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The feasibility of using lanthanide elements to mass mark hatchery-production salmon Ennevor, Bridget Carroll 1991

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T H E F E A S I B I L I T Y O F U S I N G L A N T H A N I D E E L E M E N T S TO M A S S MARK H A T C H E R Y - P R O D U C T I O N S A L M O N By BRIDGET CARROLL ENNEVOR .Sc., The U n i v e r s i t y of B r i t i s h Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Sciences) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May 1991 ® Bridget C a r r o l l Ennevor, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of /jNlfflAL SC(£AJC£ The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The development of a marking t e c h n i q u e which c o u l d be e f f e c t i v e l y and e f f i c i e n t l y used t o mark l a r g e groups of salmonids, would be a g r e a t a s s e t t o f i s h e r i e s management. S i x experiments were thus conducted t o i n v e s t i g a t e the f e a s i b i l i t y o f u s i n g t h e l a n t h a n i d e elements t o mass mark h a t c h e r y -p r o d u c t i o n salmon. The l a h t h a n i d e s , i n t r o d u c e d through the water supply, appear t o be s u i t a b l e s i n c e they demonstrate the c h a r a c t e r i s t i c s of good el e m e n t a l markers. F i s h marked wi t h t h e s e elements can be s a t i s f a c t o r i l y i d e n t i f i e d by a n a l y s i s of bony t i s s u e s by i n d u c t i v e l y coupled plasma-mass spectrometry (ICP-MS). The l a n t h a n i d e s were shown t o be s e v e r e l y t o x i c t o coho and s t e e l h e a d a l e v i n s a t c o n c e n t r a t i o n s of 100 M<?/1 i n the water supply, but were o n l y s l i g h t l y t o x i c t o coho f r y . A l e v i n and f r y appeared t o be more s e n s i t i v e t o the l i g h t - w e i g h t l a n t h a n i d e s than the h e a v i e r l a n t h a n i d e s . Coho smolts showed no adverse e f f e c t s when exposed t o lanthanum or cerium a t these c o n c e n t r a t i o n s . Lanthanides, i n t r o d u c e d i n the form of a c e t a t e s , were shown t o be absorbed from the water supply and subsequently i n c o r p o r a t e d i n the v e r t e b r a l column, o t o l i t h s and s c a l e s of coho f r y . Coho f r y exposed t o lanthanum or samarium a t c o n c e n t r a t i o n s of 100 i n the water f o r 6 weeks had d e t e c t a b l e l e v e l s of element i n t h e i r bony t i s s u e s 10% months I l l p o s t - t r e a t m e n t . When lanthanum and cerium were added t o the water s u p p l y a t c o n c e n t r a t i o n s of 100 ng/1 f o r 4 weeks, h i g h e r c o n c e n t r a t i o n s of element were accumulated i n f r y than i n smolts, a l t h o u g h smolts accumulated g r e a t e r amounts of element. I n t r o d u c t i o n o f the lanth'anide elements through the water supply, appears t o be an e f f e c t i v e method f o r the mass marking of young salmonids. i v TABLE OP CONTENTS Page ABSTRACT i i TABLE OF CONTENTS "iv LIST OF TABLES v i i i LIST OF FIGURES x i LIST OF APPENDICES x i i i ACKNOWLEDGEMENTS X T V INTRODUCTION 1 LITERATURE REVIEW 5 F i s h Tagging . 5 H i s t o r y 5 Types o f Tagging . 5 Coded-Wire Tagging 7 Chemical Marking 8 Strontium 9 Lanthanide Elements 10 Samarium 10 Dysprosium 12 Europium 14 Terbium 15 Summary of the Lanthanides . . . 15 Lanthanide Elements 17 I n t r o d u c t i o n 17 Chemical P r o p e r t i e s 20 Metabolism 22 Bony T i s s u e Development 25 Bone Development 2 6 O t o l i t h Development 27 S c a l e Development 27 T o x i c i t y 28 A n a l y t i c a l Methods 31 Neutron A c t i v a t i o n A n a l y s i s (NAA) . . . . 32 I n d u c t i v e l y C o u p l e d P l a s m a - M a s s Spectrometry (ICP-MS) 34 A n a l y s i s o f the Lanthanides by ICP-MS . . 37 V Page METHODOLOGY 43 Chemicals Used 43 Treatment C o n c e n t r a t i o n s . . 44 Sample P r e p a r a t i o n Techniques 47 Water Samples . 47 V e r t e b r a l Column E x t r a c t i o n 47 O t o l i t h E x t r a c t i o n '". . . 48 S c a l e Samples . . . 49 Sample A n a l y s i s 50 Problems With A n a l y s i s . 50 Water Standards 52 V e r t e b r a e Standards 53 EXPERIMENT 1 - INVESTIGATION OF COHO (Oncorhynchus kisutch) ALEVINS IN RECIRCULATING SYSTEMS CONTAINING A RANGE OF CONCENTRATIONS OF CERIUM, LANTHANUM, DYSPROSIUM, SAMARIUM, AND YTTERBIUM 54 I n t r o d u c t i o n 54 M a t e r i a l s and Methods 55 R e c i r c u l a t i n g U n i t s 55 Ex p e r i m e n t a l Design 57 Sampling and A n a l y t i c a l Method . . . . . . . . . 59 R e s u l t s 60 Water Q u a l i t y 60 M o r t a l i t i e s 61 Water A n a l y s i s 62 R e s i d u a l Element i n Tanks 63 A c i d R i n s i n g of Tanks 64 A l e v i n and Egg A n a l y s i s . . . . . 65 D i s c u s s i o n 67 EXPERIMENT 2 - TOXICITY STUDY USING STEELHEAD (Salmo gairdneri) ALEVINS TREATED WITH LANTHANUM AND SAMARIUM . . 71 I n t r o d u c t i o n . 71 M a t e r i a l s and Methods 71 Experimental Design 71 R e s u l t s 73 M o r t a l i t i e s 73 D i s c u s s i o n 74 v i Page EXPERIMENT 3 - INVESTIGATION USING COHO (Oncorhynchus kisutch) FRY IN STATIC TANKS CONTAINING LANTHANUM IN A RANGE OF CONCENTRATIONS 75 I n t r o d u c t i o n 75 M a t e r i a l s and Methods 76 Expe r i m e n t a l Design 76 Sampling' and A n a l y t i c a l Method 77 R e s u l t s 78 M o r t a l i t i e s 78 Water A n a l y s i s 78 Whole F r y A n a l y s i s 79 D i s c u s s i o n 81 EXPERIMENT 4 - INVESTIGATION OF COHO (Oncorhynchus kisutch) FRY IN A FLOW THROUGH TANK CONTAINING LANTHANUM . . 84 I n t r o d u c t i o n 84 M a t e r i a l s and Methods 85 Expe r i m e n t a l Design 85 Sampling and A n a l y t i c a l Method 85 R e s u l t s 87 M o r t a l i t i e s 87 Water A n a l y s i s . 87 Whole F r y A n a l y s i s 87 Bony T i s s u e A n a l y s i s 87 A n a l y s i s o f V e r t e b r a l Column 2-Months Post Treatment 88 D i s c u s s i o n 91 EXPERIMENT 5 - THE TREATMENT OF COHO (Oncorhynchus kisutch) FRY WITH LANTHANUM AND SAMARIUM AT THREE CONCENTRATIONS FOR 3 AND 6 WEEKS 95 I n t r o d u c t i o n 95 M a t e r i a l s and Methods 96 Expe r i m e n t a l Design 96 Sampling and A n a l y t i c a l Method 100 S t a t i s t i c a l A n a l y s i s 101 R e s u l t s 102 M o r t a l i t i e s 102 Growth 103 Downstream Contamination 106 Water A n a l y s i s 106 Water Standards 110 Ve r t e b r a e Standards I l l V e r t e b r a l Column A n a l y s i s 112 • O t o l i t h A n a l y s i s 119 S c a l e A n a l y s i s 122 D i s c u s s i o n 126 v i i Page EXPERIMENT 6 - THE TREATMENT OF COHO (Oncorhynchus kisutch) FRY AND SMOLTS WITH LANTHANUM AND CERIUM IN VARIOUS COMBINATIONS FOR 4 WEEKS . ' 131 I n t r o d u c t i o n 131 M a t e r i a l s and Methods 132 Expe r i m e n t a l Design 132 Sampling and A n a l y t i c a l Method 13 3 S t a t i s t i c a l A n a l y s i s . . . 134 R e s u l t s . . . . . 135 M o r t a l i t i e s 135 Water A n a l y s i s 135 Water Standards 137 V e r t e b r a e Standards 138 V e r t e b r a l Column A n a l y s i s 138 O t o l i t h A n a l y s i s 142 D i s c u s s i o n 144 CONCLUSIONS AND RECOMMENDATIONS 147 BIBLIOGRAPHY 152 APPENDICES 162 v i i i LIST OF TABLES T a b l e Page 1. C l a s s i f i c a t i o n of the l a n t h a n i d e elements and t h e i r e l e m e n t a l c h a r a c t e r i s t i c s 19 2. C h a r a c t e r i s t i c s of l a n t h a n i d e i s o t o p e s f o r d e t e r m i n a t i o n by neutron a c t i v a t i o n a n a l y s i s . . . . 33 3. ICP-MS l i m i t s of d e t e c t i o n f o r the l a n t h a n i d e s (3 S.D. o f the background). T o t a l d i s s o l v e d s o l i d s are 1 mg/g 38 4. I s o t o p i c c o m p o s i t i o n of the l a n t h a n i d e elements and t h e i r n a t u r a l r e l a t i v e abundances 39 5. Lanthanide atomic weights and m o l e c u l a r weights of the c o r r e s p o n d i n g a c e t a t e s used i n the treatment c o n c e n t r a t i o n c a l c u l a t i o n s 58 6. Lanthanide a c e t a t e (grams) added t o 16 l i t r e s o f r i v e r water. and the approximate _ t h e o r e t i c a l element c o n c e n t r a t i o n s i n each tank 59 7. Foam and p r e c i p i t a t e f o r m a t i o n , and a l e v i n m o r t a l i t i e s observed i n the tanks 4 hours and 24 hours a f t e r the a d d i t i o n of the l a n t h a n i d e a c e t a t e s . . . . . . . . 62 8. ICP-MS r e s u l t s of water a n a l y s i s of t h e tanks c o n t a i n i n g the lowest l a n t h a n i d e c o n c e n t r a t i o n s . . 63 9. O r i g i n a l treatment c o n c e n t r a t i o n and r e s i d u a l l a n t h a n i d e remaining i n the tanks a f t e r a 24-hour continuous r i n s e 64 10. Lanthanide c o n c e n t r a t i o n s i n tanks a f t e r a 0.1M or 0.5M HCl wash and a 24-hour flow through r i n s e . . . 65 11. ICP-MS a n a l y s i s of dead a l e v i n s and eggs 66 12. S t e e l h e a d a l e v i n m o r t a l i t i e s observed, i n tanks each i n i t i a l l y c o n t a i n i n g 20 a l e v i n s , d u r i n g 19-day exposure t o lanthanum and samarium a t 100 ng/1 . . . 73 13. T h e o r e t i c a l lanthanum c o n c e n t r a t i o n s , amount of lanthanum added t o each tank, and the measured c o n c e n t r a t i o n s f o r the two sample dates 79 i x T a b l e Page 14. T h e o r e t i c a l lanthanum and samarium treatment c o n c e n t r a t i o n s 97 15. L a b e l l e d coho smolts t r a n s f e r r e d t o the Department of F i s h e r i e s and Oceans West Vancouver L a b o r a t o r y sea water tank and the f i n - c l i p s used t o i d e n t i f y treatment groups " . . . 99 16. Coho f r y m o r t a l i t i e s d u r i n g the treatment p e r i o d and d u r i n g the growth p e r i o d i n tanks c o n t a i n i n g lanthanum and samarium treatments . . . . . . . . . 102 17. Mean body weights of f r y l a b e l l e d w i t h lanthanum showing the s i g n i f i c a n t e f f e c t s o f sampling time and d u r a t i o n of lanthanum exposures 104 18. Mean body weights of f r y l a b e l l e d w i t h samarium showing the s i g n i f i c a n t e f f e c t s of sampling time and d u r a t i o n o f samarium exposures 105 19. C o n c e n t r a t i o n s of lanthanum and samarium p r e s e n t i n the C a p i l a n o R i v e r a t 4 d i f f e r e n t l o c a t i o n s downstream d u r i n g the treatment p e r i o d 106 20. C o n c e n t r a t i o n o f lanthanum and samarium i n water standards a n a l y z e d by ICP-MS 110 21. C o n c e n t r a t i o n of lanthanum and samarium i n the v e r t e b r a e standards a n a l y z e d by ICP-MS 112 22. Mean lanthanum c o n c e n t r a t i o n (jug/g) i n v e r t e b r a l columns of f r y l a b e l l e d w i t h lanthanum showing the s i g n i f i c a n t e f f e c t s o f lanthanum treatment c o n c e n t r a t i o n , d u r a t i o n of lanthanum exposures, and sampling time 115 23. Mean samarium c o n c e n t r a t i o n (Mg/g) i n v e r t e b r a l columns of f r y l a b e l l e d w i t h samarium showing the s i g n i f i c a n t e f f e c t s of samarium treatment c o n c e n t r a t i o n , d u r a t i o n of samarium exposures, and sampling time 116 24. Mean amount (ng) of lanthanum i n v e r t e b r a l columns of f r y l a b e l l e d w i t h lanthanum showing the s i g n i f i c a n t e f f e c t s o f lanthanum treatment c o n c e n t r a t i o n , d u r a t i o n of lanthanum exposures, and sampling time . . . . 117 X Ta b l e Page 25. Mean amount (ng) of samarium i n v e r t e b r a l columns of f r y l a b e l l e d w i t h samarium showing t h e s i g n i f i c a n t e f f e c t s o f samarium treatment c o n c e n t r a t i o n , d u r a t i o n of samarium exposures, and sampling time 118 26. Mean lanthanum c o n c e n t r a t i o n i n o t o l i t h s o f f r y l a b e l l e d w i t h lanthanum showing t h e s i g n i f i c a n t e f f e c t s o f lanthanum treatment c o n c e n t r a t i o n , d u r a t i o n of lanthanum exposures, and sampling time . . . . 120 27. Mean samarium c o n c e n t r a t i o n i n o t o l i t h s o f f r y l a b e l l e d w i t h samarium showing the s i g n i f i c a n t e f f e c t s of samarium treatment c o n c e n t r a t i o n , d u r a t i o n of samarium exposures, and sampling time 121 28. C o n c e n t r a t i o n o f lanthanum and samarium found i n s c a l e s o f coho f r y t r e a t e d w i t h lanthanum and samarium a t 0 and 100 f o r 6 weeks showing the s i g n i f i c a n t d i f f e r e n c e s 122 29. T h e o r e t i c a l lanthanum and cerium treatment c o n c e n t r a t i o n s 13 3 30. T o t a l m o r t a l i t i e s d u r i n g the treatment p e r i o d i n tanks c o n t a i n i n g lanthanum and cerium treatments . . . . 135 31. c o n c e n t r a t i o n o f lanthanum and cerium i n water samples from tanks a n a l y z e d by ICP-MS 136 32. C o n c e n t r a t i o n o f lanthanum and cerium i n water standards a n a l y z e d by ICP-MS 137 33. C o n c e n t r a i t o n of lanthanum and cerium i n v e r t e b r a e standards a n a l y z e d by ICP-MS 138 34. Mean l a n t h a n i d e c o n c e n t r a t i o n s (/*<?/<?) i n v e r t e b r a l columns of f r y and smolts l a b e l l e d w i t h lanthanum and cerium showing the s i g n i f i c a n t e f f e c t s of lanthanum and cerium treatments 140 35. Mean l a n t h a n i d e amounts (ng) i n v e r t e b r a l columns of f r y and smolts l a b e l l e d w i t h lanthanum and cerium showing t h e s i g n i f i c a n t e f f e c t s o f lanthanum and cerium treatments 141 36. Mean l a n t h a n i d e c o n c e n t r a t i o n s (tig/g) i n o t o l i t h s of f r y and smolts l a b e l l e d w i t h lanthanum and cerium showing the s i g n i f i c a n t e f f e c t s o f lanthanum and cerium treatments 143 x i LIST OF FIGURES F i g u r e Page 1. A schematic diagram of a t y p i c a l ICP-MS showing the arrangement of the v a r i o u s components. F i g u r e redrawn from a diagram s u p p l i e d by Elemental Research Inc. MCA = M u l t i - C h a n n e l A n a l y z e r ; RF = Radio Frequency. 35 2. A t y p i c a l mass spectrum o f samarium o b t a i n e d w i t h ICP-MS i n s t r u m e n t a t i o n showing the i n d i v i d u a l peaks f o r each of the samarium i s o t o p e s . S p e c t r a o b t a i n e d from experimental data (Experiment 5) . . . . . 42 3. M o d i f i e d v e r s i o n of the M e r r i o t t b o t t l e used t o d e l i v e r the chemical s o l u t i o n s a t a co n s t a n t d r i p r a t e 46 4. Diagram of a f i s h showing the " p r e f e r r e d s c a l e sample a r e a " 50 5. U p w e l l i n g r e c i r c u l a t i n g apparatus t o r e c i r c u l a t e the water i n the treatment tanks. Arrows i n d i c a t e water flow 56 6. Lanthanum c o n c e n t r a t i o n i n the whole body of coho f r y f o l l o w i n g a 3-week exposure of lanthanum a t 2, 10, and 200 ng/1 and a 1-week or 2-week r i n s e p e r i o d . Undetectable lanthanum i n c o n t r o l tank. R e s u l t s a re r e p o r t e d as mean ± S.E. space i n fig of La per g of d r y t i s s u e 80 7. Lanthanum c o n c e n t r a t i o n s i n the water t h a t coho f r y were exposed t o over the 3-week treatment p e r i o d . Lanthanum c o n c e n t r a t i o n measured i n jug/1 89 8. Lanthanum c o n c e n t r a t i o n s i n the v e r t e b r a l column, o t o l i t h s and s c a l e s of coho f r y f o l l o w i n g a 3-week exposure t o 100 /xg/1 La and an 18-day r i n s e p e r i o d . R e s u l t s r e p o r t e d r e p r e s e n t the mean ± S.E. i n /jg o f La per g of dry bony t i s s u e 90 9. Lanthanum c o n c e n t r a t i o n s (/xg/1) i n the water t h a t coho f r y were exposed t o over the 6-week treatment p e r i o d . U n d e t e c t a b l e lanthanum i n c o n t r o l tanks 108 10. Samarium c o n c e n t r a t i o n s (/xg/1) i n the water t h a t coho f r y were exposed t o over"the 6-week treatment p e r i o d . Undetectable samarium i n c o n t r o l tanks . 109 x i i F i g u r e Page 11. C o n c e n t r a t i o n o f lanthanum p r e s e n t i n t h e bony t i s s u e s o f coho f r y 2 weeks and 10% months a f t e r t e r m i n a t i o n o f t h e t r e a t m e n t s . R e s u l t s r e p o r t e d as mean ± S.E. i n /xg o f La/kg o f v e r t e b r a e , o r i n /zg o f La/kg d r y t i s s u e ( r e l a t i v e t o c a l c i u m ) i n o t o l i t h s and s c a l e s . U n d e t e c t a b l e lanthanum i n t h e bony t i s s u e s o f u n t r e a t e d f r y 124 12. C o n c e n t r a t i o n o f samarium p r e s e n t i n t h e bony t i s s u e s o f coho f r y 2 weeks and 10% months a f t e r t e r m i n a t i o n o f t h e t r e a t m e n t s . R e s u l t s r e p o r t e d as mean ± S.E. i n /xg o f Sm/kg o f v e r t e b r a e , o r i n jug o f Sm/kg d r y t i s s u e ( r e l a t i v e t o c a l c i u m ) i n o t o l i t h s and s c a l e s . U n d e t e c t a b l e samarium i n t h e bony t i s s u e s o f u n t r e a t e d f r y 125 x i i i LIST OF APPENDICES Appendix Page 1. Acute l e t h a l doses of l a n t h a n i d e elements i n r a t s and mice • • • 1 6 3 2. ICP-MS A n a l y s i s f o r l a n t h a n i d e content o f 6% sodium h y p o c h l o r i t e b l e a c h i n g s o l u t i o n used t o d i g e s t t r a c e s of f l e s h o f f bony t i s s u e s 166 3. I n f o r m a t i o n used and r e s u l t s of computer d r i p c a l c program used f o r c a l c u l a t i o n o f l a n t h a n i d e treatments used i n Experiment 5 167 4. ICP-MS a n a l y s i s o f Oregon M o i s t P e l l e t s f e d t o coho f r y d u r i n g l a b e l l i n g p e r i o d i n Experiment 5 . . . 