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Estuarine microplankton ecology an experimental approach Spies, Annette 1984

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ESTUARINE MICROPLANKTON ECOLOGY: AN EXPERIMENTAL APPROACH by ANNETTE SPIES M . D . , - U n i v e r s i t y Of Hamburg, FRG, 1973 D r . m e d . , U n i v e r s i t y Of Hamburg, FRG, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department Of Oceanography We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA J a n u a r y 1984 © A n n e t t e S p i e s , 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h C o l u m b i a , I ag ree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head of my Department or by h i s or her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment of Oceanography The 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 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 D a t e : J a n u a r y 30 , 1984 i i A b s t r a c t The d a t a p r e s e n t e d i n t h i s t h e s i s a r e t h e r e s u l t of l a b o r a t o r y e x p e r i m e n t s , e x e c u t e d c o n c u r r e n t l y w i t h a y e a r - l o n g f i e l d s t u d y . M i c r o c o s m s (20 1 volume) were f i l l e d w i t h m i x t u r e s of n a t u r a l low and h i g h s a l i n i t y w a t e r s i n o r d e r t o g i v e a s a l i n i t y range from < 5 p p t , 10 p p t , 18 ppt t o > 26 p p t . The m i x e d w a t e r s were e n r i c h e d so as t o s i m u l a t e t h e e n t r a i n m e n t of n u t r i e n t - r i c h , s a l i n e w a t e r i n a s a l t wedge e s t u a r y . N i t r a t e - N (20 y g - a t l " 1 ) , p h o s p h a t e - P (2 nq-at 1~ 1) and s i l i c a t e - S i (50 Mg~at l " 1 ) were added t o the h i g h e s t s a l i n i t y m i c r o c o s m , p r o p o r t i o n a l l y l e s s t o l o w e r s a l i n i t i e s . A d i s t i n c t p a t t e r n of a u t o t r o p h i c and h e t e r o t r o p h i c g r o w t h d e v e l o p e d r e s e m b l i n g n a t u r a l e v e n t s i n t h e F r a s e r R i v e r e s t u a r y d u r i n g t h e p e r i o d between w i n t e r and l a t e s p r i n g . D e s p i t e s e a s o n a l v a r i a b i l i t y of the s o u r c e w a t e r s , t h e s i m u l a t e d s p r i n g bloom was r e p r o d u c i b l e under c o n s t a n t l a b o r a t o r y c o n d i t i o n s , t h u s a l l o w i n g the c o n t i n u e d p e r f o r m a n c e of e x p e r i m e n t s . S k e l e t o n e m a c o s t a t u m and T h a l a s s i o s i r a s p p . were c o n s i s t e n t l y dominant i n the s i m u l a t e d b l o o m , as t h e y a r e i n t h e S t r a i t o f G e o r g i a . The s a l i n i t y v a l u e s i n f l u e n c e d the m i c r o p l a n k t o n e c o l o g y w i t h r e s p e c t t o p h y t o p l a n k t o n s p e c i e s c o m p o s i t i o n and h e t e r o t r o p h i c a c t i v i t y . In L i g h t - and D a r k - e x p e r i m e n t s n u t r i e n t u p t a k e and g r o w t h k i n e t i c s of b a c t e r i a and a l g a e were s t u d i e d , as w e l l as t h e r o l e of h e t e r o t r o p h i c m i c r o f l a g e l l a t e s . The i n t e r a c t i o n s of t h e m i c r o p l a n k t o n were d e s c r i b e d i n n u m e r i c a l m o d e l s . I n t h e p r e s e n c e of h i g h s u b s t r a t e c o n c e n t r a t i o n s (5 mg 1~ 1 g l u c o s e = 2 mg l " 1 g l u c o s e - C ) , t h e g l u c o s e c e l l q u o t a of b a c t e r i a d e t e r m i n e d t h e t i m i n g of the h e t e r o t r o p h i c b l o o m , w h i l e g r a z i n g p a r a m e t e r s and t h e g r o s s g r o w t h e f f i c i e n c y of t h e m i c r o f l a g e l l a t e s d e t e r m i n e d t h e a b s o l u t e numbers . W i t h an e c o l o g i c a l e f f i c i e n c y of 57% between t h e two t r o p h i c l e v e l s , t h e D a r k - m o d e l r e p r e s e n t e d a sy s tem w h i c h was n e i t h e r s u b s t r a t e nor p r e d a t o r c o n t r o l l e d , but s o m e t h i n g i n b e t w e e n . The L i g h t - m o d e l gave an e s t i m a t e of n i t r o g e n r e c y c l i n g by m i c r o f l a g e l l a t e s . In p e r t u r b a t i o n e x p e r i m e n t s t h e impact of h i g h o r g a n i c l o a d ( g l u c o s e ) , s h a d i n g , C u , a heavy m e t a l m i x t u r e , and t h e h e r b i c i d e 2 , 4 - D on the m i c r o p l a n k t o n p o p u l a t i o n s was m o n i t o r e d . N a t u r a l l y o c c u r r i n g p e r t u r b a t i o n s had much g r e a t e r impact on e s t u a r i n e e c o l o g y t h a n a n t h r o p o g e n i c o n e s , even when p o l l u t a n t s were added a t c o n c e n t r a t i o n s e x c e e d i n g t h o s e i n m o d e r a t e l y p o l l u t e d e s t u a r i e s . 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 L i s t of T a b l e s v i i L i s t o f F i g u r e s v i i i A c k nowledgements x C h a p t e r I INTRODUCTION 1 C h a p t e r I I GENERAL METHODS 7 1 . INTRODUCTION 7 2. LABORATORY EXPERIMENTS 9 2.1 E x p e r i m e n t a l S e t - u p 9 2.2 S a m p l i n g D e s i g n 12 2.3 R e p l i c a t i o n E x p e r i m e n t s 13 3. ANALYSIS OF SAMPLES 14 3.1 N u t r i e n t s 14 3.2 P i g m e n t s 14 3.3 T o t a l B a c t e r i a l Numbers 15 3.4 D i s s o l v e d M o n o s a c c h a r i d e s 16 3.5 H e t e r o t r o p h i c A c t i v i t y 16 3.6 P h y t o p l a n k t o n And M i c r o z o o p l a n k t o n 18 C h a p t e r I I I THE STANDARD PATTERN 19 1 . INTRODUCTION 19 2. PATTERN OF EVENTS 21 3. VARIABILITY 24 4. EFFECTS OF SALINITY 33 5. EFFECTS OF ABIOTIC FACTORS 38 5.1 B o t t l e E f f e c t s 38 5.2 C o n s t a n t P h y s i c a l F a c t o r s 39 5.3 N u t r i e n t A d d i t i o n 41 6. INTERACTIONS OF THE BIOTA 44 6.1 D a r k - e x p e r i m e n t s 44 6.2 A u t o t r o p h s And H e t e r o t r o p h s 49 7. SUMMARY 53 V C h a p t e r IV NUMERICAL MODELING 55 1. INTRODUCTION 55 2 . MODELING OF DARK-MICROCOSMS 57 2.1 V a r i a b l e s , P a r a m e t e r s And C o n s t a n t s 57 2 . 2 S i m u l a t i o n And C o m p a r i s o n W i t h O r i g i n a l D a t a 59 2 . 3 S i m u l a t i o n Of D i f f e r e n t S a l i n i t y M i c r o c o s m s 64 2 . 4 D i s c u s s i o n Of P a r a m e t e r Changes 68 3 . MODELING OF LIGHT-MICROCOSMS . . . 7 2 3.1 V a r i a b l e s , P a r a m e t e r s And C o n s t a n t s 72 3 .2 S i m u l a t i o n Of The 10 ppt S a l i n i t y L i g h t - m i c r o c o s m .74 3 .3 S i m u l a t i o n Of O t h e r S a l i n i t i e s 78 3 .4 The D i s t r i b u t i o n Of I n o r g a n i c N i t r o g e n 82 4 . SUMMARY 89 C h a p t e r V PERTURBATION EXPERIMENTS 91 1. INTRODUCTION 91 2 . MATERIALS AND METHODS 93 3 . RESULTS AND DISCUSSION 96 3.1 E f f e c t s Of G l u c o s e 96 3 .2 E f f e c t s Of L i g h t 105 3 .3 E f f e c t s Of Heavy M e t a l s 111 3 .4 E f f e c t s Of The H e r b i c i d e 2 , 4 - D 118 4 . SUMMARY 125 C h a p t e r VI F I E L D STUDY IN THE FRASER RIVER ESTUARY 127 1. INTRODUCTION 127 2 . SAMPLING IN THE F I E L D 129 3 . RESULTS AND DISCUSSION 131 3.1 I n o r g a n i c N i t r o g e n And P h y t o p l a n k t o n 131 3 .2 D i s s o l v e d M o n o s a c c h a r i d e C o n c e n t r a t i o n s 135 3 . 3 B a c t e r i a l Numbers 138 3 .4 H e t e r o t r o p h i c A c t i v i t y 142 3 .5 I n t e r a c t i o n s Of B i o t i c And A b i o t i c F a c t o r s I n The E s t u a r y 144 4 . SUMMARY 148 v i C h a p t e r V I I DISCUSSION 150 1. INTRODUCTION 150 2 . METHODOLOGY 152 2.1 S p r i n g Bloom S i m u l a t i o n 152 2 . 2 S i m u l a t i o n Of The S a l i n i t y Range 154 2 . 3 R e p l i c a t i o n And R e p r o d u c i b i l i t y 156 2 . 4 A n a l y t i c a l Methods 157 3 . THE MERITS OF NUMERICAL MODELING 160 3.1 The D a r k - m o d e l 160 3 .2 The L i g h t - m o d e l 164 4 . PERTURBATIONS AND POLLUTANTS IN MICROCOSMS 167 4.1 H e t e r o t r o p h i c P o p u l a t i o n s 168 4 . 2 A u t o t r o p h i c P o p u l a t i o n s 170 5 . GENERAL CONCLUSIONS 173 BIBLIOGRAPHY 176 APPENDICES 1 - 22 VI 1 L i s t of T a b l e s R e l a t i v e h e t e r o t r o p h i c a c t i v i t i e s a t d i f f e r e n t s a l i n i t i e s i n t h e l a b o r a t o r y 34 C o m p o s i t i o n o f M u l t i - e l e m e n t - m i x a c c o r d i n g t o Thomas e t a l . ( 1980) 94 R e l a t i v e abundance o f s p e c i e s i n C o n t r o l and a f t e r g l u c o s e a d d i t i o n s 103 R e l a t i v e abundance o f s p e c i e s a t 2 . 5 pp t and 10 pp t s a l i n i t y a f t e r m e t a l p e r t u r b a t i o n . 112 R e l a t i v e abundance o f s p e c i e s a t 18 pp t s a l i n i t y a f t e r m e t a l p e r t u r b a t i o n 113 B a c t e r i a l numbers a t d i f f e r e n t s ea sons of t h e y e a r i n t h e F r a s e r R i v e r e s t u a r y 139 R e l a t i v e h e t e r o t r o p h i c a c t i v i t i e s a t d i f f e r e n t s ea sons of t h e y e a r i n t h e F r a s e r R i v e r e s t u a r y . . . . 1 4 2 v i i i L i s t o f F i g u r e s 1. S a m p l i n g s t a t i o n s o f f t h e F r a s e r R i v e r mouth ; s a m p l i n g s i t e s f o r l a b o r a t o r y e x p e r i m e n t s 10 2 . L a b o r a t o r y s e t - u p 11 3 . C o n c e n t r a t i o n s o f n i t r a t e and n i t r i t e , c h l o r o p h y l l a , b a c t e r i a and m i c r o f l a g e l l a t e s i n 18 pp t s a l i n i t y m i c r o c o s m s . 22 4 . The p a t t e r n of c h l o r o p h y l l a c o n c e n t r a t i o n s ; d a t a c o l l e c t e d a t d i f f e r e n t seasons 26 5 . The p a t t e r n of b a c t e r i a l numbers ; d a t a c o l l e c t e d a t d i f f e r e n t seasons 27 6 . The p a t t e r n o f n i t r a t e and n i t r i t e c o n c e n t r a t i o n s ; d a t a c o l l e c t e d a t d i f f e r e n t seasons 28 7 . N i t r a t e and n i t r i t e c o n c e n t r a t i o n s i n r e p l i c a t i o n e x p e r i m e n t s 29 8 . B a c t e r i a l numbers i n r e p l i c a t i o n e x p e r i m e n t s 30 9 . C h l o r o p h y l l a c o n c e n t r a t i o n s i n r e p l i c a t i o n e x p e r i m e n t s . .31 10. R e l a t i v e abundance of d i a t o m s p e c i e s i n m i c r o c o s m e x p e r i m e n t s 36 11 . M i c r o c o s m e x p e r i m e n t s w i t h and w i t h o u t n u t r i e n t a d d i t i o n . 42 12. B a c t e r i a l numbers , m o n o s a c c h a r i d e and n i t r o g e n c o n c e n t r a t i o n s i n D a r k - e x p e r i m e n t s 46 13. C o r r e l a t i o n between c h l o r o p h y l l a and p h a e o p h y t i n a 51 14. S i m u l a t i o n r u n w i t h o r i g i n a l p a r a m e t e r s 60 15. B a c t e r i a l and m i c r o f l a g e l l a t e numbers i n D a r k - e x p e r i m e n t s . 61 16. Two p o s s i b l e s i m u l a t i o n s o f t h e 26 pp t s a l i n i t y D a r k -m i c r o c o s m 65 17. S i m u l a t i o n r u n s of t h e 10 pp t and 18 ppt s a l i n i t y D a r k -m i c r o c o s m s 67 i x 18. M o d e l o f 10 pp t s a l i n i t y L i g h t - m i c r o c o s m 77 19. M o d e l s o f a 18 ppt and 26 ppt s a l i n i t y L i g h t - m i c r o c o s m . . . 8 1 2 0 . S i m u l a t i o n of a 5 ppt s a l i n i t y L i g h t - m i c r o c o s m 83 2 1 . B a c t e r i a l numbers , ammonium and n i t r a t e + n i t r i t e c o n c e n t r a t i o n s i n D a r k - e x p e r i m e n t s 86 2 2 . B a c t e r i a l numbers i n C o n t r o l and a f t e r one and t h r e e a d d i t i o n s o f g l u c o s e 98 2 3 . N u t r i e n t s , b a c t e r i a and c h l o r o p h y l l a a f t e r g l u c o s e p e r t u r b a t i o n a t 5 ppt s a l i n i t y 99 24 . C h l o r o p h y l l a c o n c e n t r a t i o n s a t 10 pp t and 26 pp t s a l i n i t y a f t e r g l u c o s e p e r t u r b a t i o n 100 2 5 . G l u c o s e p e r t u r b a t i o n s i n 5 ppt s a l i n i t y mic rocosms a t d i f f e r e n t seasons 101 2 6 . C h l o r o p h y l l a c o n c e n t r a t i o n s i n C o n t r o l and shaded mic rocosms 106 27 . R e l a t i v e abundance of dominant d i a t o m s p e c i e s i n C o n t r o l and shaded m i c r o c o s m s 108 2 8 . C h l o r o p h y l l a c o n c e n t r a t i o n s i n 10 ppt s a l i n i t y m i c r o c o s m a f t e r t e n - f o l d M u l t i - e l e m e n t - m i x 115 29 ; C h l o r o p h y l l a c u r v e s a t d i f f e r e n t s a l i n i t i e s a f t e r a d d i t i o n of M u l t i - e l e m e n t - m i x 116 30 . C h l o r o p h y l l a c u r v e s a t d i f f e r e n t s a l i n i t i e s a f t e r a d d i t i o n of copper 117 3 1 . C h l o r o p h y l l a c o n c e n t r a t i o n s i n 26 pp t s a l i n i t y mic rocosms a f t e r 2 , 4 - D a d d i t i o n 120 3 2 . C h l o r o p h y l l a c o n c e n t r a t i o n s i n 10 pp t s a l i n i t y m i c r o c o s m s a f t e r 2 , 4 - D a d d i t i o n 121 3 3 . N i t r a t e & n i t r i t e and C h l o r o p h y l l a c o n c e n t r a t i o n s w i t h r e s p e c t t o i n c r e a s i n g s a l i n i t i e s 132 34 . Changes i n d i s s o l v e d m o n o s a c c h a r i d e c o n c e n t r a t i o n s w i t h r e s p e c t t o s a l i n i t y 136 3 5 . S e a s o n a l c y c l e of b a c t e r i a , h e t e r o t r o p h i c a c t i v i t y , c h l o r o p h y l l a , d i s s o l v e d m o n o s a c c h a r i d e s , n i t r a t e + n i t r i t e , and t e m p e r a t u r e 145 X Acknowledgements I w i s h t o thank my s u p e r v i s o r , D r . T . R . P a r s o n s , f o r h i s a d v i c e , p a t i e n c e and s u p p o r t . I am g r a t e f u l f o r t h e c r i t i c i s m s of t h e g u i d a n c e c o m m i t t e e , D r s . P . B . C r e a n , K . J . H a l l , P . G . H a r r i s o n , P . H . L e B l o n d and A . G . L e w i s . H e l p f u l d i s c u s s i o n s and a s s i s t a n c e was g i v e n by D r . P . J . H a r r i s o n . D r . W . J . Emery and K . Thomson p r o v i d e d t h e o p p o r t u n i t y f o r the f i e l d s t u d y i n t h e F r a s e r R i v e r e s t u a r y . S p e c i a l t h a n k s t o D r . J . P a r s l o w who i n t r o d u c e d me t o computer programming and gave i n v a l u a b l e h e l p w i t h t h e n u m e r i c a l m o d e l i n g and t o D r . K. Banse , S c h o o l of O c e a n o g r a p h y , U n i v e r s i t y of W a s h i n g t o n , f o r h i s g u i d a n c e and encouragement t h a t l e d t o the c o m p l e t i o n of my s t u d i e s . T e c h n i c a l a s s i s t a n c e was g i v e n by many i n d i v i d u a l s , members of t h e DOUBC, p a r t i c u l a r l y E . Nutbrown and H . D o v e y , and v a r i o u s f e l l o w s t u d e n t s . I r e c e i v e d f i n a n c i a l s u p p o r t f rom t h e N a t u r a l S c i e n c e s and E n g i n e e r i n g R e s e a r c h C o u n c i l of Canada , Summer UBC G r a d u a t e F e l l o w s h i p s and C h e v r o n F e l l o w s h i p s i n O c e a n o g r a p h y . I am g r a t e f u l f o r t h e m o r a l s u p p o r t and g e n e r o s i t y of M r . and M r s . J . Bene of V a n c o u v e r ; t o them I d e d i c a t e t h i s t h e s i s . 1 I . INTRODUCTION E s t u a r i e s a r e of g r e a t i m p o r t a n c e t o b i o l o g i c a l o c e a n o g r a p h e r s f o r two r e a s o n s ; f i r s t , f o r t h e i r g e n e r a l l y h i g h b i o l o g i c a l p r o d u c t i v i t y and s e c o n d , because t h e y a r e c e n t r e s of human a c t i v i t y . Many of the w o r l d ' s l a r g e s t m e t r o p o l i t a n a r e a s have d e v e l o p e d near e s t u a r i e s . Through t h e i r r o l e i n t r a n s p o r t a t i o n , food p r o d u c t i o n , waste d i s p o s a l , and v a r i o u s r e c r e a t i o n a l p u r s u i t s , e s t u a r i e s a r e s i g n i f i c a n t t o human w e l f a r e . A t the same t i m e , c o m m e r c i a l f i s h e r i e s have been damaged and some c o m p l e t e l y d e s t r o y e d by p o l l u t i o n ; t h e s e i n c l u d e f i s h s p e c i e s w h i c h use t h e e s t u a r y as a m i g r a t o r y r o u t e ( e . g . P a c i f i c sa lmon) as w e l l as t h o s e w h i c h b r e e d i n e s t u a r i n e a r e a s and use the e s t u a r y as n u r s i n g grounds f o r t h e i r young ( e . g . h e r r i n g , c o d , f l a t f i s h s p e c i e s ) . S i n c e t h e m i d - 1 9 6 0 ' s , t h e need f o r a more c o m p r e h e n s i v e u n d e r s t a n d i n g o f e s t u a r i n e e c o s y s t e m s has s t i m u l a t e d c o n f e r e n c e s and sympos i a s u m m a r i z i n g t h e knowledge of t h e n a t u r a l c h a r a c t e r i s t i c s of e s t u a r i e s ( L a u f f 1967) , as w e l l as s t r e s s i n g s p e c i f i c b i o l o g i c a l t o p i c s s u c h as e s t u a r i n e m i c r o b i a l e c o l o g y ( S t e v e n s o n & C o l w e l l 1973) , p h y s i o l o g i c a l a s p e c t s ( V e r n b e r g 1975) , e s t u a r i n e p r o c e s s e s and i n t e r a c t i o n s ( e . g . C r o n i n 1975; W i l e y 1976 and 1978 ) . The impact of p o l l u t a n t s on t h e f l o r a and fauna and t h e prob lems a s s o c i a t e d w i t h e u t r o p h i c a t i o n a r e g a i n i n g i n c r e a s e d c o n s i d e r a t i o n ( P e r k i n s 1974; N e i l s o n & C r o n i n 1979 ) . Most o f the s t u d i e s a r e l a r g e l y d e s c r i p t i v e , but t h e 2 i n v e s t i g a t i o n of p r o c e s s e s i s needed f o r a b e t t e r u n d e r s t a n d i n g of t h e e s t u a r i n e e c o s y s t e m . A c o m p r e h e n s i v e d i s c u s s i o n of m i c r o b i a l e c o l o g y i n a b r a c k i s h water e n v i r o n m e n t i s p r e s e n t e d by R h e i n h e i m e r ( 1 9 7 7 ) . Based on a f i e l d s t u d y i n K i e l B i g h t ( B a l t i c S e a ) , l a b o r a t o r y i n v e s t i g a t i o n s and n u m e r i c a l m o d e l i n g , t h e r o l e of b a c t e r i a and f u n g i and t h e i r c o n t r i b u t i o n t o t o t a l p r o d u c t i o n i s e v a l u a t e d . E s t u a r i e s a r e among the most c o m p l i c a t e d a q u a t i c e c o s y s t e m s . They a r e t r a n s i t i o n zones and f r o n t a l r e g i o n s between f r e s h w a t e r and m a r i n e e n v i r o n m e n t s . T h e i r b i o l o g i c a l p r o c e s s e s a r e i n t i m a t e l y l i n k e d and i n f l u e n c e d by extreme and r a p i d f l u c t u a t i o n s of e n v i r o n m e n t a l c o n d i t i o n s , among them s a l i n i t y , l i g h t and t e m p e r a t u r e . Due t o c o n s t a n t m i x i n g and t r a n s p o r t a t i o n o f - o r g a n i s m s away from an e s t u a r i n e s t u d y a r e a , the v a l u e of e c o l o g i c a l s u r v e y s i s v e r y l i m i t e d . S u r v e y s can i n d i c a t e o n l y t h a t changes a r e o c c u r r i n g . I n o r d e r t o s t u d y p r o c e s s e s of g r o w t h , p h y s i o l o g y and i n t e r a c t i o n of t h e e s t u a r i n e b i o t a d i f f e r e n t a p p r o a c h e s a r e n e c e s s a r y . M a t h e m a t i c a l s i m u l a t i o n models of v a r y i n g c o m p l e x i t i e s have been g e n e r a t e d i n an a t t e m p t t o s y n t h e s i z e e x i s t i n g knowledge i n t o a s y s t e m a t i c and i n t e g r a t i v e scheme (Rremer & N i x o n 1978; H a m i l t o n & M a c d o n a l d 1979 ) . A l t h o u g h t h e s e model s a r e v a l u a b l e i n t h e i n t e r p r e t a t i o n of i n t e r a c t i v e components of t h e e s t u a r i n e e c o s y s t e m , t h e y s u f f e r from t h e l a c k of r e a l i s t i c r e s o l u t i o n ( V e r n b e r g e t aJL. 1978 ) . The n a t u r a l f r e q u e n c y of t h e p r o c e s s e s t o be s i m u l a t e d has o f t e n been i g n o r e d d u r i n g t h e d a t a c o l l e c t i o n . 3 E l a b o r a t e c o n t i n u o u s - f l o w a p p a r a t u s a r e n e c e s s a r y i n o r d e r t o s i m u l a t e t i d e s or s e a s o n a l changes i n r i v e r f l o w r a t e s i n a l a b o r a t o r y . A s e r i e s of c h e m o s t a t s has been used i n a s t u d y by Cooper & C o p e l a n d ( 1 9 7 3 ) . W i t h t h e i r sy s tem i t has been p o s s i b l e t o i n v e s t i g a t e t h e r e s p o n s e s of an e s t u a r i n e p l a n k t o n community t o v a r i a t i o n s i n h y d r o l o g i c a l f a c t o r s and the impact of i n d u s t r i a l e f f l u e n t w i t h changes i n f r e s h w a t e r i n p u t r a t e s . I t s t i l l has t o be p r o v e n whether c o m p l i c a t e d c o n t i n u o u s f l o w a p p a r a t u s w i l l n e c e s s a r i l y g i v e more r e a l i s t i c r e s u l t s . Some s u c c e s s i n s t u d y i n g b i o l o g i c a l p r o c e s s e s i n a water co lumn has been a c h i e v e d i n l a r g e s c a l e 'mesocosms' ( G r i c e & Reeve 1 9 8 2 ) . Up t o 1500 t o n s of s eawate r have been c a p t u r e d and t h e p r i m a r y , s e c o n d a r y and t e r t i a r y p r o d u c t i o n have been f o l l o w e d undet e x p e r i m e n t a l c o n d i t i o n s . In an e s t u a r y t h e same a p p r o a c h i s r e s t r i c t e d by the l a c k of p h y s i c a l l y s t a b l e m o o r i n g s i t e s as w e l l as s p a t i a l l i m i t a t i o n s i n the p r e s e n c e of a l l o t h e r a c t i v i t i e s on a busy e s t u a r y , such as the F r a s e r R i v e r e s t u a r y i n B r i t i s h C o l u m b i a . An a l t e r n a t i v e a p p r o a c h has been adopted i n t h e e x p e r i m e n t s of t h i s t h e s i s . The e s t u a r i n e e n v i r o n m e n t i s s i m u l a t e d w i t h r e s p e c t t o changes i n s a l i n i t y u s i n g c o n t a i n e r s w h i c h a l l o w the s t u d y of t h e m i c r o p l a n k t o n c o m m u n i t y . T h i s r e d u c e s t h e s i z e of t h e c o n t a i n e r s r e q u i r e d from 'mesocosms' t o ' m i c r o c o s m s ' ( c . 20 1 ) . I d e f i n e m i c r o c o s m s a c c o r d i n g t o L e f f l e r (1980) as s m a l l , l i v i n g model s of p r o c e s s e s r a t h e r t h a n models of s p e c i f i c n a t u r a l e c o s y s t e m s . U s u a l a q u a r i a and s i n g l e - s p e c i e s c u l t u r e s a r e n o r m a l l y e x c l u d e d . The changes i n s c a l e "between b e a k e r s 4 and b a y s " ( S t r i c k l a n d 1967) r e q u i r e the a l t e r a t i o n o f i m p o r t a n t p h y s i c a l f a c t o r s , e . g . t u r b u l e n c e and l i g h t . The consequences o f t h e s e changes a r e p o o r l y u n d e r s t o o d . T h e r e f o r e , mic rocosms and n a t u r a l e co sy s t ems p o s s e s s t h e i r own c h a r a c t e r i s t i c p r o p e r t i e s , and microcosms can s e r v e as v a l i d model s of e co sy s t ems o n l y f o r p r o p e r t i e s t h a t a r e common t o b o t h ( L e f f l e r 1 9 8 0 ) . The r e s e a r c h p r e s e n t e d h e r e i s d e s i g n e d t o answer f o u r q u e s t i o n s . F i r s t , does t h e n a t u r a l c y c l e of e v e n t s w i t h r e s p e c t t o t h e m i c r o p l a n k t o n i n t h e F r a s e r R i v e r e s t u a r y d i f f e r w i t h i n c r e a s i n g s a l i n i t y v a l u e s ? S e c o n d , i s i t p o s s i b l e t o r e p r o d u c e some c h a r a c t e r i s t i c s of e s t u a r i n e m i c r o p l a n k t o n e c o l o g y ( e . g . the d i a t o m s p r i n g bloom) i n l a b o r a t o r y s c a l e mic rocosms? T h i r d , i f t h e answer t o t h e second q u e s t i o n i s y e s , can t h e s i m u l a t e d e v e n t s be r e p l i c a t e d w i t h s u f f i c i e n t a c c u r a c y t o a l l o w f o r t h e p e r f o r m a n c e of m e a n i n g f u l p e r t u r b a t i o n e x p e r i m e n t s ? F o u r t h , i s i t p o s s i b l e t o r e p r e s e n t t h e e v e n t s o b s e r v e d i n the e x p e r i m e n t a l m i c r o c o s m s by a m a t h e m a t i c a l model w h i c h would h e l p t o u n d e r s t a n d how t h e m i c r o c o s m components i n t e r a c t ? M i c r o p l a n k t o n s t u d i e s i n t h e S t r a i t o f G e o r g i a and t h e F r a s e r R i v e r e s t u a r y have p r e v i o u s l y been p e r f o r m e d ( e . g . P a r s o n s e t a l . 1969; S t o c k n e r e t a l . 1979; A l b r i g h t 1977, 1983b) , but so f a r t h e e f f e c t s of s a l i n i t y v a r i a t i o n s have never been t h o r o u g h l y examined ( H a r r i s o n e t a_l. 1983 ) . T h e r e f o r e , i n o r d e r t o answer t h e f i r s t q u e s t i o n , a one y e a r f i e l d s u r v e y i n the F r a s e r R i v e r e s t u a r y was u n d e r t a k e n s i m u l t a n e o u s l y w i t h t h e l a b o r a t o r y e x p e r i m e n t s . The f i e l d s a m p l i n g o v e r a s a l i n i t y 5 range from < 5 ppt t o > 26 ppt i s d e s c r i b e d i n C h a p t e r V I . The r e s u l t s of t h e f i e l d s t u d y a r e compared t o l a b o r a t o r y e x p e r i m e n t s , and b e h a v i o u r a l s i m i l a r i t i e s a r e e v a l u a t e d and d i s c u s s e d . In answer t o t h e second q u e s t i o n , an e s t u a r i n e s a l i n i t y reg ime i s s i m u l a t e d w i t h r e s p e c t t o i n c r e a s i n g e n t r a i n m e n t of s a l i n e , n u t r i e n t - r i c h deep w a t e r , w h i c h p e r m a n e n t l y f e r t i l i z e s t h e e s t u a r y . The c o l l e c t i o n of h i g h and low s a l i n i t y w a t e r , the m i x i n g of t h e w a t e r s , and t h e i n i t i a t i o n of t h e microcosms a r e d e s c r i b e d i n C h a p t e r I I . The e v o l v i n g p a t t e r n p r o d u c e d by g r o w t h d i f f e r e n c e s of t h e b i o t a i s s i m i l a r t o t h a t of a n a t u r a l s p r i n g bloom ( C h a p t e r I I I ) . A u t o t r o p h i c and h e t e r o t r o p h i c g r o w t h i s s t u d i e d i n D a r k -and L i g h t - e x p e r i m e n t s . An a t t e m p t i s made t o i s o l a t e f a c t o r s w h i c h i n f l u e n c e p r o d u c t i o n i n t h e m i c r o c o s m s , t o i n v e s t i g a t e i n t e r a c t i o n s among t h e l i v i n g component s , and t o e v a l u a t e the r o l e of m i c r o f l a g e l l a t e s as p a r t of t h e e s t u a r i n e p l a n k t o n c o m m u n i t y . In answer t o the t h i r d q u e s t i o n , the r e p l i c a b i 1 i t y of mic rocosms i s s t u d i e d ( C h a p t e r I I I ) . The a im i s t o p r o v e t h a t t h e p a t t e r n o f e v e n t s g e n e r a t e d i n t h e l a b o r a t o r y i s r e p r o d u c i b l e d e s p i t e p h y s i c a l , c h e m i c a l and b i o l o g i c a l changes i n t h e p a r e n t w a t e r masses c o l l e c t e d a t d i f f e r e n t seasons o f the y e a r . I n t h i s way, p e r t u r b a t i o n s of the s i m u l a t e d p h y t o p l a n k t o n bloom can be p e r f o r m e d a t any t i m e . P e r t u r b a t i o n e x p e r i m e n t s h e l p i n d e t e r m i n i n g w h i c h f a c t o r s , n a t u r a l or man-made, have t h e g r e a t e s t impact i n a l t e r i n g t h e 6 m i c r o p l a n k t o n eco logy of the s i m u l a t e d e s tuary (Chapter V ) . In o r d e r to answer the f o u r t h q u e s t i o n , uptake and growth r a t e s of p h y t o p l a n k t o n and b a c t e r i a e s t imated i n Dark- and L i g h t - e x p e r i m e n t s are used as a b a s i s for the mathemat ica l s i m u l a t i o n s d e s c r i b e d i n Chapter I V . Numer ica l model ing i s thought to h e l p i n the i n t e r p r e t a t i o n of p r o c e s s e s under i n v e s t i g a t i o n and i n the i d e n t i f i c a t i o n of the important v a r i a b l e s i n the microcosms . Whi le a b i o l o g i c a l model such as the e s t u a r i n e microcosm c r e a t e s new d a t a , a mathemat ica l model based on these data i s an i n e x p e n s i v e way to r e p r e s e n t the p roce s se s o b s e r v e d , to p r o v i d e b e t t e r u n d e r s t a n d i n g of the same, to per form more e x t e n s i v e p e r t u r b a t i o n s and to a i d i n the t e s t i n g of h y p o t h e s e s . F i n a l l y , i t i s i n t e n d e d that these r e s u l t s w i l l a s s i s t . i n answering a l a r g e r q u e s t i o n ; what are the f o r c i n g f u n c t i o n s for p h y t o p l a n k t o n growth i n an e s t u a r y and can these be p e r t u r b e d by a n t h r o p o g e n i c e f f e c t s ? T h i s q u e s t i o n i s d i s c u s s e d i n Chapter V I I . 7 I I . GENERAL METHODS 1. INTRODUCTION The use of c o n t a i n e r s , r a n g i n g i n s i z e f rom s m a l l f l a s k s t o l a r g e p l a s t i c b a g s , i s a r e l a t i v e l y r e c e n t deve lopment i n m a r i n e e c o l o g i c a l r e s e a r c h . P i o n e e r i n g work has been done by S t r i c k l a n d & Terhune ( 1 9 6 1 ) . I n a l a r g e f l o a t i n g p l a s t i c bag t h e y were a b l e t o f o l l o w changes i n t h e p l a n k t o n p o p u l a t i o n . P i l s o n & N i x o n (1980) r e v i e w e d 21 r e p r e s e n t a t i v e m a r i n e m i c r o -and mesocosms, summarized d i f f i c u l t i e s w i t h s c a l i n g , n a t u r a l and a r t i f i c i a l l i g h t , t u r b u l e n c e , water e x c h a n g e , p r e d a t o r e x c l u s i o n and v a r i a b i l i t y . The s i z e o f c o n t a i n e r s c h o s e n f o r the e x p e r i m e n t s r e p o r t e d i n t h i s t h e s i s a l l o w s f o r t h e s t u d y of b a c t e r i a , p h y t o p l a n k t o n and m i c r o f l a g e l l a t e s . They h o l d s u f f i c i e n t water f o r f r e q u e n t s a m p l i n g o v e r a t h r e e - w e e k p e r i o d and r e p r e s e n t the upper l i m i t w h i c h can be managed by one or two p e o p l e . T h i s s i z e i s a compromise between f e a s i b i l i t y and m i n i m a l s t a t i s t i c a l r e q u i r e m e n t s . The microcosms a r e r u n as b a t c h c u l t u r e s ; t h e o p e r a t i n g t i m e i s s h o r t . C o n s t a n t p h y s i c a l f a c t o r s and a c o n s t a n t i n i t i a t i o n method improve r e p l i c a b i l i t y between microcosms and the r e p r o d u c i b i l i t y o f e x p e r i m e n t a l r e s u l t s between s e a s o n s . An i n i t i a l n u t r i e n t e n r i c h m e n t was t h o u g h t t o be n e c e s s a r y because of s e a s o n a l v a r i a b i l i t y i n the s u b s t r a t e of t h e s o u r c e 8 waters. Low n a t u r a l s u b s t r a t e l e v e l s i n batch c u l t u r e s may r e s u l t i n growth so small that s i g n i f i c a n t .' d i f f e r e n c e s of p o p u l a t i o n changes are impossible to observe. The a d d i t i o n enhances a l r e a d y high n u t r i e n t l e v e l s , as w e l l as ens u r i n g growth of the microplankton p o p u l a t i o n when l e v e l s are low. Consequently, experiments can be performed at a l l times of the year. The i n i t i a l enrichment simulates the d e c r e a s i n g entrainment of n u t r i e n t - r i c h water i n a s a l t wedge or h i g h l y s t r a t i f i e d e s t u a r y . A meaningful comparison of l a b o r a t o r y and f i e l d data r e q u i r e s the parameter measured to be the same. As such, the microplankton community i s s t u d i e d by enumeration of b a c t e r i a and m i c r o f l a g e l l a t e s and by i d e n t i f i c a t i o n of the major phytoplankton s p e c i e s . The biomass of the l a t t e r i s estimated by c h l o r o p h y l l a measurements. Nitrogen i s c o n s i d e r e d to be the l i m i t i n g f a c t o r f o r a u t o t r o p h i c growth ( A n t i a et a l . 1963; Ryther & Dunstan 1971); t h e r e f o r e , c o n c e n t r a t i o n s of n i t r a t e p l u s n i t r i t e have been measured. Glucose i s used as the s u b s t r a t e i n e v a l u a t i n g r e l a t i v e h e t e r o t r o p h i c a c t i v i t y , because almost a l l b a c t e r i a can u t i l i z e glucose (Mandelstam et a l . 1982). In the f i e l d , d e t e r m i n a t i o n of d i s s o l v e d monosaccharides g i v e s an i n d i c a t i o n of a r e a d i l y usable carbon and energy source f o r h e t e r o t r o p h s . 9 2 . LABORATORY EXPERIMENTS 2.1 E x p e r i m e n t a l S e t - u p I n the e x p e r i m e n t s d e s c r i b e d b e l o w , P y r e x f l a s k s ( c . 20 1) were f i l l e d w i t h a m i x t u r e of n a t u r a l low and h i g h s a l i n i t y w a t e r . Low s a l i n i t y s u r f a c e w a t e r (< 5 p p t ) was c o l l e c t e d from t h e banks of t h e F r a s e r R i v e r main arm c . 200-500 m ups t ream from S t e v e s t o n h a r b o r a t h i g h t i d e . The h i g h s a l i n i t y water was pumped from c . 20 m d e p t h a t t h e West V a n c o u v e r L a b o r a t o r y o f t h e D e p t . o f F i s h e r i e s and Oceans ( F i g u r e 1 ) . On c o l l e c t i o n day t h e w a t e r s were m i x e d i n o r d e r t o g i v e a s a l i n i t y range from 2-5 p p t , 10 p p t , 18 pp t t o 26-30 pp t ( F i g u r e 2 ) . F o u r f l a s k s r e p r e s e n t e d one u n i t . A t o t a l of 12 f l a s k s a l l o w e d f o r a c o n t r o l u n i t and two e x p e r i m e n t a l u n i t s . A l l e x p e r i m e n t s were p e r f o r m e d a t 12°C i n a c o n t r o l l e d e n v i r o n m e n t chamber . The f l a s k s were p o s i t i o n e d i n f r o n t of banks o f f o u r d a y l i g h t f l u o r e s c e n t t u b e s , so t h a t an i r r a d i a n c e of 200-250 /uEinst m " 2 s ' 1 was measured i n t h e m i d d l e of the empty c o n t a i n e r s . The i r r a d i a n c e was s a t u r a t i n g f o r d i a t o m s p e c i e s and s m a l l e r d i n o f l a g e l l a t e s (Chan 1978 ) . A l l e x p e r i m e n t s were p e r f o r m e d on a 12:12 L D - c y c l e . The w a t e r s i n a l l f l a s k s were m a g n e t i c a l l y s t i r r e d and a e r a t e d w i t h s u l f u r i c a c i d (3%)-washed a i r . N i t r a t e - N (20 y g - a t 1 " 1 ) , p h o s p h a t e - P (2 Mg-at l " 1 ) , and s i l i c a t e - S i (50 M g - a t l " 1 ) were added t o the h i g h e s t s a l i n i t y f l a s k , p r o p o r t i o n a l l y l e s s t o l o w e r s a l i n i t i e s . These a d d i t i o n s were made t o b o t h c o n t r o l as w e l l as e x p e r i m e n t a l m i c r o c o s m s . T o g e t h e r w i t h t h e i n i t i a l n u t r i e n t F i g u r e 1 - S h i p ' s c o u r s e o f f t h e F r a s e r R i v e r mouth d u r i n g sample c o l l e c t i o n . • S a m p l i n g s i t e s f o r l a b o r a t o r y e x p e r i m e n t s . 11 Fraser River Surface Water <5 %,S Control Unit — • Standard Pattern 18 L <5%oS J 18 L IO%oS 18 L !8%oS Water from 20 m depth >26%oS Experimental Unit ^\ S\ /\ /\ 18 L >26%oS Experimental Unit 18 L 18 L 18 L 18 L <5°-6oS IO%oS I8%»S >26%oS I. • J C 4 F i g u r e 2 - L a b o r a t o r y s e t - u p ; d a s h e d l i n e i n d i c a t e s o c c a s i o n a l m i x i n g o f s a l i n e w a t e r w i t h v e r y l o w s a l i n i t y r i v e r w a t e r . 12 e n r i c h m e n t , 1.2 p.M EDTA+iron and 1 mg 1~ 1 of g l u c o s e were added t o a l l s a l i n i t i e s i n o r d e r t o e n s u r e n o n - l i m i t i n g g r o w t h c o n d i t i o n s f o r t h e m i c r o p l a n k t o n under s t u d y . 2 .2 S a m p l i n g D e s i g n A f t e r c o l l e c t i o n and m i x i n g of t h e low and h i g h s a l i n i t y w a t e r s on day 1, t h e b i o t a was a l l o w e d t o a d j u s t t o l a b o r a t o r y c o n d i t i o n s o v e r n i g h t . I n i t i a l samples were t a k e n on day 2 p r i o r t o n u t r i e n t a d d i t i o n a n d / o r s t a r t of t h e e x p e r i m e n t a l t r e a t m e n t , 24 h l a t e r and t h e n e v e r y second day f o r two t o t h r e e weeks . A l l samples were c o l l e c t e d by s u c t i o n from t h e f l a s k s u s i n g Tygon t u b i n g of 8 mm d i a m e t e r . By t h e end of t h e e x p e r i m e n t t h e i n i t i a l volume of the mic rocosms was r e d u c e d by c . 33%. C h l o r o p h y l l a , n i t r a t e , b a c t e r i a and m i c r o z o o p l a n k t o n were sampled as d e s c r i b e d a b o v e . For t h e d e t e r m i n a t i o n of n u t r i e n t up take r a t e s and g r o w t h r a t e s of b a c t e r i a and p h y t o p l a n k t o n , a d d i t i o n a l samples were t a k e n a t 12 h i n t e r v a l s d u r i n g the e x p o n e n t i a l g r o w t h p h a s e . Depend ing on the q u e s t i o n s a s k e d i n e x p e r i m e n t s , p l a n k t o n was sampled d u r i n g t h e l a g p h a s e , d u r i n g e x p o n e n t i a l g r o w t h , a t the end of e x p o n e n t i a l g r o w t h and a f t e r n u t r i e n t e x h a u s t i o n ( s e n e s c e n c e ) . The s a m p l i n g of o t h e r p a r a m e t e r s has been d e s c r i b e d i n t h e a p p r o p r i a t e c h a p t e r s e l s e w h e r e . 1 3 2 . 3 R e p l i c a t i o n E x p e r i m e n t s R e p l i c a t i o n e x p e r i m e n t s f o c u s s e d on two q u e s t i o n s : what i s t h e v a r i a b i l i t y between d u p l i c a t e f l a s k s and how does t h e r e s p o n s e p a t t e r n of the m i c r o c o s m s change w i t h r e s p e c t t o s e a s o n a l changes i n the water c h e m i s t r y ? R e p l i c a t i o n of mic rocosms w i t h i d e n t i c a l t r e a t m e n t was t e s t e d o v e r a s a l i n i t y range from < 5 pp t t o > 26 ppt d u r i n g d i f f e r e n t seasons of t h e y e a r . The c o m p a r i s o n f o c u s s e d on v a r i a b i l i t y i n peak h e i g h t s and on r a t e s of i n c r e a s e and d e c r e a s e of p a r a m e t e r s measured . P l a n k t o n samples were compared f o r r e l a t i v e abundance of spec i e s . Data g e n e r a t e d i n m i c r o c o s m s u s u a l l y a re i n t h e form of a t i m e s e r i e s f o r each parameter m e a s u r e d . I f t h e s e r i e s from two i d e n t i c a l l y p r e p a r e d m i c r o c o s m s a r e compared , t h e y may e x h i b i t s i m i l a r f o r m , but be s h i f t e d i n p h a s e . When d a t a a r e compared a t any one i n s t a n c e , t h e a n a l y s i s of t h e v a r i a n c e w o u l d l i k e l y i n f e r t h a t t h e two m i c r o c o s m s do not r e p l i c a t e . W i t h a p p r o p r i a t e s t a t i s t i c a l t e s t s i t i s p o s s i b l e t o e l i m i n a t e t h e e f f e c t s of p h a s i n g and compare t h e r e a l t i m e d y n a m i c s (Haque e t a l . 1980) , s o m e t h i n g w h i c h was no t done i n the p r e s e n t t h e s i s . S e a s o n a l v a r i a t i o n s i n t h e r e sponse p a t t e r n of t h e mic rocosms were examined by c o m p a r i n g t h e 4 - f l a s k c o n t r o l u n i t s w h i c h were r u n c o n c u r r e n t l y w i t h a l l e x p e r i m e n t s . The t i m e s e r i e s of the c o n t r o l u n i t s r e f l e c t t h e v a r i a b i l i t y due t o b o t h s e a s o n a l changes i n the c h e m i s t r y of the d i f f e r e n t w a t e r masses and sample v a r i a t i o n . 14 3 . ANALYSIS OF SAMPLES 3.1 N u t r i e n t s A l l samples c o l l e c t e d i n t h e l a b o r a t o r y and i n the f i e l d were i m m e d i a t e l y f i l t e r e d t h r o u g h g l a s s f i b e r f i l t e r s (Whatman 9 3 4 - A H , 42 .5 mm d i a m e t e r ) , s t o r e d i n N a l g e n e b o t t l e s and f r o z e n t o - 2 0 ° C . Samples were s t o r e d f o r not more t h a n t h r e e weeks and t h e n q u i c k l y thawed b e f o r e a n a l y s i s . Automated d e t e r m i n a t i o n f o r n i t r a t e p l u s n i t r i t e was done as d e s c r i b e d by A r m s t r o n g e t a l . ( 1 9 6 7 ) . The range of c o n c e n t r a t i o n s was 0-30 y g - a t N 1 ~ 1 ; s amples w i t h h i g h e r c o n c e n t r a t i o n s were d i l u t e d w i t h a known amount of d e i o n i z e d d i s t i l l e d water b e f o r e a n a l y s i s . In one s e t of l a b o r a t o r y e x p e r i m e n t s , ammonium ( 0 t o 8 nq-a t N l " 1 ) was measured c o n c u r r e n t l y (S lawyk & M a c l s a a c 1972) . 3 .2 P i g m e n t s The a n a l y s i s of c h l o r o p h y l l a and p h a e o p h y t i n a was c a r r i e d out a c c o r d i n g t o t h e methods d e s c r i b e d by S t r i c k l a n d & P a r s o n s ( 1 9 7 2 ) . In t h e f i e l d , 500 ml t o 1000 ml of seawater was f i l t e r e d , i n l a b o r a t o r y e x p e r i m e n t s 200 ml t o 500 m l , d e p e n d i n g on t h e d e n s i t y of t h e p h y t o p l a n k t o n b l o o m . To the l a s t 100 ml of each sample 1 ml of magnesium c a r b o n a t e s u s p e n s i o n was a d d e d . The 0 .45 am f i l t e r s ( M i l l i p o r e , 47 mm d i a m e t e r ) were s t o r e d i n the d a r k i n a d e s i c c a t o r and f r o z e n t o - 2 0 ° C . S t o r a g e t i m e p r i o r t o a n a l y s i s d i d not exceed t h r e e weeks . The e x t r a c t i o n was done i n 90% a c e t o n e o v e r n i g h t . F o l l o w i n g c e n t r i f u g a t i o n , the c l e a r s u p e r n a t a n t l i q u i d was p i p e t t e d i n t o a 10 cm p a t h 15 c u v e t t e . T h e e x t i n c t i o n was m e a s u r e d i n a P e r k i n - E l m e r D o u b l e Beam S p e c t r o p h o t o m e t e r ( C o i e m a n M o d e l 124 D) a t 750 nm a n d 665 nm a g a i n s t a 90% a c e t o n e b l a n k c o n t a i n i n g a d i s s o l v e d M i l l i p o r e f i l t e r . F o r d e t e r m i n a t i o n o f p h a e o p h y t i n a , s a m p l e s w e r e a c i d i f i e d a n d r e m e a s u r e d a f t e r 5 m i n . T h e c o n c e n t r a t i o n s o f c h l o r o p h y l l a a n d p h a e o p h y t i n a w e r e c a l c u l a t e d a c c o r d i n g t o t h e e q u a t i o n s g i v e n i n S t r i c k l a n d & P a r s o n s ( 1 9 7 2 ) . 