Metabolism of Pentachlorophenol in Fish - ACS Symposium Series

8

Downloaded by KTH ROYAL INST OF TECHNOLOGY on December 7, 2015 | http://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch008

Metabolism of Pentachlorophenol in Fish KUNIO KOBAYASHI Laboratory of Fishery Chemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

Pentachlorophenol(PCP) i s a versatile pesticide widely used as an insecticide, herbicide or fungicide. In Japan, PCP was produced approximately 15,000 ton/year from 1962 to 1970 and used mostly i n the summer-time as a herbicide in paddyfields which were f i l l e d with water at a depth of few inches. The PCP applied to paddyfields easily flowed out of the fields to rivers and coastal area due to unexpected heavy rainfalls, resulting i n a high mortality of fish and shellfish. The use of PCP i n Japan was restricted i n 1971 by the Government because of i ts high toxicity to fish. However, PCP i s the second heaviest used pesticide i n the United States, although it has been mostly used for the purpose of wood preservation(1). Under such circumstances, an international symposium on "Pentachlorophenol" convened by K. Ranga Rao (University of West Florida) was held i n Pensacola, Florida, June 27-29, 1977, concerning the chemistry, pharmacology, and environmental toxicology of PCP. At the symposium, I presented a paper(2) on the metabolism of PCP i n fishes, mostly reviewing the works on the absorption, excretion and detoxification of PCP in fish and shellfish, which were done i n our laboratory. The present paper deals with a relation between toxicity and accumulation of chlorophenols i n goldfish, Carassius auratus, PCP metabolites and their amounts excreted by the three major routes (branchial, renal and biliary) i n the fish, and also with effects of pre-exposure to PCP on PCP-tolerance and on sulfate conjugation with phenol by the liver soluble fraction of the fish. Toxicity of PCP and Other Chlorophenols A study has been done regarding the acute toxicity of various chlorophenols to goldfish(av. 2 g), and their accumulation by the fish. Table I (3) shows the 24-h LC50 values of tested chlorophenols and the amounts of chlorophenols found i n the dead fish i n the media containing the chlorophenols at the concentration most close to each 24-h LC50 value, among the test 0-8412-0489-6/79/47-099-131$05.00/0 © 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 114

128

101

112

(60

(20

(10

( 8

(10

( 1.8

(0.8

( 0.2

ppm)

ppm)

ppm)

ppm)

ppm)

ppm)

ppm)

ppm)

i n the c o n c e n t r a t i o n of each

1.9

6.4

10.1

34

20

62

93

475

Concentration factor*

tested

media.

c h l o r o p h e n o l shown i n p a r e n t h e s e s most c l o s e d t o 24-h LC50 among t h e

* V a l u e s were o b t a i n e d i n f i s h which d i e d

60

9.0

Phenol

7.8

Di(2,4)

Mono(p) 16

268

10.0

Tri(2,4,6)

Mono(o)

200

1.7

Tri(2,4,5)

75

0.75

Tetra(2,3,4,6)

95

0.27

Amount f o u n d i n dead f i s h (pg/g)

Penta(2,3,4,5,6)

(ppm)

LC50

and a c c u m u l a t i o n o f c h l o r o p h e n o l s i n g o l d f i s h

24-h

The t o x i c i t y

Chlorophenols

Table I .



8.

