Biodegradation of chlorinated dibenzo-p-dioxins in batch and continuous cultures of strain JB1

Chemosphere, Vol.19, Ncs.8/9, Printed in G r e a t B r i t a i n

pp

1297-1308,

1989

0045-6535/89 $3.00 Pergamon Press plc

+

.OO

BIODKGRADATION OF CHLORINATED DIBENZO-p-DIOXINS IN BATCH AND CONTINUOUS CULTUKF.S OF STRAIN JBI

J.R. Parsons* and M.C.M.

Laboratory of Environmental University

of Amsterdam,

Storms

and Toxicological

Nieuwe Achtergracht

Amsterdam,

Chemistry, 166, 1018 WV

The Netherlands.

Abstract The biphenyl-utilizing

Alcaligenes

strain JBI cometabolizes

but is not able to grow on this compound. dibenzo-p-dioxins dichlorodioxins metabolism

of

accumulated

mixture

takes place in benzoate-grown was

induced

chemostat

the monochlorodioxin.

exposure

of chlorinated

p-dioxins

in

in cell-free extracts,

Continuous

A dihydroxylated

of a 3-methylbenzoate-grown

dibenzo-p-dioxins

and 1,2,4-trichloro-p-dioxin,

compounds

were

calculated

To whom correspondence

together

Ready cometabolism

of these

with

rate

metabolite

resulted

chemostat

of

an

increased

this

culture

in degradation

but there was no evidence The pseudo-flrst

latter

of

compound

may be addressed.

1297

of strain JBI to a

of 1,3- and 2,8-dichlorofor significant

order biodegradation

from their concentrations

which the culture was exposed.

*

batch cultures.

cultures,

2-chlorodibenzo-p-dioxin,

of 1,3-, 2,7- and 2,8-dichloro-

but was not detected in growing cultures.

tion of 1,2,3,4-tetrachloro-p-dioxln. of these

Slow cometabolism

in the culture

degrada-

rate constants and those

to

1298

Introduction Polychlorinated [I].

Despite

compounds,

all

dibenzo-p-dioxins

the

efforts

(PCDD's)

devoted

to

well

studies

of

known

the

environmental

environmental

pollutants

fate

of

these

little is known about their metabolism by microorganisms.

Metabolism salicylate,

has

but

been

not by

the dioxin.

reported

of dibenzo-p-dioxin

succlnate-grown

dihydrodibenzo-p-dioxin

and

cultures

No further metabolism

from these compounds were

thus

not

were potent degraded

strain

species

isolated

was detected.

of the dloxygenases

rate of metabolism

grown

as metabolites

Comparable

to grow on biphenyl

able

The

were

species

on

[2]. 1,2-Dihydroxy-l,2-

and a number of its mono-,

inhibitors

further.

a Pseudomonas

by

this

of these compounds

by a Beijerinckia

ted derivatives

of

1,2-dihydroxydibenzo-p-dioxin

and diols were formed from dibenzo-p-dloxin

and

are

dihydrodiols

di- and trichlorina[3].

The diols

required

of

of

the

formed

for ring fission

chlorinated

dioxins

decreased with increasing degree of chlorination. The

rate

bacteria TCDD

of

appears

metabolism

2,3,7,8-tetrachlorodibenzo-p-dioxin

of

to be very slow.

Traces

Cultures

of bacteria

isolated

from soil contaminated

for several weeks with 14C-2,3,7,8-TCDD 1-1.5%

of

the

total

radioactivity

soils and sediments: days in sediments We

here

half

lives were between

report

the

Materials

was

of

a

transformed

very

JBI

substrate.

was

Although

Cultures

number

of

in

chlorinated

dibenzo-p-dioxins

in

Alcalisenes strain JBI.

of the biphenyl-utilizlng

isolated

from soil by selective

enrichment

using biphenyl

further

glass stoppered as

experiments

nutrients

erlenmeyer

solutions

inoculated

as carbon

this strain was at first thought to be a Pseudomonas strain

for degradation

acid and

cultures

for only slowly

were formed.