3 . 3 T o t a l B a c t e r i a l N u m b e r s S u b s a m p l i n g f o r e n u m e r a t i o n o f t o t a l b a c t e r i a l n u m b e r s was a l w a y s d o n e f i r s t . U s i n g a n a u t o m a t i c p i p e t t e , 10 m l a l i q u o t s w e r e p l a c e d i n t o g l a s s v i a l s a n d i m m e d i a t e l y p r e s e r v e d w i t h f i l t e r e d ( 0 . 2 2 Mm) f o r m a l d e h y d e ( f i n a l c o n c e n t r a t i o n o f 2 % ) . S a m p l e s w e r e s t o r e d i n t h e d a r k a t 5 ° C f o r no l o n g e r t h a n t e n d a y s ( D a l e y & H o b b i e 1 9 7 5 ) . B a c t e r i a w e r e s t a i n e d w i t h a c r i d i n e o r a n g e a n d c o u n t e d on 0 . 2 um N u c l e p o r e f i l t e r s ( H o b b i e e t a l . 1 9 7 7 ) . A m i c r o s c o p e w i t h an e p i f l u o r e s c e n t u n i t i n c l u d i n g a b r o a d b a n d b l u e e x c i t a t i o n f i l t e r was u s e d f o r e n u m e r a t i o n . T e n f i e l d s a n d a t o t a l o f a t l e a s t 200 b a c t e r i a w e r e c o u n t e d . T o t a l n u m b e r s w e r e c a l c u l a t e d a c c o r d i n g t o t h e e q u a t i o n c e l l s _ s t a i n e d a r e a o f f i l t e r 1 = x . m l a r e a o f c o u n t i n g g r i d 0 . 9 5 x 2 X = mean number of c e l l s i n 10 or more f i e l d s 2 = ml of sample f i l t e r e d 0 . 9 5 = d i l u t i o n f a c t o r a f t e r f o r m a l d e h y d e p r e s e r v a t i o n s t a i n e d a r e a of f i l t e r = 2 . 0 x l 0 8 M m 2 a r e a of c o u n t i n g g r i d = 9 , 2 0 0 Mm2 16 3.4 D i s s o l v e d M o n o s a c c h a r i d e s D i s s o l v e d m o n o s a c c h a r i d e s (MCHO) were a n a l y s e d by the method d e s c r i b e d by Johnson & S i e b u r t h (1977) and Johnson e t a l . ( 1 9 8 1 ) . The e x c e s s b o r o h y d r i d e was d e s t r o y e d w i t h 0 .05 ml of 0 .7 N H C l i n s t e a d of 0 .36 N H C l . The h i g h e r a c i d i t y a s s u r e d c o m p l e t e o x i d a t i o n , t h u s i m p r o v i n g m o n o s a c c h a r i d e r e c o v e r y . O t h e r w i s e t h e o u t l i n e d p r o c e d u r e was f o l l o w e d m e t i c u l o u s l y . The t h r e e p a r t a n a l y s i s b e g i n s w i t h a b o r o h y d r i d e r e d u c t i o n t o c o n v e r t p e n t o s e s and hexose s t o t h e i r sugar a l c o h o l s . The t o t a l a l d i t o l s a r e t h e n o x i d i z e d w i t h p e r i o d a t e , t o form two moles of f o r m a l d e h y d e per mole of m o n o s a c c h a r i d e . The f o r m a l d e h y d e i s a n a l y s e d s p e c t r o p h o t o m e t r i c a l l y w i t h 3 - m e t h y l - 2 -b e n z o t h i a z o l i n o n e h y d r a z o n e h y d r o c h l o r i d e (MBTH). The method i s s u i t a b l e f o r c o n c e n t r a t i o n s as low as 40 Mg l " 1 t o t a l m o n o s a c c h a r i d e c a r b o n . S a l i n i t y has no e f f e c t on t h e t e s t . The e x c l u s i o n of c o n t a m i n a t i o n f rom the s e n s i t i v e MBTH a s s a y was f i n a l l y a c h i e v e d w i t h o u t p r e c o m b u s t i o n of the g l a s s w a r e . In a l l d e t e r m i n a t i o n s g l u c o s e was used as t h e m o n o s a c c h a r i d e s t a n d a r d . 3 .5 H e t e r o t r o p h i c A c t i v i t y The measurement of r e l a t i v e h e t e r o t r o p h i c a c t i v i t y i s ba sed on the f o l l o w i n g c o n s i d e r a t i o n s . I f one assumes t h a t V m Q ) C (maximum u p t a k e v e l o c i t y ) changes w i t h t i m e but t h e v a l u e ( K t + S n ; K t = t r a n s p o r t c o n s t a n t a t 1/2 maximum u p t a k e r a t e , S n= n a t u r a l s u b s t r a t e c o n c e n t r a t i o n ) does n o t , i t means t h a t b a c t e r i a l p o p u l a t i o n s become 'more a c t i v e ' o r ' l e s s a c t i v e ' ( P a r s o n s e t a l . 1 9 7 7 ) . A c c o r d i n g t o G r i f f i t h s e t a l . (1977) 1 7 V m a x i s c o r r e l a t e d w i t h s i n g l e s u b s t r a t e a d d i t i o n s and g i v e s a measure of r e l a t i v e m i c r o b i a l a c t i v i t y . The measurement must be r e l a t e d t o two v a r i a b l e s : ( 1 ) , t h e number of b a c t e r i a and ( 2 ) , t h e m e t a b o l i c s t a t e of t h e b a c t e r i a . S i n c e t h e number of b a c t e r i a i s d e t e r m i n e d i n d e p e n d e n t l y by c o u n t i n g , f u r t h e r v a r i a t i o n i n r e l a t i v e m e t a b o l i c a c t i v i t y can be d i a g n o s e d as b e i n g due t o t h e m e t a b o l i c s t a t e of t h e b a c t e r i a . Samples f o r h e t e r o t r o p h i c a c t i v i t y measurement were p r o c e s s e d i m m e d i a t e l y a f t e r s u b s a m p l i n g on s h i p b o a r d or i n t h e c o n t r o l l e d e n v i r o n m e n t chamber . D u p l i c a t e d a r k b o t t l e s were f i l l e d w i t h 100 ml a l i q u o t s and i n c u b a t e d w i t h u n i f o r m l y l a b e l l e d D - [ 1 4 C ( U ) ] g l u c o s e ( s p e c i f i c a c t i v i t y 4 .8 mCi m m o l " 1 , New E n g l a n d N u c l e a r ) . O n l y one c o n c e n t r a t i o n of s u b s t r a t e (165 yg g l u c o s e ) was added and t h e net u p t a k e of l a b e l l e d s o l u t e measured . F i e l d samples were i n c u b a t e d f o r two h o u r s a t ambient t e m p e r a t u r e ± 2 ° C ; l a b o r a t o r y samples were i n c u b a t e d f o r one hour a t 1 2 ° C . A f t e r f i l t e r i n g t h r o u g h 0 .22 /nm f i l t e r s ( M i l l i p o r e , 25 mm d i a m e t e r ) , f i l t e r s were p l a c e d i n s c i n t i l l a t i o n v i a l s f i l l e d w i t h 10 ml of A q u a s o l - 2 (New E n g l a n d N u c l e a r ) . A b l a n k was f i l t e r e d i m m e d i a t e l y a f t e r a d d i t i o n o f 1 " C - g l u c o s e t o 100 ml of s e a w a t e r . Samples were c o u n t e d 24 h l a t e r i n an U n i l u x I I I L i q u i d S c i n t i l l a t i o n Sys tem ( N u c l e a r C h i c a g o , Des P l a i n e s , 1 1 1 . , U S A ) . A s t a n d a r d ' " C - s o u r c e was used f o r e f f i c i e n c y c o r r e c t i o n . G i v e n the s p e c i f i c a c t i v i t y of 1 " C - g l u c o s e , t h e i n c u b a t i o n t i m e and sample s i z e , t h e u p t a k e of g l u c o s e i n uq l " 1 h ~ 1 was c a l c u l a t e d . T h i s r e l a t i v e h e t e r o t r o p h i c a c t i v i t y was t h e n r e l a t e d t o the t o t a l number o f 18 b a c t e r i a i n t h e s a m p l e , and e x p r e s s e d as g l u c o s e u p t a k e i n yg h " 1 per 10 9 b a c t e r i a . 3 .6 P h y t o p l a n k t o n And M i c r o z o o p l a n k t o n F o r a n a l y s i s of f i e l d and l a b o r a t o r y p l a n k t o n p o p u l a t i o n s , 100-200 ml samples were p r e s e r v e d w i t h 5-10 d r o p s of L u g o l ' s s o l u t i o n (200 g KI + 100 g I 2 i n 2000 ml H 2 0 + 190 ml g l a c i a l C H 3 C O O H ) . C o u n t i n g and i d e n t i f i c a t i o n was done i n a P a l m e r -Malony chamber (volume 0.1 m l ) . A t o t a l of 400 c e l l s i n a t l e a s t f o u r f i l l i n g s were c o u n t e d . H e t e r o t r o p h i c and a u t o t r o p h i c f l a g e l l a t e s were not s e p a r a t e l y e n u m e r a t e d . Based on 400 c e l l s , t h e r e l a t i v e abundance o f s p e c i e s i n the sample was c a l c u l a t e d . F l a g e l l a t e s were grouped a c c o r d i n g t o s i z e , s m a l l ones o f < 25 M m and l a r g e ones of > 25 MHI l e n g t h . To c a l c u l a t e c e l l v o l u m e , t h e d i m e n s i o n s of 20 c e l l s were ' measured ; t h e p h y t o p l a n k t e r s Ske le tonema c o s t a t u m and T h a l a s s i o s i r a s p p . were assumed t o be c y l i n d r i c a l . I n l a b o r a t o r y e x p e r i m e n t s l i v e c e l l c o u n t s f o r g rowth r a t e e s t i m a t e s o f p h y t o p l a n k t o n were done by u s i n g a F u c h s - R o s e n t h a l chamber (volume 3 . 2 x 1 0 " 3 m l ) . L i v e m i c r o z o o p l a n k t o n e s t i m a t e s were a r r i v e d a t by e m p l o y i n g t h e same chamber . I t was assumed t h a t t h e numbers of f l a g e l l a t e s l e a v i n g the f i e l d o f v i e w was e q u a l t o t h e number swimming i n t o v i e w . A g a i n g reen and c o l o u r l e s s f l a g e l l a t e s were not d i s t i n g u i s h e d . The l a r g e s t z o o p l a n k t e r s i n the m i c r o c o s m s were t i n t i n n i d s . These were enumerated a f t e r p r e s e r v a t i o n w i t h a d r o p of L u g o l ' s s o l u t i o n i n a P a l m e r - M a l o n y chamber under low m a g n i f i c a t i o n . 19 I I I . THE STANDARD PATTERN 1. INTRODUCTION The ' c a p t u r e d ' m i c r o p l a n k t o n p o p u l a t i o n i s thought t o r e p r e s e n t a v i a b l e p a r t of t h e e s t u a r i n e e c o s y s t e m . C o n t a i n m e n t e n s u r e s t h a t t h e b i o t a can be m o n i t o r e d o v e r a p e r i o d of t i m e and f a c i l i t a t e s m a n i p u l a t i o n s by the r e s e a r c h e r . The i n i t i a l n u t r i e n t e n r i c h m e n t r e s u l t s i n a d i s t i n c t p a t t e r n of a u t o t r o p h i c and h e t e r o t r o p h i c g r o w t h . T h i s s t a n d a r d p a t t e r n r e semble s a n a t u r a l d i a t o m s p r i n g b l o o m . Due t o s i m u l a t i o n o f the e s t u a r i n e s a l i n i t y and n u t r i e n t r e g i m e , t h e t o t a l b iomass w i l l i n c r e a s e w i t h h i g h e r s a l i n i t i e s , w h i l e t h e sequence of h e t e r o t r o p h i c and a u t o t r o p h i c g r o w t h remains t h e same. The s a l i n i t y r a n g e , and s e a s o n a l and e x p e r i m e n t a l v a r i a b i l i t y i n f l u e n c e the s t a n d a r d p a t t e r n as do a b i o t i c f a c t o r s due t o the e x p e r i m e n t a l s e t - u p and s o - c a l l e d b o t t l e e f f e c t s . T h i s has t o be kept i n mind when a n a l y z i n g t h e r e s u l t s and c o m p a r i n g them t o t h e f i e l d . I n o r d e r t o i n v e s t i g a t e the p r o c e s s e s w h i c h g e n e r a t e and g o v e r n the s t a n d a r d p a t t e r n , h e t e r o t r o p h i c g r o w t h i s s t u d i e d i n D a r k - e x p e r i m e n t s . F u r t h e r i n c r e a s e s i n o r g a n i c s u b s t r a t e h e l p t o e s t i m a t e b a c t e r i a l g r o w t h r a t e s and t h r e s h o l d c o n c e n t r a t i o n s f o r u p t a k e . P h y t o p l a n k t o n g r o w t h r a t e s and n i t r o g e n c e l l q u o t a a r e o b t a i n e d f rom L i g h t - e x p e r i m e n t s . The e x p e r i m e n t a l d a t a g a t h e r e d i n t h i s way a r e used t o t e s t t h e n u m e r i c a l models as shown i n C h a p t e r I V . 20 The s t a n d a r d p a t t e r n r e p r e s e n t s a s i m u l a t i o n of a p h y t o p l a n k t o n b l o o m , w h i c h i s i n i t i a l l y c h a r a c t e r i z e d by a p e r i o d of u n l i m i t e d g r o w t h and h i g h p r o d u c t i v i t y . Under t h e s e c o n d i t i o n s , t h e impact of n a t u r a l and man-made p e r t u r b a t i o n s on t h e m i c r o p l a n k t o n p o p u l a t i o n can be examined and s e p a r a t e d from o t h e r p o s s i b l e f a c t o r s w h i c h i n f l u e n c e a u t o t r o p h i c and h e t e r o t r o p h i c g r o w t h . The i m p o r t a n c e of t h e s t a n d a r d p a t t e r n w i l l become a p p a r e n t i n p e r t u r b a t i o n e x p e r i m e n t s d e s c r i b e d i n C h a p t e r V , where i t s e r v e s as a c o n t r o l . D e v i a t i o n s f rom t h e p a t t e r n w i l l be used t o show t h e impact of t h e imposed d i s t u r b a n c e s on the s i m u l a t e d b l o o m . 21 2. PATTERN OF EVENTS The i n i t i a l n u t r i e n t a d d i t i o n a s s u r e d n o n - l i m i t i n g g r o w t h c o n d i t i o n s w i t h r e s p e c t t o n i t r o g e n , p h o s p h a t e , s i l i c a t e , i r o n and o r g a n i c s u b s t r a t e . I t c r e a t e d an e n v i r o n m e n t s i m i l a r t o t h a t p r e c e d i n g a n a t u r a l p h y t o p l a n k t o n s p r i n g bloom. F i g u r e 3 shows a t y p i c a l p a t t e r n of n i t r o g e n and c h l o r o p h y l l a c o n c e n t r a t i o n s and b a c t e r i a l and m i c r o f l a g e l l a t e numbers as f o u n d i n t h e m i c r o c o s m e x p e r i m e n t s . W h i l e a b s o l u t e v a l u e s d i f f e r e d , t h e p a t t e r n o f e v e n t s was s i m i l a r a t a l l s a l i n i t i e s . The b a c t e r i a l p o p u l a t i o n r e s p o n d e d i n between 24 and 48 h t o t h e change i n t h e e n v i r o n m e n t and t o t h e n u t r i e n t ( g l u c o s e ) a d d i t i o n . Numbers p e a k e d i n a f i r s t bloom between days 3 and 5, d r o p p e d s h a r p l y by day 7, and i n c r e a s e d t o a s e c o n d h e t e r o t r o p h i c bloom f o l l o w i n g t h e p h y t o p l a n k t o n bloom. A u t o t r o p h i c g r o w t h s t a r t e d a f t e r a l a g phase o f 3 - 5 d a y s , and c h l o r o p h y l l a v a l u e s p e a k e d between d a y s 7 and 11, d e p e n d i n g on t h e s i z e o f t h e i n o c u l u m and t h e d ominant a l g a e . At day 7 n i t r o g e n ( n i t r a t e and n i t r i t e ) was n e a r l y e x h a u s t e d and b a c t e r i a l numbers r e a c h e d a minimum. I n c r e a s i n g c o n c e n t r a t i o n s of p h a e o p i g m e n t s by day 9 i n d i c a t e d d e c a y o f c e l l s as n u t r i e n t s became l i m i t i n g ( s e e F i g u r e 1 3 ) . An i n i t i a l d e c r e a s e i n m i c r o f l a g e l l a t e numbers a t i n t e r m e d i a t e s a l i n i t i e s , 10 p p t and 18 p p t , was most l i k e l y due t o t h e a b r u p t e n v i r o n m e n t a l c h a n g e s w h i c h o c c u r r e d when h i g h and low s a l i n i t y s o u r c e w a t e r s were m i x e d . In a l l e x p e r i m e n t s , m i c r o f l a g e l l a t e s seemed t o f o l l o w t h e i n i t i a l i n c r e a s e i n b a c t e r i a l b i o m a s s c l o s e l y , w h i l e a 22 F i g u r e 3 - C o n c e n t r a t i o n s o f n i t r a t e and n i t r i t e , c h l o r o p h y l l a , b a c t e r i a and m i c r o f l a g e l l a t e s i n 18 ppt s a l i n i t y m i c r o c o s m s . Mean v a l u e s of s i x e x p e r i m e n t s a r e shown. F o r s t a n d a r d e r r o r s see F i g u r e s 4, 5 , 6 . 23 c o r r e l a t i o n w i t h the second h e t e r o t r o p h i c b loom was l e s s s t r i k i n g . H i g h numbers of t i n t i n n i d s (30 t o 80 m l " 1 ) were found by day 15. The n u t r i e n t a d d i t i o n on day 2 i n c r e a s e d t h e b a c k g r o u n d l e v e l of n i t r a t e . B a c t e r i a and p h y t o p l a n k t o n need n i t r o g e n f o r p r o t e i n s y n t h e s i s , but b o t h p r e f e r t o t a k e up ammonium because i t i s l e s s e n e r g y c o s t l y (Conway 1977; M a n d e l s t a m e_t a l . 1 9 8 2 ) . T h e r e f o r e , t h e i n i t i a l c o n c e n t r a t i o n of ammonium ( a p p r o x . up t o 4 y g - a t l " 1 ) was e x h a u s t e d b e f o r e t h e n i t r a t e p l u s n i t r i t e was t a k e n up ( c f . F i g u r e 2 1 ) . N i t r o g e n compounds e x c r e t e d by t h e m i c r o z o o p l a n k t o n were assumed t o be r e c y c l e d i m m e d i a t e l y by b a c t e r i a and p h y t o p l a n k t o n , u n t i l a n o t h e r s u b s t r a t e became l i m i t i n g . The model becomes u n r e a l i s t i c f rom day 7 on w i t h r e s p e c t t o a n a t u r a l b l o o m , because i n t h e F r a s e r R i v e r e s t u a r y t h e b iomass of h e r b i v o r o u s z o o p l a n k t o n i s c l o s e l y r e l a t e d t o the p r i m a r y p r o d u c t i v i t y ( P a r s o n s 1 9 7 9 ) . These g r a z e r s , m a i n l y c o p e p o d s , were not o b s e r v e d i n t h e m i c r o c o s m s . The l a r g e s t g r a z e r s p r e s e n t were s e v e r a l s p e c i e s of t i n t i n n i d s , w h i c h seem t o f o r a g e on t h e m i c r o f l a g e l l a t e p o p u l a t i o n ( S p i t t l e r 1973; H e i n b o k e l & B e e r s 1 9 7 9 ) . 24 3 . V A R I A B I L I T Y S e a s o n a l changes i n the F r a s e r R i v e r a r e i n g e n e r a l much g r e a t e r t h a n i n the h i g h s a l i n i t y water c o l l e c t e d from 20 m d e p t h i n B u r r a r d I n l e t . Tempera ture i n the s u r f a c e water o f t h e fo rmer v a r i e s between 0 °C i n J a n u a r y and 19°C i n A u g u s t ( B e n e d i c t e_t a l . 1973) , w h i l e i n t h e deep s a l i n e water i t v a r i e s o n l y between 7 and 10°C d u r i n g t h e y e a r ( D a v i d s o n 1979) . N u t r i e n t c o n c e n t r a t i o n s showed a d i s t i n c t s e a s o n a l c y c l e . N i t r o g e n ( n i t r a t e p l u s n i t r i t e ) v a l u e s ranged from 4.1 ug-at l " 1 t o 19.6 ug-a t l " 1 i n the r i v e r w a t e r . Lowest c o n c e n t r a t i o n s were found i n September , h i g h e s t i n J a n u a r y . I n the h i g h s a l i n i t y w a t e r , v a l u e s ranged from 2 2 . 5 Mg-at 1~ 1 i n Sept t o 47 .7 /xg-at 1" 1 i n A p r i l . L i k e w i s e , ammonium c o n c e n t r a t i o n s v a r i e d w i t h t h e s e a s o n s , but were r e p o r t e d t o be l o w e r and l e s s v a r i a b l e i n m a g n i t u d e (Shim 1977; D r i n n a n & C l a r k 1980) . S e a s o n a l d i f f e r e n c e s i n t h e p l a n k t o n community w i t h r e s p e c t t o b iomass and s p e c i e s c o m p o s i t i o n c a u s e d c o n s i d e r a b l e v a r i a b i l i t y i n t h e i n o c u l u m . Water c o l l e c t e d d u r i n g t h e w i n t e r months was d o m i n a t e d by m i c r o f l a g e l l a t e s as d e s c r i b e d by T a k a h a s h i & H o s k i n s (1978) f o r w i n t e r c o n d i t i o n s i n S a a n i c h I n l e t . A c c o r d i n g l y , l o w e s t c h l o r o p h y l l a c o n c e n t r a t i o n s were found i n w a t e r s d u r i n g t h i s p e r i o d , w h i l e d u r i n g t h e o t h e r seasons p h y t o p l a n k t o n were more a b u n d a n t , t h u s i n c r e a s i n g the b iomass of t h e i n o c u l u m . B a c t e r i a l numbers were c o n s i s t e n t l y h i g h e r i n the b r a c k i s h r i v e r w a t e r , f l u c t u a t i n g between 1 . 0 4 x ! 0 6 - 2 . 0 6 x 1 0 s m l " 1 , w h i l e 25 b a c t e r i a i n t h e h i g h s a l i n i t y w a t e r v a r i e d between 0 . 1 9 X 1 0 6 -1 . 0 3 x 1 0 s m l - 1 . The numbers r e p r e s e n t t o t a l c e l l c o u n t s and no d i s t i n c t i o n was made between f r e e or p a r t i c l e - a t t a c h e d b a c t e r i a . The p r o p o r t i o n of t h e l a t t e r d e c r e a s e s w i t h i n c r e a s i n g s a l i n i t y , and i n the S t r a i t of G e o r g i a 85% of t h e b a c t e r i a a r e f r e e - l i v i n g ( B e l l & A l b r i g h t 1 9 8 1 ) . B e s i d e v a r i a t i o n s i n b i o m a s s , the m i c r o p l a n k t o n community a l s o e x p e r i e n c e s p h y s i o l o g i c a l changes d u r i n g d i f f e r e n t s e a s o n s , e . g . p h o t o s y n t h e t i c r a t e i n c r e a s e s w i t h warming o f the w a t e r i n s p r i n g ( T a k a h a s h i e t a l . 1973) and p e r i o d s of maximum h e t e r o t r o p h i c a c t i v i t y a r e found i n summer and f a l l ( A l b r i g h t 1 9 7 7 ) . D e s p i t e t h e many known and unknown s o u r c e s of v a r i a t i o n , the s i m u l a t e d p a t t e r n i n t h e microcosms rema ins t h e same. The r e s u l t i n g c h l o r o p h y l l a , b a c t e r i a and n i t r o g e n c u r v e s ( F i g u r e s 4 , 5 , and 6) r e p r e s e n t d a t a c o l l e c t e d a t d i f f e r e n t seasons of the y e a r ( w i n t e r , s p r i n g and f a l l ) , so t h a t v a r i a b i l i t y r e f l e c t s s e a s o n a l d i f f e r e n c e s i n t h e r e s p o n s e of t h e d i f f e r e n t w a t e r s as w e l l as d i f f e r e n c e s between the m i c r o c o s m s . The r e p l i c a t i o n of m i c r o c o s m s was examined i n seven e x p e r i m e n t s a t two d i f f e r e n t s e a s o n s ; r e s u l t s a r e shown i n F i g u r e s 7 , 8 , and 9 . N i t r a t e and n i t r i t e c o n c e n t r a t i o n s were not s i g n i f i c a n t l y d i f f e r e n t between r e p l i c a t e s c o n s i d e r i n g a p r e c i s i o n o f ± 1 . 1 4 uq-at l " 1 i n sample a n a l y s i s . B a c t e r i a l c o u n t s were based on e n u m e r a t i o n of t e n m i c r o s c o p i c f i e l d s and a t o t a l o f a t l e a s t 200 c e i l s . A c c o r d i n g t o S t u d e n t ' s t s t a t i s t i c the r e l a t i v e e r r o r (a=0.05) of the mean (n=l0) b a c t e r i a l numbers F i g u r e 4 - The p a t t e r n of c h l o r o p h y l l a c o n c e n t r a t i o n s ; d a t a c o l l e c t e d a t d i f f e r e n t seasons ( w i n t e r , s p r i n g , f a l l ) . B a r s i n d i c a t e mean ± 1 S . E . ; n=number o f e x p e r i m e n t s . 27 F i g u r e 5 - The p a t t e r n of b a c t e r i a l numbers ; d a t a c o l l e c t e d a t d i f f e r e n t seasons ( w i n t e r , s p r i n g , f a l l ) . B a r s i n d i c a t e mean ± 1 S . E . ; n=number of e x p e r i m e n t s . 28 F i g u r e 6 - The p a t t e r n of n i t r a t e p l u s n i t r i t e c o n c e n t r a t i o n s ; d a t a c o l l e c t e d a t d i f f e r e n t s ea sons ( w i n t e r , s p r i n g , f a l l ) . V a l u e s on day 2 r e p r e s e n t n a t u r a l c o n c e n t r a t i o n s p l u s a d d i t i o n s . B a r s i n d i c a t e mean ± 1 S . E . ; n=number o f e x p e r i m e n t s . 29 15pptS 23ppt S F i g u r e 7 - N i t r a t e and n i t r i t e c o n c e n t r a t i o n s i n r e p l i c a t i o n e x p e r i m e n t s : da shed and s o l i d l i n e s i n (a) and (b) r e p r e s e n t i d e n t i c a l t r e a t m e n t ; i n (c ) and ( d ) , wa te r o f one m i c r o c o s m (dashed) was f i l t e r e d t h r o u g h a 116 ym n e t . F i g u r e 8 - B a c t e r i a l numbers i n r e p l i c a t i o n e x p e r i m e n t s : da shed and s o l i d l i n e s i n (b) and (c ) r e p r e s e n t i d e n t i c a l t r e a t m e n t , day 9 no s a m p l e s ; i n ( a ) , w a t e r o f one m i c r o c o s m (dashed) was f i l t e r e d t h r o u g h a 116 um n e t . 31 t 3 6 7 ' l i 13 16 17 20 DAYS F i g u r e 9 - C h l o r o p h y l l a c o n c e n t r a t i o n s i n r e p l i c a t i o n e x p e r i m e n t s : dashed and s o l i d l i n e s r e p r e s e n t i n (a) and (b) i d e n t i c a l t r e a t m e n t ; i n ( a ) , b o t h m i c r o c o s m s w i t h s t a n d a r d n u t r i e n t a d d i t i o n p l u s 5 mg l " 1 g l u c o s e ; day 9 no s a m p l e s . 32 was 15% ( V e n r i c k 1978 ) . V a r i a b i l i t y was g r e a t e s t i n the c h l o r o p h y l l a c o n c e n t r a t i o n s . The p r e c i s i o n of the a n a l y s i s was ± 1.51 yg l " 1 f o r c h l o r o p h y l l a v a l u e s r a n g i n g from 0 -10 Mg l " 1 . S i n c e c h l o r o p h y l l a c o n c e n t r a t i o n s a t t h e h e i g h t o f t h e p h y t o p l a n k t o n bloom exceeded t h i s range up t o s e v e n - f o l d , d i l u t i o n of t h e a c e t o n e e x t r a c t s was n e c e s s a r y . T h e r e f o r e , the p r e c i s i o n was c o n s i d e r a b l y l o w e r i n t h e s e s a m p l e s . 33 4 . EFFECTS OF SALINITY The l a b o r a t o r y model s i m u l a t e s the e s t u a r i n e e c o s y s t e m w i t h r e s p e c t t o a s a l i n i t y r a n g e . T h e r e f o r e , t h i s e n v i r o n m e n t a l f a c t o r i s d i s c u s s e d s e p a r a t e l y from a l l t h e o t h e r s w h i c h i n f l u e n c e t h e m i c r o p l a n k t o n e c o l o g y . S e v e r a l p r o p e r t i e s a r e c l o s e l y a s s o c i a t e d w i t h s a l i n i t y , among them n u t r i e n t s , b a c t e r i a l a c t i v i t y and p h y t o p l a n k t o n s p e c i e s c o m p o s i t i o n . I n the F r a s e r R i v e r e s t u a r y , n i t r o g e n i n the s u r f a c e l a y e r i s s u p p l i e d from e n t r a i n m e n t of sea water ( T u l l y & Dodimead 1957; P a r s o n s e_t ajL. 1980) r a t h e r t h a n by i m p o r t from t h e r i v e r ( S t o c k n e r e_t a_l. 1 9 7 9 ) . N i t r a t e d a t a of t h e s o u r c e w a t e r s ( F r a s e r R i v e r and B u r r a r d I n l e t ) c o n f i r m e d the e n t r a i n m e n t mechani sm. V a l u e s i n w a t e r s of 20 m d e p t h a l w a y s exceeded t h o s e found i n the r i v e r s u r f a c e w a t e r s . S i l i c a t e i s h i g h e r i n t h e f r e s h water t h a n i n t h e sea water and s u b s t a n t i a l i n c r e a s e i n c o n c e n t r a t i o n s has been o b s e r v e d d u r i n g t h e b e g i n n i n g of t h e a n n u a l f r e s h e t ( P a r s o n s 1 9 7 9 ) . A n o t h e r n u t r i e n t t h a t a p p e a r s t o be h i g h e r i n the F r a s e r R i v e r i s v i t a m i n B 1 2 ( C a t t e l l 1 9 7 3 ) . C o n c e n t r a t i o n s of o r g a n i c c a r b o n a r e c l o s e l y a s s o c i a t e d w i t h the d e c o m p o s i t i o n of p h y t o p l a n k t o n , but a p p r e c i a b l e q u a n t i t i e s a r e l a n d - d e r i v e d v i a r u n o f f ( S tephens e t a l . 1 9 6 7 ) . The a v a i l a b i l i t y of m i c r o n u t r i e n t s ( e . g . t r a c e m e t a l s ) i s g r e a t l y i n f l u e n c e d by changes i n s a l i n i t y ( C r o s s & Sunda 1 9 7 7 ) . W h i l e n i t r o g e n and c o n s e q u e n t l y p h y t o p l a n k t o n b iomass i n c r e a s e w i t h h i g h e r s a l i n i t i e s , o r g a n i c c a r b o n and s i l i c a t e show a r e v e r s e r e l a t i o n s h i p . 34 A c c o r d i n g t o S e k i e t a l . (1969) t h e m e t a b o l i s m o f b a c t e r i a i s a f f e c t e d by s a l i n i t y c h a n g e s , and because of t h e i r s l ow g r o w t h r a t e s i n the n a t u r a l e n v i r o n m e n t due t o low l e v e l s of o r g a n i c s u b s t r a t e , b a c t e r i a need some t i m e t o adapt t o a new s a l i n i t y reg ime ( T a k a h a s h i & N o r t o n 1977) . V a l d e s (1980) has shown i n t r a n s p l a n t e x p e r i m e n t s , t h a t the m a j o r i t y of f r e s h w a t e r b a c t e r i a i s k i l l e d when p l a c e d i n m a r i n e w a t e r , but t h e i r m a r i n e c o u n t e r p a r t s r e m a i n v i a b l e i n F r a s e r R i v e r w a t e r . The same a u t h o r s u g g e s t s t h a t h e t e r o t r o p h i c a c t i v i t y of the l a t t e r i s even s t i m u l a t e d w i t h i n a c e r t a i n s a l i n i t y r a n g e . In m i c r o c o s m e x p e r i m e n t s , h i g h e r r e l a t i v e h e t e r o t r o p h i c a c t i v i t y was found a t 10 pp t and 18 ppt s a l i n i t y ( T a b l e 1 ) , i n t h o s e c o n t a i n e r s where h i g h and low s a l i n i t y s o u r c e w a t e r s were m i x e d . I t a p p e a r s t h a t t h e m i x t u r e p r o v i d e d b e t t e r g r o w t h c o n d i t i o n s f o r one or s e v e r a l b a c t e r i a l s t r a i n s of e i t h e r n a t u r a l p o p u l a t i o n . T a b l e 1 - R e l a t i v e h e t e r o t r o p h i c a c t i v i t i e s a t d i f f e r e n t s a l i n i t i e s i n d u p l i c a t e l a b o r a t o r y e x p e r i m e n t s . R a t e s a r e based on u p t a k e o f 1 " C - g l u c o s e and e x p r e s s e d as ixq g l u c o s e h " 1 per 10 9 b a c t e r i a . < 5 pp t 10 ppt 18 ppt > 26 ppt 8 . 5 6 - 1 7 . 6 3 5 7 . 3 5 - 9 1 . 3 3 5 1 . 8 2 - 6 5 . 9 8 2 7 . 6 8 - 4 2 . 8 5 ( x 1 3 . 1 0 ) ( x 7 4 . 3 4 ) ( x 5 8 . 9 0 ) ( x 3 5 . 2 6 ) I n t h e m i c r o c o s m blooms S k e l e t o n e m a c o s t a t u m and 35 T h a l a s s i o s i r a s p p . were c o n s i s t e n t l y d o m i n a n t , as t h e y a r e i n t h e a n n u a l s p r i n g p h y t o p l a n k t o n b loom i n t h e S t r a i t o f G e o r g i a . The dominant d i a t o m s showed a d i s t i n c t p a t t e r n under t h e d i f f e r e n t s a l i n i t y reg imes as shown i n F i g u r e 10. At < 5 p p t , T h a l a s s i o s i r a s p p . were d o m i n a n t , w h i l e a t 10 p p t , S . c o s t a t u m a l w a y s c o m p r i s e d more than 90% of t h e b l o o m . At > 18 p p t , t h e r e l a t i v e abundances of b o t h r e m a i n e d c o n s t a n t w i t h S. c o s t a t u m b e i n g d o m i n a n t . The i n c r e a s e d abundance of S. c o s t a t u m a t 10 p p t , a t r e l a t i v e l y low but not the l o w e s t s a l i n i t y , has a c o r r e l a t i o n i n a sed iment a s semblage a n a l y s i s i n Howe Sound, B . C . ( R o e l o f s i n p r e s s ) . Howe Sound i s a h i g h r u n o f f , e s t u a r i n e i n l e t i n w h i c h t h e Squamish R i v e r a t the head of t h e i n l e t m a r k e d l y l o w e r s t h e s a l i n i t y of t h e s u r f a c e w a t e r s . Mean m o n t h l y d i s c h a r g e of t h e Squamish R i v e r was g r e a t e r i n 1974 ( i . e . , l o w e r s a l i n i t y i n t h e s u r f a c e w a t e r s ) , when S. c o s t a t u m was the dominant b o t h i n t h e p h y t o p l a n k t o n ( S t o c k n e r e t a l . 1977) and i n a l l 24 sed iment a s s e m b l a g e s , t h a n i n 1973, when T h a l a s s i o s i r a s p p . were dominant i n the p h y t o p l a n k t o n . S a l i n i t y v a l u e s (0-20m) i n b o t h 1973 and 1974 d i d not f a l l below 10 ppt e x c e p t a t the mouth of t h e Squamish R i v e r ( S t o c k n e r e t a l . 1 9 7 7 ) . A l t h o u g h a dominance of T h a l a s s i o s i r a s p p . was never o b s e r v e d i n t h e l a b o r a t o r y mic rocosms w i t h > 10 ppt s a l i n i t y due t o the w e e d - l i k e g r o w t h of S. c o s t a t u m i n c u l t u r e s , t h e r e s u l t s of the e x p e r i m e n t s m i g h t e x p l a i n the dominance of S . c o s t a t u m i n an u n u s u a l y e a r , s u c h as 1974, when s u r f a c e water s a l i n i t y was m a r k e d l y l o w e r . A l t h o u g h s a l i n i t y i s not t h e o n l y c o n t r o l l i n g f a c t o r of t h e 3 6 <5%o 1 0 ° / c CO 187c oo >26%o - 80 o o o c <o TJ C D < CU _> JO CD r r 40 Hh n=6 n - 6 Thalassiosira spp. n = 4 n = 5 | | Skeletonema costatum F i g u r e 10 - R e l a t i v e abundance of d i a t o m s p e c i e s i n m i c r o c o s m e x p e r i m e n t s a t g i v e n s a l i n i t i e s . D a t a were c o l l e c t e d a t d i f f e r e n t s e a s o n s . B a r s r e p r e s e n t ± 1 S . E . o f mean; n=number of e x p e r i m e n t s . 37 abundance of S. c o s t a t u m , t h e s e r e s u l t s d e m o n s t r a t e a c o n s i s t e n t r e l a t i o n s h i p i n t h e m i c r o c o s m and i n t h e f i e l d s t u d i e s ( R o l o e f s & S p i e s u n p u b l . MS). S k e l e t o n e m a s p p . seem t o t h r i v e u n d e r b r a c k i s h water c o n d i t i o n s as n o t i c e d i n K i e l F j o r d , West Germany ( L e n z 1977). The main r e a s o n might be t h e a b s e n c e o f c o m p e t i t i o n i n an extreme e n v i r o n m e n t where f r e s h and s a l t w a t e r mix. A minimum o f s p e c i e s abundance i s r e a c h e d a t a b o u t 6 p p t s a l i n i t y (Remane & S c h l i e p e r 1971). The e f f e c t o f s a l i n i t y on p h y t o p l a n k t o n s p e c i e s r e q u i r e s f u r t h e r s t u d y . M o r p h o l o g i c a l c h a n g e s i n v a l v e c o n f i g u r a t i o n have been o b s e r v e d i n S. s u b s a l s u m ( P a a s c h e e t a_l. 1975). The c e l l s h a p e s of e s t u a r i n e S. c o s t a t u m a r e v e r y v a r i a b l e , b ut i t i s n ot known whether t h e r e i s a c o r r e l a t i o n w i t h c h a n g e s i n s a l i n i t y . At < 5 p p t , f r e s h w a t e r a l g a e c o n t r i b u t e d c o n s i d e r a b l y t o th e t o t a l b i o m a s s i n t h e m i c r o c o s m s , but t h e i r numbers d e c r e a s e d r a p i d l y i n h i g h e r s a l i n i t i e s . The abundance o f m i c r o z o o p l a n k t o n was a l w a y s g r e a t e r i n t h e r i v e r w a ter t h a n i n t h e deep s a l i n e w a t e r . One e x p l a n a t i o n m i g h t be t h e o c c u r r e n c e of b o t h c o l o u r e d and c o l o u r l e s s m i c r o f l a g e l l a t e s i n t h e s u r f a c e water o f t h e F r a s e r R i v e r , w h i l e a t 20 m d e p t h i n B u r r a r d I n l e t p h o t o s y n t h e t i c f l a g e l l a t e s m i g h t be l i g h t - l i m i t e d and l e s s a b u n d a n t . E n t r a i n m e n t of deep s a l i n e water i n t h e e s t u a r y m i x e s n u t r i e n t s as w e l l as c o l o u r l e s s m i c r o f l a g e l l a t e s i n t o t h e s u r f a c e l a y e r , t h u s i n c r e a s i n g t h e abundance o f m i c r o z o o p l a n k t o n . 38 5 . EFFECTS OF ABIOTIC FACTORS 5.1 B o t t l e E f f e c t s I s o l a t i n g n a t u r a l w a t e r s i n any k i n d of c o n t a i n e r w i l l have e f f e c t s on t h e g r o w t h of the b i o t a , but v e r y l i t t l e i s known of t h e c ause s and m a g n i t u d e of t h e s e b o t t l e e f f e c t s . They a r e dependent on t h e volume t o s u r f a c e r a t i o of the c o n t a i n e r , d e c r e a s e w i t h i n c r e a s i n g r a t i o , i . e . l a r g e r c o n t a i n e r s , and i n c r e a s e w i t h i n c r e a s i n g n u t r i e n t l e v e l s ( Z o b e l l 1943) . When w o r k i n g w i t h n a t u r a l m i c r o p l a n k t o n , w a l l g r o w t h of b a c t e r i a c a n n o t be p r e v e n t e d by any c u r r e n t l y known method ( p e r s o n a l c o m m u n i c a t i o n , Dr H.W. J a n n a s c h , M a r i n e B i o l o g i c a l L a b o r a t o r y , Woods H o l e ) . M a r s h a l l e t a l . (1971) have d i s c u s s e d b a c t e r i a l a t t a c h m e n t t o c o n t a i n e r w a l l s . Measurements by D i S a l v o (1973) show t h a t r a p i d i r r e v e r s i b l e a t t a c h m e n t can o c c u r w i t h i n 1 - 2 m i n u t e s . A l s o , i n c r e a s e d c o n c e n t r a t i o n s of d i s s o l v e d o r g a n i c s , such as g l u c o s e i n the m i c r o c o s m s , s t i m u l a t e p r o d u c t i o n of b a c t e r i a l s u b s t a n c e s w h i c h m e d i a t e a d h e s i o n (Corpe 1974 ) . S u c c e s s i o n i n c o l o n i z a t i o n l e a d s f rom b a c t e r i a t o p r o t o z o a and d i a t o m s . H a r t e e t a l . (1980) have s u c c e s s f u l l y r e d u c e d v i s i b l e s u r f a c e g r o w t h by p o u r i n g t h e c o n t e n t s of t h e i r c o n t a i n e r s i n t o c l e a n ones on a w e e k l y b a s i s . Because o f the s h o r t d u r a t i o n o f t h e e x p e r i m e n t s c o n c e r n e d w i t h i n t h i s t h e s i s , w a l l g r o w t h was not t a k e n i n t o c o n s i d e r a t i o n . W i t h t h e d e c l i n e of t h e p h y t o p l a n k t o n bloom v i s i b l e s u r f a c e g r o w t h o c c u r r e d i n a l l c o n t a i n e r s . At t h i s t i m e i n i t i a l g l u c o s e and n i t r o g e n c o n c e n t r a t i o n s were e x h a u s t e d and 39 w a l l g r o w t h was m a i n t a i n e d by r e c y c l e d n u t r i e n t s . C o n t a i n m e n t of n a t u r a l wate r i s o l a t e s t h e e n c l o s e d o r g a n i s m s , a n d , d e p e n d i n g on the v o l u m e , even a s h o r t i n c u b a t i o n can change the t a x o n o m i c c o m p o s i t i o n and abundance of m i c r o p l a n k t o n . C o n t a i n e r v o l u m e , i n c u b a t i o n t i m e and n u t r i e n t reg ime may a f f e c t t h e c o u r s e of changes w i t h i n c o n t a i n e d p o p u l a t i o n s . D i a t o m s a r e g e n e r a l l y more s u c c e s s f u l t h a n f l a g e l l a t e s (Smayda 1957; V e n r i c k e t a l . 1977) , a t r e n d a l s o o b s e r v e d i n t h e m i c r o c o s m e x p e r i m e n t s . Some s p e c i e s might get p h y s i c a l l y damaged d u r i n g s a m p l i n g p r o c e d u r e and m i x i n g i n the l a b o r a t o r y . A r e d u c t i o n i n s p e c i e s d i v e r s i t y can be e x p l a i n e d on a t h e o r e t i c a l l e v e l w i t h t h e h y p o t h e s i s of " contemporaneous d i s e q u i l i b r i u m " ( H u t c h i n s o n 1961; R i c h e r s o n e t a l . 1970 ) . I t s t a t e s t h a t a l a r g e number of s p e c i e s can c o e x i s t i n a homogeneous e n v i r o n m e n t c o n s i s t i n g of many water p a r c e l s , i f t h e s e p a r c e l s a r e p r e v e n t e d from coming i n t o e q u i l i b r i u m due t o a c o n t i n u o u s i n p u t of p e r t u r b a t i o n s . T h u s , i s o l a t i o n from n a t u r a l e n v i r o n m e n t a l f l u c t u a t i o n s i n c o n t a i n e r s s h o u l d move p o p u l a t i o n s towards e q u i l i b r i u m c o n d i t i o n s and r e d u c e s p e c i e s d i v e r s i t y ( V e n r i c k e_t a l . 1977) . 5 .2 C o n s t a n t P h y s i c a l F a c t o r s C o n s t a n t p h y s i c a l f a c t o r s i n t h e l a b o r a t o r y reduce v a r i a b i l i t y and improve r e p l i c a t i o n . T h i s i s an a d v a n t a g e f o r s t a t i s t i c a l a n a l y s i s , but makes any model l e s s r e a l i s t i c because the n a t u r a l e n v i r o n m e n t i s v a r i a b l e . A r t i f i c i a l l i g h t has a d i f f e r e n t s p e c t r u m t h a n s u n l i g h t and 40 a c o n s t a n t L i g h t r D a r k c y c l e e l i m i n a t e s s e a s o n a l c h a n g e s i n d a y l e n g t h , w h i c h i s l e s s i m p o r t a n t when o n l y one e v e n t o f a y e a r l y c y c l e i s s t u d i e d . In t h e l a b o r a t o r y m i c r o c o s m s i r r a d i a n c e s were r e l a t i v e l y h i g h , f a v o u r i n g d i a t o m s and s m a l l e r s i z e c l a s s e s o f c e l l s (Chan 1978). I r r a d i a n c e s were assumed t o be s a t u r a t i n g even a t t h e peak of t h e p h y t o p l a n k t o n bloom. The i n i t i a l d r o p i n c h l o r o p h y l l a c o n c e n t r a t i o n s may be c a u s e d by a d a p t a t i o n t o h i g h e r l i g h t i n t h e l a b o r a t o r y ( C o s p e r 1982) and by s e d i m e n t a t i o n o f l a r g e d i a t o m s p e c i e s , i n s p i t e of m i x i n g . At a l l s e a s o n s water o f t h e F r a s e r R i v e r was more t u r b i d t h a n t h e h i g h s a l i n i t y deep w a t e r . S e d i m e n t a t i o n o c c u r r e d d e s p i t e c o n s t a n t s t i r r i n g i n low s a l i n i t y m i c r o c o s m s . L a r g e p a r t i c l e s were removed by f i l t r a t i o n o f s o u r c e w a t e r s t h r o u g h a 116-ym-mesh N i t e x s c r e e n w i t h o u t i n f l u e n c i n g b a c t e r i a numbers, n i t r o g e n and c h l o r o p h y l l a c o n c e n t r a t i o n s ( see above F i g u r e s 7 and 8 ) . W i t h t h e d e c l i n e o f t h e p h y t o p l a n k t o n bloom, p a r t i c u l a t e m a t t e r s e t t l e d i n a l l c o n t a i n e r s . S i m i l a r o b s e r v a t i o n s were made d u r i n g s h i p b o a r d t a n k e x p e r i m e n t s (1 m 3 volume) by Smetacek e_t §_1. ( 1 9 8 0 ) . A c c o r d i n g t o t h e s e a u t h o r s , s e t t l i n g o f an i n c r e a s i n g p e r c e n t a g e of p a r t i c u l a t e m a t t e r was o b v i o u s l y c a u s e d by n i t r o g e n d e f i c i e n c y . The s e d i m e n t m a t e r i a l had c o n s i s t e n t l y h i g h e r C/N and C / C h l r a t i o s when compared t o th e s u s p e n d e d m a t t e r , i n d i c a t i n g t h e dependence o f s i n k i n g r a t e on c o m p o s i t i o n o f p a r t i c l e s . The i m p o r t a n c e of m i x i n g w i t h i n e n c l o s e d w a t e r c o l u m n s was o b s e r v e d i n C o n t r o l l e d E c o s y s t e m P o l l u t i o n E x p e r i m e n t s (CEPEX) a t S a a n i c h I n l e t , B.C.. Unmixed c o n t a i n e r s c o n s i s t e n t l y showed 41 r a p i d s e d i m e n t a t i o n of l a r g e - c e l l e d d i a t o m s and t h e i r r e p l a c e m e n t by s m a l l f l a g e l l a t e s (Thomas & S e i b e r t 1977 ) . O n l y b r i e f d a i l y s t i r r i n g m a i n t a i n e d l a r g e r p h y t o p l a n k t o n c e l l s ; t h e N : P r a t i o s were l o w e r and more s i m i l a r t o t h e ' R e d f i e l d ' r a t i o t h a n i n u n s t i r r e d c o n t a i n e r s ( E p p l e y e t a_l. 1978) . M i x i n g seemed t o f a v o u r t h e g r o w t h of d i a t o m s and i n c r e a s e d c h l o r o p h y l l a v a l u e s ( O v i a t t 1 9 8 1 ) . The l i m i t e d a i r exchange i n the microcosms was overcome by g e n t l e a i r - b u b b l i n g , w h i c h e n s u r e d a e r o b i c c o n d i t i o n s . The c o n s t a n t t e m p e r a t u r e of 12°C was about t h e mean v a l u e w h i c h p l a n k t o n e x p e r i e n c e s d u r i n g s p r i n g i n t h e F r a s e r R i v e r e s t u a r y ( B e n e d i c t et_ §_1. 1973 ) . K e e p i n g p h y s i c a l f a c t o r s c o n s t a n t f a c i l i t a t e d t h e s t u d y of s a l i n i t y e f f e c t s i n t h e e s t u a r i n e l a b o r a t o r y m o d e l . 