KOBAYASHi

Pentachlorophenol

133

media. An i n c r e a s e of the Cl-atom number i n the chlorophenols caused an abrupt increase i n t o x i c i t y , r e s u l t i n g i n the lowest 24-h LC50 value of 0.27 ppm f o r PCP, while that f o r phenol was 60 ppm. However, many small d i f f e r e n c e s were observed among the concentrations of chlorophenols found i n the dead f i s h during 24 h exposure, as compared with the d i f f e r e n c e s among t h e i r LC50 values, i . e . , the values obtained were w i t h i n a small range from 75 to 268 ug/g body weight. The c o n c e n t r a t i o n f a c t o r s of c h l o r o phenols found i n dead f i s h increased with an i n c r e a s e of the C l atom number i n the reverse of the LC50 v a l u e s , e.g., the concent r a t i o n f a c t o r s f o r phenol and PCP were 1.9 and 475, r e s p e c t i v e l y . From these results> i t seems that an i n c r e a s e of the Cl-atom number i n chlorophenols promotes an accumulation of the c h l o r o phenols by f i s h and leads t h e i r concentrations i n the f i s h to a l e t h a l l e v e l even when the f i s h were exposed to r a t h e r low conc e n t r a t i o n media, and consequently increases the f i s h - t o x i c i t y of chlorophenols. Absorption of

PCP

When g o l d f i s h were exposed to PCP-media, PCP was r a p i d l y absorbed by the f i s h at a highest c o n c e n t r a t i o n f a c t o r among the t e s t e d chlorophenols(Table I ) , u n t i l a l e t h a l l e v e l of approximately 100 ug/g body weight was reached(4). PCP absorbed by the f i s h ( a v . 40 g) from PCP-medium(0.2 ppm) was accumulated i n v a r i o u s organs, e s p e c i a l l y the g a l l bladder. Although the PCP concentration i n the g a l l bladder was the lowest among the t i s s u e s assayed at 1-h exposure, i t r a p i d l y increased with time and reached a value of 539 pg/g corresponding to a conc e n t r a t i o n f a c t o r of 2,700 at 24-h exposure. The c o n c e n t r a t i o n of PCP i n the g a l l bladder increased l i n e a r l y even a f t e r f i s h had been t r a n s f e r r e d to PCP-free running water and reached a l e v e l of 1,077 ug/g, corresponding to a c o n c e n t r a t i o n f a c t o r of 5,400 a f t e r 24-h c u l t u r e i n running water, whereas a decrease was observed i n a l l other organs examined(5). Most of the PCP found i n the g a l l bladder must have been t r a n s f e r r e d from other organs through the l i v e r a f t e r conjugation. B i l i a r y E x c r e t i o n of PCP-glucuronide The abrupt increase i n b i l i a r y c o n c e n t r a t i o n of PCP, which was observed with time a f t e r 5 h during exposure of g o l d f i s h ( a v . 90 g) to 0.1 ppm PCP, was due to accumulation of a conjugated-PCP (4.46 umol/g b i l e at 48-h exposure), whereas the amount of f r e e PCP i n the b i l e was n e g l i g i b l y small(0.09 /imol/g b i l e ) (6). The conjugated-PCP i n the b i l e ( 5 . 7 g) c o l l e c t e d from 30 g o l d f i s h (av. 110 g) exposed to 0.1 ppm PCP f o r 48 h was i s o l a t e d by t r e a t i n g the b i l e with a c t i v a t e d c h a r c o a l columns, followed by e l u t i o n with an acetone-ammonia mixture and f i n a l l y by passing

