"Land and Food Systems, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Jones, Trevor O."@en . "2010-10-07T19:43:46Z"@en . "1990"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Use of antibiotics in the aquaculture industry for the control of bacterial infections in salmon has led to public concern regarding antibiotic residues in salmon tissue and the potential health risk this poses to humans. Oxytetracycline and Romet-30 are two common antibacterial agents utilized to control disease in the local finfish aquaculture industry of British Columbia, Canada. Many fish farms in British Columbia are located within close proximity to existing shellfish stocks. The potential for antibiotic bioconcentration and the production of bacterial resistance to the drugs in bivalves is a concern. The development of a solid phase extraction technique for these two drugs from Pacific oysters, Crassostrea gigas was accomplished. Analysis of whole oysters via HPLC revealed detectable levels of oxytetracycline and Romet-30 up to 4 weeks after antibiotic treatment at the salmon farm site. Oysters suspended in the salmon net pens and surrounding waters were sampled over a 5 month period. Concentration of oxytetracycline residues in oyster tissue reached a maximum of 0.11 ppm (n=2). Levels of sulphadimethoxine in oyster tissue reached a maximum concentration of 1.27 ppm (n=2). Maximum detectable concentrations of ormetoprim in oysters were 0.26 ppm (n=2).\r\nA controlled study investigating uptake and depuration of oxytetracycline and Romet-30 in the Pacific oyster revealed results similar to those described for the salmon farm. A flow through drug delivery system for the Pacific oyster was utilized over a ten day uptake and thirty day depuration period. Levels of oxytetracycline in whole animal tissue increased in a linear fashion (r\u00B2=0.984) over the uptake period. This indicates that the drug was bioaccumulated in the oyster reaching a maximum concentration of 10.72 \u00B1 2.4 s.e. ppm (n= 3) on day 10 of uptake. Depuration of the drug was rapid. After thirteen days a concentration of 0.21 \u00B1 0.04 s.e. ppm (n=7) was detected. The detection of Romet-30 in the uptake and depuration in the oyster was more erratic. Day 7 of uptake revealed a maximum concentration of sulphadimethoxine and ormetoprim of 1.092 \u00B1 0.24 s.e. ppm (n=7) and 0.21 ppm (n=3) respectively. Depuration of Romet-30 was rapid. The concentration decreased to 0.029 ppm (n=3) by day 13.\r\nCondition indices of oysters sampled before and after antibiotic treatment indicated a significant increase (p<0.05) in condition index after treatment with medicated feed.\r\nA quantitative survey of bacteria associated with the gut of the oyster was performed. Relative numbers of endogenous bacteria were compared to oysters before and after antibiotic treatment. Numbers of bacteria significantly decreased (p< 0.05) following treatment."@en . "https://circle.library.ubc.ca/rest/handle/2429/28991?expand=metadata"@en . "U p t a k e a n d D e p u r a t i o n o f t h e A n t i b i o t i c s , O x y t e t r a c y c l i n e a n d R o m e t - 3 0 R i n t h e P a c i f i c O y s t e r , C r a s s o s t r e a g i g a s ( T h u n b e r g ) . By T r e v o r 0 . J o n e s B . S c , U n i v e r s i t y o f V i c t o r i a , 1986 A T H E S I S SUBMITTED I N P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E DEGREE OF MASTERS OF S C I E N C E i n T H E F A C U L T Y OF GRADUATE S T U D I E S ( D e p a r t m e n t o f A n i m a l S c i e n c e ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE U N I V E R S I T Y OF B R I T I S H COLUMBIA A u g u s t 1990 fj) T r e v o r Owen J o n e s , A u g u s t 2 0 , 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) i i A b s t r a c t U s e o f a n t i b i o t i c s i n t h e a q u a c u l t u r e i n d u s t r y f o r t h e c o n t r o l o f b a c t e r i a l i n f e c t i o n s i n s a l m o n h a s l e d t o p u b l i c c o n c e r n r e g a r d i n g a n t i b i o t i c r e s i d u e s i n s a l m o n t i s s u e a n d t h e p o t e n t i a l h e a l t h r i s k t h i s p o s e s t o h u m a n s . O x y t e t r a c y c l i n e a n d R o m e t - 3 0 a r e two common a n t i b a c t e r i a l a g e n t s u t i l i z e d t o c o n t r o l d i s e a s e i n t h e l o c a l f i n f i s h a q u a c u l t u r e i n d u s t r y o f B r i t i s h C o l u m b i a , C a n a d a . Many f i s h f a r m s i n B r i t i s h C o l u m b i a a r e l o c a t e d w i t h i n c l o s e p r o x i m i t y t o e x i s t i n g s h e l l f i s h s t o c k s . T h e p o t e n t i a l f o r a n t i b i o t i c b i o c o n c e n t r a t i o n a n d t h e p r o d u c t i o n o f b a c t e r i a l r e s i s t a n c e t o t h e d r u g s i n b i v a l v e s i s a c o n c e r n . T h e d e v e l o p m e n t o f a s o l i d p h a s e e x t r a c t i o n t e c h n i q u e f o r t h e s e two d r u g s f r o m P a c i f i c o y s t e r s , C r a s s o s t r e a g i g a s was a c c o m p l i s h e d . A n a l y s i s o f w h o l e o y s t e r s v i a H P L C r e v e a l e d d e t e c t a b l e l e v e l s o f o x y t e t r a c y c l i n e a n d R o m e t - 3 0 up t o 4 weeks a f t e r a n t i b i o t i c t r e a t m e n t a t t h e s a l m o n f a r m s i t e . O y s t e r s s u s p e n d e d i n t h e s a l m o n n e t p e n s a n d s u r r o u n d i n g w a t e r s w e r e s a m p l e d o v e r a 5 m o n t h p e r i o d . C o n c e n t r a t i o n o f o x y t e t r a c y c l i n e r e s i d u e s i n o y s t e r t i s s u e r e a c h e d a maximum o f 0 . 1 1 ppm ( n = 2 ) . L e v e l s o f s u l p h a d i m e t h o x i n e i n o y s t e r t i s s u e r e a c h e d a maximum c o n c e n t r a t i o n o f 1 . 2 7 ppm ( n = 2 ) . Maximum d e t e c t a b l e c o n c e n t r a t i o n s o f o r m e t o p r i m i n o y s t e r s w e r e 0 . 2 6 ppm ( n = 2 ) . A c o n t r o l l e d s t u d y i n v e s t i g a t i n g u p t a k e a n d d e p u r a t i o n o f o x y t e t r a c y c l i n e a n d R o m e t - 3 0 i n t h e P a c i f i c o y s t e r r e v e a l e d r e s u l t s s i m i l a r t o t h o s e d e s c r i b e d f o r t h e s a l m o n i i i f a r m . A f l o w t h r o u g h d r u g d e l i v e r y s y s t e m f o r t h e P a c i f i c o y s t e r was u t i l i z e d o v e r a t e n d a y u p t a k e a n d t h i r t y d a y d e p u r a t i o n p e r i o d . L e v e l s o f o x y t e t r a c y c l i n e i n w h o l e a n i m a l t i s s u e i n c r e a s e d i n a l i n e a r f a s h i o n ( r 2 = 0 . 9 8 4 ) o v e r t h e u p t a k e p e r i o d . T h i s i n d i c a t e s t h a t t h e d r u g was b i o a c c u m u l a t e d i n t h e o y s t e r r e a c h i n g a maximum c o n c e n t r a t i o n o f 1 0 . 7 2 \u00E2\u0080\u0094 2 . 4 s . e . ppm (n= 3) o n d a y 10 o f u p t a k e . D e p u r a t i o n o f t h e d r u g was r a p i d . A f t e r t h i r t e e n d a y s a c o n c e n t r a t i o n o f 0 . 2 1 \u00E2\u0080\u0094 0 . 0 4 s . e . ppm (n=7) was d e t e c t e d . T h e d e t e c t i o n o f R o m e t - 3 0 i n t h e u p t a k e a n d d e p u r a t i o n i n t h e o y s t e r was more e r r a t i c . Day 7 o f u p t a k e r e v e a l e d a maximum c o n c e n t r a t i o n o f s u l p h a d i m e t h o x i n e a n d o r m e t o p r i m o f 1 .092 \u00E2\u0080\u0094 0 . 2 4 s . e . ppm (n=7) a n d 0 . 2 1 ppm (n=3) r e s p e c t i v e l y . D e p u r a t i o n o f R o m e t - 3 0 was r a p i d . T h e c o n c e n t r a t i o n d e c r e a s e d t o 0 . 0 2 9 ppm (n=3) b y d a y 1 3 . C o n d i t i o n i n d i c e s o f o y s t e r s s a m p l e d b e f o r e a n d a f t e r a n t i b i o t i c t r e a t m e n t i n d i c a t e d a s i g n i f i c a n t i n c r e a s e ( p < 0 . 0 5 ) i n c o n d i t i o n i n d e x a f t e r t r e a t m e n t w i t h m e d i c a t e d f e e d . A q u a n t i t a t i v e s u r v e y o f b a c t e r i a a s s o c i a t e d w i t h t h e g u t o f t h e o y s t e r was p e r f o r m e d . R e l a t i v e n u m b e r s o f e n d o g e n o u s b a c t e r i a w e r e c o m p a r e d t o o y s t e r s b e f o r e a n d a f t e r a n t i b i o t i c t r e a t m e n t . Numbers o f b a c t e r i a s i g n i f i c a n t l y d e c r e a s e d (p< 0 . 0 5 ) f o l l o w i n g t r e a t m e n t . i v T a b l e o f C o n t e n t s A b s t r a c t i i T a b l e o f C o n t e n t s i v L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i A c k n o w l e d g e m e n t s i x G e n e r a l I n t r o d u c t i o n 1 C h a p t e r 1 T h e D e v e l o p m e n t o f E x t r a c t i o n M e t h o d s f o r O x y t e t r a c y c l i n e a n d R o m e t - 3 0 i n t h e P a c i f i c O y s t e r a n d t h e D e t e c t i o n o f A n t i b i o t i c R e s i d u e s i n P a c i f i c O y s t e r s S u s p e n d e d i n S a l m o n N e t - C a g e s T h r o u g h H P L C 5 I n t r o d u c t i o n 5 M e t h o d s 7 R e s u l t s 15 D i s c u s s i o n 24 C h a p t e r 2 D e t e r m i n a t i o n o f A n t i b i o t i c U p t a k e a n d D e p u r a t i o n i n t h e P a c i f i c O y s t e r 29 I n t r o d u c t i o n 29 M e t h o d s 30 R e s u l t s 36 D i s c u s s i o n 48 G e n e r a l C o n c l u s i o n s B i b l i o g r a p h y A p p e n d i c e s L i s t o f T a b l e s v i 1 . T a b l e 1. A b s o l u t e r e c o v e r i e s o f O x y t e t r a c y c l i n e , S u l p h a d i m e t h o x i n e a n d O r m e t o p r i m i n t h e P a c i f i c o y s t e r ( U B C ) . E a c h v a l u e r e p r e s e n t s a s i n g l e d e t e r m i n a t i o n a t e a c h s a m p l e c o n c e n t r a t i o n 16 2 . T a b l e 2 . A b s o l u t e r e c o v e r i e s o f O x y t e t r a c y c l i n e , S u l p h a d i m e t h o x i n e a n d O r m e t o p r i m i n t h e P a c i f i c o y s t e r ( S Y N D E L ) . E a c h v a l u e r e p r e s e n t s a s i n g l e d e t e r m i n a t i o n a t e a c h s a m p l e c o n c e n t r a t i o n 3 7 3 . T a b l e 3 . D e t e r m i n a t i o n o f b a c t e r i a l c o l o n i z a t i o n o n s e v e r a l m e d i a a s d e t e r m i n e d b y mean p r o b a b l e n u m b e r ( M P N ) . E a c h v a l u e r e p r e s e n t s t h e mean o f 6 i n d i v i d u a l o y s t e r s 46 v i i L i s t o f F i g u r e s 1 . F i g u r e 1 . L o c a t i o n o f s a l m o n f a r m s t u d y s i t e , J e r v i s I n l e t , B . C 12 2 . F i g u r e 2 . D e t a i l o f s t u d y s i t e , i n d i c a t i n g p o s i t i o n o f l a n t e r n n e t s i n r e f e r e n c e t o n e t - p e n s 13 3 . F i g u r e 3 . C a l i b r a t i o n c u r v e f o r o x y t e t r a c y c l i n e i n t h e P a c i f i c o y s t e r . E a c h d a t a p o i n t r e p r e s e n t s a s i n g l e d e t e r m i n a t i o n a t e a c h c o n c e n t r a t i o n o f d r u g ( r 2 = 0 . 9 9 8 ) 17 4 . F i g u r e 4 . C a l i b r a t i o n c u r v e f o r R o m e t - 3 0 i n t h e P a c i f i c o y s t e r . E a c h d a t a p o i n t r e p r e s e n t s a s i n g l e d e t e r m i n a t i o n a t e a c h c o n c e n t r a t i o n o f d r u g (SDM, r 2 = 0 . 9 9 0 ; OMP, r 2 = 0 . 9 9 2 ) 18 5 . F i g u r e 5 . R e p r e s e n t a t i v e c h r o m a t o g r a m s o f b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t a n d e x t r a c t s p i k e d w i t h OTC a t 2 . 0 ppm 19 6 . F i g u r e 6 . D e t e c t i o n o f o x y t e t r a c y c l i n e i n t h e P a c i f i c O y s t e r s u s p e n d e d i n t h e s a l m o n f a r m . C o n c e n t r a t i o n s a r e t h e mean o f 2 o y s t e r s f r o m e a c h s a m p l i n g p e r i o d 20 7 . F i g u r e 7 . R e p r e s e n t a t i v e c h r o m a t o g r a m s o f b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t a n d e x t r a c t s p i k e d w i t h SDM a n d OMP a t 2 . 0 ppm 22 8 . F i g u r e 8 . D e t e c t i o n o f R o m e t - 3 0 i n t h e P a c i f i c O y s t e r s u s p e n d e d i n t h e s a l m o n f a r m . C o n c e n t r a t i o n s a r e t h e mean o f 2 o y s t e r s a t e a c h s a m p l i n g p e r i o d 23 9 . F i g u r e 9 . R e p r e s e n t a t i v e c h r o m a t o g r a m s o f b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t a n d e x t r a c t s p i k e d w i t h OTC a t 2 . 0 ppm ( S y n d e l ) 38 1 0 . F i g u r e 1 0 . P l o t o f O x y t e t r a c y c l i n e u p t a k e v e r s u s t i m e i n t h e P a c i f i c o y s t e r . E a c h p o i n t r e p r e s e n t s a p o o l e d s a m p l e o f 5 a n i m a l s ( n = 3 ) , e x c e p t w h e r e d e n o t e d b y a * , t h e n n=7 39 1 1 . F i g u r e 1 1 . P l o t o f O x y t e t r a c y c l i n e d e p u r a t i o n v e r s u s t i m e i n t h e P a c i f i c o y s t e r . E a c h p o i n t r e p r e s e n t s a p o o l e d s a m p l e o f 5 a n i m a l s ( n = 3 ) , e x c e p t w h e r e d e n o t e d b y a * , t h e n n=7 39 1 2 . F i g u r e 1 2 . R e p r e s e n t a t i v e c h r o m a t o g r a m o f a b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t a n d a e x t r a c t s p i k e d w i t h SDM a t 2 . 0 ppm ( S y n d e l ) ? . 41 i x 1 3 . F i g u r e 1 3 . P l o t o f R o m e t - 3 0 (SDM a n d OMP) u p t a k e v e r s u s t i m e i n t h e P a c i f i c o y s t e r . E a c h p o i n t r e p r e s e n t s a p o o l e d s a m p l e o f 5 a n i m a l s ( n = 3 ) , e x c e p t w h e r e d e n o t e d b y a * , t h e n n=7 43 1 4 . F i g u r e 1 4 . P l o t o f R o m e t - 3 0 (SDM a n d OMP) d e p u r a t i o n v e r s u s t i m e i n t h e P a c i f i c o y s t e r . E a c h p o i n t r e p r e s e n t s a p o o l e d s a m p l e o f 5 a n i m a l s ( n = 3 ) , e x c e p t w h e r e d e n o t e d b y a * , t h e n n=7 43 1 5 . F i g u r e 1 5 . A r e p r e s e n t a t i v e c h r o m a t o g r a m o f a b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t a n d a e x t r a c t s p i k e d w i t h 2 . 0 ppm o f OMP ( S y n d e l ) 44 1 6 . F i g u r e 1 6 . H i s t o g r a m o f P a c i f i c o y s t e r c o n d i t i o n i n d e x f o r p r e a n d p o s t a n t i b i o t i c t r e a t m e n t 47 X A c k n o w l e d g e m e n t s A . F l e t c h e r , D . S a x b y , T e c h n i c i a n s a t H a r d y S e a f a r m s , D e p a r t m e n t o f O c e a n o g r a p h y , U B C , * R . A o y a m a , * J . W a l i s s e r a n d * D r . K . M c E r l a n e ( * F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s , UBC) w e r e p a r a m o u n t t o t h e c o m p l e t i o n a n d d e v e l o p m e n t o f t h e S a l m o n F a r m S t u d y . T h e c o n t r o l l e d s t u d y i n v e s t i g a t i n g a n t i b i o t i c u p t a k e a n d c l e a r a n c e i n t h e P a c i f i c o y s t e r was made p o s s i b l e b y a c o n t r a c t a w a r d e d t o S y n d e l L a b o r a t o r i e s b y t h e B . C . M i n i s t r y o f A g r i c u l t u r e a n d F i s h e r i e s . A g r a t e f u l a c k n o w l e d g e m e n t i s made o f t h e c o o p e r a t i v e w o r k d e m o n s t r a t e d b y M . L i t t l e ( S y n d e l ) a n d E . B l a c k ( B . C . M . A . F ) i n t h e d e v e l o p m e n t a n d f r u i t i o n o f t h i s s t u d y . G . O h a r a ( S y n d e l ) was r e s p o n s i b l e f o r a l l o f t h e R o m e t - 3 0 e x t r a c t i o n s f o r t h i s p o r t i o n o f t h e s t u d y , h e r e f f o r t was v e r y much a p p r e c i a t e d . T h e s u p p o r t a n d c r e a t i v e e n e r g y p r o v i d e d b y t h e s u p e r v i s i n g s c i e n t i s t , D r . G . K . Iwama was e s s e n t i a l t o t h i s w o r k . T h e a s s i s t a n c e o f N . B e r n i e r a n d J . M c G e e r i s t h o u g h t f u l l y a c k n o w l e d g e d . A g r a t e f u l a c k n o w l e d g m e n t i s made o f t h e s u p p o r t i v e g u i d a n c e p r o v i d e d b y t h e R e s e a r c h C o m m i t t e e m e m b e r s . T o my f a m i l y , who a l w a y s p r o v i d e d me w i t h a c o n s t a n t s e n s e o f h u m o u r , e n c o u r a g e m e n t a n d a b o a t l o a d f u l l o f p a t i e n c e . T h a n k y o u . 