the metabolism of chlorinated aromatic pollutants by the frog

The metabolism of chlorinated aromatic pollutants by the frog

Guelph Waterloo Centre for Graduate Work in Chemistry, Department of Chemistry, University of Guelph, Guelph, Ont., Canada NIGZWI

AND

Milieuchemie, Universio of Amsterdam, Amsterdam, The Netherlands

Received May 26, 1976

SAFE, S., D. JONES, J . KOHLI, L. 0 . RUZO, 0. HUTZINGER, and G. SUNDSTROM. 1976. The metabolism of chlorinated aromatic pollutants by the frog. Can. J. Zool. 54: 181&1823.

The metabolism of several chlorinated aromatic pollutants has been studied using the frog species Runa pipiens as a model system. The substrates were administered by intraperitoneal injection and the metabolites were isolated and identified by chromatographic and spectroscopic methods. Chlorinated biphenyls, naphthalenes, and benzenes gave a range of hydroxylated products: 4-chlorobiphenyl was converted into 4'-chloro-4-biphenylol, 4'-chloro-3,4-biphenyldiol, and 4'-chloro-3-methoxy-4-biphenylol; 4,4'-dichlorobiphenyl gave 4,4'-dichloro- 3-biphenyl01 and commercial Aroclor 1254 yielded mono-, di- and tri-chlorobiphenylols; I-chloro- and 1,4-dichloro-naphthalene gave Cchloro- and 2,4-dichloro-naphthol respectively; a series of lower chlorinated (monotetra) benzene isomers were also hydroxylated, whereas the pentachloro and hexachloro isomers did not yield any metabolic products. The results also suggested that biohydroxylation in the frog is similar to that of mammalian systems in which arene oxide intermediates are involved.

SAFE, S., D. JONES, J. KOHLI, L. 0 . RUZO, 0. HUTZINGER et G . SUNDSTROM. 1976. The metabolism of chlorinated aromatic pollutants by the frog. Can. J. Zool. 54: 1818-1823.

On a ttudie le metabolisme de plusieurs polluants aromatiques chlores, la grenouille Rana pipiens servant de systeme modele. Les substrats ont ete injectes dans la cavite abdominale; on isolait par la suite les metabolites pour les analyser par chromatographie et spectroscopie. Les biphenyles, les naphtalenes et les benzenes chlores resultent en une serie de produits hydroxyles: le Cchlorobiphenyle se transforme en 4'-chloro-4-biphenylol, 4'-chloro-3-4-biphenyldiol et 4'-chloro-3-methoxy-4-biphenylol, le 4-4'-dichlorobiphenyle se convertit en 4,4'-dichloro-3- biphenyl01 et le produit commercial Aroclor 1254 donne des mono, di et trichlorobiphenylols; le 1-chloro et le 1-4-dichloronaphtalene donnent respectivement le 4-chloro et le 2-4-dichloro- naphtol. Une serie d'isomeres chlores (mono-tetra) du benzene donnent egalement des produits hydroxyles, mais les penta et les hexachloroisom~res ne sont pas m6tabolises. Les resultats indiquent que la biohydroxylation chez la grenouille se rapproche de celle qu'on observe dans le sy steme des mammiEres chez lesquels on constate la presence d'intermediaires d'oxydes de carbures benzeniques.

[Traduit par le journal]

Introduction Halogenated aromatic chemicals are widely

used in industry and in agriculture as pest- control agents. Many of these compounds are chemically inert and also resistant to environmental degradation and have become some of the most ubiquitous and persistent environmental pollutants (Fishbein 1973). Typical of this class of chlorinated aromatic pollutants are

et al. 1974; Fishbein 1973 ; and Risebrough et al. 1968). In addition chlorinated benzenes, polychlorinated terphenyls (PCT), and polychlorinated naphthalenes have also been identified as environmental pollutants (Morita and Ohi 1975; Zitko et al. 1972; Baker and Brown 1975) and most of the chlorinated aromatic contaminants have been identified in marine and aquatic life and in marine sediments, or are present in low