169 5. Water c h e m i s t r y data f o r the ambient C a p i l a n o R i v e r water 170 6. ANOVA t a b l e s f o r m o r t a l i t i e s observed i n Experiment 5 i n lanthanum treatment tanks , . . 172 7. ANOVA t a b l e s f o r growth ( f r y weight) observed i n Experiment 5 i n lanthanum and samarium treatment tanks 173 8. ANOVA t a b l e s f o r lanthanum and samarium c o n c e n t r a t i o n i n t he v e r t e b r a l columns of f r y l a b e l l e d w i t h lanthanum and samarium i n Experiment 5 . . . . . . . 174 9. ANOVA t a b l e s f o r lanthanum and samarium amounts i n the v e r t e b r a l columns of f r y l a b e l l e d w i t h lanthanum and samarium i n Experiment 5 . 175 10. ANOVA t a b l e s f o r lanthanum and samarium c o n c e n t r a t i o n s i n the o t o l i t h s of f r y l a b e l l e d w i t h lanthanum and samarium i n Experiment 5 . 176 11. ANOVA t a b l e s f o r lanthanum and samarium c o n c e n t r a t i o n s i n t he s c a l e s , 10% months post-treatment, o f f r y l a b e l l e d w i t h lanthanum and samarium i n Experiment 5 177 12. I n f o r m a t i o n used and r e s u l t s of computer d r i p c a l c program used f o r c a l c u l a t i o n o f l a n t h a n i d e treatments used i n Experiment 6 . 178 13. ANOVA t a b l e f o r m o r t a l i t i e s observed i n Experiment 6 d u r i n g 4-week l a b e l l i n g p e r i o d 180 x i v Appendix Page 14. ANOVA t a b l e s f o r lanthanum and cerium c o n c e n t r a t i o n s and amounts i n the v e r t e b r a l columns o f f r y and smolts l a b e l l e d w i t h lanthanum and cerium i n Experiment 6 181 15. ANOVA t a b l e s f o r lanthanum and cerium c o n c e n t r a t i o n i n the o t o l i t h s o f f r y and smolts l a b e l l e d w i t h lanthanum and cerium i n Experiment 6 182 XV ACKNOWLEDGEMENTS I would l i k e t o express my a p p r e c i a t i o n t o a l l those who h e l p e d me d u r i n g my experimental work and t h e s i s p r e p a r a t i o n . In p a r t i c u l a r , I am g r a t e f u l t o my a d v i s o r Dr. "R.M. Beames f o r h i s guidance, knowledgeable a d v i c e and i d e a s d u r i n g the course of t h i s r e s e a r c h , and f o r h i s c o n s t r u c t i v e c r i t i c i s m s i n r e v i e w i n g my t h e s i s . Thanks are a l s o due t o Mr. K. P i t r e who i n i t i a t e d t h i s i p r o j e c t and f o r h i s continued support and encouragement throughout t h i s study. S p e c i a l a p p r e c i a t i o n i s extended t o Mr. E. Stone and h i s s t a f f a t C a p i l a n o Hatchery f o r t h e i r v a l u a b l e a s s i s t a n c e and e x c e l l e n t a d v i c e d u r i n g the experimental work. My thanks are a l s o due t o Dr. T. M u l l i g a n f o r h i s h e l p f u l s u g g e s t i o n s and u s e f u l i d e a s when problems arose. A l s o , I wish t o express my a p p r e c i a t i o n t o Mr. R. Brown of Elemental Research Inc. f o r h i s a n a l y t i c a l e x p e r t i s e and a s s i s t a n c e . S i n c e r e g r a t i t u d e i s extended t o the remaining members of my committee, Dr. D. Higgs, Mr. G. Berezay, and Dr. G. Iwama, f o r t h e i r a s s i s t a n c e a t v a r i o u s stages of t h i s r e s e a r c h . The f i n a n c i a l support f o r t h i s study p r o v i d e d by Department of F i s h e r i e s and Oceans i s g r a t e f u l l y acknowledged. 1 INTRODUCTION Tagging programs allow the c o l l e c t i o n of important information which can be used to evaluate the o v e r a l l e f f e c t s of f i s h e r i e s management. Juvenile salmon which have been marked before release can be i d e n t i f i e d when captured i n f i s h e r i e s and as they return to spawn. The r e s u l t s obtained from mark-recovery experiments help to improve and increase salmon production. In 1988, personnel from the Federal Department of Fisheries and Oceans approached the University of B r i t i s h Columbia to i n i t i a t e a project to develop a new tagging system. The r e s u l t s of an extensive l i t e r a t u r e search revealed that the incorporation of the lanthanide elements (rare earth elements) i n f i s h bony tissues by addition to the water supply would be worthwhile to investigate as a possible tagging method. The tagging method (coded-wire tags) currently being used f o r i d e n t i f y i n g hatchery salmon i s extremely labour intensive as i t requires the i n d i v i d u a l handling of f r y , thereby l i m i t i n g the number of marked f i s h to a small percentage of the t o t a l production. Marked f i s h are i d e n t i f i e d by an adipose f i n c l i p . A method which could greatly increase the numbers marked, and at the same time eliminate handling, would be a great asset to f i s h e r i e s management and hatchery assessment. Chemical marking offers" a favourable a l t e r n a t i v e to the labour-intensive mechanical tagging. Chemical marking would 2 enable h a t c h e r y s t a f f t o mark l a r g e groups of f r y f o r i d e n t i f i c a t i o n b e f o r e r e l e a s e . However, the marked f i s h have no e x t e r n a l i d e n t i f i e r ( i . e . no adipose f i n c l i p ) . The chemicals t o be used would have t o be: ( i ) bone seeking, and p e r s i s t i n the bony t i s s u e f o r a l o n g time; ( i i ) not harmful t o f i s h or humans; ( i i i ) d e t e c t a b l e a t very low l e v e l s ; (iv ) not found i n f r e s h water or s a l t water i n d e t e c t a b l e l e v e l s ; and (v) r e l a t i v e l y i n e x p e n s i v e . The l a n t h a n i d e s appear t o have the c h a r a c t e r i s t i c s of good elemental markers. These elements, which are i n c o r p o r a t e d i n the bony t i s s u e s , can be d e t e c t e d by I n d u c t i v e l y Coupled Plasma-Mass Spectrometry (ICP-MS) or Neutron A c t i v a t i o n A n a l y s i s (NAA) a t c o n c e n t r a t i o n s as low as 0.01 m i c r o g r a m s / l i t r e ( L o n g e r i c h e t al., 1987). D i e t i n c o r p o r a t i o n does not seem t o be the o p t i m a l method t o s u c c e s s f u l l y a d m i n i s t e r the l a n t h a n i d e elements s i n c e they are not r e a d i l y absorbed by the g a s t r o - i n t e s t i n a l t r a c t ( E l l i s , 1968; Luckey & Venugopal, 1977; and K e n n e l l y e t al., 1980) and, because of t h i s v e r y a t t r i b u t e , have been w i d e l y used as i n d i g e s t i b l e a d d i t i v e s f o r d i g e s t i b i l i t y s t u d i e s (Huston & E l l i s , 1965; E l l i s , 1968; O l b r i c h et al., 1971; Gray & Vogt, 1974; Hutcheson e t al., 1974; Luckey e t al., 1975, 1977, 1979; Young e t al., 1975, 1976; H a r t n e l l & S a t t e r , 1979; Cerasov fie 3 H e l l e r , 1980; K e n n e l l y e t al. , 1980; Uden e t al., 1980; and Arambel e t al. , 1986). However, i t was h y p o t h e s i z e d t h a t the a d d i t i o n of a c o n c e n t r a t e d aqueous s o l u t i o n of the l a n t h a n i d e s t o the water supply would be a more e f f e c t i v e method of a c h i e v i n g t i s s u e i n c o r p o r a t i o n . The use of s t r o n t i u m as a chemical marker i s c u r r e n t l y b e i n g i n v e s t i g a t e d by a r e s e a r c h group a t the P a c i f i c B i o l o g i c a l S t a t i o n , Department of F i s h e r i e s and Oceans, Nanaimo. They have demonstrated t h a t s t r o n t i u m , added t o the d i e t , i s i n c o r p o r a t e d i n t o the f i s h s c a l e s . However, s i n c e s t r o n t i u m i s r e l a t i v e l y abundant i n sea water and s i n c e a multi-element marker would a l l o w f o r a l a r g e number of d i s t i n c t combinations, the i n v e s t i g a t i o n of the l a n t h a n i d e elements, s i n g l y and i n combination, appeared t o be a more worthwhile p r o j e c t t o pursue. T h i s t h e s i s r e s e a r c h was conducted t o determine the f e a s i b i l i t y of u s i n g t h e l a n t h a n i d e elements t o c h e m i c a l l y mark c u l t u r e d salmon. Coho a l e v i n s (newly hatched j u v e n i l e salmon w i t h a y o l k sac attached) were f i r s t i n v e s t i g a t e d because a p p l i c a t i o n o f the chemical s o l u t i o n s c o u l d be more e f f i c i e n t and e a s i e r a t t h i s stage. U n f o r t u n a t e l y , the f i r s t s e t of experiments i n v o l v i n g coho and s t e e l h e a d a l e v i n s demonstrated t h a t the l a n t h a n i d e s were t o x i c , even a t low c o n c e n t r a t i o n s , t o t h i s l i f e s t a g e . The ensuing experiments were c a r r i e d out t o determine i f coho f r y and smolts were more t o l e r a n t s t a g e s t o work with, and t o determine i f these c h e m i c a l marks were 4 accumulated and r e t a i n e d i n t h e i r bony t i s s u e s . The experiments were conducted as f o l l o w s : ( i ) an i n v e s t i g a t i o n u s i n g coho a l e v i n s exposed t o a range of l a n t h a n i d e s ; ( i i ) a t o x i c i t y study u s i n g s t e e l h e a d a l e v i n s t r e a t e d with lanthanum and samarium; ( i i i ) an experiment u s i n g coho f r y h e l d i n tanks c o n t a i n i n g s t a t i c water t r e a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of lanthanum; ( i v ) an experiment u s i n g coho f r y i n a flow through tank w i t h a lanthanum c o n c e n t r a t i o n o f 100 jug/1; (v) an experiment t r e a t i n g coho f r y w i t h lanthanum and samarium a t c o n c e n t r a t i o n s of 0. 10, and 100 ug/1 f o r a 3-week p e r i o d o r a 6-week p e r i o d ; f i s h maintained and a n a l y z e d f o r l a n t h a n i d e r e t e n t i o n throughout a 10%-month p e r i o d a f t e r l a b e l l i n g ; and (v i ) combinations o f lanthanum and cerium treatments u s i n g coho f r y and smolts. 5 LITERATURE REVIEW F i s h T a g g i n g H i s t o r y S i n c e t h e l a t e n i n e t e e n t h century b i o l o g i s t s have been at t e m p t i n g t o t a g f i s h s t o c k s , based on the b e l i e f t h a t the a b i l i t y t o mark and i d e n t i f y s p e c i f i c groups of f i s h would be an important f i s h e r i e s management t o o l . Without some e f f e c t i v e way of q u a n t i f y i n g abundance, the o v e r a l l success of h a t c h e r y -p r o d u c t i o n salmon cannot be p r o p e r l y e v a l u a t e d . Much u s e f u l d a t a has been c o l l e c t e d from r e t u r n i n g marked salmon. S u r v i v a l r a t e s , growth r a t e s , m i g r a t i o n r o u t e s , p r e d a t i o n e f f e c t s , s u c c e s s based on s i z e and time of r e l e a s e , e f f e c t i v e n e s s of d i f f e r e n t r e a r i n g p r a c t i c e s , and n u t r i t i o n a l requirements are p r i n c i p a l v a r i a b l e s of i n t e r e s t . Types of Tagging; Many t e c h n i q u e s have been used t o mark f i s h . Some of these i n c l u d e : ( i ) p h y s i c a l t a g s t h a t are a t t a c h e d onto or i n s e r t e d i n t o the f i s h ; ( i i ) m u t i l a t i o n tags where the f i n s are p a r t i a l l y or c o m p l e t e l y c l i p p e d ; ( i i i ) v a r i o u s b i o l o g i c a l marks ( g e n e t i c , p a r a s i t i c , or b a c t e r i a l t a g s ) ; ( i v ) t h ermal marks where d i f f e r e n t i a l l y - s p a c e d growth r i n g s a r e l a i d down i n the o t o l i t h s ; (v) c o l d - b r a n d i n g o r t a t t o o i n g ; and (v i ) g e n e t i c markers. More r e c e n t l y , many types of chemical marks have been used. These i n v o l v e the a p p l i c a t i o n and i n c o r p o r a t i o n o f dyes, a n t i b i o t i c s , : r a d i o a c t i v e i s o t o p e s , o r s t a b l e i s o t o p e s o f elements. The use of coded-wire t a g g i n g and chemic a l marking w i t h s t r o n t i u m and the l a n t h a n i d e s w i l l be d i s c u s s e d i n d e t a i l . Some of t h e c h a r a c t e r i s t i c s o f an i d e a l mark i n c l u d e the f o l l o w i n g : ( i ) t he mark must remain w i t h the f i s h throughout i t s l i f e c y c l e a t d e t e c t a b l e l e v e l s ; ( i i ) no harmful e f f e c t s on f i s h behaviour, growth, movement or r e p r o d u c t i o n can be e v i d e n t as a r e s u l t o f the mark; ( i i i ) t he mark should be r e l a t i v e l y i n e x p e n s i v e and easy t o app l y ; ( i v ) no n a t u r a l marks s i m i l a r t o the one a p p l i e d can be p r e s e n t ; (v) the mark can be e f f e c t i v e l y and s a f e l y used f o r d i f f e r e n t ages and s p e c i e s ; and (v i ) t h e r e a re enough p o s s i b l e d i f f e r e n t combinations of marks so t h a t many groups of f i s h can be i d e n t i f i e d s e p a r a t e l y . 7 Coded-Wire Tagging The coded-wire t a g g i n g system was developed by J e f f e r t s e t a l . , (1963) and i s the method c u r r e n t l y being used i n Canada and the U n i t e d S t a t e s t o i d e n t i f y hatchery and w i l d salmon. T h i s system i n v o l v e s the i n s e r t i o n of a s m a l l coded, magnetized w i r e t a g i n t o the nose c a r t i l a g e and the c l i p p i n g of the adipose f i n f o r e x t e r n a l i d e n t i f i c a t i o n . These tags a re 1 mm long x 0.25 mm diameter and a r e marked wi t h a b i n a r y code etched a l o n g the s i d e . The tagged f i s h a re i d e n t i f i e d by the m i s s i n g adipose f i n and a s e n s i t i v e magnetic d e t e c t o r i s used t o d e t e c t the metal t a g . the t a g i s d i s s e c t e d out of the c a r t i l a g e and the code i s re a d u s i n g a microscope. Although t h i s system has been i n use s i n c e the early-1970's and has pr o v i d e d much u s e f u l i n f o r m a t i o n on the mark-recovery experiments c a r r i e d out over the l a s t 15 y e a r s , i t has not proven t o be the i d e a l method t o use i n a l l c i r c u m s t a n c e s . There are some problems a s s o c i a t e d w i t h the use of coded-wi r e t a g s . F i r s t , i t i s extremely l a b o u r - i n t e n s i v e t o ap p l y and r e c o v e r the t a g s . T h i s disadvantage, t h e r e f o r e , l i m i t s t he numbers of f i s h t h a t can be marked. Second, t h e r e a r e some problems w i t h t a g l o s s , s i n c e the t a g may work i t s way out of the nose c a r t i l a g e , o r i t i s not always d e t e c t e d i n , or re c o v e r e d from r e t u r n i n g a d u l t s ( E b e l , 1974). T h i r d , upon i n j e c t i o n o f the t a g , i t may sever the o p t i c nerve c a u s i n g impaired v i s i o n which w i l l decrease the f i s h ' s chances f o r 8 s u r v i v a l . Although the i n c i d e n c e of these problems has decreased due t o f u r t h e r improvements i n the t a g g i n g t e c h n i q u e s , t h e s e problems are s t i l l e v i d e n t . Chemical Marking A t e c h n i q u e which enables l a r g e numbers of f i s h t o be marked a t the same time without i n d i v i d u a l h a n d l i n g would be a more e f f e c t i v e marking system than the coded-wire system. Chemical marks which i n v o l v e the i n t r o d u c t i o n and subsequent i n c o r p o r a t i o n of elements i n t o bony t i s s u e s would p r o v i d e the r a p i d and r e l i a b l e method needed t o mark l a r g e batches of h a t c h e r y - p r o d u c t i o n salmonids. Many of the c h a r a c t e r i s t i c s of an i d e a l f i s h mark are s a t i s f i e d by c h e m i c a l marks. These have been d e s c r i b e d by T r e f e t h e n and Novotny (1961) as: ( i ) are not harmful t o the f i s h ; ( i i ) do not cause unusual behaviour p a t t e r n s ; ( i i i ) have no harmful e f f e c t s on metabolism; (iv ) can be a p p l i e d without h a n d l i n g the f i s h ; (v) a re e a s i l y and r e a d i l y a p p l i e d ; (vi ) can be a p p l i e d t o l a r g e numbers q u i c k l y and e a s i l y ; ( v i i ) marked and unmarked p o p u l a t i o n d i f f e r e n c e s a r e not d i s t i n g u i s h a b l e by p r e d a t o r s ; and ( v i i i ) a re i n e x p e n s i v e t o apply. Another important c o n s i d e r a t i o n i s the s a f e t y of the chemical marker i n f i s h d e s t i n e d f o r human consumption. The disadvantage o f e l e m e n t a l marks i s the f a c t t h a t they a r e not e x t e r n a l l y v i s i b l e and t h e r e f o r e the marked f i s h are not e a s i l y d i s t i n g u i s h e d . S t r o n t i u m The f e a s i b i l i t y o f u s i n g s t r o n t i u m as an e l e m e n t a l mark has been s t u d i e d e x t e n s i v e l y . S t r o n t i u m i s a bone s e e k i n g element which i s c h e m i c a l l y s i m i l a r t o c a l c i u m ( E i s e n b e r g , 1973). D i f f e r e n t groups of r e s e a r c h e r s have induced marks i n f i s h by t h e a d d i t i o n o f s t r o n t i u m t o the f