5 . 3 N u t r i e n t A d d i t i o n The i n i t i a l n u t r i e n t a d d i t i o n e n s u r e d t h a t d e s p i t e v a r y i n g c o n c e n t r a t i o n s of m a c r o - n u t r i e n t s d u r i n g t h e y e a r , a b a s i c s u p p l y of n i t r o g e n , p h o s p h a t e , s i l i c a t e and o r g a n i c s u b s t r a t e was a v a i l a b l e f o r h e t e r o t r o p h i c and a u t o t r o p h i c g r o w t h . When b a c k g r o u n d l e v e l s were h i g h , as i n w i n t e r and s p r i n g , t h e y were b o o s t e d ; a t o t h e r seasons deve lopment of t h e s t a n d a r d p a t t e r n was e n s u r e d . E x p e r i m e n t s w i t h o u t i n i t i a l n u t r i e n t a d d i t i o n showed t h e same b a s i c p a t t e r n but r e s u l t e d i n much l o w e r b iomass of b a c t e r i a as w e l l as p h y t o p l a n k t o n . An example i s shown i n F i g u r e 11 . The h e i g h t of the i n i t i a l b a c t e r i a l bloom was r e d u c e d by h a l f w i t h o u t g l u c o s e b e i n g a d d e d . The r e s i d u a l h e t e r o t r o p h i c 42 1 I I l I l l i i I 2 3 5 7 9 11 13 15 17 19 DAYS Figure 11 - Microcosm experiments with nutrient addition=control ( s o l i d lines) and without addition (dashed li n e s ) at 8 ppt s a l i n i t y ; a - chlorophyll a concentrations (uq l " 1 ) , b - b a c t e r i a l numbers ( X 1 0 6 ml" 1). 43 bloom was due t o an i n c r e a s e i n b a c t e r i a l a c t i v i t y c a u s e d by t h e change i n the e n v i r o n m e n t , i . e . c o n t a i n m e n t . In t h e s e m i c r o c o s m s , b a c t e r i a u t i l i z e d t h e o r g a n i c s u b s t r a t e p r e s e n t i n t h e s o u r c e w a t e r s . The g l u c o s e a d d i t i o n d i d not i n f l u e n c e t h e second h e t e r o t r o p h i c bloom w h i c h f o l l o w e d t h e decay of t h e p h y t o p l a n k t o n , because t h e added amount was a l r e a d y e x h a u s t e d i n t h e f i r s t b l o o m . EDTA+iron was n e c e s s a r y t o c o u n t e r a c t b o t t l e e f f e c t s . I r o n i s known t o adhere t o the w a l l o f g l a s s v e s s e l s and t o p r e c i p i t a t e i n s t o r e d w a t e r , a t w h i c h p o i n t i t i s s l o w l y or no l o n g e r a v a i l a b l e t o t h e o r g a n i s m s a t a l l (Lewin & Chen 1 9 7 1 ) . The a r t i f i c i a l c h e l a t o r keeps i r o n i n s o l u t i o n , so t h a t a l g a l g r o w t h i s not l i m i t e d by the l a c k of i t . The a d d i t i o n o f EDTA+iron was an i m p o r t a n t f e a t u r e i n the s i m u l a t i o n o f c o n d i t i o n s p r e c e d i n g t h o s e of a n a t u r a l d i a t o m s p r i n g b l o o m . 44 6 . INTERACTIONS OF THE BIOTA 6 . 1 D a r k - e x p e r imerits To s t u d y h e t e r o t r o p h i c g r o w t h w i t h o u t i n t e r f e r e n c e by a u t o t r o p h s , m i c r o c o s m s were kep t i n the d a r k ( b l a c k garbage b a g s ) . The i n i t i a l a d d i t i o n of 1 mg 1~ 1 g l u c o s e was i n c r e a s e d t o 5 mg l " 1 , and i n two microcosms even f u r t h e r t o 5 mg 1~ 1 on t h r e e c o n s e c u t i v e days i n o r d e r t o enhance b a c t e r i a l g r o w t h . P a r a l l e l t o t h e d a r k - e x p e r i m e n t s , f o u r mic rocosms w i t h s t a n d a r d t r e a t m e n t were m o n i t o r e d . B e s i d e t h e u s u a l s a m p l i n g i n t e r v a l s , samples were t a k e n e v e r y 12 h between days 3 and 7 . C o n c e n t r a t i o n s of t o t a l d i s s o l v e d m o n o s a c c h a r i d e s , n i t r a t e and n i t r i t e , as w e l l as ammonium were a n a l y s e d . The a b s o l u t e v a l u e s of the l a t t e r m i g h t be a f f e c t e d by f r e e z i n g and s t o r a g e , as w e l l as t e c h n i c a l p r o b l e m s i n the a n a l y s i s of low s a l i n i t y samples (Degobb i s 1973; E a t o n & G r a n t 1979 ) . T h e r e f o r e , t h e emphas i s i s on the g e n e r a l p a t t e r n , r a t h e r t h a n a b s o l u t e c o n c e n t r a t i o n s . The methods of a n a l y s i s a r e d e s c r i b e d i n C h a p t e r I I . I n t h e d a r k . m i c r o c o s m s c h l o r o p h y l l a c o n c e n t r a t i o n s n e v e r exceeded 5 tig l " 1 . I n i t i a l t o t a l m o n o s a c c h a r i d e s were 299 ttg l " 1 i n t h e r i v e r water and 156 Mg l " 1 i n the h i g h s a l i n i t y w a t e r . A f t e r a d d i t i o n of n u t r i e n t s i n c l u d i n g 5 mg l " 1 g l u c o s e , b a c t e r i a r e s p o n d e d d u r i n g t h e n e x t 36 h w i t h a d o u b l i n g of numbers a t < 5 p p t , a f i v e - f o l d i n c r e a s e a t 10 p p t , s i x - f o l d a t 18 ppt and n i n e - f o l d a t ^ 26 ppt s a l i n i t y . In t h e l o w e s t s a l i n i t y , n i t r o g e n was l i m i t i n g b a c t e r i a l g r o w t h (see F i g u r e 2 1 ) . By day 4 ammonium c o n c e n t r a t i o n s a t ^ 5 ppt and 45 10 ppt s a l i n i t y were below d e t e c t i o n . N i t r a t e and n i t r i t e were e x h a u s t e d by day 5 .5 a t ^ 5 ppt but a t 10 ppt c o n c e n t r a t i o n s of 2 - 3 y g - a t l " 1 were measured u n t i l t h e end of t h e e x p e r i m e n t , w h i l e i n t h e range of 10 t o > 26 ppt s a l i n i t i e s , g l u c o s e became l i m i t i n g by day 3 .5 t o 4 . 5 . The e x p e r i m e n t a l s e t - u p f a v o u r e d a e r o b i c b a c t e r i a w h i c h were a b l e t o u t i l i z e n i t r o g e n f o r p r o t e i n s y n t h e s i s and c a r b o n as s u b s t r a t e and energy s o u r c e . The u n i f o r m m o r p h o l o g i c a l c e l l shapes d u r i n g t h e f i r s t h e t e r o t r o p h i c bloom i n d i c a t e d t h a t o n l y one or two s p e c i e s c o u l d r e s p o n d t o the sudden i n c r e a s e i n s u b s t r a t e c o n c e n t r a t i o n and were a b l e t o outcompete a l l t h e o t h e r s p r e s e n t i n n a t u r a l w a t e r s . T h i s phenomenon i s not uncommon i n n a t u r e . The a u t o c h t h o n o u s or s t e a d y - s t a t e p o p u l a t i o n c o n s i s t s of s l o w l y g r o w i n g c e l l s , w h i c h a r e w e l l a d a p t e d t o t h e p r e v a i l i n g e n v i r o n m e n t a l c o n d i t i o n s . A sudden change i n t h e s e c o n d i t i o n s , e . g . s u b s t r a t e i n f l u x , g i v e s r i s e t o an e x p o n e n t i a l l y g r o w i n g p o p u l a t i o n (Brock 1 9 7 1 ) . In the microcosms w i t h 5 mg 1~ 1 g l u c o s e a d d i t i o n on t h r e e c o n s e c u t i v e days n i t r o g e n seemed t o be l i m i t i n g b e f o r e o r g a n i c s u b s t r a t e s a t 10 ppt or became l i m i t i n g a t a p p r o x . t h e same t i m e i n t h e > 26 ppt s a l i n i t y c o n t a i n e r . W i t h a t o t a l a d d i t i o n of 15 mg l " 1 g l u c o s e t h e maximum i n c r e a s e i n b a c t e r i a l numbers was s i x - f o l d a t 10 p p t , compared t o f i v e - f o l d a f t e r one a d d i t i o n , but i n c r e a s e d from n i n e - t o 2 3 - f o l d a t > 26 pp t s a l i n i t y i n the p r e s e n c e o f h i g h e r n i t r a t e and n i t r i t e c o n c e n t r a t i o n s . F i g u r e 12 shows a c o m p o s i t e p i c t u r e of b a c t e r i a l numbers , n i t r o g e n and m o n o s a c c h a r i d e c o n c e n t r a t i o n s a t 10 ppt s a l i n i t y . 46 F i g u r e 12 - B a c t e r i a l numbers , m o n o s a c c h a r i d e and n i t r o g e n c o n c e n t r a t i o n s i n D a r k - e x p e r i m e n t s a t 10 ppt s a l i n i t y ; 5 mg 1 ~ 1 g l u c o s e was added on days 2 , 3 , and 4 . 47 M i c r o f l a g e l l a t e numbers peaked t o g e t h e r w i t h t h e b a c t e r i a l bloom ( c f . F i g u r e 1 5 ) . The r a p i d i n c r e a s e i n ammonium beyond day 6 was due t o e x c r e t i o n by m i c r o z o o p l a n k t o n and e x h a u s t i o n of o r g a n i c s u b s t r a t e below t h r e s h o l d l e v e l s a n d / o r l i m i t a t i o n by o t h e r e s s e n t i a l n u t r i e n t s . When b a c t e r i a l numbers d r o p p e d , m i c r o f l a g e l l a t e s d e c r e a s e d w i t h a p p r o x . 1 - 2 days d e l a y . T o g e t h e r w i t h n u t r i e n t e x h a u s t i o n , g r a z i n g might have p r e v e n t e d b a c t e r i a l numbers from i n c r e a s i n g h i g h e r than 1 1 . 5 x 1 0 6 m l " 1 and c o n t r i b u t e d t o t h e i r d e c l i n e . B a c t e r i a l b iomass r e m a i n e d low f o r a n o t h e r 7 days and i n c r e a s e d o n l y s l i g h t l y towards t h e end of the e x p e r i m e n t . H e t e r o t r o p h i c b a c t e r i a a r e dependent on d i s s o l v e d o r g a n i c compounds f o r g r o w t h . O n l y a s m a l l f r a c t i o n of t h e DOC i n t h e n a t u r a l e n v i r o n m e n t has been i d e n t i f i e d ( W i l l i a m s 1975 ) . I n the F r a s e r R i v e r e s t u a r y v a l u e s range f rom 1 - 6 mg C 1 " 1 f o r most of t h e y e a r ( A l b r i g h t 1977 ) . C a r b o h y d r a t e s may a c c o u n t f o r 8 -24% of the t o t a l DOC ( S i e b u r t h 1979 ) . I n n a t u r e (open sea) minimum t h r e s h o l d v a l u e s f o r u p t a k e of m o n o s a c c h a r i d e s a r e a p p r o x . 50 Mg C l " 1 , w h i c h a r e much l o w e r than c r i t i c a l v a l u e s o b s e r v e d i n l a b o r a t o r y c u l t u r e s w i t h pure b a c t e r i a ( J a n n a s c h 1970) . T h i s a g r e e s w i t h f i n d i n g s i n t h e dark mic rocosms where t h r e s h o l d v a l u e s f o r u p t a k e were a p p r o x . 200 Mg C 1 " 1 m o n o s a c c h a r i d e (see F i g u r e 1 2 ) . The u t i l i z a t i o n of s o l u b l e o r g a n i c components by m i c r o o r g a n i s m s may c o n t r i b u t e a s i g n i f i c a n t f r a c t i o n t o t o t a l p r o d u c t i o n (Pomeroy 1974) . B a c t e r i a can r a p i d l y a s s i m i l a t e the more l a b i l e p o r t i o n (Azam & Hodson 1977) . By f o l l o w i n g the 48 deve lopment of n a t u r a l p h y t o p l a n k t o n and d i s s o l v e d c a r b o h y d r a t e s i n p l a s t i c t a n k s , Brockmann e t §JL. (1979) found a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between g l u c o s e and d i a t o m s , as w e l l as between d i s s o l v e d c a r b o h y d r a t e s and p h y t o p l a n k t o n b i o m a s s . D u r i n g a n a t u r a l bloom i n t h e N o r t h Sea , g l u c o s e c o m p r i s e d more t h a n 60% of t h e combined c a r b o h y d r a t e f r a c t i o n ( I t t e k k o t e_t a l . 1981) . E f f i c i e n t h e t e r o t r o p h i c u t i l i z a t i o n r e d u c e d s u b s t r a t e l e v e l s t o ambient c o n c e n t r a t i o n s w i t h i n a s h o r t t i m e ; the d e c r e a s e was found t o be i n t h e same range as b a c t e r i a l u p t a k e (Brockmann e t a_l. 1 9 7 9 ) . H e t e r o t r o p h i c m i c r o f l a g e l l a t e s i n the s i z e range 3 t o 10 ym a r e e f f e c t i v e b a c t e r i o v o r e s (Haas & Webb 1979; F e n c h e l 1982b) . T h e i r n o r m a l n u t r i t i o n a l mode i s t o i n g e s t b a c t e r i a r a t h e r t h a n d i s s o l v e d o r g a n i c m a t t e r or a c o m b i n a t i o n t h e r e o f a t l e a s t i n t h e n a t u r a l e n v i r o n m e n t , where ambient s u b s t r a t e c o n c e n t r a t i o n s a r e low (Haas & Webb 1 9 7 9 ) . When exposed t o u n u s u a l l y h i g h c o n c e n t r a t i o n s , p r o t o z o a may be a b l e t o t a k e up o r g a n i c s u b s t r a t e s d i r e c t l y (Droop 1 9 7 4 ) . I n t h e D a r k - m i c r o c o s m s d i r e c t u p t a k e of g l u c o s e by m i c r o f l a g e l l a t e s seemed t o o c c u r i n a d d i t i o n t o g r a z i n g of b a c t e r i a . No l a g phase between a b a c t e r i a l b loom and peak numbers of m i c r o f l a g e l l a t e s i s a p p a r e n t ( F i g u r e 1 5 ) , c o n t r a r y t o o b s e r v a t i o n s i n f i e l d s t u d i e s ( S o r o k i n 1977; F e n c h e l 1982b) . 49 6 . 2 A u t o t r o p h s And H e t e r o t r o p h s Some i n t e r a c t i o n s between h e t e r o t r o p h s and a u t o t r o p h s were seen i n the m i c r o c o s m e x p e r i m e n t s . The re sponse of b a c t e r i a t o a l l o c h t h o n o u s o r g a n i c s u b s t r a t e ( g l u c o s e ) i s shown i n F i g u r e 11 and i n the D a r k - e x p e r i m e n t s . I n c r e a s e d l e v e l s of d i s s o l v e d m o n o s a c c h a r i d e s (up t o 685 uq MCHO-C l " 1 ) were o b s e r v e d d u r i n g t h e d e c l i n e of t h e s i m u l a t e d p h y t o p l a n k t o n b loom as w e l l as t h e c o u p l i n g of p r i m a r y p r o d u c t i o n w i t h i n c r e a s e s i n b a c t e r i a l b iomass (see F i g u r e s 3 and 1 3 ) . Oxygen d e p l e t i o n was c o u n t e r a c t e d by a i r - b u b b l i n g . P h o t o s y n t h e s i s c a u s e d pH changes of up t o one u n i t d u r i n g the p h y t o p l a n k t o n bloom (Append ix 1 ) . C o m p e t i t i o n between b a c t e r i a and a l g a e c o u l d be o b s e r v e d and was s t u d i e d i n n u m e r i c a l s i m u l a t i o n s and p e r t u r b a t i o n e x p e r i m e n t s ( C h a p t e r IV and V ) . P h y t o p l a n k t o n i n t h e n a t u r a l e n v i r o n m e n t a r e a l w a y s a s s o c i a t e d w i t h b a c t e r i a , and t h e r e i s e v i d e n c e f o r m u t u a l i n t e r a c t i o n . A p o s i t i v e c o r r e l a t i o n between the p r i m a r y p r o d u c t i o n o f p h y t o p l a n k t o n and b a c t e r i a l abundance or m i c r o b i a l a c t i v i t y has been found (Es & M e y e r - R e i l 1982) . The r e l a t i o n s h i p can be masked by a l l o c h t h o n o u s i n p u t s of o r g a n i c m a t t e r . W h i l e t h e e x c r e t i o n of o r g a n i c compounds by h e a l t h y p h y t o p l a n k t o n i s s t i l l d e b a t e d ( H e l l e b u s t 1974; Sharp 1977; Mague e_t a l . . 1980) , s e n e s c e n t and d y i n g a l g a e a r e a ma jor s o u r c e of o r g a n i c m a t t e r , as i s g r a z i n g a c t i v i t y ( s l o p p y f e e d i n g ) by m a c r o z o o p l a n k t o n ( E p p l e y e t a l . 1981; P f a f f e n h o f e r e t a l . 1982 ) . These compounds can r e a d i l y be t a k e n up by b a c t e r i a ( L a r s s o n & Hags t rom 1979; W o l t e r 1982) , and may 50 d e t e r m i n e t h e s e a s o n a l p a t t e r n of b a c t e r i a l abundance ( c f . C h a p t e r V I ) . M i c r o b i a l a c t i v i t y might b e t t e r be c o r r e l a t e d w i t h t h e c o n c e n t r a t i o n of phaeop igment s t h a n w i t h p r i m a r y p r o d u c t i o n (Fuhrman e t a l . 1980 ) . I n m i c r o c o s m e x p e r i m e n t s , where c h l o r o p h y l l a and p h a e o p h y t i n a was measured , t h e i n c r e a s e i n b a c t e r i a l numbers t o t h e second h e t e r o t r o p h i c bloom seemed t o f o l l o w t h e appearance of p h a e o p i g m e n t s , r a t h e r t h a n t h e h e i g h t o f the p h y t o p l a n k t o n bloom ( F i g u r e 1 3 ) . B a c t e r i a may i n h i b i t a l g a l g r o w t h by m o d i f y i n g the e n v i r o n m e n t , e . g . d e p l e t i n g oxygen o r c h a n g i n g t h e p H . The l a t t e r c o n d i t i o n s a r e u n i m p o r t a n t i n t h e F r a s e r R i v e r e s t u a r y ( D r i n n a n & C l a r k 1980 ) . There a r e r e p o r t s of b a c t e r i a and v i r u s e s t h a t may cause a l g a l c e l l s t o l y s e ( C o l e 1982 ) . B a c t e r i a p r o d u c e s o l u b l e s u b s t a n c e s w h i c h can i n h i b i t o r s t i m u l a t e a l g a l g r o w t h and may be of i m p o r t a n c e i n t h e s e a s o n a l s u c c e s s i o n of p h y t o p l a n k t o n s p e c i e s ( C o l e 1 9 8 2 ) . I n the p r e s e n c e of h i g h o r g a n i c l o a d b a c t e r i a can s u c c e s s f u l l y compete w i t h a u t o t r o p h s f o r a l i m i t i n g n u t r i e n t (see C h a p t e r V ) . In t h e i r f u n c t i o n as d e c o m p o s e r s , b a c t e r i a r e m i n e r a l i z e o r g a n i c m a t t e r and r e p l e n i s h n u t r i e n t s f o r p h y t o p l a n k t o n g r o w t h . B a c t e r i a can enhance a l g a l g r o w t h by r e l e a s e of v i t a m i n s , e . g . v i t a m i n B 1 2 ( H a i n e s & G u i l l a r d 1974) and t h e poor g r o w t h of a x e n i c a l g a l c u l t u r e s i n d i c a t e s t h a t b a c t e r i a may have complex s t i m u l a t o r y e f f e c t s . P h y t o p l a n k t o n not o n l y compete w i t h b a c t e r i a f o r l i m i t i n g n u t r i e n t s , t h e y may r e l e a s e a n t i m i c r o b i a l s u b s t a n c e s ( S i e b u r t h 1 9 6 8 ) . I n l a b o r a t o r y e x p e r i m e n t s S. c o s t a t u m i n h i b i t e d some 51 F i g u r e 13 - H i s t o g r a m s show c o n c e n t r a t i o n s (ug l " 1 ) o f c h l o r o p h y l l a and p h a e o p h y t i n a ( shaded a r e a s ) d u r i n g t h e p h y t o p l a n k t o n bloom i n f o u r m i c r o c o s m e x p e r i m e n t s . S o l i d l i n e i n d i c a t e s b a c t e r i a l numbers ( X 1 0 6 m l " 1 ) . 52 s t r a i n s of b a c t e r i a w i t h t h e i n h i b i t o r y a c t i o n b e i n g s t r o n g e s t d u r i n g the e x p o n e n t i a l g r o w t h phase (Kogure e_t a l . 1979 ) . I n p e r i o d s of i n t e n s e p h o t o s y n t h e s i s t h e upward s h i f t i n pH may i n f l u e n c e t h e b a c t e r i a l a c t i v i t y and s p e c i e s c o m p o s i t i o n ( C o l e 1982) . 53 7 . SUMMARY The p a t t e r n of t h e s i m u l a t e d p h y t o p l a n k t o n bloom was d e s c r i b e d i n terms of changes i n b a c t e r i a l numbers , c h l o r o p h y l l a and n i t r o g e n c o n c e n t r a t i o n s , and m i c r o z o o p l a n k t o n abundance . The i n i t i a l n u t r i e n t a d d i t i o n was f o l l o w e d by a f i r s t h e t e r o t r o p h i c bloom w h i c h was t e r m i n a t e d by e x h a u s t i o n of g l u c o s e and m i c r o f l a g e l l a t e g r a z i n g . The h e t e r o t r o p h i c bloom was s u c c e e d e d by an i n c r e a s e i n p h y t o p l a n k t o n b i o m a s s . E x h a u s t i o n of n u t r i e n t s t e r m i n a t e d a u t o t r o p h i c g r o w t h . The decay of c e l l s gave r i s e t o a second h e t e r o t r o p h i c b l o o m . M i c r o z o o p l a n k t o n f o l l o w e d t h e changes i n b a c t e r i a l b i o m a s s . D e s p i t e the s e a s o n a l v a r i a b i l i t y of t h e s o u r c e w a t e r s t h e s t a n d a r d p a t t e r n was r e p r o d u c i b l e . T h i s way, p e r t u r b a t i o n s of t h e s i m u l a t e d p h y t o p l a n k t o n bloom c o u l d be p e r f o r m e d a t d i f f e r e n t t i m e s of t h e y e a r . The s a l i n i t y v a l u e s i n t h e e x p e r i m e n t i n f l u e n c e d the m i c r o p l a n k t o n e c o l o g y . H e t e r o t r o p h i c a c t i v i t y was h i g h e s t a t 10 ppt and 18 ppt s a l i n i t y . S k e l e t o n e m a c o s t a t u m and T h a l a s s i o s i r a s p p . had c o n s i s t e n t l y been dominant i n the s i m u l a t e d p h y t o p l a n k t o n b l o o m . The i n c r e a s e d abundance of S . c o s t a t u m a t r e l a t i v e l y l o w e r s a l i n i t i e s (10 p p t ) had a c o r r e l a t i o n t o f i e l d s t u d i e s . A b i o t i c f a c t o r s , such as b o t t l e - e f f e c t s , c o n s t a n t l a b o r a t o r y c o n d i t i o n s and t h e i n i t i a l n u t r i e n t a d d i t i o n i n f l u e n c e d t h e b i o t a of t h e mic rocosms and b i a s e d t h e c o u r s e of e v e n t s . T o t a l consequences o f t h e m u l t i p l e e f f e c t s r e s u l t i n g 54 f rom e n c l o s u r e of water i n c o n t a i n e r s were not known. D a r k - e x p e r i m e n t s f a c i l i t a t e d t h e s t u d y of b a c t e r i a , t h e i r g r o w t h k i n e t i c s and n u t r i e n t u p t a k e . The impact and r o l e of m i c r o f l a g e l l a t e s was d i s c u s s e d . A u t o t r o p h s and h e t e r o t r o p h s r e l a t e t o each o t h e r i n v a r i o u s ways ; o n l y some of t h e i r m u t u a l i n t e r a c t i o n s c o u l d be s t u d i e d under t h e g i v e n e x p e r i m e n t a l c o n d i t i o n s . 5 5 IV. NUMERICAL MODELING 1. INTRODUCTION N u m e r i c a l m o d e l i n g i s u s e d t o d e s c r i b e t h e e v e n t s w i t h i n t h e l a b o r a t o r y m i c r o c o s m s i n t e r m s of t h e i m p o r t a n t p r o c e s s e s and o r g a n i s m s . The model s h o u l d show how t h e v a r i o u s components c o u l d be i n t e r a c t i n g and what e f f e c t t h e change of v a r i a b l e s and p a r a m e t e r s h a s . The d e v e l o p m e n t o f t h e model w i l l be i n two s t e p s : f i r s t , s i m u l a t i o n of t h e D a r k - m i c r o c o s m s by d e s c r i b i n g b a c t e r i a l and m i c r o f l a g e l l a t e g r o w t h and i n t e r a c t i o n s ; s e c o n d , s i m u l a t i o n o f L i g h t - m i c r o c o s m s by d e s c r i b i n g a u t o t r o p h i c and h e t e r o t r o p h i c g r o w t h and i n t e r a c t i o n s . By s t a r t i n g o f f w i t h t h e l e s s c o m p l i c a t e d t a s k , i n s i g h t s and r e s u l t s g a i n e d i n t h e d e v e l o p m e n t of t h e D a r k - m o d e l c a n be i n c o r p o r a t e d i n t o t h e s i m u l a t i o n o f t h e more c o m p l i c a t e d L i g h t - m o d e l . The models a r e p a r t l y b a s e d on i n d e p e n d e n t s t u d i e s of p r o c e s s e s and i n t e r a c t i o n s i n t h e l a b o r a t o r y m i c r o c o s m s . Some p a r a m e t e r s and c o n s t a n t s a r e t a k e n f r om a n u m e r i c a l s i m u l a t i o n by F e n c h e l ( 1 9 8 2 b ) , w h i c h d e s c r i b e s i n t e r a c t i o n s between b a c t e r i a and m i c r o f l a g e l l a t e s i n a n a t u r a l e c o s y s t e m . O t h e r d a t a w i l l be e s t i m a t e d f r o m e x i s t i n g l i t e r a t u r e o r have t o be g u e s s e d . Due t o c o n s t a n t l a b o r a t o r y c o n d i t i o n s , t h e models w i l l have no a b i o t i c i n p u t v a r i a b l e s . P r o c e s s e s a r e d e s c r i b e d i n d i f f e r e n t i a l e q u a t i o n s and a r e i n c o r p o r a t e d i n t o FORTRAN-programmes ( c f . A p p e n d i x 2 ) . A l l r a t e s i n t h e e q u a t i o n s a r e 56 e x p r e s s e d i n u n i t s of h o u r s . A c c o r d i n g t o the s a m p l i n g s c h e d u l e o f t h e m i c r o c o s m e x p e r i m e n t s , t h e s i m u l a t i o n s t a r t s on day 2 (24 h) when i n i t i a l samples were t a k e n and a d d i t i o n s were made. The models a r e run f o r a minimum p e r i o d of a t l e a s t 300 h t o c o v e r the e x p e r i m e n t a l o b s e r v a t i o n s . V a l u e s a r e p r i n t e d out e v e r y 12 h . A l l s i m u l a t i o n i s done on the UBC Amdahl c o m p u t e r . C r i t e r i a f o r t h e s u c c e s s o f the model s w i l l be t h e c l o s e n e s s i n t i m i n g and magni tude o f e v e n t s when t h e s i m u l a t i o n run i s compared t o t h e e x p e r i m e n t a l r e s u l t s i n t h e e s t u a r i n e m i c r o c o s m s . 57 2 . MODELING OF DARK-MICROCOSMS 2.1 V a r i a b l e s , P a r a m e t e r s And C o n s t a n t s V a r i a b l e s i n the D a r k - m i c r o c o s m s were b a c t e r i a ( X ) , m i c r o f l a g e l l a t e s (Y) and g l u c o s e ( G ) . Based on r e s u l t s i n D a r k -e x p e r i m e n t s , g l u c o s e was assumed t o be the g r o w t h - l i m i t i n g s u b s t r a t e i n t h e 10 ppt t o ^ 26 ppt s a l i n i t y m i c r o c o s m s , w h i l e a t < 5 ppt n i t r o g e n was t h e l i m i t i n g f a c t o r (see F i g u r e 2 1 ) . O n l y t h e > 10 ppt s a l i n i t i e s were c o n s i d e r e d f o r t h e n u m e r i c a l s i m u l a t i o n . The p r o c e s s e s were c h a r a c t e r i z e d by M i c h a e l i s -Menton t y p e f u n c t i o n s . The changes of b a c t e r i a l numbers ( X ; 10 6 m l " 1 ) , m i c r o f l a g e l l a t e s ( Y ; 10 3 m l " 1 ) and g l u c o s e c o n c e n t r a t i o n s (G ; mg l " 1 ) o v e r t i m e ( t ) were e x p r e s s e d as dX RM • G UM • X — = • X • Y d t GK+G XK+X dY UM • X — = • Y • EY - D • Y d t XK+X dG RM • G Q — = • X • — d t GK+G EX RM = maximum g r o w t h r a t e o f b a c t e r i a GK = h a l f - s a t u r a t i o n c o n s t a n t f o r g l u c o s e (mg l " 1 ) UM = maximum c o n s u m p t i o n r a t e o f b a c t e r i a XK = h a l f - s a t u r a t i o n c o n s t a n t f o r b a c t e r i a ( 1 0 6 m l " 1 ) D = d e a t h r a t e of f l a g e l l a t e s EY = g r o s s g r o w t h e f f i c i e n c y of f l a g e l l a t e s per b a c t e r i u m Q = g l u c o s e q u o t a per b a c t e r i u m (mg per 10 9 b a c t e r i a ) EX = g r o w t h e f f i c i e n c y of b a c t e r i a 58 I n i t i a l numbers of X and Y were d e t e r m i n e d i n t h e l a b o r a t o r y m i c r o c o s m s ; the v a l u e s changed w i t h the d i f f e r e n t s a l i n i t y r e g i m e s . I n D a r k - e x p e r i m e n t s RM was c a l c u l a t e d f rom the p e r i o d of e x p o n e n t i a l g r o w t h . R a t e s of 0 .05 a t 10 pp t and 0 .06 a t 18 pp t and ^ 26 ppt s a l i n i t y were f o u n d . G , a t t h e b e g i n n i n g of t h e s i m u l a t i o n , was 5 mg l " 1 g l u c o s e , due t o a c o n s t a n t a d d i t i o n . GK was s e t t o be 0 . 3 mg l " 1 ; v a l u e s f o r a l l b a c t e r i a a r e between 0 .2 and 0 .8 mg 1~ 1 ( p e r s o n a l c o m m u n i c a t i o n , Dr J . J . R . C a m p b e l l , D e p t . of M i c r o b i o l o g y , U B C ) . I t was assumed t h a t f l a g e l l a t e s had a maximum c o n s u m p t i o n r a t e (UM) of 60 b a c t e r i a h " 1 , a g r o s s g r o w t h e f f i c i e n c y (EY) of 3 X 1 0 ~ 3 f l a g e l l a t e s per b a c t e r i u m , and t h a t t h e h a l f - s a t u r a t i o n c o n s t a n t (XK) was 5 x 1 0 s b a c t e r i a m l " 1 . A l l t h e s e v a l u e s (UM, E Y , and XK) were e x p e r i m e n t a l l y found by F e n c h e l ( 1 9 8 2 a ) . The d e n s i t y -i n d e p e n d e n t d e a t h r a t e (D=0.045) of m i c r o f l a g e l l a t e s was t a k e n from F e n c h e l ' s (1982b) m o d e l . D a t a of b a c t e r i a l g r o w t h e f f i c i e n c y on g l u c o s e range from 0 .5 t o 0 .95 i n the l i t e r a t u r e ( e . g . F e n c h e l & B l a c k b u r n 1979; L a r s s o n & Hags t rom 1979; B o l t e r 1981 ) . F o r t h e p r e s e n t s i m u l a t i o n 0 .8 was s e l e c t e d as g i v e n i n F e n c h e l & B l a c k b u r n ( 1 9 7 9 ) . F e r g u s o n & R u b l e e (1976) p r o v i d e d a t a of t h e c a r b o n c o n t e n t of a v e r a g e - s i z e m a r i n e m i c r o b e s i n c o a s t a l a r e a s . Based on t h e i r f i n d i n g s , a g l u c o s e c e l l q u o t a of 0 . 0 5 mg per 10 9 b a c t e r i a was c a l c u l a t e d . 59 2 .2 S i m u l a t i o n And C o m p a r i s o n W i t h O r i g i n a l Da ta A s i m u l a t i o n run w i t h t h e o r i g i n a l p a r a m e t e r s (RM=0.06; GK=0 .3 ; UM=0.06; XK=5 .0 ; E Y = 3 . 0 ; D=0.045; Q=0.05; E X = 0 . 8 ) , an a d d i t i o n of 5 mg 1 " 1 g l u c o s e and i n i t i a l b a c t e r i a and f l a g e l l a t e numbers as found i n the ^ 26 ppt s a l i n i t y m i c r o c o s m , i s shown i n F i g u r e 14. The s i m u l a t i o n d i d not f i t any of t h e e x p e r i m e n t a l l y d e t e r m i n e d p a t t e r n s i n t h e s a l i n i t y range of 10 ppt t o ^ 26 ppt w h i c h a r e p r e s e n t e d i n F i g u r e 15. The o r i g i n a l p a r a m e t e r s e t l e d t o a model of u n s t a b l e p r e y / p r e d a t o r o s c i l l a t i o n s , where t h e abundance of b a c t e r i a was not l i m i t e d by s u b s t r a t e c o n c e n t r a t i o n s , but by g r a z i n g . A c c o r d i n g t o e x p e r i m e n t a l d a t a ( F i g u r e 1 5 ) , b a c t e r i a l numbers peaked between days 4 (72 h) and 5 .5 (108 h) w i t h maximum numbers r a n g i n g from 8 . 8 3 x l 0 6 m l " 1 t o 1 0 . 0 2 x 1 0 s m l " 1 . G l u c o s e e x h a u s t i o n c o i n c i d e d w i t h t h e peak of t h e b a c t e r i a l b l o o m ; t h e r e f o r e g r o w t h was l i m i t e d by l a c k of o r g a n i c s u b s t r a t e r a t h e r t h a n by g r a z i n g . L i m i t a t i o n by g l u c o s e was d e m o n s t r a t e d i n m i c r o c o s m s w i t h a d d i t i o n s of up t o 15 mg l " 1 . The i n c r e a s e i n m i c r o f l a g e l l a t e s seemed t o p a r a l l e l b a c t e r i a l numbers and a peak was r e a c h e d a t a p p r o x . t h e same t ime and up t o 24 h l a t e r . M i c r o f l a g e l l a t e s were c o u n t e d a t l a r g e r t i m e i n t e r v a l s t h a n b a c t e r i a . A l s o , due t o t h e d i f f i c u l t y o f l i v e e n u m e r a t i o n , the e s t i m a t e s of a b s o l u t e numbers showed l a r g e v a r i a t i o n s i n r e p l i c a t e c o u n t s . The e x a c t t i m i n g of t h e f l a g e l l a t e b loom i s c o n s e q u e n t l y u n c e r t a i n . P r o b a b l e r e a s o n s f o r t h e f a i l u r e of t h e o r i g i n a l s i m u l a t i o n run i n c l u d e t h e f o l l o w i n g . In F i g u r e 14, i t was assumed t h a t 60 F i g u r e 14 - S i m u l a t i o n r u n w i t h o r i g i n a l p a r a m e t e r s (> 26 p p t ) . X = 1 . 0 Y = 7 . 0 G = 5 . 0 R M = 0 . 0 6 G K = 0 . 3 U M = 0 . 0 6 X K = 5 . 0 E Y = 3 . 0 D = 0 . 0 4 5 Q=0 .05 E X = 0 . 8 ( s e e t e x t , p . 5 7 , f o r k e y t o s y m b o l s ) F i g u r e 15 - B a c t e r i a l numbers ( t h i c k l i n e s ) and m i c r o f l a g e l l a t e s ( t h i n l i n e s ) i n D a r k - e x p e r i m e n t s . 62 a l l of the i n i t i a l l y p r e s e n t b a c t e r i a were g r o w i n g by u t i l i z i n g g l u c o s e . A c t i v i t y measurements of m a r i n e b a c t e r i a i n d i c a t e , h o w e v e r , t h a t the m a j o r i t y of them a r e not a c t i v e , but l i v e i n a s t a t e of dormancy ( W r i g h t 1 9 7 8 ) . I n the e u t r o p h i c K i e l B i g h t (Wes te rn B a l t i c ) the p r o p o r t i o n of m e t a b o l i c a l l y a c t i v e m i c r o b e s changes between 10 and 56% d u r i n g the y e a r (Hoppe 1 9 7 8 ) . Tempera ture and n u t r i e n t s u p p l y seem t o be the main c r i t e r i a f o r t h e b a c t e r i a l a c t i v i t y s t a t e ( A l b r i g h t 1977; Hoppe 1978 ) . An e x p e r i m e n t a l t e m p e r a t u r e of 12°C and the f a c t t h a t b a c t e r i a l numbers were unchanged o v e r 48 h ( l a g phase) i s a l s o c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t o n l y a f r a c t i o n of them was g r o w i n g . The c a l c u l a t e d g r o w t h r a t e f o r b a c t e r i a was not c o r r e c t e d f o r g r a z i n g l o s s e s and t h e r e f o r e s h o u l d be a c t u a l l y h i g h e r i n t h e m o d e l . The i n i t i a l number of m i c r o f l a g e l l a t e s was v e r y l i k e l y l o w e r . D u r i n g e n u m e r a t i o n c o l o u r l e s s and c o l o u r e d f l a g e l l a t e s were not d i s t i n g u i s h e d and p r o b a b l y not a l l were h e t e r o t r o p h s , i . e . b a c t e r i o v o r e s . C o n s i d e r i n g the s i z e of t h e f l a g e l l a t e s i n t h e D a r k - m i c r o c o s m s , the m a j o r i t y of them was s i m i l a r i n volume t o t h e s m a l l e s t s p e c i e s s t u d i e d by F e n c h e l ( 1 9 8 2 a ) . The g l u c o s e c e l l q u o t a c a l c u l a t i o n was based on a v e r a g e -s i z e n a t u r a l m a r i n e b a c t e r i a , w h i c h a r e g r o w i n g i n a s u b s t r a t e -l i m i t e d e n v i r o n m e n t . The s i m u l a t i o n w i t h t h e o r i g i n a l p a r a m e t e r s c l e a r l y showed t h a t the b a c t e r i a l g l u c o s e q u o t a (Q) was t o o l o w , s i n c e the second b a c t e r i a l bloom r e s u l t e d i n much h i g h e r numbers t h a n o b s e r v e d and g l u c o s e was s t i l l not d e p l e t e d . E n r i c h m e n t of the g rowth medium w i t h g l u c o s e r e s u l t e d i n b i g g e r 63 b a c t e r i a (Mande l s t am e t a l . 1 9 8 2 ) . A rough e s t i m a t e of t h e c o r r e c t c e l l q u o t a was c a l c u l a t e d by d i v i d i n g maximum o b s e r v e d numbers , c . 1 0 x 1 0 s m l " 1 , by 5 mg l " 1 g l u c o s e t a k e n up , w h i c h gave a q u o t a (Q) of 0 .5 mg p e r 10 9 b a c t e r i a . In o r d e r t o a c c o u n t f o r g r a z i n g l o s s e s , the g l u c o s e q u o t a (Q) had t o be a l i t t l e l o w e r t h a n 0 . 5 . I f b a c t e r i a were b i g g e r , fewer of them had t o be i n g e s t e d i n o r d e r t o g i v e an e q u i v a l e n t amount of f l a g e l l a t e b i o m a s s , o r , w i t h t h e i n g e s t i o n of one b a c t e r i u m t h e g r o s s g r o w t h e f f i c i e n c y i n c r e a s e d . I f t h e r e was i n d e e d d i r e c t g l u c o s e up take by m i c r o f l a g e l l a t e s as d i s c u s s e d i n t h e p r e v i o u s c h a p t e r , i t was not i n c l u d e d i n the model r u n . The v a r i e t y of p o s s i b i l i t i e s s u g g e s t s t h a t t h e r e might be more t h a n one s e t of p a r a m e t e r s w h i c h m i m i c s o b s e r v a t i o n s i n t h e D a r k - m i c r o c o s m s . The a im was t o f i n d the one w h i c h seemed b i o l o g i c a l l y most r e a s o n a b l e . The change o f one p a r a m e t e r v e r y l i k e l y w o u l d r e q u i r e a d j u s t m e n t o f o t h e r s t o p roduce such a r e s u l t . Changes i n the o r i g i n a l p a r a m e t e r s c o u l d be d i s c u s s e d by l i s t i n g them s e q u e n t i a l l y , o r by s t a r t i n g w i t h the f i n a l r e s u l t and d e s c r i b i n g t h e a l t e r a t i o n s w h i c h were needed t o a c h i e v e i t . The l a t t e r a p p r o a c h was chosen i n t h e subsequent d i s c u s s i o n i n s e c t i o n 2 . 3 . 64 2 . 3 S i m u l a t i o n Of D i f f e r e n t S a l i n i t y M i c r o c o s m s I n a s e r i e s of s i m u l a t i o n r u n s , i n i t i a l c o n d i t i o n s and g r a z i n g p a r a m e t e r s were changed i n o r d e r t o i n c r e a s e t o t a l numbers i n t h e f i r s t b a c t e r i a l b loom and t o c o n v e r t a l l g l u c o s e i n t o b a c t e r i a l b i o m a s s . F i g u r e 16 shows two of s e v e r a l p o s s i b l e model s of t h e > 26 ppt s a l i n i t y D a r k - m i c r o c o s m . They d i f f e r i n i n i t i a l numbers of m i c r o f l a g e l l a t e s ( Y ) , maximum c o n s u m p t i o n r a t e of b a c t e r i a (UM) and p r o p o r t i o n a l l y a d j u s t e d g r o s s g r o w t h e f f i c i e n c y ( E Y ) . In o r d e r t o d e p l e t e t h e added 5 mg 1~ 1 g l u c o s e , the c e l l q u o t a (Q) was i n c r e a s e d s i x - f o l d from 0 .05 t o 0 . 3 , w h i l e b a c t e r i a l g r o w t h e f f i c i e n c y (EX) and h a l f - s a t u r a t i o n c o n s t a n t (GK) f o r g l u c o s e r e m a i n e d unchanged w i t h r e s p e c t t o t h e o r i g i n a l s i m u l a t i o n . The i n i t i a l number of b a c t e r i a ( X ) , w h i c h were g r o w i n g a t a r a t e of 0 . 0 6 , was s e t a t 20% of t h e o b s e r v e d v a l u e . I n i t i a l f l a g e l l a t e numbers (Y) were r e d u c e d w i t h t h e d e a t h r a t e (D) unchanged . F l a g e l l a t e s were assumed t o have a l o w e r maximum c o n s u m p t i o n r a t e (UM) of 0 .015 i n s t e a d of 0 .06 and t o be l e s s e f f i c i e n t g r a z e r s . The r a t i o of maximum c o n s u m p t i o n r a t e and h a l f - s a t u r a t i o n c o n s t a n t f o r b a c t e r i a (UM/XK) was d e c r e a s e d from 0 .012 t o 0 . 0 0 5 , i . e . t h e f l a g e l l a t e s c l e a r e d l e s s volume per u n i t t i m e . Due t o t h e i n c r e a s e i n i n d i v i d u a l b a c t e r i a l b i o m a s s , the g r o s s g r o w t h e f f i c i e n c y o f f l a g e l l a t e s (EY) was a d j u s t e d from 3.0 t o 12.0 w i t h r e s p e c t t o t h e d e c r e a s e d c o n s u m p t i o n r a t e (UM). T h u s , t h e maximum c a r b o n i n t a k e by f l a g e l l a t e s was 1 . 5 - f o l d h i g h e r . The f l a g e l l a t e b loom l a g g e d 24 h b e h i n d t h e peak i n b a c t e r i a l numbers . T o t a l f l a g e l l a t e numbers were a c c e p t a b l e , c o n s i d e r i n g t h e v a r i a t i o n i n a c t u a l 65 Bocteria x!06ml'1 Flao^ellates^ |04 ml 1 Glucose mal"1 F i g u r e 16 - Two p o s s i b l e s i m u l a t i o n s o f t h e £ 26 p p t s a l i n i t y D a r k - m i c r o c o s m . I n b o t h r u n s X = 0 . 2 ; G = 5 . 0 ; R M = 0 . 0 6 ; G K = 0 . 3 ; X K = 3 . 0 ; E X = 0 . 8 ; Q = 0 . 3 ; D = 0 . 0 4 5 . a - Y = 4 . 0 ; U M = 0 . 0 1 5 ; E Y = 1 2 . 0 . b - Y = 2 . 5 ; U M = 0 . 0 2 0 ; E Y = 9 . 0 . 66 c o u n t s . The p o s s i b i l i t y of d i r e c t g l u c o s e u p t a k e c o u l d e x p l a i n t h e h i g h e r numbers i n t h e m i c r o c o s m e x p e r i m e n t s . F i g u r e 17 shows s i m u l a t i o n runs o f the 10 ppt and 18 pp t s a l i n i t y m i c r o c o s m s . A t b o t h s a l i n i t i e s the b a c t e r i a l b loom o c c u r r e d e a r l i e r than a t > 26 ppt s a l i n i t y and a b s o l u t e numbers were h i g h e r . S e v e r a l p a r a m e t e r changes were n e c e s s a r y i n o r d e r t o a d j u s t t h e m o d e l . A c t i v i t y measurements i n the F r a s e r R i v e r e s t u a r y i n d i c a t e t h a t a t i n t e r m e d i a t e s a l i n i t i e s t h e p r o p o r t i o n as w e l l as the numbers of a c t i v e b a c t e r i a i n c r e a s e ( V a l d e s & A l b r i g h t 1 9 8 1 ) . I n i t i a l b a c t e r i a l numbers were i n d e e d h i g h e r i n the 10 ppt and 18 ppt s a l i n i t y m i c r o c o s m s , t h e r e f o r e X had t o be i n c r e a s e d when t h e p r o p o r t i o n of a c t i v e b a c t e r i a was e q u a l t o t h a t a t 2: 26 pp t s a l i n i t y . B e s i d e h i g h e r i n i t i a l numbers , more b a c t e r i a (X) may a c t u a l l y grow i n t h e m i c r o c o s m s . An i n c r e a s e of X t o 0 . 6 x 1 0 6 m l " 1 and f u r t h e r t o 0 . 8 x l 0 6 m l " 1 was c o n s e q u e n t l y j u s t i f i e d . A c c o r d i n g t o V a l d e s & A l b r i g h t (1981) the mean b a c t e r i a l volume i n t h e e s t u a r y i n c r e a s e s w i t h d e c r e a s i n g s a l i n i t y . To a c c o u n t f o r b i g g e r b a c t e r i a , t h e g l u c o s e c e l l q u o t a (Q) was s e t t o 0 . 4 . W i t h an i n c r e a s i n g amount of low s a l i n i t y s u r f a c e w a t e r , i t was assumed t h a t t h e p r o p o r t i o n o f h e t e r o t r o p h i c m i c r o f l a g e l l a t e s , w h i c h consume b a c t e r i a , d e c r e a s e d , w h i l e t h e p r o p o r t i o n of p h o t o s y n t h e t i c ones became l a r g e r . I n i t i a l f l a g e l l a t e numbers (Y) were r e d u c e d t o 0 . 1 x 1 0 " m l " 1 and 0 . 0 5 x 1 0 " m l " 1 . 67 Bacteria xl06m|-1 Fla^eJlate_s_xL04m|-1 Glucose mgl"1 F i g u r e 17 - S i m u l a t i o n runs of the 10 ppt and 18 ppt s a l i n i t y Dark-microcosms. In both runs G=5.0; GK=0.3; UM=0.015; XK=3.0; EY=12.0; D=0.045; Q=0.4; EX=0.8. a - X=0.8; Y=0.5; RM=0.05. b - X=0.6; Y=1.0; RM=0.06. 68 2 .4 D i s c u s s i o n Of Parameter Changes A c c o r d i n g t o e x p e r i m e n t a l r e s u l t s , g l u c o s e e x h a u s t i o n i n t h e D a r k - m i c r o c o s m s had t o c o i n c i d e w i t h t h e b a c t e r i a l b loom because g l u c o s e was i d e n t i f i e d as t h e g r o w t h l i m i t i n g f a c t o r . W i t h a c o n s t a n t i n i t i a l s u b s t r a t e a d d i t i o n (G) and a f i x e d i n i t i a l number of b a c t e r i a ( X ) , t h e change i n g l u c o s e c o n c e n t r a t i o n s was r e l a t e d t o t h e g r o w t h r a t e (RM) of b a c t e r i a , t h e g l u c o s e c e l l q u o t a (Q) , t h e h a l f - s a t u r a t i o n c o n s t a n t f o r g l u c o s e ( G K ) , and the g r o w t h e f f i c i e n c y (EX) o f b a c t e r i a . A modest i n c r e a s e i n the g r o w t h r a t e (RM) t o a c c o u n t f o r g r a z i n g l o s s e s had l i t t l e e f f e c t on b o t h the t i m i n g of the b a c t e r i a l b loom as w e l l as a b s o l u t e numbers and d i d not l e a d t o g l u c o s e d e p l e t i o n . Changes i n the h a l f - s a t u r a t i o n c o n s t a n t f o r g l u c o s e (GK) i n t h e range of 0 .2 t o 0 .8 mg 1 " 1 d i d not a f f e c t b a c t e r i a l dynamic s s i g n i f i c a n t l y , because changes were e f f e c t i v e o n l y a t low s u b s t r a t e c o n c e n t r a t i o n s . The g r e a t e s t impac t on g l u c o s e c o n c e n t r a t i o n s (G) was a c h i e v e d by l o w e r i n g the e f f i c i e n c y (EX) or i n c r e a s i n g t h e c e l l q u o t a (Q) . But even w i t h an e f f i c i e n c y (EX) as low as 0 . 