has since been identified as an Alcaligenes strain by means of the API-2ONE

added

and incubated

which accounted

and methods

Strain

zoic

from 2,3,7,8-

130 and 400 days in soil and longer than 600

polar metabolltes

cometabolism

also

by

isolated from soil

by 2,3,7,8-TCDD

formed a polar metabollte,

[6]. 1,3,6,8-TCDD

[7]. Unidentified

chemostat and batch cultures

were produced

of Nocardiopsis and Bacillus strains

in soil and in pure cultures

[4,5].

of polar metabolltes

(2,3,7,8-TCDD)

in

flasks

I ml

once

bacterial

were

well

into

or methanol

growth

the

elsewhere

[8].

Batch

was

stationary

either

visible.

phase

at

the

Incubations

of their

growth

were

grown

in

PCDD's were

same

time

were

continued

curve

it

acid or 3-methylben-

cultures

to limit losses of PCDD's by volatilization.

acetone

or

were grown on i g/l benzoic

as reported

[8,9],

test system.

the medium

(usually

until

was the

48 or 72 h

after inoculation).

was

Continuous

cultures

limited

ca.

experiments

to were

chemost=t

system

identical

to

accounted

for by

20

grown to

avoid

the

compounds

carried

out,

not containing

those

under

which

dilution.

0.020 h "I. PCDD's were or methanol)

were ml/min

The

in the system

bacteria. bacteria cultures

were

were were

tested

grown, grown

previously

volatilization

No detectable

added to the cultures

or continuously

described

excessive

for

at dilution

Column.

[9]. The aeration PCDD's.

of

Before

volatilization

occurred

rates

either as a pulse

using a generator

their

losses the

of

under

conditions

disappearance

rates

between

(dissolved

any in a

being

0.045

and

in I ml acetone

This column contained

15 g Chromo-

1299

sorb

GAW

(45-60

mesh)

loaded

with

1,2,3,4-tetrachlorodibenzo-p-dioxins

ca.

5 mg

each

(I,3-DCDD,

of

1,3-di-,

2,8-DCDD,

2,8-di-,

1,2,4-TrCDD

1,2,4-tri-

and

and

1,2,3,4-TCDD,

resp.). Samples after

of

cultures

addition

(typically

of hexachlorobenzene

50 ml) as

were

extracted

with

internal

standard.

The

after centrifugation and reduced in volume up by being eluted

equal hexane

volumes layer

through a column containing ca.

of

the

PCDD's

were

removed

i g each of 100-120 mesh silica + 40% (Tracor 550,

GC on 160-180 mesh Chromosorb 750, I m x 2 mm; Hewlett-Packard 5890, DB-5, recoveries

was

to ca. I ml. This extract then underwent clean-

w/w H2SO 4 and silica + 33% w/w I N NaOH. Analysis was by GC-ECD

The

of hexane

determined

by

analyzing

samples

2% Dexil 300

30 m x 0.33 nun). containing

known

quantities of these compounds and were routinely above 90%. Control experiments were carried out under sterile but otherwise identical conditions. Crude cell extract was used in experiments designed to yield metabolites. was

obtained by

treating

with a Branson sonifier and incubated

for

Acidification

with

a concentrated

cell suspension

H2SO4,

2-chlorodibenzo-p-dioxin extraction

with hexane

The sample was redissolved

I ml

were

added and

(2-CDD)

(added as acetone

and evaporation

in 20 ml acetone,

the mixture was

filtered and again evaporated to dryness. gave after filtration

cultures)

(75 W, i0 x 20 sec). The extract was suspended in phosphate buffer

5 h with

dryness followed. iodomethane

The extract

(from 300 ml chemostat

refluxed

solution).

of the hexane

layer

to

i g potassium carbonate and

for 8 h. The mixture

was

then

Dissolution in toluene or 2,2,4-trimethylpentane

a sample which was analyzed on a Finnigan 1020 GC-MS in the E1 mode

at 70 eV (Supelco SP2331 column). The chlorodioxins were synthesized as reported [i0,ii].

