Uptake and elimination of polychlorinated arokatic ethers by fish: chloroanisoles

Chemosphere, Vol.16, No.5, P r i n t e d in G r e a t B r i t a i n

pp

953-962,

UPTAKE AND ELIMI~ATT0~ O~ P O L ~ L O R I N A T E D

1987

AR~TIC

0 0 4 5 - 6 5 3 5 / 8 7 $ 3 . 0 0 + .OO P e r g a m o n J o u r n a l s Ltd.

~

BY FISH: C~,ORDRNI~OLE~

P31toon Opl~rhul~en" and Paul I. Voor~

LaboratorF of E n v i E o ~ n t a l Nieuwe ~ t e l ~ J E a c ~ t

and ~ c o l o ~ i c a l

Ch~si~tz~, b~li~z~ity of ~ t e z d a ~ . The H e t h e r l a J ~ .

166, 1018 tflr I M ~ t ~ l ~ J ~ ,

P3LST~T

Uptake rate oonstants of I0 ~hloroanlsoles by flab az~ cmqpezable to those of other hy~rophobic cbemlc~Is. Since fast elimination is found for all o m ~ e n e r a the resultlr~ bioconcentratio~ face,ors aze lower than those predicted f z ~ their h l ~ i ~ i t i e s . Elimination is ~ainly due to ~s4~olim at the ether bond and the rate ~ m s t a n t s are Inde1~n~ent of hy~rogt~bicity. The pz~m~ o f t h e ether ~ s dk~es not Influeno~ the octan-l-Ol/watar p a r t i t i o n ooeffiQlent of the c h l o r l n a t ~ al~a~tIc l ~ E ~ . a E b o n s significantly.

~

In

mt~ltes

of

the aw2~mina

polychlorinated used 1 , 2 .

This

m t t c

I

O

M

or kinttl~

of

btoacct~lation

of

hy~rooaz~mms, such as p o l ~ h l o r l n a t e d

may be

justified

by

the

high

environmental

hT~rophobic

bemnes

C h m L lC a ] j

and - b i p h e n y l s

gerslsten¢e

of

such

in

fish,

are often

chemicals,

in

combination wlth t h e i r h~zo1~obic nature. Hithex~o lens a t t e n t i o n l s paid t o the bloaocm~lation o f p o l ~ l o r l n a t e d aromatic ~ z o ~ s z ~ o n a which oontaln i~m~tlonal gzouI~ o f considerably l m ~ r stability. In

a

meries

of

studies

the

lnfluen~

of

ether

bondl

on

the

bioconcentratlon

of

polyphenyl

stz~ctums by f i s h w i l l be investigated. Although I t i s o f t e n a m ~

t h a t the ether gzoul~ are

unstable

the

under

various

l~ly~lghenylethers

is

c~Mitlons,

it

ham

been

shown t h a t

~ x q p e z a b l e t o t h a t o f g o l T c h l o z ~ b l g h e n y l s 3 . ~en~e,

bioacc~mulat ion it

of

was c o n c l u d e d t h a t

the e t h e r bond~ between Ix~en~l gz~xq:s a r e n o t z e a d i l y attached, Whet~er t h i s a3~o bo1~s f o ~ o t h e ~ azo~atic

~

which

-~ibe~sofuz~m~, w i l l I n the p r e ~ e n t

contain

bonds,

ether

much

as

ch l o z ~ d i b e n z o - p - d i o x l n s ,

be the subject o f f u z t h e r I n v e s t l ~ a t l o n s .

study the zole o f t ~ e ether bond between p h e n y l 953

and methyl groul~

of

or

954

polychloricated biotic

anlsoles

is

samples. 4-6 Slnce

investigated.

anthropogenic

assumed that these com|xmnd8

Chlorinated

production

anisoles

are

of chlorinated

in

found

anlsole8

various

types

18 not high,

are products of the methylation of corresponding

of

it is

chlorophenols by

fungi and m i c r o o r g a n i m a s . 6 - 1 2 Although

it can be assumed that chloroanisolas

bioaccumulation

and

bioaccumulation

factor

pentachlorophenol, In

the

present

studied

and

metabollsm of

psntachloroanisole. 13

pentachloroanisole

which is caused by a s l m ~ r

study

the

of

the

results

are hydrophobic,

hydro~K)bicity are

compared

is

this

than

study

that

it

of

shG~n that

is

the

less

~he

hydrophoblc

elimination rate.

and with

In

higher

only one study reports on the

bloaccumulation

kinetics

those

hydrophobIc

of

other

of

chloroanlsoles chemicals

are

such

U

chlorobenzenes.

