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The effect of manganese on the concentration of biologically available copper to the diatom, Thalassiosira… Kazumi, Junko 1985

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THE EFFECT OF MANGANESE THE CONCENTRATION OF BIOLOGICALLY AVAILABLE COPPER TO THE DIATOM, THALASSIOSIRA PSEUDONANA by JUNKO KAZUMI B . S c , 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 , 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t s o f O c e a n o g r a p h y and. Z o o l o g y ) 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 t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r 1985 © J u n k o Kazumi, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Oceanography/Zoology The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 r w . October, 1985 DE-6(3/81) ABSTRACT Mn was f o u n d t o r e d u c e t h e t o x i c i t y o f Cu t o t h e m a r i n e d i a t o m T h a l a s s i o s i r a pseudonana ( c l o n e 3H) i n t h e c h e m i c a l l y w e l l d e f i n e d medium A q u i l ( M o r e l e t a l . , 1979), v e r i f y i n g t h e r e s u l t s o f Sunda and Huntsman ( 1 9 8 3 ) . A c a t i o n - e x c h a n g e r e s i n t e c h n i q u e d e v e l o p e d by Z o r k i n (1983) was m o d i f i e d f o r use i n n a t u r a l s e a w a t e r s a m p l e s t o e s t i m a t e t h e b i o l o g i c a l l y a c t i v e Cu and Mn. S eawater samples t a k e n from t h e b o t t o m w a t e r s o f a l o c a l f j o r d were f o u n d t o s u p p o r t b e t t e r g r o w t h of t h e t e s t o r g a n i s m t h a n samples f r o m s h a l l o w w a t e r s , a l t h o u g h t h e c o n c e n t r a t i o n of t h e b i o l o g i c a l l y a c t i v e Cu a s e s t i m a t e d by t h e r e s i n t e c h n i q u e was s i m i l a r f o r s a m p l e s c o l l e c t e d from a l l d e p t h s . The b i o l o g i c a l l y a c t i v e Mn c o n c e n t r a t i o n was f o u n d t o be h i g h e r i n t h e b o t t o m w a t e r s a m p l e s , i n d i c a t i n g t h a t t h e b i o a s s a y o r g a n i s m was p r o b a b l y r e s p o n d i n g t o t h e i n t e r a c t i o n between t h e i o n i c forms of Cu and Mn, r a t h e r t h a n t o c h a n g e s i n t h e b i o l o g i c a l l y a c t i v e Cu c o n c e n t r a t i o n . i i i TABLE OF CONTENTS L I S T OF TABLES v i L I S T OF FIGURES v i i ACKNOWLEDGEMENTS i x GENERAL INTRODUCTION 1 I . THE EFFECT OF Mn ON Cu TOXICITY TO A MARINE DIATOM . 4 INTRODUCTION 4 MATERIALS AND METHODS 6 1. B i o a s s a y s u s i n g a w e l l d e f i n e d medium 6 2. Media p r e p a r a t i o n 7 (a) S t a n d a r d Ocean Water 7 (b) B i o a s s a y medium 8 (c) T r a c e m e t a l s t o c k s o l u t i o n s 9 3. P r o c e d u r e 9 4. Growth r a t e c a l c u l a t i o n s 10 RESULTS AND DISCUSSION 12 1. Respo n s e o f t h e b i o a s s a y o r g a n i s m t o Cu ... 12 2. Response o f t h e b i o a s s a y o r g a n i s m t o Cu and Mn 16 I I . TESTING OF A CATION-EXCHANGE RESIN TECHNIQUE TO ESTIMATE THE BIOLOGICALLY ACTIVE Cu AND Mn 22 INTRODUCTION 22 MATERIALS AND METHODS 26 1. Sample p r e p a r a t i o n 26 2. Column o p e r a t i o n 26 (a) M a t e r i a l s 26 i v (b) P r e p a r a t i o n o f t h e r e s i n 29 ( c ) Column p r o c e d u r e 29 (d) E l u t i o n of t h e c o l u m n s 30 3. D e t e r m i n a t i o n o f e l u t e d m e t a l s w i t h GFAAS . 31 RESULTS AND DISCUSSION 33 1. C h a r a c t e r i z a t i o n of t h e r e s i n Dowex AG 50W-X12 33 (a) E q u i l i b r a t i o n o f t h e r e s i n t o Cu and Mn .. 33 (b) Cu and Mn a d s o r p t i o n c u r v e s 39 ( c ) E f f e c t of Cu on Mn a d s o r p t i o n 41 (d) E f f e c t of n u t r i e n t s 44 I I I . THE EFFECT OF AMBIENT LEVELS OF Mn ON Cu TOXICITY IN NATURAL WATER SAMPLES 52 INTRODUCTION 52 MATERIALS AND METHODS 55 1 . C h a r a c t e r i s t i c s o f I n d i a n Arm 55 2. C o l l e c t i o n o f n a t u r a l s e a w a t e r 55 3. A n a l y s i s o f n a t u r a l w a ter samples f o r t o t a l d i s s o l v e d Cu and Mn 57 4. A d a p t a t i o n o f t h e r e s i n method f o r n a t u r a l w a t e r s a m p l e s 58 5. B i o a s s a y s and r e s i n e x p e r i m e n t s u s i n g n a t u r a l w a ter 58 RESULTS AND DISCUSSION 62 1. A d a p t a t i o n o f t h e r e s i n method f o r n a t u r a l w a t e r samples 62 2. B i o a s s a y s 63 3. R e s i n e x p e r i m e n t s 67 4. C o m p a r i s o n o f b i o a s s a y a n d r e s i n r e s u l t s .. 71 GENERAL SUMMARY 76 REFERENCES CITED 78 APPENDIX I 86 v i L I S T OF TABLES T a b l e 1. E f f e c t of A q u i l n u t r i e n t s on t h e a d s o r p t i o n o f Cu and Mn t o Dowex AG-50W-X12 r e s i n 46 T a b l e 2. Dowex AG 50W-X12 r e s i n column t e s t s e r i e s f o r e s t i m a t i n g e f f e c t i v e m e t a l c o n c e n t r a t i o n s 61 T a b l e 3. Growth r a t e o f T h a l a s s i o s i r a p s e u d o n a n a i n I n d i a n Arm water 66 T a b l e 4. E f f e c t i v e Cu and Mn c o n c e n t r a t i o n s i n c o p p e r - e n r i c h e d I n d i a n Arm water 69 v i i L I S T OF FIGURES F i g u r e 1. Growth r a t e v e r s u s t o t a l Cu added t o A q u i l w i t h o u t EDTA . 13 F i g u r e 2. The e f f e c t o f Cu on c e l l c o n c e n t r a t i o n o v e r t i m e 15 F i g u r e 3. The e f f e c t o f Cu on c e l l c o n c e n t r a t i o n i n t h e p r e s e n c e of Mn 17 F i g u r e 4. The e f f e c t of v a r y i n g Mn c o n c e n t r a t i o n s on Cu t o x i c i t y i n E D T A - f r e e A q u i l 19 F i g u r e 5. R e s i n column 28 F i g u r e 6. E l u a t e Mn v e r s u s sample volume r e q u i r e d f o r e q u i l i b r a t i o n 34 F i g u r e 7. E l u a t e Cu v e r s u s sample volume (50-500ml) r e q u i r e d f o r e q u i l i b r a t i o n 35 F i g u r e 8. E l u a t e Cu v e r s u s sample volume ( 5 0 - l 0 0 0 m l ) r e q u i r e d f o r e q u i l i b r a t i o n 36 F i g u r e 9. E l u a t e Cu v e r s u s sample volume (50-800ml) r e q u i r e d f o r e q u i l i b r a t i o n 38 F i g u r e 10. E l u a t e Mn v e r s u s t o t a l Mn added 40 F i g u r e 11. E l u a t e Cu v e r s u s t o t a l Cu added 42 F i g u r e 12. The e f f e c t of Mn on Cu a d s o r p t i o n t o t h e r e s i n 43 F i g u r e 13. E l u a t e Mn v e r s u s t o t a l Mn added i n t h e p r e s e n c e o f Cu 45 F i g u r e 14. The e f f e c t o f Fe on Cu a d s o r p t i o n t o t h e r e s i n (10-500 nM Fe) 48 F i g u r e 15. The e f f e c t of Fe on Cu a d s o r p t i o n t o t h e r e s i n (100-5000 nM Fe) 49 F i g u r e 16. The e f f e c t o f Fe on Mn a d s o r p t i o n t o t h e r e s i n 51 F i g u r e 17. L o c a t i o n o f sample c o l l e c t i o n 56 v i i i F i g u r e 18. E l u a t e Cu v e r s u s sample volume r e q u i r e d f o r e q u i l i b r a t i o n f o r Dowex AG 50W-X8 r e s i n 64 F i g u r e 19. E l u a t e Mn v e r s u s sample volume r e q u i r e d f o r e q u i l i b r a t i o n f o r Dowex AG 50W-X8 r e s i n 65 F i g u r e 20. The e f f e c t of v a r y i n g EMnC v a l u e s on Cu t o x i c i t y i n n a t u r a l w a t e r s a m p l e s 72 i x ACKNOWLEDGEMENTS I would s i n c e r e l y l i k e t o thank my s u p e r v i s o r , D r . A.G. L e w i s f o r h i s s u p p o r t and encouragement d u r i n g t h e s e p a s t few y e a r s . I am a l s o d e e p l y g r a t e f u l t o Dr. N.G. Z o r k i n f o r h i s i n v a l u a b l e a d v i c e on my r e s e a r c h . I e x t e n d my a p p r e c i a t i o n t o my r e s e a r c h c ommittee members, D r s . K . J . H a l l and E.V. G r i l l f o r t h e i r i n p u t i n t h e p r e p a r a t i o n of t h i s t h e s i s . . 1 GENERAL INTRODUCTION Copper (Cu) and manganese (Mn) are required nutrients for the growth of phytoplankton because they are esse n t i a l components in a variety of enzyme systems and serve as cofactors in photosynthesis. Mn is especially important for electron transport and oxygen evolution in photosystem II (Cheniae and Martin, 1970; Diner and J o l i o t , 1976), while Cu is important as a component of plastocyanin, a protein that is involved in electron transfer in photosynthetic reactions (Rains, 1976). In contrast to Mn, Cu can be detrimental to phytoplankton at r e l a t i v e l y low concentrations. C e l l response to elevated Cu le v e l s includes enlargement of c e l l s due to a reduction in c e l l d i v i s i o n (Fisher et al.,1981) and a decrease in growth rate (Sunda and G u i l l a r d , 1976). Physiological processes, such as nitra t e uptake, photosynthetic carbon assimilation, i n h i b i t i o n of ni t r a t e reductase synthesis (Harrison e_t a_l. , 1977) and s i l i c i c acid uptake (Goering et a l . , 1977; Rueter e_t a l . , 1981), are also affected by elevated Cu l e v e l s . In addition, the net phosphorous n u t r i t i o n of phytoplankton may be influenced since a l k a l i n e phosphatase a c t i v i t y has been shown to be inh i b i t e d by Cu (Rueter, 1983). High levels of Cu have been suggested to i n h i b i t the storage of l i p i d s . S h i f r i n and Chisolm (1981) observed that, under nitrogen stress, copper tolerant strains had higher l i p i d fractions when compared to 2 i n t o l e r a n t s t r a i n s o f m a r i n e and f r e s h water p h y t o p l a n k t o n . The d e t r i m e n t a l e f f e c t s of e l e v a t e d Cu l e v e l s t o p h y t o p l a n k t o n have been r e c e n t l y shown t o be r e d u c e d by t h e p r e s e n c e o f Mn (Sunda e t a l . , 1981; Sunda and Huntsman, 1983). Sunda e t a l . (1981) have f o u n d t h a t t h e a d d i t i o n o f a s l i t t l e a s 1 nM Mn t o S a r g a s s o s e a w a t e r c o n t a i n i n g an e s t i m a t e d 3-10 nM Cu c a u s e d an i n c r e a s e i n g r owth r a t e of C h a e t o c e r o u s s o c i a l i s . Sunda and Huntsman (1983) have a l s o f o u n d t h a t t h e a d d i t i o n o f Mn a l l e v i a t e d t h e t o x i c i t y of Cu t o T h a l a s s i o s i r a p s e u d o n a n a and a r e l a t e d o c e a n i c d i a t o m , T h a l a s s i o s i r a o c e a n i c a . U s u a l l y i n t h e open o c e a n , a m b i e n t l e v e l s o f Cu a r e t h o u g h t t o have a d e l e t e r i o u s e f f e c t on o c e a n i c s p e c i e s o f p h y t o p l a n k t o n ( G a v i s e t a_l. , 1981), w h i l e n a t u r a l c o n c e n t r a t i o n s o f Mn a r e t h o u g h t t o be d e f i c i e n t t o b o t h o c e a n i c and n e r i t i c s p e c i e s of a l g a e ( B r a n d e t a l . , 1983). In t h e s u r f a c e w a t e r s o f t h e c e n t r a l N o r t h P a c i f i c , t o t a l d i s s o l v e d Cu has been r e p o r t e d t o r a n g e from 1.5 t o 3.0 nM ( B o y l e et a l . , 1977), w h i l e t o t a l d i s s o l v e d Mn has been f o u n d t o r a n g e from 0.3 t o 3.0 nM (Klinkhammer and B e n d e r , 1980). However, i n c e r t a i n c o a s t a l a r e a s w i t h r e s t r i c t e d w a ter c i r c u l a t i o n ( s u c h as c e r t a i n f j o r d s ) , d i s s o l v e d Mn i s o f t e n e n r i c h e d . The h i g h Mn c o n c e n t r a t i o n s a r e u s u a l l y t h e r e s u l t o f d e p l e t e d oxygen l e v e l s , w h i c h o f t e n o c c u r i n t h e b o t t o m w a t e r s (Emerson e t a l . , 1979). Some o f t h e f j o r d s o f B r i t i s h C o l u m b i a can o f f e r an i d e a l 3 s i t u a t i o n i n w h i c h t h e e f f e c t o f n a t u r a l l y o c c u r r i n g c o n c e n t r a t i o n s of Mn c a n be t e s t e d on r e d u c i n g Cu t o x i c i t y t o p h y t o p l a n k t o n . In t h i s s t u d y , t h e r e s p o n s e o f t h e b i o a s s a y o r g a n i s m T h a l a s s i o s i r a p seudonana t o v a r i o u s c o n c e n t r a t i o n s of b o t h Cu and Mn was f i r s t d e t e r m i n e d i n t h e a r t i f i c i a l s e a w a t e r medium, A q u i l ( M o r e l e t a_l . , 1979). A r e s i n t e c h n i q u e d e v e l o p e d by Z o r k i n (1983) and Z o r k i n e t a l . ( i n p r e s s ) t o e s t i m a t e t h e b i o l o g i c a l l y a v a i l a b l e f r a c t i o n s of Cu and Mn was t h e n t e s t e d w i t h A q u i l . F i n a l l y , b o t h t h e r e s i n t e c h n i q u e and t h e b i o a s s a y s were c o n d u c t e d w i t h n a t u r a l w a ter s a m p l e s c o l l e c t e d f r o m a l o c a l m a r i n e f j o r d t o i n v e s t i g a t e whether a m b i e n t l e v e l s o f Mn c a n r e d u c e Cu t o x i c i t y t o - T . p s e u d o n a n a . 4 I . THE EFFECT OF MN ON CU TOXICITY TO A MARINE DIATOM INTRODUCTION The b i o l o g i c a l e f f e c t s of Cu and Mn a r e g o v e r n e d by t h e m e t a l s ' s p e c i a t i o n ( F l o r e n c e , 1982a; F l o r e n c e and B a t l e y , 1980). E x p e r i m e n t s u s i n g w e l l d e f i n e d a r t i f i c i a l media s u c h as A q u i l ( M o r e l e t a_ l . , 1979) have l e d t o t h e c o n c l u s i o n t h a t i t i s t h e c o n c e n t r a t i o n of t h e f r e e i o n i c f o r m of t h e m e t a l r a t h e r t h a n t h e t o t a l m e t a l c o n c e n t r a t i o n , w h i c h a f f e c t s t h e g r o w t h o f p h y t o p l a n k t o n (Sunda and G u i l l a r d , 1976; A n d e r s o n and M o r e l , 1978). The c o n c e n t r a t i o n of t h e f r e e i o n i c f o r m w i l l be i n f l u e n c e d by f a c t o r s s u c h as t h e n a t u r e and t h e c o n c e n t r a t i o n of l i g a n d s a v a i l a b l e t o b i n d t h e m e t a l and t h e l e v e l of p a r t i c u l a t e s a v a i l a b l e t o a d s o r b th e m e t a l . In a d d i t i o n t o i o n i c a c t i v i t i e s , m e t a l r a t i o s i n s o l u t i o n w i l l a l s o a f f e c t t h e r e s p o n s e of p h y t o p l a n k t o n . M e t a l - m e t a l i n t e r a c t i o n s can o c c u r i n wh i c h a p o t e n t i a l l y t o x i c m e t a l , s u c h a s Cu, can compete w i t h a m e t a l s u c h as Mn f o r an u p t a k e s i t e e i t h e r i n o r on t h e c e l l . Sunda e t a l . (1981) f o u n d t h a t low Cu:Mn r a t i o s were not a s d e t r i m e n t a l t o t h e g r o w t h of. C h a e t o c e r o u s s o c i a l i s a s were h i g h m e t a l r a t i o s . T h e y s u g g e s t t h a t by c o m p e t i n g w i t h Mn a t t h e Mn n u t r i t i o n a l s i t e s , Cu may be t o x i c t o p h y t o p l a n k t o n a t t h e h i g h Cu:Mn r a t i o s . 5 The e f f e c t of m e t a l - m e t a l i n t e r a c t i o n s c a n v a r y between s p e c i e s . F o r example, Braek e t a_l . (1980) i n v e s t i g a t e d t h e i n t e r a c t i o n of Zn and Cd on p h y t o p l a n k t o n and f o u n d t h a t t h e s e m e t a l s a c t e d s y n e r g i s t i c a l l y on T h a l a s s i o s i r a  p s eudonana (Huds.) H a s l e , b u t a n t a g o n i s t i c a l l y on S k e l e t o n e m a c o s t a t u m , c l o n e S k e l - 0 . W i t h Cu and Mn, A n d e r s o n and M o r e l (1978) have f o u n d t h a t a d r o p i n Mn l e v e l s d i d not c a u s e a f u r t h e r d e c r e a s e i n t h e m o t i l i t y of G o n y a u l a x t a m a r e n s i s i n d u c e d by i n c r e a s e d c u p r i c i o n a c t i v i t i e s . However, Sunda and Huntsman (1983) have f o u n d t h a t an i n c r e a s e i n c u p r i c i o n a c t i v i t y a t low Mn i o n a c t i v i t i e s s t o p p e d t h e growth o f T h a l a s s i o s i r a p seudonana (WHOI c l o n e 3H) and a r e l a t e d o c e a n i c d i a t o m , T h a l a s s i o s i r a  o c e a n i c a (WHOI c l o n e 13-1), b u t t h a t t h e e f f e c t was c o m p l e t e l y r e v e r s e d by i n c r e a s i n g t h e Mn i o n a c t i v i t y . The e f f e c t of a m e t a l - m e t a l i n t e r a c t i o n on a p a r t i c u l a r b i o a s s a y o r g a n i s m must be d e t e r m i n e d s i n c e t h e r e s p o n s e o f p h y t o p l a n k t o n t o t h e i n t e r a c t i o n c a n v a r y between s p e c i e s . T h i s c h a p t e r d i s c u s s e s t h e r e s p o n s e of T h a l a s s i o s i r a  p s e u d o n a n a ( c l o n e 3H) t o v a r i o u s c o m b i n a t i o n s of Cu and Mn c o n c e n t r a t i o n s i n a r t i f i c i a l s e a w a t e r . The f i r s t s e r i e s of e x p e r i m e n t s was c o n d u c t e d w i t h d i f f e r e n t Cu c o n c e n t r a t i o n s t o c h a r a c t e r i z e t h e r e s p o n s e of t h e o r g a n i s m t o Cu a l o n e . The s e c o n d s e r i e s of b i o a s s a y s examined t h e a b i l i t y o f Mn t o r e d u c e Cu t o x i c i t y . 