1 , l , l - trichloro - 2,2 - b i ~ ( ~ - chlorophenyl)ethane concentration in diverse water samples (Jensen (DDT) and 1,l-dichloro-2,2-bis@-chloropheny1)- et al. 1969; Koeman et al. 1969; DeLong et al. ethane (DDE) as well as the polychlorinated 1973; Homet al. 1974; Baker and Brown 1975). biphenyls (PCB), which have been identified in The biological impact of chlorinated aromatic almost all areas ofthe global ecosystem (Hutzinger pollutants has received considerable attention

Can

. J. Z

ool.

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

4/14

For

pers

onal

use

onl

y.

SAFE ET AL.

TABLE 1. A summary of the metabolism of chlorinated aromatic substrates by frogs

Chlorinated aromatic Metabolite % conversion into substratea (molecular ion) metabolitea

4,4'-dichlorobiphenyl Aroclor 1254

1-chloronaphthaleneb 1 ,4-dichloronaphthaleneb Halowax 1031b Chlorobenzene 1,4dichlorobenzene 1,2,3-dichlorobenzene 1,3,5-trichlorobenzene 1,2,4-trichlorobenzene 1,2,3,4-tetrachlorobenzene 1,2,3,5-tetrachlorobenzene 1,2,4,5-tetrachlorobenzene Pentachlorobenzene Hexachlorobenzene

NOTE: The chlorinated aromatic substrates were purchased from the Aldrich Chemical Company. .Eighty milligrams of substrate administered equally to four frogs: yield is based on the 80 mg of substrate;

remarnder of substrate and metabol~tes were not excreted into the water. bChloronaphthalene results have been reported in part by Sundstrom et 01. (1975).

(Kimbrough 1974; Fishbein 1973) and their metabolism in diverse systems has also been studied. The metabolism of polychlorinated biphenyls, naphthalenes, and benzenes gives a range of hydroxylated products (Safe et al. 1976; Ruzo et al. 1975; Kohli et al. 1976). This bio- chemical modification of the lipophilic pollutants into a more polar metabolite (or conjugate metabolite) is a detoxification procedure that can result in the subsequent excretion of the polar product. More recent work has shown that the biohydroxylation proceeds via arene oxide intermediates (Jerina and Daly 1974) and these activated aromatic molecules are associated with chemical carcinogenicity (Heidelberger 1975).

Creaven and co-workers, (1965) have shown that biphenyl is metabolized in vitro by both rainbow trout (Salmo gairdinerii) and frogs, al- though at a much slower rate than mammals. In addition, drug metabolism by marine vertebrates indicates a whole range of metabolic pathways that are comparable with those observed in mammals (Adamson 1967). This paper describes the use of the frog (Rana pipiens) as a model amphibian in the study of the metabolism of chlorinated aromatic pollutants that are associated with the aquatic environment. Previous

works has indicated that trout did not metabolize chlorinated aromatic components (Hutzinger, Nash et al. 1972) and it is therefore of interest to determine if this property is typical of aquatic amphibians.

Materials and Methods Substrates

A summary of the model substrates used in the experiments is given in Table 1 and their commercial sources are also indicated. The deuterated substrates 4-chIoro-4'- deuterobiphenyl and 1-chloro4deuteronaphthalene were synthesized by reductive deiodination of khloro-4'- iodobiphenyl and 1-chloro-4-iodonaphthalene respectively with lithium aluminum deuteride as described pre- viously (Safe et al. 1975; Ruzo et al. 1976). The isotopic purity of the deuterated chlorobiphenyl and chloronaphthalene substrates were determined by mass spectrometric analysis to be 90 and 85% respectively.

