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UBC Theses and Dissertations

The direct examination of biologically active Cu in seawater Zorkin, N. R. 1983

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THE DIRECT EXAMINATION OF BIOLOGICALLY ACTIVE CU IN SEAWATER by NICHOLAS R. GIUNIO-ZORKIN B . S c , U n i v e r s i t y of V i c t o r i a , V i c t o r i a , 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Departments of Oceanography And Zoology) We accept t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA @ August 1983 (c) N i c h o l a s R. G i u n i o - Z o r k i n , l 9 8 3 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^^-C* O L O 0—V The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date fiotCy. 1 ~L / f r g DE-6 (3/81) i i ABSTRACT An a n a l y t i c a l t e c h n i q u e f o r the d i f f e r e n t i a t i o n of b i o l o g i c a l l y a c t i v e copper (Cu) i n seawater was d e v e l o p e d . The proced u r e i n v o l v e s p a s s i n g a seawater sample t h r o u g h an i o n exchange r e s i n of the s u l p o n a t e type u n t i l complete b r e a k t h r o u g h of m e t a l i o n i s a c h i e v e d . The sorbed Cu i s then e l u t e d and i t s t o t a l c o n c e n t r a t i o n i s d e t e r m i n e d by a n o d i c s t r i p p i n g v o ltammetry. Comparison w i t h Cu a d s o r p t i o n from s t a n d a r d seawater samples of s i m i l a r c o m p o s i t i o n , pH, and i o n i c s t r e n g t h y i e l d s a Cu e q u i v a l e n t measurement t h a t i s r e l a t e d t o the f r e e c u p r i c i o n a c t i v i t y of the sample. S i n c e the c u p r i c i o n i s b e l i e v e d t o be the t o x i c form of the m e t a l , the Cu e q u i v a l e n t measurement can be r e l a t e d t o the b i o l o g i c a l l y a c t i v e f r a c t i o n of Cu. The measurement of b i o l o g i c a l l y a c t i v e Cu by t h e r e s i n t e c h n i q u e was v e r i f i e d by comparing the a n a l y t i c a l r e s u l t s w i t h r e s u l t s from p h y t o p l a n k t o n b i o a s s a y s . T e s t s were f i r s t c o n d u cted i n a r t i f i c i a l seawater t h a t had i t s c h e m i s t r y w e l l d e f i n e d and where model o r g a n i c l i g a n d (EDTA, NTA, h i s t i d i n e and g l u t a m i c a c i d ) were used t o c o n t r o l the s p e c i a t i o n of the m e t a l . In the e x p e r i m e n t s u s i n g the o r g a n i c l i g a n d s EDTA, NTA, or g l u t a m i c a c i d added t o Cu s p i k e d a r t i f i c i a l s eawater, a s t r o n g r e l a t i o n s h i p between the Cu e q u i v a l e n t v a l u e s and growth r a t e s of t he b i o a s s a y o r g a n i s m was found (r=0.92). However, i n ex p e r i m e n t s w i t h h i s t i d i n e , t h i s r e l a t i o n s h i p was much weaker and was a t t r i b u t e d t o 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 Cu-h i s t i d i n e complexes onto the r e s i n . The a d s o r p t i o n of the s e complexes r e s u l t s i n o v e r e s t i m a t i n g the amount of b i o l o g i c a l l y a c t i v e Cu p r e s e n t i n the sample. The few s t u d i e s on the e l e c t r o c h e m i c a l n a t u r e of o r g a n i c c o m p l e x i n g agents i n seawater s u g g e s t s , however, t h a t most a r e n e g a t i v e l y charged. Thus the t e c h n i q u e would be s u i t a b l e i n many seawater systems. The a n a l y t i c a l and b i o a s s a y t e c h n i q u e s were then a p p l i e d t o n a t u r a l seawater samples c o l l e c t e d from f i v e depths i n a l o c a l f j o r d . A d i s c r e p a n c y was found between some of the b i o a s s a y and r e s i n t e s t r e s u l t s . However, the d i s c r e p a n c y was a t t r i b u t e d t o a p h y s i o l o g i c a l Cu-Mn i n t e r a c t i o n i n the b i o a s s a y o r g a n i s m and not t o a problem w i t h the r e s i n t e c h n i q u e . i v TABLE OF CONTENTS ABSTRACT . i i LIST OF TABLES i x LIST OF FIGURES x i ACKNOWLEDGEMENTS x i v I . INTRODUCTION 1 I I . ANALYTICAL METHODS FOR STUDYING CU SPECIATION 8 A. ELECTROCHEMICAL METHODS 8 1. S p e c i f i c Ion P o t e n t i o m e t r y 8 2. Voltammetry 9 c (a) Anodic s t r i p p i n g voltammetry ., 10 (b) Chemical forms measured by ASV 11 (c) Measurement of b i o l o g i c a l l y a c t i v e Cu 12 B. ANALYTICAL METHODS BASED ON PRELIMINARY CHEMICAL AND PHYSICAL SEPARATION 16 1. S e p a r a t i o n Based on O r g a n i c S o l v e n t S o l u b i l i t y ... 16 2. M o l e c u l a r S i z e S e p a r a t i o n 18 3. S e p a r a t i o n Based on Charge and L a b i l i t y of Complexes ...... 20 I I I . THE MEASUREMENT OF BIOLOGICALLY ACTIVE CU BY A MARINE DIATOM 22 A. INTRODUCTION 22 B. MATERIALS AND METHODS ... 26 1. Medium P r e p a r a t i o n 26 2. B i o a s s a y s 29 V 3. Test of Mode- of Fe A d d i t i o n 32 4. Growth Measurements 33 5. Chelex-100 as an Ion-Exchanger .. 35 C. COMPUTER MODELLING OF CU SPECIATION IN AQUIL ... 38 D. RESULTS 43 1. Model L i g a n d Study 43 2. Growth Rate as a F u n c t i o n of C u p r i c Ion A c t i v i t y . 55 3. E f f e c t of pH 62 4. Fe A d d i t i o n s 63 D. DISCUSSION 69 1. Growth Rate as a F u n c t i o n of pCu* 69 2. E x p e r i m e n t a l C o n s i d e r a t i o n s 71 3. E n v i r o n m e n t a l C o n s i d e r a t i o n 74 IV. THE MEASUREMENT OF BIOLOGICALLY ACTIVE CU BY A STRONGLY ACIDIC CATION EXCHANGER 76 A. INTRODUCTION . 76 B. THEORETICAL CONSIDERATIONS 78 1. I n t r o d u c t i o n t o Ion-Exchange R e s i n s 78 2. Theory 79 3. A p p l i c a t i o n of the Ion-Exchange P r o c e d u r e t o the D e t e r m i n a t i o n of C a t i o n i c Cu S p e c i e s i n Seawater . 82 C. MATERIALS AND METHODS 86 1. Column P r e p a r a t i o n 86 (a) M a t e r i a l s 86 (b) P r e p a r a t i o n of and column p r o c e d u r e .. 87 (c) P r o c e d u r e f o r e l u t i o n of column p r i o r t o ASV . 90 2. ASV P r o c e d u r e f o r M e a s u r i n g T o t a l Cu .............. 91 (a) Equipment 91 (b) P r e - p l a t i n g and p r e - c o n d i t i o n i n g of the e l e c t r o d e 92 (c) G e n e r a l p r o c e d u r e 93 (d) C a l i b r a t i o n 94 (e) P rocedure f o r measuring t o t a l Cu i n the e l u a t e s of the r e s i n a n a l y s i s 96 3. C h a r a c t e r i z a t i o n of the R e s i n AG 50W-X12 97 (a) Column e q u i l i b r a t i o n 97 (b) P r e c i s i o n 98 (c) E f f e c t of pH on the a d s o r p t i o n of Cu by the r e s i n 98 (d) E f f e c t of s a l i n i t y on the a d s o r p t i o n of Cu by the r e s i n 99 (e) E f f e c t s of n u t r i e n t s on a d s o r p t i o n of Cu by the r e s i n 99 ( f ) A d s o r p t i o n c u r v e s f o r Cu i n SOW w i t h no o r g a n i c l i g a n d s p r e s e n t 101 4. Model L i g a n d Study 101 RESULTS 104 1. C h a r a c t e r i z a t i o n of the R e s i n i n A r t i f i c i a l Seawater 104 (a) E q u i l i b r a t i o n of the r e s i n t o Cu 104 (b) P r e c i s i o n of the ASV and column a d s o r p t i o n t e c h n i q u e 107 (c) E f f e c t of pH 110 (d) N u t r i e n t e f f e c t s 110 (e) I o n i c s t r e n g t h e f f e c t s 115 ( f ) A d s o r p t i o n c u r v e s f o r Cu .........115 3. Model L i g a n d Study .. . 119 4. Comparison of the Ch e m i c a l Assay and B i o a s s a y R e s u l t s .126 E. DISCUSSION 132 V. APPLICATION OF THE RESIN TECHNIQUE TO NATURAL SEAWATER .138 A. INTRODUCTION 138 B. COLLECTION OF NATURAL SEAWATER .140 C. MATERIALS AND METHODS 144 1. P r e l i m i n a r y Sample P r e p a r a t i o n ...144 2. B i o a s s a y s .............144 3. C a l i b r a t i o n of the R e s i n i n Low S a l i n i t y SOW ......145 4. A p p l i c a t i o n of the Ion-Exchange Method t o N a t u r a l Seawater 147 5. Manganese-Copper I n t e r a c t i o n ... .148 6. N a t u r a l Seawater S o l u b l e Agents E x t r a c t e d from Sediments 149 D. RESULTS .151 1. P r e l i m i n a r y T e s t s 151 2. B i o a s s a y s ...153 3. A p p l i c a t i o n of the R e s i n Technique t o N a t u r a l Seawater .155 4. Comparison of the R e s i n and B i o a s s a y R e s u l t s .....160 4. Study of Water S o l u b l e Agents from Sediments ......164 E. DISCUSSION ...168 V I . GENERAL CONCLUSIONS 174 V I I . REFERENCES CITED APENNDIX A. BIOASSAY DATA LIST OF TABLES T a b l e I . Summary of t e c h n i q u e s used t o study t r a c e m e t a l s p e c i a t i o n i n n a t u r a l waters 17 T a b l e I I . P r e p a r a t i o n of A q u i l 28 T a b l e I I I . S t a b i l i t y c o n s t a n t s f o r the more i m p o r t a n t Cu complexes 40 T a b l e IV. Summary of b i o a s s a y r e s u l t s 44 T a b l e V. V a r i a t i o n s i n pH of c u l t u r e s w i t h and w i t h o u t the a d d i t i o n of Cu over a f i v e day p e r i o d 63 T a b l e V I . L i g a n d s , l i g a n d c o n c e n t r a t i o n s and Cu c o n c e n t r a t i o n s s t u d i e d . ..................102 T a b l e V I I . The e f f e c t of f l o w r a t e on the a d s o r p t i o n of Cu by t h e r e s i n ....106 T a b l e V I I I . P r e c i s i o n of the o v e r a l l r e s i n a n a l y s i s ...108 T a b l e IX. P r e c i s i o n of t h e ASV a n a l y s i s 109 T a b l e X. The e f f e c t of pH on the a d s o r p t i o n of Cu by the r e s i n .110 T a b l e X I . The e f f e c t of the A q u i l n u t r i e n t s on the a d s o r p t i o n of Cu 111 T a b l e X I I . The E f f e c t of Fe on the a d s o r p t i o n of Cu 112 T a b l e X I I I . The E f f e c t of Aged Fe S t o c k s on the a d s o r p t i o n of Cu 113 T a b l e XIV. Cu e q u i v v a l u e s i n SOW i n the presence of EDTA, GLU and NTA f o r S e r i e s I 121 T a b l e XV. Cu e q u i v v a l u e s i n SOW i n t h e presence of EDTA,. GLU and NTA f o r s e r i e s I I ...123 T a b l e XVI. The a d s o r p t i o n of Cu i n the presence of HIS ....124 T a b l e X V I I . Cu e q u i v v a l u e s from SOW i n the presence of HIS .125 T a b l e X V I I I . pKs of H i s t i d i n e 126 T a b l e XIX. Growth r a t e s , and pCu as e s t i m a t e d by the r e s i n a n a l y s i s and by c a l c u l a t i o n .........127 T a b l e XX. Growth r a t e s of the b i o a s s a y organism i n seawater t a k e n from f i v e depths i n I n d i a n Arm 156 T a b l e XXI. R e s u l t s of the r e s i n a n a l y s i s conducted on the n a t u r a l water samples ...,.159 T a b l e X X I I . H y d r o g r a p h i c and t r a c e m e t a l d a t a from water samples c o l l e c t e d from f i v e depths a t s t a t i o n IND-2 ........ 163' T a b l e X X I I I . R e s i n and b i o a s s a y r e s u l t s from the sediment s t u d y 1 66 LIST OF FIGURES Figure 1. P o s s i b l e chemical forms of Cu i n seawater 5 Figure 2. The model organic l i g a n d s used i n the bioassays . 31 Figure 3. Growth of the bioassay organism i n A q u i l i n the presence of Cu and with no organic l i g a n d s added 48 Figure 4. Growth ra t e (% of c o n t r o l ) versus the - l o g of the added Cu c o n c e n t r a t i o n (Cu T) i n A q u i l w i t h no organics added 49 Figure 5. E f f e c t of Cu upon growth ( d i v s day" 1) during the 24-96 hr p e r i o d 50 Figure 6. Growth rate (% of c o n t r o l ) versus the - l o g of the Cu co n c e n t r a t i o n (Cu T) i n the presence of GLU 51 Figu r e 7. Growth rate (% of c o n t r o l ) versus the - l o g of the Cu co n c e n t r a t i o n (Cu T) i n the presence of HIS 52 Figure 8. Growth rate (% of c o n t r o l ) versus the - l o g of the Cu concen t r a t i o n (Cu T) i n the presence of NTA 53 Figure 9. Growth rate (% of c o n t r o l ) versus the - l o g of the Cu concen t r a t i o n (Cu T) i n the presence of EDTA 54 Figure 10. Growth ra t e (% of c o n t r o l ) versus the c a l c u l a t e d pCu* i n the presence of GLU 57 Figu r e 11. Growth rate (% of c o n t r o l ) versus the c a l c u l a t e d pCu* i n the presence of HIS 58 Figure 12. Growth ra t e (% of c o n t r o l ) versus the c a l c u l a t e d pCu* i n the presence of NTA 59 Figu r e 13. Growth rate (% of c o n t r o l ) versus the c a l c u l a t e d pCu* i n the presence of EDTA 60 F i g u r e 14. Growth r a t e (% of c o n t r o l ) v e r s u s the c a l c u l a t e d pCu* f o r a l l the b i o a s s a y r e s u l t s 61 F i g u r e 15. C e l l growth w i t h f r e s h Fe s t o c k s and Fe-EDTA s t o c k s added b e f o r e and a f t e r a u t o c l a v i n g 66 F i g u r e 16. C e l l growth w i t h a u t o c l a v e d Fe s t o c k added t o the medium 67 F i g u r e 17. C e l l growth w i t h aged Fe s t o c k s added t o the medium .... 68 F i g u r e 18. Econocolumn 88 F i g u r e 19. E f f l u e n t Cu (% of i n f l u e n t ) v e r s u s the e f f l u e n t volume . 1 05 F i g u r e 20. Change i n the a d s o r p t i o n of Cu w i t h s a l i n i t y ...116 F i g u r e 21. A d s o r p t i o n c u r v e s f o r Cu i n SOW w i t h and w i t h o u t t h e a d d i t i o n of Fe 118 F i g u r e 22. Growth r a t e (% of c o n t r o l ) v e r s u s the n e g a t i v e l o g of the Cu e q u i v v a l u e s ......129 F i g u r e 23. Growth r a t e (% of c o n t r o l ) v e r s u s pCu e s t i m a t e d from the r e s i n r e s u l t s ....130 F i g u r e 24. Growth r a t e (% of c o n t r o l ) v e r s u s pCu* c a l c u l a t e d by MINEQL ...131 F i g u r e 25. L o c a t i o n of sample c o l l e c t i o n 142 F i g u r e 26. E l u a t e Cu v e r s u s the e f f l u e n t volume f o r the e q u i l i b r i u m experiment 152 F i g u r e .27. A d s o r p t i o n c u r v e s f o r Cu u s i n g low s a l i n i t y SOW w i t h and w i t h o u t the a d d i t i o n of Fe 154 F i g u r e 28. Growth r a t e (% of c o n t r o l ) v e r s u s the t o t a l Cu x i i i c o n c e n t r a t i o n i n the n a t u r a l water samples 157 F i g u r e 29. Growth r a t e (% of c o n t r o l ) v e r s u s the - l o g of the Cu e q u i v v a l u e s 161 F i g u r e 30. The e f f e c t of Mn on r e d u c i n g the t o x i c i t y of Cu 165 x i v 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 , Dr.. A.G. L e w i s , f o r h i s s u g g e s t i o n s , a d v i c e and c o n t i n u e d a s s i s t a n c e t h r o u g h o u t my s t u d i e s . My deep a p p r e c i a t i o n i s extended t o Dr. E.V. G r i l l f o r h i s i n p u t and p a t i e n c e w i t h my work. I am g r a t e f u l t o my r e s e a r c h committee members,- D r s . H a l l , Thompson, G o s l i n e and F l e t c h e r f o r t h e i r a d v i c e and s u g g e s t i o n s f o r i m p r o v i n g the m a n u s c r i p t . I w i s h t o acknowledge my f e l l o w g r a d u a t e s t u d e n t s , p a r t i c u l a r l y Mark W e l l s , f o r t h e i r s u p p o r t and a s s i s t a n c e . F i n a l l y , I would e s p e c i a l l y l i k e t o thank my mother and f a t h e r f o r t h e i r m oral support throughout my e d u c a t i o n and Robin f o r her adherence. 1 I . INTRODUCTION Copper (Cu) i s an e n v i r o n m e n t a l c o n s t i t u e n t of c o n s i d e r a b l e i m p o r t a n c e . Not o n l y i s i t n e c e s s a r y f o r the proper f u n c t i o n i n g of many p h y s i o l o g i c a l p r o c e s s e s i n marine p h y t o p l a n k t o n , i t has a l s o been found t o be t o x i c t o many s p e c i e s a t the c o n c e n t r a t i o n s n o r m a l l y encountered i n c o a s t a l seawater. The forms of Cu which can be u t i l i z e d by p h y t o p l a n k t o n a r e not d e f i n i t e l y known but t h e r e i s c o n s i d e r a b l e e v i d e n c e t o suggest t h a t i t s b i o a v a i l a b i l i t y i s c o n t r o l l e d by the a c t i v i t y of the f r e e c u p r i c i o n and t h a t b oth the i n o r g a n i c and o r g a n i c c o m p l e x i n g agents t h a t a l t e r the i o n i c a c t i v i t y a l s o a f f e c t the m e t a l ' s b i o l o g i c a l a v a i l a b i l i t y . T h i s has g e n e r a t e d c o n s i d e r a b l e i n t e r e s t i n the q u e s t i o n of the c o m p l e x a t i o n of Cu by n a t u r a l o r g a n i c s and the importance of t h i s p r o c e s s i n d e t o x i f y i n g heavy m e t a l s i n n a t u r a l systems. Thus the s p e c i a t i o n of the m e t a l , which i s c o n t r o l l e d by water c h e m i s t r y , must be c o n s i d e r e d when a s s e s s i n g p o t e n t i a l uptake and t o x i c i t y . U n f o r t u n a t e l y , t h e r e a r e p r e s e n t l y no a n a l y t i c a l methods a v a i l a b l e t h a t have demonstrated an a b i l i t y t o measure b i o l o g i c a l l y a c t i v e Cu i n seawater and thus i t s e n v i r o n m e n t a l impact cannot be r e a d i l y a s s e s s e d except by t e d i o u s b i o a s s a y t e c h n i q u e s . The major g o a l of t h i s t h e s i s was t o d e v e l o p a r a p i d a n a l y t i c a l p r o c e d u r e t h a t would be c a p a b l e of e s t i m a t i n g the 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 n a t u r a l seawater. S p e c i f i c a l l y , the h y p o t h e s i s t e s t e d was t h a t the response of a marine d i a t o m t o Cu was p r e d i c t a b l e from the amount of Cu adsorbed by 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 . 2 The e f f e c t of s y n t h e t i c and n a t u r a l c h e l a t o r s i n r e d u c i n g the 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 has been w e l l documented ( e . g . , Spencer, 1957; F i t z g e r a l and F a u s t , 1963; E r i c k s o n e_t a l . , 1970; M o r r i s and R u s s e l l , 1973; McKnight and M o r e l , 1980; C a n t e r f o r d and C a n t e r f o r d , 1980). Steeman N i e l s e n and Wium-Andersen (1970) suggested t h a t the e f f e c t of o r g a n i c c h e l a t o r s i n s t i m u l a t i n g the growth of a l g a e , as was o b s e r v e d by Joh n s t o n (1963, 1964) and Barber and R y t h e r (1969), was due t o the c o m p l e x a t i o n of the c u p r i c i o n ( C u 2 + ) a l t h o u g h such s t i m u l a t i o n has sometimes been a t t r i b u t e d t o an i n c r e a s e i n a v a i l a b i l i t y of t r a c e m e t a l s ( J o h n s t o n , 1964). Sunda and G u i l l a r d (1976) have p r o v i d e d d i r e c t e v i d e n c e t h a t c o r r e l a t e s the growth of p h y t o p l a n k t o n w i t h the c u p r i c i o n a c t i v i t y ( a c t i v i t y = c o n c e n t r a t i o n x a c t i v i t y c o e f f i c i e n t ) of the medium. U s i n g an e s t u a r i n e d i a t o m and green a l g a i n seawater i n which the t r a c e m e t a l s were c h e l a t e d w i t h t r i s h y d r o x y m e t h y l a m i n o m e t h a n e ( T R I S ) , they demonstrated t h a t growth r a t e i n h i b i t i o n and Cu c o n t e n t of c e l l s was not r e l a t e d t o the t o t a l Cu c o n c e n t r a t i o n but t o the a c t i v i t y of the u n c h e l a t e d c u p r i c i o n . S i n c e the c h e m i s t r y of the medium was w e l l d e f i n e d , the c u p r i c i o n a c t i v i t y c o u l d be c a l c u l a t e d by a computer e q u i l i b r i u m model. A s i m i l a r s tudy conducted by Anderson and M o r e l (1978) w i t h the d i n o f l a g e l l a t e Gonyaulax t a m a r e n s i s demonstrated t h a t the s h o r t term Cu s e n s i t i v i t y of the organism was a unique f u n c t i o n of the c u p r i c i o n a c t i v i t y . Davey e t a l . (1973) p r e s e n t e d p l o t s of growth i n h i b i t i o n of t he d i a t o m T h a l a s s i o s i r a pseudonana v s . Cu c o n c e n t r a t i o n i n 3 media c o n t a i n i n g 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 (EDTA) or h i s t i d i n e ( H I S ) , and found t h a t the p l o t s resembled p o t e n t i o m e t r i c t i t r a t i o n c u r v e s ; i . e . , a sharp i n f l e c t i o n i n the growth c u r v e o c c u r r e d a t the p o i n t where the Cu c o n c e n t r a t i o n exceeded t h a t of the c h e l a t o r . A c h e m i c a l s p e c i a t i o n model was a p p l i e d t o Davey's r e s u l t s by J a c k s o n and Morgan (1978) t o compute the c o n c e n t r a t i o n of f r e e c u p r i c i o n i n t h e i r s o l u t i o n s . I t was c o n c l u d e d t h a t the f r e e c u p r i c i o n was the t o x i c form because, even w i t h d i f f e r e n t c h e l a t o r s p r e s e n t , i d e n t i c a l c a l c u l a t e d c o n c e n t r a t i o n s of c u p r i c i o n c o r r e s p o n d e d t o i d e n t i c a l growth r a t e s . The s t u d i e s above used s y n t h e t i c c h e l a t o r s t o c o n t r o l the c o n c e n t r a t i o n of the c u p r i c i o n i n c u l t u r e media. In a p p l y i n g t h e s e r e s u l t s t o the n a t u r a l environment i t would seem r e a s o n a b l e t o assume t h a t p h y t o p l a n k t o n w i l l respond i n the same manner t o Cu c h e l a t e d by n a t u r a l o r g a n i c m a t t e r . Sunda and Lewis (1978) s t u d i e d the r e l a t i o n s h i p between b i n d i n g of Cu by o r g a n i c m a t t e r p r e s e n t i n r i v e r water and the t o x i c i t y of Cu t o the a l g a l s p e c i e s M o n o c h r y s i s l u t h e r i . F i l t e r e d r i v e r water c o n t a i n i n g h i g h c o n c e n t r a t i o n s of o r g a n i c m atter was added i n d i f f e r e n t p r o p o r t i o n s t o t h e i r c u l t u r e media t o v a r y the l e v e l of Cu c o m p l e x a t i o n . They found t h a t an i n c r e a s e i n the c o n c e n t r a t i o n of n a t u r a l l i g a n d s 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 t o x i c i t y and t h a t t h i s d e c r e a s e c o u l d be r e l a t e d t o the c u p r i c i o n a c t i v i t y of the medium as was measured by an i o n -s e l e c t i v e e l e c t r o d e . However, i t may not o n l y be the c u p r i c i o n t h a t i s t o x i c . 4 In s t u d i e s w i t h a l g a e i n f r e s h w a t e r , Wagemann and B a r i c a (1979) found t h a t the a b i l i t y of Cu t o a c t as an a l g i c i d e was b e s t e x p l a i n e d by c o n s i d e r i n g the t o x i c f r a c t i o n t o be comprised of C u 2 + , CuOH + and Cu(OH) 2. U n f o r t u n a t e l y , d a t a i n the l i t e r a t u r e a r e t o o sp a r s e t o prove or d i s p r o v e the t o x i c i t y of CuOH + or Cu(OH) 2. In t h e s t u d i e s t h a t have demonstrated the c u p r i c i o n t o be the t o x i c form i n seawater, the t o x i c i t y of the h y d r o x i d e s p e c i e s has not been c o n s i d e r e d . The q u e s t i o n of the t o x i c form of Cu cannot be examined w i t h o u t f i r s t r e v i e w i n g the c h e m i c a l forms of Cu t h a t have been p r e d i c t e d t o e x i s t i n seawater. Copper can t h e o r e t i c a l l y appear i n t h r e e phases which may or may not be s e p a r a b l e ; i . e . , p a r t i c u l a t e , c o l l o i d a l and s o l u b l e . In a d d i t i o n , t h e s e may be s u b d i v i d e d i n t o o r g a n i c and i n o r g a n i c f r a c t i o n s ( F i g . 1 ) . Due t o i n t e r a c t i o n s between the i n o r g a n i c c o n s t i t u e n t s i n seawater, Cu w i l l o c cur not o n l y as the f r e e h y d r a t e d i o n but a l s o i n complexes formed w i t h the a n i o n i c s p e c i e s such as OH", C I " , C 0 3 2 " and S0<,2". The c o n c e n t r a t i o n of the s e complexes, which i n c l u d e s s p e c i e s such as [ C u 2 + ( H 2 0 ) 6 1 2 + , CuOH +, Cu(OH) 2, CuC0 3, Cu(C0 3 ) 2 2 ", C u C l + , C u C l 2 , and CuSOj,, has been e s t i m a t e d u s i n g c h e m i c a l e q u i l i b r i u m models (Dyrssen and Wedborg, 1974; Stumm and B r a u n e r , 1975). The p r e d i c t e d v a l u e s d i f f e r c o n s i d e r a b l y , however. For example, Z i r i n o and Yamamoto (1972) p r e d i c t e d t h a t , f o r seawater of 35 ppt s a l i n i t y , 25°C and a pH of 8.0, the p e r c e n t a g e s of the major s p e c i e s were 90% Cu(OH) 2, 7.7% CuC0 3, and 1.0% CuOH +. Morgan and S i b l e y (1975), on t h e o t h e r hand, p r e d i c t e d 95.5% C u C l + , 3.5% CuHC0 3 +, and 3.5% CuC0 2 Simple hydrated metal Simple inorganic complexes Simple organic complexes Stable organic complexes Adsorbed onto inorganic and organic colloids Particulate Cu(H 20)f CuOH Cu(OH)2 CuC0 3° Cu(C0 3) 2 CuCl + CuSO. 2-Cu-SR Cu-OOCR Cu-histidine -CH —C=0 / 2 \ NH2 0 x^Cu 0=C-CH„ 2+ Cu -Fe 20 3 2+ Cu -MnC>2 ~2+ „ Cu -Humate Remains of living organisms Organic particles Inorganic particles Approximate diameter (nm) 0.8 1-10 1-10 10-500 450 R= Organic molecule Figure 1. Possible chemical forms of Cu in seawater. 6 t o be the p e r c e n t a g e d i s t r i b u t i o n . More r e c e n t c a l c u l a t i o n s by a computer e q u i l i b r i u m model have p r e d i c t e d the major s p e c i e s t o be CuC03 ( W e s t a l l et a l . , 1976) "The wide v a r i a t i o n i n t h e s e c a l c u l a t i o n s (see Nordstrom et a l . (1979) f o r a comparison of t h i r t e e n computer models) i s the r e s u l t of u s i n g d i f f e r e n t p u b l i s h e d v a l u e s of the s t a b i l i t y c o n s t a n t s , p a r t i c u l a r l y those of the hydroxo and c a r b o n a t o s p e c i e s ( L e c k i e and D a v i s , 1979). In a d d i t i o n t o i n o r g a n i c c o m p l e x a t i o n , the p r e s ence of Cu-o r g a n i c complexes i n seawater i s h i g h l y l i k e l y because Cu has a g r e a t a f f i n i t y f o r amino, i m i n o , s u l f h y d r y l , c a r b o x y l and h y d r o x y l groups and thus bonds r e a d i l y t o o r g a n i c m o l e c u l e s . But, as P o c k l i n g t o n (1977) p o i n t e d o u t , no one has y e t i s o l a t e d and c h a r a c t e r i z e d a C u - o r g a n i c complex from seawater. The p r e s e n c e of Cu a s s o c i a t e d w i t h o r g a n i c s has been i n f e r r e d from measurements of Cu b e f o r e and a f t e r samples have undergone t r e a t m e n t s d e s i g n e d t o i s o l a t e or d e s t r o y the o r g a n i c f r a c t i o n of the sample. Slowey et a_l. (1967) found t h a t when samples of f i l t e r e d seawater were e x t r a c t e d w i t h s m a l l p o r t i o n s of c h l o r o f o r m , 10-60% of the t o t a l Cu was t r a n s f e r r e d i n t o the o r g a n i c phase. A c c o r d i n g t o C o r c o r a n and A l e x a n d e r (1964), Cu e x i s t s i n seawater p r i m a r i l y i n a s o l u b l e n o n i o n i c s t a t e and p r o b a b l y as an o r g a n i c complex. They base t h e i r c o n c l u s i o n on i o n i c Cu a n a l y s i s b e f o r e and a f t e r d i g e s t i o n w i t h p e r c h l o r i c a c i d ; a f t e r d i g e s t i o n i o n i c Cu l e v e l s were g r e a t l y i n c r e a s e d . In samples c o l l e c t e d o f f the c o a s t of Southern C a l i f o r n i a by W i l l i a m s (1969), from 5 t o 28% of the t o t a l Cu was o r g a n i c a l l y a s s o c i a t e d , based upon o x i d a t i o n by u l t r a v i o l e t (UV) 7 i r r a d i a t i o n . O r g a n i c a l l y a s s o c i a t e d Cu has a l s o been i n f e r r e d from v a r i o u s t o x i c i t y and growth e x p e r i m e n t s ( J o h n s t o n , 1964; Barber and R y t h e r , 1 9 6 9 ; Steeman N i e l s e n and Wium Andersen, 1970; Lewis et a l . , 1973). A review of m e t a l l o - o r g a n i c i n t e r a c t i o n s i n n a t u r a l waters has been w r i t t e n by Mantoura (1981) . Copper may a l s o be p r e s e n t adsorbed t o c o l l o i d a l or m a c r o s c o p i c s i z e d p a r t i c u l a t e m a t t e r . I n o r g a n i c s o l i d s such as hydrous o x i d e s of Mn and Fe have been shown t o adsorb Cu (Vuceta and Morgan, 1978; Swallow e_t a l . , 1 980) and such m a t e r i a l s have been suggested t o a c t as a p o s s i b l e e n v i r o n m e n t a l s i n k f o r heavy m e t a l s such as Cu (Jenne, 1968; S h o l k o v i t z , 1978). A r e v i e w on Cu i n oceans and e s t u a r i e s has been w r i t t e n by Lewis and Cave (1982) . 8 I I . ANALYTICAL METHODS FOR STUDYING CU SPECIATION A. ELECTROCHEMICAL METHODS There a re many i n s t r u m e n t a l t e c h n i q u e s c a p a b l e of d i r e c t l y measuring d i s s o l v e d m e t a l l e v e l s i n s o l u t i o n w i t h o u t i n i t i a l c h e m i c a l s e p a r a t i o n and/or p r e c o n c e n t r a t i o n ( e . g . , n e u t r o n a c t i v a t i o n , atomic a b s o r p t i o n , X-ray f l u o r e s c e n c e ) . However, such methods g i v e o n l y the t o t a l m e t a l c o n c e n t r a t i o n and p r o v i d e no i n f o r m a t i o n on the p a r t i c u l a r forms of m e t a l p r e s e n t . Voltammetry and s p e c i f i c i o n p o t e n t i o m e t r y , by c o n t r a s t , a r e t e c h n i q u e s whose response i s dependent on t r a c e m e t a l s p e c i a t i o n a l t h o u g h t h e i r a p p l i c a t i o n t o seawater i s f r a u g h t w i t h problems. 1. S p e c i f i c Ion P o t e n t i o m e t r y I o n - s e l e c t i v e e l e c t r o d e s (ISE) d e v e l o p a p o t e n t i a l which i s dependent s o l e l y on i o n i c a c t i v i t y . Thus, i n a sense, they a r e c a p a b l e of d i r e c t l y d i s t i n q u i s h i n g between the f r e e and bound m e t a l i o n s . The c u p r i c i o n e l e c t r o d e has been used t o de t e r m i n e t h e a c t i v i t y of the c u p r i c i o n i n n a t u r a l w a t e r s (Smith and Manahan, 1973; S t e l l a and G a n z e r i i - V a l e n t i n i , 1979) and seawater samples ( J a v i n s k i e t a l , 1974; W i l l i a m s and B a l d w i n , 1976). However, t h e use of t h e s e e l e c t r o d e s i n seawater has been c r i t i s e d because of c h l o r i d e i n t e r f e r e n c e ( M i d g l e y , 1976). Oglesby e t a^. (1977), u s i n g d i f f e r e n t background e l e c t r o l y t e s , found t h a t the response of the e l e c t r o d e was n o n - N e r n s t i a n i n a c h l o r i d e medium w h i l e the response was N e r n s t i a n i n a l l the 9 o t h e r e l e c t r o l y t e s s t u d i e d . A c c o r d i n g t o W e s t a l l et a l . (1979), C u ( l l ) from the b u l k s o l u t i o n i s reduced a t -the e l e c t r o d e s u r f a c e t o C u ( l ) which i s s t a b i l i z e d by c h l o r i d e c o m p l e x a t i o n w i t h subsequent o x i d a t i o n of the mixed s u l p h i d e e l e c t r o d e m a t e r i a l . T h i s r e s u l t s i n n o n - N e r n s t i a n and u n p r e d i c t a b l e b e h a v i o u r . The b e h a v i o r of the e l e c t r o d e has been sug g e s t e d t o resemble t h a t of a p o l a r i z e d sensor r a t h e r t h a t a s t a n d a r d ISE ( Z i r i n o and S e l i g m a n , 1981). Thus i t i s not p o s s i b l e t o make a b s o l u t e measurements of c u p r i c i o n i n seawater w i t h the c u p r i c i o n e l e c t r o d e a l t h o u g h measurements of r e l a t i v e d i f f e r e n c e s i n the c u p r i c i o n a c t i v i t y of seawater samples has been a t t e m p t e d ( J a v i n s k i e t a_l, 1974; W i l l i a m s and B a l d w i n , 1976). 2. Voltammetry V o l t a m m e t r i c t e c h n i q u e s t h a t have been d e v e l o p e d f o r the d e t e r m i n a t i o n of m e t a l s i n n a t u r a l waters i n c l u d e p u l s e p o l a r o g r a p h y (Fonds e t a l . , 1964), d i f f e r e n t i a l p u l s e p o l a r o g r a p h y ( B a r k e r and G a r d i n e r , 1960), a n o d i c s t r i p p i n g voltammetry ( C h r i s t i a n , 1969) as w e l l as many o t h e r s . Anodic s t r i p p i n g voltammetry, because of i t s a b i l i t y t o p r e c o n c e n t r a t e the m e t a l , i s the most s e n s i t i v e t e c h n i q u e and i s c a p a b l e of d i r e c t l y measuring Cu c o n c e n t r a t i o n s a t the l e v e l s e n c o u n t e r e d i n n a t u r a l seawater. There has been i n c r e a s i n g use of t h i s t e c h n i q u e due t o i t s a b i l i t y t o s i m u l t a n e o u s l y d e t e r m i n e s e v e r a l e l e m e n t s , n o n d e s t r u c t i v e l y , a t sub-ug 1~ 1 c o n c e n t r a t i o n s and w i t h r e l a t i v e l y i n e x p e n s i v e i n s t r u m e n t a t i o n . Because of the 10 p o t e n t i a l of ASV f o r s t u d y i n g Cu s p e c i a t i o n and because i t was used i n the p r e s e n t s t u d y , a d e t a i l e d d e s c r i p t i o n of the t e c h n i q u e i s g i v e n . (a) Anodic s t r i p p i n g voltammetry Anodic s t r i p p i n g voltammetry (ASV) i s a two-step t e c h n i q u e . The f i r s t s t e p i n v o l v e s the c a t h o d i c d e p o s i t i o n of a p o r t i o n of the d i s s o l v e d m e t a l i o n onto a s u i t a b l e e l e c t r o d e . T h i s w o r k i ng e l e c t r o d e i s e i t h e r a hanging mercury drop e l e c t r o d e (HMDE) or a t h i n mercury f i l m e l e c t r o d e (TFE) d e p o s i t e d onto a s u b s t r a t e such as carbon ( g l a s s y c a r b o n , wax impregnated g r a p h i t e , carbon p a s t e ; E l v i n g e_t a J . , 1964), Pt or Ag. T h i s s t e p p r e c o n c e n t r a t e s the metal i n t o a s m a l l volume. In the second s t e p the me t a l i s s t r i p p e d out of t h e amalgam by the a p p l i c a t i o n of an an o d i c scan. C u r r e n t and p o t e n t i a l measurements are made d u r i n g the second s t e p and t h e p o s i t i o n and h e i g h t of the s t r i p p i n g peaks a r e c h a r a c t e r i s t i c of the type and c o n c e n t r a t i o n of m e t a l i o n s o r i g i n a l l y i n s o l u t i o n (Siegerman and O'Dom, 1972). The d e t e c t i o n l i m i t i s dependent on the d e p o s i t i o n t i m e , the s t i r r i n g r a t e and p h y s i c a l parameters of c e l l d e s i g n used d u r i n g t h e d e p o s i t i o n s t e p . To i n c r e a s e the s e n s i t i v i t y of ASV a d i f f e r e n t i a l p u l s e wave-form can be a p p l i e d d u r i n g the s t r i p p i n g s t e p (DPASV). In DPASV, the m e t a l s i n the amalgam a r e r e o x i d i z e d u s i n g a slow l i n e a r a n o d i c p o t e n t i a l ramp upon which a r e superimposed f i x e d h e i g h t v o l t a g e p u l s e s . Measurement of the r e s u l t i n g c u r r e n t i n c r e a s e i s d e l a y e d u n t i l t he l a t t e r s t a g e s of the p u l s e - l i f e 11 when the c a p a c i t i v e component has l a r g e l y decayed t h e r e b y i n c r e a s i n g the s i g n a l t o n o i s e r a t i o . In the p o l a r o g r a p h i c a n a l y z e r used i n the p r e s e n t s t u d y , the p u l s e d u r a t i o n was 56.7 msec w i t h the f i r s t 40 msec b e i n g used t o a l l o w the c a p a c i t i v e c u r r e n t t o decay t o a n e g l i g i b l e v a l u e w h i l e the l a s t 16.7 msec of the p u l s e e s s e n t i a l l y was used t o measure the f a r a d a i c c u r r e n t . C u r r e n t samples are t a k e n j u s t p r i o r t o the p u l s e a p p l i c a t i o n and a g a i n j u s t b e f o r e the p u l s e i s completed. I t i s the d i f f e r e n c e between the two c u r r e n t s t h a t i s d i s p l a y e d as a peak shaped r e a d - o u t . The o v e r a l l s e n s i t i v i t y of the a n a l y s i s i s i n c r e a s e d one t o two o r d e r s of magnitude over c o n v e n t i o n a l ASV by u s i n g the d i f f e r e n t i a l p u l s e wave-form (Siegerman and O'Dom, 1972). (b) C h e m i c a l forms measured by ASV ASV has been used e x t e n s i v e l y t o study t r a c e m e t a l s i n n a t u r a l waters ( e . g . , A l l e n e t a _ l . , 1970; A b d u l l a h and R o y l e , 1972; D u i n k e r and Kramer, 1977; F l o r e n c e and B a t l e y , 1977a,b; Sugai and H e a l y , 1978; Nygaard and H i l l , 1979; F i q u r a and M c D u f f i e , 1979). The t e c h n i q u e i s q u i t e unique i n t h a t i t can d i s c r i m i n a t e between ' l a b i l e ' and 'bound' m e t a l s p e c i e s (Chau, 1973; Chau et a l . , 1974a,b; L a z a r e t a l . , 1981; P l a v s i c e t a l . , 1982); the l a b i l e f r a c t i o n i s measureable by ASV and t h i s f r a c t i o n i n c l u d e s the f r e e h y d r a t e d i o n , i n o r g a n i c complexes such as c h l o r o , hydroxo and c a r b o n a t o s p e c i e s , and o r g a n i c complexes t h a t d i s s o c i a t e a t t h e e l e c t r o d e i n t h e time s c a l e of the measurement. The e x p e r i m e n t a l approach t a k e n i s t o measure 12 the m e t a l a t the n a t u r a l pH of the sample or a f t e r making i t a c i d i c ( c a . pH 5) w i t h C0 2 (Nygaard and H i l l , 1979) or a c e t a t e b u f f e r ( A b d u l l a h e t a l . , 1976), and then remeasure i t a f t e r a c i d d i g e s t i o n of the s o l u t i o n . The two measurements are then used t o d e t e r m i n e l a b i l e and bound metal ( A l l e n e_t a l . , 1970; G a r d i n e r and S t i f f , 1975). (c) Measurement of b i o l o g i c a l l y a c t i v e Cu S i n c e ASV i s s e n s i t i v e t o the c h e m i c a l form of the m e t a l i n s o l u t i o n , i t has been suggested t h a t the p r o c e d u r e might be a b l e t o i d e n t i f y the f r a c t i o n t h a t i s b i o l o g i c a l l y a v a i l a b l e (Mancy and A l l e n , 1977; W h i t f i e l d and T u r n e r , 1979). A r e v i e w of the l i t e r a t u r e , however, s u g g e s t s t h a t t h e r e i s l i t t l e hope of a c h i e v i n g t h i s g o a l . To s u c c e s s f u l l y e s t i m a t e the 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, o n l y those s p e c i e s t h a t are a v a i l a b l e t o the organism s h o u l d c o n t r i b u t e t o the ASV s i g n a l . F i q u r a and M c D u f f i e (1979) measured the p e r c e n t l a b i l e Cu i n an a c e t a t e medium i n the p r e s e n c e of EDTA, NTA, humic a c i d s and g l y c i n e ( a l l of which a r e known t o form Cu complexes t h a t a r e not b i o l o g i c a l l y a c t i v e ) . In t h e presence of 1 x 10" 3M g l y c i n e (1 ^ = 8.1), Cu was found t o be 100% l a b i l e . Even i n the presence of EDTA, where the f r e e Cu c o n c e n t r a t i o n was e x t r e m e l y low, some l a b i l e Cu was measured. Only w i t h the a d d i t i o n of humic a c i d s d i d Cu become e n t i r e l y i n a c t i v e . G a c h t e r e t a l . (1973), u s i n g a c o m p l e x i n g c a p a c i t y t e c h n i q u e d e v e l o p e d by Chau et a l . (1974b), performed ASV measurements on l a k e waters a t pH 7.0 u s i n g a s e r i e s of l i g a n d s 13 of d i f f e r e n t s t a b i l i t y c o n s t a n t s ( t a r t r a t e , c i t r a t e , g l y c i n e , NTA, EDTA). They found t h a t when the c o n c e n t r a t i o n of Cu was l e s s than the c o n c e n t r a t i o n o f t o t a l added l i g a n d those complexes h a v i n g a l o g c o n d i t i o n a l s t a b i l i t y c o n s t a n t l e s s t h a t c a . 