated Pesticide Residues” (D. D. Kaufman, G. G. Still, G. D. Paulson, and S. K. Bandall, Eds.), p. 1, Amer. Chem. Sot., Washington, D.C., 1976. 18. T. S. Hsu and R. Bartha, Biodegradation of chloroaniline-humus complexes in soil and in culture solution, Soil Sci 118, 213 (1974). 19. S. U. Khan and K. C. Ivarson, Microbiological release of unextracted (bound) residues from an organic soil treated with prometryn, J. Agric. Food Chem. 29, 1301 (1981). 20. T. W. Fuhremann and E. P. Lichtenstein, Release of soil-bound methyl [14C] parathion residues and their uptake by earthworms and oat plants, J. Agric. Food Chem. 26, 605 (1978).
BIOCHEMISTRY
AND
PHYSIOLOGY
Copolymerization
23, 261-272 (1985)
of Halogenated
JEAN-MARCBOLLAG Laboratory
of Soil Microbiology,
The Pennsylvania
Phenols and Syringic Acid
ANDSHU-YEN State
University,
LIU University
Park,
Pennsylvania
16802
Received February 23, 1984; accepted July 5, 1984 Enzyme-catalyzed copolymerization of phenols with one to five chlorines (4chlorophenol; 2.4and 2,bdichlorophenol; 4-bromo-2-chlorophenol; 2,3,6- and 2,4,5-trichlorophenol; 2,3,5,6-tetrachlorophenol; and pentachlorophenol) and syringic acid was studied with an extracellular lactase of the fungus Rhizoctonia praticola. This reaction is of interest since it presents a model for explaining ‘the incorporation of anthropogenic compounds into humic substances. When the laccase was incubated together with the halogenated phenols and syringic acid, two types of hybrid products were found: (1) phenols covalently bound to an orthoquinone product of syringic acid resulting in the formation of quinonoid oligomers, and (2) phenols covalently bound to decarboxylated products of syringic acid resulting in the formation of phenolic oligomers. Mass spectra of hybrid oligomers gave typical chlorine isotopic patterns which coincided with their respective chlorophenol monomer. It was concluded that all hybrid products contained only one halogenated phenol molecule and that no dehalogenation took place. o 1985 Academic press, I~C. INTRODUCTION
beled 2,4-D was associated in the soil in a nonextractable form after 35 days. They concluded that it was not 2,4-D but its degradation products which were incorporated into soil organic matter. They did not find 2,4-dichlorophenol and assumed that-if it is formed-it must be rapidly degraded, but they did not consider the possibility of its binding to humic material. McCall et al. (7) also showed the incorporation of 2035% of some form of the ring carbon of 2,4D and 2,4,5-trichlorophenoxyacetic acid into humic substances of the soil. Evidence of the incorporation of pentachlorophenol (PCP) into soil constituents was recently provided by Weiss et al. (8). They found that the major residues of PCP in soil were bound residues which accounted for 28.6% of the applied radioactivity. Previously, we have shown that syringic acid can be polymerized by a lactase from the fungus Rhizoctonia praticola with the formation of a variety of oligomers ranging from dimers to hexamers (9). In the present study we further demonstrate that the enzyme can copolymerize syringic acid products and various chlorophenols. The occurrence of similar reactions in soil may lead
Chlorophenols are major intermediates of phenoxyalkanoate herbicides and other pesticides. They have also been found in pesticide preparations and industrial wastes. Their fate and behavior in the soil environment are of great concern for their toxic properties, particularly since they can easily form soil-bound residues. In soil, many compounds possessing phenolic characteristics and originating from lignin, other organic material, or through synthesis by microorganisms are essential constituents of humus (1, 2). Therefore, it is conceivable that halogenated and other xenobiotic phenols may also be incorporated into organic matter during the humification process (3, 4). Wolf and Martin (5) showed that 14C-ringlabeled 2,4-D (2,4-dichlorophenoxyacetic acid) and chlorpropham (isopropyl 3-chlorophenylcarbamate) or their transformation products were incorporated into humic-like polymers produced by fungi. Smith and Muir (6) found that approximately 15% of the radioactivity derived from carboxyl-labeled [14C]2,4-D and 30% from i4C-ring-la261
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D 1985 by Academic Press, Inc. of reproduction in any form reserved.