Administration of Substrates The chlorinated aromatic substrate (80 mg) was dis-

solved in a minimum volume of vegetable oil (4-5 ml) and administered equally to four frogs by intraperitoneal injection. The frogs were kept without feeding in large Erlenmeyer flasks (4 1) containing 250 ml of water. After 4 days the water was replaced and the experiments termi- nated at the 8th day. The combined water samples were analyzed for metabolites as described.

Extraction and Isolation of Metabolites The water containing t t e frog excreta was extracted

Can

. J. Z

ool.

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

4/14

For

pers

onal

use

onl

y.

CAN. J . ZOOL. VOL. 54, 1976

TABLE 2. Nuclear magnetic resonance spectra of chlorinated aromatic metabolites

Metabolite (substrate) Proton chemical shifts, ppm

4'-chloro-4-biphenyl01 7.55(d,J = 8.2Hz,2H),7.48(d,J= 8.2Hz, (4-chlorobiphenyl) 2H), 7.42(d, J = 8.2Hz, 2H), 7.18

(d, J = 8.2 Hz), 2H) 4'-chloro-3-methoxy-4- 7.47 (d, J = 8.2Hz, 2H), 7.38 (d, J = 8.2

biphenyl01 Hz, 2H),7.08 (q, J = 8.2,2.2Hz, lH), 7.04 (4-chlorobiphenyl) (d, J = 2.2 Hz, lH), 6.99 (d, J = 8.2 Hz,

lH), 3.92 (s, 1H) 4'-chloro-3,4-biphenyldiol 7.43 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.2

(4-chlorobiphenyl) Hz, 2H), 7.08 (d, J = 1.9 Hz, lH), 7.01 (q, J = 8.2, 1.9 Hz, lH), 6.93 (d, J = 8.2 Hz, 1H)

4,4'-dichloro-3-biphenyl01 7.47 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.2 (4,4'-dichlorobiphenyl) Hz, 2H), 7.36 (d, J = 8.2 Hz, lH), 7.21

(d, J = 8.2 Hz, lH), 7.05 (q, J = 8.2, 2.2 Hz, 1H)

4-chloronaphthol 8.23, 7.57 (m, 4H), 7.37 (d, J = 8.2Hz, 1H) (I-chloronaphthalene) 6.70 (d, J = 8.2 Hz, 1H).

(two times) with an equal volume of chloroform. The dried chloroform extracts were concentrated and the residue purified by preparative thin-layer chromatography (tlc) using silica gel HF,,, (Merck) as the sorbent. The plates were eluted in chloroform or hexane:ether (7:3) and bands that were fluorescent under ultraviolet light were removed, extracted with ether, and analyzed by mass spectrometry. Those bands that exhibited ions containing the familiar isotopic distribution characteristic of chlorinated species (Hutzinger, Safe et a / . 1972) were further purified by tlc and their structures were determined by comparative gas-liquid chromatography (glc) withauthen- tic standards and by spectroscopic techniques.

The extracted aqueous material was carefully acidified with sulfuric acid to give a 4 N solution then heated at 100 "C for 2 h. The solution was cooled, extracted with ether, and the organic residues examined by tlc. Only trace quantities of metabolites were observed in the hydrolized extracts.

Gas Chromatographic and Spectroscopic Methods Gas liquid chromatographic analysis was carried out

on a Hewlett-Packard model 5710 chromatograph equipped with a flame ionization detector and a glass column (0.3 cm x 2 m) packed with 3% OV 225 on Gas Chrom Q. Operating conditions were: helium carrier gas flow, 30 ml/min; hydrogen flow, 60 ml/min; air flow, 200 ml/min; inlet and detector temperatures 300°C; column temperatures, variable as required.

Mass spectra were obtained using a low resolution Varian MAT CH-7 spectrometer at 70 eV equipped with electrical detection. The samples were inserted at ion source temperatures of 10 "C and the temperature was increased as required. Nuclear magnetic resonance (nmr) spectra were recorded on a Varian HR220 spectrometer using deuterochloroform as solvent and a summary of nmr data is given in Table 2.