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


134

PESTICIDE AND XENOBIOTIC

M E T A B O L I S M IN AQUATIC ORGANISMS

t h e c o n c e n t r a t e d e l u a t e t h r o u g h a Sephadex G-10 c o l u m n w i t h w a t e r . The i s o l a t e d P C P - c o n j u g a t e ( c a . 13 u m o l e s ) was i d e n t i f i e d as p e n t a c h l o r o p h e n y l - p - g l u c u r o n i d e by h y d r o l y s i s on i n c u b a t i o n w i t h { 3 - g l u c u r o n i d a s e , by t h i n - l a y e r and g a s - l i q u i d c h r o m a t o g r a p h y and by d e t e r m i n a t i o n o f t h e m o l a r r a t i o o f PCP t o g l u c u r o n i c a c i d . No o t h e r c o n j u g a t e s i n c l u d i n g t h e s u l f a t e - c o n j u g a t e w e r e d e t e c t e d i n the b i l e ( 6 ) . G l i c k m a n e_t a l . h a v e a l s o r e p o r t e d t h a t P C P - g l u c u r o n i d e i s e x c r e t e d i n t h e b i l e o f r a i n b o w t r o u t e x p o s e d t o PCP and p e n t a c h l o r o a n i s o l e m e d i a ( 7 ) . The b i l i a r y e x c r e t i o n a f t e r g l u c u r o n i d e c o n j u g a t i o n must be one o f g e n e r a l d e t o x i f i c a t i o n mechanisms f o r PCP i n f i s h . E x c r e t i o n of P C P - s u l f a t e i n t o S u r r o u n d i n g Water When g o l d f i s h w e r e t r a n s f e r r e d f r o m PCP-media t o P C P - f r e e w a t e r , t h e PCP a b s o r b e d by t h e f i s h was q u i c k l y e x c r e t e d i n t o s u r r o u n d i n g w a t e r w i t h a h a l f - l i f e o f c a . 10 h(4_), m o s t l y i n a c o n j u g a t e d - f o r m a c c o m p a n i e d w i t h a s m a l l amount o f f r e e - f o r m ( 8 ) . A f t e r 15-h e x p o s u r e t o 560 l i t e r s o f 0.5 ppm PCP, 240 g o l d f i s h ( a v . 35 g) w e r e t r a n s f e r r e d t o 560 l i t e r s o f P C P - f r e e w a t e r and c u l t u r e d f o r 24 h. A c o n j u g a t e d - P C P a m o u n t i n g t o 0.36 mmoles was e x c r e t e d i n t h e w a t e r d u r i n g t h e 24 h c u l t u r e p e r i o d . The c o n j u g a t e was i s o l a t e d by a p r o c e d u r e s i m i l a r t o t h a t u s e d f o r t h e i s o l a t i o n o f t h e P C P - c o n j u g a t e f r o m b i l e , as m e n t i o n e d a b o v e . The i s o l a t e d c o n j u g a t e was i d e n t i f i e d as p e n t a c h l o r o p h e n y l s u l f a t e by p r e c i p i t a t i o n w i t h B a C l 2 , by c o l u m n and t h i n - l a y e r c h r o m a t o g r a p h y , by U V - a b s o r p t i o n s p e c t r a , and by d e t e r m i n a t i o n o f t h e m o l a r r a t i o o f PCP t o S O 4 . PCP-glucuronide which i s excreted i n b i l e was n o t d e t e c t e d ( 8 ) . A P C P - c o n j u g a t e e x c r e t e d by s h o r t - n e c k e d c l a m , Tapes p h i l i p p i n a r u m , i n t o s u r r o u n d i n g w a t e r has b e e n a l s o i d e n t i f i e d as t h e sulfate-conjugate(9). Renal E x c r e t i o n of P C P - s u l f a t e The d i r e c t e x c r e t i o n o f PCP by f i s h i n t o s u r r o u n d i n g w a t e r must be m o s t l y f r o m b o t h r o u t e s , i . e . g i l l s and k i d n e y . An e x p e r i m e n t h a s b e e n done t o c o n f i r m t h e p a r t i c i p a t i o n o f r e n a l r o u t e i n PCP e x c r e t i o n by g o l d f i s h , u s i n g a u r i n e c o l l e c t i n g a p p a r a t u s shown i n F i g u r e 1. The a p p a r a t u s was s e p a r a t e d i n t o two c o m p a r t m e n t s ( A , B ) w i t h a s i l i c o n e r u b b e r sponge s h e e t w h i c h h a d a h o l e i n t h e m i d d l e t o h o l d f i s h a t j u s t the back of p e c t o r a l f i n . B o t h t h e h e a d and r e a r p a r t s o f f i s h w e r e c o v e r e d w i t h two s m a l l b o x e s w h i c h had many h o l e s and w e r e f i x e d t o a c o v e r p l a t e o f t h e a p p a r a t u s . P C P - f r e e w a t e r was s u p p l i e d i n t o t h e h e a d c o m p a r t m e n t ( A ) a t a f l o w r a t e o f 1 l i t e r / h and t h e o v e r f l o w f r o m (A) was r e s e r v e d i n an i c e - c o o l e d t a n k f o r t h e a n a l y s i s o f PCP e x c r e t e d f r o m g i l l s . F i s h u r i n e was l e d by a c a n n u l a t o an i c e - c o o l e d f l a s k t h r o u g h