262 to the incorporation organic matter.
BOLLAG
of xenobiotics
into soil
AND
LIU
column (10 Frn) using a radial compression module (RCM-100, Waters Associates, Inc., Milford, Mass.). Isocratic elution was MATERIALS AND METHODS made with a solvent composition of acetoEnzyme. An extracellular fungal lactase nitrile:methanol:water (3:5:2, v/v) at a flow was isolated from the culture filtrate of R. rate of 1 mllmin. Quantitative analysis was praticola and the number of enzyme units carried out with a Waters’ data module used were established in an assay with the using the external standard method. Triplisubstrate 2,6-dimethoxyphenol as previcate samples were evaluated for each treatously described (10). ment. The calibration curves were linear in Substrate incubation and product re- the range between 0 and 20 Fg. covery. Ten micromoles of a halogenated Product characterization was based on phenol dissolved in 1 ml 0.1 N NaOH, 25 electron impact mass spectrometric analpmol syringic acid in 0.8 ml 70% ethanol, ysis (70 eV) with sample introduction by and 12.5 units of R. praticola lactase were direct insertion probe on an AEI MS-902 or mixed with 0.1 M phosphate buffer (pH 6.8) Kratos MS-50 mass spectrometer at temto make a 25ml reaction mixture. All as- peratures varying from 220” to 300°C. says were incubated in the dark for 3 hr at Proton NMR spectra were taken on a 30°C. Boiled enzyme preparations were Bruker 360 MHz instrument with a Fourier used as controls. transform system using deuteriochloroform Precipitates formed in the reaction mix- as a solvent. tures were collected by filtering through a Chemicals. Syringic acid, 2,6-dichloroGelman GN-6 metricel membrane (pore phenol, 2,3,5,6-tetrachlorophenol, 2,4,5size, 0.45 pm). The precipitates on the trichlorophenol, 2,3,6&chlorophenol, and membrane were washed with hexane to re- pentachlorophenol were purchased from move surface contaminants and then sub- Fluka Chemical Corporation (Hauppauge, jected to mass spectrometric analysis. The N.Y.). 4-Chlorophenol, 2,4-dichlorophenol, filtrate was extracted three times with equal and 2-chloro-4-bromophenol were obtained volumes of hexane to recover unreacted from Aldrich Chemical Company (Milchlorophenols and products. The combined waukee, Wise.). Pentachlorophenol and hexane extract and samples from the 2,3,6-trichlorophenol were treated with achexane washing were evaporated to dry- tive charcoal and recrystallized from benness and redissolved in an aliquot of ace- zene before use. The chemical purity of all tonitrile for chromatographic analysis. reagents was analyzed by TLC or HPLC. Analytical methods. Thin-layer chromaRESULTS tography (TLC) was carried out using silica gel F-254 plates (Brinkman Instruments, When the halogenated phenols 4-chloroInc., Westbury, N.Y.) with layer thickness phenol; 2,4- and 2,6-dichlorophenol; 4of 0.25 mm. Products from the hexane ex- bromo-2-chlorophenol; 2,3,6- and 2,4,5tract were separated in a solvent system of trichlorophenol; 2,3,5,6-tetrachlorophenol; ethyl acetate: hexane (1: 1, v/v). and pentachlorophenol were incubated for High-performance liquid chromato3 hr individually with syringic acid in the graphy (HPLC) was performed with a Model presence of a lactase from R. praticola, the 6000A pump, U6K injector, and a 440 uv color of the reaction mixture changed and detector operating at a wavelength of 254 the formation of a precipitate was obnm (Waters Associates, Inc., Milford, served. The liquid phase and the precipitate were separately analyzed. In subsequent Mass.). All samples were passed through experiments it was established that the Sep-Pak C’* cartridges before separation by an 8 mm x 10 cm reverse-phase C-18 products of the two phases had different