Results Chlorobenzene Metabolism

Chlorobenzene, 1 ,4-dichlorobenzene, 1,2,3-,

1,3,5-, and 1,2,4-trichlorobenzenes, 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-tetrachlorobenzenes, pentachlorobenzene, and hexachlorobenzene were used as model substrates for this study and a summary of the metabolites that were identified i; given in Table 1. The hydroxylated products were all identified by comparative glc with authentic standards (Safe et al. 1976) and the structures confirmed by mass spectrometry. The mono-, di-, tri-, and tetra-chlorobenzenes all yielded phenolic metabolities and dechlorinated and rearranged products were identified only for the tetrachloroisomers. The higher chlorinated penta- and hexa-chlorobenzene isomers did not give any metabolic products.

Chloronaphthalene Metabolism 4-Chloronaphthol was identified as the sole

metabolite of 1-chloronaphthalene (Table 1) and the structure was confirmed by comparison of the chromatographic and spectroscopic data (Table 2) with an authentic sample of this compound (Ruzo et al. 1975). The only metabolite of 1,4-dichloronaphthalene was a mono- hydroxylated product (M' 212) and the 220 MHz nmr spectrum and glc retention time were identical to a commercial sample of 2,4-dichloro- naphthol (Eastman). The direct hydroxylation product, 1,4-dichloro-2-naphthol, was not de- tected in the water extracts. The commercial chlorinated naphthalene, Halowax 103 1, is pri- marily a mixture of 1- and 2-chloronaphthalene. Metabolism of Halowax 1031 followed by clean- up and mass spectrometric analysis indicated the formation of chloronaphthol products (M+ 178).

Can

. J. Z

ool.

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

4/14

For

pers

onal

use

onl

y.

SAFE ET AL. 1821

The metabolism of 1-chlor0-4-[~H]naphthalene (90% isotopic purity) also gave the 4-chloronaphthol as the sole metabolite and mass spectrometric analysis indicated 75% retention of the deuterium in the metabolite.

Chlorinated Biphenyl Metabolism Thin-layer chromatography of the extract

containing the 4-chlorobiphenyl metabolites gave three products. The least polar component (Mf 234) corresponded to a chloromethoxybiphenylol and the nmr spectrum indicated the presence of a single isomer with proton resonances as shown in Table 2. Previous results with rabbits (Safe et al. 1975) gave a mixture of the two possible chloromethoxybiphenylols, 4'-chloro-3-methoxy- 4 - biphenyl01 and 4 ' - chloro - 4 - methoxy - 3 - bi - phenylol, whereas only one of these two isomers is observed as a frog metabolite. The major metabolite (Mf 204) gave nmr signals at 6 7.55 (d, J = 8.2 Hz), 7.48 (d, J = 8.2 Hz), 7.42 (d, J = 8.2 Hz), and 7.18 (d, J = 8.2 Hz) ppm, which was identical with the spectrum of 4'- chloro-4-biphenyl01 (Safe et al. 1975). The most polar metabolite exhibited an Mf at m/e 220 and the nmr spectrum gave 6 7.43 (d, J = 8.2 Hz), 7.37 (d, J = 8.2 Hz), 7.08 (d, J = 1.9 hz), 7.01 (q, J = 8.2 and 1.9 Hz), and 6.93 (d, J = 8.2 Hz) ppm. The spectrum was identical to that of 4'-chloro-3,4-biphenyldiol (Safe et al. 1975). The sole metabolite of 4,4'-dichlorobiphenyl gave a M+ at m/e 238 and the nmr spectrum is given in Table 2. The spectral data were identical to the results reported for 4,4'-dichloro-3-biphenyl01 (Safe et al. 1976). Thin-layer chromatographic purification of the Aroclor 1254 metabolites gave several bands and the mass spectrum of the crude extract exhibited molecular ions cor- responding to chlorobiphenylols (Mf 204), di- chlorobiphenylols (M + 238), and trichlorobiphenylols (M+ 272).