5 , g l u c o s e s t i l l d i d not l i m i t b a c t e r i a l g r o w t h . O n l y an i n c r e a s e i n c e l l q u o t a (Q) , making b a c t e r i a b i g g e r , c a u s e d s u b s t r a t e e x h a u s t i o n t o g e t h e r w i t h t h e peak i n b a c t e r i a l numbers , w h i c h were , h o w e v e r , e x c e e d i n g t h o s e found i n t h e m i c r o c o s m s . I n o r d e r t o s i m u l a t e the D a r k - m i c r o c o s m s w i t h r e s p e c t t o a b s o l u t e b a c t e r i a l numbers and p r o p e r t i m i n g of t h e b l o o m , p a r a m e t e r s c o n c e r n i n g b a c t e r i a l g r o w t h on g l u c o s e o r t h o s e c o n c e r n i n g g r a z i n g a c t i v i t y by f l a g e l l a t e s c o u l d be a l t e r e d . 69 R e a s o n a b l e changes i n g r o w t h r a t e (RM) were l i m i t e d t o a s m a l l i n c r e a s e i n t h e v a l u e and t h e r e f o r e d i d not have much e f f e c t . A s s u m i n g t h a t o n l y a f r a c t i o n of t h e m i c r o b i a l p o p u l a t i o n was a c t u a l l y g r o w i n g , i . e . by l o w e r i n g t h e i n i t i a l b a c t e r i a l numbers ( X ) , t h e t i m i n g of t h e b a c t e r i a l b loom was a d j u s t e d as w e l l as a b s o l u t e numbers . When X was s e t a t 5% of the o b s e r v e d v a l u e and the o r i g i n a l g l u c o s e q u o t a of 0 .05 was not i n c r e a s e d , a b s o l u t e b a c t e r i a l numbers exceeded t h o s e found e x p e r i m e n t a l l y more than f o u r - f o l d and t h e bloom was too l a t e . An i n c r e a s e i n g l u c o s e q u o t a (Q) t o 0 .3 r e s u l t e d i n l o w e r b a c t e r i a l numbers and by v a r y i n g t h e i n i t i a l v a l u e of b a c t e r i a ( X ) , t h e t i m i n g of t h e b loom was c h a n g e d ; h i g h e r i n i t i a l numbers (X) r e s u l t e d i n an e a r l i e r b l o o m , w h i l e l o w e r ones d e l a y e d the b l o o m . The r e d u c t i o n of X i n c o m b i n a t i o n w i t h a g l u c o s e q u o t a (Q) of 0 . 3 w i t h o u t changes i n g r a z i n g p a r a m e t e r s r e s u l t e d i n too few f l a g e l l a t e s . I n t h e o r i g i n a l s i m u l a t i o n , f l a g e l l a t e s c o n t r o l l e d b a c t e r i a l numbers . W i t h a l o w e r g r a z i n g a c t i v i t y due t o a s m a l l e r number of b a c t e r i a - i n g e s t i n g f l a g e l l a t e s (Y) or due t o a l o w e r maximum c o n s u m p t i o n r a t e (UM), b a c t e r i a l numbers c o u l d be i n c r e a s e d a t t h e h e i g h t of the b l o o m . A r e d u c e d maximum c o n s u m p t i o n r a t e (UM) w i t h o u t a change i n t h e h a l f - s a t u r a t i o n c o n s t a n t f o r b a c t e r i a (XK) r e s u l t e d i n l e s s e f f i c i e n t f l a g e l l a t e g r a z i n g , i . e . h i g h e r b a c t e r i a l numbers and a d e l a y e d b l o o m . In a d d i t i o n , t h e t i m e - l a g between b a c t e r i a l and m i c r o f l a g e l l a t e bloom became t o o l o n g . A l t e r a t i o n s of t h e h a l f - s a t u r a t i o n c o n s t a n t (XK) a l o n e d i d not i n f l u e n c e t h e t i m i n g of the 70 b a c t e r i a l b loom s i g n i f i c a n t l y , but r e s u l t e d i n l o w e r peak numbers . By d e c r e a s i n g t h e g r a z i n g a c t i v i t y w i t h o u t c h a n g i n g t h e g r o s s g r o w t h e f f i c i e n c y (EY) of t h e f l a g e l l a t e s , t h e i r t o t a l numbers were t o o l o w . The d e n s i t y i n d e p e n d e n t d e a t h r a t e (D) of m i c r o f l a g e l l a t e s was not a l t e r e d a t a l l , because t h e r e was no e x p e r i m e n t a l v a l u e a v a i l a b l e . By i n c r e a s i n g t h e g l u c o s e c e l l q u o t a (Q) s i x - f o l d , making b a c t e r i a b i g g e r , and w i t h no change i n the maximum c o n s u m p t i o n r a t e (UM), t h e c a r b o n uptake r a t e p e r f l a g e l l a t e would i n c r e a s e s i x - f o l d , r e s u l t i n g i n f a r t o o l a r g e and t o o few f l a g e l l a t e s . I n o r d e r t o s i m u l a t e a p p r o x . f l a g e l l a t e numbers i n t h e D a r k -m i c r o c o s m s , fewer b a c t e r i a had t o be i n g e s t e d t o g i v e an e q u i v a l e n t amount of f l a g e l l a t e b i o m a s s . C o n s e q u e n t l y , t h e g r o s s g rowth e f f i c i e n c y of f l a g e l l a t e s (EY) had t o be i n c r e a s e d . W i t h a g l u c o s e q u o t a (Q) of 0 . 3 and a f o u r - f o l d i n c r e a s e i n E Y , f l a g e l l a t e s were 1.5 t i m e s as b i g i n terms of g l u c o s e q u o t a . As suming a g l u c o s e q u o t a (Q) of 0 . 3 and a b a c t e r i a l e f f i c i e n c y (EX) of 0 . 8 , an i n i t i a l g l u c o s e c o n c e n t r a t i o n of 5 mg l " 1 wou ld y i e l d a maximum number of b a c t e r i a of 1 3 . 3 x 1 0 s m l " 1 . . Numbers o b s e r v e d i n t h e D a r k - m i c r o c o s m s were about 36% l o w e r . In s u b s t r a t e - ( g l u c o s e ) c o n t r o l l e d e x p e r i m e n t s , i t a p p e a r s t h a t t h e g l u c o s e c e l l q u o t a (Q) d e t e r m i n e s s u b s t r a t e l i m i t a t i o n , and t h u s t h e t i m i n g of t h e b a c t e r i a l b l o o m , w h i l e t h e g r o s s g r o w t h e f f i c i e n c y (EY) d e t e r m i n e s t h e number of g r a z e r s and the h e i g h t of t h e b l o o m . In c o n c l u s i o n , the s i m u l a t i o n of the e v e n t s i n t h e 10 ppt t o ^ 26 ppt s a l i n i t y D a r k - m i c r o c o s m s r e q u i r e d changes of s e v e r a l 71 p a r a m e t e r s of t h e o r i g i n a l r u n . The g l u c o s e q u o t a per b a c t e r i u m had t o be i n c r e a s e d i n o r d e r t o c o n v e r t a l l added g l u c o s e i n t o b a c t e r i a l b i o m a s s . I n i t i a l b a c t e r i a l and f l a g e l l a t e numbers were l o w e r e d , because not a l l o f t h e former were assumed t o be a c t i v e and g r o w i n g , and p r o b a b l y no t a l l o f t h e l a t t e r were b a c t e r i o v o r e s . The maximum c o n s u m p t i o n r a t e of b a c t e r i a had t o be l o w e r e d and a d e c r e a s e d r a t i o of maximum c o n s u m p t i o n r a t e t o t h e h a l f - s a t u r a t i o n c o n s t a n t made g r a z i n g l e s s e f f i c i e n t . W i t h f l a g e l l a t e s g r a z i n g on b i g g e r b a c t e r i a , t h e g r o s s g r o w t h e f f i c i e n c y of t h e former had t o be i n c r e a s e d i n o r d e r t o g i v e the p r o p e r number of g r a z e r s . 72 3. MODELING OF LIGHT-MICROCOSMS 3.1 V a r i a b l e s , Parameters And Cons tan t s V a r i a b l e s i n the L i g h t - m i c r o c o s m s were p h y t o p l a n k t o n ( P ) , n i t r o g e n (N) , b a c t e r i a (X) , m i c r o f l a g e l l a t e s (Y)', and g lucose ( G ) . A u t o t r o p h i c growth was expres sed as changes i n c h l o r o p h y l l a c o n c e n t r a t i o n s . Forms of n i t r o g e n i n c l u d e d ammonium, n i t r a t e and n i t r i t e ; i n i t i a l c o n c e n t r a t i o n s of a l l forms were added and expressed as a s i n g l e v a r i a b l e N . D i s s o l v e d o r g a n i c n i t r o g e n (DON) and d i s s o l v e d o r g a n i c carbon (DOC) were not measured i n the microcosm e x p e r i m e n t s . C o n s e q u e n t l y , the uptake of DON and DOC other than g lucose was not c o n s i d e r e d i n the s i m u l a t i o n , and n e i t h e r were the r e l e a s e of o r g a n i c s u b s t r a t e s by p h y t o p l a n k t o n or r e m i n e r a l i s a t i o n p roce s se s by b a c t e r i a . Thus , the model was r e s t r i c t e d to the i n i t i a l growth p e r i o d . Due to the l a ck of g r a z e r s , p h y t o p l a n k t o n c e l l s sank to the bottom of the c o n t a i n e r f o l l o w i n g n u t r i e n t d e p l e t i o n . The l o s s of c e l l s was not d e s c r i b e d . A l s o not i n c l u d e d i n the model was the e x c r e t i o n of n i t r o g e n compounds by m i c r o f l a g e l l a t e s . Based on r e s u l t s of the L i g h t - e x p e r i m e n t s ( s t andard pattern=1 mg 1~1 g l u c o s e ) , i t was assumed tha t b a c t e r i a l growth was l i m i t e d by g lucose and p h y t o p l a n k t o n growth by n i t r o g e n i n the < 5ppt to ^ 26 ppt s a l i n i t y microcosms . Only i n g lucose p e r t u r b a t i o n s and D a r k - e x p e r i m e n t s , the a d d i t i o n of 5-15 mg l " 1 of g lucose r e s u l t e d i n n i t r o g e n l i m i t a t i o n of b a c t e r i a l growth at low s a l i n i t i e s . H e t e r o t r o p h s grow on o r g a n i c c a r b o n , but i n 73 a d d i t i o n r e q u i r e n i t r o g e n compounds f o r p r o t e i n s y n t h e s i s . The uptake of b o t h c a r b o n and n i t r o g e n was assumed t o o c c u r i n a f i x e d p r o p o r t i o n ; t h u s c a r b o n and n i t r o g e n q u o t a s f o r b a c t e r i a were c o n s i d e r e d t o be c o n s t a n t . The g r o w t h e f f i c i e n c y on n i t r o g e n was t a k e n as 100% f o r b a c t e r i a (Azam et_ a_l. 1983) and p h y t o p l a n k t o n . A l g a l c e l l s were assumed t o have c o n s t a n t n i t r o g e n and c h l o r o p h y l l a q u o t a per c e l l d u r i n g t h e e n t i r e e x p e r i m e n t . The g r o w t h of p h o t o s y n t h e t i c m i c r o f l a g e l l a t e s was i g n o r e d , because t h e i r t o t a l b iomass was s m a l l i n c o m p a r i s o n t o t h e b iomass of the dominant a l g a e . The p r o c e s s e s i n t h e L i g h t - m i c r o c o s m s were c h a r a c t e r i z e d by M i c h a e l i s - M e n t e n type f u n c t i o n s . The e q u a t i o n s f o r changes i n b a c t e r i a l numbers ( X ) , m i c r o f l a g e l l a t e s (Y) and g l u c o s e (G) were t h e same as i n the s i m u l a t i o n o f the D a r k - m i c r o c o s m s (see s e c t i o n 2.1 i n t h i s C h a p t e r ) . E q u a t i o n s r e p r e s e n t i n g changes i n p h y t o p l a n k t o n b iomass (P) and changes i n n i t r o g e n c o n c e n t r a t i o n s (N) over t i m e ( t ) were e x p r e s s e d as dP SM * N — = • p d t NK+N dN SM • N QP RM • G — = • p • — - • X-QN d t NK+N QC GK+G SM = maximum g r o w t h r a t e of p h y t o p l a n k t o n NK = h a l f - s a t u r a t i o n c o n s t a n t f o r n i t r o g e n ( y g - a t l " 1 ) QP = n i t r o g e n q u o t a per p h y t o p l a n k t o n c e l l ( p g - a t ) QC = c h l o r o p h y l l a q u o t a per p h y t o p l a n k t o n c e l l (pg) RM = maximum g r o w t h r a t e of b a c t e r i a GK = h a l f - s a t u r a t i o n c o n s t a n t f o r g l u c o s e (mg 1~ 1) QN = n i t r o g e n q u o t a per b a c t e r i u m ( y g - a t per 10 9 b a c t e r i a ) 74 I n i t i a l v a l u e s of P , N , X , and Y were d e t e r m i n e d i n t h e l a b o r a t o r y m i c r o c o s m s ; t h e v a l u e s changed w i t h d i f f e r e n t s a l i n i t y r e g i m e s . G , a t t h e b e g i n n i n g of t h e s i m u l a t i o n was 1 mg 1 " ' g l u c o s e ( s t a n d a r d p a t t e r n ) , due t o a c o n s t a n t a d d i t i o n . P a r a m e t e r s of b a c t e r i a l and f l a g e l l a t e g r o w t h were t a k e n f rom t h e D a r k - m i c r o c o s m s i m u l a t i o n of t h e a p p r o p r i a t e s a l i n i t y v a l u e . 3 .2 S i m u l a t i o n Of The 10 ppt S a l i n i t y L i g h t - m i c r o c o s m In t h e 10 pp t s a l i n i t y m i c r o c o s m s , a s i n g l e d i a t o m s p e c i e s , S k e l e t o n e m a c o s t a t u m , c o m p r i s e d more t h a n 90% of t h e p h y t o p l a n k t o n b l o o m , w h i l e a t h i g h e r and l o w e r s a l i n i t i e s a l g a l p o p u l a t i o n s were more d i v e r s e . The p a r a m e t e r s d e s c r i b i n g b a c t e r i a l and f l a g e l l a t e g r o w t h were the same as i n the s i m u l a t i o n run shown i n F i g u r e 16a. Based on d a t a o b t a i n e d from D a r k - e x p e r i m e n t s , a n i t r o g e n q u o t a f o r b a c t e r i a (QN) o f 2 . 0 Mg~at p e r 10 9 b a c t e r i a was c a l c u l a t e d . W i t h a g l u c o s e q u o t a of 0 .4 mg ( 2 . 2 uM) per 1 0 s b a c t e r i a , t h e g l u c o s e / N r a t i o p e r b a c t e r i a l c e l l was 1 . 1 , w h i c h was i n agreement w i t h t h e C / N r a t i o g i v e n i n F e n c h e l & B l a c k b u r n ( 1 9 7 9 ) . The c a l c u l a t i o n o f t h e maximum g r o w t h r a t e (SM) of S . c o s t a t u m was based on c e l l c o u n t s d u r i n g t h e e x p o n e n t i a l g r o w t h p h a s e . The v a l u e of 0 .055 was e q u a l t o maximum r a t e s r e p o r t e d by Smayda (1973) and H i t c h c o c k (1980a) i n h i g h l i g h t i n t e n s i t y c u l t u r e s a t t e m p e r a t u r e s of 10 - 1 2 ° C , and was e q u a l t o t h e maximum g r o w t h r a t e s o f b a c t e r i a found i n t h e D a r k -m i c r o c o s m s . W h i l e p h y t o p l a n k t o n and b a c t e r i a l g r o w t h r a t e s were e q u a l , a l g a e had a l a g phase about t h r e e t i m e s as l o n g as 75 b a c t e r i a . A c o m b i n a t i o n of f a c t o r s c o u l d be r e s p o n s i b l e f o r the d e l a y e d o n s e t of p h y t o p l a n k t o n g r o w t h . D u r i n g t h e i n i t i a l h e t e r o t r o p h i c b l o o m , b a c t e r i a may have p r e v e n t e d a l g a l g r o w t h by p r o d u c i n g i n h i b i t o r y s u b s t a n c e s ( C o l e 1982) . A l s o , changes i n e n v i r o n m e n t a l c o n d i t i o n s , such as i r r a d i a n c e , p h o t o p e r i o d , o s m o t i c c h a n g e s , t e m p e r a t u r e and a i r - b u b b l i n g may have i n d u c e d a u x o s p o r e f o r m a t i o n i n the d i a t o m p o p u l a t i o n b e f o r e v e g e t a t i v e g r o w t h resumed, as Drebes (1977) s u g g e s t e d . F o r S. c o s t a t u m E p p l e y ejt a l . ( 1 969) g i v e a h a l f - s a t u r a t i o n c o n s t a n t (NK) of 0 .5 M M f o r the u p t a k e of n i t r a t e . M a c l s a a c & Dugda le (1969) c a l c u l a t e d a v a l u e of 1.0 f o r n a t u r a l p o p u l a t i o n s i n c o a s t a l w a t e r , w h i c h i s the same as i n l a b o r a t o r y c u l t u r e s . The h i g h e r v a l u e was used i n t h e m o d e l . The c h l o r o p h y l l a c o n t e n t p e r t e l l was c a l c u l a t e d t o be 0 . 5 pg i n t h e 10 ppt s a l i n i t y m i c r o c o s m . The same v a l u e has been r e p o r t e d by Cosper (1982) f o r S. c o s t a t u m a t s i m i l a r i r r a d i a n c e s . The n i t r o g e n q u o t a was s e t a t 3 .0 pg ( 0 . 2 p g - a t N) per c e l l (Cosper 1982 ) . The r e s u l t i n g N / C h l r a t i o was 6 ; a v a l u e , e q u a l t o t h a t g i v e n by P a r s o n s e t a l . (1961) f o r n u t r i e n t s u f f i c i e n t c e l l s . A s i m u l a t i o n run w i t h b a c t e r i a l and f l a g e l l a t e p a r a m e t e r s as i n F i g u r e 16a, w i t h p r o p o r t i o n a l l y a d j u s t e d i n i t i a l b a c t e r i a l (X) and f l a g e l l a t e numbers ( Y ) , w i t h an a d d i t i o n , of 1 mg 1"' g l u c o s e (G) and w i t h c h l o r o p h y l l a (P) and n i t r o g e n c o n c e n t r a t i o n s (N) as found i n a 10 ppt s a l i n i t y L i g h t -m i c r o c o s m , r e s u l t e d i n a p r e m a t u r e p h y t o p l a n k t o n b l o o m . A l g a l g r o w t h s t a r t e d w i t h o u t a l a g phase r e s u l t i n g i n e a r l y n i t r o g e n d e p l e t i o n . B a c t e r i a l numbers matched t h o s e found 76 e x p e r i m e n t a l l y ; r e l a t i v e l y low t o t a l numbers r e s u l t e d i n a s m a l l p o p u l a t i o n of h e t e r o t r o p h i c f l a g e l l a t e s . A c c o r d i n g t o t h e model c . 90% of the m i c r o f l a g e l l a t e s were p h o t o s y n t h e t i c . W h i l e a t t h e h e i g h t of the p h y t o p l a n k t o n bloom more t h a n 90% of t h e a l g a l c e l l s were i d e n t i f i e d as S. c o s t a t u m , t h e i r r e l a t i v e abundance a t t h e b e g i n n i n g o f t h e L i g h t - e x p e r i m e n t s v a r i e d between 8.5% and 20%. T h u s , t h e i n i t i a l f r a c t i o n of c h l o r o p h y l l a o r i g i n a t i n g f rom S. c o s t a t u m c e l l s was s m a l l . In o r d e r t o s i m u l a t e a b loom of t h i s s p e c i e s , a r e d u c t i o n of the e x p e r i m e n t a l l y found c h l o r o p h y l l a v a l u e seemed t o be j u s t i f i e d . When the i n i t i a l v a l u e (P) was r e d u c e d t o a p p r o x . 5%, c h l o r o p h y l l a c o n c e n t r a t i o n s showed a s h a r p i n c r e a s e a f t e r a 2 .5 day l a g p h a s e . In F i g u r e 18, t h e s i m u l a t i o n o f a 10 ppt s a l i n i t y L i g h t - m i c r o c o s m (X=0 .95 ; P=0 .075 ; N=19.66 ; SM=0.055; NK=1 .0 ; QC=0.5; QP=0.2; QN=2.0) i s . shown and compared t o e x p e r i m e n t a l r e s u l t s of seven L i g h t - e x p e r i m e n t s ( s t a n d a r d p a t t e r n ) . The t i m i n g of the bloom and the c h l o r o p h y l l a peak v a l u e were i n agreement w i t h r e s u l t s i n the L i g h t - m i c r o c o s m . Because t h e s i n k i n g of p h y t o p l a n k t o n c e l l s or l o s s e s due t o g r a z i n g were not i n c l u d e d i n the m o d e l , p igment v a l u e s remained h i g h . N i t r o g e n d e p l e t i o n c o i n c i d e d w i t h the p h y t o p l a n k t o n b l o o m . 77 Bacteria x10 6 ml"1 Chlaygr1 NPi + NO i ug-at r1 20-10-': I '. i i I'-. i i i 48 96 144 h F i g u r e 18 - a - M o d e l o f 10 p p t s a l i n i t y L i g h t - m i c r o c o s m , b - Mean v a l u e s o f 7 e x p e r i m e n t s ( s t a n d a r d p a t t e r n ) a t t h e same s a l i n i t y , b u t d i f f e r e n t s e a s o n s . 78 3 .3 S i m u l a t i o n Of O t h e r S a l i n i t i e s The s i m u l a t i o n of the 18 pp t and > 26 pp t s a l i n i t y m i c r o c o s m s i s d e s c r i b e d and d i s c u s s e d t o g e t h e r , because o f b e h a v i o r a l s i m i l a r i t i e s . The h i g h e r s a l i n i t y mic rocosms d i f f e r e d from t h e 10 ppt one w i t h r e s p e c t t o a s h o r t e r l a g phase b e f o r e the o n s e t of p h y t o p l a n k t o n g r o w t h , i n t h e i r s p e c i e s c o m p o s i t i o n and i n h i g h e r n i t r o g e n , c o n s e q u e n t l y h i g h e r a l g a l b i o m a s s . W h i l e a t 10 ppt s a l i n i t y , n u t r i e n t d e p l e t i o n c o i n c i d e d w i t h t h e peak i n c h l o r o p h y l l a c o n c e n t r a t i o n s , i . e . the peak of t h e p h y t o p l a n k t o n b l o o m , p igment v a l u e s i n the h i g h e r s a l i n i t y mic rocosms i n c r e a s e d by about 30% f o r a n o t h e r 24 h f o l l o w i n g n u t r i e n t d e p l e t i o n . C e l l s c o n t i n u e d t o d i v i d e and s y n t h e s i z e c h l o r o p h y l l a by u s i n g n i t r o g e n from i n t e r n a l c e l l p o o l s . The a s s u m p t i o n of c o n s t a n t n i t r o g e n and c h l o r o p h y l l a q u o t a o b v i o u s l y does not h o l d . C e l l q u o t a s v a r y w i t h g rowth r a t e ( H a r r i s o n et_ a_l. 1976; Goldman & M c C a r t h y 1978) and d e c r e a s e w i t h i n c r e a s i n g n u t r i e n t d e f i c i e n c y . When a l g a l c e l l d i v i s i o n c o n t i n u e s f o l l o w i n g n u t r i e n t d e p l e t i o n , i n d i v i d u a l c e l l s become p r o g r e s s i v e l y s m a l l e r . The use of c o n s t a n t q u o t a s cause s a d i s c r e p a n c y between t h e s i m u l a t i o n run and t h e e x p e r i m e n t a l d a t a . When t h e e x a c t c h l o r o p h y l l a c o n c e n t r a t i o n i s m o d e l e d , t h e n i t r o g e n d e p l e t i o n i s t o o l a t e . W i t h the n i t r o g e n d e p l e t i o n p r o p e r l y t i m e d , t h e c h l o r o p h y l l a c o n c e n t r a t i o n i s bound t o be t o o h i g h . I n s p i t e of t h i s l i m i t a t i o n , c o n s t a n t q u o t a s were assumed f o r c o n v e n i e n c e s a k e . A t 18 ppt and ^ 26 ppt s a l i n i t y , t h e p h y t o p l a n k t o n bloom was c o n s i s t e n t l y d o m i n a t e d by S. c o s t a t u m and T h a l a s s i o s i r a 79 s p p . ; they a c c o u n t e d f o r about 85% (by numbers) of a l l a l g a l c e l l s . The r e m a i n i n g 15% were made up by C h a e t o c e r o s s p p . , s e v e r a l pennate d i a t o m forms and f l a g e l l a t e s . S . c o s t a t u m and T h a l a s s i o s i r a s p p . seemed t o o c c u r i n a f i x e d r a t i o of a p p r o x . 3 : 1 . The l a t t e r s p e c i e s were not f u r t h e r d i s t i n g u i s h e d , but c e l l s were g rouped i n t o t h r e e d i f f e r e n t s i z e c l a s s e s . The s m a l l e s t T h a l a s s i o s i r a c e l l s had about t w i c e t h e volume of one S. c o s t a t u m c e l l . W h i l e t h e r e l a t i v e abundance of s p e c i e s a t 18 ppt and ^ 26 ppt s a l i n i t y were t h e same, the p r o p o r t i o n of l a r g e T h a l a s s i o s i r a c e l l s i n t h e h i g h e s t s a l i n i t y was d o u b l e of t h a t a t 18 p p t . Growth r a t e (SM) c a l c u l a t i o n s were based on c e l l c o u n t s w i t h no d i s t i n c t i o n between t h e d i f f e r e n t s p e c i e s . W h i l e S . c o s t a t u m grew f a s t e r , t h e r a t e of g r o w t h of T h a l a s s i o s i r a s p p . was s l o w e r , r e s u l t i n g i n a mean maximum g r o w t h r a t e (SM) of a l l c e l l s o f 0 .040 and 0 .038 i n t h e 18 ppt and > 26 ppt s a l i n i t y m i c r o c o s m s . The mean r a t e was l o w e r t h a n i n t h e 10 ppt s a l i n i t y e x p e r i m e n t s . Based on e x p e r i m e n t a l d a t a , an average c h l o r o p h y l l a c o n t e n t per c e l l (QC) of 0 .4 pg was c a l c u l a t e d f o r the 18 ppt and ^ 26 ppt s a l i n i t i e s . As o u t l i n e d a b o v e , t h e n i t r o g e n q u o t a showed l a r g e v a r i a t i o n d u r i n g t h e d i f f e r e n t phases of g r o w t h . A c a l c u l a t e d v a l u e based on e x p e r i m e n t a l d a t a f rom the o n s e t of e x p o n e n t i a l g r o w t h p r o v e d t o be f a r t o o h i g h , w h i l e v a l u e s a t a l a t e r phase were v i r t u a l l y z e r o . C o n s e q u e n t l y , q u o t a had t o be gues sed and a d j u s t e d i n o r d e r t o g i v e the r i g h t amount of c h l o r o p h y l l a a t t h e h e i g h t of t h e p h y t o p l a n k t o n b l o o m . A t 80 18 ppt s a l i n i t y a n i t r o g e n q u o t a (QP) of 0 .25 p g - a t per c e l l was most f i t t i n g , w h i l e a t ^ 26 ppt 0 . 3 p g - a t was c h o s e n . The h a l f -s a t u r a t i o n c o n s t a n t f o r n i t r a t e (NK) r e m a i n e d unchanged w i t h r e s p e c t t o t h e 10 ppt s a l i n i t y s i m u l a t i o n . I n i t i a l c h l o r o p h y l l a v a l u e s (P) used i n the model s were about 30% of t h e o r i g i n a l v a l u e . B a c t e r i a l and f l a g e l l a t e p a r a m e t e r s were t a k e n from t h e D a r k - m o d e l s shown i n F i g u r e s 16a (26 p p t ) and 17b (18 p p t ) . I n i t i a l b a c t e r i a l (X) and f l a g e l l a t e numbers (Y) were a d j u s t e d p r o p o r t i o n a l l y , w h i l e t h e g l u c o s e a d d i t i o n was c o n s t a n t a t 1 mg l " 1 . In F i g u r e 19 t h e s i m u l a t i o n runs of one 18 ppt (X=0 .6 ; Y = 2 . 0 ; G=1 .0 ; P=0 .5 ; N=33 .72 ; SM=0.04; NK=1 .0 ; QC=0.4; QP=0.25; QN=2.0) and one > 26 ppt L i g h t - m i c r o c o s m (X=0 .2 ; Y = 4 . 0 ; G=1 .0 ; P=0 .75 ; N=47.87 ; SM=0.04; NK=1.0 ; QC=0.4; QP=0.3; QN=2.0) a r e shown. The models a r e compared t o mean v a l u e s of b a c t e r i a l , c h l o r o p h y l l a and n i t r o g e n c o n c e n t r a t i o n s o f s e v e r a l e x p e r i m e n t s ( s t a n d a r d p a t t e r n ) . A s i m u l a t i o n of the l o w e s t 5 p p t ) s a l i n i t y L i g h t -m i c r o c o s m was hampered by t h e l a c k of d a t a f o r b a c t e r i a l and m i c r o f l a g e l l a t e g r o w t h . A D a r k - m o d e l of t h i s s a l i n i t y was not c o n s i d e r e d because b a c t e r i a were l i m i t e d by b o t h n i t r o g e n as w e l l as g l u c o s e . A t < 5 ppt s a l i n i t y , the p h y t o p l a n k t o n bloom o c c u r r e d l a t e r t h a n a t 10 ppt and was composed of s m a l l T h a l a s s i o s i r a s p p . and g r e e n a l g a e . The p r e s e n c e of t h e l a t t e r s p e c i e s was r e f l e c t e d i n t h e h i g h c h l o r o p h y l l a q u o t a (QC) of 1.2 p g . The mean maximum g r o w t h r a t e (SM) f o r a l l c e l l s was 0 . 0 4 3 . A s i m u l a t i o n run w i t h b a c t e r i a l and f l a g e l l a t e p a r a m e t e r s as i n the 10 ppt s a l i n i t y D a r k - m o d e l , p r o p e r l y 61 F i g u r e 19 - Top - model s of a 18 ppt and a £ 26 ppt s a l i n i t y L i g h t - m i c r o c o s m . Bot tom - mean v a l u e s o f 6 (18 p p t ) and 7 (£ 26 p p t ) e x p e r i m e n t s a t d i f f e r e n t s e a s o n s . ( B a c t e r i a X 1 0 6 m l ' 1 ; C h l a uq l " 1 ; N 0 3 + N 0 2 n g - a t l " 1 ) 82 a d j u s t e d i n i t i a l b a c t e r i a l (X) and f l a g e l l a t e numbers ( Y ) , and an i n i t i a l c h l o r o p h y l l a c o n c e n t r a t i o n (P) of c . 7% of t h e e x p e r i m e n t a l l y found v a l u e , r e s u l t e d i n a model w h i c h s i m u l a t e d t h e ^ 5 ppt s a l i n i t y L i g h t - m i c r o c o s m w e l l . But when t h i s s i m u l a t i o n was compared t o t h e mean v a l u e s o f 7 e x p e r i m e n t s ( s t a n d a r d p a t t e r n ) , t h e r e s u l t s were more d i v e r g e n t ( F i g u r e 2 0 ; X = 1 . 0 ; Y = 2 . 0 ; G=1 . 0 ; P=0 .075 ; N=9 .55 ; SM=0.043; NK=1 .0 ; QC=1.2; QP=0.5; QN=1.8) . W h i l e a t 10 pp t and h i g h e r s a l i n i t i e s the s i m u l a t i o n of one m i c r o c o s m a g r e e d w e l l w i t h e x p e r i m e n t a l d a t a c o l l e c t e d a t d i f f e r e n t seasons of t h e y e a r , t h e ^ 5 ppt s a l i n i t y L i g h t - m o d e l d i d not compare e q u a l l y w e l l w i t h e x p e r i m e n t a l d a t a w h i c h i n c l u d e d s e a s o n a l v a r i a b i l i t y . T h i s might be due t o t h e l a c k of d a t a f o r b a c t e r i a l and f l a g e l l a t e g r o w t h a t < 5 p p t s a l i n i t y o r t o a g r e a t e r v a r i a t i o n i n t h e l o w e s t s a l i n i t y m i c r o c o s m s . 3 .4 The D i s t r i b u t i o n Of I n o r g a n i c N i t r o g e n B a c t e r i a and p h y t o p l a n k t o n need i n o r g a n i c n i t r o g e n f o r g r o w t h . In most m i c r o c o s m e x p e r i m e n t s the up take by one or t h e o t h e r c o u l d not be d i s t i n g u i s h e d , because a l g a l c e l l s s t a r t e d t o grow w h i l e t h e i n i t i a l h e t e r o t r o p h i c bloom was s t i l l i n p r o g r e s s . I n a few e x p e r i m e n t s , h o w e v e r , p h y t o p l a n k t o n d i d no t e n t e r t h e e x p o n e n t i a l g r o w t h phase u n t i l b a c t e r i a l numbers r e a c h e d a minimum a f t e r t h e b l o o m . The n i t r o g e n u p t a k e due t o b a c t e r i a r a n g e d from between 3 . 5 and 4 .7 y g - a t 1"* i n t h e s e m i c r o c o s m s , w h i c h was the same amount t h e L i g h t - m o d e l p r e d i c t e d f o r s i m u l a t i o n s w i t h 1 mg 1~ 1 g l u c o s e ( s t a n d a r d p a t t e r n ) and n i t r o g e n u p t a k e was not s i g n i f i c a n t l y d i f f e r e n t o v e r t h e 8 3 F i g u r e 20 - a - S i m u l a t i o n of a £ 5 ppt s a l i n i t y L i g h t -m i c r o c o s m ; b - Mean v a l u e s of 7 e x p e r i m e n t s ( s t a n d a r d p a t t e r n ) a t the same s a l i n i t y , but a t d i f f e r e n t s e a s o n s . ( B a c t e r i a X 1 0 6 m l " 1 ; C h l a (ig l ' 1 ; N 0 3 + N 0 2 ug -a t l ' 1 ) 84 s a l i n i t y range o f < 5 ppt t o > 26 ppt ( 3 . 6 - 5 . 3 y g - a t N l " 1 ; x = 4 . 2 ) . Due t o t h e i n c r e a s e i n n i t r a t e p l u s n i t r i t e c o n c e n t r a t i o n s w i t h i n c r e a s i n g s a l i n i t y , b a c t e r i a l u p t a k e a c c o u n t e d f o r 38% of the t o t a l i n o r g a n i c n i t r o g e n a t ^ 5 p p t , but o n l y f o r 11% a t > 26 ppt s a l i n i t y . I n the f o l l o w i n g an a t t e m p t i s made t o b a l a n c e i n o r g a n i c n i t r o g e n i n o r d e r t o e v a l u a t e the i m p o r t a n c e of n i t r o g e n r e c y c l i n g w h i c h was not c o n s i d e r e d i n t h e m o d e l . When b a c t e r i a l g r o w t h was enhanced by i n c r e a s i n g the g l u c o s e a d d i t i o n from 1 mg t o 5 mg 1 ~ 1 , w i t h a l l o t h e r v a r i a b l e s and p a r a m e t e r s unchanged , the model p r e d i c t e d a f i v e - f o l d n i t r o g e n uptake by b a c t e r i a o f a p p r o x . 20 /ug-at N 1" 1 . The s i m u l a t e d i n c r e a s e d b a c t e r i a l uptake t o t a l l y d e p l e t e d n i t r o g e n c o n c e n t r a t i o n s a t ^ 5 ppt and 10 p p t ; no p h y t o p l a n k t o n bloom c o u l d d e v e l o p . At h i g h e r s a l i n i t i e s , t h e r e m a i n i n g n i t r o g e n p r o v i d e d f o r an a l g a l bloom w h i c h was r e d u c e d by about 50% from t h e 1 mg l e v e l . Peak v a l u e s of c h l o r o p h y l l a were r e a c h e d 24 h e a r l i e r , due t o n i t r o g e n e x h a u s t i o n . The t i m i n g of t h e b a c t e r i a l b loom was not d i f f e r e n t f rom the D a r k - m o d e l , but numbers were l o w e r a t s a l i n i t i e s where n i t r o g e n became l i m i t i n g . The p a t t e r n p r e d i c t e d by the model d i f f e r e d f rom e x p e r i m e n t a l l y found d a t a of g l u c o s e p e r t u r b a t i o n s as d e s c r i b e d i n C h a p t e r V , s e c t i o n 3 . 1 . In microcosms t h e i n c r e a s e i n o r g a n i c s u b s t r a t e t o 5 mg 1~ 1 g l u c o s e c a u s e d a d e l a y and d e p r e s s i o n of the p h y t o p l a n k t o n bloom a t < 5 ppt (see F i g u r e 2 3 ) , w h i l e the impact a t h i g h e r s a l i n i t i e s was l e s s p r o n o u n c e d . The t i m i n g of the bloom was not a l t e r e d a t 18 ppt 85 and > 26 ppt s a l i n i t y , but peak v a l u e s were s l i g h t l y r e d u c e d ( F i g u r e 2 4 ) . The d i s c r e p a n c y between model p r e d i c t i o n and e x p e r i m e n t a l o b s e r v a t i o n s m i g h t be c a u s e d by two f a c t o r s : t h e c a l c u l a t e d n i t r o g e n q u o t a p e r 10 9 b a c t e r i a (QN) m i g h t be t o o h i g h ( i . e . t h e q u o t a b e i n g d i f f e r e n t when b a c t e r i a a r e g r o w i n g i n h i g h o r g a n i c n u t r i e n t c o n c e n t r a t i o n s ) , o r the model d i d not t a k e i n t o a c c o u n t n i t r o g e n r e c y c l i n g by m i c r o z o o p l a n k t o n . W i t h a c a l c u l a t e d n i t r o g e n q u o t a (QN) of 2 .0 uq-at per 10 9 b a c t e r i a , t h e L i g h t - m o d e l a g r e e d w e l l w i t h b a c t e r i a l up take as o b s e r v e d i n m i c r o c o s m s under s t a n d a r d c o n d i t i o n s . The g l u c o s e / N r a t i o was e q u i v a l e n t t o t h e a v e r a g e C / N r a t i o i n t h e m a j o r i t y of b a c t e r i a ( F e n c h e l & B l a c k b u r n 1979 ) . However , when b a c t e r i a l g r o w t h was enhanced by t h e a d d i t i o n of 5 mg 1~ 1 g l u c o s e , t h e up take o f n i t r o g e n ranged between 16 - 18 ug~at N 1 " 1 i n D a r k -microcosms w h i c h was l o w e r t h a n p r e d i c t e d by the model ( F i g u r e 2 1 ) . The d i s c r e p a n c y m i g h t be e x p l a i n e d by a c t u a l l y l o w e r n i t r o g e n q u o t a i n the D a r k - e x p e r i m e n t s o r , i f n i t r o g e n q u o t a were u n c h a n g e d , t h a t the m i s s i n g 2-4 jug - at N 1~ 1 were s u p p l i e d by m i c r o z o o p l a n k t o n e x c r e t i o n , s o m e t h i n g not i n c l u d e d i n the s i m u l a t i o n . Ammonium c o n c e n t r a t i o n s i n D a r k - m i c r o c o s m s showed a r a p i d i n c r e a s e d u r i n g t h e d e c l i n e of t h e h e t e r o t r o p h i c bloom ( F i g u r e - 2 1 ) , w h i l e f l a g e l l a t e numbers r e a c h e d a maximum a t or s h o r t l y a f t e r t h e b l o o m . The n i t r o g e n q u o t a p e r b a c t e r i a l c e l l (QN) may be d i f f e r e n t when c e l l s grow i n r i c h medium, because up take mechanisms f o r d i f f e r e n t s u b s t a n c e s a r e not c o m p l e t e l y i n d e p e n d e n t (Fuhs e t 86 Figure 21 - Bac t e r i a l numbers X 1 0 6 ml" 1 (a), ammonium (b) and n i t r a t e + n i t r i t e (c) concentrations in uq-at l " 1 in Dark-experiments at d i f f e r e n t s a l i n i t i e s . 87 a l . 1 9 7 2 ) . A v a r i e t y o f b a c t e r i a a c c u m u l a t e s t o r a g e p r o d u c t s , e . g . PHB ( P o l y - ^ S - h y d r o x y b u t y r a t e ) i n t h e p r e s e n c e o f a n e x c e s s c a r b o n o r e n e r g y s o u r c e ( p e r s o n a l c o m m u n i c a t i o n , D r H .W. J a n n a s c h , M a r i n e B i o l o g i c a l L a b o r a t o r y , W o o d s H o l e ) . I n a m o d e l s i m u l a t i n g g l u c o s e p e r t u r b a t i o n , a l o w e r q u o t a s e e m e d t o b e j u s t i f i e d . I n o r d e r t o m a t c h t h e m o d e l a n d b a c t e r i a l u p t a k e o f 16 - 18 / x g - a t N 1~ 1 a s f o u n d i n D a r k - e x p e r i m e n t s , t h e q u o t a h a d t o b e l o w e r e d b y 2 0 - 3 0 % . T o c o n c l u d e , w i t h a n i t r o g e n q u o t a (QN) o f 2 . 0 uq-at p e r l O 9 b a c t e r i a t h e L i g h t - m o d e l c o m p a r e d f a v o u r a b l y w i t h e x p e r i m e n t a l r e s u l t s a s f o u n d i n C o n t r o l - m i c r o c o s m s ( s t a n d a r d p a t t e r n = 1 mg l " 1 g l u c o s e ) . A f t e r i n c r e a s i n g t h e i n i t i a l a m o u n t o f g l u c o s e t o 5 mg 1 ~ 1 , t h e m o d e l p r e d i c t e d a m u c h l o w e r p h y t o p l a n k t o n b i o m a s s t h a n a c t u a l l y o b s e r v e d i n g l u c o s e p e r t u r b a t i o n s . A s d i s c u s s e d a b o v e , t h e n i t r o g e n q u o t a m i g h t h a v e b e e n t o o h i g h i n t h e p r e s e n c e o f l a r g e a m o u n t s o f o r g a n i c s u b s t r a t e . W i t h a c e l l q u o t a (QN) r e d u c e d b y 2 0 % t o 1.6 y g - a t p e r 1 0 9 b a c t e r i a , t h e m o d e l s t i l l p r e d i c t e d a r e d u c t i o n o f t h e p h y t o p l a n k t o n b l o o m b y 7 5 % a t 10 p p t a n d 2 5 % a t > 26 p p t s a l i n i t y , w h i l e t h e a d d i t i o n o f 5 mg l " 1 g l u c o s e t o L i g h t -m i c r o c o s m s d i d n o t r e s u l t i n a s i g n i f i c a n t r e d u c t i o n a t 10 p p t a n d o n l y a 16% r e d u c t i o n a t t h e h i g h e s t s a l i n i t y . V a l u e s f o r 18 p p t s a l i n i t y c a m e i n b e t w e e n t h e s e e x t r e m e s . T h e d i s c r e p a n c y b e t w e e n m o d e l a n d e x p e r i m e n t a l o b s e r v a t i o n s c a n b e a t t r i b u t e d t o n u t r i e n t r e c y c l i n g b y m i c r o f l a g e l l a t e s , w h i c h w a s n o t i n c l u d e d i n t h e s i m u l a t i o n . W h i l e a t > 26 p p t s a l i n i t y a b o u t 10% o f t h e b l o o m m i g h t b e d u e t o " r e g e n e r a t e d p r o d u c t i o n " ( D u g d a l e & 88 G o e r i n g 1967) , the p r o p o r t i o n i n c r e a s e s up t o 66% a t ^ 5 ppt s a l i n i t y . Not s u r p r i s i n g l y , r e c y c l i n g of n u t r i e n t s i s of i n c r e a s i n g i m p o r t a n c e a t low ambient n i t r o g e n c o n c e n t r a t i o n s . R e c y c l i n g of n u t r i e n t s c o u l d e x p l a i n t h e s m a l l e r impac t of g l u c o s e p e r t u r b a t i o n s i n t h e m i c r o c o s m s . 89 4. SUMMARY The p r o c e s s e s i n D a r k - and L i g h t - m i c r o c o s m s were d e s c r i b e d i n n u m e r i c a l m o d e l s . The o r i g i n a l d a t a s e t based on e x p e r i m e n t a l r e s u l t s , l i t e r a t u r e d a t a and a model of b a c t e r i a and m i c r o f l a g e l l a t e i n t e r a c t i o n s p r o p o s e d by F e n c h e l (1982b) d i d not s i m u l a t e the p r o c e s s e s i n D a r k - m i c r o c o s m s a t any of t h e g i v e n s a l i n i t i e s . S e v e r a l p a r a m e t e r changes were n e c e s s a r y t o a d j u s t the model t o e x p e r i m e n t a l o b s e r v a t i o n s , where b a c t e r i a l c e l l q u o t a and t h e g r o s s g r o w t h e f f i c i e n c y of m i c r o f l a g e l l a t e s had the dominant e f f e c t i n d e t e r m i n i n g t h e t i m i n g and a b s o l u t e numbers i n t h e b a c t e r i a l b l o o m . There was no u n i v e r s a l s e t of p a r a m e t e r s f i t t i n g a l l s a l i n i t i e s . The L i g h t - m o d e l , based on r e s u l t s of t h e D a r k - s i m u l a t i o n , e x p e r i m e n t a l and l i t e r a t u r e d a t a , was r e s t r i c t e d t o t h e i n i t i a l p h y t o p l a n k t o n g r o w t h p h a s e , because n e i t h e r r e c y c l i n g of n u t r i e n t s nor any p r o c e s s e s f o l l o w i n g the a l g a l b loom were i n c l u d e d . The L i g h t - m o d e l a g r e e d w e l l w i t h the p r o c e s s e s o b s e r v e d i n t h e 10 ppt t o £ 26 ppt s a l i n i t y m i c r o c o s m s , w h i l e a t t h e l o w e s t s a l i n i t y the s i m u l a t i o n was l e s s c l o s e , p r o b a b l y due t o t h e l a c k o f p r o p e r d a t a and g r e a t e r s e a s o n a l v a r i a b i l i t y i n the e x p e r i m e n t s . A budget of i n o r g a n i c n i t r o g e n r e v e a l e d t h e p a r t i t i o n i n g of s u b s t r a t e between b a c t e r i a and a l g a e . A t low ambient n u t r i e n t c o n c e n t r a t i o n s b a c t e r i a l u p t a k e may a c c o u n t f o r a c o n s i d e r a b l e p r o p o r t i o n o f t o t a l i n o r g a n i c n i t r o g e n . The i m p o r t a n c e of n u t r i e n t r e c y c l i n g by m i c r o z o o p l a n k t o n was d e m o n s t r a t e d i n the 90 d i s c r e p a n c y between t h e b e h a v i o r of t h e L i g h t - m o d e l when i n i t i a l g l u c o s e c o n c e n t r a t i o n s were i n c r e a s e d t o 5 mg 1~ 1 and the e x p e r i m e n t a l r e s u l t s o b t a i n e d i n g l u c o s e - p e r t u r b e d m i c r o c o s m s . In t h e s e e x p e r i m e n t s up t o 66% of t h e p h y t o p l a n k t o n bloom was a t t r i b u t e d t o r e g e n e r a t e d n i t r o g e n . 91 V . PERTURBATION EXPERIMENTS 1. INTRODUCTION E s t u a r i e s g e n e r a l l y a r e of h i g h b i o l o g i c a l p r o d u c t i v i t y and a r e t h e r e f o r e r a t h e r v u l n e r a b l e t o major changes i n t h e i r b a l a n c e . The impact of man's a c t i v i t i e s and p e r t u r b a t i o n s i s t h u s of g r e a t i n t e r e s t . The a d d i t i o n of p o l l u t a n t s ( m e t a l s , o i l , p e s t i c i d e s ) have been shown t o be h a r m f u l i n numerous r e s p o n s e s , y e t , many e s t u a r i e s have not changed a p p r e c i a b l y d e s p i t e t h e s e p o l l u t a n t s b e i n g p r e s e n t ( e . g . M a r s h a l l 1982 ) . In t h e S t r a i t o f G e o r g i a the g e n e r a l l e v e l o f p r o d u c t i v i t y does not r e f l e c t p o l l u t i o n of t h i s e n v i r o n m e n t , e i t h e r by p o i s o n s or e n r i c h m e n t ( P a r s o n s 1972; H a r r i s o n e t a l . 1983; W a l d i c h u k 1 9 8 3 ) . D e s p i t e p o l l u t i o n , e s t u a r i e s a r e r e s i l i e n t , a l t h o u g h i n t h e m s e l v e s h i g h l y v a r i a b l e e n v i r o n m e n t s . T i d e s , f l o o d s , e r o s i o n and s e d i m e n t a t i o n cause changes on a t i m e s c a l e f rom h o u r s t o c e n t u r i e s . I t i s h y p o t h e s i z e d t h a t n a t u r a l and u n n a t u r a l p e r t u r b a t i o n s a p p l i e d t o an e s t u a r y do not r e s u l t i n a breakdown of the e c o s y s t e m , but may cause s h i f t s i n the f o o d c h a i n . The change i n e s t u a r i n e food c h a i n s , e . g . from an a u t o t r o p h i c t o a h e t e r o t r o p h i c s y s t e m , might e n t a i l h i g h e r s e c o n d a r y and t e r t i a r y p r o d u c t i o n under t h e l a t t e r ( P a r s o n s et_ a_l. 1 9 8 1 ) . O b v i o u s l y , t h e e s t u a r y i t s e l f c a n n o t be m a n i p u l a t e d t o t e s t t h e impact of v a r i o u s p e r t u r b a t i o n s . F o r t e s t i n g t h e h y p o t h e s i s , a b i o l o g i c a l model of an e s t u a r y , c o n s i s t i n g of a s e r i e s of c o n t r o l l e d a q u a t i c m i c r o c o s m s , has been c o n s t r u c t e d . 