Disposition of Toxic Substances in Mussels (Mytilus califorianus

16 Disposition of Toxic Substances in Mussels (Mytilus

Downloaded by FUDAN UNIV on April 7, 2017 | http://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch016

califorianus): Preliminary Metabolic and Histologic Studies ROBERT I. KRIEGER, SHIRLEY J. GEE, LORI O. LIM, JOHN H. ROSS, and ANNE WILSON Department of Environmental Toxicology, University of California, Davis, CA 95616 CHARLES ALPERS and SEFTON R. WELLINGS Department of Medical Pathology, University of California, Davis, CA 95616 Deaths of target organisms associated with intentional pesticide applications to insect-infested crops, weed-choked roadsides, and nematode-laced fields are predictable, desirable, and relatively easy to measure. Likewise, catastropic releases of chlorine from ruptured tank cars or of crude oil from scuttled supertankers may produce a spectrum of biological effects including toxicity. These events are easily associated with exposures to toxic substances and particular environmental circumstances. In both the intentional and the catastrophic cases, when death is the end-point, the establishment of cause-and-effect relationships is straightforward. Altered growth and development, reduced vigor, body burdens of persistent chemicals, genetic defects, and impaired reproduction are harmful effects which are more difficult to detect and evaluate. One means to detect such harmful effects i s use of monitoring organisms. The concept was explored by Haldane who advocated i n the late 19th century (1) that small animals such as mice or birds be used to warn miners of toxic levels of carbon monoxide. The small animals' rapid respiration being very sensitive to carbon monoxide inhibition, symptoms of poisoning gave timely warning of harmful proportions of CO in the workers environment. More r e c e n t l y , e n v i r o n m e n t a l m o n i t o r i n g h a s b e e n accomp l i s h e d u s i n g b a c t e r i a , m o l l u s c s , f i s h , a n d b i r d s (.2*3)· B i o accumulation o f mercurials ( 4 ) , p o l y c h l o r i n a t e d biphenyls ( 5 ) , and o r g a n o c h l o r i n e p e s t i c i d e s ( 6 , 7 ) h a s b e e n u s e d t o f a c i l i t a t e environmental sampling. An EPA-sponsored "Mussel Watch" program has u t i l i z e d b i v a l v e s f r o m b o t h c o a s t s a n d t h e G u l f o f M e x i c o a s s e n t i n e l organisms i n m o n i t o r i n g f o r halogenated hydrocarbons, a r t i f i c i a l r a d i o n u c l i d e s , p e t r o l e u m components a n d h e a v y m e t a l s (8). R e s u l t s o f such s t u d i e s have been used b y r e g u l a t o r y a g e n c i e s , e n v i r o n m e n t a l a d v o c a t e s , and o t h e r s c o n c e r n e d b o t h w i t h

0-8412-0489-6/79/47-099-259$05.00/0 © 1979 American Chemical Society Khan et al.; Pesticide and Xenobiotic Metabolism in Aquatic Organisms ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PESTICIDE AND


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m i c r o c o n s t i t u e n t s o f t h e f o o d s u p p l y and changes i n e n v i r o n m e n t a l quality. B i o l o g i c a l c o n s e q u e n c e s o f l o w l e v e l body b u r d e n s o f p e r s i s t e n t p o l l u t a n t s a r e more p o o r l y d e f i n e d t h a n i s t h e c e r t a i n t y o f e x p o s u r e . Whether o r n o t e x p o s u r e t o p e r s i s t e n t c h e m i c a l s i s a s s o c i a t e d w i t h t o x i c i t y w i l l be d e t e r m i n e d by c o m p l e x i n t e r a c t i o n o f a number o f p r o c e s s e s w h i c h c a n be c o n v e n i e n t l y d e s i g nated d i s p o s i t i o n processes. These i n c l u d e u p t a k e ( a b s o r p t i o n ) , d i s t r i b u t i o n , receptor i n t e r a c t i o n , deposition (storage), b i o t r a n s f o r m a t i o n ( m e t a b o l i s m ) , and e l i m i n a t i o n ( e x c r e t i o n , d e p u r a t i o n ) w h i c h d e t e r m i n e t h e b i o l o g i c a l f a t e and e f f e c t s o f t o x i c s u b s t a n c e s . H i s t o l o g i c a l s t u d y o f s t r e s s e d o r g a n i s m s may r e v e a l c o r r e s p o n d i n g c h a n g e s i n t i s s u e f o r m and f u n c t i o n . These c o n s i d e r a t i o n s l e n d t h e m s e l v e s t o e x p e r i m e n t a l s t u d y , and r e s u l t s of s u c h s t u d i e s may be v a l u a b l e i n p r e d i c t i v e t o x i c o l o g y . H i s t o l o g i c a l s t u d i e s and i n v e s t i g a t i o n s o f d i s p o s i t i o n p r o c e s s e s are being coupled i n order to assess the c o n d i t i o n of p o p u l a t i o n s o f C a l i f o r n i a m u s s e l s M y t i l u s c a l i f o r n i a n u s . Whether r e s u l t s o f f u t u r e s t u d i e s o f t h i s s o r t c a n be u s e d d i a g n o s t i c a l l y to r e v e a l the presence of c h e m i c a l s t r e s s o r s i n the environment and c o n t r i b u t e t o e v a l u a t i o n o f t h e i r i m p a c t i s a c o n c e r n w h i c h m o t i v a t e s much o f o u r e x p e r i m e n t a l work. M e t h o d s and