Administration of 4-chlor0-4'[~H]biphenyl (85% purity by mass spectrometric analysis) gave the three expected metabolites, which were analyzed for deuterium content by mass spectrometric analysis. 4'-Chloro-4-biphenyl01 gave molecular ions at m/e 204 and 205 and 79% of the deuterium was retained in the metabolites; the mass spectrum of 4'-chloro-3,4-biphenyl diol gave a molecular ion at mle 220 and 221 with 39% retention of deuterium and similarly the molecular ion species of the chloromethoxybiphenylol indicated 39% retention of deuterium in this metabolite. The results were analogous

to those observed for the metabolism of Cchloro- 4'-[2~]biphenyl in the rabbit (Safe et al. 1975).

Discussion Industrial chemicals and pest-control agents

are translocated throughout the aquatic environment and their impact on marine and aquatic life is not yet fully understood. Persistent pollutants such as the halogenated aromatic compounds are not readily degraded in the environment and are known to accumulate in higher trophic levels of the food chain (Fishbein 1973). It was therefore of interest to determine the metabolic fate of such chemicals by an aquatic species since detoxification and excretion serve a useful function in protecting animals from bioaccumulation.

In vitro studies on the hydroxylation of biphenyl by trout and frog liver microsomal enzymes indicated the formation of a 4-hydroxybiphenyl metabolite in both species, and in addition 2-hydroxybiphenyl was also identified in the frog experiments (Creaven et al. 1965). The microsomal enzyme activity required reduced nicotinamide adenine dinucleotide phosphate (NADPH) and was similar to mammalian liver microsomal preparations but with markedly reduced enzyme activity (Creaven et al. 1965).

The summary given in Table 1 clearly indicates that the frog is an ideal aquatic organism to study the metabolism of chlorinated aromatics. Chlori- nated benzene, naphthalene, and biphenyl isomers are converted into a range of hydroxylation products and both commercial PCB (Aroclor 1254) and polychlorinated naphthalene (Halo- wax 1031) are also hydroxylated. Administration of both DDT and DDE to frogs as described in this paper did not yield any identifiable metabolic products in the water; these experiments are currently being reinvestigated (Sundstrom, un- published observations).

The metabolism of the mono-, di-, and trichlorobenzene isomers all gave direct hydroxylation products; however, 1,2,3,4-tetrachlorobenzene yielded 2,3,4,6-tetrachlorophenol as the sole phenolic metabolite. The formation of this product requires a 1,2-chlorine migration from the site of hydroxylation to an adjacent carison atom (i.e. NIH shift) and suggests the intermediacy of an arene oxide (Jerina and Daly 1974). The 1,2,3,5- and 1,2,4,5-tetrachlorobenzene both gave hydroxylation-dechlorination metabolites and these products are also typically formed from arene oxide intermediates. The higher chlorinated

Can

. J. Z

ool.

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

4/14

For

pers

onal

use

onl

y.

1822 CAN. J. ZOOL. VOL. 54, 1976

penta- and hexa-chlorobenzene did not yield any measurable quantities of metabolite, thus com- plementing previous results in which higher chlorinated aromatics were more resistant to biodegradation (Kohli et al. 1976). Presumably these lipophilic substrates are stored in fatty tissue and are only slowly metabolized after re- lease from this tissue.