KOBAYASHi

Pentachlorophenol

Air PCP-free ι water

11

/-Silicone rubber sponge sheet /

A

^Silicone cannula

II Acrylic plastic box Reservoir Figure 1.

Thermos bottle Apparatus for determination of excretion routes of PCP in fish



136

PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS

a h o l e i n t h e compartment (B)· A f t e r 24-h e x p o s u r e t o 0.1 ppm PCP, g o l d f i s h ( a v . 70 g) w e r e t r a n s f e r r e d i n t o t h e u r i n e c o l l e c t i n g a p p a r a t u s and a c a t h e t e r i n s e r t e d i n t o the u r i n a r y bladder from the u r o g e n i t a l c a v i t y . A p p r o x i m a t e l y 13 m l o f u r i n e was c o l l e c t e d f r o m e a c h f i s h f o r 24 h. The u r i n e c o l l e c t e d f r o m 14 f i s h was p o o l e d and u s e d f o r t h e d e t e r m i n a t i o n o f PCP. More t h a n 95 % o f t h e PCP e x c r e t e d i n t h e u r i n e was f o u n d i n a conjugate-form. The i s o l a t i o n and i d e n t i f i c a t i o n o f t h e conjugated-PCP were c a r r i e d out a c c o r d i n g t o t h e p r o c e d u r e p r e v i o u s l y u s e d f o r t h e P C P - c o n j u g a t e s e x c r e t e d i n b i l e and i n s u r r o u n d i n g w a t e r . The P C P - c o n j u g a t e e x c r e t e d i n t h e u r i n e was i d e n t i f i e d as p e n t a c h l o r o p h e n y l s u l f a t e . P C P - g l u c u r o n i d e was n o t d e t e c t e d i n the u r i n e ( 1 0 ) . I t i s very i n t e r e s t i n g from the comparative b i o c h e m i c a l view p o i n t t h a t g o l d f i s h e x c r e t e PCP i n t h e u r i n e as t h e s u l f a t e and i n t h e b i l e as t h e g l u c u r o n i d e , w h i l e r a b b i t s w h i c h w e r e o r a l l y a d m i n i s t e r e d w i t h PCP-Na e x c r e t e d P C P - g l u c u r o n i d e a c c o m p a n i e d by a l a r g e amount o f f r e e - P C P i n t h e u r i n e , T a s h i r o e t a l . ( 1 1 ) . Whole V i e w o f M a j o r D e t o x i f i c a t i o n P a t h w a y s f o r