COPOLYMERIZATION
OF HALOGENATED
chemical properties. The supematant of the reaction mixture was extracted with hexane and the analysis of this extract is described under Quinonoid Hybrid Oligomers. The precipitate was removed by membrane filtration and washed thoroughly with hexane; the chemical characteristics of the precipitate are described under Phenolic Hybrid Oligomers. If the enzyme assay with the various substrates was done with a boiled enzyme preparation or without enzyme, no color change or product formation (as determined by TLC and HPLC) could be observed. Quinonoid Hybrid Oligomers
The liquid phase of the enzyme assays was extracted with hexane. The red color of the hexane fraction indicated the presence of quinonoid products which could be separated from the chlorophenols by TLC (Table 1). The colored spots were extracted with ethyl acetate and subsequently analyzed by mass spectroscopy. Evaluation of the spectra indicated the formation of dimerit and trimeric quinonoid hybrid products. The mass spectra revealed that all products gave identical chlorine patterns as compared to the starting chlorophenol. These results indicated that only one molecule of chlorophenol is incorporated into the hybrid quinonoid dimers and trimers, as shown in the mass spectra of syringic acid and 2,ddichlorophenol, 4-bromo-2-chloro-
phenol, or 2,4,Strichlorophenol (Fig. 1) and in Table 2. The increase in M + 2 shown in the mass spectra was due to the reduction of the quinone molecule to a phenolic compound in the ionization chamber. We could not obtain quinonoid hybrid oligomers from higher chlorinated phenols such as 2,3,5,6-tetrachlorophenol and pentachlorophenol, and syringic acid. However, it is possible that the amount of quinones produced was insufficient for detection with our technique. Phenolic Hybrid Oligomers
Phenolic oligomers which appeared as precipitates in the reaction mixtures were collected by membrane filtration. Mass spectral analysis of the precipitates obtained from the combination of syringic acid and various chlorophenols indicated the presence of dimeric to pentameric hybrid oligomers (Table 2; Figs. 2, 3, and 4). The chlorine isotopic cluster pattern shown on the mass spectra enabled us to determine the number of chlorine atoms present in each hybrid oligomer and thus the type of chlorophenol molecule incorporated. Hybrid oligomers originating from various chlorophenols gave typical chlorine cluster patterns identical to their respective chlorophenol monomers, indicating that only one molecule of chlorophenol was present in the various oligomers and that there was no loss of chlorine. In our earlier studies we established that
TABLE Thin-Layer
Chromatographic
Syringic acid combined with
0 Solvent system: hexane-ethyl
1
Separation of Quinonoid Hybrid Various Chlorophenols
Chlorophenol Monomer (R,)
4-Chlorophenol 2,4-Dichlorophenol 2,6-Dichlorophenol 4-Bromo-2-chlorophenol 2,4,5-Trichlorophenol
0.700 0.86 0.88 0.84 0.93 acetate (1: 1, v/v).
263
PHENOLS
Products
qf Syringic
Acid
Quinonoid product Dimer (I$)
Trimer W,1
0.44 0.62 0.67 0.56 0.77
0.62 0.67 0.56 -
and
a
Y c
p z-
v, z
> I-
0
50
FIG. 1. Mass
50
49
50
50
spectra
69
100
I,
of quinonoid
84
100
loo
200
hybrid
products
150
132 I, Ill,++& ,,,,,.
150
150
of syringic
200
250
acid
200
350
and 2,&dichlorophenol,
m/z
250
+5-v-d~~r-yr.
300
344
Dimrr
250
300
L
ACID
350
ACID
300
ACID
+
+
450
+
4-bromo-2-chlorophenol,
SYRINGIC
400
SYRlNGlC
SYRINGIC
2,4,5-
-450
550
or 2,4,5-trichloropheaol.