6 MATERIALS AND METHODS 1. B i o a s s a y s u s i n g a w e l l d e f i n e d medium The m a r i n e c e n t r i c d i a t o m T h a l a s s i o s i r a pseudonana (WHOI c l o n e 3H) was u s e d a s t h e b i o a s s a y o r g a n i s m b e c a u s e i t i s s e n s i t i v e t o c o p p e r and has a c o n s i s t e n t s i z e ( a p p r o x i m a t e l y 4.5 urn i n d i a m e t e r ) a n d shape w h i c h makes i t s u i t a b l e f o r e l e c t r o n i c p a r t i c l e c o u n t i n g . The b i o a s s a y s were c o n d u c t e d u s i n g A q u i l ( M o r e l e_t a l _ . , 1979), a w e l l d e f i n e d a r t i f i c i a l medium f o r w h i c h c h e m i c a l s p e c i a t i o n c a n be e s t i m a t e d by t h e computer model MINEQL ( W e s t a l l e t a l . , 1976). I n o c u l a f o r t h e b i o a s s a y m edia were o b t a i n e d f r o m e x p o n e n t i a l l y g r o w i n g s t o c k c u l t u r e s o f T. p s e u d o n a n a . The c u l t u r e s were i n i t i a l l y a x e n i c , and r e a s o n a b l e c a r e was t a k e n t o m a i n t a i n them i n t h i s s t a t e . The c u l t u r e s o l u t i o n c o n t a i n e d a l l A q u i l n u t r i e n t s , v i t a m i n s and t r a c e m e t a l s b u t w i t h r e d u c e d l e v e l s o f Mn and w i t h o u t t h e c h e l a t i n g a g e n t e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D T A ) . P r e l i m i n a r y e x p e r i m e n t s w i t h T. p s e u d o n a n a i n d i c a t e d t h a t Mn l e v e l s c o u l d be l o w e r e d t o 1 nM w i t h o u t t h e c e l l s r e a c h i n g Mn l i m i t a t i o n . T h i s c o n c e n t r a t i o n was a d o p t e d f o r t h e s t o c k c u l t u r e t o r e d u c e Mn c a r r y o v e r t o t h e t e s t f l a s k s . EDTA was not a d d e d t o s t o c k s o l u t i o n s n o r t o any o f t h e t e s t m e d i a b e c a u s e i t s s t r o n g c o m p l e x i n g c a p a c i t y f o r Cu ( S i l l e n and M a r t e l l , 1964) c o u l d mask t h e e f f e c t o f Mn on r e d u c i n g Cu 7 t o x i c i t y . Enough stock culture was added to the flasks to obtain an i n i t i a l c e l l concentration of approximately 1000 - 2000 c e l l s / m l . The organisms were grown in an environmentally cont r o l l e d room at 16°C with an in-fla s k l i g h t intensity of 100 uEin m"2 sec" 1 on a 16:8 hour light:dark cycle. The c e l l concentrations were measured d a i l y using a Coulter Counter (model Zf) over a period of 5 days. 2. Media preparation (a) Standard Ocean Water Standard ocean water (SOW) (Morel et a_l. , 1979) was made by dissolving a l l major seawater s a l t s except MgCl 2.6H 20 in 12 l i t e r s of glass d i s t i l l e d water (GDW) measured into a 20 l i t e r glass carboy. The magnesium chloride (previously dried for 2 days at 60°C) was then added and the volume made up to 20 l i t e r s with GDW. A l l the sa l t s used were reagent grade. The SOW was bubbled with acid-cleaned (1N H2SO,,), f i l t e r e d (0.4 urn Nuclepore) a i r to allow complete mixing and e q u i l i b r a t i o n . The f i n a l pH of the medium was 8.0±0.05. The SOW was then passed through an ion-exchange column (Chelex-100®, 100-200 mesh, Bio-Rad Laboratories) to remove trace metal impurities. Chelexed SOW had undetectable levels of Mn and Cu, as measured by di r e c t i n j e c t i o n graphite furnace atomic absorption 8 s p e c t r o p h o t o m e t r y (GFAAS). The d e t e c t i o n l e v e l s of t h i s t e c h n i q u e a r e a p p r o x i m a t e l y 10 nM f o r Mn and 15 nM f o r Cu. (b) B i o a s s a y medium The b i o a s s a y medium was p r e p a r e d i n a 10 l i t e r N a l g e n e p o l y c a r b o n a t e c a r b o y . The medium c o n s i s t e d of 10 l i t e r s of SOW t o w h i c h a p p r o p r i a t e amounts o f A q u i l v i t a m i n s and n u t r i e n t s ( p r e v i o u s l y p a s s e d t h r o u g h C h e l e x - 1 0 0 ) were added. The c o n c e n t r a t i o n of t h e A q u i l s a l t s , n u t r i e n t s and v i t a m i n s a r e g i v e n i n A p p e n d i x I , T a b l e I . The medium was t h e n b u b b l e d w i t h a c i d - c l e a n e d and f i l t e r e d C 0 2 u n t i l t h e pH r e a c h e d a p p r o x i m a t e l y 6.5. T h i s s t e p was n e c e s s a r y t o p r e v e n t p r e c i p i t a t i o n d u r i n g a u t o c l a v i n g . The c a r b o y w i t h t h e medium was t h e n a u t o c l a v e d f o r 1 h r a t 121°C and 15 p s i , and c o o l e d t o room t e m p e r a t u r e . F i n a l l y , t h e A q u i l t r a c e m e t a l mix ( w i t h Cu, b u t w i t h o u t EDTA and Mn) and f r e s h l y p r e p a r e d Fe s t o c k were a d d e d . The Fe s t o c k was a l l o w e d t o e q u i l i b r a t e f o r s e v e r a l h o u r s a f t e r p r e p a r a t i o n t o a l l o w t h e f o r m a t i o n of f e r r i c h y d r o x i d e t o o c c u r b e f o r e a d d i t i o n t o t h e A q u i l . The pH o f t h e A q u i l was i n c r e a s e d t o 8.0, i f n e c e s s a r y , by b u b b l i n g w i t h a c i d - c l e a n e d and f i l t e r e d a i r . The f u l l A q u i l medium d i f f e r s from t h a t o f M o r e l e t a l . (1979) i n t h a t n e i t h e r EDTA n o r Mn was added, and t h a t t h e Fe was a d d e d as f r e s h l y p r e c i p i t a t e d Fe h y d r o x i d e . B e c a use of t h e low s o l u b i l i t y o f Fe i n s e a w a t e r , EDTA has o f t e n been a d d e d t o c u l t u r e media t o keep t h e Fe s o l u b i l i z e d . However, 9 t h i s p r o c e d u r e was n o t s u i t a b l e i n t h i s s t u d y b e c a u s e of t h e s t r o n g a f f i n i t y EDTA has f o r Cu. S i n c e f r e s h l y p r e c i p i t a t e d f e r r i c h y d r o x i d e has been f o u n d t o be as e f f e c t i v e a s t h e A q u i l Fe-EDTA mix i n s u p p l y i n g Fe t o p h y t o p l a n k t o n ( W e l l s e t a l . , 1983), t h i s mode o f Fe a d d i t i o n was u s e d . (c ) T r a c e m e t a l s t o c k s o l u t i o n s Cu s t o c k s were p r e p a r e d d a i l y from a s t o c k s o l u t i o n of 1 x 10~ 3M C u C l 2 i n 1% r e a g e n t g r a d e HN0 3. L i k e w i s e , Mn s t o c k s were f r e s h l y p r e p a r e d from a s t o c k s o l u t i o n of 1 x 10" 3M M n C l 2 i n 1% HN0 3. A l l g l a s s and p l a s t i c w a r e u s e d i n t h e e x p e r i m e n t s were i n i t i a l l y r i n s e d 3 t i m e s w i t h GDW, s o a k e d i n 1N r e a g e n t g r a d e HC1 f o r a week, t h e n r i n s e d 3 t i m e s a g a i n w i t h GDW. F o r s u b s e q u e n t u s e s , t h e g l a s s and p l a s t i c w a r e were r i n s e d 3 t i m e s w i t h GDW, s o a k e d i n 1N HC1 f o r a b o u t 12 h r s , and r i n s e d w i t h GDW 3 t i m e s . 3. P r o c e d u r e A l l m a n i p u l a t i o n s were done i n a c l a s s 100 l a m i n a r f l o w hood w i t h a l l p o s s i b l e p a r t s r e p l a c e d by p o l y p r o p y l e n e t o m i n i m i z e t r a c e m e t a l c o n t a m i n a t i o n . A l i q u o t s (250 ml) of a u t o c l a v e d A q u i l were p o u r e d i n t o p r e v i o u s l y a u t o c l a v e d 500 ml p o l y c a r b o n a t e e r l e n m e y e r f l a s k s w i t h p o l y p r o p y l e n e s c r e w c a p s . A p p r o p r i a t e s p i k e s o f Cu and Mn were added and 10 a l l o w e d t o e q u i l i b r a t e o v e r n i g h t b e f o r e i n o c u l a t i o n w i t h T. p s e u d o n a n a . E a c h t e s t was r u n i n t r i p l i c a t e w h i c h g a v e a r e l a t i v e s t a n d a r d d e v i a t i o n o f t h e g r o w t h r a t e o f a p p r o x i m a t e l y 5%. pH b u f f e r s were n o t a d d e d t o t h e b i o a s s a y medium a s t h e y a r e o f t e n s t r o n g m e t a l c h e l a t o r s . However, t r a c e m e t a l s p e c i a t i o n and t h e b i o l o g i c a l l y a v a i l a b l e f r a c t i o n o f t h e m e t a l , i s h i g h l y i n f l u e n c e d by pH ( e . g . P e t e r s o n e t a l . , 1 9 8 4 ) . The pH d u r i n g t h e b i o a s s a y c h a n g e d f r o m 8.00±0.05 on t h e d a y o f i n o c u l a t i o n t o a maximum o f 8.43±0.07 f o u r d a y s l a t e r . S i n c e t h e pH c h a n g e was s i m i l a r i n t h e c o n t r o l c u l t u r e s , any d i f f e r e n c e b e t w e e n c o n t r o l a n d t e s t pH was a s s u m e d n o t t o p r o d u c 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 m e t a l s p e c i a t i o n . 4. G r o w t h r a t e c a l c u l a t i o n s The e f f e c t o f Cu a n d Mn on T. p s e u d o n a n a was e x p r e s s e d i n t e r m s o f g r o w t h r a t e o v e r t h e p e r i o d day 1 t o day 4, w h i c h was c a l c u l a t e d a s f o l l o w s : g r o w t h r a t e ( d i v d a y 1 ) = l o g (N,/N 0) x 3.322 t w h e r e N 0 and N, a r e c e l l c o n c e n t r a t i o n s a t t h e s t a r t a n d e n d , r e s p e c t i v e l y , o f a t i m e p e r i o d , t ( G u i l l a r d , 1 9 7 3 ) . S i n c e l o w g r o w t h r a t e s were o b t a i n e d i n t h e f i r s t 24 h o u r s o f t h e b i o a s s a y ( p r o b a b l y due t o t h e b i o a s s a y o r g a n i s m 11 acc l imat iz ing to the medium), day 0 values were excluded from the growth rate c a l c u l a t i o n s . In some instances, growth rates were expressed as a percentage of the contro l c u l t u r e s . This was defined by the r e l a t i o n s h i p : growth rate (% of contro l ) = growth rate test x 100 growth rate cont ro l 12 RESULTS AND DISCUSSION 1. R e s p o n s e o f t h e b i o a s s a y o r g a n i s m t o Cu As p h y t o p l a n k t o n s p e c i e s may r e s p o n d d i f f e r e n t l y t o e l e v a t e d Cu c o n c e n t r a t i o n s , t h e r e s p o n s e o f T h a l a s s i o s i r a  p seudonana t o v a r i o u s Cu l e v e l s forms a b a s e l i n e a g a i n s t w h i c h t h e e f f e c t of Mn c a n be r e l a t e d . In g e n e r a l , e s t u a r i n e s p e c i e s a r e more r e s i s t a n t than o c e a n i c s p e c i e s . F u r t h e r m o r e , some c l o n e s o f even p o s s i b l y t h e same s p e c i e s of p h y t o p l a n k t o n o b t a i n e d f r o m n e r i t i c e n v i r o n m e n t s have been f o u n d t o be more h a r d y t h a n t h o s e from o c e a n i c r e g i o n s ( G a v i s e t a l . , 1981). Copper s p i k e s r a n g i n g f r o m 20-120 nM, i n 20 nM i n c r e m e n t s , were added t o A q u i l c o n t a i n i n g t h e A q u i l c o n c e n t r a t i o n of Mn (28.3 nM) t o c h a r a c t e r i z e t h e r e s p o n s e o f T h a l a s s i o s i r a pseudonana t o Cu. The e f f e c t o f i n c r e a s i n g l e v e l s o f Cu i s shown by a d e c r e a s e i n g r o w t h r a t e o f T. pse u d o n a n a ( F i g . 1 ) . A l t h o u g h a c o n c e n t r a t i o n o f 100 nM was r e q u i r e d f o r a 50% d e c r e a s e i n growth r a t e , Cu l e v e l s of 20 nM were f o u n d t o be d e l e t e r i o u s under t h e e x p e r i m e n t a l c o n d i t i o n s . The g r e a t e s t change i n growth r a t e o f t h e t e s t o r g a n i s m o c c u r r e d i n t h e r a n g e between 40 and 100 nM Cu, where 20 nM i n c r e a s e s i n Cu l e v e l s r e s u l t e d i n a r a p i d l i n e a r d e c r e a s e i n g r o w t h r a t e . R e d u c t i o n i n g r o w t h r a t e s by e l e v a t e d Cu c o n c e n t r a t i o n s has been p r e v i o u s l y r e p o r t e d f o r t h i s c l o n e o f T. pse u d o n a n a by R u e t e r e t a l . ( 1 9 8 1 ) , 13 F i g u r e 1. Growth r a t e v e r s u s t o t a l Cu added t o A q u i l w i t h o u t EDTA. B a r s r e p r e s e n t ±2 s t a n d a r d d e v i a t i o n s . 1 4 G a v i s e t a l . (1 98 1 ) , and Z o r k i n ( 1 9 8 3 ) . Changes i n g r o w t h r a t e due t o added Cu were e v i d e n t 2 d a y s a f t e r i n o c u l a t i o n and became more marked a f t e r 4 d a y s ( F i g . 2 ) . As t h e c o n c e n t r a t i o n of Cu and e x p o s u r e t i m e i n c r e a s e d t h e d e c r e a s e i n g r o w t h r a t e became more p r o n o u n c e d . Such a d e l a y e d r e s p o n s e t o a d d e d Cu has a l s o been p r e v i o u s l y shown f o r t h i s c l o n e by Sunda and G u i l l a r d ( 1 9 7 6 ) . M o r e l and M o r e l - L a u r e n s (1983) s u g g e s t t h a t t h e d e l a y i n t h e d e t r i m e n t a l e f f e c t of a m e t a l c o u l d be due t o t h e e x h a u s t i o n o f an i m p o r t a n t n u t r i e n t p o o l w i t h i n t h e c e l l . In t h i s c a s e , Cu may be a c t i n g t o i n h i b i t t h e u p t a k e a n d / o r a s s i m i l a t i o n o f an e s s e n t i a l n u t r i e n t s u c h as s i l i c i c a c i d ( R u e t e r and M o r e l , . 1 9 8 1 ) . The d e t r i m e n t a l e f f e c t s o f Cu t o any o r g a n i s m a r e c a u s e d by i t s a b i l i t y t o b i n d t o o r g a n i c l i g a n d s and c hange i t s f u n c t i o n a l n a t u r e . I n p h y t o p l a n k t o n s u c h as T h a l a s s i o s i r a p s e u d o n a n a , Cu r e a c t s w i t h f u n c t i o n a l g r o u p s ( e . g . c a r b o x y l , amino and s u l f h y d r y l g r o u p s ) on t h e o u t e r s u r f a c e of t h e c e l l ( D a v i e s , 1978). F i s h e r and J o n e s (1981) e x a m i n e d t h e r e l a t i v e t o x i c i t y of Cu (among o t h e r m e t a l s ) t o t h e m a r i n e d i a t o m A s t e r i o n e l l a j a p o n i c a and c o r r e l a t e d t h e s e d a t a w i t h t h e s u l f u r r e a c t i v i t y o f t h e m e t a l s . Cu, w h i c h i s one o f t h e most s u l f u r r e a c t i v e m e t a l s , was f o u n d t o be t h e most t o x i c t o t h e d i a t o m . Once bound t o t h e s u l f h y d r y l g r o u p s , Cu i s t h o u g h t t o d e c o u p l e c e l l d i v i s i o n and t h e p h o t o s y n t h e t i c a p p a r a t u s ( F i s h e r e t a l . , 1981). O v e r n e l l 15 F i g u r e 2. The e f f e c t o f Cu on c e l l c o n c e n t r a t i o n o v e r t i m e A 160 nM Cu. B a r s a r e p r e s e n t when ±2 s t a n d a r d d e v i a t i o n s e x c e e d t h e s i z e o f t h e s y m b o l . 16 (1975) has f o u n d t h a t once i n s i d e t h e c e l l , Cu c a n l i m i t -b o t h t h e H i l l and m o d i f i e d M e h l e r r e a c t i o n s w h i c h measures p h o t o s y s t e m I I and p h o t o s y s t e m I a c t i v i t i e s . 2. Response o f t h e b i o a s s a y o r g a n i s m t o Cu and Mn In t h e f i r s t s e r i e s o f e x p e r i m e n t s , t h e r e s p o n s e of t h e t e s t o r g a n i s m t o Cu i n t h e p r e s e n c e of a f i x e d l e v e l of Mn was d e t e r m i n e d . Cu was added t o A q u i l i n two s e t s o f f l a s k s t o o b t a i n a f i n a l c o n c e n t r a t i o n of 100 nM. No Mn was added t o one s e t of f l a s k s w h i l e i n a s e c o n d s e t , Mn was added t o g i v e a f i n a l l e v e l of 1000 nM. A c o n t r o l s e t c o n t a i n e d A q u i l l e v e l s o f Mn (28.3 nM) and Cu (1 nM). The r e s p o n s e of T. pseudonana t o Cu i n t h e p r e s e n c e , and a b s e n c e of Mn i s shown as a d i f f e r e n c e i n g r owth r a t e ( F i g . 3 ) . The c u l t u r e s w i t h o u t t h e Mn s p i k e showed a r e d u c t i o n i n g r o w t h r a t e a f t e r a d e l a y o f 2 d a y s , whereas t h e c u l t u r e s w i t h t h e Mn s p i k e and t h e c o n t r o l s showed s i m i l a r g r o w t h r a t e s . The e f f e c t o f Mn on r e d u c i n g Cu t o x i c i t y t o T. pseudonana was f u r t h e r i n v e s t i g a t e d i n t h e s e c o n d s e r i e s o f e x p e r i m e n t s i n w h i c h b o t h t h e Cu and Mn c o n c e n t r a t i o n s were v a r i e d . F o r e a c h e x p e r i m e n t , 4 l i t e r s of A q u i l were p r e p a r e d : 3 l i t e r s were u s e d t o t e s t t h e Mn c o n c e n t r a t i o n , and 1 l i t e r was u s e d as a c o n t r o l i n w h i c h t h e Mn c o n c e n t r a t i o n was a t A q u i l l e v e l s (28.3 nM). Mn was added t o t h e 3 l i t e r s o f medium, w h i c h was d i v i d e d i n t o t h e f l a s k s p r i o r t o t h e a d d i t i o n o f Cu s p i k e s . S i x Mn c o n c e n t r a t i o n s 17 F i g u r e 3. The e f f e c t of Cu on c e l l c o n c e n t r a t i o n i n t h e p r e s e n c e o f Mn Symbols: © c o n t r o l (0.1 nM Cu, V l O O ^ M C u ^ C U W l t h 1 0 0 0 n M M n ; 18 ( 1 , 10, 50, 100, 500 and 1000 nM) a t e a c h of s e v e n Cu c o n c e n t r a t i o n s ("0", 20, 40, 60, 80, 100 and 120 nM) were t e s t e d . C o m p a r i s o n of t h e g r o w t h r a t e s between t h e c o n t r o l s and t h e b i o a s s a y s w i t h o u t t h e e x t r a Cu added e n s u r e d t h a t t h e Mn l e v e l t e s t e d was n o t l i m i t i n g t o t h e g r o w t h o f t h e t e s t o r g a n i s m . The a l l e v i a t i o n of Cu t o x i c i t y by Mn seems t o be d e p e n d e n t on t h e amount o f Mn p r e s e n t ( F i g . 