PCP

A f t e r 24-h e x p o s u r e t o 0.1 ppm PCP, two m a l e g o l d f i s h ( 6 6 and 70 g) were t r a n s f e r r e d e a c h i n t o a u r i n e c o l l e c t i n g a p p a r a t u s and a c a t h e t e r i n s e r t e d i n t o t h e u r i n a r y b l a d d e r , as d e s c r i b e d a b o v e . A f t e r h o l d i n g f o r 24 h i n t h e a p p a r a t u s , t h e f i s h w e r e removed and d i s s e c t e d a f t e r c o l l e c t i n g b l o o d by c u t t i n g t h e t a i l o f f . A l i q u o t s o f t h e d i s s e c t e d o r g a n s w e r e a s s a y e d f o r PCP c o n t e n t . The amounts o f f r e e - and c o n j u g a t e d - P C P c o n t a i n e d i n t h e o v e r f l o w f r o m t h e c o m p a r t m e n t ( A ) , t h e w a t e r i n t h e compartment(Β), t h e u r i n e and t h e b i l e c o l l e c t e d f r o m t h e f i s h w e r e a l s o d e t e r m i n e d . T a b l e H (12) shows a c h a r a c t e r i s t i c a c c u m u l a t i o n o f PCP i n t h e g a l l b l a d d e r among t h e o r g a n s , as p r e v i o u s l y o b s e r v e d i n an e x p e r i m e n t on t h e t u r n o v e r o f a b s o r b e d PCP i n t h e f i s h ( 5 ) . T a b l e TJI shows t h e amounts o f f r e e - and c o n j u g a t e d - P C P e x c r e t e d f r o m e a c h o f t h e b i l i a r y , r e n a l and b r a n c h i a l r o u t e s . As m e n t i o n e d a b o v e , t h e amount o f PCP a c c u m u l a t e d i n g a l l b l a d d e r i n c r e a s e d l i n e a r l y f o r 24 h e v e n a f t e r f i s h had b e e n t r a n s f e r r e d f r o m PCP-medium t o P C P - f r e e w a t e r ( 5 ) . T h e r e f o r e , t h e amount o f PCP e x c r e t e d i n t h e b i l e must be r e d u c e d t o h a l f t h e v a l u e o b t a i n e d , as compared w i t h t h o s e i n o t h e r e x c r e t i o n r o u t e s . The amounts o f PCP e x c r e t e d f r o m t h e b r a n c h i a l , r e n a l and b i l i a r y r o u t e s c o r r e s p o n d e d t o a p p r o x i m a t e l y 5 2 , 24 and 22 % o f t h e w h o l e amount o f PCP e x c r e t e d by t h e f i s h , r e s p e c t i v e l y . On t h e o t h e r h a n d , t h e amount o f PCP i n t h e w a t e r o f compartment(Β), w h i c h may be due t o e x c r e t i o n f r o m t h e body s u r f a c e and l e a k o f u r i n e f r o m t h e u r o g e n i t a l c a v i t y , was n e g l i g i b l y s m a l l compared w i t h t h o s e e x c r e t e d f r o m t h e above t h r e e r o u t e s . A p p r o x i m a t e l y 30 % o f t h e PCP e x c r e t e d f r o m t h e g i l l s was i n a f r e e - f o r m , w h e r e a s a l m o s t a l l t h e PCP e x c r e t e d i n b o t h t h e b i l e


In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 72 46 1260 16 39 45 28 40 9 22 30

90 95 520 48 113 20 8 47 115 968 2082

1.256 2.058 0.413 2.952 2.877 0.439 0.265 1.150 12.910 43.498 69.813

55 65 1675 11 33 36 19 38 7 26 33

80 83 633 27 50 12 3 40 86 1092 2158

1.450 1.281 0.378 2.452 1.504 0.333 0.168 1.053 12.849 42.475 65.913

Blood

Digestive

G a l l bladder

Testis

Liver

Kidney

Spleen

Other v i s c e r a

Muscle

Remainder

Whole body

tract

30

60

1.996

26

52

1.972

Gills

(pg/g)

(pg)

(g)

(ug/g)

Cone.

(pg)

PCP

(g)

Weight Cone.

PCP

Fish-2

Total

Weight

Fish-1

Amounts o f PCP r e t a i n e d i n v a r i o u s t i s s u e s o f g o l d f i s h w h i c h w e r e h e l d i n t h e a p p a r a t u s shown i n F i g u r e 1 f o r 24 h , a f t e r 24 h e x p o s u r e t o 0.1 ppm PCP

Total

Tissue

Table H .