400
400
CHLOROPHENOL
TRICHLOROPHENOL
500
4- BROMO-E-
350
2-G - DlCHLOROPHENOL
of Hybrid
Oligomers
of Syringic Acid
C,,H,Wl,
-
-
-
332
-
478 C,,H,,O,ClBr -
342 Ct3Hs0,ClBr 332
CnH704Cl3
C21H1606C~2
434
C,,W',Cl,
298
C2lH,606C12
434
C,,H,O,Clb 298
G3%04C12
Trimer -
Dimer 264”
Quinonoid oligomers (hexane extract)
after Incubation
TABLE
2
C,,W'4C15
G&,oWl, 416
382
C,,H,,O,Cl,
348
C,,H,,O,Cl,
358 C,,H,,O,BrCl 348
CMH,204C12
314
C,,H,204Cl2
‘%HnWl 314
Dimer 280
and Chlorophenols
of a Lactase
from
Rhizoctonia
C22H,707C1,
568
C22H,807C14
534
C22H,,O,Cl3
500
C22H,,07C1,
510 C22H2007BrCI 500
C22H2007CI2
466
C22H2007C12
C22H2,07C1 466
Trimer 432
720 C,OH~SO,OC~S
C,oH26O,oCl4
686
C30H270,0C13
652
C,&,,O,,Cl,
812
C38H34013C14
804 W-W&h 804 GdMWl, 838
652 C30H27010C13
770 W-W,~C4 -
C,BH,~O,,C~,
C3&370,3Cl 770
Pentamer 136
praticola
618 C,o%@,oCh. -
C,oH2tP,oCl2
C,o%O,oCl 618
Tetramer 584
Phenolic oligomers (precipitate)
in the Presence
a m/z value of molecular ion. b Molecular composition based on number of chlorine atoms and m/z value of a given compound.
Pentachlorophenol
2,3,5,6-Tetrachlorophenol
2,4,5-Trichlorophenol
2,3,6-Trichlorophenol
4-Bromo-2chlorophenol
2,6-Dichlorophenol
2,4-Dichlorophenol
Reaction of syringic acid with 4Chlorophenol
Formation
s z
8
F s m” ?
%
2
% z g ij 5
BOLLAG
266 SYRINGIC
AND LIU
ACID
+
4 - CHLOROPHENOL
28o DIMER
TETRAMER 584
TRIMER
100
200
300
400
m/z 100
s
1
SYRINGIC
ACID
+
4 - BROMO
- 2 - CHLOROPHENOL
I
I
TRIMER 510
100
200
l,...,l.,~"~'~~l 400
300
i' 500
600
m/z FIG. 2. Mass spectra of phenolic hybrid products of syringic acid and I-chlorophenol 2-chlorophenol.
oligomers of syringic acid are coupled by aryl-oxygen-aryl (C-O) linkages (9). The binding of PCP to syringic acid products had to be by C-O linkage exclusively at the position of l-0-4’, since this is the only way to achieve a molecular ion which will fit a
or I-bromo-
given molecular composition containing five chlorine atoms. It can be concluded that PCP-syringic acid hybrid oligomers consist of one molecule of PCP bound to one to several molecules of decarboxylated syringic acid prod-
COPOLYMERIZATION SYRINGIC
400
OF ACID
HALOGENATED ‘2.6
+
500
267
PHENOLS
DICHLOROPHENOL
600
700
800
m ./z
SYRINGIC
ACID
+
2.4.5
-TRICHLOROPHENOL DIMER
FIG. 3. Mass trichlorophenol.