4 ) . The h i g h e r t h e l e v e l o f Mn added t o t h e b i o a s s a y medium, t h e l e s s d e t r i m e n t a l t h e e f f e c t e x c e s s Cu had on t h e g r o w t h r a t e . However, a t h i g h c o n c e n t r a t i o n s of Cu (120 nM), t h e e f f e c t of t h e Mn seems t o be r e d u c e d . Such an e f f e c t has a l r e a d y been r e p o r t e d f o r C h a e t o c e r o u s s o c i a l i s (Sunda e t a l . , 1981), T h a l a s s i o s i r a pseudonana and T. o c e a n i c a (Sunda and Huntsman, 1983). Sunda e t a l . (1981) and Sunda and Huntsman (1983) b e l i e v e t h a t t h e r e d u c t i o n o f Cu t o x i c i t y by Mn i s p u r e l y a p h y s i o l o g i c a l r e s p o n s e o f t h e o r g a n i s m and n o t due t o t h e a d s o r p t i o n o f Cu o n t o manganese o x i d e s . Mn i s t h o u g h t t o r e d u c e t h e t o x i c i t y o f Cu by e i t h e r p r o t e c t i n g p o t e n t i a l t a r g e t s i t e s w i t h i n t h e c e l l o r c o m p e t i n g f o r t r a n s p o r t s i t e s a c r o s s t h e c e l l membrane. F o r example, Mn has been f o u n d t o r e v e r s e Cu i n h i b i t i o n o f t h e H i l l and M e h l e r r e a c t i o n s i n i s o l a t e d c h l o r o p l a s t s (Habermann, 1969). The r e v e r s a l was b e l i e v e d t o be c a u s e d by Mn p r o t e c t i n g t h e e n z y m a t i c s i t e s i n t h e c h l o r o p l a s t s from Cu. Mn may a l s o compete w i t h Cu f o r t h e same u p t a k e s i t e on t h e s u r f a c e o f 19 o o .0 48.0 72.0 TOTAL CU ADDED (NM) F i g u r e 4. The e f f e c t of v a r y i n g Mn c o n c e n t r a t i o n s on Cu t o x i c i t y i n E D T A - f r e e A q u i l . S y m b o l s : A 1000 nM Mn; * 500 nM Mn; O 100 nM Mn; X 50 nM Mn; + 1 0 nM Mn; © 1 nM Mn. V e r t i c a l b a r s p r e s e n t when ±2 s t a n d a r d d e v i a t i o n s e x c e e d t h e s i z e of t h e s y m b o l . 20 t h e c e l l . A s i m i l a r c o m p e t i t i o n f o r u p t a k e s i t e s has been f o u n d f o r Cu and Zn, i n w h i c h an i n c r e a s e i n z i n c a c t i v i t y i n t h e medium c a u s e d a d r o p i n t h e amount o f Cu t a k e n up p e r c e l l i n c u l t u r e s o f T h a l a s s i o s i r a p s e u d o n a n a ( R u e t e r and M o r e l , 1981). S i m i l a r l y , t h e a d d i t i o n of Fe t o C d - s t r e s s e d c u l t u r e s of T h a l a s s i o s i r a w e i s f l o q i i was f o u n d t o r e v e r s e th e i n h i b i t i o n o f g r o wth r a t e i n i t i a l l y c a u s e d by Cd t o x i c i t y ( F o s t e r and M o r e l , 1982). The c o m p e t i t i o n among m e t a l s f o r u p t a k e s i t e s e i t h e r i n t h e c e l l , o r on t h e c e l l s u r f a c e , may be p a r t i a l l y due t o th e n a t u r e of t h e u p t a k e s i t e and t o t h e m e t a l ' s c h e m i c a l c h a r a c t e r i s t i c s . N i e b o e r and R i c h a r d s o n (1980) p l o t t e d a c o v a l e n t i n t e r a c t i o n p a r a m e t e r a g a i n s t an e l e c t r o s t a t i c i n t e r a c t i o n p a r a m e t e r t o g r o u p t h e e l e m e n t s a c c o r d i n g t o t h e i r b o n d i n g c h a r a c t e r i s t i c s . B o t h Cu ( I I ) and Mn ( I I ) were t e r m e d " m i c r o n u t r i e n t " m e t a l s w h i c h form weak e l e c t r o s t a t i c and c o v a l e n t bonds t o O-donors s u c h as c a r b o x y l a t e and p h o s p h o d i e s t e r g r o u p s . Cu ( I ) was termed a " t o x i c and n o n - e s s e n t i a l " m e t a l w h i c h forms s t r o n g , p r e d o m i n a n t l y c o v a l e n t bonds t o S- or N - d o n o r s , s u c h as s u l f h y d r y l g r o u p s and amino a c i d s . Sunda e t a_l. (1981) b e l i e v e t h a t a t h i g h Cu:Mn r a t i o s , t h e d e t r i m e n t a l e f f e c t o f Cu t o p h y t o p l a n k t o n i s c a u s e d by t h e p r e s e n c e o f two t y p e s of u p t a k e s i t e s on t h e c e l l ; t h e f i r s t i s t h o u g h t t o be a f f e c t e d by b o t h Mn and Cu w h i l e t h e s e c o n d i s a f f e c t e d s o l e l y by Cu. The f i r s t t y p e o f u p t a k e s i t e c h a r a c t e r i s e d 21 by Sunda e t a l . (1981) may p o s s i b l y have O - d o n a t i n g f u n c t i o n a l g r o u p s t o w h i c h b o t h Cu ( I I ) and Mn ( I I ) can b i n d . A t e l e v a t e d l e v e l s o f Cu, t h e Cu ( I I ) may d i s p l a c e t h e Mn, w h i c h u s u a l l y b i n d s more w e a k l y t o t h e f u n c t i o n a l g r o u p s . S i m i l a r l y , t h e s e c o n d t y p e of u p t a k e s i t e may have S- o r N - d o n a t i n g f u n c t i o n a l g r o u p s t o w h i c h Cu ( I ) can s t r o n g l y b i n d ; a t h i g h c o n c e n t r a t i o n s of Cu, Cu ( I ) may b l o c k o r m o d i f y t h e f u n c t i o n a l g r o u p s on t h e s u r f a c e o f t h e c e l l . The d e t r i m e n t a l e f f e c t s o f Cu t o p h y t o p l a n k t o n a t h i g h Cu:Mn r a t i o s may t h e r e f o r e be c a u s e d by t h e Cu d i s p l a c i n g t h e Mn a t t h e f i r s t s i t e , and by p o s s i b l y a l t e r i n g t h e f u n c t i o n a l g r o u p s a t t h e s e c o n d s i t e . 22 I I . TESTING OF A CATION-EXCHANGE RESIN TECHNIQUE TO ESTIMATE THE BIOLOGICALLY ACTIVE CU AND MN INTRODUCTION Io n - e x c h a n g e r e s i n s have been u s e d e x t e n s i v e l y f o r t h e a n a l y t i c a l measurements of t r a c e m e t a l s i n n a t u r a l w a t e r s ( e . g . F i l b y e t a l . , 1974; F l o r e n c e and B a t l e y , 1976). An a d v a n t a g e o f t h e t e c h n i q u e , e s p e c i a l l y when w o r k i n g w i t h v e r y low m e t a l l e v e l s , i s t h e a b i l i t y of t h e r e s i n t o c o n c e n t r a t e t h e m e t a l s i n a g i v e n s a mple. When a l a r g e volume of a d i l u t e m e t a l s o l u t i o n i s p a s s e d t h r o u g h t h e r e s i n , t h e a d s o r b e d m e t a l s c a n be r e c o v e r e d i n a r e l a t i v e l y s m a l l volume of e l u a t e and r e d u c t i o n s i n .volume o f s e v e r a l o r d e r s o f m a g n i t u d e a r e t h e r e f o r e p o s s i b l e . The t e c h n i q u e a l s o i n v o l v e s l i t t l e h a n d l i n g o f t h e s a m p l e s , t h u s t h e c h a n c e s f o r c o n t a m i n a t i o n by t r a c e m e t a l s c a n be m i n i m i z e d . The a d s o r p t i o n o f m e t a l s o n t o t h e i o n - e x c h a n g e r e s i n i s a f f e c t e d by t h e d e g r e e o f c r o s s - l i n k a g e , t h e i o n - e x c h a n g e c a p a c i t y and t h e mesh s i z e o f t h e r e s i n ( D o r f n e r , 1972). I t i s a l s o i n f l u e n c e d by t h e s i z e and c h a r g e of t h e m e t a l i o n as w e l l as t h e c o n c e n t r a t i o n and t h e n a t u r e of l i g a n d s i n t h e sample ( I n c z e d y , 1966). As t h e c o n c e n t r a t i o n o f l i g a n d s i n s o l u t i o n i n c r e a s e s , more m e t a l s w i l l t e n d t o be c p m p l e x e d and l e s s w i l l be a v a i l a b l e f o r a d s o r p t i o n o n t o t h e r e s i n . U s u a l l y , t h o s e m e t a l s n o t bound o r w e a k l y bound by l i g a n d s a r e more l i k e l y t o be a d s o r b e d by t h e i o n - e x c h a n g e r e s i n . 2 3 C o n s e q u e n t l y , r e s i n s s u c h as C h e l e x - 1 0 0 have been u s e d t o d e t e r m i n e m e t a l c o m p l e x a t i o n c a p a c i t y ( e . g . B a c c i n i and S u t e r , 1979) and t h e t r a c e m e t a l s p e c i a t i o n ( e . g . F i g u r a and M c D u f f i e , 1979; F l o r e n c e , 1982b) of n a t u r a l w a t e r s . However, i t has been r e c e n t l y r e p o r t e d t h a t t r a c e metal-EDTA c h e l a t e s c a n a d s o r b o n t o C h e l e x - 1 0 0 (Sunda, 1984) and m e t a l - l i g a n d c o m p l e x e s w i t h a r e s i d u a l p o s i t i v e c h a r g e ( e . g . C u - h i s t i d i n e ) have a l s o been f o u n d t o a d s o r b o n t o a c a t i o n - e x c h a n g e r e s i n ( Z o r k i n , 1983; Z o r k i n e t a l . , i n p r e s s ) . T h i s c o u l d p o s s i b l y l e a d t o an o v e r e s t i m a t i o n of t h e l e v e l s o f f r e e m e t a l i o n , w h i c h a r e t h o u g h t t o be t h e most b i o l o g i c a l l y a v a i l a b l e . E q u i l i b r a t i o n between t h e t e s t s o l u t i o n and a s t r o n g l y a c i d i c c a t i o n - e x c h a n g e r e s i n (Dowex AG 50W-X12) was u s e d i n t h e p r e s e n t s t u d y t o e s t i m a t e t h e c o n c e n t r a t i o n of b i o l o g i c a l l y a v a i l a b l e Cu and Mn i n a r t i f i c i a l and n a t u r a l s e a w a t e r . The t h e o r y and t e c h n i q u e f o r t h e use o f t h i s r e s i n a r e g i v e n i n Z o r k i n (1983) and Z o r k i n e t a l . ( i n p r e s s ) . To e n s u r e t h a t t h e r e s i n a d s o r b e d o n l y t h e i o n i c s p e c i e s , Z o r k i n (1983) added o r g a n i c l i g a n d s s u c h a s EDTA, GLU and NTA t o s e a w a t e r samples c o n t a i n i n g Cu and a n a l y s e d t h e f i n a l e l u a t e f o r l e v e l s o f i o n i c m e t a l . As t h e l i g a n d c o n c e n t r a t i o n i n t h e s e a w a t e r s a m p l e s o r t h e s t a b i l i t y c o n s t a n t s o f t h e Cu b i n d i n g l i g a n d s i n c r e a s e d , t h e amount of Cu a d s o r b e d o n t o t h e r e s i n was d e c r e a s e d . The a d s o r p t i o n of Cu t o t h e r e s i n was t h e r e f o r e c o n c l u d e d t o be c o n t r o l l e d by 24 t h e f r a c t i o n o f t h e t o t a l m e t a l c o n c e n t r a t i o n n o t c o m p l e x e d by o r g a n i c l i g a n d s . M o r e o v e r , a s t r o n g r e l a t i o n s h i p ( r = 0.92, p<0.05) between t h e b i o l o g i c a l l y e f f e c t i v e Cu c o n c e n t r a t i o n (ECC) (an e x p r e s s i o n of t h e amount of Cu a d s o r b e d o n t o t h e r e s i n ) and g r o w t h r a t e s o f T h a l a s s i o s i r a  p s e u d o n a n a was f o u n d t o o c c u r f o r a l l c o m b i n a t i o n s of Cu and l i g a n d c o n c e n t r a t i o n s s t u d i e d e x c e p t l i g a n d s w h i c h were w e a k l y c h a r g e d and d i d not c o m p l e t e l y c a n c e l t h e c h a r g e o f t h e c u p r i c i o n . The amount of i n o r g a n i c Cu i n s o l u t i o n (Cu i n o r g ) and 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 amount of Cu a d s o r b e d o n t o t h e r e s i n ( [ C u ] ) c a n be e x p r e s s e d by t h e e q u a t i o n : [Cu i n o r g ] = [ C u ] x X i n o r g where X i n o r g i s t h e v a l u e of t h e d i s t r i b u t i o n c o e f f i c i e n t o f Cu d e r i v e d f r o m measurements on a r t i f i c i a l s e a w a t e r of t h e same pH and s a l i n i t y ( Z o r k i n e_t a l . , i n p r e s s ) . T h e r e i s a l i m i t a t i o n t o t h e t e c h n i q u e , however, as p o s i t i v e l y c h a r g e d Cu c o m p l e x e s ( s u c h as t h o s e formed w i t h HIS) were a l s o f o u n d t o a d s o r b o n t o t h e r e s i n . In n a t u r a l w a t e r s , w e a k l y c h a r g e d l i g a n d s ( e . g . ammonia and amino a c i d s ) c a n form p o s i t i v e l y c h a r g e d c o m p l e x e s w i t h Cu and may be i m p o r t a n t i n some p l a c e s s u c h a s sewer o u t f a l l s ( C l a r k e t a l . , 1972). However, i n most a r e a s t h e m a j o r i t y o f t h e m e t a l l o - o r g a n i c c o m p l e x e s i n n a t u r a l w a t e r s has been f o u n d t o have a n e g a t i v e c h a r g e ( F l o r e n c e and B a t l e y , 1980). C o n s e q u e n t l y , 25 t h e a d s o r p t i o n of p o s i t i v e l y c h a r g e d c o m p l e x e s o n t o t h e r e s i n i s c o n s i d e r e d t o be r e l a t i v e l y u n i m p o r t a n t . In t h i s c h a p t e r , t h e a d s o r p t i o n of Cu and Mn t o t h e r e s i n (Dowex AG 50W-X12) i n a r t i f i c i a l s e a w a t e r i s e xamined t o d e t e r m i n e t h e sample volume r e q u i r e d f o r e q u i l i b r a t i o n , t h e c o n c e n t r a t i o n o f m e t a l s t o be added t o t h e s e a w a t e r s a m p l e s , and t h e e f f e c t of n u t r i e n t s ( i n p a r t i c u l a r Fe) on t h e e f f i c i e n c y o f t h e r e s i n . 26 MATERIALS AND METHODS 1. Sample p r e p a r a t i o n Cu and Mn s t o c k s were p r e p a r e d d a i l y f r o m s t o c k s o f 1 x 10" 3M C u C l 2 i n 1% HN0 3 and 1 x 10" 3M M n C l 2 i n 1% HN0 3. An i r o n s t o c k of 5 x 10"*M, when needed, was made by d i s s o l v i n g 0.135 g of F e C l 3 . 6 H 2 0 i n 1 l i t e r o f GDW, t h e n l e f t t o e q u i l i b r a t e f o r 2-3 h o u r s b e f o r e u s e . The a r t i f i c i a l s e a w a t e r s a m p l e s were composed of SOW (made i n t h e same manner as d e s c r i b e d p r e v i o u s l y ) t o w h i c h t h e a p p r o p r i a t e m e t a l s were a d d e d . The p r e p a r e d s a m p l e s were a l w a y s l e f t f o r 2-4 h o u r s t o e q u i l i b r a t e b e f o r e p a s s i n g them t h r o u g h t h e r e s i n c o l u m n s . 2. Column o p e r a t i o n (a) M a t e r i a l s A Dowex-50 c a t i o n - e x c h a n g e r e s i n ( B i o - R a d L a b o r a t o r i e s , AG 50W-X12, 200-400 mesh) i n t h e h y d r o g e n f o r m was u s e d i n t h e s e e x p e r i m e n t s . T h i s r e s i n i s composed o f a s t y r e n e d i v i n y l b e n zene c o p o l y m e r l a t t i c e t o w h i c h s u l f o n i c a c i d g r o u p s (R-SO3-) a r e a t t a c h e d . The i o n e x c h a n g e c a p a c i t y , when c o m p l e t e l y d r y , i s 5.0 m i l l e q u i v a l e n t s / g r a m (Dow C h e m i c a l Company, 1958). 27 The a p p a r a t u s c o n s i s t e d o f r e s i n c o l u m n s made f r o m 12 x 75 mm p o l y p r o p y l e n e t e s t t u b e s w i t h t h e bottoms c u t o f f and b o t h ends f i t t e d w i t h p o l y p r o p y l e n e column t i p s . The g r i d s u p p o r t f o r t h e r e s i n was made of f r i t t e d p o l y e t h y l e n e . B o t h t h e column t i p s and t h e g r i d s u p p o r t were f r o m d i s a s s e m b l e d E c o n o - c o l u m n s ® ( B i o - R a d L a b o r a t o r i e s , Richmond, Ca., U.S.A.) of 1.0 cm ( i . d . ) x 5.0 cm s i z e . P o l y p r o p y l e n e s y r i n g e s (60 c c ) , u s e d f o r a c i d c l e a n i n g o f t h e s y s t e m and t h e e q u i l i b r a t i o n w i t h SOW, were c o n n e c t e d t o t h e columns w i t h p o l y p r o p y l e n e 3-way L u e r L o c k s ( F i g . 5 ) . The samp l e s were d e l i v e r e d t o t h e r e s i n v i a p o l y p r o p y l e n e m i c r o t u b i n g ( i . d . 