e e d . Ophel & Judd (1968) , Behrens Yamada e t al. (1979), Behrens Yamada & M u l l i g a n (1982), and G u i l l o u & de l a Noiie (1987) have used feeds c o n t a i n i n g s t r o n t i u m f o r long-term marking of f i s h . They have s u c c e s s f u l l y demonstrated t h a t s t r o n t i u m i s absorbed from t h e g a s t r o -i n t e s t i n a l t r a c t and subsequently d e p o s i t e d i n t o t h e bony t i s s u e . These experiments have a l s o shown t h a t t h e r e a r e no adverse e f f e c t s of s t r o n t i u m on f i s h s u r v i v a l , metabolism or growth. S t r o n t i u m appears t o r e p l a c e c a l c i u m d i r e c t l y i n the bony t i s s u e and t h e r e i s no evidence of d i s t u r b a n c e i n c a l c i u m metabolism. Furthermore, h i g h s t r o n t i u m c o n t e n t i n the f r e s h water r e g i o n o f the s c a l e s (the c e n t r a l area) i s d e t e c t a b l e up t o 18 months a f t e r t h e s t r o n t i u m - e n r i c h e d d i e t has been f e d (Behrens Yamada e t al., 1979; and Behrens Yamada & M u l l i g a n , 1982) . However, s t r o n t i u m i s r e l a t i v e l y abundant i n seawater a t approximately 8 mg per l i t r e (Hummel & Smales, 1956). Once the f i s h m igrate t o sea f o r the a d u l t stage o f the l i f e c y c l e , s t r o n t i u m from the seawater i s a l s o i n c o r p o r a t e d i n t o the s c a l e s i n the seawater growth r e g i o n (the o u t e r r i n g s ) . T h i s a d d i t i o n a l s t r o n t i u m i s of a l a r g e enough c o n c e n t r a t i o n t o mask the mark o r i g i n a l l y l a i d , down d u r i n g the freshwater stage. The use o f a m i c r o a n a l y t i c a l technique, which i s capable o f a n a l y z i n g o n l y the c e n t r a l p o r t i o n of the s c a l e , may a l l o w the d i f f e r e n t i a t i o n between marked f i s h and unmarked f i s h (Behrens Yamada e t al., 1979; and Behrens Yamada, 1982). Another c o n s i d e r a t i o n i s t h a t multi-element combinations, r a t h e r than the use of a s i n g l e element, enables the i d e n t i f i c a t i o n o f a g r e a t e r number of d i f f e r e n t groups of h a t c h e r y - p r o d u c t i o n salmon. However, a s i n g l e element c o u l d be used t o c h e m i c a l l y mark f i s h i f the f i s h a r e exposed t o the element a t d i f f e r e n t time i n t e r v a l s . T h i s type of marking would r e s u l t i n growth r i n g s w i t h an enhanced c o n c e n t r a t i o n o f the elements s p a t i a l l y arranged. Although t h i s would i n c r e a s e the number of p o s s i b l e marks, the a n a l y t i c a l t e c h n i q u e ( l a s e r a b l a t i o n by ICP-MS) s t i l l needs t o be developed f o r t h e a n a l y s i s of s m a l l areas i n the f i s h s c a l e s . Lanthanide Elements  Samarium A very s m a l l number of r e s e a r c h e r s have i n v e s t i g a t e d the p o s s i b i l i t y of u s i n g the l a n t h a n i d e elements t o mass mark f i s h . Attempts t o use samarium were r e p o r t e d by M i c h i b a t a & H o r i (1981) and M i c h i b a t a (1981). These r e s e a r c h e r s s t a t e t h a t samarium would be a s u i t a b l e element t o use s i n c e i t o f f e r s the f o l l o w i n g advantages: ( i ) t h e apparent absence of harmful e f f e c t s on the f i s h ; (ii.) (the s m a l l p r o b a b i l i t y of i n t e r f e r e n c e from n a t u r a l l y o c c u r r i n g samarium; ( i i i ) t h e l o n g b i o l o g i c a l h a l f - l i f e of t h e d e p o s i t e d samarium; (i v ) no r a d i o a c t i v e c o n t a m i n a t i o n of the f i s h or o t h e r organisms; and (v) s a f e t y i n h a n d l i n g . In the f i r s t s e t of experiments, M i c h i b a t a & H o r i (1981), i n j e c t e d doses of samarium i n t o the abdomen of medaka (Oryzias latipes) and g o l d f i s h (Carassius auratus). The e l e m e n t a l mark was d e t e c t a b l e f o r up t o two y ears a f t e r the l a s t i n j e c t i o n . Samarium was accumulated i n the k i d n e y s , i n t e s t i n e , l i v e r , v e r t e b r a e , g i l l s , s c a l e s , and muscle. Although t h i s method was s u c c e s s f u l i n i n t r o d u c i n g the element i n t o the f i s h , i t r e q u i r e d i n d i v i d u a l h a n d l i n g and had t o be r e s t r i c t e d t o f i s h l a r g e enough t o i n j e c t . S i n c e a mass marking method would be more e f f i c i e n t , M i c h i b a t a (1981) c a r r i e d out an experiment i n which samarium was i n c l u d e d i n the d i e t of the two s p e c i e s of f i s h p r e v i o u s l y used. They found t h a t the l e v e l of samarium decreased w i t h i n 30 days of completion of f e e d i n g , but then remained c o n s t a n t over the next year. The t i s s u e s i n which.the samarium was d e t e c t e d were the f i f t h b r a n c h i a l a r c h , the s c a l e s , 12 and t h e g i l l s . I n i t i a l l y , t h e r e was some samarium p r e s e n t i n t h e l i v e r and i n t e s t i n e , but a f t e r 90 days the samarium was u n d e t e c t a b l e i n t h e s e organs. T h i s i n d i c a t e d t h a t s h o r t term s t o r a g e t a k e s p l a c e i n the s o f t t i s s u e s and l o n g term s t o r a g e o c c u r s i n t h e bony t i s s u e s . * M i c h i b a t a proposed t h a t the r o u t e o f e n t r y was though the water, not through the d i e t . In these experiments, samarium c o n c e n t r a t i o n i n the water supply c o u l d have been e l e v a t e d as a r e s u l t of d i s s o l v e d s p i l l e d f e e d or excrement. In a d d i t i o n , d i f f e r e n t groups of r e s e a r c h e r s w i t h v a r i o u s animal s p e c i e s have shown t h a t the l a n t h a n i d e elements ar e not absorbed from the g a s t r o - i n t e s t i n a l t r a c t ( E l l i s , 1968; Luckey & Venugopal, 1977; and K e n n e l l y e t al., 1980). Samarium does appear t o be a f e a s i b l e l a n t h a n i d e t o use as a c h e m i c a l mark f o r f i s h . Samarium a d m i n i s t e r e d t o f i s h through th e d i e t or by i n j e c t i o n remains i n the bony t i s s u e s f o r an extended p e r i o d of time ( M i c h i b a t a , 1981; and M i c h i b a t a & H o r i , 1981). Dysprosium Babb e t al. (1967) attempted t o i n t r o d u c e dysprosium i n t o chinook salmon f i n g e r l i n g s u s i n g a v a r i e t y of methods. These i n c l u d e d weekly i n t r a m u s c u l a r i n j e c t i o n s , 24 hour d i p s i n dysprosium s o l u t i o n once a week f o r f i v e weeks, f o r c e d f e e d i n g of dysprosium-enriched d i e t s , and v o l u n t a r y f e e d i n g of dysprosium-enriched d i e t s . The a n a l y t i c a l method used t o d e t e c t the dysprosium was neutron a c t i v a t i o n a n a l y s i s . T h i s method was 13 not s e n s i t i v e enough t o d e t e c t any dysprosium i n the f i s h f e d the e n r i c h e d d i e t s or p l a c e d i n the s o l u t i o n (< 1 /xg/ml) , but i t d i d d e t e c t the dysprosium i n the i n j e c t e d f i s h . Perhaps a more s e n s i t i v e a n a l y t i c a l method would have been a b l e t o d e t e c t the s m a l l e r amounts of element p r e s e n t i n the treatment groups. From t h e s e r e s u l t s , the i n t r a m u s c u l a r i n j e c t i o n s appeared t o be the most e f f e c t i v e of the f o u r methods used. However, s i n c e i n j e c t i o n s r e q u i r e i n d i v i d u a l h a n d l i n g , they a r e time consuming and r e s u l t i n i n c r e a s e d m o r t a l i t i e s . S i m i l a r r e s u l t s were found by M i l l e r (1963). In t h i s experiment t h e treatments i n c l u d e d the f e e d i n g of dysprosium-e n r i c h e d d i e t s , submergence of f i s h i n 100 nq/1 dysprosium s o l u t i o n , and i n t r a m u s c u l a r i n j e c t i o n s ; the a n a l y t i c a l method used was neutron a c t i v a t i o n a n a l y s i s . The chinook f i n g e r l i n g s f e d t h e dysprosium-enriched d i e t s and those submerged i n the s o l u t i o n d i d not d e p o s i t d e t e c t a b l e amounts of dysprosium i n t o the bony t i s s u e . In c o n t r a s t , i n t r a m u s c u l a r i n j e c t i o n s r e s u l t e d i n the v a r i a b l e a b s o r p t i o n and d e p o s i t i o n of dysprosium (up t o 368 M9J/?;) i n t h e bones of f i s h sampled a t the end of f i v e weeks. The dysprosium p e r s i s t e d i n the bones f o r up t o f i v e months a f t e r the i n j e c t i o n s , i n d i c a t i n g t h a t long term s t o r a g e i s i n the bony t i s s u e s . From thes e data, i t appears t h a t the i n j e c t i o n method i s the most e f f e c t i v e way of i n d u c i n g a d e t e c t a b l e mark. The f i s h i n t h i s experiment were o n l y submerged i n the dysprosium s o l u t i o n f o r 24-hour p e r i o d s once a week f o r f i v e weeks w i t h no d e t e c t a b l e mark l a i d down. I t i s 14 p o s s i b l e t h a t a l o n g e r exposure time would have r e s u l t e d i n d e t e c t a b l e amounts of dysprosium i n the t i s s u e s . Europium There a r e v a r y i n g r e p o r t s i n the l i t e r a t u r e on- the success of europium as a f i s h mark. Japanese r e s e a r c h e r s (Anon., 1974) induced europium marks through the f e e d i n g of europium-enriched d i e t s t o j u v e n i l e chum salmon (Oncorhynchus keta). The d i e t s f e d t o the f i s h c o n t a i n e d 1,000 and 15,000 nq/q of europium. Neutron a c t i v a t i o n a n a l y s i s showed d e t e c t a b l e amounts of europium i n the s c a l e s and o t o l i t h s f o r up t o 1 y e a r a f t e r the l a b e l l i n g . Another r e s e a r c h e r , Kato (1985), a l s o succeeded i n i n d u c i n g a europium mark i n chum salmon through t h e d i e t . He found the element t o be p r e s e n t i n the f i s h s c a l e s f o r up t o 2 y e a r s a f t e r f e e d i n g of the europium-treated d i e t s was d i s c o n t i n u e d . H i s r e s u l t s a l s o i n d i c a t e l o n g term s t o r a g e of europium i n the bony t i s s u e s . M i c h i b a t a & H o r i (1981) d i s c u s s e d the experiment c a r r i e d out by Shibuya i n 1979 which i n v o l v e d the i n t r o d u c t i o n of europium through the d i e t . Shibuya d i s c o v e r e d t h a t the element was accumulated i n the s c a l e s , but the mark l a s t e d o n l y 3 months a f t e r l a b e l l i n g . The c o n c l u s i o n s reached from t h e r e s u l t s o b t a i n e d i n t h e s e experiments v a r y c o n s i d e r a b l y . U n f o r t u n a t e l y , the paper w r i t t e n by Shibuya (1979) was i n Japanese and a t r a n s l a t i o n was not a v a i l a b l e . Consequently, the v a l i d i t y of t h e s e r e s u l t s cannot be. assessed. However, the o t h e r two 15 r e s e a r c h e r s have c l e a r l y demonstrated t h a t europium was taken up by the f i s h and was i n c o r p o r a t e d i n t o the s c a l e s . Terbium Muncy and D ' S i l v a (1981) conducted an experiment u s i n g w a l l e y e (Stizostedion vitreum) eggs and f r y i n which v a r y i n g c o n c e n t r a t i o n s of l a n t h a n i d e s o l u t i o n s were a p p l i e d f o r d i f f e r e n t p e r i o d s . The s o l u t i o n s added t o the water supply were terb i u m c h l o r i d e , sodium terbium c i t r a t e , t e r b i u m d i c i t r a t e , europium c h l o r i d e , and neodymium d i c i t r a t e . These r e s e a r c h e r s determined t h a t terbium d i c i t r a t e appeared t o be s u i t a b l e f o r marking the w a l l e y e eggs d u r i n g the water-hardening p r o c e s s . The other_ terbium s o l u t i o n s appeared t o be i n e f f e c t i v e i n marking the eggs and f r y . In a d d i t i o n , t h e r e were no d e t e c t a b l e l e v e l s of neodymium or europium found i n e i t h e r the eggs or the f r y . Summary of the Lanthanides The r e s u l t s of a l l t h e s e marking experiments i n v o l v i n g the use of the l a n t h a n i d e elements show t h a t under c e r t a i n c o n d i t i o n s these elements are taken up by the f i s h and are subsequently i n c o r p o r a t e d i n t o the bony t i s s u e s . Furthermore, l o n g term s t o r a g e o c c u r s i n the bony t i s s u e s w h i l e the s h o r t term s t o r a g e i s i n the s o f t t i s s u e s . Of the v a r i o u s methods used, i n c o r p o r a t i o n of the l a n t h a n i d e s i n the d i e t does not appear t o be a s u i t a b l e method 16 s i n c e t h e s e elements are not absorbed through the g a s t r o -i n t e s t i n a l t r a c t a t l e v e l s which c o u l d be d e t e c t e d u s i n g the a n a l y t i c a l t e c h n i q u e a v a i l a b l e . I n j e c t i o n s of samarium and dysprosium d i d r e s u l t i n c o n s i d e r a b l e c o n c e n t r a t i o n s d e p o s i t e d i n , and r e t a i n e d by the bone. However, a mass marking method would be p r e f e r a b l e t o i n j e c t i o n . A p o s s i b l e a l t e r n a t i v e i s submergence i n a l a n t h a n i d e s o l u t i o n f o r extended p e r i o d s of time. T h i s may r e s u l t i n a s u f f i c i e n t amount of t h e l a n t h a n i d e elements b e i n g absorbed and s t o r e d by the f i s h f o r these elements t o be used as d e t e c t a b l e markers. 17 Lanthanide Elements I n t r o d u c t i o n The l a n t h a n i d e elements are a l s o r e f e r r e d t o as the lanthanons o r t h e r a r e e a r t h elements. They are a s e r i e s of f i f t e e n elements s t a r t i n g w i t h lanthanum (atomic number, 57) and ending w i t h l u t e t i u m (atomic number, 71) . T h e i r e l e c t r o n c o n f i g u r a t i o n p l a c e s them i n t o subgroup I I I B o f t h e p e r i o d i c t a b l e o f the elements. A l l l a n t h a n i d e s are c l o s e l y r e l a t e d elements w i t h v e r y s i m i l a r chemical and p h y s i c a l p r o p e r t i e s (Kyker, 1961). The t h r e e c l a s s i f i c a t i o n s o f these elements are as f o l l o w s : ( i ) l i g h t l a n t h a n i d e s : lanthanum (La), cerium (Ce) , praseodymium (Pr) . neodymium (Nd). promethium (Pm) . and samarium (Sm); ( i i ) medium l a n t h a n i d e s : europium (Eu), ga d o l i n i u m (Gd), te r b i u m (Tb), dysprosium (Dy), and holmium (Ho); and ( i i i ) heavy l a n t h a n i d e s : erbium ( E r ) , t h u l i u m (Tm), y t t e r b i u m (Yb), and l u t e t i u m (Lu). i The o n l y r a d i o a c t i v e l a n t h a n i d e i s promethium. Y t t r i u m (Y) i s a group I I I A element and, although not a t r u e r a r e e a r t h , i t i s u s u a l l y i n c l u d e d w i t h the r a r e e a r t h s s i n c e i t has s i m i l a r p r o p e r t i e s t o t h e heavy l a n t h a n i d e s ( V i c k e r y , 1961; and Kyker, 1962) . The c l a s s i f i c a t i o n s and atomic weights of the l a n t h a n i d e s and y t t r i u m are p r o v i d e d i n T a b l e 1. The t i t l e " r a r e e a r t h " i s both m i s l e a d i n g and i n a c c u r a t e . Although c o n c e n t r a t e d d e p o s i t s of the l a n t h a n i d e s are r a r e , 18 th e s e elements are r e l a t i v e l y abundant i n the e a r t h ' s c r u s t . Cerium, f o r example, oc c u r s i n g r e a t e r amounts than do t i n , c o b a l t , g o l d , and s i l v e r ( T a y l o r , 1964; and Luckey and Venugopal, 1977). Although these elements are metals, they were named e a r t h s because they were f i r s t i d e n t i f i e d as a mixture of oxi d e s t h a t c l o s e l y resembled the a l k a l i n e e a r t h oxides ( M o e l l e r , 1963). The major m i n e r a l source f o r the l a n t h a n i d e o x i d e s i s monazite sand, where 90% of the l a n t h a n i d e p o r t i o n i s the l i g h t elements. Due t o advanced s e p a r a t i o n and p u r i f i c a t i o n methodology, l a n t h a n i d e compounds are a v a i l a b l e i n h i g h l y p u r i f i e d forms from s e v e r a l commercial s o u r c e s . The odd-numbered and h e a v i e r l a n t h a n i d e s are g e n e r a l l y the most p r e c i o u s and d i f f i c u l t t o p u r i f y , t h e r e f o r e t h e s e tend t o be more expensive. 19 T a b l e 1. C l a s s i f i c a t i o n of the l a n t h a n i d e elements and t h e i r e l emental c h a r a c t e r i s t i c s . Element C r u s t a l Abundance 1 C l a s s i f -i c a t i o n Atomic Number Atomic Weight N a t u r a l Isotopes Y t t r i u m (Y) 33 . 0 L i g h t 39 88.905 1 Lanthanum (La) 30.0 L i g h t 57 138.91' 2 Cerium (Ce) 60.0 L i g h t 58 140.12 4 Praseodymium (Pr) 8.2 L i g h t 59 140.907 1 Neodymium (Nd) 28.0 L i g h t 60 144.24 7 Promethium (Pm) 2 0.0 L i g h t 61 (145) Samarium (Sm) 6.0 L i g h t 62 150.35 7 Europium (Eu) 1.2 Medium 63 151.96 2 Gadolinium (Gd) 5.4 Medium 64 157.25 7 Terbium (Tb) 0.9 Medium 65 158.924 1 Dysprosium (Dy) 3 . 0 Medium 66 162.50 7 Holmium (Ho) 1.2 Medium 67 164.930 1 Erbium (Er) 2.8 Heavy 68 167.26 6 Thulium (Tm) 0.5 Heavy 69 168.934 1 Yt t e r b i u m (Yb) 3.0 Heavy 70 173.04 7 Lutetium (Lu) 0.5 Heavy 71 174.97 2 1 C r u s t a l abundance measured i n /xg/g ( T a y l o r , 1964) . 