Results The strain used the

carbon

strain

source,

was

in the experiments and

screened

cometabolizes

for

its

ability

described was isolated from soil with biphenyl as a number

of

to degrade

chlorinated

other

growth was observed on chlorinated dibenzo-p-dioxins. this strain were The

results,

degradation

of

1,3-,

dichlorodioxins cultures,

in

Table 2,7-

i,

and

indicated 2,8-DCDD.

ready

degradation

However,

[8]. When

aromatic

this

compounds

no

Benzoic acid-grown batch cultures of

then tested for their ability to cometabolize

shown

biphenyls

chlorinated

this

mono- and dichlorodioxins. of

2-CDD

apparently

but

slow

much

slower

degradation

of

may have been caused by the conditions under which bacteria grow in batch

rather

than

only

reflecting

an

intrinsic

poor

degradability

of

these

latter

compounds. Typical

results

benzoate-grown acetone

obtained

chemostat

solution

in two experiments with mixtures of 2-CDD and 2,8-DCDD in a

culture

of these

of

strain JBI

compounds was

are

injected

shown

in Fig.

i. In experiment

i an

into a culture which had not previously

been exposed to PCDD's.

2-CDD disappeared from the culture at a rate much higher than that

attributed

(dilution

to

dilution

first-order

degradation.

24 h

the

place.

after

start

of

rate constant

In contrast, the

D - 0.045 h'l),

and apparently

underwent

no degradation of 2,8-DCDD was detected until about

experiment,

when

induction

of degradation

appeared

to take

1300

100

0

0.1

I

0

6

I

I

I

12

18

24

time

Fig. I.

90

(h)

Repeated experiments with mixtures of 2-CDD and 2,8-DCDD in a chemostat of strain JBI. The line marked D indicates the dilution rate. Experiment i: o 2-CDD; ~ 2,8-DCDD. Experiment 2: --e-- 2-CDD; A 2,8-DCDD.

10

131

J o

o

0.1 0

i

J

J

J

5

10

15

20

time 0

Fig. 2.

2.7-E)(=E~3

Experiment with 2,7-DCDD

(h) ....... 11)=0.045

h- 1

in a chemostat culture of strain JBI.

25

culture

1301

TABLE 1 DEGRADATION OF PCDD's IN BATCH CULTURES OF STRAIN JBI PCDD

Incubation period (h)

Initial conc.(~g/l)

Final conc, Culture

2-CDD

70 48 66 66 48 24

14.0 321 290 315 27.5 252

nd** 7.3 200 210 Ii.0 322

1,3-DCDD 2,7-DCDD 2,8-DCDD

_+1.3 ±6 _+I0 _+14 _+1.9 _+12

(~Ii) Control 10.3 213 260 315 17.6 330

±4.1 ±20 _+30 _+0.2 ±3

±2.4 _+12 ±20 ±5 ±1.6 _+9

*_+SD. **Not detected.

When this experiment degradation g/i)

of

after

2-CDD

6 h.

Initial exposure culture,

was repeated

was

There

rapid, was

now

of the culture

presumably

also

the

evidence

for

by induction

of degradative

first-order

enzymes,

growth

observed

in

chemostat increased

batch

of

batch

of

2),

(ca.

i

2,8-DCDD.

led to adaptation of the

and thus to increased degradation

cultures,

cultures.

However,

degradation

degradation

which

the

of dichlorodioxins

in batch and chemostat

and

may

fact of

thus

of 2,7-DCDD

that

explain

batch

(Table 2) indicates

cultures has a dominating

ON DEGRADATION

(Fig.

2) and

206 17.2 18.8 9.4 0.59

49.1 17.4 20.6 10.2 0.61

A chemostat h -I,

was

1,2,3,4-TCDD.

from a chemostat

exposed

continuously

generator

to the culture.

degradation

of chlorinated

of this experiment

column

_+15.2 _+0.9 _+0.3 +0.3 -+0.15

5.1 16.3 15.9 9.4 0.64

_+0.3 -+1.5 _+1.6 -+0.8 +0.05

228 21.2 19.8 9.0 0.60

culture exposed continuously

to a mixture

was

A similar biphenyls

used

to

in the culture

(Cc) were However,

from

not

a

show

in growth

system was

in chemostat

cultures

significantly

to PCDD's.