1.19 mm) c o n n e c t e d t o t h e c a p s o f 500 ml N a l g e n e p o l y p r o p y l e n e s c r e w c a p b o t t l e s i n t o w h i c h t h e samples were p l a c e d . A n o t h e r p i e c e of p o l y p r o p y l e n e t u b i n g l e a d from t h e c a p s o f t h e b o t t l e s t o t h e N i t r o g e n gas l i n e . The f l o w o f t h e s a m p l e s was m a i n t a i n e d u n d e r p r e s s u r e o f ab o u t 1 p s i f i l t e r e d ( g l a s s f i b e r f i l t e r ) N 2 g a s . A l l p l a s t i c w a r e u s e d i n t h e e x p e r i m e n t s was i n i t i a l l y s o a k e d i n 1N r e a g e n t g r a d e HC1 f o r 3 d a y s t h e n r i n s e d 3 t i m e s w i t h GDW. S u b s e q u e n t c l e a n i n g c o n s i s t e d of a c i d w a s h i n g w i t h 1N r e a g e n t g r a d e HC1 f o r a few h o u r s , p r i o r t o r i n s i n g 3 t i m e s w i t h GDW. 28 chamber for ocid cleaning and equilibration of resin sample tube delivery tube resin Figure 5. Resin column 29 (b) P r e p a r a t i o n of t h e r e s i n To remove any o r g a n i c s t h e r e s i n was f i r s t washed w i t h m e t h a n o l t h e n r i n s e d 3 t i m e s w i t h GDW. The r e s i n was t h e n s l u r r i e d w i t h 1 N i s o p i e s t i c a l l y d i s t i l l e d HC1 t o c o n v e r t t h e r e s i n i n t o t h e p r o t o n a t e d form, and t h e n r i n s e d 3 t i m e s w i t h GDW. F i n a l l y , t h e r e s i n was d r i e d a t 100°C u n t i l no change i n w e i g h t was d e t e c t e d , t h e n s t o r e d i n a p o l y p r o p y l e n e s c r e w c a p p e d b o t t l e i n a d e s s i c a t o r u n t i l u s e . A p p r o x i m a t e l y 40 g o f r e s i n was p r e p a r e d a t one t i m e . F o r t h e e x p e r i m e n t s , t h e d r i e d r e s i n was w e i g h e d (0.75 o r 1.0 g ) , s l u r r i e d i n a b o u t 5 ml of GDW, and p o u r e d i n t o i n d i v i d u a l c o l u m n s . (c) Column p r o c e d u r e Any a i r b u b b l e s i n t h e column were removed by a d d i n g a b o u t 0.5 ml GDW and s t i r r i n g t h e r e s i n w i t h a g l a s s r o d . The r e s i n c o l u m n s were c l e a n e d o f t r a c e m e t a l s and c o n v e r t e d t o t h e h y d r o g e n form by f i r s t p a s s i n g 5 ml o f 3N i s o p i e s t i c a l l y d i s t i l l e d H C l t h r o u g h t h e c o l u m n s . T h i s was f o l l o w e d by a 5 ml r i n s e of GDW. F i f t y ml o f SOW (pH = 8.0±0.05) was t h e n p a s s e d t h r o u g h t h e columns t o r a i s e t h e pH t o 8.0±0.05 and t o a l l o w t h e s a l t s t o come i n t o e q u i l i b r i u m w i t h t h e r e s i n . The samples (500 ml) were t h e n p a s s e d t h r o u g h t h e columns a t a f l o w r a t e o f a b o u t 6 ml/min. A l l o f t h e e f f l u e n t p a s s i n g t h r o u g h t h e r e s i n was d i s c a r d e d . 30 • (d) E l u t i o n o f t h e co l u m n s EDTA and n i t r i l o t r i a c e t i c a c i d (NTA) s o l u t i o n s were u s e d t o e l u t e t h e c o l u m n s . T h e s e were made w i t h A n a l a r g r a d e EDTA ( d i s o d i u m s a l t ) o r NTA ( t r i s o d i u m s a l t , m o n o h y d r a t e ) added t o GDW t o make s o l u t i o n s w i t h a f i n a l c o n c e n t r a t i o n of 1 x 10" 2M. When needed, an EDTA s o l u t i o n of 3 x 10" 2M was p r e p a r e d i n t h e same manner. P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d t h a t t h e s e s o l u t i o n s were more e f f e c t i v e i n e l u t i n g t h e m e t a l s from t h e r e s i n a t low pH. C o n s e q u e n t l y , t h e pH of t h e NTA was a d j u s t e d t o 5.5-6.5 ( f r o m an o r i g i n a l pH o f 11.0) u s i n g i s o p i e s t i c a l l y d i s t i l l e d 3N HC1. The pH o f t h e EDTA s o l u t i o n was l e f t u n a d j u s t e d a t 4.8. Mn To measure t h e amount of Mn a d s o r b e d t o t h e r e s i n , 20 ml a l i q u o t s o f I x t O " 2 M EDTA were u s e d t o e l u t e t h e c o l u m n s . The Mn c o n c e n t r a t i o n i n t h e e l u a t e was t h e n d e t e r m i n e d by G r a p h i t e f u r n a c e a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y (GFAAS). ( P r e l i m i n a r y e x p e r i m e n t s showed t h a t most of t h e Mn was removed a f t e r t h e f i r s t 10 ml of EDTA had p a s s e d t h r o u g h t h e r e s i n . ) 31 Cu P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d more s e n s i t i v e GFAAS Cu r e a d i n g s when t h e r e s i n was e l u t e d w i t h 1 x 10" 2M NTA t h a n when i t was e l u t e d w i t h 1 x 10" 2M EDTA. To measure t h e amount o f Cu a d s o r b e d t o t h e r e s i n , 20 ml o f 1 x 10" 2M NTA was p a s s e d t h r o u g h t h e r e s i n . (The f i r s t 10 ml o f NTA t h a t p a s s e d t h r o u g h t h e r e s i n was f o u n d t o remove most o f t h e a d s o r b e d Cu.) The NTA was a l s o e f f e c t i v e f o r r e m o v i n g Mn f r o m t h e r e s i n a n d was l a t e r u s e d f o r e l u t i o n o f b o t h m e t a l s . The e l u a t e s a m p l e s were s t o r e d i n 30 ml p o l y p r o p y l e n e N a l g e n e s c r e w c a p b o t t l e s and a n a l y z e d by GFAAS. 3. D e t e r m i n a t i o n o f e l u t e d m e t a l s w i t h GFAAS The amount o f m e t a l s i n t h e e l u a t e was d e t e r m i n e d by d i r e c t i n j e c t i o n o f t h e e l u a t e i n t o a P e r k i n - E l m e r 560 a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e r , w i t h a P e r k i n - E l m e r HGA g r a p h i t e f u r n a c e , c o u p l e d t o a P e r k i n - E l m e r HGA 400 p r o g r a m m e r . Cu- a n d M n - e n r i c h e d EDTA o r NTA s o l u t i o n s f o r m e d t h e s t a n d a r d s o l u t i o n s , w i t h t h e same EDTA and NTA c o n c e n t r a t i o n a s u s e d f o r e l u t i n g t h e c o l u m n s . The Cu and Mn s t a n d a r d s were u s e d t o draw c a l i b r a t i o n c u r v e s f r o m w h i c h t h e c o n c e n t r a t i o n s o f t h e s a m p l e s c o u l d be e s t i m a t e d u s i n g l i n e a r r e g r e s s i o n a n a l y s i s . T h r e e a b s o r b a n c e l e v e l s were 32 o b t a i n e d f o r e a c h sample and s t a n d a r d . To d e t e c t any d e t e r i o r a t i o n o f t h e g r a p h i t e t u b e d u r i n g sample a n a l y s i s , t h e f i r s t two r e a d i n g s were t a k e n i n s u c c e s s i o n , w h i l e t h e t h i r d was t a k e n a f t e r a t l e a s t one a n a l y s i s o f t h e s a m p l e s . Mn A sample volume of 10 u l was i n j e c t e d i n t o a p y r o l y t i c a l l y c o a t e d g r a p h i t e t u b e . The 279.5 nm r e s o n a n c e l i n e o f a V a r i a n T e c h t r o n Mn h o l l o w c a t h o d e lamp run a t a c u r r e n t of 12 mA was u s e d . The sample was s l o w l y d r i e d a t t e m p e r a t u r e s of 100, 120 and 500°C f o r 35 s e c , c h a r r e d a t 1000°C f o r 15 s e c , t h e n a t o m i z e d a t 2700°C f o r 5 s e c . Cu An i n j e c t i o n volume of 25 u l was used on t h e GFAAS t o d e t e r m i n e Cu l e v e l s i n t h e s a m p l e s . The 324.7 nm r e s o n a n c e l i n e of a V a r i a n T e c h t r o n h o l l o w c a t h o d e Cu lamp ru n a t a c u r r e n t o f 13 mA was u s e d . The sample was d r i e d a t t e m p e r a t u r e s o f 110 and 150°C f o r 30 s e c , c h a r r e d a t 1000°C f o r 10 s e c , t h e n f i n a l l y a t o m i z e d a t 2700°C f o r 5 s e c . 33 RESULTS AND,DISCUSSION 1. C h a r a c t e r i z a t i o n o f t h e r e s i n Dowex AG 50W-X12 (a) E q u i l i b r a t i o n o f t h e r e s i n t o Cu and Mn An i m p o r t a n t c h a r a c t e r i s t i c t h a t must be d e t e r m i n e d i s t h e sample volume n e c e s s a r y f o r t h e r e s i n t o come i n t o e q u i l i b r i u m w i t h t h e m e t a l s i n t h e sample. D i f f e r e n t sample volumes o f SOW c o n t a i n i n g 100 nM Mn were p a s s e d t h r o u g h t h e r e s i n t o d e t e r m i n e t h e volume r e q u i r e d f o r e q u i l i b r a t i o n . The sample volumes were i n c r e a s e d from 50 t o 500 ml i n 50 ml i n c r e m e n t s . E l u a t e Mn l e v e l s were f o u n d t o r i s e ( F i g . 6) up t o 300 ml, w i t h l i t t l e c hange t o 500 m l , i n d i c a t i n g t h a t r e s i n - M n e q u i l i b r a t i o n had been a c h i e v e d . To d e t e r m i n e t h e sample volume n e c e s s a r y f o r t h e r e s i n t o come i n t o e q u i l i b r i u m w i t h Cu, two e x p e r i m e n t s w i t h 35 p p t SOW c o n t a i n i n g 50 nM Cu were c o n d u c t e d . I n t h e f i r s t t e s t , t h e sample v o l u m e s were i n c r e a s e d f r o m 50 t o 500 ml i n 50 ml i n c r e m e n t s , w h i l e i n t h e s e c o n d t e s t , sample v o l u m e s i n c r e a s e d t o 1000 ml i n 100 ml i n c r e m e n t s . E l u a t e Cu c o n c e n t r a t i o n s were f o u n d t o r i s e up t o 450-500 m l , ( F i g s . 7 & 8 ) , i n d i c a t i n g t h a t r e s i n - C u e q u i l i b r a t i o n had been a c h i e v e d . 34 F i g u r e 6. E l u a t e Mn v e r s u s sample volume r e q u i r e d f o r e q u i l i b r a t i o n . (100 nM Mn i n 35 p p t SOW.) 35 Figure 7. Eluate Cu versus sample volume (50-500 ml) SVired f ° r e ( 3 u i l i b r a t i o n (50 nM Cu in 35 ppt 36 F i g u r e 8. E l u a t e Cu v e r s u s sample volume ( 5 0 - 1 0 0 0 ml) r e q u i r e d f o r e q u i l i b r a t i o n ( 5 0 nM Cu i n 3 5 p p t SOW). 37 The amount of m e t a l a d s o r b e d o n t o t h e r e s i n was fo u n d t o i n c r e a s e a t s a l i n i t i e s below t h e 35 p p t f o u n d i n SOW, p r e s u m a b l y b e c a u s e of r e d u c e d c o m p e t i t i o n w i t h t h e major c a t i o n s f o r b i n d i n g s i t e s on t h e r e s i n ( Z o r k i n , 1983). S i n c e n a t u r a l s e a w a t e r samples t o be u s e d i n t h i s s t u d y have s a l i n i t i e s of a p p r o x i m a t e l y 25 p p t , t h e sample volume r e q u i r e d f o r t h e r e s i n t o come i n t o e q u i l i b r i u m w i t h Cu was t e s t e d a t t h e lo w e r s a l i n i t y . D i f f e r e n t sample volumes between 50 and 800 ml were t e s t e d u s i n g 6 l i t e r s of 25 ppt SOW (35 p p t SOW d i l u t e d w i t h GDW) c o n t a i n i n g 58.3 nM Cu. E l u a t e Cu c o n c e n t r a t i o n s were f o u n d t o i n c r e a s e t o sample v o l u m e s of 500 ml, w i t h l i t t l e change i n a d s o r b e d Cu, i n d i c a t i n g r e s i n - C u e q u i l i b r a t i o n a t a sample volume of a p p r o x i m a t e l y 500 ml ( F i g . 9 ) . C o n s e q u e n t l y , sample volumes of 500 ml were us e d i n a l l o f t h e e x p e r i m e n t s . The sample volume r e q u i r e d f o r t h e e q u i l i b r a t i o n of t h e r e s i n c a n be i n f l u e n c e d by t h e f l o w r a t e o f t h e sample d e l i v e r e d t o t h e r e s i n . A r e l a t i v e l y s m a l l sample volume may be r e q u i r e d f o r e q u i l i b r a t i o n when t h e f l o w r a t e i s f a s t , a s i n c r e a s e d f l o w may e r o d e t h e t h i c k n e s s o f t h e l i q u i d f i l m s u r r o u n d i n g t h e r e s i n b e a d. T h i s w o u l d r e d u c e t h e d i s t a n c e t h a t t h e m o l e c u l e s have t o t r a v e l i n o r d e r t o r e a c t w i t h t h e r e s i n and t h e e q u i l i b r a t i o n o f t h e r e s i n w i t h t h e m e t a l s i n t h e sample c a n t h u s be a c c o m p l i s h e d q u i c k l y and w i t h a s m a l l sample volume ( I n c z e d y , 1966). However, Z o r k i n (1983) f o u n d t h a t f l o w r a t e s e q u a l t o o r above 2.0 38 F i g u r e 9. E l u a t e Cu v e r s u s sample volume (50-800 ml) r e q u i r e d f o r e q u l i b r a t i o n (58.3 nM Cu i n 25 p p t SOW). 39 ml/min did not influence the sample volume required for the e q u i l i b r a t i o n of Dowex AG 50W-X12 (the same resin used in th i s study) with the metals in the sample. Nevertheless, the flow rates in t h i s study were kept f a i r l y constant at approximately 5 to 6 ml/min to reduce the p o s s i b i l i t y of channeling of the solution through the columns at very high flow rates. (b) Cu and Mn adsorption curves In order to determine the amount of Cu and Mn adsorbed onto the resin when natural water samples are used, the resin has to be c a l i b r a t e d with standards of a known composition. The number of standards required for c a l i b r a t i o n can be reduced i f metal levels in the standards and metal levels adsorbed onto the resin show a linear r e l a t i o n s h i p over the range of metal concentrations to be tested. The l i n e a r i t y of the Mn-resin relationship was tested using Mn concentrations of 50, 75, 100, 250 and 500 nM in 35 ppt SOW, with two r e p l i c a t e s run for each concentration. The Mn adsorption curve was found to be l i n e a r , even when 5000 nM Mn was present (Figs. 10 & 13). S i m i l a r l y , the l i n e a r i t y of the Cu-resin relationship was tested using Cu concentrations of 10, 50, 100, 250 and 500 nM in 25 ppt SOW, with two r e p l i c a t e s run for each concentration. The Cu adsorption curve was found to be linear at the low concentrations although the slope of the 40 F i g u r e 10. E l u a t e Mn v e r s u s t o t a l Mn added ( i n 35 p p t SOW). 41 a d s o r p t i o n c u r v e d e c r e a s e d a f t e r 250 nM ( F i g . 1 1 ) , i n d i c a t i n g t h a t t h e r e s i n was n o t as s e n s i t i v e t o Cu a t t h e h i g h e r c o n c e n t r a t i o n s . However, t h e d i f f e r e n t s l o p e of t h e c u r v e a t h i g h Cu c o n c e n t r a t i o n s was e x p e c t e d t o be of l i t t l e p r a c t i c a l i m p o r t a n c e b e c a u s e o f t h e low l e v e l s o f Cu t o be u s e d i n t h i s s t u d y . The s l o p e s of t h e a d s o r p t i o n c u r v e s have a l s o been f o u n d t o d i f f e r s l i g h t l y even under s e e m i n g l y i d e n t i c a l o p e r a t i n g c o n d i t i o n s ( Z o r k i n , 1983). C o n s e q u e n t l y , t h e s t a n d a r d s o l u t i o n s were a l w a y s p a s s e d t h r o u g h t h e columns a t t h e same t i m e and under t h e same o p e r a t i n g c o n d i t i o n s as t h e n a t u r a l water s a m p l e s . ( c ) E f f e c t of Cu on Mn a d s o r p t i o n S i n c e t h e c a t i o n - e x c h a n g e r e s i n w i l l be u s e d t o e s t i m a t e t h e i o n i c s p e c i e s of Cu and Mn i n n a t u r a l w a t e r s a m p l e s , i t was n e c e s s a r y t o t e s t whether Cu and Mn i n t e r f e r e d w i t h one a n o t h e r f o r a d s o r p t i o n o n t o t h e r e s i n . To d e t e r m i n e t h e e f f e c t of Mn on Cu a d s o r p t i o n , 5 l i t e r s o f 35 ppt SOW c o n t a i n i n g 50 nM Cu was d i v i d e d i n t o 500 ml a l i q u o t s and Mn s p i k e s of 100, 500, 1000 and 5000 nM were adde d . E a c h Mn c o n c e n t r a t i o n was r u n i n d u p l i c a t e . The e l u a t e Cu l e v e l s d i d n o t change much o v e r t h e r a n g e o f Mn c o n c e n t r a t i o n s t e s t e d a l t h o u g h t h e r e was a s l i g h t d e c r e a s e i n t h e e l u a t e Cu c o n c e n t r a t i o n when 5000 nM Mn was p r e s e n t ( F i g . 1 2 ) . However, t h i s was c o n s i d e r e d n o t t o be s i g n i f i c a n t a s ±2 s t a n d a r d d e v i a t i o n s f o r a l l t h e d a t a 42 F i g u r e 11. E l u a t e Cu v e r s u s t o t a l Cu added ( i n 25 p p t SOW). 43 (M _ cn" <_D C J l i l m t—°P (M' 1 0.0 ~~l 1 1 0 0 . 0 2 0 0 . 0 TOTRL MN ADDED — i 1 1 3 0 0 . 0 . 4 0 0 . 0 5 0 0 . 0 (NM) (X10 1 ) F i g u r e 12. The e f f e c t o f Mn on Cu a d s o r p t i o n t o t h e r e s i n (100-5000 nM Mn, 50 nM Cu i n 35 p p t SOW). V e r t i c a l b a r s p r e s e n t when ±2 s t a n d a r d d e v i a t i o n s e x c e e d t h e s i z e of t h e symb o l . 44 p o i n t s were f o u n d t o o v e r l a p . When t h e same e l u a t e samples were a n a l y s e d f o r Mn, t h e r e l a t i o n s h i p between t h e amount of Mn a d s o r b e d o n t o t h e r e s i n and t h e t o t a l Mn added was f o u n d t o be l i n e a r , i n d i c a t i n g t h a t t h e p r e s e n c e of 50 nM Cu had l i t t l e e f f e c t on t h e a d s o r p t i o n o f Mn o n t o t h e r e s i n ( F i g . 