10 c o u l d be c o n s i d e r e d ASV l a b i l e ; however, as D a v i s o n (1978) p o i n t e d o u t , t h i s e f f e c t i v e e l e c t r o a c t i v i t y of Cu complexes c o u l d be based on k i n e t i c r a t h e r than thermodynamic c o n s i d e r a t i o n s and, t h u s , t h a t the s t a b i l i t y c o n s t a n t may not be a good e s t i m a t e of e l e c t r o a c t i v i t y . S i n c e ASV has been shown t o measure Cu i n o r g a n i c complexes which a r e known not t o be b i o l o g i c a l l y a c t i v e w i t h r e s p e c t t o p h y t o p l a n k t o n ( g l y c i n e , t a r t r a t e , EDTA, NTA), the a b i l i t y of the ASV t e c h n i q u e t o measure b i o l o g i c a l l y a c t i v e Cu i s i n q u e s t i o n . The magnitude of t h e l a b i l e m e t a l f r a c t i o n a l s o changes when the ASV parameters a r e changed. D e p o s i t i o n p o t e n t i a l and d e p o s i t i o n time have been used t o d i s c r i m i n a t e between d i f f e r e n t s p e c i e s of Cu (Matson, 1968). Schonberger and P i c k e r i n g (1980) demonstrated t h a t , when d e t e r m i n i n g Cu i n s o l u t i o n s c o n t a i n i n g EDTA, the h e i g h t of the c u r r e n t peak changed as the d e p o s i t i o n p o t e n t i a l was v a r i e d . In a d d i t i o n , s i n c e l a b i l i t y i s dependent on k i n e t i c as w e l l as thermodynamic f a c t o r s , i t seems u n l i k e l y t h a t t h e r e i s a unique s e t of o p e r a t i n g parameters f o r the ASV a n a l y s i s s u i t a b l e f o r measuring o n l y b i o l o g i c a l l y a c t i v e Cu under a l l c i r c u m s t a n c e s . I t i s w e l l known t h a t the degree of i o n i z a t i o n and the a s s o c i a t e d a b i l i t y of many o r g a n i c s t o form m e t a l - l i g a n d complexes a r e a f f e c t e d by pH. To change the pH of a sample from 1 4 i t s n a t i v e v a l u e f o r a n a l y s i s t h e n may r e s u l t i n e i t h e r i n c r e a s e d or de c r e a s e d i o n i z a t i o n and metal b i n d i n g c a p a b i l i t y . T h e r e f o r e , t o reduce the r i s k of a l t e r i n g the m e t a l - o r g a n i c a s s o c i a t i o n , the n a t i v e pH of a sample must be used. However, t h i s can pose a problem when ASV i s t o be used a t the n a t u r a l pH of seawater ( c a . 8.2). In a l k a l i n e s o l u t i o n s , two Cu peaks a r e o f t e n o b s e r v e d i n the a n a l y s i s . I t has been suggested t h a t the more a n o d i c peak i s due t o the f o r m a t i o n of C u ( I ) s p e c i e s a t the s u r f a c e d u r i n g the o x i d a t i o n of Cu(Hg) or an a d s o r p t i o n of C u ( l l ) hydroxy compounds ( S i n k o and D o l e z a l , 1970). S i e b e r t and Hume (1981), u s i n g a mercury c o a t e d g r a p h i t e e l e c t r o d e i n raw seawater (pH 8.4), found t h a t r e p e a t e d c y c l e s of p l a t i n g and s t r i p p i n g caused a c o n t i n u a l d e c r e a s e i n peak h e i g h t w i t h each c y c l e . T h i s d i d not o c c u r i n a c i d i f i e d samples. They suggested t h a t C u ( l ) i o n s form an i n s o l u b l e or h i g h l y a d s o r b a b l e s p e c i e s which i s e l e c t r o - i n a c t i v e and adheres t o the e l e c t r o d e s u r f a c e . Z i r i n o and Kounaves (1980) suggested t h a t the o v e r a l l r e d u c t i o n of C u ( I l ) a t the n a t u r a l pH i s k i n e t i c a l l y h i n d e r e d and thus i s i r r e v e r s i b l e . With such i n t e r f e r e n c e s w i t h the e l e c t r o d e , t he c a l i b r a t i o n of Cu l e v e l s i n a sample of pH 8.0 becomes d i f f i c u l t . S i e b e r t and Hume (1981) c o n c l u d e d t h a t , from a p r a c t i c a l s t a n d p o i n t , t he ASV d e t e r m i n a t i o n of Cu i n seawater a t i t s n a t u r a l pH s h o u l d be a v o i d e d . In c o n c l u s i o n , ASV i s not s u i t a b l e f o r s t u d y i n g Cu s p e c i a t i o n i n seawater p a r t i c u l a r l y when t r y i n g t o e s t i m a t e b i o l o g i c a l l y a c t i v e m e t a l . The t e c h n i q u e , however, i s a s e n s i t i v e and r e l i a b l e method f o r measuring t o t a l d i s s o l v e d 15 m e t a l c o n c e n t r a t i o n s i n w e l l d e f i n e d and a c i d i c s o l u t i o n s and, f o r t h i s r e a s o n , ASV was used to measure t o t a l Cu c o n c e n t r a t i o n s i n the a r t i f i c i a l seawater used i n the p r e s e n t s t u d y . 16 B. ANALYTICAL METHODS BASED ON PRELIMINARY CHEMICAL AND PHYSICAL SEPARATION Trace metal s p e c i a t i o n i s normally studied using methods that i n v o l v e a p r e l i m i n a r y chemical or p h y s i c a l separation step whereby s p e c i f i c metal forms are i s o l a t e d from the sample. The metal i s o l a t e d i s then q u a n t i f i e d by an appropriate a n a l y t i c a l technique, such as atomic absorption spectrophotometry. A comparison of l e v e l s before and a f t e r treatment i s often used to separate s e v e r a l c l a s s e s of metal species. A b r i e f o u t l i n e of the methods that have been used i n n a t u r a l waters and seawater samples w i l l be given below. A b r i e f summary of techniques used to study t r a c e metal s p e c i a t i o n i n n a t u r a l waters i s presented i n Table I. 1. Separation Based on Organic Solvent S o l u b i l i t y Solvent e x t r a c t i o n techniques have been used to d i s t i n q u i s h between organic and inorganic bound Cu. The technique simply in v o l v e s mixing a seawater sample with an organic solvent such as chloroform, hexanol or carbon t e t r a c h l o r i d e . The amount of Cu i n the solvent f r a c t i o n i s then equal to the Cu a s s o c i a t e d with organics. However, as Florence and Batley (1980) pointed out, the method probably gives low r e s u l t s because charged Cu complexes could not be e x t r a c t e d and Cu adsorbed onto organic c o l l o i d a l matter may only be p a r t i a l l y e x t r a c t e d . Solvent e x t r a c t i o n has been a p p l i e d to seawater (Slowey et a_l., 1 967) to measure o r g a n i c a l l y bound Cu. 1 7 Table I. Analytical methods for studying trace metal speciation in natural waters. Electro-Chemical Methods Specific Ion Potentiometry: Voltammetry: e.g. Cupric ion electrode Pulse Polarography Differential Pulse Polarography Differential Pulse Anodic Stripping Voltammetry Methods Based on Preliminary  Chemical and Physical Separation Organic-Inorganic Separation: Molecular Size Separation: Separation based on charge: Calculation Methods Solvent extraction Chelation-solvent extraction Membrane f i l t r a t i o n U l t r a f i l t r a t i o n Dialysis Gel chromatography Centrifugation Conventional ion-exchange resins Chelex-100 Computer Models: e.g. Comics Haltafall Mineql Redeql2 18 2. M o l e c u l a r S i z e S e p a r a t i o n Techniques t h a t f r a c t i o n a t e t r a c e m etal s p e c i e s on the b a s i s of s i z e d i f f e r e n c e s i n c l u d e f i l t r a t i o n , g e l f i l t r a t i o n , d i a l y s i s and c e n t r i f u g a t i o n . U l t r a f i l t r a t i o n , d i a l y s i s and c e n t r i f u g a t i o n g e n e r a l l y a r e used t o s e p a r a t e m e t a l p r e s e n t i n t r u e s o l u t i o n from t h a t a s s o c i a t e d w i t h c o l l o i d a l m a t e r i a l . Membrane f i l t e r s w i t h 0.45 um pore d i a m e t e r are n o r m a l l y used t o d i s t i n g u i s h between d i s s o l v e d and p a r t i c u l a t e forms of m e t a l . However, such a pore s i z e i s s t i l l l a r g e enough t o a l l o w c o l l o i d a l m a t t e r t o pass t h r o u g h and o t h e r p r o c e d u r e s must be used t o i s o l a t e the s p e c i e s i n t r u e s o l u t i o n . U l t r a f i l t r a t i o n i s s i m p l y a form of f i l t r a t i o n i n which the pore s i z e of the f i l t e r i s of the same magnitude as m o l e c u l a r or i o n i c s p e c i e s i n t r u e s o l u t i o n . Pore s i z e s can range from 1 t o 10 nm, p r o v i d i n g f i l t e r s c a p a b l e of r e j e c t i n g m o l e c u l e s w i t h m o l e c u l a r w e i g h t s i n the range of 500 t o 300,000 (Schmidt, 1978). G e n e r a l l y the membranes have no net e l e c t r i c a l c harge a l t h o u g h some have i o n i c s i t e s . U l t r a f i l t r a t i o n has been used i n the i n v e s t i g a t i o n of t r a c e m e t a l forms i n n a t u r a l and model a q u a t i c systems ( S c h i n d l e r , e t a l . , 1972; Andren and H a r r i s , 1975; Smith, 1976). D i s a d v a n t a g e s of the t e c h n i q u e i n c l u d e the c o s t , the time t a k e n f o r f i l t r a t i o n , the a d s o r p t i o n of m e t a l s from the s o l u t i o n onto the f i l t e r s , and a problem of i n i t i a l m e t a l c o n t a m i n a t i o n of the f i l t e r s ( H art and D a v i e s , 1978). In d i a l y s i s , the pore s i z e of the membrane i s such (1 nm) t h a t o n l y the f r e e m e t a l i o n and the s m a l l e s t complexed s p e c i e s a r e e x p e c t e d t o d i f f u s e t h r o u g h the membrane. I t i s thus used 19 to d i f f e r e n t i a t e m e tal bound -to c o l l o i d a l m a tter and t h a t i n t r u e s o l u t i o n (Hart and D a v i e s , 1978). However, t h e r e are problems when d i a l y s i s membranes are used f o r metal s p e c i a t i o n s t u d i e s . The membranes u s u a l l y have a n e g a t i v e charge a s s o c i a t e d w i t h them and n e g a t i v e s p e c i e s e x p e r i e n c e a s m a l l e r e f f e c t i v e pore s i z e because of e l e c t r o s t a t i c r e p u l s i o n and thus they r e q u i r e a l o n g time t o a t t a i n e q u i l i b r i u m (Benes and S t e i n n e s , 1974). In a d d i t i o n , c o n s i d e r a b l e d i s s o c i a t i o n of some me t a l complexes may occur a t the membrane s u r f a c e because of the n e g a t i v e charge (Guy and C h a k r a b a r t i , 1975). From a p r a c t i c a l s t a n d p o i n t , d i a l y s i s i s not c o n v e n i e n t because of the time needed f o r the a n a l y s i s (Benes and S t e i n n e s , 1975) and because e l i m i n a t i o n of me t a l c o n t a m i n a t i o n from the d i a l y s i s membrane i s e x t r e m e l y d i f f i c u l t . G e l f i l t r a t i o n chromatography i s based upon the i n c l u s i o n and subsequent e l u t i o n of the s o l u t e t h r o u g h a s t a t i o n a r y phase c o n s i s t i n g of a h e t e r o p o r o u s , c r o s s - l i n k e d p o l y m e r i c g e l . M o l e c u l e s e x c e e d i n g the s i z e of the e x c l u s i o n l i m i t of the g e l a r e e l u t e d i n i t i a l l y w h i l e s m a l l e r m o l e c u l e s , which permeate the g e l p a r t i c l e s t o v a r y i n g degrees depending on t h e i r shape and s i z e , a r e e l u t e d from the g e l column i n o r d e r of d e c r e a s i n g m o l e c u l a r s i z e . G e l f i l t r a t i o n has been used p a r t i c u l a r l y t o s t u d y m e t a l -o r g a n i c i n t e r a c t i o n s i n n a t u r a l waters ( G j e s s i n g and Lee, 1967; Mantoura and R i l e y , 1975; Sugai and H e a l y , 1978). The d i s a d v a n t a g e s of the t e c h n i q u e a r e t h a t i t i s g e n e r a l l y n e c e s s a r y t o p r e c o n c e n t r a t e the sample, which may a l t e r the 20 c o m p o s i t i o n o f t h e s a m p l e , a n d t h a t t h e h i g h r a t i o o f s o l v e n t t o s a m p l e v o l u m e c a n . r e s u l t i n l a r g e b l a n k s ( P l u m b a n d L e e , 1 9 7 3 ; B e n e s e t a l , 1 9 7 6 ) . . 3 . S e p a r a t i o n B a s e d o n C h a r g e a n d L a b i l i t y o f C o m p l e x e s C h e l a t i n g r e s i n s , s u c h a s C h e l e x - 1 0 0 , h a v e b e e n a p p l i e d t o t h e c o n c e n t r a t i o n a n d d e t e r m i n a t i o n o f t r a c e m e t a l s i n s e a w a t e r ( R i l e y a n d T a y l o r , 1 9 6 8 a , b ; R i l e y a n d T a y l o r , 1 9 7 2 ; F l o r e n c e a n d B a t l e y , 1 9 7 5 , 1 9 7 6 ) . T h e s e r e s i n s s h o w a n u n u s u a l l y h i g h p r e f e r e n c e f o r t h e t r a n s i t i o n m e t a l s o v e r t h o s e o f t h e a l k a l i o r a l k a l i n e e a r t h m e t a l s . F i g u r a a n d M c D u f f i e ( 1 9 8 0 ) d e v e l o p e d a n a n a l y t i c a l s c h e m e t o d i f f e r e n t i a t e t r a c e m e t a l s p e c i e s o n t h e i r r e l a t i v e l a b i l i t i e s w i t h r e s p e c t t o C h e l e x - 1 0 0 u s e d i n c o n j u n c t i o n w i t h A S V . S p e c i e s w e r e c l a s s i f i e d a s ' v e r y l a b i l e ' , ' m o d e r a t e l y l a b i l e ' , ' s l o w l y l a b i l e ' o r i n e r t d e p e n d i n g o n t h e c h a r a c t e r i s t i c t i m e s c a l e o f t h e m e a s u r e m e n t . B a t l e y a n d F l o r e n c e ( I 9 7 6 a , b ) a l s o u s e d C h e l e x - 1 0 0 a n d A S V t o q u a n t i t a t i v e l y a s s a y s e v e n d i f f e r e n t h e a v y m e t a l s p e c i e s i n n a t u r a l w a t e r s . I n b o t h s t u d i e s , C h e l e x - 1 0 0 w a s f o u n d t o d i s s o c i a t e a l a r g e r p e r c e n t a g e o f t h e c o m p l e x e s i n t h e s a m p l e s t h a n A S V ; t h u s t h e m o s t l a b i l e c l a s s o f m e t a l w a s t h a t w h i c h w a s A S V l a b i l e . S i n c e t h e A S V m e a s u r e m e n t c a n n o t b e u s e d a s a m e a s u r e o f b i o l o g i c a l l y a c t i v e C u i n s e a w a t e r , t h e i o n - e x c h a n g e m e t h o d , w h i c h d i s s o c i a t e s e v e n s t r o n g e r c o m p l e x e s , w o u l d a l s o n o t b e e x p e c t e d t o m e a s u r e b i o l o g i c a l l y a c t i v e m e t a l . C o n v e n t i o n a l c a t i o n a n d a n i o n e x c h a n g e r e s i n s a r e c a p a b l e o f d i s t i n q u i s h i n g p a r t i c u l a r m e t a l s p e c i e s b a s e d u p o n t h e i r .21 charge. A few studies have used these ion-exchangers to study metal speciation in sewage (Cantwell et a l . , 1982), freshwaters (Filby et a l . , 1974; Benes and Steinnes, 1975; Benes -et a l . , 1976; Shuman and Dempsey, 1977) and seawater samples (Marchand, 1974). The present study used a conventional cation exchange resin to study Cu speciation in seawater and a description of cation-exchange resins i s given in Section IV. 22 I I I . THE MEASUREMENT OF BIOLOGICALLY ACTIVE CU BY A MARINE DIATOM A. INTRODUCTION A l t h o u g h Cu i s a r e q u i r e d t r a c e element f o r p h y t o p l a n k t o n , e n v i r o n m e n t a l c o n c e n t r a t i o n s as low as 1 ug l " 1 can be t o x i c (Steeman N i e l s e n et aJL., 1969; M a n d e l l i , 1969; M a r t i n and O l a n d e r , 1971; E r i c k s o n , 1972). The s e n s i t i v i t y t o Cu can v a r y c o n s i d e r a b l y , not o n l y among a l g a l groups but a l s o among a l g a l s p e c i e s of the same group ( E r i c k s o n et a l . , 1970). The d i n o f l a g e l l a t e s and cyanophytes seem t o be the most s e n s i t i v e w h i l e the green f l a g e l l a t e s the most r e s i s t a n t ( M a n d e l l i , 1969; E r i c k s o n e t a l . , 1970). Diatoms, on the o t h e r hand, a r e found t o be both q u i t e s e n s i t i v e and r e s i s t a n t t o Cu. Steeman N i e l s e n and Wium-Andersen (1971) showed t h a t as l i t t l e as 1.2 ug 1~ 1 Cu i n s y n t h e t i c seawater i n h i b i t e d t he growth of the d i a t o m , N i t z s c h i a p a l e a , w h i l e C a n t e r f o r d e t a l . (1978) found t h a t i n e n r i c h e d n a t u r a l seawater Cu c o n c e n t r a t i o n s as h i g h as 150 ug l " 1 d i d not i n h i b i t the growth of the d i a t o m D i t y l u m  b r i q h t w e l l i . A wide range of m o r p h o l o g i c a l a b n o r m a l i t i e s a r e found when marine diatoms a r e exposed t o t o x i c l e v e l s of Cu. The most p r e v a l e n t e f f e c t s a re changes i n c e l l s i z e or e x t e r n a l morphology, and reduced growth r a t e s ( E r i c k s o n , 1972; F i s h e r e t a l . , 1981; Thomas e t a l . , 1980). Other e f f e c t s i n c l u d e a p r o l o n g e d l a g phase (M o r e l e t a l . , 1978) and changes i n the i n t e r n a l appearance of the c e l l ; when c e l l s of T h a l a s s i o s i r a 23 a e s t i v a l i s were exposed t o h i g h l e v e l s of Cu the c y t o p l a s m became g r a n u l a r and y e l l o w i s h i n c o l o r , the c h l o r o p l a s t i n t e g r i t y was d i s r u p t e d , and more d e l i c a t e s p i n e s were e x t r u d e d from the m a r g i n a l p r o c e s s e s (Thomas e_t a l . , 1980). P h y s i o l o g i c a l p r o c e s s e s shown t o be i n h i b i t e d by Cu have i n c l u d e d n i t r a t e uptake, p h o t o s y n t h e t i c carbon a s s i m u l a t i o n , and n i t r a t e r e d u c t a s e s y n t h e s i s ( H a r r i s o n e t a l . , 1977). In a d d i t i o n , s i l i c i c a c i d uptake has been shown t o be reduced i n the presence of Cu which was a t t r i b u t e d t o a h y p o t h e t i c a l Cu-s e n s i t i v e Si(OH)„ t r a n s p o r t s i t e ( G o e r i n g et aL. , 1977; Rueter e t a l . , 1981) . I t i s b e l i e v e d t h a t Cu produces i t s t o x i c e f f e c t by e n t e r i n g i n t o s t r o n g complexes w i t h o r g a n i c l i g a n d s such as c a r b o x y l , s u l f h y d r y l , p h o s p h a t i d i c , amino and o t h e r groups p r e s e n t on the s u r f a c e of the c e l l ( D a v i e s , 1978). At low a c t i v i t i e s Cu r e a c t s p r i m a r i l y w i t h the s u r f a c e of the c e l l and d i s r u p t s membrane a c t i v i t i e s such as c e l l d i v i s i o n and p e r m e a b i l i t y ( E r i c k s o n e t a l . , 1970; M o r e l e t a l . , 1978). Once Cu i s w i t h i n the c e l l i t may i n a c t i v a t e numerous enzymes t h r o u g h d i s p l a c e m e n t of the a c t i v a t i n g m e t a l , or t h r o u g h b i n d i n g t o s u l f h y d r y l groups ( R o t h s t e i n , 1959) or o t h e r f u n c t i o n a l groups ( E i c h h o r n , 1975). In f r e s h w a t e r a l g a e , Cu has been shown t o i n a c t i v a t e b o t h the H i l l r e a c t i o n , a measure of photosystem I I , and the m o d i f i e d Mehler r e a c t i o n which measures photosystem I a c t i v i t y ( O v e r n e l l , 1975). F i s h e r e t a l . (1981) found t h a t when the 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 was t r e a t e d w i t h Cu i t showed an above normal p h o t o s y n t h e t i c r a t e on a per c e l l b a s i s 2 4 but the e x c r e t i o n of p h o t o s y n t h e t i c a l l y f i x e d carbon was d e p r e s s e d . T h i s i n d i c a t e d an u n c o u p l i n g of p h o t o s y n t h e s i s from c e l l d i v i s i o n w i t h the c e l l s becoming e n l a r g e d when the f i x e d c a rbon c o u l d not be e x c r e t e d or u t i l i z e d i n c e l l d i v i s i o n . In r e c e n t y e a r s numerous s t u d i e s have demonstrated t h a t the 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 i s c o n t r o l l e d by the c u p r i c i o n a c t i v i t y of the medium and not the t o t a l m e t a l c o n c e n t r a t i o n (Sunda and G u i l l a r d , 1976; Anderson and M o r e l , 1978; J a c k s o n and Morgan, 1978; Sunda and L e w i s , 1978; C a n t e r f o r d and C a n t e r f o r d , 1980). T h i s was demonstrated by e x p e r i m e n t s conducted i n c h e m i c a l l y w e l l - d e f i n e d media where the c u p r i c i o n a c t i v i t y was c a l c u l a t e d by computer m o d e l l i n g t e c h n i q u e s and model o r g a n i c l i g a n d s were used t o c o n t r o l the c u p r i c i o n a c t i v i t y . B i o a s s a y t e s t s showed t h a t the growth r a t e of the p h y t o p l a n k t o n organism was c o r r e l a t e d t o the c a l c u l a t e d c u p r i c i o n a c t i v i t y but not t o the t o t a l Cu c o n c e n t r a t i o n . In t h e f o l l o w i n g s e c t i o n , d a t a a r e p r e s e n t e d on the growth of the p h y t o p l a n k t o n organism, T h a l a s s i o s i r a pseudonana (WHOI, c l o n e 3H), which was used t o measure the amount of b i o l o g i c a l l y a c t i v e Cu i n the a r t i f i c i a l seawater medium A q u i l (Morel e t a l . , 1979). The organism was c a l i b r a t e d over a range of Cu c o n c e n t r a t i o n s and t h e c h e m i c a l a c t i v i t y of the m e t a l was, f o l l o w i n g the approach i n the s t u d i e s mentioned above, c o n t r o l l e d w i t h v a r i o u s model l i g a n d s . The l i g a n d s were chosen on the b a s i s of the s t a b i l i t y of t h e i r complexes w i t h Cu and t h e i r e f f e c t on the a c t i v i t y of Cu was e s t i m a t e d u s i n g the computer e q u i l i b r i u m model MINEQL ( W e s t a l l e t a l . , 1976). The 2 5 purpose of these e x p e r i m e n t s was t o determine the r e l a t i o n s h i p between the growth r a t e of the assay organism and the c u p r i c i o n a c t i v i t y of the medium. These d a t a w i l l be s u b s e q u e n t l y used t o t e s t the a n a l y t i c a l method d e v e l o p e d f o r the measurement of b i o l o g i c a l l y a c t i v e Cu i n seawater. 26 B. MATERIALS AND METHODS 1 . Medium P r e p a r a t i o n V a r i a t i o n s of the medium ' A q u i l ' ( Morel et a_l. , 1979) were used throughout the p r e s e n t s t u d y f o r both the s t o c k and e x p e r i m e n t a l c u l t u r e s . A q u i l was chosen because the t r a c e m etal s p e c i a t i o n i s w e l l d e f i n e d . C u l t u r e medium p r e p a r a t i o n i n v o l v e d the p r e p a r a t i o n of major s a l t , major n u t r i e n t , t r a c e m e t a l and v i t a m i n s o l u t i o n s s e p a r a t e l y and then combining t h e s e j u s t b e f o r e use. A l l c h e m i c a l s used i n the medium p r e p a r a t i o n were of r e agent grade. Any g l a s s w a r e used was i n i t i a l l y r i n s e d 3X w i t h g l a s s d i s t i l l e d water (GDW), soaked i n 6N HCL f o r 24 h r , and then r i n s e d 3X w i t h GDW. The same g l a s s w a r e was used f o r a p a r t i c u l a r s o l u t i o n throughout the term of the s t u d y . Major seawater s a l t s (SOW): The major s a l t s were p r e p a r e d i n 20 l i t e r b a t c h e s . The s a l t s ( T a ble I I ) , e x c l u d i n g M g C l 2 , were added t o 18 1 of GDW i n a 20-1 g l a s s c arboy and bubbled w i t h a c i d c l e a n e d (6N H 2SO„), f i l t e r e d (0.45 Mm N u c l e p o r e ) a i r u n t i l t he m i x t u r e was c o m p l e t e l y d i s s o l v e d . M g C l 2 was then added t o the s a l t s o l u t i o n which was brought up t o 20 1 w i t h GDW (M g C l 2 was d r y e d f o r 2 days a t 70°C b e f o r e use because of i t s h y g r o s c o p i c n a t u r e ) . The medium was then bubbled w i t h a c i d c l e a n e d , f i l t e r e d a i r o v e r n i g h t t o a l l o w e q u i l i b r a t i o n and t o a d j u s t the pH t o 8.0±.05. The s o l u t i o n was then passed t h r o u g h an ion-exchange r e s i n ( C h e lex-100, 100-200 mesh) and s t o r e d i n a c i d c l e a n e d 6-1 b o r o s i l i c a t e f l a s k s u n t i l use. The p r e p a r a t i o n of the ion-exchange r e s i n i s d e s c r i b e d i n S e c t i o n I I I . B . 5 . 27 Background Cu l e v e l s i n the c h e l e x t r e a t e d SOW were below d e t e c t i o n l i m i t ( 1 . 5 7 X 1 0 " 9 M Cu) as measured by 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 . N u t r i e n t s ; Phosphate, n i t r a t e and s i l i c a t e s t o c k s were p r e p a r e d s e p a r a t e l y a t 1000X the f i n a l c o n c e n t r a t i o n . The s t o c k s were p r e p a r e d as g i v e n i n Table I I . Each s o l u t i o n was a d j u s t e d t o a p p r o x i m a t e l y pH 8.0 and then passed through an i o n -exchange r e s i n ( C h e l e x - 1 0 0 ) . To a l l o w the use of the same i o n -exchange column f o r a l l s o l u t i o n s , NaCl was added t o the phosphate and s i l i c a t e s t o c k s t o p r o v i d e the same i o n i c s t r e n g t h i n a l l s t o c k s . V i t a m i n s : S t o c k s of b i o t i n and B 1 2 were p r e p a r e d from c r y s t a l l i n e compounds. These were then d i l u t e d and thiamine«HCl was added t o g i v e a v i t a m i n mix a t 5000X the f i n a l c o n c e n t r a t i o n ( T a b l e I I ) . The f i n a l s o l u t i o n was f r o z e n i n 20 ml screw cap v i a l s a f t e r heat s t e r i l i z a t i o n (121°C, 15 m i n ) . Trace M e t a l s ; Two s e p a r a t e s t o c k s o l u t i o n s , were p r e p a r e d from t h e s a l t s g i v e n i n T a b l e I I , the f i r s t c o n s i s t i n g of Cu, Mo p l u s Co and the second of Mn p l u s Zn. A l i q u o t s of each were then combined t o g i v e a t r a c e m e t a l mix a t 1000X the f i n a l c o n c e n t r a t i o n . The Fe s t o c k was p r e p a r e d a t 1000X the f i n a l c o n c e n t r a t i o n by a d d i n g F e C l 3 t o GDW. The s t o c k was made up f r e s h and then 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 hours b e f o r e b e i n g added t o the c u l t u r e s . S p e c i a l c o n s i d e r a t i o n was g i v e n t o the method of Fe p r e p a r a t i o n and a d d i t i o n (see S e c t i o n I I I . B . 3 ) . A q u i l p r e p a r a t i o n ; G e n e r a l l y , 4 1 of A q u i l were needed f o r each b i o a s s a y . A q u i l was p r e p a r e d by a d d i n g 1 ml of the t r a c e 28 Table I I . Preparation of A q u i l SOW; NaCl MgCl 2.6H 20 Na 2S0 a CaCl 2,2H 20 KC1 NaHC03 KBr H 3 B O 3 S r C l 2 . 6 H 2 0 NaF NUTRIENTS: NaH2PO« NaCl NaNO, NaSi0 3.9H 20 NaCl 490.6 g 222.0 81 .9 30.8 14.0 > 20 1 > Chelex > Storage 4.0 2.0 0.6 0.34 0.06 1 .38 g 5.26 8.5 3.55 4.38 pH adjusted to 8 NaOH —> 1 1 —> Chelex —> Storage 4°C NaOH —> 1 1 —> Chelex —> Storage 4°C HCL —> 1 1 —> Chelex —> Storage 4°C TRACE METALS: CuCl 2.5H 20 (NH«) 6Mo 70 2„.4H 20 CoCl 2.6H 20 MnCl 2.4H 20 ZnSO,.7H20 FeCl 3.6H 20 VITAMIN MIX: B 1 2 B i o t i n Thiamine 1 ml .249 g —> 1 1 > .265 1 ml .595 —> 1 1 > Mixed metal Stock 1 L i t e r .455 1 ml .115 —> 1 1 > . 122 —> 1 1 > Fe Stock .1 ml 11.0 mg —>.01 1 > 1ml Vitamin 10.0 —> .1 1 > Stock 0.1 L i t e r 20.0 > 29 m e t a l mix, 1 ml of each of the n u t r i e n t s o l u t i o n s , and 0 . 5 ml of v i t a m i n mix t o each 1 of SOW. The A q u i l was then t r a n s f e r e d t o an a c i d c l e a n e d 4 - 1 Rimax a s p i r a t o r b o t t l e , bubbled w i t h C 0 2 t o a pH of 5 . 7 and a u t o c l a v e d ( 1 2 1 ° C ) f o r 3 0 min ( b u b b l i n g w i t h C 0 2 p r e v e n t e d 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 medium was then c o o l e d and, i f n e c e s s a r y , bubbled w i t h a c i d c l e a n e d , f i l t e r e d a i r u n t i l i t had a pH of 8 . 0 ± . 0 5 . At t h i s p o i n t , 1 ml of Fe s t o c k was added t o each 1 of A q u i l t o g i v e a f i n a l c o n c e n t r a t i o n of 4 . 5 x 1 0 " 7 M . The A q u i l was then ready t o be used i n the b i o a s s a y s . 2 . B i o a s s a y s In p r e l i m i n a r y e x p e r i m e n t s the s e n s i t i v i t y of the b i o a s s a y o r g a n i s m t o Cu was d e t e r m i n e d i n A q u i l when no o r g a n i c l i g a n d s were added. Three b i o a s s a y s were performed u s i n g the Cu c o n c e n t r a t i o n ranges of 0 . 1 - 6 . 3 0 x 1 0 " 8 M a t 1 . 5 7 x 1 0 " 8 M i n c r e m e n t s ; . 0 . 1 t o 1 5 . 7 X 1 0 " 8 M a t 3 . 9 0 x 1 0 " 8 M i n c r e m e n t s ; and 0 . 1 - 4 7 . 1 x 1 0 " 8 M a t 1 5 . 7 x 1 0 " 8 M i n c r e m e n t s . The same b i o a s s a y p r o c e d u r e was f o l l o w e d as w i l l be g i v e n below f o r the o r g a n i c l i g a n d b i o a s s a y s . B i o a s s a y s were conducted w i t h L - g l u t a m i c a c i d (GLU), L— h i s t i d i n e ( H I S ) , n i t r i l o t r i a c e t i c a c i d (NTA) and 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 (EDTA) ( F i g . 2 ) . These were used t o change the c o n c e n t r a t i o n of the c u p r i c i o n w h i l e m a i n t a i n i n g a c o n s t a n t c o n c e n t r a t i o n of t o t a l Cu. L i g a n d s t o c k s were p r e p a r e d a t c a . 1 0 0 0 X the f i n a l c o n c e n t r a t i o n and s t o r e d i n a c i d c l e a n e d p o l y p r o p y l e n e b o t t l e s a t 4°C i n the d a r k . The 30 GLU s t o c k was d i s s o l v e d i n GDW heated t o 80°C. The b i o a s s a y p r o c e d u r e i n v o l v e d a d d i n g a l i q u o t s (250 ml) of the a u t o c l a v e d A q u i l t o 15 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 erlenmeyer f l a s k s . (Of the 15 f l a s k s , 3 were used as c o n t r o l s w h i l e the r e m a i n i n g 12 were used t o run 3 r e p l i c a t e s a t each of 4 d i f f e r e n t Cu c o n c e n t r a t i o n s ) . The a p p r o p r i a t e l i g a n d a t the prop e r c o n c e n t r a t i o n was added t o a l l f l a s k s . Then, depending on the ex p e r i m e n t , Cu was added i n one of two c o n c e n t r a t i o n ranges. The v a l u e s c o m p r i s i n g t h e s e two ranges were 0.789, 1.57, 2.36, and 3.15 x 1 0 " 7 M C U ( i . e . , 5, 10, 15, and 20 ug l" 1 Cu) or 0.393, 0.789, 1.18, and 1.57 x 1 0 " 7 M Cu ( i . e . , 2.5, 5.0, 7.5, and 10 ug l " 1 Cu). The medium was l e f t c o v e r e d f o r 2-3 hours b e f o r e b e i n g i n n o c u l a t e d w i t h the t e s t o r ganism. The c e n t r i c d iatom T h a l a s s i o s i r a pseudonana ( = C y c l o t e l l a  nana H u s t e d t ) H a s l e and Heimdal (WHOI c l o n e 3H) was used as the b i o a s s a y organism because of i t s s e n s i t i v i t y t o Cu, i t s u n i f o r m s i z e and shape ( f a c t o r s which makes i t amenable t o measurement by 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 ) , and i t s f a s t growth r a t e (up t o 2.5 d o u b l i n g s d a y " 1 ) . The organism v a r i e s from 2.5 t o 10 txm i n s i z e and n o r m a l l y appears s i n g l y or i n p a i r s i n l a b o r a t o r y c u l t u r e s . The e x p e r i m e n t a l c u l t u r e s were i n n o c u l a t e d w i t h enough s t o c k c u l t u r e (1-2 ml) t o p r o v i d e a c e l l c o n c e n t r a t i o n of 1000-2000 c e l l m l " 1 . A l l c u l t u r e s were grown a t a temperature of 15±1°C a t an i n - f l a s k l i g h t i n t e n s i t y of 95 juEin m" 2s" 1 on a 16:8 hour l i g h t - d a r k c y c l e . C e l l c o n c e n t r a t i o n s were m o n i t o r e d 31 O O C C H . C H ^ O O " C H 2 C H — N • O O C C H 2 ^ C H 2 C O O " EDTA , C H 2 C O O ' N — C H 2 C O O_ ' C H . C O O -NTA H H C = C C H — C C O O -H K HIS H H " O O C C H 2 C H — C C O O-N H 3 G L U F i g u r e 2 . The model or g a n i c l i g a n d s used i n the b i o a s s a y s . 32 d a i l y over the t e s t p e r i o d w i t h a C o u l t e r Counter (model Z f ) . The pH was measured i n i t i a l l y and on the t h i r d day of growth. A l l t e s t s were run i n t r i p l i c a t e u n l e s s o t h e r w i s e s t a t e d . A x e n i c s t o c k c u l t u r e s were i n i t i a l l y o b t a i n e d from the N o r t h e a s t P a c i f i c C u l t u r e C o l l e c t i o n ( U n i v e r s i t y of B.C., Vancouver, B.C.) and m a i n t a i n e d i n A q u i l p r e p a r e d as above except t h a t an Fe-EDTA mix (EDTA = 5.0 x 10" 7M) was added b e f o r e a u t o c l a v i n g . I n i t i a l l y , EDTA was added because i t was b e l i e v e d n e c e s s a r y t o keep Fe s o l u b l e d u r i n g the a u t o c l a v i n g s t e p . I t was l a t e r found t h a t t h i s a d d i t i o n was not n e c e s s a r y but the a d d i t i o n of EDTA was c o n t i n u e d so as t o m a i n t a i n s i m i l a r c o n d i t i o n s i n a l l b i o a s s a y s . A l l t r a n s f e r s and i n n o c u l a t i o n s were conducted i n a c l a s s 100 l a m i n a r f l o w hood i n which a l l p o s s i b l e m e t a l p a r t s had been r e p l a c e d by p o l y p r o p y l e n e . P r e c a u t i o n s were taken t o m i n i m i z e b a c t e r i a l and t r a c e m e t a l c o n t a m i n a t i o n . 3. T e s t of Mode of Fe A d d i t i o n I r o n i s v e r y i n s o l u b l e i n seawater and i n c u l t u r i n g p r o c e d u r e s i t i s g e n e r a l l y complexed w i t h a c o m p l e x i n g agent t o keep i t s o l u b i l i z e d . Because the a d d i t i o n of c o m p l e x i n g agents t o the c u l t u r e s f o r t h i s purpose was not d e s i r a b l e b i o a s s a y t e s t s were performed t o de t e r m i n e (1) i f f r e s h l y p r e c i p i t a t e d Fe c o u l d s u p p l y the organism w i t h Fe as r e a d i l y as a Fe-EDTA mix, (2) i f a u t o c l a v i n g of the medium c o n t a i n i n g Fe or a u t o c l a v i n g of the Fe s t o c k s c o u l d change the m e t a l ' s a v a i l a b i l i t y , and (3) i f a g e i n g of the Fe s t o c k s c o u l d change i t s a v a i l a b i l i t y . Two 33 b i o a s s a y s were performed u s i n g the A q u i l medium where the Fe was added i n the manner o u t l i n e d below. Fe was added t o g i v e a f i n a l c o n c e n t r a t i o n of 4 . 