The mechanism of the biohydroxylation was also investigated using I-chlor0-4-[~H]naphthalene and 4-chlor0-4'-[~H]biphenyl as model substrates. Isolation of their respective hydroxy metabolites and analysis by mass spectrometry indicated 75 and 79% retention of deuterium in their respective metabolities. These results are consistent with metabolism via arene oxide intermediates (Fig. I), which typically rearrange with a 1,2 H (or 2H) transfer and this rearrangement or NIH shift (Jerina and Daly 1974) accounts for the retention of the deuterium in the metabolic products. The 1,2-C1 shift has also been observed in the metabolism of 1,4-dichloronaphthalene and this result is also consistent with an arene oxide intermediate. Mass spectrometric analysis of 4'-chloro-3,4-biphenyldiol and the chloromethoxybiphenylol indicated that only 39% of the deuterium is retained. This result indicates that the second hydroxyl group is introduced after the initial hydroxylation; since the catechol- like metabolites retain only one-half the 'H observed in the phenol, 4'-chloro-4-biphenylol, the second hydroxylation step occurs via a direct hydroxylation procedure. This sequence of hydroxylation of chlorobiphenyl was also observed in the rabbit (Safe et al. 1975) and is comparable with the stepwise hydroxylation of phenylalanine to give 3,4-dihydroxyphenylalanine (Guroff et al. 1966) and butamoxane to give 6,7-dihydroxy- butamoxane (Murphy et al. 1974).

The in vivo results clearly show that the frog

is an excellent model aquatic species for metabolic studies. Workup and isolation of the metabolities is much easier than typical animal studies using rats and rabbits since the excreta contain far less coextractive material. The data also show that a series of typical chlorinated aromatic pollutants give metabolic products similar to those observed in other mammals and the metabolic pathway (i.e. arene oxide intermediates) is also comparable with mammalian systems. The data also suggest that by metabolism the frog more readily detoxifies chlorinated aromatic pollutants than do trout. This may result in more pollutant bioaccumulation in the latter species; however, this would have to be confirmed by additional experiments and tissue analyses over extended time periods.

Acknowledgements The financial assistance of Environment

Canada and the National Research Council of Canada is gratefully acknowledged and the assistance of Dr. A. Grey and the Canadian 220 MHz NMR Centre was also appreciated.

ADAMSON, R. H. 1967. Drug metabolism in marine vertebrates. Fed. Proc. 26: 1047-1055.

BAKER, R. A., and E. BROWN. 1975. Proceedings, First Chemical Congress of North America, Environmental Division. Abstr. 78.

CREAVEN, P. J., D. V. PARKE, and R. T. WILLIAMS. 1965. A fluonmetric study of the hydroxylation of biphenyl in vitro by liver preparations of various species. Biochem. J. 96: 879-885.

DELONG, R. L., W. G. GILMARTIN, and J. G. SIMPSON. 1973. Premature births in California sea lions: associa- tion with high organochlorine pollutant residue levels. Science, 181: 1168-1170.

FISHBEIN, L. 1973. Mutagens and potential mutagens in the biosphere. Sci. Total Environ. 4: 305-340.

GUROFF, G., C. A. REIFSNYDER, and J. DALY. 1966. Re- tention of deuterium in p-tyrosine formed enzymatically from p-deuterophenylalanine. Biochem. Biophys. Res. Comm. 24: 720-724.

HEIDELBERGER, C. 1975. Chemical carcinogenesis. In Annual reviews of biochemistry. Vol. 44. Annual Re- views Inc., Palo Alto, California. pp. 79-121.

HOM, W., R. W. RISEBROUGH, A. SOUTAR, and D. R. H(X' YOUNG. 1974. De~osition of DDE and polychlorinated

x OH biphenyls in datedsediments of the ~ a n t a ~ i r b a r a basin. Science, 184: 1197-1 199.

HUTZINGER, O., D. M. NASH, S. SAFE, A. S. W. DEFREITAS, R. J. NORSTROM, D. J. WILDISH, and V. ZITKO. 1972. Polychlorinated biphenyls: metabolic be- haviour of pure isomers in pigeons, rats and brook trout. Science, 178: 312-3 14.