1 3 ) . C o n s e q u e n t l y , f o r t h e l e v e l s of m e t a l s i n t h e s a m p l e s , i t was c o n c l u d e d t h a t Cu and Mn d i d not i n t e r f e r e w i t h one a n o t h e r f o r a d s o r p t i o n t o t h e r e s i n . (d) E f f e c t of n u t r i e n t s S i n c e t h e n a t u r a l w ater s a m p l e s a r e t o be amended w i t h A q u i l 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 , t h e i n f l u e n c e of add e d n u t r i e n t s was t e s t e d on t h e a d s o r p t i o n o f Cu and Mn t o t h e r e s i n . Cu and Mn were added t o 5 l i t e r s o f 25 p p t SOW t o o b t a i n l e v e l s o f 50 nM Cu and 100 nM Mn. The SOW was t h e n d i v i d e d i n t o f i v e 1 l i t e r a l i q u o t s , w h i c h were t r e a t e d a s f o l l o w s : 1) no a d d i t i o n o f n u t r i e n t s ; 2) a d d i t i o n o f a l l n u t r i e n t s a t A q u i l c o n c e n t r a t i o n s ( s e e A p p e n d i x I , T a b l e 1) e x c e p t f o r s i l i c a t e ( S i ) and i r o n ( F e ) ; 3) a d d i t i o n of o n l y S i a t t h e A q u i l c o n c e n t r a t i o n (1.25 x 10" 5M); 4) a d d i t i o n o f o n l y S i a t 5 t i m e s t h e A q u i l c o n c e n t r a t i o n (6.25 x 10~ 5M); 5) a d d i t i o n o f o n l y Fe a t A q u i l c o n c e n t r a t i o n (4.51 x 10" 7M). Two r e p l i c a t e s were r u n f o r e a c h a d d i t i o n . S i a nd Fe were added t o t h e s e a w a t e r s a m p l e s t o d e t e r m i n e whether t h e amount o f Cu and Mn a d s o r b e d o n t o t h e r e s i n w ould be a f f e c t e d , a s b o t h s i l i c i c a c i d and h y d r a t e d Fe F i g u r e 13. E l u a t e Mn v e r s u s t o t a l Mn added i n t h e o f Cu (50 nM Cu i n 35 p p t SOW). 46 ( I I I ) o x i d e s have been d e m o n s t r a t e d t o d e c r e a s e t h e A S V - l a b i l e Cu i n s e a w a t e r due t o a d s o r p t i o n o n t o c o l l o i d a l S i and Fe p a r t i c l e s (Lumsden and F l o r e n c e , 1983; F l o r e n c e e t a l . , 1983). T a b l e 1 shows t h a t e l u a t e Cu c o n c e n t r a t i o n s d i d n o t d i f f e r when n u t r i e n t s and v a r i o u s l e v e l s o f S i were added t o t h e s a m p l e s . An i n c r e a s e i n e l u a t e Cu was however, o b s e r v e d when t h e n u t r i e n t s t o g e t h e r w i t h A q u i l l e v e l s of Fe (4.51 x 10" 7M) were a d d e d . T a b l e 1. E f f e c t of A q u i l n u t r i e n t s on t h e a d s o r p t i o n of Cu and Mn (50 nM Cu, 100 nM Mn i n 25 ppt SOW) t o Dowex AG 50W-X12 r e s i n . See A p p e n d i x , T a b l e 1 f o r A q u i l n u t r i e n t l e v e l s . Where a p p r o p r i a t e , () = ±2 s t a n d a r d d e v i a t i o n s . T e s t Cu (nM/g r e s i n ) Mn (nM/g r e s i n ) No n u t r i e n t s 7.90 (0.49) 4.27 (0.25) A l l n u t r i e n t s e x c e p t S i and Fe 8.42 (0.25) 3.92 (0.07) A q u i l l e v e l s of S i (1.25 x 10" 5 M) 8.48 (0.33) 3.22 (0.92) 5 t i m e s A q u i l l e v e l s o f S i (6.25 x 10" 5 M) 8.93 (0.16) 3.69 (0.57) A q u i l l e v e l s of Fe (4.51 x 10" 7 M) 11.11 (1.94) 3.87 (0.00) To f u r t h e r t e s t t h e e f f e c t o f Fe on e l u a t e Cu l e v e l s , Cu was added t o 5 l i t e r s o f 25 p p t SOW t o o b t a i n a Cu c o n c e n t r a t i o n o f 50 nM. In t h e f i r s t e x p e r i m e n t , Fe c o n c e n t r a t i o n s o f 10, 50, 100 and 500 nM were t e s t e d , w h i l e 47 i n t h e s e c o n d e x p e r i m e n t , Fe c o n c e n t r a t i o n s of 100, 500, 1000 and 5000 nM were t e s t e d . Two r e p l i c a t e s were run f o r e a c h c o n c e n t r a t i o n . Low l e v e l s o f Fe (10-100 nM) were f o u n d t o have l i t t l e e f f e c t on t h e e l u a t e Cu l e v e l s ( F i g . 14), w h i l e Fe c o n c e n t r a t i o n s between 100 and 1000 nM were f o u n d t o i n c r e a s e t h e e l u a t e Cu l e v e l s d r a m a t i c a l l y ( F i g s . 14 & 1 5 ) . Beyond 1000 nM Fe however, f u r t h e r i n c r e a s e s i n Fe l e v e l s r e s u l t e d i n l i t t l e c hange i n e l u a t e Cu c o n c e n t r a t i o n s ( F i g . 15), i n d i c a t i n g t h a t t h e r i s e i n e l u a t e Cu was p r o b a b l y not due t o Cu c o n t a m i n a t i o n from t h e Fe s t o c k . The r i s e i n e l u a t e Cu a t h i g h Fe l e v e l s i s r a t h e r u n e x p e c t e d s i n c e , a t h i g h Fe c o n c e n t r a t i o n s , Cu i s e x p e c t e d t o a d s o r b o n t o c o l l o i d a l f e r r i c h y d r o x i d e s (Swallow e t a l . , 1980), and made l e s s a v a i l a b l e f o r a d s o r p t i o n o n t o t h e r e s i n . As w e l l , B e n j a m i n and L e c k i e (1980) r e p o r t t h a t w i t h more c o l l o i d s i n t r o d u c e d t o a s o l u t i o n , more m e t a l s were removed from s o l u t i o n by a d s o r p t i o n o n t o c o l l o i d s . The f l o w r a t e s were v i s i b l y s l o w e r i n t h e samples w i t h h i g h (500-5000 nM) Fe c o n c e n t r a t i o n s , p o s s i b l y i n d i c a t i n g t h a t c o l l o i d a l Fe o x i d e s may have become t r a p p e d i n t h e i n t e r s t i c e s between t h e t i g h t l y p a c k e d r e s i n b eads ( I n c z e d y , 1966). T h e s e t r a p p e d Fe o x i d e s may have Cu a d s o r b e d o n t o them, w h i c h may a l s o have been e l u t e d when t h e NTA was p a s s e d t h r o u g h t h e r e s i n . E l l i o t and Huang (1979) f o u n d t h a t u nder n o r m a l s u r f a c e w a t e r c o n d i t i o n s , l e s s Cu ( I I ) was a d s o r b e d o n t o A l 2 0 3 p a r t i c l e s i f t h e NTA:Cu r a t i o i n 48 F i g u r e 14. The e f f e c t of Fe on Cu a d s o r p t i o n t o t h e r e s i n (10-500 nM Fe, 50 nM Cu i n 25 p p t SOW). 49 F i g u r e 15. The e f f e c t o f Fe on Cu a d s o r p t i o n t o t h e r e s i n (100-5000 nM Fe, 50 nM Cu i n 25 p p t SOW). 50 s o l u t i o n was g r e a t e r t h a n 1.0. T h e r e f o r e , t h e h i g h c o n c e n t r a t i o n o f t h e NTA us e d i n t h i s s t u d y may have removed t h e Cu a d s o r b e d o n t o t h e Fe o x i d e s , a s w e l l a s t h e Cu a d s o r b e d o n t o t h e r e s i n , r e s u l t i n g i n t h e h i g h e l u a t e Cu l e v e l s when e l e v a t e d c o n c e n t r a t i o n s of Fe were add e d . S i n c e Fe has s u c h a d r a m a t i c e f f e c t on e l u a t e Cu l e v e l s , t h e e f f e c t o f Fe on e l u a t e Mn l e v e l s was t e s t e d even t h o u g h the l e v e l o f Mn a d s o r b e d o n t o t h e r e s i n d i d not change i n t h e p r e s e n c e of n u t r i e n t s and v a r i o u s amounts of S i and Fe ( T a b l e 1 ) . Mn was add e d t o 5 l i t e r s of 35 p p t SOW t o o b t a i n a c o n c e n t r a t i o n o f 100 nM, and Fe s p i k e s of 10, 50, 100 and 500 nM were add e d . The e l u a t e Mn c o n c e n t r a t i o n s were f o u n d t o r e m a i n unchanged o v e r t h e Fe l e v e l s t e s t e d , e v e n i n t h e p r e s e n c e of 500 nM Fe ( F i g . 16). However, a d e c r e a s e i n t h e f l o w r a t e was a g a i n o b s e r v e d w i t h s a m p l e s c o n t a i n i n g h i g h Fe c o n c e n t r a t i o n s and, as n o t e d p r e v i o u s l y , t h i s was t h o u g h t t o be due t o t h e Fe o x i d e s becoming t r a p p e d by t h e network o f t h e r e s i n . U n l i k e e l u a t e Cu l e v e l s , e l u a t e Mn l e v e l s do n o t i n c r e a s e i n t h e p r e s e n c e o f h i g h c o n c e n t r a t i o n s o f F e . One p o s s i b l e e x p l a n a t i o n i s t h a t a l t h o u g h Mn a d s o r b s o n t o Fe o x i d e s , t h e k i n e t i c s f o r t h e r e a c t i o n between Mn-NTA i s s l o w e r t h a n f o r Cu-NTA. T h e r e f o r e , when t h e NTA i s p a s s e d t h r o u g h t h e r e s i n , t h e e l u a t e Mn l e v e l s w i l l n o t be a f f e c t e d a s o n l y a r e l a t i v e l y s m a l l amount of Mn w i l l be e l u t e d from t h e Fe o x i d e s . 51 o co" CD <\i" CD - — ^ —o U J — c r - ' ZD , — I 1 1 I 0 0 10.0 20.0 30.0 5 0 0 TOTAL FE ADDED (NM) (X10 1 ) F i g u r e 16. The e f f e c t of Fe on Mn a d s o r p t i o n t o t h e r e s i n (10-500 nM Fe, 100 nM Cu i n 35 p p t SOW). 52 I I I . THE EFFECT OF AMBIENT LEVELS OF MN ON CU TOXICITY IN NATURAL WATER SAMPLES INTRODUCTION The b i o l o g i c a l e f f e c t of a m e t a l i n s e a w a t e r has been w e l l e s t a b l i s h e d t o be i n f l u e n c e d by i t s c h e m i c a l s p e c i a t i o n ( e . g . Sunda and G u i l l a r d , 1976; A n d e r s o n and M o r e l , 1978). The s p e c i a t i o n of a m e t a l i s u s u a l l y e s t i m a t e d t h r o u g h e i t h e r t h e o r e t i c a l computer models ( e . g . MINEQL, MINTEQ) or d i r e c t a n a l y s i s of n a t u r a l water s a m p l e s . The 'computer models e s t i m a t e c h e m i c a l s p e c i a t i o n by u s i n g thermodynamic d a t a f o r most of t h e e l e m e n t s and t h e m a j o r r e a c t i o n s o c c u r r i n g between t h e v a r i o u s c h e m i c a l s p e c i e s under s p e c i f i e d c o n d i t i o n s of p r e s s u r e , t e m p e r a t u r e and i o n i c s t r e n g t h ( F l o r e n c e and B a t l e y , 1980). However, t h e thermodynamic d a t a ( e . g . e q u i l i b r i u m c o n s t a n t s ) a r e o f t e n d e t e r m i n e d i n s o l u t i o n s l e s s complex t h a n s e a w a t e r and have t o be a d j u s t e d i n o r d e r f o r them t o be a p p l i e d t o s e a w a t e r , and e r r o r s i n c a l c u l a t i n g t h e c h e m i c a l s p e c i a t i o n may t h u s r e s u l t (Stumm and Morgan, 1981). Most models s u c h a s MINEQL ( W e s t a l l e t a _ l . , 1976) a l s o do n o t i n c l u d e t h e e f f e c t of a d s o r p t i o n o f f r e e i o n s , l i g a n d s and c o m p l e x e s t o o r on s o l i d s s u c h as c o l l o i d a l h y d r a t e d Fe ( I I I ) o x i d e s and s i l i c i c a c i d b e c a u s e t h e r e l e v a n t e q u i l i b r i u m c o n s t a n t s a r e unknown ( R u e t e r e t a l . , 1981). D e s p i t e t h e s e drawbacks however, e q u i l i b r i u m models a r e u s e f u l f o r e s t i m a t i n g t h e 53 c h e m i c a l s p e c i a t i o n i n a c h e m i c a l l y w e l l d e f i n e d s y s t e m , and have o f t e n been u s e d i n s t u d i e s a s s o c i a t i n g t h e s p e c i a t i o n o f a m e t a l w i t h i t s b i o a v a i l a b i l i t y t o o r g a n i s m s ( e . g . A n d e r s o n and M o r e l , 1978; 1982). The c h e m i c a l s p e c i a t i o n of m e t a l s can a l s o be e s t i m a t e d t h r o u g h d i r e c t a n a l y s i s of t h e water sample by e l e c t r o c h e m i c a l t e c h n i q u e s ( u s u a l l y A n o d i c s t r i p p i n g v o l t a m m e t r y ) or c h e m i c a l s e p a r a t i o n methods ( e . g . u l t r a f i l t r a t i o n , i o n - e x c h a n g e and d i a l y s i s ) , or v a r i o u s c o m b i n a t i o n s of t h e s e t e c h n i q u e s ( F l o r e n c e and B a t l e y , 1976; H a r t and D a v i e s , 1981; F l o r e n c e e_t a_l . , 1983). The c h e m i c a l s p e c i e s d e t e r m i n e d by s u c h methods a r e n o r m a l l y o p e r a t i o n a l l y d e pendent on t h e method o f d e t e r m i n a t i o n , c o n s e q u e n t l y i t i s d i f f i c u l t t o compare t h e s e r e s u l t s t o e q u i l i b r i u m p r e d i c t i o n s . However, t h e d i r e c t a n a l y s i s o f w a t e r samples when compared t o t h e o r e t i c a l e q u i l i b r i u m m o d e l s , may g i v e a more r e a l i s t i c e s t i m a t e of c h e m i c a l s p e c i a t i o n and t h e b i o a v a i l a b i l i t y of a m e t a l i n n a t u r a l w a t e r s . T h i s c h a p t e r d i s c u s s e s r e s u l t s f r o m t h e use of a c a t i o n - e x c h a n g e r e s i n t e c h n i q u e , d e v e l o p e d by Z o r k i n (1983) and d e s c r i b e d i n c h a p t e r 2, t o e s t i m a t e t h e b i o l o g i c a l l y a v a i l a b l e Cu i n n a t u r a l water s a m p l e s . The t e c h n i q u e was m o d i f i e d i n t h i s s t u d y f o r a s s e s s i n g Mn i n n a t u r a l w a t e r s . B i o a s s a y s w i t h T h a l a s s i o s i r a p seudonana were a l s o c o n d u c t e d on t h e same water s a m p l e s , and t h e r e s u l t s compared t o t h e 54 amount of Cu and Mn a d s o r b e d o n t o t h e r e s i n t o d e t e r m i n e whether ambient Mn l e v e l s c o u l d a f f e c t t h e c o n c e n t r a t i o n o f b i o l o g i c a l l y a v a i l a b l e Cu and t h u s i n f l u e n c e t h e t o x i c i t y of Cu t o p h y t o p l a n k t o n . The n a t u r a l w ater s a m p l e s were c o l l e c t e d f r o m I n d i a n Arm, a l o c a l m a r i n e f j o r d w i t h h i g h c o n c e n t r a t i o n s of d i s s o l v e d Mn, e s p e c i a l l y i n d eep water ( W h i t f i e l d , 1976). The h i g h c o n c e n t r a t i o n of d i s s o l v e d Mn i n t h e i n l e t i s due p r i m a r i l y t o t h e d e p l e t i o n o f oxygen i n t h e b o t t o m w a t e r s . At low oxygen c o n c e n t r a t i o n s , p a r t i c u l a t e , i n s o l u b l e Mn (IV) o x i d e s u n d ergo a change i n o x i d a t i o n s t a t e and a r e c o n v e r t e d t o s o l u b l e Mn ( I I ) i o n s (Emerson e_t a_l . , 1979). S i n c e I n d i a n Arm c o m p l e t e l y t u r n s o v e r o n l y o c c a s i o n a l l y ( B u r l i n g , 1983), t h e s o l u b l e Mn a c c u m u l a t e s i n t h e d eep w a t e r s of t h e f j o r d , r e s u l t i n g i n t h e e x t r e m e l y h i g h c o n c e n t r a t i o n s of d i s s o l v e d Mn. 55 MATERIALS AND METHODS 1. C h a r a c t e r i s t i c s of I n d i a n Arm The s e a w a t e r s a m p l e s were t a k e n from I n d i a n Arm, a s h a l l o w - s i l l e d f j o r d l o c a t e d n o r t h e a s t o f V a n c o u v e r , B.C. ( F i g . 1 7). The s i l l a t t h e mouth of t h e i n l e t i s o n l y 26 m deep, a l t h o u g h t h e maximum d e p t h of t h e b a s i n i s o v e r 200 m ( D a v i d s o n , 1976). A m u l t i - l a y e r e s t u a r i n e c i r c u l a t i o n o c c u r s i n I n d i a n Arm, w i t h a t h i n o u t f l o w i n g l a y e r of b r a c k i s h w a t e r above an i n f l o w i n g d e n s e r , s a l t i e r l a y e r ( B u r l i n g , 1983). C o m p l e t e m i x i n g o f t h e f j o r d , w h i c h i s i n f l u e n c e d by t h e volume o f f r e s h w a t e r r u n o f f , t i d a l m i x i n g and d e n s i t y o f t h e i n c o m i n g S t r a i t of G e o r g i a w a t e r , o c c u r s o n l y o c c a s i o n a l l y . The p h y s i c a l c h a r a c t e r i s t i c s of I n d i a n Arm have been w e l l documented by D a v i d s o n (1976) and G i l m a r t i n ( 1 9 6 2 ) . 2. C o l l e c t i o n of n a t u r a l s e a w a t e r Seawater from f i v e d e p t h s (10, 50, 100, 150 and 200 m) were c o l l e c t e d a t s t a t i o n IND-2 (49° 23.5' N, 122° 52.5' W) , l o c a t e d a t t h e d e e p e s t p o i n t o f t h e i n l e t . F o r t y l i t e r s f rom e a c h d e p t h were g a t h e r e d u s i n g 8 l i t e r N i s k i n b o t t l e s w i t h t h e e l a s t i c r e p l a c e d by one made from s i l i c o n e r u b b e r . The s a m p l e s were f i l t e r e d t h r o u g h Gelman 196 mm, 0.45 urn f i l t e r s w i t h i n 24 h o u r s a f t e r c o l l e c t i o n . The i n i t i a l 2 56 F i g u r e 17. L o c a t i o n of sample c o l l e c t i o n . 5 7 l i t e r s of f i l t e r e d sample was u s e d as a r i n s e a n d . d i s c a r d e d b e f o r e e a c h c a r b o y was f i l l e d w i t h t h e s a m p l e . The samples were s t o r e d i n 2 0 l i t e r p o l y e t h y l e n e c a r b o y s a t 16°C i n t h e d a r k , and were u s e d w i t h i n 2 weeks t o 3 months a f t e r c o l l e c t i o n . The N i s k i n b o t t l e s , f i l t e r s , f i l t e r h o l d e r s and c a r b o y s were i n i t i a l l y r i n s e d w i t h 6N HC1, s o a k e d i n 1 N HC1 f o r a day, t h e n r i n s e d 3 t i m e s w i t h GDW. 3. A n a l y s i s of n a t u r a l w a t e r s a m p l e s f o r t o t a l d i s s o l v e d Cu and Mn T o t a l d i s s o l v e d Mn i n t h e w ater s a m p l e s c o l l e c t e d f r o m f i v e d e p t h s was d e t e r m i n e d by d i r e c t i n j e c t i o n GFAAS a f t e r a c i d i f y i n g t h e s a m p l e s t o pH 2 w i t h 1 N i s o p i e s t i c a l l y d i s t i l l e d HC1. The Mn c o n c e n t r a t i o n s i n t h e samples were e s t i m a t e d from a s t a n d a r d c u r v e d e r i v e d f r o m GFAAS a n a l y s i s of M n - e n r i c h e d 2 5 p p t SOW. T o t a l d i s s o l v e d Cu i n t h e w a t e r s a m p l e s was d e t e r m i n e d by d i f f e r e n t i a l p u l s e a n o d i c s t r i p p i n g v o l t a m m e t r y (ASV) a f t e r a c i d i f y i n g t h e samples t o pH 2 . 5 w i t h 1N i s o p i e s t i c a l l y d i s t i l l e d HC1. The ASV measurements were made w i t h a P r i n c e t o n A p p l i e d R e s e a r c h C o r p o r a t i o n model 3 7 4 p o l a r o g r a p h i c a n a l y s e r . A m e r c u r y f i l m was p l a t e d o n t o t h e r o t a t i n g T e f l o n g r a p h i t e e l e c t r o d e by u s i n g a p o t e n t i a l between - 0 . 