5 x 10~ 7M i n a l l f l a s k s . I n the f i r s t t e s t s e r i e s , the methods of Fe a d d i t i o n employed were as f o l l o w s : 1) F r e s h l y p r e p a r e d Fe was added b e f o r e and a f t e r a u t o c l a v i n g of the medium; 2) The medium was a u t o c l a v e d but t h e r e was no a d d i t i o n of Fe; 3) An Fe-EDTA mix (EDTA=5.Ox 10" 7M) was added t o the medium b e f o r e and a f t e r the medium was a u t o c l a v e d ; and 4) An Fe s t o c k was a u t o c l a v e d and added t o a u t o c l a v e d medium. In the second t e s t s e r i e s , methods 1 and 2 were r e p e a t e d t o g e t h e r w i t h the f o l l o w i n g a d d i t i o n a l methods: 5) A one week o l d Fe s t o c k was added t o an a u t o c l a v e d medium; and 6) A t h r e e month o l d Fe s t o c k was added t o an a u t o c l a v e d medium. F r e s h l y p r e p a r e d Fe s t o c k was added t o the c u l t u r e f l a s k s a t the end of the b i o a s s a y p e r i o d t o demonstrate t h a t growth was l i m i t e d by a Fe d e f i c i e n c y . The aged Fe s t o c k s were s t o r e d i n p o l y p r o p y l e n e b o t t l e s a t room temperature and a l l s t o c k s were s t o r e d a t t h e i r h y d r o l y s i s pH. Innoculum c u l t u r e s were grown i n a medium t o which no Fe was added so as t o p r e c o n d i t i o n the organism t o Fe l i m i t a t i o n and t o reduce Fe c a r r y o v e r from the innoculum medium. 4.. Growth Measurements C e l l number was used t o e x p r e s s the amount of growth. Both c e l l y i e l d and growth r a t e were used. C e l l y i e l d i s s i m p l y the t o t a l p o p u l a t i o n a t the end of an e x p e r i m e n t a l run minus t h a t a t 3 4 the s t a r t . I t r e p r e s e n t s the t o t a l growth t h a t o c c u r r e d d u r i n g t h e measurement p e r i o d and f a i l s t o show what i s happening a t any p a r t i c u l a r t i m e . Growth r a t e , -on the o t h e r hand, r e f l e c t s any changes o c c u r r i n g i n the growth p r o c e s s a t v a r i o u s times t h r o u g h o u t th e e x p e r i m e n t . T h e r e f o r e , depending on the sampling f r e q u e n c y , p h y s i o l o g i c a l changes i n the p o p u l a t i o n of d i f f e r e n t s c a l e l e n g t h s can be measured. I f growth r a t e s a r e averaged over the e n t i r e experiment the i n f o r m a t i o n o b t a i n e d i s the same as t h a t o b t a i n e d from c e l l y i e l d . I n t h e p r e s e n t s t u d y , growth r a t e s were e x p r e s s e d u s i n g a ' l o g a r i t h m - t o - b a s e - 2 * s c a l e t h a t i n d i c a t e s the number of d i v i s i o n s per day, K where: l o g ( N, / N 0 ) K ( d i v i s i o n s d a y " 1 ) = x 3.322 t N 0 and N, were the c e l l c o n c e n t r a t i o n s a t the b e g i n n i n g and end, r e s p e c t i v e l y , of a p e r i o d of t i m e , t . Both d a i l y growth r a t e s and growth r a t e s averaged over many days were used. R e l a t i v e growth r a t e s , which e x p r e s s the amount of growth r e l a t i v e t o t h a t of the c o n t r o l c u l t u r e s , were a l s o used. T h i s l a t t e r q u a n t i t y i s d e f i n e d by the r e l a t i o n s h i p : 35 K t e s t K % = x 100 K c o n t r o l where K % i s the r e l a t i v e growth r a t e . 5. Chelex-100 as an Ion-Exchanger Chelex-100 (Bio-Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a ) was used to remove tra c e metal i m p u r i t i e s i n both the major s a l t s o l u t i o n and the n u t r i e n t stocks. I t i s a c h e l a t i n g c a t i o n exchanger comprised of a styrene divinylbenzene copolymer matrix c o n t a i n i n g iminodiacetate a c i d f u n c t i o n a l groups with c r o s s -l i n k a g e of 1-2% and a pore s i z e of approximately 25 %. The s e l e c t i v i t y of the r e s i n f o r trace metals i s based on c h e l a t e formation rather than on c a t i o n charge, s i z e , or other p h y s i c a l c h a r a c t e r i s t i c s and i t i s c o n t r o l l e d by the p r o p e r t i e s of the r e s i n ' s IDA groups; t h i s f u n c t i o n a l group binds Cu and other heavy metals much more s t r o n g l y than the a l k a l i n e earth and a l k a l i metal c a t i o n s . (R represents the styrene-divinylbenzene copolymer matrix) 36 Chelex-100 p r e p a r a t i o n : Due to a low degree of cross l i n k a g e , Chelex was subject to large changes i n volume as the c a t i o n i c form of the r e s i n was changed. Therefore, r e s i n preparation and regeneration was performed i n the batch mode p r i o r to being added to a column. Chelex-100 was s u p p l i e d i n the Na + form and was converted to the i o n i c composition and pH of the s o l u t i o n to be p u r i f i e d so as to avoid any a l k a l i n i t y changes that might occur i n the medium i f the r e s i n gained or l o s t protons as the s o l u t i o n was passed through. The method of new r e s i n preparation was d i f f e r e n t from the method used when the r e s i n was to be regenerated. New Chelex: A 40 g p o r t i o n of the r e s i n was placed i n a 500 ml polypropylene disposable beaker ( a c i d cleaned), r i n s e d with 100 ml of methanol to remove any r e s i d u a l o r g a n i c s , and r i n s e d 3X with 200 ml of GDW. The r e s i n was then s l u r r i e d i n 300 ml of the s o l u t i o n to be cleaned and t i t r a t e d with a c i d (6N HC1) to a pH of ca. 8.0. The s o l u t i o n was decanted, f r e s h s o l u t i o n added and the t i t r a t i o n procedure repeated. Fresh s o l u t i o n was added four or f i v e times or u n t i l the pH was s t a b l e a f t e r the a d d i t i o n of f r e s h s o l u t i o n . The r e s i n was then ready to be poured i n t o the column. Regenerated Chelex: The columns were regenerated a f t e r ca. 60 1 of SOW was p u r i f i e d . The top 5 cm of the r e s i n was g e n e r a l l y d i s c o l o r e d and removed before regeneration. The r e s i n was removed from the column, r i n s e d with 200 ml GDW, then s l u r r i e d i n 200 ml of 1N HC1 and s t i r r e d for 30 min. The a c i d was then decanted and the r e s i n was r i n s e d 3X with 200 ml of 3 7 GDW. To c o n v e r t the r e s i n t o the Na + form, 200 ml of 0.5 NaOH was added, s t i r r e d f o r 30 min and r i n s e d 3X w i t h GDW. The r e s i n then underwent the same pr o c e d u r e as f o r new c h e l e x . The f i n a l s t e p i n the r e s i n p r e p a r a t i o n was the p r e p a r a t i o n of the columns. A 5.0 x 60.0 cm g l a s s column f i t t e d w i t h g l a s s wool was f i l l e d w i t h the s o l u t i o n t o be c l e a n e d . While the column was d r i p p i n g , a s l u r r y of the r e s i n was poured i n t o the column at a c o n s t a n t r a t e . A s l u r r y was used so as t o p r e v e n t any b u b b l e s from b e i n g t r a p p e d as the r e s i n was packed. A f l u i d head of 25 cm was m a i n t a i n e d over the r e s i n a t a l l t i m e s . 38 C. COMPUTER MODELLING OF CU SPECIATION IN AQUIL In the p a s t , s t a b i l i t y c o n s t a n t s have been used s i n g l y or i n p a i r s t o c a l c u l a t e the e q u i l i b r i u m c o n c e n t r a t i o n of a p a r t i c u l a r m e t a l - l i g a n d s p e c i e s i n aqueous systems.. However, i n a complex medium such as seawater many m e t a l s a r e c a p a b l e of r e a c t i n g w i t h each l i g a n d and many l i g a n d s can r e a c t w i t h each m e t a l . Thus t o compute the c o r r e c t e q u i l i b r i u m c o n c e n t r a t i o n of any s p e c i e s , a l l the competing e q u i l i b r i a must be taken i n t o a ccount s i m u l t a n e o u s l y . A l t h o u g h the t a s k i s t o o l a b o r i o u s f o r manual c a l c u l a t i o n , computer programs have become a v a i l a b l e f o r s o l v i n g such problems ( e . g . , P e r r i n , 1965; P e r r i n and Sayce, 1967; M o r e l and Morgan, 1972). More than a dozen programs a p p l i c a b l e t o n a t u r a l water c h e m i s t r y have been r e v i e w e d by Nordstrom et a l . (1979). The machine computation e f f i c i e n c y of some of t h e s e programs i s examined by Legget (1977). One of t h e g e n e r a l methods used i n s o l v i n g the complex a r r a y of s i m u l t a n e o u s e q u a t i o n s t h a t govern the s p e c i e s c o m p o s i t i o n i n seawater i s termed th e e q u i l i b r i u m c o n s t a n t a p proach. A c c o r d i n g l y t o Nordstrom et a l . (1979), t h i s a pproach i s " s u b j e c t t o the c o n d i t i o n s of 1) mass b a l a n c e and 2) c h e m i c a l e q u i l i b r i u m . The mass b a l a n c e c o n d i t i o n s r e q u i r e s t h a t t h e computed sum of the f r e e and d e r i v e d (complexes) s p e c i e s be e q u a l t o the g i v e n t o t a l c o n c e n t r a t i o n . C hemical e q u i l i b r i u m r e q u i r e s t h a t t h e most s t a b l e arrangement f o r a g i v e n system be f o u n d , as d e f i n e d by the e q u i l i b r i u m c o n s t a n t s f o r a l l mass a c t i o n e x p r e s s i o n s of the system." In the p r e s e n t s t u d y , the d i s t r i b u t i o n of Cu s p e c i e s i n the 39 c u l t u r e media was e s t i m a t e d u s i n g the computer program MINEQL ( W e s t a l l et a l . , 1976). T h i s model, which uses the e q u i l i b r i u m c o n s t a n t approach, i s s u p p l i e d w i t h a d a t a f i l e c o n s i s t i n g of e q u i l i b r i u m c o n s t a n t s s e l e c t e d m a i n l y from the t a b u l a t i o n s of S i l l e n and M a r t e l l (1964,1971). The model e s t i m a t e s the f r e e m e t a l i o n c o n c e n t r a t i o n i n s o l u t i o n when the t o t a l m e tal c o n c e n t r a t i o n and the thermodynamic c o n s t a n t s f o r a l l the p o s s i b l e m e t a l complexes a r e g i v e n . The s t a b i l i t y c o n s t a n t s f o r the more i m p o r t a n t Cu-complexes used i n the computer model a r e g i v e n i n Ta b l e I I I . Boron-Cu complexes were e l i m i n a t e d from the c a l c u l a t i o n s because of the u n c e r t a i n t y of t h e i r s t a b i l i t y c o n s t a n t s i n seawater. The thermodynamic c o n s t a n t s used i n the model a r e f i r s t c o r r e c t e d t o the a p p r o p r i a t e i o n i c s t r e n g t h of the medium, i n t h i s case seawater, by means of a c t i v i t y c o e f f i c i e n t s e s t i m a t e d from the D a v i e s a p p r o x i m a t i o n ( D a v i e s , 1962). I o n i c s t r e n g t h c o r r e c t i o n s ; An a c t i v i t y c o e f f i c i e n t , f , of an i o n i n a medium of i o n i c s t r e n g t h , I , can be e s t i m a t e d by: I 1 / 2 Log f = Z 2 e T ( -0.21 ) [1] 1 + I 1 / 2 where: Z i o n i c c h a r g e , e d i e l e c t r i c c o n s t a n t of the s o l v e n t T temperature Table I I I . S t a b i l i t y constants f o r the important Cu complexes used i n the computer model. Inorganic Complexes P 2 = K i X K 2 E q u i l i b r i u m l o g K, l o g (S2 Cu 2 + + OH" = Cu(OH) +Cu 2 + + 20H" = Cu(OH)2° Cu 2 + + c o 3 2 - = CuC0 3° Cu 2 + + 2C0 3 2 " = Cu(C0 3 6.0 16.68 6.70 3 / 2 2 - 9.90 Organic Complexes Glutamic A c i d H i s t i d i n e NTA EDTA 7.9 14.4 11.1 19.40 14.5 17.10 20.6 41 I = i o n i c s t r e n g t h I o n i c s t r e n g t h i s d e f i n e d by: I = 1/2 I Zj 2 Cj i : a l l s p e c i e s i n s o l u t i o n where: Zf = i o n i c charge of s p e c i e s i C; = c o n c e n t r a t i o n of s p e c i e s i The thermodynamic e q u i l i b r i u m c o n s t a n t , K 1 f f o r a r e a c t i o n M 2 * + T.' - ^ . N ML can be w r i t t e n : { ML } [ ML ] f M L K [2] { M } { L } [ M ] [ L ] f f, where { } r e p r e s e n t s a c t i v i t y , [ ] r e p r e s e n t s c o n c e n t r a t i o n and f r e p r e s e n t s the a c t i v i t y c o e f f i c i e n t . Charges a r e o m i t t e d f o r the sake of s i m p l i c i t y . Upon rearrangement: [ ML ] = K, . [ M ] [ L ] [3] fML 42 The q u a n t i t y f M f L K' . K [4] M L i s termed an apparent e q u i l i b r i u m constant since i t has a constant value i n a l l s o l u t i o n s of given i o n i c s t r e n g t h . 43 D. RESULTS 1. Model L i g a n d Study T a b l e IV g i v e s the e x p e r i m e n t a l parameters and r e s u l t s of the b i o a s s a y t e s t s . I n c l u d e d a r e one t e s t run u s i n g the A q u i l medium w i t h v a r i o u s c o n c e n t r a t i o n s of Cu and s i x t e s t s u s i n g A q u i l w i t h v a r i o u s l i g a n d a d d i t i o n s (EDTA, GLU, HIS, NTA), l i g a n d c o n c e n t r a t i o n s and Cu c o n c e n t r a t i o n s . Growth r a t e s i n the f i r s t 24 hours were g e n e r a l l y lower than the maximum p o s s i b l e growth due t o the organism becoming c o n d i t i o n e d t o the medium (see Appendix A) and, d u r i n g t h i s time p e r i o d , t h e growth of t h e c o n t r o l c u l t u r e s was u s u a l l y i d e n t i c a l t o the t e s t c u l t u r e s except when h i g h Cu l e v e l s were used. Because of t h i s , the f i r s t 24 hours of growth was not i n c l u d e d i n t he growth r a t e c a l c u l a t i o n s and growth r a t e s r e p o r t e d were averaged over the next 72 ho u r s . T e s t run 1 d e t e r m i n e d the e f f e c t of Cu on the growth r a t e of T. pseudonana when no o r g a n i c l i g a n d s were added. The organis m was found t o be h i g h l y s e n s i t i v e t o Cu under t h e s e e x p e r i m e n t a l c o n d i t i o n s ( F i g . 3 ) . Cu began t o i n h i b i t growth when i t was added i n s l i g h t e x c e s s of 1.57 x 10" 8M Cu a l t h o u g h a c o n c e n t r a t i o n of g r e a t e r than 4.71 x 10" 7M was needed t o o b t a i n t o t a l growth i n h i b i t i o n . The growth response was not l i n e a r over the t o x i c range of the m e t a l but appeared t o be a 'two-s t e p ' response ( F i g . 4 ) . A r a p i d l i n e a r d e c r e a s e i n growth was seen i n i t i a l l y w i t h i n c r e a s i n g Cu l e v e l s u n t i l growth r a t e s d e c r e a s e d t o j u s t below 50% of the c o n t r o l . At t h i s p o i n t , a T a b l e IV. Growth r a t e d a t a from b i o a s s a y s u s i n g the model o r g a n i c l i g a n d s EDTA, NTA, HIS and GLU. L i g a n d L i g a n d T o t a l Cu Growth Rate % of TEST Conc.(M) (x10" 8M) ( d i v d a y 1 ) C o n t r o l 2 EDTA 5.0x10" 8 1.0x10- 7 2.5X10" 5 0.1 c o n t r o l 1 . 49±.01 1 15.7 0.49±.01 3212 31 .4 0.391.01 2613 47. 1 0 . 2 1 ±. 0 1 1 411 0.1 c o n t r o l 2.12±.01 3.9 1.59±.03 7511 7.9 1.011.01 4714 11.8 1.01±.03 4810 1 5.7 0.921.03 4311 0.1 c o n t r o l 2.021.01 1 .57 2.011.02 1 0011 3.15 1.691.11 8415 4.72 1.251.02 6212 6.30 1.061.02 5211 0.1 c o n t r o l 1 .521.05 3.9 1 .611.03 1 0012 7.9 1.451.10 9517 11.8 1.021.06 6714 15.7 0 . 881.03 5812 0.1 c o n t r o l 1.801.00 3.9 1 .821.03 10111 7.9 1.861.01 1 0411 11.8 1.741.04 9712 25.7 1.131.05 6312 0.1 c o n t r o l 1.761.03 7.9 1.371.05 7813 15.7 0.991.05 5613 23.6 0.881.05 5010 31.5 0.811.01 4611 0.1 c o n t r o l 1 .861.09 7.9 1 .791.05 9613 15.7 1.291.04 6912 23.6 1 .031.01 5511 31.5 0.93+05 5013 T a b l e IV. ( c o n ' t ) L i g a n d L i g a n d T o t a l Cu Growth Rate % of TEST C o n c . ( M ) ( X 1 0 " 8 M ) ( d i v d a y ' 1 ) C o n t r o l 7. 5x1 0" 5 0. 1 c o n t r o l 1 .98±. 01 7. 9 1 .97±. 02 1 0 0 1 1 15. 7 1 . 9 1 1 . 09 9715 23. 6 1 .621. 10 8215 31 . 5 1 .521. 05 77 + 2 1 . 0x1 0" 5 0. 1 c o n t r o l 1 .931. 02 7. 9 1 .351. 07 7013 15. 7 0 .961. 00 5010 2. 5x1 0" 7 7. 9 1 .931. 05 10012 1 5. 7 1 .321. 03 6812 5. 0x1 0" 5 0. 1 c o n t r o l 2 . 001. 04 7. 9 2 .021. 04 1 0112 15. 7 1 .751. 02 8711 7. 5x1 0" 5 7. 9 2 . 0 1 1 . 01 1 0 0 1 1 15. 7 1 .911. 02 9511 1 . 0x1 0- 7 0.1 c o n t r o l 1.971.01 7.9 1.171.04 5912 15.7 0.871.01 4411 23.6 0.861.05 4413 31.5 0.701.02 3511 2. 5x1 0" 7 0.1 c o n t r o l 1 .891.02 7.9 1.501.05 7913 15.7 1.051.01 5511 23.6 0 . 881.02 4711 31 .5 0.841.04 4512 5. 0x1 0- 7 0.1 c o n t r o l 2.041.03 7.9 1.891.04 9312 15.7 1.311.03 6411 23.6 1.091.08 5414 31 .5 1.071.04 5212 7. 5x1 0" 7 0.1 c o n t r o l 1.811.04 7.9 1.841.03 10211 15.7 1.401.02 7711 23.6 1.131.03 6312 31 .5 0.971.02 5411 Table IV. (con't) Ligand Ligand T o t a l Cu Growth Rate % of TEST C o n c . ( M ) ( X 1 0 " 8 M ) (div day" 1) Control 5 HIS 1.0X10' 7 0.1 c o n t r o l 1.90±.01 7.9 1.871.03 99±2 15.7 0.89±.02 47±1 23.6 0.521.01 2711 31.5 0.271.07 1413 2.5X10" 7 0.1 c o n t r o l 2.101.01 7.9 2.111.04 10012 15.7 2.141.04 10212 23.6 1.471.06 70+3 31.6 1.021.06 4913 5.0X10" 6 0.1 c o n t r o l 1.961.01 15.7 1.981.01 10110 31.5 2.051.04 10512 47.2 1.981.01 10111 63.0 1.931.03 9912 1Mean 11 s.d. based on 2 r e p l i c a t e s . 2NL No l i g a n d a d d i t i o n . 47 p l a t e a u was seen i n the c u r v e and p r o p o r t i o n a t e l y h i g h e r Cu l e v e l s were needed t o reduce the growth r a t e f u r t h e r . A s i m i l a r t w o - s t e p p r o c e s s has been d e s c r i b e d f o r t h i s c l o n e of T. pseudonana by G a v i s et a l . (1981). Growth r a t e measurements d u r i n g the 24-48, 48-72 and 72-96 hour p e r i o d s r e v e a l e d a g e n e r a l response t o Cu w i t h r e s p e c t t o exposure time ( F i g . 5 ) . As exposure time i n c r e a s e d the t o x i c e f f e c t of the m e t a l was more pronounced and the l o w e s t growth r a t e s were observed a f t e r 72 h o u r s . Such a d e l a y e d response 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 organism by Sunda and G u i l l a r d (1976) (see a l s o G a v i s et a l . , 1981). T e s t runs 2 t h r o u g h 5 examined the a b i l i t y of s p e c i f i c o r g a n i c l i g a n d s t o reduce the t o x i c i t y of Cu. The growth p a t t e r n s of t h e o r g a n i s m o b t a i n e d i n the p r e s e n c e of t h e s e l i g a n d s a r e shown i n F i g s . 6-9. A l t h o u g h a l l f o u r l i g a n d s t e s t e d reduced the t o x i c i t y of Cu, t h e c o n c e n t r a t i o n r e q u i r e d t o reduce the t o x i c i t y t o a g i v e n l e v e l d i f f e r e d f o r each compound. The l o w e s t c o n c e n t r a t i o n of each ' l i g a n d t h a t a l l o w e d f u l l growth i n the presence of 7.87 x 10" 8M Cu was: EDTA, 5.0 x 10" 8M; HIS, 1.0 x 10" 7M; NTA, 7.5 x 10" 7M; and GLU, 2.5 x 10" 5M. EDTA appeared t o b i n d Cu i n a 1:1 Cu:EDTA r a t i o as was suggested by a sharp d e c l i n e i n the growth r a t e when Cu was added i n s l i g h t l y g r e a t e r c o n c e n t r a t i o n s than the l i g a n d c o n c e n t r a t i o n ( T able IV, Test 2 ) . The a b i l i t y of HIS t o reduce Cu t o x i c i t y was g r e a t e r t h a n t h a t of NTA even though i t has a lower s t a b i l i t y c o n s t a n t f o r Cu. However, i f one examines a l l the competing e q u i l i b r i a i n 48 F i g u r e 3. Growth of the b i o a s s a y organism i n A q u i l i n the presence of Cu and w i t h no o r g a n i c l i g a n d s added. Symbols: • C o n t r o l ; A 1 . 5 7 X 1 0 " 8 M ; 0 3 . 1 5 X 1 0 " 8 M ; V 4 . 7 2 X 1 0 " 8 M ; O 6 . 2 9 X 1 0 " 8 M added Cu. Bars a r e ±1 s.d. 49 F i g u r e 4. Growth r a t e (% of c o n t r o l ) v e r s u s t h e - l o g of the added Cu c o n c e n t r a t i o n (Cu T) i n A q u i l w i t h no o r g a n i c s added. Bars a r e ±1 s.d. 100 _ 50 F i q u r e 5. E f f e c t of Cu upon growth ( d i v s d a y ' 1 ) d u r i n g the 24-96 hr p e r i o d . Symbols: • 24-48 h r . ; A 48-72hr.; + 72-96 h r . p e r i o d . B a r s are ±1 s.d. 2.5 2.0 51 Figure 6. Growth rate (% of c o n t r o l ) versus the - l o g of the Cu conce n t r a t i o n (Cu T) i n the presence of GLU. Symbols: • 1.0X10" 5 M: • 2.5X10" 5 M; and + 7 . 5 x l O ' 5 M c o n c e n t r a t i o n s . Bars are ±1 s.d. 6.4 6.6 6.8 7.0 7.2 - l o g C u T 52 Figure 7. Growth rate (% of c o n t r o l ) versus the - l o g of the Cu conce n t r a t i o n (Cu T) i n the presence of HIS. Symbols: • 1.0X10- 7 M; • 2.5X10" 7 M; and + 5 . 0X 1 0 " 7 M c o n c e n t r a t i o n s . Bars are ±1 s.d. 53 Figure 8. Growth rate (% of c o n t r o l ) versus the - l o g of the Cu c o n c e n t r a t i o n (Cu T) i n the presence of NTA. Symbols: • 1.0X10" 7 M; • 2 . 5X 1 0 ' 7 M ; 4 5.0X10- 7 M; and • 7 . 5 X 1 0 _ 7 M c o n c e n t r a t i o n s . Bars are ±1 s.d. 54 Figure 9. Growth rate (% of c o n t r o l ) versus the - l o g of added Cu con c e n t r a t i o n (Cu T) i n the presence of EDTA. Symbols: • 5.0X10~ b M; and A 1 . 0 X 1 0 _ 7 M c o n c e n t r a t i o n s . Bars are ±1 s.d. •*—» — p O 20 -6.6 6.8 7.0 7.2 7.4 " l o g C u T 55 the a r t i f i c i a l seawater by the computer model, i t becomes apparent t h a t a l a r g e p e r c e n t a g e of NTA i s bound i n Ca and Mg complexes w h i l e HIS i s n o t . Thus the amount of f r e e l i g a n d a v a i l a b l e f o r Cu c o m p l e x a t i o n was g r e a t e r f o r HIS than f o r NTA. When EDTA was added i n exce s s of Cu, s l i g h t l y lower growth r a t e s were found i n the c o n t r o l c u l t u r e s than i n c u l t u r e s c o n t a i n i n g the lower Cu c o n c e n t r a t i o n s ( T a b l e IV, T e s t 2 ) . T h i s e f f e c t was a l s o seen i n c u l t u r e s t h a t had h i g h c o n c e n t r a t i o n s of HIS added (Table IV, Test 5 ) . T h i s may be due t o t h e s e l i g a n d s b i n d i n g many of the n u t r i t i o n a l t r a c e m e t a l s i n the A q u i l medium (Mn, Co, Mo, and Zn) which may cause some of t h e s e m e t a l s t o become l i m i t i n g . W i th the a d d i t i o n of Cu t h e n , a p r o p o r t i o n of the m e t a l s w i l l be d i s p l a c e d from the l i g a n d t h u s f r e e i n g them f o r uptake by the organism. A second p o s s i b i l i t y i s t h a t the a d d i t i o n of EDTA reduces the c u p r i c i o n c o n c e n t r a t i o n t o a p o i n t where i t i s n u t r i t i o n a l l y d e f i c i e n t . W i t h the a d d i t i o n of Cu the c u p r i c i o n a c t i v i t y s i m p l y i n c r e a s e s t o a p o i n t where i t i s no l o n g e r d e f i c i e n t . The c u l t u r e s whose growth r a t e s were improved w i t h t h e a d d i t i o n of low c o n c e n t r a t i o n s of Cu were b e l i e v e d t o be more r e p r e s e n t a t i v e of a h e a l t h y p o p u l a t i o n and were used as t h e c o n t r o l c u l t u r e s f o r t h a t p a r t i c u l a r t e s t . 2. Growth Rate as a F u n c t i o n of C u p r i c Ion A c t i v i t y In F i g s . 10-13, growth r a t e s a r e p l o t t e d as a f u n c t i o n of the n e g a t i v e l o g t o the base 10 of the c u p r i c i o n a c t i v i t y (pCu* where * i n d i c a t e s i t i s a computed v a l u e ) . C u p r i c i o n 5 6 c o n c e n t r a t i o n s were c a l c u l a t e d u s i n g the computer model MINEQL and c o n v e r t e d t o a c t i v i t i e s by m u l t i p l i c a t i o n by an a c t i v i t y c o e f f i c i e n t e s t i m a t e d from the D a v i e s a p p r o x i m a t i o n (eqn. 1 ) . When examining each l i g a n d s e p a r a t e l y . , a s t r o n g l i n e a r r e l a t i o n s h i p was seen between growth r a t e and pCu* l e v e l s of 8.4 and' 10.0. S l i g h t d e v i a t i o n s from t h i s l i n e a r r e l a t i o n s h i p were seen i n growth r a t e s of c u l t u r e s c o n t a i n i n g h i g h c o n c e n t r a t i o n s of GLU ( F i g . 10). Above a pCu* l e v e l of 10.0 c e l l growth was no l o n g e r i n h i b i t e d w h i l e the p l a t e a u of the growth c u r v e , d e s c r i b e d p r e v i o u s l y , was reached a t a pCu* of a p p r o x i m a t e l y 8.4. The e x c e p t i o n was t h a t of HIS where the growth r a t e s - i n the pCu* range of t h i s p l a t e a u were g e n e r a l l y lower than the o t h e r l i g a n d s . The r e l a t i o n s h i p between growth r a t e and pCu* was q u i t e good when examining the l i g a n d s s e p a r a t e l y . However, when c u r v e s f o r each l i g a n d were p r e s e n t e d t o g e t h e r ( F i g . 14) the s c a t t e r i n the cu r v e was much g r e a t e r a l t h o u g h a l i n e a r r e l a t i o n s h i p was s t i l l a p p a r e n t . T h i s s c a t t e r c o u l d be due t o v a r i a b i l i t y not taken i n t o account i n the b i o a s s a y t e c h n i q u e , d i f f e r e n c e s i n b i o l o g i c a l l y a c t i v e Cu not a s s o c i a t e d w i t h the c u p r i c i o n , or t o e r r o r s i n the c a l c u l a t i o n of pCu* by the computer model. The reason f o r the v a r i a b i l i t y w i l l be d i s c u s s e d i n S e c t i o n I I I . D . 1 . In the computer model, i t was c a l c u l a t e d t h a t the maximum pCu* p o s s i b l e b e f o r e the p r e c i p i t a t i o n of m a l a c h i t e ( C u 2 ( O H ) 2 C 0 3 ) would occur was 8.13. W i t h no o r g a n i c l i g a n d s p r e s e n t , p r e c i p i t a t i o n of m a l a c h i t e would occur i n A q u i l when a 57 Figure 10. Growth rate (% of c o n t r o l ) versus the c a l c u l a t e d pCu* i n the presence of GLU. Symbols: • 1.0X10" 5 M ; • 2.5X10" 5 M; and + 7. 5X 1 0 " 5 M added GLU. Bars are ±1 s.d. 100 80 c o ° 60 03 40 H 2 O 20-] 8.4 9.0 9.6 pCu 10 .2 10.8 Figure 11. Growth rate (% of c o n t r o l ) versus the c a l c u l a t e d pCu* i n the presence of HIS. Symbols: • 1 . 0 X 1 0 _ 7 M ; and A 2 . 5 X 1 0 _ 7 M added HIS. Bars are ±1 s.d. 59 Figure 12. Growth rate (% of c o n t r o l ) versus the c a l c u l a t e d pCu* i n the presence of NTA. Symbols: • 1 . 0 X 1 0 _ 7 M ; • 2.5X10" 7 M; + 5.0X10' 7 M; and • 7.5X10- 7 M added NTA. Bars are ±1 s.d. 8.4 9.0 9.6 pCu 10 .2 10.8 Figure 13. Growth rate (% of c o n t r o l ) versus the c a l c u l a t e d pCu* i n the presence of EDTA. Symbols: • 5.0x10 " 8 M ; and A 1 . 0 X 1 0 _ 7 M added EDTA. Bars are ±1 s.d. 61 F i g u r e 14. Growth r a t e (% of c o n t r o l ) v e r s u s the c a l c u l a t e d pCu* f o r a l l the b i o a s s a y r e s u l t s . Symbols: O EDTA; • NTA; 0 HIS; and A GLU. Bars a r e ±1 s.d. ~ ~ l — 9.0 8.4 9.6 pCu< 10 .2 10.8 62 t o t a l Cu c o n c e n t r a t i o n of 11.9 x 10~ 7M was exceeded. S i n c e the org a n i s m s h o u l d o n l y respond t o the c u p r i c i o n a c t i v i t y of the s o l u t i o n , no f u r t h e r decrease i n growth s h o u l d be seen when Cu i s added above t h i s s o l u b i l i t y l i m i t . However, t h i s d i d not seem t o be the c a s e . Cu c o n c e n t r a t i o n s above 11.9 x 10 _ 7M d i d cause a f u r t h e r r e d u c t i o n i n growth ( F i g . 3 ) . T h i s phenomenon might be e x p l a i n e d i n two ways. F i r s t , i t i s p o s s i b l e t o o b t a i n a m e t a s t a b l e s t a t e w i t h r e s p e c t t o the f r e e c u p r i c i o n whereby s u p e r s a t u r a t i o n of the s o l u t i o n does o c c u r (E.V. G r i l l p e r s . comm.) or second, the computer model may o v e r e s t i m a t e the e x t e n t t o which the c a r b o n a t e i o n complexes the c u p r i c i o n ( e r r o r i n the Cu-carbonate s t a b i l i t y c o n s t a n t s ) . I n e i t h e r case the c u p r i c i o n a c t i v i t y of the s o l u t i o n c o u l d then exceed the s o l u b i l i t y p r e d i c t e d by the model. Because of t h i s , p r e c i p i t a t i o n was not c o n s i d e r e d when c a l c u l a t i n g pCu* l e v e l s i n the c u l t u r e medium. 3. E f f e c t of pH S i n c e pH i s an i m p o r t a n t f a c t o r when c o n s i d e r i n g Cu c h e m i s t r y , the pH changes t h a t occur over the c o u r s e of the b i o a s s a y were examined. The pH was m o n i t o r e d d a i l y over a s i x -day p e r i o d i n c u l t u r e s t h a t had no Cu added and i n c u l t u r e s c o n t a i n i n g 1.57 x 10" 7M Cu. Copper was added t o one s e t of c u l t u r e s t o l i m i t growth t o d e t e r m i n e i f a pH change was s o l e l y due t o growth. The pH i n c u l t u r e s c o n t a i n i n g 1.57 x 1 0 _ 7 M Cu i n c r e a s e d m a r g i n a l l y over the s i x day p e r i o d w h i l e the pH i n c u l t u r e s 63 w i t h o u t any Cu i n c r e a s e d s u b s t a n t i a l l y i n the l a s t two days of the b i o a s s a y (Table V ) . The pH was r e l a t e d t o the growth of the organism because of p h o t o s y n t h e t i c removal of C0 2; s i g n i f i c a n t pH changes were seen o n l y a f t e r c e l l numbers exceeded 50,000 c e l l m l " 1 . Tab l e V. V a r i a t i o n s i n pH of c u l t u r e s w i t h and w i t h o u t the a d d i t i o n of Cu over a f i v e day p e r i o d No Cu added 1.57X10" 7 M Cu added pH c e l l s m l _ 1 pH c e l l s m l _ Day 0 8 .02±. 05 1 17911166 8.021.05 16201104 Day 1 8 .061. 03 58791294 8.071.02 32971102 Day 2 8 .08±. 03 151271792 8.071.03 5475182 Day 3 8 . 17±. 02 4772612254 8.091.02 77271188 Day 4 8 .38±. 03 12470415519 8.111.03 90451307 Day 5 . 8 .58±. 07 238597114488 8.111.03 101561467 1Mean 11 s.d based on 3 r e p l i c a t e s . 4. Fe A d d i t i o n s Due t o the low s o l u b i l i t y of Fe i n seawater, s p e c i a l c o n s i d e r a t i o n was g i v e n t o the p r e p a r a t i o n and mode of Fe a d d i t i o n . EDTA has o f t e n been added t o c u l t u r e media t o keep Fe s o l u b i l i z e d (Droop, 1961; J o h n s t o n , 1964). T h i s p r a c t i c e was not s u i t a b l e f o r my a p p l i c a t i o n s i n c e EDTA has a s t r o n g a f f i n i t y f o r Cu and would d r a s t i c a l l y a l t e r t he Cu s p e c i a t i o n i n the 64 e x p e r i m e n t a l c u l t u r e s . T h e r e f o r e , t e s t s were performed t o dete r m i n e the o p t i m a l method of Fe p r e p a r a t i o n and a d d i t i o n t h a t would maximize the growth of the organism but e l i m i n a t e the need f o r an Fe c h e l a t o r . In t h e f i r s t e x p e r i m e n t , the b e n e f i c i a l e f f e c t of the Fe-EDTA mix was t e s t e d over t h a t of a f r e s h l y p r e p a r e d Fe s t o c k . C u l t u r e s h a v i n g an Fe-EDTA mix as an Fe source grew a t the same r a t e and had the same c e l l y i e l d as d i d c u l t u r e s h a v i n g o n l y 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 added ( F i g . 15). The f r e s h l y p r e p a r e d f e r r i c h y d r o x i d e s t o c k c o u l d be added e i t h e r b e f o r e or a f t e r a u t o c l a v i n g w i t h no change i n the r e s u l t s . A u t o c l a v i n g of the Fe s t o c k b e f o r e i t s a d d i t i o n t o the medium d i d , however, cause a s u b s t a n t i a l d e c r e a s e i n growth. Both the growth r a t e and f i n a l c e l l y i e l d were c o n s i d e r a b l y reduced as compared t o the c u l t u r e s w i t h f r e s h l y p r e p a r e d Fe and c u l t u r e s h a v i n g no Fe added ( F i g . 16). Ag e i n g of the s t o c k s had a s i m i l a r e f f e c t as t h a t of a u t o c l a v i n g . When a s t o c k was aged f o r 3 months and added t o the medium, both the growth r a t e and f i n a l c e l l y i e l d of the organism was g r e a t l y reduced ( F i g . 17). Ageing of the Fe s t o c k s f o r one week was not s u f f i c i e n t t o cause any r e d u c t i o n i n the growth r a t e of the organism. Because the or g a n i s m grew f o r 3-4 days i n t e s t c u l t u r e s c o n t a i n i n g no added Fe, r e s i d u a l Fe must have been p r e s e n t i n the medium, even though the Fe l e v e l was below 1.79x10 _ 8M as measured by g r a p h i t e f u r n a c e AAS. T h i s Fe may be a r e s u l t of c a r r y o v e r from the innoculum, Fe i n the c e l l p o o l of the 65 organism, or Fe c o n t a m i n a t i o n i n the sample. F u r t h e r s t u d i e s i n t h i s l a b have been conducted on the c h e m i s t r y of Fe i n A q u i l and i t s a v a i l a b i l i t y t o p h y t o p l a n k t o n ( W e l l s -et a l . , i n p r e s s ) . 66 Figure 15. C e l l growth with f r e s h Fe stocks ( • ) and Fe-EDTA stocks added before ( A ) and a f t e r ( 0 ) a u t o c l a v i n g . D a y s 67 F i g u r e 16. C e l l growth w i t h f r e s h Fe ( A ), no Fe ( • ) and a u t o c l a v e d Fe s t o c k s { 0 ) added t o the c u l t u r e medium. 2 3 4 5 D a y s Figure 17. C e l l growth with f r e s h Fe stocks ( A ) and Fe stocks aged for one week ( 0 ) and three months ( • ) added to the c u l t u r e medium. 1 2 3 4 D a y s 69 D. DISCUSSION 1 . Growth Rate as a F u n c t i o n of pCu* In c u l t u r e s c o n t a i n i n g no o r g a n i c l i g a n d s , growth i n h i b i t i o n of T. pseudonana was found when Cu exceeded a c o n c e n t r a t i o n of 1.57 x 10" 8M. However, upon the a d d i t i o n of EDTA, GLU, HIS, or NTA, much h i g h e r Cu l e v e l s c o u l d be added w i t h no d e l e t e r i o u s e f f e c t . T h i s i n d i c a t e d a r e d u c t i o n i n b i o l o g i c a l l y a c t i v e Cu i n the presence of the s e c o m p l e x i n g a g e n t s . The de c r e a s e i n t o x i c i t y was a t t r i b u t e d t o a de c r e a s e i n the c u p r i c i o n a c t i v i t y of the medium by c o m p l e x a t i o n of the c u p r i c i o n . Other workers have p r e s e n t e d e v i d e n c e t h a t the e f f e c t of Cu on the growth of T. pseudonana (Davey, 1975; Sunda and G u i l l a r d , 1976; G a v i s e t a l . , 1981) as w e l l as o t h e r p h y t o p l a n k t o n s p e c i e s (Anderson and M o r e l , 1978; C a n t e r f o r d and C a n t e r f o r d , 1980; J a c k s o n and Morgan, 1978) can be c o r r e l a t e d b e t t e r w i t h the c u p r i c i o n c o n c e n t r a t i o n than w i t h the t o t a l Cu c o n c e n t r a t i o n . An e s t i m a t i o n of the pCu* i n the medium was made u s i n g the computer model MINEQL. From th e s e r e s u l t s , growth i n h i b i t i o n of T. pseudonana was seen below a pCu* of 10.0 w h i l e t o t a l growth i n h i b i t i o n o c c u r r e d a t a pCu* below 8.0. Sunda and G u i l l a r d (1976) found s i m i l a r r e s u l t s ; i . e . , growth i n h i b i t i o n of T. pseudonana was found below a pCu* of 10.5 and t o t a l growth i n h i b i t i o n was seen below 8.3. They c a l c u l a t e d pCu* v a l u e s i n the c u l t u r e s of Davey e t a l . (1973) and found t h a t t o t a l growth i n h i b i t i o n of t h e i r t e s t organism a l s o o c c u r r e d a t a pCu* below 70 8.0. The c a l c u l a t i o n of pCu* i n seawater has i t s l i m i t a t i o n s . V a r i a t i o n s i n p u b l i s h e d s t a b i l i t y c o n s t a n t data and, t o a l e s s e r e x t e n t , problems w i t h a c t i v i t y c o e f f i c i e n t c o r r e c t i o n s s e r i o u s l y compromise the a c c u r a c y w i t h which pCu* can be computed. Most s t a b i l i t y c o n s t a n t s a re d e r i v e d i n s o l u t i o n s u n l i k e t h a t of seawater and a r e det e r m i n e d a t a d i f f e r e n t i o n i c s t r e n g t h . The e x t r a p o l a t i o n of these s t a b i l i t y c o n s t a n t s t o a seawater m a t r i x and the c o n c u r r e n t i o n i c s t r e n g t h c o r r e c t i o n s needed may l e a d t o i n a c c u r a t e r e s u l t s . The r e l a t i o n s h i p between the c a l c u l a t e d pCu* and growth r a t e was q u i t e s t r o n g when any one l i g a n d was examined ( F i g s . 10-13). However, when the pCu* v a l u e s f o r a l l the l i g a n d s and l i g a n d c o n c e n t r a t i o n s were combined, the r e l a t i o n s h i p was not n e a r l y as s t r o n g ( F i g . 14) and t h i s weakness was a t t r i b u t e d p r i m a r i l y t o i n a c c u r a c i e s g e n e r a t e d i n the computer model. When c o n s i d e r i n g each l i g a n d , even though i n a c c u r a t e thermodynamic d a t a may be used t o det e r m i n e the pCu* of t h e medium, the b i a s i n t h e c a l c u l a t i o n s would be c o n s i s t e n t over a l l the Cu and l i g a n d c o n c e n t r a t i o n s used. However, the b i a s i n the c a l c u l a t i o n s w i l l d i f f e r f o r each l i g a n d . The r e s u l t w i l l then be a g r e a t e r v a r i a b i l i t y i n the e s t i m a t i o n of pCu* i n a m u l t i - l i g a n d p l o t w i t h a subsequent weaker r e l a t i o n s h i p between pCu* and growth r a t e . The growth r a t e d a t a w i l l be used f o r comparison purposes w i t h an a n a l y t i c a l t e c h n i q u e d e s i g n e d t o e s t i m a t e b i o l o g i c a l l y a c t i v e m e t a l . Presumably growth r a t e i s an a b s o l u t e measure of t 71 pCu even though the l a t t e r v a l u e cannot be c a l c u l a t e d p r e c i s e l y i n the c u l t u r e medium. 