spectra
of
phenolic
hybrid
products
ucts. The possible structural configurations of PCP-syringic acid hybrid oligomers are presented in Fig. 5. The molecular compositions of the hybrid oligomers from other chlorophenols and syringic acid (listed in Table 2) can be derived when the chlorine content and molecular ion are taken into consideration. According to the molecular compositions, each hybrid oli-
of syringic
acid and 2,6-dichlorophenol
or 2,4-S-
gomer consists of one chlorophenol and one to several molecules of syringic acid derivatives. Therefore, it appears reasonable to assume that the hydroxyl group of the chlorophenols is the reactive site. Consequently, the binding of chlorophenols other than PCP to syringic acid products should take place via C-O linkage as in the case of the PCP-syringic acid complex. A
268
BOLLAG SYRINGIC
350
400
ACID
+
AND
LIU
2.3.5.6 - TETRACHLOROPHENOL
450
560
m/z
SYRINGIC
350
400
ACID
450
+
PENTACHLOROPHENOL
500
550
600
650
700
m/z
FIG. 4. Mass pentachlorophenol.
spectra
of phenoiic
hybrid
products
model is proposed in Fig. 6 in which the structural configurations of phenolic and quinonoid oligomers from a reaction of syringic acid and 2,4,5-TCP is presented. It is most likely that hybrid oligomers higher than hexamers were formed but that they could not be detected since the mass spectrometer was only programmed to a mass range of 800.
of syringic
acid and 2,3,5,6-tetrachlorophenol
or
Zdentzjkation of 3-Methoxy-5-(2’,4’,5’trichlorophenoxy)benzoquinone(l,2) (MTPQ), a Hybrid Product of 2,4,5TCP and Syringic Acid The quinone dimer from the combination of 243TCP and syringic acid was isolated by TLC and further purified by HPLC for chemical characterization. High resolution mass spectrometric analysis of the quinone
COPOLYMERIZATION
OF
HALOGENATED
269
PHENOLS
m/z 416
PENTACnLOROPHENOL
FIG.
SYRINGIC
5. Proposed
structures
ACID
of hybrid
qf
products
hybrid dimer yielded an elemental composition of C,,H,O,Cl, (calculated, 333.9567; found, 333.9575). The increase in M + 2 could be explained by the reduction of the quinone molecule to a phenolic compound PUINONOID
syringic
m/z 332 OCH3
2.4,5-TRICHLOROPHENOL
SYRINOIC
ACID
m/r
m/z 196
FIG.
346
6. Proposed
mh
structures
of hybrid
pentachlorophenol
OLIOOYER
Cl
PHENOLIC
and
in the ionization chamber. NMR analysis further verified the structural configuration of the molecule (Table 3). The results clearly indicated that 2,4,Strichlorophenol was covalently bound to a monomethoxy
Cl&,+-$,
m/r
acid
196
OLIQOYER
500
products
m/t 652
of
syringic
acid
and
2.4,5-trichlorophenol.
BOLLAG
270
AND LIU
TABLE Proton
NMR
Data
for
a Quinonoid
Hybrid product
3
Hybrid Dimer, 3-Methoxy-5-(2’,4’,5’-trichlorophenoxy)benzoquinone(l,2), of 2,4,5-Trichlorophenol and Syringic Acid
Chemical shift (w-d
Splitting integral
7.64 7.33 6.05 5.18 3.89
orthoquinone derived from syringic acid through a C-O linkage. Consequently, the product was identified as 3-methoxy-5(2’,4’,5’ - trichlorophenoxy) benzoquinone(12).
Quantitative Analysis of the Formation of MTPQ The decrease in 2,4,5-TCP and the formation of the hybrid quinone dimer (MTPQ) were monitored at different time intervals for a period of 3.5 hr. As shown in Fig. 7, the concentration of the hybrid quinone dimer gradually increased during the 2.5-hr reaction period while the concentration of 2,4,5-TCP decreased. Approximately 42% of the metabolized 2,4,5-TCP was recovered as the hybrid quinone dimer after 2 hr of incubation.