In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 0.00

24

Body s u r f a c e (including leaked urine)

1.06 1.41 0.09

0.01 0.66 0.03

1.22 2.22 0.06

0.965*

0.015*

1.175*

1.93

Conjugated-PCP (pmole)

g)

0.03

Free-PCP (umole)

(70

2.35

Conjugated-PCP (μπιοΐβ)

Fish-2

* V a l u e s w e r e c a l c u l a t e d a s h a l f t h e amount o f PCP f o u n d i n g a l l b l a d d e r , b e c a u s e t h e amount o f PCP a c c u m u l a t e d i n g a l l b l a d d e r i n c r e a s e d l i n e a r l y f o r 24 h , a f t e r f i s h h a d b e e n t r a n s f e r r e d f r o m PCP-medium t o P C P - f r e e w a t e r .

0.88

24

Branchial

0.07

0.015*

24*

24

0.03

Free-PCP (umole)

F i s h -1 (66 g)

48

Excretion time (h)

Amounts o f f r e e - and c o n j u g a t e d - P C P e x c r e t e d f r o m e a c h r o u t e o f g o l d f i s h , d u r i n g h o l d i n g f o r 24 h i n t h e a p p a r a t u s shown i n F i g u r e 1

Renal

Biliary

Excretion route

T a b l e ΠΕ.



8.

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Pentachlorophenol

139

and u r i n e was i n a c o n j u g a t e d - f o r m . As m e n t i o n e d a b o v e , t h e c o n j u g a t e d - P C P e x c r e t e d f r o m b o t h t h e b r a n c h i a l and r e n a l r o u t e s i s P C P - s u l f a t e , w h i l e t h a t excreted i n the b i l e i s PCP-glucuronide. Therefore, the t o t a l amount o f t h e s u l f a t e e x c r e t e d by t h e f i s h f o r 24 h was 2.47-3.44 μιηοΐββ, w h i l e t h a t o f t h e g l u c u r o n i d e was 0.97-1.17 u m o l e s , namely t h e e x c r e t i o n r a t i o o f t h e s u l f a t e t o t h e g l u c u r o n i d e i n t h e f i s h was a p p r o x i m a t e l y 2.7. A p p r o x i m a t e l y 60 % o f t h e s u l f a t e c o n j u g a t e e x c r e t e d by t h e f i s h was due t o d i f f u s i o n i n t o t h e s u r r o u n d i n g w a t e r t h r o u g h t h e g i l l s and t h e r e s t was e x c r e t e d i n the u r i n e . The b i l e c o n t a i n i n g P C P - g l u c u r o n i d e , h o w e v e r , must be s e c r e t e d f r o m g a l l b l a d d e r i n t o i n t e s t i n e when f i s h had f e d . F o r t h i s r e a s o n , a f u r t h e r e x p e r i m e n t was p e r f o r m e d on t h e h y d r o l y s i s o f P C P - g l u c u r o n i d e c o n t a i n e d i n b i l e on i n c u b a t i o n w i t h i n t e s t i n a l mucus o f g o l d f i s h . As shown i n T a b l e IV ( 1 2 ) , a c o n s i d e r a b l e amount o f t h e PCPg l u c u r o n i d e i n b i l e was h y d r o l y z e d by t h e i n t e s t i n a l mucus. This i n d i c a t e s t h a t t h e g l u c u r o n i d e c o n j u g a t i o n p l a y s an i m p o r t a n t r o l e i n r e d u c t i o n of the c o n c e n t r a t i o n of free-PCP i n the f i s h b o d y , b u t n o t i n e l i m i n a t i o n o f PCP f r o m t h e f i s h b o d y as com p a r e d w i t h t h e s u l f a t e c o n j u g a t i o n , b e c a u s e t h e PCP r e l e a s e d f r o m t h e g l u c u r o n i d e i n t h e i n t e s t i n e must be r e a b s o r b e d t h e r e . E f f e c t o f P r e - e x p o s u r e t o PCP gation Activity