2. E x p e r i m e n t a l C o n s i d e r a t i o n s The b i o a s s a y t e c h n i q u e was used t o measure b i o l o g i c a l l y a c t i v e Cu i n a c u l t u r e medium c o n t a i n i n g a p a r t i c u l a r c o m b i n a t i o n of l i g a n d and Cu c o n c e n t r a t i o n s . Many f a c t o r s must be c o n s i d e r e d when u s i n g t h i s t e c h n i q u e . The i m p o r t a n t c o n s i d e r a t i o n s w i l l be d i s c u s s e d below. Changes i n Organism Response t o C u p r i c Ion A c t i v i t i e s : W h i l e the d i f f i c u l t i e s i n a c c u r a t e l y c a l c u l a t i n g pCu* have been d i s c u s s e d , the r e p r o d u c i b i l i t y of the growth r a t e of the organism a t a g i v e n pCu has not been c o n s i d e r e d . To use growth r a t e t o e s t i m a t e pCu, i t must be assumed t h a t the organism's response t o a s p e c i f i c pCu v a l u e does not change w i t h the a d d i t i o n of any o t h e r component i n the medium. However, r e c e n t s t u d i e s have i n d i c a t e d t h a t the a d d i t i o n of S i ( O H ) 4 (Rueter e t a l . , 1981) and M n 2 + (Sunda e_t a_l. , 1981) can i n c r e a s e the growth of p h y t o p l a n k t o n w h i l e a g i v e n pCu l e v e l i s m a i n t a i n e d i n the medium. Rueter e t a l . (1981) h y p o t h e s i z e d t h a t Cu i n h i b i t s the f u n c t i o n i n g of a Si(OH) f l t r a n s p o r t s i t e on the c e l l and t h a t b o t h a r e t r a n s p o r t e d i n t o the c e l l a t the same s i t e . I n a d d i t i o n , he suggested t h a t the pCu i n the medium c o n t r o l s the r a t e of Cu uptake and the c o n c e n t r a t i o n of i n t r a c e l l u l a r Cu, which i n t u r n c o n t r o l s the growth r a t e . T h e r e f o r e , i n c r e a s i n g the c o n c e n t r a t i o n of S i ( O H ) 4 a l l o w s i t t o compete more 72 s u c c e s s f u l l y f o r t h i s common uptake s i t e and reduces Cu upta k e . To e x p l a i n the Mn e f f e c t , Sunda e t a l . (1981) proposed a s i m p l e c e l l u l a r b i n d i n g model i n which Cu competes w i t h Mn a t an i n t e r n a l b i n d i n g s i t e , such as an enzyme. When the enzyme i s bound t o Cu i t would be i n a c t i v a t e d and growth would be d e c r e a s e d . An i n c r e a s e i n the Mn c o n c e n t r a t i o n a l l o w s Mn t o d i s p l a c e Cu from t h i s s i t e and l e s s e r amounts of the enzyme w i l l be d e a c t i v a t e d . T h i s Mn-Cu i n t e r a c t i o n w i l l be d i s c u s s e d i n more d e t a i l i n S e c t i o n V. In the case of Mn, the c o n c e n t r a t i o n added t o the c u l t u r e medium was much lower than the c o n c e n t r a t i o n of Cu needed t o cause t o x i c i t y . At such a r a t i o i t was assumed t h a t the c o m p e t i t i o n of Mn was a t a minimum. T h e r e f o r e , l i t t l e or no manganese i n t e r f e r e n c e was e x p e c t e d . Changes i n pH over the term of the b i o a s s a y : In seawater, the d i s t r i b u t i o n of c u p r i c i o n and i t s complexes v a r i e s w i t h pH ( Z i r i n o and Yamamoto, 1972). In the presence of o n l y i n o r g a n i c l i g a n d s , c u p r i c i o n i s thought t o be m a i n l y complexed by OH" and C 0 3 2 " i o n s i n seawater a t a pH of 8.0 ( F l o r e n c e and B a t l e y , 1976; K e s t e r e t a l . , 1975; Z i r i n o and Yamamoto, 1972). An i n c r e a s e i n pH r e s u l t s i n i n c r e a s i n g the c o n c e n t r a t i o n of thes e l i g a n d s and hence an i n c r e a s e i n the c o m p l e x a t i o n of Cu and a de c r e a s e i n the f r e e c u p r i c i o n c o n c e n t r a t i o n . In the presence of o r g a n i c l i g a n d s , t h e i r a b i l i t y t o complex Cu i s dependent on t h e i r i n h e r e n t s t a b i l i t y c o n s t a n t and the pH of the s o l u t i o n . As the pH i n c r e a s e s t h e r e i s l e s s p r o t o n c o m p e t i t i o n f o r the l i g a n d and more f r e e l i g a n d i s a v a i l a b l e f o r c o m p l e x a t i o n . (The 73 e f f e c t of pH i s dependent on the pK of the f u n c t i o n a l group t h a t complexes the m e t a l ) . Thus an i n c r e a s e i n pH w i l l not o n l y i n c r e a s e the c o n c e n t r a t i o n of both OH - and C 0 3 2 _ i o n s , but a l s o may i n c r e a s e the c o n c e n t r a t i o n of the a c t i v e l i g a n d c o n c e n t r a t i o n . For t h e s e r e a s o n s , i t i s i m p o r t a n t t o m a i n t a i n a stea d y pH th r o u g h o u t the p e r i o d of the ex p e r i m e n t . B u f f e r s a re a way of a c c o m p l i s h i n g t h i s but were not c o n s i d e r e d because such a g e n t s g e n e r a l l y complex m e t a l i o n s . I n the pH e x p e r i m e n t , major pH changes were seen i n r a p i d l y growing c u l t u r e s a t the end of the e x p e r i m e n t a l p e r i o d ( T a b l e V) and the pH was r e l a t e d t o t h e c o n c e n t r a t i o n of c e l l s i n the medium and not t o the time span of the e x p e r i m e n t . An i n c r e a s e i n pH became apparent when the medium c o n t a i n e d 5 0 , 0 0 0 c e l l s m l * 1 or g r e a t e r . However, i n t h e b i o a s s a y e x p e r i m e n t s , c u l t u r e s i n h i b i t e d by Cu i n f r e q u e n t l y r e a c h e d the c e l l c o n c e n t r a t i o n s t h a t were found t o a f f e c t pH. In some i n s t a n c e s though, h i g h e r c e l l c o n c e n t r a t i o n s were o b t a i n e d but o n l y on the l a s t day of the t e s t . Because of t h i s , t h e pH changes t h a t d i d occur i n the c u l t u r e s were not b e l i e v e d t o have a s i g n i f i c a n t e f f e c t . A l t h o u g h t h e r e was a l a r g e r i n c r e a s e i n pH i n the c o n t r o l c u l t u r e s , a h i g h e r pH d i d not a f f e c t growth i n such c u l t u r e s . B a c t e r i a l C o n s i d e r a t i o n s : The presence of b a c t e r i a c o u l d a f f e c t the r e s u l t s of the b i o a s s a y by p o s s i b l e m o d i f i c a t i o n of the c u l t u r e medium. A l t h o u g h p r e c a u t i o n s were taken t o e l i m i n a t e b a c t e r i a from the c u l t u r e medium, p o s s i b l e c o n t a m i n a t i o n c o u l d r e s u l t from the u n s t e r i l i z e d t r a c e m e t a l 74 s t o c k s added t o the medium. E r i c k s o n (1972) examined the e f f e c t of b a c t e r i a on Cu t o x i c i t y t o T. pseudonana and found t h a t the growth response t o Cu was the same i n both b a c t e r i a f r e e and b a c t e r i a c o n t a i n i n g seawater. I t was noted t h a t i f the seawater he employed had o n l y a s m a l l amount of o r g a n i c s u b s t r a t e p r e s e n t then l a r g e numbers of b a c t e r i a were p r o b a b l y not p r e s e n t . Because of E r i c k s o n ' s work, the p r e c a u t i o n s taken t o reduce b a c t e r i a , and the low amounts of o r g a n i c m a t e r i a l p r e s e n t i n the A q u i l medium, b a c t e r i a were not b e l i e v e d t o a f f e c t the r e s u l t s i n a s i g n i f i c a n t manner. 3. E n v i r o n m e n t a l C o n s i d e r a t i o n To d e t e r m i n e the r e l e v a n c e of the pCu* v a l u e s found t o be t o x i c i n t h i s s tudy comparisons were made w i t h pCu* v a l u e s c a l c u l a t e d f o r n a t u r a l w a t e r s . Sunda (1975) o b t a i n e d a r a t i o f o r the a c t i v i t y of c u p r i c i o n t o the t o t a l Cu c o n c e n t r a t i o n of 10" 1*' 8 i n seawater f r e e of o r g a n i c l i g a n d s h a v i n g a c h l o r i n i t y of 19 p p t , a temperature of 25°C and a pH of 8.2. Sunda and G u i l l a r d (1976), u s i n g Sunda's r a t i o and C h e s t e r and S t o n e r ' s (1974) e s t i m a t i o n s of Cu l e v e l s i n open ocean wa t e r s (2-60 x 1 0 _ 9 M ) , c a l c u l a t e d a seawater pCu* range of 10.5-9.0 w i t h a mean pCu* of 9.7. T h i s range o v e r l a p s the pCu* range found t o be i n h i b i t o r y t o T. pseudonana. T h i s i m p l i e s t h a t n a t u r a l Cu l e v e l s c o u l d be t o x i c t o t h i s p h y t o p l a n k t o n s p e c i e s i n seawater c o n t a i n i n g l i t t l e or no o r g a n i c c o m p l e x i n g a g e n t s . B i n d i n g of Cu t o o r g a n i c m o l e c u l e s has been proposed as a means of r e d u c i n g Cu t o x i c i t y i n n a t u r a l w a t e r s (Steeman N i e l s e n 75 and Wium-Andersen, 1970). S t u d i e s i n v o l v i n g Cu t i t r a t i o n s of r i v e r w a t e r , l a k e water' or c o a s t a l seawater u t i l i z i n g i o n -s e l e c t i v e e l e c t r o d e s ( S t i f f , 1971; Sunda and Hansen, 1979), t o x i c i t y b i o a s s a y s (Gachter e t a_l. , 1978; G i l l e s p i e and V a c c a r o , 1978) and Cu a d s o r p t i o n by Mn0 2 (van den Berg and Kramer, I979a,b) a l l p r e d i c t e d t h a t d i s s o l v e d Cu i s p r i m a r i l y bound by o r g a n i c s . I f a p h y t o p l a n k t o n organism o n l y responds t o f r e e Cu and not t o o r g a n i c a l l y bound Cu, as i n d i c a t e d by the p r e s e n t s t u d y , then t o measure the t o t a l c o n c e n t r a t i o n of the m e t a l i n n a t u r a l w a t e r s as an e s t i m a t e of t o x i c i t y i s m e a n i n g l e s s . 76 IV. THE MEASUREMENT OF BIOLOGICALLY ACTIVE CU BY A STRONGLY ACIDIC CATION EXCHANGER A. INTRODUCTION None of the a n a l y t i c a l methods developed to date have been shown to measure the l e v e l of b i o l o g i c a l l y a c t i v e Cu i n seawater. However, c e r t a i n a n a l y t i c a l methods have the p o t e n t i a l f o r measuring the chemical (and hence the b i o l o g i c a l ) a c t i v i t y of metals i n s o l u t i o n . One of these methods i s i o n -exchange ( A l l e n et a l , 1975; Mancy and A l l e n , 1977; T r e i t et a l . , 1983). Ion-exchange can be used to separate c a t i o n s or anions using, r e s p e c t i v e l y , s t r o n g l y a c i d i c or s t r o n g l y basic r e s i n s . F i l b y et. a l . (1974) passed f i l t e r e d r i v e r water through both an anion-exchange and a cation-exchange r e s i n and d i v i d e d the d i s s o l v e d metals i n t o a n i o n i c and c a t i o n i c adsorbed species. N e u t r a l species d i d not adsorb on e i t h e r r e s i n . In the three r i v e r s s t u d i e d , Zn was found to be mostly cation-exchangeable. Shuman and Dempsey (1977) a l s o used a s t r o n g l y a c i d i c cation-exchange r e s i n to pre-concentrate c a t i o n i c metal species i n r i v e r waters before the metals were determined by atomic absorbtion spectrophotometry. C a t i o n i c species of Cd, Cr, Cu, Pb and Zn were observed with some ani o n i c species of Cr, Cu and Zn a l s o being present. Using n a t u r a l seawater, Marchand (1974) determined various physico-chemical forms of f i v e r a d i o a c t i v e metal isotopes by cation-exchange chromatography. An a l i q u o t of seawater was spiked with the appropriate r a d i o n u c l i d e and added 77 t o the t o p of a c a t i o n - e x c h a n g e column. The column was then e l u t e d w i t h seawater. A n i o n i c , c a t i o n i c , n e u t r a l and c o l l o i d a l s p e c i e s were e s t i m a t e d from the r a t e of m e t a l e l u t i o n . The m e t a l s p e c i e s t h a t r a p i d l y e l u t e d were grouped as n e u t r a l and n e g a t i v e l y c h a r g e d and the s l o w l y e l u t e d s p e c i e s as p o s i t i v e l y c h a r g e d . In t h i s s e c t i o n , the p o t e n t i a l of a s t r o n g l y a c i d i c c a t i o n -exchange r e s i n t o measure the c o n c e n t r a t i o n of the f r e e c a t i o n i c s p e c i e s of Cu i n seawater was examined. An e q u i l i b r i u m approach u s i n g a column procedure was u t i l i z e d . The model o r g a n i c l i g a n d s used i n the b i o a s s a y s were a l s o used t o c o n t r o l the c h e m i s t r y of Cu i n the r e s i n e x p e r i m e n t s . To d e t e r m i n e i f the r e s i n measurement c o u l d be used as an e s t i m a t e of b i o l o g i c a l l y a c t i v e m e t a l , growth r a t e s from the b i o a s s a y s were compared t o the r e s u l t s of t h e r e s i n a n a l y s i s . 78 B. THEORETICAL CONSIDERATIONS 1 . I n t r o d u c t i o n t o Ion-Exchange R e s i n s The most w i d e l y used i o n - e x c h a n g e r s are s y n t h e t i c o r g a n i c r e s i n s . Adams and Holmes (1935) o r i g i n a l l y d e s c r i b e d the p r e p a r a t i o n of s y n t h e t i c ion-exchange r e s i n s . S y n t h e t i c r e s i n s o f f e r advantages over n a t u r a l i o n - e x c h a n g e r s i n t h a t (1) s y n t h e t i c r e s i n s have g r e a t e r p h y s i c a l and c h e m i c a l s t a b i l i t y , (2) they can be p r e p a r e d as spheres of u n i f o r m s i z e , (3) they have much g r e a t e r exchange c a p a c i t i e s , (4) the r a t e of exchange i s f a s t e r , and (5) the r e s i n can be made-to-order f o r v a r i o u s needs (Rieman and Walton, 1970). Modern c a t i o n - e x c h a n g e r s a r e c o n s i d e r e d t o be porous s a l t s c o n t a i n i n g an i n s o l u b l e a n i o n and m o b i l e c a t i o n s t h a t can be exchanged f o r i o n s of e q u a l charge from the s u r r o u n d i n g medium. The r e s u l t i n g ion-exchange r e a c t i o n i s r e v e r s i b l e , s t o i c h i o m e t r i c and f o l l o w s the law of mass a c t i o n . T h e i r exchange c h a r a c t e r i s t i c s a r e m a i n l y d e t e r m i n e d by the a c i d i c groups a t t a c h e d t o the o r g a n i c m a t r i x such as p h e n o l a t e , s u l p h o n a t e or c a r b o x y l a t e groups ( S c h u b e r t , 1948). Many f a c t o r s i n f l u e n c e the d i f f e r e n t i a l uptake of i o n s onto a c a t i o n - e x c h a n g e r e s i n . The a d s o r p t i o n of i o n s i s i n f l u e n c e d by t h e i r s i z e and c h a r g e , the i n t r i n s i c p r o p e r t i e s of the r e s i n ( s u c h as mesh s i z e , c a p a c i t y , degree of c r o s s - l i n k a g e ) , the r e l a t i v e c o n c e n t r a t i o n s of the i o n s c a p a b l e of exchange, and the r e a c t i o n time ( D o r f n e r , 1972). R u l e s i n d i c a t i n g how each of t h e s e f a c t o r s a f f e c t s s e l e c t i v i t y have been found e m p i r i c a l l y 79 a n d t h e s e c a n b e f o u n d i n H e l f f e r i c h ( 1 9 6 2 ) a n d D o r f n e r ( 1 9 7 2 ) . C o n v e n t i o n a l c a t i o n - e x c h a n g e r e s i n s h a v e b e e n u s e d t o d i s t i n g u i s h f r e e m e t a l i o n s f r o m t h o s e b o u n d t o o r g a n i c l i g a n d s f o r m a n y d e c a d e s . T h e m e a s u r e m e n t o f t h e c o n c e n t r a t i o n o f f r e e m e t a l i n e q u i l i b r i u m w i t h a c a t i o n - e x c h a n g e r e s i n h a s b e e n u s e d t o e x a m i n e s t a b i l i t y c o n s t a n t s f o r d i f f e r e n t m e t a l - o r g a n i c c o m p l e x e s i n s o i l s ( S c h n i t z e r a n d S k i n n e r , 1 9 6 6 ; M a t s u d a a n d I t o , 1 9 7 0 ; A r d a k a n i a n d S t e v e n s o n , 1 9 7 2 ) a n d i n f r e s h w a t e r ( A l l e n e t a l , 1 9 7 5 ) . 2 . T h e o r y I f a c a t i o n - e x c h a n g e r e s i n o f t h e s t r o n g a c i d t y p e t h a t i s l o a d e d w i t h s o d i u m i o n s , N a + ., i s b r o u g h t i n c o n t a c t w i t h a s o l u t i o n c o n t a i n i n g a d i v a l e n t m e t a l i o n , M 2 + , t h e e x c h a n g e r e a c t i o n c a n b e e x p r e s s e d b y t h e e q u a t i o n M 2 + + 2 N a + R " = M 2 + ( R - ) 2 + 2 N a + [ 5 ] w h e r e R - r e p r e s e n t s a n e g a t i v e s i t e i n t h e i o n - e x c h a n g e m a t r i x . T h u s a p p l y i n g t h e l a w o f m a s s a c t i o n o n e o b t a i n s t h e f o l l o w i n g e x p r e s s i o n { M R } { N a } 2  K i = [ 6 ] { M } { N a R } 2 w h e r e { } d e n o t e s a c t i v i t y , K , i s t h e t h e r m o d y n a m i c e q u i l i b r i u m 8 0 constant and ionic charges are omitted for the sake of s i m p l i c i t y . Rewritting [ 6 ] in terms of concentrations and appropriate a c t i v i t y c o e f f i c i e n t s we obtain f M R f N Q [ MR ] [ Na ] 2 m [ 7 ] f M f N a R f M 1 [ NaR ] 2 where the a c t i v i t y c o e f f i c i e n t s are denoted by f and [ ] denotes the metal.concentration in the solution and on the resin. Although the a c t i v i t y c o e f f i c i e n t for an ion in the solution outside the resin bead can be estimated (e.g., using the Davies approximation; see eqn. 1, Section I I I . B . C ) , that of the ions inside the beads cannot be determined e a s i l y . Because of t h i s , K, i s usually combined with the a c t i v i t y c o e f f i c i e n t s to obtain an apparent equilibrium constant [ MR ] [ Na ] 2 f f ' M NaR R. = . K i [ 8 ] [ M 1 i NaR ] 2 f M R f N Q which has a value e n t i r e l y dependent on a n a l y t i c a l l y measureable qua n t i t i e s . It must be kept in mind that, unlike a thermodynamic constant, K' varies with the nature and concentration of the e l e c t r o l y t e ; i . e . , K' w i l l remain constant only as long as the a c t i v i t y c o e f f i c i e n t r a t i o s of the species undergoing exchange remain constant. 81 When the metal i o n i s of a c o n s i d e r a b l y lower c o n c e n t r a t i o n than the c a t i o n of the background e l e c t r o l y t e ([Na] >> [ M ] ) , any ion-exchange r e a c t i o n i n v o l v i n g the metal i o n w i l l not have a s i g n i f i c a n t e f f e c t on the c o m p o s i t i o n of the s o l u t i o n or r e s i n phases. As a r e s u l t , the a c t i v i t y c o e f f i c i e n t r a t i o s of the i o n s i n the r e s i n and s o l u t i o n phases w i l l remain e s s e n t i a l l y c o n s t a n t . Moreover, s i n c e the c o n c e n t r a t i o n of Na + i n both the s o l u t i o n and r e s i n phases undergoes o n l y a n e g l i g i b l e change, e q u a t i o n [8] can be r e w r i t t e n i n the form [MR] [ N a R ] 2 X = = K, [9] [M] [ N a ] 2 where X i s termed the d i s t r i b u t i o n c o e f f i c i e n t f o r M. The v a l u e of the d i s t r i b u t i o n c o e f f i c i e n t , X, i s c l e a r l y dependent on the c h a r a c t e r i s t i c s of the m e t a l and s o l u t i o n f o r which i t i s measured. S c h u b e r t ' s Approach: As demonstrated by Schubert (1948), ion-exchange can be used t o e s t i m a t e s t a b i l i t y c o n s t a n t s of m e t a l complexes by measuring t h e i r a d s o r p t i o n s onto a c a t i o n -exchange r e s i n . That i s , by f i r s t measuring the d i s t r i b u t i o n c o e f f i c i e n t , X, i n the absence of a c o m p l e x i n g agent and then d e t e r m i n i n g the amount of m e t a l on the r e s i n i n the presence of a c o m p l e x i n g agent, i t i s p o s s i b l e t o e s t i m a t e the amount of m e t a l bound by the c o m p l e x i n g agent. T h i s can be done because, even w i t h the c o m p l e x i n g agent p r e s e n t (compare S e c t i o n 8 2 I I I . B . C . ) , the r e s i n i s s t i l l i n e q u i l i b r i u m with the f r e e metal ion as expressed by equation [ 9 ] ; thus the c o n c e n t r a t i o n of the fr e e metal ion in s o l u t i o n i s given by [ M R ] [ M ] = , [10] X and that c o n t a i n e d i n the complex i s j u s t the d i f f e r e n c e between the t o t a l and f r e e metal c o n c e n t r a t i o n s . 3. A p p l i c a t i o n of the Ion-Exchange Procedure to the Determination of C a t i o n i c Cu Species i n Seawater In complex media such as seawater, where Cu occurs not only as the f r e e ion but a l s o i n v a r i o u s c a t i o n i c complexes such as C u C l + and Cu(OH) +, the amount of Cu that binds to a c a t i o n -exchange r e s i n w i l l be determined by the c o n c e n t r a t i o n and d i s t r i b u t i o n c o e f f i c i e n t of a l l such s p e c i e s . However, as long as the v a r i o u s s o l u t i o n parameters ( i . e . , s a l i n i t y , pH, a l k a l i n i t y ) remain constant, then, due to e q u i l i b r i a that e x i s t between the v a r i o u s s p e c i e s , t h e i r c o n c e n t r a t i o n s are a l l p r o p o r t i o n a l to the a c t i v i t y of the f r e e Cu i o n s . The t o t a l amount of Cu adsorbed by the r e s i n i s t h e r e f o r e a f i x e d f u n c t i o n of the a c t i v i t y and c o n c e n t r a t i o n of f r e e Cu and, moreover, of the t o t a l Cu present i n d i s s o l v e d i n o r g a n i c s p e c i e s . T h e r e f o r e , equation [9] can be r e w r i t t e n using the t o t a l i n o r g a n i c Cu c o n c e n t r a t i o n i n s t e a d of the f r e e c u p r i c i o n : 8 3 [MR] X, = [11] [M i n o r g ] where X, i s a c o n d i t i o n a l d i s t r i b u t i o n c o e f f i c i e n t d e f i n e d i n terms of [M i n o r g ] , the t o t a l amount of Cu c o n t a i n e d i n i n o r g a n i c s p e c i e s . As a consequence of the above r e l a t i o n s h i p , i f seawater i s passed t h r o u g h a column of c a t i o n - e x c h a n g e r e s i n u n t i l the l a t t e r has a c h i e v e d e q u i l i b r i u m w i t h the f e e d , the amount of Cu a dsorbed w i l l be d i r e c t l y r e l a t e d t o the t o t a l i n o r g a n i c Cu c o n c e n t r a t i o n and , hence, the pCu of the sample. B e f o r e t h e s e v a l u e s can be d e t e r m i n e d , however, i t i s f i r s t n e c e s s a r y t o c a l i b r a t e the r e s i n . T h i s can be done by d e t e r m i n i n g how the amount of Cu adsorbed from seawater f r e e of o r g a n i c l i g a n d s and h a v i n g a g i v e n s a l i n i t y and pH v a r i e s as a f u n c t i o n of the t o t a l Cu c o n c e n t r a t i o n . The d i s t r i b u t i o n c o e f f i c i e n t , X,, which i s g i v e n by the s l o p e of t h i s r e l a t i o n s h i p , can then be used t o c a l c u l a t e the c o n c e n t r a t i o n of i n o r g a n i c Cu from v a l u e s of a dsorbed Cu o b s e r v e d f o r seawater samples c o n t a i n i n g v a r i o u s o r g a n i c l i g a n d s ( p r o v i d i n g t h a t the complexes formed w i t h the l a t t e r a r e n e u t r a l or n e g a t i v e l y c h a r g e d ) . : To f u r t h e r e s t i m a t e the pCu of seawater, a l l the c o m p l e x i n g l i g a n d s , t h e i r c o n c e n t r a t i o n s , and the t o t a l Cu c o n c e n t r a t i o n must be known. W i t h t h e s e , the c o n c e n t r a t i o n of the f r e e c u p r i c i o n can be c a l c u l a t e d by computer m o d e l l i n g t e c h n i q u e s ( e . g . , 84 W e s t a l l e t a l . 1976; MINEQL). An a c t i v i t y c o e f f i c i e n t , e s t i m a t e d by the D a v i e s a p p r o x i m a t i o n , can then be a p p l i e d t o t h i s f r e e m e t a l i o n c o n c e n t r a t i o n t o o b t a i n the pCu f o r t h a t p a r t i c u l a r t o t a l Cu c o n c e n t r a t i o n . Once a r a t i o between t o t a l i n o r g a n i c Cu and pCu i s e s t a b l i s h e d t h i s can be a p p l i e d t o o t h e r t o t a l i n o r g a n i c Cu c o n c e n t r a t i o n s t o d etermine t h e i r a p p r o p r i a t e pCu. ( T h i s r a t i o i s c o n s t a n t as l o n g as the p r e c i p i t a t i o n of Cu does not o c c u r , the c o n c e n t r a t i o n of the i n o r g a n i c l i g a n d s does not change, and the s a l i n i t y and pH remain c o n s t a n t . ) S i n c e the r a t i o of the c u p r i c i o n a c t i v i t y t o the t o t a l i n o r g a n i c c o n c e n t r a t i o n as c a l c u l a t e d by MINEQL at an i o n i c s t r e n g t h of 0.7, i s 1 0 " 1 « 8 3 , m u l t i p l i c a t i o n of [M i n o r g ] by t h i s f a c t o r p r o v i d e s an e s t i m a t e of the pCu of the s o l u t i o n . Thus one can w r i t e an e q u a t i o n of the form [MR] [MR] X 2 = = [12] [M i n o r g ] x 10" 1 • 8 3 { Cu } where X 2 r e p r e s e n t s a d i s t r i b u t i o n c o e f f i c i e n t d e f i n e d i n terms of pCu. Thus t o e s t i m a t e the pCu of an unknown sample, i t i s p a s s e d t h r o u g h the r e s i n column u n t i l e q u i l i b r i u m i s a c h i e v e d w i t h the f e e d s o l u t i o n , and then the c o n c e n t r a t i o n of Cu bound t o the r e s i n i s measured and the pCu of the sample e v a l u a t e d by d i v i d i n g the c o n c e n t r a t i o n of adsorbed Cu by X 2. I t s h o u l d be noted t h a t f o r the purpose of e s t i m a t i n g t r a c e m e t a l a c t i v i t i e s i n seawater, the above method w i l l o n l y work i f 85 the r e s i n has s i m i l a r s e l e c t i v i t i e s f o r a l k a l i , a l k a l i n e e a r t h and t r a n s i t i o n m e t a l s . I f a r e s i n t h a t i s h i g h l y s e l e c t i v e f o r t r a n s i t i o n m e t a l s , such as C h e l e x - 1 0 0 , i s used, the amount of Cu adsorbed w i l l be a f f e c t e d by c o m p e t i t i o n f o r s i t e s between the d i f f e r e n t t r a n s i t i o n m e t a l s i n the sample. C o n s e q u e n t l y , the amount of Cu adsorbed would be a f u n c t i o n of the t r a c e m e t a l c o m p o s i t i o n which, i n c o n t r a s t t o the major seawater c a t i o n s , i s h i g h l y v a r i a b l e and not r e l a t e d t o the s a l i n i t y . 8 6 C. MATERIALS AND METHODS 1. Column P r e p a r a t i o n (a) M a t e r i a l s A r t i f i c i a l seawater (SOW) was used as the e x p e r i m e n t a l medium i n a l l t e s t s . I t s p r e p a r a t i o n i s d e s c r i b e d i n S e c t i o n I I I . B . 1 . The SOW was passed t h r o u g h a Chelex-100 column b e f o r e use t o remove t r a c e m e t a l c o n t a m i n a n t s . For each b a t c h of SOW, the pH was measured and, i f n e c e s s a r y , a d j u s t e d t o pH 8.0±.05. Copper s t o c k s were p r e p a r e d d a i l y from a 1000 mg 1" 1 s t a n d a r d of C u C l 2 made up i n n i t r i c a c i d . To o b t a i n u l t r a p u r e a c i d , 12N HC1 was i s o t h e r m a l l y d i s t i l l e d t o g i v e a f i n a l c o n c e n t r a t i o n of p u r i f i e d 5N HCL. A c i d i f i e d SOW was used i n the e l u t i o n of the r e s i n columns. T h i s s o l u t i o n was p r e p a r e d by a d d i n g 2 ml of 5N HCL t o each l i t e r of SOW. Resin-column e x p e r i m e n t s were performed w i t h 1.0 cm ( i . d . ) x 5.0 cm Econocolumns® (Bio-Rad l a b o r a t o r i e s , Richmond, CA. U.S.A.) which c o n s i s t e d of a g l a s s tube, a p o l y e t h y l e n e bed s u p p o r t , and a p o l y p r o p y l e n e r e s e r v o i r and column t i p ; both ends of the column were equipped w i t h easy c o n n e c t o r l u e r f i t t i n g s ( F i g . 18). P a r t i c l e s up t o 35 <xm were r e t a i n e d by the bed s u p p o r t . P r e c i s e f l o w r a t e s i n the columns were m a i n t a i n e d by a Technicon® a u t o a n a l y z e r pump used i n c o n j u n c t i o n w i t h L a n c e r m a n i f o l d pump t u b i n g ( L a n c e r , S t . L o u i s , Mo. U.S.A.). The f l o w r a t e s were d e t e r m i n e d by the d i a m e t e r of the tube used. 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) was used t o connect the 87 pump tubes t o both the columns and sample b o t t l e s . Eppendorf p i p e t t e t i p s ( c l e a r , 10-100 ixl) were used as c o n n e c t o r s between the m i c r o t u b i n g , columns and pump t u b i n g . S t r o n g l y a c i d i c Dowex-50 c a t i o n - e x c h a n g e r e s i n was used (Bio-Rad L a b o r a t o r i e s AG 50W-X12). The s u l p h o n i c a c i d r e s i n was o b t a i n e d i n the hydrogen form as 200-400 mesh beads: B a t c h No. 10572. The r e s i n , which i s composed of s u l p h o n i c a c i d groups a t t a c h e d t o a s t y r e n e d i v i n y l b e n z e n e polymer l a t t i c e (R-S 0 3 " ) , had an exchange c a p a c i t y of 5.0 meq g" 1 (dry w t . ) , 12% c r o s s l i n k a g e and an apparent pK < 1 ( H e l f f e r i c h , 1962, p.86). V a r i o u s s i z e s of Nalgene p o l y p r o p y l e n e b o t t l e s were used th r o u g h o u t the st u d y . To reduce t r a c e m e t a l c o n t a m i n a t i o n , the b o t t l e s were soaked i n 6N HCL f o r 3 days, r i n s e d 3X w i t h GDW, and then soaked i n 0.5N HCL f o r one week. F i n a l l y , they were r i n s e d , f i l l e d w i t h GDW, and s t o r e d u n t i l use. (b) P r e p a r a t i o n of and column proc e d u r e A l l column m a n i p u l a t i o n was c a r r i e d out i n the Laminar f l o w hood d e s c r i b e d e a r l i e r . One gram of r e s i n ( d i r e c t from the b o t t l e ) was weighed o u t , s l u r r i e d i n 4-5 ml of GDW and poured i n t o a column. The r e s i n was washed w i t h 50 ml of GDW and then a l l o w e d t o e q u i l i b r a t e w i t h the GDW f o r 1-2 days p r i o r t o use. A s o l u b l e r e d compound was e v i d e n t i n the i n i t i a l wash water and was assumed t o be an o r g a n i c by-product of the m a n u f a c t u r i n g p r o c e s s . I n i t i a l l y , methanol was used t o remove any r e s i d u a l o r g a n i c s p r e s e n t i n the r e s i n ; however, t h e r e d i d not seem t o be any b e n e f i c i a l e f f e c t of the methanol r i n s e o v er t h a t of s i m p l y Figure 18. Econocolumn. 89 washing w i t h GDW so t h i s s t e p was e l i m i n a t e d . One t o ten columns were p r e p a r e d a t any one t i m e . To remove any t r a c e m e t a l c o n t a m i n a n t s p r e s e n t , the r e s i n was washed w i t h 50 ml of a c i d i f i e d SOW (pH c a . 1.0). I t was then c o n v e r t e d t o the a p p r o p r i a t e i o n i c c o m p o s i t i o n and pH by r e a c t i n g i t w i t h 50 ml of SOW a t pH 8.01.05. D u r i n g the c o n v e r s i o n s t e p , the pH of the e f f l u e n t began t o r i s e s h a r p l y a f t e r o n l y 10-15 ml of SOW and was the same as the i n f l u e n t a f t e r 35-40 ml. A f l u i d head of 1 cm was kept above the r e s i n a t a l l t i m e s . Due t o the h i g h degree of r e s i n c r o s s - l i n k a g e , t h e r e was o n l y a s m a l l amount of s h r i n k i n g or s w e l l i n g w i t h a change i n i o n i c form. A l s o , a s l i g h t d a r k e n i n g of the r e s i n was apparent as the r e s i n l o s t H+ and began t o e q u i l i b r a t e w i t h the SOW. At t h i s p o i n t the a p p r o p r i a t e s o l u t i o n was ready t o be passed t h r o u g h the column. The t e s t s o l u t i o n was pumped v i a the a u t o a n a l y z e r pump a p p a r a t u s t o the econocolumn a t a c o n s t a n t f l o w rate.. The s o l u t i o n was d e l i v e r e d t o j u s t above the r e s i n bed by a p o l y p r o p y l e n e m i c r o t u b e (see F i g . 18). A f t e r the samples had p a s s e d t h r o u g h the r e s i n , any sample p r e s e n t i n the i n t e r s t i a l spaces of the r e s i n or i n the l u e r f i t t i n g of the column was removed b e f o r e the e l u t i o n s t e p was begun. T h i s was a c c o m p l i s h e d by a l l o w i n g a s l i g h t p o s i t i v e p r e s s u r e t o b u i l d up i n the columns by c o n t i n u i n g the pumping a c t i o n of the f l o w system a f t e r the d e l i v e r y tubes had run d r y . ( A p p r o x i m a t e l y 0.3 ml of r e s i d u a l s o l u t i o n was e x p e l l e d . ) Because the t r a n s f e r tubes f o r f e e d i n g the sample s o l u t i o n 90 were a l s o used f o r the e l u e n t , the tubes were c l e a n e d b e f o r e the e l u e n t was passed t h r o u g h . T h i s i n v o l v e d d i s c o n n e c t i n g the tubes from the columns, f l u s h i n g them w i t h GDW, and w i t h a c i d i f i e d SOW. F i n a l l y , the tubes were r e c o n n e c t e d t o the columns and the e l u t i o n p rocedure was begun. (c) P r o c e d u r e f o r e l u t i o n of column p r i o r t o ASV To de t e r m i n e the amount of Cu bound t o the r e s i n a t the end of an a n a l y s i s , the column was e l u t e d w i t h a c i d i f i e d SOW (pH ca . 1.0) and the Cu c o n c e n t r a t i o n i n the e l u a t e d etermined by ASV. The f i r s t 25 ml of the e l u a t e was c o l l e c t e d i n 50 ml p o l y c a r b o n a t e c e n t r i f u g e tubes c o n t a i n i n g p o l y p r o p y l e n e c a p s . P r e l i m i n a r y s t u d i e s i n d i c a t e d t h a t most of the Cu was removed w i t h t h e f i r s t 15 ml of the e l u a t e . However, .25 ml was c o l l e c t e d t o ensure maximum Cu r e c o v e r y a t a l l Cu c o n c e n t r a t i o n s . The same a p p a r a t u s used t o pump the sample t h r o u g h the column was a l s o used t o pump the e l u e n t . When the e l u t i o n p r o c e d u r e was completed the r e s i n was i m m e d i a t e l y r e g e n e r a t e d by p a s s i n g a f u r t h e r 15 ml of a c i d i f i e d SOW t h r o u g h the r e s i n and then r e a c t i n g i t w i t h 50 ml of SOW a t pH 8.0±.05. The r e s i n was s t i r r e d w i t h a g l a s s r o d t o remove any b u b b l e s t r a p p e d i n the r e s i n d u r i n g the e l u t i o n p r o c e d u r e . The columns were s t o r e d i n t h i s i o n i c form u n t i l f u t u r e use. 91 2. ASV Procedure f o r Measuring T o t a l Cu (a) Equipment The c u r r e n t - v o l t a g e 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 esearch C o r p o r a t i o n (PAR) model 374 p o l a r o g r a p h i c a n a l y s e r . The e l e c t r o l y s i s c e l l c o n s i s t e d of a PAR K66 c e l l t o p and a PAR K60 b o r o s i l i c a t e g l a s s c e l l bottom. The g l a s s c e l l s were c o a t e d w i t h a 0.5% c h l o r o s i l a n e s o l u t i o n t o reduce Cu a d s o r p t i o n onto the w a l l of the v e s s e l . Sample s o l u t i o n s were s t i r r e d w i t h an 8 x 13 mm t e f l o n - c o v e r e d s t i r r i n g bar c o u p l e d t o a c o n s t a n t speed magnetic s t i r r e r . A PAR K77 s a t u r a t e d c a l o m e l r e f e r e n c e e l e c t r o d e (SCE) was p l a c e d i n a PAR K65 r e f e r e n c e e l e c t r o d e b r i d g e tube w i t h a v y c o r t i p t o form a d o u b l e - j u n c t i o n r e f e r e n c e e l e c t r o d e . The o u t e r j u n c t i o n was f i l l e d w i t h a r t i f i c i a l sea water (SOW). A bare p l a t i n u m w i r e s e r v e d as the a u x i l i a r y e l e c t r o d e . G l a s s y carbon e l e c t r o d e s were p r e p a r e d by s e a l i n g w a f e r s of 0.63 cm d i a m e t e r g l a s s y carbon ( B e c k w i t h Carbon Corp.) i n t o a 112 cm l e n g t h of Pyrex g l a s s t u b i n g w i t h epoxy r e s i n . The g l a s s y carbon was then p o l i s h e d m e t a l l o g r a p h i c a l l y w i t h a 0.05 Mm a l u m i n a s l u r r y b e i n g used i n the f i n a l p o l i s h i n g s t e p t o a t t a i n a m i r r o r - l i k e f i n i s h . A s m a l l q u a n t i t y of mercury was p l a c e d i n s i d e the g l a s s tube t o make an e l e c t r i c a l c o n t a c t between a p l a t i n u m w i r e and the g l a s s y carbon d i s c . Wax was poured i n t o the tube t o p r e v e n t the l o s s of mercury. F i n a l l y , the e l e c t r o d e was soaked i n d i l u t e HCL f o r 24 hr t o remove c o n t a m i n a n t s from the p o l i s h i n g p r o c e s s . P o l i s h i n g of the 92 e l e c t r o d e was n e c e s s a r y i n t e r m i t t e n t l y and t h i s s i m p l y i n v o l v e d a l i g h t s c r u b b i n g of the e l e c t r o d e w i t h a 0.05 Mm alumina s l u r r y on a p i e c e of f e l t . (b) P r e - p l a t i n g and p r e - c o n d i t i o n i n g of the e l e c t r o d e A s e p a r a t e e l e c t r o l y s i s c e l l and ASV u n i t was used i n the p r e - p l a t i n g of the e l e c t r o d e . A PAR model 174A p o l a r o g r a p h i c a n a l y s e r was used i n c o n j u n c t i o n w i t h a 200 ml t e f l o n e l e c t r o l y s i s c e l l machined from a t e f l o n r o d . S t i r r i n g was by a t e f l o n s t i r r i n g bar used i n c o n j u n c t i o n w i t h a magnetic s t i r r e r . B e f o r e b e g i n n i n g any a n a l y s e s , the e l e c t r o d e was p r e - p l a t e d t o produce a mercury f i l m . T h i s s t e p i n v o l v e d p l a c i n g the e l e c t r o d e i n a s o l u t i o n of 500 mg l - 1 H g C l 2 , made s l i g h t l y a c i d i c w i t h n i t r i c a c i d . A f t e r p u r g i n g w i t h N 2 f o r 10 min, a p o t e n t i a l of -0.2 V vs SCE was a p p l i e d t o the e l e c t r o d e f o r 50 seconds. The c u r r e n t t o the e l e c t r o d e was kept between 0.2 and 0.3 ma by adjustment of the s t i r r i n g r a t e . The Hg f i l m t h i c k n e s s was c o n t r o l l e d by the s t i r r i n g r a t e and the d e p o s i t i o n t i m e . Once the p l a t i n g s t e p had been c o m p l e t e d , the e l e c t r o d e was t r a n s f e r e d t o the e l e c t r o l y s i s c e l l of the model 374 p o l a r o g r a p h i c a n a l y s e r where i t was then p l a c e d i n a s o l u t i o n of a c i d i f i e d SOW and s u b j e c t e d t o 3-4 p l a t i n g - s t r i p p i n g sequences under the same o p e r a t i n g c o n d i t i o n s used i n the subsequent a n a l y s i s . The e l e c t r o d e was p r e v e n t e d from d r y i n g d u r i n g i t s t r a n s f e r by keeping a l a r g e drop of s o l u t i o n a t the end of the e l e c t r o d e . 