MkTERIALS AND METHODS

Chemlcals :

Chlorinated

anlsoles

were

synthesized

by

methylation

of

the

corresponding

chlorophenols with iodomethane in acetone in the presence o f K2CO 3 .14 Pentachlorobenzene

and

2,3,5,6

tetrachlorophenol

were

obtained

from Aldrich,

while

all

other

chlorophenols were obtained from Fluka. All chem/cals were ) 98% pure as confirmed by GC-E(2). As solvents,

re-distilled n-haxane,

toluene and acetone were used (Merck).

For derivatization iodomethane and iodoethane (Merck) were used.

Chemical analysis: masured,

After being killed in liquid nitrogen,

the fish (3 fishes each sample) were

weighed and homogenized in a mortar. To the homoger~te I00 NL redistilled n--hexane and

25 mL demineralized water was added and the sample was heated under reflux for 90 minutes. After cooling

, addition of i0 mL 1 N-NaOH iolution and centrifugation,

by evaporation and concentrated

the hexane was removed

to 1 mL. After elutlon of the extract through activated silica

columns using I0 mL hexane these samples were analyzed by GC-ECD and GC/MS. To the residual water

fraction 5 mL of 4N-H2SO 4 solution was added together with an additional

100 mL of redistilled

n-hexane

hexane

layer

was

separated

and

to extract phenols. concentrated

to

After

1 mL.

To

20 minutes the

heating

extract

i00

under mL

reflux the

acetone,

5 mL

iodcethane and 5 g K2SO 4 were added and this solution was heated under reflux for 15 hours. After removal of the acetone by evaporation and addition of 1 mL n-hexane, through

activated

silica

using

10 mL

solvent.

Then

the

extracts

the extracts were eluted

were

concentrated

to

i mL

followed by analysis by GC-ECD and GC/MS. Samples of 1oo mL water form the aquarium were analyzed similarly in two steps. For these smqples the heating under reflux was replaced by duplicate extraction at room temperature with 2 x 25 all n-hexane.

Gas

chromatography:

equipped

with

For

quantification

63Ni-Electron

Capture

of all

Dector

samples

a Packard-Backer

and on-colmnn

injector was

428

used.

gas

The

chrQmatograph detector warn

955

connected with a Shlmadzu

Chrumatopac C-R2AX integrator.

Injection

(0.2 ~i) was

at 55 C in a

CP-SII-5 column (25m x 0.22 ram) with Ar/CR 4 95%/5% as carrier gas. A Hewlett Packard, model 2982 A, GC-MS with electron impact ionization at 70 eV was used. 50m x 0.22

me CP-Sil-5

column.

Helium was

used

Injection was splitless (I~L) in a

as carrier

gas.

These

GC/MS

conditions

were

comparable to those described previously 15. After spiking clean fish samples with 1 mL of a solution of a mixture of the chloroanisoles hexane,

r~overies

in

between 62% for 2,3,5,6 trichloroa~isole and 93% for pentachloroanisole were

found for all congeners.

Spiking of water samples always showed a 1OO% recovery.

After spiking

clean fish samples with a mixture of chlorophenols (detected as chlorophenetoles ), the recoveries ranged from 71% for 2,4,6-tri to 84% for pentachlorophenol. 84% for 2,4,5 tri- to 89% for 2,3,4,5 tetrachlorophenol.

In water these recoveries ran~ed from

After spiking the clean fish and water

samples with chlorinated anisoles only traces of chlorinated phenols were found (c 0.01%)

Experiments weight were

96 one year old male 9uppies ( P o e c $ ~ a

98 mg and mean fat content 5% were placed in 30 L aquaria.

similar to those described

continuous

previously 16 . Test coa~ounds were

The experimental

conditions

added to the water,

with a

flow aqueous saturation system. 16 ContaDtination of the water was stopped before the

fish were added. After placesmnt, clean

re~$cu~a~a) with mean length of 15 ram, mean

water 16

to

study

the

the fish were exposed for seven days and then transferred into

elimination

rates.