1 G E N E R A L INTRODUCTION T h e u s e o f a n t i b i o t i c s i n t h e a q u a c u l t u r e i n d u s t r y f o r t h e c o n t r o l o f b a c t e r i a l i n f e c t i o n s i n s a l m o n h a s l e d t o p u b l i c c o n c e r n r e g a r d i n g a n t i b i o t i c r e s i d u e s i n s a l m o n t i s s u e a n d t h e r e s u l t i n g p o t e n t i a l h e a l t h r i s k t h i s p o s e s t o h u m a n s . Due t o t h e n a t u r e o f s a l m o n c u l t u r e i n B r i t i s h C o l u m b i a ( B . C . ) many o f t h e n e t p e n s y s t e m s a r e l o c a t e d w i t h i n c l o s e p r o x i m i t y t o e x i s t i n g s h e l l f i s h s t o c k s . A common m e t h o d o f d r u g d e l i v e r y u t i l i z e d a t a s a l m o n f a r m s i t e i s t h e u s e o f p r e m e d i c a t e d f e e d . T h i s p r a c t i c e h a s b e e n t h e f o c u s o f much a t t e n t i o n m a i n l y p r o m p t e d b y t h e f a c t t h a t t h e d e s t i n y o f many a n t h r o p o g e n i c s u b s t a n c e s i n t h e m a r i n e e c o s y s t e m i s n o t w e l l u n d e r s t o o d . T h e p o t e n t i a l f o r c h e m o t h e r a p e u t a n t s t o e v e n t u a l l y b i o a c c u m u l a t e i n s h e l l f i s h i s t h e f o c u s i n t h i s s t u d y . I n r e c e n t s t u d i e s , l e v e l s o f o x y t e t r a c y c l i n e w e r e d e t e c t e d i n t h e s e d i m e n t f o r u p t o n i n e t y d a y s p o s t t r e a t m e n t a t a s a l m o n c u l t u r e s i t e ( S a m u e l s e n e t a l . , 1 9 8 8 ; J a c o b s e n a n d B e r g l i n d , 1 9 8 8 ) . O x y t e t r a c y c l i n e a n d R o m e t - 3 0 R a r e two a n t i b i o t i c s t h a t h a v e b e e n a p p r o v e d f o r u s e i n t h e a q u a c u l t u r e i n d u s t r y i n B . C . O x y t e t r a c y c l i n e h a s b e e n u s e d a s a n a n t i b a c t e r i a l a g e n t f o r o v e r 25 y e a r s ( J a c o b s e n , 1 9 8 9 ) , i t i s a w i d e s p e c t r u m a n t i b i o t i c e f f e c t i v e a g a i n s t b o t h g r a m p o s i t i v e a n d g r a m n e g a t i v e b a c t e r i a . I t i s w a t e r s o l u b l e a n d i s q u i c k l y d i s t r i b u t e d t h r o u g h o u t t h e t i s s u e s a n d b o d i l y f l u i d s o f f i s h when a d m i n i s t e r e d o r a l l y ( H e r w i g , 1 9 7 9 ) . O x y t e t r a c y c l i n e h a s b e e n u s e d i n t h e t r e a t m e n t o f F u r u n c u l o s i s , V i b r i o s i s , 2 E n t e r i c r e d m o u t h d i s e a s e a n d C o l u m n a r i s d i s e a s e . R o m e t - 3 0 i s a p o t e n t i a t e d s u l p h o n a m i d e c o n t a i n i n g s u l p h a d i m e t h o x i n e a n d o r m e t o p r i m i n a 5 :1 r a t i o . T h i s a n t i b i o t i c i s l i p i d s o l u b l e a n d i s e f f e c t i v e a g a i n s t many b a c t e r i a l p a t h o g e n s o f w h i c h a r e i n c l u d e d V i b r i o o r d a l i a n d V i b r i o a n g u i l l a r u m , t h e c a u s a t i v e a g e n t s o f V i b r i o s i s , a d i s e a s e common t o f a r m e d r a i s e d s a l m o n ( B r o w n a n d G r a t z e k , 1 9 8 0 ) . T h e o b j e c t i v e i n t h i s s t u d y was t o d e t e r m i n e t h e e x t e n t o f a n t i b i o t i c a c c u m u l a t i o n i n t h e P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s ( T h u n b e r g ) s u s p e n d e d i n a s a l m o n n e t p e n s y s t e m a n d i n a c o n t r o l l e d e x p e r i m e n t t o d e t e r m i n e t h e u p t a k e a n d c l e a r a n c e r a t e s o f o x y t e t r a c y c l i n e a n d R o m e t - 3 0 . T o a c h i e v e t h i s , m o d i f i c a t i o n o f e x i s t i n g a n t i b i o t i c e x t r a c t i o n m e t h o d s f o r s a l m o n i d s w e r e n e c e s s a r y t o d e t e c t d r u g r e s i d u e s i n w h o l e o y s t e r t i s s u e . A p a s t s t u d y f o c u s e d o n t h e e f f e c t s o f i n t e n s i v e f i s h c u l t u r e o n o y s t e r p e r f o r m a n c e ( J o n e s a n d Iwama, I n p r e s s ) . S a m p l e s c o l l e c t e d d u r i n g t h a t s t u d y p e r i o d w e r e u t i l i z e d t o c o n f i r m t h e s u c c e s s o f e x t r a c t i o n m e t h o d s d e v e l o p e d h e r e . T h e d e t e c t i o n o f a n t i b i o t i c r e s i d u e s i n t h e s e o y s t e r s c o m p r i s e d t h e f i e l d e x p e r i m e n t s f o r t h i s s t u d y . V a r i o u s m e t h o d s f o r t h e a n a l y s i s o f a n t i b i o t i c s i n f i s h t i s s u e h a v e b e e n r e p o r t e d . Two b i o a s s a y s h a v e b e e n r e p o r t e d f o r t h e d e t e r m i n a t i o n o f t r i m e t h o p r i m i n t h e t i s s u e s o f t r o u t ( M c C a r t h y e t a l . , 1 9 7 4 ; M c C r a c k e n e t a l . , 1 9 7 6 ) . M i c r o b i o l o g i c a l m e t h o d s o f t e n l a c k t h e s e n s i t i v i t y r e q u i r e d t o q u a n t i f y a n t i b i o t i c c o n c e n t r a t i o n s i n f i s h t i s s u e a n d a r e 3 u s u a l l y n o n s p e c i f i c . T h e B r a t t o n - M a r s h a l l , ( B r a t t o n a n d M a r s h a l l , 1939) c o l o u r i m e t r i c a s s a y h a s b e e n u s e d e x t e n s i v e l y t o m e a s u r e s u l p h a d i m e t h o x i n e r e s i d u e l e v e l s i n t h e b l o o d a n d t i s s u e s o f d i f f e r e n t t r o u t s p e c i e s ( H a r a a n d I n o u e , 1 9 6 7 ; H a r a e t a l . , 1 9 6 7 ; S n i e s z k o a n d F r i d d l e , 1 9 5 1 ) . C h r o m a t o g r a p h i c m e t h o d s o f f e r t h e a d v a n t a g e s o f s p e c i f i c i t y a n d i n c r e a s e d s e n s i t i v i t y o v e r many o t h e r a n a l y t i c a l p r o c e d u r e s . T r i m e t h o p r i m c o n c e n t r a t i o n s i n s k i n , m u s c l e a n d b l o o d o f r a i n b o w t r o u t h a v e b e e n s u c c e s s f u l l y m e a s u r e d b y H P L C ( J a c o b s e n , 1 9 8 9 ) . O x y t e t r a c y c l i n e c o n c e n t r a t i o n s h a v e a l s o b e e n s u c c e s s f u l l y m e a s u r e d b y H P L C u s i n g s o l i d p h a s e e x t r a c t i o n t e c h n i q u e s i n r a i n b o w t r o u t a n d A t l a n t i c s a l m o n ( R o g s t a d e t a l . , 1 9 8 8 ; J a c o b s e n , 1 9 8 9 ) . R o m e t - 3 0 c o n c e n t r a t i o n s h a v e r e c e n t l y b e e n m e a s u r e d i n P a c i f i c s a l m o n b y H P L C u s i n g a s o l i d p h a s e e x t r a c t i o n t e c h n i q u e ( W a l l i s e r e t a l . , 1 9 9 0 ) . T o d a t e t h e r e h a v e n o t b e e n a n y m e t h o d s r e p o r t e d f o r t h e a n a l y s i s o f a n t i b i o t i c s i n o y s t e r t i s s u e u s i n g H P L C . T h e t e c h n i q u e s p o r t i o n o f t h i s s t u d y a t t e m p t e d t o d e v e l o p a s o l i d p h a s e e x t r a c t i o n t e c h n i q u e f o r O T C , SDM a n d OMP f r o m w h o l e o y s t e r t i s s u e . T h e m e t h o d s d e s c r i b e d h e r e w e r e m o d i f i e d f r o m e x t r a c t i o n s s u c c e s s f u l l y d e v e l o p e d a n d p e r f o r m e d o n s a l m o n t i s s u e . I n s h e l l f i s h , i n v e s t i g a t i o n s i n t o t h e u p t a k e a n d c l e a r a n c e o f a r a n g e o f e n v i r o n m e n t a l c o n t a m i n a n t s h a v e p r i n c i p a l l y f o c u s e d o n t h e common m u s s e l M y t i l u s e d u l i s ( F o n g , 1 9 7 6 ; F o s s a t o a n d C a n z o n i e r , 1976 ; F r i e d r i c h a n d F i l i c e , 1 9 7 6 ; S i m p s o n , 1 9 7 9 ) . U p t a k e a n d c l e a r a n c e r a t e s f o r 4 a v a r i e t y o f c h e m i c a l s i n f i n f i s h h a v e a l s o b e e n d e t e r m i n e d ( K e c k e t a l . f 1 9 8 4 ; S a l t e a n d L i e s t o l , 1 9 8 3 ) . T h e u p t a k e a n d c l e a r a n c e r a t e s o f a n t i b i o t i c c h e m i c a l s i n s h e l l f i s h h a v e n o t b e e n p r e v i o u s l y d e t e r m i n e d . T h i s n e c e s s i t a t e d t h e n e e d f o r a c o n t r o l l e d d r u g u p t a k e a n d c l e a r a n c e s t u d y f o r s h e l l f i s h . 5 Chapter 1: The Development of Extrac t ion Methods for Oxytetracyc l ine and Romet-30 i n the P a c i f i c Oyster and the Detection of A n t i b i o t i c Residues i n P a c i f i c Oysters Suspended i n Salmon Net-Cages Through HPLC. Introduct ion I t h a s b e e n n e c e s s a r y f o r t h e s a l m o n f a r m i n g i n d u s t r y i n B r i t i s h C o l u m b i a t o p e r i o d i c a l l y t r e a t f i s h w i t h a n t i b i o t i c s t o c o n t r o l b a c t e r i a l d i s e a s e s . T h e u s e o f o x y t e t r a c y c l i n e a n d R o m e t - 3 0 a s a n t i b a c t e r i a l a g e n t s i n f i n f i s h c u l t u r e h a v e p r o v e n t o b e t h e m o s t w i d e l y i m p l e m e n t e d ( L i t t l e , 1 9 9 0 ) . A r e s u l t a n t e f f e c t o f w i d e s p r e a d u s e o f c h e m o t h e r a p e u t i c s i n t h e m a r i n e e n v i r o n m e n t h a s b e e n t h e d e v e l o p m e n t o f r e s i s t a n c e s t r a i n s o f b a c t e r i a a s s o c i a t e d w i t h t h e s e d i m e n t a n d i n some i n s t a n c e s t h e g u t o f b i v a l v e s . S t r a i n s r e s i s t a n t t o o x y t e t r a c y c l i n e a n d R o m e t - 3 0 h a v e b e e n i d e n t i f i e d f r o m t h e s t o m a c h c o n t e n t s o f t h e P a c i f i c o y s t e r ( K e l l y , 1 9 9 0 ) . C o r r e l a t i o n s b e t w e e n t h e u s e o f d r u g s a n d t h e f r e q u e n c y o f r e s i s t a n t b a c t e r i a i s o l a t e d f r o m f i s h h a v e a l s o b e e n r e p o r t e d ( A o k i e t a l . f 1 9 8 1 ; T a k a s h i m a e t a l . , 1 9 8 5 ) . I n r e s p o n s e t o t h e s e n e g a t i v e s i d e e f f e c t s , t h e n e e d f o r t h e d e v e l o p m e n t o f a s i m p l e , r a p i d e x t r a c t i o n m e t h o d f o r a n t i b i o t i c s i n P a c i f i c o y s t e r s , C r a s s o s t r e a g i g a s , was p r o p o s e d . O x y t e t r a c y c l i n e c o n c e n t r a t i o n s h a v e b e e n s u c c e s s f u l l y m e a s u r e d b y H P L C u s i n g s o l i d p h a s e e x t r a c t i o n t e c h n i q u e s i n r a i n b o w t r o u t a n d A t l a n t i c s a l m o n ( R o g s t a d e t 6 a l . , 1988 ; J a c o b s e n , 1 9 8 9 ) . R o m e t - 3 0 c o n c e n t r a t i o n s h a v e a l s o r e c e n t l y b e e n m e a s u r e d i n P a c i f i c s a l m o n b y H P L C u s i n g a s o l i d p h a s e e x t r a c t i o n t e c h n i q u e ( W a l l i s e r e t a l . , 1 9 9 0 ) . T o d a t e t h e r e h a v e n o t b e e n a n y m e t h o d s r e p o r t e d f o r t h e a n a l y s i s o f R o m e t - 3 0 a n d o x y t e t r a c y c l i n e i n o y s t e r t i s s u e u s i n g H P L C . A l i m i t e d number o f o y s t e r s p r e v i o u s l y s u s p e n d e d i n a s a l m o n f a r m n e t - p e n ( J o n e s a n d Iwama, 1990) w e r e o b t a i n e d . T r e a t m e n t w i t h a n t i b i o t i c s h a d o c c u r r e d a t t h e f a r m s i t e a n d t h e t r e a t m e n t s c h e d u l e h a d b e e n r e c o r d e d . T h e s e s a m p l e s a l l o w e d f o r s u c c e s s f u l c o n f i r m a t i o n o f t h e d e v e l o p e d e x t r a c t i o n t e c h n i q u e s . 7 M e t h o d s a n d M a t e r i a l s S o l i d p h a s e e x t r a c t i o n t e c h n i q u e s f o r o x y t e t r a c y c l i n e a n d R o m e t - 3 0 w e r e m o d i f i e d f r o m e x i s t i n g m e t h o d s d e v e l o p e d f o r s a l m o n i d s i n t h e F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s , U . B . C . ( W a l i s s e r e t a l . 1990) O x y t e t r a c y c l i n e (OTC) O x y t e t r a c y c l i n e was o b t a i n e d f r o m H o f f m a n n - L a R o c h e L t d . T h e e q u i p m e n t u t i l i z e d was a Beckman s y s t e m w h i c h i n c l u d e d : H P L C pump, m o d e l 110A s o l v e n t m e t e r i n g s y s t e m ; a m o d e l 160 d e t e c t o r ; a m o d e l 210 s a m p l e i n j e c t i o n v a l v e e q u i p p e d w i t h a 20 u L l o o p . U l t r a v i o l e t d e t e c t i o n was s e t a t 365 nm f o r a l l a n a l y s e s . T h e m o b i l e p h a s e c o n s i s t e d o f 0 . 0 2 M N a 2 H P 0 4 pH 2 . 2 5 a n d 25% MeOH. T h e H P L C c o l u m n was a 250 mm x 4 . 6 mm I . D . U l t r a s p h e r e I o n - P a i r 5 urn c o l u m n . F i l t r a t i o n o f t h e m o b i l e p h a s e p r i o r t o u s e was d o n e u s i n g a H P L C s o l v e n t c l e a n - u p a s s e m b l y a n d F P V e r i c e l 47 mm, 0 . 4 5 urn membrane f i l t e r s . P e a k i n t e g r a t i o n s w e r e c a l c u l a t e d m a n u a l l y . E x t r a c t i o n P r o c e d u r e I n d i v i d u a l o y s t e r s a m p l e s w e r e t h a w e d a t r o o m t e m p e r a t u r e a n d t h e t i s s u e r e m o v e d a s e p t i c a l l y a n d t o w e l l d r i e d . W h o l e a n i m a l s a m p l e s w e r e g r o u n d i n a b l e n d e r a n d a 5 . 0 g s u b s a m p l e o f t h e s l u r r y was r e m o v e d . T o e a c h 5 . 0 g s a m p l e w e r e a d d e d 1 5 . 0 mL p h o s p h a t e b u f f e r a n d 1 .0 mL 50% T C A ( t r i c h l o r o a c e t i c a c i d ) . E a c h s a m p l e was h o m o g e n i z e d i n a 5 0 . 0 mL p l a s t i c c e n t r i f u g e t u b e f o r t h i r t y s e c o n d s a t medium 8 s p e e d u s i n g a B r i n k r a a n P o l y t r o n h o m o g e n i z e r M o d e l PT 1 0 / 3 5 . T h e s a m p l e s w e r e c e n t r i f u g e d f o r t w e n t y f i v e m i n u t e s a t 7800 g a n d 4 . 0 \u00C2\u00B0 C ( J A - 2 0 r o t o r ) . T h e s u p e r n a t a n t was t r a n s f e r r e d t o a 5 0 . 0 mL t u b e , a n d a n o t h e r 1 5 . 0 mL p h o s p h a t e b u f f e r a n d 1 .0 mL T C A a d d e d t o t h e r e m a i n i n g r e s i d u e . T h e s a m p l e was h o m o g e n i z e d a n d c e n t r i f u g e d a g a i n u n d e r t h e same c o n d i t i o n s , t h e s u p e r n a t a n t was t r a n s f e r r e d a n d c o m b i n e d w i t h t h e f i r s t a l i q u o t . T h e c o m b i n e d e x t r a c t s w e r e t h e n p a s s e d t h r o u g h a n a c t i v a t e d S e p - P a k C 1 8 c a r t r i d g e . A c t i v a t i o n o f t h e S e p - P a k c a r t r i d g e p r i o r t o l o a d i n g t h e s a m p l e was a c c o m p l i s h e d b y p a s s i n g 5 . 0 mL o f m e t h a n o l f o l l o w e d by 5 . 0 mL o f w a t e r . 7 . 0 mL o f m e t h a n o l was u s e d t o e l u t e t h e C-*- 8 c o l u m n a n d t h e e l u a t e s c o l l e c t e d . T h e e l u a t e s w e r e e v a p o r a t e d u n d e r n i t r o g e n i n a 4 0 . 0 \u00C2\u00B0 C w a t e r b a t h . T h e r e s i d u e was r e c o n s t i t u t e d i n 1 .0 mL o f m e t h a n o l . Romet 30 (SDM & OMP) S u l p h a d i m e t h o x i n e (SDM) a n d o r m e t o p r i m (OMP) w e r e o b t a i n e d f r o m H o f f m a n - L a R o c h e L t d . E q u i p m e n t u t i l i z e d was a Beckman s y s t e m w h i c h i n c l u d e d : H P L C pump, m o d e l 110A s o l v e n t m e t e r i n g s y s t e m ; a m o d e l 210 s a m p l e i n j e c t i o n v a l v e e q u i p p e d w i t h a 20 u l l o o p , a H i t a c h i M o d e l 155 v a r i a b l e w a v e l e n g t h d e t e c t o r w i t h a S h i m a d z u C - R I A C h r o m a t o p a c d a t a p r o c e s s o r . U l t r a v i o l e t d e t e c t i o n was s e t a t 280 nm f o r a l l a n a l y s e s . T h e H P L C c o l u m n was a 250 mm x 4 . 6 mm I . D . U l t r a s p h e r e I o n -P a i r 5 um c o l u m n . T h e m o b i l e p h a s e , a c e t o n i t r i l e / m e t h a n o l / 0 . 1 M p h o s p h a t e b u f f e r , pH 4 . 0 , was d e l i v e r e d i s o c r a t i c a l l y 9 a t 1 . 0 l a L / m i n . F i l t r a t i o n o f t h e m o b i l e p h a s e p r i o r t o u s e was d o n e u s i n g a H P L C s o l v e n t c l e a n - u p a s s e m b l y a n d F P V e r i c e l 47 mm, 0 . 4 5 urn membrane f i l t e r s . E x t r a c t i o n P r o c e d u r e T h i s p r o c e d u r e h a s b e e n m o d i f i e d f r o m W a l i s s e r e t a l . ( 1 9 9 0 ) . I n d i v i d u a l o y s t e r s a m p l e s w e r e t h a w e d a t r o o m t e m p e r a t u r e a n d t h e t i s s u e r e m o v e d a s e p t i c a l l y a n d t o w e l l d r i e d . W h o l e a n i m a l s a m p l e s w e r e g r o u n d up i n a b l e n d e r a n d a 5 . 0 g s u b s a m p l e o f t h e s l u r r y was r e m o v e d . T o e a c h 5 . 0 g s a m p l e w e r e a d d e d 1 5 . 0 mL a c e t o n i t r i l e a n d 0 . 5 mL 50% T C A . E a c h s a m p l e was h o m o g e n i z e d i n a 50 mL p l a s t i c c e n t r i f u g e t u b e f o r 30 s e c o n d s a t medium s p e e d u s i n g a B r i n k m a n P o l y t r o n h o m o g e n i z e r M o d e l PT 1 0 / 3 5 . T h e s a m p l e s w e r e c e n t r i f u g e d ( J A - 2 0 r o t o r ) f o r 25 m i n u t e s a t 7800 g a n d 4 . 0 \u00C2\u00B0 C . T h e s u p e r n a t a n t was t r a n s f e r r e d t o a 5 0 . 0 mL t u b e , a n d e v a p o r a t e d u n d e r n i t r o g e n i n a 4 0 . 0 \u00C2\u00B0 C w a t e r b a t h . T h e r e s i d u e was r e c o n s t i t u t e d i n 5 . 0 mL o f d i s t i l l e d w a t e r a n d v o r t e x - m i x e d f o r 30 s e c o n d s . T h e e x t r a c t s w e r e p a s s e d d i r e c t l y i n t o a 5 . 0 mL L u e r - l o k M u l t i f i t B - D g l a s s s y r i n g e w i t h a n a c t i v a t e d S e p - P a k C 1 8 c a r t r i d g e ( W a t e r s A s s o c i a t e s ) a t t a c h e d . A c t i v a t i o n o f t h e S e p - P a k c a r t r i d g e p r i o r t o l o a d i n g t h e s a m p l e was a c c o m p l i s h e d b y p a s s i n g 5 . 0 mL o f m e t h a n o l f o l l o w e d b y 5 . 0 mL o f w a t e r . T h e e x t r a c t s w e r e t h e n p a s s e d t h r o u g h t h e S e p - P a k c a r t r i d g e a n d t h e e l u e n t was d i s c a r d e d . SDM a n d OMP w e r e s u b s e q u e n t l y e l u t e d f r o m t h e c a r t r i d g e i n t o a 1 5 . 0 mL c u l t u r e t u b e w i t h 5 . 0 mL o f 10 m e t h a n o l . S a m p l e s w e r e d r i e d u n d e r n i t r o g e n i n a w a t e r b a t h a t 40 \u00C2\u00B0 C a n d s t o r e d a t - 2 0 \u00C2\u00B0 C u n t i l r e q u i r e d f o r a n a l y s i s . J u s t p r i o r t o a n a l y s i s e a c h s a m p l e was r e c o n s t i t u t e d w i t h 1 .0 mL o f t h e m o b i l e p h a s e a n d v o r t e x m i x e d f o r t h i r t y s e c o n d s . A 2 0 . 0 u L a l i q u o t was i n j e c t e d o n t o t h e H P L C c o l u m n . T h e i n j e c t i o n v a l v e was f l u s h e d w i t h 1 . 0 mL m o b i l e p h a s e b e t w e e n e a c h a n a l y s i s . C a l i b r a t i o n C u r v e a n d R e c o v e r y A c a l i b r a t i o n c u r v e was p r e p a r e d f r o m o y s t e r t i s s u e s a m p l e s (5g) s p i k e d w i t h 1 .0 mL o f t h e a p p r o p r i a t e SDM/OMP, OTC s t a n d a r d s o l u t i o n s t o g i v e f i n a l c o n c e n t r a t i o n s o f 0 . 2 , 0 . 4 , 0 . 5 , 0 . 7 , 1 . 0 , 2 . 0 , 5 . 0 a n d 1 0 . 0 u g / g o f t i s s u e f o r Romet a n d 0 . 2 5 , 0 . 5 , 1 . 5 , 2 . 5 a n d 5 . 0 u g / g o f t i s s u e f o r O T C . C a l i b r a t i o n c u r v e s w e r e p l o t t e d . T h e r e c o v e r y o f R o m e t - 3 0 a n d OTC f r o m o y s t e r t i s s u e was d e t e r m i n e d b y t h e a d d i t i o n o f 1 . 0 m l o f e a c h o f t h e p r e p a r e d s t a n d a r d s o l u t i o n s c o n t a i n i n g 1 . 2 5 , 2 . 5 , 5 . 0 , 1 0 . 0 , 2 5 . 0 u g o f d r u g t o 5 . 0 g s a m p l e s o f c o n t r o l o y s t e r t i s s u e . T h e s a m p l e s w e r e e x t r a c t e d a n d a n a l y z e d a s d e s c r i b e d a b o v e . R e c o v e r y was d e t e r m i n e d b y c a l c u l a t i n g t h e a r e a o f O T C , SDM a n d OMP f o r e x t r a c t e d t i s s u e s a m p l e s a n d c o m p a r i n g t h e s e t o t h e a r e a s o b t a i n e d f r o m u n e x t r a c t e d s t a n d a r d s o l u t i o n s o f i d e n t i c a l q u a n t i t i e s . 11 F i e l d S t u d y S a m p l e s o f o y s t e r s u t i l i z e d i n t h e d e v e l o p m e n t o f t h e a n t i b i o t i c a s s a y s w e r e o b t a i n e d f r o m a p r e v i o u s s t u d y o n t h e e f f e c t s o f i n t e n s i v e f i s h f a r m i n g o n o y s t e r p e r f o r m a n c e . T h i s s t u d y was p e r f o r m e d i n J e r v i s I n l e t , B . C . s i t u a t e d 110 Km. N . W . o f V a n c o u v e r ( F i g . 1 ) . T h e r e w e r e two a r e a s o f s t u d y ; t h e e x p e r i m e n t a l a r e a a n d t h e c o n t r o l a r e a ( F i g . 