2,8-DCDD,

these

used previously

_+44 _+3.1 +3.0 +1.5 +0.14

acid at a dilution rate of

of 1,3-DCDD,

dissolve

are shown in Fig. 3. The concentrations

(ANOVA test, P
did

(±SD) Control

culture of strain JBI, grown on 3-methylbenzoic

A

inoculated

induced,

OF PCDD's IN BATCH CULTURE

21,32,81,2,41,2,3,4-

inoculated

degradation

that the difference

Final concentration (#g/l) Pre-exposed* Unexposed

*Cultures

was

is comparable

limited

influence.

Initial conc. (~g/1) (±SD) +8 +-0.2 +0.i -+0.i +0.01

the

cultures,

dichlorodioxins

PCDD

TrCDD

degradation

for the induction of degradation

in

TABLE 2 INFLUENCE OF PRE-EXPOSURE

supplied

level

induction period for the degradation of the dichlorodioxins

phase

culture

conditions

0.044

(experiment

the detection

(data not shown).

The apparent to

falling below

to these compounds had apparently

rates. There was similar evidence 1,3-DCDD

14 days later with the same culture

concentrations

in

to determine

of strain JBI of 1,3-DCDD,

lower than those

1,2,4-TrCDD

compounds

the

the rates

of

[9]. The results

2,8-DCDD and 1,2,4-

in the influent medium

this was not the case for 1,2,3,4-TCDD

and

medium

(P>O.IO).

(Cm)

1302

1,3-DCDD 10

d 0 O

2

0 0

2

4

6

8

10

8

10

t,rne (d) o

Cm

+

Cc

2,8-DCDD 10

(]

2

0 0

2

4

6 time (d)

Cm

+

Ge

1303

1,2,4-TrCDD 10

T d o o

E

2

0 0

I

I

[

4

6

8

10

time (el) o

+

On

Cc

1,2,3,4-TCDD 1.00

0.80

0.60

0

0.40

O-20

0.00 0

1

I

I

I

2

4

6

8

time On

Fig. 3,

10

(d) +

Cc

Continuous exposure of a 3-methylbenzoate-grown chemostat culture of strain JBI to a mixture of 1,3-DCDD, 2,8-DCDD, 1,2,4-TrCDD and 1,2,3,4-TCDD. Cm= concentration in the influent medium; Cc= concentration in the culture (±SD).

1304

Pseudo

first-order

biodegradation

rate constants

were

calculated

with

the

following

steady state mass balance equation:

D

where

D

is

the

dilution

Cm

rate

volatilization rate constant.

k b' C c

=

constant,

+

k b'

D Cc

+

kv C c

and k v

the

For the compounds considered here, k v is not significant,

the biodegradation

rate

constant

and

thus:

D(Cm kb '

Cc)

-

Cc

The values of k b' thus calculated are given in Table 3.

TABLE 3 BIODEGRADATION RATE CONSTANTS OF PCDD's IN A CHEMOSTAT CULTURE OF STRAIN JBI PCDD

Concentrations Cm**

1,3-DCDD 2,8-DCDD 1,2,4-TrCDD

2.82 ±i.ii 5.80 ±2.20 4.84 ±1.87

;(b (h- I ) (±SD)

( ~ / I ) ~$~D) Cc 0.636 ±0.174 0.195 ±0.054 2.03 ±0.95

*First-order biodegradation rate constant. ***Concentration in culture.

0o180 ±0.106 1.37 ±0.62 0.075 ±0.058

**Concentration in influent medium.

The isolation of metabolites of 2-CDD was carried out by incubating this compound with crude were

cell

detected

retention had

extract

obtained

by GC-MS

from an adapted

analysis

time had a molecular

a molecular

ion of m/z

chemostat

of a methylated ion of m/z 218

278,

culture.

extract.

of a methoxy group in a lateral (e.g.