Singlet Singlet Doublet Doublet Singlet
Proton assignments
Coupling (H-4
Ha Hb Hc Hd Methoxy
(1H) (1H) (1H) (1H) (3H)
2.5 2.5
the hybrid oligomers (e.g., Fig. 4), suggesting that these products are not involved in the copolymerization reaction with chlorophenols. On the other hand, the products with m/z 290, 306, and their corresponding trimers and tetramers (9) are no longer detected in the present mass spectra, indicating their reaction with chlorphenols. Previously, we reported that mono- and disubstituted chlorinated phenols such as 2,4-dichlorophenol or 4-bromo-2-chloro-
2,4,5-TRICHLOROPHENOL
DISCUSSION
Syringic acid is a representative of the numerous humus monomers. It is easily oxidized when exposed to phenoloxidases (9, 11). In our laboratory, a number of investigations have been undertaken on the laccase-catalyzed polymerization and copolymerization of syringic acid (9, 12, 13). In the present study if was found that decarboxylated syringic acid products and monomethoxy-orthoquinone are able to complex with differently substituted halogenated phenols. Syringic acid dimers with ml z 350 and 364, and their corresponding trimers and tetramers with m/z 502, 516 and m/z 654, 668, respectively (9), usually appeared in the mass spectrum together with
;t
.I
c
HYBRID
1 .----.-.-. ,*----
.A*’
0 0
PRODUCT
120
60
180
MINUTES
FIG. 7. Disappearance of 2,4,5-trichlorophenol and formation of a quinonoid hybrid dimer [3-methoxy-5(2’,4’,5’-trichlorophenoxy)benzoquinone(l,2)] during incubation of 2,4,5-trichlorophenol and syringic acid in the presence of a lactase of R. praticola.
COPOLYMERIZATION
OF HALOGENATED
phenol can be oxidatively coupled to higher oligomers (14), but higher chlorinated phenols such as trichlorophenols, 2,3,5,6tetrachlorophenol, and pentachlorophenol were not transformed by the R. praticola lactase (S.-Y. Liu, unpublished data). Konishi and Inoue (15) found that a lactase from Trametes versicolor can transform pentachlorophenol to quinonoid products. In their experiments, the reactions were carried out with high concentrations of pentachlorophenol (500 ppm) at pH 6.2, 30°C for 2 days. Repeating their experiment under the same conditions, we obtained similar results with a lactase from T. versicolor, but not with the R. praticola laccase (unpublished data). When chlorophenols, syringic acid, and the lactase were incubated together, the primary reaction appeared to be the copolymerization of the two substrates. In our previous experiments with chlorinated phenols as the only substrate, oligomers were clearly determined (16), while in the present study only hybrid products and oligomers of syringic acid were found in the mass spectral analysis. Apparently, the formation of oligomers from chlorophenols alone was suppressed. It is suggested that a sequence of reactions is involved in the copolymerization of chlorophenols with syringic acid products. It appears that oxidation of syringic acid is a prerequisite for this copolymerization. Our previous studies, in which the formation of multiple products of syringic acid has been established, support this view (9). The evidence presented in this paper clearly demonstrates the chemical binding between chlorophenols and syringic acid products. These findings imply the potential incorporation of xenobiotics into soil organic matter during the humitication process. Such products are known as bound residues (4, 17), and are a cause of concern because of their environmental impact. Particularly, phenols are of concern due to their inherent toxicity and ever-increasing prevalence as byproducts in industry. Sev-
PHENOLS
271