2 ) . I n t h e e x p e r i m e n t a l a r e a , 3 s t a t i o n s w e r e s i t u a t e d i n t h e n e t c a g e s a n d 1 s t a t i o n was l o c a t e d a d j a c e n t t o t h e n e t c a g e s . Two f u r t h e r s t a t i o n s w e r e s i t u a t e d 200m f r o m t h e p e n s y s t e m a n d a n o t h e r a t 500m. T h e c o n t r o l a r e a c o n s i s t e d o f 2 c o m m e r c i a l o f f b o t t o m o y s t e r o p e r a t i o n s . One c o n t r o l s t a t i o n , 6 k i l o m e t r e s e a s t o f t h e s a l m o n f a r m , was a n e s t a b l i s h e d o y s t e r l e a s e w i t h e x i s t i n g b a s e l i n e g r o w t h d a t a . T h e o t h e r c o n t r o l , 4 k i l o m e t r e s s o u t h e a s t o f t h e f a r m was a n e w l y e s t a b l i s h e d o p e r a t i o n w h e r e p e r f o r m a n c e was u n k n o w n . L a n t e r n n e t s w e r e s t o c k e d w i t h 2 0 - 1 y e a r o l d o y s t e r s a n d s u s p e n d e d t o a d e p t h o f 2 m. S a m p l e s w e r e c o l l e c t e d e v e r y two weeks o v e r t h e c o u r s e o f f i v e m o n t h s b e g i n n i n g i n J u n e , 1 9 8 9 . T h e a n t i b i o t i c t r e a t m e n t s c h e d u l e a t t h e f a r m s i t e was r e c o r d e d . O x y t e t r a c y c l i n e was a d m i n i s t e r e d f o r a p e r i o d o f t e n d a y s c o m m e n c i n g o n 0 6 / 0 7 / 8 9 a n d e n d i n g o n 1 5 / 0 7 / 8 9 . R o m e t - 3 0 was a d m i n i s t e r e d i n a p r e m e d i c a t e d f e e d f o r t e n d a y s c o m m e n c i n g o n 2 0 / 0 6 / 8 9 a n d e n d i n g o n 3 0 / 0 6 / 8 9 . O y s t e r s w e r e s a m p l e d a t b o t h t h e c o n t r o l a n d e x p e r i m e n t a l s i t e s e v e r y f o u r t e e n d a y s c o m m e n c i n g 0 1 / 0 7 / 8 9 a n d e n d i n g 1 5 / 0 9 / 8 9 . 12 F i g u r e 1 . L o c a t i o n o f s a l m o n f a r m s t u d y s i t e , J e r v i s I n l e t , B . C . 13 Study Site C o n t r o l S i t e s 0 \u00C2\u00A9 4 Kn 6 Kn LI Salmon Farm H 500 m +-\u00C2\u00BB 200 m S t n . l Stn.2 Stn.3 Stn.4 Stn.6 Stn.6 F i g u r e 2 . D e t a i l o f S t u d y s i t e , i n d i c a t i n g p o s i t i o n o f l a n t e r n n e t s i n r e f e r e n c e t o n e t p e n s . 14 S t a t i s t i c s A n a l y s i s o f v a r i a n c e was c o m p u t e d f o r a l l a n t i b i o t i c d a t a c o l l e c t e d a t t h e f a r m s i t e . T u k e y s t e s t s w e r e p e r f o r m e d o n a l l d a t a t o d i s c e r n d i f f e r e n c e s , d i f f e r e n c e s w e r e j u d g e d t o be s i g n i f i c a n t a t t h e 95% l e v e l ( p < 0 . 0 5 ) . 15 R E S U L T S C a l i b r a t i o n c u r v e a n d R e c o v e r y T h e c a l i b r a t i o n c u r v e f o r o x y t e t r a c y c l i n e ( F i g . 3) i n d i c a t e s a l i n e a r r e l a t i o n s h i p ( r 2 = 0 . 9 9 8 ) o v e r t h e d r u g c o n c e n t r a t i o n r a n g e o f 0 . 2 5 , 0 . 5 , 1 . 5 , 2 . 5 a n d 5 . 0 u g / g o f t i s s u e a n d p e a k a r e a . T h e mean r e c o v e r y f o r o x y t e t r a c y c l i n e o v e r t h e same c o n c e n t r a t i o n r a n g e was 9 2 . 4 \u00E2\u0080\u0094 1 .54 s . e . % ( n = 5 ) . C a l i b r a t i o n c u r v e s f o r SDM a n d OMP a r e p r e s e n t e d i n F i g u r e 4 . T h e a r e a r a t i o m e a s u r e m e n t s a p p r o x i m a t e d a l i n e a r r e l a t i o n s h i p f o r OMP ( r 2 = 0 . 9 9 2 ) a n d SDM ( r 2 = 0 . 9 9 0 ) b e t w e e n t h e c o n c e n t r a t i o n r a n g e 0 . 2 - 10 u g / g t i s s u e a n d p e a k a r e a . T h e mean p e r c e n t r e c o v e r y o f SDM a n d OMP o v e r t h e c o n c e n t r a t i o n r a n g e 0 . 5 - 6 . 0 ppm was 4 7 . 8 \u00E2\u0080\u0094 0 . 8 5 s . e . % (n=5) f o r SDM a n d 6 6 . 8 \u00C2\u00B1 0 . 3 9 s . e . % (n=5) f o r OMP. O x y t e t r a c y c l i n e (OTC) A r e p r e s e n t a t i v e c h r o m a t o g r a m o f a n o y s t e r t i s s u e e x t r a c t c o n t a i n i n g OTC i s shown i n F i g u r e 5 . S a m p l e s o f o y s t e r s c o l l e c t e d d u r i n g t h e m o n t h o f J u l y r e v e a l e d d e t e c t a b l e l e v e l s o f O T C . F i g u r e 6 i n d i c a t e s t h e s t a t i o n s a n d s a m p l i n g p e r i o d s a t w h i c h d r u g was i d e n t i f i e d . T h e c o n c e n t r a t i o n o f d r u g i d e n t i f i e d a t s t a t i o n 2 o n 1 5 / 0 7 / 8 9 was 0 . 1 1 ppm ( n = 2 ) . A p p r o x i m a t e l y two weeks l a t e r ( 0 1 / 0 8 / 8 9 ) o y s t e r s s a m p l e d f r o m s t a t i o n 1 a n d 2 w e r e f o u n d t o c o n t a i n OTC a t 0 . 0 8 ppm ( n = 2 ) . O y s t e r s a t s t a t i o n 3 o n t h e same d a t e w e r e d e t e r m i n e d t o c o n t a i n OTC a t 0 . 0 6 ppm a n d a t s t a t i o n 4 o y s t e r s w e r e d e t e c t e d w i t h OTC a t 0 . 0 5 4 ppm. T h e s e l e v e l s o f a n t i b i o t i c 16 i n o y s t e r s c o i n c i d e d w i t h d a y t e n o f t r e a t m e n t t o t h e s a l m o n a t t h e f a r m s i t e a n d f o u r t e e n d a y s p o s t t r e a t m e n t r e s p e c t i v e l y . No d e t e c t a b l e l e v e l s o f OTC w e r e i d e n t i f i e d a t s t a t i o n s 5 a n d 6 o r a t t h e c o n t r o l s i t e s a t a n y t i m e d u r i n g t h e s t u d y p e r i o d . T A B L E 1. RECOVERY OF O T C , OMP AND SDM FROM WHOLE P A C I F I C OYSTER T I S S U E ( V a l u e s r e p r e s e n t a s i n g l e d e t e r m i n a t i o n ) S a m p l e OTC OMP SDM C o n c e n t r a t i o n % R e c o v e r y % R e c o v e r y % R e c o v e r y (ppm) 0 . 2 5 0 . 5 1 .0 2 . 0 5 . 0 8 0 . 2 9 0 . 1 9 5 . 0 9 8 . 3 9 8 . 4 6 4 . 0 7 8 . 0 . 9 6 . 0 9 6 . 1 0 4 8 . 0 5 0 . 0 6 4 . 0 7 5 . 2 0 Mean R e c o v e r y 9 2 . 4 6 6 . 8 4 7 . 4 17 CALIBRATION CURVE FOR OXYTETRACYL1NE IN THE PACIFIC OYSTER DRUG CONCENTRATION (ppm) Figure 3. C a l i b r a t i o n curve for oxyte tracyc l ine i n the P a c i f i c Oyster . Each data po int represents a s i n g l e determination at each concentrat ion of drug. ( r 2 = 0.998) 18 CALIBRATION CURVE FOR ROMET-30 IN THE PACIFIC OYSTER CONCENTRATION OF DRUG (ppm) Figure 4. C a l i b r a t i o n curve for Romet-30 i n the P a c i f i c Oyster . Each data point represents a s ing l e determination at each concentrat ion of drug. (OMP, r 2 = 0.992; SDM, r 2 = 0.990) 19 i V F i g u r e 5 . R e p r e s e n t a t i v e c h r o m a t o g r a m s o i b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t a n d e x t r a c t s p i k e d w i t h OTC a t 2.0 p p m . 20 SAMPLING DATES 1983: A-15/07 DRUG: OXYTETRACYCLINE ( n = E ) -B-01/08 C-15/08 B B B B PPM _ . 100 050 A 500 +-\u00E2\u0080\u0094200 Stn . l Stn.2 Stn.3 Stn.4 Stn.5 Stn.B Controls Figure 6. Detect ion of oxyte tracyc l ine i n the P a c i f i c oyster suspended i n the salmon farm. Concentrations are the mean of 2 oysters from each sampling p e r i o d . 21 Romet-30 S u l p h a d i m e t h o x i n e (SDM) F i g u r e 7 i s a r e p r e s e n t a t i v e c h r o m a t o g r a m o f R o m e t - 3 0 s p i k e d o y s t e r t i s s u e . F i g u r e ' 8 i n d i c a t e s t h e s t a t i o n s a n d s a m p l i n g p e r i o d s a t w h i c h R o m e t - 3 0 was d e t e c t e d i n o y s t e r s . A maximum c o n c e n t r a t i o n o f 1 . 2 7 ppm was d e t e c t e d f o r SDM a t s t a t i o n 4 o n 0 1 / 0 7 / 8 9 . SDM was s t i l l p r e s e n t i n r e l a t i v e l y h i g h c o n c e n t r a t i o n s i n t h e o y s t e r s (n=2) s a m p l e d two weeks l a t e r ( 1 5 / 0 7 / 8 9 ) . F o u r weeks p o s t t r e a t m e n t r e v e a l e d s i g n i f i c a n t l e v e l s o f SDM i n t h e o y s t e r s s a m p l e d f r o m s t a t i o n 4 ( 1 . 2 3 p p m ) . O r m e t o p r i m (OMP) OMP i n c o m p a r i s o n was d e t e c t e d i n s m a l l c o n c e n t r a t i o n s i n t h e o y s t e r s t h r o u g h o u t t h e s t u d y ( F i g u r e 8 ) . O y s t e r s s a m p l e d a t s t a t i o n 4 o n J u l y 1 r e v e a l e d l e v e l s o f OMP o f 0 . 5 ppm ( n = 2 ) . On J u l y 15 o y s t e r s s a m p l e d f r o m s t a t i o n 5 h a d d e t e c t a b l e l e v e l s o f OMP o f 0 . 2 6 ppm ( n = 2 ) . F o u r weeks p o s t t r e a t m e n t r e v e a l e d o n l y t r a c e c o n c e n t r a t i o n s o f t h e d r u g i n o y s t e r s f r o m S t a t i o n 1 ( 0 . 1 0 p p m ) . 22 BL\u00C2\u00A3NK EXTRAZT F i g u r e 7 . R e p r e s e n t a t i v e c h r o m a t o g r a m s o f b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t s a n d e x t r a c t s p i k e d w i t h SDM a n d OMP a t 2 . 0 p p m . 23 SAMPLING DATES 1989: A-01/07 B-15/07 DRUG: ROMET-30 ( OMP SDM ) ( n = 2 ) B C B C- 01/08 PPM 1 .00 -f- 0.50 A 500 -H 200 m Stn . l Stn.2 Stn.3 Stn.4 Stn.5 Stn.6 Contro ls Figure 8. Detection of Romet-30 i n the p a c i f i c oyster suspended i n the salmon farm. Concentrations are the mean of 2 oysters from each sampling p e r i o d . 24 D I S C U S S I O N T h e s o l i d p h a s e e x t r a c t i o n t e c h n i q u e f o r OTC was m o d i f i e d f r o m a m e t h o d d e v e l o p e d f o r s a l m o n i d s a t t h e F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s , U n i v e r s i t y o f B r i t i s h C o l u m b i a . T h e m e t h o d p r o v e d t o be r a p i d a n d r e p e a t a b l e . R e c o v e r y o f a n t i b i o t i c f r o m s p i k e d o y s t e r t i s s u e was g r e a t e r t h a n 9 2 . 0 % . . D u r i n g t h e d e v e l o p m e n t o f t h e e x t r a c t i o n a s m a l l i m m i s c i b l e l a y e r w o u l d f o r m a f t e r t h e s a m p l e was p a s s e d t h r o u g h t h e s e p p a k c a r t r i d g e . T h i s l a y e r was b e l i e v e d t o b e a c o m b i n a t i o n o f p i g m e n t a n d l i p i d n a t u r a l l y f o u n d w i t h i n t h e o y s t e r t i s s u e . P u r i f i c a t i o n o f t h e e l u e n t by c h a r c o a l f i l t r a t i o n p r o v e d u n s u c c e s s f u l . T h e i m m i s c i b l e l a y e r was i n j e c t e d a l o n e o n t h e HPLC t o d e t e r m i n e i f t h i s l a y e r c o n t a i n e d a n y a n t i b i o t i c r e s i d u e s . No p e a k s w e r e i d e n t i f i e d a n d r e s u l t a n t l y t h e r e m o v a l o f t h i s l a y e r f r o m t h e r e m a i n i n g e x t r a c t i o n s o c c u r r e d . T h i s g r e a t l y i m p r o v e d t h e c l e a n l i n e s s o f t h e c h r o m a t o g r a m r e d u c i n g t h e i n t e r f e r e n c e c o n s i d e r a b l y . T h e e x t r a c t i o n f o r R o m e t - 3 0 was m o d i f i e d f r o m t h e m e t h o d d e v e l o p e d b y W a l l i s e r e t a l . (1990) f o r P a c i f i c s a l m o n . T h i s e x t r a c t i o n a l l o w e d f o r t h e s i m u l t a n e o u s q u a n t i f i c a t i o n o f s u l p h a d i m e t h o x i n e a n d o r m e t o p r i m i n w h o l e P a c i f i c o y s t e r t i s s u e . T h e e x t r a c t i o n e f f i c i e n c i e s f o r b o t h SDM a n d OMP f r o m o y s t e r t i s s u e a r e c o m p a r a b l e t o t h o s e r e p o r t e d f o r s a l m o n t i s s u e ( W a l l i s e r e t a l . 1 9 9 0 ) . T h e p r e s e n c e o f p i g m e n t s a n d l i p i d s a s s o c i a t e d w i t h t h e o y s t e r t i s s u e may be r e s p o n s i b l e f o r r e l a t i v e l y l o w e r r e c o v e r i e s . 25 I t h a s b e e n p r e v i o u s l y d o c u m e n t e d t h a t i n c r e a s e s i n t o t a l l i p i d c o n t e n t o f t i s s u e may a d v e r s e l y a f f e c t a n t i b i o t i c e x t r a c t i o n e f f i c i e n c i e s ( W a l l i s e r e t a l . f 1 9 9 0 . T h e minimum q u a n t i f i a b l e c o n c e n t r a t i o n o f R o m e t - 3 0 d e t e c t e d i n w h o l e o y s t e r t i s s u e was 0 . 1 0 ppm. T h i s m e t h o d was d e t e r m i n e d t o s u c c e s s f u l l y q u a n t i f y c o n c e n t r a t i o n s o f b o t h SDM a n d OMP t h r o u g h a s i n g l e e x t r a c t i o n . Many s t u d i e s h a v e d e m o n s t r a t e d t h a t s u s p e n s i o n f e e d e r s i n t h e m a r i n e e n v i r o n m e n t c o n c e n t r a t e a w i d e r a n g e o f a n t h r o p o g e n i c s u b s t a n c e s ( S t e n n e r a n d N i c k l e s s , 1 9 7 4 ; P h i l l i p s , 1976 ; P r i n g l e e t a l . , 1 9 6 8 ; B o y d e n , 1 9 7 7 ; B r o o k s a n d R u m s b y , 1 9 6 5 ) . B a s e d o n t h e c o n c e n t r a t i o n s o f o x y t e t r a c y c l i n e i n t h e o y s t e r s s a m p l e d a t t h e f a r m s i t e i t w o u l d a p p e a r t h a t u p t a k e o f t h e d r u g was q u i t e r a p i d i n r e s p o n s e t o t h e a d m i n i s t r a t i o n o f t h e p r e m e d i c a t e d f e e d t o t h e s a l m o n . T r e a t m e n t a t m o s t s a l m o n f a r m s i t e s t y p i c a l l y l a s t s f o r a p e r i o d o f t e n d a y s b u t may v a r y d e p e n d i n g o n t y p e o f d r u g a n d s p e c i e s o f f i s h b e i n g t r e a t e d . J u l y 1 5 , 1989 c o i n c i d e d w i t h d a y 3 o f t r e a t m e n t a t t h e f a r m s i t e , t h e h i g h e s t c o n c e n t r a t i o n ( O . l l p p m ) o f o x y t e t r a c y c l i n e was d e t e c t e d a t s t a t i o n 2 o n t h e f a r m s i t e ( r e f e r t o F i g . 6 ) . S t a t i o n 2 was t h e c l o s e s t i n p r o x i m i t y t o t h e p e n o f f i s h b e i n g t r e a t e d . O x y t e t r a c y c l i n e d i s s o l v e s r e a d i l y i n s e a w a t e r , i s w a t e r s o l u b l e a n d i s v e r y s u s c e p t i b l e t o o x i d a t i o n ( L i t t l e , 1 9 9 0 ) . O t h e r s t u d i e s h a v e shown t h a t o x y t e t r a c y c l i n e i n t h e m a r i n e e n v i r o n m e n t i s s l o w t o d e g r a d e a n d e x h i b i t s a c h r o n i c 26 p e r s i s t e n c e i n t h e s e d i m e n t ( J a c o b s e n a n d B e r g l i n d , 1 9 8 8 ) . T h e s e s t u d i e s h a v e n o t i n d i c a t e d t h e c h e m i c a l f o r m o f t h e a n t i b i o t i c . Due t o t h e d r u g ' s h i g h a f f i n i t y f o r c o m p l e x a t i o n w i t h a v a i l a b l e i o n s , l e v e l s o f OTC may be d e t e c t e d b u t r e m a i n u n a v a i l a b l e f o r u p t a k e b y o t h e r b i o t a ( J a c o b s e n a n d B e r g l i n d , 1 9 8 8 ) . I t i s e v i d e n t f r o m t h i s s t u d y t h a t a f t e r 7 d a y s p o s t t r e a t m e n t ( A u g u s t 1 , 1989) t h e r e w e r e s t i l l d e t e c t a b l e l e v e l s o f o x y t e t r a c y c l i n e i n t h e o y s t e r s s u s p e n d e d w i t h i n t h e n e t c a g e s . I n a r e c e n t s t u d y ( J a c o b s e n , 1 9 8 9 ) c l e a r a n c e t i m e s o f f r e s h w a t e r r a i n b o w t r o u t a f t e r t r e a t m e n t w i t h o x y t e t r a c y c l i n e w e r e d e t e r m i n e d t o b e ; 90 d a y s f o r a m b i e n t w a t e r t e m p e r a t u r e s b e l o w 6 \u00C2\u00B0 C , 70 d a y s f o r a m b i e n t w a t e r t e m p e r a t u r e s b e t w e e n 6 a n d 12 \u00C2\u00B0 C a n d 60 d a y s f o r t e m p e r a t u r e s a b o v e 12 \u00C2\u00B0 C . F r o m t h e s e s t u d i e s i t i s e v i d e n t t h a t a n t i b i o t i c s a r e c o n t i n u a l l y b e i n g r e l e a s e d t h r o u g h t h e common r o u t e s o f m e t a b o l i c e x c r e t i o n i n s a l m o n i d s ( g i l l s , f a e c e s , m o u t h ) . T h e p h a r m a c o - k i n e t i c s o f t h e d r u g i n t h e f i s h may p r o d u c e t h i s t i m e r e l e a s e m e c h a n i s m o f d r u g i n t r o d u c t i o n i n t o t h e e n v i r o n m e n t . T h i s m e c h a n i s m i s h y p o t h e s i z e d t o b e a p o s s i b l e s o u r c e o f t h e p r o l o n g e d d r u g l e v e l s i n t h e o y s t e r s o n A u g u s t 1 , 1 9 8 9 . R o m e t - 3 0 was i d e n t i f i e d i n t h e o y s t e r s o n J u l y 1 , 1989 i n s t a t i o n s 3 , 4 a n d 5 . T h i s c o i n c i d e d w i t h d a y 1 o f p o s t t r e a t m e n t . C o n c e n t r a t i o n s o f SDM w e r e a t t h e i r maximum d e t e c t a b l e l e v e l s . S t a t i o n 4 , t h e p e n c l o s e s t t o t h e t r e a t e d f i s h e x h i b i t e d t h e h i g h e s t c o n c e n t r a t i o n o f 1 . 2 7 ppm (n=2, F i g . 8 ) . I n a r e c e n t s t u d y t h e r a t e o f t i s s u e u p t a k e a n d 27 d e c l i n e o f SDM a n d OMP was shown t o d i f f e r i n s a l m o n i d s ( W a l i s s e r e t a l . , 1 9 9 0 ) . SDM was d e t e c t a b l e i n s a l m o n t i s s u e f o r up t o 7 d a y s a f t e r c e s s a t i o n o f t h e t r e a t m e n t , w h i l e OMP c o u l d be d e t e c t e d u p t o d a y f o u r t e e n p o s t t r e a t m e n t . A p p r o x i m a t e l y two weeks a f t e r c e s s a t i o n o f a n t i b i o t i c t r e a t m e n t t o t h e s a l m o n , d e t e c t a b l e l e v e l s o f b o t h OMP a n d SDM w e r e i s o l a t e d f r o m o y s t e r s i n s t a t i o n s 4 a n d 5 . B o t h o f t h e s e s t a t i o n s w e r e o u t s i d e o f t h e n e t c a g e s y s t e m a n d a s F i g u r e 5 i n d i c a t e s , s t a t i o n 5 was l o c a t e d 200 m away f r o m t h e f i s h f a r m . By A u g u s t 1 5 , 4 weeks p o s t t r e a t m e n t , d e t e c t a b l e l e v e l s o f SDM w e r e r e c o r d e d i n o y s t e r s s a m p l e d f r o m s t a t i o n s 1 a n d 4 . T h e h i g h ( 1 . 2 3 ppm) c o n c e n t r a t i o n d e t e c t e d i n t h e o y s t e r s a t s t a t i o n 4 i s o f n o t e . A s was i n d i c a t e d i n t h e s t u d y b y W a l l i s s e r e t a l . ( 1 9 9 0 ) , c l e a r a n c e o f R o m e t - 3 0 i n e x p e r i m e n t a l f i s h was up t o two weeks p o s t t r e a t m e n t . T h e l e v e l s o f d r u g i n t h e o y s t e r s w e r e n o t s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) f r o m t h o s e c o n c e n t r a t i o n s d e t e c t e d o n J u l y 1 5 , o n e d a y p o s t t r e a t m e n t . T h i s w o u l d s u g g e s t t h a t e i t h e r t h e o y s t e r i s s l o w t o d e p u r a t e SDM f r o m i t s t i s s u e s o r t h a t a s o u r c e o f R o m e t - 3 0 o t h e r t h a n t h a t d i r e c t l y e x c r e t e d f r o m t h e f i s h i s p r e s e n t . I t i s c o n c e i v a b l e t h a t m e d i c a t e d f e e d was c a u g h t i n t h e mesh o f t h e s a l m o n n e t a n d o r d o c k s t r u c t u r e a n d was r e l e a s e d i n t h e w a t e r c o l u m n o v e r t i m e , no m e t h o d was d e v i s e d t o t e s t t h i s t h e o r y . U n l i k e t h e s a l m o n i d s i n t h e u p t a k e e x p e r i m e n t ( W a l i s s e r e t a l . f 1990) SDM was d e t e c t a b l e i n t h e g r e a t e s t c o n c e n t r a t i o n s i n o y s t e r s o v e r t h e s t u d y p e r i o d . A l t h o u g h 28 n o t e v i d e n t i n t h e r e s u l t s o f t h i s s t u d y o n o y s t e r s , t h e l e v e l s o f OMP t h a t m i g h t h a v e b e e n e x c r e t e d f r o m t h e f i s h c o u l d h a v e b e e n s u f f i c i e n t l y l o w e n o u g h t o e s c a p e d e t e c t i o n b y t h e e x t r a c t i o n m e t h o d u s e d . V e r y l o w l e v e l s o f OMP w e r e d e t e c t e d u p t o 4 weeks p o s t t r e a t m e n t ( F i g . 