92 S i m i l a r a p p r o a c h e s t o e x p e r i m e n t s on the b e n t h i c boundary l a y e r ( e . g . Z e i t z s c h e l & D a v i s 1978) and t o p o l l u t i o n of f r e s h w a t e r mic rocosms ( e . g . Taub & Crow 1978) have been r e p o r t e d . T h i s t h e s i s i s n o v e l i n i t s a p p r o a c h t o s i m u l a t e e s t u a r i n e s a l i n i t y v a l u e s and c e r t a i n t y p e s of p e r t u r b a t i o n s . The r e s p o n s e of t h e m i c r o p l a n k t o n t o imposed p e r t u r b a t i o n s m i g h t r e s u l t i n d e v i a t i o n s f rom t h e s t a n d a r d p a t t e r n and t h i s w i l l t h e n be t a k e n as i n d i c a t i o n of a s h i f t i n t h e food c h a i n of t h e e s t u a r y . Some b a c k g r o u n d d a t a on the n a t u r a l e c o l o g y of t h e m i c r o p l a n k t o n of the F r a s e r R i v e r e s t u a r y a r e a v a i l a b l e . The a r e a i s g e n e r a l l y d o m i n a t e d by a d i a t o m s p r i n g bloom and by a d i a t o m community of h i g h e r d i v e r s i t y d u r i n g t h e summer (S tephens e t a l . 1969; P a r s o n s e t a l . 1969; Shim 1 9 7 6 ) . H e t e r o t r o p h i c a c t i v i t y of b a c t e r i o p l a n k t o n may domina te t h e m i c r o p l a n k t o n d u r i n g t h e w i n t e r months and c o n s t i t u t e an i m p o r t a n t p a r t of t h e community a t o t h e r t i m e s of t h e y e a r ( A l b r i g h t I 9 8 3 a , b ) . D a t a on t h e water q u a l i t y of t h e F r a s e r R i v e r have been r e v i e w e d by D r i n n a n & C l a r k ( 1 9 8 0 ) . 93 2 . MATERIALS AND METHODS The l a b o r a t o r y s e t - u p , s a m p l i n g d e s i g n and a n a l y s i s of samples were d e s c r i b e d i n C h a p t e r I I . A t o t a l of 10 d i f f e r e n t p e r t u r b a t i o n s o v e r a s a l i n i t y range f rom < 5 ppt t o ^ 26 ppt were p e r f o r m e d . W i t h each p e r t u r b a t i o n e x p e r i m e n t , a c o n t r o l , i n form of a s t a n d a r d p a t t e r n , was r u n . In p e r t u r b a t i o n e x p e r i m e n t s t h e g l u c o s e a d d i t i o n was i n c r e a s e d from 1 mg 1~ 1 ( s t a n d a r d a d d i t i o n ) t o 5 mg l " 1 , i n o r d e r t o s i m u l a t e h i g h o r g a n i c l o a d . S i n g l e a d d i t i o n s . were compared w i t h r e p e t i t i v e ones on t h r e e c o n s e c u t i v e d a y s . W i t h n e u t r a l d e n s i t y s c r e e n s , t h e i n c i d e n t l i g h t was r e d u c e d by 90 %. An i r r a d i a n c e of c . 2 2 . 5 y E i n s t n r 2 s~ 1 was measured i n s i d e the empty and shaded f l a s k s . The impact of heavy m e t a l s was t e s t e d i n p e r t u r b a t i o n s i n c l u d i n g t h e a d d i t i o n o f 30 uq l " 1 o f Cu as w e l l as a heavy m e t a l m i x t u r e ( c f . T a b l e 2) a t c o n c e n t r a t i o n s of f i v e and t e n t i m e s the amount found i n a m o d e r a t e l y p o l l u t e d e s t u a r y such as N a r r a g a n s e t t B a y , Rhode I s l a n d , USA ( G o l d b e r g e t a l . 1977) . The same a p p r o a c h e m p l o y i n g t h e M u l t i - e l e m e n t - m i x was used by Thomas e t a l . (1980) i n a CEPEX s t u d y . T a b l e 2 shows t h e c o m p o s i t i o n and c o n c e n t r a t i o n s of t h e t e n heavy m e t a l s i n t h e o r i g i n a l m i x t u r e . S i n c e t h e s t a n d a r d a d d i t i o n of EDTA+iron would a f f e c t the a v a i l a b i l i t y of t h e heavy m e t a l s , changes i n t h e e x p e r i m e n t a l s e t - u p were n e c e s s a r y . The EDTA+iron was r e p l a c e d by a d d i t i o n o f F e C l 3 i n t h e same m o l a r c o n c e n t r a t i o n i n the e x p e r i m e n t a l u n i t as w e l l as i n the c o n t r o l . 94 T a b l e 2 - C o m p o s i t i o n o f M u l t i - e l e m e n t - m i x a c c o r d i n g t o Thomas e t a l . ( 1980) . M e t a l Compound jug l - 1 As V N a 2 H A s O , 5 . 0 Cd C d C l 2 0 .75 Cr K 2 C r 2 0 7 3.0 Cu C u S O „ • 5 H 2 0 3 .0 Hg H g C l 2 0 .15 N i N i C l 2 5 .0 Pb P b C l 2 3.0 Sb K ( S b O ) C « , H a 0 6 • 1/2 H 2 0 1 .5 Se S e 0 2 1 .5 Zn Z n C l 2 5 .0 In B r i t i s h C o l u m b i a , t h e h e r b i c i d e 2 , 4 - D i s used i n a e r i a l s p r a y i n g of w o o d l a n d s . A p p l i c a t i o n o f t h e same i n c o a s t a l a r e a s might a f f e c t t h e water q u a l i t y of e s t u a r i e s and i n l e t s . The h e r b i c i d e 2 , 4 - D , d i c h l o r o p h e n o x y a c e t i c a c i d , m o l . w t . 2 2 1 . 0 , (D 2128-SIGMA, C h e m i c a l Company) , was k i n d l y s u p p l i e d by Dr M . K . Upadhyaya of t h e P l a n t S c i e n c e D e p a r t m e n t , UBC. The p u r e compound was used i n t h e p e r t u r b a t i o n e x p e r i m e n t s r a t h e r t h a n c o m m e r c i a l f o r m u l a t i o n s w h i c h c o n t a i n o t h e r c h e m i c a l s b e s i d e 2 , 4 - D . The a c i d form of 2 , 4 - D i s o n l y s l i g h t l y s o l u b l e i n w a t e r , a t 2 5 ° C 0 .09 g 100 m l - 1 , but h i g h l y s o l u b l e i n e t h a n o l ( H e r b i c i d e Handbook, Weed S c i e n c e S o c i e t y of A m e r i c a 1 9 7 4 ) . On 95 day 3 2 , 4 - D was added t o t h e m i c r o c o s m s r e s u l t i n g i n f i n a l c o n c e n t r a t i o n s of 10 " " M and 10~ 3 M . The 2 , 4 - D was d i s s o l v e d i n s u b - s a m p l e s of wate r f rom t h e m i c r o c o s m s . The 1 0 " 3 M c o n c e n t r a t i o n was m i x e d w i t h 0 .5 ml e t h a n o l (95%) t o a s s i s t d i s s o l u t i o n , b e f o r e b e i n g combined w i t h the subsample and p o u r e d back i n t o t h e m i c r o c o s m s . The e t h a n o l i n t h e h i g h e s t 2 , 4 - D c o n c e n t r a t i o n i n c r e a s e d t h e t o t a l c a r b o n added t o 107.5 mg l " 1 (96 mg C l " 1 f rom 2 , 4 - D and 11 .5 mg C l " 1 f rom e t h a n o l ) , t h u s i n c r e a s i n g the c a r b o n s o u r c e f o r b a c t e r i a l g r o w t h . 96 3 . RESULTS AND DISCUSSION 3.1 E f f e c t s Of G l u c o s e P r o d u c t i v i t y of an e s t u a r y i s a r e s u l t of b a l a n c e s between a u t o t r o p h y and h e t e r o t r o p h y , and between i m p o r t and e x p o r t ( S i b e r t & Naiman 1980) . E s t u a r i n e c i r c u l a t i o n i n t r o d u c e s n u t r i e n t s f rom t h e o c e a n ; r i v e r s i n t r o d u c e o r g a n i c m a t t e r , s e d i m e n t s and n u t r i e n t s . These i m p o r t s s t i m u l a t e b o t h a u t o t r o p h i c and h e t e r o t r o p h i c g r o w t h . D i s s o l v e d o r g a n i c m a t t e r i n e s t u a r i e s i n c l u d e s l a r g e amounts of s u g a r s ( S e k i e t a_l. 1969) as w e l l as s m a l l q u a n t i t i e s of amino a c i d s ( e . g . H o b b i e e t a l . 1968 ) . W h i l e c o n c e n t r a t i o n s of t h e l a t t e r a r e l o w , measurements of f l u x r a t e s i n d i c a t e t h a t the c o n t r i b u t i o n t o p r i m a r y p r o d u c t i o n might be as h i g h as 10% (Hobbie e t a l . 1968; B i l l e n e t a l . 1980 ) . H e t e r o t r o p h i c p o p u l a t i o n s have c o n s i d e r a b l e p o t e n t i a l f o r o x i d i z i n g s m a l l o r g a n i c m o l e c u l e s . W i t h i n a p e r i o d of 30 h , b a c t e r i a a r e c a p a b l e of r e s p o n d i n g and a d a p t i n g t o a 1 0 0 - f o l d i n c r e a s e i n amino a c i d c o n c e n t r a t i o n ( W i l l i a m s & Gray 1 9 7 0 ) . G l u c o s e a d d i t i o n s t o m i c r o c o s m s of a s a l i n i t y range from < 5 p p t , 10 p p t , 18 ppt t o > 26 pp t r e s u l t e d i n a 25 t o 7 5 - f o l d i n c r e a s e i n d i s s o l v e d m o n o s a c c h a r i d e c o n c e n t r a t i o n s compared t o n a t u r a l l e v e l s found i n the F r a s e r R i v e r e s t u a r y . The e n r i c h m e n t i n o r g a n i c c a r b o n gave r i s e t o h i g h e r b a c t e r i a l numbers and l e d t o l o w e r c h l o r o p h y l l a c o n c e n t r a t i o n s a t the h e i g h t of t h e s i m u l a t e d p h y t o p l a n k t o n b l o o m . When 5 mg 1~ 1 of g l u c o s e were added on day 2 t o g e t h e r w i t h i n o r g a n i c n u t r i e n t s , 97 t h e b a c t e r i a r e sponded w i t h i n 24 t o 48 h w i t h an e x p o n e n t i a l i n c r e a s e i n numbers as shown i n F i g u r e s 22 and 2 3 . The f a s t e s t r e s p o n s e was o b s e r v e d a t i n t e r m e d i a t e s a l i n i t i e s , 10 ppt and 18 p p t , w h i l e a b s o l u t e numbers were h i g h e s t a t 18 ppt and > 26 p p t . W i t h t h r e e a d d i t i o n s of 5 mg 1~ 1 o f g l u c o s e on t h r e e c o n s e c u t i v e days (day 2 , 3 and 4 ) , a f u r t h e r i n c r e a s e i n b a c t e r i a l numbers was o b s e r v e d a t s a l i n i t i e s > 10 ppt ( F i g u r e 2 2 ) , w h i l e a t S 5 ppt a b s o l u t e numbers were v i r t u a l l y t h e same a f t e r one and t h r e e a d d i t i o n s ( F i g u r e 2 3 ) . In g l u c o s e - p e r t u r b e d mic rocosms (see F i g u r e 2 3 ) , n u t r i e n t s ( N 0 3 + N 0 2 ) were e x h a u s t e d 2 - 6 days e a r l i e r t h a n i n t h e c o n t r o l s . The h i g h e r o r g a n i c s u b s t r a t e c o n c e n t r a t i o n s s t i m u l a t e d m i c r o b i a l g r o w t h , r e s u l t i n g i n enhanced uptake of i n o r g a n i c n i t r o g e n by t h e b a c t e r i a . B e s i d e an i n c r e a s e i n m e t a b o l i c a c t i v i t y , t h e s t i m u l a t i o n was p r o b a b l y a l s o due t o a change i n s p e c i e s c o m p o s i t i o n , namely t h e r a p i d g r o w t h of a zymogenous m i c r o b i a l p o p u l a t i o n . N u t r i e n t c o n c e n t r a t i o n s i n F i g u r e 23 show the p a r t i t i o n i n g of s u b s t r a t e between h e t e r o t r o p h s and a u t o t r o p h s . The i n i t i a l d e c r e a s e was due t o b a c t e r i a l u p t a k e , t h e second due t o a s s i m i l a t i o n by p h y t o p l a n k t o n . The p a t t e r n was s i m i l a r a t a l l s a l i n i t i e s . F i g u r e s 23 and 24 show examples of c h l o r o p h y l l a c u r v e s a f t e r one and m u l t i p l e a d d i t i o n s of 5 mg l " 1 o f g l u c o s e . W i t h i n c r e a s i n g c o n c e n t r a t i o n s of g l u c o s e the p h y t o p l a n k t o n bloom was d e l a y e d a n d / o r peak v a l u e s of c h l o r o p h y l l a r e d u c e d . A t < 5 pp t s a l i n i t y t h e impact was g r e a t e s t . One s i n g l e a d d i t i o n caused a d e l a y i n t h e s i m u l a t e d p h y t o p l a n k t o n bloom by two days and a F i g u r e 22 - C o n c e n t r a t i o n s of b a c t e r i a i n c o n t r o l ( s o l i d l i n e ) m i c r o c o s m s and a f t e r one (dashed l i n e ) and t h r e e ( d o t t e d l i n e ) a d d i t i o n s o f 5 mg 1 _ 1 o f g l u c o s e . 20H 21 DAYS F i g u r e 23 - C o n c e n t r a t i o n s of n u t r i e n t s , b a c t e r i a and c h l o r o p h y l l a a f t e r g l u c o s e p e r t u r b a t i o n a t ^ 5 p p t ; c o n t r o l ( s o l i d l i n e ) , 5 mg 1 _ 1 g l u c o s e on day 2 (dashed l i n e ) , 5 mg l * 1 g l u c o s e on day 2 , 3 and 4 ( d o t t e d l i n e ) . F i g u r e 24 - C h l o r o p h y l l a c o n c e n t r a t i o n s a t 10 ppt and 26 ppt s a l i n i t y : c o n t r o l ( s o l i d l i n e ) , 5 mg l " 1 o f g l u c o s e on day 2 (dashed l i n e ) , 5 mg l " 1 o f g l u c o s e on day 2 , 3 and 4 ( d o t t e d l i n e ) . 101 F i g u r e 25 - C h l o r o p h y l l a c o n c e n t r a t i o n s i n < 5 ppt s a l i n i t y m i c r o c o s m s : a - s t a n d a r d p a t t e r n ( c o n t r o l ) , b - G l u c o s e p e r t u r b a t i o n (5mg 1 _ 1 on day 2 ) . B a r s i n d i c a t e mean ± 1 S . E . ; n=number of e x p e r i m e n t s . E x p e r i m e n t s were p e r f o r m e d a t d i f f e r e n t s e a s o n s . 102 r e d u c t i o n i n p h y t o p l a n k t o n b i o m a s s . T h i s d e l a y was o b s e r v e d c o n s i s t e n t l y a t d i f f e r e n t seasons of t h e y e a r ( F i g u r e 2 5 ) , w h i l e t h e r e d u c t i o n of t h e bloom d i d no t seem t o be s i g n i f i c a n t when mean c h l o r o p h y l l a v a l u e s i n c l u d e d s e a s o n a l v a r i a b i l i t y . A f t e r t h r e e a d d i t i o n s o f 5 mg l " 1 g l u c o s e , p h y t o p l a n k t o n g r o w t h was t o t a l l y s u p p r e s s e d i n t h e ^ 5 ppt s a l i n i t y m i c r o c o s m ( F i g u r e 23) . T a b l e 3 i n d i c a t e s t h e c o m p o s i t i o n of t h e p l a n k t o n samples c o l l e c t e d on day 9 . A t s a l i n i t i e s of 10 ppt and h i g h e r , t h e r e v i r t u a l l y was no d i f f e r e n c e between the g l u c o s e - p e r t u r b e d microcosms and the c o n t r o l s . At ^ 5 ppt s a l i n i t y t h e p r o p o r t i o n of s m a l l f l a g e l l a t e s (< 20 (im) was o v e r 10 t i m e s g r e a t e r t h a n i n t h e c o n t r o l m i c r o c o s m . A f t e r g l u c o s e p e r t u r b a t i o n of 5 mg t o a t o t a l of 15 mg l " 1 t h e y c o n t r i b u t e d n e a r l y 50% of t h e p l a n k t o n . The low c h l o r o p h y l l a v a l u e s i n t h e p e r t u r b e d mic rocosms ( 0 . 5 8 and 1.39 uq 1 ~ 1 ) s u g g e s t e d t h a t t h e m a j o r i t y of f l a g e l l a t e s were c o l o u r l e s s . The c h l o r o p h y l l a c o n c e n t r a t i o n was about 10% of the v a l u e i n the c o n t r o l m i c r o c o s m . A l g a e and b a c t e r i a compete f o r o r g a n i c s u b s t r a t e s of low m o l e c u l a r w e i g h t , e . g . u r e a (Remsen e t a l . 1972 ) . E v i d e n c e p o i n t s t o t h e s u c c e s s of b a c t e r i a , but o p p o s i t e f i n d i n g s have been r e p o r t e d as w e l l (Saks & Kahn 1 9 7 9 ) . The dominant d i a t o m s p e c i e s , S. c o s t a t u m and T h a l a s s i o s i r a , p r e s e n t i n t h e m i c r o c o s m e x p e r i m e n t s a r e not known t o grow on g l u c o s e ( H e l l e b u s t & L e w i n 1977 ) . C o m p e t i t i o n f o r i n o r g a n i c s u b s t r a t e s , such as p h o s p h a t e and i n o r g a n i c n i t r o g e n compounds has been d e s c r i b e d and under c e r t a i n c o n d i t i o n s a l g a l g r o w t h can be s e v e r e l y l i m i t e d 103 ( e . g . Rhee 1972; Thayer 1974 ) . T a b l e 3 - R e l a t i v e abundance of s m a l l f l a g e l l a t e s , d i a t o m s and g reen a l g a e on day 9 i n C o n t r o l and a f t e r one and t h r e e i n i t i a l g l u c o s e a d d i t i o n s of 5 mg 1 ~ 1 . Means ± S . E . ( i n b r a c k e t s ) a r e g i v e n ; n=4. SPECIES CONTROL 5 mg l " 1 3x5 mg 1 - 1 ^ 5 ppt S a l i n i t y D i a t o m s & Green A l g a e 9 6 . 0 ( ± 1 . 6 3 ) 5 2 . 7 ( ± 3 . 0 2 ) 5 2 . 7 ( ± 1 . 5 0 ) S m a l l F l a g e l l a t e s 4 . 0 ( ± 1 . 6 3 ) 4 7 . 3 ( ± 3 . 0 2 ) 4 7 . 5 ( ± 1 . 5 0 ) 10 ppt S a l i n i t y D ia toms & Green A l g a e 9 8 . 8 ( ± 0 . 4 8 ) 9 9 . 5 ( ± 0 . 2 9 ) 9 7 . 5 ( ± 0 . 8 7 ) S m a l l F l a g e l l a t e s 1 . 2 ( ± 0 . 4 8 ) 0 . 5 ( ± 0 . 2 9 ) 2 . 5 ( ± 0 . 8 7 ) 18 pp t S a l i n i t y D i a t o m s & Green A l g a e 100.0 9 9 . 5 ( ± 0 . 5 0 ) 9 7 . 5 ( ± 0 . 4 8 ) S m a l l F l a g e l l a t e s 0 .0 0 . 5 ( ± 0 . 5 0 ) 3 . 2 ( ± 0 . 4 8 ) > 26 pp t S a l i n i t y D i a t o m s 9 9 . 2 ( ± 0 . 4 8 ) 9 9 . 5 ( ± 0 . 5 0 ) 9 6 . 8 ( ± 1 . 1 9 ) S m a l l F l a g e l l a t e s 0 . 8 ( ± 0 . 4 8 ) 0 . 5 ( ± 0 . 5 0 ) 1 . 5 ( ± 1 . 1 9 ) I n t h e F r a s e r R i v e r e s t u a r y low s a l i n i t i e s a r e a s s o c i a t e d w i t h low i n o r g a n i c n u t r i e n t l e v e l s ( C h a p t e r V I ) . In the p r e s e n c e of h i g h o r g a n i c s u b s t r a t e c o n c e n t r a t i o n s b a c t e r i a may exhaus t ambient i n o r g a n i c n i t r o g e n . Under t h e s e c o n d i t i o n s 1 04 n u t r i e n t r e c y c l i n g by m i c r o z o o p l a n k t o n becomes i n c r e a s i n g l y i m p o r t a n t as shown i n C h a p t e r I V , s e c t i o n 3 . 4 . F o l l o w i n g t h e a d d i t i o n of 5 mg l " 1 g l u c o s e , up t o 66% of t h e b iomass i n t h e d e l a y e d and r e d u c e d p h y t o p l a n k t o n bloom might be due t o " r e g e n e r a t e d p r o d u c t i o n " (Dugda le & G o e r i n g 1 9 6 7 ) . H e t e r o t r o p h i c and a u t o t r o p h i c c o m p e t i t i o n f o r n i t r a t e has been s u g g e s t e d by P a r k e r e t §_1. (1975) i n a s t r a t i f i e d e s t u a r y . In t h e p r e s e n c e of o r g a n i c c a r b o n - r i c h and n i t r o g e n - d e f i c i e n t p u l p m i l l e f f l u e n t , h e t e r o t r o p h i c g r o w t h was e n h a n c e d , w h i l e p r i m a r y p r o d u c t i v i t y was i n h i b i t e d by l a c k of n i t r o g e n . In s p i t e of v e r y low p r i m a r y p r o d u c t i v i t y , l a r g e s t a n d i n g c r o p s o f b o t h z o o p l a n k t o n and f i s h i n d i c a t e d t h e p e r s i s t a n c e of a w e l l d e v e l o p e d f o o d c h a i n ( S i b e r t & Brown 1975 ) . P a r s o n s e t a l . (1981) e n r i c h e d CEPEX e n c l o s u r e s w i t h g l u c o s e and s t u d i e d t h e e f f e c t s on t h e p r o d u c t i v i t y of t h e s y s t e m . Enhancement o f h e t e r o t r o p h i c g r o w t h c a u s e d d e p r e s s i o n of p h o t o s y n t h e t i c p r o d u c t i o n by a l g a e but r e s u l t e d i n h i g h e r s e c o n d a r y and t e r t i a r y p r o d u c t i o n . C o m p e t i t i o n between b a c t e r i a and p h y t o p l a n k t o n f o r a l i m i t i n g s u b s t r a t e was c o n f i r m e d i n t h e m i c r o c o s m e x p e r i m e n t s . In t h e p r e s e n c e of h i g h o r g a n i c l o a d and low n i t r o g e n c o n c e n t r a t i o n s b a c t e r i a outcompete p h y t o p l a n k t o n , c a u s i n g a d e p r e s s i o n o r c o m p l e t e s u p p r e s s i o n o f a u t o t r o p h s , as o b s e r v e d i n t h e < 5 ppt s a l i n i t y m i c r o c o s m ( F i g u r e 2 3 ) . H e t e r o t r o p h i c a c t i v i t y was measured on day 3 , when b a c t e r i a were i n t h e e x p o n e n t i a l g r o w t h p h a s e . Due t o s u b s t r a t e e n r i c h m e n t , g l u c o s e up take r a t e s were 1-2 o r d e r s of magni tude 105 h i g h e r t h a n i n samples f rom t h e S t r a i t of G e o r g i a a t c o r r e s p o n d i n g s a l i n i t i e s (see T a b l e 1 i n C h a p t e r I I I and T a b l e 7 i n C h a p t e r V I ) . The h i g h e s t up take r a t e s were found a t i n t e r m e d i a t e s a l i n i t i e s , 10 ppt and 18 p p t . 3 .2 E f f e c t s Of L i g h t Among t h e e n v i r o n m e n t a l f a c t o r s w h i c h i n f l u e n c e p h o t o s y n t h e s i s of p h y t o p l a n k t o n a r e l i g h t , n u t r i e n t s and t e m p e r a t u r e . I n the F r a s e r R i v e r e s t u a r y , t e m p e r a t u r e i s l i m i t i n g o n l y near the s u r f a c e d u r i n g t h e w i n t e r and t h e s p r i n g ( T a k a h a s h i e_t aJL. 1973) . N u t r i e n t l i m i t a t i o n i s i m p o r t a n t d u r i n g t h e summer, w h i l e near s u r f a c e l i g h t i s t h e main l i m i t i n g f a c t o r t h r o u g h o u t t h e r e s t of t h e y e a r . D u r i n g t h e a n n u a l f r e s h e t , May t o J u n e , the F r a s e r R i v e r c a r r i e s a huge amount of s i l t . T h i s s i l t l o a d i n c r e a s e s t h e t u r b i d i t y of t h e water ( h i g h e r k T v a l u e ) , t h u s r e d u c e s the l i g h t a v a i l a b l e f o r p h o t o s y n t h e s i s . D r e d g i n g has the same e f f e c t , w h i l e the c o n s t r u c t i o n of dams i n the r i v e r r e d u c e s t h e s i l t l o a d ; t h u s k T becomes s m a l l e r . The i n f l u e n c e of l i g h t on t h e g r o w t h of n a t u r a l p l a n k t o n p o p u l a t i o n s was s t u d i e d a t d i f f e r e n t s a l i n i t i e s 5 pp t t o ^ 26 p p t ) a t a c o n s t a n t t e m p e r a t u r e of 1 2 ° C . The a c t u a l amount of l i g h t r e c e i v e d by t h e p h y t o p l a n k t o n i s not known because no measurement was t a k e n i n t h e w a t e r - f i l l e d m i c r o c o s m s , but an i r r a d i a n c e of c . 2 2 . 5 y E i n s t n r 2 s " 1 i n t h e empty c o n t a i n e r s l e d t o t h e a s s u m p t i o n t h a t l i g h t l e v e l s were i n d e e d l i m i t i n g . F i g u r e 26 shows the c h l o r o p h y l l a c u r v e s a t ^ 5 p p t , 10 p p t , 18 ppt and £ 26 ppt s a l i n i t i e s . The r e d u c t i o n of l i g h t 106 F i g u r e 26 - C h l o r o p h y l l a c o n c e n t r a t i o n s of t h e c o n t r o l ( s o l i d l i n e ) and 1 0 % - l i g h t ( d o t t e d l i n e ) mic rocosms from < 5 pp t t o > 26 pp t s a l i n i t i e s . 107 by 90% c a u s e d a d e l a y of 2 - 6 days i n t h e p h y t o p l a n k t o n b l o o m . The s h o r t e s t d e l a y (2 days ) was o b s e r v e d a t 10 ppt s a l i n i t y and the l o n g e s t (6 days ) a t < 5 p p t . At b o t h 18 ppt and > 26 ppt s a l i n i t y t h e d e l a y was f o u r d a y s . Maximum c h l o r o p h y l l a c o n c e n t r a t i o n s were a l w a y s g r e a t e r i n t h e shaded m i c r o c o s m s . D i e l v a r i a t i o n s i n t h e c h l o r o p h y l l a c o n t e n t of m a r i n e p h y t o p l a n k t o n a r e w e l l documented ( S o u r n i a 1974; H i t c h c o c k 1980b) . I n o r d e r t o e l i m i n a t e t h e s e v a r i a t i o n s , s a m p l i n g was a l w a y s done a t t h e same t i m e of the day between 10:00 and 1 2 : 0 0 . V a r i o u s f a c t o r s might have been r e s p o n s i b l e f o r t h e h i g h e r c h l o r o p h y l l a peak v a l u e s i n the shaded m i c r o c o s m s : the v a r i a t i o n s i n c h l o r o p h y l l a c o n t e n t i n d i f f e r e n t s p e c i e s g r o w i n g a t d i f f e r e n t s a l i n i t i e s , o r h i g h e r c h l o r o p h y l l a c o n t e n t per c e l l as a d a p t a t i o n t o low l i g h t ( B e a r d a l l & M o r r i s 1976 ) . The o b s e r v e d d i f f e r e n c e s were m o s t l y due t o t h e l a t t e r . C o n s i d e r i n g the s p e c i e s c o m p o s i t i o n and s u c c e s s i o n of the dominant d i a t o m s , T h a l a s s i o s i r a s p p . seemed t o be i n h i b i t e d when t h e i n c i d e n t l i g h t was r e d u c e d by 90%. T h i s e f f e c t was most p r o n o u n c e d i n the 18 ppt. and > 26 pp t s a l i n i t y mic rocosms where t h e p h y t o p l a n k t o n bloom was a l w a y s composed of the two dominant s p e c i e s S k e l e t o n e m a c o s t a t u m and T h a l a s s i o s i r a s p p . . F i g u r e 27 shows t h e r e l a t i v e abundance of b o t h s p e c i e s based on a t o t a l of 400 c e l l s c o u n t e d . I n t h e shaded microcosms S. c o s t a t u m outgrew T h a l a s s i o s i r a s p p . ; the c e l l numbers of the l a t t e r h a v i n g been r e d u c e d by more t h a n 30%. D u r i n g e n u m e r a t i o n T h a l a s s i o s i r a s p p . were g rouped i n t o t h r e e d i f f e r e n t s i z e c l a s s e s ( s m a l l c e l l s o f 1,889 y 3 v o l . ; medium c e l l s of 4 ,942 M 3 108 F i g u r e 27 - R e l a t i v e abundance of dominant d i a t o m s p e c i e s i n c o n t r o l ( c . 225 u E i n s t n r 2 s e c " 1 ) and 10% l i g h t ( c . 2 2 . 5 jxEins t m " 2 s e c " 1 ) m i c r o c o s m s a t s a l i n i t i e s £ 18 p p t . B a r s i n d i c a t e x ± S . E . ; n = 2 109 v o l . ; l a r g e c e l l s of 23 ,268 u3 v o l . ) . W h i l e a l l s i z e c l a s s e s were found i n t h e c o n t r o l m i c r o c o s m s , s m a l l T h a l a s s i o s i r a s p e c i e s d o m i n a t e d i n t h e shaded c o n t a i n e r s w h i l e l a r g e c e l l s were a l t o g e t h e r a b s e n t . A t ^ 5 ppt s a l i n i t y t h e dominant d i a t o m s were T h a l a s s i o s i r a s p p . i n t h e c o n t r o l as w e l l as i n t h e low l i g h t m i c r o c o s m , w h i l e a t 10 p p t , S . c o s t a t u m a l w a y s c o m p r i s e d > 90% of the b loom s p e c i e s i n d e p e n d e n t of v a r i a t i o n s i n i r r a d i a n c e s . T h i s s u g g e s t e d t h a t s a l i n i t y was o f g r e a t e r i m p o r t a n c e t h a n l i g h t . The i n c r e a s e d abundance of S . c o s t a t u m under low l i g h t c o n d i t i o n s i s a l s o seen i n B . C . c o a s t a l w a t e r s , where i n some y e a r s T h a l a s s i o s i r a s p p . , i n o t h e r y e a r s S. c o s t a t u m , i s t h e f i r s t i n t h e d i a t o m s p r i n g bloom ( T a k a h a s h i e t a l . 1973; S t o c k n e r e t a l . 1977 ) . S t o c k n e r e t a l . (1977) n o t e d t h a t , l a r g e l y as a r e s u l t of l o w e r e d l i g h t l e v e l s i n 1974, when S . c o s t a t u m d o m i n a t e d b o t h t h e p h y t o p l a n k t o n and t h e sed iment a s semblages i n Howe Sound ( R o e l o f s i n p r e s s ) t h e s p r i n g bloom was l a t e r i n 1974 t h a n i n 1973, when T h a l a s s i o s i r a s p p . d o m i n a t e d t h e p h y t o p l a n k t o n . A n o t h e r example f rom t h e F r a s e r R i v e r a r e a showed a T h a l a s s i o s i r a s p . dominant i n t h e e a r l y s p r i n g b loom of 1966, w h i l e S. c o s t a t u m was the e a r l y dominant i n 1967; t h e l i g h t l e v e l s were l ower i n e a r l y 1967 t h a n i n 1966 ( T a k a h a s h i • e_t §_1. 1 9 7 3 ) . In y e a r s when l e s s l i g h t was a v a i l a b l e t h a n i n ' n o r m a l ' y e a r s , S . c o s t a t u m outcompeted T h a l a s s i o s i r a s p p . . The r e s u l t s of s h a d i n g i n t h e p r e s e n t e x p e r i m e n t s s u p p o r t t h e s u g g e s t i o n t h a t i r r a d i a n c e p l a y s a ma jor r o l e i n the 110 d e t e r m i n a t i o n o f s p e c i e s s u c c e s s i o n and dominance i n t h e s p r i n g bloom. Low i r r a d i a n c e s seem t o f a v o u r s m a l l e r s p e c i e s , w h i l e l a r g e r ones a r e e l i m i n a t e d . E f f e c t s o f t e m p e r a t u r e have been e x c l u d e d under t h e c o n t r o l l e d e x p e r i m e n t a l c o n d i t i o n s a t 12°C. The e f f e c t o f l o w e r i r r a d i a n c e s upon b a c t e r i a was n o t c o n c l u s i v e . The s e c o n d h e t e r o t r o p h i c bloom was d e l a y e d by two d a y s a t < 5 p p t , 10 ppt and £ 26 ppt s a l i n i t i e s , w h i l e a t 18 p p t the t i m i n g d i d not d i f f e r s i g n i f i c a n t l y ( c f . A p p e n d i x 3 ) . No d i f f e r e n c e was o b s e r v e d i n m i c r o f l a g e l l a t e numbers i n e i t h e r , c o n t r o l n o r s h a d e d m i c r o c o s m s b e c a u s e o f t h e l a r g e v a r i a b i l i t y i n c o u n t s ( c f . A p p e n d i x 3 ) . 111 3 .3 E f f e c t s Of Heavy M e t a l s A c h e m i c a l i n t e r p r e t a t i o n of t h e r e s u l t s of t h e m e t a l p e r t u r b a t i o n e x p e r i m e n t s i s d i f f i c u l t because t h e i d e n t i t y and c o n c e n t r a t i o n of c h e l a t o r s , w h i c h d e t e r m i n e t h e a c t i v i t y of t h e f r e e m e t a l i o n s , a r e not known i n n a t u r a l w a t e r s . In t h i s e m p i r i c a l a p p r o a c h , h o w e v e r , t h e r e l a t i v e t o x i c i t i e s of v a r i o u s heavy m e t a l s can be i n v e s t i g a t e d w i t h r e s p e c t t o t h e i r e c o l o g i c a l impac t on n a t u r a l e s t u a r i n e m i c r o p l a n k t o n p o p u l a t i o n s . In t h e p r e s e n c e of the s t a n d a r d a d d i t i o n of E D T A + i r o n , t h e f i v e - f o l d c o n c e n t r a t i o n of t h e b a s i c M u l t i - E l e m e n t - m i x (see T a b l e 2 , S e c t i o n 2) had no s i g n i f i c a n t e f f e c t on b a c t e r i a and m i c r o z o o p l a n k t o n numbers and c h l o r o p h y l l a c o n c e n t r a t i o n s ( c f . A p p e n d i x 4 ) . However , p l a n k t o n a n a l y s i s r e v e a l e d s i g n i f i c a n t d i f f e r e n c e s between t h e c o n t r o l and p e r t u r b e d m i c r o c o s m s . A t ^ 5 ppt s a l i n i t y , T h a l a s s i o s i r a s p p . , t h e dominant d i a t o m s , were s i g n i f i c a n t l y r e d u c e d i n numbers a f t e r heavy m e t a l a d d i t i o n i n f a v o u r of a g r e a t e r p r o p o r t i o n of s m a l l f l a g e l l a t e s (< 20 nm). The r e s u l t s a r e p r e s e n t e d i n T a b l e 4 . At 10 ppt s a l i n i t y , where S. c o s t a t u m was d o m i n a n t , no d i f f e r e n c e s c o u l d be s e e n . At 18 pp t and > 26 ppt s a l i n i t y S. c o s t a t u m seemed t o grow b e t t e r t h a n T h a l a s s i o s i r a s p p . i n t h e p r e s e n c e of heavy m e t a l s , but o n l y t h e r e s u l t s a t 18 pp t s a l i n i t y were s i g n i f i c a n t ( T a b l e 5 ) . The i n i t i a l h i g h v a l u e s of s m a l l f l a g e l l a t e s i n the low and h i g h s a l i n i t y water i s a s e a s o n a l e f f e c t . The water was c o l l e c t e d i n l a t e J a n u a r y , 1982. Table 4 : Relative abundance of dominant diatoms and small f l a g e l l a t e s i n co n t r o l and heavy metal perturbed microcosms. Means 1 S.E. are given of a t o t a l of 400 c e l l s counted; n - 4. Species Day 5 Day 11 Day 15 2.5 %o S Control T h a l a s s i o s i r a spp. Small F l a g e l l a t e s 1.0(±0.71) 99.0(±0.71) 84.0(10.41) 1.8(11.11) 68.0(13.03) 10.0(11.47) 5x ME-mix Th a l a s s i o s i r a spp. Small F l a g e l l a t e s 1.5(±0.64) 98.2(±0.75) 53.2(13.54) 30.0(12.34) 48.8(14.35) 28.2(13.20) 10 %.S Control S. costatum Small F l a g e l l a t e s 5.8(±2.32) 94.0(±2.34) 95.5(11.55) 1.8(11.11) 83.8(12.17) 13.0(11.87) 5x ME-mix S. costatum Small F l a g e l l a t e s 1.5(10.96) 98.5(10.96) 91.5(10.50) 1.2(10.63) 78.0(12.55) 15.0(13.24) Table 5: Relative abundance of dominant diatoms and small flagellates in control and heavy metal perturbed microcosms. Means ± S.E. n - 4. are given of a total of 400 cells counted; Species Day 5 Day 7 Day 9 Day 15 18 %. S S. costatum 4.8(13.45) 25.2(±5.26) 53.0(18.03) 55.0(12.61) Control Thalassiosira spp. 2.0(10.91) 36.8(±4.25) 32.2(14.68) 18.0(12.27) Small Flagellates 91.0(±3.03) 34.2(12.46) 9.2(11.11) 21.2(11.49) S. costatum 8.2(12.39) 66.8(14.92) 76.0(11.08) 79.5(12.22) 5x ME-mix Thalassiosira spp. 2.0(10.91) 10.0(11.87) 12.8(12.29) 5.2(12.50) Small Flagellates 85.5(±2.99) 17.2(13.09) 8.8(11.80) 14.5(12.33) 1 1 4 A f t e r t h e EDTA+iron was r e p l a c e d by F e C l 3 , t h e a d d i t i o n o f the f i v e - and t e n - f o l d M u l t i - E l e m e n t - m i x or 30 uq 1 " ' Cu c a u s e d a d e l a y i n t h e p h y t o p l a n k t o n bloom and a s u p p r e s s i o n of t h e c h l o r o p h y l l a peak v a l u e s a t a l l s a l i n i t i e s e x c e p t i n the 10 p p t m i c r o c o s m . Here t h e t i m i n g o f the c h l o r o p h y l l a peak was no t a l t e r e d and t h e h e i g h t not s i g n i f i c a n t l y r e d u c e d ( F i g u r e 2 8 ) . F i g u r e s 29 and 30 show c h l o r o p h y l l a c u r v e s a t d i f f e r e n t s a l i n i t i e s and w i t h v a r y i n g c o n c e n t r a t i o n s of heavy m e t a l s . When the p h y t o p l a n k t o n bloom was d e l a y e d , n u t r i e n t e x h a u s t i o n o c c u r r e d l a t e r . B a c t e r i a l g r o w t h seemed t o be s t i m u l a t e d f o l l o w i n g the a d d i t i o n of the t e n - f o l d M u l t i - E l e m e n t - m i x as w e l l as 30 uq 1~ 1 Cu a l o n e ( c f . A p p e n d i x 5 ) . T h i s s t i m u l a t i o n m i g h t have been due t o the l e a k a g e of o r g a n i c s u b s t r a t e s t h r o u g h a damaged p h y t o p l a n k t o n c e l l membrane ( B e n t i e y - M o w a t & R e i d 1 9 7 7 ) , or due t o the e x c r e t i o n of o r g a n i c compounds from c e l l s under s t r e s s (Steemann N i e l s e n e t a l . 1969 ) . W i t h o u t t h e a r t i f i c i a l c h e l a t o r EDTA, t h e e f f e c t of heavy m e t a l p e r t u r b a t i o n s on the s p e c i e s c o m p o s i t i o n and s u c c e s s i o n was much more p r o n o u n c e d . The t o x i c i t y of the ME-mix was d e t e r m i n e d by Cu and Hg a c c o r d i n g t o H o l l i b a u g h et. a_l. ( 1 9 8 0 ) . B e s i d e s the i n c r e a s e i n s m a l l f l a g e l l a t e s a t ^ 5 ppt s a l i n i t y , a change i n t h e dominant s p e c i e s was a l s o o b s e r v e d . I n s t e a d of a b loom of the c e n t r i c d i a t o m s T h a l a s s i o s i r a s p p . as i n t h e c o n t r o l m i c r o c o s m , the pennate d i a t o m s s u c h as T h a l a s s i o n e m a s p p . were dominant i n the p e r t u r b e d c o n t a i n e r s ( c f . A p p e n d i x 5 ) . A f t e r Cu a d d i t i o n s N i t z s c h i a s p p . were found i n g r e a t e r numbers , w h i c h a g r e e s w i t h t h e f i n d i n g s of Thomas & S e i b e r t (1977) d u r i n g F i g u r e 28 - C o n c e n t r a t i o n of c h l o r o p h y l l a i n 10 ppt m i c r o c o s m a f t e r t e n - f o l d M u l t i - E l e m e n t - m i x . 1 1 6 F i g u r e 29 - C h l o r o p h y l l a c u r v e s a t d i f f e r e n t s a l i n i t i e s a f t e r a d d i t i o n of M u l t i - E l e m e n t - M i x . 117 F i g u r e 30 - C h l o r o p h y l l a c u r v e s a t d i f f e r e n t s a l i n i t i e s a f t e r t h e a d d i t i o n of 30 ug l " 1 C u . 118 a CEPEX s t u d y . A g a i n , a t 18 ppt and >: 26 ppt s a l i n i t i e s S. c o s t a t u m grew b e t t e r t h a n T h a l a s s i o s i r a s p p . i n t h e p r e s e n c e of heavy m e t a l s ( c f . A p p e n d i x 5 ) . At 10 ppt s a l i n i t y , t h e p h y t o p l a n k t o n b loom was a l w a y s composed of S . c o s t a t u m , a s p e c i e s w h i c h was l e a s t a f f e c t e d by m e t a l a d d i t i o n s . M o r e l e t a l . (1978) t e s t e d the c o p p e r t o x i c i t y t o S. c o s t a t u m and found i t t o be most r e s i s t a n t . T h u s , t h e o c c u r r e n c e and d o m i n a t i o n of S . c o s t a t u m i n w a t e r s of c . 10 ppt s a l i n i t y i s an i m p o r t a n t b u f f e r a g a i n s t t h e d e s t a b i l i z a t i o n of the e s t u a r i n e e c o s y s t e m . T h i s e f f e c t i s not o b s e r v e d w i t h p h y t o p l a n k t o n s p e c i e s t h a t a r e more abundant a t l o w e r and h i g h e r s a l i n i t i e s , s u c h as T h a l a s s i o s i r a s p p . . 3 .4 E f f e c t s Of The H e r b i c i d e 2 , 4 - D The h e r b i c i d e 2 , 4 - D has been i n use now f o r about 40 y e a r s as a s y s t e m i c h e r b i c i d e t o c o n t r o l b r o a d l e a f weeds , b r o a d l e a f p e r e n n i a l h e r b a c e o u s and woody p l a n t s , as w e l l as a q u a t i c weeds . The e f f e c t s of 2 , 4 - D have been e x t e n s i v e l y s t u d i e d i n h i g h e r p l a n t s . I t c a u s e s i n c r e a s e d and abnorma l g r o w t h due t o i t s s i m i l a r i t y t o n a t u r a l l y o c c u r r i n g a u x i n , a f f e c t s r e s p i r a t i o n , d e p l e t e s s t a r c h and sugar r e s e r v e s and i n t e r f e r e s w i t h RNA, DNA and p r o t e i n s y n t h e s i s ; but t h e p r i m a r y mode of a c t i o n i s s t i l l not known. C o n c e n t r a t i o n s of 1 0 " " - 1 0 " 3 M have been shown t o be i n h i b i t o r y i n h i g h e r p l a n t s ( A s h t o n & C r a f t s 1981 ) . When s p r a y e d from a i r p l a n e s o v e r l a r g e a r e a s , r i v e r s , e s t u a r i e s and i n l e t s a r e not s p a r e d . A c c o r d i n g t o t h e recommendat ions i n the B . C . F I E L D CROP GUIDE 1983, 2 , 4 - D may be s p r a y e d i n s o l u t i o n s of 0 . 5 M maximum c o n c e n t r a t i o n . T h i s 119 c o n c e n t r a t i o n r e s u l t s when t h e maximum recommended amount of 2 , 4 - D h a " 1 i s d i s s o l v e d i n t h e s m a l l e s t recommended v o l u m e . I n r u n n i n g o r h i g h l y mixed w a t e r s t h i s i n i t i a l c o n c e n t r a t i o n i s i m m e d i a t e l y d i l u t e d . S t r a t i f i c a t i o n of 2 , 4 - D w i t h d e p t h has been r e p o r t e d i n s t a g n a n t w a t e r s where h i g h e s t c o n c e n t r a t i o n s were found near the s u r f a c e and p h y t o t o x i c a c t i v i t y showed l i t t l e d e c r e a s e f o r p e r i o d s of s e v e r a l months ( W o j t a l i k e t a l . 1971; Bovey & Young 1 9 8 0 ) . D e g r a d a t i o n of 2 , 4 - D depends on oxygen c o n c e n t r a t i o n and t e m p e r a t u r e . W h i l e the t o x i c i t y of 2 , 4 - D on f i s h , i n c l u d i n g s a l m o n i d s (Meehan ejb a_l. 1974) , f r e s h w a t e r and e s t u a r i n e i n v e r t e b r a t e s and f r e s h w a t e r a l g a e have been s t u d i e d , no such i n f o r m a t i o n has been a s s e m b l e d w i t h r e s p e c t t o m a r i n e m i c r o p l a n k t o n . The h e r b i c i d e 2 , 4 - D was added on day 3 j u s t b e f o r e the s t a r t of t h e p h y t o p l a n k t o n b l o o m . The a d d i t i o n of t h e 1 0 " 3 M s o l u t i o n c a u s e d a d r o p i n pH i n t h e l o w e r s a l i n i t y w a t e r s (2 u n i t s a t ^ 5 p p t ; 1 u n i t a t 10 p p t ) . I n t h e s e mic rocosms the pH never r o s e above 7 d u r i n g t h e e n t i r e e x p e r i m e n t . At 18 ppt and ^ 26 p p t s a l i n i t y , t h e a d d i t i o n of 2 , 4 - D d i d not change t h e pH because of t h e b u f f e r i n g q u a l i t i e s of s eawater ( c f . A p p e n d i x 1 ) . A f t e r t h e a d d i t i o n of 2 , 4 - D a t 10 " * M f i n a l c o n c e n t r a t i o n , t h e c h l o r o p h y l l a c u r v e s i n t h e < 5 ppt and ^ 26 pp t s a l i n i t y microcosms were not s i g n i f i c a n t l y d i f f e r e n t f rom t h e c o n t r o l s ( F i g u r e 3 1 ) . At 10 ppt ( F i g u r e 32) the c h l o r o p h y l l a peak was d e l a y e d by 2 days but of the same peak h e i g h t , w h i l e a t 18 ppt t h e d e l a y was l e s s t h a n 2 days and t h e peak h e i g h t s l i g h t l y F i g u r e 31 - C h l o r o p h y l l a c o n c e n t r a t i o n s i n > 26 pp t s a l i n i t y microcosms a f t e r a d d i t i o n of 2 , 4 - D . Arrow i n d i c a t e s second n u t r i e n t a d d i t i o n . o I O % » s Control 1 0 " 4 M 2.4D IO - 3 M 2.4D F i g u r e 32 - C h l o r o p h y l l a c o n c e n t r a t i o n s i n 10 ppt s a l i n i t y microcosms a f t e r a d d i t i o n of 2 , 4 - D . Arrow i n d i c a t e s second n u t r i e n t a d d i t i o n . 122 d e c r e a s e d . A f t e r a d d i t i o n o f 2 , 4 - D a t 10~ 3 M f i n a l c o n c e n t r a t i o n no p h y t o p l a n k t o n bloom o c c u r r e d a t any s a l i n i t y u n t i l day 11 . On day 15 a second n u t r i e n t a d d i t i o n (same c o n c e n t r a t i o n s as i n i n i t i a l s t a n d a r d a d d i t i o n ) was added t o 3 m i c r o c o s m s ( 5 5 p p t , 10 ppt and >: 26 ppt s a l i n i t y ) w h i c h p r e v i o u s l y had r e c e i v e d 1 0 " 3 M 2 , 4 - D . W h i l e c h l o r o p h y l l a l e v e l s a t 18 ppt (no second n u t r i e n t a d d i t i o n ) r e m a i n e d low ( c . 2 M g l " 1 ) , a b loom of g reen f l a g e l l a t e s s t a r t e d on day 21 i n t h e < 5 ppt and 10 ppt s a l i n i t y mic rocosms ( F i g u r e 3 2 ) . In t h e > 26 ppt s a l i n i t y c o n t a i n e r c h l o r o p h y l l a v a l u e s were o v e r 8 M g l " 1 on day 17, h o v e r e d a r o u n d 10 M g l " 1 , b e f o r e d r o p p i n g t o 1 (ig l " 1 on day 25 ( F i g u r e 3 1 ) . A v i s i b l e d i f f e r e n c e i n c o l o u r between t h e ^ 5 p p t , t h e 10 ppt and t h e > 26 ppt s a l i n i t y microcosms was o b s e r v e d . The former two were g r e e n , w h i l e the l a t t e r y e l l o w i s h - b r o w n . I n b o t h low s a l i n i t y c o n t a i n e r s the bloom was composed of g r e e n f l a g e l l a t e s ( Chlamydomonas s p . , - i d e n t i f i e d and i s o l a t e d as #440 by J . A c r e m a n , c u r a t o r of NEPCC) w h i l e a t > 26 ppt s a l i n i t y g reen f l a g e l l a t e s and d i a t o m s were p r e s e n t . The s p e c i e s c o m p o s i t i o n was not s i g n i f i c a n t l y a l t e r e d by 1 0 " ° M 2 , 4 - D a t < 5 p p t , 10 ppt and £ 26 ppt s a l i n i t i e s , e x c e p t f o r an a p p a r e n t i n c r e a s e i n numbers of C h a e t o c e r o s s p p . a t t h e s e s a l i n i t i e s ( c f . A p p e n d i x 6 ) . However , a t 18 ppt s a l i n i t y numbers of t h e l a t t e r were s i g n i f i c a n t l y h i g h e r ( 2 5 . 0 % ± 9 . 2 ) t h a n i n the c o n t r o l s ( 6 . 5 % ± 5 . 