2 R a d i o a c t i v e l a n t h a n i d e . The uses of the l a n t h a n i d e s are d i v e r s e and widespread. They i n c l u d e : ceramics, carbon a r c s , c a t a l y s t s , l i g h t e r f l i n t s , t e l e v i s i o n tubes, m i r r o r s , t e x t i l e w a t e r p r o o f i n g compounds, p a i n t d r i e r s , e l e c t r o n i c equipment, and many o t h e r s (Kyker, 1961; and Luckey & Venugopal, 1977). As r e s e a r c h i n t o these elements c o n t i n u e s , i t i s l i k e l y t h a t more a p p l i c a t i o n s of the l a n t h a n i d e s w i l l be d i s c o v e r e d . Chemical P r o p e r t i e s The l a n t h a n i d e elements are i n n e r - t r a n s i t i o n a l elements which share s i m i l a r chemical and m e t a b o l i c p r o p e r t i e s . The atomic weights of the l a n t h a n i d e s i n c r e a s e i n v e r y s m a l l increments. V a r i o u s c h a r a c t e r i s t i c s t h a t are c o r r e l a t e d w i t h t h e i n c r e a s e i n atomic number ar e : ( i ) i n c r e a s i n g a c i d i t y , c o v a l e n t c h a r a c t e r i s t i c s and s t a b i l i t y of complex i o n s , and ( i i ) d e c r e a s i n g b a s i c i t y , i o n i c c h a r a c t e r i s t i c s , thermal s t a b i l i t y and s o l u b i l i t y . Atomic r a d i i decrease w i t h subsequent i n c r e a s e s i n atomic number from 1.2 angstroms f o r lanthanum t o 0.95 angstroms f o r l u t e t i u m . A l l of the elements e x i s t i n a t r i v a l e n t (Ln 3 +) s t a t e , however lanthanum, samarium, europium and y t t e r b i u m can a l s o e x i s t i n a d i v a l e n t (Ln 2 +) s t a t e , and cerium, praseodymium, terbium i n a t e t r a v a l e n t (Ln 4 +) s t a t e (Kyker, 1961). A wide range of l a n t h a n i d e compounds can be formed, each w i t h a v a r y i n g degree of s o l u b i l i t y . In g e n e r a l , the n i t r a t e s , h a l i d e s , p e r c h l o r a t e s , t h i o c y a n a t e s and a c e t a t e s are a l l r e l a t i v e l y s o l u b l e . The s u l p h a t e s are moderately s o l u b l e , w h i l e the o x i d e s , h y d r o x i d e s , f l u o r i d e s , o x a l a t e s , carbonates, and phosphates are r e l a t i v e l y i n s o l u b l e (Topp, 1965). In aqueous s o l u t i o n s , the h a l i d e s h y d r o l y s e r e a d i l y t o produce i n s o l u b l e oxide h a l i d e s : LnX 3 + H20 -> LnOX + 2HX, where Ln r e p r e s e n t s any l a n t h a n i d e and X r e p r e s e n t s any h a l i d e , f o r example: L a C l 3 + H20 -> LaOCl + 2HC1. Lanthanides w i t h o r g a n i c a c i d s , such as a c e t a t e s , are r e l a t i v e l y s t a b l e i n aqueous s o l u t i o n s and do not tend t o h y d r o l y s e . The a c e t a t e compounds are moderately s o l u b l e i n water a t room temperature (Topp, 1965). From t h i s , i t would appear t h a t the a c e t a t e form i s p r e f e r a b l e over the s a l t form t o use i n the i n t r o d u c t i o n of t h e l a n t h a n i d e s t o aqueous s o l u t i o n s . The c h e m i c a l p r o p e r t i e s of the l a n t h a n i d e elements determine t h e i r behaviour i n l i v i n g systems. Once l a n t h a n i d e c a t i o n s are i n t r o d u c e d i n t o the g a s t r o - i n t e s t i n a l t r a c t , they e i t h e r undergo a h y d r o l y s i s r e a c t i o n or r e a c t w i t h the normal b i o c h e m i c a l c o n s t i t u e n t s t o form complexes of i n s o l u b l e compounds. At the p h y s i o l o g i c a l pH of l i v i n g organisms, h y d r o l y s i s of the l a n t h a n i d e s i s h i g h l y favoured. T h i s i o n i c r e a c t i o n proceeds v e r y q u i c k l y and the r e s u l t i n g l a n t h a n i d e h y d r o x i d e s and phosphates formed i n the g a s t r o - i n t e s t i n a l t r a c t a re i n s o l u b l e and p r e c i p i t a t e out. The h y d r o l y s i s r e a c t i o n i s as f o l l o w s : L n 3 + + 3H20 -> Ln(OH) 3 + 3H +. S i n c e the a f f i n i t y of l a n t h a n i d e s towards phosphate i s h i g h , i n s o l u b l e phosphate complexes tend t o form. C h e l a t i n g agents such as c i t r a t e s or l a c t a t e s may be p r e s e n t i n the t i s s u e s . The l a n t h a n i d e c a t i o n s show a s t r o n g tendency t o complex w i t h t h e s e compounds, thus keeping them i n s o l u t i o n . The r e s u l t i n g complexes are s t a b l e and are not s u b j e c t t o h y d r o l y s i s i n b i o l o g i c a l f l u i d s . Lanthanides i n t r o d u c e d i n t o the g a s t r o - i n t e s t i n a l t r a c t as l a n t h a n i d e c i t r a t e 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) complexes appear t o be absorbed. S i n c e t h e s e l a r g e complexes r e s i s t h y d r o l y s i s r e a c t i o n s , i n s o l u b l e l a n t h a n i d e compounds do not form (Luckey & Venugopal, 1977). S i n c e these complex compounds a r e not r e a d i l y a v a i l a b l e , t h i s r o u t e was not i n v e s t i g a t e d f u r t h e r . The l a n t h a n i d e elements tend t o adhere t o p a r t i c u l a t e matter and s u r f a c e s w i t h which they come i n t o c o n t a c t . They e x h i b i t t h e i r a d s o r p t i v e p r o p e r t i e s a t v e r y low c o n c e n t r a t i o n s i n aqueous s o l u t i o n s (Luckey & Venugopal, 1977). The a d s o r p t i o n of l a n t h a n i d e s t o i n g e s t a p a r t i c l e s i s p a r t i a l l y r e s p o n s i b l e f o r the passage of the l a n t h a n i d e s through the g a s t r o - i n t e s t i n a l t r a c t . Adherence t o s u r f a c e s i s sometimes r e f e r r e d t o as a d s o r p t i o n or " p l a t i n g - o u t " . Lanthanides a l s o i n t e r a c t w i t h v a r i o u s t i s s u e components, which can i n c l u d e nucleo p r o t e i n s , plasma p r o t e i n s , amino a c i d s , p h o s p h o l i p i d s , enzymes, and i n o r g a n i c anions (Kyker, 1961; and Luckey & Venugopal, 1977). The heavy l a n t h a n i d e s tend t o complex w i t h complete p r o t e i n s w h i l e the l i g h t l a n t h a n i d e s tend t o complex w i t h i n d i v i d u a l amino a c i d s (Graca e t al., 1962). Although t h e r e a re some d i f f e r e n c e s between the l i g h t and the heavy l a n t h a n i d e s , the o v e r a l l chemical r e a c t i o n s a re q u i t e s i m i l a r f o r the s e r i e s . Consequently, t h e r e i s a tendency t o group the p r o p e r t i e s of the l a n t h a n i d e s t o g e t h e r . Metabolism The b i o l o g i c a l and chemical c h a r a c t e r i s t i c s of the l a n t h a n i d e s a f f e c t how they are me t a b o l i z e d by l i v i n g organisms. No l a n t h a n i d e has been proven t o be e s s e n t i a l or t o have a r o l e i n t he metabolism of any p l a n t s or animals. These elements are not p r e s e n t i n p l a n t or animal ' t i s s u e (Kyker, 1961). T h e i r absence i n p l a n t t i s s u e suggests t h a t p l a n t s d i s c r i m i n a t e a g a i n s t t h e a b s o r p t i o n of l a n t h a n i d e s from s o i l . T h i s e f f e c t i v e l y b l o c k s the d i e t a r y t r a n s f e r of l a n t h a n i d e s from s o i l t o animals (Luckey & Venugopal, 1977). In mammals the g a s t r o - i n t e s t i n a l (Gl) a b s o r p t i o n of s o l u b l e l a n t h a n i d e s a l t s i s n e g l i g i b l e - l e s s than 0.05% ( E l l i s , 1968; Luckey & Venugopal, 1977; and K e n n e l l y e t al., 1980). The l a n t h a n i d e o x i d e s are e x c e l l e n t markers f o r use i n d i g e s t i b i l i t y and r a t e of passage s t u d i e s s i n c e v i r t u a l l y a l l the element passes through the t r a c t . Poor a b s o r p t i o n of l a n t h a n i d e s has been r e p o r t e d i n a l l s p e c i e s s t u d i e d : r a t s and mice (Cochran e t al., 1950; Marcus & Lengemann, 1962; Haley, 1965; Hutcheson e t al., 1975; and Luckey e t al. , 1975), c a t t l e (Garner e t al. , 1960; Huston & E l l i s , 1965; O l b r i c h e t al., 1971; Young e t al., 1975, 1976; H a r t n e l l & S a t t e r , 1979; Uden e t al. , 1980; and Arambel e t al., 1986), monkeys (Hutcheson e t al., 1974), sheep ( E l l i s , 1968; and Pond e t al. , 1985), llamas (Cerasov & H e l l e r , 1980), swine (Kenne l l y e t al. , 1980), and humans (Hayes e t al. , 1964; and Luckey e t al., 1977, 1979). Once the simple l a n t h a n i d e s a l t s e n t e r t h e g a s t r o -i n t e s t i n a l (Gl) t r a c t , they form i n s o l u b l e h y d r o x i d e s and phosphates which p r e c i p i t a t e out as d e s c r i b e d i n the c h e m i s t r y s e c t i o n of t h i s review (Kyker, 1961; E l l i s , 1968; and Luckey & Venugopal, 1977) . The h e a v i e r l a n t h a n i d e s tend t o s e p a r a t e out more than the l i g h t l a n t h a n i d e s and are even l e s s w e l l absorbed. They can a l s o combine w i t h o r g a n i c matter t o form i n s o l u b l e phosphates which a l s o pass completely through the G l t r a c t . In a d d i t i o n , the l a n t h a n i d e hydroxides have v e r y s t r o n g a d s o r p t i v e p r o p e r t i e s t h a t cause them t o adhere t o p a r t i c u l a t e food matter. Once bound t o t h i s m a t e r i a l , they move through the t r a c t a l o n g w i t h t h e i n g e s t a (Kyker, 1961; and Hutcheson e t al., 1975). The l a n t h a n i d e s are o f t e n d e s c r i b e d as bone-seeking elements. Small doses of l a n t h a n i d e s have r e g u l a r l y been shown t o accumulate i n the bony t i s s u e (Durbin e t al., 1956; Jowsey e t al., 1958; Kyker, 1961; M i c h i b a t a , 1981; and M i c h i b a t a & H o r i , 1981). The elements are d i s t r i b u t e d r a p i d l y i n t o the l i v e r and ki d n e y s , w i t h g r a d u a l uptake and r e t e n t i o n by t h e s k e l e t o n (Luckey & Venugopal, 1977). Durbin e t al. (1956) found t h a t a p p r o x i m a t e l y 55% of the dysprosium a d m i n i s t e r e d i n t r a m u s c u l a r l y t o r a t s was d e p o s i t e d i n the bone. Approximately 50% of the a d m i n i s t e r e d l a n t h a n i d e s were d e p o s i t e d i n t h e l i v e r , and the m a j o r i t y of t h i s was e x c r e t e d w i t h i n 2 months. Furthermore, young animals absorb and r e t a i n the i n j e c t e d l a n t h a n i d e s b e t t e r than o l d e r animals (Luckey & Venugopal, 1977). The h i g h l y m i n e r a l i z e d non-growing bone s u r f a c e , not the o s t e o i d t i s s u e , i s the s i t e o f l a n t h a n i d e d e p o s i t i o n i n mammals. Bone m i n e r a l i s r e s p o n s i b l e f o r l a n t h a n i d e uptake from the plasma and f o r the subsequent d e p o s i t i o n i n t o the bone (Durbin e t al., 1956; and Jowsey e t al., 1958). The t r a n s p o r t o f l a n t h a n i d e s from bl o o d t o s o f t t i s s u e s and bone depends on the c o n c e n t r a t i o n , the n a t u r e of the l a n t h a n i d e compound, and the mode of a d m i n i s t r a t i o n (Luckey & Venugopal, 1977). In f i s h , t he g i l l s a r e the main c o n t a c t a r e a w i t h the e x t e r n a l environment, which i m p l i e s t h a t the g i l l s a l s o serve as the main uptake s i t e f o r d i s s o l v e d compounds, i n c l u d i n g heavy metals and c a l c i u m ( P a r t & Svanberg, 1981; and P e r r y & Wood, 1985). T h e o r e t i c a l l y , c a l c i u m i o n s d i s s o l v e d i n t h e water are taken up by t h e c h l o r i d e c e l l s i n the g i l l s , t hen t r a n s p o r t e d t o ot h e r t i s s u e s by the c i r c u l a t o r y system (Payan e t al., 1981). In a review a r t i c l e , Weiss (1974) s t a t e s t h a t the L a 3 + i o n i s a s p e c i f i c a n t a g o n i s t o f Ca 2 + i o n s , i n b i o l o g i c a l systems. Lanthanum i o n s have been found t o d i s p l a c e o r r e p l a c e c a l c i u m i n d i f f e r e n t c e l l f u n c t i o n s , and they prevent the i n f l u x o f Ca 2* i o n s by competing f o r a v a i l a b l e b i n d i n g s i t e s (Das e t al., 1988). L a 3 + i o n s p r o b a b l y e n t e r the f i s h by the same r o u t e as the Ca 2 + i o n s . Bony T i s s u e Development S i n c e the l a n t h a n i d e elements are bone-seekers, the development o f the bony t i s s u e s i s very important i n the success of the ch e m i c a l l a b e l l i n g . The development and growth of the v e r t e b r a l column, o t o l i t h s and s c a l e s may have an e f f e c t on the l e n g t h of time the element remains i n the t i s s u e . L i t t l e i s known about t h e p h y s i o l o g y o f c a l c i f i e d m a t e r i a l i n t o t he f i s h ' s bony t i s s u e s . However, i t has been demonstrated t h a t once the bony t i s s u e s a re formed, the c a l c i f i e d m a t e r i a l appears t o remain i n t h e same l o c a t i o n and s t a t e as i t was when i n i t i a l l y d e p o s i t e d . In g e n e r a l , the bony t i s s u e s grow by the continuous, c o n c e n t r i c a l d e p o s i t i o n o f c a l c i f i e d t i s s u e . Although t h e r e are some d i f f e r e n c e s i n c a l c i f i c a t i o n between the v e r t e b r a e , o t o l i t h s and s c a l e s , the c a l c i u m metabolism i s c o - o r d i n a t e d . The growth of thes e bony t i s s u e s i s p a r a l l e l t o one another, but the o t o l i t h s a r e the f i r s t t o develop ( P a n n e l l a , 1980). A c h e m i c a l l a b e l which i s d e p o s i t e d a t s i t e s of c a l c i f i c a t i o n p r o v i d e s an elem e n t a l mark i n the t i s s u e . In t h i s way t e t r a c y c l i n e has been used t o l a b e l v a r i o u s t y p e s o f hard t i s s u e s such as: s c a l e s , o p e r c u l a , v e r t e b r a e , s p i n e s , and o t o l i t h s . T h i s a n t i b i o t i c remains i n the bony t i s s u e , without any harmful e f f e c t on the f i s h , as a r e s u l t of the c o n c e n t r i c a l growth and development (Casselman, 1987). Bone Development Moss (1961) d e s c r i b e s bone as c o n s i s t i n g of a c o l l a g e n o u s f i b r e o r g a n i c m a t r i x w i t h a s s o c i a t e d mucoproteins which i s c a l c i f i e d by h y d r o x y a p a t i t e s a l t s . The bone i s made from a c a l c i u m phosphate m a t r i x . The d e p o s i t i o n of bony t i s s u e i s a s s o c i a t e d w i t h the presence of o s t e o b l a s t s which are the bone-forming c e l l s . As w i t h the o t o l i t h s and s c a l e s , the v e r t e b r a l column i s l a i d down c o n c e n t r i c a l l y . N o r r i s e t al. (1963) r e p o r t e d t h a t Ca 4 5 d e p o s i t i o n o c c u r s between the o s t e o i d l a y e r s and p r e v i o u s l y 2 7 m i n e r a l i z e d bone. T h i s f u r t h e r demonstrates the c o n c e n t r i c n ature of the bony t i s s u e development. O t o l i t h Development There are t h r e e p a i r s of f i s h o t o l i t h s : l a p i l l u s , s a g i t t a , and a s t e r i c u l u s . The l a r g e s t i s the s a g i t t a ( p i . s a g i t t a e ) , and because o f i t s r e l a t i v e l y l a r g e s i z e i t i s most o f t e n s t u d i e d . An o t o l i t h , u n l i k e r e g u l a r bone, i s composed of c a l c i u m carbonate (CaC0 3) c r y s t a l s , i n the a r a g o n i t e form, and a p r o t e i n a c e o u s o r g a n i c m a t e r i a l (Gauldie, 1986; and P a n n e l l a , 1980). P a n n e l l a (1971) f i r s t d e s c r i b e d the presence of d a i l y growth r i n g s i n f i s h o t o l i t h s . Subsequently, a number of r e s e a r c h e r s have observed these growth r i n g s . The o t o l i t h s have been r e p o r t e d t o grow c o n c e n t r i c a l l y as a r e s u l t of the continuous c a l c i u m carbonate d e p o s i t i o n (Mugiya e t al., 1979; W i l s o n & L a r k i n , 1980; and N e i l s o n & Geen, 1982). The CaC0 3 d e p o s i t e d i n the o t o l i t h s i s not r e c y c l e d and the o t o l i t h s remain unchanged throughout the l i f e o f the f i s h . A l s o , any elements i n c o r p o r a t e d i n t o the o t o l i t h s from the ambient water supply would be r e f l e c t e d i n the elemental a n a l y s i s of the growth r i n g s (Campana & N e i l s o n , 1985). S c a l e Development The f o r m a t i o n o f s c a l e s begins i n the area between the d o r s a l f i n and the adipose f i n , along the l a t e r a l l i n e . They 28 then extend a n t e r i o r l y towards the operculum, and p o s t e r i o r l y towards the c a u d a l f i n . As a r e s u l t , the s c a l e s along the p o s t e r i o r p o r t i o n of the l a t e r a l l i n e are the l a r g e s t , d e c r e a s i n g i n s i z e away from t h i s r e g i o n ( J o l l i e , 1984; and Witkowski e t al., 1984). The c e n t r e of the s c a l e i s commonly r e f e r r e d t o as the focus and the r i n g s t h a t surround i t are c a l l e d c i r c u l i . The s c a l e s a r e composed of c a l c i u m phosphate as are the bones. The c a l c i f i e d m a t e r i a l i s l a i d down c o n c e n t r i c a l l y as i n