8 ) . I n s u m m a r y , t h e HPLG m e t h o d s d e v e l o p e d f o r b o t h o x y t e t r a c y c l i n e a n d R o m e t - 3 0 a l l o w f o r s u c c e s s f u l q u a n t i f i c a t i o n i n P a c i f i c o y s t e r s . T h e H P L C a n a l y s e s f o r b o t h d r u g s r e p o r t e d i n t h i s s t u d y w e r e r e l i a b l e a n d r e s u l t e d i n t h e d e t e c t i o n o f O T C , SDM a n d OMP a t l e v e l s down t o 0 . 0 5 a n d 0 . 1 0 ppm r e s p e c t i v e l y . T h e r e s u l t s o f e x t r a c t i o n s p e r f o r m e d o n t h e o y s t e r s s u s p e n d e d a t t h e s a l m o n f a r m s u g g e s t s t h a t u p t a k e o f t h e d r u g s o c c u r r e d . T h e s e p r e l i m i n a r y r e s u l t s d i c t a t e d t h e n e e d f o r a c o n t r o l l e d u p t a k e a n d c l e a r a n c e o f OTC a n d R o m e t - 3 0 i n t h e P a c i f i c o y s t e r . 29 C h a p t e r 2: D e t e r m i n a t i o n o f A n t i b i o t i c U p t a k e a n d D e p u r a t i o n i n t h e P a c i f i c O y s t e r . I n t r o d u c t i o n I n v e s t i g a t i o n s i n t o t h e u p t a k e a n d c l e a r a n c e o f a r a n g e o f e n v i r o n m e n t a l c o n t a m i n a n t s h a v e p r i n c i p a l l y f o c u s e d o n t h e common m u s s e l M y t i l u s e d u l i s ( F o n g , 1 9 7 6 ; F o s s a t o a n d C a n z o n i e r , 1 9 7 6 ; F r i e d r i c h a n d F i l i c e , 1 9 7 6 ; S i m p s o n , 1 9 7 9 ) . T h e s e o r g a n i s m s c a n a l s o c o n c e n t r a t e a v a r i e t y o f c h l o r i n a t e d h y d r o c a r b o n s ( R i s e b r o u g h e t a l . , 1 9 7 6 ) , a r o m a t i c h y d r o c a r b o n s ( D i S a l v o e t a l . . 1 9 7 5 ; F o s s a t o a n d C a n z o n i e r , 1976) a n d m e t a l i o n s (Lowe a n d M o o r e , 1979) w i t h i n t h e i r t i s s u e s , a n d t h e r e f o r e t h e y r e p r e s e n t g o o d i n d i c a t o r s o f x e n o b i o t i c b i o a c c u m u l a t i o n . I n v i e w o f t h e p r e l i m i n a r y r e s u l t s o b s e r v e d a t t h e s a l m o n f a r m , a more c o n t r o l l e d e x p e r i m e n t was u n d e r t a k e n t o d i s t i n g u i s h t h e p o s s i b l e e x t e n t o f d r u g u p t a k e a n d c l e a r a n c e i n o y s t e r s . I t i s e s s e n t i a l t h a t t h e u p t a k e a n d c l e a r a n c e r a t e s o f t h e a n t i b i o t i c s u n d e r i n v e s t i g a t i o n b e d e t e r m i n e d f o r o y s t e r s t o a i d i n t h e e s t a b l i s h m e n t o f h a r v e s t i n g g u i d e l i n e s . T o d a t e t h e r e h a v e b e e n n o p u b l i s h e d s t u d i e s r e g a r d i n g u p t a k e a n d c l e a r a n c e o f a n t i b i o t i c s i n s h e l l f i s h . A n e f f o r t was a l s o u n d e r t a k e n t o d i f f e r e n t i a t e t h e e f f e c t s o f s u c h c h e m i c a l s , o n a m e a s u r e o f o y s t e r c o n d i t i o n a n d o n e n d o g e n o u s l e v e l s o f b a c t e r i a a s s o c i a t e d w i t h t h e g u t o f t h e o y s t e r . 30 METHODS AND M A T E R I A L S A m a r i n e h o l d i n g f a c i l i t y e q u i p p e d w i t h a f l o w t h r o u g h s e a w a t e r s y s t e m was u t i l i z e d a t E . V . S . C o n s u l t a n t s L t d . i n N o r t h V a n c o u v e r , B . C . N i n e 25 l i t r e h o l d i n g t a n k s w e r e s t o c k e d w i t h 70 - one y e a r o l d o y s t e r s s u p p l i e d b y F a n n y B a y O y s t e r s L t d . T h e o y s t e r s w e r e d i v i d e d i n t o two t r a y s t h a t w e r e s u s p e n d e d one o v e r t h e o t h e r i n t h e t a n k . T h e t r a y s w e r e r e v e r s e d e a c h d a y s o a s t o r e d u c e p o s i t i o n i n g e f f e c t s . E a c h t a n k was s u p p l i e d w i t h f i l t e r e d a m b i e n t s e a w a t e r a t a f l o w r a t e o f 0 . 7 5 L / m i n . A d j u s t m e n t o f f l o w r a t e s t o e a c h t a n k was a c c o m p l i s h e d b y i n d i v i d u a l v a l v e s . O x y t e t r a c y c l i n e was d e l i v e r e d v i a a c o m m e r c i a l l y p r e p a r e d f i s h f e e d t h a t was g r o u n d t o a f i n e mash ( 0 . 0 5 mm d i a m . ) . T h e c o n c e n t r a t i o n o f o x y t e t r a c y c l i n e a d m i n i s t e r e d was 1 .33 gm / t a n k / 2 4 h r s . R o m e t - 3 0 was a l s o d e l i v e r e d v i a a m a s h e d c o m m e r c i a l l y p r e p a r e d p r e m e d i c a t e d f e e d , d e l i v e r e d a t a d o s a g e o f 1 . 1 1 3 gm / t a n k / 2 4 h r s . U n m e d i c a t e d f i s h f e e d was u s e d f o r t h e c o n t r o l . T h e f e e d was i n t r o d u c e d c o n t i n u o u s l y o v e r e a c h t w e n t y f o u r h o u r p e r i o d i n t o s e p a r a t e h e a d e r t a n k s b y a u t o m a t e d f e e d e r s . F e e d d e l i v e r y t o t h e i n d i v i d u a l e x p e r i m e n t a l a n d c o n t r o l t a n k s was b y s u s p e n s i o n i n w a t e r . T h e a n t i b i o t i c t r e a t m e n t a n d c o n t r o l w e r e r u n i n t r i p l i c a t e . T o d i f f e r e n t i a t e a n y v a r i a t i o n i n h e r e n t i n t h e s y s t e m p r e l i m i n a r y t e s t s w e r e p e r f o r m e d o n t h e p h y s i c a l a s p e c t s o f t h e d r u g d e l i v e r y s y s t e m . I t was d e t e r m i n e d t h a t t h e m e d i c a t e d f e e d h a d a r e s i d e n t t i m e o f a p p r o x i m a t e l y 30 s e c o n d s i n t h e h e a d e r t a n k s . T h e u p t a k e p e r i o d l a s t e d t e n d a y s a n d s a m p l e s w e r e c o l l e c t e d o n d a y s 1 , 3 , 7 a n d 1 0 . F i v e o y s t e r s w e r e r a n d o m l y c h o s e n f r o m e a c h t a n k , s t o r e d i n p l a s t i c z i p l o c k b a g s , l a b e l l e d a n d i m m e d i a t e l y f r o z e n o n d r y i c e . W a t e r t e m p e r a t u r e (\u00E2\u0080\u0094 0 . 0 5 0 C ) , s a l i n i t y \u00E2\u0080\u00A2(\u00E2\u0080\u0094 0 . 0 5 \u00C2\u00B0 / o o ) , d i s s o l v e d o x y g e n (\u00E2\u0080\u0094 0 . 0 5 m g / L ) a n d f l o w r a t e s ( \u00E2\u0080\u0094 5 . 0 m L / m i n . ) w e r e r e c o r d e d d a i l y o v e r t h e s t u d y p e r i o d . S a m p l i n g f o r t h e d u r a t i o n o f t h e d e p u r a t i o n p e r i o d was s e t f o r d a y s 1 , 3 , 23 a n d 30 p o s t u p t a k e . HPLC a n d E x t r a c t i o n T e c h n i q u e s A n a l y s i s f o r o x y t e t r a c y c l i n e a n d R o m e t - 3 0 i n o y s t e r t i s s u e s was p e r f o r m e d f o r t h i s p a r t o f t h e s t u d y a t S y n d e l L a b o r a t o r i e s L t d . i n V a n c o u v e r , B . C . T h e e x t r a c t i o n t e c h n i q u e f o r o x y t e t r a c y c l i n e was m o d i f i e d f r o m Oka e t a l . ( 1 9 8 3 ) . T h e e x t r a c t i o n m e t h o d u t i l i z e d f o r R o m e t - 3 0 was d e v e l o p e d b y a n d i s t h e c o m m e r c i a l p r o p e r t y o f S y n d e l L a b o r a t o r i e s L t d . O x y t e t r a c y c l i n e (OTC) O x y t e t r a c y c l i n e was o b t a i n e d f r o m H o f f m a n n - L a R o c h e L t d . T h e e q u i p m e n t u t i l i z e d was a M i l l i p o r e s y s t e m i n c l u d i n g : H P L C pump, m o d e l 501 s o l v e n t m e t e r i n g s y s t e m ; u n i v e r s a l l i q u i d c h r o m a t o g r a p h i n j e c t o r , m o d e l U 6 K a ; d e t e c t o r m o d e l 4 5 0 ; B a s e l i n e 810 c h r o m a t o g r a p h y i n t e g r a t i o n s o f t w a r e . C h r o m a t o g r a p h y was p e r f o r m e d w i t h a c o m m e r c i a l l y p r e p a r e d U B o n d a p a k C18 c o l u m n . T h e i n j e c t i o n v o l u m e was 2 5 u L . 32 U l t r a v i o l e t d e t e c t i o n was s e t a t 340 nm f o r a l l a n a l y s e s . T h e m o b i l e p h a s e c o n s i s t e d o f 80 % 50 mM N a H 2 P 0 4 pH 2 . 5 a n d 20% a c e t o n i t r i l e . F i l t r a t i o n o f t h e m o b i l e p h a s e p r i o r t o u s e was d o n e u s i n g a H P L C s o l v e n t c l e a n - u p a s s e m b l y a n d F P V e r i c e l 47 mm, 0 . 4 5 um membrane f i l t e r s . E x t r a c t i o n P r o c e d u r e A s o l i d p h a s e e x t r a c t i o n m e t h o d was m o d i f i e d f r o m t h a t o f Oka e t a l . ( 1 9 8 3 ) . I n d i v i d u a l o y s t e r s a m p l e s w e r e t h a w e d a t room t e m p e r a t u r e a n d t h e t i s s u e r e m o v e d a s e p t i c a l l y a n d t o w e l l d r i e d . A t * t i m e s ( d e n o t e d i n r e s u l t s ) t h e 5 a n i m a l s f r o m e a c h r e p l i c a t e w e r e c o l l e c t i v e l y h o m o g e n i z e d a n d t h e s a m p l e t r e a t e d a s a p o o l e d r e s u l t ( N = l ) . T o d e t e r m i n e t h e v a r i a t i o n w i t h i n t h e p o p u l a t i o n w i t h r e s p e c t t o u p t a k e a n d d e p u r a t i o n o f d r u g , e x t r a c t i o n s o f i n d i v i d u a l a n i m a l s w e r e a l s o p e r f o r m e d . W h o l e a n i m a l s a m p l e s w e r e g r o u n d i n a b l e n d e r a n d a 2 0 . 0 g s u b s a m p l e o f t h e s l u r r y was r e m o v e d . T o e a c h 2 0 . 0 g s a m p l e , 50 m l o f b u f f e r was a d d e d . T h e b u f f e r u s e d was 38.5% 2 8 . 4 1 g / L N a 2 H P o 4 a n d 61.5% 1 9 . 2 1 g / L c i t r i c a c i d f i x e d t o a pH o f 4 . 0 . E a c h s a m p l e was h o m o g e n i z e d i n a 75 m l p l a s t i c c e n t r i f u g e t u b e f o r 30 s e c o n d s a t medium s p e e d u s i n g a B r i n k m a n P o l y t r o n h o m o g e n i z e r M o d e l P T 1 0 / 3 5 . T h e s a m p l e s w e r e c e n t r i f u g e d f o r t w e n t y f i v e m i n u t e s a t 6500 g a n d 4 . 0 \u00C2\u00B0 C ( J A - 2 0 r o t o r ) . T h e s u p e r n a t a n t was t r a n s f e r r e d t o a 50 m l t u b e a n d p a s s e d t h r o u g h a n a c t i v a t e d S e p - P a k C 1 8 c a r t r i d g e . A c t i v a t i o n o f t h e S e p - P a k c a r t r i d g e p r i o r t o l o a d i n g t h e s a m p l e was a c c o m p l i s h e d b y p a s s i n g 5 . 0 m l o f 33 m e t h a n o l f o l l o w e d b y 5 . 0 m l o f w a t e r . 2 0 . 0 m l o f m e t h a n o l was u s e d t o e l u t e t h e C 1 8 c o l u m n . T h e e l u a t e s w e r e c o l l e c t e d i n 100 m l r o u n d b o t t o m f l a s k s a n d r o t o - e v a p o r a t e d a t room t e m p e r a t u r e . T h e r e s i d u e s w e r e t h e n f i x e d t o 1 .0 m l w i t h m e t h a n o l a n d i n j e c t e d . R o m e t - 3 0 (SDM & OMP) T h e H P L C c o n d i t i o n s a n d e q u i p m e n t a r e s i m i l a r t o t h o s e f o r O T C . U l t r a v i o l e t d e t e c t i o n was s e t a t 270 nm f o r a l l a n a l y s e s . T h e m o b i l e p h a s e f o r t h e S u l p h a d i m e t h o x i n e e x t r a c t i o n s c o n s i s t e d o f 75% 50 mM N a H 2 P 0 4 pH 2 . 5 a n d 25% a c e t o n i t r i l e . T h e m o b i l e p h a s e f o r o r m e t o p r i m e x t r a c t i o n s was 88% 50 mM N a H 2 P 0 4 pH 2 . 5 a n d 12% a c e t o n i t r i l e . E x t r a c t i o n P r o c e d u r e T h e R o m e t - 3 0 e x t r a c t i o n i s a n o r g a n i c l i q u i d - l i q u i d e x t r a c t i o n a n d i s t h e c o m m e r c i a l p r o p e r t y o f S y n d e l L a b o r a t o r i e s . T h e two a n t i b i o t i c c o m p o n e n t s i n t h e c o m m e r c i a l p r e p a r a t i o n o f R o m e t - 3 0 a r e o r m e t o p r i m a n d s u l p h a d i m e t h o x i n e . E a c h c o m p o n e n t was e x t r a c t e d a n d i n j e c t e d s e p a r a t e l y . R e c o v e r y T h e r e c o v e r i e s o f o x y t e t r a c y c l i n e , s u l p h a d i m e t h o x i n e a n d o r m e t r o p r i m f r o m o y s t e r t i s s u e w e r e d e t e r m i n e d b y t h e a d d i t i o n o f 1 mL o f e a c h o f t h e p r e p a r e d s t a n d a r d s o l u t i o n s c o n t a i n i n g 2 . 0 , 5 . 0 , 1 0 . 0 a n d 3 0 . 0 u g o f d r u g t o 20 g 34 s a m p l e s o f c o n t r o l o y s t e r t i s s u e . T h e s a m p l e s w e r e e x t r a c t e d a n d a n a l y z e d a s d e s c r i b e d a b o v e . R e c o v e r y ( T a b l e 2) was d e t e r m i n e d b y c a l c u l a t i n g t h e a r e a o f O T C , SDM a n d OMP f o r e x t r a c t e d t i s s u e s a m p l e s a n d c o m p a r i n g t h e s e t o t h e a r e a s o b t a i n e d f r o m u n e x t r a c t e d s t a n d a r d s o l u t i o n s o f i d e n t i c a l q u a n t i t i e s . S e n s i t i v i t y T h e s e n s i t i v i t y o r l o w e r d e t e c t i o n l i m i t was d e t e r m i n e d b y t h e s o f t w a r e i n t e g r a t i o n s y s t e m . I t a s s u m e s a l i n e a r c a l i b r a t i o n c u r v e f o r a l l a n t i b i o t i c s t e s t e d . C o n d i t i o n I n d e x O y s t e r s w e r e s a m p l e d a t r a n d o m a t t h e s t a r t o f t h e u p t a k e p e r i o d a n d c o n d i t i o n i n d i c e s d e t e r m i n e d (n=10) ( A l d r i c h a n d C r o w l e y , 1986) b y t h e e q u a t i o n : DRY MEAT WEIGHT g DRY S H E L L WEIGHT g X 1000 A t t h e c o m p l e t i o n o f t h e t e n d a y u p t a k e p e r i o d o y s t e r s w e r e s a m p l e d f r o m t h e e x p e r i m e n t a l g r o u p (n=12) t o d e t e r m i n e i f a d i e t s u p p l e m e n t e d w i t h m e d i c a t e d f e e d s i g n i f i c a n t l y c h a n g e d c o n d i t i o n o f t h e a n i m a l . B a c t e r i o l o g i c a l A n a l y s i s C o m p a r i s o n o f t h e r e l a t i v e b a c k g r o u n d c o m p o s i t i o n o f t h e n a t u r a l b a c t e r i a l f l o r a o f t h e e x p e r i m e n t a l p o p u l a t i o n o f o y s t e r s was d e t e r m i n e d a c c o r d i n g t o 35 m e t h o d s a d o p t e d f r o m K e l l y , ( 1 9 9 0 ) . S i x o y s t e r s e a c h , f r o m t h e c o n t r o l , OTC a n d R o m e t - 3 0 e x p e r i m e n t a l t a n k s w e r e a n a l y z e d . P l a t i n g m e d i a a n d m e t h o d s e m p l o y e d i n t h e q u a n t i t a t i v e d e t e r m i n a t i o n s w e r e a s f o l l o w s : B l o o d a g a r b a s e n o . 2 w i t h 5% s h e e p b l o o d M a k c o n c h e y a g a r T h i o s u l p h a t e c i t r a t e b i o s a l t s s u c r o s e a g a r ( T C B S ) T h e q u a n t i t a t i v e d e t e r m i n a t i o n s w e r e e m p l o y e d u s i n g a l l t h r e e m e d i a . S t r e a k i n g o f t h e p l a t e s was p e r f o r m e d a s e p t i c a l l y w i t h a g a s t r i c c r o s s s e c t i o n o f o y s t e r t i s s u e . A c o m p a r i s o n o f t h e number o f c o l o n i e s o b t a i n e d o n c o u n t p l a t e s p r e p a r e d f r o m t h e s h e l l f i s h a t r o o m t e m p e r a t u r e ( c i r c a 20 t o 25 \u00C2\u00B0 C ) was made . S t a t i s t i c a l A n a l y s e s One way a n a l y s i s o f v a r i a n c e was u s e d t o a n a l y z e a l l d a t a f o r s t a t i s t i c a l d i f f e r e n c e s . S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n mean v a l u e s o f d r u g c o n c e n t r a t i o n s f o r b o t h u p t a k e a n d d e p u r a t i o n s a m p l i n g p e r i o d s w e r e d i s c e r n e d u s i n g T u k e y s t e s t s . T u k e y s t e s t s w e r e a l s o e m p l o y e d t o d i s t i n g u i s h s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n c o n d i t i o n i n d i c e s a n d b a c t e r i a l c o u n t s b e f o r e a n d a f t e r m e d i c a t i o n . T h e l e v e l o f r e j e c t i o n was s e t a t p < 0 . 0 5 f o r a l l d e t e r m i n a t i o n s . 36 R E S U L T S W a t e r p a r a m e t e r s A v e r a g e w a t e r p a r a m e t e r s f o r t h e s t u d y p e r i o d w e r e : t e m p e r a t u r e , 1 2 . 0 \u00C2\u00B0 C \u00C2\u00B1 1 .3 s . e . ( n = 4 0 ) ; s a l i n i t y , 2 8 . 0 \u00C2\u00B0 / o o ( n = 4 0 ) ; d i s s o l v e d 0 2 , 8 . 7 m g / L (n=40) a n d f l o w r a t e 0 . 7 5 L / m i n . \u00E2\u0080\u0094 0 . 2 s . e . ( n = 3 6 0 ) . C a l i b r a t i o n C u r v e a n d R e c o v e r y T h e c o m p u t e r s o f t w a r e \" B a s e l i n e 810\" i n t e g r a t e s t h e p e a k a r e a s a n d a s s u m e s a l i n e a r c a l i b r a t i o n c u r v e b a s e d o n e n t e r e d c o n c e n t r a t i o n r a n g e s . T h e r e c o v e r i e s o f t h e t h r e e d r u g s a r e p r e s e n t e d i n T a b l e 2 . F r o m t h i s a s s u m p t i o n o f l i n e a r i t y t h e d e t e c t i o n l i m i t s o r s e n s i t i v i t i e s a r e c a l c u l a t e d a s t h e f o l l o w i n g ; 0 . 2 ppm f o r O T C , 0 . 0 5 ppm f o r O r m e t o p r i m a n d 0 . 0 3 ppm f o r S u l p h a d i m e t h o x i n e . O x y t e t r a c y c l i n e U p t a k e F i g u r e 9 i s a r e p r e s e n t a t i v e c h r o m a t o g r a m o f P a c i f i c o y s t e r t i s s u e e x t r a c t s p i k e d w i t h 2 . 0 ppm o f o x y t e t r a c y c l i n e . S a m p l i n g o f o y s t e r s d u r i n g t h e t e n d a y u p t a k e t r i a l i n d i c a t e d i n c r e a s i n g c o n c e n t r a t i o n s o f a n t i b i o t i c , r e a c h i n g a maximum o f * 1 0 . 7 2 ppm + 2 . 4 s . e . (n=7) o n d a y 1 0 . F i g u r e 10 i n d i c a t e s t h e c o n c e n t r a t i o n s o f o x y t e t r a c y c l i n e f r o m o y s t e r s s a m p l e d o n d a y s ; 1 , 3 , 7 , a n d 1 0 . T A B L E 2 . RECOVERY OF O T C , OMP AND SDM FROM WHOLE P A C I F I C O Y S T E R T I S S U E ( V a l u e s r e p r e s e n t a s i n g l e d e t e r m i n a t i o n ) S a m p l e C o n c e n t r a t i o n (ppm) OTC % R e c o v e r y OMP % R e c o v e r y SDM % R e c o v e r y 0 . 2 3 8 . 0 3 6 . 0 0 . 5 4 2 . 0 4 2 . 0 1 .0 4 0 . 0 4 0 . 0 5 4 . 0 2 . 0 5 9 . 0 5 2 . 0 5 . 0 5 3 . 0 1 0 . 0 6 4 . 0 Mean R e c o v e r y 5 2 . 3 4 4 . 8 4 6 . 0 38 BUNK EXTRACT \ \ \ .00 2.00 3.00 4.00 ninu-tes 5.00 2.20 2.00 H 1.80 -j 1.S0 1 .AO 1 .20 -i 1.00-1 0.00 1.00 0 0 3.00 4.00 ' 5.00' F i g u r e 9 . A r e p r e s e n t a t i v e c h r o m a t o g f a m o f b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t a n d a e x t r a c t s p i k e d w i t h 2 . 