HUTZINGER, O., S. SAFE, and V. ZITKO. 1972. Analysis of chlorinated aromatic hydrocarbons by exhaustive

FIG. 1. Metabolism via arene oxide intermediates. chlorination: qualitative and structural aspects of per-

Can

. J. Z

ool.

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

4/14

For

pers

onal

use

onl

y.

SAFE ET AL. I823

chloro derivatives of biphenyl naphthalene, terphenyl, dibenzofuran, dibenzodioxin and DDE. Inter. J . Envi- ron. Anal. Chem. 2: 95-106.

1974. The chemistry .of PCBs. Chemical Rubber Co., Press, Cleveland, Ohio, U.S.A.

JENSEN, S., A. C. JOHNELS, M. OLSSON, and G. OTTERUND. 1969. DDTand PCB in marine animals from Swedish waters. Nature, 224: 247-250.

JERINA, D. M., and J. W. DALY. 1974. Arene oxides a new aspect of drug metabolism. Science, 185: 573-582.

KIMBROUGH, R. D. 1974. The toxicity of polychlorinated polycyclic compounds and related chemicals. In CRC critical reviews in toxicology. Chemical Rubber Co. Press, Cleveland. pp. 445-498.

KOEMAN, J. H., M. C. TEN NOEVER DE BRAUW, and R. H. DEVOS. 1969. Chlorinated biphenyls in fish mussels and birds from the river Rhine and the Netherlands coastal area. Nature, 221: 11261128.

KOHLI, J., D. JONES, and S. SAFE. 1976. The metabolism of higher chlorinated benzene isomers. Can. J. Biochem. 54: 203-208.

MORITA, M., and G. OHI. 1975. p-Dichlorobenzene in human tissue and atmosphere in Tokyo metropolitan area. Environ. Pollut. 8: 269-274.

MURPHY, P. J., J. R. BERNSTEIN, and R. E. MCMAHON. 1974. The formation of catechols by consecutive hy- droxylations: a study of the microsomal hydroxylation of Butamoxane. Mol. Pharmacol. 10: 634-639.

RISEBROUGH, R. W., P. RIECHE, D. B. PEAKALL, S. G. HERMAN, and M. N. KIRVEN. 1968. Polychlorinated biphenyls in the global ecosystem. Nature, 2 0 : 1098-1 102.

Ruzo, L. O., D. JONES, S. SAFE, and 0. HUTZINGER. 1976. The metabolism of chlorinated naphthalenes. J. Agric. Food Chem. 24: 581-583.

Ruzo, L. O., S. SAFE, 0. HUTZINGER, N. PLATONOW, and D. JONES. 1975. Hydroxylated metabolites of chloronaphthalenes (Halowax 1031) in pig urine. Chemosphere. pp. 121-123.

SAFE, S., 0. HUTZINGER, and D. JONES. 1975. The mechanism of chlorobiphenyl metabolism. J. Agric. Food Chem. 23: 851-853.

SAFE, S., D. JONES, and 0. HUTZINGER. 1976. Metabolism of 4,4'-dihalogenobiphenyls. J. Chem. Soc., Perkin Trans. 1. pp. 357-359.

SUNDSTROM, G., 0. HUTZINGER, S. SAFE, L. RUZO, and D. JONES. 1975. Methods for the study of toxic and persistent chemicals in aquatic organisms as exemplified by chloronaphthalenes. In Sublethal effects of toxic chemicals on aquatic animals. Edited by J. H. Koeman and J . J. T. W. A. Strik. Elsevier, Amsterdam. pp. 177-188.

ZITKO, V. , 0. HUTZINGER, W. D. JAMIESON, and P. M. K. CHOI. 1972. Polychlorinated terphenyls in the environment. Bull. Environ. Contam. Toxicol. 7: 200-201.

Can

. J. Z

ool.

Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

Uni

vers

ity o

f P.

E.I

. on

11/1

4/14

For

pers

onal

use

onl

y.

the metabolism of chlorinated aromatic pollutants by the frog

Documents