the v e r t e b r a l column and the o t o l i t h s . Any elements i n c o r p o r a t e d i n t o the c e n t r a l r e g i o n of the s c a l e s s h o u l d remain t h e r e throughout the f i s h ' s l i f e h i s t o r y ( L a p i & M u l l i g a n , 1981). T o x i c i t y When i n t r o d u c i n g any f o r e i g n chemicals i n t o a b i o l o g i c a l system, p o s s i b l e t o x i c o l o g i c a l e f f e c t s need t o be c o n s i d e r e d . U n f o r t u n a t e l y , no s t u d i e s on the d i r e c t e f f e c t s of the l a n t h a n i d e s on f i s h have been r e p o r t e d i n the l i t e r a t u r e . C u r r e n t knowledge on the t o x i c e f f e c t s of the l a n t h a n i d e s i s l i m i t e d t o s e v e r a l acute t o x i c o l o g i c a l measurements i n mammals. The t o x i c i t y s t u d i e s reviewed here d e s c r i b e the e f f e c t s of d i f f e r e n t l a n t h a n i d e compounds a d m i n i s t e r e d by v a r i o u s methods i n t o r o d e n t s . When compared w i t h o t h e r elements, the l a n t h a n i d e s are c o n s i d e r e d t o be r e l a t i v e l y n o n - t o x i c or o n l y s l i g h t l y t o x i c . The method of a d m i n i s t r a t i o n of the l a n t h a n i d e compounds i s a d e t e r m i n i n g f a c t o r i n the t o x i c i t y e f f e c t s found i n mammals. Some o f the d i f f e r e n t modes of i n t r o d u c t i o n used by Venugopal & Luckey (1974) i n c l u d e : ( i ) o r a l a d m i n i s t r a t i o n and a b s o r p t i o n from the g a s t r o -i n t e s t i n a l t r a c t (PO); ( i i ) i n t r a v e n o u s i n j e c t i o n w i t h f a s t d i s t r i b u t i o n t o t i s s u e s ( I V ) ; ( i i i ) l u n g i n h a l a t i o n ; ( i v ) subcutaneous a d m i n i s t r a t i o n (SC); (v) i n t r a p e r i t o n e a l i n j e c t i o n ( I P ) ; ( v i ) i n t r a m u s c u l a r i n j e c t i o n (IM); and ( v i i ) a b s o r p t i o n through the s k i n . Intravenous i n j e c t i o n appeared t o l e a d t o g r e a t e s t t o x i c i t y whereas o r a l a d m i n i s t r a t i o n appeared t o have the l e a s t e f f e c t . I n t r a p e r i t o n e a l and subcutaneous a d m i n i s t r a t i o n a l s o r e s u l t e d i n c o m p a r a t i v e l y low t o x i c i t y ( L a s z l o e t al., 1952). O r a l t o x i c i t y of the l a n t h a n i d e s i s v e r y low due t o t h e i r n e g l i g i b l e a b s o r p t i o n from the g a s t r o - i n t e s t i n a l t r a c t , which has been shown by many r e s e a r c h e r s . As d e s c r i b e d e a r l i e r , the l a n t h a n i d e s a l t s undergo a h y d r o l y s i s r e a c t i o n i n l i v i n g organisms which r e s u l t s i n the fo r m a t i o n o f i n s o l u b l e hydroxides or phosphates. These compounds are not absorbed from the G l t r a c t (Cochran e t al., 1950; Haley, 1965; and Luckey & Venugopal, 1977). A l s o , the l a n t h a n i d e s tend t o adsorb t o p a r t i c u l a t e food i n g e s t a and pass d i r e c t l y through the t r a c t (Venugopal & Luckey, 1974). Mice f e d d i e t s c o n t a i n i n g g r e a t e r than 5000 ug/g of l a n t h a n i d e oxides showed normal g e n e r a l appearance, growth, maturation, and r e p r o d u c t i o n (Luckey & Venugopal, 1977). Haley e t al. (1961) found t h a t o r a l doses of up t o 2 g/kg of d i e t produced no m o r t a l i t i e s i n r a t s and t h e r e was no evidence of i n t e r n a l damage from the i n g e s t i o n of samarium and gadolinium. A c c o r d i n g t o the LD 5 0 v a l u e s of the l a n t h a n i d e s , the medium weight l a n t h a n i d e s are the l e a s t t o x i c , and the l i g h t l a n t h a n i d e s are s l i g h t l y more t o x i c than the heavy l a n t h a n i d e s . The t o x i c i t y of the l a n t h a n i d e s g e n e r a l l y decreases w i t h i n c r e a s i n g atomic weight (Kyker & Cress, 1957; and Luckey & Venugopal, 1977). Lanthanum i s c o n s i d e r e d t o be the most t o x i c l a n t h a n i d e because of i t s e l e c t r o - c h e m i c a l c h a r a c t e r i s t i c s of h i g h e l e c t r o p o s i t i v i t y , h i g h charge d e n s i t y , and h i g h tendency t o form complexes through e l e c t r o s t a t i c bonding (Luckey & Venugopal, 1977). The t o x i c o l o g i c a l data (LD 5 0 values) of d i f f e r e n t l a n t h a n i d e compounds a d m i n i s t e r e d t o r a t s and mice u s i n g v a r i o u s methods are summarized i n Appendix 1. A c c o r d i n g t o d i f f e r e n t groups of r e s e a r c h e r s , the symptoms of acute l a n t h a n i d e i n t o x i c a t i o n i n rodents i n c l u d e : w r i t h i n g , a t a x i a , s e d a t i o n , laboured r e s p i r a t i o n , and immediate d e f e c a t i o n (Haley, 1965; Luckey & Venugopal, 1977; and B e l i l e s , 1978). Some of the c l i n i c a l symptoms i n c l u d e : s o f t t i s s u e c a l c i f i c a t i o n a t s i t e s of i n j e c t i o n , l i v e r edema and n e c r o s i s , pulmonary edema, and hyperaemia (Luckey & Venugopal, 1977). S p e c i f i c i n j u r i e s have been r e p o r t e d i n e x p e r i m e n t a l animals. L a s z l o e t al. (1952) observed l i v e r damage i n rodents which had r e c e i v e d lanthanum c h l o r i d e by in t r a v e n o u s i n j e c t i o n i n the t o x i c dose range. I n j e c t i o n s o f the l a n t h a n i d e s r e s u l t e d i n the p r o d u c t i o n o f f a t t y l i v e r s , which d i d not occur a f t e r o r a l a d m i n i s t r a t i o n (Haley, 1965). T h i s a g a i n demonstrates t h a t the mode of i n t r o d u c t i o n o f these elements has a s u b s t a n t i a l e f f e c t on t h e i r t o x i c i t y . The above d i s c u s s i o n on the t o x i c i t y of the' l a n t h a n i d e elements has been l i m i t e d t o the e f f e c t s observed i n s m a l l mammals. S i n c e no work has been c a r r i e d out s p e c i f i c a l l y on f i s h , any i n f e r e n c e s made assume t h a t the e f f e c t s would be the same i n f i s h as i n mammals. T h i s i s not a v a l i d comparison s i n c e t h e r e a re d i f f e r e n t methods t h a t can be used i n the i n t r o d u c t i o n o f the l a n t h a n i d e s i n t o the f i s h . For example, l a n t h a n i d e s may be i n t r o d u c e d i n t o f i s h by submerging them i n a l a n t h a n i d e s o l u t i o n o r by c a n n u l a t i o n o f a bl o o d v e s s e l . However, one of the comparisons t h a t can be made between modes of a d m i n i s t r a t i o n between f i s h and mammals i s o r a l a d m i n i s t r a t i o n . U s i n g t h i s method, the l a n t h a n i d e s were not absorbed by the i n t e s t i n a l t r a c t o f e i t h e r animal, t h e r e f o r e no t o x i c e f f e c t s were observed. A n a l y t i c a l Methods Very few c l a s s i c i n s t r u m e n t a l methods are a v a i l a b l e f o r the d e t e r m i n a t i o n and q u a n t i f i c a t i o n of the l a n t h a n i d e elements. Although the s p e c t r a o f the l a n t h a n i d e s i s w e l l known and documented, s p e c t r o s c o p i c methods are g e n e r a l l y not s e n s i t i v e enough f o r t i s s u e a n a l y s i s (Kyker, 1961). Methods which l a c k t h e n e c e s s a r y s e n s i t i v i t y i n c l u d e Atomic A b s o r p t i o n Spectrophotometry, Emission Spectroscopy and X-ray F l u o r e s c e n c e Spectroscopy (Topp, 1965). Two methods t h a t do o f f e r h i g h s e n s i t i v i t y f o r l a n t h a n i d e d e t e c t i o n are Neutron A c t i v a t i o n A n a l y s i s (NAA) and I n d u c t i v e l y Coupled Plasma-Mass Spectrometry (ICP-MS). Although each o f these methods w i l l be d i s c u s s e d , ICP-MS was the method chosen f o r t h i s e x p e r i m e n t a l work. Neutron A c t i v a t i o n A n a l y s i s (NAA) The l a n t h a n i d e elements e x h i b i t c e r t a i n c h a r a c t e r i s t i c s which enables neutron a c t i v a t i o n a n a l y s i s t o d e t e c t them a t ve r y low l e v e l s i n t i s s u e samples. These p r o p e r t i e s i n c l u d e : a l a r g e thermal neutron c r o s s - s e c t i o n , a h i g h i s o t o p i c abundance, and a s u i t a b l e decay scheme and h a l f - l i f e (Table 2 ) . A c t i v a t i o n a n a l y s i s c o n s i s t s of exposing a s t a b l e i s o t o p i c sample t o a neutron source and the subsequent measurement of c h a r a c t e r i s t i c e n e r g i e s e m i t t e d from the r a d i o a c t i v e i s o t o p e s produced as a r e s u l t of the n u c l e a r t r a n s f o r m a t i o n s . The n u c l e a r r e a c t i o n t h a t i s important i s the neutron, gamma r e a c t i o n . T h i s i n v o l v e s the a b s o r p t i o n of a neutron by the nucleus of a s t a b l e i s o t o p e , f o l l o w e d by the emi s s i o n o f a gamma photon. The n u c l i d e produced i s one mass u n i t h i g h e r than the i s o t o p e t h a t absorbed the neutron (Lyon, 1964; Rakovic, 1970; and Kruger, 1971). 33 T a b l e 2. C h a r a c t e r i s t i c s o f l a n t h a n i d e i s o t o p e s f o r de t e r m i n a t i o n by neutron a c t i v a t i o n a n a l y s i s . 1 Isotope Produced S t a b l e Isotopes, % Abundance H a l f - L i f e Decay Energy (keV) Neutron C r o s s -S e c t i o n (barns) 90y 8 9Y = 100 58.8 d 1.545 1.4 ± 0.3 b 1 4 0 L a * 1 3 9 L a = 99.91 40.22 h 3.769 2.8 ± 0.3 b 1 4 1Ce * 1 4 0Ce = 88.48 33 d 0.581 29 ± 3 b 1 4 2 p r 1 4 1 P r = 100 19.2 h 2.16 18 ± 3 b 1 4 7Nd 1 4 6Nd = 17.62 11.1 d 0.91 1.4 ± 0.2 b 153Sm * 152Sm = 26.72 46.8 h 0.801 210 ± 10 b 1 5 4Eu * 1 5 3Eu = 52.18 16 y 1.97 1500 ± 400 b 1 5 9Gd 1 5 8Gd = 24.87 18 h 0.95 3.5 ± 1.0 b 1 6 0 T b * 1 5 9Tb = 100 73 d 1.72 525 ± 100 b 1 6 5Dy * 1 6 4Dy = 28.18 2.3 h 1.30 2600 ± 200 b 1 6 6Ho 1 6 5Ho = 100 26.9 h 1.847 61.2 ± 2.0 b 1 6 9 £ r I 6 8 £ r = 27.07 9.4 d 0.34 1.9 ± 0.2 b 1 7 0Tm 1 6 9Tm = 100 128.6 d 0.967 92 ± 4 b 1 7 5Yb * 1 7 4Yb = 31.84 101 h 0.467 46 ± 4 b 1 7 6 L u 1 7 5 L u = 97.41 3.7 h 1.3 21 ± 3 b 1 CRC Handbook of Chemistry and P h y s i c s , 60th E d i t i o n (1980). * Represents the i s o t o p e s w i t h t he most s u i t a b l e c h a r a c t e r i s t i c s f o r d e t e r m i n a t i o n by neutron a c t i v a t i o n a n a l y s i s . Neutron a c t i v a t i o n a n a l y s i s , based on the a c t i v a t i o n o f . s t a b l e i s o t o p e s o f the elements of i n t e r e s t , i d e n t i f i e s elements which were a n a l y z e d . The use and a n a l y s i s of s t a b l e i s o t o p e s , i n c o n t r a s t t o the use of r a d i o a c t i v e i s o t o p e s , e l i m i n a t e s the p o s s i b i l i t y o f r a d i o a c t i v e contamination of f a c i l i t i e s and l i v i n g organisms. U n f o r t u n a t e l y , t h e r e a r e a few drawbacks t o the f e a s i b i l i t y of u s i n g NAA f o r t h i s work. For example, o p e r a t i o n a l n u c l e a r r e a c t o r s needed f o r the neutron sources are not w i d e l y a v a i l a b l e , the a n a l y t i c a l time may be lengthy depending on the decay scheme of the r e s u l t i n g i s o t o p e , the s i z e of sample r e q u i r e d i s r e l a t i v e l y l a r g e , and l i q u i d samples cannot be a n a l y z e d by NAA. In a d d i t i o n , ICP-MS i s more p r e c i s e and a c c u r a t e than NAA (Turnlund, 1989). I n d u c t i v e l y Coupled Plasma-Mass Spectrometry (ICP-MS) I n d u c t i v e l y Coupled Plasma-Mass Spectrometry i s a newly a v a i l a b l e t e c hnology f o r elemental and i s o t o p i c a n a l y s e s . Houk & Thompson (1988) i n t r o d u c e d ICP-MS as a method f o r the d e t e r m i n a t i o n of i s o t o p e s of t r a c e elements. The combination of the c h a r a c t e r i s t i c s of the two components of t h i s instrument are r e s p o n s i b l e f o r the h i g h s e n s i t i v i t y . The ICP i s b a s i c a l l y used f o r a t o m i z a t i o n and i o n i z a t i o n of elements c a r r i e d i n an aqueous a e r o s o l from l i q u i d samples, and the MS i s f o r s e n s i t i v e and s e l e c t i v e measurement of a l l element i s o t o p e s (Houk & Thompson, 1988) . The f i r s t instrument f o r commercial use was i n t r o d u c e d by SCIEX Inc. i n 1983. SCIEX and VG Isotopes are the two l e a d i n g manufacturers of ICP-MS i n s t r u m e n t a t i o n . The f o l l o w i n g f i g u r e i s a schematic diagram of a t y p i c a l ICP-MS instrument. 35 RF/DC QUAD RF CONTROLLER SUPPLY ELECTRODE BIAS SUPPLIES QUADRUPOLE RF HEAD l a c PULSE AMPLIFIER L MCA A QUADRUPOLE CONTROL I VACUUM STAGES COMPUTER ] • JIU in iinirr/}= PLASMA RF POWER SUPPLY PLASMA RF MATCHING UNIT GAS CONTROL DRAIN SAMPLE F i g u r e 1. A schematic diagram of a t y p i c a l ICP-MS showing the arrangement of the v a r i o u s components. F i g u r e redrawn from a diagram s u p p l i e d by Elemental Research Inc. MCA = M u l t i - C h a n n e l A n a l y z e r ; RF = Radio Frequency. There a r e some advantages and disadvantages t o u s i n g ICP-MS i n s t r u m e n t a t i o n as d e s c r i b e d by Douglas & Houk (1985), Gray (1986), T a y l o r (1986), Selby & H i e f t j e (1987) and Turnlund (1989). Some of the unique, b e n e f i c i a l f e a t u r e s o f ICP-MS i n c l u d e : ( i ) r a p i d , multi-elemdnt a n a l y s i s f o r the simultaneous d e t e c t i o n o f a wide range o f elements; ( i i ) h i g h s e n s i t i v i t y r e s u l t i n g i n low d e t e c t i o n l i m i t s i n the range of 0.02 t o 0.7 ng/1; ( i i i ) r e l a t i v e l y low background l e v e l s ; ( i v ) simple s p e c t r a i n which t h e r e i s one peak f o r each ele m e n t a l i s o t o p e ; and (v) i s o t o p e r a t i o d e t e r m i n a t i o n s are p o s s i b l e . U n f o r t u n a t e l y , as w i t h a l l a n a l y t i c a l t e c h n i q u e s , t h e r e are some disadvantages t o u s i n g ICP-MS: ( i ) h i g h i n i t i a l purchase and maintenance c o s t s ; h i g h sample a n a l y s i s c o s t s ; ( i i ) a few m a t r i x and s p e c t r a l i n t e r f e r e n c e s ; ( i i i ) v a r i a t i o n i n instrument performance; ( i v ) l i m i t e d t o l e r a n c e f o r a n a l y s i s o f d i s s o l v e d s o l i d s ; and (v) samples are d i g e s t e d and d i l u t e d which reduces s e n s i t i v i t y . Because t h i s i s a r e l a t i v e l y new technique, r e s e a r c h s t i l l needs t o be c a r r i e d out t o s o l v e some of these problems. To o b t a i n maximum s e n s i t i v i t y f o r the d e t e c t i o n of a s p e c i f i c element, the plasma and mass spectrometer parameters s h o u l d be a d j u s t e d t o the optimum mass range s e t t i n g f o r t h a t element. When d e t e c t i o n of a range of elements i s r e q u i r e d , t h e r e i s some l o s s i n s e n s i t i v i t y f o r s p e c i f i c elements s i n c e a wider mass range i s used. However, t h i s decrease i s v e r y minor and has not been shown t o be a s i g n i f i c a n t problem ( T a y l o r , 1986). The d e t e c t i o n l i m i t s a c h i e v e d by ICP-MS are f a r lower than those a c h i e v e d by other t e c h n i q u e s ; even those of g r a p h i t e furnace-atomic a b s o r p t i o n spectroscopy (Houk & Thompson, 1988). D e t e c t i o n l i m i t s are p a r t i a l l y determined by the s e n s i t i v i t y o f the instrument t o the element and by the background n o i s e of ot h e r elements (Douglas & Houk, 1985). The samples are i n t r o d u c e d i n the form of s o l u t i o n s and are sprayed i n t o the ICP w i t h a n e b u l i z e r . The argon ICP i s an i o n i z a t i o n source which heats the a e r o s o l o f the l i q u i d sample t o approximately 7,000°K. At t h a t temperature the d i s s o l v e d s o l i d s are va p o u r i z e d , atomized and i o n i z e d . The ICP can e f f e c t i v e l y and e f f i c i e n t l y i o n i z e almost a l l elements s i n c e the average energy of the argon plasma (13.6 eV) i s h i g h e r than the f i r s t i o n i z a t i o n p o t e n t i a l of most elements ( T a y l o r , 1986). A f r a c t i o n o f the i o n s are d i r e c t e d i n t o a quadrupole mass spectrometer which s e p a r a t e s the i o n s a c c o r d i n g t o t h e i r mass t o charge r a t i o . The i o n s are then counted by a p u l s e counter where each count r e p r e s e n t s one d e t e c t e d i o n (Douglas & Houk, 1985; and Houk & Thompson, 1988) . The data are then r e c o r d e d by a d a t a h a n d l i n g system and c a l c u l a t e d t o a c o n c e n t r a t i o n through the a p p r o p r i a t e software. S o l i d s can be analyzed by e i t h e r l a s e r a b l a t i o n or e l e c t r o t h e r m a l v a p o r i z a t i o n , without having f i r s t t o be d i s s o l v e d (Houk & Thompson, 1988). A n a l y s i s of the Lanthanides by ICP-MS ICP-MS i s an e s p e c i a l l y " s e n s i t i v e method f o r d e t e c t i o n o f the l a n t h a n i d e s . The d e t e c t i o n l i m i t s f o r these elements a r e 38 ve r y low, r a n g i n g from 0.01 t o 0.3 ug/g (Table 3 ) . Although the s e n s i t i v i t y and l i m i t s are e x c e l l e n t f o r a l l the l a n t h a n i d e s , t h e r e i s some v a r i a t i o n between elements (Long e r i c h e t a l . , 1987; and Houk & Thompson, 1988). T a b l e 3. ICP-MS l i m i t s o f d e t e c t i o n f o r the l a n t h a n i d e s (3 S.D. of the background). T o t a l d i s s o l v e d s o l i d s a re 1 mg/g.1 Element D e t e c t i o n L i m i t (Mg/g s o l i d ) Element D e t e c t i o n L i m i t (Mg/g s o l i d ) Y 0. 