0 ppm o f O T C . UPTAKE OF OXYTETRACYCLINE IN THE PACIFIC OYSTER, C. gigas ( + / - 1 s.e.) 14.0 39 12.0-2 a. a. 1 u. o z o 10.0 8.0 J 6.0 - I T* 1 c z o o 4.0 2.0 0.0 t r 3 DAYS i i i i 7 10 F i g u r e 10. P l o t o f o x y t e t r a c y c l i n e u p t a k e v e r s u s t i m e i n t h e P a c i f i c o y s t e r . E a c h p o i n t r e p r e s e n t s a p o o l e d sample o f .5 a n i m a l s (n=3) , e x c e p t where d e n o t e d by a * , t h e n n=7. DEPURATION OF OXYTETRACYCLINE IN THE PACIFIC OYSTER, C. gigas ( + / - 1 s.e.) 8.O-1 7.0-s 6.0-a. 5.0-o \u00E2\u0080\u00A2 u. o 1 o 4.0-z /\u00E2\u0080\u0094\ E | c a S> 3.0-LLI o 2.0-z 1.0-0.0-I. 1 3 13 DAYS 30 F i g u r e 11 . P l o t o f o x y t e t r a c y c l i n e d e p u r a t i o n v e r s u s t i m e i n t h e P a c i f i c o y s t e r . E a c h p o i n t r e p r e s e n t s a p o o l e d sample o f 5 a n i m a l s (n=3) , e x c e p t where d e n o t e d by a * , t h e n n=7. 40 D e p u r a t i o n F o l l o w i n g a t h i r t y d a y d e p u r a t i o n p e r i o d d u r i n g w h i c h t i m e no m e d i c a t e d f e e d was a d m i n i s t e r e d , a n a s y m p t o t i c d e p u r a t i o n c u r v e r e s u l t e d , F i g u r e 1 1 . T h e c o n c e n t r a t i o n o f a n t i b i o t i c d e t e c t e d i n w h o l e a n i m a l t i s s u e d e c l i n e d s t e a d i l y f r o m a maximum o f 6 . 0 7 ppm \u00E2\u0080\u0094 1 .08 s . e . (n=3) o n d a y 1 o f d e p u r a t i o n t o a min imum q u a n t i f i a b l e l e v e l o f * 0 . 2 1 ppm \u00E2\u0080\u0094 0 . 0 4 s . e . (n=7) o n d a y 13 o f d e p u r a t i o n . S a m p l i n g o n d a y t h i r t y o f d e p u r a t i o n r e s u l t e d i n no d e t e c t a b l e l e v e l o f OTC i n a n y o f t h e o y s t e r s s a m p l e d . R o m e t - 3 0 S u l p h a d i m e t h o x i n e (SDM) U p t a k e F i g u r e 12 i s a r e p r e s e n t a t i v e c h r o m a t o g r a m o f o y s t e r t i s s u e e x t r a c t s p i k e d w i t h SDM. D u r i n g t h e t e n d a y u p t a k e p e r i o d , SDM r e a c h e d a maximum c o n c e n t r a t i o n i n w h o l e a n i m a l t i s s u e s o f 1 . 0 9 ppm \u00E2\u0080\u0094 0 . 2 4 s . e . (n=3) o n d a y 7 a n d d e c r e a s e d t o * 0 . 5 0 3 ppm \u00C2\u00B1 0 . 2 1 s . e . (n=7) o n d a y 1 0 , s e e F i g u r e 1 3 . 41 BL2NK ECIR^Cr 1.50 -l .40 -1.30-s a 1.00 !.00 3.00 4.00 Minutes 5.00 1 .20 -T \" : : J - - - - ' ^ \u00E2\u0080\u0094 i I i, 0.00 0.20 0.40 0.60 0.30 1.00 1.20 x ID1 ninutes F i g u r e 1 2 . R e p r e s e n t a t i v e c h r o m a t o g r a m o f a b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t a n d e x t r a c t s p i k e d w i t h SDM a t 2 . 0 ppm. 42 D e p u r a t i o n T h e c o n c e n t r a t i o n o f SDM o n d a y 1 o f d e p u r a t i o n was 0 . 0 3 ppm \u00E2\u0080\u0094 0 . 0 0 5 s . e . ( n = 3 ) . T h e d e t e c t a b l e l e v e l s o f t h e d r u g t h e n i n c r e a s e d o n d a y 3 o f t h e s t u d y t o 0 . 1 1 ppm + 0 . 0 0 2 s . e . ( n = 3 ) . On d a y t h i r t y o f d e p u r a t i o n no d e t e c t a b l e l e v e l s o f d r u g w e r e i s o l a t e d f r o m t h e e x p e r i m e n t a l g r o u p ( F i g . 1 4 ) . O r m e t o p r i m (OMP) U p t a k e OMP was d e t e c t e d i n s m a l l c o n c e n t r a t i o n s o n d a y t h r e e i n one o f t h e t h r e e e x p e r i m e n t a l r e p l i c a t e s . A s F i g u r e 13 i n d i c a t e s 0 . 0 3 ppm ( n = l ) was t h e minimum c o n c e n t r a t i o n d e t e c t e d a n d o n d a y 7 o f u p t a k e o y s t e r s s a m p l e d f r o m one o f t h e t h r e e r e p l i c a t e s r e v e a l e d l e v e l s o f OMP o f 0 . 2 1 ppm ( n = l ) . F i g u r e 15 i s a r e p r e s e n t a t i v e c h r o m a t o g r a m o f o y s t e r t i s s u e e x t r a c t s p i k e d w i t h 2 . 0 ppm o f OMP. D e p u r a t i o n A t no p o i n t i n t h e t h i r t y d a y d e p u r a t i o n p e r i o d was OMP d e t e c t e d i n t h e o y s t e r s s a m p l e d f r o m t h e e x p e r i m e n t a l p o p u l a t i o n s . UPTAKE OF ROMET-30 IN THE PACIFIC OYSTER, C. gigas 2 a. a. o pi o | i s _ 54 0.6 UJ o z o o 1.6 1.4-1.2-1.0 0.8H 0.4 0.2 H 0.0 - i r (+/- 1 M.) ~i 1 r DAYS T 1 1 1 10 > SDM A OMP 43 Figure 13. P l o t of Romet-30 (SDM&OMP) uptake versus time i n the P a c i f i c oys ter . Each po int represents a pooled sample of 5 animals (n=3), except where denoted by a * , then n=7. DEPURATION OF ROMET-30 IN THE PACIFIC OYSTER, C. gigas (+/- 1 \u00E2\u0080\u00A2\u00E2\u0080\u00A2\u00C2\u00AB-) 0.12 2 &. 0.09 o O o o z o o 0.03-0.00 1 3 13 DAYS 30 SDM OMP not detected Figure 14. P l o t of Romet-30 (SDM&OMP) depuration versus time i n the P a c i f i c oys ter . Each po int represents a pooled sample of 5 animals (n=3), except where denoted by a * , then n=7. 44 .00 3.00 n i n u i s s I . I S H 1 .10 1.05 -4 0.95 .'.... \u00E2\u0080\u0094 \u00E2\u0080\u00A2 r \u00E2\u0080\u00A2 I ' ' ' I 1 ' 1 I ' ' 1 I ' ' ! I ' I 0.00, 0.20 0.10 0.60 O.aO l.OO 1.20 1.40 x 10* ninutes F i g u r e 1 5 . A r e p r e s e n t a t i v e c h r o m a t o g r a m o f a b l a n k P a c i f i c o y s t e r t i s s u e e x t r a c t a n d a e x t r a c t s p i k e d w i t h 2 . 0 ppm o f OMP. 45 C o n d i t i o n I n d e x F i g u r e 16 i n d i c a t e s t h e c h a n g e i n c o n d i t i o n i n d e x f o r t h e t e n d a y u p t a k e s t u d y . T h e i n d e x a t t h e commencement o f t h e t e n d a y s t u d y was 3 5 . 0 \u00E2\u0080\u0094 4 . 2 2 s . e . (n=10) a n d u p o n c o m p l e t i o n o f t h e u p t a k e s t u d y was 4 1 . 7 \u00E2\u0080\u0094 1 .94 s . e . ( n = 1 2 ) . T h e s e v a l u e s w e r e d e t e r m i n e d t o b e s i g n i f i c a n t l y d i f f e r e n t ( p < 0 . 0 5 ) . B a c t e r i o l o g i c a l Q u a n t i f i c a t i o n T a b l e 3 s u m m a r i z e s t h e a v e r a g e c o u n t s o f 6 i n d i v i d u a l o y s t e r s b e f o r e a n d a f t e r t r e a t m e n t w i t h b o t h a n t i b i o t i c s . T h e b l o o d a g a r medium a p p e a r e d t o g i v e t h e maximum c o u n t i n t h e c o n t r o l g r o u p . T h e b a c t e r i a o n t h e s e p l a t e s w e r e v e r y m u c o i d a n d t e n d e d t o p r o d u c e v e r y c o n f l u e n t c o l o n i e s . T h e r e was a s i g n i f i c a n t d e c r e a s e i n c o l o n i e s c o u n t e d o n t h e R o m e t -30 b l o o d a g a r medium p o s t t r e a t m e n t . T h i s was t h e g r e a t e s t d e c r e a s e i n g r o w t h o b s e r v e d . M a k c o n c h e y a g a r w h i c h s e l e c t i v e l y c u l t u r e s g r a m p o s i t i v e b a c t e r i a a s s o c i a t e d w i t h sewage o u t f a l l p r o d u c e d 145 c o l o n i e s i n t h e c o n t r o l o y s t e r s a n d f a i l e d t o p r o d u c e a n y c o l o n i e s p o s t t r e a t m e n t . T h i o s u l f a t e C i t r a t e B i o s a l t s S u c r o s e a g a r ( T C B S ) w h i c h s e l e c t i v e l y c u l t u r e s t h o s e s p e c i e s o f b a c t e r i a a s s o c i a t e d w i t h s a l t w a t e r a n d i n p a r t i c u l a r V i b r i o c a u s a t i v e b a c t e r i a , p r o d u c e d a p p r o x i m a t e l y 398 c o l o n i e s i n t h e c o n t r o l o y s t e r s . T C B S medium p r o d u c e d a s i g n i f i c a n t l y l o w e r number ( p < 0 . 0 5 ) o f c o l o n i e s i n t h e R o m e t - 3 0 ( 1 2 3 . 6 ) a n d O x y t e t r a c y c l i n e ( 1 7 8 . 2 ) t r e a t e d o y s t e r s c o m p a r e d t o c o n t r o l s . T A B L E 3 . D e t e r m i n a t i o n o f b a c t e r i a l c o l o n i z a t i o n o n s e v e r a l m e d i a a s d e t e r m i n e d b y mean p r o b a b l e number ( M P N ) . MEDIUM CONTROL R O M E T - 3 0 OTC Mean c o u n t o f 6 o y s t e r s B l o o d a g a r c o n f l u e n t 9 3 . 7 * 1 5 9 . 0 * M a c k o n c h e y 1 4 5 . 0 0* 0* a g a r T C B S a g a r 3 9 8 . 4 1 2 3 . 6 * 1 7 8 . 0 * * D e n o t e s s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l v a l u e (p< 0 . 0 5 ) 47 CONDITION INDEX OF PACIFIC OYSTERS P R E AND POST TREATMENT (+ / - 1 s.e.) 50-n o o o X x ^ Z \u00C2\u00A9 \u00E2\u0080\u0094 JC O \u00C2\u00A3 O * O o E T3 40 A 30 -4 20 J 10H PRE TREATMENT POST TREATMENT F i g u r e 1 6 . H i s t o g r a m o f P a c i f i c o y s t e r c o n d i t i o n i n d e x f o r p r e a n d p o s t a n t i b i o t i c t r e a t m e n t . ( * d e n o t e s s i g n i f i c a n t s t a t i s t i c a l d i f f e r e n c e ) 48 D I S C U S S I O N I n v i e w o f t h e p r e l i m i n a r y r e s u l t s o b s e r v e d a t t h e s a l m o n f a r m , a more c o n t r o l l e d e x p e r i m e n t was u n d e r t a k e n t o d i s t i n g u i s h t h e p o s s i b l e e x t e n t o f d r u g u p t a k e a n d c l e a r a n c e i n o y s t e r s . A n e f f o r t was a l s o u n d e r t a k e n t o d i f f e r e n t i a t e t h e e f f e c t s o f s u c h c h e m i c a l s , o n a m e a s u r e o f o y s t e r c o n d i t i o n a n d o n e n d o g e n o u s l e v e l s o f b a c t e r i a a s s o c i a t e d w i t h t h e g u t o f t h e o y s t e r . I n t h i s c o n t r o l l e d e x p e r i m e n t t h e u p t a k e o f a n t i b i o t i c s i n o y s t e r s r e v e a l e d r e s u l t s t h a t w e r e n o t u n l i k e t h o s e f r o m t h e s a l m o n f a r m s i t e . W i t h r e s p e c t t o o x y t e t r a c y c l i n e t h e u p t a k e r a t e o v e r a p e r i o d o f 10 d a y s r e s e m b l e d a l i n e a r r e l a t i o n s h i p ( F i g u r e 1 0 , r 2 = 0 . 9 7 5 ) . T h i s i n d i c a t e s t h a t a b i o a c c u m u l a t i o n o f OTC o c c u r r e d i n t h e P a c i f i c o y s t e r . D u r i n g t h e u p t a k e p e r i o d , t h e r e was no d a t a t o i n d i c a t e t h a t a s a t u r a t i o n t h r e s h o l d was r e a c h e d f o r OTC i n t h e P a c i f i c o y s t e r . D e p u r a t i o n i m m e d i a t e l y f o l l o w e d t h e u p t a k e p e r i o d a n d c o n c l u d e d o n d a y 30 when no d e t e c t a b l e r e s i d u e s w e r e i d e n t i f i e d . D e p u r a t i o n s y s t e m s f o r s h e l l f i s h h a v e b e e n u t i l i z e d , b y many c o u n t r i e s whose w a t e r s a r e c h r o n i c a l l y p o l l u t e d b y f e c a l c o l i f o r m s ( N e i l s o n e t a l . , 1 9 7 8 ; S o u n e s s e t a l . , 1 9 7 9 ; L e d o e t a l . , 1 9 8 3 ) . T h e r a p i d r a t e o f b a c t e r i a l d e p u r a t i o n i n s h e l l f i s h i n e n g i n e e r e d d e p u r a t i o n f a c i l i t i e s , s o m e t i m e s o c c u r r i n g w i t h i n 48 h o u r s f o r some s p e c i e s o f b a c t e r i a , h a s b e e n d e m o n s t r a t e d ( T h i S o n a n d F l e e t , 1 9 8 0 ) . W i t h i n 13 d a y s o f d e p u r a t i o n t h e mean c o n c e n t r a t i o n o f o x y t e t r a c y c l i n e i n t h e o y s t e r s h a d 49 s i g n i f i c a n t l y d e c r e a s e d b y 1 0 . 5 ppm. T h i s w o u l d i n d i c a t e a r a p i d c l e a r a n c e o f d r u g f r o m t h e t i s s u e s o f t h e o y s t e r . T h e f i l t r a t i o n r a t e s o f o y s t e r s h a v e b e e n d e t e r m i n e d i n p r e v i o u s s t u d i e s f o r a s p e c t r u m o f s e a w a t e r c o n d i t i o n s ( L o o s a n o f f a n d T o m m e r s , 1948 ; W a l n e , 1 9 7 2 ; B r a n d a n d T a y l o r , 1974; B a y n e e t a l . , 1 9 7 7 ) . T h e a v e r a g e t e m p e r a t u r e f o r t h e 30 d a y d e p u r a t i o n p e r i o d was 12 \u00C2\u00B0 C a n d t h e mean d r y w e i g h t o f t h e e x p e r i m e n t a l g r o u p o f o y s t e r s was 2 . 0 g ( b a s e d o n c o n d i t i o n i n d e x d a t a , n = 1 2 ) . A c c o r d i n g l y a r e p r e s e n t a t i v e amount o f w a t e r f i l t e r e d b y a n o y s t e r w o u l d be e s t i m a t e d a s 60 mLs p e r m i n u t e ( W a l n e , 1 9 7 2 ) . By e x t r a p o l a t i o n t h e mean amount o f t i m e r e q u i r e d b y t h e e x p e r i m e n t a l p o p u l a t i o n t o d e p u r a t e a c o n c e n t r a t i o n o f OTC o f 1 0 . 5 ppm i n 13 d a y s c a n b e t h e o r e t i c a l l y d e t e r m i n e d . A c c o r d i n g t o t h e f i l t r a t i o n r a t e r e p o r t e d b y W a l n e , (1972) t h e amount o f d r u g d e p u r a t e d i n t h i s t i m e i n t e r v a l w o u l d b e 0 . 0 0 9 p p m / L i t r e . B a s e d o n t h e same p a r a m e t e r s o f d e t e r m i n a t i o n t h e c o n c e n t r a t i o n o f OTC f i l t e r e d a n d c o n c e n t r a t e d f o r t h e t e n d a y u p t a k e was 0 . 0 1 2 p p m / L i t r e . I t a p p e a r s t h a t t h e r a t e o f u p t a k e i s q u i t e c l o s e l y r e l a t e d t o t h e r a t e o f d e p u r a t i o n f o r t h e P a c i f i c o y s t e r ( F i g . 1 1 ) . S u l p h a d i m e t h o x i n e (SDM) was one o f t h e two c o m p o n e n t s o f R o m e t - 3 0 t h a t was p r e s e n t i n s u f f i c i e n t l e v e l s t h r o u g h o u t t h e u p t a k e a n d d e p u r a t i o n p h a s e s o f t h e s t u d y t o p r o d u c e a d e s c r i p t i v e t r e n d . OMP was n o t a s p r e d i c t a b l e i n i t s u p t a k e a n d c l e a r a n c e b y t h e o y s t e r s . I n c o m p a r i s o n t o o x y t e t r a c y c l i n e , s u l p h a d i m e t h o x i n e i s much more s t a b l e i n 50 t h e m a r i n e e n v i r o n m e n t ( L i t t l e , 1 9 9 0 ) . R o m e t - 3 0 i s a l i p i d s o l u b l e d r u g a n d t h e r e f o r e m u s t be d i g e s t e d b y t h e a n i m a l f o r a b s o p r t i o n t o o c c u r . T h i s c h a r a c t e r i s t i c may be r e s p o n s i b l e f o r p r o d u c i n g t h e v a r i e d r a t e s o f u p t a k e a n d c l e a r a n c e o b s e r v e d f o r R o m e t - 3 0 i n t h e P a c i f i c o y s t e r . A n o t h e r p o s s i b l e f a c t o r i n p r o d u c i n g t h i s a n o m a l y may b e t h a t SDM a n d OMP w e r e n o t e v e n l y d i s t r i b u t e d i n t h e h e a d e r t a n k a n d d r u g d e l i v e r y s y s t e m . U p o n i n t r o d u c t i o n o f R o m e t - 3 0 m a s h e d f e e d p e l l e t s t o t h e h e a d e r t a n k a r e s i d u e was o b s e r v e d t o f o r m o n t h e w a t e r s u r f a c e , t h i s may h a v e b e e n t h e d r u g c o m i n g o u t o f s o l u t i o n . I n t h e c a s e o f OTC no s u c h o b s e r v a t i o n was r e c o r d e d . T h e o y s t e r s u s e d i n t h i s s t u d y w e r e s u p p l i e d b y a c o m m e r c i a l g r o w e r who s e l e c t e d o y s t e r s w i t h i n a one y e a r o l d s i z e r a n g e . T h i s p r o d u c e d a p o p u l a t i o n o f o y s t e r s t h a t w e r e f a i r l y u n i f o r m i n s i z e . I t h a s b e e n d e m o n s t r a t e d t h a t t h e r a t e s o f m o s t p h y s i o l o g i c a l p r o c e s s e s a r e d e p e n d e n t o n i n d i v i d u a l b o d y s i z e ( r e v i e w e d b y B a y n e e t a l . , 1 9 7 6 a ) . T h e e f f e c t s o f s i z e a r e r e m o v e d f r o m p h y s i o l o g i c a l m e a s u r e m e n t s b y a p p l i c a t i o n o f t h e a l l o m e t r i c m o d e l r e l a t i n g t h e r a t e o f t h e p r o c e s s (F) t o b o d y mass ( W ) . A c c o r d i n g t o W a l n e , (1972) t h e r e l a t i o n b e t w e e n t h e s p e c i f i c f i l t r a t i o n r a t e , F , a n d t h e d r y meat w e i g h t , W, c a n b e d e s c r i b e d b y t h e e q u a t i o n : F = a W b a i s t h e i n t e r c e p t o f t h e l i n e a n d b t h e s l o p e . T h e v a l u e o f b s h o u l d a p p r o x i m a t e 0 . 6 6 7 when W i s d r y meat w e i g h t . T h i s f o r m u l a a s s u m e s t h a t d r y meat w e i g h t i s 20% o f t h e l i v e wet 51 w e i g h t . D r y meat w e i g h t was d e t e r m i n e d f o r a s u b s a m p l e o f 2 2 i n d i v i d u a l s . A c c o r d i n g t o t h e a b o v e e q u a t i o n , t h e f i l t r a t i o n r a t e s o f t h e e x p e r i m e n t a l o y s t e r s w e r e d e t e r m i n e d t o be q u i t e v a r i a b l e . I f t h e two c o m p o n e n t s o f R o m e t - 3 0 (OMP a n d SDM) w e r e n o t e q u a l l y s u s p e n d e d i n t h e d e l i v e r y s y s t e m t h e n c o u p l e d w i t h t h e v a r i a t i o n o b s e r v e d i n o y s t e r s i z e a n d f i l t r a t i o n r a t e s may h a v e b e e n r e s p o n s i b l e i n p r o d u c i n g s u c h v a r i e d d r u g c o n c e n t r a t i o n s w i t h i n t h e a n i m a l s . T h e d e g r e e t o w h i c h c l e a r a n c e r a t e i s m a i n t a i n e d a t a s t e a d y s t a t e i s l a r g e l y d e p e n d e n t o n t h e s p e c i e s b e i n g s t u d i e d . I n many i n t e r t i d a l b i v a l v e s , p u m p i n g r a t e i s m a i n t a i n e d a t a c o n s t a n t l e v e l o v e r c o n s i d e r a b l e p e r i o d s o f t i m e ( B a y n e e t a l . , 1 9 7 7 ; B r a n d a n d T a y l o r , 1 9 7 4 ) . Y e t i n c o n t r a s t , s u b t i d a l b i v a l v e s h a v e b e e n shown t o e x h i b i t a v e r y i n t e r m i t t e n t p a t t e r n o f p u m p i n g a c t i v i t y ( B r a n d a n d T a y l o r , 1 9 7 4 ) . B a s e d o n a mean w a t e r t e m p e r a t u r e o f 12 \u00C2\u00B0 C a n d mean f i l t r a t i o n r a t e o f 60 m l / m i n u t e , t h e o y s t e r s i n e a c h t a n k (n=70) w o u l d f i l t e r a p p r o x i m a t e l y 252 l i t r e s p e r h o u r . W a t e r was f l o w i n g t o t h e t a n k s a t a r a t e o f 750 m l / m i n u t e , i n one h o u r , 45 l i t r e s o f w a t e r f l o w e d t h r o u g h e a c h t a n k . What i s e v i d e n t h e r e i s t h a t d e p e n d e n t o n t h e o y s t e r s p o s i t i o n i n t h e t a n k some a n i m a l s w o u l d b e f i l t e r i n g t h e same b o d y o f w a t e r o r a m i x t u r e t h e r e o f , a s much a s 7 t i m e s . I f t h e d r u g s b e i n g a d m i n i s t e r e d w e r e n o t u n i f o r m i n t h e i r d i s t r i b u t i o n t h e n i t i s r e a s o n a b l e t o a s sume t h a t some i n d i v i d u a l s c o u l d p o s s i b l y f i l t e r a n d come i n t o c o n t a c t w i t h v a r i a b l e c o n c e n t r a t i o n s o f d r u g . 