In repeated

experiment,

a metabolite

of a monomethoxymonochlorodioxin

and was

with

2-CDD.

compounds

the

shortest

The second peak

to a dimethoxymonochlorodioxin.

spectrum contained M + - 15, M + - 43 and M + - 58 fragments characteristic

The compound

(35Gi peak)

corresponding

Two chlorinated

Its mass

(Fig. 4). The M + - 15 fragment is

2 or 3) position

[12].

with m/z 248 and a mass spectrum characteristic

was detected

(Fig.

5). The mass spectrum of this compound

also contained both M + - 15 and M + - 43 peaks, again indicating that the methoxy group was in a

lateral

(100:47:57) [12],

position.

differ

suggesting

compound dioxin.

is

from

that

either

The

relative

those

intensities

reported

the methoxy

group

for

of

the M +,

M+

- 15

and M +

2-chloro-3-methoxydlbenzo-p-dioxin

is in either

the 7 or 8 positions,

2-chloro-7-methoxydibenzo-p-dioxin

or

- 43

peaks

(100:70:93

i.e.

that the

2-chloro-8-methoxydibenzo-p-

1305

I00-

50

M+-15 M*.58

~..:.:..,~.~,~:,,~,.,..~J.,..,;,,.~,L,,,..:?,..~.,:,L 100

Fig. 4,

i

M~_/.3

,I:, I,,,L . !. : ~.., .:......... ,~I, ...... .:..,.[.:.

200

3O0

m/z

Mass spectrum of the dimethoxychlorodioxin incubations of strain JBI with 2-CDD.

isolated

after

methylation

from

100

I M÷_~3 50

M*_15

..,'!.:

I

,,ill.

. , . ,

. . . .

t ,I.,,..,,...,.!'

! . . . .

.

.... ,. ........

,

I,.,

,. •

IO0

Fig. 5.

150

mlz

200

Mass spectrum of the monomethoxychlorodioxin incubations of strain JBI with 2-CDD.

'

. . . .

I ; ' ' ' ,

. . . .

I. 250

I . . . .

'

isolated after

. . . .

methylation

from

1306

Klecka

and

Gibson

described

the

isolation

of

1,2-dihydrodiols

metabolites of dibenzo-p-dloxin and its monochlorlnated derivatives

and

1,2-dihydroxy

[2,3]. 1,2-Dihydroxy-

1,2-dihydrodibenzo-p-dloxin underwent acid-catalyzed dehydration to yield 2-hydroxydibenzop-dioxin,

as did the chlorinated derivatives.

1,2-Dihydroxylated metabolltes

(catechols)

were formed by further metabolism of the dihydrodlols. The results reported by Klecka and Gibson suggest that the monomethoxychlorodioxin described above

is the methylated derivative of a monohydroxychlorodioxin formed during

work-up by acid-catalyzed dehydration of a dihydrodlol and that the dimethoxychlorodioxin is the methylated derivative of the dihydroxylated metabolite of the dihydrodiol (Fig. 6). These metabolites cultures,

could not be detected when 2-CDD was degraded in batch or chemostat

which may mean that growing cells are capable of further metabolism,

although

identification of other metabolites is required before firm conclusions can be drawn.

Ct4 L , ~ / ~

"

C

t

~

H

1,2-dihydrodiol

2 -CDD

1. + C

Fig. 6.

I

~

H

Proposed pathway for the initial degradation of 2-CDD by strain JBI.