05 Tb 0.05 La 0.03 Dy 0.20 Ce 0.03 Ho 0. 05 Pr 0. 03 Er 0.20 Nd 0.14 Tra 0. 03 Sm 0.13 Yb 0.20 Eu 0.05 Lu 0.05 Gd 0.30 1 L o n g e r i c h e t al. (1987). i The f a c t o r s t h a t a f f e c t the s e n s i t i v i t y of ICP-MS are the i s o t o p i c abundances and p o s s i b l e i n t e r f e r e n c e from mass o v e r l a p . The l a r g e r the i s o t o p i c abundance, the g r e a t e r the s e n s i t i v i t y t o t h a t element - p r o v i d e d t h a t t h e r e are no i n t e r f e r e n c e s from o t h e r e l e m e n t a l i s o t o p e s . T a b l e 4 shows the i s o t o p i c weights and per cent abundances of the v a r i o u s l a n t h a n i d e s . 39 Ta b l e 4. I s o t o p i c composition of the l a n t h a n i d e elements and t h e i r n a t u r a l r e l a t i v e abundances. 1 Ln # of Isotopes I s o t o p i c Weight % R e l a t i v e Abundance Y 1 89 100 La 2 138, 139 0.09, 99.91 Ce 4 136, 138, 140, 142 0.19, 0.25, 88.48, 11.07 Pr 1 141 100 Nd 7 142, 143, 144, 145, 146, 148, 150 27.11, 12.14, 23.83, 8.29, 17.26, 5.74, 5.63 Sm 7 144, 147, 148, 149, 150, 152, 154 3.09, 14.97, 11.24, 13.83, 7.44, 26.72, 22.71 Eu 2 151, 153 47.77, 52.23 Gd 7 152, 154, 155, 156, 157, 158, 160 0.20, 2.15, 14.73, 20.47, 15.68, 24.87, 21.90 Tb 1 159 100 Dy 7 156, 158, 160, 161, 162, 163, 164 0.05, 0.09, 2.29, 18.88, 25.53, 24.97, 28.19 Ho 1 165 100 Er 6 162, 164, 166, 167, 168, 170 0.14, 1.56, 33.41, 22.94, 27.07, 14.88 Tm 1 169 100 Yb 7 168, 170, 171, 172, 173, 174, 176 0.14, 3.03, 14.31, 21.82, 16.13, 31.84, 12.73 Lu 2 175, 176 97.40, 2.60 1 T a b l e o f Isotopes, 7th E d i t i o n (1978). The monoisotopic l a n t h a n i d e s t h a t have 100% abundance of one i s o t o p e a re y t t r i u m , praseodymium, terbium, holmium, and th u l i u m . Other l a n t h a n i d e s may have two i s o t o p e s , but one of the i s o t o p e s w i l l have the g r e a t e r abundance ( 1 3 8La = 0.09% and 1 3 9 L a = 99.91%) . Some elements have as many as seven i s o t o p e s ; 40 t h e s e i n c l u d e , neodymium, samarium, gadolinium, dysprosium, and y t t e r b i u m (Table 4 ) . In the s e l e c t i o n of the i s o t o p e t o analyz e , both the abundance and the p o s s i b l e i n t e r f e r e n c e s need t o be c o n s i d e r e d . For the monoisotopic elements t h e r e i s no problem. For elements w i t h more than one i s o t o p e , t he most abundant i s o t o p e w i t h the l e a s t i s o b a r i c o v e r l a p i s the one which i s analyzed. Some examples o f p r e f e r a b l e i s o t o p e s have been p r e s e n t e d by L o n g e r i c h e t al., 1987: (i ) 8 9Y - monoisotopic; ( i i ) u 1 P r - monoisotopic; ( i i i ) 1 5 9Tb - monoisotopic, w i t h a s m a l l i n t e r f e r e n c e from 1 4 3Nd 1 60 + (12.14%); (iv ) 1 6 5Ho - monoisotopic, w i t h a s m a l l i n t e r f e r e n c e from 1 4 9Sm 1 60 + (13.8%); (v) 1 6 9Tm - monoisotopic, w i t h a s m a l l i n t e r f e r e n c e from l 5 3Eu 1 60 + (52.2%); (vi ) 1 3 9 L a - the most abundant i s o t o p e a t 99.91%, and the o n l y i s o t o p e without i n t e r f e r e n c e ; ( v i i ) 1 4 0Ce - the most abundant i s o t o p e a t 88.48%, and the o n l y i s o t o p e without i n t e r f e r e n c e ; ( v i i i ) 1 4 7Sm - the most abundant i s o t o p e a t 15.0% without i n t e r f e r e n c e . The second c h o i c e would be 149Sm a t 13.8% abundance. The t h i r d c h o i c e would be t h e most abundant i s o t o p e , but i t has i n t e r f e r e n c e from 1 5 2Gd 1 60 + (0.20%); ( i x ) 1 6 3Dy - the f o u r i s o t o p e s t h a t have s i m i l a r abundances of 18.88, 25.53, 24.97, and 28.19%, but the 1 6 3Dy (24.97%) i s o t o p e has the l e a s t i n t e r f e r e n c e s ; and (x) 1 7 3Yb - t h i s i s o t o p e s has o n l y one i n t e r f e r e n c e from 1 5 7Gd 1 60 + (15.7%), which i s l e s s than the i n t e r f e r e n c e found i n 1 7 1Yb from 1 5 5Gd 1 60 + (14.9%). Any i n t e r f e r e n c e caused by these oxides are minimal as the r e l a t i v e abundance of the o x i d e s , on a w e l l - t u n e d ICP-MS, i s approximately 0.1% or l e s s of the element i s o t o p e p r e s e n t i n the sample. An example u s i n g the samarium spectrum has been g i v e n by L o n g e r i c h e t al. (1986). The seven i s o t o p e s of samarium are found i n the mass range of 144 t o 154. The s p e c t r a of t h i s l a n t h a n i d e i s c l e a r l y unique and each peak r e p r e s e n t s one i s o t o p e . The s p e c t r a shows the d i f f e r e n t r e l a t i v e abundances by the v a r y i n g magnitudes. As can be seen i n F i g u r e 2, the ICP-MS s p e c t r a are r e l a t i v e l y simple. 42 142 144 146 148 150 152 154 F i g u r e 2. A t y p i c a l mass spectrum o f samarium o b t a i n e d w i t h ICP-MS i n s t r u m e n t a t i o n showing t h e i n d i v i d u a l peaks f o r each o f t h e samarium i s o t o p e s . S p e c t r a o b t a i n e d from e x p e r i m e n t a l d a t a (Experiment 5 ) . METHODOLOGY A series of experiments was conducted to investigate the f e a s i b i l i t y of using the lanthanide elements to l a b e l juvenile coho salmon. Two experiments investigated the t o x i c i t y of these elements to coho and steelhead alevins; and four experiments investigated the f e a s i b i l i t y of marking coho f r y and smolts using the lanthanides. Experiments were conduced at Capilano Hatchery, North Vancouver. A l l experimental f i s h were exposed to concentrated aqueous solutions of lanthanide acetates added to the ambient r i v e r water. After the completion of the l a b e l l i n g , bony tissues were extracted and analyzed for lanthanide accumulation. Chemicals Used For a l l treatments c a r r i e d out, the lanthanide elements used were acetates i n powder or c r y s t a l l i n e form and a l l were 99.9% reagent q u a l i t y . The acetate form was chosen because lanthanide s a l t s with organic acids have no tendency to hydrolyse. Lanthanide halides ( i . e . chlorides) are very soluble i n water at room temperature and hydrolyse r e a d i l y i n solution to produce oxide halides (Topp, 1965). However, when incorporated at low concentrations (100 fig/1) i n the water, neither the chlorides nor the acetates should hydrolyse and the s a l t s should di s s o c i a t e into the Ln 3 +, CI", and CH3C02" ions to dissolve and become hydrated ions - Ln(H 20) x 3 +. Because of these p r o p e r t i e s , both the c h l o r i d e and a c e t a t e forms appear t o be s u i t a b l e f o r marking f i s h . The l a n t h a n i d e s used and the s u p p l i e r s a re as f o l l o w s : y t t r i u m a c e t a t e t e t r a h y d r a t e , Y(C 2H 30 2) 3- 4H20 ( A l f a C a t a l o g Chemicals, Davers); lanthanum ( III) a c e t a t e hydrate, La(C 2H 30 2) 3- 1%H20 ( A l d r i c h Chemical Company Inc., Milwaukee); cerium ( I I I ) a c e t a t e hydrate, Ce (C 2H 30 2) 3- 1%H20 ( A l f a C a t a l o g Chemicals, Davers); samarium ( III) a c e t a t e hydrate, Sm(C 2H 30 2) 3- 3H20 ( A l d r i c h Chemical Company Inc., Milwaukee); dysprosium ( I I I ) a c e t a t e hydrate, Dy (C 2H 30 2) 3- 4H20 ( A l d r i c h Chemical Company Inc., Milwaukee); and y t t e r b i u m (III) a c e t a t e t e t r a h y d r a t e , Yb (C 2H 30 2) 3- 4H20 ( A l d r i c h Chemical Company Inc., Milwaukee). The p r i c e of the l a n t h a n i d e a c e t a t e s v a r i e d g r e a t l y from $18/100g f o r lanthanum (III) a c e t a t e h y d r a t e t o $170/100g f o r y t t e r b i u m (III) a c e t a t e t e t r a h y d r a t e . The b l e a c h i n g s o l u t i o n used t o d i g e s t t h e t r a c e s o f t i s s u e o f f the v e r t e b r a l column and o t o l i t h s was a 6% sodium h y p o c h l o r i t e s o l u t i o n (BDH Chemicals Inc., T o r o n t o ) . A n a l y s i s on t h i s b l e a c h i n g s o l u t i o n f o r l a n t h a n i d e c o n t e n t a re r e p o r t e d i n Appendix 2. Treatment C o n c e n t r a t i o n s The c o n c e n t r a t i o n s i n the s t a t i c tanks were made by adding a measured amount of the c o n c e n t r a t e d l a n t h a n i d e a c e t a t e s t o c k s o l u t i o n d i r e c t l y i n t o a measured q u a n t i t y of ambient r i v e r water i n each tank. Throughout the treatment p e r i o d the water i n t h e t a n k s was n o t changed and no a d d i t i o n a l w a t e r was added. F o r t h e d u r a t i o n o f t h e t r e a t m e n t , w a t e r samples were c o l l e c t e d f o r e l e m e n t a l a n a l y s i s t o ensure t h e c o n c e n t r a t i o n s were c o r r e c t and c o n s t a n t . L a n t h a n i d e s added t o a f l o w - t h r o u g h system had t o be d r i p p e d i n a t a c o n s t a n t r a t e w h i l e t h e ambient r i v e r w a t e r f l o w e d i n a t a s e t r a t e . The c o n c e n t r a t i o n o f t h e l a n t h a n i d e a c e t a t e s t o c k s o l u t i o n and t h e r e q u i r e d d r i p r a t e s were d e t e r m i n e d u s i n g a computer program d e v e l o p e d s p e c i f i c a l l y f o r t h e s e t y p e s o f c a l c u l a t i o n s . The r i v e r w a t e r was s e t a t a f l o w r a t e o f one l i t r e p e r minute. The c o n c e n t r a t e d l a n t h a n i d e s t o c k s o l u t i o n was mixed w i t h t h e r i v e r w a t e r i n a f u n n e l b e f o r e b e i n g d e l i v e r e d t o t h e t a n k . A m o d i f i e d v e r s i o n o f t h e M e r r i o t t b o t t l e was used t o d e l i v e r t h e c o n c e n t r a t e d l a n t h a n i d e s o l u t i o n i n t o t h e f u n n e l ( F i g u r e 3 ) . Once t h e b o t t l e had been c l o s e d t i g h t l y and t h e d r i p r a t e s e t , a i r e n t e r e d t h e b o t t l e t h r o u g h t h e r i g i d t u b i n g and was c o l l e c t e d a t t h e t o p o f t h e b o t t l e . The n e g a t i v e p r e s s u r e w h i c h was c r e a t e d e l i m i n a t e d t h e e f f e c t o f g r a v i t y , t h e r e f o r e t h e d r i p r a t e remained c o n s t a n t . 46 - R i g i d T ubing •Plug With H o l e P l a s t i c Cap S o l u t i o n L e v e l 10-1 P l a s t i c B o t t l e A i r L i n e T u b i n g Flow Adjustment Figure 3. Modified version of the Merriott b o t t l e used to d e l i v e r the chemical solutions at a constant drip rate. 47 Sample P r e p a r a t i o n Techniques Water Samples For the d u r a t i o n of the treatment p e r i o d element c o n c e n t r a t i o n i n the tanks was monitored. Depending on the p a r t i c u l a r experiment, the water samples were taken every few days or on a weekly b a s i s . For each sample, 10 ml of tank water was c o l l e c t e d i n a v i a l and immediately a c i d - f i x e d w i t h 3 drops of c o n c e n t r a t e d n i t r i c a c i d . The samples were shaken and s t o r e d i n a c o o l l o c a t i o n u n t i l they were submitted f o r a n a l y s i s by ICP-MS. To minimize any " p l a t e out" e f f e c t s , the samples were c o l l e c t e d i n the morning and d e l i v e r e d t o the l a b o r a t o r y (Elemental Research Inc., North Vancouver) f o r prompt a n a l y s i s . The water samples were analyzed without m o d i f i c a t i o n . V e r t e b r a l Column E x t r a c t i o n Feed was w i t h h e l d f o r the 24-hour p e r i o d p r i o r t o sampling. T h i s helped t o reduce s t r e s s and decrease the g a s t r o - i n t e s t i n a l t r a c t c o n t e n t s , thus m i n i m i z i n g v a r i a t i o n i n body weight. A f t e r the f i s h were k i l l e d by a s p h y x i a t i o n , the weights of i n d i v i d u a l f i s h were r e c o r d e d and the f i s h were d e c a p i t a t e d . The heads were immediately f r o z e n and s t o r e d f o r o t o l i t h removal a t a l a t e r d ate. F i n s , t a i l and i n t e r n a l organs were sep a r a t e d and d i s c a r d e d . The m a j o r i t y of the f l e s h was d i s s e c t e d out from the v e r t e b r a l column and the remaining t r a c e s of f l e s h were then d i g e s t e d w i t h a 6% sodium h y p o c h l o r i t e s o l u t i o n . S i n c e t h i s b l e a c h i n g s o l u t i o n would have d i s s o l v e d the bony t i s s u e as w e l l 48 as t h e f l e s h , t h e d i g e s t i o n p r o c e s s had t o be c l o s e l y m o n i t o r e d . The l e n g t h o f t i m e r e q u i r e d f o r complete s o f t t i s s u e d i g e s t i o n i n c r e a s e d w i t h t h e s i z e o f f i s h . Once t h e f l e s h was c o m p l e t e l y d i g e s t e d , t h e v e r t e b r a l column was removed from t h e d i g e s t i o n s o l u t i o n , r i n s e d w i t h d i s t i l l e d w a t e r and p l a c e d i n a sample v i a l . The c l e a n backbones were d r i e d o v e r n i g h t a t 70°C and t h e w e i g h t s r e c o r d e d . The samples were t h e n s u b m i t t e d t o E l e m e n t a l R e s e a r c h I n c . (ERI) f o r ICP-MS a n a l y s i s . B e f o r e t h e samples were a n a l y z e d , t h e v e r t e b r a l columns f i r s t were d i g e s t e d i n c o n c e n t r a t e d n i t r i c a c i d and t h e n d i l u t e d w i t h d i s t i l l e d w a t e r t o make a 10 ml sample. Once a n a l y z e d , t h e r e s u l t s were r e p o r t e d as a c o n c e n t r a t i o n r e l a t i v e t o t h e sample w e i g h t . O t o l i t h E x t r a c t i o n F o r r e m o v a l o f t h e o t o l i t h s , t h e e n t i r e f i s h head was p l a c e d i n a p e t r i d i s h w i t h a 6% sodium h y p o c h l o r i t e s o l u t i o n . As t h e o t o l i t h s a r e t h e d e n s e s t s t r u c t u r e s i n t h e f i s h ' s body and a r e composed o f c a l c i u m c a r b o n a t e , t h e y a r e a b l e t o w i t h s t a n d t h e d i g e s t i o n p r o c e d u r e (Tre a c y & C r a w f o r d , 1981). A f t e r o v e r n i g h t d i g e s t i o n , o n l y t h e o t o l i t h s remained i n t h e d i s h . The s a g i t t a e , t h e l a r g e s t o f t h e t h r e e p a i r s o f o t o l i t h s , were removed and r i n s e d w i t h d i s t i l l e d w a t e r , p l a c e d i n a v i a l and d r i e d o v e r n i g h t . S i n c e t h e s a g i t t a e a r e s m a l l , a few were p l a c e d i n each v i a l . The sample w e i g h t was n o t r e c o r d e d . The p r e p a r e d samples were t h e n s u b m i t t e d f o r a n a l y s i s by ICP-MS. The o t o l i t h s were d i g e s t e d i n c o n c e n t r a t e d n i t r i c a c i d and t h e n d i l u t e d p r i o r t o a n a l y s i s . The r e s u l t s were r e p o r t e d as a c o n c e n t r a t i o n w h i c h was c a l c u l a t e d r e l a t i v e t o t h e i n t e r n a l c a l c i u m c o n c e n t r a t i o n o f t h e o t o l i t h s . The c a l c i u m c o n c e n t r a t i o n o f t h e o t o l i t h s was s e t n o m i n a l l y a t 10% by ERI. S c a l e Samples U s i n g a s h a r p b l a d e , s c a l e s were s c r a p e d from b o t h s i d e s o f t h e f i s h ' s body i n t h e " p r e f e r r e d s c a l e sample a r e a " ( F i g u r e 4 ) . T h i s i s t h e a r e a between t h e l a t e r a l l i n e and t h e d o r s a l f i n . The s c a l e s were t h e n p l a c e d i n a s m a l l p e t r i d i s h f i l l e d w i t h d i s t i l l e d w a t e r and soaked f o r up t o 24 h o u r s . T h i s s e r v e d t o wash most o f t h e mucus from t h e s u r f a c e o f t h e s c a l e s , making i t e a s i e r t o remove t h e i n d i v i d u a l s c a l e s from t h e d i s h . Once t h e s c a l e s were r e l a t i v e l y c l e a n , t h e y were examined under a d i s s e c t i n g m i c r o s c o p e and were t h e n s e l e c t e d t o make t h e sample. The c l e a n e s t s c a l e s were p l a c e d i n a sample v i a l and a i r - d r i e d o v e r n i g h t . R e g e n e r a t e d s c a l e s were a v o i d e d . F o r a f i s h w e i g h i n g a p p r o x i m a t e l y 10 grams, 80 s c a l e s were c o l l e c t e d . S i n c e t h e s c a l e s were so s m a l l and t h e r e was a p r o b l e m w i t h s t a t i c e l e c t r i c i t y , t h e w e i g h t o f each sample was n o t r e c o r d e d . The samples were t h e n s u b m i t t e d f o r ICP-MS a n a l y s i s . As w i t h t h e o t h e r s o l i d samples, t h e s c a l e samples had t o be d i g e s t e d and d i l u t e d p r i o r t o a n a l y s i s . The r e s u l t s were t h e n r e p o r t e d 50 as a c o n c e n t r a t i o n c a l c u l a t e d r e l a t i v e t o the s c a l e s ' i n t e r n a l c a l c i u m c o n c e n t r a t i o n , s e t a t 10%. Preferred Scale Sample Area F i g u r e 4. Diagram of a f i s h showing the " p r e f e r r e d s c a l e sample a r e a " . Sample A n a l y s i s Problems With A n a l y s i s A few problems arose w i t h r e