0 ) . The bloom i n d u c e d a f t e r a second n u t r i e n t a d d i t i o n i n t h e > 26 ppt s a l i n i t y m i c r o c o s m was composed of T h a l a s s i o s i r a s p p . (69.5%) and of g r e e n f l a g e l l a t e s 1 23 (20 .8%) . B a c t e r i a l numbers i n c r e a s e d i n the p r e s e n c e of 10 " " M and 1 0 " 3 M 2 , 4 - D i n m i c r o c o s m s , where pH was not a l t e r e d . The h i g h e r numbers i n 1 0 " 3 M 2 , 4 - D ' might be p a r t i a l l y a t t r i b u t e d t o t h e a d d i t i o n of e t h a n o l w h i c h a c c o u n t e d f o r 10.7% of the c a r b o n t o t a l ( c f . A p p e n d i x 6 ) . A c c o r d i n g t o s t u d i e s of b a c t e r i a i n t h e s o i l , some m i c r o b e s a r e i n h i b i t e d w h i l e o t h e r s a r e s t i m u l a t e d by t h e p r e s e n c e o f 2 , 4 - D . F a c u l t a t i v e a n a e r o b e s a r e more t o l e r a n t , gram n e g a t i v e s l e s s i n h i b i t e d t h a n gram p o s i t i v e ones and 2 , 4 - D i s more t o x i c i n a c i d i c s o i l s t h a n i n a l k a l i n e s o i l s (Bovey & Young 1980 ) . T h i s might e x p l a i n the r e d u c e d numbers of b a c t e r i a a t ^ 5 pp t and 10 ppt s a l i n i t y w h e r e , i n t h e p r e s e n c e of h i g h 2 , 4 - D c o n c e n t r a t i o n s , t h e pH was l o w e r e d by 1 t o 2 u n i t s . The d i s a p p e a r a n c e of i n o r g a n i c n i t r o g e n i n the c o n t a i n e r s w i t h 1 0 " 3 M 2 , 4 - D , where no p h y t o p l a n k t o n bloom o c c u r r e d , r e m a i n s u n e x p l a i n e d . B a c t e r i a l numbers were o n l y s l i g h t l y h i g h e r t h a n i n the c o n t r o l s . H e t e r o t r o p h i c a c t i v i t y was not measured , but c o u n t s of m i c r o z o o p l a n k t o n d i d not sugges t h i g h e r g r a z i n g p r e s s u r e i n 2 , 4 - D p e r t u r b e d m i c r o c o s m s ( c f . A p p e n d i x 6 ) . The e f f e c t of 2 , 4 - D on p h y t o p l a n k t o n has been v a r i o u s l y r e p o r t e d i n t h e l i t e r a t u r e . I n l a b o r a t o r y b i o a s s a y s of n a t u r a l f r e s h w a t e r p h y t o p l a n k t o n a s semblages a s t i m u l a t i o n o f p h o t o s y n t h e s i s a t 2 mg 1 " ' 2 , 4 - D has been r e p o r t e d , w h i l e 10 mg l " 1 and > 22 mg l " 1 have been r e p o r t e d not t o k i l l 18 genera of a l g a e ( B o y l e 1980 ) . T h i s i s i n agreement w i t h my r e s u l t s o b t a i n e d i n the e s t u a r i n e m i c r o c o s m s , where 1 0 " " M 124 (22.1 mg l " 1 ) 2 , 4 - D showed no s i g n i f i c a n t a l t e r a t i o n of c h l o r o p h y l l a peak v a l u e s . Poorman (1973) r e p o r t e d g r o w t h i n h i b i t i o n of E u g l e n a q r a c i 1 i s a f t e r a 7 day e x p o s u r e t o 100 mg l " 1 2 , 4 - D . In t h e p r e s e n t e x p e r i m e n t s 10~ 3 M (221 mg l " 1 ) 2 , 4 - D p r e v e n t e d a p h y t o p l a n k t o n b loom e n t i r e l y . O b s e r v a t i o n s a f t e r the second n u t r i e n t a d d i t i o n on day 15 seem t o i n d i c a t e t h a t 2 , 4 - D c o n c e n t r a t i o n s had been s u b s t a n t i a l l y l o w e r e d due t o b i o l o g i c a l and c h e m i c a l d e c o m p o s i t i o n , t o a l l o w the g r o w t h of g reen f l a g e l l a t e s and d i a t o m s , e s p e c i a l l y T h a l a s s i o s i r a s p p . . 125 4 . SUMMARY An e x p e r i m e n t a l a p p r o a c h was used t o d e t e r m i n e the f a c t o r s , n a t u r a l or man-made, w h i c h have t h e g r e a t e s t impac t on e s t u a r i n e m i c r o p l a n k t o n e c o l o g y . In m i c r o c o s m s f i l l e d w i t h n a t u r a l w a t e r of < 5 p p t , 10 p p t , 18 p p t , and > 26 pp t s a l i n i t y , i n i t i a l n u t r i e n t a d d i t i o n r e s u l t e d i n a s i m u l a t e d p h y t o p l a n k t o n s p r i n g b l o o m . The i n f l u e n c e of h i g h o r g a n i c l o a d ( g l u c o s e ) , s h a d i n g , p o l l u t a n t s such as heavy m e t a l s and t h e h e r b i c i d e 2 , 4 - D on t h e m i c r o p l a n k t o n p o p u l a t i o n s were m o n i t o r e d . The a d d i t i o n of g l u c o s e s t i m u l a t e d m i c r o b i a l g r o w t h , and a t low i n o r g a n i c n u t r i e n t c o n c e n t r a t i o n s , b a c t e r i a competed s u c c e s s f u l l y w i t h p h y t o p l a n k t o n f o r the l i m i t i n g s u b s t r a t e , t h u s c a u s i n g a d e p r e s s i o n or t o t a l s u p p r e s s i o n of t h e s i m u l a t e d b l o o m . Low i r r a d i a n c e s d e l a y e d t h e bloom and c a u s e d a change i n the d i a t o m s p e c i e s c o m p o s i t i o n . Heavy m e t a l s , added as a m i x t u r e or Cu a l o n e , a l t e r e d p h y t o p l a n k t o n g r o w t h o n l y when t h e i r c o n c e n t r a t i o n s exceeded f i v e t o t e n t i m e s t h o s e found i n m o d e r a t e l y p o l l u t e d e s t u a r i e s . A t 10 ppt s a l i n i t y even t h e h i g h e s t t e s t c o n c e n t r a t i o n employed d i d not s i g n i f i c a n t l y r educe t h e b l o o m , because the dominant d i a t o m a t t h i s s a l i n i t y was S k e l e t o n e m a c o s t a t u m , a s p e c i e s w h i c h i s most r e s i s t a n t t o heavy m e t a l p o l l u t i o n . The h e r b i c i d e 2 , 4 - D had no s i g n i f i c a n t impac t on the p h y t o p l a n k t o n bloom a t 10 " * M c o n c e n t r a t i o n , but a t 1 0 " 3 M the g r o w t h of a l g a e was t o t a l l y s u p p r e s s e d . Such h i g h c o n c e n t r a t i o n s a r e u n l i k e l y t o o c c u r i n the e s t u a r i n e e n v i r o n m e n t because of r a p i d m i x i n g and d i l u t i o n , and 126 d e c o m p o s i t i o n o f 2 , 4 - D . The impact of n a t u r a l and man-made p e r t u r b a t i o n s c o u l d be seen as an a l t e r a t i o n of t h e s p e c i e s c o m p o s i t i o n p r i o r t o changes i n the t i m i n g and t h e magni tude of t h e s i m u l a t e d p h y t o p l a n k t o n b l o o m . The g r e a t e s t e f f e c t of p e r t u r b a t i o n s was o b t a i n e d i n t h e s e e x p e r i m e n t s by m a n i p u l a t i o n s w h i c h c o u l d be c a u s e d by n a t u r a l e v e n t s , s u c h as s h a d i n g f rom s i l t l o a d o r i n c r e a s e s i n o r g a n i c s u b s t r a t e due t o r u n - o f f . W h i l e t h e s e e v e n t s c o u l d a l s o be g e n e r a t e d by man i n an e s t u a r y , the heavy m e t a l and h e r b i c i d e p e r t u r b a t i o n , w h i c h s h o u l d be r e g a r d e d e n t i r e l y as a n t h r o p o g e n i c , c a u s e d l e s s change i n t h e p h y t o p l a n k t o n b l o o m . 127 V I . F I E L D STUDY IN THE FRASER RIVER ESTUARY 1. INTRODUCTION P r i t c h a r d (1967) d e f i n e d an e s t u a r y as a s e m i - e n c l o s e d c o a s t a l body of water w i t h a f r e e c o n n e c t i o n t o t h e open sea and w i t h i n w h i c h s e a water i s m e a s u r a b l y d i l u t e d w i t h f r e s h w a ter d e r i v e d from l a n d d r a i n a g e . F u r t h e r c l a s s i f i c a t i o n s a r e b a s e d on g e o m o r p h o l o g i c a l s t r u c t u r e s , t h e b a l a n c e between f r e s h w a t e r i n f l o w and e v a p o r a t i o n , as w e l l as e s t u a r i n e c i r c u l a t i o n p a t t e r n s . W i t h r e s p e c t t o t h e l a t t e r , t h e F r a s e r R i v e r e s t u a r y i s c h a r a c t e r i z e d by a t i d a l l y p u l s e d s a l t wedge i n t h e l o w e r r e a c h o f t h e r i v e r i t s e l f and a h i g h l y s t r a t i f i e d 'plume' t h a t e x t e n d s o v e r t h e c e n t r a l and s o u t h e r n p a r t o f t h e S t r a i t o f G e o r g i a ( L e B l o n d 1983). R e c e n t r e v i e w s on t h e p h y s i c a l o c e a n o g r a p h y of t h e a r e a a r e p r e s e n t e d by Thomson (1981) and L e B l o n d ( 1 9 8 3 ) . F r e s h w a t e r r u n o f f from t h e F r a s e r R i v e r d e t e r m i n e s t h e d i s t r i b u t i o n o f s a l i n i t y , hence d e n s i t y i n t h e e s t u a r y and t h e i r s e a s o n a l v a r i a t i o n s . M e l t w a t e r from snow and g l a c i e r s c o n s t i t u t e more t h a n t w o - t h i r d s o f t h e t o t a l r u n o f f w i t h a maximum i n l a t e May and e a r l y J u n e . R i v e r f l o w and t i d a l a m p l i t u d e d e t e r m i n e t h e p r o p a g a t i o n of t h e s a l t wedge. The i n t r u s i o n o f s a l t water r e a c h e s f u r t h e s t under low f l o w c o n d i t i o n s , b e f o r e b e i n g swept o u t a g a i n w i t h t h e ebb t i d e . From t h e r i v e r s ' s mouth, a s h a l l o w , b r a c k i s h s u r f a c e l a y e r 1 28 (plume) spreads i n t o the S t r a i t of G e o r g i a . Sur face s a l i n i t i e s vary between 0.5 ppt and 27 ppt d u r i n g the annual c y c l e . Lowest v a l u e s are found d u r i n g the f r e s h e t i n the v i c i n i t y of the mouth, w h i l e i n w i n t e r , when runof f i s s m a l l , s a l i n i t i e s i n the c e n t r a l and southern S t r a i t of G e o r g i a are u n i f o r m l y h i g h . Entra inment of n u t r i e n t - r i c h deep water and runof f seem to enhance m i c r o p l a n k t o n growth i n the e s t u a r y (Parsons et_ a l . 1970; S tockner et a l . 1980; A l b r i g h t 1983b), w h i l e the l a r g e sediment l o a d of the plume water r e s t r i c t s l i g h t p e n e t r a t i o n , thus a f f e c t i n g p h o t o s y n t h e s i s . In a recent review by H a r r i s o n et a l . (1983) the b i o l o g i c a l oceanography of the S t r a i t of G e o r g i a , i n c l u d i n g the F r a s e r R i v e r e s t u a r y , i s summarized. Freshwater runo f f causes l a r g e v a r i a t i o n s in s a l i n i t y , but the e f f e c t s on the m i c r o p l a n k t o n community have yet to be examined. In the pre sent s tudy b i o l o g i c a l l y important f a c t o r s are r e l a t e d to changes i n s a l i n i t y which r e s u l t when f r e s h and s a l t water mix i n the F r a s e r R i v e r e s t u a r y . The f i n d i n g s are compared to data from l a b o r a t o r y microcosms , i n which a s a l i n i t y range was s i m u l a t e d i n order to study i t s i n f l u e n c e on the m i c r o p l a n k t o n community under e x p e r i m e n t a l c o n d i t i o n s . 129 2 . SAMPLING IN THE FIELD S u r f a c e water samples (1 m d e p t h ) were c o l l e c t e d on a m o n t h l y b a s i s f rom J u l y 1981 t o J u l y 1982 i n t h e s o u t h e r n p a r t of t h e c e n t r a l S t r a i t o f G e o r g i a . From May t o J u l y and i n September s a m p l i n g was done t w i c e a month . F i g u r e 1 shows t h e s h i p ' s c o u r s e w i t h i t s 42 f i x e d s t a t i o n s . I t t o o k a p p r o x . 18 h t o v i s i t a l l s t a t i o n s d u r i n g one c r u i s e . The c r u i s e s were p a r t of an e x t e n s i v e S T D - s u r v e y u n d e r t a k e n by Dr W . J . Emery ( D e p t . of O c e a n o g r a p h y , U B C ) . The c r u i s e t r a c k c o v e r e d an a r e a o f f t h e F r a s e r R i v e r mouth and p r o v i d e d t h e o p p o r t u n i t y t o t a k e samples o f d i f f e r e n t s a l i n i t i e s a c c o r d i n g t o t h e s a l i n i t y v a l u e s i n the l a b o r a t o r y e x p e r i m e n t s . I t d i d n o t , h o w e v e r , a l l o w f o r t h e c o l l e c t i o n of < 5 p p t , 10 p p t , 18 p p t , and > 26 pp t s a l i n i t y w a t e r d u r i n g e v e r y month of t h e y e a r , because t h e e x t e n t and d i r e c t i o n of t h e F r a s e r R i v e r plume changes s e a s o n a l l y , as w e l l as w i t h t i d e s and w i n d . D u r i n g t h e w i n t e r months o n l y h i g h e r s a l i n i t i e s c o u l d be s a m p l e d , w h i l e i n summer, d u r i n g a p e r i o d of h i g h e s t r u n - o f f , o n l y l o w e r s a l i n i t i e s up t o 18 ppt were e n c o u n t e r e d . A t s e l e c t e d s t a t i o n s water samples were t a k e n w i t h a 5-1 N i s k i n b o t t l e . On s h i p b o a r d , t e m p e r a t u r e and s a l i n i t y of the water were measured w i t h a YSI M o d e l 33 p o r t a b l e s a l i n i t y -t e m p e r a t u r e - c o n d u c t i v i t y meter ( Y e l l o w S p r i n g s I n s t r u m e n t C o . , Y e l l o w S p r i n g s , O h i o , U S A ) . I f s a l i n i t i e s were < 5 p p t , 10 ± 1 p p t , 18 ± 1 p p t , o r > 26 ppt subsamples were t a k e n f o r c h l o r o p h y l l a , n i t r a t e and d i s s o l v e d m o n o s a c c h a r i d e 130 d e t e r m i n a t i o n , f o r t o t a l b a c t e r i a l numbers , h e t e r o t r o p h i c a c t i v i t y measurements , and p h y t o p l a n k t o n i d e n t i f i c a t i o n . U s u a l l y t r i p l i c a t e samples were c o l l e c t e d f o r each s a l i n i t y v a l u e . For l a b o r a t o r y a n a l y s i s , samples were i m m e d i a t e l y f i l t e r e d , f r o z e n a n d / o r p r e s e r v e d . Subsamples f o r measurement o f h e t e r o t r o p h i c a c t i v i t y were p r o c e s s e d on t h e s h i p . The a n a l y s e s of a l l samples were done as d e s c r i b e d i n C h a p t e r I I . 131 3. RESULTS AND DISCUSSION 3.1 I n o r g a n i c N i t r o g e n And P h y t o p l a n k t o n N i t r o g e n , phospha te and s i l i c a t e a r e e s s e n t i a l m a c r o -n u t r i e n t s f o r p h y t o p l a n k t o n , e s p e c i a l l y d i a t o m s . Among t h e ma jor i n o r g a n i c n u t r i e n t s , t h e o n l y one t o be s u p p l i e d i n abundance by t h e F r a s e r R i v e r i s s i l i c a t e . S m a l l amounts of n i t r o g e n and p h o s p h o r u s a r e p r e s e n t as w e l l ( D r i n n a n & C l a r k 1 9 8 0 ) , but t h e s e e l e m e n t s a r e found i n much l a r g e r amounts i n s eawate r w h i c h i s e n t r a i n e d i n t o t h e s u r f a c e l a y e r . D u r i n g the summer month i t i s b e l i e v e d t h a t n i t r o g e n becomes l i m i t i n g ( H a r r i s o n e t a l . 1983 ) . By m e a s u r i n g ambient n i t r a t e and n i t r i t e c o n c e n t r a t i o n s i n t h e F r a s e r R i v e r e s t u a r y one w o u l d e x p e c t c o n c e n t r a t i o n s t o i n c r e a s e w i t h i n c r e a s i n g s a l i n i t y . F i e l d d a t a i n F i g u r e 33 show t h a t t h i s was i n d e e d t r u e f o r the g r e a t e r p a r t of t h e y e a r . F o r a t l e a s t e i g h t months , f rom O c t o b e r t o May, t h e p r i m a r y s o u r c e of n i t r a t e and n i t r i t e was deep s a l i n e water r a t h e r than l a n d -d e r i v e d i n p u t . I n t h e summer, f rom June t o Sep tember , n i t r o g e n c o n c e n t r a t i o n s were low o v e r t h e e n t i r e s a l i n i t y range due t o i n c r e a s e d u p t a k e by m i c r o p l a n k t o n . Under t h e s e c o n d i t i o n s n i t r o g e n r e c y c l i n g by z o o p l a n k t o n and i n p u t f rom l a n d (sewage) becomes i n c r e a s i n g l y i m p o r t a n t i n a d d i t i o n t o e n t r a i n m e n t . S h o r t p e r i o d s of s t r o n g w i n d s can a l s o r e p l e n i s h n i t r o g e n i n t h e e u p h o t i c z o n e , r e s u l t i n g i n enhanced p h y t o p l a n k t o n g r o w t h ( T a k a h a s h i e t a l . 1977 ) . 132 35" 25 15-5-N O " + N O ; Oc t -May -| Jun-Sept 12 10-8 4-2-CHL —r-18 Jun-Sept Oc t -May £10 * 26 ppt F i g u r e 33 - C o n c e n t r a t i o n s of n i t r a t e + n i t r i t e (jxg-at 1" ' ) and c h l o r o p h y l l a {uq l " 1 ) w i t h r e s p e c t t o i n c r e a s i n g s a l i n i t i e s a t d i f f e r e n t seasons of t h e y e a r . B a r s r e p r e s e n t ± 1 S . E . o f mean. 1 33 W i t h r e s p e c t t o n i t r o g e n c o n c e n t r a t i o n s , the n u t r i e n t a d d i t i o n t o t h e l a b o r a t o r y mic rocosms s i m u l a t e d t h e range found i n t h e F r a s e r R i v e r e s t u a r y d u r i n g t h e g r e a t e r p a r t of the y e a r . W h i l e i n t h e l a b o r a t o r y e x p e r i m e n t s h i g h e s t n i t r o g e n c o n c e n t r a t i o n s , hence h i g h e s t s a l i n i t i e s , gave r i s e t o t h e l a r g e s t amount of p h y t o p l a n k t o n b i o m a s s , the c o r r e l a t i o n i n t h e f i e l d i s masked by f a c t o r s t h a t were e x c l u d e d from t h e m i c r o c o s m s . Most i m p o r t a n t among t h e s e b e i n g l i g h t l i m i t a t i o n , g r a z i n g and p a t c h i n e s s . As shown i n F i g u r e 3 3 , t h e r e seems t o be a c o r r e l a t i o n between n i t r o g e n ( i . e . s a l i n i t y ) and c h l o r o p h y l l a p igment v a l u e s , when c o n c e n t r a t i o n s f o r t h e p e r i o d O c t o b e r t o May a r e compared . The v a l u e s of b o t h n i t r o g e n and c h l o r o p h y l l a showed an i n c r e a s e w i t h s a l i n i t y . S t a t i s t i c a l l y , however , p igment c o n c e n t r a t i o n s were not s i g n i f i c a n t l y d i f f e r e n t over t h e s a l i n i t y r a n g e . In t h e p e r i o d from June t o September g r e a t e s t p h y t o p l a n k t o n b iomass c o i n c i d e d w i t h v e r y low n i t r a t e and n i t r i t e c o n c e n t r a t i o n s i n the e s t u a r y . Under t h e s e c o n d i t i o n s a l a r g e p r o p o r t i o n was p r o b a b l y " r e g e n e r a t e d p r o d u c t i o n " (Dugda le & G o e r i n g 1 9 6 7 ) . The c r u i s e i n m i d - A p r i l 1982 c o i n c i d e d w i t h t h e d i a t o m s p r i n g b loom i n t h e S t r a i t o f G e o r g i a . The s p e c i e s c o m p o s i t i o n of t h e p l a n k t o n (26 ppt s a l i n i t y ) showed t h a t i n t h e 400 c e l l s c o u n t e d , o n l y d i a t o m s were p r e s e n t and of t h e s e , 93.8% were S k e l e t o n e m a c o s t a t u m and T h a l a s s i o s i r a s p p . t o t h e r a t i o of 3 :2 r e s p e c t i v e l y . W h i l e t h e former d o m i n a t e d on a q u a n t i t a t i v e b a s i s , t h e l a t t e r c o n t r i b u t e d most t o the b iomass of t h e bloom because of i t s l a r g e r c e l l s i z e . I n a l l m i c r o c o s m e x p e r i m e n t s 134 t h e s i m u l a t e d p h y t o p l a n k t o n bloom was composed of the same two s p e c i e s a s s e m b l a g e s , but under l a b o r a t o r y c o n d i t i o n s S. c o s t a t u m d i d b e t t e r t h a n T h a l a s s i o s i r a s p p . , r e s u l t i n g i n a 3:1 r a t i o a t £ 18 ppt s a l i n i t i e s . Throughout t h e summer, S. c o s t a t u m and T h a l a s s i o s i r a s p p . m a i n t a i n e d a s t r o n g p r e s e n c e , o c c a s i o n a l l y even f o r m i n g a n o t h e r bloom ( c f . A p p e n d i x 7 ) . The a l m o s t a b s o l u t e dominance of S. c o s t a t u m a t 10 pp t s a l i n i t y as found i n t h e s i m u l a t e d m i c r o c o s m blooms was not seen i n t h e f i e l d . D u r i n g t h e n a t u r a l s p r i n g b loom i n t h e F r a s e r R i v e r e s t u a r y t h e c r u i s e t r a c k d i d not a l l o w f o r s u f f i c i e n t s a m p l i n g of t h e l o w e r s a l i n i t i e s and the summer months were c h a r a c t e r i z e d by g r e a t e r d i v e r s i t y i n d i a t o m s p e c i e s . In m i d -J u n e , 1982, s m a l l T h a l a s s i o s i r a s p p . d o m i n a t e d t h e p h y t o p l a n k t o n i n ttte l o w e s t s a l i n i t y sample (< 5 p p t ) and t h r e e weeks l a t e r t h e y a c c o u n t e d f o r about 70% r e l a t i v e abundance a t < 5 ppt and 10 ppt s a l i n i t i e s . At t h e same t i m e , S. c o s t a t u m was t h e dominant s p e c i e s a t h i g h e r s a l i n i t i e s . I n t h e p r e v i o u s y e a r (August 1981) , S. c o s t a t u m formed a summer bloom a t 18 ppt s a l i n i t y , w h i l e o v e r t h e e n t i r e s a m p l i n g p e r i o d i t s r e l a t i v e abundance a t < 5 ppt s a l i n i t y d i d not exceed 6%. T h i s f i n d i n g a g r e e d w i t h r e s u l t s o f m i c r o c o s m e x p e r i m e n t s , where a t t h e l o w e s t s a l i n i t y T h a l a s s i o s i r a s p p . were a l w a y s dominant i n t h e l a b o r a t o r y b l o o m s , w h i l e S. c o s t a t u m was f o u n d a t 10 ppt and h i g h e r s a l i n i t i e s . C h a e t o c e r o s s p p . were of m i n o r i m p o r t a n c e i n m i c r o c o s m e x p e r i m e n t s , but were b l o o m - f o r m i n g s p e c i e s i n t h e f i e l d d u r i n g t h e summer months . A p p a r e n t l y , t h e l a b o r a t o r y m a n i p u l a t i o n s r e s u l t e d i n a s e l e c t i o n of a s p r i n g bloom d i a t o m 1 35 a s s e m b l a g e , r a t h e r than c o n d i t i o n s f a v o u r a b l e t o a summer bloom s p e c i e s l i k e C h a e t o c e r o s s p p . i n t h e F r a s e r R i v e r e s t u a r y . 3 .2 D i s s o l v e d M o n o s a c c h a r i d e C o n c e n t r a t i o n s T o t a l d i s s o l v e d o r g a n i c c a r b o n (DOC) i n t h e S t r a i t o f G e o r g i a v a r i e s between 1.5 mg l " 1 i n w i n t e r and about 3 .0 mg l " 1 i n summer ( P a r s o n s 1979) . D i s s o l v e d m o n o s a c c h a r i d e s (MCHO) may c o n s t i t u t e 8-24% of t h e t o t a l DOC ( S i e b u r t h 1979) , t h u s r e p r e s e n t i n g a c o n s i d e r a b l e amount of r e a d i l y a v a i l a b l e s u b s t r a t e f o r h e t e r o t r o p h s . S o l u b l e c a r b o h y d r a t e s , measured by t h e p h e n o l s u l f u r i c a c i d method of up t o 3 mg l " 1 , have been r e p o r t e d i n t h e Nanaimo R i v e r e s t u a r y d u r i n g J u l y ( S e k i e t a l . 1969) . Throughout the f i e l d s t u d y i n t h e F r a s e r R i v e r e s t u a r y , h i g h e s t v a l u e s of d i s s o l v e d m o n o s a c c h a r i d e s r anged between 500 and 620 M g MCHO-C 1 " 1 . The p r e c i s i o n of the a n a l y s i s was ± 33 M g MCHO-C l " 1 f o r v a l u e s up t o 500 M g l " 1 . C o n c e n t r a t i o n s d i d not show a d i s t i n c t m o n t h l y maximum, but r a t h e r a c o r r e l a t i o n w i t h s a l i n i t y , i . e . d i s t a n c e from l a n d and p r i m a r y p r o d u c t i o n . As shown i n F i g u r e 34a mean c o n c e n t r a t i o n s of d i s s o l v e d MCHO-C were about d o u b l e a t low s a l i n i t i e s d u r i n g summer when compared t o v a l u e s a t 18 ppt and > 26 ppt s a l i n i t i e s . Average c o n c e n t r a t i o n s a t the h i g h e s t s a l i n i t y were comparab le t o v a l u e s found i n N a r r a g a n s e t t Bay , R . I . , d u r i n g w i n t e r e m p l o y i n g the same a n a l y t i c a l method ( Johnson & S i e b u r t h 1977) , w h i l e peak v a l u e s a t low s a l i n i t i e s i n t h e F r a s e r R i v e r e s t u a r y were s i g n i f i c a n t l y h i g h e r d u r i n g the summer. At t h e h i g h e s t s a l i n i t y (> 26 p p t ) , samples f rom a l l 136 F i g u r e 34 - Changes of d i s s o l v e d m o n o s a c c h a r i d e c o n c e n t r a t i o n s w i t h s a l i n i t y , a - mean v a l u e s of a l l c r u i s e s a m p l e s ; b - mean v a l u e s b e f o r e and d u r i n g t h e f r e s h e t ; n=number of c r u i s e s . 1 37 seasons showed l i t t l e v a r i a b i l i t y ( F i g u r e 3 4 ) , w h i l e a t low s a l i n i t i e s l o c a l h y d r o g r a p h i c and b i o l o g i c a l f a c t o r s c a u s e d c o n s i d e r a b l e v a r i a t i o n i n t h e d i s s o l v e d MCHO d a t a . In F i g u r e 34b t h e r e s u l t s of two c r u i s e s b e f o r e and a t t h e o n s e t of t h e f r e s h e t a r e compared t o d a t a found d u r i n g t h e p e r i o d of h i g h e s t r u n o f f . B e f o r e the f r e s h e t , h i g h e s t MCHO-C c o n c e n t r a t i o n s were a s s o c i a t e d w i t h l o w e s t s a l i n i t i e s c l e a r l y i n d i c a t i n g l a n d d e r i v e d i n p u t , w h i l e w i t h i n c r e a s i n g d i s t a n c e from l a n d , i . e . h i g h e r s a l i n i t i e s , c o n c e n t r a t i o n s d r o p p e d s i g n i f i c a n t l y . D u r i n g t h e f r e s h e t , MCHO-C v a l u e s were low a t l o w e s t s a l i n i t y p r o b a b l y due t o d i l u t i o n . The maximum c o n c e n t r a t i o n s a t 10 ppt and the l a r g e v a r i a b i l i t y a t 18 ppt s a l i n i t y seemed t o i n d i c a t e t h a t a t t h i s t i m e d i s s o l v e d m o n o s a c c h a r i d e s were g e n e r a t e d _in s i t u r a t h e r t h a n s u p p l i e d by r u n o f f . B e f o r e t h e o n s e t of t h e f r e s h e t , c h l o r o p h y l l a c o n c e n t r a t i o n s were l o w , w h i l e d u r i n g t h e peak f l o w p h y t o p l a n k t o n b iomass i n c r e a s e d about t e n - f o l d s u g g e s t i n g a c o r r e l a t i o n between MCHO-C and h i g h e r p r i m a r y p r o d u c t i o n and g r a z i n g a c t i v i t i e s d u r i n g t h i s p e r i o d of t h e y e a r . W h i l e the main s o u r c e s o f d i s s o l v e d m o n o s a c c h a r i d e s seemed t o v a r y w i t h the seasons as does t o t a l DOC, t h e p e r c e n t a g e of MCHO-C i n t h e c e n t r a l and s o u t h e r n S t r a i t of G e o r g i a a p p e a r e d t o be c o n s t a n t t h r o u g h o u t t h e y e a r a t a p p r o x . 14%. I n t h e l a b o r a t o r y c o n t r o l - e x p e r i m e n t s 1 mg 1~ 1 g l u c o s e was added t o a l l c o n t a i n e r s o v e r t h e e n t i r e s a l i n i t y r a n g e . W h i l e t h e t o t a l amount was about d o u b l e t h e n a t u r a l c o n c e n t r a t i o n s of d i s s o l v e d m o n o s a c c h a r i d e s a t low s a l i n i t i e s b e f o r e t h e o n s e t of 138 t h e f r e s h e t , t h e d e c r e a s e of MCHO-C w i t h i n c r e a s i n g s a l i n i t i e s was not s i m u l a t e d . The p r e s e n c e of d i f f e r e n t m i c r o b i a l p o p u l a t i o n s i n t h e low and h i g h s a l i n i t y w a t e r s and d u r i n g t h e d i f f e r e n t s e a s o n s , was r e f l e c t e d i n t h e v a r i a b l e r e s p o n s e t o the s u b s t r a t e a d d i t i o n , as seen i n t h e t i m i n g of the i n i t i a l h e t e r o t r o p h i c bloom and i n a b s o l u t e numbers a t the peak (see C h a p t e r I I I ) . W h i l e the i n i t i a l a d d i t i o n of g l u c o s e was d e p l e t e d d u r i n g the f i r s t h e t e r o t r o p h i c b l o o m , i n c r e a s e d MCHO-C c o n c e n t r a t i o n s were found d u r i n g the d e c l i n e of the a u t o t r o p h i c bloom (up t o 685 ug MCHO-C l " 1 ) . E x t r a c e l l u l a r d i s s o l v e d m o n o s a c c h a r i d e t o c h l o r o p h y l l a r a t i o s i n the mic rocosms were 17 a t < 5 p p t , 28 a t 10 p p t , 24 a t 18 p p t , and a g a i n 28 a t > 26 ppt s a l i n i t i e s . The s m a l l e r r a t i o a t the l o w e s t s a l i n i t y might r e f l e c t the p r e s e n c e of g reen a l g a e d u r i n g the p h y t o p l a n k t o n b l o o m , w h i c h have an i n t r a c e l l u l a r c a r b o h y d r a t e t o c h l o r o p h y l l a r a t i o about 2 /3 of t h a t of Ske le tonema c o s t a t u m ( r a t i o = 2 1 ) , t h e dominant s p e c i e s a t h i g h e r s a l i n i t i e s ( P a r s o n s e t a_l. 1 9 6 1 ) . In t h e f i e l d , MCHO-C c o n c e n t r a t i o n s seemed t o f o l l o w peaks i n c h l o r o p h y l l a w i t h a 2 - 4 week t i m e - l a g ( c f . F i g u r e 3 5 ) . 3 .3 B a c t e r i a l Numbers B a c t e r i a l b iomass i n e s t u a r i e s i s r e p o r t e d t o v a r y w i t h seasons and s a l i n i t y ( A l b r i g h t 1977; Palumbo & F e r g u s o n 1978; Ducklow & K i r c h m a n 1983) . D u r i n g t h e one y e a r s a m p l i n g p e r i o d i n the F r a s e r R i v e r e s t u a r y a t o t a l of 75 samples were a n a l y s e d f o r b a c t e r i a l numbers . T a b l e 6 shows s i g n i f i c a n t s e a s o n a l d i f f e r e n c e s i n b a c t e r i a l a b u n d a n c e . In the p e r i o d between l a t e 139 s p r i n g and summer b a c t e r i a l b iomass was t w o - t o f o u r - f o l d h i g h e r t h a n d u r i n g t h e r e s t of t h e y e a r . T a b l e 6 - B a c t e r i a l numbers ( X 1 0 5 m l " 1 ) a t d i f f e r e n t s ea sons of t h e y e a r (mean ± 1 S . E . ) ; n = number of c r u i s e s . S a l i n i t y 29/30 S e p t . - 8 F e b r . 13 A p r i l - 1 6 / 1 7 S e p t . < 5 pp t no samples 21 .02 ± 2 . 8 3 (n = 4) 10 ppt no samples 17.52 ± 1 . 5 5 (n = 5) 18 ppt 11 .83 ± 2 . 4 5 19.87 ± 2 . 7 2 (n = 3) (n = 6) > 26 pp t 5 .82 ± 1 . 6 9 21 .12 ± 2 . 6 5 (n = 6) (n = 6) Numbers seemed t o i n c r e a s e f o l l o w i n g t h e d i a t o m s p r i n g bloom and r e m a i n e d h i g h d u r i n g t h e summer month i n t h e p r e s e n c e of l a r g e r p h y t o p l a n k t o n b iomass ( F i g u r e 3 5 ) . T o t a l b a c t e r i a l numbers i n t h e F r a s e r R i v e r e s t u a r y v a r i e d between 1 . 8 x 1 0 s m l " 1 ( l o w e s t c o u n t ) i n December and 4 . 3 6 x 1 0 s m l " 1 ( h i g h e s t c o u n t ) i n Augus t ( c f . A p p e n d i x 2 ) . Numbers were l o w e r t h a n t h o s e found by Palumbo & F e r g u s o n (1978) i n t h e s h a l l o w marsh e s t u a r y of t h e Newport R i v e r , N . C . , but were c o m p a r a b l e t o b a c t e r i a l c o u n t s r e p o r t e d by A l b r i g h t (1983a) i n t h r e e e s t u a r i e s of t h e B . C . c o a s t . W h i l e t o t a l b a c t e r i a l numbers of 75 samples v a r i e d l i t t l e more t h a n one o r d e r of m a g n i t u d e o v e r t h e one y e a r s a m p l i n g p e r i o d , t h e v a r i a t i o n was 1 40 c o n s i d e r a b l y l o w e r t h a n i n c o u n t s g i v e n by B e l l & A l b r i g h t ( 1 9 8 1 ) . E m p l o y i n g t h e same e n u m e r a t i o n t e c h n i q u e t h e i r s t u d y r e p o r t e d numbers o f more t h a n 10 7 or even 10 8 b a c t e r i a m l " 1 . In l a b o r a t o r y e x p e r i m e n t s , a f t e r s t a n d a r d n u t r i e n t a d d i t i o n , b a c t e r i a l numbers v a r i e d i n t h e same ran g e a s i n t h e f i e l d . Somewhat h i g h e r t o t a l numbers were f o u n d f o l l o w i n g t h e a u t o t r o p h i c bloom ( h i g h e s t c o u n t 5.4x10 6 m l " 1 ) . O n l y i n p e r t u r b a t i o n e x p e r i m e n t s , a f t e r t h e a d d i t i o n of 5-15 mg l " 1 g l u c o s e , d i d b a c t e r i a l numbers i n c r e a s e t o a maximum o f a p p r o x . 2 . 4 x l 0 7 m l " 1 . In t h e F r a s e r R i v e r e s t u a r y a c o r r e l a t i o n between b a c t e r i a l numbers and s a l i n i t y was f o u n d o n l y d u r i n g f a l l and w i n t e r a t 18 p p t and > 26 p p t s a l i n i t i e s , t h e o n l y s a l i n i t y v a l u e s e n c o u n t e r e d d u r i n g t h e s e months; t o t a l b a c t e r i a l c o u n t s d e c r e a s e d w i t h h i g h e r s a l i n i t y ( T a b l e 6 ) . Palumbo & F e r g u s o n (1978) have e x p l a i n e d t h e i n v e r s e l i n e a r d i s t r i b u t i o n w i t h s a l i n i t y by c o n s e r v a t i v e m i x i n g o f b a c t e r i a . In p r e v i o u s s t u d i e s o f t h e F r a s e r R i v e r e s t u a r y , d e c r e a s i n g b a c t e r i a l numbers as m easured by e p i f l u o r e s c e n c e m i c r o s c o p y have been r e p o r t e d w i t h i n c r e a s i n g s a l i n i t i e s ( B e l l & A l b r i g h t 1981; V a l d e s & A l b r i g h t 1981; A l b r i g h t 1983a), but t h e . r e s u l t s f r o m t h e r i v e r and h i g h s a l i n i t y w a t e r s a r e s t a t i s t i c a l l y 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 a n d t h e v a r i a b i l i t y o f t h e s a m p l e s i s not i n d i c a t e d . A c c o r d i n g t o r e s u l t s i n T a b l e 6 f o r t h e s pring/summer p e r i o d , t h e main p h y t o p l a n k t o n g r o w i n g s e a s o n s , t o t a l b a c t e r i a l c o u n t s d i d not s i g n i f i c a n t l y d i f f e r o v e r t h e e n t i r e s a l i n i t y r ange of < 5 p p t t o > 26 p p t . 141 The s l i g h t d e c r e a s e i n b a c t e r i a ( T a b l e 6) from < 5 ppt t o 10 ppt s a l i n i t y d u r i n g t h e p e r i o d of g r e a t e s t r u n o f f might have been p a r t i a l l y c a u s e d by d i l u t i o n a n d / o r s e d i m e n t a t i o n . The F r a s e r R i v e r c a r r i e s a l a r g e s i l t l o a d and t h e m a j o r i t y of b a c t e r i a l c e l l s seemed t o be a t t a c h e d t o p a r t i c l e s ( B e l l & A l b r i g h t 1 9 8 1 ) . G o u l d e r (1976) found a p o s i t i v e c o r r e l a t i o n between a t t a c h e d c e l l s and the number of p a r t i c l e s i n the Humber R i v e r . A t t a c h m e n t o f b a c t e r i a may r e s u l t i n t o o low c e l l c o u n t s d u r i n g e p i f l u o r e s c e n t e n u m e r a t i o n because not a l l t h e c e l l s might be v i s i b l e , b e i n g h i d d e n u n d e r n e a t h p a r t i c l e s . Chances of u n d e r e s t i m a t i o n become p r o g r e s s i v e l y l e s s w i t h h i g h e r s a l i n i t i e s . I n c r e a s e d s e d i m e n t a t i o n of p a r t i c l e s o c c u r s when f r e s h and s a l t water meet and the s u r f a c e c h a r g e of p a r t i c u l a t e s c h a n g e s . I n t h i s p r o c e s s , p a r t i c l e s as w e l l as t h e a t t a c h e d b a c t e r i a a r e r e d u c e d i n numbers . W h i l e s e d i m e n t a t i o n c o u l d e x p l a i n the d e c r e a s e i n b a c t e r i a l numbers d u r i n g f a l l and w i n t e r and from < 5 ppt t o 10 ppt s a l i n i t y i n s p r i n g and summer, t h e subsequent i n c r e a s e i n b a c t e r i a l c e l l s a t h i g h e r s a l i n i t i e s d u r i n g the l a t t e r seasons must be due t o o t h e r f a c t o r s , such as i n c r e a s e s i n s u b s t r a t e c o n c e n t r a t i o n s r e s u l t i n g from p r i m a r y p r o d u c t i o n and p o s s i b l y h i g h e r t e m p e r a t u r e s . In l a b o r a t o r y e x p e r i m e n t s t h e second h e t e r o t r o p h i c bloom f o l l o w e d t h e d e c l i n e of t h e s i m u l a t e d p h y t o p l a n k t o n b l o o m . A c o r r e l a t i o n between the i n c r e a s e i n b a c t e r i a l numbers and the p r e s e n c e of phaeop igment s was s u g g e s t e d ( c f . F i g u r e 1 3 ) , but a r e l a t i o n s h i p between peak c h l o r o p h y l l a c o n c e n t r a t i o n s and the subsequent i n c r e a s e i n b a c t e r i a l b iomass c o u l d not be 142 e s t a b l i s h e d on a q u a n t i t a t i v e b a s i s . The p r e v a i l i n g b a c t e r i a l p o p u l a t i o n s might have changed i n the source waters d u r i n g d i f f e r e n t seasons and the d i f f e r e n t n a t u r a l p o p u l a t i o n s might have needed v a r i e d times to adapt to changing experimental c o n d i t i o n s . 3.4 H e t e r o t r o p h i c A c t i v i t y A l l measurements of r e l a t i v e h e t e r o t r o p h i c a c t i v i t y were r e l a t e d to b a c t e r i a l biomass in the sample and expressed as glucose uptake, Mg h" 1 per 10 9 b a c t e r i a . While b a c t e r i a l biomass v a r i e d roughly one order of magnitude between the spring/summer and the f a l l / w i n t e r seasons, r e l a t i v e h e t e r o t r o p h i c a c t i v i t i e s were a l s o s i g n i f i c a n t l y d i f f e r e n t over the same p e r i o d s of the year as shown in Table 7. Table 7 - R e l a t i v e h e t e r o t r o p h i c a c t i v i t i e s (Mg glucose h " 1 per 10 3 b a c t e r i a ) at d i f f e r e n t seasons of the year (mean ±1 S.E.); n = number of c r u i s e s S a l i n i t y 29/30 Sept.-8 Febr. 13 April-16/17 Sept. < 5 ppt no samples 0.26 ±0.03 (n = 4) 10 ppt no samples 0.25 ±0.06 (n = 5) 18 ppt 0.72 ±0.06 0.23 ±0.08 (n=3) (n=5). > 26 ppt 0.65 ±0.04 0.19 ±0.07 (n=6) (n=5) 1 43 R e l a t i v e h e t e r o t r o p h i c a c t i v i t y was a b o u t t h r e e - f o l d h i g h e r d u r i n g t h e f a l l and w i n t e r when b a c t e r i a were l e s s a b u n d a n t , t h a n i n t h e s p r i n g and summer. W h i l e h e t e r o t r o p h i c a c t i v i t i e s showed s i g n i f i c a n t s e a s o n a l v a r i a t i o n s , a c o r r e l a t i o n w i t h s a l i n i t y c o u l d n o t be e s t a b l i s h e d i n t h e p r e s e n c e o f s e a s o n a l and sample v a r i a t i o n . Among t h e f a c t o r s w h i c h i n f l u e n c e h e t e r o t r o p h i c a c t i v i t y and g r o w t h , n u t r i e n t s and t e m p e r a t u r e seemed t o be t h e most i m p o r t a n t ones (Hoppe 1978). A s t u d y f r o m N a r r a g a n s e t t Bay, R . I . , shows t h a t s e a s o n a l c h a n g e s i n t h e w a t e r t e m p e r a t u r e were s u f f i c i e n t t o s e l e c t t h e r m a l and t a x o n o m i c t y p e s ( S i e b u r t h 1967). The d i f f e r e n t b a c t e r i a l p o p u l a t i o n s d u r i n g t h e y e a r l y c y c l e were f o u n d t o have d i s t i n c t g r o w t h maxima a t a c e r t a i n t e m p e r a t u r e . In t h e F r a s e r R i v e r e s t u a r y , c o l d e r t e m p e r a t u r e s seem t o s e l e c t b a c t e r i a l p o p u l a t i o n s w i t h h i g h h e t e r o t r o p h i c a c t i v i t y ( c f . T a b l e 7 and F i g u r e 35) w h i l e i n summer p o p u l a t i o n s h e t e r o t r o p h i c a c t i v i t y seems t o be l o w e r . W i t h sudden c h a n g e s i n o r g a n i c s u b s t r a t e c o n c e n t r a t i o n s , y e t a n o t h e r p o p u l a t i o n m i g h t g a i n dominance ( c f . F i g u r e 3 5 ) . I t seems t h a t g l u c o s e h e t e r o t r o p h i c a c t i v i t y c h a r a c t e r i z e s d i s t i n c t b a c t e r i a l p o p u l a t i o n s , t h r i v i n g a t p a r t i c u l a r e n v i r o n m e n t a l c o n d i t i o n s . In l a b o r a t o r y e x p e r i m e n t s , i n c o n t a i n e r s w i t h a m i x t u r e o f h i g h and low s a l i n i t y w a t e r s , h i g h e r h e t e r o t r o p h i c a c t i v i t y was f o u n d a t 10 p p t and 18 p p t s a l i n i t i e s ( c f . C h a p t e r I I I , T a b l e 1 ) . Most l i k e l y , t h e m i x t u r e p r o v i d e d b e t t e r growth c o n d i t i o n s f o r a p a r t i c u l a r b a c t e r i a l p o p u l a t i o n p r e s e n t i n t h e p a r e n t w a t e r s . W i t h r e s p e c t t o h e t e r o t r o p h i c a c t i v i t y , an 144 a n a l o g y between t h e l a b o r a t o r y r e s u l t s and o b s e r v a t i o n s i n t h e e s t u a r y i s p u r e l y s p e c u l a t i v e w i t h o u t f u r t h e r i n v e s t i g a t i o n s . Based on s e v e r a l t r a n s e c t s t h r o u g h the F r a s e r R i v e r e s t u a r y , a s t i m u l a t i o n of h e t e r o t r o p h i c a c t i v i t i e s a t i n t e r m e d i a t e s a l i n i t i e s has been s u g g e s t e d by V a l d e s & A l b r i g h t (1981) and A l b r i g h t ( 1 9 8 3 a ) . These a u t h o r s e x p l a i n t h e s t i m u l a t i o n by "a more n u t r i t i o u s m i l i e u " i n t h e e s t u a r y , w h i c h may be p a r t i a l l y due t o e n t r a i n m e n t of deep s a l i n e water r i c h i n n i t r a t e and p h o s p h a t e . But i n o r g a n i c n u t r i e n t s by t h e m s e l v e s a r e of l i m i t e d s t i m u l a t i n g i n f l u e n c e on h e t e r o t r o p h i c b a c t e r i a ; o r g a n i c s u b s t r a t e i s o f g r e a t e r i m p o r t a n c e ( c f . C h a p t e r I I I & I V ; S e k i e t a l . 1969 ) . As shown i n F i g u r e 34 , d i s s o l v e d m o n o s a c c h a r i d e c o n c e n t r a t i o n s d e c r e a s e w i t h i n c r e a s i n g s a l i n i t y d u r i n g t h e ma jor p a r t of t h e y e a r . The h i g h e r h e t e r o t r o p h i c a c t i v i t i e s a t i n t e r m e d i a t e s a l i n i t i e s might be due t o v a r i o u s f a c t o r s , e . g . one p a r t i c u l a r p o p u l a t i o n of h e t e r o t r o p h s w i t h p r e f e r e n c e f o r g l u c o s e u p t a k e t h r i v i n g under the g i v e n e n v i r o n m e n t a l c o n d i t i o n s and c h a n g i n g c o n d i t i o n s might f a v o u r a d i f f e r e n t p o p u l a t i o n a t any one t i m e ( S i e b u r t h 1 9 6 7 ) . 3 .5 I n t e r a c t i o n s Of B i o t i c And A b i o t i c F a c t o r s I n The E s t u a r y F i g u r e 35 summarizes s e a s o n a l v a r i a t i o n s i n t h e m i c r o p l a n k t o n , s u b s t r a t e c o n c e n t r a t i o n s , and t e m p e r a t u r e as o b s e r v e d d u r i n g the one y e a r f i e l d s t u d y i n t h e F r a s e r R i v e r e s t u a r y . Because the t i m i n g of e v e n t s a t £ 10 pp t s a l i n i t i e s d i d not d i f f e r , the d a t a were lumped t o g e t h e r and mean v a l u e s were p l o t t e d . I n t h e p e r i o d from December t o A p r i l , o n l y > 26 ppt s a l i n i t i e s were e n c o u n t e r e d and d e p i c t e d . 145 F i g u r e 35 - S e a s o n a l c y c l e of b a c t e r i a ( X 1 0 6 m l " 1 ) , h e t e r o t r o p h i c a c t i v i t y (jig g l u c o s e h " 1 p e r 10 9 b a c t e r i a ) , c h l o r o p h y l l a (ug l " 1 ) , d i s s o l v e d m o n o s a c c h a r i d e s (mg MCHO-C l " 1 ) , n i t r a t e + n i t r i t e ( y g - a t - N l " 1 ) , and t e m p e r a t u r e ( ° C ) . 