Materials

M u s s e l s . M y t i l u s c a l i f o r n i a n u s were u s u a l l y c o l l e c t e d a t l o w t i d e i n t h e r o c k y , i n t e r t i d a l zone o f S c h o o l h o u s e B e a c h n e a r Bodega Bay (Sonoma Co.) on t h e c e n t r a l C a l i f o r n i a c o a s t . Other s i t e s h a v e i n c l u d e d S a l t P o i n t , Bodega Head, and F o r t B r a g g . A n i m a l s w e r e u s e d on t h e c o a s t o r t r a n s p o r t e d t o D a v i s where t h e y were m a i n t a i n e d i n a e r a t e d a q u a r i a c o n t a i n i n g I n s t a n t Ocean^ S y n t h e t i c Sea S a l t (1.025 s p e c i f i c g r a v i t y , 11°C, 12/12 L/D). E x c e p t as n o t e d , m u s s e l s w e r e u s e d w i t h i n 2 w e e k s . C o n d i t i o n Index. S h e l l volume, determined v o l u m e t r i c a l l y , and t i s s u e mass w e r e measured f o r d e t e r m i n a t i o n o f t h e c o n d i t i o n i n d e x ( 9 ) . T i s s u e (g) d i v i d e d by s h e l l v o l u m e (ml) y i e l d e d t h e i n d e x w h i c h r a n g e d b e t w e e n 0.45 and 0.60. Histology. Whole m u s s e l s were f i x e d f o r 1-2 d a y s i n H e l l y ' s f l u i d (10) and s t o r e d i n 70% e t h a n o l . T i s s u e was b l o c k e d a t 2 mm t h i c k n e s s , embedded i n p a r a f f i n , s e c t i o n e d a t a b o u t 7 urn, s t a i n e d w i t h h e m a t o x y l i n and e o s i n , and mounted on g l a s s s l i d e s u s i n g standard procedures. I n V i v o E x p o s u r e s . A l d r i n (200 yg/200 μΐ DMSO o r m e t h a n o l ) was a d m i n i s t e r e d by i n j e c t i o n i n t o t h e p o s t e r i o r a d d u c t o r m u s c l e o r s l o w l y r e l e a s e d f r o m a s y r i n g e (27 gauge, 1/2 i n c h ) d i r e c t l y o n t o v i s c e r a . M u s s e l s i n s i t u i n t h e i r n a t u r a l h a b i t a t and o t h e r s c o n t a i n e d i n s m a l l c h e e s e c l o t h bags were a l s o t r e a t e d . After

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

16.

KRiEGER E T A L .

Mussels

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( Mytilus califorianus )

d e s i g n a t e d i n t e r v a l s , m u s s e l s were q u i c k - f r o z e n l a t e r s t o r e d a t -15°C p r i o r t o a n a l y s i s .