g a r d t o the a n a l y s i s of samples c o l l e c t e d from the f i r s t s e t s of marking experiments. The s e v e r i t y and nature of the problems were d i v e r s e and v a r i a b l e . Some of t h e s e w i l l be d i s c u s s e d b r i e f l y and g e n e r a l l y here, w i t h d e t a i l s i n c l u d e d i n the r e s u l t s s e c t i o n s of the a p p r o p r i a t e experiments. Values markedly lower than t h e o r e t i c a l c o n c e n t r a t i o n s were r e p o r t e d f o r water samples submitted t o Elemental Research Inc. (ERI). The company had a problem w i t h the secondary standards b e i n g used t o c a l i b r a t e t h e ICP-MS i n s t r u m e n t . The v i a l s i n w h i c h t h e s t a n d a r d s were b e i n g s t o r e d were n o t a i r t i g h t . As a r e s u l t , t h e s o l v e n t g r a d u a l l y e v a p o r a t e d , l e a v i n g a more c o n c e n t r a t e d s t a n d a r d i n t h e v i a l as t i m e p r o g r e s s e d . Because t h i s e r r o r was n o t d e t e c t e d u n t i l t h e t h i r d e x p e r i m e n t on coho f r y ( E xperiment 5) , no c o r r e c t i o n s were made p r i o r t o t h a t t i m e . C o n s e q u e n t l y , t h e e x p e r i m e n t a l v a l u e s r e p o r t e d f o r t h e f i r s t two e x p e r i m e n t s u s i n g coho f r y were lo w e r t h a n t h e t r u e v a l u e s . U n f o r t u n a t e l y , no r e c o r d s were k e p t on t h e age o r d a t e s o f use o f t h e f a u l t y s t a n d a r d s , t h e r e f o r e t h e r e was no way o f c o r r e c t i n g f o r t h e s e e r r o r s . The v a l u e s were e s t i m a t e d t o be 10 t o 3 0% t o o low. When t h e s e problems w i t h t h e sec o n d a r y s t a n d a r d s were d i s c o v e r e d t h e v i a l s used t o s t o r e t h e s t a n d a r d s were r e p l a c e d . S i n c e t h a t t i m e t h e s t a n d a r d s were r e g u l a r l y c h ecked f o r a c c u r a c y by t h e a n a l y t i c a l company ( E R I ) . A n o t h e r problem was some h i g h anomalous v a l u e s i n some o f t h e s u b m i t t e d samples. ERI s u g g e s t e d t h a t t h i s e r r o r had r e s u l t e d from c o n t a m i n a t i o n i n t h e e x p e r i m e n t a l t a n k s , t h e d i s s e c t i o n l a b o r t h e a n a l y t i c a l l a b . Upon f u r t h e r e x a m i n a t i o n , i t was r e v e a l e d t h a t one o f t h e s e c o n t a m i n a t e d samples c o n t a i n e d t h e whole range o f l a n t h a n i d e s r a t h e r t h a n j u s t t h e one element b e i n g used. However, o t h e r c o n t a m i n a t e d samples c o n t a i n e d o n l y t h e s i n g l e element w h i c h was b e i n g i n v e s t i g a t e d . F o r t u n a t e l y , t h e s e a n o m a l i e s were low i n f r e q u e n c y and were o b v i o u s , and t h u s e a s i l y r e c o g n i z e d . No o t h e r p o s s i b l e e x p l a n a t i o n s f o r t h e s e a n o m a l i e s were s u g g e s t e d o r i d e n t i f i e d . Subsequent to the detection of these problems, b l i n d standards were included with samples submitted for analysis. Either water or vertebrae standards were used, to correspond with the type of sample ( l i q u i d or s o l i d ) . The d e t a i l s of the standards submitted are described below. Water Standards Water standards were prepared by the author to check the accuracy of the analysis of regular samples. The concentrations covered the range expected i n the samples. The standards were prepared from aliquots of solutions having known concentrations of lanthanides. The f i n a l volume of the standards were then made to 10 ml by the addition of d i s t i l l e d water. Three drops of concentrated n i t r i c acid were added to each v i a l to a c i d - f i x the standards. These standards were then analyzed by ICP-MS together with the experimental water samples. The p o s s i b i l i t y of adsorption of lanthanides onto the sides of the sample v i a l s was investigated. Teflon containers and v i a l s have been shown not to be subject to adsorption of these elements. Comparable r e s u l t s were obtained from standards submitted i n both t e f l o n and polypropylene v i a l s . Therefore, the adsorption of lanthanides onto the sides of the polypropylene v i a l s must have been n e g l i g i b l e . V e r t e b r a e Standards V e r t e b r a e standards submitted w i t h s e l e c t e d v e r t e b r a e samples were prepared u s i n g one of two d i f f e r e n t methods. The standards f o r Experiment 5 were made by s p i k i n g a v i a l c o n t a i n i n g a d r i e d c o n t r o l ( l a n t h a n i d e - f r e e ) v e r t e b r a l column of known weight w i t h an a l i q u o t of the co n c e n t r a t e d l a n t h a n i d e a c e t a t e s t o c k s o l u t i o n . The v i a l was then d r i e d o v e r n i g h t . The r e s u l t s o b t a i n e d from these standards were lower than the c a l c u l a t e d v a l u e s . T h i s p r e p a r a t i o n method was thus d i s c o n t i n u e d because i t was b e l i e v e d t h a t p o s s i b l y t h e n i t r i c a c i d was not a b l e t o d i g e s t the l a n t h a n i d e d r i e d onto the s i d e of t he v i a l , w i t h the r e s u l t t h a t a v a r i a b l e p o r t i o n of the l a n t h a n i d e p r e s e n t was not going i n t o s o l u t i o n , and t h e r e f o r e c o u l d not be d e t e c t e d . A new method was then used t o prepare the v e r t e b r a e standards f o r Experiment 6. T h i s i n v o l v e d the c r u s h i n g o f s e v e r a l d r i e d v e r t e b r a l columns i n t o a f i n e powder u s i n g a mortar and p e s t l e . The v e r t e b r a e powder was weighed onto a watch g l a s s and a known volume of the l a n t h a n i d e a c e t a t e s p i k e was added. The r e s u l t i n g s l u r r y was then mixed t h o r o u g h l y and oven d r i e d . The d r i e d s p i k e d powder was then mixed a g a i n and d i v i d e d i n t o v i a l s f o r a n a l y s i s . EXPERIMENT 1 - INVESTIGATION OF COHO (Oncorhynchus kisutch) ALEVINS I N RECIRCULATING SYSTEMS CONTAINING A RANGE OF CONCENTRATIONS OF CERIUM, LANTHANUM, DYSPROSIUM, SAMARIUM, AND YTTERBIUM I n t r o d u c t i o n T h i s study was undertaken i n an attempt t o develop an e f f e c t i v e mass marking technique t o i d e n t i f y h a t c h e r y - p r o d u c t i o n salmon. The f e a s i b i l i t y of u s i n g the l a n t h a n i d e elements t o l a b e l coho a l e v i n was i n v e s t i g a t e d as a f i r s t s t e p . There are f o u r reasons why a l e v i n were i n v e s t i g a t e d f i r s t : ( i ) m i n e r a l uptake from the water supply o c c u r s a t the a l e v i n stage; ( i i ) a l e v i n r e q u i r e a much s m a l l e r volume of water per f i s h than f r y ; t h e r e f o r e l e s s water and l e s s l a n t h a n i d e element s o l u t i o n would be needed per tank; ( i i i ) r e c i r c u l a t i n g systems c o u l d be designed t o re-use the l a n t h a n i d e s o l u t i o n s , so t h a t they would not have t o be c o n t i n u a l l y added t o the system; and (iv ) an a l e v i n - l a b e l l i n g procedure c o u l d be c o n v e n i e n t l y i n c o r p o r a t e d i n t o a r o u t i n e hatchery o p e r a t i o n . The purpose of t h i s experiment was t o compare v a r i o u s l a n t h a n i d e s a t v a r y i n g c o n c e n t r a t i o n s w i t h r e s p e c t t o uptake and t o x i c i t y . F i v e l a n t h a n i d e s , each a t t h r e e c o n c e n t r a t i o n s , were i n t r o d u c e d i n t o the water supply of the a l e v i n . The experimental tanks were s e t up as u p w e l l i n g r e c i r c u l a t i n g u n i t s . M a t e r i a l s and Methods R e c i r c u l a t i n g U n i t s U p w e l l i n g r e c i r c u l a t i n g systems were s e t up f o r these marking experiments t o l a b e l coho (Oncorhynchus kisutch) a l e v i n s w i t h the l a n t h a n i d e elements ( F i g u r e 5) . The system i n c o r p o r a t e d a submersible pump which p r o p e l l e d the water up a p i p e t o the r e s e r v o i r and t o the i n c u b a t i o n u n i t . The flow r a t e out o f the pump was non-a d j u s t a b l e and was c o n t r o l l e d by a v a l v e p o s i t i o n e d below both the r e s e r v o i r and the i n c u b a t i o n u n i t . The water was s e t t o flow through the i n c u b a t i o n u n i t a t approximately one l i t r e per minute w i t h the s u r p l u s being d i r e c t e d t o t h e r e s e r v o i r . T h i s p r o v i d e d the a l e v i n s w i t h a continuous water flow w h i l e m i n i m i z i n g the d i s t u r b a n c e t o the a l e v i n s . The r e s e r v o i r had t h r e e important f u n c t i o n s : ( i ) t o p r o v i d e an o u t l e t f o r the s u r p l u s water; ( i i ) t o a e r a t e the water by a g i t a t i o n r e s u l t i n g from s p i l l i n g over the s i d e o f the r e s e r v o i r ; and ( i i i ) t o ensure t h a t the i n c u b a t i o n u n i t would never completely d r a i n . The r e c i r c u l a t i n g system had two s a f e t y checks i n case o f a power f a i l u r e , where the pump would cease working: ( i ) a check v a l v e t o prevent back flow; and ( i i ) a r e s e r v o i r p l a c e d h i g h e r than the i n c u b a t i o n u n i t . Together, these would l i m i t t he back flow of water and ensure t h a t the water would never completely d r a i n out o f the i n c u b a t i o n u n i t , thereby l e a v i n g the a l e v i n s without water. 56 Reservoir Incubation Unit (with alevin) Flow Valve Tank Check Valve Water Level L i t t l e Giant Pump (Model 2E-38N) F i g u r e 5. U p w e l l i n g r e c i r c u l a t i n g apparatus t o r e c i r c u l a t e the water i n the treatment tanks. Arrows i n d i c a t e water flow. 57 Experimental Design F i f t y coho a l e v i n s and f i v e coho eggs were p l a c e d i n the i n c u b a t i o n u n i t of the r e c i r c u l a t i n g apparatus i n each of the 14 experimental tanks. A t o t a l o f s i x t e e n l i t r e s o f ambient r i v e r water (10°C) was added t o each tank and a p e r i o d of two days passed b e f o r e the chemical treatments were added t o the tanks (February, 1989). The treatments c o n s i s t e d of cerium, lanthanum, dysprosium, samarium and y t t e r b i u m a l l a t c o n c e n t r a t i o n s of 20, 100 and 300 /xg/ml, except y t t e r b i u m which d i d not have a 300 ng/xal treatment (Table 6). Because a l i m i t e d number of r e c i r c u l a t i n g a p p a r a t i were a v a i l a b l e , t h e r e were no r e p l i c a t e s f o r any of the treatments and t h e r e were no n e g a t i v e c o n t r o l s . The a l e v i n s and eggs used i n t h i s experiment were coho salmon from C a p i l a n o R i v e r (B.C.) brood s t o c k . A t the s t a r t of the experiment, the a l e v i n s appeared h e a l t h y and normal. They r e s t e d on the bottom of the i n c u b a t i o n u n i t , becoming a c t i v e when d i s t u r b e d by l i g h t o r touch, then s e t t l i n g back a t the bottom of the f u n n e l soon a f t e r the d i s t u r b a n c e was d i s c o n t i n u e d . Although a l e v i n were of primary i n t e r e s t i n t h i s experiment, a s m a l l number of eyed eggs were a l s o used. For each treatment group, a measured amount of l a n t h a n i d e a c e t a t e c r y s t a l s was added t o 500 ml of r i v e r water and heated u n t i l the c r y s t a l s were completely d i s s o l v e d . The s o l u t i o n was then c o o l e d i n a bucket o f i c e t o 10°C, and poured i n t o the r e s e r v o i r of the r e c i r c u l a t i n g u n i t . T h i s method ensured t h a t 58 t h e l a n t h a n i d e s s l o w l y mixed w i t h t h e t a n k w a t e r t h r o u g h t h e r e c i r c u l a t i n g a c t i o n o f t h e pump. A p p r o x i m a t e l y 1 ml o f c o n c e n t r a t e d sodium h y d r o x i d e was t h e n added t o each t a n k t o i n c r e a s e t h e pH on t h e a s s u m p t i o n t h a t t h i s would h e l p t o compensate f o r any a c i d i t y caused by t h e l a n t h a n i d e a c e t a t e s . T a b l e 5. L a n t h a n i d e a t o m i c w e i g h t s and m o l e c u l a r w e i g h t s o f t h e c o r r e s p o n d i n g a c e t a t e s used i n t h e t r e a t m e n t c o n c e n t r a t i o n c a l c u l a t i o n s . Element (Symbol) Ato m i c Weight S a l t M o l . Wt. o f S a l t Mol Wt/ A t Wt 1 Cerium (Ce) 140.12 Ce a c e t a t e 317.25 2.2623 Lanthanum (La) 138.91 La a c e t a t e 316.05 2.2753 Dysprosium (Dy) 162.50 Dy a c e t a t e 339.64 2.0901 Samarium (Sm) 150.36 Sm a c e t a t e 327.48 2.1780 Y t t e r b i u m (Yb) 173.00 Yb a c e t a t e 422.24 2.4407 1 M o l e c u l a r Weight / A t o m i c Weight. The a t o m i c w e i g h t o f each t h e l a n t h a n i d e e lements and t h e m o l e c u l a r w e i g h t s o f t h e c o r r e s p o n d i n g a c e t a t e s a r e shown i n T a b l e 5. T a b l e 6 shows t h e amount o f l a n t h a n i d e a c e t a t e added t o each t a n k and t h e t h e o r e t i c a l l a n t h a n i d e t r e a t m e n t c o n c e n t r a t i o n . Sample Treatment C a l c u l a t i o n Grams S a l t Used / L i t r e s — X 1000 M o l e c u l a r Wt. / A t o m i c Wt. = /xg l a n t h a n i d e / m l 59 T a b l e 6. L a n t h a n i d e a c e t a t e (grams) added t o 16 l i t r e s o f r i v e r w a t e r , and t h e appr o x i m a t e t h e o r e t i c a l element c o n c e n t r a t i o n s i n each t a n k . Tank Compound S a l t Added (g) T h e o r e t i c a l C o n c e n t r a t i o n 1 Ce a c e t a t e 0.675 20 /ig/ml 2 Ce a c e t a t e 3.393 100 ng/ml 3 Ce a c e t a t e 10.181 300 nq/ml 4 La a c e t a t e 0.682 20 fig/ml 5 La a c e t a t e 3.409 100 ng/ml 6 La a c e t a t e 10.235 300 Lig/ml 7 Dy a c e t a t e 0.623 20 u.g/ml 8 Dy a c e t a t e 3.132 100 jug/ml 9 Dy a c e t a t e 9.422 300 ng/ml 10 Sm a c e t a t e 0.655 20 /xg/ml 11 Sm a c e t a t e 3.264 100 jug/ml 12 Sm a c e t a t e 9.806 300 ng/ml 13 Yb a c e t a t e 0.733 20 ng/Tal 14 Yb a c e t a t e 3.653 100 (ig/ml S a m p l i n g and A n a l y t i c a l Method Dead a l e v i n s and eggs were sampled from t h e t a n k s c o n t a i n i n g 100 and 300 fig/ml o f c e r i u m , lanthanum, d y s p r o s i u m and samarium. The a l e v i n samples c o n s i s t e d o f t h r e e whole a l e v i n s p e r v i a l and one v i a l w i t h o n l y t h e c o n t e n t s o f y o l k s a c s f rom t h r e e a l e v i n s . The egg samples c o n s i s t e d o f t h r e e whole eggs p e r v i a l and one v i a l w i t h o n l y t h r e e empty egg s h e l l s ( c a s e s ) . A l l samples were o v e n - d r i e d o v e r n i g h t , d r y w e i g h t r e c o r d e d , and a n a l y z e d by ICP-MS f o r l a n t h a n i d e c o n t e n t . R e s u l t s Water Q u a l i t y W i t h i n f o u r hours of the a d d i t i o n of the l a n t h a n i d e s o l u t i o n s and NaOH, a foamy substance developed i n the tanks c o n t a i n i n g the h i g h e s t c o n c e n t r a t i o n s . The amount of foam p r e s e n t v a r i e d from none ( i n the tanks w i t h the lowest c o n c e n t r a t i o n s o f a l l elements and the tank w i t h samarium a t 3 00 jug/ml) t o a t h i c k l a y e r of bubbles over the s u r f a c e ( i n the tanks w i t h the h i g h e s t c o n c e n t r a t i o n s of cerium, lanthanum, and dysprosium) . A white p r e c i p i t a t e a l s o formed i n the 100 and 300 jig/ml tanks f o r a l l elements. There was a s t r o n g c o r r e l a t i o n between h i g h l a n t h a n i d e c o n c e n t r a t i o n and presence o f foam and p r e c i p i t a t e . A f t e r 24 hours, the foam i n a l l tanks had d i s s i p a t e d , but the p r e c i p i t a t e was s t i l l p r e s e n t i n the tanks c o n t a i n i n g 100 and 300 /xg/ml of a l l elements (Table 7) . The water pH i n a l l tanks, a f t e r t he a d d i t i o n of the l a n t h a n i d e s and NaOH, ranged from 6.20 t o 6.67 u n i t s . The water i n t he tanks c o n t a i n i n g the lowest c o n c e n t r a t i o n s of a l l elements had the h i g h e s t pH v a l u e s , w h i l e the water i n the tanks c o n t a i n i n g the 100 iig/ml c o n c e n t r a t i o n s of a l l elements had the lowest pH v a l u e s (Table 7) . No other water parameters were t e s t e d . M o r t a l i t i e s F i s h m o r t a l i t y was noted i n the tanks c o n t a i n i n g the h i g h e s t l a n t h a n i d e c o n c e n t r a t i o n s w i t h i n f o u r hours of the a d d i t i o n of c h e m i c a l s . For the 100 /xg/ml c o n c e n t r a t i o n s of a l l elements, w i t h the e x c e p t i o n of ytterbium, a l l a l e v i n were dead a f t e r f o u r hours. At the 300 /xg/ml c o n c e n t r a t i o n , m o r t a l i t i e s a f t e r f o u r hours were p r e s e n t o n l y i n the tanks c o n t a i n i n g cerium and lanthanum (Table 7 ) . A l l a l e v i n s i n a l l tanks were dead a f t e r a 24-hour p e r i o d . M o r t a l i t y c h a r a c t e r i s t i c s of the a l e v i n s i n c l u d e d : arched s p i n e , congealed y o l k sac, open mouth, and decomposing f l e s h w i t h white t i p s on the f i n s and t a i l . A f t e r 48 hours, a l l eggs i n a l l tanks were dead. 62 T a b l e 7. Foam and p r e c i p i t a t e f o r m a t i o n , and a l e v i n m o r t a l i t i e s o b s e r v e d i n t h e t a n k s 4 hours and 24 h o u r s a f t e r t h e a d d i t i o n o f t h e l a n t h a n i d e a c e t a t e s . A t Time o f Treatment A f t e r 4 Hours A f t e r 24 Hours Ln /ig/ml pH Mo r t s Foam 1 p p t 2 M o r t s Foam p p t Ce - 20 6.30 0 % 0% — 100 % 0% -Ce - 100 6.20 100 % 25-75% + 100 % 0% + Ce - 300 6.29 100 % 25-75% + 100 % 0% + L a - 20 6.57 0 % <25% - 100 % 0% -La - 100 6.33 100 % >75% + 100 % 0% + L a - 300 6.36 100 % >75% + 100 % 0% + Dy - 20 6.67 0 % 0% - 100 % 0% -Dy - 100 6.24 100 % <25% + 100 % 0% + Dy - 300 6.34 0 % 25-75% + 100 % 0% + Sm - 20 6.62 0 % <25% - 100 % 0% -Sm - 100 6.27 100 % <25% + 100 % 0% + Sm - 300 6.38 0 % 0% + 100 % 0% + Yb - 20 6.37 0 % 0% - 100 % 0% -Yb - 100 6.23 0 % <25% + 100 % 0% + 1 s u r f a c e a r e a c o v e r e d by foam e x p r e s s e d as a p e r c e n t . 