on P C P - t o l e r a n c e

and

S u l f a t e Conju

One h u n d r e d g o l d f i s h ( a v . 1.6 g) w e r e d i v i d e d i n t o two g r o u p s o f 50 f i s h e a c h ; one group was e x p o s e d t o 0.1 ppm PCP e v e r y o t h e r day f o r 4 d a y s and a n o t h e r was p l a c e d i n P C P - f r e e w a t e r as c o n t r o l , before a t o x i c i t y test. On t h e t h i r d day a f t e r t h e end o f p r e - e x p o s u r e , each t e n f i s h of b o t h groups were t r a n s f e r r e d t o 0.1, 0.2, 0.3, 0.4 and 0.5 ppm P C P - m e d i a , r e s p e c t i v e l y . F i g u r e 2 (13) shows an o b v i o u s i n c r e a s e i n P C P - t o l e r a n c e o f t h e f i s h p r e - e x p o s e d t o PCP when compared w i t h t h e c o n t r o l g r o u p . I n o u r p r e v i o u s s t u d i e s on t h e s u l f a t e c o n j u g a t i o n o f p h e n o l s by f i s h l i v e r s , a l l t h e l i v e r s l i c e s o f t h e t e s t f i s h and s h e l l f i s h exhibited s u l f a t e conjugation a c t i v i t i e s with phenol(14), and among v a r i o u s l i v e r c e l l f r a c t i o n s s e p a r a t e d by ultracentrif u g a t i o n , only the s o l u b l e f r a c t i o n d i s p l a y e d the s u l f a t e c o n j u g a t i o n a c t i v i t y f o r p h e n o l and v a r i o u s p h e n o l i c c o m p o u n d s ( 1 5 ) . A f u r t h e r s t u d y has b e e n made o f an e f f e c t o f P C P - e x p o s u r e on t h e s u l f a t e c o n j u g a t i o n a c t i v i t y f o r p h e n o l by f i s h l i v e r soluble fraction. Twenty g o l d f i s h ( a v . 66 g) w e r e d i v i d e d i n t o two g r o u p s o f 10 f i s h e a c h ; one group was e x p o s e d t o 0.1 ppm PCP e v e r y o t h e r day f o r 4 d a y s and a n o t h e r was c u l t u r e d i n P C P - f r e e r u n n i n g w a t e r . On t h e t h i r d day a f t e r t h e e x p o s u r e , t h e l i v e r s were t a k e n o u t f r o m t h e f i s h , h o m o g e n i z e d by a P o t t e r ' s homogen i z e r and f r a c t i o n a t e d u s i n g an u l t r a c e n t r i f u g e . A l l the s o l u b l e f r a c t i o n s prepared from the i n d i v i d u a l l i v e r s were s u b j e c t e d t o


In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. mucus o f f i s h .

500

Samples w e r e i n c u b a t e d f o r 5 h a t 20 °C w i t h c o n t i n u o u s s h a k i n g .

10 m l o f b i l e - 0 . 9 % N a C l ( 1 : 1 0 0 ) and 0.69 g o f i n t e s t .

No. 2:

NaCl(1:100).

10 m l o f b i l e - 0 . 9 %

282

218

No. 2

496

492

4

(nmole)

(nmole)

(nmole)

T o t a l PCP

Conjugated-PCP

Free-PCP

No. 1

Sample

mucus

H y d r o l y s i s o f P C P - g l u c u r o n i d e i n b i l e o f g o l d f i s h by i t s i n t e s t i n a l

No. 1:

Table IV.



8.