u s i n g d r y i c e and

Uptake and E l i m i n a t i o n . S i x m u s s e l s were p l a c e d i n t o 600 m l b e a k e r s c o n t a i n i n g 300 m l a e r a t e d I n s t a n t O c e a n (10-12°C) f o r a 1/2 h r a c c l i m a t i o n p e r i o d b e f o r e a d d i t i o n o f a n t i p y r i n e (3 u C i / b e a k e r ) ( o r o t h e r t e s t compound). A l i q u o t s (1.0 m l ) w e r e t a k e n a t i n t e r v a l s a n d p l a c e d d i r e c t l y i n t o s c i n t i l l a t i o n f l u i d (10 m l , 3a70B, R e s e a r c h P r o d u c t s I n t e r n a t i o n a l C o r p . , E l k G r o v e V i l l a g e , IL). I n i t i a l e x p e r i m e n t s showed t h a t u p t a k e was c o m p l e t e w i t h i n 2 h o u r s . A t t h e end o f 2 h o u r s (uptake) t h e m u s s e l s were r i n s e d , a n t i p y r i n e s o l u t i o n was r e p l a c e d w i t h f r e s h I n s t a n t O c e a n , and s a m p l i n g a t i n t e r v a l s c o n t i n u e d . P o r t i o n s o f t h e w a t e r w e r e a l s o a n a l y z e d f o r a n t i p y r i n e and m e t a b o l i t e s . Data expressed i n c o u n t s p e r m i n u t e p e r 1.0 m l w e r e u s e d t o r e p r e s e n t u p t a k e a n d elimination of antipyrine. E x p e r i m e n t s w e r e r o u t i n e l y done i n triplicate. Graphs r e l a t i n g a n t i p y r i n e c o n c e n t r a t i o n s and time were used to c a l c u l a t e clearance r a t e s . A r e l a t i o n s h i p between apparent a n t i p y r i n e s t e a d y s t a t e c o n c e n t r a t i o n s a t 120 a n d 240 m i n u t e s ( a p i 2 0 > ap2»+o) and m u s s e l b o d y w a t e r and m a n t l e c a v i t y w a t e r was a l s o d e t e r m i n e d ( k ) . M a n t l e c a v i t y water i s t h a t volume h e l d b e t w e e n t h e v a l v e s when t h e m u s s e l s a r e c l o s e d , e . g . , when t r a n s f e r r e d f r o m t h e u p t a k e s o l u t i o n (300 m l ) t o t h e e l i m i n a t i o n s o l u t i o n (300 m l ) . T h e i n i t i a l a n t i p y r i n e c o n c e n t r a t i o n ( a p o ) was d e t e r m i n e d a t t h e b e g i n n i n g o f t h e e x p e r i m e n t . A s s u m i n g no l o s s o f a n t i p y r i n e , complete m i x i n g o f t h e s o l u t i o n s , and i t s d i s t r i b u t i o n i n t o t o t a l m u s s e l b o d y w a t e r , when a n a p p a r e n t steady state i s achieved, the following r e s u l t s : R


R

300 a p = (300 + k i o ) 0

k

1 2 0

2

apo = 300 ( — -

api2o

(api o) 2

- 1)

S i m i l a r l y , a t achievement o f the second apparent s t e a d y s t a t e f o l l o w i n g t r a n s f e r o f t h e a n t i p y r i n e t r e a t e d m u s s e l t o 300 m l w a t e r , t h e f o l l o w i n g e q u a t i o n can be w r i t t e n : k2t+o a p

1 2

o

= (300 + k i i o ) 2

ap2t+o

300 ap2t*o k 2 i+ ο

=

api2o

- ap2»+o

w h e r e k2i+o i s t h e b o d y and r e s i d u a l w a t e r c o n s t a n t d e r i v e d f r o m steady state a n t i p y r i n e concentrations. I n i d e n t i c a l s y s t e m s , u p t a k e o f a l d r i n ( 3 0 , 300, 3000 yg/300 ml) has been demonstrated. S i m i l a r l y , b o t h u p t a k e and e l i m i n a t i o n


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of p - n i t r o a n i s o l e measured.