At

regular

intervals

during

the

exposure

and

elimination periods both fish and water were sam~Ind to allow chemical analysis.

RESULTS

During the period of exposure of the fish all ehloroanisoles

showed a rapid uptake.

Throughout

the uptake perlod chloroanisoles concentrations in water all decreased significantly. For none of these comEx~unds steady-state concentrations were observed at the end of the experiment

in both

fish

of

all

In figure I this is shown for 2,3,5 tri-, 2,3,6 tri and pentachloroanisole.

The

and

water.

chloroanisoles.

This

is

caused

by

a

continuous

decrease

in

aqueous

concentrations

decreasing chloroanisole concentrations in water did not result in a simultaneous increase of the Concentrations

in

fish.

For

2,3,6-tri

and

2,4,6-trichloroanisoles

a

simultaneous

decrease

in

concentrations in both fish and water is even observed after two days exposure. For several other congeners,the concentrations in fish at their maximum values were two days after the start of the exposure and remained almost Constant during the rest of the uptake period. The mass balance for each chloroanisole congeners, calculated from the data of the fish and water samplod during the exposure period, seven

days

introduced

exposure amount

is

to

all

is shown in table I. From this table it is clear that after

chloroanisole

recovered.

These

congeners

low

coa~9ound from the system by (co-)evaporation, for

instance

by

metabolism.

Whereas

the

only

recoveries

do

a

small

indicate

fraction either

of

the

removal

initially

of the

test

or removal by transformation of the test cou~ound,

total

aRount

of

test

compound

exposure period ranged frol 6% to 40% for the chlorinated anisoles,

recovered

during

nearly 75 % of the

the

956

T a b l e i. M a s s b a l a n c e for t e o t c o m p o u n d s in a q u a r l u m d u r i n g e x p o s u r e period.

,%

B

2462

47

2,3,5

1526

2,3,6

450

2,4,5

1170

2,3,4

TCA

C

D

E

F

G

516

54

71

1474

40.2%

21

278

24

35

1168

23.5%

4

40

2

2

402

10.5%

18

342

19

22

769

34.4%

2,4,6

350

3

17

i

2

327

6,4%

3,4,5

1079

11

122

14

18

914

15.3%

2595

32

457

163

242

1700

34.5%

832

7

99

7

24

695

16.5%

2,3,4,5

~

2,3,4,6 2,3,5,6

783

6

75

28

46

629

19.8%

2,3,4,5,6

PC,.A

492

4

36

32

40

380

22.8%

1,2,3,4,5

~

209

3

58

40

54

54

74.1%

* all

amounts

are

e ~ r ~

in

~j

A : estimated amount i n w a t e r a t t - 0 B : removed by Sampling o f w a t e r C : estimated amount i n w a t e r a t t

- 7 daym

D : removed by 8ampling o f f i s h E : estimated amount i n l i v i n g

F

: unexplained,

G : • recovery

~:

i.e. A - ( B ~ = + D + E )

, i.e. 1 0 0 % * ( B ~ = ÷ D + E ) / A

trichloroanlsole : tetrachloroanisole :

f i s h a t t - 7 days

pentachloroanisole

l ~ 3 z : pentachlorobenzene

957

2

tri

2

2,3,6 tri

t

2

penta

t

t

Piqurs I. Concentrations of 2,3,5 tri-, 2,3,6 trl- and pentachloroanlsole fish ( ~ J / g , m )

in water ( ~ / L , O )

and

durir~ the period of e ~ o ~ u r e ,

1~ntachlorobenzene was recovered. Values comi~Lrable to the latter value were found previo,sly for chlorinated bi1~henyls and n e 1 = h t ~ e ~ m Prom

table 2,

placement

in comparable ex~erlmente 16 .

it is clear that the c~11ozoanlmoles were elimlnated rapidly from the fish. After

in clean water biological half liwe8 for all trichloro congeners are less than 1 day.

Por the tetra- and pentachlozoanimole8 half lives between i and 4 dalm are found. Since throughout the uptake period a r a p ~ ac/uarium is found for all Qongenerm,

decreale of the total amount of test com~our~m in the

no ~ l a t i o n m

of bioconcentration

factors are possible.