era1 studies indicate that xenobiotics bound to humic substances can later be released by the activity of microorganisms (18, 19) or earthworms (20), thus posing a delayed health hazard. ACKNOWLEDGMENTS The preparation of this article was supported in part by a research grant from the Environmental Protection Agency (R-808165) and by the Pennsylvania Agriculturaf Experiment Station (Journal Series No. 6830). The views expressed herein do not necessarily reflect the opinion of the Environmental Protection Agency and no endorsement of that agency should be inferred. REFERENCES 1. K. Haider, J. P. Martin, and Z. Filip, Humus biochemistry, in “Soil Biochemistry” (E. A. Paul and A. D. McLaren, Ed.), Vol4, p. 195, Marcel Dekker, New York, 1975. 2. W. Flaig, H. Beutelspacher, and E. Rietz, Chemical composition and physical properties of humic substances, in “Soil Components” (J. E. Gieseking, Ed.), Vol. 1, p. 1. Springer-Verlag. New York, 1975. 3. R. Bartha, Pesticide residues in humus. ASM News 46, 356 (1980). 4. J.-M. Bollag and M. J. Loll, Incorporation of xenobiotics into soil humus, Experientiu 39. 1221 (1983). 5. D. C. Wolf and J. P. Martin, Decomposition of fungal mycelia and humic-type polymers containing carbon-r4 from ring and sidechain labeled 2.4-D and chlorpropham, Soil Sci. Sot. Amer. J. 40, 700 (1976). 6. A. E. Smith and D. C. G. Muir, Determination of extractable and non-extractable radioactivity from prairie soils treated with carboxyl- and ring-labelled [14C] 2.4-D. Weed RPS. 20, 123 (1980). 7. P. J. McCall, S. A. Vrona, and S. S. Kelley, Fate of uniformly carbon-14 ring labeled 2.4.5trichlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid, J. Agric. Food Chem. 29, loo (1981). 8. U. M. Weiss, I. Scheunert, W. Klein, and E Korte, Fate of pentachlorophenol-r4C in soil under controlled conditions, J. Agric. Food Chem. 30, 1191 (1982). 9. S.-Y. Liu, R. D. Minard, and J.-M. Bollag, Oligomerization of syringic acid, a lignin derivative, by a phenoloxidase. Soil Sci. Sot. Amer. J. 45, 1100 (1981). 10. J.-M. Bollag, R. D. Sjoblad. and S.-Y. Liu. Characterization of an enzyme from Rhizocronia praticola which polymerizes phenolic compounds, Canad. .I. Microbial. 2.5, 229 (1979).
272
BOLLAG
11. T. Ishihara and M. Ishihara, Oxidation of syringic acid by fimgal lactase, Mokuzai Gakkaishi 22, 371 (1976). 12. J.-M. Bollag, S.-Y. Liu, and R. D. Minard, Crosscoupling of phenolic humus constituents and 2,4-dichlorophenol, Soil Sci. Sot. Amer. J. 44, 52 (1980). 13. J.-M. Bollag, R. D. Minard, and S.-Y. Liu, Crosslinkage between anilines and phenolic humus constituents, Environ. Sci. Technol. 17, 72 (1983). 14. J.-M. Bollag, R. D. Sjoblad, and R. D. Minard, Polymerization of phenolic intermediates of pesticides by a fungal enzyme, Experientia 33, 1564 (1977). 15. K. Konishi and Y. Inoue, Detoxification mechanism of pentachlorophenol by the lactase of Coriolus versicolor, Wood Res. 18, 463 (1972).
AND LIU 16. R. D. Minard, S.-Y. Liu, and J.-M. Bollag, Oligomers and quinones from 2,4-dichlorophenol, J. Agric. Food Chem. 29, 250 (1981). 17. D. D. Kaufman, Bound and conjugated pesticide residues, in “Bound and Conjugated Pesticide Residues” (D. D. Kaufman, G. G. Still, G. D. Paulson, and S. K. Bandall, Eds.), p. 1, Amer. Chem. Sot., Washington, D.C., 1976. 18. T. S. Hsu and R. Bartha, Biodegradation of chloroaniline-humus complexes in soil and in culture solution, Soil Sci 118, 213 (1974). 19. S. U. Khan and K. C. Ivarson, Microbiological release of unextracted (bound) residues from an organic soil treated with prometryn, J. Agric. Food Chem. 29, 1301 (1981). 20. T. W. Fuhremann and E. P. Lichtenstein, Release of soil-bound methyl [14C] parathion residues and their uptake by earthworms and oat plants, J. Agric. Food Chem. 26, 605 (1978).