( i n i t i a l c o n c e n t r a t i o n 1.5 mg/300 m l ) have b e e n


T i s s u e Homogenates and M i c r o s o m e s . P r e p a r a t i o n s were made f r o m g i l l , m a n t l e , g r e e n g l a n d , and v i s c e r a ( a l l t i s s u e e x c e p t a d d u c t o r m u s c l e s ) i n e i t h e r i c e - c o l d 1.15% KC1 (w/v) o r 0.05M p h o s p h a t e b u f f e r , pH 7.4. Homogenates (10 o r 20% w/v) were p r e p a r e d u s i n g e i t h e r a l l - g l a s s , hand o p e r a t e d t i s s u e g r i n d e r s o r a W a r i n g B l e n d o r (10 s e c a t h i g h s p e e d ) . M i c r o s o m a l f r a c t i o n s w e r e p r e p a r e d by c e n t r i f u g a t i o n o f t h e 10,000 g max χ 30 m i n s u p e r n a t a n t a t 100,000 g max χ 60 m i n . P e l l e t s were resuspended i n b u f f e r u s i n g t i s s u e g r i n d e r w i t h a t e f l o n p e s t l e and f i n a l p r o t e i n (11) c o n c e n t r a t i o n r a n g e d b e t w e e n 3 and 10 mg/ml. C y t o c h r o m e D e t e r m i n a t i o n s . M i c r o s o m a l s u s p e n s i o n s (1-5 mg p r o t e i n / m l ) w e r e a s s a y e d f o r c y t o c h r o m e s bs and P-450 (12) u s i n g a C a r y 15 s p e c t r o p h o t o m e t e r o p e r a t e d a t room t e m p e r a t u r e ( 2 0 23°C). S u s p e n s i o n s i n 0.05M p h o s p h a t e b u f f e r , pH 7.4, w e r e c o n t a i n e d i n 3 m l c u v e t t e s w i t h a 1 cm p a t h l e n g t h . Sodium d i t h i o n i t e was t h e r e d u c t a n t . The e x t i n c t i o n c o e f f i c i e n t o f 171 mM cm was a p p l i e d t o t h e 428-490 nm a b s o r b a n c e i n c r e m e n t . 1

1

Monooxygenase A s s a y s . I n c u b a t i o n media c o n t a i n e d the f o l l o w ing ( f i n a l concentrations): 0.05M p h o s p h a t e b u f f e r , pH 7.4, g l u c o s e - 6 - p h o s p h a t e (G-6-P, 2.3 mM), G-6-P d e h y d r o g e n a s e (3 u n i t s ) , NADP (0.23 mM), and KC1 (2.8 mM), and v a r i o u s t i s s u e preparations. S u b s t r a t e s w e r e added i n s m a l l v o l u m e s (25 y l o r l e s s ) o f MeOH. Samples ( 1 . 1 m l ) w e r e s h a k e n i n a t h e r m o s t a t e d ( u s u a l l y a t 22°C) w a t e r b a t h and r e a c t i o n s t e r m i n a t e d by enzyme dénaturâtion. S p e c i f i c a n a l y t i c a l p r o c e d u r e s f o r a l d r i n e p o x i d a t i o n ( 1 3 ) , ^ C H a O - p - n i t r o a n i s o l e O - d e m e t h y l a t i o n ( 1 4 ) , and H - b e n z o ( a ) p y r e n e o x i d a t i o n (15) h a v e b e e n d e s c r i b e d . 1

3

C h e m i c a l s . A n t i p y r i n e , c a r b o n m o n o x i d e ( M a t h e s o n , Coleman and B e l l , L o s A n g e l e s , C A ) , and ^ C H a - N - a n t i p y r i n e (11.1 mCi/mM, ICN, I r v i n e , CA) w e r e p u r c h a s e d . Aldrin (1,8,9,10,11,11-hexachloro-2,3-7,6-endo-2,1-7,8-exo-tetracyclo (6.2.1.1 ' .0 ' ) d o d e c a - 4 , 9 - d i e n e ) and i t s e p o x i d e , d i e l d r i n were g i f t s o f S h e l l D e v e l o p m e n t Co. ( M o d e s t o , C A ) . E a c h was r e c r y s t a l l i z e d f r o m m e t h a n o l - w a t e r s o l u t i o n s and was g r e a t e r t h a n 99% p u r e as d e t e r m i n e d b y gas c h r o m a t o g r a p h y . C H 0 - p - N i t r o a n i s o l e (1.9 mCi/mmole) was s y n t h e s i z e d (14) and H - b e n z o ( a ) p y r e n e (8.3 C i / mmole) was p u r c h a s e d (Amersham-Searle Co., A r l i n g t o n H e i g h t s , IL). S o l v e n t s w e r e n a n o g r a d e ( M a l l i n c k r o d t , S t . L o u i s , MO). Other c h e m i c a l s were a t l e a s t r e a g e n t grade. 1

3

6

2

7

3

3

Results U p t a k e and E l i m i n a t i o n . M a g n i t u d e s o f e x p o s u r e o f l a m e l l i b r a n c h s t o t o x i c s u b s t a n c e s w i l l be d e t e r m i n e d by r a t e s o f f l o w of water through the g i l l s , c o n c e n t r a t i o n s of the t o x i c substance,



16.

KRiEGER E T A L .