146 As t h e F r a s e r R i v e r i s l o c a t e d i n t e m p e r a t e l a t i t u d e s , s e a s o n a l c h a n g e s have, a major i n f l u e n c e on t h e m i c r o p l a n k t o n e c o l o g y . A d d i t i o n a l v a r i a t i o n i s i n t r o d u c e d by s p e c i f i c h y d r o l o g i c a l f a c t o r s , e .g. t h e f r e s h e t . T e m p e r a t u r e i n s u r f a c e w a t e r s v a r i e d between 7°C i n F e b r u a r y and 21°C i n A u g u s t . Low t e m p e r a t u r e s a r e shown t o i n h i b i t p h y t o p l a n k t o n g r o w t h i n e a r l y s p r i n g ( T a k a h a s h i e t a_l. 1973). The warming o f t h e w a t e r s and i n c r e a s i n g i r r a d i a n c e s r e s u l t e d i n a d i a t o m s p r i n g bloom i n A p r i l , w h i c h was f o l l o w e d by a h e t e r o t r o p h i c bloom i n May ( c f . A p p e n d i x 7 ) . In p l a n k t o n s a m p l e s s m a l l f l a g e l l a t e s i n c r e a s e d i n r e l a t i v e abundance t o a b o u t 80%. W i t h t h e o n s e t o f th e f r e s h e t , c h l o r o p h y l l a c o n c e n t r a t i o n s d r o p p e d f u r t h e r , b e c a u s e of d i l u t i o n a nd l i g h t l i m i t a t i o n i n t h e s i l t - l a d e n w a t e r s . R i v e r r u n o f f and h i g h e r t e m p e r a t u r e s c a u s e d s t r a t i f i c a t i o n , and a n o t h e r r i s e i n c h l o r o p h y l l a c o n c e n t r a t i o n s l e d t o n i t r a t e d e p l e t i o n a t t h e h e i g h t of t h e summer bloom. An i n c r e a s e i n i n o r g a n i c n i t r o g e n i n September r e s u l t e d i n a f a l l bloom, b e f o r e t e m p e r a t u r e s d r o p p e d t o w i n t e r v a l u e s . Between O c t o b e r and F e b r u a r y , c h l o r o p h y l l a c o n c e n t r a t i o n s h o v e r e d a r o u n d 2 uq l " 1 w h i l e t h e p l a n k t o n was d o m i n a t e d by s m a l l f l a g e l l a t e s . T e m p e r a t u r e c h a n g e s seemed t o a f f e c t b a c t e r i a l p o p u l a t i o n s as w e l l . A t l e a s t t h r e e d i f f e r e n t dominant p o p u l a t i o n s c o u l d be d i s t i n g u i s h e d . In September t h e summer p o p u l a t i o n w i t h r e l a t i v e low h e t e r o t r o p h i c a c t i v i t y was r e p l a c e d by a w i n t e r p o p u l a t i o n of low b i o m a s s b ut h i g h h e t e r o t r o p h i c a c t i v i t y . W i t h t h e warming of t h e w a t e r s i n s p r i n g , b a c t e r i a l b i o m a s s i n c r e a s e d 147 w h i l e h e t e r o t r o p h i c a c t i v i t y d e c r e a s e d , m a r k i n g t h e t a k e o v e r by a d i f f e r e n t p o p u l a t i o n . Y e t a n o t h e r b a c t e r i a l g r o u p seemed t o be a b l e t o t a k e a d v a n t a g e of i n c r e a s e s i n o r g a n i c s u b s t r a t e i n t h e p r e s e n c e o f t h e summer a u t o t r o p h bloom and h i g h t e m p e r a t u r e s . T h e r e might have been many more d i s t i n c t b a c t e r i a l p o p u l a t i o n s w h i c h c o u l d n o t be d i s t i n g u i s h e d i n t h e w i d l y - s p a c e d s a m p l i n g p a t t e r n f o l l o w e d and t h e s e t o f a n a l y s e s p e r f o r m e d . I n c r e a s e s i n b a c t e r i a l b i o m a s s seemed t o c o i n c i d e w i t h h i g h e r p r i m a r y p r o d u c t i o n i n f a l l and s p r i n g , w h i l e d u r i n g t h e summer bloom b a c t e r i a l numbers d r o p p e d , p e r p h a p s p a r t i a l l y due t o i n t e n s e g r a z i n g . The low b a c t e r i a l b i o m a s s i n w i n t e r i n t h e p r e s e n c e of l a r g e m i c r o z o o p l a n k t o n p o p u l a t i o n s m ight a l s o be e x p l a i n e d by g r a z i n g p r e s s u r e , w h i l e t h e h i g h h e t e r o t r o p h i c a c t i v i t y might* be i n d i c a t i v e o f r e l a t i v e l y h i g h p r o d u c t i o n . N i t r a t e and n i t r i t e c o n c e n t r a t i o n s showed an i n v e r s e r e l a t i o n s h i p w i t h c h l o r o p h y l l a v a l u e s , b e i n g h i g h i n t h e w i n t e r and low i n t h e summer. D i s s o l v e d m o n o s a c c h a r i d e c o n c e n t r a t i o n s seemed t o l a g b e h i n d p e a k s i n p h y t o p l a n k t o n b i o m a s s by 2-4 weeks. But t h e c o r r e l a t i o n was o b s c u r e d by l a r g e v a r i a b i l i t y and v e r y l i k e l y by t h e f a s t r e s p o n s e o f b a c t e r i a l p o p u l a t i o n s t o i n c r e a s e s i n o r g a n i c s u b s t r a t e (Brockmann e_t a l . 1979). 148 4 . SUMMARY The r e s u l t s o f a y e a r l o n g f i e l d s u r v e y i n the F r a s e r R i v e r e s t u a r y have been p r e s e n t e d . S u r f a c e w a t e r samples c o v e r i n g a s a l i n i t y range f rom < 5 pp t t o > 26 ppt were a n a l y z e d f o r n i t r a t e and n i t r i t e , d i s s o l v e d m o n o s a c c h a r i d e s , c h l o r o p h y l l a , b a c t e r i a l numbers , r e l a t i v e h e t e r o t r o p h i c a c t i v i t y and m i c r o p l a n k t o n s p e c i e s . S e a s o n a l and h y d r o g r a p h i c changes i n t h e e s t u a r y a r e shown t o a f f e c t the m i c r o p l a n k t o n e c o l o g y . Two major p e r i o d s were d i s t i n g u i s h e d , f a l l / w i n t e r and s p r i n g / s u m m e r , d u r i n g w h i c h d a t a d i f f e r e d s i g n i f i c a n t l y . Throughout t h e g r e a t e r p a r t of the y e a r , e n t r a i n m e n t of deep s a l i n e w a t e r was i d e n t i f i e d as t h e major s o u r c e of n i t r o g e n . W i t h r e s p e c t t o t h i s p r o c e s s , the l a b o r a t o r y microcosms s i m u l a t e d f i e l d c o n d i t i o n s , i n c l u d i n g t h e dominance of c e r t a i n p h y t o p l a n k t o n s p e c i e s found i n b o t h the n a t u r a l and s i m u l a t e d s p r i n g b l o o m . D i s s o l v e d m o n o s a c c h a r i d e s were s u p p l i e d e i t h e r by r u n o f f or a u t o t r o p h i c p r o d u c t i o n . When s u p p l y by r u n o f f d o m i n a t e d , c o n c e n t r a t i o n s d e c r e a s e d w i t h d i s t a n c e from l a n d , i . e . s a l i n i t y , w i t h _in s i t u p r o d u c t i o n d o m i n a n t , h i g h e s t v a l u e s were found a t i n t e r m e d i a t e s a l i n i t i e s . An a p p a r e n t i n c r e a s e i n MCHO-C f o l l o w e d peaks i n c h l o r o p h y l l a c o n c e n t r a t i o n s i n b o t h the f i e l d and i n l a b o r a t o r y m i c r o c o s m s . B a c t e r i a l numbers v a r i e d between the same range i n the f i e l d and i n t h e l a b o r a t o r y a f t e r t h e s t a n d a r d n u t r i e n t 1 49 a d d i t i o n s . I n s p r i n g and summer, numbers i n t h e f i e l d were s i g n i f i c a n t l y h i g h e r i n t h e p r e s e n c e of enhanced p r i m a r y p r o d u c t i o n and numbers i n c r e a s e d f o l l o w i n g t h e s i m u l a t e d b loom i n t h e m i c r o c o s m s . A c o r r e l a t i o n w i t h s a l i n i t y was o b s e r v e d d u r i n g t h e f a l l and the w i n t e r p e r i o d s i n t h e e s t u a r y . H e t e r o t r o p h i c a c t i v i t i e s seemed t o v a r y w i t h changes i n t h e s e a s o n a l l y dominant b a c t e r i a l p o p u l a t i o n . H i g h v a l u e s were found i n w i n t e r a s s o c i a t e d w i t h low b i o m a s s , w h i l e d u r i n g t h e summer p o p u l a t i o n s seemed t o be more d i v e r s e . H i g h e r r e l a t i v e h e t e r o t r o p h i c a c t i v i t y i n t h e f i e l d and i n t h e l a b o r a t o r y might be i n d i c a t i v e of a b a c t e r i a l p o p u l a t i o n b e s t a d a p t e d t o a p r e v a i l i n g e n v i r o n m e n t a l s i t u a t i o n . The s e a s o n a l c y c l e i n the F r a s e r R i v e r e s t u a r y has been summarized w i t h r e s p e c t t o t h e sample a n a l y s e s p e r f o r m e d . The i n t e r a c t i o n of a b i o t i c and b i o t i c f a c t o r s r e s u l t e d i n a d i s t i n c t p a t t e r n of m i c r o p l a n k t o n s u c c e s s i o n . 1 50 V I I . DISCUSSION 1 . INTRODUCTION More t h a n 15 y e a r s ago S t r i c k l a n d (1967) a d v o c a t e d c o n t r o l l e d e n v i r o n m e n t a l e c o s y s t e m e x p e r i m e n t s t o f i l l the gap between t h e " s u p e r - n a t u r a l i s t s " and t h e b e a k e r - e n v i r o n m e n t a l i s t s i n a q u a t i c e c o l o g y . W i t h new t e c h n i q u e s , such as remote s e n s i n g from s p a c e , t h e former g roup a c q u i r e d a p o w e r f u l t o o l i n s t u d y i n g l a r g e s c a l e p r o c e s s e s i n t h e o c e a n s , w h i l e t h e l a t t e r c o n t i n u e d t o r e v e a l p h y s i o l o g i c a l r e s p o n s e s of s i n g l e or m u l t i p l e s p e c i e s c u l t u r e s t o c h a n g i n g l a b o r a t o r y c o n d i t i o n s . C o n t r o l l e d e c o s y s t e m r e s e a r c h does no t r e p l a c e t h e need f o r c u l t u r e e x p e r i m e n t s , nor does i t make ocean s u r v e y s u n n e c e s s a r y ( P a r s o n s 1 9 8 1 ) . I t d o e s , h o w e v e r , a l l o w g r e a t e r s p e c i e s c o m b i n a t i o n s t o o c c u r t h a n can be o b t a i n e d under n o r m a l c u l t u r e p r o c e d u r e s ; t h u s , a c o m b i n a t i o n of a l l t h r e e a p p r o a c h e s might b e s t promote o n e ' s u n d e r s t a n d i n g of m a r i n e a q u a t i c e c o s y s t e m s . Wherever e n c l o s u r e s have been used i n o r d e r t o o b t a i n an e x a c t r e p l i c a o f n a t u r e , t h e r e s u l t s have been d i s a p p o i n t i n g ( M e n z e l & S t e e l e 1978) . The g o a l of m i c r o c o s m s t u d i e s i s not t o squeeze a n a t u r a l e c o s y s t e m i n t o c o n t a i n e r s of v a r y i n g s i z e and a t t e m p t t o s t u d y ' t h e r e a l w o r l d ' i n m i n i a t u r e ; t h i s g o a l i s n e i t h e r a t t a i n a b l e o r n e c e s s a r y ( O v i a t t e t a l . 1977 ) . M i c r o c o s m s a r e u s e f u l as b i o l o g i c a l m o d e l s ; t h e s c a l e s a r e d i f f e r e n t from t h e n a t u r a l e c o s y s t e m , but n a t u r a l e v e n t s a r e 151 more c l o s e l y s i m u l a t e d t h a n i n a s i n g l e s p e c i e s c u l t u r e ( P a r s o n s 1 9 7 8 ) . The advantage of e n c l o s u r e s i s l a r g e l y i n the s t u d y of i n t e r a c t i o n s , r a t e p r o c e s s e s o v e r t i m e , s p e c i e s a s semblages and i n t h e a c h i e v e m e n t of a mass b a l a n c e f o r d i s t r i b u t i o n of c e r t a i n e l e m e n t s ( M e n z e l & S t e e l e 1978; P a r s o n s 1 9 8 1 ) . B i o l o g i c a l as w e l l as m a t h e m a t i c a l models a l l o w m a n i p u l a t i o n s so t h a t e f f e c t s and l i m i t s o f v a r i o u s f o r c i n g f u n c t i o n s and b i o l o g i c a l c o e f f i c i e n t s may be b e t t e r u n d e r s t o o d ( P a r s o n s 1 9 7 8 ) . 152 2 . METHODOLOGY In s u m m a r i z i n g the e x p e r i m e n t a l r e s u l t s of t h e CEPEX p r o j e c t , M e n z e l (1977) c o n c l u d e d t h a t s m a l l - s c a l e l a b o r a t o r y e x p e r i m e n t s w i t h n a t u r a l p o p u l a t i o n s can be used w i t h some c o n f i d e n c e t o p r e d i c t e v e n t s a t t h e m i c r o p l a n k t o n l e v e l i n much l a r g e r s y s t e m s . The a im of t h i s t h e s i s was t o d e m o n s t r a t e t h e p o t e n t i a l of mic rocosms i n e s t u a r i n e m i c r o p l a n k t o n r e s e a r c h and t o e v a l u a t e t h e i r u s e f u l n e s s i n t e s t i n g the impac t of n a t u r a l and man-made p e r t u r b a t i o n s . 2.1 S p r i n g Bloom S i m u l a t i o n A s i m u l a t i o n of s p r i n g b loom c o n d i t i o n s was chosen f o r two r e a s o n s : f i r s t , t h e d i a t o m s p r i n g bloom marks t h e b e g i n n i n g of t h e a n n u a l g r o w i n g season and d e t e r m i n e s t h e abundance of z o o p l a n k t o n , p r i m a r i l y h e r b i v o r o u s copepods w h i c h a r e the f o o d of l a r v a l and j u v e n i l e f i s h l a t e r i n the season ( P a r s o n s 1 9 7 6 ) . Any v a r i a t i o n of the e x t e n t and t i m i n g of t h e s p r i n g b l o o m , as w e l l as any change from a d i a t o m based food c h a i n t o one based on b a c t e r i a and m i c r o z o o p l a n k t o n , might r e s u l t i n l o w e r y i e l d t o t h e f i s h e r y (Greve & P a r s o n s 1977) . S e c o n d , r e s e m b l a n c e s between t h e dynamic b e h a v i o u r of microcosms and t h a t of n a t u r a l sys tems have p r e v i o u s l y been shown i n f r e s h w a t e r l a k e m i c r o c o s m s , w h e r e , f o l l o w i n g a w e l l d e f i n e d i n i t i a t i o n p r o c e d u r e , a s p r i n g - l i k e bloom o c c u r r e d ( H a r t e e t a l . 1 9 8 0 ) . I n m i c r o c o s m e x p e r i m e n t s a sequence of n a t u r a l c o n d i t i o n s was s i m u l a t e d comparab le t o t h o s e i n the f i e l d between w i n t e r 1 53 and l a t e s p r i n g ( c f . C h a p t e r s I I I and V I ) . I n i t i a l l y , t h e m i c r o p l a n k t o n was d o m i n a t e d by s m a l l m i c r o f l a g e l l a t e s , not u n l i k e t h e w i n t e r p o p u l a t i o n s ( T a k a h a s h i & H o s k i n s 1978 ) . The n u t r i e n t a d d i t i o n c r e a t e d an e n v i r o n m e n t s i m i l a r t o t h a t p r e c e d i n g t h e n a t u r a l s p r i n g b l o o m , and l a b o r a t o r y c o n d i t i o n s as d e s c r i b e d i n C h a p t e r I I f a v o u r e d the g r o w t h o f d i a t o m s . D e s p i t e c h a n g i n g s p e c i e s c o m p o s i t i o n i n t h e i n o c u l u m , the s i m u l a t e d bloom was a l w a y s composed of S k e l e t o n e m a c o s t a t u m and T h a l a s s i o s i r a s p p . , a f i n d i n g c o n s i s t e n t w i t h the n a t u r a l d i a t o m s p r i n g b loom of t h e F r a s e r R i v e r e s t u a r y ( T a k a h a s h i e t a_l. 1973; Shim 1 9 7 6 ) . I n the l a b o r a t o r y as w e l l as i n t h e f i e l d , the d e c l i n e i n p r i m a r y p r o d u c t i o n was f o l l o w e d by an i n c r e a s e i n b a c t e r i a l numbers ( F i g u r e s 13, 35) and m i c r o z o o p l a n k t o n abundance ( C h a p t e r I I I , S e c t i o n 2 ; C h a p t e r V I ) . By c h o o s i n g o n l y one event f o r s i m u l a t i o n , the d i a t o m s p r i n g b l o o m , i t i s not p o s t u l a t e d t h a t o t h e r e v e n t s a r e l e s s i m p o r t a n t or not w o r t h w h i l e s t u d y i n g o r t h a t the e f f e c t s of t h e p e r t u r b a t i o n s d u r i n g t h e s p r i n g - l i k e bloom a r e the same as t h r o u g h o u t t h e y e a r . The l a b o r a t o r y s e t - u p and e x p e r i m e n t a l c o n d i t i o n s c h o s e n were not s u i t a b l e t o s i m u l a t e summer c o n d i t i o n s when t e m p e r a t u r e s a r e h i g h e r , s t r a t i f i c a t i o n i s p r e s e n t and a d i f f e r e n t n u t r i e n t reg ime p r e v a i l s - a l l f a c t o r s f a v o u r i n g a d i f f e r e n t m i c r o p l a n k t o n p o p u l a t i o n ( C h a p t e r V I ) . 1 54 2 .2 S i m u l a t i o n Of The S a l i n i t y Range W h i l e the s p r i n g bloom s i m u l a t i o n r e p r e s e n t s a s t u d y o v e r t i m e , t h e s a l i n i t y range d e s c r i b e s t h e s p a t i a l d i m e n s i o n i n an e s t u a r y ; i t s t a n d s f o r i n c r e a s i n g d i s t a n c e from t h e f r e s h w a t e r s o u r c e . Due t o e s t u a r i n e c i r c u l a t i o n , t i d a l and w i n d m i x i n g , s a l i n i t y and t e m p e r a t u r e a r e v e r y dynamic e n v i r o n m e n t a l p r o p e r t i e s and t h e i r r e p r e s e n t a t i o n i n s t a t i c c o n t a i n e r s and under c o n s t a n t l a b o r a t o r y c o n d i t i o n s may seem i n a d e q u a t e . S i n c e e s t u a r i n e eco sy s tems a r e " o p e n " s y s t e m s , g o v e r n e d by i m p o r t and e x p o r t , M a r g a l e f (1967) and Cooper & C o p e l a n d (1973) c o n s i d e r e d c o n t i n u o u s c u l t u r e methodo logy more a p p r o p r i a t e t h a n b a t c h t y p e c u l t u r e s because of t h e c l o s e r r e s e m b l a n c e of t h e former t o e s t u a r i n e d y n a m i c s . The d i f f e r e n c e i n a p p r o a c h i s a m a t t e r of m i c r o c o s m p h i l o s o p h y , as t o whether t h e g o a l i s a m i c r o e c o s y s t e m a l a n a t u r e , or a s t u d y - a i d t o i n v e s t i g a t e p r o c e s s e s , i n t e r a c t i o n s and s p e c i e s a s s e m b l a g e s . W h i l e even t h e most s o p h i s t i c a t e d a p p a r a t u s - s t i l l t o be d e v e l o p e d - w i l l no t g i v e an e x a c t r e p l i c a of a n a t u r a l e s t u a r y , the b a t c h c u l t u r e mic rocosms p r o v e d adequate f o r t h e p u r p o s e of t h i s s t u d y i n o r d e r t o i n v e s t i g a t e the i n f l u e n c e of s a l i n i t y and a s s o c i a t e d f a c t o r s on an e s t u a r i n e m i c r o p l a n k t o n c o m m u n i t y . Due t o e n t r a i n m e n t of n u t r i e n t - r i c h deep w a t e r not o n l y s a l i n i t y i n c r e a s e s , but i n o r g a n i c n i t r o g e n and phospha te as w e l l . Many o t h e r e n v i r o n m e n t a l f a c t o r s a r e e i t h e r p o s i t i v e l y or n e g a t i v e l y c o r r e l a t e d w i t h the i n c r e a s i n g amount of seawater m i x e d i n t o f r e s h w a t e r ( c f . C h a p t e r s I I I and V I ) . L o c a l c o n d i t i o n s d e t e r m i n e the i n d i g e n o u s p l a n k t o n community and t h e 155 s e a s o n a l p r o g r e s s i o n i s r e g u l a t e d by changes i n t h e s e c o n d i t i o n s (Smayda 1980) . E x p e r i m e n t a l and f i e l d o b s e r v a t i o n s show t h a t p h y t o p l a n k t o n s p e c i e s t o l e r a t e wide r anges of s a l i n i t i e s ( e . g . B r a a r u d 1951; M c L a c h l a n 1 9 6 1 ) , t h e r e f o r e s a l i n i t y per se i s not c o n s i d e r e d t o be a s t r o n g r e g u l a t o r y f a c t o r (Smayda 1980 ) . However , i n e s t u a r i n e e n v i r o n m e n t s , Remane & S c h l i e p e r (1971) found a s p e c i e s minimum a t 6 p p t , w i t h i n c r e a s i n g abundance of f r e s h w a t e r and m a r i n e s p e c i e s a t l o w e r and h i g h e r v a l u e s , r e s p e c t i v e l y . The m i c r o c o s m s were s p e c i a l l y s u i t e d t o s t u d y t h e e f f e c t of s a l i n i t y because o t h e r i m p o r t a n t f a c t o r s , s u c h as l i g h t and t e m p e r a t u r e , were kept c o n s t a n t . The d i f f e r e n t s a l i n i t i e s were m i x e d from two s o u r c e w a t e r s and t h e n u t r i e n t a d d i t i o n was w e l l d e f i n e d . Over t h e e x p e r i m e n t a l s a l i n i t y r a n g e , s i g n i f i c a n t d i f f e r e n c e s i n s p e c i e s c o m p o s i t i o n were found ( F i g u r e 1 0 ) . I n the l o w e s t s a l i n i t y m i c r o c o s m s , g r o w t h of S . c o s t a t u m seemed t o be i n h i b i t e d , an o b s e r v a t i o n c o n s i s t e n t w i t h t h e d i m i n i s h e d abundance o f t h i s s p e c i e s i n low s a l i n i t y f i e l d samples ( c f . A p p e n d i x 7 ) . Paasche (1975) found t h e g r o w t h r a t e of an O s l o f j o r d c l o n e of S. c o s t a t u m r e d u c e d by h a l f a t 4 ppt s a l i n i t y and o b s e r v e d no g r o w t h a t ^ 2 p p t . T h u s , s a l i n i t y might a f f e c t p h y t o p l a n k t o n s p e c i e s c o m p o s i t i o n by c o m p e t i t i v e e x c l u s i o n . The v i r t u a l d o m i n a t i o n of S. c o s t a t u m a t 10 ppt has a p a r a l l e l i n a sed iment a s semblage o b s e r v e d i n Howe Sound , B . C . ( R o e l o f s i n p r e s s ) . C o n s e q u e n t l y , w h i l e t h e s a l i n i t y range employed h e r e d i f f e r e d from t h e more dynamic v a l u e s found i n n a t u r e , t h e r e s u l t s f rom t h e f i e l d and m i c r o c o s m e x p e r i m e n t s complemented 156 each o t h e r and i n d i c a t e d t h a t s a l i n i t y might have a s e l e c t i v e i n f l u e n c e where ex t reme s a l i n i t y f l u c t u a t i o n s p r e v a i l . S i n c e d i s t i n c t p h y t o p l a n k t o n p o p u l a t i o n s d o m i n a t e d d i f f e r e n t s a l i n i t i e s , p e r t u r b a t i o n s might have a d i f f e r e n t impac t d e p e n d i n g on the s p e c i e s p r e s e n t . R e s u l t s of t h e p e r t u r b a t i o n e x p e r i m e n t s a r e d i s c u s s e d below ( S e c t i o n 4 ) . I t w o u l d be i n t e r e s t i n g t o u n d e r t a k e a t a x o n o m i c s t u d y of b a c t e r i a i n o r d e r t o e s t a b l i s h whether d i f f e r e n t p o p u l a t i o n s grow i n t h e d i f f e r e n t s a l i n i t y m i c r o c o s m s , whether t h e s e match the f i e l d p o p u l a t i o n s , how t h e zymogenous p o p u l a t i o n d i f f e r s from t h e a u t o c h t h o n o u s and whether t h e zymogenous s p e c i e s d i f f e r w i t h the s a l i n i t y v a l u e s . Such a s t u d y m i g h t u n r a v e l t h e s t i l l l a r g e l y u n e x p l a i n e d i n c r e a s e i n h e t e r o t r o p h i c a c t i v i t i e s a t i n t e r m e d i a t e s a l i n i t i e s ( A l b r i g h t 1983b) . 2 . 3 R e p l i c a t i o n And R e p r o d u c i b i l i t y By i s o l a t i n g a v i a b l e p a r t of a n a t u r a l e c o s y s t e m i n l a b o r a t o r y m i c r o c o s m s , t h e v a r i a b i l i t y and p a t c h i n e s s of n a t u r e due t o t h e p h y s i c a l d i s s i m i l a r i t y of wate r masses i s e x c l u d e d . N e v e r t h e l e s s , s t a t i s t i c a l r e p l i c a t i o n i s p r o b l e m a t i c a l i n sys tems w h i c h t e n d towards random b e h a v i o u r . However , d e s p i t e t h e d i f f i c u l t i e s of r e p l i c a b i l i t y , mic rocosms have shown t o be u s e f u l e x p e r i m e n t a l t o o l s i n e c o l o g i c a l r e s e a r c h and p o l l u t i o n s t u d i e s ( G i e s y , J r . 1980) . The s i m u l a t i o n of o n l y one n a t u r a l e v e n t , a c a r e f u l m i x i n g of h i g h and low s a l i n i t y s o u r c e w a t e r s t o enhance an even d i s t r i b u t i o n of c h e m i c a l and b i o l o g i c a l p r o p e r t i e s between 1 57 m i c r o c o s m s , and a w e l l d e f i n e d n u t r i e n t a d d i t i o n , r e s u l t e d i n good r e p l i c a t i o n of d u p l i c a t e e x p e r i m e n t s as shown i n C h a p t e r I I I . A f t e r a l l , t h e e f f e c t of p e r t u r b a t i o n s i s d i f f i c u l t t o e v a l u a t e i f t h e v a r i a n c e between r e p l i c a t e s e x c e e d s t h a t between t r e a t m e n t s . E f f e c t s of p h a s i n g between two i d e n t i c a l l y t r e a t e d microcosms were p r e s e n t , but were t h o u g h t t o be n e g l i g i b l e i n t h e s h o r t - t e r m e x p e r i m e n t s ( F i g u r e s 8 and 9 ) . T h e r e f o r e , no s t a t i s t i c a l t e s t s were p e r f o r m e d t o e l i m i n a t e t h e e f f e c t s of p h a s i n g . On a s e a s o n a l b a s i s , t h e emphas i s i n t h e m i c r o c o s m a p p r o a c h was not on s t a t i s t i c a l r e p l i c a t i o n , but r a t h e r on o b t a i n i n g r e p r o d u c i b l e r e s u l t s w i t h r e s p e c t t o the s t a n d a r d p a t t e r n . Thus t h e impact o f p e r t u r b a t i o n s c o u l d be a p p r a i s e d r e l a t i v e t o t h e s t a n d a r d p a t t e r n . 2 . 4 A n a l y t i c a l Methods The sample a n a l y s e s p e r f o r m e d were t h o u g h t t o be i m p o r t a n t w i t h r e s p e c t t o the emphas i s o f t h i s t h e s i s , but d i d not c o v e r a l l a n a l y s e s c o n s i d e r e d t o be i n t e r e s t i n g and w o r t h w h i l e s t u d y i n g , due t o l i m i t a t i o n s on the a b s o l u t e t i m e a v a i l a b l e t o a s i n g l e r e s e a r c h e r . F o r n u t r i e n t measurements and in v i t r o c h l o r o p h y l l a d e t e r m i n a t i o n , s t a n d a r d methods as d e s c r i b e d by S t r i c k l a n d & P a r s o n s (1972) were employed ( c f . C h a p t e r I I ) . The e x t r a c t e d c h l o r o p h y l l a was t a k e n as an e s t i m a t e of p h y t o p l a n k t o n b i o m a s s . W h i l e under s t a n d a r d c o n d i t i o n s and i n t h e f i e l d , p igment a n a l y s i s g i v e s a c c e p t a b l e e s t i m a t e s , i n . p e r t u r b a t i o n e x p e r i m e n t s , c h l o r o p h y l l a , as t h e o n l y measure f o r b i o m a s s , 158 might l e a d t o e r r o n e o u s r e s u l t s . T h i s became a p p a r e n t i n t h e shaded m i c r o c o s m s where the i n c r e a s e d p igment c o n c e n t r a t i o n r e f l e c t e d a p h y s i o l o g i c a l r e s p o n s e t o t h e l o w e r i r r a d i a n c e r a t h e r t h a n an i n d i c a t i o n of a h i g h e r b i o m a s s . U s i n g e p i f l u o r e s c e n c e m i c r o s c o p y as d e s c r i b e d by H o b b i e e t a l . ( 1 9 7 7 ) , t o t a l b a c t e r i a l numbers c o u l d be s u b j e c t t o u n d e r e s t i m a t i o n : b a c t e r i a might be h i d d e n u n d e r n e a t h p a r t i c l e s and v e r y s m a l l ones might be m i s s e d a l t o g e t h e r . A l t h o u g h , Zimmermann (1977) c a l c u l a t e d t h a t c a . 10% of t h e t o t a l b a c t e r i a c o u l d pas s t h r o u g h 0 .2 jum p o r e s i z e N u c l e p o r e f i l t e r s , the same f i l t e r s were used h e r e . The measurement of r e l a t i v e h e t e r o t r o p h i c a c t i v i t y by u p t a k e of s m a l l o r g a n i c m o l e c u l e s d e s c r i b e s o n l y one a s p e c t of m i c r o b i a l a c t i v i t y (Es & M e y e r - R e i l l 1982) but a l l o w s f o r a c o m p a r i s o n o f h e t e r o t r o p h i c a c t i v i t y between samples and s e a s o n s . T e s t i n g one s u b s t r a t e ( g l u c o s e ) might i n f l u e n c e t h e r e s u l t s i n f a v o u r of t h o s e b a c t e r i a p r e f e r r i n g g l u c o s e as a c a r b o n and e n e r g y s o u r c e . The method i s not s u i t a b l e f o r t h e measurement of t o t a l a c t i v i t y (Hanson 1980) and does not a l l o w f o r c o n c l u s i o n s t o be drawn c o n c e r n i n g b a c t e r i a l g r o w t h . The d i s s o l v e d m o n o s a c c h a r i d e a s s a y d e s c r i b e d by Johnson & S i e b u r t h (1977) i s v e r y s e n s i t i v e and c o n t a m i n a t i o n i n a r o u t i n e l a b o r a t o r y c a n cause c o n s i d e r a b l e p r o b l e m s . The t h r e e p a r t a n a l y s i s b e g i n s w i t h a b o r o h y d r i d e r e d u c t i o n t o c o n v e r t p e n t o s e s and hexose s t o t h e i r sugar a l c o h o l s . The t o t a l a l d i t o l s a r e t h e n o x i d i z e d w i t h p e r i o d a t e , t o form two moles of f o r m a l d e h y d e per mole of m o n o s a c c h a r i d e . The f o r m a l d e h y d e i s a n a l y s e d 159 s p e c t r o p h o t o m e t r i c a l l y w i t h 3 - m e t h y l - 2 - b e n z o t h i a z o l i n o n e h y d r a z o n e h y d r o c h l o r i d e (MBTH). The a n a l y s i s i n c l u d e s sugar a l c o h o l s but no t p o l y s a c c h a r i d e s . W i t h r e s p e c t t o t h e l a t t e r , i t i s u n i q u e among t h e t e s t s f o r c a r b o h y d r a t e s i n seawater and i t i s s u p e r i o r t o o t h e r methods i n t h a t c a l i b r a t i o n c u r v e s f o r e q u i m o l a r c o n c e n t r a t i o n s of d i f f e r e n t c a r b o h y d r a t e s a r e s i m i l a r . The v a r i a t i o n i s o n l y due t o s m a l l d i f f e r e n c e s i n m o l e c u l a r w e i g h t between p e n t o s e s and h e x o s e s . W i t h r e s p e c t t o o t h e r o r g a n i c compounds p r e s e n t i n s e a w a t e r , o n l y s e r i n e i n t e r f e r e s w i t h t h e r e a c t i o n , but a t c o n c e n t r a t i o n s f a r e x c e e d i n g t h o s e i n s eawate r ( Johnson & S i e b u r t h 1 9 7 7 ) . The e n u m e r a t i o n of s m a l l f l a g e l l a t e s was p r o b l e m a t i c a l . L i v e c o u n t s were t e d i o u s and had t o be p e r f o r m e d w i t h i n a s h o r t t i m e a f t e r s a m p l i n g . R e p l i c a t e c o u n t s showed l a r g e v a r i a b i l i t y , because c e l l s were moving a r o u n d v e r y f a s t . C o l o u r e d and c o l o u r l e s s f l a g e l l a t e s c o u l d not be d i s t i n g u i s h e d under t h e s e c i r c u m s t a n c e s . In p r e s e r v e d s a m p l e s , a b e t t e r f i x a t i o n method may reduce d e f o r m a t i o n of c e l l s and l o s s of f l a g e l l a (van der V e e r 1 9 8 2 ) . I n o r d e r t o d i s t i n g u i s h a u t o t r o p h i c and h e t e r o t r o p h i c m i c r o f l a g e l l a t e s , t h e f l u o r o c h r o m e , f l u o r e s c e i n i s o t h i o c y a n a t e (FITC) s t a i n i n g has been s u g g e s t e d as b e i n g s u p e r i o r t o a c r i d i n e orange ( S h e r r & S h e r r 1 9 8 3 ) . 1 60 3. THE MERITS OF NUMERICAL MODELING N u m e r i c a l m o d e l i n g was u s e d t o d e s c r i b e t h e p r o c e s s e s w i t h i n t h e D a r k - and L i g h t - m i c r o c o s m s w i t h r e s p e c t t o t h e g r o w th of m i c r o p l a n k t o n a s w e l l as i n t e r a c t i o n s between t h e two t r o p h i c l e v e l s , b a c t e r i a and h e t e r o t r o p h i c f l a g e l l a t e s . The models were u s e d t o t e s t t h e v a l i d i t y o f t h e measurements and t h e a s s u m p t i o n s d e r i v e d from t h e s e d a t a . I t was n e c e s s a r y t o summarize a l l knowledge a b o u t t h e m i c r o c o s m s , as w e l l as t h e u n d e r l y i n g a s s u m p t i o n s ; t h e l a t t e r had t o be c l e a r l y and q u a n t i t a t i v e l y e x p r e s s e d . T h i s p r o c e s s showed where u n d e r s t a n d i n g and d a t a were m i s s i n g . In s p i t e of t h e r a t h e r s i m p l i s t i c m o d e l i n g a t t e m p t , u s e f u l i n f o r m a t i o n was g a i n e d w i t h r e s p e c t t o t h e p r o c e s s e s w i t h i n t h e m i c r o c o s m s . 3.1 The D a r k - m o d e l The o r i g i n a l p a r a m e t e r s e t d e r i v e d f r o m e x p e r i m e n t a l o b s e r v a t i o n s , l i t e r a t u r e d a t a and F e n c h e l ' s (1982b) model, d i d n o t r e p r e s e n t t h e e v e n t s i n t h e D a r k - m i c r o c o s m s . By a d j u s t i n g t h e p a r a m e t e r s , i t became a p p a r e n t t h a t t h e m o d e l ' s b e h a v i o u r was more s e n s i t i v e t o some c o e f f i c i e n t s t h a n t o o t h e r s . R e a s o n a b l e c h a n g e s i n b a c t e r i a l g r o w t h r a t e and h a l f - s a t u r a t i o n c o n s t a n t s f o r b a c t e r i a and g l u c o s e had l i t t l e i n f l u e n c e on t h e m o d e l ' s o u t p u t , w h i l e c h a n g e s i n t h e i n i t i a l v a l u e s o f t h e s t a t e v a r i a b l e s , b a c t e r i a and m i c r o f l a g e l l a t e s , as w e l l as b a c t e r i a l e f f i c i e n c y and c e l l q u o t a were o f much g r e a t e r i m p o r t a n c e . R e d u c i n g b a c t e r i a l e f f i c i e n c y had a s i m i l a r e f f e c t t o an 161 i n c r e a s e i n t h e c e l l q u o t a , but the d e p l e t i o n of t h e i n i t i a l l y added 5 mg l " 1 g l u c o s e i n c o m b i n a t i o n w i t h e x p e r i m e n t a l l y o b s e r v e d numbers of b a c t e r i a c o u l d not be a c h i e v e d w i t h o u t an i n c r e a s e i n t h e q u o t a . The h i g h e r g l u c o s e c e l l q u o t a of b a c t e r i a was most l i k e l y a r e s p o n s e t o s u b s t r a t e e n r i c h m e n t (Mandel s tam e t a l . 1982; Es & M e y e r - R e i l l 1982) , and a l t h o u g h h i g h , r e p r e s e n t e d a v a l u e not unknown i n c o a s t a l a r e a s (Azam e t a l . 1983) . The i n c r e a s e i n g l u c o s e c e l l q u o t a r e q u i r e d changes i n o t h e r p a r a m e t e r s i n o r d e r t o d e v e l o p a b i o l o g i c a l l y r e a s o n a b l e m o d e l . W i t h o u t a d j u s t m e n t of t h e maximum c o n s u m p t i o n r a t e and g r o s s g r o w t h e f f i c i e n c y of t h e f l a g e l l a t e s , t h e i r c a r b o n i n t a k e w o u l d i n c r e a s e s i x - f o l d and w o u l d r e s u l t i n a s i x - f o l d i n c r e a s e i n t h e i r b i o m a s s , w i t h t o t a l numbers b e i n g f a r t o o l o w . F o l l o w i n g a 75% r e d u c t i o n i n c o n s u m p t i o n r a t e and a p r o p o r t i o n a l i n c r e a s e i n g r o s s g r o w t h e f f i c i e n c y , t h e f l a g e l l a t e s i n t h e D a r k - m o d e l were 1.5 t i m e s b i g g e r i n terms of t h e i r g l u c o s e q u o t a compared t o t h o s e i n F e n c h e l ' s (1982b) m o d e l , w h i l e o n l y 42% as e f f i c i e n t as g r a z e r s . F e n c h e l ' s model of p r e y / p r e d a t o r c y c l e s i s based on a f i e l d s t u d y c a r r i e d out d u r i n g summer i n s h a l l o w L i m f j o r d e n , Denmark, and on e x p e r i m e n t a l l y d e t e r m i n e d g r o w t h and c o n s u m p t i o n r a t e s of h e t e r o t r o p h i c f l a g e l l a t e s . B a c t e r i a l numbers i n t h e f i e l d f l u c t u a t e d between 1 . 5 - 3 x l 0 6 m l " 1 . As suming a c o n s t a n t b a c t e r i a l g r o w t h r a t e , h i s model showed an i n h e r e n t t e n d e n c y f o r c y c l i c a l b e h a v i o u r w h i c h c o u l d h e l p t o e x p l a i n the r e l a t i v e l y c o n s t a n t and low b a c t e r i a l c o n c e n t r a t i o n s u s u a l l y found i n t h e 162 m a r i n e e n v i r o n m e n t . However, F e n c h e l (1982b) s u g g e s t s t h a t t h e c y c l e s were most l i k e l y i n i t i a t e d by a summer peak i n p r i m a r y p r o d u c t i o n and t h a t t h e a m p l i t u d e s would t e n d t o d e c r e a s e o v e r t i m e . H i s model does not s u p p o r t s u c h s u g g e s t i o n s . On t h e c o n t r a r y , t h e a m p l i t u d e s t e n d t o i n c r e a s e o v e r t i m e , due t o t h e a s s u m p t i o n of a c o n s t a n t b a c t e r i a l g r o w t h r a t e . I f t h e c y c l e s a r e i n d e e d i n i t i a t e d by an i n c r e a s e i n o r g a n i c s u b s t r a t e , h i s model does not a c c o u n t f o r s u c h c h a n g e s . In F e n c h e l ' s model as w e l l as i n t h e D a r k - m o d e l , b a c t e r i a l g r o w t h r a t e s were assumed t o be c o n s t a n t . But t h e f o r m e r model p r e d i c t e d p r e y / p r e d a t o r c y c l e s , w h i l e t h e l a t t e r r e s u l t e d i n s i n g l e b a c t e r i a l and f l a g e l l a t e b l ooms. The d i s s i m i l a r i t y was due t o d i f f e r e n t i n i t i a l c o n d i t i o n s . W h i l e i n F e n c h e l ' s m o d el, a l l b a c t e r i a p r e s e n t were assumed t o be g r o w i n g , i n t h e D a r k -m i c r o c o s m s , o n l y a s m a l l f r a c t i o n was c o n s i d e r e d t o be a c t i v e and d i v i d i n g . The s u b s t r a t e e n r i c h m e n t d i d n o t o n l y r e s u l t i n b i g g e r i n d i v i d u a l b a c t e r i a , but p r e s u m a b l y c a u s e d c h a n g e s i n t h e b a c t e r i a l p o p u l a t i o n . F a s t e r g r o w i n g s p e c i e s were s e l e c t e d ; t h u s f l a g e l l a t e s , were no l o n g e r a b l e t o m a i n t a i n g r a z i n g c o n t r o l . The r e s u l t i n g numbers of b a c t e r i a were two t o t h r e e -f o l d h i g h e r t h a n t h o s e o c c u r r i n g i n t h e f i e l d and i n F e n c h e l ' s m o d e l . S i m i l a r l y , not a l l f l a g e l l a t e s i n i t i a l l y p r e s e n t i n t h e D a r k - m i c r o c o s m s were h e t e r o t r o p h s , i . e . b a c t e r i o v o r e s . W i t h o u t t h e s u b s t a n t i a l r e d u c t i o n i n t h e i n i t i a l v a l u e s o f b a c t e r i a and m i c r o f l a g e l l a t e s , t h e D a r k - m o d e l l e d t o p r e y / p r e d a t o r o s c i l l a t i o n s i n s t e a d of t h e o b s e r v e d l a r g e s i n g l e p e a k s . In t h e p r e s e n c e of h i g h s u b s t r a t e c o n c e n t r a t i o n s , t h e 163 g l u c o s e c e l l q u o t a d e t e r m i n e d the t i m i n g of the b a c t e r i a l bloom and g r a z i n g t h e peak numbers . The impact of g r a z i n g was r e g u l a t e d by t h e i n i t i a l number o f f l a g e l l a t e s , the maximum c o n s u m p t i o n r a t e and t h e g r o s s g rowth e f f i c i e n c y of t h e f l a g e l l a t e s . W h i l e s u b s t r a t e c o n c e n t r a t i o n s were c l e a r l y of ma jor i m p o r t a n c e , the D a r k - m o d e l p r e d i c t e d the c o n v e r s i o n of 57% of t h e b a c t e r i a l c a r b o n i n t o f l a g e l l a t e b i o m a s s . W i t h such h i g h e c o l o g i c a l e f f i c i e n c y , g r a z i n g was s i g n i f i c a n t i n t h e t r a n s f e r of e n e r g y t o h i g h e r t r o p h i c l e v e l s . No u n i v e r s a l pa rameter s e t f i t t e d a l l s a l i n i t i e s , i n d i c a t i n g t h a t the p r o c e s s e s i n t h e microcosms d i f f e r e d . But w i t h m i n o r a d j u s t m e n t s i n t h e g l u c o s e q u o t a and i n the i n i t i a l v a l u e of b a c t e r i a l and f l a g e l l a t e numbers , the D a r k - m o d e l c o u l d be a d a p t e d t o e x p e r i m e n t a l o b s e r v a t i o n s a t a l l s a l i n i t i e s , such as d i f f e r e n c e s i n t h e l a g phase o f b a c t e r i a r e s u l t i n g i n an up t o 36 h s h i f t i n the t i m i n g of t h e b a c t e r i a l b l o o m . The D a r k - m o d e l was u s e f u l i n i d e n t i f y i n g t h e i m p o r t a n t c o e f f i c i e n t s and the n a t u r e of i n t e r a c t i o n s between p a r a m e t e r s . The d y n a m i c s d e s c r i b e d r e p r e s e n t e d n e i t h e r a c l a s s i c a l p r e y / p r e d a t o r nor a t o t a l l y s u b s t r a t e - c o n t r o l l e d s y s t e m , but r a t h e r s o m e t h i n g i n b e t w e e n . The model was v a l i d a t e d by s i m u l a t i o n and c o m p a r i s o n w i t h r e s p e c t t o t h e b a c t e r i a l d y n a m i c s , w h i l e the v a l i d i t y of f l a g e l l a t e dynamics c o u l d not be e s t a b l i s h e d , due t o the l a c k o f p r o p e r d a t a . The model was i n s e c u r e i n some a s s u m p t i o n s and d i d not l e a d t o a s i n g l e s o l u t i o n ; f u r t h e r r e f i n e m e n t was t h e r e f o r e abandoned . 164 3.2 The L i g h t - m o d e l The s i m u l a t i o n o f t h e L i g h t - m i c r o c o s m s was r e s t r i c t e d t o t h e i n i t i a l g r o w t h p h a s e b e c a u s e t h e model i g n o r e d t h e r e l e a s e o f o r g a n i c s u b s t a n c e s by p h y t o p l a n k t o n , t h e e x c r e t i o n o f n i t r o g e n o u s compounds by m i c r o f l a g e l l a t e s and r e m i n e r a l i z a t i o n . The b a c t e r i a l p a r a m e t e r s a r r i v e d a t i n f i t t i n g t h e D a r k - m o d e l were a l s o f o u n d t o be a p p r o p r i a t e i n t h e s i m u l a t i o n o f t h e L i g h t - m i c r o c o s m s . U s i n g t h e same p a r a m e t e r s , t o t a l b a c t e r i a l numbers, t h e t i m i n g o f t h e f i r s t h e t e r o t r o p h i c bloom and b a c t e r i a l n i t r o g e n u p t a k e compared f a v o u r a b l y w i t h e x p e r i m e n t a l r e s u l t s , a t l e a s t f o r t h e > 10 p p t s a l i n i t i e s ( F i g u r e s 18, 19). P h y t o p l a n k t o n g r o w t h was s i m u l a t e d as a f u n c t i o n of e x t e r n a l n u t r i e n t c o n c e n t r a t i o n s , as p r e v i o u s l y done i n many o t h e r models ( e . g . S t e e l e 1974; W i n t e r e t a _ l . 1975). B e c a u s e of t h e above m e n t i o n e d a s s u m p t i o n , t h e L i g h t - m o d e l c o u l d not p r e d i c t an i n c r e a s e i n c h l o r o p h y l l a f o l l o w i n g n u t r i e n t d e p l e t i o n . O n l y more s o p h i s t i c a t e d n u t r i e n t g r o w t h m o d e l s , a l l o w i n g f o r v a r i a b l e c e l l q u o t a s and l u x u r y c o n s u m p t i o n , would be a b l e t o do s o . The L i g h t - m o d e l , however, a l l o w e d one t o b u d g e t i n o r g a n i c n i t r o g e n i n c o n t r o l - and g l u c o s e - p e r t u r b e d m i c r o c o s m s . By c o m p a r i n g the e x p e r i m e n t a l l y f o u n d r e s u l t s w i t h t h o s e p r e d i c t e d by t h e model, i t was p o s s i b l e t o q u a n t i f y t h e amount o f p h y t o p l a n k t o n b i o m a s s b a s e d on r e c y c l e d n i t r o g e n . J o h a n n e s (1968) h y p o t h e s i z e d t h a t c o n t r a r y t o t e r r e s t r i a l e c o s y s t e m s , where b a c t e r i a and f u n g i d o m i n a t e n u t r i e n t r e g e n e r a t i o n p r o c e s s e s , d i r e c t r e g e n e r a t i o n by b a c t e r i a 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 s t i t u t e s o n l y a m i n o r f r a c t i o n and under 1 65 c e r t a i n c o n d i t i o n s , b a c t e r i a and p h y t o p l a n k t o n may even compete f o r i n o r g a n i c n u t r i e n t s . The l a t t e r has been c o n f i r m e d i n many s t u d i e s ( e . g . Rhee 1972; P a r k e r e t a_l. 1975; P a r s o n s et_ a l . 