3 V a n d - 0 . 1 5 V w i t h a d e p o s i t i o n t i m e o f 6 0 s e c . Ten ml o f t h e s e a w a t e r sample was p l a c e d i n a c h l o r o s i l a n e - c o a t e d b o r o s i l i c a t e c e l l ( t o r e d u c e m e t a l a d s o r p t i o n t o w a l l s ) and t h e t o t a l Cu was 58 d e t e r m i n e d by p a s s i n g a p o t e n t i a l between - 0 . 9 V and - 0 . 1 5 V t h r o u g h t h e e l e c t r o d e s . The Cu c o n c e n t r a t i o n s i n t h e samples were d e t e r m i n e d by t h e s t a n d a r d a d d i t i o n method. 4. A d a p t a t i o n o f t h e r e s i n method f o r n a t u r a l water s a m p l e s The Dowex AG 50W-X8 r e s i n ( B i o - R a d L a b o r a t o r i e s , 200-400 mesh) u s e d i n t h e e x p e r i m e n t s w i t h n a t u r a l s e a w a t e r samples was p r e p a r e d i n t h e same manner as d e s c r i b e d p r e v i o u s l y f o r Dowex AG 50W-X12. A 20 ml a l i q u o t of 3 x 10" 2M EDTA was u s e d t o remove t h e a d s o r b e d Cu and Mn from t h e r e s i n . The EDTA s o l u t i o n and t h e Cu and Mn s t o c k s were a l l p r e p a r e d i n t h e manner d i s c u s s e d i n c h a p t e r 2. 5 . B i o a s s a y s and r e s i n e x p e r i m e n t s u s i n g n a t u r a l water F o r e a c h e x p e r i m e n t , 15 l i t e r s o f w a t e r from one d e p t h was t r a n s f e r r e d t o a 20 l i t e r p o l y e t h y l e n e c a r b o y . The pH of t h e water was measured and a d j u s t e d t o 8 . 0 0±0.05 by b u b b l i n g f o r 6-12 h o u r s ( d e p e n d i n g on t h e sample) w i t h a c i d - c l e a n e d (1 N H 2 S O „ ) and f i l t e r e d (0.4 urn N u c l e o p o r e ) a i r . A l l p l a s t i c w a r e ( i n c l u d i n g b o t t l e s and c a r b o y s ) was a c i d c l e a n e d i n t h e same manner a s d e s c r i b e d p r e v i o u s l y . The n a t u r a l w ater samples were amended w i t h A q u i l n u t r i e n t s and t r a c e m e t a l mix ( b u t w i t h o u t EDTA o r Mn) t o e n s u r e t h a t t h e y w o u l d n o t become d e f i c i e n t t o t h e b i o a s s a y o r g a n i s m . F r e s h l y p r e p a r e d Fe s t o c k , a l l o w e d t o e q u i l i b r a t e 59 f o r s e v e r a l h o u r s , was a l s o added a t 4.51 x 10' 8M (10% of t h e A q u i l Fe c o n c e n t r a t i o n ) . The s m a l l e r Fe s p i k e u s e d i n t h e s e e x p e r i m e n t s was t o m i n i m i z e t h e i n f l u e n c e Fe may have had on e l u a t e Cu l e v e l s i n t h e r e s i n e x p e r i m e n t s ( e . g . F i g s . 14 & 1 5 ) . D u r i n g t h e s h o r t t e r m o f t h e b i o a s s a y s , t h e r e d u c e d l e v e l of added Fe was not e x p e c t e d t o i n f l u e n c e c e l l numbers o r growth r a t e s o f T h a l a s s i o s i r a pseudonana (B. M u e l l e r , pers.comm.). A f t e r t h e a d d i t i o n o f t h e n u t r i e n t s , t h e water was l e f t t o e q u i l i b r a t e f o r 2-3 h o u r s , and t h e n Cu s p i k e s of 40, 60, 80, 120 and 160 nM were added. The samples were t h e n l e f t o v e r n i g h t t o e q u i l i b r a t e p r i o r t o use i n t h e b i o a s s a y and r e s i n e x p e r i m e n t s . A p o r t i o n o f t h e p r e p a r e d n a t u r a l w ater s a m p l e s was u s e d i n t h e b i o a s s a y s , w h i c h were c o n d u c t e d i n t h e same manner as d e s c r i b e d p r e v i o u s l y . However, t h e water samples were not a u t o c l a v e d p r i o r t o i n o c u l a t i o n w i t h T h a l a s s i o s i r a  p seudonana t o r e d u c e p r e c i p i t a t i o n and t o p r e s e r v e any n a t u r a l o r g a n i c s t h a t may be p r e s e n t . A t o t a l of 15 p o l y c a r b o n a t e e r l e n m e y e r f l a s k s , e a c h c o n t a i n i n g 250 ml of sample, were p r e p a r e d : 3 f l a s k s were u s e d f o r t h e c o n t r o l s w h i l e t h e r e s t were u s e d f o r t e s t i n g t h e Cu c o n c e n t r a t i o n s . T h r e e r e p l i c a t e s were r u n f o r e a c h Cu c o n c e n t r a t i o n . The samples were i n o c u l a t e d w i t h T. pseudonana t o o b t a i n i n i t i a l c e l l c o n c e n t r a t i o n s between 1000-2000 c e l l s / m l . C e l l numbers were c o u n t e d o v e r a p e r i o d of 5 d a y s u s i n g a C o u l t e r C o u n t e r . 60 The r e m a i n d e r of t h e p r e p a r e d n a t u r a l water sample was u s e d i n t h e r e s i n e x p e r i m e n t s . The sample was d i v i d e d i n t o 500 ml a l i q u o t s and p l a c e d i n t o 500 ml p o l y p r o p y l e n e b o t t l e s . The Cu and Mn s t a n d a r d s (pH = 8.00±0.05) used t o c a l i b r a t e t h e r e s i n columns were p r e p a r e d a t t h e same t i m e a s t h e n a t u r a l s e a w a t e r s a m p l e s . The s t a n d a r d s (made from SOW w i t h t h e a p p r o p r i a t e Cu o r Mn s p i k e s ) were a l s o d i v i d e d i n t o 500 ml a l i q u o t s and p l a c e d i n t o 500 ml p o l y p r o p y l e n e b o t t l e s . T h r e e r e p l i c a t e s were p r e p a r e d f o r e a c h s t a n d a r d and C u - e n r i c h e d n a t u r a l s e a w a t e r s a m p l e . The r e s i n columns were r u n i n t h e same manner as d e s c r i b e d p r e v i o u s l y , w i t h 12 c o l u m n s p r e p a r e d a t one t i m e . One r e p l i c a t e of a sample o r a s t a n d a r d was p a s s e d t h r o u g h a column as shown i n T a b l e 2. S i n c e e a c h sample and s t a n d a r d had 3 r e p l i c a t e s , t h e co l u m n s were r u n 3 t i m e s i n s u c c e s s i o n . The c o l u m n s were e l u t e d w i t h 20 ml of 3 x 10" 2M EDTA p r i o r t o a n a l y s i s of t h e e l u a t e f o r Cu and Mn w i t h GFAAS. 61 T a b l e 2. Dowex AG 50W-X12 r e s i n column t e s t . s e r i e s f o r e s t i m a t i n g e f f e c t i v e m e t a l c o n c e n t r a t i o n s . Column No. M e t a l C o n c e n t r a t i o n S o l u t i o n 1 None ( b l a n k ) D i l u t e d SOW 2 40 nM Cu Cu s t a n d a r d s (SOW) 3 80 nM Cu IT 4 120 nM Cu f f 5 No added Cu N a t u r a l s e a w a t e r 6 40 nM Cu 7 60 nM Cu 8 80 nM Cu i i 9 120 nM Cu 10 160 nM Cu »i 1 1 100 nM Mn Mn s t a n d a r d s (SOW) 12 300 nM Mn 62 RESULTS AND DISCUSSION 1. A d a p t a t i o n o f t h e r e s i n method f o r n a t u r a l w a t e r s a m p l e s P r e l i m i n a r y e x p e r i m e n t s w i t h n a t u r a l s e a w a t e r s a m p l e s i n d i c a t e d t h a t n e i t h e r 1 x 10" 2M NTA nor 1 x 10" 2M EDTA were e f f e c t i v e i n e l u t i n g a l l of t h e Cu and Mn f r o m t h e Dowex AG 50W-X12 r e s i n . O t h e r e l u e n t s were t h e r e f o r e t e s t e d f o r t h e i r e f f e c t i v e n e s s i n r e m o v i n g t h e m e t a l s from t h e r e s i n . The e l u e n t s i n c l u d e d a 2 x 10" 2M EDTA:NTA (50:50) s o l u t i o n ; h o t (80°C) and warm (40°C) 3 x 10" 2M EDTA; h i g h pH (9.5) and low pH (5.0) w i t h 3 x 10" 2M NTA; 1 x 10"ftM, 1 x 10" 3M, 1 x 10' 2M 1 - P y r r o l i d i n e c a r b o d i t h i o i c a c i d (as t h e ammonium s a l t ) ; 3 N r e a g e n t g r a d e n i t r i c , a c e t i c , s u l f u r i c a c i d s , and 1 N, 3 N, 5 N i s o p i e s t i c a l l y d i s t i l l e d h y d r o c h l o r i c a c i d . A 20 ml a l i q u o t of 3 N HC1 was e f f e c t i v e i n r e m o v i n g t h e m e t a l s from t h e r e s i n a l t h o u g h any a d s o r b e d m a j o r s e a w a t e r c a t i o n s were a l s o e l u t e d by t h e a c i d . The p r e s e n c e o f s e a w a t e r c a t i o n s i n t h e e l u a t e , e s p e c i a l l y i n h i g h c o n c e n t r a t i o n s , has been f o u n d t o r e d u c e t h e s e n s i t i v i t y of t h e GFAAS r e a d i n g s , a s a h i g h b a c k g r o u n d t o s i g n a l r a t i o i s p r o d u c e d when t h e c a t i o n s a r e v o l a t i l i z e d d u r i n g t h e a t o m i z a t i o n s t e p ( K i n g s t o n e t a l . , 1978). C o n s e q u e n t l y , t o m i n i m i z e t h e b a c k g r o u n d s a l t i n t e r f e r e n c e , c h e l a t o r s s u c h as EDTA o r NTA ( w h i c h remove t h e t r a c e m e t a l s f r o m t h e r e s i n but not a l l of t h e s e a w a t e r c a t i o n s ) were c o n s i d e r e d t o be more s u i t a b l e f o r e l u t i n g t h e c o l u m n s . 63 Since various concentrations and eluent volumes of EDTA and NTA did not remove the metals from the resin Dowex AG 50W-X12, a diff e r e n t resin with a lower cross-linkage (Dowex AG 50W-X8) was tested. With the Dowex AG 50W-X8, 20 ml of 3 x 10"2M EDTA was found to be e f f e c t i v e in eluting the metals adsorbed onto the resin. It was presumed that the resin with the lower cross-linkage has larger pores through which the EDTA molecules could d i f f u s e in and the metal-EDTA complex could diffuse out more e a s i l y . To determine the sample volume required for the Dowex AG 50W-X8 to achieve eq u i l i b r a t i o n with Cu and Mn, d i f f e r e n t sample volumes of 25 ppt SOW containing 50 nM Cu and 100 nM Mn were passed through the columns. The sample volumes tested increased from 100 to 1000 ml in 100 ml increments. Eluate Cu and Mn concentrations were found to r i s e up to approximately 400 ml, with r e l a t i v e l y l i t t l e change to 1000 ml, indicating that the resin had attained equilibrium with Cu and Mn (Figs. 18 & 19). Consequently, sample volumes of 500 ml were used for these resin experiments. 2. Bioassays Cu was added to 6 l i t e r s of natural seawater c o l l e c t e d at each depth to obtain concentrations of 40, 60, 80, 120 and 160 nM. With more Cu added to the medium, the growth rates of Thalassiosi ra pseudonana were found to decrease (Table 3). However, the detrimental effect of Cu at 64 Figure 18. Eluate. Cu versus sample volume required for e q u i l i b r a t i o n for Dowex AG 50W-X8 resin (50 nM Cu in 35 ppt SOW). 65 F i g u r e 19. E l u a t e Mn v e r s u s sample volume r e q u i r e d f o r e q u i l i b r a t i o n f o r Dowex AG 50W-X8 r e s i n (100 nM Mn i n 2 5 ppt SOW). 6 6 T a b l e 3. Growth r a t e of T h a l a s s i o s i r a p seudonana i n I n d i a n Arm w a t e r . Where a p p r o p r i a t e , 0 = ± 2 s t a n d a r d d e v i a t i o n s . D e p t h Copper added Growth r a t e % C o n t r o l (m) (nM) ( d i v i s i o n s / d a y ) 10 0 1 .89 ( 0 . 0 2 ) 4 0 1 . 5 2 ( 0 . 0 2 ) 81 (1 ) 6 0 1 . 2 9 < 0 . 0 3 ) 6 8 ( 2 ) 8 0 1 .24 ( 0 . 0 1 ) 6 6 (1 ) 1 2 0 1.17 ( 0 . 0 2 ) 6 2 ( 2 ) 1 6 0 0 . 8 9 ( 0 . 0 2 ) 47 ( 2 ) 5 0 0 1 .80 ( 0. 1 4 ) 40 1 .50 ( 0 . 0 4 ) 8 3 ( 3 ) 6 0 1 .30 < 0.01 ) 72 ( 1 ) 8 0 1.32 ( 0 . 0 5 ) 7 3 ( 4 ) 1 2 0 1. 1 5 I 0 . 0 5 ) 64 ( 4 ) 1 6 0 0 . 8 9 < 0 . 0 2 ) 4 9 ( 2 ) 1 0 0 0 2 . 2 0 0 . 0 3 ) 40 2 . 0 5 , 0 . 0 2 ) 9 3 (1 ) 6 0 2.01 < R 0 . 0 2 ) 91 (1 ) 8 0 1 . 8 9 1 0 . 0 1 ) 8 6 ( 1 ) 1 2 0 1 . 5 3 [ 0 . 0 2 ) 7 0 (1 ) 160 1.31 [ 0 . 0 2 ) 6 0 ( 2 ) 1 5 0 0 2 . 1 6 [ 0 . 0 4 ) 40 2 . 1 6 [ 0 . 0 2 ) 1 0 0 (1 ) 6 0 1 . 9 5 [ 0 . 1 1 ) 9 0 ( 6 ) 8 0 1 . 9 6 [ 0 . 0 6 ) 91 ( 3 ) 1 2 0 1 . 7 6 [ 0 . 1 0 ) 81 ( 6 ) 1 6 0 1 . 3 7 ( 0 . 0 6 ) 6 3 ( 4 ) 2 0 0 0 1 .88 ( 0 . 0 4 ) 4 0 1 .84 ( 0 . 0 5 ) 98 ( 3 ) 6 0 1 .88 ( 0 . 0 4 ) 1 0 0 ( 2 ) 8 0 1 .80 ( 0 . 0 2 ) 9 6 (1 ) 1 2 0 1 .60 ( 0 . 0 2 ) 8 5 ( 1 ) 1 6 0 1 .22 ( 0 . 0 1 ) 6 5 (1) e l e v a t e d c o n c e n t r a t i o n s was f o u n d t o be r e d u c e d when t h e t e s t o r g a n i s m was grown i n wa t e r s a m p l e s c o l l e c t e d f r o m t h e d e e p e s t d e p t h s ( 1 5 0 and 2 0 0 m). F o r example, t h e g r o w t h r a t e ( as compared t o t h e c o n t r o l ) was n o t a f f e c t e d when a Cu 67 s p i k e of 60 nM was added t o t h e 200 m water sample, w h i l e t h e same Cu s p i k e added t o t h e 10 m water sample r e d u c e d t h e g r o w t h r a t e by 32%. One p o s s i b l e e x p l a n a t i o n i s t h a t d eep w a t e r s c o n t a i n more n a t u r a l l y o c c u r r i n g l i g a n d s ( e . g . amino a c i d s , f u l v i c and humic a c i d s ) t h a n s h a l l o w w a t e r s . A h i g h c o n c e n t r a t i o n of s u c h l i g a n d s has been shown p r e v i o u s l y t o r e d u c e t h e amount o f b i o a v a i l a b l e Cu ( e . g . Borgmann and R a l p h , 1983) and t h e r e f o r e may e x p l a i n t h e b e t t e r g r o w t h r a t e s of T. pseudonana i n t h e deep w a t e r s a m p l e s . 3. R e s i n e x p e r i m e n t s The r e s i n e x p e r i m e n t s were c o n d u c t e d u s i n g 9 l i t e r s o f n a t u r a l s e a w a t e r from e a c h d e p t h a s i n d i c a t e d i n T a b l e 2. The samples were p r e p a r e d i n t h e same manner as d e s c r i b e d p r e v i o u s l y f o r t h e b i o a s s a y s . I n o r d e r t o d e t e r m i n e t h e amount o f m e t a l a d s o r b e d o n t o t h e r e s i n from t h e s e a w a t e r sample, t h e r e s i n had t o be c a l i b r a t e d w i t h s t a n d a r d s o f a known c o m p o s i t i o n . T h e s e s t a n d a r d s were made from Cu or Mn s p i k e s added t o SOW o f t h e same s a l i n i t y as t h a t o f t h e samples ( a p p r o x i m a t e l y 25-27 p p t ) . The d i l u t e d SOW was made from f u l l s t r e n g t h SOW and GDW. S t a n d a r d s of 40, 80 and 120 nM Cu, and 100 and 300 nM Mn were p a s s e d t h r o u g h t h e columns a t t h e same t i m e and u n d e r t h e same o p e r a t i n g c o n d i t i o n s as t h e s a m p l e s . The amount o f Cu o r Mn a d s o r b e d o n t o t h e r e s i n when a sample and a s t a n d a r d a r e p a s s e d t h r o u g h t h e columns c a n be r e l a t e d , and t h i s r e l a t i o n s h i p can be e x p r e s s e d as 68 t h e e f f e c t i v e m e t a l c o n c e n t r a t i o n (EMC) (see Z o r k i n et. a l . , i n p r e s s ) . The EMC v a l u e s a r e d e p e n d e n t on b o t h t h e t o t a l Cu and Mn c o n c e n t r a t i o n and t h e e x t e n t t o w h i c h t h e s e m e t a l s a r e bound by v a r i o u s l i g a n d s . The EMC f o r Cu o r Mn w i l l e q u a l t h e t o t a l Cu o r Mn c o n c e n t r a t i o n f o r a n a t u r a l s e a w a t e r sample o n l y i f t h e l i g a n d s p r e s e n t i n t h e sample a r e e x a c t l y t h e same as t h o s e l i g a n d s p r e s e n t i n t h e s t a n d a r d s . The o n l y major i o n s t h o u g h t t o i n f l u e n c e Cu and Mn s p e c i a t i o n i n t h e SOW us e d f o r t h e s t a n d a r d s a r e t h e h y d r o x i d e and c a r b o n a t e i o n s f o r Cu and c h l o r i d e i o n s f o r Mn. C o n s e q u e n t l y , t h e EMC f o r Cu and Mn f o r n a t u r a l s e a w a t e r s a m p l e s a r e c o n s i d e r e d t o be a measure o f t h e e x t e n t o f m e t a l c o m p l e x a t i o n by l i g a n d s o t h e r t h a n c a r b o n a t e and h y d r o x i d e i o n s f o r Cu and by l i g a n d s o t h e r t h a n c h l o r i d e i o n s f o r Mn. The same Cu s p i k e s (40, 60, 80, 120 and 160 nM) t e s t e d i n t h e b i o a s s a y s were t e s t e d i n t h e r e s i n e x p e r i m e n t s . W i t h more Cu a d d e d t o t h e s e a w a t e r s a m p l e s , t h e e f f e c t i v e Cu c o n c e n t r a t i o n s (ECC) were f o u n d t o i n c r e a s e ( T a b l e 4 ) , i n d i c a t i n g t h a t more Cu was a d s o r b e d o n t o t h e r e s i n . However, n o t a l l of t h e add e d Cu was a d s o r b e d o n t o t h e r e s i n , a s t h e a d d i t i o n o f a 40 nM Cu s p i k e t o a 50 m w a t e r sample p r o d u c e d an ECC o f o n l y 43.8 nM, w h i l e t h e same water sample w i t h o u t t h e a d d i t i o n o f Cu had a b a c k g r o u n d ECC of 17.3 nM. T h i s s u g g e s t s t h a t t h e s e a w a t e r s a m p l e s p r o b a b l y c o n t a i n e d some n a t u r a l l y o c c u r r i n g l i g a n d s , w h i c h w o u l d t e n d 69 T a b l e 4. E f f e c t i v e Cu and Mn c o n c e n t r a t i o n s i n c o p p e r -e n r i c h e d I n d i a n Arm w a t e r . Where a p p r o p r i a t e , () = ±2 s t a n d a r d d e v i a t i o n s . [ ] = Co p p e r p r e s e n t i n u n e n r i c h e d w a t e r . ECC = e f f e c t i v e c o p p e r c o n c e n -t r a t i o n , EMnC = e f f e c t i v e manganese c o n c e n t r a t i o n . Manganese p r e s e n t = t o t a l d i s s o l v e d manganese. D e p t h Copper ECC Manganese EMnC (m) Added C o n c e n t r a t i o n P r e s e n t C o n c e n t r a t i o n (nM) (nM) (nM) (nM) 10 [14.2 (1 . 