Discussion

Bacteria growing in batch cultures are exposed to an excess of nutrients for almost the whole growth phase. This, in which

cometabolism

and the limited length of the growth phase,

is possible,

i.e. the period

may make cometabollsm of some xenobiotic

chemicals

unfavourable in batch cultures. In the environment, bacteria are always under the influence of a nutrient limitation. This is also the case in a chemostat culture, in which bacteria can be grown under different nutrient limitations [13]. Therefore, we consider a chemostat

1307

culture

a more

more,

the

culture

realistic

use

of

a

chemostat

to xenobiotic

adaptation

of

the

The

culture.

these compounds and 2). thus

is

differences

under

systems

1,3-,

is almost

not

expose

and

a

of chemostat

I).

cultures

faster

in

grown

suited

degradation

batch

in batch

cultures

of

is that

the constant order.

is slow

cultures

of the batch

pre-induced

the

bacteria

carbon

limitation

well

to systematic

was probably

defined studies

cultures

conditions

that

indicates

that

cultures are

influence.

chemostats,

of the influence

and may

fact

used in this work were

of great in

of

(Figs. i

cultures

However,

under which bacteria grow in batch and chemostat

under

in benzoic

degradation

to the cultures

cultures.

the fact that the chemostat

growing

induction and

to pseudo-first

In chemostat

Further-

enzyme

2,8-dichlorodibenzo-p-dioxins (Table

studies.

continuously

thus favouring

as long as the growth phase

slow

In particular,

be

well

to

periods,

advantage

strain JBI

apparently

a continuous

can

it possible

the kinetics of biodegradation 2,7-

of

in the conditions

most important.

bacteria

makes

for biodegradation

is induced about 20-24 hours after their addition

the

degradation

grown

of

cultures

for extended

Another

cultures

This period

explain

also

simplifies

degradation batch

than batch

chemicals

biomass concentration

acid-grown

system

Cultures

which

of environmental

makes

of

these

parameters

on

biodegradation. Klecka

and Gibson

bacteria

grown

on

by the Pseudomonas this compound indicate

Degradative

[2,3].

or

in

derivatives

and dichlorodioxins

in the degradation

are

presence

of

strain,

(Beije-

hiphenyl

for its degradation

strain were induced by

able to induce degradative

systems

chlorinated

but not by dichlorodioxins.

as an Alcaligenes

of aromatic

its

These compounds were metabolized

the

in the Beijerinckia

enzymes

and its monochlorinated

which we have now identified

involved

strains

and

was not able to induce the enzymes required

strain.

that mono-

of dibenzo-p-dioxin

(Pseudomonas)

salicylate

rinckia). Dibenzo-p-dioxin

JPBI,

the metabolism

by Pseudomonas and Beijerinckia

derivatives by

described

Our results

enzymes

in strain

although

some of the enzymes

are of course already

induced in cultures

growing on benzoate. As

expected,

dibenzo-p-dioxins benzoate-grown 1,3-DCDD,

2-CDD

is degraded

(e.g.

Fig.

chemostat

2,8-DCDD,

detection

level

0.i

dioxins tions,

values

are not but

degradation

by

rates

The

much

1,2,3,4-TCDD

the

membranes

(Table

influenced

other

slower

of

and

that

has

This

by steric

factors,

trace

than of

the more

this

such

unsubstituted

transport

effects

molecules

in

a

and

In fact, 2,8-DCDD rates

of these

on the metabolic

to consider

would

reacof the

that the value of

to a half llfe of 0.5 h.

of 1,2,4-TrCDD

with

ring,

As an indication

and the apparent

lack of degradation

have a completely

unsubstituted

attack would be expected to occur quite readily.

for

found

to a mixture of 2-CDD,

aromatic

processes.

it may be of interest

molecules

chlorinated

was

that the degradation

and electronic

as

since both structures

hydrophohlc

highly

compound

(data not shown: C c of 2-CDD ca. i00 ~g/l; an

suggests

(Table 3) corresponds

degradation

is suprising,

size

rapidly

no

much more readily than 2,8-DCDD.