So, only estimates of the concentration ratio between fish and water are made, which are llsted in table 2. Based on the ratiol between the concentratlonm in fish and water at the end of the period

of

( table

exposure

2 ).

The

previously 16 . chlorinated factors. eluent.

(cfish/Cwater)

accumulation In

anisoles

and

data

listed

An octadecyl modified

clearaun~e rates,

pentachlom0benzene

of

the

a~k~ition v a l u e s are

the

for

in table

uptak~ were

octan-l-ol/water

2. These values

silica column was u~ed

rates

have

comparable

partition

are estimated

been to

estimated

tho/~

coefficients

found of

the

from RP-MPLC capacity

in combination with aqueous methanol as

The capacity factors of the chlorinated anlsoles were compared to those of chlorinated

and alkylated henzenes of which l o ~ , o c

t values axe reported. This method to estimate log Kd,oc t

v a l u e s has been reported prevlously 17 .

Metabolism. contained ohlorinated

In table 3 the results of GC/MS anallmms are listed. a

number

of

phenol8

in

chlorlnated the

flmh

1~termtoles.

ar~ wlter

These

laR~lea.

tetrachloro~henol were not found in the fish samples.

As is shown,

phenetoles

Only

2,3,%

zeprement

tri-,

2,4,6

the samples all the tri-

premenoe and

of

2,3,5,6

958

Table 2. Values of log Kd,oc t , log K c, k I and k 2 foz the test ~ d s .

10g K d , o c t *

l o g Kc

kl(mL/g*d )

k2(d -1)

2 , 3 , 4 T CA

3.74

3.09

1450

1.9

2,3,5

3.93

3.05

1480

1.2

2,3,6

3.64

2.52

1610

4.0

2,4,5

3.85

2.81

1430

2.4

2,4,6

4.11

2.86

1600

2.5

3,4,5

4.22

3.09

1240

0.92

2,3,4,5 TeCA

4.51

3.67

940

0.42

2,3,4,6

4,75

3.34

1860

0.37

2,3,5,6

4.68

3.69

1480

0.44

2,3,4,5,6

PC]%

5.45

3.96

1710

0.32

1,2s3,4,5

PCBz

5.16

4.08

1490

0.14

DISCU~I~

The octan-l-ol/water partition coefficient from figure 2, that the bloooncentration

is used as a measure of hydrop~obicity.

It is clear

factors of highly chlorinated animoles are higher than

%hose of the less chlorinated cor~enere. Principally this is consistent with the bioconcentration factors of chlorinate(] benzenes, it may be

clear

that

there

naphthalenes and bIphenyls 16. From the data In table 2 however,

Is no

llmear

relationship

between

ells/nation

rate

constants

and

Kd,oc t values as has been found for the other compounds. It

must

be

noted

chlorobenzenes.

Kd,oc t

values

for

chloroanisoles

are

close

to

the

values

for

This ir~icates that the ether bond only slightly influence the octan-l-ol/water

partitioning, solvent/water

that

This

is

transfer

considerations

in

agreement

with

the

observations

l)y

Riebensehl

on

the

organic

free energies of anlsole and henzene 18 . It is also consistent with

of the h ~ r o p h o b l c

fragmental

(f) - and substltuents

group contribution

the

to log

Kd,oc t as proposed for methoxF groups 19. The

rough estimates

of the uptake

rate constants

seem to be consistent

between log Kd,oc t and log k 1, as obtained for chlorobenzerms, the low bioconcentration test compounds.

Baud

the

chloroanisoles.

This In

the relationship

coefficients may primarily be explained by high clearance rates of the

on the observed high loss of the test compounds

formation of metabolltes, we

pentachloroanisole

with

naphthalenes aml biphenyls. Hence,

from the system and the

suggest that the total clearance is dominated by transformations of

is

Rainbow

in

agreement

Trout

( Salmo

with

the

observations

Gardnlerl )12 •

where

of

GlicJuRn

oblerved

et

al.

transfommatlon

with of

959

Table 3. Gas chromatography

compound

and mass spectra data for chlorinated

Retention

Masses

Found in:

anisoles and phenetoles.

Fish

Water

Time ( r a i n )

2,3,4

T CA

15.80

210,195,168

X

X

2,3,4

~=P

16.92

224,196

n.d.

n.d.