5 ) ] < 10 < 1 0 16.52 (0.23) Tf 40 43.33 (1.79) II fl 60 62.61 (0.62) II f! 80 77.68 (0.71 ) II ff 1 20 120.14 (1.18) ft 1 60 154.98 (1.81) II IT 50 [16.5 (2 .4)] 60.4 (0.6) 29.4 (0.72) 0 17.29 (0.24) n ti 40 43.81 (2.53) n ti 60 67.40 (0.24) »i n 80 79.70 (4.62) ft 1 20 126.30 (5.39) II ii 160 152.97 (4.62) II II 100 [54.5 (5 .3)] 633.5 (9.4) 369.9 (6.4) 0 14.83 (0.85) H n 40 43.45 (1.49) n II 60 67.27 (0.65) H 80 78.35 (2.49) n n 120 129.74 (3.42) ft ti 160 159.70 (3.43) TI ft 150 [13.2 (3 .9)] 1443.5 (20.1 ) 1281.7 (23.6) 0 12.85 (0.45) n 40 40.62 (0.89) »i 60 59.35 (2.53) ii ii 80 77.26 (1.56) ft II 1 20 120.61 (3.97) n II 160 153.43 (3.57) n ft 200 [17.9 (1 .3 ) ] 2504.1 (26.7) 1671.7 (18.1) 0 16.96 (0.63) » ! 40 43.62 (1.08) It « 60 62. 18 (1.02) fl ft 80 78.26 (1.18) fl tt 120 130.72 (2.95) fl it 160 154.49 (5.27) ft 70 t o r e d u c e t h e amount of added Cu a v a i l a b l e t o a d s o r b o n t o t h e r e s i n . The v e r t i c a l p r o f i l e of ECC f o r samples w i t h o u t Cu a d d i t i o n and t h e t o t a l d i s s o l v e d Cu c o n c e n t r a t i o n were a l s o f o u n d t o d i f f e r , s u g g e s t i n g t h e p r e s e n c e of n a t u r a l l y o c c u r r i n g l i g a n d s . F o r example, t h e 100 m water had o v e r t w i c e t h e l e v e l of t o t a l d i s s o l v e d Cu compared t o t h e o t h e r d e p t h s , a l t h o u g h a c o r r e s p o n d i n g peak was n o t e v i d e n t i n t h e ECC ( T a b l e 4 ) . T h i s s u g g e s t s t h a t a r e l a t i v e l y h i g h c o n c e n t r a t i o n of l i g a n d s i s p r e s e n t i n t h e 100 m w ater sample and t h a t a h i g h d e g r e e of c o m p l e x a t i o n of Cu may be o c c u r r i n g . F o r e a c h o f t h e f i v e Cu s p i k e s added t o t h e n a t u r a l w a t e r s a m p l e s , t h e ECC was f o u n d t o be i n d e p e n d e n t of t h e d e p t h from which t h e samples were c o l l e c t e d ( T a b l e 4 ) . I n c o n t r a s t , t h e e f f e e t i v e Mn c o n c e n t r a t i o n s (EMnC) were f o u n d t o i n c r e a s e from u n d e t e c t a b l e l e v e l s i n t h e 10 m sample t o a p p r o x i m a t e l y 1650 nM i n t h e 200 m sample ( T a b l e 4 ) . The o v e r a l l v e r t i c a l p r o f i l e s of t h e EMnC and t h e d i s s o l v e d Mn a r e s i m i l a r , a l t h o u g h t h e l a t t e r v a l u e s were f o u n d t o be somewhat g r e a t e r . T h i s i n d i c a t e s t h a t l i k e Cu, some c o m p l e x a t i o n of Mn may be o c c u r r i n g by n a t u r a l l y o c c u r r i n g l i g a n d s . The i n c r e a s e i n d i s s o l v e d Mn l e v e l s w i t h d e p t h i s i n c o n t r a s t t o t h e open o c e a n s where Mn i s u s u a l l y e n r i c h e d a t t h e s u r f a c e . T h i s e n r i c h m e n t i s p a r t l y due t o t h e e x t r e m e l y slow o x i d a t i o n k i n e t i c s o f s o l u b l e Mn ( I I ) t o i n s o l u b l e Mn 71 ( I V ) ( A h r l a n d , 1975) and t h e p h o t o r e d u c t i o n of i n s o l u b l e manganese o x i d e s by l i g h t i n t h e p r e s e n c e of d i s s o l v e d o r g a n i c s u b s t a n c e s (Sunda e t a l . , 1983). In some f j o r d s s u c h a s I n d i a n Arm, d i s s o l v e d Mn i s f o u n d t o i n c r e a s e w i t h d e p t h due t o t h e d e p l e t i o n of oxygen and t h e r e d u c t i o n o f manganese o x i d e s . Such a v e r t i c a l p r o f i l e o f d i s s o l v e d Mn has p r e v i o u s l y been d e s c r i b e d f o r I n d i a n Arm a t t h e same s t a t i o n (IND-2) by W h i t f i e l d ( 1 9 7 4 ) . 4. C o m p a r i s o n of b i o a s s a y and r e s i n r e s u l t s The growth r a t e s o f T h a l a s s i o s i r a pseudonana were f o u n d t o d e c r e a s e w i t h h i g h e r ECC v a l u e s when p l o t t e d as a f u n c t i o n of t h e ECC e s t i m a t e d by t h e r e s i n a n a l y s i s ( F i g . 2 0 ) . T h i s i n d i c a t e d t h a t t h e amount o f Cu a v a i l a b l e t o t h e o r g a n i s m c o u l d be r e l a t e d t o t h e amount of Cu a d s o r b e d o n t o t h e r e s i n , as p r e v i o u s l y shown by Z o r k i n (1983) and Z o r k i n e t a l . ( i n p r e s s ) . The r e l a t i o n s h i p between t h e g r o w t h r a t e s and t h e ECC was not as p r o n o u n c e d however, i n t h e water samples c o l l e c t e d f r o m deep w a t e r s . From t h e b i o a s s a y r e s u l t s , more c o m p l e x a t i o n of Cu ( i . e . low ECC) a p p e a r p o s s i b l e i n t h e d eep water s a m p l e s . However, t h e r e s i n a n a l y s i s i n d i c a t e d s i m i l a r ECC v a l u e s i n b o t h t h e s h a l l o w and deep water s a m p l e s . In c o n t r a s t , t h e EMnC v a l u e s i n c r e a s e d d r a m a t i c a l l y w i t h d e p t h . T h i s s u g g e s t s t h a t Mn i s r e s p o n s i b l e f o r r e d u c i n g t h e Cu t o x i c i t y t o t h e t e s t o r g a n i s m i n t h e d e e p water s a m p l e s . Z o r k i n (1983) a l s o 72 © o i 1 1 1 1 1 0.0 40.0 80.0 120.0 160.0 200.0 ECC (NM) Figure 20. The effect of varying EMnC values on Cu t o x i c i t y in natural water samples. Symbols: X 200 m water (EMnC = 1671.7 nM); <J> 150 m water (EMnC = 1281.7 nM); * 100 m water (EMnC = 369.9 nM); A 50 m water (EMnC = 29.4 nM); © 10m water (EMnC = < 1 nM). 73 f o u n d t h a t T. pseu d o n a n a grew b e t t e r i n t h e deep water s a m p l e s from t h e same s t a t i o n i n I n d i a n Arm, and s u g g e s t e d t h a t t h e h i g h e r g r o w t h r a t e s m ight be due t o t h e p r e s e n c e of h i g h c o n c e n t r a t i o n s o f Mn, a l t h o u g h t h e EMnC was not me a s u r e d . In t h i s s t u d y , t h e h i g h EMnC v a l u e s o b t a i n e d w i t h t h e d eep water s a m p l e s i n d i c a t e t h a t most o f t h e Mn was i n t h e b i o l o g i c a l l y a v a i l a b l e i o n i c f o r m and not i n t h e p a r t i c u l a t e f o r m . I f t r u e , t h e e f f e c t o f Mn on r e d u c i n g Cu t o x i c i t y does not a p p e a r t o be due t o t h e a d s o r p t i o n of Cu o n t o manganese o x i d e s w h i c h would make Cu l e s s a v a i l a b l e t o o r g a n i s m s . F u r t h e r m o r e , Sunda e t a_l. (1981) and Diem and Stumm (1984) have v e r i f i e d t h a t t h e o x i d a t i o n k i n e t i c s of Mn ( I I ) under n a t u r a l s e a w a t e r c o n d i t i o n s a r e e x t r e m e l y s l o w . The e f f e c t of Mn on r e d u c i n g Cu t o x i c i t y t o t h e b i o a s s a y o r g a n i s m i s t h e r e f o r e c o n s i d e r e d t o be due n o t t o t h e r e d u c t i o n i n t h e b i o l o g i c a l l y a c t i v e Cu c o n c e n t r a t i o n by r e m o v a l t h r o u g h a d s o r p t i o n p r o c e s s e s , b u t r a t h e r t h e p h y s i o l o g i c a l r e s p o n s e o f t h e o r g a n i s m t o t h e i o n i c forms of Cu and Mn. Sunda and Huntsman (1983) i n v e s t i g a t e d t h e b i o c h e m i c a l mechanism of t h e Cu-Mn i n t e r a c t i o n and f o u n d t h a t an i n c r e a s e i n c u p r i c i o n a c t i v i t y a t low Mn i o n a c t i v i t i e s (1 x 1 0 " 1 0 - 5 M) s t o p p e d t h e g r o w t h of T h a l a s s i o s i r a p s e u d o n a n a ( 3 H ) . However, t h e d e t r i m e n t a l e f f e c t of Cu was shown t o be r e d u c e d when t h e Mn i o n a c t i v i t y was i n c r e a s e d . The a u t h o r s t h e n a d d e d 5*Mn t o 74 d e t e r m i n e t h e c e l l u l a r Mn c o n t e n t of t h e c e l l s and f o u n d t h a t t h e c e l l u l a r Mn c o n t e n t d e c r e a s e d when t h e c u p r i c i o n a c t i v i t y was i n c r e a s e d . The d e c r e a s e i n g r o w t h r a t e was a l s o a s s o c i a t e d w i t h a d e c r e a s e i n c e l l u l a r Mn l e v e l s w h i c h , i n t u r n , was a s s o c i a t e d w i t h a d e c r e a s e i n Mn i o n a c t i v i t y . However, a f t e r a c e r t a i n c o n c e n t r a t i o n , f u r t h e r a d d i t i o n s o f Mn d i d not a f f e c t c e l l u l a r Mn nor t h e growth r a t e s . Thus, Mn and Cu i o n a c t i v i t i e s were t h o u g h t t o a f f e c t g r o w t h r a t e by t h e i r i n f l u e n c e on c e l l u l a r Mn l e v e l s . The manganese t r a n s p o r t r a t e i n t o t h e c e l l has s u b s e q u e n t l y been f o u n d t o be r e l a t e d t o t h e f r e e Mn i o n c o n c e n t r a t i o n r a t h e r t h a n t h e t o t a l Mn c o n c e n t r a t i o n (Sunda and Huntsman, 1985). In a d d i t i o n , t h e c e l l u l a r Mn i s t h o u g h t t o be r e g u l a t e d t h r o u g h a n e g a t i v e f e e d b a c k c o n t r o l , a l t h o u g h t h e e x a c t mechanism i s s t i l l u n c e r t a i n . When a b i o a s s a y o r g a n i s m i s u s e d t o e s t i m a t e t h e b i o l o g i c a l a v a i l a b l i t y o f a m e t a l s u c h as Cu, t h e main a s s u m p t i o n i s t h a t t h e o r g a n i s m i s r e s p o n d i n g o n l y t o t h e m e t a l b e i n g t e s t e d . However, Sunda e_t a l . ( 1 9 8 1 ) , Sunda and Huntsman (1983;1985) have d e m o n s t r a t e d t h a t Mn c a n r e d u c e t h e t o x i c i t y o f Cu t o p h y t o p l a n k t o n . The p r e s e n t s t u d y v e r i f i e s t h i s f i n d i n g and i n d i c a t e s t h a t i t i s a c h a r a c t e r i s t i c of water from a n a t u r a l e n v i r o n m e n t . O t h e r m e t a l s s u c h as Zn and Fe a r e t h o u g h t t o be i m p o r t a n t when Cu t o x i c i t y i s c o n s i d e r e d i n n a t u r a l w a t e r s . Murphy e t a l . (1984) u s e d an u n c h e l a t e d s y s t e m t o s t u d y t h e combined 75 e f f e c t s of Mn and Fe d e f i c i e n c i e s (a l i k e l y c o m b i n a t i o n under n a t u r a l c o n d i t i o n s ) on Cu s e n s i t i v i t y o f o c e a n i c and n e r i t i c s p e c i e s o f T h a l a s s i o s i r a . The o c e a n i c s p e c i e s were more s e n s i t i v e t o c h a n g e s i n m e t a l l e v e l s t h a n t h e n e r i t i c s p e c i e s . The Fe r e q u i r e m e n t was f o u n d t o i n c r e a s e i n t h e p r e s e n c e of Cu o r t h e a b s e n c e o f Mn, w h i l e t h e Mn r e q u i r e m e n t was f o u n d t o i n c r e a s e i n t h e p r e s e n c e of Cu o r t h e a b s e n c e of F e . I t i s b e c o m i n g a p p a r e n t t h a t t h e n u t r i t i o n a l m e t a l s t a t u s of p h y t o p l a n k t o n i s complex and must be c o n s i d e r e d when m e t a l t o x i c i t y i s c o n s i d e r e d i n n a t u r a l s y s t e m s . 76 GENERAL SUMMARY Cu and Mn a r e e s s e n t i a l i n t r a c e amounts f o r p h y s i o l o g i c a l p r o c e s s e s i n p h y t o p l a n k t o n . At h i g h c o n c e n t r a t i o n s , Mn r a r e l y becomes t o x i c , w h i l e Cu can have a d v e r s e e f f e c t s . The t o x i c i t y o f Cu t o some p h y t o p l a n k t o n s p e c i e s has r e c e n t l y been shown t o be r e d u c e d i n t h e p r e s e n c e of Mn (Sunda e_t a l . , 1981; Sunda and Huntsman, 198.3; 1985). However, not a l l a l g a e a r e a f f e c t e d i n t h e same manner ( e . g . A n d e r s o n and M o r e l , 1978), t h u s i t was n e c e s s a r y t o d e t e r m i n e t h e e f f e c t of Mn on Cu t o x i c i t y t o t h e b i o a s s a y o r g a n i s m , T h a l a s s i o s i r a pseudonana u s e d i n t h i s s t u d y . The t e s t o r g a n i s m was grown i n A q u i l , a w e l l - d e f i n e d s e a w a t e r medium, i n t h e a b s e n c e o f a r t i f i c i a l c h e l a t i n g a g e n t s . W i t h more Mn added t o t h e t e s t media, Cu t o x i c i t y t o T. pseudonana was f o u n d t o be r e d u c e d , v e r i f y i n g t h e f i n d i n g s of Sunda e t a_l. (1981) and Sunda and Huntsman ( 1 9 8 3 ; 1 9 8 5 ) . To e s t i m a t e t h e b i o l o g i c a l l y a c t i v e Cu and Mn, a c a t i o n - e x c h a n g e t e c h n i q u e d e v e l o p e d by Z o r k i n (1983) and Z o r k i n e t a_l. ( i n p r e s s ) was t e s t e d i n A q u i l . The sample volume r e q u i r e d f o r e q u i l i b r a t i o n of t h e r e s i n , t h e l i n e a r i t y of a d s o r p t i o n o f m e t a l s t o t h e r e s i n , and t h e e f f e c t o f A q u i l n u t r i e n t s on t h e e f f i c i e n c y o f t h e r e s i n was t e s t e d . 77 M o d i f i c a t i o n s t o t h e t e c h n i q u e were n e c e s s a r y b e f o r e i t c o u l d be a p p l i e d t o n a t u r a l s e a w a t e r s a m p l e s , as t h e m e t a l s c o u l d n o t be c o m p l e t e l y r e c o v e r e d f r o m t h e Dowex AG 50W-X12 r e s i n . The use o f a r e s i n w i t h a l o w e r c r o s s - l i n k a g e , as i s Dowex AG 50W-X8, e l i m i n a t e d t h i s p r o b l e m , p r e s u m a b l y by a l l o w i n g t h e metal-EDTA complex t o d i f f u s e i n and out more e a s i l y t h r o u g h t h e l o o s e r n e twork o f t h e r e s i n . The m o d i f i e d r e s i n t e c h n i q u e was t h e n a p p l i e d t o n a t u r a l s e a w a t e r samples t a k e n f r o m I n d i a n Arm, a l o c a l f j o r d w i t h h i g h ambient l e v e l s o f Mn. The r e s i n was u s e d t o d e t e r m i n e whether i o n i c Mn l e v e l s c o u l d a f f e c t t h e c o n c e n t r a t i o n of b i o l o g i c a l l y a v a i l a b l e Cu. B i o a s s a y s w i t h T h a l a s s i o s i r a pseudonana were c o n d u c t e d on t h e same water s a m p l e s and t h e s e r e s u l t s were compared t o t h e r e s u l t s f r o m t h e r e s i n t e c h n i q u e . T. p s e u d o n a n a was f o u n d t o grow b e t t e r i n t h e samples c o l l e c t e d f r o m deep w a t e r s , w h i c h c o n t a i n h i g h l e v e l s of b i o a v a i l a b l e Mn a s e s t i m a t e d by t h e r e s i n t e c h n i q u e . The i o n i c Cu c o n c e n t r a t i o n s a s e s t i m a t e d by t h e method was not a f f e c t e d by t h e p r e s e n c e o f Mn, i n d i c a t i n g t h a t t h e o r g a n i s m was r e s p o n d i n g t o c h a n g e s i n b i o l o g i c a l l y a c t i v e Mn. The t o x i c i t y of m e t a l s s u c h as Cu t o o r g a n i s m s c a n t h e r e f o r e be complex, and t h e e f f e c t s of o t h e r m e t a l s s u c h a s Mn must a l s o be c o n s i d e r e d when n a t u r a l s y s t e m s a r e c o n s i d e r e d . 78 REFERENCES CITED A h r l a n d , S. ( 1 9 7 5 ) . M e t a l c o m p l e x e s p r e s e n t i n s e a w a t e r , I n : G o l d b e r g , E.D. ( e d . ) , The n a t u r e o f s e a w a t e r , p. 219-244, Dahlem Workshop R e p o r t , B e r l i n . A n d e r s o n , D.M. and F.M.M. M o r e l ( 1 9 7 8 ) . C o p p e r s e n s i t i v i t y of G o n y a u l a x t a m a r e n s i s . L i m n o l . O c e a n o g r . 23:283-295. A n d e r s o n , M.A. and F.M.M. M o r e l ( 1 9 8 2 ) . The i n f l u e n c e of aqueous i r o n c h e m i s t r y on t h e up t a k e o f i r o n by t h e c o a s t a l d i a t o m T h a l a s s i o s i r a w e i s s f l o g i i . L i m n o l . O c e a n o g r . 27:789-813. B a c c i n i , P. and U. S u t e r ( 1 9 7 9 ) . MELIMEX, an e x p e r i m e n t a l heavy m e t a l p o l l u t i o n s t u d y : c h e m i c a l s p e c i a t i o n and b i o l o g i c a l a v a i l a b i l i t y of c o p p e r i n l a k e w a t e r . S c h w e i z . Z. H y d r o l . 4J_:291-314. B e n j a m i n , M.M. and J.O. L e c k i e ( 1 9 8 0 ) . A d s o r p t i o n of m e t a l s a t o x i d e i n t e r f a c e s . E f f e c t s of t h e c o n c e n t r a t i o n s of a d s o r b a t e and c o m p e t i n g m e t a l s , I n : B a k e r , R.A. ( e d . ) , C o n t a m i n a n t s and s e d i m e n t s , V.2, A n a l y s i s , c h e m i s t r y , b i o l o g y , p. 305-322. Ann A r b o r S c i e n c e , Ann A r b o r , M i c h i g a n . Borgmann, U. and K.M. R a l p h ( 1 9 8 4 ) . Copper c o m p l e x a t i o n and t o x i c i t y t o f r e s h w a t e r z o o p l a n k t o n . A r c h . E n v i r o n . Contam. T o x i c o l . 