93 The t h i c k n e s s of the Hg f i l m i s an i m p o r t a n t parameter. The s o l u b i l i t y of Cu i n Hg i s r e p o r t e d t o be o n l y 0.002% at 20°C (Stephen and Stephen, 1963). T h e r e f o r e , i f the f i l m i s too t h i n the Hg can become s u p e r s a t u r a t e d w i t h Cu a t low s o l u t i o n c o n c e n t r a t i o n s , a phenomenon which w i l l r e s u l t i n a change of s l o p e i n the response c u r v e . T h i c k f i l m s were a l s o not d e s i r a b l e because t h e r e i s a b r o a d e n i n g of the Cu peak, presumably due t o a l o n g e r time r e q u i r e d f o r d i f f u s i o n of the Cu out of the Hg f i l m . (c) G e n e r a l p r o c e d u r e Once the e l e c t r o d e was p r e - p l a t e d and p r e - c o n d i t i o n e d a sample was p l a c e d i n the e l e c t r o l y s i s c e l l . I n i t i a l l y , the sample was purged w i t h N 2 f o r 5 min t o remove a l l the 0 2 from the sample (the N 2 was deoxygenated by p a s s i n g i t t h r o u g h a vanadous c h l o r i d e s o l u t i o n as d e s c r i b e d by PAR a p p l i c a t i o n note No. 108). A f t e r p u r g i n g , a p o t e n t i a l of -0.7 V vs SCE was a p p l i e d t o the e l e c t r o d e f o r a p r e s e l e c t e d time t o c o n c e n t r a t e the m e t a l i n t o the mercury f i l m . D u r i n g t h i s time the s o l u t i o n was s t i r r e d a t a c o n s t a n t r a t e and a N 2 atmosphere was m a i n t a i n e d i n the c e l l . A f t e r p l a t i n g , s t i r r i n g was ceased and the s o l u t i o n was a l l o w e d t o come t o r e s t f o r 15 sec w h i l e m a i n t a i n i n g the a p p l i e d p o t e n t i a l . F i n a l l y , an a n o d i c scan from -0.7 V t o -0.15 V was made u s i n g the d i f f e r e n t i a l p u l s e mode ( p u l s e h e i g h t , 50 mv) a t 5 mv s " 1 t o s t r i p the m e t a l out of the mercury f i l m . The d e p o s i t i o n time was dependent upon the 9 4 c o n c e n t r a t i o n of Cu i n the sample. In p r e l i m i n a r y s t u d i e s , i t was found t h a t the h e i g h t of the c u r r e n t peak f o r Cu i n the f i r s t p l a t i n g - s t r i p p i n g sequence was always lower than the peak h e i g h t of the second and f u r t h e r sequences. T h e r e f o r e , two p l a t i n g - s t r i p p i n g sequences were performed on the sample w i t h the f i r s t sequence used t o c o n d i t i o n the e l e c t r o d e and the second sequence used t o q u a n t i f y the m e t a l . (d) C a l i b r a t i o n There a r e a t l e a s t two methods of r e l a t i n g the v o l t a m m e t r i c response t o the metal c o n c e n t r a t i o n i n the sample. These a r e t h r o u g h the use of a w o r k i n g c u r v e o b t a i n e d from a n a l y s i s of s t a n d a r d s o l u t i o n s and t h r o u g h the method of s t a n d a r d a d d i t i o n s . C a l i b r a t i o n u s i n g a w o r k i n g c u r v e , whereby th e v o l t a m m e t r i c r e s p o n s e s from a s e t of s t a n d a r d s are compared t o the v o l t a m m e t r i c response from the sample i n q u e s t i o n , i s the s i m p l e s t method. However, c a r e must be taken t o s i m u l a t e the same c o n d i t i o n s i n the w o r k i n g s t a n d a r d s as t h o s e found i n the s o l u t i o n of i n t e r e s t as peak h e i g h t can be dependent on f a c t o r s i n the s o l u t i o n s o t h e r than the c o n c e n t r a t i o n of the m e t a l b e i n g measured. Such f a c t o r s i n c l u d e pH, the p r e s e n c e of s u r f a c e a c t i v e a g e n t s , the i o n i c s t r e n g t h , and the n a t u r e of the s u p p o r t i n g e l e c t r o l y t e . To compensate f o r the e f f e c t of the sample m a t r i x t h e method of s t a n d a r d a d d i t i o n s i s o f t e n used. I t i n v o l v e s f i r s t an i n i t i a l d e t e r m i n a t i o n of the h e i g h t of the c u r r e n t peak of 95 the m e t a l i n s o l u t i o n and t h e n , a f t e r a d d i n g a s p i k e of the same m e t a l , d e t e r m i n i n g the i n c r e a s e i n h e i g h t of the c u r r e n t peak.. I f the added metal p r o p o r t i o n s i t s e l f amongst a l l the forms of the metal i n the sample the s t a n d a r d a d d i t i o n method measures the t o t a l m e t a l c o n t e n t of the sample. I f , however, the s t a n d a r d o n l y p r o p o r t i o n s i t s e l f amongst the ASV l a b i l e s p e c i e s , then the a n a l y s i s g i v e s o n l y the l a b i l e m e t a l c o n c e n t r a t i o n . I n t e r p r e t a t i o n of the ASV r e s u l t s from the s t a n d a r d a d d i t i o n method i s thus d i f f i c u l t when the d i s t r i b u t i o n of the added m e t a l i s u n c e r t a i n . For ASV t o unambiguously determine the ' t o t a l ' m e t a l i n a sample, a l l the metal must be c o n v e r t e d t o ASV l a b i l e forms. In the case of t o t a l d i s s o l v e d m e t a l i n seawater, the sample i s u s u a l l y f i r s t f i l t e r e d t h r o u g h a c o n v e n t i o n a l 0.45 nm f i l t e r and then a c i d i f i e d t o c a . pH 2 w i t h HCL or HN0 3. Under the a c i d c o n d i t i o n s , the metal i s b e l i e v e d t o be q u a n t i t a t i v e l y c o n v e r t e d t o l a b i l e forms. Other more severe t r e a t m e n t s such as u l t r a v i o l e t i r r a d i a t i o n or n i t r i c a c i d d i g e s t i o n a r e sometimes used t o attempt complete c o n v e r s i o n ( F l o r e n c e and B a t l e y , 1977a). Throughout the p r e s e n t s t u d y , o n l y measurements of t o t a l Cu were made. Thus a l l samples were a c i d i f i e d w i t h i s o t h e r m a l l y d i s t i l l e d HCL t o a t t a i n a pH of c a . 2.0. Both the wo r k i n g c u r v e and s t a n d a r d a d d i t i o n methods were used t o c a l i b r a t e the m e t a l l e v e l i n a sample. In most c a s e s the w o r k i n g c u r v e was used when measuring Cu l e v e l s 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 o l u t i o n such as SOW w h i l e the s t a n d a r d a d d i t i o n method was used i n the 9 6 a n a l y s i s of n a t u r a l seawater samples so as t o t a k e i n t o account any m a t r i x i n t e r f e r e n c e s . (e) P r o c e d u r e f o r measuring t o t a l Cu i n the e l u a t e s of the r e s i n a n a l y s i s In the r e s i n t e c h n i q u e , Cu was e l u t e d from the r e s i n u s i n g 25 ml of a c i d i f i e d SOW and the t o t a l Cu c o n c e n t r a t i o n i n the e l u a t e s was det e r m i n e d by ASV u s i n g the wo r k i n g c u r v e method. The g e n e r a l p r o c e d u r e i n v o l v e d (1) p r e p a r i n g and p r e -c o n d i t i o n i n g an e l e c t r o d e , (2) g e n e r a t i n g two v o l t a m m e t r i c t r a c e s f o r each sample, and (3) g e n e r a t i n g a s t a n d a r d c u r v e . The same o p e r a t i n g c o n d i t i o n s and p r o c e d u r e s as o u t l i n e d i n S e c t i o n IV.C.2.C were used when a n a l y z i n g t h e s e samples. G e n e r a l l y , an a l i q u o t of 10 ml was n e c e s s a r y f o r each ASV measurement. S t a n d a r d c u r v e s were g e n e r a t e d from the a c i d i f i e d SOW t h a t was used t o e l u t e the columns. An a l i q u o t (10 ml) of t h i s s o l u t i o n was added t o the e l e c t r o l y s i s c e l l and s u b s e q u e n t l y s p i k e d w i t h i n c r e a s i n g Cu c o n c e n t r a t i o n s . The v o l t a m m e t r i c r esponse f o r each Cu s p i k e was then measured. The h e i g h t of the v o l t a m m e t r i c c u r r e n t peak f o r Cu of the samples was then compared t o t h a t of the s t a n d a r d s t o e s t i m a t e the Cu c o n c e n t r a t i o n i n the samples. The c o n c e n t r a t i o n range of the s t a n d a r d c u r v e was d e t e r m i n e d by the c o n c e n t r a t i o n range of the samples under s t u d y . The s e n s i t i v i t y of the e l e c t r o d e would i n f r e q u e n t l y change over the c o u r s e of the a n a l y s i s . When t h i s o c c u r r e d , s t a n d a r d 97 c u r v e s were run d u r i n g the sample a n a l y s i s which reduced the e f f e c t of any s e n s i t i v i t y changes. An experiment t o determine the p r e c i s i o n of the ASV pro c e d u r e was conducted. A 500 ml s o l u t i o n of a c i d i f i e d SOW (pH c a . 2.0) was s p i k e d w i t h Cu t o g i v e a t o t a l Cu c o n c e n t r a t i o n of 2.36 x 10" 7M. The sample was a l l o w e d t o e q u i l i b r a t e f o r 2 hr b e f o r e a l i q u o t s (25 ml) were t r a n s f e r r e d t o 10 a c i d c l e a n e d 50 ml p o l y c a r b o n a t e t u b e s . These samples then underwent t h e same a n a l y s i s p rocedure used t o e s t i m a t e the Cu l e v e l s i n the e l u a t e s of the r e s i n a n a l y s i s . 3. C h a r a c t e r i z a t i o n of the R e s i n AG 50W-X12 (a) Column e q u i l i b r a t i o n To d e t e r m i n e the volume of sample needed t o a t t a i n column e q u i l i b r a t i o n , SOW c o n t a i n i n g a known c o n c e n t r a t i o n of Cu was passed t h r o u g h the r e s i n and the Cu i n the i n f l u e n t and e f f l u e n t was measured. One 1 of SOW was s p i k e d w i t h Cu t o g i v e a c o n c e n t r a t i o n of 7.89 x 10" 8M and l e f t f o r 2 hr t o a l l o w e q u i l i b r a t i o n . Two columns were p r e p a r e d and 300 ml of the sample was passed t h r o u g h each column a t a f l o w r a t e of 1.7±0.1 ml m i n " 1 . The e f f l u e n t was c o l l e c t e d i n 50 ml a l i q u o t s and bo t h the i n f l u e n t and e f f l u e n t was measured f o r t o t a l Cu by ASV. The e f f e c t of f l o w r a t e on the e q u i l i b r a t i o n of the r e s i n was s t u d i e d . Copper was added t o t h r e e 500 ml p o r t i o n s of SOW to g i v e a c o n c e n t r a t i o n of 7.89 x 10" 8M and l e f t f o r 12 hr p r i o r 98 t o use. S i x columns were p r e p a r e d and 250 ml of SOW were passed t h r o u g h the a p p r o p r i a t e columns a t 0 .8 ml m i n - 1 , 2.0 ml m i n - 1 or 2 .9 ml m i n " 1 . • Each f l o w r a t e was run i n d u p l i c a t e . "When the s o l u t i o n s had passed through the r e s i n the columns were e l u t e d and the e l u a t e s measured f o r Cu by ASV. (b) P r e c i s i o n P r e c i s i o n was de t e r m i n e d by r e p l i c a t e a n a l y s i s of a sample of SOW c o n t a i n i n g 7 .89 x 10" 8M Cu. Two a l i q u o t s of r e s i n were t e s t e d under d i f f e r e n t o p e r a t i n g and c h e m i c a l c o n d i t i o n s . For the f i r s t a l i q u o t , e i g h t columns were p r e p a r e d and 250 ml of SOW c o n t a i n i n g 7 .89 x 10" 8M Cu was passe d t h r o u g h each column a t a fl o w r a t e of 1.7 ml m i n " 1 . The e i g h t columns were then e l u t e d and the e l u a t e s a n a l y s e d f o r Cu by ASV. In the second a l i q u o t , t e n columns were p r e p a r e d and 250 ml of SOW c o n t a i n i n g 7 .89 x 10" 8M Cu was passed t h r o u g h each column a t a f l o w r a t e of 2 . 9 ml m i n - 1 . The columns were e l u t e d and the e l u a t e s a n a l y s e d f o r Cu by ASV. In t h i s e x p e r i m e n t , Fe was added t o a l l t e s t s o l u t i o n s a t a c o n c e n t r a t i o n of 4.5 x 10" 7M t o determine the e f f e c t of Fe on the p r e c i s i o n . (c) E f f e c t of pH on the a d s o r p t i o n of Cu by the r e s i n Ten columns were p r e p a r e d f o r t h i s e x p e r i m e n t . F i v e 500 ml a l i q u o t s of SOW c o n t a i n i n g 7 .89 x 10" 8M Cu were a d j u s t e d t o d i f f e r e n t pH v a l u e s (7.53, 7 . 9 6 , 8.13, 8.15, 8.42) by the a d d i t i o n of 0.1N HCL or 0.1N NaOH. These samples were then 99 passed t h r o u g h the a p p r o p r i a t e column a t a f l o w r a t e of 1.7 ml m i n " 1 . A l l the columns were e l u t e d and the e l u a t e s a n a l y s e d f o r t o t a l Cu by ASV. Samples f o r each pH were run i n d u p l i c a t e . (d) E f f e c t of s a l i n i t y on the a d s o r p t i o n of Cu by the r e s i n Four 500 ml a l i q u o t s of SOW of 5, 15, 25 and 35 ppt s a l i n i t y were p r e p a r e d by d i l u t i o n of SOW a t 35 ppt w i t h GDW. Copper was added t o a l l samples t o g i v e a f i n a l c o n c e n t r a t i o n of 7.89 x 10" 8M. The pH of the samples was measured and, i f n e c e s s a r y , a d j u s t e d t o c a . pH 8.0 w i t h the a d d i t i o n of 0.1N NaOH. Ten columns were p r e p a r e d and 250 ml of the v a r i o u s s a l i n i t y samples were passed t h r o u g h e i g h t columns a t a f l o w r a t e of 2.9 ml m i n " 1 . Two a d d i t i o n a l columns had 250 ml of SOW (35 p p t ) , w i t h o u t added Cu, passed through them. A l l t e s t s were run i n d u p l i c a t e . A l l columns were e l u t e d and the e l u a t e s a n a l y s e d f o r Cu by ASV. (e) E f f e c t s of n u t r i e n t s on a d s o r p t i o n of Cu by the r e s i n In the f i r s t e x p e r i m e n t , two 500 ml samples of SOW were p r e p a r e d . In the f i r s t sample, Cu was added t o g i v e a c o n c e n t r a t i o n of 7.89 x 10" 8M w h i l e i n the second, Cu was added i n the same c o n c e n t r a t i o n b u t , i n a d d i t i o n , a l l the 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 s were added a t t h e i r A q u i l c o n c e n t r a t i o n s . Then 250 ml of b o t h samples were passed t h r o u g h s e p a r a t e columns a t a f l o w r a t e of 1.7 ml m i n " 1 . D u p l i c a t e s of 100 each sample were r u n . The columns were e l u t e d and the e l u a t e s a n a l y s e d f o r Cu by ASV. In the second e x p e r i m e n t , s i x 500 ml samples were p r e p a r e d as f o l l o w s : 1) SOW c o n t a i n i n g 7.89 x 10" 8M Cu; 2) SOW c o n t a i n i n g 7.89 x 10" 8M Cu p l u s the 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 s except f o r Fe; 3) SOW c o n t a i n i n g 7.89 x 10" 8M Cu w i t h Fe a t the A q u i l c o n c e n t r a t i o n (4.5 x 10" 7M); 4) same as 3 but 2x the Fe c o n c e n t r a t i o n ; 5) same as 3 but 3x the Fe c o n c e n t r a t i o n ; 6) same as 3 but 4x the Fe c o n c e n t r a t i o n . The samples were l e f t f o r 12 hr t o e q u i l i b r a t e a f t e r which 250 ml of each sample was passed t h r o u g h the a p p r o p r i a t e columns a t a f l o w r a t e of 2.9 ml m i n " 1 . The columns were then e l u t e d and the e l u a t e s a n a l y s e d f o r Cu by ASV. In a d d i t i o n , the t o t a l Cu c o n c e n t r a t i o n i n a l l samples was d e t e r m i n e d by ASV. In the t h i r d and f i n a l e x p e r i m e n t , Fe s t o c k s were p r e p a r e d i n d i f f e r e n t manners. Three Fe s t o c k s were p r e p a r e d a t t h e i r A q u i l c o n c e n t r a t i o n as f o l l o w s : 1) F e C l 3 was added t o GDW and l e f t f o r 5 min b e f o r e i t s a d d i t i o n ; 2) as i n 1 but the Fe s t o c k was a l l o w e d t o p r e c i p i t a t e and age f o r 6 h r ; 3) as i n 2 but the Fe s t o c k was aged f o r 72 h r . The Cu and Fe s t o c k s were then added t o SOW t o g i v e s o l u t i o n s h a v i n g 1) o n l y 7.89 x 10" 8M Cu; 2) o n l y a 5 min aged Fe s t o c k ; 3) 7.89x 10" 8M Cu p l u s a 5 min aged Fe s t o c k ; 4) 7.89 x 10" 8M Cu p l u s a 6 hr aged Fe s t o c k ; and 5) 7.87 x 10" 8M Cu and a 72 hr aged Fe s t o c k . The s o l u t i o n s were a l l o w e d t o e q u i l i b r a t e f o r 12 hr and then 250 ml of each sample was passed t h r o u g h the a p p r o p r i a t e column a t a f l o w r a t e of 2.9 ml m i n - 1 . The columns were e l u t e d and the e l u a t e s 101 a n a l y s e d f o r Cu by ASV. ( f ) A d s o r p t i o n c u r v e s f o r Cu i n SOW w i t h no o r g a n i c l i g a n d s p r e s e n t A d s o r p t i o n c u r v e s f o r Cu were g e n e r a t e d under d i f f e r e n t f l o w r a t e s , w i t h and w i t h o u t the a d d i t i o n of Fe. In the f i r s t e x p e r i m e n t , e i g h t columns were p r e p a r e d and 250 ml p o r t i o n s of SOW c o n t a i n i n g 3.93, 7.87, 11.9 or 15.7 x 10" 8M Cu were passed t h r o u g h the a p p r o p r i a t e column at a f l o w r a t e of 1.7 ml m i n - 1 . Each Cu c o n c e n t r a t i o n was run i n d u p l i c a t e . The columns were e l u t e d and the e l u a t e a n a l y s e d f o r Cu by ASV. In the second t e s t , n i n e columns were p r e p a r e d and 250 ml p o r t i o n s of SOW c o n t a i n i n g Cu from 0 t o 31.5 x 10" 8M Cu a t 3.93 x 10' 8M i n c r e m e n t s were passed t h r o u g h the a p p r o p r i a t e column at 2.9 ml m i n " 1 . I r o n was added t o a l l of t h e s e samples a t a c o n c e n t r a t i o n of 4.5 x 10" 7M. Once a g a i n the columns were e l u t e d and t h e e l u a t e s a n a l y s e d f o r Cu by ASV. 4. Model L i g a n d Study The same l i g a n d s used i n the b i o a s s a y s (EDTA, GLU, HIS, and NTA) were used i n these r e s i n e x p e r i m e n t s . L i g a n d s t o c k s were p r e p a r e d from reagent grade c h e m i c a l s and were made up a t c a . 1000X the f i n a l l i g a n d c o n c e n t r a t i o n . The l i g a n d and Cu c o n c e n t r a t i o n s used a r e shown i n T a b l e V I . Two s e r i e s of e x p e r i m e n t s were performed u s i n g t h e same l i g a n d s and Cu c o n c e n t r a t i o n s except t h a t some o p e r a t i o n a l parameters were 1 0 2 changed. A l l Fe s t o c k s were p r e p a r e d f r e s h and 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 hours b e f o r e use. T a b l e V I . The l i g a n d s , l i g a n d c o n c e n t r a t i o n s and Cu c o n c e n t r a t i o n s s t u d i e d (+). L i g a n d A d d i t i o n Cu A d d i t i o n ( X 1 0 " 8 M ) L i g a n d ( X 1 0 " 7 M ) 3 . 9 3 7 . 8 9 1 1 . 8 1 5 . 7 EDTA 0 . 5 + + . + + 1 . 0 + + + + 2 . 5 + + 5 . 0 . + + GLU 1 0 0 . + + 2 5 0 . + + 5 0 0 . + + 7 5 0 . + + HIS 1 . 0 + 2 . 5 + + + + 5 . 0 + NTA 1 . 0 + + 2 . 5 + + 5 . 0 + + 7 . 5 + + 1 0 . 0 •+ •+ In the f i r s t s e r i e s of e x p e r i m e n t s , e i g h t columns were p r e p a r e d and were run s i m u l t a n e o u s l y . Of t h e s e e i g h t , s i x columns were used f o r the t e s t s o l u t i o n s and two columns were used f o r s t a n d a r d Cu s o l u t i o n s . The t e s t s o l u t i o n s were made up i n SOW by a d d i n g a c o n c e n t r a t i o n of Cu ( e i t h e r 7 . 8 9 x 1 0 " 8 M or 1 5 . 7 x 1 0 " 8 M Cu) and the a p p r o p r i a t e c o n c e n t r a t i o n of l i g a n d . The s t a n d a r d Cu s o l u t i o n s were made up i n SOW by a d d i n g e i t h e r 7 . 8 9 x 1 0 " 8 M or 1 5 . 7 x 1 0 " 8 M Cu, w i t h no l i g a n d s added. Three c o n c e n t r a t i o n s of the same l i g a n d and one s t a n d a r d were a l l run 103 a t the same t i m e . Both the t e s t and s t a n d a r d Cu s o l u t i o n s were made up i n 500 ml p o r t i o n s 12 hr p r i o r t o use t o a l l o w e q u i l i b r a t i o n of the l i g a n d w i t h Cu.. Once the samples had e q u i l i b r a t e d , 250 ml of the s t a n d a r d and each of the t e s t s o l u t i o n s were passed t h r o u g h the a p p r o p r i a t e column a t a f l o w r a t e of 1.7 ml m i n - 1 . D u p l i c a t e s of both the t e s t and s t a n d a r d s o l u t i o n s were r u n . The columns were then e l u t e d and the e l u a t e s a n a l y s e d f o r Cu by ASV. The Cu c o n c e n t r a t i o n bound t o the r e s i n from the s t a n d a r d Cu s o l u t i o n was used t o c a l i b r a t e the amount of Cu bound t o the r e s i n from the t e s t samples. Except f o r EDTA, the same l i g a n d c o n c e n t r a t i o n s and Cu c o n c e n t r a t i o n s were s t u d i e d i n the second s e r i e s as i n the f i r s t but some o p e r a t i o n a l and c h e m i c a l c o n d i t i o n s were changed. The f l o w r a t e was changed from 1.7 ml m i n - 1 t o 2.9 ml m i n " 1 and Fe was added t o a l l the samples t o g i v e a f i n a l c o n c e n t r a t i o n of 4.5 x 10" 7M. Ten columns were p r e p a r e d and run s i m u l t a n e o u s l y w i t h o u t d u p l i c a t i o n t o a l l o w two c o n c e n t r a t i o n s of Cu (7.89 x 10" 8M or 15.7 x 10" 8M), f o u r c o n c e n t r a t i o n s of one l i g a n d , and two s t a n d a r d s o l u t i o n ( e i t h e r 7.89 x 10~ 8M or 15.7 x 10" 8M Cu) t o be a n a l y s e d a t the same t i m e . Samples were p r e p a r e d i n 250 ml p o r t i o n s and l e f t t o e q u i l i b r a t e f o r 12 h r . The samples were then passed t h r o u g h the a p p r o p r i a t e column. F i n a l l y , the columns were e l u t e d and the e l u a t e s a n a l y s e d f o r Cu by ASV. Wi t h EDTA, f o u r c o n c e n t r a t i o n s of Cu (3.93, 7.89, 11.8 and 15.7 x 10" 8M) and two l i g a n d c o n c e n t r a t i o n s (5.0 x 10" 8M and 10.0 x 10" 8M) were used a t one t i m e . 104 D. RESULTS 1. C h a r a c t e r i z a t i o n of the R e s i n i n A r t i f i c i a l Seawater (a) E q u i l i b r a t i o n of the r e s i n t o Cu I t i s i m p o r t a n t t h a t the ion-exchanger a t t a i n complete e q u i l i b r i u m w i t h the i n p u t s o l u t i o n b e f o r e d e t e r m i n i n g the amount of Cu bound t o the r e s i n . To d e t e r m i n e the volume of sample needed t o e q u i l i b r a t e one gram of the r e s i n , the column e f f l u e n t was m o n i t o r e d f o r Cu as 300 ml of SOW c o n t a i n i n g 7.87 x 10" 8M Cu was passed t h r o u g h i t . F i g . 19 shows the e f f l u e n t Cu c o n c e n t r a t i o n c a l c u l a t e d as a p e r c e n t a g e of the i n f l u e n t p l o t t e d a g a i n s t the volume of e f f l u e n t . A l a r g e p e r c e n t a g e of t h e Cu i n s o l u t i o n was removed by the r e s i n from t h e f i r s t 200 ml of sample. Beyond 200 m l , however, the d i f f e r e n c e between the i n f l u e n t and e f f l u e n t c o n c e n t r a t i o n s was l e s s than the s t a n d a r d d e v i a t i o n of the ASV t e c h n i q u e . C o n s e q u e n t l y , a sample volume of 250 ml was used th r o u g h o u t the r e s i n e x p e r i m e n t s . The volume of sample needed t o e q u i l i b r a t e the r e s i n p o t e n t i a l l y changes w i t h v a r i a t i o n s i n the f l o w r a t e . As the f l o w r a t e i n c r e a s e s the time t h a t a u n i t volume of s o l u t i o n i s i n c o n t a c t w i t h the r e s i n d e c r e a s e s l e a v i n g l e s s t ime f o r e q u i l i b r a t i o n . On the o t h e r hand, i n c r e a s i n g the f l o w r a t e d e c r e a s e s the f i l m d i f f u s i o n l a y e r around the r e s i n bead, p o s s i b l y r e s u l t i n g i n f a s t e r e q u i l i b r a t i o n t i m e s ( D o r f n e r , 1972). In o r d e r t o t e s t the e f f e c t of f l o w r a t e on column 105 F i g u r e 19. E f f l u e n t Cu (% of i n f l u e n t ) v e r s u s the e f f l u e n t volume. V a l u e s a r e a mean of 2 r e p l i c a t e s ±1 s.d. 106 e q u i l i b r a t i o n , SOW c o n t a i n i n g 7.87 x 10" 8M Cu was passed t h r o u g h the r e s i n a t 0.8, 2.0 and 2.9 ml m i n " 1 . W h i l e l e s s Cu was adsorbed by the r e s i n a t the l o w e s t f l o w r a t e (0.8 ml m i n " 1 ) , above 2.0 ml m i n " 1 the d i f f e r e n c e was not s i g n i f i c a n t ( T a ble V I I ) . Thus, i t appears t h a t f l o w r a t e s e q u a l t o or e x c e e d i n g 2.0 ml min" 1 do not have a s i g n i f i c a n t i n f l u e n c e on the volume needed f o r e q u i l i b r a t i o n . The o b s e r v e d d i f f e r e n c e a t the l o w e s t f l o w r a t e may, i n p a r t , be due t o p r e s s u r e e f f e c t s caused by pumping the samples t h r o u g h the columns. Flow r a t e s much above 2.9 ml min" 1 were not p o s s i b l e because of the back p r e s s u r e d e v e l o p e d , w h i l e v e r y low f l o w r a t e s were not p r a c t i c a l because of the time needed f o r the a n a l y s i s . T a b l e V I I . The e f f e c t of f l o w r a t e on the a d s o r p t i o n of Cu by the r e s i n . . SOW c o n t a i n i n g 7.87 x 10" 8M Cu was passed t h r o u g h the r e s i n a t t h r e e f l o w r a t e s . Flow r a t e E l u a t e Cu (x!0 " 9 m o l g" 1) 0.8 ml min - i 5.04±0.31 1 2.0 ml min 1 6.14+0.00 2.9 ml min - 1 5.98+0.47 1Mean 11 s.d. based on 2 r e p l i c a t e s 107 (b) P r e c i s i o n of the ASV and column a d s o r p t i o n t e c h n i q u e The p r e c i s i o n of the a d s o r p t i o n t e c h n i q u e was d e t e r m i n e d u s i n g two s e p a r a t e b a t c h e s of r e s i n . SOW c o n t a i n i n g 7.87 x 10" 8M Cu was passed t h r o u g h a s e r i e s of columns and the amount of Cu adsorbed by the r e s i n then measured. The 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 of the a n a l y s i s , which averaged 8% ( T a b l e V I I I ) , was s i m i l a r i n b o t h c a s e s . A l t h o u g h , the mean Cu c o n c e n t r a t i o n bound t o the r e s i n was d i f f e r e n t i n the two t e s t s , t h e s e d i f f e r e n c e s a r e a t t r i b u t a b l e t o the d i f f e r e n t b a t c h e s of r e s i n used and v a r i a t i o n s a r i s i n g from t h e i r p r e p a r a t i o n . However, the p r e c i s i o n of the column method i n c l u d e s the v a r i a b i l i t y of the ASV t e c h n i q u e used t o q u a n t i f y the Cu i n the column e l u a t e s . To s e p a r a t e the v a r i a b i l i t y of the ASV t e c h n i q u e from t h a t of the v a r i a b i l i t y due t o the r e s i n , the p r e c i s i o n of the ASV p r o c e d u r e was d e t e r m i n e d . Ten a l i q u o t s (25 ml) of SOW, h a v i n g 2.36 x 10" 7M Cu added, were t r a n s f e r e d t o the p o l y c a r b o n a t e tubes used t o c o l l e c t the column e l u a t e s . The Cu l e v e l s i n t h e s e tube were then d e t e r m i n e d by u s i n g the normal ASV p r o c e d u r e . The p r e c i s i o n of the ASV p r o c e d u r e was the same as r e p o r t e d f o r the o v e r a l l column method (RSD 8%, T a b l e I X ) . Thus i t was c o n c l u d e d t h a t the v a r i a b i l i t y between the r e s i n columns was, a t l e a s t , w i t h i n the v a r i a b i l i t y of the ASV p r o c e d u r e . T a b l e V I I I . P r e c i s i o n of the r e s i n a n a l y s i s d e t e r m i n e d by r e p l i c a t e a n a l y s i s of SOW c o n t a i n i n g 7.87 x 10" 8M Cu. A and B a r e a n a l y s e s on two s e p a r a t e b a t c h e s of r e s i n . Cu adsorbed t o r e s i n ( x l 0 " 9 m o l g" 1) Column A B 1 5.51 4.41 2 6.14 4.41 3 5.82 4.41 4 5.98 4.72 5 5.82 4.56 6 5.82 4.25 7 5.19 4.41 8 4.88 • 4.72 9 5.04 10 4.41 Mean 5.67 4.56 S t a n d a r d 0.47 0.32 D e v i a t i o n R e l a t i v e 8 % 7 % S t a n d a r d D e v i a t i o n T a b l e IX. P r e c i s i o n of the ASV p r o c e d u r e . Determined by r e p l i c a t e a n a l y s i s of SOW c o n t a i n i n g 2.36 x 10" 7M Cu. Tube Number Cu Cone. ( X 1 0 " 7 M ) 1 2.64 2 2.68 3 2.47 4 2.29 5 2.27 6 2.20 7 2.38 8 2.25 9 2.24 10 2.15 Mean 2.36 St a n d a r d .183 D e v i a t i o n R e l a t i v e S t a n d a r d 8 % D e v i a t i o n 110 (c) E f f e c t of pH The response of the r e s i n was t e s t e d over the e n t i r e pH range t h a t would be en c o u n t e r e d i n the s e e x p e r i m e n t s . There were no s i g n i f i c a n t d i f f e r e n c e s i n the uptake of Cu by the r e s i n between pH v a l u e s of 7.57 and 8.15 (Table X ) . However, an i n c r e a s e of pH t o 8.42, d i d cause a r e d u c t i o n i n the amount of Cu bound t o the r e s i n . T a b l e X. The e f f e c t of pH on the a d s o r p t i o n of Cu by the r e s i n . SOW c o n t a i n i n g 7.87 x 10" 8M Cu was passed t h r o u g h the r e s i n a t v a r i o u s pH v a l u e s . pH E l u a t e Cu ( x l 0 " 9 m o l g" 1) 7.53 6.77±0.02 1 7.96 7.08±0.11 8.13 7.08±0.26 8.15 6.92±n.a.2 8.42 5.51±0.33 1Mean ±1 s.d. based 2 n . a . not a v a i l a b l e . on 2 r e p l i c a t e s . (d) N u t r i e n t e f f e c t s The 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 were added t o SOW t o determine i f t h e i r p r e s e nce a f f e c t e d the a d s o r p t i o n of Cu by the r e s i n . A p r e l i m i n a r y s t u d y i n d i c a t e d t h a t t h e i r a d d i t i o n 111 i n c r e a s e d the amount of Cu bound t o the r e s i n as compared t o when they were not p r e s e n t (Table X I ) . F u r t h e r t e s t s were performed t o determine which n u t r i e n t s caused t h i s r e s p o n s e . T a b l e X I . The e f f e c t of the 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 s on the a d s o r p t i o n of Cu by the r e s i n . SOW c o n t a i n i n g 7.87 x 10~ 8M Cu w i t h and w i t h o u t the a d d i t i o n of 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 s was passed t h r o u g h the r e s i n . E l u a t e Cu (x10- 9mol g- 1) SOW a l o n e 6.3010.35 1 SOW + N u t r i e n t s 7.71±0.06 " + Trace M e t a l s 'Mean ±1 s.d. based on 2 r e p l i c a t e s A d s o r p t i o n of Cu by f e r r i c h y d r o x i d e i s w e l l documented ( D a v i s and L e c k i e , 1978; Swallow et. a l . , 1980), and i t s a d d i t i o n t o SOW might t h e r e f o r e be e x p e c t e d t o e f f e c t the response of the r e s i n . C o n s e q u e n t l y , Fe was the f i r s t n u t r i e n t t e s t e d . In the f i r s t e x p e r i m e n t , the a d s o r p t i o n of Cu i n the pr e s e n c e of Fe a l o n e was compared w i t h t h a t i n the presence of a l l the o t h e r n u t r i e n t s e x cept Fe. SOW w i t h o u t n u t r i e n t s or Fe a d d i t i o n was used as a c o n t r o l . I t was found t h a t s i m i l a r amounts of Cu were bound t o t h e r e s i n from both the c o n t r o l samples and the samples w i t h the A q u i l n u t r i e n t s minus Fe (Ta b l e X I I ) . When Fe was added t o SOW, however, the amount of Cu bound t o the r e s i n i n c r e a s e d , a l t h o u g h the a d d i t i o n of up t o 4X the i n i t i a l Fe c o n c e n t r a t i o n caused no f u r t h e r i n c r e a s e . 1 12 I n c r e a s e d Cu uptake i n the presence of Fe was not due Cu c o n t a m i n a t i o n from the Fe s t o c k . There was no i n c r e a s e i n the t o t a l Cu c o n c e n t r a t i o n s of the samples upon the a d d i t i o n of the Fe s t o c k , as measured by ASV (Table X I I ) and , i n c r e a s i n g the Fe c o n c e n t r a t i o n up t o 4X d i d not i n c r e a s e the Cu uptake s i g n i f i c a n t l y as would be e x p e c t e d i f the Fe s t o c k was c o n t a m i n a t i n g the sample. T a b l e X I I . The e f f e c t of Fe on the a d s o r p t i o n of Cu. SOW c o n t a i n i n g 7.87 x 1 0 " B M Cu w i t h v a r i o u s A q u i l n u t r i e n t and Fe a d d i t i o n s passed t h r o u g h the r e s i n . A l s o the t o t a l Cu l e v e l s i n the samples b e f o r e a n a l y s i s a r e p r e s e n t e d . E l u a t e Cu Sample Cu Treatment (x10" 9mol g" 1) ( X 1 0 " 8 M ) SOW a l o n e 6.45 7.08 SOW + N u t r i e n t s -FE 6.92 7.08 SOW + Fe 8.18 6.92 SOW + 2X Fe 8.50 8.03 SOW + 3X Fe 8.81 6.92 SOW + 4X Fe 8.50 6.92 To f u r t h e r t e s t the e f f e c t of Fe on the r e s i n , Fe s t o c k s aged f o r d i f f e r e n t l e n g t h s of time were added t o SOW c o n t a i n i n g 7.87 x 1 0 " 8 M Cu. Fe s t o c k s f r e s h l y p r e p a r e d (added b e f o r e any p r e c i p i t a t e was e v i d e n t ) , aged f o r 6 hr and aged f o r 72 hr were added t o the SOW and passed t h r o u g h the r e s i n . The h i g h e s t Cu c o n c e n t r a t i o n s bound t o the r e s i n were w i t h the SOW c o n t a i n i n g 1 13 the f r e s h l y p r e p a r e d Fe s t o c k (Table X I I I ) . The 6 hr and 72 hr Fe samples had l e s s a d s o r p t i o n of Cu than d i d the 5 min Fe s t o c k sample but they s t i l l had h i g h e r l e v e l s than the SOW h a v i n g no Fe added. When f r e s h l y p r e p a r e d Fe s t o c k was added t o SOW c o n t a i n i n g no Cu and passed t h r o u g h the r e s i n , t h e r e were n o n - d e t e c t a b l e l e v e l s of Cu bound t o the r e s i n . T h i s was a f u r t h e r i n d i c a t i o n t h a t t h e r e was no Cu c o n t a m i n a t i o n from the Fe s t o c k s . T h i s s u g g e s t s t h a t the i n c r e a s e d a d s o r p t i o n of Cu by the r e s i n was c o r r e l a t e d w i t h an a d s o r p t i o n of c o l l o i d a l f e r r i c h y d r o x i d e . T a b l e X I I I . The e f f e c t of aged Fe s t o c k s on the a d s o r p t i o n of Cu. SOW c o n t a i n i n g 7.87 x 10" 9M Cu w i t h the a d d i t i o n of Fe s t o c k s aged f o r 5 min, 6 hr and 72 hr was passed t h r o u g h t h e r e s i n . E l u a t e Cu (x10- 9mol g" 1) No Cu-5 min Fe n.d. 2 No Fe ( c o n t r o l ) 5.8210.63 1 Cu-5 min Fe 11.80±0.47 Cu-6 hr Fe 8.97±0.47 Cu-72 hr Fe 8.81±0.31 'Mean ±1 s.d. based 2 n . d . n o n - d e t e c t a b l e on 2 r e p l i c a t e s A mechanism t h a t might account f o r a d s o r p t i o n of hydrous f e r r i c o x i d e s by the r e s i n i s sug g e s t e d by the o b s e r v a t i o n of P a r f i t t and Smart (1978). They found a s t r o n g a d s o p t i o n of 1 14 s u l p h a t e i o n s by Fe o x i d e s which they a t t r i b u t e d t o a d i s p l a c e m e n t of s u r f a c e h y d r o x y l groups and c o o r d i n a t i o n of the adsorbed SO,,2" i o n by s u r f a c e F e 3 + i o n s . However, a c c o r d i n g t o the s i t e - b i n d i n g model of D a v i s and L e c k i e (1978) on a d s o r p t i o n onto Fe o x i d e s u r f a c e s , the s u l p h a t e i o n i s bound a t p r o t o n a t e d s u r f a c e s i t e s f o r m i n g Fe-OH 2-SO f l" or Fe-OH2-HSO<, groups, a model a l s o a p p l i e d by B a l i s t r i e r i and Murray (1981) t o d e s c r i b e the a d s o r p t i o n of s u l p h a t e by g o e t h i t e i n seawater. Thus the a d s o r p t i o n of F e - o x i d e by the ion-exchange r e s i n might be e x p l a i n e d by a s i m i l a r i n t e r a c t i o n between the s u l p h o n a t e groups of the r e s i n and s u r f a c e FeOH 2 + groups. The p o p u l a t i o n of p r o t o n a t e d FeOH 2 + s u r f a c e s i t e s , and hence t h e a d s o r p t i o n of s u l f a t e i o n s , i s n o r m a l l y o n l y s i g n i f i c a n t a t pH v a l u e s below 8. However, under weakly b a s i c c o n d i t i o n s , a c o a d s o r p t i o n of Cu and Fe o x i d e s by the r e s i n might o c c u r as a r e s u l t of a mechanism s i m i l a r t o t h a t o b s e r v e d by T i p p i n g (1981), who found t h a t n e g a t i v e l y c h a r g e d humates were a d s o r b e d onto n e g a t i v e l y c h a r g e d i r o n o x i d e s u r f a c e s i n s o l u t i o n s c o n t a i n i n g d i v a l e n t c a t i o n s . E l e c t r o p h o r e t i c measurements i n d i c a t e d t h a t t h e r e was a c o a d s o r p t i o n of d i v a l e n t c a t i o n s t h a t presumably d e c r e a s e d the e l e c t r o s t a t i c r e p u l s i o n between t h e adsor b e n t and a d s o r b a t e . Thus, the a d s o r p t i o n of n e g a t i v e l y charged F e - o x i d e s by the r e s i n might be brought about by a s i m i l a r c o a d s o r p t i o n of d i v a l e n t c a t i o n s i n c l u d i n g C u 2 + . Ano t h e r 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 t h e r e may be t r a p p i n g of c o l l o i d a l Fe h y d r o x i d e by the r e s i n . However, the aged Fe s t o c k s showed the l e a s t a c t i v i t y and they were e x p e c t e d t o have 115 the l a r g e s t c o l l o i d a l p a r t i c l e s . A l s o , because of the pore s i z e of the r e s i n beads, c o l l o i d a l p a r t i c l e s a r e b e l i e v e d t o be e x c l u d e d from the r e s i n . F l o r e n c e ( 1 9 7 7 ) showed t h a t s o l u t i o n s of c o l l o i d a l h y d r a t e d f e r r i c o x i d e and l a r g e o r g a n i c dyes were q u a n t i t a t i v e l y r e j e c t e d by an ion-exchange r e s i n ( C h e l e x - 1 0 0 ) . (e) I o n i c s t r e n g t h e f f e c t s The s a l i n i t y of the SOW used i n the l a b o r a t o r y study was 35 p p t . However, as t h i s t e c h n i q u e might be used i n f u t u r e s t u d i e s a t o t h e r s a l i n i t i e s , i t was d e s i r a b l e t o d etermine how the a d s o r p t i o n of Cu v a r i e s w i t h s a l i n i t y . As the s a l i n i t y of a sample d e c r e a s e s the c o n c e n t r a t i o n of c a t i o n s competing w i t h Cu f o r b i n d i n g s i t e s on the r e s i n a l s o d e c r e a s e s and, hence, the t o t a l amount of Cu taken up by the r e s i n s h o u l d i n c r e a s e . T h i s e x p e c t a t i o n i s c o n f i r m e d by F i g . 