3).

in this experiment,

ring where enzymatic that

1,3-DCDD

to be metabolized

1.37 for k b' of 2,8-DCDD

more

fact,

and 1,2,3,4-TCDD

pg/l).

of k b'

only

also

much

In

culture of strain JBI exposed continuously

1,2,4-TrCDD

ca.

therefore be expected had a higher

i).

influences a cross

their

section

diffusion

larger

than

of

aromatic

It has been suggested through 9.5

A

biological

diffusion

is

1308

blocked [14]. This was proposed to explain the lack of uptake by

fish of such hydrophobic

chemicals as hexabromobenzene, which has an octanol-water partition coefficient higher than 10 6 and a cross section of 9.6 A. The cross sections of 1,2,4-TrCDD and 1,2,3,4-TCDD (9.8 [14] are even larger than that of hexabromobenzene and may restrict their rate of diffusion into microbial cells, and thus their biodegradation rate. The metabolites extract

suggest

that

dibenzo-p-dioxins were

not

of 2-CDD isolated from incubations of this compound with crude cell its

degradation

follows

a similar

pathway

to

that of

chlorinated

in Pseudomonas and Beijerinckia strains [2,3] (Fig. 6). These metabolites

identified

in

growing

cultures

degradation under such conditions,

exposed

to

2-CDD,

which

may

indicate

further

although further work is obviously required to clarify

this.

Acknowledgements The authors thank M. van den Berg and J.M.D. van der Steen for technical assistance, and O.M. Neijssel and A. Opperhuizen for stimulating discussions. References 1 O. Hutzinger, M.J. Blumich, M. van den Berg and K. Olie, Sources and fate of PCDD's and PCDF's: an overview, Chemosphere, 14(1985)581-600. 2 G.M. Klecka and D.T. Gibson, Metabolism of dibenzo[l,4]dioxan by a Pseudomonas species, Biochem. J., 180(1979)639-645. 3 G.M. Klecka and D.T. Gibson, Metabolism of dlbenzo-p-dloxin and chlorinated dibenzo-pdioxins by a Beijerinckia species, Appl. Environ. Microblol., 39(1980)639-645. 4 F. Matsumara and H.J. Benezet, Studies on the bioaccumulation and microbial degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxln, Environ. Health Perspect., 5(1973)253258. 5 J.F. Quensen, III, and F. Matsumara, Oxidative degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin by microorganisms, Environ. Toxlcol. Chem., 2(1983)261-268. 6 M. Philippi, J. Schmid, H.K. Wipf and R.A. HOtter, A microbial metabolite of TCDD, Experientia, 38(1982)659-661. 7 D.C.G. Muir, A.L. Yarechewski, R.L. Corbet, G.R.B. Webster and A.E. Smith, Laboratory and field studies on the fate of l,,3,6,8-tetrachlorodibenzo-p-dioxin in soil and sediments, J. Agric. Food Chem., 33(1985)518-523. 8 J.R. Parsons, D.T.H.M. SiJm, A. van Laar and O. Hutzinger, Biodegradation of chlorinated biphenyls and benzoic acids by a Pseudomonas strain, Appl. Microbiol. Biotechnol., 29(1988)81-84. 9 J.R. Parsons and D.T.H.M. Slim, Biodegradation kinetics of polychlorinated biphenyls in continuous cultures of a Pseudomonas species, Chemosphere, 17(1988)1755-1766. I0. A.E. Pohland and G.C. Young, Preparation and characterization of chlorinated dibenzop-dioxins, J. Agric. Food Chem., 20(1972)1093-1099. ii. A.P. Gray, S.P. Cepa, I.J. Solomon and O. Aniline, Synthesis of specific polychlorinated dlbenzo-p-dioxins, J. Org. Chem., 41(1976)2435-2437. 12. M.Th.M. Tulp and O. Hutzinger, Identification of hydroxylated chlorodibenzo-p-dioxins, chlorodibenzofurans, chlorodlphenyl ethers and chloronaphthalenes as their methyl ethers by gas chromatography mass spectrometry, Biomed. Mass Spectrom., 5(1978)224-231. 13. H.G. Schlegel, General Microbiology, Sixth ed., Cambridge University Press, Cambridge, 1986. 14. A. Opperhuizen, E.W. v.d. Velde, F.A.P.C. Gobas, D.A.K. Liem, J.M.D. v.d, Steen and O. Hutzinger, Relationship between bioeoncentration in fish and steric factors of hydrophobic chemicals in fish, Chemosphere, 14(1985)1871-1896. (Received

in G e r m a n y

26 M a y

1989;

accepted

27 J u n e

1989)