52 T h e e f f e c t s o f p o o l i n g s a m p l e s o n d r u g d e t e r m i n a t i o n i n t h e o y s t e r w a r r a n t s d i s c u s s i o n . F i g u r e s 13 a n d 14 r e p r e s e n t t h e u p t a k e a n d d e p u r a t i o n o f R o m e t - 3 0 i n t h e o y s t e r a n d r e f e r e n c e i s made t o t h e s a m p l e s i z e s i n t h e p l o t s . D u r i n g u p t a k e t h e g r e a t e s t mean c o n c e n t r a t i o n s w e r e o b s e r v e d i n t h e o y s t e r s t h a t w e r e n o t p o o l e d . T h e r e l a t i v e l y l a r g e s t a n d a r d e r r o r s a s s o c i a t e d w i t h t h o s e d a t a w h e r e t h e s a m p l e s i z e i s 7 , i n d i c a t e a s i g n i f i c a n t v a r i a t i o n o f d r u g c o n c e n t r a t i o n s w i t h i n t h e i n d i v i d u a l a n i m a l s . When t h e s a m p l e s a r e p o o l e d t h e s t a n d a r d e r r o r i s g r e a t l y r e d u c e d . I t may be t h a t t h e p o o l e d s a m p l e s mask t h e t r u e c o n c e n t r a t i o n o f c h e m i c a l s w i t h i n i n d i v i d u a l o y s t e r s . F o r a n a n t i b i o t i c l i k e OTC t h a t i s w a t e r s o l u b l e a n d u b i q u i t o u s i n i t s d i s p e r s i o n i n t h e w a t e r c o l u m n , t h e p o o l i n g o f s a m p l e s may n o t h a v e s i g n i f i c a n t l y a f f e c t e d i t s d e t e c t i o n . Y e t i n t h e c a s e o f r o m e t - 3 0 t h a t was d e t e c t e d i n much l o w e r c o n c e n t r a t i o n s , t h e e f f e c t s o f p o o l i n g may h a v e s i g n i f i c a n t l y r e d u c e d i t s d e t e c t i o n . C o n d i t i o n i n d e x a s a d e t e r m i n a t i o n o f e c o p h y s i o l o g i c a l s t a t u s h a s b e e n w e l l d o c u m e n t e d i n b i v a l v e s ( M a n n , 1 9 7 8 ; L u c a s a n d B e r r i n g e r , 1 9 8 5 ; Brown a n d H a r t w i c k , 1 9 8 8 ) . T h e e f f e c t s o f v a r y i n g f e e d l e v e l s a n d c h a n g e s i n e n v i r o n m e n t a l w a t e r p a r a m e t e r s o n t h e b i o l o g i c a l p e r f o r m a n c e o f b i v a l v e s i s a n a r e a t h a t h a s b e e n i n t e n s e l y s t u d i e d ( L o o s a n o f f a n d T o m e r s , 1 9 4 9 ; L i p o v s k y a n d Chew, 1 9 7 2 ; T e n o r e a n d D u n s t a n , 1 9 7 3 ; M o r i , 1 9 7 9 ) . T h e m a s h e d f i s h f e e d ( a p p r o x . 0 . 0 0 5 mm d i a m . ) i n t r o d u c e d i n t o t h e o y s t e r h o l d i n g t a n k s was 53 p o s t u l a t e d t o i n c r e a s e t h e c o n c e n t r a t i o n o f s u s p e n d e d o r g a n i c s made a v a i l a b l e t o t h e o y s t e r a s a n e n e r g y s o u r c e . I n b i v a l v e s , t h e g i l l s f i l t e r a n d r e m o v e f r o m t h e v e n t i l a t i o n c u r r e n t a l l s u s p e n d e d p a r t i c l e s g r e a t e r t h a n 2 - 5 urn i n d i a m e t e r w i t h 100% r e t e n t i o n e f f i c i e n c y ( J o r g e n s e n , 1 9 7 5 ; M o h l e n b e r g a n d R i i s g a r d , 1 9 7 8 ) . T h e i n c r e a s e d o r g a n i c l o a d i n g a p p e a r e d t o s i g n i f i c a n t l y i n c r e a s e (p< 0 . 0 5 ) c o n d i t i o n i n d i c e s o f e x p e r i m e n t a l o y s t e r s w i t h i n a p e r i o d o f 10 d a y s . T h e r e s u l t s f r o m t h i s s t u d y a r e i n a g r e e m e n t w i t h p r e v i o u s s t u d i e s o n b i v a l v e s ( B a y n e a n d T h o m p s o n , 1 9 7 0 ; G a b b o t t a n d S t e p h e n s o n , 1 9 7 4 ; J o n e s a n d Iwama, 1 9 9 0 ) . S h e l l f i s h i n m o s t p a r t d u e t o t h e i r i n h e r e n t a b i l i t y t o a c c u m u l a t e a v a r i e t y o f o r g a n i s m s c o u p l e d w i t h t h e i r i m p o r t a n c e a s a c o m m e r c i a l f o o d f i s h e r y h a v e b e e n t h e f o c u s o f many b a c t e r i o l o g i c a l s t u d i e s ( C o l w e l l a n d L i s t o n , 1 9 7 1 ; B a r o s s a n d L i s t o n , 1 9 7 0 ; L i p o v s k y , 1 9 7 1 ; L i p o v s k y a n d Chew, 1 9 7 2 ; M e t c a l f e t a l . , 1 9 7 9 ) . I t h a s b e e n a s s u m e d b y many b a c t e r i o l o g i s t s t h a t t h e b a c t e r i a l p o p u l a t i o n s a s s o c i a t e d w i t h a q u a t i c a n i m a l s s u c h a s o y s t e r s , a r e d i r e c t l y r e l a t e d t o t h e b a c t e r i a l c o n t e n t o f t h e s u r r o u n d i n g w a t e r ( C o l w e l l a n d L i s t o n , 1 9 7 1 ) . A c c o r d i n g l y , t h e t y p e s o f b a c t e r i a o c c u r r i n g i n o y s t e r s w i l l v a r y d e p e n d i n g o n t h e f l o r a o f t h e e n v i r o n m e n t . I n t h e c a s e o f f i n f i s h e s i t i s now b e l i e v e d t h a t a d e f i n i t e \" f i s h c o m m e n s a l f l o r a \" e x i s t s . T h e w o r k b y C o l w e l l a n d L i s t o n , (1971) s u p p o r t s t h e v i e w t h a t o y s t e r s do i n f a c t h a r b o r b a c t e r i a l c o m m e n s a l f l o r a , w h i c h i s c o m p o s e d o f t y p i c a l l y m a r i n e p s y c h r o p h y l i c b a c t e r i a w e l l a d a p t e d 54 p h y s i o l o g i c a l l y t o l i f e w i t h i n t h e m i c r o e n v i r o n m e n t o f t h e s h e l l f i s h . I n t h i s s t u d y , t h e b a c t e r i a t h a t f o r m e d t h e g r e a t e s t number o f c o l o n i e s w e r e t h o s e t h a t showed a p a r t i a l o r c o m p l e t e s a l t d e p e n d e n c e d e m o n s t r a t e d b y t h e g r o w t h s t i m u l a t i n g e f f e c t o f t h e T C B S m e d i u m . T h e number o f b a c t e r i a l c o l o n i e s a s s o c i a t e d w i t h a l l t h r e e m e d i a u s e d s i g n i f i c a n t l y d e c r e a s e d ( p < 0 . 0 5 ) a f t e r t h e 10 d a y a n t i b i o t i c u p t a k e p e r i o d . T h e M a c k o n c h e y medium ( a s e l e c t i v e medium f o r b a c t e r i a a s s o c i a t e d w i t h sewage) i n p a r t i c u l a r p r o d u c e d no b a c t e r i a l g r o w t h a f t e r t h e t r e a t m e n t p e r i o d . T h i s w o u l d s u g g e s t t h a t i n i t i a l l y t h e a n t i b i o t i c s i n t r o d u c e d i n t o t h e w a t e r c o l u m n c o u l d h a v e some d e g r e e o f e f f e c t o n t h e c o m m e n s a l f l o r a o f t h e o y s t e r . R e c e n t s t u d i e s h a v e i n d i c a t e d t h a t t h e b a c t e r i a a s s o c i a t e d w i t h s h e l l f i s h w i l l d i s p l a y r e s i s t a n c e t o a n t i b i o t i c s ( L e d o e t a l . , 1 9 8 3 ; K e l l y , 1 9 9 0 ) . 55 GENERAL CONCLUSIONS AND RECOMMENDATIONS T h e H P L C m e t h o d s d e v e l o p e d i n t h i s i n t h i s s t u d y a l l o w f o r s u c c e s s f u l q u a n t i f i c a t i o n o f b o t h o x y t e t r a c y c l i n e a n d R o m e t - 3 0 i n P a c i f i c o y s t e r t i s s u e . T h e a s s a y f o r R o m e t - 3 0 i s p a r t i c u l a r l y a d v a n t a g e o u s i n t h a t i t r e q u i r e s o n l y a s i n g l e e x t r a c t i o n f o r t h e d e t e r m i n a t i o n o f b o t h SDM a n d OMP i n o y s t e r t i s s u e . T h e r e c o v e r i e s o f b o t h SDM a n d OMP f r o m o y s t e r t i s s u e w e r e s i m i l a r t o t h o s e r e p o r t e d f o r s a l m o n i d s . I t i s b e l i e v e d t h a t t h e h i g h p i g m e n t a n d l i p i d c o n t e n t a s s o c i a t e d w i t h t h e o y s t e r t i s s u e was r e s p o n s i b l e f o r t h e l o w e r r e c o v e r y . W i t h r e s p e c t t o t h e w h o l e a n i m a l c o n c e n t r a t i o n s o f a n t i b i o t i c s d e t e c t e d i n t h e o y s t e r s a t t h e s a l m o n f a r m , i t w o u l d a p p e a r t h a t t h e e f f e c t s w e r e l o c a l i z e d t o w i t h i n 200 m o f t h e f a r m s i t e . T h e d i s p e r s a l o f c h e m i c a l s i n t h e m a r i n e e n v i r o n m e n t a r e m o s t l i k e l y s u b j e c t t o s i t e s p e c i f i c h y d r o g r a p h i c c o n d i t i o n s . T h e c o n t r o l l e d u p t a k e a n d c l e a r a n c e s t u d y c o n f i r m e d t h e p r e m i s e o f d r u g u p t a k e a n d b i o a c c u m u l a t i o n i n t h e P a c i f i c o y s t e r . T h e u p t a k e o f o x y t e t r a c y c l i n e s i g n i f i c a n t l y i n c r e a s e d o v e r t i m e p r o d u c i n g a l i n e a r r e l a t i o n s h i p . T h e d e p u r a t i o n o f t h e d r u g i n t h e o y s t e r i s b e s t d e s c r i b e d b y a n a s y m p t o t i c c u r v e , w i t h t h e g r e a t e s t c l e a r a n c e o f d r u g o c c u r r i n g w i t h i n t h e f i r s t 13 d a y s . R o m e t - 3 0 d i d n o t p r o d u c e s u c h a d e f i n e d r e l a t i o n s h i p o f u p t a k e a n d c l e a r a n c e w i t h i n t h e o y s t e r . T h e u p t a k e o f b o t h c o m p o n e n t s o f R o m e t - 3 0 , SDM a n d OMP was s p o r a d i c i n i t s d e t e c t a b l e c o n c e n t r a t i o n s . SDM 56 was c l e a r l y t h e more d e t e c t a b l e o f t h e two t h r o u g h o u t u p t a k e a n d c l e a r a n c e . C l e a r a n c e o f R o m e t - 3 0 was c o m p l e t e b y d a y t h i r t e e n . I t i s r ecommended t h a t i f s h e l l f i s h a r e t o b e h a r v e s t e d w i t h i n 500 m o f a s a l m o n g r o w o u t s i t e t h a t a r e c o r d o f t h e s a l m o n i d d r u g t r e a t m e n t s c h e d u l e be c o n s u l t e d s o a s t o a s s u r e o f s u f f i c i e n t (30 d a y s ) a n t i b i o t i c c l e a r a n c e i n t h e b i v a l v e s . I n a p o l y c u l t u r e s i t u a t i o n , a s i m i l a r p e r i o d o f c l e a r a n c e s h o u l d be o b s e r v e d a n d / o r movement o f t h e s h e l l f i s h t o a c l e a n s i t e t o a l l o w f o r a c o m p l e t e d e p u r a t i o n p e r i o d . 57 R e f e r e n c e s A l d r i c h , J . C . a n d M . C r o w l e y . 1 9 8 6 . C o n d i t i o n a n d v a r i a b i l i t y i n M y t i l u s e d u l i s ( L . ) f r o m d i f f e r e n t h a b i t a t s i n I r e l a n d . A q u a c u l t u r e 5 2 : 2 7 3 - 2 8 6 . A o k i , T . , K i a t o , T . a n d K . K a w a n o . 1 9 8 1 . C h a n g e s i n d r u g r e s i s t a n c e o f V i b r i o a n g u i l l a r u m i n c u l t u r e d a y u , P l e c o g l o s s u s a l t i v e l i s T e m m i n c k a n d S c h l e g e l , i n J a p a n . J . F i s h D i s e a s e s 4 : 2 2 3 - 2 3 0 . B a r o s s , J . a n d J . L i s t o n . 1 9 7 0 . O c c u r e n c e o f V i b r i o p a r a h a e m o l y t i c u s a n d r e l a t e d h e m o l y t i c v i b r i o s i n m a r i n e e n v i r o n m e n t s o f W a s h i n g t o n S t a t e . A p p l . M i c r o b i o l 2 0 : 1 7 9 - 1 8 6 . B a y n e , B . L . , L i v i n g s t o n e , D . R . , M o o r e , M . N . a n d J . W i d d o w s . 1976 a . A C y t o c h e m i c a l a n d B i o c h e m i c a l I n d e x o f S t r e s s i n M y t i l u s e d u l i s . M a r . P o l l . B u l l . 7 : 2 2 1 - 2 2 4 . B a y n e , B . L . a n d R . J . Thompsom. 1 9 7 0 . Some p h y s i o l o g i c a l c o n s e q u e n c e s o f k e e p i n g M y t i l u s e d u l i s i n t h e l a b o r a t o r y . H e l g o l a n d e r w i s s . M e e r e s u n t e r s . 2 0 : 5 2 6 -5 5 2 . 58 B a y n e , B . L . , W i d d o w s , J . W . a n d R . I . E . N e w e l l . 1 9 7 7 . P h y s i o l o g i c a l m e a s u r e m e n t s o n e s t u a r i n e b i v a l v e m o l l u s c s i n t h e f i e l d . I n : B i o l o g y o f b e n t h i c o r g a n i s m s , E d . B . K . K e e g a n , P . O ' C e i d i g h a n d P . J . S . B o a d e n , Pergammon, N . Y . : 5 7 - 6 8 . B o y d e n , C . R . 1 9 7 7 . E f f e c t o f s i z e u p o n m e t a l c o n t e n t o f s h e l l f i s h . J . M a r . B i o l . A s s o c . U K . 5 7 : 6 7 5 - 7 1 4 . B r a n d , A . R . a n d A . C . T a y l o r . 1 9 7 4 . P u m p i n g a c t i v i t y o f A r t i c a i s l a n d i c a a n d some o t h e r common b i v a l v e s . M a r . B e h a v . P h y s i o l . 3 : 1 - 1 5 . B r a t t o n , A . C . a n d E . K . M a r s h a l l . 1 9 3 9 . A new c o u p l i n g c o m p o n e n t f o r s u l f a n i l a m i d e d e t e r m i n a t i o n . J . B i o l . Chem. 1 2 8 : 5 3 7 - 5 5 0 . B r o o k s , R . R . a n d M . G . R u m s b y . 1 9 6 5 . T h e b i o g e o c h e m i s t r y o f t r a c e e l e m e n t u p t a k e b y some New Z e a l a n d b i v a l v e s . L i m n o l . O c e a n o g r . 1 0 : 5 2 1 - 5 2 8 . B r o w n , E . E . a n d J . B . G r a t z e k . 1 9 8 0 . F i s h F a r m i n g H a n d b o o k . T h e A V I P u b l i s h i n g C o . I n c . , C o n n e t i c u t . 3 9 1 p p . 59 B r o w n , J . R . a n d E . B . H a r t w i c k . 1 9 8 8 b . I n f l u e n c e s o f t e m p e r a t u r e , s a l i n i t y a n d a v a i l a b l e f o o d u p o n s u s p e n d e d c u l t u r e o f t h e P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s : I I . C o n d i t i o n i n d e x a n d s u r v i v a l . A q u a c u l t u r e 7 0 : 2 5 3 - 2 6 7 . C o l w e l l , R . R . a n d J . L i s t o n . 1 9 7 1 . M i c r o b i o l o g y o f S h e l l f i s h . B a c t e r i o l o g i c a l s t u d y o f t h e n a t u r a l f l o r a o f P a c i f i c O y s t e r s ( C r a s s o s t r e a g i g a s ) . A p p l . M i c r o b i o l . 8 : 1 0 4 - 1 0 9 . D i S a l v o , L . H . , G u a r d , H . E . a n d L . H u n t e r . 1 9 7 5 . T i s s u e h y d r o c a r b o n b u r d e n o f m u s s e l s a s p o t e n t i a l m o n i t o r s o f e n v i r o n m e n t a l h y d r o c a r b o n i n s u l t . E n v i r o n . S c i . T e c h n o l . 9 : 2 4 7 - 2 5 1 . F o n g , W . C . 1 9 7 6 . U p t a k e a n d R e t e n t i o n o f K u w a i t C r u d e o i l a n d i t s E f f e c t s o n O x y g e n U p t a k e b y t h e S o f t S h e l l C l a m , Mya a r e n a r i a . J . F i s h R e s . b d . C a n . 3 3 : 2 7 7 4 - 2 7 8 0 . F o s s a t o , V . U . a n d W . J . C a n z o n i e r . 1 9 7 6 . H y d r o c a r b o n u p t a k e a n d l o s s b y t h e m u s s e l M y t i l u s e d u l i s . M a r . B i o l . 3 6 : 2 4 3 - 2 5 0 . F r i e d r i c h , A . R . a n d F . P . F i l i c e . 1 9 7 6 . U p t a k e a n d a c c u m u l a t i o n o f t h e n i c k e l i o n b y M ; y t i l u s e d u l i s . B u l l . E n v i r o n . C o n t a m . T o x i c o l . 1 6 : 7 5 0 - 7 5 5 . 60 G a b b o t t , P . A . a n d R . R . S t e p h e n s o n . 1 9 7 4 . A n o t e o n t h e r e l a t i o n s h i p b e t w e e n t h e d r y w e i g h t c o n d i t i o n i n d e x a n d t h e g l y c o g e n c o n t e n t o f a d u l t o y s t e r s ( O s t r e a e d u l i s L ) k e p t i n t h e l a b o r a t o r y , j . C o n s . i n t . E x p l o r . M e r . 3 5 : 3 5 9 - 3 6 1 . H a r a , T . a n d S . I n o u e . 1 9 6 7 . C o n c e n t r a t i o n s o f V a r i o u s S u l f o n a m i d e s i n t h e T i s s u e s o f R a i n b o w T r o u t a f t e r O r a l D o s a g e . B u l l . J p n . S o c . S c i . F i s h . 3 3 : 6 1 8 . H a r a , T . , I n o u e , S . a n d N . S a i t o . 1 9 6 7 . C o n c e n t r a t i o n s o f V a r i o u s S u l f o n a m i d e s i n t h e T i s s u e s o f R a i n b o w T r o u t a f t e r O r a l D o s a g e o f t h e S o d i u m S a l t s . B u l l . J p n . S o c . S c i . F i s h . , 3 3 : 6 2 4 . H e r w i g , N . 1 9 7 9 . H a n d b o o k o f D r u g s a n d C h e m i c a l s U s e d i n T r e a t m e n t o f F i s h D i s e a s e s . C h a r l e c C . T h o m a s , I l l i n o i s . 2 7 2 p p . J a c o b s e n , M . D . 1 9 8 9 . W i t h d r a w l t i m e s o f f r e s h w a t e r r a i n b o w t r o u t , S a l m o g a i r d n e r i R i c h a r d s o n , a f t e r t r e a t m e n t w i t h o x o l i n i c a c i d , o x y t e t r a c y c l i n e a n d t r i m e t o p r i m . J o u r n . o f F i s h D i s e a s e s 1 2 : 2 9 - 3 6 . J a c o b s e n , P . a n d L . B e r g l i n d . 1 9 8 8 . P e r s i s t e n c e o f O x y t e t r a c y c l i n e i n S e d i m e n t s f r o m F i s h F a r m s . A q u a c u l t u r e 7 0 : 3 6 5 - 3 7 0 . 61 J o n e s , T . O a n d G . K . Iwama, 1 9 9 0 . P o l y c u l t u r e o f t h e P a c i f i c O y s t e r , C r a s s o s t r e a g i g a s ( T h u n b e r g ) w i t h C h i n o o k S a l m o n , O n c o r h y n c h u s t s h a w y t s c h a . A q u a c u l t u r e : I n P r e s s J o r g e n s e n , C . B . 1 9 7 5 . On g i l l f u n c t i o n i n t h e m u s s e l , M y t i l u s e d u l i s . O p h e l i a , 1 3 : 1 8 7 - 2 3 2 . K e c k , G . , M a r t e l l , C . a n d J . P . G e r a r d . 1 9 8 4 . P h a r m a c o l o g i e d e s p o i s s o n s . R e v u e de M e d e c i n e V e t e r i n a i r e , 1 3 3 : 2 1 7 -2 2 8 . K e l l y , M . 1 9 9 0 . P e r s o n a l c o m m u n i c a t i o n . D e p a r t m e n t o f M i c r o b i o l o g y , S h a u g h n e s s y H o s p i t a l , U n i v e r s i t y o f B r i t i s h C o l u m b i a . L e d o , A . , G o n z a l e z , E . , B a r j a , J . L . a n d A . E . T o r a n z o . 1 9 8 3 . E f f e c t o f D e p u r a t i o n o n t h e R e d u c t i o n o f B a c t e r i o l o g i c a l I n d i c a t o r s i n C u l t u r e d M u s s e l s ( M y t i l u s e d u l i s ( L i n n a e u s ) ) . J o u r n . o f S h e l l f i s h R e s . 3 : 5 9 - 6 4 . L i p o v s k y , V . P . 1 9 7 1 . M o r t a l i t y o f P a c i f i c o y s t e r s , C r a s s o s t r e a g i g a s ( T h u n b e r g ) . I . T h e i n f l u e n c e o f T e m p e r a t u r e a n d E n r i c h e d S e a w a t e r o n t h e O y s t e r -B a c t e r i a R e l a t i o n s h i p . M a s t e r s T h e s i s U n i v . W a s h . , C o l l . F i s h . , S e a t t l e , W a h i n g t o n : 4 5 p p . 62 L i p o v s k y , V . P . a n d K . K . Chew. 1 9 7 2 . M o r t a l i t y o f P a c i f i c O y s t e r s ( C r a s s o s t r e a g i g a s ) : T h e I n f l u e n c e o f T e m p e r a t u r e a n d E n r i c h e d S e a w a t e r o n O y s t e r s u r v i v a l . P r o c . N a t . S h e l l F i s h . A s s o c . 6 2 : 7 2 - 8 0 . L i t t l e , M . 1 9 9 0 . P e r s o n a l c o m m u n i c a t i o n . S y n d e l L a b o r a t o r i e s L t d . V a n c o u v e r , B . C . L o o s a n o f f , V . L . a n d F . D . T o m m e r s . 1 9 4 8 . E f f e c t o f s u s p e n d e d s i l t a n d o t h e r s u b s t a n c e s o n t h e r a t e o f f e e d i n g o f o y s t e r s . S c i e n c e 1 0 7 : 6 9 - 7 0 . L o w e , D . M . a n d M . N . M o o r e . 1 9 7 9 . T h e c y t o c h e m i c a l d i s t r i b u t i o n o f z i n c ( Z n l l ) a n d i r o n ( F e l l l ) i n t h e common m u s s e l , M y t i l u s e d u l i s . a n d t h e i r r e l a t i o n s h i p w i t h l y s o s o m e s . J . M a r . B i o l . A s s . U . K . 