20, which shows t h a t t h e r e was almost a d o u b l i n g of the amount of Cu adsorbed from 5 ppt SOW as compared t o 35 ppt SOW. I t i s e v i d e n t from t h e s e r e s u l t s t h a t c a l i b r a t i o n of the r e s i n must be made a t each s a l i n i t y s t u d i e d . ( f ) A d s o r p t i o n c u r v e s f o r Cu The r e s i n was t o be c a l i b r a t e d i n the model l i g a n d s t u d y by comparing th e amount of Cu adsorbed t o the r e s i n from the t e s t s o l u t i o n s w i t h t h a t adsorbed from s t a n d a r d Cu s o l u t i o n s c o n t a i n i n g no o r g a n i c l i g a n d s . To m i n i m i z e the number of s t a n d a r d Cu s o l u t i o n s needed f o r c a l i b r a t i o n a l i n e a r F i g u r e 20. Change i n the a d s o r p t i o n of Cu w i t h s a l i n i t y V a l u e s a r e a mean of 2 r e p l i c a t e s ±1 s.d. 300H Salinity (%) 1 17 r e l a t i o n s h i p between the amount of Cu bound t o the r e s i n and the t o t a l Cu c o n c e n t r a t i o n i n the s t a n d a r d s o l u t i o n was d e s i r e d . The r e l a t i o n s h i p between the amount of Cu bound by the r e s i n and i t s c o n c e n t r a t i o n i n s o l u t i o n between 0 and 15.7 x 10" 8M ( a t 3.93 x 10" 8M i n c r e m e n t s ) was found t o be l i n e a r ( F i g . 2 1 ) . Between 0 and 31.5 x 10" 8M (at 3.93 x 10" 8M i n c r e m e n t s ) , on the o t h e r hand, the r e l a t i o n s h i p was o n l y l i n e a r up t o a c o n c e n t r a t i o n of a p p r o x i m a t e l y 19.67 x 10" 8M Cu ( F i g . 21) b u t , a t h i g h e r Cu c o n c e n t r a t i o n s , the s l o p e of the a d s o r p t i o n c u r v e began t o d e c l i n e . The s l o p e of the a d s o r p t i o n c u r v e s d i f f e r e d between the two t e s t s . An i n c r e a s e d a d s o r p t i o n of Cu was seen i n the second t e s t due t o the presence of Fe i n the s e samples. The i n c r e a s e d a d s o r p t i o n due t o Fe has been p r e v i o u s l y d i s c u s s e d ( S e c t i o n IV.D.1.d). In t h e o r y , the a d s o r p t i o n of Cu by the r e s i n i s dependent on the c u p r i c i o n a c t i v i t y of the sample. T h e r e f o r e , a l i n e a r c a l i b r a t i o n r e l a t i o n s h i p can be assumed i n the case of samples h a v i n g a t o t a l m e t a l c o n c e n t r a t i o n i n the range of n o n - l i n e a r i t y as l o n g as t h e i r c u p r i c i o n a c t i v i t i e s a r e i n the l i n e a r range. C u p r i c i o n a c t i v i t y can be d e c r e a s e d w h i l e m a i n t a i n i n g a h i g h t o t a l Cu c o n c e n t r a t i o n by the presence of o r g a n i c c o m p l e x i n g a g e n t s . I t has been shown t h a t the c a l i b r a t i o n r e l a t i o n s h i p i s l i n e a r w i t h i n the l i m i t s i n d i c a t e d above; however, the s l o p e s of t h i s r e l a t i o n s h i p may d i f f e r w i t h d i f f e r e n t o p e r a t i n g c o n d i t i o n s . A l s o , t h e r e was some s l i g h t v a r i a b i l i t y from one F i g u r e 2 1 . A d s o r p t i o n c u r v e s f o r Cu i n SOW w i t h ( • ) w i t h o u t ( A ) t h e a d d i t i o n o f F e . 119 s e r i e s of t e s t s t o the next even under a p p a r e n t 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 . Hence, s t a n d a r d s o l u t i o n s were always run at the same time and under the same c o n d i t i o n s as the sample s o l u t i o n . 3. Model L i g a n d Study The a b i l i t y of the r e s i n t e c h n i q u e t o measure the c o m p l e x a t i o n of Cu by EDTA, GLU, HIS and NTA was examined. The r e s u l t s of t h e s e e x p e r i m e n t s were r e p o r t e d as 'Cu e q u i v a l e n t s ' (Cu e q u i v ) which r e p r e s e n t s the c o n c e n t r a t i o n of Cu t h a t , when p r e s e n t i n o r g a n i c l i g a n d f r e e SOW (S=35 p p t , pH=8.0), r e s u l t s i n t he a d s o r p t i o n of an amount of Cu e q u a l t o t h a t adsorbed from the t e s t s o l u t i o n ; e.g., a Cu e q u i v v a l u e of 2.5 x 10" 8M means t h a t the t e s t s o l u t i o n had adsorbed the same amount of Cu onto the r e s i n as would be adsorbed from a 2.5 x 10" 8M s t a n d a r d Cu s o l u t i o n . A Cu e q u i v i s a v a l u e p r o p o r t i o n a l t o t h e pCu of a sample, a q u a n t i t y which can be e s t i m a t e d by the method d e s c r i b e d i n S e c t i o n IV.B.3. However, the a c c u r a c y of the pCu e s t i m a t e i s dependent on the a c c u r a c y of c a l c u l a t i n g t h e pCu of the s t a n d a r d Cu s o l u t i o n s used t o c a l i b r a t e the r e s i n . U n f o r t u n a t e l y , the c a l c u l a t i o n of pCu, even i n w e l l d e f i n e d a r t i f i c i a l s eawater, i s q u e s t i o n a b l e . I f the Cu e q u i v v a l u e of a t e s t sample was d i f f e r e n t from i t s t o t a l Cu c o n c e n t r a t i o n t h i s i n d i c a t e d t h a t the c o m p l e x a t i o n of Cu i n t h i s sample was g r e a t e r than i n a Cu s t a n d a r d of the same t o t a l Cu c o n c e n t r a t i o n . S i n c e Cu i s a l r e a d y complexed by the C 0 3 2 " and OH" i o n s i n the s t a n d a r d s , reduced Cu e q u i v v a l u e s 120 i n d i c a t e d 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 than t h e s e i n o r g a n i c l i g a n d s . Cu e q u i v v a l u e s can t h e r e f o r e be used t o i n d i c a t e the c o m p l e x a t i o n of Cu by the model o r g a n i c l i g a n d s . In t he f i r s t s e r i e s of e x p e r i m e n t s , the a d d i t i o n of EDTA, GLU or NTA was found t o reduce the Cu e q u i v v a l u e s of the t e s t s o l u t i o n s ( T a b l e s XIV, XV) thus i n d i c a t i n g the r e s i n ' s a b i l i t y t o d e t e c t c o m p l e x a t i o n of Cu by t h e s e l i g a n d s . As t h e l i g a n d ' s c o n c e n t r a t i o n i n c r e a s e d the Cu e q u i v v a l u e of the sample d e c r e a s e d and the e x t e n t of r e d u c t i o n was r e l a t e d t o the l i g a n d ' s s t a b i l i t y c o n s t a n t f o r Cu. EDTA, w i t h the h i g h e s t s t a b i l i t y c o n s t a n t ( l o g K 1=20.6), reduced the Cu e q u i v v a l u e s markedly a t a p p r o x i m a t e l y the same c o n c e n t r a t i o n as the t o t a l Cu c o n c e n t r a t i o n l e v e l s of the s o l u t i o n . G l u t a m i c a c i d ( l o g k.f=7.B7)r on the o t h e r hand, had t o be added i n c o n c e n t r a t i o n s two o r d e r s of magnitude h i g h e r than EDTA t o g i v e a s i m i l a r r e d u c t i o n i n the Cu e q u i v v a l u e . In a second s e r i e s of e x p e r i m e n t s the same l i g a n d s , l i g a n d c o n c e n t r a t i o n s and Cu c o n c e n t r a t i o n s were s t u d i e d but the p r o c e d u r e and some o p e r a t i o n a l c o n d i t i o n s were m o d i f i e d . The main p r o c e d u r a l change was t h a t a l l t e s t samples f o r a p a r t i c u l a r l i g a n d were used i n one e x p e r i m e n t a l r u n . T h i s approach was ta k e n t o e l i m i n a t e i n t e r - t e s t v a r i a t i o n s by u s i n g the same Cu s t a n d a r d s t o c a l i b r a t e a l l the samples. Two o p e r a t i o n a l c o n d i t i o n s were a l s o m o d i f i e d . F i r s t , a h i g h e r f l o w r a t e was used (2.9 ml m i n - 1 as opposed t o 1.7 ml m i n - 1 ) t o reduce the a n a l y s i s t i m e . Second, Fe was added t o a l l samples a t the A q u i l c o n c e n t r a t i o n t o s i m u l a t e c o n d i t i o n s i n the c u l t u r e 121 T a b l e XIV. Cu e q u i v v a l u e s i n SOW i n the pre s e n c e of EDTA GLU and NTA f o r S e r i e s I . V a l u e s a r e Cu e q u i v ( X 1 0 " 8 M ) L i g a n d L i g a n d Cone. ( X 1 0 " 7 M ) Cu A d d i t i o n 7.87x10- BM 15.7X10- 8M EDTA 0.5 1 .0 2.5 5.0 3.93±0.32 1 2.52±0.32 1 .26+0.47 1 . 10±0.47 10.90±0.00 6.30±0.15 0.47±0.02 n. a 2 GLU 1 00. 250. 500. 750. 6.77±0. 16 5.19±0.06 3.30±0.47 n .a 10.54±0.00 7.87±0.47 5.04±0.47 3.93±0.32 NTA 1.0 2.5 5.0 7.5 10.0 5.19±0.16 4.56±0.16 3.93±0.47 3.46±0.00 3.46±0.63 11 .49±0.15 7.24±0.47 5.67±0.15 4.72±0.47 n .a. 1Mean ±1 s.d. based on 2 r e p l i c a t e s . 2 n . a . not a v a i l a b l e . 122 media used i n the b i o a s s a y s . Once a g a i n the a d d i t i o n of EDTA, GLU and NTA reduced the Cu e q u i v v a l u e s over t h a t of the t o t a l Cu c o n c e n t r a t i o n but t h e r e were some d i f f e r e n c e i n the a b s o l u t e v a l u e s as compared t o s e r i e s one. When u s i n g NTA, the Cu e q u i v v a l u e s were a l l h i g h e r than the e s t i m a t e s i n s e r i e s one; a l t h o u g h the l a r g e s t d i f f e r e n c e was o n l y 23%. When u s i n g GLU the o p p o s i t e was t r u e , the r e s u l t s i n s e r i e s two were, on the a v e r a g e , 15% lower than s e r i e s one w i t h a maximum d i f f e r e n c e of 32% found i n the h i g h e s t c o n c e n t r a t i o n of GLU used. For EDTA, the r e s u l t s between the s e r i e s were v e r y s i m i l a r and any d i f f e r e n c e s were w i t h i n the range of e x p e r i m e n t a l e r r o r . The second s e r i e s of e x p e r i m e n t s were b e l i e v e d t o be more r e p r e s e n t i t i v e of the c o n d i t i o n s found i n the b i o a s s a y c u l t u r e medium because of the presence of Fe. Because of t h i s , the r e s u l t s of s e r i e s two were used f o r comparison w i t h the b i o a s s a y r e s u l t s . The a d d i t i o n of HIS produced r e s u l t s v e r y d i f f e r e n t from t h a t of the o t h e r l i g a n d s . When HIS was added t o SOW ( c o n t a i n i n g 7.87 x 10" 8M Cu), a t c o n c e n t r a t i o n s h i g h enough t o complex a s u b s t a n t i a l f r a c t i o n of the Cu, t h e r e was a g r e a t e r amount of Cu adsorbed t o the r e s i n than when u s i n g H I S - f r e e SOW ( T a b l e X V I ) . Only a t the h i g h e s t l i g a n d c o n c e n t r a t i o n d i d the amount of Cu on the r e s i n b e g i n t o d e c r e a s e . The experiment w i t h HIS was then r e p e a t e d u s i n g one l i g a n d c o n c e n t r a t i o n (2.5 x 10" 7M) and f o u r Cu c o n c e n t r a t i o n s (3.93, 7.87, 11.8, 15.7 x 10" 8M). Once a g a i n , t h e r e was a g r e a t e r 123 Tab l e XV. Cu e q u i v v a l u e s i n SOW i n the p r e s e n c e of EDTA, GLU and NTA f o r S e r i e s I I . V a l u e s a r e Cu e q u i v ( x l Q - 8 M ) . L i g a n d Cone. L i g a n d (x 10" 7M) 3.93 Cu A d d i t i o n (x10" 8M) 7.87 11.9 15.7 EDTA 0.5 1 .0 2.52 1 .73 4.09 2.83 8.03 3.93 12.27 6.45 NTA 1 .0 2.5 5.0 7.5 6.61 5.51 3.78 3.62 14.16 9.29 6. 14 5.51 GLU 100. 250. 500. 750. 6. 14 4.41 3.15 2.68 10.70 5.67 4.41 2.68 124 Table X V I . The a d s o r p t i o n of Cu i n the presence of HIS. S o l u t i o n s of SOW c o n t a i n i n g 7.87 x 10~ 8M Cu and v a r i o u s c o n c e n t r a t i o n of HIS were passed t h r o u g h the r e s i n . L i g a n d Cone. (x1 0" 7M) E l u a t e Cu ( x l 0 " 9 m o l g" 1) 0 . 0 4.72±0.161 1 .0 5.19±0.16 2.5 5.35±0.16 5.0 3.78±0.47 1Mean ±1 s.d. based on 2 r e p l i c a t e s c o n c e n t r a t i o n of Cu adsorbed up by the r e s i n when HIS was p r e s e n t ( T a b l e X V I I ) . I n c r e a s e d Cu l e v e l s on the r e s i n , as compared t o the s t a n d a r d s , a r e r e f l e c t e d i n Cu e q u i v v a l u e s b e i n g g r e a t e r than the t o t a l Cu c o n c e n t r a t i o n p r e s e n t i n the sample. These h i g h e r l e v e l s c o u l d not be a t t r i b u t e d t o c o n t a m i n a t i o n from the HIS s t o c k as t h e r e was no measurable i n c r e a s e i n the Cu c o n c e n t r a t i o n of the sample upon the a d d i t i o n of HIS as measured by ASV. These r e s u l t s can be e x p l a i n e d by the uptake of p o s i t i v e l y c h a r g e d Cu-HIS complexes by the r e s i n . At the pH of seawater HIS e x i s t s m a i n l y as n e u t r a l m o l e c u l e s w i t h a s m a l l p e r c e n t a g e e x i s t i n g as p o s i t i v e l y c h a r g e d s p e c i e s (pK's a r e g i v e n i n T a b l e X V I I I ) . C o m p l e x a t i o n of the l i g a n d w i t h Cu w i l l t h e r e f o r e r e s u l t i n complexes w i t h a charge of +2 or +3 which, because of t h e i r c h a r g e , s h o u l d be adsorbed by the r e s i n . A f f i n i t y of t h i s t ype of r e s i n f o r charge d o r g a n i c s has been r e p o r t e d i n the l i t e r a t u r e . . Moore and S t e i n (1951) found a 125 T able X V I I . Cu e q u i v v a l u e s from SOW i n the presence of 2.5 x 1 0 " 7 M HIS. Cu Cone, i n SOW E l u a t e Cu Cu equiv.. ( X 1 0 " b M ) (x10- 9mol g - 1 ) ( X 1 0 - 8 M ) 3.93 3..7810.161 5.04±0.16 1 7.87 6.61±0.16 8.66±0.16 11.8 9.13±0.16 12.12±0.32 15.7 11.65101.2 15.42101.4 NO HIS ( s t a n d a r d ) 7.87 5.9810.03 1Mean 11 s.d. based on 2 r e p l i c a t e s s t r o n g a f f i n i t y f o r HIS by p o l y s u l p h o n a t e d p o l y s t y r e n e r e s i n s when exa m i n i n g the s e p a r a t i o n of amino a c i d s . They found reduced r e c o v e r i e s of b a s i c amino a c i d s , such as HIS, from th e r e s i n as compared w i t h the o t h e r e l e v e n amino a c i d s t e s t e d . 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 o r g a n i c s have a l s o been shown f o r o t h e r r e s i n s . P a k a l n s e t a l . (1978) found t h a t 13% of the t o t a l exchange c a p a c i t y of Chelex-100 r e s i n was used i n the a d s o r p t i o n of c a t i o n i c d e t e r g e n t s where o n l y 2 and 3% of the t o t a l exchange c a p a c i t y was used f o r a n i o n i c and n o n - i o n i c d e t e r g e n t s , r e s p e c t i v e l y . The uptake of the Cu-HIS complex c o u l d not o n l y e x p l a i n t h e r e s i n ' s l a c k of response t o the c o m p l e x a t i o n of Cu by the l i g a n d , but a l s o the i n c r e a s e d a d s o r p t i o n of Cu by the r e s i n . An i n c r e a s e i n a d s o r p t i o n c o u l d occur because of the r e s i n r e s p o n d i n g t o b oth the a c t i v i t y of the i n o r g a n i c Cu s p e c i e s as w e l l as the a c t i v i t y of the Cu-HIS complex. 126 Table X V I I I . pK's of H i s t i d i n e pK of H 3 L 2 + H 2 L + HL 1.82 6.05 9.17 Taken from S i l l e n and M a r t e l (1971). 4. Comparison of the Chemical Assay and B i o a s s a y R e s u l t s The growth r a t e of the b i o a s s a y o r g a n i s m f o r each c o m b i n a t i o n of l i g a n d and Cu c o n c e n t r a t i o n a n a l y s e d was compared t o 1) the Cu e q u i v v a l u e s d e t e r m i n e d by the r e s i n t e c h n i q u e , 2) the e s t i m a t e of pCu c a l c u l a t e d from the r e s i n r e s u l t s u s i n g the method d e s c r i b e d i n S e c t i o n IV.B.3 (eqn. 12) and 3) the e s t i m a t e 1 of pCu* as c a l c u l a t e d w i t h the computer model MINEQL. Growth r a t e s and both e s t i m a t e s of pCu a r e p r e s e n t e d i n T a b l e XIX. In F i g . 22, growth r a t e s a r e p l o t t e d as a f u n c t i o n of the n e g a t i v e l o g of the Cu e q u i v v a l u e s . There was a s t r o n g c o r r e l a t i o n between the - l o g Cu e q u i v e s t i m a t e d by the r e s i n a n a l y s i s and the growth r a t e of the organism. A l i n e a r r e l a t i o n s h i p was seen between the v a l u e s of 7.5 and 6.85 which c o r r e s p o n d e d t o c o n c e n t r a t i o n s of 3.16 x 10~ 8M and 14.1 x 10~ 8M Cu, r e s p e c t i v e l y . A p l a t e a u i n the growth c u r v e was apparent a t - l o g Cu e q u i v v a l u e s l e s s than 7.15. Growth r a t e s were a l s o p l o t t e d as a f u n c t i o n of pCu as e s t i m a t e d by the r e s i n a n a l y s i s ( F i g . 2 3 ) . A s t r o n g l i n e a r r e l a t i o n s h i p was seen between a pCu of 8.7 and 9.3 w h i l e , above 9.3, t h e r e was no f u r t h e r i n h i b i t i o n of growth. A c o r r e l a t i o n 127 Table XIX. Growth r a t e s , and pCu as estimated by the r e s i n a n a l y s i s and by c a l c u l a t i o n . Ligand Ligand T o t a l Cu Resin Mineql Growth rate Conc.(M) (X 1 0 - 8 M ) pCu pCu* (% Control) 5.0X10" b 3.9 9.44 10.25 100 7.9 9.21 9.33 90±7 1 1 .8 8.92 8.99 63±4 15.7 8.74 8.79 55±2 1.0X10- 7 3.9 9.61 10.91 98±1 7.9 9.39 10.21 100±1 11.8 9.23 9.44 94±1 15.7 9.02 9.05 61 ±2 1.OxlO" 5 7.9 9.04 9.04 78±3 15.7 8.80 8.74 56±3 2.5x1fJ- 5 7.9 9.19 9.26 96±3 15.7 9.08 8.97 69±2 5.0X10" 5 7.9 9.34 9.63 1 0 1 ±2 15.7 9.19 9.33 87±1 7.5X10" 5 7.9 9.42 9.90 100±1 15.7 9.36 9.60 97±5 1.0x10" 7 7.9 9.01 9.17 59±2 15.7 8.68 8.82 44±1 2.5X10" 7 7.9 9.09 9.43 79±3 15.7 8.86 9.06 55±1 5.0X10" 7 7.9 9.25 9.69 93±2 15.7 9.04 9.34 64±1 7.5X10" 7 7.9 9.28 9.85 1 02±1 15.7 9.09 9.51 77±1 1Mean ±1 s.d. based on 3 r e p l i c a t e s . 128 c o e f f i c i e n t ( r ) f o r the c u r v e , e x c l u d i n g p o i n t s where the growth r a t e s were 100% of the c o n t r o l s , was 0.92. When the same growth d a t a were p l o t t e d as a f u n c t i o n of pCu* as e s t i m a t e d by the computer model ( F i g . 2 4 ) , the c o r r e l a t i o n was not as s t r o n g . The c o r r e l a t i o n c o e f f i c i e n t ( r ) f o r t h i s c u r v e was 0.77. T h i s s u g g e s t s t h a t t h e r e may have been e i t h e r i n t e r a c t i o n s i n the media a f f e c t i n g the s p e c i a t i o n of Cu which the model f a i l s t o a c c o u n t f o r o r , as p r e v i o u s l y d i s c u s s e d , the use of i n a c c u r a t e s t a b i l i t y c o n s t a n t s . 129 F i g u r e 22. Growth r a t e (% of c o n t r o l ) v e r s u s the n e g a t i v e l o g of the Cu e q u i v v a l u e s . Data from t e s t s u s i n g • EDTA, A GLU and 0 NTA as the model o r g a n i c l i g a n d s . i 1 1 1 1 1 1 1 1 6.7 7.0 7.3 7.6 7.9 -log Cu . 0 equiv 130 F i g u r e 23. Growth r a t e (% of c o n t r o l ) v e r s u s pCu e s t i m a t e d from t h e r e s i n r e s u l t s . Data from t e s t s u s i n g • EDTA, A GLU and <> NTA as the model o r g a n i c l i g a n d s . Bars a r e ±1 s.d. 8.5 8.8 9.1 9.4 9.7 pCu 131 F i g u r e 24. Growth r a t e (% of c o n t r o l ) v e r s u s pCu* c a l c u l a t e d by MINEQL. Data from t e s t s u s i n g • EDTA, A GLU and 0 NTA as the model o r g a n i c l i g a n d s . B a r s a r e ±1 s.d. 132 E. DISCUSSION In the p r e l i m i n a r y e x p e r i m e n t s i n which the a d s o r p t i o n of Cu by the r e s i n was s t u d i e d i n o r g a n i c l i g a n d f r e e a r t i f i c i a l seawater (SOW) of w e l l - d e f i n e d c o m p o s i t i o n , the o v e r a l l p r e c i s i o n of the t e c h n i q u e , i n d i c a t e d by the 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 , was found t o be 8% which i n c l u d e s the e r r o r i n the ASV a n a l y s i s . The s e n s i t i v i t y of the t e c h n i q u e was shown t o be l i m i t e d o n l y by the q u a n t i t y of r e s i n used ( c a . 3.1 x I 0 " 9 m o l g" 1 of r e s i n ) ; however, as the q u a n t i t y of r e s i n d e t e r m i n e s the volume of sample needed f o r the a n a l y s i s the l a t t e r may i n f a c t be the l i m i t i n g f a c t o r . The a d s o r p t i o n of Cu by the r e s i n was found t o be l i n e a r up t o a s o l u t i o n Cu c o n c e n t r a t i o n of a p p r o x i m a t e l y 19.7 x 10" 8M. Above t h i s the r e s i n s t i l l a d sorbed Cu but the s l o p e of the a d s o r p t i o n c u r v e was reduced. However, as t h e r e g i o n of l i n e a r response encompassed the c o n c e n t r a t i o n range t h a t caused s u b l e t h a l e f f e c t s i n the b i o a s s a y organism and the range of c o n c e n t r a t i o n s t h a t c o u l d be e x p e c t e d i n c o a s t a l marine waters (see F o r s t n e r and Wittman, 1979, p.83), t h i s d e p a r t u r e from l i n e a r i t y i s of l i t t l e p r a c t i c a l s i g n i f i c a n c e . At a c o n s t a n t Cu c o n c e n t r a t i o n , the a d d i t i o n of the model l i g a n d s EDTA, GLU and NTA reduced the Cu e q u i v l e v e l s of the samples r e f l e c t i n g a r e d u c t i o n i n the a d s o r p t i o n of Cu by the r e s i n i n t h e i r p r e s e n c e . The e x t e n t of the r e d u c t i o n was r e l a t e d t o the l i g a n d c o n c e n t r a t i o n and the s t a b i l i t y of i t s complexes w i t h Cu. As the c o n c e n t r a t i o n of the l i g a n d or i t s s t a b i l i t y c o n s t a n t i n c r e a s e d , the Cu e q u i v v a l u e of the sample d e c r e a s e d . From t h i s i t was e v i d e n t t h a t a d s o r p t i o n by the 1 33 r e s i n was c o n t r o l l e d not by the t o t a l Cu c o n c e n t r a t i o n but by the f r a c t i o n of the t o t a l l e v e l t h a t was not complexed by the o r g a n i c l i g a n d s . To determine i f ' the a d s o r p t i o n of Cu by the r e s i n c o u l d be r e l a t e d t o the response of the b i o a s s a y t e s t organism, growth r a t e s of the organism were p l o t t e d as a f u n c t i o n of the n e g a t i v e l o g of the Cu e q u i v v a l u e s as de t e r m i n e d by the r e s i n technique.. For a l l the 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 , t h e r e was a s t r o n g r e l a t i o n s h i p between the Cu e q u i v v a l u e s and growth r a t e s . T h i s i n d i c a t e s t h a t the r e s i n t e c h n i q u e can be used t o e s t i m a t e the t o x i c i t y of Cu i n thes e t e s t s o l u t i o n s . S i n c e the c u p r i c i o n has been shown t o be the p r i n c i p a l t o x i c form of the metal f o r many p h y t o p l a n k t o n s p e c i e s (Sunda and G u i l l a r d , 1976; Anderson and M o r e l , 1978; J a c k s o n and Morgan, 1978; G a v i s e t a l . , 1981), a s t r o n g r e l a t i o n s h i p between growth r a t e and the r e s u l t s of the r e s i n t e c h n i q u e would i n f e r t h a t the response of the r e s i n was t o the a c t i v i t y of the c u p r i c i o n , a l t h o u g h the a c t i v i t y of the o t h e r p o s i t i v e l y charged Cu s p e c i e s found i n seawater ( C u C l + , CuOH +) may a l s o c o n t r i b u t e t o the amount of Cu adsorbed onto the r e s i n . However, a t a g i v e n pH and s a l i n i t y , the c o n c e n t r a t i o n of C u C l + , CuOH + and a l l o t h e r Cu complexes formed w i t h major c o n s t i t u e n t s i s d i r e c t l y p r o p o r t i o n a l t o the a c t i v i t y of C u 2 + , and thus t h e r e s i n i s s t i l l r e s p o n d i n g t o the c u p r i c i o n a c t i v i t y . Growth r a t e s were a l s o p l o t t e d as a f u n c t i o n of the pCu* d e t e r m i n e d by c a l c u l a t i o n and pCu as e s t i m a t e d from the r e s i n 134 a n a l y s i s . The r e l a t i o n s h i p was much s t r o n g e r w i t h the pCu e s t i m a t e d by the r e s i n a n a l y s i s than w i t h pCu* c a l c u l a t e d by the computer model. The weak r e l a t i o n s h i p between growth r a t e s and c a l c u l a t e d pCu* v a l u e s can be a t t r i b u t e d t o c a l c u l a t i o n i n a c c u r a c i e s g e n e r a t e d by e r r o r s i n the thermodynamic d a t a or e l s e t o undocumented i n t e r a c t i o n s . The r e s i n d i d not always respond p r o p e r l y t o the c o m p l e x a t i o n of Cu by the a d d i t i o n of an o r g a n i c c o m p l e x i n g a g e n t . Upon the a d d i t i o n of HIS t o a sample, a t c o n c e n t r a t i o n s known t o complex Cu, the amount of Cu bound t o the r e s i n was g r e a t e r than w i t h a s i m i l a r sample t h a t l a c k e d HIS, a r e s u l t a p p a r e n t l y due the a d s o r p t i o n of c a t i o n i c Cu-HIS complexes. T h i s a d s o r p t i o n of p o s i t i v e l y charged Cu-complexes i n d i c a t e s a p o t e n t i a l l i m i t a t i o n t o the t e c h n i q u e ' s a b i l i t y t o measure b i o l o g i c a l l y a c t i v e m e t a l i n n a t u r a l w a t e r s . The e x i s t e n c e of such a l i m i t a t i o n depends on the presence of t h e s e t y p e s of complexes i n the water body under s t u d y . I t i s t h u s u s e f u l t o examine the type of Cu-complexes t h a t may be e n c o u n t e r e d i n n a t u r a l w a t e r s . Many n a t u r a l w a t e r s can p a r t i a l l y mask the p r e s e n c e of m e t a l i o n s by r e n d e r i n g them u n a v a i l a b l e f o r measurement by c o n v e n t i o n a l c h e m i c a l (Chau et a _ l . , 1 974a,b) and b i o l o g i c a l (Davey et a l . , 1973; G i l l e s p i e and V a c c a r o , 1978) m e t a l s e n s i n g t e c h n i q u e s ; e v i d e n c e s u p p o r t s the concept t h a t t h i s masking i s due t o o r g a n i c c o m p l e x a t i o n of t h e s e m e t a l s (Duinker and Kramer, 1977). The r e p o r t e d d i s t r i b u t i o n of o r g a n i c a l l y a s s o c i a t e d Cu i n marine and f r e s h w a t e r has ranged from 0-100% ( e . g . , Slowey e t 135 a l . , 1967; W i l l i a m s , 1969; B a t l e y and Gardner, 1978). However, Mantoura (1981) r e p o r t s t h a t a c o n s i s t e n t average of c a . 20% of the t o t a l Cu i n seawater appears t o be i n an o r g a n i c form a l t h o u g h the number of m e t a l l o - o r g a n i c complexes a c t u a l l y i d e n t i f i e d i s v e r y s m a l l . T h e r e f o r e , the p r e s ence of m e t a l l o -o r g a n i c complexes i s g e n e r a l l y i n f e r r e d from the presence of o r g a n i c m a t e r i a l t h a t has the a p parent p o t e n t i a l t o complex m e t a l s . Many r e s e a r c h e r s have i m p l i c a t e d i n t e r m e d i a t e and h i g h m o l e c u l a r weight o r g a n i c compounds i n m e t a l l o - o r g a n i c i n t e r a c t i o n s (Ramamoorthy and Kushner, 1975a,b; Benes et a l . , 1976; G i l l e s p i e and V a c c a r o , 1978); w i t h humic compounds b e i n g most f r e q u e n t l y i n d i c a t e d because of t h e i r abundance i n n a t u r a l w a t e r s ( S c h n i t z e r and Khan, 1972). Low m o l e c u l a r weight o r g a n i c l i g a n d s (<200 AMU) are p o t e n t i a l l y c a p a b l e of b i n d i n g m e t a l s ( S i l l e n and M a r t e l , 1971) but the c o n c e n t r a t i o n of t h e s e compounds i s b e l i e v e d t o be v e r y low making i t u n l i k e l y t h a t they would complex m e t a l s t o any g r e a t degree (Duursma, 1970; Stumm and Morgan, 1970). In a d d i t i o n , the s t a b i l i t y c o n s t a n t s of t h e s e compounds f o r Cu ( e . g . , amino a c i d s ) a r e not p a r t i c u l a r l y l a r g e . The major f a c t o r d e t e r m i n i n g the a d s o r b a b i l i t y of Cu-o r g a n i c complexes i s the type and amount of e l e c t r i c a l c harge c a r r i e d by the o r g a n i c l i g a n d because t h i s w i l l g e n e r a l l y d etermine the o v e r a l l charge of the m e t a l l o - o r g a n i c complex. In the few r e p o r t s t h a t a r e a v a i l a b l e the d i s s o l v e d o r g a n i c m a t t e r (DOM) i n n a t u r a l water has been g e n e r a l l y found t o have a net 136 n e g a t i v e charge (Packlam, 1964). For example, N e i h o f and Loeb (1974) found t h a t when t e s t p a r t i c l e s of q u a r t z , germanium and an anion-exchange r e s i n were exposed t o n a t u r a l seawater, they a l l d i s p l a y e d n e g a t i v e s u r f a c e c h a r g e s . When t h e s e p a r t i c l e s were immersed i r i the same n a t u r a l seawater f o l l o w i n g i t s exposure t o u l t r a v i o l e t i r r a d i a t i o n t o p h o t o - o x i d i z e o r g a n i c m a t t e r , the s u r f a c e c h a r g e s were n e a r l y the same as when they were exposed t o a r t i f i c i a l seawater c o n t a i n i n g no o r g a n i c m a t t e r . They a t t r i b u t e d t h i s n e g a t i v e charge t o adsorbed o r g a n i c c o n s t i t u e n t s and l i s t e d p r o t e i n s , humic a c i d s and o t h e r s u b s t a n c e s d e r i v e d from the d e g r a d a t i o n of n a t u r a l p r o d u c t s of marine and t e r r e s t r i a l o r i g i n as p o s s i b l e a gents because they are s u r f a c e - a c t i v e polymers t h a t c a r r y a net n e g a t i v e charge a t the pH of seawater (from Duursma, 1965). R a s h i d (1971) found t h a t when b o t h d i v a l e n t and t r i v a l e n t m e t a l s r e a c t e d w i t h humic a c i d s of marine o r i g i n t he m e t a l s appeared t o l o s e t h e i r i o n i c c h a r a c t e r i s t i c s . When u s i n g a c a t i o n exchange r e s i n , P i l l a i e t a l . (1971) found t h a t the major f r a c t i o n of A l , Fe, Cu and Zn i n humic a c i d e x t r a c t s of marine sediments escaped t h r o u g h the r e s i n a l t h o u g h some s m a l l f r a c t i o n of Cu d i d undergo some exchange. S i n c e the Cu-humate complex was not adsorbed by the r e s i n t h i s i n d i c a t e d t h a t the complex d i d not c a r r y a net p o s i t i v e c h a r g e . The d a t a a v a i l a b l e suggest t h a t much or most of the m e t a l l o - o r g a n i c complexes i n n a t u r a l waters bear a n e g a t i v e c h a r g e . The presence of p o s i t i v e l y charged Cu-complexes i s , n e v e r t h e l e s s , s t i l l p o s s i b l e and, t h u s , must be c o n s i d e r e d when 137 a p p l y i n g the r e s i n t e c h n i q u e t o n a t u r a l w a t e r s . Another p o t e n t i a l problem when a p p l y i n g the r e s i n t e c h n i q u e t o n a t u r a l water samples i s w i t h r e s p e c t t o Fe. The a d d i t i o n of Fe t o samples i n c r e a s e d the uptake of Cu by the r e s i n a l t h o u g h a l l o t h e r f a c e t s of the a n a l y s i s appeared t o be u n a f f e c t e d . I t was a l s o found t h a t f r e s h l y p r e p a r e d Fe s t o c k s were more r e a c t i v e than aged Fe s t o c k s . I f Fe i s p r e s e n t i n a n a t u r a l seawater sample then i t s h o u l d be added t o the Cu s t a n d a r d s so the c o n d i t i o n s of a d s o r p t i o n w i l l be the same i n both the sample and the .standard. However, i f the Fe i n the sample i s u n r e a c t i v e w i t h r e s p e c t t o the r e s i n , the a d d i t i o n of f r e s h l y p r e p a r e d Fe t o the s t a n d a r d s , which i s r e a c t i v e , would b i a s the r e s u l t s . O b v i o u s l y , t h i s problem can be r e s o l v e d o n l y by f u r t h e r study of the n a t u r e of the Fe i n n a t u r a l seawater and i t s r e a c t i v i t y w i t h r e s p e c t t o the r e s i n . 138 V. APPLICATION OF THE RESIN TECHNIQUE TO NATURAL SEAWATER A. INTRODUCTION Many s t u d i e s have examined the c o m p l e x i n g c a p a c i t y of n a t u r a l w a t e r s ( e . g . , Chau et. a_l. , 1974a, b; D u i n k e r and Kramer, 1977; G u r t i s e n et a l . , 1977). T h i s ' c a p a c i t y ' r e p r e s e n t s the e x t e n t t o which the o r g a n i c or i n o r g a n i c c o n s t i t u e n t s of n a t u r a l waters can b i n d a m e t a l i o n by c o m p l e x a t i o n or a d s o r p t i o n (Chau and Wong, 1976). The c h e m i c a l s t r u c t u r e s of the n a t u r a l compounds r e s p o n s i b l e f o r the s t r o n g i n t e r a c t i o n s w i t h metal i o n s have not y e t been e l u c i d a t e d (Srna e_t a l . , 1980), a l t h o u g h r e c e n t work ( T r i c k e t a l . , 1983) s u g g e s t s t h a t p h y t o p l a n k t o n m e t a b o l i t e s may p l a y a r o l e i n the c o m p l e x i n g c a p a c i t y of n a t u r a l w a t e r s . A number of b i o l o g i c a l (Davey e t a l . , 1973; G i l l e s p i e and V a c c a r o , 1978) and c h e m i c a l t e c h n i q u e s (Chau et. a l . , 1974a; Shuman and Woodward, 1977; S t o l z b e r g and R o s i n , 1977; Sugai and H e a l y , 1978) have been d e v e l o p e d t o q u a n t i f y the c o m p l e x i n g c a p a c i t y of n a t u r a l w a t e r s . The v a l u e s v a r y c o n s i d e r a b l y but Mantoura (1981) e s t i m a t e s an average of 0.27 iM Cu l " 1 f o r e s t u a r i n e and c o a s t a l marine w a t e r s . I t has been suggested t h a t the o b s e r v e d v a r i a t i o n s i n Cu t o x i c i t y (Davey et a l . , 1973; Anderson and M o r e l , 1978) and s u c c e s s i o n of p h y t o p l a n k t o n s p e c i e s (Gachter e t a l . , 1973) c o u l d be due t o d i f f e r e n c e s i n the c o m p l e x a t i o n c a p a c i t i e s of n a t u r a l w a t e r s . Sunda and Lewis (1978) found t h a t by i n c r e a s i n g the q u a n t i t y of r i v e r water, which c o n t a i n e d a h i g h c o n c e n t r a t i o n of o r g a n i c m a t t e r , t o t h e i r c u l t u r e medium, the t o x i c i t y of Cu was 1 39 p r o g r e s s i v e l y reduced. Srna et a l . (1980) demonstrated a s t r o n g n e g a t i v e c o r r e l a t i o n between c o m p l e x a t i o n c a p a c i t y and Cu t o x i c i t y f o r v a r i o u s b o d i e s of water t h a t i n c l u d e d a f r e s h w a t e r l a k e , open ocean w a t e r s , s u r f zones, bays and c o a s t a l l a g o o n s . In the p r e s e n t s t u d y , a l o c a l marine i n l e t was chosen as a sa m p l i n g s i t e f o r seawater c o l l e c t i o n because p r e v i o u s r e p o r t s i n d i c a t e d t h a t i t had a measurable c o m p l e x i n g c a p a c i t y . E r i c k s o n (1973) i n f e r r e d from ASV measurements t h a t the Cu i n the i n l e t was p a r t i a l l y complexed by n a t u r a l l y o c c u r r i n g d i s s o l v e d o r g a n i c s o l u t e s . Q u a n t i t a t i v e i n f o r m a t i o n on the. co m p l e x i n g c a p a c i t y of the i n l e t was p r e s e n t e d by W h i t f i e l d and Lewis (1976). They r e p o r t e d c o m p l e x i n g c a p a c i t i e s as EDTA e q u i v a l e n t v a l u e s and found v a l u e s of up t o 5.6 x 10" 7M. They c o n c l u d e d t h a t t h e i n l e t ' s c o m p l e x i n g a b i l i t y v a r i e d t h r o u g h the year and, subsequent t o w i n t e r i n t r u s i o n s , t h e r e was an i n c r e a s e i n t he c o m p l e x a t i o n c a p a c i t y due t o the i n t r u s i o n of o r g a n i c m a t e r i a l . To t e s t t h e a b i l i t y of the r e s i n t e c h n i q u e t o measure the c o m p l e x a t i o n of Cu by n a t u r a l l y o c c u r r i n g c o m p l e x i n g a g e n t s , seawater w i t h a c o m p l e x i n g c a p a c i t y was n e c e s s a r y . Seawater from I n d i a n Arm was c o l l e c t e d from a s e r i e s of depths i n the e x p e c t a t i o n t h a t i t had a c o m p l e x i n g a b i l i t y and t h a t t h i s a b i l i t y would change w i t h d e pth ( W h i t f i e l d and L e w i s , 1976). In a d d i t i o n , t o v e r i f y t h a t the r e s i n was r e s p o n d i n g t o b i o l o g i c a l l y a c t i v e m e t a l , b i o a s s a y s w i t h T. pseudonana were conducted on the same water samples. Assays were a l s o c a r r i e d out u s i n g seawater s o l u b l e c o m p l e x i n g agents e x t r a c t e d from 1 40 marine sediments. These agents had p r e v i o u s l y been shown t o reduce Cu t o x i c i t y (Lewis et. aJL. , 1973). B. COLLECTION OF NATURAL SEAWATER Seawater was c o l l e c t e d i n 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 near Vancouver, B.C. ( F i g . 