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( Mytilus califorianus )

263

and t h e e x t e n t o f u p t a k e . R a t e s o f v e n t i l a t i o n , i . e . v o l u m e o f water f l o w i n g t h r o u g h g i l l s ( l i t e r s w a t e r / h r / m u s s e l ) have been m e a s u r e d d i r e c t l y and i n d i r e c t l y ( 1 7 ) . R a t e s o f s o l u t e r e m o v a l per u n i t t i m e a r e termed c l e a r a n c e . General reviews include t h o s e by Owen ( 1 8 ^ 1 9 ) , J o r g e n s e n ( 2 0 ) a n d A l i ( 2 1 ) . M o s t i n d i r e c t measurements a r e made u s i n g p a r t i c u l a t e s u s p e n s i o n s o f m a t e r i a l s u c h a s c o l l o i d a l g r a p h i t e , s i l t , a n d a l g a e . Bayne t a b u l a t e d v e n t i l a t i o n r a t e d a t a f o r f o u r s p e c i e s i n c l u d i n g M. californianus (17). Q u a n t i t a t i v e e s t i m a t i o n o f v e n t i l a t i o n b y i n d i r e c t methods i n mussels r e q u i r e s f o u r assumptions (16): a) r e d u c t i o n o f c o n c e n t r a t i o n r e s u l t s from u p t a k e , b) c o n s t a n t v e n t i l a t i o n (pumping) r a t e , c ) u p t a k e o f a c o n s t a n t p e r c e n t a g e o f c o n c e n t r a t i o n ( f i r s t o r d e r p r o c e s s ) , d) homogeneity o f t h e t e s t s o l u t i o n a t a l l t i m e s . Our t r a n s p o r t s t u d i e s h a v e u t i l i z e d a n t i p y r i n e ( 2 2 , 23) a w a t e r s o l u b l e , s t a b l e c h e m i c a l o f l o w a c u t e t o x i c i t y t o mussels. I t i s r e a d i l y d i s s o l v e d i n ocean water o r I n s t a n t Ocean a n d i s n e i t h e r a d s o r b e d n o r v o l a t i l i z e d f r o m t h e 300 m l t e s t s y s t e m . M u s s e l s pump t h r o u g h o u t t h e 4 h o u r t e s t p e r i o d a n d t h i s a c t i o n i s a p p a r e n t l y s u f f i c i e n t t o i n s u r e homo geneity of the solution. I n s p e c t i o n o f e a r l y u p t a k e and e l i m i n a t i o n curves ( a n t i p y r i n e c o n c e n t r a t i o n as a f u n c t i o n of time) prompted u s e o f C o u g h l a n ' s e q u a t i o n ( 1 6 ) f o r w a t e r t r a n s p o r t . A n t i p y r i n e u p t a k e r a t e s o b t a i n e d by m e a s u r i n g d e c l i n i n g c o n c e n t r a t i o n s o f t h e s o l u t e may b e o b t a i n e d f r o m t h e f o l l o w i n g equation: -dC _ dt "

,mn.

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—dC where t h e u p t a k e r a t e , was t h e r a t e o f d e c r e a s e o f C^; M was the v o l u m e o f t h e t e s t s y s t e m ; n. was t h e number o f a n i m a l s ; m was the c l e a r a n c e r a t e o f a s i n g l e a n i m a l . T h i s may b e s o l v e d a s follows : 1ηΟ InC M 0 t m = — χ n t I n d i r e c t measurements o f c l e a r a n c e h a v e t h e a d v a n t a g e o f r e q u i r ing l i t t l e m a n i p u l a t i o n o f t h e m u s s e l s , b u t s u f f e r from t h e f a c t t h a t r a t e s may b e a f f e c t e d b y c o n c e n t r a t i o n s o f s u s p e n d e d p a r t i c u l a t e s and s o l u t e s . A n t i p y r i n e f r o m 300 m l s o l u t i o n s c o n t a i n i n g 0.6, 6, o r 60μΜ was r e a d i l y t a k e n up by m u s s e l s . W i t h i n 40-80 m i n u t e s a n a p p a r ent s t e a d y s t a t e was a c h i e v e d . U p t a k e e x p e r i m e n t s w e r e r o u t i n e l y c o n d u c t e d f o r 120 m i n u t e s ( F i g u r e 1 ) . A n a n a l o g o u s a n t i p y r i n e e l i m i n a t i o n c u r v e i s shown i n t h e l o w e r p o r t i o n o f t h e f i g u r e . A t t h e h i g h e s t d o s e (60 μΜ) m u s s e l s sometimes c l o s e d , b u t o t h e r s v e n t i l a t e d w i t h no a p p a r e n t i l l e f f e c t s . The i n t e r m e d i a t e c o n c e n t r a t i o n (6 μΜ) was, t h e r e f o r e , u s e d i n a l l s u b s e q u e n t e x p e r i ments. Λ


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

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Figure 1. Antipyrine uptake and elimination data taken from 3 experiments. Six mussels in 300 mL Instant Ocean® were used in each. The 1 mL aliquots were taken at the end of each interval.