13:403-409. B o y l e , E.A., F.R. S c l a t e r and J.M. Edmond ( 1 9 7 7 ) . The d i s t r i b u t i o n o f d i s s o l v e d c o p p e r i n t h e P a c i f i c . E a r t h P l a n e t S c i . L e t t . 37:38-54. B u r l i n g , R.W. ( 1 9 8 2 ) . Deep c i r c u l a t i o n s i n i n l e t s : I n d i a n Arm as a c a s e s t u d y , I n : E l l i s , D.V. ( e d . ) , M a r i n e T a i l i n g s D i s p o s a l , p. 85-131. Ann A r b o r S c i e n c e , Ann A r b o r , M i c h i g a n . 79 B r a e k , G.S., D. M a l n e s and A. J e n s e n ( 1 9 8 0 ) . Heavy m e t a l t o l e r a n c e of m a r i n e p h y t o p l a n k t o n IV. Combined e f f e c t o f z i n c and cadmium on growth and u p t a k e i n some m a r i n e d i a t o m s . J . e x p . mar. B i o l . E c o l . 42:39-54. B r a n d , L . E . , W.G. Sunda and R.R.L. G u i l l a r d ( 1 9 8 3 ) . L i m i t a t i o n o f m a r i n e p h y t o p l a n k t o n r e p r o d u c t i v e r a t e s by z i n c , manganese, and i r o n . L i m n o l . O c e a n o g r . 28: 1182-1198. C h e n i a e , G.M. and I . F . M a r t i n ( 1 9 7 0 ) . S t u d i e s on t h e f u n c t i o n of manganese i n p h o t o s y n t h e s i s , I n : H i l l m a n , W.S. ( e d . ) , P a p e r s i n p l a n t p h y s i o l o g y , p. 245-256, H o l t , R i n e h a r t and W i n s t o n , New Y o r k , New Y o r k . C l a r k , M.E., G.A. J a c k s o n and W.J. N o r t h ( 1 9 7 2 ) . D i s s o l v e d f r e e amino a c i d s i n s o u t h e r n C a l i f o r n i a c o a s t a l w a t e r s . L i m n o l . O c e a n o g r . 17:749-758. D a v i d s o n , L.W. ( 1 9 7 9 ) . P h y s i c a l o c e a n o g r a p h y of B u r r a r d I n l e t and I n d i a n Arm, B r i t i s h C o l u m b i a . M.Sc. T h e s i s , D e p t . of P h y s i c s , 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 . D a v i e s , A.G. ( 1 9 7 8 ) . P o l l u t i o n s t u d i e s w i t h m a r i n e p l a n k t o n . P a r t I I . Heavy M e t a l s , I n : R u s s e l , F.S. and M. Younge ( e d s . ) , A d v a n ces i n M a r i n e B i o l o g y , 15:381-508. D i n e r , B.A. and P. J o l i o t ( 1 9 7 6 ) . Oxygen e v o l u t i o n and manganese, I n : T r e b s t , A. and M. A v r o n ( e d s . ) , P h o t o s y n t h e s i s I , p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t and p h o t o p h o s p h o r y l a t i o n , E n c y c l o p e d i a of p l a n t p h y s i o l o g y , V.5, p. 187-205, S p r i n g e r - V e r l a g , New Y o r k , New Y o r k . D o r f n e r , K. ( e d . ) , ( 1 9 7 2 ) . Ion e x c h a n g e r s , Ann A r b o r S c i e n c e , Ann A r b o r , M i c h i g a n . Dow C h e m i c a l Company ( 1 9 5 8 ) . Dowex: Ion e x c h a n g e , L a k e s i d e P r e s s , C h i c a g o , I l l i n o i s . 80 Emerson, S., R.E. C r a n s t o n and P.S. L i s s ( 1 9 7 9 ) . Redox s p e c i e s i n a r e d u c i n g f j o r d : e q u i l i b r a t i o n and k i n e t i c c o n s i d e r a t i o n s . Deep Sea R e s . 26:859-878. F i g u r a , P. and B. M c D u f f i e ( 1 9 7 9 ) . Use o f c h e l e x r e s i n f o r d e t e r m i n a t i o n o f l a b i l e t r a c e m e t a l f r a c t i o n s i n aqueous l i g a n d media and c o m p a r i s o n o f t h e method w i t h a n o d i c s t r i p p i n g v o l t a m m e t r y . A n a l . Chem. 51:120-125. F i l b y , R.H., K.R. Shah and W.H. Funk ( 1 9 7 4 ) . I n : V o g t , J.R. and W. Meyer ( e d s . ) , P r o c e e d . 2nd I n t e r n . C o n f . on N u c l e a r Methods i n E n v i r o n m e n t a l R e s e a r c h , NITS 1974-10, U n i v . M i s s o u r i , C o l u m b i a , M i s s o u r i . F i s h e r , N.S. and G.J. J o n e s ( 1 9 8 1 ) . Heavy m e t a l s and m a r i n e p h y t o p l a n k t o n : c o r r e l a t i o n o f t o x i c i t y and s u l f h y d r y l b i n d i n g . J . P h y c o l . 17:108-111 . F i s h e r , N.S., G.J. J o n e s and D.M. N e l s o n ( 1 9 8 1 ) . E f f e c t s o f c o p p e r and z i n c on g r o w t h , m o r p h o l o g y , and m e t a b o l i s m of A s t e r i o n e l l a j a p o n i c a C l e v e . J . exp. mar. B i o l . E c o l . 51:37-56. F l o r e n c e , T.M. (1982 a ) . The s p e c i a t i o n o f t r a c e e l e m e n t s i n w a t e r s . T a l a n t a 29:345-364. F l o r e n c e , T.M. (1982 b ) . Development o f p h y s i c o - c h e m i c a l s p e c i a t i o n p r o c e d u r e s t o i n v e s t i g a t e t h e t o x i c i t y of c o p p e r , l e a d , cadmium and z i n c t o w a r d s a q u a t i c b i o t a . A n a l . Chim. A c t a 141:73-94. F l o r e n c e , T.M. and G.E. B a t l e y ( 1 9 7 6 ) . T r a c e m e t a l s s p e c i e s i n s e a w a t e r - I Removal of t r a c e m e t a l s from s e a w a t e r by a c h e l a t i n g r e s i n . T a l a n t a 23:179-186. F l o r e n c e , T.M. and G.E. B a t l e y ( 1 9 8 0 ) . C h e m i c a l s p e c i a t i o n i n n a t u r a l w a t e r s , CRC C r i t i c a l r e v i e w s i n a n a l y t i c a l c h e m i s t r y . 9:219-296. 81 F l o r e n c e , T.M., B.G. Lumsden and J . J . F a r d y ( 1 9 8 3 ) . E v a l u a t i o n o f some p h y s i c o - c h e m i c a l t e c h n i q u e s f o r t h e d e t e r m i n a t i o n of t h e f r a c t i o n of d i s s o l v e d c o p p e r t o x i c t o t h e m a r i n e d i a t o m N i t z s c h i a c l o s t e r i u m . A n a l . Chim. A c t a 15:281-295. F o s t e r , P.L. and F.M.M. M o r e l ( 1 9 8 2 ) . R e v e r s a l o f cadmium t o x i c i t y i n a d i a t o m : An i n t e r a c t i o n between cadmium and i r o n . L i m n o l . O c e a n o g r . 27:745-752. G a v i s , J . , R.R.L. G u i l l a r d and B.L. Woodward ( 1 9 8 1 ) . C u p r i c i o n a c t i v i t y and t h e g r o w th of p h y t o p l a n k t o n c l o n e s i s o l a t e d f r o m d i f f e r e n t m a r i n e e n v i r o n m e n t s . J . Mar. Res. 39:315-333. G i l m a r t i n , M. ( 1 9 6 2 ) . A n n u a l c y c l i c c h a n g e s i n t h e p h y s i c a l o c e a n o g r a p h y of a B r i t i s h C o l u m b i a f j o r d . J . F i s h . Res. Bd. Can. 19:921-974. G o e r i n g , J . J . , D. B o i s s e a u and A. H a t t o r i ( 1 9 7 7 ) : E f f e c t s of c o p p e r on s i l i c i c a c i d u p t a k e by a m a r i n e p h y t o p l a n k t o n p o p u l a t i o 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 . 27:58-65. G u i l l a r d . R.R.L. ( 1 9 7 3 ) . D i v i s i o n r a t e s , I n : S t e i n , J.R. ( e d . ) , Handbook of P h y c o l o g i c a l Methods: C u l t u r e Methods and Growth Measurements, Cambridge U n i v e r s i t y P r e s s , C a m b r i d g e . Habermann, H.M. ( 1 9 6 9 ) . R e v e r s a l o f c o p p e r i n h i b i t i o n i n c h l o r o p l a s t r e a c t i o n s by manganese. P l a n t P h y s i o l . 44: 331-336. H a r r i s o n , W.G., R.W. E p p l e y and E.H. Renger ( 1 9 7 7 ) . P h y t o p l a n k t o n n i t r o g e n m e t a b o l i s m , n i t r o g e n b u d g e t s , and o b s e r v a t i o n s on c o p p e r t o x i c i t y : 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 . B u l l . Mar. S c i . 27: 44-57. H a r t , B.T. and S.H.R. D a v i e s ( 1 9 8 1 ) , T r a c e m e t a l s p e c i a t i o n i n t h e f r e s h w a t e r and e s t u a r i n e r e g i o n s o f t h e Y a r r a R i v e r , V i c t o r i a . E s t . C o a s t . S h e l f S c i . 12:353-374. 82 Inczedy, J . (1966). A n a l y t i c a l appl icat ions of ion exchangers, Pergamon Press, New York, New York. Kingston, H . M . , I . L . Barnes, T . J . Brady, T . C . Rains and M.A. Champ (1978). Separation of eight t r a n s i t i o n elements from a l k a l i and a l k a l i n e earth elements in estuarine and seawater with a chela t ing res in and t h e i r determination by Graphite furnace atomic absorption spectroscopy. Ana l . Chem. 50:2064-2070. Klinkhammer, G.P. and M.L . Bender (1980). The d i s t r i b u t i o n of manganese in the P a c i f i c Ocean. Earth Planet S c i . L e t t . 46:361-384. Lumsden, B .G. and T .M. Florence (1983). A new a l g a l assay procedure for the determination of the t o x i c i t y of copper species in seawater. Env. Tech. L e t t . 4: 271-276. Morel , F .M.M. and N.M.L . Morel-Laurens (1983). Trace metals and plankton in the oceans: facts and speculat ions , In : Wong, C . S . , E . Boyle, K.W. Bruland, J . D . Burton and E .D . Goldberg (eds . ) , Trace metals in seawater, NATO conf. ser. 4, Mar. S c i . V . 9 , p. 841-869, Plenum Press, New York, New York. Morel , F . M . M . , J . G . Rueter, D.M. Anderson and R . R . L . G u i l l a r d (1979). A q u i l : a chemically defined phytoplankton cul ture medium for trace metal s tudies . J . Phycol . j_5:135-1 41 . Morel , N . M . L . , J . G . Rueter and F.M.M. Morel (1978). Copper t o x i c i t y to Skeletonema costatum (Bac i l l a r iophyceae) . J . Phycol . J_4:43-48. Nieboer, E . and D.H.S. Richardson (1980). The replacement of the nondescript term 9heavy metals' by a b i o l o g i c a l l y and chemically s i g i n i f i c a n t c l a s s i f i c a t i o n of metal ions . Environ. P o l l . Ser. B. 1:3-26. 8 3 O v e r n e l l , J . ( 1 9 7 5 ) . T h e e f f e c t o f some h e a v y m e t a l i o n s o n p h o t o s y n t h e s i s i n a f r e s h w a t e r a l g a . P e s t . B i o c h e m . P h y s i o l . 5 : 1 9 - 2 6 . P e t e r s o n , H . G . , F . P . H e a l y a n d F . Wagemann ( 1 9 8 4 ) . M e t a l t o x i c i t y t o a l g a e : A h i g h l y p H d e p e n d e n t p h e n o m e n o n . C a n . J . F i s h . A q u a t . S c i . 4 J _ : 9 7 4 - 9 7 9 . R a i n s , D . W . ( 1 9 7 6 ) . M i n e r a l m e t a b o l i s m , I n : B o n n e r , J . a n d J . E . V a r n e r , ( e d s . ) , P l a n t b i o c h e m i s t r y ( t h i r d e d . ) , p . 5 6 1 - 5 9 7 , A c a d e m i c P r e s s , New Y o r k , New Y o r k . R u e t e r , J . G . , J r . ( 1 9 8 3 ) . A l k a l i n e p h o s p h a t a s e i n h i b i t i o n b y c o p p e r : I m p l i c a t i o n s t o p h o s p h o r u s n u t r i t i o n a n d u s e a s a b i o c h e m i c a l m a r k e r o f t o x i c i t y . L i m n o l . O c e a n o g r . 2 8 : 7 4 3 - 7 4 8 . R u e t e r , J . G . , J r . a n d F . M . M . M o r e l ( 1 9 8 1 ) . T h e i n t e r a c t i o n b e t w e e n z i n c d e f i c i e n c y a n d c o p p e r t o x i c i t y a s i t a f f e c t s t h e s i l i c i c a c i d u p t a k e m e c h a n i s m s i n T h a l a s s i o r a p s u e d o n a n a . L i m n o l . O c e a n o g r . 2 6 : 6 7 - 7 3 . R u e t e r , J . G . , J r . , S . W . C h i s o l m a n d F . M . M . M o r e l ( 1 9 8 1 ) . T h e * e f f e c t o f c o p p e r t o x i c i t y i n s i l i c i c a c i d u p t a k e a n d g r o w t h i n T h a l a s s i o s i r a p s e u d o n a n a ( B a c i l l a r i o p h y c e a e ) . J . P h y c o l . _ T 7 : 2 7 0 - 2 7 8 . S h i f r i n , N . S . a n d S . W . C h i s o l m ( 1 9 8 1 ) . P h y t o p l a n k t o n l i p i d s : i n t e r s p e c i f i c d i f f e r e n c e s a n d e f f e c t s o f n i t r a t e , s i l i c a t e a n d l i g h t - d a r k c y c l e s . J . P h y c o l . 1 7 : 3 7 4 - 3 8 4 . S i l l e n , L . G . a n d A . E . M a r t e l l ( 1 9 6 4 ) . S t a b i l i t y c o n s t a n t s o f m e t a l i o n c o m p l e x e s . C h e m . S o c . L o n d . S p e c . P u b l . 1 7 . S t u m m , W. a n d J . J . M o r g a n ( 1 9 8 1 ) . A q u a t i c c h e m i s t r y , ( s e c o n d e d . ) , W i l e y a n d S o n s , New Y o r k , New Y o r k . S u n d a , W . G . ( 1 9 8 4 ) . M e a s u r e m e n t o f m a n g a n e s e , z i n c a n d c a d m i u m c o m p l e x a t i o n i n s e a w a t e r u s i n g c h e l e x i o n - e x c h a n g e e q u i l i b r i a . M a r . C h e m . 1 4 : 3 6 5 - 3 7 8 . 84 Sunda, W.G. and R.R.L. G u i l l a r d ( 1 9 7 6 ) . The r e l a t i o n s h i p between c u p r i c i o n a c t i v i t y a nd t h e t o x i c i t y of c o p p e r t o p h y t o p l a n k t o n . J . Mar. R e s . 34:511-529. Sunda, W.G. and S.A. Huntsman ( 1 9 8 3 ) . E f f e c t o f c o m p e t i t i v e i n t e r a c t i o n s between manganese and c o p p e r on c e l l u l a r manganese and g r o w t h i n e s t u a r i n e and o c e a n i c s p e c i e s o f t h e d i a t o m T h a l a s s i o s i r a . L i m n o l . O c e a n o g r . 28: 924-934. Sunda, W.G. and S.A. Huntsman ( 1 9 8 5 ) . R e g u l a t i o n of c e l l u l a r manganese and manganese t r a n s p o r t r a t e s i n t h e u n i c e l l u l a r a l g a Chlamydomonas. L i m n o l . O c e a n o g r . 30: 71-80. Sunda, W.G. and S.A. Huntsman and G.R. Ha r v e y ( 1 9 8 3 ) . P h o t o r e d u c t i o n o f Mn o x i d e s i n s e a w a t e r and i t s g e o c h e m i c a l and b i o l o g i c a l i m p l i c a t i o n s . N a t u r e 301: 234-236. Sunda, W.G., R.T. B a r b e r and S.A. Huntsman ( 1 9 8 1 ) . P h y t o p l a n k t o n g r o w t h i n n u t r i e n t r i c h s e a w a t e r : I m p o r t a n c e o f copper-manganese c e l l u l a r i n t e r a c t i o n s . J . Mar. Res. 39:567-586. S w a l l o w , K . C , D.M. Hume and F.M.M. M o r e l ( 1 9 8 0 ) . S o r p t i o n o f c o p p e r and l e a d by h y d r o u s f e r r i c o x i d e . Env. S c i . T e c h n o l . 14:1326-1331. W e l l s , M.L., N.G. Z o r k i n and A.G. L e w i s ( 1 9 8 3 ) . The r o l e of c o l l o i d c h e m i s t r y i n p r o v i d i n g a s o u r c e o f i r o n t o p h y t o p l a n k t o n . J . Mar. Res. 41:731-746. W e s t a l l , J . C . , J . L . Z a c h a r y and F.M.M. M o r e l ( 1 9 7 6 ) . MINEQL. a computer p r o g r a m f o r t h e c a l c u l a t i o n of c h e m i c a l e q u i l i b r i u m c o m p o s i t i o n o f aqueous s y s t e m s . T e c h n i c a l N o te No.18, Water Q u a l i t y L a b . , D e p t . o f C i v i l Eng., M.I.T., C a m b r i d g e , M a s s a c h u s e t t s . W h i t f i e l d , P.H. ( 1 9 7 4 ) . S e a s o n a l c h a n g e s i n h y d r o g r a p h i c and c h e m i c a l p r o p e r t i e s o f I n d i a n Arm and t h e i r e f f e c t on t h e c a l a n o i d c o p e p o d E u c h a e t a j a p o n i c a . M.Sc. T h e s i s , D e p t . o f Z o o l o g y , 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 . 85 Z o r k i n , N.G. ( 1 9 8 3 ) . The d i r e c t e x a m i n a t i o n of b i o l o g i c a l l y a c t i v e Cu i n s e a w a t e r , Ph.D. T h e s i s , D e p a r t m e n t o f O c e a n o g r a p h y , U 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 . Z o r k i n , N.G., E.V. G r i l l and A.G. L e w i s ( 1 9 8 5 ) . An i o n - e x c h a n g e method f o r m e a s u r i n g t h e c o n c e n t r a t i o n of b i o l o g i c a l l y a c t i v e Cu i n s e a w a t e r . A n a l . Chim. A c t a ( i n p r e s s ) . 8 6 A P P E N D I X I 87 Table I. Composition of Aquil medium modified from Morel et a l . (1979). I n i t i a l weight . volume Stock concentration Final concentration Substance (g) ( l i t e r s ) (M) (K) Aquil salts NaCl 490. 6 20 4, .20 X 10" -1 4. 20 X 10' -1 (SOW) Na 2S0 4 81. e 20 2. .88 X 10' •2 2. 88 X 10" -2 CaCl 2.2H z0 30. e 20 1. .05 X 10" -2 1. 05 X ic" -2 KC1 14. 0 20 9, .39 X 10' -4 9. 39 X 10" -4 NaHCO 4. 0 20 2. ,38 X 10' -3 2. 36 X 10" •3 KBr 2. 0 20 8. .40 X 10" •4 8. 40 X 10" -4 H 3 B 0 3 0. 6 20 1*. ,85 X 10' •4 4. 85 X 10" -4 SrCl 2.6H 20 0. 314 20 6. ,38 X 10" •5 6. 3e X 10" •5 NaF 0. 06 20 7. ,14 X 10' •5 7. 14 X 10" -5 MgCl2.6H20 222. 0 20 5. 46 X 10" •2 5. 46 X 10' •2 Nutrients NaHjPC^.HjO 1. 38 1 1. 00 X 10" •2 1. 00 X 10" -5 NaNO 8. 50 1 1. 00 X 10" •1 1. 00 X 10' •4 • 2 Na„£iO_.9K.0 z 55 1 1. 25 X 10" 1. 25 X 10" Trace Metals CuSO^.5H?0 0. 249 1 9. 97 X 10" •4 S. 97 X 10" •10 (NH 4) 6Mo 70 2 u.4H 20 0. 265 1 1. 50 X 10" •3 1. 50 X ic" •9 CoCl 2.6H 20 0. 59 5 1 2. 50 X 10" •3 2. 50 X 10" •9 MnCl2.4H20 0. 199 0. 1 1. 00 X 10" •3 1. 00 X 10" •9 ZnSO^.VH 0 0. 115 0. 1 4. 00 X 10" •3 4 . 00 X 10" •9 .4 • 7 FeCl 3.6K 20 0. 122 1 14. 51 X 10" 4. 51 X i c " 0 7 g/l g/l Vitamins B12 Biotin 0. 0. 011 01 0. 0. 01 1 1. 1. 10 00 X X 10 10" 1 g/l g/l 5 5 . 5 .0 X X 10" 10" 7 Thiamine HC1 0. 020 0. 1 2. 00 X 10" 1 g/l 1 .0 X 10" 4 g/l 88 T a b l e I I . H y d r o g r a p h i c d a t a f o r I n d i a n Arm. C r u i s e d a t e = A u g u s t 24, 1984. D e p t h (m) T e m p e r a t u r e (°C) S a l i n i t y ( p p t ) Oxygen (ml/1) pH 1 0 1 1 .40 24.783 4.17 7.63 50 1 1 .34 26.044 3.87 7.62 100 8.35 26.873 1 .68 7.39 1 50 8.35 26.970 1 .47 7.29 200 8.35 26.983 1 .38 7.32 

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