2 5 ) , which i s c o n n e c t e d t o the S t r a i t of G e o r g i a t h r o u g h B u r r a r d I n l e t and the p o r t of Vancouver. The s i l l a t the e n t r a n c e of the I n l e t i s o n l y 26 meters (m) deep w h i l e the depth of the c e n t r a l b a s i n i s over 200 m. There i s a c h a r a c t e r i s t i c two 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 w i t h a t h i n l a y e r of r e l a t i v e l y b r a c k i s h water f l o w i n g out a t the s u r f a c e and a compensating i n f l o w of d e n s e r , more s a l i n e water below t h i s ( G i l m a r t i n , 1962). S i g n i f i c a n t exchange and o v e r t u r n of the d e e p o w a t e r o c c u r s o n l y i n t e r m i t t e n t l y w i t h the exchange b e i n g p a r t i a l l y c o n t r o l l e d by the volume of f r e s h water 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 of the S t r a i t of G e o r g i a water ( D a v i d s o n , 1979). 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 the I n l e t and h y d r o g r a p h i c d a t a a r e g i v e n i n g r e a t d e t a i l by G i l m a r t i n (1962) and Davidson (1979). Water samples from f i v e depths were t a k e n a t s t a t i o n IND-2 (49°23.5'N, 122°52.5'W) which i s l o c a t e d a t the s o u t h e r n end of the deepest p o r t i o n of the b a s i n . F o r t y l i t e r s of seawater from each depth were c o l l e c t e d w i t h a 96-1 f i b e r g l a s s and l u c i t e sampler ( W h i t f i e l d and L e w i s , 1976). The samples were f i l t e r e d w i t h i n 6 hr of c o l l e c t i o n t h r o u g h a Gelmann 293 mm, 0.45 ixm f i l t e r . F i v e l i t e r s of sample were passed t h r o u g h the f i l t e r as a r i n s e which was d i s c a r d e d b e f o r e each sample was f i l t e r e d . 141 The f i l t e r e d samples were s t o r e d i n 23-1 p o l y e t h y l e n e c o n t a i n e r s a t 4°C i n the dark. P r i o r t o use, the s t o r a g e c a r b o y s were r i n s e d w i t h 6N HCL, soaked i n 0.5N HCL f o r 3 days, r i n s e d 3X w i t h GDW and f i n a l l y , f i l l e d w i t h GDW. H y d r o g r a p h i c d a t a were c o l l e c t e d a t the s p e c i f i c depths u s i n g NIO sampling b o t t l e s ( N a t i o n a l I n s t i t u t e of Oceanography, Wormley, U.K.). Temperatures were d e t e r m i n e d w i t h r e v e r s i n g thermometers (±0.01°C) and water samples were drawn f o r s a l i n i t y d e t e r m i n a t i o n s . Oxygen d e t e r m i n a t i o n s were done on board s h i p u s i n g a m o d i f i e d W i n k l e r t e c h n i q u e ( C a r r i t t and C a r p e n t e r , 1966). In a d d i t i o n , a c o n t i n u o u s p r o f i l e of te m p e r a t u r e and c o n d u c t i v i t y was completed u s i n g a CTD. Figure 25. Location of sample c o l l e c t i o n . 144 C. MATERIALS AND METHODS 1.. P r e l i m i n a r y Sample P r e p a r a t i o n For the r e s i n and b i o a s s a y t e s t s conducted on each d e p t h , a 10-1 a l i q u o t of sample was removed from the s t o r a g e c a r b o y and put i n a 12-1 p o l y p r o p y l e n e c a r b o y . To reduce 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 l b o t t l e s and sample c a r b o y s went t h r o u g h the same c l e a n i n g procedure as d e s c r i b e d i n the p r e v i o u s s e c t i o n . Once t r a n s f e r e d t o the 12-1 carboy the pH of the sample was measured and then a d j u s t e d t o 8.05±.05 by b u b b l i n g the sample w i t h a c i d c l e a n e d (HzSO,,), f i l t e r e d (0.45 Mm) a i r . A l l samples were below pH 8.05 i n i t i a l l y and b u b b l i n g t i m e s ranged from 6-12 hours depending on the sample. A f t e r b u b b l i n g , the 10-1 sample was passed t h r o u g h a S a r t o r i u s membrane f i l t e r (0.45 Mm) t o remove any p a r t i c u l a t e matter formed d u r i n g the b u b b l i n g p r o c e d u r e or d u r i n g the s t o r a g e p e r i o d ( a l g a l g r o w t h ) . The f i l t r a t e was c o l l e c t e d i n a 12-1 p o l y c a r b o n a t e c a r b o y . The f i r s t l i t e r of f i l t r a t e was d i s c a r d e d t o reduce c o n t a m i n a t i o n from the f i l t e r . The 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 i n the sample was d e t e r m i n e d by ASV. The sample was then ready t o be used i n b o t h t h e b i o a s s a y t e s t s and the r e s i n a n a l y s i s . 2. B i o a s s a y s The b i o a s s a y p r o c e d u r e f o l l o w e d t h a t used i n S e c t i o n I I I , w i t h the e x c e p t i o n t h a t the a u t o c l a v i n g p r o c e d u r e was e l i m i n a t e d because of the e f f e c t heat may have on the o r g a n i c s p r e s e n t i n 145 the water. An o u t l i n e of the bioassay procedure i s given below. For each bioassay, 4 1 of a prepared sample was t r a n s f e r r e d to a 4-1 Pyrex a s p i r a t o r b o t t l e . To ensure adequate n u t r i e n t c o n c e n t r a t i o n s over the term of the bioassay, the A q u i l n u t r i e n t s and t r a c e metals were added at t h e i r A q u i l c o n c e n t r a t i o n s with the Fe stock being f r e s h l y prepared and allowed t o p r e c i p i t a t e f o r s e v e r a l hours before i t s a d d i t i o n . A l i q u o t s (250 ml) of the sample were then t r a n s f e r r e d to 15 p r e v i o u s l y a u t o c l a v e d 500 ml polycarbonate Erylenmeyer f l a s k s and Cu was added at c o n c e n t r a t i o n s of 0.0, 3.93, 7.87, 11.8 or 15.7 x 10" 8M. The samples were l e f t f o r three hours to e q u i l i b r a t e . (Each Cu c o n c e n t r a t i o n was run i n t r i p l i c a t e . ) F i n a l l y , the medium was i n n o c u l a t e d with enough t e s t organism to o b t a i n an i n i t i a l c e l l number between 1000-2000 c e l l m l " 1 . C e l l numbers were monitored over a f i v e day p e r i o d by a C o u l t e r Counter In a d d i t i o n to the b i o a s s a y s with the n a t u r a l water samples, a bioassay u s i n g low s a l i n i t y SOW was performed. GDW was added to SOW u n t i l the s a l i n i t y was the same as the Indian Arm 200 m sample (S=27.5 p p t ) . The pH was measured and a d j u s t e d to pH 8.01.05. The same bioassay procedure was then f o l l o w e d as g i v e n above. 3. C a l i b r a t i o n of the Resin i n Low S a l i n i t y SOW Since s a l i n i t y has been p r e v i o u s l y shown to i n f l u e n c e the r e s i n a n a l y s i s , the r e s i n was c h a r a c t e r i z e d i n a r t i f i c i a l seawater (SOW) that had been d i l u t e d to the s a l i n i t y of the 146 n a t u r a l w a t e r s a m p l e s . T h r e e t e s t s w e r e c o n d u c t e d . T h e f i r s t t e s t w a s t o d e t e r m i n e t h e v o l u m e n e e d e d t o b r i n g t h e r e s i n i n t o e q u i l i b r i u m w i t h t h e s a m p l e . T h e s e c o n d w a s t o t e s t i f t h e a d s o r p t i o n o f C u b y t h e r e s i n w a s s t i l l l i n e a r a t t h e s e l o w e r s a l i n i t i e s . T h e f i n a l t e s t e x a m i n e d t h e a d s o r p t i o n o f C u b y t h e r e s i n i n t h e p r e s e n c e o f t h e A q u i l n u t r i e n t s a n d t r a c e m e t a l s . C o l u m n p r e p a r a t i o n , c o l u m n p r o c e d u r e , a n d t h e A S V t e c h n i q u e u s e d t o q u a n t i f y t h e C u i n t h e e l u a t e w e r e a l l t h e s a m e a s d e s c r i b e d i n S e c t i o n I V . B . E x c e p t t h a t , a f l o w r a t e o f 2 . 9 m l m i n " 1 w a s u s e d . I n a r e s i n e q u i l i b r i u m e x p e r i m e n t , S O W a t a s a l i n i t y o f 2 7 . 5 p p t w a s p r e p a r e d b y d i l u t i n g S O W ( 3 5 p p t ) w i t h G D W . T h e p H w a s m e a s u r e d a n d a d j u s t e d t o 8 . 0 5 ± . 0 5 w i t h 0 . 1 N N a O H . C o p p e r w a s t h e n a d d e d t o a 2 - 1 a l i q u o t t o g i v e a f i n a l C u c o n c e n t r a t i o n o f 7 . 8 7 x - 1 0 " 8 M . T e n c o l u m n s w e r e p r e p a r e d a n d 5 0 , 1 0 0 , 1 5 0 , 2 0 0 , 2 5 0 o r 3 0 0 m l w a s p a s s e d t h r o u g h t h e a p p r o p r i a t e c o l u m n w i t h t h e c o l u m n s b e i n g r u n i n d u p l i c a t e . F i n a l l y , t h e c o l u m n s w e r e e l u t e d w i t h a c i d i f i e d S O W ( p H c a . 1 . 0 ) a n d t h e e l u a t e s a n a l y s e d f o r C u b y A S V . I n a s a l i n i t y e x p e r i m e n t , t h r e e a d s o r p t i o n c u r v e s w e r e g e n e r a t e d u s i n g S O W a t 2 4 . 0 , 2 5 . 5 a n d 2 7 . 5 p p t t o t e s t t h e l i n e a r i t y o f a d s o r p t i o n a t t h e a p p r o x i m a t e s a l i n i t i e s o f t h e n a t u r a l w a t e r s a m p l e s . S a m p l e s w e r e p r e p a r e d b y d i l u t i n g S O W w i t h G D W . S O W o f e a c h s a l i n i t y w a s r u n i n a s e p a r a t e e x p e r i m e n t . F o r e a c h e x p e r i m e n t , f i v e 5 0 0 m l s a m p l e s w e r e p r e p a r e d i n 5 0 0 m l p o l y p r o p y l e n e b o t t l e s . T o t h e s e s a m p l e s , t h e A q u i l n u t r i e n t s a n d t r a c e m e t a l s i n c l u d i n g F e w e r e a d d e d a t 147 t h e i r A q u i l c o n c e n t r a t i o n . Each sample was then s p i k e d w i t h 0.0, 3.93, 7.87, 11.8, or 15.7 x 10" 8M Cu and l e f t f o r two hours to e q u i l i b r a t e . F i n a l l y , 250 ml of each sample were passed t h r o u g h the a p p r o p r i a t e column w i t h each sample b e i n g run i n d u p l i c a t e . The columns were e l u t e d w i t h a c i d i f i e d SOW (pH ca . 1.0) and the e l u a t e s a n a l y s e d f o r Cu by ASV. In a d d i t i o n , the e xperiment u s i n g the 25.5 ppt SOW was r e p e a t e d except t h a t the 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 s were not added. 4. A p p l i c a t i o n of the Ion-Exchange Method t o N a t u r a l Seawater In most c a s e s , the r e s i n and b i o a s s a y t e s t s were conducted on the same b a t c h of p r e p a r e d seawater. For the r e s i n a n a l y s i s , f o u r 500 ml a l i q u o t s of a p r e p a r e d seawater sample were t r a n s f e r e d 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 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 s were added a t t h e i r A q u i l c o n c e n t r a t i o n s t o a l l samples. The Fe s t o c k was f r e s h l y p r e p a r e d and a l l o w e d t o p r e c i p i t a t e f o r s e v e r a l hours b e f o r e use. Each sample was then s p i k e d w i t h 3.93, 7.87, 11.8 or 15.7 x 10 - 8M Cu. The samples were l e f t f o r 12 hr t o a l l o w e q u i l i b r a t i o n between Cu and the s o l u t i o n . E i g h t r e s i n columns were p r e p a r e d and 250 ml of each of the f o u r s o l u t i o n s were run t h r o u g h the a p p r o p r i a t e column w i t h each sample b e i n g run i n d u p l i c a t e . The columns were e l u t e d w i t h a c i d i f i e d SOW and the e l u a t e s a n a l y s e d f o r Cu by ASV. To c a l i b r a t e the sample columns, a s t a n d a r d SOW s o l u t i o n was p r e p a r e d a t the same t i m e , i n the same manner, and then run th r o u g h a s e t of columns under e x a c t l y the same c o n d i t i o n s used 148 f o r t he sample columns. The s t a n d a r d s o l u t i o n s were made up i n SOW (35 ppt) d i l u t e d w i t h GDW t o the same s a l i n i t y as the sample under study.. The same n u t r i e n t s and t r a c e m e t a l s added t o the the n a t u r a l water samples were added t o the s t a n d a r d s . I f n e c e s s a r y , the pH of the s t a n d a r d was a d j u s t e d t o the pH of the samples. In most t e s t s Cu was added t o g i v e a f i n a l c o n c e n t r a t i o n of 7.87 x 10 _ 8M i n the s t a n d a r d s o l u t i o n . In another e x p e r i m e n t , the r e s i n a n a l y s i s of the 50 m seawater sample was r e p e a t e d w i t h o u t n u t r i e n t or t r a c e m e t a l a d d i t i o n . The r e s u l t s of the a n a l y s i s w i t h and w i t h o u t n u t r i e n t s were then compared t o i n d i c a t e the e f f e c t of the A q u i l n u t r i e n t s . The s t a n d a r d s used f o r c a l i b r a t i o n of the columns i n t h i s experiment were made up i n SOW d i l u t e d t o the s a l i n i t y of the 50 m sample, w i t h o u t the a d d i t i o n of n u t r i e n t s , but w i t h the a d d i t i o n of 7.87 x 10" 8M Cu. 5. Manganese-Copper I n t e r a c t i o n S i n c e d i s s o l v e d Mn c o n c e n t r a t i o n s a r e h i g h i n I n d i a n Arm wat e r s ( W h i t f i e l d , 1974), the Mn c o n c e n t r a t i o n s i n the water samples were 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 i n t o the g r a p h i t e f u r n a c e of an atomic a b s o r p t i o n spectrophotometer.. I n j e c t i o n volumes were 50 /il. A 1000 mg 1" 1 Mn s t o c k was p r e p a r e d by the a d d i t i o n of MnCl 2«4H 20 t o GDW. Mn s t a n d a r d s were made up from t h i s s t o c k i n SOW d i l u t e d t o a s a l i n i t y of 25 ppt w i t h GDW. The s t a n d a r d s were a c i d i f i e d w i t h 0.05 ml of i s o t h e r m a l l y d i s t i l l e d 5N HCL. The samples t o be a n a l y s e d were a c i d i f i e d t o pH 2 by the a d d i t i o n of 5N HC1 and l e f t f o r two days. A s t a n d a r d c u r v e 149 was g e n e r a t e d and the Mn c o n c e n t r a t i o n s of the samples were d e t e r m i n e d by i n t e r p o l a t i o n from t h i s graph. To t e s t the e f f e c t of Mn on t h e response of the organism t o Cu, two b i o a s s a y s were run s i m u l t a n e o u s l y u s i n g the seawater from the 50 m d e p t h , where t h e ambient Mn c o n c e n t r a t i o n was l o w e s t . The b i o a s s a y p r o c e d u r e was the same as d e s c r i b e d f o r the o t h e r n a t u r a l water samples except t h a t , i n one of the b i o a s s a y s , 0.05 ml of a 1000 mg 1~ 1 Mn s t o c k was added, which gave a f i n a l Mn c o n c e n t r a t i o n of 3.64 x 10" 6M. In the second of the two b i o a s s a y s Mn was o n l y p r e s e n t i n the A q u i l c o n c e n t r a t i o n p l u s t h e background c o n c e n t r a t i o n (3.9 x 10" 8M). The b i o a s s a y s were run f o r f i v e days and the growth of t h e organism was m o n i t o r e d d a i l y w i t h a C o u l t e r C o u n t e r . 6. N a t u r a l Seawater S o l u b l e Agents E x t r a c t e d from Sediments The r e s i n and b i o a s s a y t e c h n i q u e s were a l s o used t o d e t e c t the c o m p l e x a t i o n of Cu by n a t u r a l o r g a n i c s e x t r a c t e d from s e d i m e n t s . Sediment (1000 g) from I n d i a n Arm was added t o 8 1 of SOW (35 ppt) i n a 10-1 round bottom f l a s k . The m i x t u r e was s t i r r e d f o r two days u s i n g a l a r g e t e f l o n c o a t e d s t i r r i n g bar i n c o m b i n a t i o n w i t h a magnetic s t i r r e r and then a l l o w e d t o s e t t l e . The SOW was pumped out of the f l a s k w i t h a p e r i s t a l t i c pump and passed t h r o u g h a 192 mm S a r t o r i u s membrane f i l t e r (0.45 nm) to remove p a r t i c u l a t e m a t t e r . The f i n a l f i l t r a t e was c o l l e c t e d i n a 12-1 p o l y c a r b o n a t e c a r b o y . The pH of SOW d i d not change w i t h a d d i t i o n of the sediment and the t o t a l Cu c o n c e n t r a t i o n i n the sample was below the ASV d e t e c t i o n l i m i t of 1.57 x 10" 9M. 150 B e f o r e the b i o a s s a y or r e s i n a n a l y s e s were conducted the f i l t r a t e was d i l u t e d w i t h GDW t o a s a l i n i t y of 27.5 p p t . Four l i t e r s were then used i n the b i o a s s a y and 2.5 l i t e r s i n the r e s i n a n a l y s e s . The pH was measured but no adjustment was n e c e s s a r y . In the b i o a s s a y , f o u r Cu c o n c e n t r a t i o n s (3.93, 7.87, 11.8 and 15.7 x 10~ 8M) were used. The same b i o a s s a y p r o c e d u r e was f o l l o w e d as i n S e c t i o n V.B.2. C e l l numbers were m o n i t o r e d over a f i v e day p e r i o d by a C o u l t e r C o u n t e r . For the r e s i n a n a l y s i s , f i v e 500 ml a l i q u o t s of the p r e p a r e d sample were t r a n s f e r r e d 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 o which the 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 s were then added a t t h e i r A q u i l c o n c e n t r a t i o n . The samples were s p i k e d w i t h 3.93, 7.87, 11.8 or 15.7 x 10" 8M Cu. The samples were a l l o w e d t o e q u i l i b r a t e f o r 12 hr b e f o r e 250 ml of each was passed t h r o u g h the a p p r o p r i a t e column. Each sample was run i n d u p l i c a t e . The columns were then e l u t e d w i t h a c i d i f i e d SOW and the e l u a t e s a n a l y s e d f o r Cu by ASV. Two s t a n d a r d columns were used t o c a l i b r a t e the sample columns. The s t a n d a r d s o l u t i o n was made up i n SOW d i l u t e d t o the same s a l i n i t y and w i t h the same n u t r i e n t a d d i t i o n as the samples. Cu was added t o g i v e a c o n c e n t r a t i o n of 7.87 x 10" 8M. 151 D. RESULTS 1. P r e l i m i n a r y T e s t s The s a l i n i t y of the I n d i a n Arm samples was lower than t h a t of the medium used t o c h a r a c t e r i z e the r e s i n i n S e c t i o n IV. S i n c e s a l i n i t y has been shown t o a f f e c t the r e s i n , p r e l i m i n a r y t e s t s were conducted t o c h a r a c t e r i z e the r e s i n a t the s a l i n i t y of the n a t u r a l samples. In the f i r s t e x p e r i m e n t , the volume of sample needed t o a c h i e v e column e q u i l i b r a t i o n was d e t e r m i n e d . As i n SOW of s a l i n i t y 35 p p t , a sample volume of 250 ml appeared t o be the minimum volume n e c e s s a r y t o b r i n g the r e s i n i n t o e q u i l i b r i u m w i t h the s o l u t i o n ( F i g . 2 6 ) . The amount of Cu bound t o the r e s i n a f t e r 300 ml was s l i g h t l y h i g h e r than a f t e r 250 ml, but t h e d i f f e r e n c e was w i t h i n the e x p e r i m e n t a l e r r o r of the t e c h n i q u e . Once a g a i n , 250 ml was chosen as the sample volume f o r a l l e x p e r i m e n t s i n t h i s s e c t i o n . In the second e x p e r i m e n t , t h r e e Cu a d s o r p t i o n c u r v e s were g e n e r a t e d u s i n g SOW d i l u t e d t o 24.0, 25.5 and 27.5 ppt t o d e t e r m i n e i f the a d s o r p t i o n of Cu by the r e s i n was l i n e a r a t t h e s e lower s a l i n i t i e s . F i g . 27 shows a l i n e a r a d s o r p t i o n c u r v e a t t h e s e s a l i n i t i e s a l t h o u g h the l i n e of b e s t f i t f o r a l l c u r v e s does not go t h r o u g h the o r i g i n . T h i s i s a t t r i b u t e d t o the a d s o r p t i o n of Cu by the r e s i n b e i n g l e s s a t a low s o l u t i o n Cu c o n c e n t r a t i o n (3.93 x 10* 8M) than a t h i g h e r Cu l e v e l s . For example, a t a s a l i n i t y of 25.5 ppt the a d d i t i o n of 3.93 x 10" 8M Cu g i v e s an a d s o r p t i o n of 2.97 x I 0 ~ 9 m o l Cu per gram of r e s i n . 152 F i g u r e 26. E l u a t e Cu v e r s u s the e f f l u e n t volume f o r the e q u i l i b r i u m e x p e r i m e n t . V a l u e s a r e a mean of 2 r e p l i c a t e s ±1 s.d. 1.0-1 Eff luent Volume (mis) 153 However, w i t h an a d d i t i o n a l 3.93 x 10" 8M s p i k e of Cu t o SOW, the amount of Cu bound t o the r e s i n i s 6.88 x I 0 " 9 m o l which c o r r e s p o n d s t o a 3.91 x I 0 " 9 m o l i n c r e a s e . T h i s i s an i n c r e a s e of a p p r o x i m a t e l y 1.0 x I 0 " 9 m o l Cu over t h a t of the f i r s t s p i k e and such an i n c r e a s e a c c o u n t s f o r the change i n s l o p e of the a d s o r p t i o n c u r v e s . These r e s u l t s suggest t h a t p o s s i b l y not a l l Cu was b e i n g e l u t e d and t h a t a c o n s t a n t amount was r e t a i n e d (or l o s t i n some o t h e r f a s h i o n ) by each column. The d i f f e r e n c e i n the a d s o r p t i o n of Cu a t low and h i g h s o l u t i o n Cu c o n c e n t r a t i o n s i s not g r e a t but i n d i c a t e s the a d v i s a b i l i t y of u s i n g s t a n d a r d s t h a t c l o s e l y resemble the Cu l e v e l s i n the samples t o be a n a l y s e d . In the experiment t o d e t e r m i n e the a d s o r p t i o n of Cu i n the pr e s e n c e of the 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 s , a d s o r p t i o n c u r v e s were g e n e r a t e d i n 25.5 ppt s a l i n i t y SOW w i t h and w i t h o u t the a d d i t i o n of n u t r i e n t s or t r a c e m e t a l s . From F i g . 27, i t i s c l e a r t h a t the s l o p e of the c u r v e i n c r e a s e s when a l l the s t o c k s were added. The i n c r e a s e in' s l o p e was e x p e c t e d and was a t t r i b u t e d t o the Fe a d d i t i o n ; the e f f e c t of which has been p r e v i o u s l y d e t e r m i n e d i n SOW a t 35 ppt ( F i g . 2 1 ) . 2. B i o a s s a y s At a l l the added Cu l e v e l s , the samples from the 100 and 200 m depths s u p p o r t e d b e t t e r growth than d i d the samples from 10 or 50 m. The g r e a t e s t d i f f e r e n c e i n growth was seen between the 50 and 200 m samples ( T a b l e X X ) . For example, when Cu was added t o the 50 m water a t 11.8 x 10" 8M the growth r a t e of the 154 F i g u r e 27. A d s o r p t i o n c u r v e s f o r Cu u s i n g low s a l i n i t y SOW w i t h and w i t h o u t the a d d i t i o n of Fe. Symbols: • 24 p p t ; A 25.5 p p t ; and 0 27.5 ppt SOW. • 25.5 ppt SOW w i t h no Fe a d d i t i o n . V a l u e s a r e a mean of 2 r e p l i c a t e s ±1 s.d. 1 5 n 155 organism was reduced by 66% but, with the same addition of Cu to the 200 m water, the growth rate was reduced by only 13%. The 75 m water also supported better growth than did the shallower waters but not to the same extent as the deeper waters. The differences found in the growth rates were i n i t i a l l y interpreted as differences in the concentration of natural complexing agents in the samples. It f i r s t appeared that the complexation of Cu by natural complexing agents was much greater in the deep waters of 100 and 200 m than in the shallower waters of 10, 50 and 75 m. The growth patterns found in these bioassays were then compared to the growth pattern obtained in a bioassay using SOW that was di l u t e d to the s a l i n i t y of the natural water samples (Fig. 28). The growth pattern of the 10 and 50 m seawater most clo s e l y resembled the pattern found in the di l u t e d SOW, although better growth was s t i l l seen at the Cu levels in the natural waters. This was an indication that there was s t i l l some reduction in the b i o l o g i c a l l y active Cu by natural complexing agents or some other mechanism in the shallower water samples. 3 . Application of the Resin Technique to Natural Seawater The results of the resin analysis are reported as Cu equiv as described in Section IV.D.2. To r e i t e r a t e , the Cu equiv value of a sample i s dependent on both the t o t a l Cu concentration and the extent to which free Cu i s complexed by the ligands in solution. This value w i l l be equivalent to the t o t a l Cu concentration of the sample only when the ligands T a b l e XX. Growth r a t e s of the b i o a s s a y o r g a n i s m i n seawater t a k e n from f i v e depths i n I n d i a n Arm. A l s o growth r a t e s i n SOW a t 27.5 ppt a r e p r e s e n t e d . Depth Cu added Growth Rate % of C o n t r o l m (X10" 8M) ( d i v d a y " 1 ) SOW 0.1 1 .7510.03 1 (27.5 3.9 1.34±0.05 77±3 PP t ) 7.9 1.07±0.06 61 ±4 11.8 0.70±0.03 40±2 15.7 0.39±0.03 22±2 10 0.1 2.33±0.03 3.9 2.23±0.07 96±3 7.9 1.55±0.02 66±1 11.8 0.98±0.03 42±1 15.7 0.86±0.07 37±3 50 0. 1 2.07±0.04 3.9 1.82±0.02 88±1 7.9 0.86±0.02 42±1 11.8 0.77±0.03 34±1 15.7 0.71±0.03 34±1 75 0.1 2.36±0.01 3.9 2.34±0.02 99±1 7.9 1.95±0.01 82±0 11.8 1.34±0.02 57±1 15.7 1.10±0.02 47±1 100 0.1 2.07±0.02 3.9 1.97±0.04 95±2 7.9 1.93±0.01 93±0 11.8 1.75±0.02 85±1 15.7 1.53±0.10 74±5 200 0.1 2.42±0.01 3.9 2.33±0.02 96±1 7.9 2.27±0.00 94±0 11.8 2.11±0.01 87±1 15.7 1.73±0.06 7 1 ±3 Mean ±1 s.d. based on 3 r e p l i c a t e s . 157 F i g u r e 28. Growth r a t e (% of c o n t r o l ) v e r s u s the t o t a l Cu c o n c e n t r a t i o n i n the n a t u r a l water samples. Symbols: • 10 meter; A 50 meter; 0 7 5 meter; O 100 meter; and V 200 meter samples; • SOW a t 27.5 p p t . Bars a r e ±1 s.d. 158 p r e s e n t i n the sample a r e the same as those p r e s e n t i n the s t a n d a r d SOW used f o r the c a l i b r a t i o n of the sample. In SOW, the h y d r o x i d e and c a r b o n a t e i o n s a r e the o n l y major i o n s thought t o a f f e c t Cu s p e c i a t i o n . T h e r e f o r e , a d i f f e r e n c e between the Cu eq u i v v a l u e and the t o t a l Cu c o n c e n t r a t i o n w i l l g i v e an i n d i c a t i o n of the e x t e n t of Cu 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 than the h y d r o x i d e and c a r b o n a t e i o n s . The Cu e q u i v v a l u e s f o r the v a r i o u s samples a r e p r e s e n t e d i n T a b l e XXI. The h i g h e s t v a l u e s a r e found i n the 100 m seawater. In f a c t , t h e s e v a l u e s a r e h i g h e r than the c o n c e n t r a t i o n of Cu added t o the samples. However, t h i s i s due t o t h e h i g h background c o n c e n t r a t i o n of Cu i n the water from t h i s d e pth (3.95 x 10~ 8M), and when the Cu e q u i v l e v e l i s compared t o the t o t a l Cu c o n c e n t r a t i o n the v a l u e s a r e a c t u a l l y found t o be lower (Table X X I ) . The 75 m water has the lo w e s t v a l u e s but the d i f f e r e n c e s between i t and the 10, 50 and 200 m samples a r e m a r g i n a l . The d i f f e r e n c e between the t o t a l Cu c o n c e n t r a t i o n and the Cu e q u i v v a l u e s of a sample can be used t o i n d i c a t e the e x t e n t of Cu c o m p l e x a t i o n by n a t u r a l l y o c c u r r i n g c o m p l e x i n g a g e n t s . The Cu e q u i v v a l u e s f o r a l l d e p t h s were lower than the t o t a l Cu c o n c e n t r a t i o n p r e s e n t i n the samples. A l s o from the Cu e q u i v v a l u e s i t was i n f e r r e d t h a t the 100 m water had the l e a s t c o m p l e x i n g a b i l i t y w h i l e the 75 m had the h i g h e s t . The d i f f e r e n c e s found between the 10, 50 and 200 m water were w e l l w i t h i n the e x p e r i m e n t a l e r r o r of the t e c h n i q u e . T a b l e X X I . R e s u l t s of r e s i n a n a l y s i s c o n ducted on the n a t u r a l water samples. Depth Cu added T o t a l Cu Cu e q u i v % A c t i v e 2 (m) (X10" 8M) (X10" 8M) (x10" 8M) 10 3.9 5.46 3.01±.25 1 55 7.9 9.40 5.821.41 62 11.8 13.33 9.731.13 73 15.7 17.23 12.921.17 75 50 3.9 5.36 2.601.01 49 7.9 9.30 6.121.13 66 11.8 13.23 9.661.25 73 15.7 17.13 14.701.01 86 75 3.9 5.54 2.311.06 42 7.9 9.48 5.491.27 58 11.8 13.41 8.231.43 61 15.7 17.31 12.381.61 72 100 3.9 7.88 5.631.35 71 7.9 11.82 9.081.06 77 11.8 15.75 13.011.30 66 15.7 19.65 17.041.00 87 200 3.9 5.74 3.211.33 56 7.9 9.68 6.221.22 64 11.8 13.61 9.991.01 73 15.7 17.51 13.791.14 79 1Mean 11 s.d. based on 2 r e p l i c a t e s . 2Cu e q u i v / T o t a l Cu cone, x 100. 160 4. Comparison of the R e s i n and B i o a s s a y R e s u l t s The r e s u l t s of the r e s i n a n a l y s i s were compared t o the r e s u l t s of the b i o a s s a y s c o nducted on the same samples t o dete r m i n e i t s a b i l i t y t o e s t i m a t e b i o l o g i c a l l y a c t i v e Cu i n n a t u r a l seawater. In F i g . 29 the growth r a t e s of the b i o a s s a y o r g a n i s m a r e p l o t t e d as a f u n c t i o n of the - l o g of the Cu e q u i v v a l u e s as e s t i m a t e d by the r e s i n a n a l y s i s . I f the a d s o r p t i o n of Cu by the r e s i n was r e l a t e d t o the same f r a c t i o n of metal s p e c i e s t h a t were t o x i c t o the organism then a s i n g l e c u r v e would d e s c r i b e b oth s e t s of d a t a . From F i g . 29 i t i s e v i d e n t t h a t t h e r e i s o n l y a weak a s s o c i a t i o n between growth r a t e and the Cu e q u i v v a l u e s . I t a l s o appears t h a t the r e l a t i o n s h i p i s dependent on the depth from which the water sample was o b t a i n e d . G e n e r a l l y , as the d e p t h i n c r e a s e s the Cu e q u i v v a l u e needed t o cause a s p e c i f i c d e c r e a s e i n growth a l s o i n c r e a s e s . The p l o t , i n f a c t , c l o s e l y resembles the p a t t e r n found when growth r a t e s were p l o t t e d as a f u n c t i o n of the t o t a l Cu c o n c e n t r a t i o n ( F i g . 2 8 ) . I t i s apparent from t h e s e r e s u l t s t h a t the r e s i n a n a l y s i s cannot be used t o e s t i m a t e t h e t o x i c i t y of t h i s p a r t i c u l a r s e t of n a t u r a l water samples. W i t h the s u c c e s s of the t e c h n i q u e under l a b o r a t o r y c o n d i t i o n s , however, o t h e r p o s s i b l e reasons t o e x p l a i n t h e s e r e s u l t s were examined. In the r e s i n t e c h n i q u e i t i s assumed t h a t the r e s i n responds t o the f r e e c u p r i c i o n a c t i v i t y or t h a t the response i s p r o p o r t i o n a l t o t h i s v a l u e as l o n g as p o s i t i v e l y c h a r g e d Cu-o r g a n i c complexes, such as HIS, a r e not p r e s e n t . I f p o s i t i v e l y 161 F i g u r e 29. Growth r a t e (% of c o n t r o l ) v e r s u s the - l o g of the Cu e q u i v v a l u e s . Symbols: A 10 meter; • 50 meter; <> 75 meter; V 100 meter; and O 200 meter samples. Bars a r e ±1 s.d. 6.7 7.0 7.3 7.6 7.9 - log Cu equiv 1 62 charged Cu-complexes a r e p r e s e n t , the r e s i n i s l i k e l y t o adsorb t h e s e complexes which would r e s u l t i n an o v e r e s t i m a t i o n of the c u p r i c i o n a c t i v i t y ( h i g h e r Cu e q u i v v a l u e s ) of the sample. The r e s i n a n a l y s i s would then i n d i c a t e the medium t o be more t o x i c than i t a c t u a l l y i s and the growth r a t e s found i n t h i s medium would be h i g h e r than e x p e c t e d from the r e s i n a n a l y s i s . T h i s i s a p o s s i b l e e x p l a n a t i o n f o r the r e s u l t s o b t a i n e d i n the n a t u r a l samples. I f p o s i t i v e l y c h a r g e d o r g a n i c s were p r e s e n t i n the 100 and 200 m samples then the r e s i n a n a l y s i s would p r e d i c t t h e s e samples t o be much more t o x i c than the b i o a s s a y s would show and a poor r e l a t i o n s h i p between Cu e q u i v v a l u e s and growth r a t e would be found. Another e x p l a n a t i o n f o r the d i s c r e p a n c y between the r e s i n and b i o a s s a y t e c h n i q u e s c o n c e r n s the assumptions made i n the b i o a s s a y t e c h n i q u e . The p r i n c i p a l a s sumption i s t h a t the response of the organi s m i s c o n s t a n t f o r a s p e c i f i c c u p r i c i o n a c t i v i t y . However, a p h y s i o l o g i c a l i n t e r a c t i o n between Cu and Mn has r e c e n t l y been d e s c r i b e d (Sunda et. a l . , 1981) which i n d i c a t e s t h a t w i t h i n c r e a s i n g Mn, the t o x i c i t y of Cu d e c r e a s e s . I f t h i s o c c u r r e d i n the I n d i a n Arm samples, the change i n t o x i c i t y c o u l d have been due t o changes i n the Mn c o n c e n t r a t i o n and not t o any c o m p l e x a t i o n r e a c t i o n s a f f e c t i n g the Cu c h e m i s t r y . W h i t f i e l d (1974) found t h a t Mn i n c r e a s e d c o n s i d e r a b l y i n the deeper waters of I n d i a n Arm, a t the same s t a t i o n where the water samples were c o l l e c t e d f o r the p r e s e n t study (IND-2). He found Mn l e v e l s 6 t o 10 ti m e s g r e a t e r i n the deep water than i n 163 the s u r f a c e w a t e r . Because of t h i s t he Mn c o n c e n t r a t i o n s i n the I n d i a n Arm water samples were d e t e r m i n e d . A d r a m a t i c d i f f e r e n c e was seen i n the Mn c o n c e n t r a t i o n between the s u r f a c e and deep waters ( T a b l e X X I I ) . The 200 m water had a Mn c o n c e n t r a t i o n a p p r o x i m a t e l y two o r d e r s of magnitude g r e a t e r than d i d the 10, 50 and 75 m w a t e r s ; the 200 m water had a Mn c o n c e n t r a t i o n of 3.64 x 10" 6M w h i l e the l o w e s t Mn l e v e l was found i n the seawater from 50 m a t 1.63 x 10" 8M. T a b l e X X I I . H y d r o g r a p h i c and t r a c e m e t a l d a t a from water samples c o l l e c t e d from f i v e depths a t s t a t i o n IND-2. Depth Temp. S a l i n i t y Oxygen T o t a l Cu T o t a l Mn pH (M) (°c) (ppt) (ml l " 1 ) ( X 1 0 " 8 M ) (x10" 7M) 10 1 1 .89 23.432 5.81 1.53±.161 0.601.06' 7.96 50 9.24 25.961 4.60 1.43±.08 0.161.01 7.67 75 8.28 26.886 2.88 1.61±.11 1.301.11 7.65 100 8.10 27.200 2.28 3.951.22 5.491.44 7.39 200 8.05 27.503 0.70 1.81±.07 36.4011.5 7.58 'Mean 11 s.d. based on 3 r e p l i c a t e s S i n c e a l a r g e d i f f e r e n c e was found i n the Mn c o n c e n t r a t i o n s of the n a t u r a l samples, t h e e f f e c t of Mn on the growth response of the b i o a s s a y organism t o Cu was t e s t e d . Two b i o a s s a y s were performed on the water c o l l e c t e d from the 50 m de p t h where the Mn l e v e l was l o w e s t . In t h e f i r s t b i o a s s a y , Mn was added t o the 164 sample a t t h e Mn c o n c e n t r a t i o n of the 200 m seawater (3.64 x 10~ 6M), w h i l e i n the second b i o a s s a y , Mn was added o n l y i n the A q u i l c o n c e n t r a t i o n . Both b i o a s s a y s were run s i m u l t a n e o u s l y . The growth p a t t e r n s of the organism o b t a i n e d i n the b i o a s s a y s w i t h and w i t h o u t the a d d i t i o n of Mn a r e p r e s e n t e d i n F i g . 30. I t i s e v i d e n t t h a t the a d d i t i o n of Mn has an impact on the growth r a t e of the organism a t a l l the Cu c o n c e n t r a t i o n s s t u d i e d . The medium w i t h the h i g h c o n c e n t r a t i o n of Mn had much h i g h e r growth r a t e s than d i d the medium h a v i n g low Mn. In f a c t , the growth p a t t e r n observed i n the medium c o n t a i n i n g h i g h Mn c o n c e n t r a t i o n s c l o s e l y resembles the growth p a t t e r n observed i n the n a t u r a l water samples from 100 and 200 m, both of which had h i g h Mn c o n c e n t r a t i o n s . I t a ppears t h a t the response of the organism t o Cu changes w i t h the Mn c o n c e n t r a t i o n of the sample. In the n a t u r a l water samples t h e n , i n c r e a s e d growth r a t e s a r e e x p e c t e d i n the 100 and 200 m samples as compared t o the s h a l l o w e r w a t e r s because of the h i g h Mn c o n c e n t r a t i o n p r e s e n t even though the r e s i n a n a l y s i s e s t i m a t e d 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 m e t a l i n the deep wat e r s t o be g r e a t e r than i n the s h a l l o w e r w a t e r s . 