16.

KRiEGER E T A L .

Mussels

(Mytilus califorianus)

265

From c u r v e s i n c l u d i n g t h e o n e shown i n F i g u r e l antipyrine c l e a r a n c e r a t e s were c a l c u l a t e d u s i n g Coughlan's f o r m u l a ( 1 6 ) . Rates were c a l c u l a t e d f o r 3 i n t e r v a l s : 0-5 m i n , 0-20 m i n , a n d 520 m i n ( F i g u r e 2 ) , E a c h p a r t i c u l a r i n t e r v a l y i e l d e d a p p a r e n t r a t e s w h i c h were r e m a r k a b l y p r e c i s e ( T a b l e I ) . Data a r e p r e s e n t e d f o r two e x p e r i m e n t s i n w h i c h 3 s e t s o f m u s s e l s w e r e u s e d a f t e r different intervals i n the laboratory. This s e r i e s of mussels was t h e s o u r c e o f most o f t h e a n i m a l s u s e d i n d i s p o s i t i o n s t u d i e s t o d a t e . The l a r g e s t c h a n g e s i n a n t i p y r i n e c o n c e n t r a t i o n w e r e observed during the f i r s t 5 min i n t e r v a l . The change r e s u l t e d b o t h from d i l u t i o n o f t h e a n t i p y r i n e s o l u t i o n by w a t e r w i t h i n the m a n t l e c a v i t y a n d f r o m a n t i p y r i n e u p t a k e . D u r i n g t h e 5-20 m i n i n t e r v a l , m u s s e l pumping p r o v i d e s c o n t i n u o u s e x p o s u r e a n d u p take i s f i r s t order. The h i g h e r r a t e s f o r t h e f i r s t two t i m e i n t e r v a l s ( 0 - 5 , 0-20 m i n ) a r e i n f l u e n c e d b y t h e m a n t l e c a v i t y d i l u t i o n f a c t o r . A n t i p y r i n e c l e a r a n c e r a t e s , e.g. 110-150 m l / h r b e tween 5 a n d 20 m i n , a r e c a l c u l a t e d r a t e s ( T a b l e I ) a r e l e s s t h a n published v e n t i l a t i o n rates (17), indicating that antipyrine i s not 1 0 0 % a b s o r b e d d u r i n g a s i n g l e p a s s o f w a t e r t h r o u g h t h e mussels. N e u t r a l r e d clearance, an i n d i c a t o r of v e n t i l a t i o n ( 1 9 ) , y i e l d e d r a t e s o f 136 t o 1181 m l / h r i n u n p u b l i s h e d p r e l i m i n a r y studies. C l e a r l y , a n t i p y r i n e c l e a r a n c e i s n o t l i m i t e d by v e n t i l a t i o n per se.


t

From t h e same u p t a k e a n d e l i m i n a t i o n d a t a t h e m a n t l e c a v i t y and body w a t e r v o l u m e ( k ) w e r e c a l c u l a t e d f o r m u s s e l s a t e a c h storage i n t e r v a l (Table I I ) . S i n c e a l l measurements o f u p t a k e and e l i m i n a t i o n a r e made w i t h t h e same t e s t o r g a n i s m s , i t i s h y p o t h e s i z e d t h a t k i s a c o n s t a n t that can be c a l c u l a t e d from t h e i n i t i a l a n t i p y r i n e (apg) a n d t h e s t e a d y s t a t e v a l u e s ( a p i 2 0 ^ P24o) · mean (+S.D,) c a l c u l a t e d c o n s t a n t s ( k i 2 0 * ^2*+o) w e r e 114+24 m l a n d 109+17. The v a l u e s w e r e n o t s t a t i s t i c a l l y d i f f e r e n t ( S t u d e n t s t - t e s t , p

Disposition of Toxic Substances in Mussels (Mytilus califorianus

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