1981) as w e l l as i n t h i s t h e s i s ( C h a p t e r s I V , V ) . B a c t e r i a f a c i l i t a t e n u t r i e n t r e c y c l i n g by b r e a k i n g down o r g a n i c m a t t e r and by a s s i m i l a t i n g o r g a n i c as w e l l as i n o r g a n i c s u b s t r a t e s . They a r e t h e m s e l v e s then consumed by b a c t e r i o v o r e s and n u t r i e n t s a r e r e c y c l e d by e x c r e t i o n ( e . g . ammonium and p h o s p h a t e compounds ) . Recent f i n d i n g s of a more r a p i d r e c y c l i n g of p a r t i c u l a t e m a t t e r i n n a t u r e t h a n by m i c r o b i a l a c t i v i t y a l o n e , s u p p o r t t h e c o n c e p t t h a t h e t e r o t r o p h i c f l a g e l l a t e s and m i c r o z o o p l a n k t o n p l a y a ma jor r o l e i n t h e r e m i n e r a l i z a t i o n i n t h e sea ( H a r r i s o n 1980; Mann 1982) . Azam e t a l . (1983) sugge s t a ' m i c r o b i a l l o o p ' , i n c l u d i n g m i c r o z o o p l a n k t o n , t o a c c o u n t f o r r a p i d n u t r i e n t r e c y c l i n g i n t h e e u p h o t i c z o n e . M i c r o h e t e r o t r o p h s seem t o a c c o u n t f o r t h e l a r g e s t f r a c t i o n of r e g e n e r a t e d n i t r o g e n i n many a r e a s , up t o 90% and more i n c o a s t a l r e g i o n s ( e . g . H a r r i s o n 1978 ) . The e x c r e t i o n of m i c r o z o o p l a n k t o n has not been measured i n t h e f i e l d because of t h e d i f f i c u l t y i n s e p a r a t i n g t h e m i c r o p l a n k t o n components ( H a r r i s o n 1 9 8 0 ) . The m i c r o c o s m e x p e r i m e n t s i n c o m b i n a t i o n w i t h n u m e r i c a l m o d e l i n g made i t p o s s i b l e t o i n d i r e c t l y a s s e s s t h e i m p o r t a n c e of r e c y c l i n g ( C h a p t e r I V ) . W i t h e x c l u s i o n of m a c r o z o o p l a n k t o n , the d i s c r e p a n c y between p h y t o p l a n k t o n b iomass p r o d u c t i o n as p r e d i c t e d by t h e L i g h t - m o d e l and the a c t u a l l y o b s e r v e d v a l u e i n the g l u c o s e - p e r t u r b a t i o n s can be e x p l a i n e d by m i c r o f l a g e l l a t e 166 e x c r e t i o n . Up t o 66% of t h e p h y t o p l a n k t o n b iomass f o l l o w i n g the a d d i t i o n of 5 mg 1~ 1 g l u c o s e might be a t t r i b u t e d t o " r e g e n e r a t e d p r o d u c t i o n " (Dugdale & G o e r i n g 1 9 6 7 ) , the p e r c e n t a g e b e i n g h i g h e s t i n the p r e s e n c e of low n i t r a t e and n i t r i t e c o n c e n t r a t i o n s w h i c h a g r e e s w i t h f i n d i n g s i n t h e f i e l d ( H a r r i s o n 1980) . In t h e m i c r o c o s m e x p e r i m e n t s , g r o w t h r a t e s of b a c t e r i a and d i a t o m s (Ske le tonema c o s t a t u m ) were i d e n t i c a l . A s i m i l a r o b s e r v a t i o n was made i n a s t u d y of m i c r o b i a l p l a n k t o n dynamics o f f H a w a i i ( L a n d r y e t a_l. u n p u b l . M S ) . The c o m p e t i t i v e advantage o f b a c t e r i a o v e r p h y t o p l a n k t o n seems t o be based on a f a s t e r r e s p o n s e t o e n v i r o n m e n t a l changes r a t h e r t h a n t o d i f f e r e n c e s i n g r o w t h r a t e s of the dominant p o p u l a t i o n s . The l a g phase of b a c t e r i a i n t h e m i c r o c o s m s was 1/3 of t h a t of the a l g a e . In the p r e s e n c e of a p l e n t i f u l c a r b o n s o u r c e , b a c t e r i a m i g h t have a l r e a d y d e p l e t e d a l i m i t i n g n i t r o g e n s o u r c e b e f o r e the d i a t o m s e n t e r e d t h e e x p o n e n t i a l g r o w t h p h a s e . 167 4. PERTURBATIONS AND POLLUTANTS IN MICROCOSMS I n o r d e r t o p r e s e r v e the b i o l o g i c a l i n t e g r i t y of a v a l u a b l e e s t u a r i n e e c o s y s t e m , a b e t t e r u n d e r s t a n d i n g of t h e n a t u r a l e c o l o g y , as w e l l as of the e f f e c t s of p e r t u r b a t i o n s i s n e c e s s a r y . The p u r p o s e of t h i s s t u d y was t o e v a l u a t e t h e p o s s i b l e c o n t r i b u t i o n m i c r o c o s m r e s e a r c h can make t o t h i s e n d . R e s u l t s i n C h a p t e r V i n d i c a t e t h a t p e r t u r b a t i o n s w h i c h c o u l d be c a u s e d by n a t u r a l e v e n t s or man's a c t i v i t y had much g r e a t e r impact on t h e m i c r o p l a n k t o n p o p u l a t i o n t h a n t h o s e of e x c l u s i v e l y a n t h r o p o g e n i c o r i g i n . W i t h r e s p e c t t o t h e l a t t e r , t h e m i c r o c o s m e x p e r i m e n t s r e f l e c t s h o r t - t e r m o b s e r v a t i o n s ; t o x i c e f f e c t s a t t r i b u t a b l e t o l o n g - t e r m exposure a r e not s e e n , but t h e s e might be of i m p o r t a n c e , as w e l l as t h e t r a n s f e r of p o l l u t a n t s t o h i g h e r t r o p h i c l e v e l s . The i n i t i a l n u t r i e n t e n r i c h m e n t of t h e n a t u r a l w a t e r s i n t h e m i c r o c o s m e x p e r i m e n t s - c e r t a i n l y a p e r t u r b a t i o n i n i t s e l f -enhanced the n a t u r a l n u t r i e n t l e v e l s i n a s a l t wedge e s t u a r y and f a c i l i t a t e d t h e e s t a b l i s h m e n t of a r e p r o d u c i b l e p a t t e r n not u n l i k e t h a t of a n a t u r a l d i a t o m s p r i n g b l o o m . The p e r t u r b a t i o n by n u t r i e n t e n r i c h m e n t was t h o u g h t t o be n e c e s s a r y t o s e t a ' ba se l i n e ' f o r f u r t h e r m a n i p u l a t i o n s . The s i m i l a r i t y between m i c r o c o s m and n a t u r a l e v e n t s , e . g . t h e sequence of a u t o t r o p h i c and h e t e r o t r o p h i c g r o w t h and the s p e c i e s c o m p o s i t i o n , j u s t i f i e d t h i s a p p r o a c h . 1 68 4.1 H e t e r o t r o p h i c P o p u l a t i o n s A l l p e r t u r b a t i o n s p e r f o r m e d a f f e c t e d t h e m i c r o p l a n k t o n c o m m u n i t y . G l u c o s e as w e l l as any o t h e r o r g a n i c compound, s u c h as 2 , 4 - D , were most l i k e l y the p r i m a r y r e g u l a t o r s of b a c t e r i a l a c t i v i t y , as shown by t h e two o r d e r o f magn i tude h i g h e r g l u c o s e u p t a k e f o l l o w i n g t h e 5 mg l " 1 a d d i t i o n ( T a b l e 1, C h a p t e r I I I ) compared t o f i e l d samples ( T a b l e 7 , C h a p t e r V I ) . V a c c a r o (1969) p o s t u l a t e d t h a t as a r e s u l t of o r g a n i c e n r i c h m e n t a r e d u c t i o n o f f u n c t i o n a l s p e c i e s o c c u r s , w h i l e the t o t a l b a c t e r i a l p o p u l a t i o n i n c r e a s e s . The g r e a t d i v e r s i t y i n a n a t u r a l m i c r o b i a l community e n a b l e s b a c t e r i a t o adapt t o c o n s t a n t l y c h a n g i n g n u t r i t i o n a l e n v i r o n m e n t s ( L e l o n g e t a l . 1980 ) . P r e v a i l i n g p h y s i c a l and s u b s t r a t e c o n d i t i o n s s e l e c t the most s u i t a b l e s p e c i e s a s semblage and subsequent changes i n c o n d i t i o n s d e t e r m i n e t h e s u c c e s s i o n . In t h e p r e s e n c e of h i g h o r g a n i c c o n c e n t r a t i o n s , b a c t e r i a outcompete p h y t o p l a n k t o n f o r a l i m i t i n g n u t r i e n t because t h e former o p t i m i z e the s p e c i e s c o m p o s i t i o n more r a p i d l y . G l u c o s e a d d i t i o n i n c r e a s e d b a c t e r i a l p r o d u c t i o n and r e s u l t e d i n a l a r g e r m i c r o f l a g e l l a t e p o p u l a t i o n . P a r s o n s e t a_l. ( 1 9 8 1 ) , i n r e p o r t i n g on e n r i c h m e n t s t u d i e s i n CEPEX bags , s u g g e s t e d a d i r e c t energy t r a n s f e r from b a c t e r i a t o z o o p l a n k t o n . S i z e s p e c t r u m a n a l y s i s d i d not i n d i c a t e an i n t e r m e d i a r y s t e p ; h i g h e r s e c o n d a r y and t e r t i a r y p r o d u c t i o n was o b s e r v e d . I n t h e mic rocosms h o w e v e r , i n t h e absence of n e t -z o o p l a n k t o n , g r a z i n g by m i c r o f l a g e l l a t e s was i m p o r t a n t w i t h r e s p e c t t o c a r b o n t r a n s f e r as w e l l as r e c y c l i n g of n u t r i e n t s ( C h a p t e r I V ) . A p o s s i b l e change i n the food c h a i n from an 169 a u t o t r o p h i c t o a h e t e r o t r o p h i c s y s t e m was i n d i c a t e d i n t h e l o w e s t s a l i n i t y m i c r o c o s m , where f l a g e l l a t e s a c c o u n t e d f o r a b o u t 50% o f t h e p l a n k t o n ( T a b l e 3 ) . I f , i n s t e a d of a d i r e c t e n e r g y t r a n s f e r f r o m b a c t e r i a t o z o o p l a n k t o n , m i c r o f l a g e l l a t e s r e p r e s e n t t h e l i n k between t h e t r o p h i c l e v e l s , t h e y i e l d of t h e s y s t e m i s v e r y l i k e l y r e d u c e d . L i g h t m i g h t have a s t i m u l a t o r y e f f e c t on b a c t e r i a l u p t a k e o f s u g a r s and amino a c i d s , but r e s u l t s do not seem t o be c o n c l u s i v e (Es & M e y e r - R e i l l 1982). A r e d u c t i o n i n i r r a d i a n c e a f f e c t e d b a c t e r i a i n d i r e c t l y . The a u t o t r o p h i c bloom, t h u s t h e a v a i l a b i l i t y o f o r g a n i c s u b s t r a t e s , was d e l a y e d and t h e s e c o n d h e t e r o t r o p h i c bloom a p p e a r e d t o be p o s t p o n e d . An i n c r e a s e i n b a c t e r i a l numbers f o l l o w i n g heavy m e t a l a d d i t i o n has been o b s e r v e d i n CEPEX e x p e r i m e n t s ( V a c c a r o e t a l . 1977) as w e l l as i n t h e m i c r o c o s m s . The s t i m u l a t i o n of b a c t e r i a l g r o w t h was most l i k e l y due t o t h e r e l e a s e o f o r g a n i c s u b s t r a t e by p h y t o p l a n k t o n under s t r e s s ; a view f a v o u r e d by t h e r e s u l t s o f t h e CEPEX s t u d y ( V a c c a r o e t a l . 1977). In CEPEX e x p e r i m e n t s h e t e r o t r o p h i c a c t i v i t y was i n c r e a s e d f o l l o w i n g c o p p e r a d d i t i o n s but t h e v a l u e s r e t u r n e d t o p r e -e x p o s u r e l e v e l s a f t e r a few d a y s ( M e n z e l 1977); t h u s t h e method seems t o have l i t t l e m e r i t as an i n d i c a t o r f o r p o l l u t i o n . T h e r e f o r e , measurements o f h e t e r o t r o p h i c a c t i v i t y were not p e r f o r m e d d u r i n g m e t a l - p e r t u r b a t i o n s i n t h e m i c r o c o s m s . However, b i o a s s a y e x p e r i m e n t s w i t h p r o p e r c o n t r o l s and p o l l u t a n t c o n c e n t r a t i o n s w e l l above a m b i e n t may i n d i c a t e whether t o l e r a n c e has been d e v e l o p e d and t h u s s u g g e s t i f t h e p o p u l a t i o n t e s t e d had 170 been exposed e a r l i e r t o t h a t p o l l u t a n t ( M e n z e l 1 9 7 7 ) . 4 .2 A u t o t r o p h i c P o p u l a t i o n s F o l l o w i n g g l u c o s e e n r i c h m e n t , c o m p e t i t i o n o f b a c t e r i a w i t h a l g a e f o r l i m i t i n g n i t r o g e n l e d t o a d e p r e s s i o n , even t o t a l s u p p r e s s i o n , of the p h y t o p l a n k t o n bloom ( F i g u r e s 2 3 , 2 4 ) . The impact was r e d u c e d due t o n u t r i e n t r e c y c l i n g by m i c r o z o o p l a n k t o n ( C h a p t e r I V ) . L i g h t r e d u c t i o n , whether c a u s e d by a n a t u r a l s i l t - l o a d or i n c r e a s e d p a r t i c u l a t e m a t t e r from sewage or i n d u s t r i a l e f f l u e n t s , might have a l a r g e impact on t h e e s t u a r i n e p h y t o p l a n k t o n bloom and c o n s e q u e n t l y h i g h e r t r o p h i c l e v e l s . I n shaded m i c r o c o s m s the a l g a l b loom was d e l a y e d by 2-6 d a y s . L i g h t l i m i t a t i o n r e d u c e s a l g a l g r o w t h r a t e s and a s m a l l i n o c u l u m of c e l l s m i g h t be c o n s t a n t l y f l u s h e d out of t h e e s t u a r y . A bloom may t a k e much l o n g e r t o d e v e l o p and m i g h t o c c u r e v e n t u a l l y f u r t h e r s e a w a r d . The r e s u l t i n g mi smatch of p r i m a r y and s e c o n d a r y p r o d u c t i o n w i t h l a r v a l f i s h deve lopment might have d e l e t e r i o u s i m p l i c a t i o n s f o r t h e f i s h e r y . L i g h t may a l s o p l a y a r o l e i n the d e t e r m i n a t i o n of t h e s p e c i e s c o m p o s i t i o n ( F i g u r e 2 7 ) . Lower i r r a d i a n c e s seemed t o f a v o u r s m a l l e r s p e c i e s and c e l l s , an i m p o r t a n t a s p e c t i n s i z e -s e l e c t e d f e e d i n g of the dominant z o o p l a n k t e r s . In t h e m i c r o c o s m s , w i t h heavy m e t a l s a d d e d , t h e c e n t r i c d i a t o m s T h a l a s s i o s i r a s p p . were r e p l a c e d by a p o p u l a t i o n of p e n n a t e s , and f o l l o w i n g c o p p e r a d d i t i o n s , N i t z s c h i a s p p . seemed t o be more a b u n d a n t . B o t h o b s e r v a t i o n s ag ree w i t h f i n d i n g s d u r i n g the CEPEX e x p e r i m e n t s , where t h e s e l e c t i o n of r e s i s t e n t 171 s p e c i e s r e s u l t e d i n a l a r g e r abundance o f p e n n a t e d i a t o m s , w h i c h might not be p r e f e r r e d by z o o p l a n k t o n (Thomas et_ a l . 1977). N i t z s c h i a spp. a r e known t o e x c r e t e l a r g e amounts of o r g a n i c s u b s t a n c e s (Steemann N i e l s e n & Wium-Andersen 1971) w h i c h may p l a y a r o l e i n d e t o x i f i c a t i o n . W h i l e t h e a c t u a l r e d u c t i o n i n a l g a l b i o m a s s d i d not seem s i g n i f i c a n t , c h a n g e s i n s p e c i e s d ominance m i g h t have c o n s i d e r a b l e i m p l i c a t i o n s . A l g a l s p e c i e s c o m p o s i t i o n was shown t o be a more s e n s i t i v e i n d i c a t o r o f m e t a l p e r t u r b a t i o n s t h a n c h a n g e s i n b i o m a s s ( C h a p t e r V, S e c t i o n 3 ) . C o pper a d d i t i o n s a r e known t o r e d u c e p h o t o s y n t h e s i s (Thomas e t a l . 1977), but t h e e f f e c t s w i l l depend on t h e s h o r t - t e r m p o l l u t i o n h i s t o r y o f t h e o r i g i n a l e n v i r o n m e n t w h i c h w i l l d e t e r m i n e t h e s e n s i t i v i t y of a p a r t i c u l a r a l g a l c l o n e (Murphy & B e l a s t o c k 1980). The t o x i c i t y of m e t a l s , as shown i n c u l t u r e e x p e r i m e n t s w i t h c o p p e r , i s r e l a t e d t o t h e i o n a c t i v i t y r a t h e r t h a n t h e t o t a l c o n c e n t r a t i o n . The i o n a c t i v i t y i n t u r n c a n be a l t e r e d i n d e p e n d e n t l y o f t o t a l m e t a l c o n c e n t r a t i o n s by v a r i a t i o n s i n t h e amount of c h e l a t o r and pH (Sunda & G u i l l a r d 1976). The pH i n t h e F r a s e r R i v e r v a r i e s between 7 and 8, but m i g h t be as low as 6 ( D r i n n a n & C l a r k 1980). The l o w e r t h e pH, t h e s m a l l e r t h e c o p p e r i o n a c t i v i t y and t h e e f f e c t o f t h e heavy m e t a l p o l l u t a n t . M e t a l b i n d i n g t o t h e m i c r o c o s m s u r f a c e s can a l s o a f f e c t t h e a p p a r e n t t o x i c i t y by l o w e r i n g t h e t o t a l c o n c e n t r a t i o n of t h e i o n s i n s o l u t i o n ( S t r u e m p l e r 1973). W i t h o u t knowing t h e c h e l a t o r c o n c e n t r a t i o n s and i n t h e p r e s e n c e o f s e a s o n a l l y and b i o l o g i c a l l y i n d u c e d pH c h a n g e s , t h e e v a l u a t i o n o f heavy m e t a l 1 72 p e r t u r b a t i o n s becomes d i f f i c u l t , and the impact i s b e t t e r a s s e s s e d by u s i n g i n d i c a t o r s p e c i e s ( P h i l l i p s 1977 ) . 173 5. GENERAL CONCLUSIONS The use o f m i c r o c o s m s a s e x p e r i m e n t a l t o o l s i n e s t u a r i n e r e s e a r c h a p p e a r s t o be j u s t i f i e d and i s i n k e e p i n g w i t h e a r l i e r c o n c l u s i o n s by ( M e n z e l 1977) and M a r s h a l l & M e l l i n g e r (1980) who showed t h a t s m a l l m i c r o c o s m s were a good r e p r e s e n t a t i o n o f e v e n t s i n l a r g e r mesocosms, a t l e a s t a t t h e l e v e l o f m i c r o p l a n k t o n . The e x p e r i m e n t s d e m o n s t r a t e t h a t a c a r e f u l i n i t i a t i o n method r e s u l t s i n good s t a t i s t i c a l r e p l i c a t i o n o f d u p l i c a t e m i c r o c o s m s . F u r t h e r m o r e , t h e t e c h n i q u e c an be u s e d t o g i v e r e p r o d u c i b l e r e s u l t s i n terms o f m i c r o p l a n k t o n p r o d u c t i o n o v e r a s a l i n i t y r a n g e f r o m < 5 p p t t o > 26 p p t d e s p i t e s e a s o n a l v a r i a t i o n s i n t h e p a r e n t w ater masses. The same l a b o r a t o r y c o n d i t i o n s ( c o n s t a n t l i g h t , t e m p e r a t u r e and n u t r i e n t a d d i t i o n ) p r o d u c e a c o n s t a n t r e s p o n s e w i t h r e s p e c t t o t h e m i c r o p l a n k t o n p a t t e r n t h r o u g h o u t t h e y e a r . The d i a t o m s p r i n g bloom g e n e r a t e d i n t h e l a b o r a t o r y , t h e s o - c a l l e d ' s t a n d a r d p a t t e r n 1 , r e p r e s e n t s a r e a l i s t i c s i m u l a t i o n of n a t u r a l blooms as r e c o r d e d i n t h e F r a s e r R i v e r e s t u a r y . M i c r o c o s m s seem e s p e c i a l l y s u i t e d f o r p h y s i o l o g i c a l s t u d i e s and e c o l o g i c a l r e s e a r c h , when t h e l a t t e r i s v e r i f i e d by f i e l d o b s e r v a t i o n s o r t h e f o r m e r a r e examined i n t o t a l l y c o n t r o l l e d l a b o r a t o r y c u l t u r e e x p e r i m e n t s . In c o m b i n a t i o n w i t h n u m e r i c a l m o d e l i n g , t h e v a l i d i t y o f measurements and a s s u m p t i o n s d e r i v e d f r o m t h e d a t a c a n be t e s t e d . By r e p r e s e n t i n g t h e b i o l o g i c a l p r o c e s s e s i n t h e m i c r o c o s m s i n m a t h e m a t i c a l m o d e l s , i m p o r t a n t 174 c o e f f i c i e n t s c a n be i d e n t i f i e d and by c h a n g i n g t h e c o e f f i c i e n t s , t h e i r s e n s i t i v i t y w i t h r e s p e c t t o t h e e c o s y s t e m p r o c e s s e s c a n be e v a l u a t e d . B a c t e r i a l g l u c o s e q u o t a and t h e g r o s s g r o w t h e f f i c i e n c y o f m i c r o f l a g e l l a t e s a r e shown t o have a dom i n a n t e f f e c t on t h e h e t e r o t r o p h i c d y n a m i c s i n t h e m i c r o c o s m s . The n a t u r e o f i n t e r a c t i o n s between m i c r o p l a n k t o n can be i d e n t i f i e d and i t i s p o s s i b l e t o e s t i m a t e t h e e c o l o g i c a l e f f i c i e n c y between t r o p h i c l e v e l s . M i c r o c o s m e x p e r i m e n t s , i n c o m b i n a t i o n w i t h n u m e r i c a l m o d e l i n g , make i t p o s s i b l e t o i n d i r e c t l y a s s e s s i m p o r t a n t p r o c e s s e s , e .g. n i t r o g e n r e c y c l i n g , w h i c h would be d i f f i c u l t o r i m p o s s i b l e t o measure d i r e c t l y . The e x p e r i m e n t s d e m o n s t r a t e t h a t a t e c h n i q u e c an be u s e d t o a s s e s s t h e e f f e c t s of n a t u r a l and man-made p e r t u r b a t i o n s . W h i l e p e o p l e a r e l a r g e l y c o n c e r n e d a b o u t t h e l a t t e r , n a t u r e a c t u a l l y c o n t r o l s t h e e c o s y s t e m b e h a v i o u r more t h a n man, i n s p i t e of t h e l a t t e r ' s p o l l u t a n t s . The g r e a t e s t impact o f a p e r t u r b a t i o n on t h e ' s t a n d a r d p a t t e r n ' was o b t a i n e d i n t h e s e e x p e r i m e n t s by m a n i p u l a t i o n s w h i c h c o u l d be c a u s e d by n a t u r a l e v e n t s , s u c h a s s h a d i n g from s i l t l o a d o r i n c r e a s e d o r g a n i c s u b s t r a t e due t o r u n o f f . W h i l e t h e s e e v e n t s c o u l d a l s o be g e n e r a t e d by man i n an e s t u a r y , t h e heavy m e t a l p e r t u r b a t i o n e x p e r i m e n t s , w h i c h s h o u l d be r e g a r d e d e n t i r e l y a s a s i m u l a t e d e f f e c t o f man, c a u s e d l e s s change i n t h e ' s t a n d a r d p a t t e r n ' . W h i l e n a t u r e seems t o c o n t r o l t h e e c o s y s t e m , p o l l u t a n t s may s t i l l be i m p o r t a n t i n o t h e r ways, i . e . i n c a u s i n g d i s e a s e i n man. The use o f m i c r o c o s m s i n m o n i t o r i n g s h o r t - t e r m e f f e c t s has 175 been d e m o n s t r a t e d . F o r t h e a s se s sment of l o n g - t e r m changes c a u s e d by a n t h r o p o g e n i c p o l l u t i o n o t h e r t e c h n i q u e s might be s u p e r i o r . 176 BIBLIOGRAPHY A l b r i g h t , L . J . 1977. H e t e r o t r o p h i c b a c t e r i a l dynamics i n t h e l o w e r F r a s e r R i v e r , i t s e s t u a r y and G e o r g i a S t r a i t , B r i t i s h C o l u m b i a , Canada . M a r . B i o l . 3 9 : 2 0 3 - 1 1 . A l b r i g h t , L . J . 1983a. I n f l u e n c e o f r i v e r - o c e a n plumes upon b a c t e r i o p l a n k t o n p r o d u c t i o n of the S t r a i t o f G e o r g i a , B r i t i s h C o l u m b i a . M a r . E c o l . P r o g . S e r . 1 2 : 1 0 7 - 1 3 . A l b r i g h t , L . J . 1983b. H e t e r o t r o p h i c b a c t e r i a l b i o m a s s e s , a c t i v i t i e s and p r o d u c t i v i t i e s w i t h i n t h e F r a s e r R i v e r p l u m e . C a n . J . F i s h . A q u a t . S c i . 40 ( S u p p l . 1):216-20. A n t i a , N . J . , C D . M c A l l i s t e r , T . R . P a r s o n s , K . S tephens and J . D . H . S t r i c k l a n d . 1963. F u r t h e r measurements of p r i m a r y p r o d u c t i o n u s i n g a l a r g e - v o l u m e p l a s t i c s p h e r e . L i m n o l . O c e a n o g r . 8 : 1 6 6 - 8 3 . A r m s t r o n g , F . A . J . , C . R . S t e a r n s and J . D . H . S t r i c k l a n d . 1967. The measurement of u p w e l l i n g and subsequent b i o l o g i c a l p r o c e s s e s by means of t h e T e c h n i c o n A u t o a n a l y z e r and a s s o c i a t e d e q u i p m e n t . Deep-Sea R e s . 1 4 : 3 8 1 - 8 9 . A s h t o n , F . M . and A . S . C r a f t s . 1981. Mode of A c t i o n of H e r b i c i d e s . 2nd e d . , J . W i l e y & Sons , New Y o r k 525 p . Azam, F . and R . E . Hod son . 1977. S i z e d i s t r i b u t i o n and a c t i v i t y of m a r i n e m i c r o h e t e r o t r o p h s . L i m n o l . O c e a n o g r . 2 2 : 4 9 2 -501 . Azam, F . f T . F e n c h e l , J . G . F i e l d , J . S . G r a y , L . A . M e y e r - R e i l and F . T h i n g s t a d . 1983. The e c o l o g i c a l r o l e of w a t e r - c o l u m n m i c r o b e s i n t h e s e a . M a r . E c o l . P r o g . S e r . 1 0 : 2 5 7 - 6 3 . B e a r d a l l , J . and I . M o r r i s . 1976. The c o n c e p t s of l i g h t i n t e n s i t y a d a p t a t i o n i n m a r i n e p h y t o p l a n k t o n : some e x p e r i m e n t s w i t h P h a e o d a c t y l u m t r i c o r n u t u m . M a r . B i o l . 3 7 : 3 7 7 - 8 7 . B e l l , C . R . and L . J . A l b r i g h t . 1981. 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B a c t e r i o l . 4 6 : 3 9 - 5 9 . 193 LIST OF APPENDICES Appendix 1: pH - Measurementes 1 Appendix 2: FORTRAN-programmes of Models 3 Appendix 3: Shading Experiments 5 Appendix 4: Metal-perturbations with EDTA 6 Appendix 5: Metal-perturbations without EDTA 8 Appendix 6: 2,4-D Perturbations 12 Appendix 7: Cruise Data 16 APPENDIX 1 pH - Measurements i n Control-experiments and 2,4-D Perturbations Day C* c * * 4 5 ppt s a l i n i t y IO"* M IQ-3 M 2,4-D C* c * * 10 ppt s a l i n i t y 10" 4 M -3 10 1 2 6.39 7.65 7.71 7.73 6.88 7.73 7.73 7.75 3 6.59 6.91 5 6.78 7.49 7.52 5.69 6.69 7.53 7.31 6.70 7 6.70 6.72 7.00 6.58 7.00 7.00 7.59 6.55 9 7.00 7.16 7.39 6.42 7.20 7.75 7.68 6.40 11 6.73 6.96 7.52 6.59 6.84 7.31 7.25 6.62 13 6.89 7.02 7.69 6.91 6.99 7.41 7.45 6.81 15 6.78 7.06 7.39 6.69 6.90 7.20 7.40 6.70 17 6.70 6.99 7.42 6.73 6.90 7.29 7.45 6.75 19 6.75 7.05 7.51 6.91 6.95 7.38 7.40 6.88 21 6.92 7.15 6.56 7.18 7.14 6.65 23 6.00 6.41 25 6.22 6.81 c * c * * Control-experiment (12.11.82-30.11.82) Control-experiment concurrent with 2,4-D perturbations pH - Measurements i n Control-experiments and 2,4-D Perturbations Day 18 ppt s a l i n i t y Z26 ppt s a l i n i t y C * c * * 10" 4 M 10" 3 M 2,4-D C* C * * l O " 4 M 1 0 - 3 1 2 6.96 7.79 7.76 7.76 7.01 7.72 7.72 7.72 3 7.00 7.18 5 7.10 7.34 7.60 7.31 7.32 7.60 7.66 7.30 7 7.25 8.21 8.29 7.15 7.95 8.41 8.16 7.20 9 8.00 7.61 8.20 7.25 8.12 8.19 7.91 7.20 11 7.90 7.37 8.28 7.40 8.00 7.95 7.71 7.39 13 7.98 6.71 7.78 7.10 7.82 7.55 7.57 7.25 15 7.60 7.55 7.97 7.49 7.55 7.81 7.77 7.58 17 7.35 7.63 7.76 7.49 7.35 7.72 7.69 7.87 19 7.15 7.53 7.84 7.58 7.25 7.70 7.79 8.05 21 7.22 7.40 7.15 7.39 7.47 7.81 23 6.71 7.00 25 6.85 7.00 C* » Control-experiments (12.11.82-30.11.82) C**= Control-experiment concurrent with 2,4-D perturbations APPENDIX 2 C Program to Model Bacteria and Flagellates (Dark-model) C I n i t i a l i z e READ(5,20) X.Y.G 20 FORMAT(10F10.4) REA0(5.20) RM.GK.UM.XK.EY.D.Q.EX READ(5.20) TIME,DT NSTP=TIME/DT+.1 NS12»12/DT+. 1 T = 24 WRITE(6,10) 10 FORMAT(1X.4X,'RM',8X.'GK',8X.'UM'. 8X,'XK'.8X,'EV,9X,'D',9X,'0',8X,'EX') WRITE(S,40) RM.GK.UM.XK.EY.D.Q.EX WRITE(6,40) TIME,DT 40 FORMAT(1X.8F10.3) WRITE(6.30) T.X.Y.G DO 500 I=1,NSTP R=RM*G/(GK+G) U=UM*X/(XK+X) DX=R*X-Y*U DY=Y*EY*U-D*Y DG=-R*X*0/EX X=X+DX*DT Y=Y+OY*DT G=G+DG*DT T»I*0T+24 J-I/NS12 J=I-d*NS12 IF(d.NE.O) GO TO 500 WRITE(6,30) T.X.Y.G 30 F0RMAT(4F15.3) 50O CONTINUE STOP END VO C PROGRAM TO MODEL LIGHT MICROCOSMS C INITIALIZE REAL N.NK READ(5.20) X.Y.G,P.N 20 FORMAT(10F10.4) READ(5,20) RM.GK.UM.XK.EY.D.O.EX.SM.NK READ(5,20) OC.OP.ON READ(5.20) TIME,DT NSTP=TIME/DT+.1 NS12=12/DT+.1 T = 24 WRITE(6.10) 10 FORMAT(1X.4X.'RM',6X,'GK',GX,'UM',6X,'XK',6X,'EY',6X,'D',6X, '0'.GX, 'EX',6X. 'SM',6X.'NK',6X,'OC,6X.'OP',6X,'ON') WRITE(6,40) RM,GK,UM,XK,EY,D.0.EX.SM.NK,OC,OP.ON WRITE(6,40) TIME,0T 40 FORMAT(1X.13F8.3) WRITE(6,30) T,X.Y.G.P.N DO 5CO 1 = 1 ,NSTP R=RM*G/(GK+G) U=UM*X/(XK+X) S=SM*N/(NK+N) DX=R*X-Y*U DY=Y*EY*U-D*Y 0G=-R*X*Q/EX DP=S*P DN=-S*P*OP/OC-R*X*ON X=X+DX*DT Y=Y+DY*DT G=G+DG*DT P=P+DP*DT N=N+DN*DT T=I*DT+24 J=I/NS12 J=I-J*NS12 IF (<J . NE .O) GO TO 500 WRITE(6,30) T.X.Y.G.P.N 30 F0RMAT(6F15.3) 500 CONTINUE STOP END VO — J 198 APPENDIX 3 A - B a c t e r i a l numbers ( x l 0 6 m l - 1 ) i n Control- and 10%-light-experiments B - Microzooplankton counts (log numbers ml -^) i n Control- and 10%-light-experiments 2.5 ppt 10 ppt 18 ppt 26 ppt Salinity Day C S C S C S C S 2 A 1.31 1.42 1.16 1.11 0.65 0.82 0.48 0.58 3 3.18 3.20 4.24 4.64 4.04 4.49 1.10 2.90 5 0.89 0.89 0.22 0.36 0.21 0.42 2.40 1.01 7 0.96 0.75 0.20 0.34 0.28 0.25 0.54 0.39 9 0.91 0.94 1.21 0.87 1.27 1.46 4.02 1.68 11 2.21 1.10 1.64 1.71 1.13 2.12 2.94 3.06 13 1.60 1.65 0.99 2.31 2.73 2.29 0.90 0.81 15 1.93 1.38 1.52 0.76 1.18 1.12 0.80 0.88 17 1.98 1.48 1.14 0.88 0.54 0.65 0.69 1.23 19 1.53 1.37 1.79 1.41 0.86 0.62 1.07 1.38 21 1.07 1.24 1.91 1.19 0.40 0.76 1.45 1.50 2 B 4.97 4.99 4.94 4.65 4.57 4.54 3.94 4.00 3 5.18 4.99 4.86 4.76 4.05 4.21 4.21 4.05 5 5.03 4.81 4.14 4.24 4.44 4.48 4.24 4.35 7 4.78 4.81 3.94 4.14 4.03 3.40 4.60 3.10 9 4.60 4.81 4.00 3.88 4.24 3.49 4.27 4.21 11 4.21 4.30 3.94 3.88 3.88 4.00 4.21 4.44 15 4.51 4.27 4.00 4.00 3.70 3.88 4.00 3.94 17 4.14 4.60 4.00 3.94 3.40 3.40 3.49 3.94 19 4.05 4.63 3.40 3.70 3.40 3.57 3.27 3.40 21 4.48 4.48 4.10 4.18 3.40 3.88 3.49 3.57 C = Control (225 juEinst m s ) S = 10%-light (22.5 uEinst m~2 s" 1) 1 APPENDIX 4 A - B a c t e r i a l numbers (xlO^ml""*) i n Control- and 5xME-perturbation-experiments i n presence of EDTA-Fe. B - Chlorophyll a^  (ug l - 1 ) i n Control- and 5xME-perturbation-experi-ments i n presence of EDTA-Fe. 2.5 ppt 10 ppt 18 ppt 26 ppt Salinity Day C M C M C M C M 2 A 1.31 1.15 1.16 1.12 0.65 0.62 0.48 0.48 3 3.18 3.11 4.24 4.04 4.04 3.65 1.10 0.86 5 0.89 1.39 0.22 0.28 0.21 0.67 2.40 2.65 7 0.96 0.63 0.20 0.24 0.28 0.33 0.54 0.30 9 0.91 0.79 1.21 1.00 1.27 0.96 4.02 0.97 11 2.21 1.69 1.64 2.08 1.13 1.71 2.94 3.24 13 1.60 1.44 0.99 1.07 2.73 2.12 0.90 1.97 15 1.93 1.75 1.52 0.84 1.18 1.96 0.80 1.43 17 1.98 1.54 1.14 0.90 0.54 0.85 0.69 0.76 19 1.53 1.70 1.79 1.05 0.86 0.47 1.07 0.67 21 1.07 1.36 1.91 0.84 0.40 0.17 1.45 1.06 2 B 5.73 5.38 1.71 5.19 6.03 2.27 1.71 1.94 3 2.73 0.41 2.22 3.29 3.94 0.0 0.64 3.71 5 2.45 0.0 0.0 3.66 4.03 0.92 3.52 5.15 7 6.12 3.66 18.97 16.14 33.52 57.18 76.90 46.51 9 16.87 13.16 40.19 41.35 54.40 16.99 42.16 50.05 11 17.80 16.70 21.05 23.83 37.75 40.36 30.97 23.89 13 9.86 6.38 3.48 5.22 13.92 19.14 13.92 18.56 15 7.54 5.80 5.22 6.38 11.02 11.60 7.54 10.44 17 6.96 10.44 3.48 5.80 11.02 11.02 5.80 5.80 19 2.90 4.06 2.32 2.90 5.22 4.06 0.58 3.48 21 3.48 2.32 1.16 1.74 5.22 6.96 1.16 4.06 C = Control M = 5xMulti-element-mix + EDTA-Fe 200 Microzooplankton counts (log numbers ml ) i n Control- and 5xME-perturbation-experiments i n presence of EDTA-Fe 2.5 ppt 10 ppt 18 ppt 26 ppt Salinit; Day C M C M C H C M 2 4.97 5.03 4.94 4.92 4.57 4.51 3.94 4.00 3 5.18 5.18 4.86 4.83 4.05 3.80 4.21 4.10 5 5.03 5.15 4.14 4.00 4.44 4.60 4.24 3.94 7 A.78 4.61 3.94 4.24 4.03 3.64 4.60 4.81 9 4.60 4.74 4.00 4.18 4.24 4.10 4.27 4.33 11 4.21 4.70 3.94 3.94 3.8B 3.57 4.21 4.18 15 4.51 4.44 4.00 4.18 3.70 4.14 4.00 3.70 17 4.14 4.60 4.00 3.88 3.40 3.94 3.49 3.64 19 4.05 4.40 3.40 3.88 3.40 4.00 3.27 3.88 21 4.48 4.35 4.10 4.10 3.40 3.57 3.49 3.10 C = Control M = 5xMulti-element-mix + EDTA-Fe APPENDIX 5 Bacterial Numbers in Control- and Metal-Perturbation-experiments (xlfj6 ml - 1) 2.7 ppt 10 ppt 18 ppt 26 ppt Salinity Day C Cu M* C Cu M** C Cu M* C Cu M** 2 1.39 1.75 1.74 1.28 1.35 1.29 1.26 1.67 1.12 0.61 0.62 0.62 3 2.49 2.20 1.72 2.08 2.45 1.50 1.98 2.04 1.68 1.15 1.25 0.88 5 3.56 4.91 5.03 1.03 1.94 4.42 3.72 5.44 4.06 4.13 10.74 5.83 7 0.66 0.91 1.05 0.30 0.84 0.31 1.18 1.52 1.17 1.62 1.64 3.33 9 0.29 0.42 0.66 0.42 0.64 0.45 1.75 1.92 1.97 2.64 2.61 2.94 11 0.45 0.44 0.68 1.11 0.59 1.37 1.83 1.76 1.52 1.65 2.43 0.84 13 0.74 0.44 0.70 2.33 0.64 1.28 2.79 1.73 1.48 3.97 2.39 0.58 15 0.60 0.60 0.46 2.38 2.17 0.81 1.84 1.00 1.68 3.99 2.47 0.35 In a l l microcosms EDTA-Fe was replaced by equimolar solution of FeCl C • Control Cu » 30 ug Cu l - 1 M* • 5xMulti-element-mix M**»10xMulti-element-mix Species composition i n Control-experiments and a f t e r Metal-perturbation (5xME) at 5 5 ppt s a l i n i t y . EDTA-iron was replaced by F e C ^ . Numbers represent r e l a t i v e abundance (%) at the height of the phytoplankton bloom. cont r o l 5xME Small f l a g e l l a t e s 19.0 44.0 T h a l a s s i o s i r a spp. 70.0 21.0 Thalassionema spp. 6.0 30.0 S. costatum 3.0 Freshwater species <1.0 Pennate diatoms 1.0 4.0 Species composition in Control-experiments and after copper addition (30ug . Numbers represent relative abundance (Z); FeCl, instead of EDTA-iron addition. Salinity 2.7 ppt CONTROL Small flagellates Thalassiosira spp. Thalassionema spp. S_. costatum  Witzschia spp. COPPER Small Flagellates Thalassiosira spp. Thalassionema spp. S_. costatum  Nltzschia spp. Salinity 10 ppt CONTROL Small flagellates Thalassiosira spp. Thalassionema spp. S. costatum COPPER Small flagellates Thalassiosira epp. Thalassionema spp. S. costatum Day 2 Day 5 Day 7 Day 9 Day 11 87.0 92.0 19.0 15.0 15.0 3.0 5.0 70.0 80.0 74.0 2.0 1.0 6.0 3.5 8.5 6.0 *1.0 3.0 1.0 1.0 1.0 1.0 2.0 87.5 63.0 85.5 40.0 13.0 9.5 24.0 5.0 51.0 61.0 1.0 10.5 5.0 5.0 16.0 <1.0 '-I.O 1.0 <1.0 2.0 3.5 77.0 25.0 7.5 10.0 7.0 2.0 1.0 1.0 2.0 11.5 72.0 91.5 89.0 80.5 26.0 6.0 19.0 7.0 1.0 1.0 3.0 8.5 73.5 94.0 80.5 Species composition in Control-experiments and after copper addition (30ug l - 1 ) . Numbers represent relative abundance (Z); FeCl, instead of EDTA-iron addition. Salinity 18 ppt Day 2 Day 5 Day 7 Day 9 Day 11 Small flagellates 68.0 23.5 8.0 8.0 5.0 Thalassiosira spp. 6.5 19.0 9.0 17.0 11.0 Thalassionema spp. 2.5 S. costatum 20.0 54.5 82.0 71.5 78.5 Nitzschia spp. 1.5 1.5 1.0 2.0 4.0 COPPER small flagellates 62.5 46.5 11.0 17.0 Thalassiosira spp. 10.0 5.0 11.0 6.0 Thalassionema spp. 4.0 S. costatum 18.0 47.0 76.5 71.0 Nitzschia spp. 2.0 2.0 1.0 5.5 Salinity 26 ppt CONTROL Small flagellates 68.0 20.5 20.5 15.0 18.5 Thalassiosira spp. 10.0 20.0 21.0 36.5 35.0 S. costatum 19.0 56.0 56.5 46.0 44.5 Nitzschia spp. 1.0 1.0 1.5 2.0 1.0 COPPER Small flagellates 63,0 29.0 9.0 10.0 Thalassiosira spp. 12.5 9.0 6.0 8.0 S. costatum 23.5 62.0 83.0 77.5 Nitzschia spp. 1.0 2.5 5.0 APPENDIX 6 Plankton composition after 2,4-D perturbations; numbers represent relative abundance of Bpecies (Z). Salinity 5 ppt Flagellates Green algae S_. costatum  Thalassiosira spp. Pennate diatoms Day 2: Control 80.0 4.0 14.5 1.0 0.5 IP - 4 M 2,4-D 69.0 9.5 21.5 10 M 2,4-D 81.2 4.5 6.8 0.5 0.5 Flagellates Green algae S^. costatum  Thalassiosira spp. Pennate diatoms Day 11: 6.8 25.5 51.2 13.8 1.8 10.5 17.2 62.2 0.8 3.5 Day 25: 96.0 3.8 0.2 Salinity 10 ppt Day 2: Control Flagellates Green algae S_. costatum  Thalassiosira spp. Chaetoceros spp. Pennate diatoms 70.0 11.5 16.0 1.0 1.5 10"4 M 2,4-D 73.5 4.0 20.0 1.5 0.5 0.5 10"3 M 2,4-D 69.0 11.0 17.0 2.0 1.0 Day 7: Day 9: Day 25: Flagellates Green algae S^. costatum  Thalassiosira spp. Chaetoceros spp. Pennate diatoms 38.5 58.8 0.5 1.2 1.0 43.8 44.8 3.8 6.0 1.7 99.2 0.5 0.2 Salinity IB ppt Day 2: Control Flagellates Green algae S_. costatum  Thalassiosira spp. Chaetoceros spp. Pennate diatoms 73.0 4.0 14.5 3.0 1.0 Day 7: Flagellates Green algae S_. costatum  Thalassiosira spp. Chaetoceros spp. Pennate diatoms 26.5 0.5 58.2 5.0 6.5 3.2 10"4 W 2,4-D 10"3 M 2,4-D 67.0 10.0 20.5 2.0 62.5 11.5 18.5 1.0 0.5 0.5 Day 9: 16.5 Day 19: 86.8 38.5 13.8 25.0 5.8 1.2 2.5 0.2 9.5 Salinity 26 ppt Day 2: Flagellates 71.0 60.0 61.0 Green algae 8.2 23.0 15.0 S_. costatum 15.5 15.5 17.5 Thalassiosira spp. 2.0 1.0 2.0 Chaetoceros spp. Pennate diatoms 0.5 0.5 1.0 Day 7: Day 7: Day 19: Flagellates 35.2 48.2 20.8 Green algae S. costatum 43.0 22.0 0.8 Thalassiosira spp. 3.8 6.8 69.5 Chaetoceros spp. 5.2 12.5 1.0 Pennate diatoms 10.3 6.2 8.0 B a c t e r i a l numbers (xlO^/ml) i n Control-microcosms and a f t e r addition of 2,4-D. 5 ppt S 10 ppt S 18 ppt S 26 ppt S Day C lO"* l O " 3 C l O " 4 l O - 3 C l O " 4 10-3 C 10" 4 l O " 3 2 1.46 1.27 1.19 0.98 1.37 1.24 1.20 1.22 1.17 1.25 1.23 1.04 3 2.01 1.82 2.51 1.89 2.22 2.69 2.18 2.28 2.60 1.71 1.56 1.93 5 3.49 3.96 2.85 3.29 4.87 3.87 2.62 3.60 5.15 4.83 5.25 6.20 7 0.56 0.56 2.56 0.12 0.24 1.80 0.53 0.26 1.87 1.15 1.11 2.46 9 0.74 0.43 1.51 0.19 0.43 1.62 2.11 3.12 2.65 4.67 7.58 4.13 11 1.58 0.67 0.63 1.13 2.11 1.46 2.33 4.61 0.62 4.44 5.75 2.50 13 1.93 0.70 0.28 2.29 1.81 2.14 3.49 1.97 2.82 1.66 3.00 2.42 15 2.75 0.98 0.71 3.19 1.60 1.51 1.13 0.75 2.23 1.00 3.09 1.67 17 3.06 1.59 1.14 2.31 1.36 1.35 1.80 1.06 1.43 1.14 1.08 1.61 19 1.69 1.74 1.10 1.29 0.66 0.86 2.58 0.99 1.47 2.17 1.35 2.97 21 1.16 1.97 1.35 1.51 0.25 1.76 3.27 0.84 1.84 2.19 0.78 3.08 23 1.31 1.60 1.45 1.52 25 1.51 2.63 1.31 1.13 C = Control 1 0 - 4 = 10" 4 M 2,4-D 10" 3 -> 10" 3 M 2,4-D Microzooplankton counts (log numbers) i n Control-microcosms and a f t e r a d d i t i o n of 2,4-D 5 ppt S 10 ppt S 18 ppt S 26 ppt S Day C i o - 4 10-3 C IO" 4 IO" 3 C IO" 4 10-3 C IO"* I O - 3 2 4.99 4.89 4.05 3 5.49 4.69 4.65 4.24 5 4.72 4.68 4.86 4.60 4.57 4.35 4.69 4.54 3.94 4.69 4.57 3.39 7 4.44 4.63 4.60 4.44 4.44 4.79 4.35 4.30 4.76 4.60 4.60 5.29 9 4.18 4.39 4.39 4.10 4.51 4.63 4.00 4.30 4.18 3.88 4.44 4.30 11 3.69 3.57 3.88 4.18 4.30 4.10 3.39 3.88 4.10 3.69 3.69 3.94 13 4.18 4.05 3.57 4.10 4.44 4.18 4.18 4.24 3.69 3.69 4.30 3.39 15 4.10 3.88 3.57 4.18 4.00 4.54 3.39 3.27 4.18 3.69 3.69 3.69 17 3.94 4.18 4.00 3.79 3.69 3.94 2.79 3.57 3.94 3.79 3.69 4.30 19 3.39 3.49 3.64 2.79 3.79 3.57 3.10 3.39 3.88 3.75 3.69 3.79 21 2.79 3.88 4.00 3.69 3.75 3.39 3.39 3.69 3.75 3.49 3.94 4.27 23 4.10 4.48 3.88 4.54 25 4.44 4.39 3.79 3.94 C - Control 10-4 - 1 0 - A M 2,4-D 10" 3 - IO" 3 M 2,4-D F i e l d Data from the S t r a i t of Georgia (1981/1982) Cruises S T Bacteria Het.Activ. Chl N0 2&N0 3 MCHO (Date) (ppt) (°C) (xl0 6/ml) (ug/h/10 9) (UR/1) (ug-at/1) OUR/ 21.07.81 9.0 16.4 1.51 0.27 2.11 1.50 9.8 16.8 1.81 — 3.76 0.51 470 10.08.81 18.0 20.0 4.36 0.12 9.26 0.48 517 18.0 20.0 1.82 0.30 2.69 0.61 350 17.5 20.0 1.81 0.24 2.97 0.28 283 10.0 22.0 1.84 0.38 5.20 0.33 368 10.0 22.0 2.08 0.48 9.86 0.66 455 10.0 22.0 1.93 0.26 3.43 1.80 — 16.09.81 26.0 13.0 1.79 0.30 7.77 15.00 150 17.09.81 25.7 16.5 3.31 0.18 1.22 0.18 — 27.0 16.5 3.32 0.24 9.28 b.d. — 26.8 16.0 4.02 0.08 4.87 b.d. 210 29.09.81 26.2 13.5 1.50 — 7.96 12.84 245 26.8 14.0 1.45 — 6.95 15.38 234 27.0 13.0 1.37 — 6.26 16.54 619 30.09.81 26.4 13.0 1.07 0.67 4.13 19.88 184 1.10.81 18.7 14.0 1.67 1.26 2.58 11.92 — 18.9 14.0 1.53 0.38 3.27 11.78 — F i e l d Data from the S t r a i t of Georgia (1981/1982) Cruises S T Bacteria Het.Activ. Chl N0 2&N0 3 MCHO (Date) (ppt) (°C) (xl0 6/ml) (ug/h/10 9) (JUR/1) ((ug-at/1) (MB/ 15.10.81 19.0 12.3 1.48 0.58 1.76 16.10 331 18.4 11.9 1.14 0.75 1.60 16.57 128 17.5 12.2 1.19 0.60 1.37 15.47 — 18.2 12.1 1.26 0.46 b.d. 15.77 299 25.5 12.0 0.87 0.70 3.46 22.26 167 26.5 12.0 0.59 — 1.30 26.28 — 10.11.81 25.5 14.8 0.57 — 1.67 21.47 — 26.0 11.5 0.55 0.62 2.27 22.20 158 26.0 12.3 0.34 0.64 2.81 23.88 79 11.11.81 18.0 11.4 0.68 0.63 2.25 17.36 279 18.5 11.5 0.77 0.65 1.32 18.43 — 18.0 12.0 0.80 0.94 2.25 19.50 252 7.12.81 26.8 10.1 0.18 0.89 1.18 32.16 — 27.0 10.0 0.22 1.16 2.39 30.63 228 27.0 10.0 0.25 0.26 1.32 32.51 332 12.01.82 27.0 8.7 0.28 0.94 2.37 28.58 131 27.0 8.0 0.28 0.41 0.56 27.69 258 27.0 10.0 0.21 3.48 27.90 195 F i e l d Data from the S t r a i t of Georgia (1981/1982) Cruises S T Bacteria Het.Activ. Chl N0 2&N0 3 MCHO-C (Date) (ppt) (°C) (xl0 6/ml) (ug/h/10 9) (ug/1) (ug-at/1) (MR/1) 8.02.82 26.0 7.0 0.55 0.58 2.22 27.05 230 25.0 6.9 0.41 — 1.67 27.26 270 25.2 7.2 0.39 0.34 1.85 27.80 580 13.04.82 25.0 9.0 2.70 0.32 9.48 20.16 183 25.0 9.0 2.63 0.26 9.30 18.14 302 26.5 9.0 2.53 0.07 0.95 12.66 231 12.05.82 18.5 12.5 3.06 0.14 7.37 9.64 402 27.0 12.0 2.41 0.04 2.92 10.56 150 27.0 11.5 2.11 0.05 4.10 9.89 308 27.0 12.0 2.20 0.04 3.80 10.82 18 13.05.82 10.0 9.0 2.24 0.22 1.62 7.82 589 4.7 10.0 2.10 0.25 2.22 8.45 569 18.2 11.1 2.96 0.14 2.50 9.92 192 1.06.82 18.5 14.0 2.02 — 1.51 10.69 217 18.5 14.0 2.08 — 1.95 10.23 368 26.0 13.0 2.19 0.06 3.06 12.03 — 26.0 13.0 2.59 0.02 2.04 11.78 82 26.0 13.0 2.53 0.10 1.35 12.29 144 18.0 13.6 1.81 0.12 1.02 10.03 260 F i e l d Data from the S t r a i t of Georgia (1981/1982) Cruises S T Bacteria Het.Activ. Chl N0 2&N0 3 MCHO-C (Date) (ppt) (°C) (xl0 6/ml) (pg/h/10 9) (ug/D (ug-at/1) Oug/D 1.06.82 10.3 12.5 1.78 0.04 1.35 9.95 444 9.3 12.0 1.55 0.06 0.97 9.82 — 2.8 12.0 1.87 0.19 1.21 8.55 498 1.5 12.0 2.07 0.11 1.67 9.27 398 3.0 13.0 2.01 0.26 0.60 9.46 527 15.06.82 5.0 16.0 3.66 — 41.99 0.48 — 18.8 18.5 2.89 — 2.02 0.23 221 17.5 18.2 2.03 0.10 3.09 0.20 — 5.1 20.2 2.25 0.34 2.09 2.21 263 4.7 21.0 2.64 — 2.97 3.16 168 5.07.82 5.0 15.0 1.71 0.09 7.52 0.16 261 10.8 15.5 1.82 0.16 11.20 1.39 400 10.0 16.0 1.58 0.41 7.68 0.71 190 10.1 16.5 1.24 0.49 8.93 0.46 367 24.5 15.3 1.29 0.37 9.54 1.61 355 25.0 15.1 0.73 0.36 10.95 0.42 125 24.5 14.9 1.44 0.62 16.24 0.51 129 4.8 15.1 1.26 0.41 24.50 5.49 230 19.0 14.5 0.97 0.80 4.80 1.02 114 17.2 14.5 1.35 0.37 9.84 4.72 134 Plankton Analyses of Cruise Samples: 5 ppt s a l i n i t y 13.5.82 1.6.82 15.6.82 5.7.82 Small f l a g e l l a t e s 77.5 78.5 34.5 23.5 Large f l a g e l l a t e s 1.5 2.5 0.3 Green algae 12.0 7.0 2.0 Th a l a s s i o s i r a spp. 5.0 2.5 57.5 69.5 S. costatum 3.0 6.0 0.5 0.8 Pennate diatoms 2.5 3.5 0.2 Others 1.0 4.0 0.2 Dino f l a g e l l a t e s 1.0 2.0 Chaetoceros spp. 1.5 10 ppt s a l i n i t y 21.7.81 10.8.81 13.5.82 1.6.82 5.7.82 Small f l a g e l l a t e s 60.8 86.5 73.5 21.8 Large f l a g e l l a t e s 4.0 1.0 Green algae 14.7 8.2 7.0 7.0 Th a l a s s i o s i r a spp. 8.8 1.5 1.5 74.0 S. costatum 15.1 2.5 9.3 Chaetoceros spp. 58.1 0.5 0.5 Pennate diatoms 4.2 1.5 4.3 others 1.0 2.2 1.0 1.5 Dinoflage l l a t e s 14.7 10.5 1.2 Ti n t i n n i d s 1.7 ro Plankton Analyses of Cruise Samples: 18 ppt s a l i n i t y 10.8.81 1.10.81 15.10.81 11.11.81 12.5.82 1.6.82 Small f l a g e l l a t e s 1.7 75.0 71.0 66.0 82.5 79.5 Large l a g e l l a t e s 1.5 6.0 1.0 5.0 Green algae 1.9 7.5 15.0 9.5 1.0 0.5 T h a l a s s i o s i r a spp. 4.9 8.5 5.0 12.0 7.0 5.0 S. costatum 77.5 6.0 5.0 Chaetoceros spp. 11.2 2.0 1.0 Pennate diatoms 2.5 1.0 1.5 1.0 1.5 Others 1.0 4.0 1.5 2.5 Dino f l a g e l l a t e s 1.8 Tin t i n n i d s 5.5 4.0 3.0 15.6.82 5.7.82 Small f l a g e l l a t e s 54.5 15.5 Large f l a g e l l a t e s 2.5 1.2 Green algae T h a l a s s i o s i r a spp. 16.5 5.0 S. costatum 15.0 70.0 Chaetoceros spp. 3.0 3.5 Pennate diatoms 1.5 3.2 Others 6.5 1.0 Dino f l a g e l l a t e s 0.5 0.5 Plankton Analysis of Cruise Samples: 26 ppt 16.9.81 29.9.81 Small f l a g e l l a t e s 50.7 19.1 Large f l a g e l l a t e s 7.5 1.5 Green algae 13.4 11.2 T h a l a s s i o s i r a spp. 5.0 11.8 S. costatum 11.0 8.4 Chaetoceros spp. 2.5 37.4 Pennate diatoms 6.0 9.7 Others 0.4 0.9 Ti n t i n n i d s 3.5 8.2.82 13.4.82 Small f l a g e l l a t e s 94.5 Large f l a g e l l a t e s 2.8 Green algae T h a l a s s i o s i r a spp. 1.8 41.5 S. costatum 56.8 Chaetoceros spp. Pennate diatoms 1.8 Others s a l i n i t y 15.10.81 10.11.81 7.12.81 12.1.82 73.0 59.0 67.5 70.0 2.5 5.0 1.5 6.5 16.0 13.5 23.0 16.5 6.5 13.5 7.0 5.0 2.0 2.0 1.0 4.0 1.0 1.0 1.0 1.5 2.5.82 1.6.82 5.7.82 91.8 48.5 10.0 4.8 1.5 1.5 2.2 0.5 2.0 5.0 0.5 23.0 52.0 11.5 19.2 0.2 12.5 11.8 1.5 0.5 

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