4. Study of Water S o l u b l e Agents from Sediments The. a b i l i t y of the r e s i n a n a l y s i s t o d e t e c t any c o m p l e x a t i o n of Cu by h i g h l e v e l s of n a t u r a l c o m p l e x i n g agents was t e s t e d . N a t u r a l seawater s o l u b l e agents were e x t r a c t e d from a sediment taken from I n d i a n Arm by m i x i n g the sediment w i t h a r t i f i c i a l seawater (SOW) f o r a s p e c i f i e d p e r i o d of t i m e . There 165 F i g u r e 30. The e f f e c t of Mn on r e d u c i n g the t o x i c i t y of Cu. Symbols: • 50 meter sample w i t h no Mn added; • 50 meter sample w i t h 3.64 x 10" 6M Mn added. Bars are ±1 s .d. 100 _ 6.6 6.8 7.0 log Cu 7.2 7.4 166 was no attempt t o i d e n t i f y the type or determine the q u a n t i t y of any o r g a n i c agents p r e s e n t . The sample was t i t r a t e d w i t h Cu and the amount of r e s i n a c t i v e m e t a l a t each Cu c o n c e n t r a t i o n was measured. A b i o a s s a y was then conducted on the sample u s i n g the same Cu c o n c e n t r a t i o n s as the r e s i n a n a l y s i s and the amount of b i o l o g i c a l l y a c t i v e m e t a l was e s t i m a t e d . The r e s u l t s of the sediment study a re p r e s e n t e d i n Ta b l e X X I I I . T a b l e X X I I I . R e s i n and b i o a s s a y r e s u l t s from the sediment study.. Cu added Cu e q u i v % A c t i v e 3 Growth r a t e % of (x 10" 8M) (x 10" 8M) ( d i v s d a y 1 ) C o n t r o l 0. 1 n.d. 1 .79±0.02 1 3.93 1 .29±0.04 2 32 1 •75±0.06 98 7.87 2.31±0.01 29 1 .85±0.02 1 02 11.8 3.30±0.08 28 1 .85±0.02 104 15.7 4.66±0. 1 1 30 1 .89±0.01 1 06 1Mean ±1 s.d. based on 3 r e p l i c a t e s 2Mean ±1 s.d. based on 2 r e p l i c a t e s 3Cu e q u i v / T o t a l Cu cone, x 100 In the b i o a s s a y t e s t s i t was found t h a t Cu c o u l d be added i n c o n c e n t r a t i o n s of up t o 15.7 x 10" 8M w i t h no d e l e t e r i o u s e f f e c t . H i g h growth r a t e s were seen i n c u l t u r e s a t a l l Cu c o n c e n t r a t i o n throughout the f o u r days of growth; a f t e r the f o u r t h day a l l c u l t u r e s had reached scenescence. The h i g h growth r a t e s i n d i c a t e d t h a t t h e r e was c o n s i d e r a b l e c o m p l e x a t i o n of Cu i n t h i s medium such t h a t the t o x i c form of the me t a l was 167 reduced t o below the c o n c e n t r a t i o n t h a t a f f e c t s the organism. The growth r a t e i n t h e c u l t u r e s h a v i n g h i g h e r Cu l e v e l s were s l i g h t l y g r e a t e r than f o r low Cu c o n c e n t r a t i o n s . The e x p l a n a t i o n f o r t h i s may be the same as t h a t g i v e n f o r the r e s u l t s o b t a i n e d when u s i n g h i g h EDTA or HIS c o n c e n t r a t i o n s (see S e c t i o n I I I . D . 1 ) . The r e s i n a n a l y s i s , c o n ducted on s i m i l a r samples as the b i o a s s a y , a l s o i n d i c a t e d s t r o n g c o m p l e x a t i o n of Cu i n t h i s medium. The Cu e q u i v v a l u e s d e t e r m i n e d f o r each Cu c o n c e n t r a t i o n was a t most 32% of the t o t a l Cu c o n c e n t r a t i o n as r e p r e s e n t e d by the % a c t i v e v a l u e i n Ta b l e X X I I I . A l s o , the v a l u e s were below the l e v e l s t h a t would cause a t o x i c response i n the b i o a s s a y organism e x c e p t f o r the sample h a v i n g the l a r g e s t Cu s p i k e (15.7 x 10~ BM) where a Cu e q i v v a l u e of 4.66 x 10" 8M Cu was found i n the sample. As compared t o the e x p e r i m e n t s w i t h model o r g a n i c l i g a n d s i n S e c t i o n I I I , t h e r e s h o u l d be a r e d u c t i o n i n growth r a t e a t the Cu e q u i v l e v e l found i n the sample w i t h the l a r g e s t Cu s p i k e b u t , as was i n d i c a t e d by the b i o a s s a y , t h e r e was no t o x i c r e s p o n s e . T h i s c o u l d be e x p l a i n e d , once a g a i n , by the presence of h i g h Mn c o n c e n t r a t i o n s found i n the samples; l e v e l s above 1 mg l " 1 were found i n t h e samples as measured by g r a p h i t e f u r n a c e AAS. 168 E. DISCUSSION In S e c t i o n IV, i t was demonstrated t h a t the ion-exchange e q u i l i b r i u m method c o u l d be used t o e s t i m a t e b i o l o g i c a l l y a c t i v e Cu i n a r t i f i c i a l seawater when the s p e c i a t i o n of Cu was a f f e c t e d by v a r i o u s o r g a n i c l i g a n d s . In t h i s s e c t i o n , a s i m i l a r e x p e r i m e n t a l approach was t a k e n e x c e p t t h a t n a t u r a l seawater c o n t a i n i n g n a t u r a l c o m p l e x i n g agents was used t o t e s t the i o n -exchange t e c h n i q u e . Seawater was c o l l e c t e d a t v a r i o u s depths from a l o c a l f j o r d whose wat e r s had p r e v i o u s l y been shown t o have a c o m p l e x i n g c a p a c i t y and then t e s t e d by t h e proposed b i o a s s a y and r e s i n t e c h n i q u e s . In t h e b i o a s s a y s , the growth p a t t e r n s o b t a i n e d f o r the range of Cu c o n c e n t r a t i o n s used were d i f f e r e n t f o r each n a t u r a l water sample a l t h o u g h the 100 and 200 m samples were v e r y s i m i l a r . The 100 and 200 m samples appeared t o s u p p o r t much b e t t e r growth than d i d the samples from the 10, 50 or 75 m depths ( F i g . 2 8 ) . T h i s was i n i t i a l l y i n t e r p r e t e d as an a b i l i t y of the deep waters t o s e q u e s t e r Cu due t o the p r e s e n c e of n a t u r a l c o m p l e x i n g a g e n t s . In a d d i t i o n , i t appeared t h a t the s h a l l o w e r w a t e r s s e q u e s t e r e d Cu t o a l i m i t e d degree as was i n d i c a t e d by t h e s e waters s u p p o r t i n g b e t t e r growth than d i d a r t i f i c i a l seawater (SOW) h a v i n g no o r g a n i c l i g a n d s p r e s e n t . C o m p l e x a t i o n of Cu i n t h e n a t u r a l water samples was a l s o i n d i c a t e d by the r e s i n a n a l y s i s . T h i s was shown by the Cu e q u i v v a l u e s of a l l samples b e i n g lower than the t o t a l Cu c o n c e n t r a t i o n . The l o w e s t p e r c e n t a g e of complexed m e t a l was seen i n the 100 m seawater ( h i g h % a c t i v e v a l u e s ) w h i l e s i m i l a r 169 c o m p l e x a t i o n was found i n the 10, 50, 75 and 200 m. A l t h o u g h the Cu e q u i v v a l u e s of the n a t u r a l water samples were lower than t h e i r c o r r e s p o n d i n g t o t a l Cu c o n c e n t r a t i o n , t h e s e v a l u e s were such t h a t a t o x i c response i n the organism was e x p e c t e d . From t h e s e v a l u e s i t appeared t h a t the 100 m sample s h o u l d have the l o w e s t growth r a t e s w h i l e b e t t e r growth was e x p e c t e d i n the 10, 50, 75 and 200 m samples. However, t h e s e c o n c l u s i o n s were c o n t r a d i c t e d by the a c t u a l b i o a s s a y d a t a . The 100 and 200 m samples had the h i g h e s t growth r a t e s w h i l e the 10 and 50 m waters were found t o have the l o w e s t . The weak r e l a t i o n s h i p between the r e s i n a n a l y s i s and the b i o a s s a y s was b e s t i l l u s t r a t e d when the - l o g of the Cu e q u i v v a l u e s were p l o t t e d as a f u n c t i o n of the growth r a t e s ( F i g . 2 9 ) . T h i s weak r e l a t i o n s h i p was not a t t r i b u t e d t o an. e r r o r i n the r e s i n a n a l y s i s but t o a p h y s i o l o g i c a l Cu-Mn i n t e r a c t i o n found i n the b i o a s s a y organism. Such an i n t e r a c t i o n has been r e c e n t l y d i s c u s s e d i n d e t a i l f i r s t by Sunda e t a l . (1981) and then Sunda and Huntsman ( i n p r e p . ) . Sunda e_t a l . (1981) found t h a t the a d d i t i o n of Mn t o t h e i r c u l t u r e s of the d i a t o m C h a e t o c e r o s s o c i a l i s c o u l d r e v e r s e the t o x i c e f f e c t of Cu. They d i s c u s s e d the p o s s i b i l i t y of Cu a d s o r p t i o n t o manganese o x i d e s formed by the o x i d a t i o n of M n ( l l ) t o e x p l a i n t h i s antagonism. The a d s o r p t i o n of Cu would reduce the c u p r i c i o n a c t i v i t y of the medium and s u b s e q u e n t l y reduce i t s t o x i c i t y . However, they p r e s e n t e d e v i d e n c e t o show t h a t the k i n e t i c s of o x i d a t i o n of M n ( l l ) t o manganese o x i d e s a r e e x c e e d i n g l y slow a t the pH of seawater. They added 2 x 10" 5M M n C l 2 t o seawater c o l l e c t e d a t 170 800 m and then s t o r e d the s o l u t i o n a t room temperature f o r seven y e a r s . At the end of t h i s p e r i o d , they c o n c l u d e d t h a t the manganese was a l l i n the s o l u b l e manganous form and t h a t no d e t e c t a b l e o x i d a t i o n had o c c u r r e d . To f u r t h e r s u p p o r t t h e i r a ssumption they found t h a t e x c e e d i n g l y low c o n c e n t r a t i o n s of manganese were r e q u i r e d t o r e v e r s e the t o x i c n a t u r e of Cu t o t h e i r t e s t organism. They doubted t h a t a t such low Mn c o n c e n t r a t i o n s , even i f a l l the M n ( l l ) was o x i d i z e d t o manganese o x i d e s , enough Cu c o u l d be absorbed t o r e v e r s e Cu t o x i c i t y t o the e x t e n t found i n t h e i r t e s t s . The presence of reduced Mn i s a l s o l i k e l y i n n a t u r a l s eawater. Sunda et. a l . ( 1 983) p r e s e n t e d e v i d e n c e t h a t p h o t o r e d u c t i o n of manganese o x i d e s by 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 o c c u r s i n seawater. They s t a t e d t h a t such r e a c t i o n s appeared t o be i m p o r t a n t i n m a i n t a i n i n g manganese i n a d i s s o l v e d reduced form i n p h o t i c w a t e r s . W i t h the a d s o r p t i o n of Cu by Mn u n l i k e l y , Sunda e_t a l . . (1981) proposed t h a t the growth s t i m u l a t i o n by Mn must be p h y s i o l o g i c a l . The a b i l i t y of Mn t o r e v e r s e Cu t o x i c i t y was e x p l a i n e d by a c o m p e t i t i v e i n t e r a c t i o n between the two m e t a l s a t c e l l u l a r s i t e s i n v o l v e d i n manganese n u t r i t i o n . The s i t e s c o u l d be e i t h e r s u r f a c e or i n t r a c e l l u l a r s i t e s . F u r t h e r , they d e s c r i b e d a c e l l u l a r model t o e x p l a i n the r e l a t i o n s h i p between the growth r a t e of t h e i r t e s t organism and the i o n i c a c t i v i t i e s of Mn and Cu. A t w o - s i t e c e l l u l a r b i n d i n g model was dev e l o p e d i n which Cu competes w i t h Mn a t one of the s i t e s but o n l y Cu r e a c t s a t the second. A two s i t e model was d e v e l o p e d because 171 they found t h a t growth r a t e was dependent on the r a t i o of manganous i o n a c t i v i t y t o c u p r i c i o n a c t i v i t y a t low c u p r i c i o n a c t i v i t i e s b u t , a t h i g h e r c u p r i c i o n a c t i v i t i e s , the dependence of Cu t o x i c i t y on manganous a c t i v i t y was not n e a r l y as pronounced. The exact mechanism of the Cu-Mn growth r a t e .antagonism has not y e t been c l e a r l y e l u c i d a t e d . In the p r e s e n t study the c o m p e t i t i o n between Cu and Mn f o r n u t r i t i o n a l s i t e s o n / i n the c e l l c o u l d e x p l a i n the weak r e l a t i o n s h i p between the Cu e q u i v v a l u e s and growth r a t e s . W i t h t o x i c l e v e l s of Cu, growth r a t e s would i n c r e a s e as the Mn c o n c e n t r a t i o n i n c r e a s e d even though the c u p r i c i o n a c t i v i t y of the s o l u t i o n remained c o n s t a n t . T h i s t y p e of response was found i n the n a t u r a l water samples of I n d i a n Arm. As the Mn c o n c e n t r a t i o n i n c r e a s e d i n the samples t h e i r t o x i c i t y d e c r e a s e d even though the r e s i n a n a l y s i s e s t i m a t e d the samples t o have s i m i l a r 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 s . F u r t h e r e v i d e n c e t o s u p p o r t the Cu-Mn antagonism was found i n the b i o a s s a y experiment u s i n g Cu s p i k e d 50 m seawater samples w i t h and w i t h o u t the a d d i t i o n of Mn. When Mn was added t o the 50 m water a t the same Mn c o n c e n t r a t i o n found i n the 200 m seawater ( 3 . 6 4 x 1 0 _ 6 M ) , the growth r a t e s were h i g h e r , a t a l l the Cu c o n c e n t r a t i o n s used, than t h a t of the 50 m water h a v i n g Mn added a t the A q u i l c o n c e n t r a t i o n . In f a c t , the growth p a t t e r n found at h i g h Mn l e v e l s c l o s e l y resembled the growth p a t t e r n found i n the b i o a s s a y s of the 100 and 200 m seawater which a l s o had h i g h Mn l e v e l s . Because of t h i s Mn i n t e r f e r e n c e , the b i o a s s a y t e c h n i q u e 172 c o u l d not be used t o e s t i m a t e the b i o l o g i c a l a c t i v i t y of the c u p r i c i o n i n samples e n r i c h e d w i t h Mn u n l e s s the r e l a t i o n s h i p between Mn and Cu was known and s u b s e q u e n t l y the measurement of b i o l o g i c a l l y a c t i v e m e t a l by the r e s i n c o u l d not be v e r i f i e d . E x a m i n a t i o n of the Cu-Mn i n t e r a c t i o n was beyond the scope of the p r e s e n t s t u d y but f u r t h e r work i s s t r o n g l y suggested as t h i s i s such an i m p o r t a n t c o n s i d e r a t i o n i n t o x i c i t y s t u d i e s . However, the measurements from the r e s i n a n a l y s i s d i d i n d i c a t e c o m p l e x a t i o n of Cu by c o m p l e x i n g agents i n the samples and t h e r e f o r e the t e c h n i q u e showed a p o t e n t i a l f o r measuring the c o m p l e x i n g c a p a c i t y of n a t u r a l w a t e r s . To f u r t h e r t e s t the p o t e n t i a l of the r e s i n f o r s t u d y i n g c o m p l e x i n g c a p a c i t y , the a d s o r p t i o n c u r v e f o r Cu i n the presence of c o m p l e x i n g agents e x t r a c t e d from sediments was examined i n low s a l i n i t y SOW. On a v e r a g e , o n l y 30% of each of the s p i k e d Cu c o n c e n t r a t i o n s was r e s i n a c t i v e , thus s u g g e s t i n g c o n s i d e r a b l e c o m p l e x a t i o n of the m e t a l . The b i o a s s a y performed on the same sample a l s o i n d i c a t e d s t r o n g c o m p l e x a t i o n of Cu. Growth r a t e s were a t t h e i r maximum f o r a l l the Cu c o n c e n t r a t i o n s used. However, the Cu e q u i v v a l u e i n the sample h a v i n g the l a r g e s t Cu s p i k e (15.7X10" 8M) was a t a l e v e l t h a t was e x p e c t e d t o cause t o x i c i t y . T h i s d i s c r e p a n c y c a n , a t l e a s t p a r t l y , be e x p l a i n e d by the presence of h i g h background Mn c o n c e n t r a t i o n s found i n the samples t h e r e b y r e d u c i n g the t o x i c i t y of Cu t o the organism. In c o n c l u s i o n , i t appears t h a t c o m p l e x a t i o n of Cu by n a t u r a l seawater s o l u b l e a gents d i d o c c u r , as demonstrated by the r e s u l t s of the r e s i n a n a l y s i s and measurement of 173 b i o l o g i c a l l y a c t i v e Cu by the b i o a s s a y t e c h n i q u e . The i n t e r p r e t a t i o n of the r e s u l t s , however, has some l i m i t a t i o n s because of the Cu-Mn i n t e r f e r e n c e . . 174 V I . GENERAL CONCLUSIONS The a d s o r p t i o n of Cu onto 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 of the s u l p h o n a t e type (AG 50W X12) can be used t o d i f f e r e n t i a t e Cu i n c a t i o n i c s p e c i e s from t h a t of a n i o n i c or uncharged s p e c i e s p r e s e n t i n seawater. The p r o c e d u r e i n v o l v e s b r i n g i n g the r e s i n i n t o e q u i l i b r i u m w i t h a seawater sample and then measuring the c o n c e n t r a t i o n of adsorbed Cu. Comparison w i t h Cu a d s o r p t i o n from s t a n d a r d Cu seawater s o l u t i o n s of s i m i l a r c o m p o s i t i o n , pH, and i o n i c s t r e n g t h y i e l d s a Cu e q u i v a l e n t measurement t h a t can be r e l a t e d t o the c o n c e n t r a t i o n (or a c t i v i t y ) of the f r e e c u p r i c i o n . A Cu e q u i v a l e n t v a l u e r e p r e s e n t s the c o n c e n t r a t i o n of Cu t h a t , when p r e s e n t i n o r g a n i c l i g a n d f r e e a r t i f i c i a l seawater, r e s u l t s i n the a d s o r p t i o n of an amount of Cu e q u a l t o t h a t adsorbed from the t e s t s o l u t i o n . In a r t i f i c i a l seawater (SOW), t h e a d d i t i o n of model o r g a n i c l i g a n d s (EDTA, NTA, GLU) d e c r e a s e s the a d s o r p t i o n of Cu by the r e s i n and the e x t e n t of t h i s d e c r e a s e i s r e l a t e d t o the c o n c e n t r a t i o n and the s t a b i l i t y of the C u - l i g a n d complex. Not a l l Cu c o m p l e x i n g a g e n t s shows such a re s p o n s e , however. When HIS i s added t o SOW, Cu a d s o r p t i o n i s a c t u a l l y h i g h e r than i n s i m i l a r samples l a c k i n g HIS. T h i s i n c r e a s e i s a t t r i b u t e d t o the a d s o r p t i o n of the p o s i t i v e l y c h a r g e d Cu-HIS complexes. From t h i s i t i s e v i d e n t t h a t c a r e must be taken when s t u d y i n g n a t u r a l water samples t h a t may c o n t a i n p o s i t i v e l y c harged o r g a n i c c o m p l e x i n g a g e n t s . A b i o a s s a y p r o c e d u r e was de v e l o p e d t h a t measured the b i o l o g i c a l l y a c t i v e f r a c t i o n of Cu i n seawater. B i o a s s a y s were 175 c o n d u c t e d on s i m i l a r a r t i f i c i a l seawater samples t o those used i n the r e s i n a n a l y s i s . A s t r o n g r e l a t i o n s h i p between the growth r a t e s and the Cu e q u i v a l e n t v a l u e s was found t h u s i n d i c a t i n g the a b i l i t y of the ion-exchange t e c h n i q u e t o measure b i o l o g i c a l l y a c t i v e Cu. The b i o a s s a y and r e s i n t e c h n i q u e s were then used on n a t u r a l seawater samples c o l l e c t e d from a l o c a l f j o r d . In the b i o a s s a y s , growth r a t e s were h i g h e r i n the samples from the 100 and 200 meter depths than i n the s h a l l o w e r water samples. However, as c o n c l u d e d from the Cu e q u i v a l e n t v a l u e s , the b i o l o g i c a l l y a c t i v e f r a c t i o n of Cu i n the water samples was s i m i l a r or even h i g h e r i n the deep waters ( t h e 100 meter water had the h i g h e s t Cu e q u i v v a l u e s ) . P l o t t i n g growth r a t e s as a f u n c t i o n of the - l o g of the Cu e q u i v v a l u e s demonstrated the weak r e l a t i o n s h i p between the r e s u l t s of the r e s i n and b i o a s s a y p r o c e d u r e s . However, the weak r e l a t i o n s h i p i s due t o a p h y s i o l o g i c a l Cu-Mn i n t e r a c t i o n t h a t a f f e c t s the b i o a s s a y org a n i s m and not t o a problem w i t h the r e s i n t e c h n i q u e . B i o a s s a y s i n the 50 meter water w i t h and w i t h o u t the a d d i t i o n of Mn a t the c o n c e n t r a t i o n found i n the 200 meter water (3.64x10" 6M), showed a d e c r e a s e i n Cu t o x i c i t y w i t h the a d d i t i o n of the Mn. In f a c t , the growth found i n the 50 meter water p l u s Mn c l o s e l y resembled the growth found i n the 200 meter sample. A l t h o u g h the measurement of b i o l o g i c a l l y a c t i v e Cu by the r e s i n t e c h n i q u e c o u l d not be v e r i f i e d by the b i o a s s a y t e s t s , i t was e v i d e n t t h a t the t e c h n i q u e was r e s p o n d i n g t o c o m p l e x a t i o n of Cu by n a t u r a l c o m p l e x i n g a g e n t s . T h i s was i n d i c a t e d by the Cu 176 e q u i v v a l u e s of a l l the n a t u r a l water samples b e i n g lower than t h e i r t o t a l Cu c o n c e n t r a t i o n . F u r t h e r m o r e , a h i g h l e v e l of c o m p l e x a t i o n was i n d i c a t e d by the r e s i n a n a l y s i s of SOW t h a t had been mixed w i t h a sediment p r e v i o u s l y shown t o c o n t a i n seawater s o l u b l e c o m p l e x i n g a g e n t s . The p o t e n t i a l of the ion-exchange method f o r s t u d y i n g Cu s p e c i a t i o n i n seawater has been demonstrated. The a n a l y t i c a l scheme can be a p p l i e d t o the d i r e c t measurement of b i o l o g i c a l l y a c t i v e Cu or may be i n c o r p o r a t e d i n t o o t h e r t y p e s of measurement, such as the d e t e r m i n a t i o n of the c o m p l e x i n g c a p a c i t y of n a t u r a l or p o l l u t e d w a t e r s . The t e c h n i q u e i s not o n l y l i m i t e d t o Cu but i s a p p l i c a b l e t o many of the o t h e r t r a c e m e t a l s p r e s e n t i n seawater. P r a c t i c a l advantages i n c l u d e low equipment c o s t s , r e l a t i v e l y f a s t a n a l y s i s t i m e , good s e n s i t i v i t y and b e i n g c o n d u c i v e t o a u t o m a t i o n . 177 V I I . REFERENCES CITED A b d u l l a h , M.I. and R o y l e , L.G., (1972). The d e t e r m i n a t i o n of copper, l e a d , cadmium, n i c k e l , z i n c and c o b a l t i n n a t u r a l waters by p u l s e p o l a r o g r a p h y . A n a l . Chim. A c t a 58:283-288. 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A note on the p o l a r o g r a p h i c b e h a v i o u r of the C u ( I I ) i o n s e l e c t i v e e l e c t r o d e i n seawater. Mar. Chem. 10:249-255. Z i r i n o , A. and Yamamoto, S., (1972). A pH-dependent model f o r the c h e m i c a l s p e c i a t i o n of co p p e r , z i n c , cadmium and l e a d i n seawater. L i m n o l . and Oceanogr. 17:661-671. APENNDIX A. BIOASSAY DATA 197 H i s t i d i n e L - H i s t i d i n e (1.0X10" 7M) (Sep 7/81) Cu C o n c e n t r a t i o n ( X 1 0 _ 8 M ) Days C o n t r o l 7.9 15.7 23.6 31.5 0-1 1 .00( .40) 1 .07 ( .18) 0.96( .06) 0.31 ( .10) 0. 17( . 1.2) 1-2 2 . 10( .03) 1 .97 ( .08) 1 .29( .07) 0.58( .05) 0.08( .06) 2-3 2 .04( .03) 1 .74( .02) 0.76( .05) 0.57( .07) 0.53( .38) 3-4 1 .56( .01 ) 1 .91 ( .02) 0.63( .01 ) 0.40( .05) 0. 19( .18) X 1-4 1 .90( .01 ) 1 .87( .03) 0.89( .02) 0.52( .01 ) 0.27( .07) % Of C o n t r o l 99 ( 2 ) 47 ( 1 ) 27 ( 1 ) 14 ( 3 ) L - H i s t i d i n e (2.5X10" 7M) (Aug 17/81) Cu C o n c e n t r a t i o n ( X 1 0 _ 8 M ) Days C o n t r o l 7.9 15.7 23.6 31.5 0-1 0 .24( .04) 0.30( .05) 0.27( .07) 0. 18( .06) 0.30( .11) 1-2 2 .45( .04) 2.37( .08) 2.46( .07) 2.08( .04) 1 .72( .11) 2-3 1 .98( .01 ) 2.06( .03) 2.08( .05) 1 . 19( .04) 0.79( .05) 3-4 i .86( .03) 1 .92 ( .09) 1 .87( .06) 1 . 16( .11) 0.56( .10) X 1-4 2 . 10( .01 ) 2. 1 1 ( .04) 2. 14( .04) 1 .47( .06) 1 .02 ( .06) % Of C o n t r o l 100 ( 2 ) 102 ( 2 ) 70 ( 3 ) 49 ( 3 ) 'Mean of K ±1 s.d. based on 3 r e p l i c a t e s . 1 9 8 H i s t i d i n e ( c o n ' t ) L - H i s t i d i n e ( 5 . 0 X 1 0 " 6 M ) ( J u l 2 7 / 8 1 ) Cu C o n c e n t r a t i o n ( x l O " 8 M ) Days C o n t r o l 7 . 9 1 5 . 7 2 3 . 6 3 1 . 5 0 - 1 0 . 1 8 ( . 0 4 ) 0 . 2 3 ( . 0 6 ) 0 . 1 6 ( . 0 7 ) 0 . 2 9 ( . 0 7 ) 0 . 3 0 ( . 0 4 ) 1 - 2 1 . 8 4 ( . 0 3 ) 1 . 8 8 ( . 0 2 ) 1 . 8 9 ( . 0 2 ) 1 . 8 6 ( . 0 3 ) 1 . 8 4 ( . 0 2 ) 2 - 3 2 • 0 8 ( . 0 4 ) 2 . 0 8 ( . 0 4 ) 2 . 1 9 ( . 0 1 ) 2 . 1 0 ( . 0 4 ) 2 . 0 0 ( . 1 3 ) 3 - 4 1 . 9 6 ( . 0 4 ) 1 . 9 8 ( . 0 3 ) 2 . 0 6 ( . 1 4 ) 1 . 9 9 ( . 0 1 ) 1 . 9 6 ( . 0 3 ) X 1 - 4 1 . 9 6 ( . 0 1 ) 1 . 9 8 ( . 0 1 ) 2 . 0 5 ( . 0 4 ) • 1 . 9 8 ( . 0 1 ) 1 . 9 3 ( . 0 3 ) % of C o n t r o l 199 NTA NTA (1..0X10" 7M) (Sep 28/82) Cu C o n c e n t r a t i o n ( x l O ' 8 M ) Days C o n t r o l 7.9 15.7 23.6 31 .5 0-1 0.83(.02) 0.93( .25) 0.68(. 03) 0.40( .11) 0. 37( .13) 1-2 2.55(.02) 1 .84( .04) 1 . 65(. ,07) 1 .68 ( .18) 1 . 14( .03) 2-3 2. 13(.06) 0.88( .04) 0.62(. 05) 0.61 ( .04) 0. 69( .02) 3-4 1 .22(.07) 0.78( .09) 0.35(. ,05) 0.31 ( .01 ) 0. 26( .01 ) X 1-4 1.97(.01) 1 . 17( .04) 0.87(. ,01 ) 0.86( .05) 0. 7 ( .02) % of C o n t r o l 59 ( 2 ) 44 ( 1 ) 44 ( 3 ) 35 ( 1 ) NTA (2 . 5 X 1 0 - 7 M ) (Oct 5/82) Cu C o n c e n t r a t i o n (X10 - 8 M ) Days C o n t r o l 7.9 i s . : 7 23. 6 31 . 5 0-1 1 . 25(.14) 1 .32 ( .03) 1.28(. .06) 1 .26( .12) 1 . 10( .07) 1-2 2.42(.14) 2. 1 1 ( .05) 1 . 84(. 06) 1 .67 ( .06) 1 . 78( .00) 2-3 2 . 0 9 ( . 1 4) 1 .24( .06) 0.74(. .05) 0.58( .07) 0. 46 ( .06) 3-4 1.16(.05) 1 .17( .04) 0.56(, .03) 0.40( .06) 0. 29( .04) X 1-4 1.89(.02) 1 .5 ( .05) 1 . 05(. .01 ) 0.88( .02) 0. 84( .04) % of C o n t r o l 79 ( 3 ) 55 ( 1 ) 47 ( 1 ) 45 i ( 2 ) 200 NTA (con't) NTA ( 5 . 0 X 1 0 " 7 M ) (Oct 19/82) Cu C o n c e n t r a t i o n ( X 1 0 " 8 M ) Days C o n t r o l 7.9 15.7 23.6 31.5 0-1 1 . O K . 0 7 ) 0 . 9 0 ( . 05 ) 0 . 9 8 ( . 1 4 ) 0 . 9 5 ( . 1 5 ) 1.01 ( . 1 0 ) 1-2 2 • 22 ( . 0 6 ) 2.32(1 . 7 3 2 . 0 7 ( . 1 3 ) 1 .85 ( . 0 8 ) 1 .87 ( . 1 3 ) 2 - 3 1 , 9 7 ( . 0 3 ) 1 . 73(. 02) 1 .01 ( . 0 3 ) 0 . 8 9 ( . 0 5 ) 0 .81 ( . 0 9 ) 3 - 4 1 . 9 2 ( .01 ) 1 . 6 1 ( . 05 ) 0 . 8 6 ( . 0 2 ) 0 . 6 5 ( . 0 5 ) 0 . 5 2 ( . 1 3 ) X 1-4 2 . 0 4 ( . 0 3 ) 1 .89 (. 04) 1 .31 ( . 0 3 ) 1 .09( . 0 8 ) 1 .07 ( . 0 4 ) % of C o n t r o l 93 ( 2 ) 64 ( 1 ) 54 ( 4 ) 52 ( 2 ) NTA ( 7 . 5 X 1 0 " 7 M ) (Jan 11/82) Cu C o n c e n t r a t i o n ( X 1 0 ' 8 M ) Days C o n t r o l 7.9 15.7 23.6 31.5 0-1 2 .43( .37) 2.47( .16) 2.13( .22) 2.71 ( .24) 2.56( .27) 1-2 1 .80( .07) 1 .90( .05) 1 .68 ( .04) 1 .62 ( .11) 1 .55 ( .06) 2-3 1 .82( .05) 1 .83( .09) 1 .37 ( .02) 1.01 ( .05) 0.81 ( .05) 3-4 1 .80( .01 ) 1 .80( .12) 1 -1 3 ( .01 ) 0.76( .05) 0.56( .07) X 1-4 1 .81 ( .04) 1 .84( .03) 1 .40 ( .02) 1 . 13( .03) 0.97( .02) % of C o n t r o l 102 ( 1 ) 77 ( 1 ) 63 ( 2 ) 54 ( 1 ) 201 G l u t a m i c A c i d L - G l u t a m i c A c i d (1..0x10 _ 5M) (Mar .21/82) Cu C o n c e n t r a t i o n (x10" 8M) Days C o n t r o l 7.9 15.7 23.6 31.5 0-1 1 .86( .07) 1 .84(.12) 1 .76( .15) 1 .63 ( .08) 1 .63 ( .05) 1-2 2 .32( .09) 2.11(.06) 1 .69( .07) 1 .65( .05) 1 .48 ( .03) 2-3 1 .53( .04) 1.23(.06) 0.89( .05) 0.72( .03) 0.68( .03) 3-4 1 .44( .05) 0.76(0.03 0.39( .03) 0.27( .05) 0.27( .07) X 1-4 1 .76( .03) 1.37(.05) 0.99( .05) 0.88( .01 ) 0.81 ( .02) % of C o n t r o l 78 ( 3 ) 56 ( 3 ) 50 ( 0 ) 46 ( 1 ) L - G l u t a m i c A c i d ( 2 . 5 X 1 0 " 5 M ) (Mar 15/82) Cu C o n c e n t r a t i o n ( X 1 0 ~ 8 M ) Days C o n t r o l 7.9 15.7 23.6 31.5 0-1 1 .60( .23) 1 .64 ( .10) 1 .55( .18) 1 .33 ( .15) 1 .20 ( .09) 1-2 2 .52( .13) 2.42( .14) 2.04( .08) 1 .80 ( .05) 1 .66( .04) 2-3 1 .43( .02) 1 .42( .12) 1 .01 ( .05) 0.79( .06) 0.75( .01 ) 3-4 1 .64( .17) 1 .55 ( .16) 0.81 ( .06) 0.48( .04) 0.40( .16) X 1-4 1 .86( .09) 1 .79 ( .05) 1 .29( .04) 1 .03 ( .01 ) 0.93( .05) % of C o n t r o l 96 ( 3 ) 69 ( 2 ) 55 ( 1 ) 50 ( 3 ) 202 G l u t a m i c A c i d ( c o n ' t ) L - G l u t a m i c A c i d (7..5X10" 5 M) (Sep 14/82) Gu C o n c e n t r a t i o n ( x l O ~ 8 M ) Days C o n t r o l 7.9 15.7 23.6 31.5 0-1 0.92( .05) 0.88(.06) 0.89(.08) 0.89( .13) 0.87( .04) 1-2 2.32( .03) 2.30(.09) 2 . 1 5 (. 1 2 ) 2.02( .08) 1 .95 ( .08) 2-3 2.06( .02) 2.08(.04) 1 .73(.08) 1 .50 ( .11) 1 .34( .05) 3-4 1 .54( .02) 1.53(.05) 1.82(.08) 1 .35( .11) 1 . 2 9 ( .07) X 1-4 1 .98( .01 ) 1.97(.02) 1.91(.09) 1 .62 ( .10) 1 .52( .05) % of C o n t r o l 100 ( 1 ) 97 ( 5 ) 82 ( 5 ) 77 ( 2 ) L - G l u t a m i c Ac i d ( 1 . 0 x 1 0 " 5 M and 2.5x10" 5 M ) (Jun 7/82) Cu C o n c e n t r a t i o n ( X 1 0 " 8 M ) 1.0X10" 5 2 . 5 X 1 0 " 5 M Days C o n t r o l 7.9 15.7 7.9 15.7 0-1 1 .71( .25) 1 .49( .07) 1 .75( .09) 1 ,89( .13) 1 .99( .17) 1-2 2 .21 ( .05) 1 .89 ( .07) 1 .62( .03) 2.37( .06) 1 .84( .04) 2-3 1 .81 ( .04) 1 .22 ( .09) 0.84( .05) 1 .79 ( .03) 1 .21 ( .05) 3-4 1 • 76( .04) 0.95( .06) 0.43( .03) 1 .62 ( .08) 0.91 ( .03) X 1-4 1 .93( .02) 1 .35( .07) 0.96( .00) 1 .93( .05) 1 .32 ( .03) % of C o n t r o l 70 ( 3 ) 50 ( 0 ) 100 ( 2 ) 68 ( 2 ) 203 G l u t a m i c A c i d ( c o n ' t ) L - G l u t a m i c A c i d ( 5 . 0 X 1 0 _ 5 M a n d 7 . 5 X 1 0 ~ 5 M ) ( J u n 1 4 / 8 2 ) C u C o n c e n t r a t i o n ( X 1 0 _ 8 M ) 5 . 0 X 1 0 " 5 M 7 . 5 X 1 0 " 5 M D a y s C o n t r o l 7 . 9 1 5 . 7 7 . 9 1 5 . 7 0 - 1 1 . 1 8 ( . 1 2 ) 1 . 3 0 ( . 0 4 ) 1 . 3 5 ( . 0 8 ) 1 . 3 4 ( . 1 9 ) 1 . 3 2 ( . 0 3 ) 1 - 2 2 . 2 1 ( . 0 6 ) 2 . 3 2 ( . 1 2 ) 2 . 1 6 ( . 0 4 ) 2 . 1 5 ( . 1 1 ) 2 . 2 8 ( . 2 1 ) 2 - 3 1 . 7 8 ( . 0 3 ) 1 . 7 9 ( . 0 5 ) 1 . 6 8 ( . 0 9 ) 1 . 9 3 ( . 1 0 ) 1 . 5 3 ( . 2 3 ) 3 - 4 2 . 0 3 ( . 0 4 ) 1 . 9 4 ( . 0 4 ) 1 . 4 1 ( . 0 3 ) 1 . 9 4 ( . 0 7 ) 1 . 9 1 ( . 0 3 ) X 1 - 4 2 . 0 0 ( . 0 4 ) 2 . 0 2 ( . 0 4 ) 1 . 7 5 ( . 0 2 ) 2 . 0 1 ( . 0 1 ) 1 . 9 1 ( . 0 2 ) % o f C o n t r o l 1 0 1 ( 2 ) 8 7 ( 1 ) 1 0 0 ( 1 ) 9 5 ( 1 ) 204 EDTA EDTA (5.0x10" 8M) (May 24/82) Cu C o n c e n t r a t i o n ( X 1 0 " 8 M ) Days C o n t r o l 3.9 7.9 1 1 . 8 15. 7 0-1 0.54(.02) 0.66( .08) 0.64( .13) 0.54( .18) 0.52( .05) 1-2 1 . 58(.13 ) 1 .58 ( .06) 1 .54 ( .08) 1 .31 ( .13) 1 .21 ( .07) 2-3 1 .56(.06) 1 .71 ( .06) 1 .48( .11) 1 .06( .02) 0.91 ( .03) 3-4 1 .41(.09) 1 .55( .09) 1 .32 ( .14) 0.69( .10) 0.53( .02) X 1-4 1 .52(.05) 1 .61 ( .03) 1 .45( .10) 1 .02( .06) 0.88( .03) % of C o n t r o l 106 ( 2 ) 95 ( 7 ) 67 ( 4 ) 58 ( 2 ) EDTA ( 1 . 0 X 1 0 " 7 M ) (May 31/82) Cu C o n c e n t r a t i o n (x10" 8M) Days C o n t r o l 3.9 7.9 11.8 15.7 0-1 1 .72( .25) 2.00( .04) 2.04( .2 ) 1 .97( .04) 1 .86( .33) 1-2 1 .78( .01 ) 1 .85( .03) 1 .83 ( .03) 1 .74( .02) 1 .49( .05) 2-3 1 .90( .00) 1 .93 ( .01 ) 1 .93( .01 ) 1 .80( .01 ) 1 .02 ( . 13) 3-4 1 • 72( .01 ) 1 ,69( .13) 1 .83( .04) 1 .68( .11) 0.88( .07) X 1-4 1 .80( .00) 1 . 8 2 { .03) 1 .86( .01 ) 1 .74( .04) 1 . 13( .05) % of C o n t r o l 101 ( 1 ) 1 04 ( 1 ) 97 ( 2 ) 63 ( 2 ) 205 No L i g a n d A d d i t i o n No A d d i t i o n (Low S a l i n i t y ) ( J u l 27/82) Cu C o n c e n t r a t i o n ( x l 0 _ 8 M ) Days C o n t r o l 3.9 7.9 11.8 15.7 0-1 1 • 40( .05) 1 .40 ( .21 ) 0. 99( .05) 0.23( .04) 0.02( .05) 1-2 2 .15( .01 ) 1 .94( .04) 1 . 75( .19) 0.89( .05) 0.42( .32) 2-3 2 .07( .05) 1 .09( .07) 0. 94( .05) 0.80( .15) 0.28( .09) 3-4 1 .03( .02) 1 .00 ( .08) 0. 51 ( .03) 0.42( .04) 0. 16( .26) 4-5 0 • 05( .03) 0.83( .01 ) 0. 37( .06) 0.23( .07) -.25( .26) X 1-4 1 .75( .03) 1 .34( .05) 1 . 07( .06) 0.70( .03) 0.39( .03) % of C o n t r o l 77 ( 3 ) 61 ( 4 ) 40 ( 2 ) 22 ( 2 ) No A d d i t i o n (Jun 21/82) Cu C o n c e n t r a t i o n . ( x 1 0" 8M) Days C o n t r o l 3.9 7.9 11.8 15.7 0-1 1 • 68( .14) 1 .85( .08) 1 .54 ( .04) 1 .18( .10) 1 .03 ( .13) 1-2 2 .38( .02) 2.09( .03) 1 .86( .03) 1 .68( .03) 1 .40 ( .05) 2-3 2 . 16( .03) 1 .37 ( .06) 0.90( .04) 0.96( .02) 0.95( .03) 3-4 1 .82( .01 ) 1 .31 ( .05) 0.20( .35) 0.39( .04) 0.41 ( .07) X 1-4 2 . 12( .01 ) 1 .59( .03) 1 .01 ( .01 ) 1.01 ( .03) 0.92( .03) % of C o n t r o l 75 ( 1 ) 47 ( 4 ) 48 ( 0 ) 43 ( 1 ) 2 0 6 No L i g a n d A d d i t i o n ( c o n ' t ) No A d d i t i o n Cu C o n c e n t r a t i o n ( X 1 0 ~ 8 M ) Days C o n t r o l 1 . 0 2 . 0 3 . 0 4 . 0 0 - 1 0 . 8 6 ( . 0 1 ) 0 .93C . 0 3 ) 0 . 9 2 ( . 0 7 ) 0 . 8 7 ( . 0 7 ) 0.90C . 1 0 ) 1 - 2 2 . 1 6 ( . 0 4 ) 2 . 0 3 ( . 0 4 ) 1 . 9 0 ( . 1 4 ) 1 . 6 9 ( . 0 4 ) 1 . 5 8 ( . 0 5 ) 2 - 3 2 . 0 9 ( . 0 3 ) 2 . 1 2 ( . 0 5 ) 1 . 5 4 ( . 0 6 ) 1 . 0 7 ( . 0 5 ) 0 . 8 9 ( . 0 7 ) 3 - 4 1 . 8 0 ( . 0 3 ) 1 . 8 8 ( . 0 4 ) 1 . 6 3 ( . 1 7 ) 0 . 9 8 ( . 0 8 ) 0 . 7 1 ( . 0 2 ) X 1 - 4 2 . 0 2 ( . 0 1 ) 2 . 0 1 ( . 0 2 ) 1 . 6 9 ( . 1 1 ) 1 . 2 5 ( . 0 2 ) 1 . 0 6 ( . 0 2 ) % of C o n t r o l 1 0 0 ( 1 ) 8 4 ( 5 ) 6 2 ( 1 ) 5 2 ( 1 ) 207 N a t u r a l Water Samples 10 meters ( J u l 7/82) Cu C o n c e n t r a t i o n ( X 1 0 _ 8 M ) Days C o n t r o l 3.89 7.87 11.8 15.7 0-1 0 .74( .02) 0.64( .10) 0.69( .14) 0.61 ( .12) 0.48( .13) 1-2 2 .40( .05) 2.38( .18) 1 .92( .21 ) 1 .76( .13) 1 .64( .23) 2-3 2 .27( .08) 1 .88 ( .15) 1 .24( .09) 0.75( .05) 0.73( .08) 3-4 2 .31 ( .08) 2.42( .05) 1 .50 ( .09) 0.44( .07) 0.20( .03) 4-5 0 .04( .01 ) 0.43( .10) 2. 13( .13) 0.37( .08) 0. 15( .05) X 1-4 2 .33( .03) 2.23( .07) 1 .55 ( .02) 0.98( .03) 0.86( .07) % of C o n t r o l 96 ( 3 ) 66 ( 1 ) 42 ( 1 ) 37 ( 3 ) 50 M e t e r s ( J u l 12/82) Cu C o n c e n t r a t i o n ( X 1 0 _ 8 M ) Days C o n t r o l 7.9 15.7 23.6 31.5 0-1 1 .59( .14) 1 .87 ( .11) 1 .70 ( .04) 1 .50 ( .09) 1 .24( .12) 1-2 2 .46( .15) 1 .99 ( .12) 1 .71 ( .08) 1 .42( .07) 1 .23( .06) 2-3 2 .51 ( .08) 1 .64 ( .04) 1 .08 ( .24) 0.71 ( .05) 0.91 ( .14) 3-4 1 .88( .21 ) 2.36( .20) 1 .30 ( .53) 0.57( .04) 0.38( .17) 4-5 0 .03( .07) 0.75( .33) 1 .34 ( .81 ) 0.08( .01 ) 0. 12( .04) X 1-4 2 .29( .05) 1 .99( .11) 1 .36( .28) 0.90( .01 ) 0.84( .03) % of c o n t r o l 87 ( 5 ) 59 ( 12) 39 ( 0 ) 37 ( 1 ) 208 N a t u r a l Water Samples ( c o n ' t ) 75 M e t e r s ( J u l 14/82) Cu C o n c e n t r a t i o n ( X 1 0 _ 8 M ) Days C o n t r o l 3.9 7.9 11.8 15.7 0-1 2. 00( .14) 2. 1 1 ( .05) 2 . 1 6 ( .08) 1 .97 ( .16) 1 .51 ( .08) 1-2 2. 65( .03) 2.54( .05) 2. 1 4( .08) 1 .80( .02) 1 .55( .07) 2-3 2. 47( .07) 2.26( .05) 1 .68( .06) 1 . 14( .05) 1 .09 ( .02) 3-4 1 . 96( .06) 2.21 ( .03) 2.03( .06) 1 .08 ( .02) 0.65( .04) 4-5 0. 17( .05) 0.31 ( .09) 1 .38 ( .04) 1 .41 ( .00) 0.52( .04) X 1-4 2. 36( .01 ) 2.34C .02) 1 .95 ( .01 ) 1 .34 ( .02) 1 . 1 0 ( .02) % of C o n t r o l 99 ( 1 ) 82 ( 0 ) 57 ( 1 ) 47 ( 1 ) 200 Meters ( J u l 19/82) Cu C o n c e n t r a t i o n ( X 1 0 " 8 M ) Days C o n t r o l 3.9 7.9 11.8 15.7 0-1 2. 13( .04) 2. 12( .05) 2.03( .02) 1 .84( .02) 1 .57 ( .02) 1-2 2. 69( .06) 2.46( .04) 2.36( .05) 2.09( .07) 1 .88 ( .02) 2-3 2. 50( .05) 2.50( .01 ) 2.52( .05) 2.35( .03) 2.13( .05) 3-4 1 . 62( .06) 1.81 ( .10) 1 .90 ( .01) 2.07( .03) 2.05( .03) 4-5 0. 03( .02) 0 . 01 ( .07) 0. 12( .12) 0.54( .12) 1 .35( .09) X 1-4 2. 27( .03) 2.26( .03) 2.26( .03) 2 .1 7 ( .02) 2.02( .02) % of C o n t r o l 100 ( 1 ) 100 ( 1 ) 96 ( 1 ) 89 ( 1 ) 209 N a t u r a l Water Samples ( c o n ' t ) Mn Experiment 50 Me t e r s ( r e p e a t ) w i t h o u t Mn a d d i t i o n (Aug 16/82) Cu C o n c e n t r a t i o n ( X 1 0 ~ 8 M ) Days C o n t r o l 3.9 7.9 11.8 15.7 0-1 1 . 53( .24) 1 .65 ( .06) 1 .51 ( .13) 1 . 3 9 ( .02) 1 .33 ( .06) 1-2 2. 38( .10) 2. 12( .03) 1 .89( .00) 1 .71 ( .06) 1 .54 ( .09) 2-3 2. 04( .02) 1 .60 ( .04) 0.63( .05) 0.60( .11) 0.55( .04) 3-4 1 . 79( .04) 1 .74( .04) 0.02( .13) 0.02( .02) -.02( .09) 4-5 • 19( .17) 0.38( .18) -.03( .07) -.14( .04) 0.00( .19) X 1-4 2. 07( .04) 1 .82 ( .02) 0.86( .02) 0.77( .02) 0.71 ( .03) % of C o n t r o l 88 ( 1 ) 42 ( 1 ) 37 ( 1 ) 34 ( 1 ) 50 M e t e r s ( w i t h 3.64xl0" 6M Mn) (Aug 16/82) Cu C o n c e n t r a t i o n ( X 1 0 _ 8 M ) Days C o n t r o l 3.9 7.9 11.8 15.7 0-1 1 . 91( .05) 1 .84 ( .11) 1 .79( .01 ) 1 . 75(.20) 1 .37 ( .09) 1-2 2. 20( .09) 2. 16( .01 ) 1 .99( .07) 1 . 66(.10) 1 .49 ( .05) 2-3 2. 15( .02) 2. 13( .02) 1 .88( .05) 1 . 82(.05) 1 ..56 ( .08) 3-4 1 . 91 ( .07) 1 .89( .04) 1 .70( .07) 1 . 58(.07) 1 .45( .06) 4-5 0. OK .03) 0.03( .02) 0.36( .13) 0. 65(12) 1 .47( .12) X 1-4 2. 08( .02) 2.06( .02) 1 .86( .03) 1 . 69(.02) 1 .50 ( .04) % Of C o n t r o l 99 ( 1 ) 89 ( 1 ) 81 ( 1 ) 72 ( 2 ) 210 Natural Water Samples (con't) 200 Meters (repeat) (Jul 28/82) Cu Concentration (x10~8M) Days Control 3.9 7.9 11.8. 15.7 0-1 1 . 16( .04) 1 .31 ( .11) 1 .15( .07) 0.87( .13) 0.74( .13) 1-2 2. 26( .01 ) 2. 17( .01 ) 2. 10( .06) 1 .77 ( .05) 1 .13( .04) 2-3 2. 57( .05) 2.48( .04) 2.45( .02) 2.25( .04) 1 .86( .08) 3-4 2. 42( .01 ) 2.34( .01 ) 2.27( .04) 2.29( .03) 2 . 2 0 ( .06) 4-5 0. 71( .06) 1 .02 ( .08) 1 .20( .01 ) 1 .77 ( .08) 2. 1 1 ( .06) X 1-4 2. 42( .01 ) 2.33( .02) 2.27( .00) 2. 1 1 ( .01 ) 1 .73 ( .06) % O f Control 97 ( 1 ) 94 ( 0 ) 88 ( 1 ) 72 ( 2 ) 21 1 Sediment Study Mud Sample ( J u l 26/82) Cu C o n c e n t r a t i o n (xlO' 8M) Days C o n t r o l 3.9 7.9 11.8 15.7 0-1 2 .03( .16) 2.22( .17) 2. 12( .07) 2.08( .05) 2.07( .01 ) 1-2 1 • 60( .06) 1 .54( .10) 1 .66 ( .09) 1 .63 ( .05) 1 .59( .05) 2-3 2 .35( .04) 2.39( .04) 2.35( .01 ) 2 . 3 6 ( .01 ) 2.33( .06) 3-4 1 .42( .13) 1 .33 ( .04) 1 .48( .06) 1 .56( .07) 1 .74( .07) 4-5 - .04( .00) -.07( .03) -.02( .01 ) -.01 ( .02) -.04( .03) X 1-4 1 .79( .02) 1 .75( .06) 1 .83( .01 ) 1 .85( .02) 1 .89( .01 ) % of C o n t r o l 98 ( 3 ) 102 ( 1 ) 104 ( 1 ) 106 ( 0 ) 

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