2,3,5

~

12.71

210,195,168

X

X

2,3,5

TCP

13.48

224,196

X

n.d.

2,3,6

'rcA

210,195,168

x

x

2,3,6

~

224,196

x

x

9.05 10.04

2,4,5

TC~

12.35

210,195,168

x

x

2,4,5

TCP

13.29

224,196

x

x

2,4,6

~

7.05

210,195,168

x

x

2,4,6

~

7.84

3,4,5

TC~

3,4,5

TCP

224,196

n.d.

n.d.

13.37

210,195,168

x

x

14.35

224.196

x

x

2,3,4,5

Te(~

24.19

244.229,201

x

x

2,3,4,5

TeCI)

25.65

258.230

x

x

2,3,4,6

TeCA

18.46

244.229,201

x

n.d.

2,3,4,6

~

19.99

258.230

x

n.d.

2,3,5,6

TeCA

17.96

244,229,201

x

x

2,3,5,6

TeCP

19.26

258 230

x

n.d.

2,3,4,5,6

]PCR

26.57

278,263,235

x

x

2,3,4,5,6

PCP

28.13

292,264

x

n.d.

1,2,3,4,5

PCBz

17.54

248,213

x

x

x: compound i s found n.d. : compound i s not detected

TCP:

trichlorophenetole, representing trlchlorophenol

TeCP: t e t r a c h l o r o p h e n e t o l e , r e p r e s e n t i n g t e t r a c h l o x ~ p h e n o l PCP:

pentaohlorophenetole, reprementlng pentachlorophenol

960

5 •

•

I

o

•

• ~%

nm

~g

.

O~ o ...I

3

nm m lle •

2

12

~

4i

~

et

Log Kd,oc t

Figure

2. ;telatlonshlp between the 1ogazlttmm o£ o o t a n - l - o l / v a t e r p a r t i t i o n ooe££iolentB and

£ish/wmter p a r t i t i o n c o e f f i c i e n t s f o r chlozoanlmole8 (1) and chloz~ben=enee(o)

4 m•

==== L = l m ~ % • 3

O~

o

..J

oo

2

I

i

2

Figure 3. Re1~tionmhip ~

n

i

4

3Log Kd,oct

|

5

n

6

7

the logarlttmm o f the ootan-l-o]~water p a r t i t i o n coe££iclenta and

the uptake rate constants f o r chloz~aniaoles (1) and chlo~benzenes (0).

961

into p s n t a c h l o r o p h e n o l was found.

psntachloroani8ole chlorophenols

have significantly

lower ancuBulation

In

addition

factors than

it has been found that for instance

the

chlorobenzenes°

due to the presence of the hydroxyl group. Since it has been shown e ~ r e

that the bioconcentration

of chlorinated diphemll ethers

is

(xxaparable to that of chlorinated blphenyls 3, it may be o l e a r that ether bonds in anisolee and in diphenyl ethers h a v e v e r y different suB~eptibilltie8 towards biotic transformation processes.

CONCLUSIONS

Clearance

rates

of

chlorlnated

estiwated hy~rophobicity.

anis01es

in

fish

are

much

higher

than

expected

from

their

%~nis may be explained by ~etabollc hydrolysis of the ether bonds into

oorresponding hydroxyl groups. Due to these high elimination rates bioconcentration

factors are

relatively low, omepared to those of chlorobenzenes and other hydrophobic chemicals. The presence of the methoxy groups result in a m ~ l l 1~ether

presence

the

significantly

could

of

change of the Kd,oc t value relative to that of benzene.

groups

aethoxy

not be confirmed,

also

influence

elr~e metabolimn

the

fish

lipid/water

made meamurements

partitioning

of actual partition

coefficients impossible.

REFERENCES

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4. MiyaJwki,T.,

Kooiman,D.,

Voors,P.I. ; CheBoaphere,

3. Opperhuizen,A.,

Keneko,S.,

Hutzlnger,o.;c~lemosphere,

1113-1115. 10,

(1981),

811-932.

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Horri,S., Yamagishi,T.;

Bull.Environ.Contam.Toxicol.,

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(Received

in G e r m a n y

5 November

1986;

accepted

5 December

1986)

in press.