KOBAYASHi

Pentachlorophenol

Time of exposure (hr) Figure 2. Increase in PCP-tolerance of goldfish pre-exposed to PCP: ( ), survival patterns of goldfish which were pre-exposed to 0.1 ppm PCP every other day for 4 days, before the toxicity test; ( ), survival patterns of goldfish which were not pre-exposed to PCP before the test. The temperature was kept at 20°C


141

PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC


142

Exposed to PCP >

500

•

-J 200

Control

•

-• . · · · • •·

ORGANISMS

•·

•

-

ο ο

Ε 100 Ν

ι5

ο

1

I

I

ι

8

I

10

I

2

I

1

1

4

6

8

1_

10

Fish number Figure 3. Increase in sulfate conjugation activity of liver-soluble fractions of goldfish exposed to PCP. The fish were exposed to 0.1 ppm PCP every other day for 4 days at20°C.



8.

KOBAYAsm

Pentachlorophenol

143

the assay o f s u l f a t e c o n j u g a t i o n a c t i v i t y f o r phenol. F i g u r e 3 (13) shows a p p r o x i m a t e l y 26 % a u g m e n t a t i o n i n t h e sulfate conjugation a c t i v i t y of l i v e r soluble fractions of the f i s h e x p o s e d t o PCP f o r 4 d a y s , when compared w i t h t h e c o n t r o l group. The p r e - e x p o s u r e t o PCP i n c r e a s e d b o t h t h e P C P - t o l e r a n c e and t h e s u l f a t e c o n j u g a t i o n a c t i v i t y i n g o l d f i s h . This suggests t h a t f i s h h a v e some a b i l i t y t o i n c r e a s e t h e i r P C P - t o l e r a n c e when the f i s h had been exposed t o s u b l e t h a l PCP-media, and a l s o t h a t the s u l f a t e c o n j u g a t i o n a c t i v i t y i s an important f a c t o r d e t e r mining the PCP-tolerance o f f i s h .

Literature Cited 1. Cirelli, D.P., In "Pentachlorophenol", pp. 13-18(K.R. Rao, Ed.), Plenum Press, New York, Ν. Y., 1978. 2. Kobayashi, Κ., In "Pentachlorophenol", pp. 89-105(K.R. Rao, Ed.), Plenum Press, New York, Ν. Y., 1978. 3. Kobayashi, Κ., H. Akitake, and K. Manabe, Bull. Japan. Soc. Sci. Fish., submitted. 4. Kobayashi, Κ., and H. Akitake, Bull. Japan. Soc. Sci. Fish., (1975), 41 (1), 87-92 5. Kobayashi, Κ., and H. Akitake, Bull. Japan. Soc. Sci. Fish., (1975), 41 (1), 93-99. 6. Kobayashi, K., S. Kimura, and E. Shimizu, Bull. Japan. Soc. Sci. Fish., (1977), 43 (5), 601-607. 7. Glickman, A.H., C.N. Statham, A. Wu, and J.J. Lech, Toxicol. Appl. Pharmacol., (1977), 41 (3), 649-658. 8. Akitake, H., and K. Kobayashi, Bull. Japan. Soc. Sci. Fish., (1975), 41 (3), 321-327. 9. Kobayashi, Κ., H. Akitake, and T. Tomiyama, Bull. Japan. Soc. Sci. Fish., (1970), 36 (1), 103-108. 10. Kobayashi, Κ., and N. Nakamura, Bull. Japan. Soc. Sci. Fish., submitted. 11. Tashiro, S., T. Sasamoto, T. Aikawa, S. Tokunaga, E. Taniguchi and M. Eto, J. Agr. Chem. Soc. Japan, (1970), 44 (3), 124-129. 12. Kobayashi, Κ., and Ν. Nakamura, Bull. Japan. Soc. Sci. Fish., submitted. 13. Kobayashi, Κ., and K. Higuma, Bull. Japan. Soc. Sci. Fish., submitted. 14. Kobayashi, K., S. Kimura, and H. Akitake, Bull. Japan. Soc. Sci. Fish., (1976), 42 (2), 171-177. 15. Kimura, S., and K. Kobayashi, Bull. Japan. Soc. Sci. Fish., submitted. RECEIVED

January 2, 1979.


Metabolism of Pentachlorophenol in Fish - ACS Symposium Series

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