5 9 : 8 5 1 - 8 5 8 . L u c a s , A . a n d P . G . B e r r i n g e r . 1 9 8 5 . T h e u s e o f p h y s i o l o g i c a l c o n d i t i o n i n d i c e s i n m a r i n e b i v a l v e a q u a c u l t u r e . A q u a c u l t u r e 4 4 : 1 8 7 - 2 0 0 . M a n n , R . , 1 9 7 8 . Some b i o c h e m i c a l a n d p h y s i o l o g i c a l a s p e c t s o f g r o w t h a n d g a m e t o g e n e s i s i n C r a s s o s t r e a g i g a s a n d O s t r e a e d u l i s g r o w n a t s u s t a i n e d e l e v a t e d t e m p e r a t u r e s . J . M a r . B i o l . A s s o c . U . K . 5 8 : 9 5 - 1 1 0 . 63 M c C a r t h y , D . H . , S t e v e n s o n , J . P . a n d A . W . S a l s b u r y . 1 9 7 4 . A C o m p a r a t i v e P h a r m a c o - K i n e t i c S t u d y o f t h e S u l p h o n a m i d e s a n d a S u l p h o n a m i d e P o t e n t i a t o r , T r i m e t h o p r i m i n R a i n b o w T r o u t . A q u a c u l t u r e 4 : 2 9 9 - 3 0 3 . M c C r a c k e n , A . , F i d g e o n , S . , O ' B r i e n , J . J . a n d D . A n d e r s o n . 1 9 7 6 . A n I n v e s t i g a t i o n o f A n t i b i o t i c a n d D r u g R e s i d u e s i n F i s h . J o u r n a l A p p l . B a c t . 4 0 : 6 1 . M e t c a l f , T . G . , B . M u l l i n , D . E r i c k s o n , E . M o u l t o n a n d E . L a r k i n . 1 9 7 9 . B i o a c c u m u l a t i o n a n d d e p u r a t i o n o f e n t e r o v i r u s e s b y t h e s o f t - s h e l l e d c l a m Mya a r e n a r i a . A p p l . E n v i r o n . M i c r o b i o l . 3 8 : 2 7 5 - 2 8 2 . M o h l e n b e r g , F . a n d H . U . R i i s g a r d . 1 9 7 8 . E f f i c i e n c y o f p a r t i c l e r e t e n t i o n i n 13 s p e c i e s o f s u s p e n s i o n f e e d i n g b i v a l v e s . O p h e l i a 1 7 : 2 3 9 - 2 4 6 . M o r i , K . 1 9 7 9 . E f f e c t s o f A r t i f i c i a l E u t r o p h i c a t i o n o n t h e M e t a b o l i s m o f t h e J a p a n e s e o y s t e r C r a s s o s t r e a g i g a s . M a r i n e B i o l o g y . 5 3 : 3 6 1 - 3 6 9 . 64 N e i l s o n , B . J . , H a v e n , D . S . , P e r k i n s , F . O . , M o r a l e s - a l a m o , R . , a n d M . W . R h o d e s . 1 9 7 8 . B a c t e r i a l d e p u r a t i o n b y t h e A m e r i c a n o y s t e r C r a s s o s t r e a v i r g i n i c a u n d e r c o n t r o l l e d c o n d i t i o n s . I I . P r a c t i c a l c o n s i d e r a t i o n s a n d p l a n t d e s i g n . V a . I n s t . M a r . S c i . S p e c . R e p . N o . 8 8 : 4 8 p p . O k a , H . , U n o , K . , H a r a d a , K . a n d M . S u z u k i . 1 9 8 3 . I m p r o v e m e n t o f C h e m i c a l a n a l y s i s o f A n t i b i o t i c s . I I . E v a l u a t i o n o f P r e p a c k e d C 1 8 C a r t r i d g e f o r E x t r a c t i o n o f T e t r a c y c l i n e s . Y a k u g a k u Z a s s h i . 5 : 5 3 1 - 5 3 7 . P h i l l i p s , D . J . H . 1 9 7 6 . T h e common m u s s e l , M y t i l u s e d u l i s , a s a n i n d i c a t o r o f p o l l u t i o n b y z i n c , c a d m i u m , l e a d a n d c o p p e r . I . E f f e c t s o f e n v i r o n m e n t a l v a r i a b l e s o n u p t a k e o f m e t a l s . M a r . B i o l . 3 8 : 5 9 - 6 9 . P r i n g l e , B . H . , H i s s o n g , D . E . , K a t z , E . L . a n d M . G . M u l a w k a . 1 9 6 8 . T r a c e m e t a l a c c u m u l a t i o n b y e s t u a r i n e m o l l u s k s . J . S a n i t . E n g . D i v . , Am. S o c . C i v i l E n g . 9 4 : 4 5 5 - 4 7 5 . R i s e b r o u g h , R . W . , D e L a p p e , B . W . a n d T . T . , S c h m i d t . 1 9 7 6 . B i o a c c u m u l a t i o n f a c t o r s o f c h l o r i n a t e d h y d r o c a r b o n s b e t w e e n m u s s e l s a n d s e a w a t e r . M a r . P o l l . B u l l . 7 : 2 2 5 -2 2 8 . R o g s t a d , A . , H o r m a z a b a l , V . a n d M . Y n d e s t a d . 1 9 8 8 . O p t i m i z a t i o n o f s o l i d p h a s e e x t r a c t i o n o f o x y t e t r a c y c l i n e f r o m f i s h t i s s u e a n d i t s d e t e r m i n a t i o n b y H P L C . J o u r n a l o f L i q u i d C h r o m a t o g r a p h y 1 1 : 2 3 3 7 - 2 3 4 7 S a l t e , R . a n d K . L i e s t o l . 1 9 8 3 . D r u g w i t h d r a w a l f r o m f a r m e d f i s h . A c t a V e t e r i n a r i a S c a n d i n a v i c a 2 4 : 4 1 8 - 4 3 0 . S a m u e l s e n , O . , T o r s v i k , V . , H a n s e n , P . K . , P i t t m a n , K . a n d A E r v i k . 1 9 8 8 . O r g a n i c W a s t e a n d A n t i b i o t i c s F r o m A q u a c u l t u r e . I C E B S , C M . 1988 F : 1 4 , S e s s . W. M a r i c u l t u r e C o m m i t t e e . S i m p s o n , R . D . 1 9 7 9 . U p t a k e a n d L o s s o f Z i n c a n d L e a d b y M u s s e l ( M y t i l u s e d u l i s ) a n d R e l a t i o n s h i p s w i t h b o d y W e i g h t a n d R e p r o d u c t i v e c y c l e . M a r . P o l l . B u l l . 1 0 : 7 4 -7 8 . S n i e s z k o , S . F . a n d S . B . F r i d d l e . 1 9 5 1 . F u r t h e r S t u d i e s o n F a c t o r s D e t e r m i n i n g T i s s u e L e v e l s o f S u l f a m e r a z i n e i n T r o u t . T r a n s . Am. F i s h . S o c . 8 1 : 1 0 1 . S o u n e s s , R . A . , B o w r e y , R . G . a n d G . H . F l e e t . 1 9 7 9 . C o m m e r c i a l d e p u r a t i o n o f t h e S y d n e y r o c k o y s t e r C r a s s o s t r e a c o m m e r c i a l i s . F o o d T e c h n o l . A u s t . 3 1 : 5 3 1 - 5 3 7 . 66 S t e n n e r , R . D . a n d G . N i c k l e s s . 1 9 7 4 . D i s t r i b u t i o n o f some h e a v y m e t a l s i n o r g a n i s m s i n H a r d a n g e r f j o r d a n d S k j e r s t a d f j o r d , N o r w a y . W a t e r A i r S o i l P o l l u t . 3 : 2 7 9 -2 9 1 . T a k a s h i m a , N . , A o k i , T . a n d T . K i a t o . 1 9 8 5 . E p i d e m i o l o g i c a l s u r v e i l l a n c e o f d r u g - r e s i s t a n t s t r a i n s o f P a s t e u r e l l a p i s c i c i d a . F i s h P a t h o l o g y 2 0 : 2 0 9 - 2 1 7 . T e n o r e , K . R . a n d W . M . D u n s t a n . , 1 9 7 3 . C o m p a r i s o n o f f e e d i n g a n d b i o d e p o s i t i o n o f t h r e e b i v a l v e s a t d i f f e r e n t f o o d l e v e l s . M a r . B i o l . 2 1 : 1 9 0 - 1 9 5 . T h i S o n , N . a n d G . H . F l e e t , 1 9 8 0 . B e h a v i o u r o f p a t h o g e n i c b a c t e r i a i n t h e o y s t e r , C r a s s o s t r e a c o m m e r c i a l i s d u r i n g d e p u r a t i o n , r e l a y i n g a n d s t o r a g e . A p p l . E n v i r o n . M i c r o b i o l . 4 0 : 9 9 4 - 1 0 0 2 . W a l n e , P . R . 1 9 7 2 . T h e i n f l u e n c e o f c u r r e n t s p e e d , b o d y s i z e a n d w a t e r t e m p e r a t u r e o n t h e f i l t r a t i o n r a t e o f f i v e s p e c i e s o f b i v a l v e s . J . M a r . B i o l . A s s o c . U . K . 5 2 : 3 4 5 -3 7 4 . W a l i s s e r , J . A . , H . M . B u r t , D . D . K i t t s , T . A . V a l g a n d K . M . M c E r l a n e . 1 9 9 0 . H P L C A n a l y s i s o f R o m e t - 3 0 R i n s a l m o n f o l l o w i n g a d m i n i s t r a t i o n o f m e d i c a t e d f e e d . J o u r n a l o f C h r o m a t o g r a p h y : I n P r e s s . 67 A P P E N D I X 68 A p p e n d i x 1 OTC E x t r a c t i o n s - U.B.C. 69 70 71 72 7 73 74 75 76 77 78 79 80 Appendix 2 ROMET-30 E x t r a c t i o n s - U.B.C. 82 -2ER0 iCfcO 2.\u00C2\u00AB\u00C2\u00BB U ? tftl i 86 89 \u00E2\u0080\u00A2 Si 1 \u00C2\u00A3 ! O 6 O \u00C2\u00BB 2 I T \u00E2\u0080\u0094 \u00C2\u00BBs. I , 2 i iliiiiiiiiiiliii III I - \u00E2\u0080\u0094 - = \u00C2\u00AB - - \u00E2\u0080\u00A2 - = ?0 91 r 92 I 4.8 CHROMA TOG RAN i ncnOfti2CD CHfcOtlATOPAC C-ftfcA TILE 0 no b DC THOli iCFOfcT no 169 PKHO Tint AREA UK IDHO CUMC 1 7.6809 2 6.872 1 1622 0.4324 3 9.638 2147650 83.3901 4 212649 8.2767 TOTAL 2369263 180 START 6A/0I/90STOP 06i401 01 CHROnAIOGRAfl 2 flEMORUEB UAfcntMTttO PEftK TirtEI-'UMhOlOdi * AITCH<8>-3 J ANAL I . / |08^ 01/90 16t 06i 38 3 9.630 94 X t W M u w a a. 95 CHROtlf lTOPAC C - R 6 A S A M P L E no a R E P O R T MO 112 PKNO I 2 3 T i n e 5.633 9. 632 12.187 13.928 22.732 A R E A NK I D N O F I L E METHOD C O M C 41 M A R E T O T A L -TEN<3>=3 AMAL 00 '0' /99 219324 2263413 13234 13324 225567 2724336 16:39:34 6. 998 7. 825 7.7835 83. 1472 3.3764 9.439 8.2739 199 3. 667 \"] 9 . 6 5 3 =\u00C2\u00BB 22 .775 CHROflfrTOPAC C-R6A SANPLE (10 9 REPORT NO 113 \u00E2\u0080\u0094 F I L E \" \"' METHOD 41 P K N O TIME A R E A MK IDNO CONC N A M E 667 998 633 775 197211 12169 2147367 212442 7. 676 9.4733 33.5313 3.2639 96 START STftRT 98 26 . a CHROHATOPAC c -SAtiPLE NO e REPORT HO 638 F 1LE IIETHOb PKNO T 1IIC AREA IIK 1 tillO 1 4 . 9 3 3 5278 7 9851 2 \u00C2\u00A3 . 0 2 9 9722 14 7882 3 6 . 9 8 3 16623 V 25 1483 4 7 . 6 0 2 7742 V 1 1 7123 5 10 .142 9812 14 844 6 1 1 . 7 5318 8 8451 ? 2 6 . 6 3 1 1605 17 55? TOTAL 66098 108 STOP.Th<8 )=3 l START 9 6 / 8 5 / 6 ? . 928 5? 888 1 7 . 6 3 5 VO REPORT HO 63c. AREA It K IDHO 1 4 .952 3979 18. 0o67 2 6 .862 7*54 : r. 9> ^ \"\u00C2\u00BB \u00C2\u00BB-) r> \u00C2\u00BB is, ff> \u00E2\u0080\u00A2\u00C2\u00BB \u00E2\u0080\u0094 \u00E2\u0080\u0094 .7. f> o i> r> \u00C2\u00AB r\u00C2\u00BB \u00C2\u00AB I \u00E2\u0080\u00A2 \u00C2\u00AB i .j J ^ K- ^ U X \u00E2\u0080\u00A2_>\" o X > > > > > w \u00E2\u0080\u0094 \"J - 0> \u00E2\u0080\u0094 T III t I\I \u00C2\u00AB i \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 \u00E2\u0080\u0094 _ \u00E2\u0080\u0094 _ T =* ^ ir>'T><\u00C2\u00BBfs,isirtrvto'\u00C2\u00AB\u00C2\u00BB ^ O X X - - - i: 1 , n \u00C2\u00BB to 11 2 3 II 14 12 13 14 . IS TINE S. 192 3.627 6.82 7.412 8.17 .383 .313 267 387\u00E2\u0080\u009E 112 .588 .327 .723 .6 .86 TOTAL START 88/88/81 AREA UK IDNO 14372 6462 19115 24423 15372 3565 4411 19678 \u00E2\u0080\u009E,I3143 36378 13671 5369 21683 28565 I 1252 \u00E2\u0080\u00A2 i o l i t '\u00E2\u0080\u00A2 CONC 6.6327 2.7214 8.8499 18.2833 . 6.4736 > 1.5614 1.8573 8.2873 , 3.5332,, 15.317 5.7576 2.2612 9.1317 12.6297 4.7383 NANE 237451 188 92144123 .733 o 00 \u00E2\u0080\u00A2 1H. 11 <\u00C2\u00AB -.- - S t ' i ! i l l \u00E2\u0080\u00A2i,l ' \u00E2\u0080\u00A2ii.iM-?i CHROHATOPAC C-R6A SAMPLE REPORT NO 0 NO 658 METHOD 41 PKHO TIME AREA MK IDNO CONC NAME 1 e 3 6.218 6.717 21.937 3336 7743 V 219336 1 . 4 4 4 3.3373 9 3 . 1 9 8 3 : TOTAL START 98/08/08 236629 2 3 1 3 4 1 5 9 108 i 28.373 v O VO 110 WWW '?\u00C2\u00BB If! I N 1 : \u00E2\u0080\u00A2m i i \u00E2\u0080\u00A2 . , , , 1 - . , . , CHRONATOPAC j 1 SAMPLE NO 0 NETHOD r *. REPORT NO 632 >\u00E2\u0080\u00A2 J. PKNO TINE AREA NK \u00E2\u0080\u00A2UNO CONC 1 3.233 18693 9.3887 .' \u00C2\u00A3 3.92 7823 V 6.8694 ; ' i 3 6.927 18418 16.1614 4 7.483 9822 V 8.6221 5 8.268 71 IS 6.2463 : 6 11.383 6843 6.6667 i i 7 12.467 6531 3.7333 9 13.34 12699 V 11.3233 9 18.612 24467 21.4791 i s 24.863 9304 8.1673 TOTAL 113912 186 INANE START e e / B O ' e i IN .#r,an 11(U-am i i si.ii , i ; i 6 S I 5 8 1 4 3 CHRONATOPAC C-R6A SAHPLE NO 8 REPORT NO 657 FILE METHOD 41 1 5. 15? 3843 4.3414 2 6.63 16366 18.4211 3 7.382 2486 V 2.717? 4 8.18 3233 3.6521 5 8.645 5167 V 5. 8378 6 11.367 13827 15.6281 7 13.892 3963 4.412 8 21.623 39831 44.9978 TOTAL 88517 START 86/88/61 03148132 \u00C2\u00A3 3 M 5- f! - - f l i 7 i \u00C2\u00BB \u00C2\u00AB 0 \u00C2\u00BB 1 \ \u00C2\u00BB l | O ( I ) -n 4 \u00C2\u00ABi n \u00C2\u00AB> m IS. rtj \u00E2\u0080\u0094 \u00E2\u0080\u0094 4\u00C2\u00BB< \u00C2\u00AB C 0. E 115 l i s A p p e n d i x 3 OTC E x t r a c t i o n s - S y n d e l 117 Sample: o t c - 5 Day 7 C h a n n e l : 353 ni A c q u i r e d : 09-APR-90 13:59 Method: C:\BASE\DATA\OTC P i l e n a o e : o t c - 5 - \" O p e r a t o r : DDV 1.97 2.35 3.33 \u00C2\u00AB.38 \u00C2\u00A9 i t e r a t e . 118 S a a p l e : :an A c h a n \u00E2\u0080\u009E e J . 3 5 3 A c q u i r e d : 09-JPB-90 14:40 Method: C:\BASE\DATAVOTC hj S. 1 .3' 3 t u - 2.43 P i l e n a i e : conA O p e r a t o r : DDV 3.31 c 119 o S a n p l e : con 3 C h a n n e l : 153 an P i l e n a i e : confl S q u i r e d : 09-APS-90 14:49 Method: C:\BASE\DATJ\OTC O p e r a t o r : DOV C o i i e n t s : O i y t e t . C o n t r o l 5< 1 Q \" 4 v c l t s 0.97 - 1.96 - 2.43 S a i p l e : con G C h a n n e l : 353 ta P i l e n a u e : conG A c q u i r e d : 09-APB-90 14:58 Method: C:\BASE\DATA\OTC O p e r a t o r : DDV C o n t e n t s : O i y t e t . C o n t r o l i< i O - 1 WO I tS Sample: con L C h a n n e l : 353 m U'l a 1.36 SaopJe: can .1 C h a n n e l : P i l e n a u e : conN O p e r a t o r : DDV - 0.75 g g to\" .\"o 1 t s = , - 1.96 .\".--=\u00E2\u0080\u00A2 - 2 . 4 3 \u00E2\u0080\u00A2 = \u00E2\u0080\u00A2 - 3.04 3 -I \u00E2\u0080\u00A2s 1 123 C h a n n e l : 353 nil -;l , ... Hethod: C M B m i M W O T C O p i r a t o r l D M ne l f i \" l j j _ M .4. \u00E2\u0080\u009E 124 S a n p i e : otc-5/3-0-2 C h a n n e l : 353 n i A c q u i r e d : 09-AP8-90 13:31 Method: C:\8ASE\DATA\0TC F i l e n a m e : o i - 5 - 1 0 2 O p e r a t o r : ODV - 1 \j 1 t s 0.62 r n a 1.96 2.43 3.03 -,_ 4.39 in a 125 S a i g l e : o i y t s t r a c y e ' , i n e A c q u i r e d : 19 - *P R-90 t : i < I n j V e t : 25.00 C h a n n e l : 3S3 m Method: C:\BASE\DATA\0TC F i l e n a i e : o t c s t d O p e r a t o r : TOJ >< l O -1 I t s O ' O 03 o 03 Ul i CO o CO Ul o o o 1.36 3 \u00C2\u00A3 a 4. l.M ___ * ~ 4.56 o t c 01 O\" O S a i p l e : o t c 3 C h a o a e l : 353 n i F i l e n a a e : o t c B A c q u i r e d : 09-AP9-90 13:11 Method: C:\3ASS\DATA\0TC O p e r a t o r : DDV N * U f i j S \u00C2\u00A3 2 5 '10 in - 0.44 1.97 ' _ 4.40 o a o [ i i e : s i s '. A c q u i r e d : 09-At-K- Pinnae:: ;jj \ia S e t o o d : C:>3AS\u00C2\u00A3\a*T4\i)IC 127 r u e n a i e : occ'\u00C2\u00BB O p e r a t o r : DDV :T3 '-> _ 1. 2.44 3.04 4.43 128 S a i o l e : a t e J C h a n n e l : :53 ns A c q u i r e d : \u00E2\u0080\u00A2H-APMQ U:*4 Method: C: 13ASE\"-OATAVQTC F i l e n a i e : H:E O p e r a t o r : ODV 2.44 iti '-> Ml 4.43 01 S a a p l e : o t c ? C h a n n e l : 353 .ia A c q u i r e d : 09-APR-9I) 12:03 Hethod: C:\BASE\DATAWC ^ P i l e n a i e : o t c ? O p e r a t o r : ODV 0.38 . - 1.97 2.43 so S3 _ 4.41 S a o p i e : nz I o r J Channel! 133 am \u00C2\u00BB>i.\u00C2\u00BB.., \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 A c q u i r e d : 09-APR-90 11:34 Method: C:\BASE\DATA\OTC J ! ! \u00C2\u00B1 ' C o a i e n t s : S a i o l e I o r J u p e r a t o r . DPV S a i p i e : o t c I C h a n n e l : 353 na F i l e n a u e : o t s J A c q u i r e d : Q9-APR-90 11:54 Method: C:\3ASE\DATA\0TC O p e r a t o r : DDV I D ' \" 1 . e a t s ^ r* r-j hJ ixi \"lij \u00C2\u00BB\u00E2\u0084\u00A2i I in j f\ jaw i Q O 5 O S O 132 Sampler see - \u00C2\u00AB J C h a n n e l : 353 u ' i l e n a a e 1 o c c ' o r ' A c q u i r e d : 09-APP.-90 11:34 Method: C : \u00C2\u00BB M S S \u00C2\u00BB 0 \u00C2\u00BB T \u00C2\u00BB \u00C2\u00BB O T C O p e r a t o r : C o m e n t s : S a n n l e L o r J % \u00E2\u0080\u0094 % o-t\" i r 1.90 . 2.33 - 2. 4.34 133 Sample: o t c M C h a n n e l : 353 n i A c q u i r e d : Q9-APR-90 13:02 Method: C:\8ASE\DATA\OTC P i l e n a u e : otcM O p e r a t o r : DOV tjj 9' ;*~t \" r\u00E2\u0084\u00A21 3 Its O 0.62 a 1.97 - 2.43 ._ 4.40 3.02 S a BPie: O - l - i O l ' C h a n n e l : !53 m A c q u i r e d : 16-APR-90 3:5? Method: C:\BASt\0ATA\OTC In j V o l : 25.00 f i i t n a i e : O - l - l O l O p e r a t o r : IOJ x l O \" 1 u o l t s >-\u00C2\u00BB H \u00C2\u00BB NJ M W b In b In b o o o o o _l , I , I 1 1 i L . S a i p U : 0-1 -201 C h a n n e l : 353 n i A c q u i r e : t l - A H - M Him Method: C:\3ASE\0ATA\OTC I o j V o l : 25.30 F i U n a i e : 0-1-201 O p e r a t o r : T0J x 1 Q \" 1 u o l t s a c a ro O a 05 O 03 a ro a o _ L _ - 0.13 M ( i 3 C 136 S a a p l e : 0 -1 - J01 C h a n n e l : !S3 n i A c q u i r e d : :S-APft-90 1 0 : U K e t h o d : C:\BASE\OATA\QTC I n j V o l : 25.00 F i l e n a i e : 0-1-301 O p e r a t o r : T0J l O - 1 v o 1 t s a \u00E2\u0080\u00A2 a a a in a a a _ i _ M in a b a in a 0.23 M a-a c i x Lr \u00E2\u0080\u00A2- 1M 3.0J 2 % a-\u00C2\u00AB..*! oic CD b -S a i p l e : D - l - k O I A c q u i r e d : 16-APB-90 I n j V o l : 25.90 10:27 C h a n n e l : !S3 na Method: C:\8ASE\0ATA\QTC F i l e n a i e : 0 - l - t O l O p e r a t o r : TOJ S a i p l e : 0-1-501 C h a n n e l : !S3 n i f i l e n a i e : 0-1-501 A c q u i r e d : I5-APJ-9D 10:35 Method: C:\oASt\0ATA\0TC O p e r a t o r : TOJ I n j V o l : 25.00 x 1 Q \" 1 u o l t s >* M rO b In b In o o a o j , i , 1 . 1 139 S a i p l e : 0 - 0 - 5 ' A c q u i r e d : I S-AP8-90 In) v o l : 25.00 C h a n n e l : 1 5 2 n i 3 : 2 5 Method: C : \ B A S E \ 0 A T A \ O T C F i l e n a i e : 0 O S O p e r a t o r : TOJ l O - 1 u o I t s O a ui o r j a a _ L _ In o 2.07 3 . 1 9 -~j - k.85 o t c r \ 140 S a i p i e : 0-O-C A c q u i r e d : l6-APE-30 3:36 I n j V o l : li.it a a a \u00E2\u0080\u00A2 a O 1 o Ln o _ i _ C h a n n e l : 3S3 n i K e t h o d : C:\BASE\DATA\OTC l O \" 1 u o l t s b o in o _ L _ F i l e n a i e : DOC O p e r a t o r : I0J a .- 3.15 3 3 A 5. o-05 b \u00E2\u0080\u00A2 a K 11/ I S - \u00C2\u00BB.57 o t e i; I 141 S u p ! * : o-0-0 A c q u i r e d : tS-APR-30 I n j V o l : 25.00 C h a n n e l : 353 ,m Method: C : \ B A S E \ 0 A T A \ 0 T C F i l e n a i e : 000 O p e r a t o r : TOJ l O - 1 U Q 1 -ts O ' o rj a-a a a in o a a rj 0 1 o 3 ^ ro O 05 a \u00E2\u0080\u00A2 o 3.10 1.52 o t c 142 S a i p l e : 0-O-E A c q u i r e d : I J - A P I -I n j V o l : 25.00 90 9:05 C h a n n e l : 153 i t i Method: C:\SASE\0ATA\0TC l O - 1 <~>o I t s f i l e n a i e : Out O p e r a t o r : TOJ O \" a a a in a w a o In a 143 S a i o i e : 0-13-0-2\u00E2\u0080\u00941 A c q u i r e d : 19-APB-90 In] V o l : 2S.0O 10:23 C h a n n e l : 353 n i Method: C:\BASE\0ATA\0TC l O - 1 v o 1 t s F i l e n a i e : 013-O21 O p e r a t o r : T0J a a in a a a a\" a r o In a to a a rj a 9.40 7 1 . 3 8 > - 2 . U . - 3 . 0 3 ( - k.52 o t e 144 S a i p l e : 0-13-0-2-2 A c q u i r e d : I9-APR-90 10:33 I n j v o l : 25.00 C h a n n e l : 353 \u00C2\u00AB\u00E2\u0080\u00A2 Method: C : \ B A S E \ D A T A \ 0 T C F i l e n a i e : 013-022 O p e r a t o r : TOJ x l O rO - 1 in a a a u o 1 t s rO in a i CO a a 10 in a _ L _ 1 . 9 1 > - 2 . H _ 3.03 Hi'~ k.ss o t c 145 S a i p l e : 0-13-0-2-3 C h a n n e l : 353 n i F i l e n a i e : 013-023 A c q u i r e d : IS-API-JO I 0 : U Method: C:\8ASE\0ATA\0TC O p e r a t o r : TOJ I n j V o l : 25.00 x l O - 1 u o l t s \u00E2\u0080\u00A2* >\u00E2\u0080\u00A2\u00C2\u00BB M IM CJ a 03 b -a S a i p l e : 0 - U - 0 - 2 - * C h a n n e l : !53 m A c q u i r e d : 19-APR-90 10:55 Method: C:\BASE\0ATA\0TC I o j V o l : 25.00 F i l e n a i e : D13-02k O p e r a t o r : iOJ 147 Sample: 0-13-0-2-5 C h a n n e l : 353 n i A c q u i r e d : I9-APP.-90 11:05 Method: C:\BASE\0ATA\OTC I n j V o l : 25.00 F i l e n a i e : 013-025 O p e r a t o r : TOJ x l O - 1 \"JO 1 t s O \" O rj a-a 3 \u00E2\u0080\u00A2=\u00C2\u00BB to o Ul ~ ~ 1.91 s Ul O a a ro In a _ L _ to b a 0.9S 3.05 K o t e 03 b -a 148 a ~ a M a -a S a i p l e : 5 - I 3 - 0 - ! A c q u i r e d : 19-APR-90 I n j V o l : 25.00 Q O 3:J0 in O C h a n n e l : Method: l O C:\8ASE\0ATA\0TC - 1 <~>o 1 t s a a _ i_ In a _ i_ F i l e n a i e : 513-0-1 O p e r a t o r : TOJ CO a a if \u00C2\u00B0 in ._ 1 . 3 7 - 3 . 0 3 o t c 0) 149 S a i p i e : 0-13-0-3 C h a n n e l : 353 na 'inTvol- itu'** 9:53 Utii0i'' : : m i ^ m \ ^ f i l e n a i e : 013-0-3 O p e r a t o r : TOJ Q a in a M a a in a _ L _ 1 h ii 1 . 3 9 :.\u00C2\u00ABs 3 . 0 3 i / \u00C2\u00BB.S\u00C2\u00AB o t c 150 S a i o l e : 0-13-C-2 A c q u i r e d : I9-AP9-90 I n j V o l : 25.00 10:13 C h a n n e l : Method: l O 353 n i C:\8ASE\0AIA\0TC F i l e n a i e : 013-O p e r a t o r : T0J - 1 v o 1 t s o a a O o _1_ b a _ i _ a a _i b -a v . 1.98 \u00E2\u0080\u00A2 2.4! 3.9* 5. o- 1 cn 151 S a i p l e : 0 -I3-C-! A c q u i r e d : I9-AP8-90 I n j V o l : 25.JO 10:03 C h a n n e l : 353 n i Method: C : \ 8 A S E \ D A T A \ 0 T C F i l e n a i e : 0 1 3 - C - i O p e r a t o r : TOJ l O - 1 <_>o I t s a a a' o M a 3 if \u00C2\u00B0 Ml b a CO b a _ i _ a a 1 . 3 8 . _ 3 . 0 3 152 S a i o l e : 0-30-0-3-1 C h a n n e l : 353 n\u00C2\u00BB A c q u i r e d : 0\u00C2\u00BB-\u00C2\u00ABAT-30 i i : U Method: C : \ M S f \ G A U \ 3 T C I n j V o l : :5.00 f i l e n a i e : D-30 O p e r a t o r : 70J IM LT1 3 O \u00E2\u0080\u00A23 Ln i3 o o 1.33 \u00E2\u0080\u00A2 > - 2.3\u00C2\u00BB 1 J i i i 153 S a i p l e : 0-30-0-3-2 A c q u i t : H-*A'f-90 Inj V o l : 25.30 0:19 C h a n n e l : 3S3 nr Method: C : \ E A S c \ 0 A r A \ 0 i C t o - 1 w o 1 t s F i i s n a i e : 0-30 O p e r a t o r : TOJ c \u00E2\u0080\u00A2 o o o M O O IJ1 O a a o \u00E2\u0080\u00A2 a ~ I . S 3 1.36 = \u00E2\u0080\u0094 3.3.; a cn :.3 a 154 S a i p l e : 0-20-0-3-3 C h a n n e l : 353 .n A c q u i r e d : 3k-:c l O - 1 y o l t s F i l e n a i e : JOMET-O p e r a t o r : go r O 1.93 164 i a i p l e : 3BH-C C h a n n e l : 213 n i A c q u i r e d : 30-AF.1-9O U : 2 2 Method: C:'.8-iic\OAr*,:0,t I n j V o l : 25.30 it) - 1 >.tO I t 5 f i l e n a i e : :M-O p e r a t o r : O ' a a 01 o \u00E2\u0080\u00A23 _ 1 _ o o .'O \u00E2\u0080\u00A24 \u00E2\u0080\u00A2 -J J .1 J < o - 1 A - I o J \" t . U - 5.43 \u00E2\u0080\u00A2 $.5t 3.32 ) ~ 9.3? sit - u . 2 2 -ftH-'l - 11.38 1 r 1.3 J 165 S \u00C2\u00BB p l \u00C2\u00AB : :0H-\u00C2\u00A3 r S a n n s i \u00E2\u0080\u00A2 O o e r a t o r : 30 i t s O O Jl O O O \" O O O c r H-T+-1:0 ~ \u00E2\u0080\u00A2=> .. H \u00E2\u0080\u00A23 -O i 1 \" | M ~ O i f -j :3 -\"> ~ 5.38 ~ 5.53 - 9.59 s d l - I t . 0 1 a o o r = \u00E2\u0080\u0094 u ; o J l a i s . is 55 as-s I \" I 12 2.20 -1.20 0 -i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094|\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094|\u00E2\u0080\u0094i\u00E2\u0080\u0094' 1 r 00 0.20 0.40 0.60 x iO* Minutes ' I ' 1 1 I 1.00 120 80 60-l 0.00 0.20 0.40 0.60 0.80 1.00 1.20 x 10* ninutes 168 S a i p l e : RQKET-H C h a n n e l : 210 n i A c q u i r e d : 2J-APH-90 10:25 Method: C:\6ASE\0AIA\SOM I n j V o l : 25.00 x 1 Q - 1 u o l t s F i l e n a i e : B0KE7-H O p e r a t o r : go O ' C3 c c _ L _ to a o 2.33 J * a a 1.39 a S.H a 1 05 ' - C ' a Z. o 3 c ID in CO \" a a ' a to' a r*-\ - S.59 J - 8.99 > , - 9.98 i d l I I. I I . 30 S a i p l e : HOKET-K C h a n n e l : 270 na A c q u i r e d : 20-APS-90 15:17 Method: C:\8ASE\DATA\SDH I n j V o l : 25.00 x i O - 1 u o l t s F i l e n a a e : ROM ET - It O p e r a t o r : 90 O U O O in c a \u00E2\u0080\u00A2 o o. 'cn \u00E2\u0080\u00A2 2.k0 . H 2.00 A 1.80 \u00C2\u00AB1 0 3 l 1.60 0 1 .40 1 .20 H 0.00 J... \ \u00C2\u00AB i -T M A i i -i\u00E2\u0080\u0094|\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094|\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094' i r 0.20 0.40 O.GO 0.80 x 10 ninutes T T 1.00 1.20 ' I ' 1.40 S a i p l e : JOKCQN-0 C h a n n e l : 210 n i F i l e n a i e : ROMCOH-0 A c q u i r e d : 27-APB-90 11:25 Method: C:\SASE\0ATA\S0II O p e r a t o r : 30 I n j V o l : 25.00 x l O - 1 u o l t s M CO i 0 0 0 0.13 - s s . _ 2 . U J > J , _ M 9 / j - 5 . 3 7 1.93 S a i p l e : SOHCON-H C h a n n e l : 270 n i F i l e n a i e : RflHCON-H A c q u i r e d : 27-APR-90 12:07 Method: C:\BASE\DATA\SDM O p e r a t o r : go I n j V o l : 25.00 x i O \" 1 u o l t s C _ L _ C a a 1.93 ^ ' \u00E2\u0080\u00A2 - 3 . 9 5 ~ k.50 5.13 6.S3 5.38 1 i ' l _ 11.27 | ) - 12.15 174 S a i p l e : oap s t d C h a n n e l : 270 na F i l e n a i e : o i o s t d A c q u i r e d : 27-APR-90 13:02 Method: C:\SASE\DATA\THP10HP O p e r a t o r : TOJ I n j V o l : 25.00 x l O \" \" u o l t s O N \u00C2\u00BB H* o in o ui S a i p l e : o i p A C h a n n e l : 270 n i A c q u i r e d : 27-AP8-30 14:22 Method: C:\aASE\QATA\TSPSOHP I n j V o l : 25.00 F i l e n a i e : o i p - A O p e r a t o r : TOJ x l O - 1 v o 1 t s rO C o 03 a a\" a ro\" \u00E2\u0080\u00A2 - 2 . U - r 3 \" cn \u00E2\u0080\u00A2 a i 5.22 r CO o ro\" a o . 15.U 176 S a i p l e : 5\u00C2\u00ABD-.\" C h a n n e l : 210 l a f i l e n a a e : s s s A c q u i r e d : <12-t4AV-30 10:35 Method: C : \ S A 2 J \ 0 \u00C2\u00BB T J \ T \u00C2\u00BB H f l H \u00C2\u00BB O p e r a t o r : !0v Inj V o l : 25.30 O ' 1 \" J o \u00E2\u0080\u00A2Jj - J in o 3 1 J j a -i c en 5.23 - 3.: 'O.iO t \u00C2\u00BB I it-3 1 177 S a i p l e : ; M - \u00C2\u00A3 C h a n n e l : !70 \u00E2\u0080\u00A2>\u00C2\u00BB f i l e n a i e : 0 1 0 -t c q u i r e d : 5 ) ' \u00C2\u00BB \u00C2\u00BB H I t\u00C2\u00AB:!S Method: \u00E2\u0080\u00A2::\ei*?'0Ar\u00C2\u00AB.\;M?v:SP S o e r s t o r : TJC i n j v o l : :;.:3 x t O ~ 1 ' J O i t\u00C2\u00BB 3 n o o a -H, \u00E2\u0080\u00A23 ^ i a J - i . s o \u00E2\u0080\u00A23 1 : !' 5.1? \u00E2\u0080\u00A2\u00E2\u0080\u00A2A \u00E2\u0080\u0094 1 \u00E2\u0080\u00A2; - 3.;.) .23 :x 15.!t cao 93 s B I \u00E2\u0080\u00A2 3 ^ 12 2.40 2.20 2.00 H i-t \u00C2\u00A7 1.80 O n X 1.60 1 .40 1 .20H V \ T T -1\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u00941\u00E2\u0080\u0094\"\u00E2\u0080\u0094'\u00E2\u0080\u00941 I ' 1 \u00E2\u0080\u00A2 I 1 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1 40 x 10* ninutes S a i p l e : o i o i l U C h a n n e l : 170 na F i l e n a i e : o i c ! K ! A c q u i r e d : 0 2 - H A f - 9 0 H : 5 2 Method: C:\6A5c\OArA\WPIOW O p e r a t o r : T ; J I n j V o l : 25.03 \u00E2\u0080\u0094 - \u00C2\u00BB r O r O r O .6. 'J) CO o r o o o a a c o f O a a \u00E2\u0080\u00A2 ,\u00E2\u0080\u009E J i \u00C2\u00B0 \ :': - \u00C2\u00BB.32 a J )-*\u00E2\u0080\u00A2\u00E2\u0080\u00A2 .32 a \"1 1 ' - :.33 - 3.:? ) - 10.32 t i p 180 S a i p l e : o i p H C h a n n e l : 270 n i A c q u i r e d : 27-AP8-30 13:kS Method: C:\8A5E\DATA\THPtOttP I n j V o l : 25.00 x l O \" 1 u o l t s F i l e n a i e : o i p - H O p e r a t o r : TOJ O in o Q O ITI o o -o 2.03 O -c\" a a \" - J - 5.27 r .En ' a - U.3J c 181 S a i p l e : oap J A c q u i r e d : 27-APR-90 H : 0 i I n j V o l : 25.00 C h a n n e l : 210 n i Method: C:\8ASE\DATA\TKPi0ltP l O - 1 u o 1 t s F i l e n a i e : o i p - J O p e r a t o r : TOJ o \u00E2\u0080\u00A2 o o 1. o c i A \" o o s.os - 5.28 O 1 8 \" J o 9.17 O U1 o o o M In a CO o a . _ 2.03 U . 3 I 0*p 00 S3-. as as l . G O -s I g 32 3 3 ^ l .50 !! 1.40 0 3 1 .30 1 .20 1 .10 . A t i \u00E2\u0080\u0094 r - r - j 0.00 0.20 T\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094|\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094\"\u00E2\u0080\u0094[\u00E2\u0080\u0094>\u00E2\u0080\u0094' ' | r 0.40 O.BO 0.80 K 10* ninutes t I i i '\u00E2\u0080\u00A2 | ' \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 I \u00E2\u0080\u00A2 1.00 1 2 0 1-40 183 S a i p l e : oap c o n t r o l 0 C h a n n e l : 270 m A c q u i r e d : 27-APR-90 14:41 Method: C:\BASE\DATA\THPtCHP I n j V o l : 25.00 F i l e n a i e : o i p c o n D O p e r a t o r : TOJ x l O \" KJO 1 t S C O cn o a\" a a a . _ 2.03 = - 1.42 ) - 5.21 cn \u00E2\u0080\u00A2 CO ro O - 9.30 i! - 10.32 t i p PJ \" a a 15.40 184 Supls: JOUK-COS-? CSuiri: 270 U Aecralred: 20-APJ.-90 12:07 Sltlod: C:\8ASS\DATl\7HrHllP U] 70l: 25.00 x l O \" 1 u o l t s I l i u m : lO-COB-f OftritOT: TOJ to o J. a 0? a _ L _ CO a _ i _ IO a a to to c a\" o . a -to-o A\" a x g\" (.12 - IM . - 2.03 a -c O\" a A - 7.32 to' a i n oo o o \u00C2\u00ABn 2.50 0 5 2.00 0 X 1.50-- i \u00E2\u0080\u0094 I \u00E2\u0080\u0094 i \u00E2\u0080\u0094 | \u00E2\u0080\u0094 i \u00E2\u0080\u0094 I \u00E2\u0080\u0094 i \u00E2\u0080\u0094 | \u00E2\u0080\u0094 r 0.00 0.20 0.40 1 I ' 0.60 1 I 1 1 1 I 1 0.80 1.00 ' I 1 1 ' I 1 1.20 1.40 x 10* ninutes 186 S a a p l e : oascon-S C h a n n e l : 2)5 as A c q u i r e d : 0 2 - M T - t t :\u00C2\u00BB:51 Method: C:\8AiJ\0ArA\TS-U0M? I n j v o l : 25.JO F i U n a a e : s a o e o v : O p e r a t o r : r o . O \u00E2\u0080\u00A2 O a o 3 O JJ -i a -i 5 - 5.23 -4-! j i, :! - 13.2! t a p c J I _ :5.26 187 5 a i p l e : OODCC.TH c h a n n e l : 213 - j A c q u i r e d : 32-\u00C2\u00ABAY 30 <1:!2 Method: C:\eASE\0JtrA\~5tPtC-HP I n j V o l : 25.00 f i l e n a i e : caocon-.H O p e r a t o r : \"Oj 3 O \u00E2\u0080\u00A2-4 f 3 1 35 \" 5.1: a 1 3 1 I > J \"1 ! 3 J \u00E2\u0080\u00A2\u00E2\u0080\u00A2 >< 1 o 1 w o 1 \"t r a' a 3 \u00E2\u0080\u00A2o c c 3 1-2.:2 i a i o l e : onoccn-K A c q u i r e d : 02-*\u00C2\u00BB*T-90 12: Inj V o l ; 25.00 C h a n n e l : 210 n i Method: C:\8*S\u00C2\u00A3\0.\u00C2\u00ABn\ntMO\u00C2\u00BBP t o - t VO I f i l e n a i e : c i p e c i v * O p e r a t o r : TGJ O O O a . 3 3 O o o o ro 3 3 \u00E2\u0080\u00A2 ~ A . 8 ! \u00E2\u0080\u00A271 a i.tk 3 3 1 - 1.1} i a i p l e : SGH-O-1-S-! C h a n n e l : :;c .11 F i l e n a i e : A c q u i r e d : 0S-HAY -9J Method: 0:\\u00C2\u00A3iJ\u00C2\u00A3VC1TA\iDM O p e r a t o r : I n j V o l : 25.30 >\u00E2\u0080\u00A2 t o \" 1 * r o r o r o :;! - 3.5S l - i 3 . : : s d i S U P W : S O H - 0 3 - 8 3 C h a o n a l : 2 7 0 n i F i l e a i i i : S D H - 0 3 8 3 A c q o i r t d : 0 3 - H A T - 9 0 1 1 : 3 5 U t t h o d : C : \ S A S E \ 0 A T A \ S 0 I 4 O p t n t o r : 90 U j V o l : 2 5 . 0 0 x l O - 1 u o l t s \u00E2\u0080\u0094 l>J M C i l bi In a in a In 0 0 0 0 0 1 . 1 . 1 . 1 . 1 - 0.33 193 S a i p l e : S O H - d a v M C h a n n e l : 210 n\u00C2\u00BB f i l e n a i e : 5 0 \u00C2\u00BB -D ; A c q u i r e d : OI-HAV-90 3:51 Method: C : t f A $ C \ Q A T A \ J M O p e r a t o r : ;o I n j V o l : 25. oo \u00C2\u00AB l O \" ' u o I t s o \u00E2\u0080\u00A2 o a a a 1 3 \u00C2\u00B0 3.12 4.21 5.12 1 - 5.53 CO ' - 1 3 \u00E2\u0080\u00A2 a 9.33 ; d i o I Ik c in a 3.30 194 S a i p l e : : 0 I H a y - ? - 2 C h a n n e l : 212 n i A c q u i r e d : 33-\u00C2\u00BBA\u00C2\u00BB-90 i O : ; s Method: c:\8ASI\OATi\501i I n j V o l : 25.30 f i l e n a i e : ::\u00C2\u00BB-:\u00E2\u0080\u00A2'-! O p e r a t o r : :c a 1 CD - S.S3 3.39 -| \u00E2\u0080\u00A2\u00E2\u0080\u00A2') - 9.91 :dJ o J y I t -\u00C2\u00B0 1 12.19 \u00C2\u00AB t o 1 ; t s O ' a I i a ! = \u00E2\u0080\u00A2 - 2.3 S M B W : : 0 i c c 5 ' - i - : C h a n n e l : 11': at A c q u i r e d : 0 : - s t ' - 9 G ;\u00C2\u00BB:.. Method: : : \ ( ; \u00C2\u00AB \ g * W r ; : ! i I n j V o l : 35.c: F i l e n a i e : -Oh-D? O p e r a t o r : as 1.0 ' C O --- \" 3.35 5.58 i i . 5 : 4-3.9: . ' _ 2.3k sar-r : : ' ! ! 196 \u00E2\u0080\u00A2 a n o U A c q u i r e d I n j v o l ;;.:c Channel: : : j K s t h o d : ::\tx:i\U F i U n a i e : -SH-O p e r a t o r : jo 1 J 1 If.-. 1 t .: a c _ L _ I S 5.1,; H i I J ? . s : ;_ J ,: - i.li sit -i i a ! I 197 : a a o i e : \u00E2\u0080\u00A2 *C3U!r=d: :2 -\u00C2\u00ABiv - J0 i n j v o l : C h a n n e l : VI \"it , - : : 3 Method: : : \ S J ? : \ C ' i ' ; \ i : . i f i l e n a i e : SC\u00C2\u00BB-O p e r a t o r : r r i CO cn 1 .80 1 .60 I O X 140 1.20 \ - i 1 r -0.00 1 r 0.20 - , 1 r 0.40 - i 1 1 1 r -0.60 0.80 1 I 1 1.00 x 10 Minutes 199 Sample: D-13-R-2 Channel: 270 nm lename: D13-R-2 A c q u i r e d : n-m-ii 13:50 n e t t e d : C:\MSE\DAU\SOII I n j v o l : 25.00 O p e r a t o r : go x l O - 1 u o 1 -ts M O J * a 3 c ID a w o o 05 ' o C O ' o O ' o w o 1 - 3 . 1 3 k.SA \\t- 5.2k \ - 5.51 05 a CO a rO b a rO rO a ro a - 10.53 s d i _ 11.31 - t2.U Lit 200 F i l e n a i e : :0Kl O p e r a t o r : :t 201 S a i p l e : SOH-030-83 C h a n n e l : 270 m A c q u i r e d : 09-IIAI-90 15:58 l e t h o d : C:\8ASE\0ATA\S0lt I n j V o l : 25.00 l O - 1 9 Mtiiot: ::>.:JitV;-if;r4H0iiS! \u00E2\u0080\u00A2iWaa\u00C2\u00BBe Oper a t o r i\5 \u00E2\u0080\u00A23 a u'* \u00E2\u0080\u00A2 3 3 ' 3 3 i J - 1 . 8 8 S . - 3 3 - 3 . : 9 ! - : 9 . 2 8 t\u00C2\u00BB3 205 Saiple: OHP-O-t-t-2 Ci iat i l : 270 ni Acquired: 09-HAT-90 U : ! l Method: C:\8ASE\0ATA\TllPiOaP Inj Vol: 35.00 x tO\"1 u o l t s F i l e n a i e : 0HP-0IP.J O p e r a t o r : TOJ O \u00E2\u0080\u00A2 a in a o a In a co a a CO In a a i o \u00E2\u0080\u00A2 - 2.2t 1.30 O \u00E2\u0080\u00A2 A' O a -05 \u00E2\u0080\u00A2 a X \" M 3 1\ _ 5.51 S.80 3 o-Si 3 \u00C2\u00B0 C ro u i \u00E2\u0080\u0094 b -a 1.03 I\" 9.31 a a 13.71 S a i p l e : 0HP-0-3-R-1 C h a n n e l : 210 m A c q u i r e d : O S - H M O U : 5 I Method: C:\BASE\OATA\THPtOHP I n j V o l : 25.00 F i l e n a n : 0MP-03tt 1 O p e r a t o r : TOJ x lO - 1 KJO 1 tS a rj o a a \u00E2\u0080\u00A2 to ' a o \u00E2\u0080\u00A2 a o 1 CO' a CO ' a O\" to -a ro In a i \u00E2\u0080\u00A2- 2.22 1.92 5.60 9.37 10.53 t i p - 13.93 \u00E2\u0080\u00A2 ; j \u00C2\u00BB U : \u00E2\u0080\u00A2>\u00C2\u00AB\u00C2\u00BB\u00E2\u0080\u009423-Sl C a i n n e l : 2'3 \u00E2\u0080\u00A2.\u00C2\u00BB m i i m : m * A c o g i r e d : ::-\u00C2\u00BB.>?-j8 t ls\u00C2\u00AB Method: ::\JsU\0\u00C2\u00BB.*sUiiFiC-JI\u00C2\u00BB O s i r j t o r : <:: Inj V o l : 3S.J3 3.50 3 1 ^ 1 1 l - s M ' \" J ' is, cn ca ~ M c o c o a 3 2.03 207 cn i S.J:-I i l -ea co 8.25 1 - 3.C3 a 1 I -r-13.25 tap CO o G .00 \u00C2\u00A5 0 3 0 5.00 4.00 3.00 -2.00 -i T 1 .00 - J \u00E2\u0080\u0094 i 1\u00E2\u0080\u0094r\u00E2\u0080\u0094j\u00E2\u0080\u0094i\u00E2\u0080\u0094I r 0.00 0.20 0.40 T T T 1 1 1 1 1 1 | 1 1 1 | 1 0.60 0.80 1-00 1.20 1.40 x 10* Minutes 209 S a i p l e : OHP-0-3-C-2 C h a n n e l : 270 m A c q u i r e d : 09-NAT-90 15:27 Method: C:\8ASC\0ATA\TMPtOMP I n j V o l : 25.30 x l O - 1 v o l t s F i l e n a i e : QMP-03C2 O p e r a t o r : I 0 J O ' a hJ a _ L _ \u00C2\u00B11 a cn o 03 O b a i a \u00E2\u0080\u00A2 k l \" a 1 . 3 1 O O \" 05 ' a 3 03-3 \u00C2\u00B0 C rr ro in O \" o > - 3.23 3.97 ~ U l \" 5.59 V5,25 1.1(3 D - 8.13 i 10.73 t i p a 1 3 . 5 1 o i p 210 S a i o l e : omp-day-7 -1 C h a n n e l : 270 nt A c q u i r e d : 07-KAY-3O It:2k Method: C:\BASE\GATA\TMPsaMP I n j V o l : 25.00 F i l e n a i e : o i p 0 7 -O p e r a t o r : TOJ x i O * u o l t s o c M O 03 o - t . o a cn o o 1 o J = - - 2.AS 2.03 O -: I _ 5.09 3.07 r I i 211 S a i p l e : o m - d a r ' - I C h a n n e l : 270 n i A c q u i r e d : 07-HAY-90 l l : W Method: C:\8ASc\0ATA\TitPiOKP I n j V o l : 25.00 F i l e n a i e o i p 0 7 - 2 O p e r a t o r : TOJ x l O - 1 < J O 1 t s a b' a o a a . _ 2.04 2.48 O -Jh i ' a o -; J * 8\" ._ 5.27 .22 a -Z. & 3 \u00C2\u00B0 c - r S a i p l e : 0 * 0 - J a v - ? C h a n n e l : m .11 F i l e n a i e : A c q u i r e d : C7-WAt-S3 -2:09 Method: C:--JA$5\0\u00C2\u00BBri\TK?JC!l\u00C2\u00BB O p e r a t o r : I n j V o l : 2:.00 x 10 \" 1 J O 1 s 213 S a a p l e : : a o - d a y - 7 - 4 C h a n n e l : 2 1 0 n\u00C2\u00BB A c q u i r e d : 3 7 - M A Y - S O 1 2 : 3 0 Method: \u00E2\u0080\u00A2 : : \ 8 ' . S \u00C2\u00A3 \ 0 A T A \ : . i P \u00C2\u00BB c \u00C2\u00BB P I n j V o l : 2 5 . 0 0 X lO\" 1 'JOlt'5 F i l e n a a e : o s o C O p e r a t o r : \"0J O\" a o 2 . 3 ; 73 - ! 1 . 3 3 -) - 5.SO 31 [ .\u00E2\u0080\u00A2\u00E2\u0080\u00A2 \" 5.S3 T 1 - 7 . 4 1 i'j- 3 . 1 3 - 1 0 . 3 8 t i s O 214 S a i p l e : o i p - d a y - 1 ' 5 C h a n n e l : 210 n i A c q u i r e d : 07-HAT-90 13:2k Method: C:\8ASE\0ATA\TKPSOItP I n j V o l : 25.00 x 1 0 _ 1 u o l t s F i l e n a i e : o i p D l - 5 O p e r a t o r : TOJ _ 2.03 _ 2.58 215 a -a Supli: D-U-M lanjirid: 2 0 - m - M I B J Vol: 2 5 . 0 0 1 1 : 4 9 in a _ i _ Ciuu i : Xttaod: lO 270 U C : \ B i S X \ U n \ T D U n - 1 wo 1 \u00C2\u00B1\u00C2\u00BB rO a a i M u m : D-lJ-l-1 Optxitor: WJ in o ? 1-a . - 2 . 0 1 O x. 8\" a - o 3 CO\" 3 \u00C2\u00B0 C t b\" a ^ 5.13 ) S a i p l e : o i p 0-13-8-2 C h a n n e l : 270 n i A c q u i r e d : 27-API-90 13:20 Method: C:\BASE\DATA\TMPiOMP I o j V o l : 25.00 x t O \" 1 u o l t s F i l e n a i e : oapQ13R2 O p e r a t o r : TOJ a a a b' o ui o ro b a Ul a CO b a in a rO\" a 2.03 a 4 1 - k.82 * a 8.20 217 S a e o l e : oip-O-13-8-3 C h a n n e l : 239 r.i A c q u i r e d : V-m-it ; 3 : A i Method: C : \ 8 A J E \ 0 A m T H f M H P I n j V o l : 25.30 x i O \" 1 v o I t s F i l e n a i e : mi'W. O p e r a t o r : \"0J a o a o o a a a a . o a j ' \" J - 5.2? a CO ' O\" 1 1 10.23 t i p to \" a 218 l a i p l e : 3 i a - 0 - U c o n - ! C h a n n e l : 2'0 M F i l e n a i e : c i o O U c ! A c q u i r e d : 37-t*Ar-30 i k : 3 S Method: C:\BASE\CATA\T24P1C-MF O p e r a t o r : /Q.; I n j V o l : 2S.00 x t O \" 1 w o I t s c o o _ 1 _ w a a -i 2.3; JJ a . a so ' 2.k! \u00E2\u0080\u00A2 I I a j | ,'\u00E2\u0080\u00A2 ~ '0.22 t i p 4-\u00C2\u00B0 1 -i 15.1k - i p o 220 Sawli: OXP-O-30-8-2 Ciaanil: 270 nt Acquire: JS-NAT-90 13:32 Netbod: C:\SASE\DArA\TWtOIIP Inj Vol: 25.Ot x l O \" 1 v o l t s Filsnm: OKP030I2 Optritor: TOJ O\" o ro b a i CO b o o o rO\" O . A' o - 2.20 1.80 0 5 ' o 5.57 O \u00E2\u0080\u00A2 CO' a 8.01 ) ~ 9.10 o rO -O \u00C2\u00BB - 13 . 8 2 221 S a i p l e : OHP-0-30-R-3 C h a a n e l : 270 m A c q u i r e d : 09-MAY-90 13:5k Method: C:\BASE\0ATA\T14PtOMP I n j V o l : 25.00 x l O - 1 <_>o l t s f i l e a e i e : OMP03D83 O p e r a t o r : TOJ O \u00E2\u0080\u00A2 O o 1 fO\" a to b a L_ CO b o \u00E2\u0080\u00A2 - 2.22 a o t.ot a 2. b\" 3 \u00C2\u00B0 C r\u00C2\u00A5 n b' o - 8.I2 9.\u00C2\u00BBl - II.61 tO' O a 13.93 "@en . "Thesis/Dissertation"@en . "10.14288/1.0098133"@en . "eng"@en . "Animal Science"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Uptake and depuration of the antibiotics, oxytetracycline and Romet-30 in the Pacific oyster, Crassostrea gigas (Thunberg)"@en . "Text"@en . "http://hdl.handle.net/2429/28991"@en .