2 p r e c i p i t a t e p r e s e n t (+) p r e c i p i t a t e a b s e n t (-). Water A n a l y s i s Water samples from t h e t a n k s c o n t a i n i n g t h e l o w e s t l a n t h a n i d e c o n c e n t r a t i o n s were a n a l y z e d f o r element c o n c e n t r a t i o n . I n each c a s e t h e r e s u l t s were l o w e r t h a n t h e c a l c u l a t e d v a l u e s ( T a b l e 8 ) . 63 T a b l e 8. ICP-MS r e s u l t s of water a n a l y s i s o f the tanks c o n t a i n i n g the lowest l a n t h a n i d e c o n c e n t r a t i o n s . Lanthanide C a l c u l a t e d C o n c e n t r a t i o n A n a l y s i s R e s u l t s Mg/ml Ce 18.6 15.2 La 18.7 12.5 Dy 18.6 15.4 Sm 18.8 13.5 Yb 18.7 16.6 R e s i d u a l Element i n Tanks A f t e r the a l e v i n s had a l l d i e d , the tanks and r e c i r c u l a t i n g u n i t s were r i n s e d f o r 24 hours by pumping f r e s h water through the apparatus a t the maximum r a t e of the pump t o remove the l a n t h a n i d e s . Then 16 l i t r e s of r i v e r water was added t o each tank, the r e c i r c u l a t i n g u n i t s were r e - s e t up and a l e v i n s were p l a c e d i n t o the i n c u b a t i o n u n i t s . No a d d i t i o n a l l a n t h a n i d e s were added. A l l a l e v i n s i n the tanks were dead w i t h i n 48 hours and water samples were then taken from each tank. I t would appear t h a t t h e r e was s t i l l enough element remaining i n each tank a f t e r the 24-hour r i n s e because m o r t a l i t i e s s t i l l o c c u r r e d . C o n c e n t r a t i o n o f l a n t h a n i d e i n each tank was between 0.424 and 1.2 50 Mg/ml, r e g a r d l e s s o f the s t r e n g t h of the o r i g i n a l treatment c o n c e n t r a t i o n (Table 9 ) . 64 T a b l e 9. O r i g i n a l t r e a t m e n t c o n c e n t r a t i o n and r e s i d u a l l a n t h a n i d e r e m a i n i n g i n t h e t a n k s a f t e r a 24-hour c o n t i n u o u s r i n s e . Tank L a n t h a n i d e O r i g i n a l C o n c e n t r a t i o n R e s i d u a l C o n c e n t r a t i o n 1 Cerium 20 ug/ml 0.745. Mg/ml 2 Cerium 100 ug/ml 0.998 Mg/ml 3 Cerium 300 Mg/ml 0.941 Mg/ml 4 Lanthanum 20 Mg/ml 0.707 Mg/ml 5 Lanthanum 100 Mg/ml 1.030 Mg/ml 6 Lanthanum 300 Mg/ml 0.731 Mg/ml 7 Dysprosium 20 Mg/ml 1.070 Mg/ml 8 Dysprosium 100 Mg/ml 1.250 Mg/mi 9 Dysprosium 300 Mg/ml 0.586 Mg/ml 10 Samarium 20 Mg/ml 0.424 Mg/ml 11 Samarium 100 Mg/ml 0.433 Mg/ml 12 Samarium 300 Mg/ml 0.708 Mg/mi 13 Y t t e r b i u m 20 Mg/ml 0.498 Mg/ml 14 Y t t e r b i u m 100 Mg/ml 0.499 Mg/ml A c i d R i n s i n g o f Tanks The 24-hour c o n s t a n t f l o w - t h r o u g h r i n s e w i t h f r e s h w a t e r was n o t e f f e c t i v e i n c o m p l e t e l y removing t h e l a n t h a n i d e s from t h e r e c i r c u l a t i n g a p p a r a t u s and t h e t a n k s . A c i d washes w i t h 0.1M and 0.5M h y d r o c h l o r i c a c i d (HC1) were a t t e m p t e d . A f t e r t h e equipment was t r e a t e d w i t h t h e a c i d , i t was a g a i n r i n s e d w i t h f r e s h w a t e r . The r e c i r c u l a t i n g systems were s e t - u p i n each o f t h e t a n k s a f t e r t h e was h i n g was complete and w a t e r samples were t a k e n 24 hours l a t e r . The 0.1M and 0.5M HCl appeared t o be e f f e c t i v e i n removing the r e s i d u a l l a n t h a n i d e s from the system (Table 10). Ta b l e 10. Lanthanide c o n c e n t r a t i o n s i n tanks a f t e r a 0.1M o r 0.5M HCl wash and a 24-hour flow through r i n s e . R e s i d u a l Lanthanide A c i d Wash A n a l y s i s R e s u l t s Ce 0.75 /xg/ml 0.1M HCl <0.01 /xg/ml Ce 0.94 /xg/ml 0.5M HCl <0.01 /xg/ml La 0.73 /xg/ml 0.1M HCl <0.01 /xg/ml Dy 0.59 /xg/ml 0.5M HCl 0.01 /xg/ml Sm 0.71 /xg/ml None 0.07 /xg/ml Yb 0.50 /xg/ml None 0.09 /xg/ml A l e v i n and Egg A n a l y s i s Some o f the dead a l e v i n s and eggs were sent f o r a n a l y s i s by ICP-MS. The r e s u l t s of these analyses are shown i n Tabl e 11. The v i a l c o n t a i n i n g the t h r e e y o l k s had the lowest c o n c e n t r a t i o n of l a n t h a n i d e p r e s e n t (4.9 /xg Dy/g of dry t i s s u e or 0.3 /xg D y / v i a l ) . The a l e v i n s t r e a t e d w i t h lanthanum, cerium, and dysprosium a t 100 /xg/ml had between 34.2 and 76.7 ng l a n t h a n i d e / g of dry t i s s u e (5.5 t o 11.6 /xg/vial) , w h i l e those t r e a t e d w i t h samarium a t 100 /xg/ml had o n l y 5.9 /xg Sm/g (0.8 /xg/vial) . A l e v i n exposed t o 300 /xg/ml of cerium had the h i g h e s t c o n c e n t r a t i o n of cerium p r e s e n t (242 /xg Ce/g) . The r e s u l t s o b t a i n e d f o r the a n a l y s i s of the dead eggs and the empty egg s h e l l s showed approximately equal amounts of the l a n t h a n i d e s p r e s e n t i n each v i a l (707 t o 1110 /x g / v i a l ) , b u t t h e c o n c e n t r a t i o n s p r e s e n t were v e r y d i f f e r e n t between t h e two t y p e s o f samples. The whole eggs t r e a t e d w i t h d y s p r o s i u m a t 300 ug/ml had a c o n c e n t r a t i o n o f 3891 /xg Dy/g d r y w e i g h t , w h i l e t h e empty egg s h e l l s , t r e a t e d i n t h e same manner had n e a r l y f i f t e e n t i m e s t h e c o n c e n t r a t i o n o f dy s p r o s i u m . The eggs were exposed t o a l l e l e m e n t s f o r 48 hours and c o n s e q u e n t l y had more element p r e s e n t t h a n t h e a l e v i n s w h i c h were exposed f o r 4 ho u r s ( T a b l e 1 1 ) . T a b l e 11. ICP-MS a n a l y s i s o f dead a l e v i n s and eggs. A l e v i n and Eggs A n a l y z e d L a n t h a n i d e , C o n c e n t r a t i o n , and D u r a t i o n Sample Weight (g) R e s u l t s Mg/g /xg Ln Pe r V i a l 3 y o l k s Dy 100 /xg/ml (4 h r s ) 0.061 4.9 0.3 3 a l e v i n La 100 /xg/ml (4 h r s ) 0.116 55.2 6.4 3 a l e v i n Ce 100 /xg/ml (4 h r s ) 0.161 34.2 5.5 3 a l e v i n Sm 100 ug/ml (4 h r s ) 0.135 5.9 0.8 3 a l e v i n Dy 100 /xg/ml (4 h r s ) 0.151 76.7 11.6 3 a l e v i n Ce 300 /xg/ml (4 h r s ) 0.162 242.3 39.3 3 eggs La 300 /xg/ml (48 h r s ) 0.239 4654.1 1110 3 eggs Sm 300 /xg/ml (48 h r s ) 0.236 3791.8 896 3 eggs Dy 300 /xg/ml (48 h r s ) 0.192 3891.5 746 3 c a s e s 1 Dy 3 00 /xg/ml (48 h r s ) 0.012 57479.7 707 1 c a s e s = empty egg s h e l l s . 67 D i s c u s s i o n A l l c o n c e n t r a t i o n s of l a n t h a n i d e s t e s t e d were 100% t o x i c t o the a l e v i n s . The l i g h t l a n t h a n i d e s appeared t o be more t o x i c than the h e a v i e r ones. T h i s t o x i c e f f e c t c o u l d p o s s i b l y be a t t r i b u t e d t o the s m a l l e r s i z e and atomic weight of lanthanum and cerium than of samarium, dysprosium and y t t e r b i u m . I t i s p o s s i b l e t h a t the l i g h t e r elements may be t r a n s p o r t e d a c r o s s the g i l l e p i t h e l i a and/or the y o l k sac membrane a t an a c c e l e r a t e d r a t e w i t h a r e s u l t a n t e a r l i e r onset of t o x i c i t y , a lthough t h e r e are no p u b l i s h e d r e s u l t s t o s u b s t a n t i a t e t h i s h y p o t h e 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 t o x i c e f f e c t s observed i n the a l e v i n s c o u l d be the l a n t h a n i d e s were accumulated i n the a l e v i n s a t an a c c e l e r a t e d r a t e and were not e x c r e t e d . The e l e v a t e d l e v e l s of element e v e n t u a l l y caused m o r t a l i t y . The absence of m o r t a l i t i e s , a f t e r 4 hours, i n the tanks c o n t a i n i n g 300 /xg/ml of dysprosium and samarium, w h i l e t h e r e were no s u r v i v o r s i n the tanks c o n t a i n i n g 100 /xg/ml of these elements, c o u l d have r e s u l t e d from a r e v e r s a l of the treatments. I t i s not l i k e l y t h a t h i g h ammonia l e v e l s caused the m o r t a l i t i e s s i n c e the s t o c k i n g d e n s i t y was so low and s i n c e the a l e v i n s were i n the tanks f o r such a s h o r t p e r i o d . A l s o , a e r a t i o n of the water would have decreased ammonia l e v e l s and i n c r e a s e d d i s s o l v e d oxygen l e v e l s . I t was h y p o t h e s i z e d t h a t the a d d i t i o n of the l a n t h a n i d e a c e t a t e s would i n c r e a s e the a c i d i t y of the water and t h a t a base would need t o be added t o n e u t r a l i z e the water. Because the 68 e x t e n t t o which the v a r i o u s c o n c e n t r a t i o n s of l a n t h a n i d e s added would a f f e c t the pH was unknown, a constant amount of NaOH was added t o each tank. Because the r e s u l t a n t v a r i a b l e pH may have confounded r e s u l t s and because markedly lower c o n c e n t r a t i o n s of l a n t h a n i d e s were t o be used i n subsequent experiments, i t was d e c i d e d t h a t NaOH a d d i t i o n would not be p r a c t i s e d i n subsequent experiments. The foam and p r e c i p i t a t e t h a t developed i n the h i g h e s t c o n c e n t r a t i o n tanks may have r e s u l t e d from the chemical r e a c t i o n between the l a n t h a n i d e s and the c o n c e n t r a t e d NaOH or between the l a n t h a n i d e s and o r g a n i c s i n the r i v e r water. The t u r b u l e n c e caused by the a c t i v i t y of the pumps c o u l d have f u r t h e r e d the development of the foam. The foam had completely d i s s i p a t e d w i t h i n 24 hours. The p r e c i p i t a t e t h a t formed c o u l d have been l a n t h a n i d e hydroxide r e s u l t i n g from the f o l l o w i n g chemical r e a c t i o n between the l a n t h a n i d e a c e t a t e s and the NaOH: Ln(C 2H 30 2) 3 + 3NaOH -> 3NaC 2H 30 2 + Ln(OH) 3. The water samples taken from the 20 /xg/ml treatment tanks r e s u l t e d i n lower l a n t h a n i d e c o n c e n t r a t i o n s than those which were c a l c u l a t e d . P o s s i b l e reasons f o r these low a n a l y t i c a l v a l u e s are not known. Even w i t h a 24-hour flow-through r i n s e , t h e r e was c o n s i d e r a b l e r e t e n t i o n of l a n t h a n i d e s on the s i d e s of t h e tanks and on the apparatus as evidenced by the l a n t h a n i d e c o n c e n t r a t i o n s i n the water i n the tanks when they were recharged. A known c h a r a c t e r i s t i c of the l a n t h a n i d e elements i s t h e i r tendency t o adhere t o s u r f a c e s a t very low c o n c e n t r a t i o n s i n aqueous s o l u t i o n s (Luckey & Venugopal, 1977). F l u s h i n g w i t h water alone was not e f f e c t i v e i n removing the adhered elements. Regardless of the o r i g i n a l treatment c o n c e n t r a t i o n i n the tank, the r e s i d u a l l a n t h a n i d e c o n c e n t r a t i o n s i n the r i n s i n g water were approximately equal, thus i n d i c a t i n g an upper l i m i t t o the amount of l a n t h a n i d e t h a t can adhere t o the s u r f a c e s of the tank and apparatus. However, i t was found t h a t t h i s r e s i d u e c o u l d be removed by r i n s i n g the tank and apparatus w i t h 0.1M HCl. During t h i s r i n s i n g , the i n s o l u b l e hydroxide form was converted t o the s o l u b l e c h l o r i d e . /Analysis of dead a l e v i n s and eggs showed very h i g h c o n c e n t r a t i o n s of l a n t h a n i d e s . T h i s c o u l d have o c c u r r e d as a r e s u l t of these elements adhering t o the mucus c o a t i n g of the a l e v i n s and eggs, r a t h e r than being absorbed i n t o the t i s s u e s . I f t h i s were the case, the r e s u l t i n g c o n c e n t r a t i o n would not have been r e p r e s e n t a t i v e of the elements which were a c t u a l l y i n c o r p o r a t e d i n t o the a l e v i n s or eggs. T h i s t h e o r y was v e r i f i e d by the comparison of l a n t h a n i d e content i n the whole a l e v i n and i n the y o l k s alone, and by the l a n t h a n i d e content found i n the whole eggs and i n the empty egg s h e l l s alone (Table 11). As a r e s u l t of these f i n d i n g s i t was d e c i d e d t h a t , f o r f u t u r e experiments, c o l l e c t i o n of b i o l o g i c a l samples would be made two weeks post-experiment, d u r i n g which time u n t r e a t e d water would be p r o v i d e d . I t was h y p o t h e s i z e d t h a t such a d e l a y would a l l o w the r i n s i n g away of adhered l a n t h a n i d e s and the d e p o s i t i o n of the absorbed l a n t h a n i d e s i n the bony t i s s u e . Because a l l c o n c e n t r a t i o n s t e s t e d i n t h i s experiment proved t o be t o x i c , i t was d e c i d e d t h a t much lower c o n c e n t r a t i o n s would be used f o r f u t u r e t r i a l s . A 10-day t r i a l was conducted t o compare the m o r t a l i t y r a t e s of coho f r y and a l e v i n s i n s t a t i c tanks c o n t a i n i n g cerium a t c o n c e n t r a t i o n s of 0, 0.02, 0.2 and 2 Mg/ml. A l l of the f r y and a l e v i n s i n the tanks w i t h 2 Mg/ml d i e d w i t h i n 24 hours. A f t e r 4 days, a l l of the a l e v i n s and o n l y 1 f r y were dead i n the tank c o n t a i n i n g 0.2 Mg/ml. At the end of the 10-day t r i a l , 4 f r y were s t i l l a l i v e i n the 0.2 Mg/ml tank and 5 f r y and 2 a l e v i n s were s t i l l a l i v e i n the 0.02 ug/ml tank. S i n c e the a l e v i n s were more s e n s i t i v e than the f r y t o cerium a t t h e s e c o n c e n t r a t i o n s , f r y were chosen f o r subsequent i n v e s t i g a t i o n s . EXPERIMENT 2 - TOXICITY STUDY USING STEELHEAD (Salmo gairdneri) ALEVINS TREATED WITH LANTHANUM AND SAMARIUM I n t r o d u c t i o n Experiment 1 showed t h a t coho a l e v i n s were s e n s i t i v e t o the l a n t h a n i d e elements. As a r e s u l t of the t o x i c e f f e c t s observed, the a l e v i n stage was determined t o be u n s u i t a b l e f o r these marking s t u d i e s . However, f u r t h e r i n v e s t i g a t i o n i n t o the t o x i c e f f e c t s of these elements on a d i f f e r e n t s p e c i e s of salmonid would be u s e f u l . T h i s experiment was designed t o i n v e s t i g a t e the s e n s i t i v i t y o f s t e e l h e a d a l e v i n s t o lanthanum and samarium a t c o n c e n t r a t i o n s of 0, 10 and 100 jug/1 i n the water supply. The c o n c e n t r a t i o n s employed i n t h i s study were markedly lower than those used i n Experiment 1. M a t e r i a l s and Methods Experimental Design T h i s a l e v i n study was c a r r i e d out c o n c u r r e n t l y w i t h the t h i r d coho f r y l a b e l l i n g study (Experiment 5) . The d e s i g n i n v o l v e d a f a c t o r i a l arrangement of two l a n t h a n i d e s each a t t h r e e c o n c e n t r a t i o n s . The treatments used were lower than those of Experiment 1 and c o n s i s t e d of 0, 10 or 100 nq/1 of lanthanum o r samarium. There were 2 r e p l i c a t e s of each treatment. Twenty s t e e l h e a d (Salmo gairdneri) a l e v i n s were p l a c e d i n mesh c o n t a i n e r s i n each of 10 experimental tanks c o n t a i n i n g 100 coho f r y i n July, 1989. The tanks were set up with flow-through systems. At the s t a r t of the experiment, the a l e v i n had absorbed the majority of t h e i r yolk sacs and, they had 552 accumulated thermal units (ATUs). Dead alevins were removed and recorded on a d a i l y basis. Because t h i s was a t o x i c i t y study, no samples were taken for analysis by ICP-MS. 73 Results M o r t a l i t i e s A l l steelhead alevins i n the tanks containing lanthanum at 100 jug/1 died within 8 days, and a l l those i n tanks containing samarium at 100 Mg/1 died within 19 days (Table 12) . No m o r t a l i t i e s were observed i n any other tanks during t h i s period. Dead alevins were found i n an arched po s i t i o n with months open, a congealed yolk sac, and a white colouring. Table 12. Steelhead a l e v i n m o r t a l i t i e s observed, i n tanks each i n i t i a l l y containing 20 alevins, during 19-day exposure to lanthanum and samarium at 100 Mg/1-Time1 La 100 iig/1 (Tank 1) La 100 Mg/1 (Tank 2) Sm 100 Mg/1 (Tank 1) Sm 100 Mg/1 (Tank 2) 5 15 18 0 0 "6 3 2 0 0 7 1 - 7 7 8 1 - 4 3 9 - - 6 6 10 - - 2 2 19 - - 1 2 Total 20 20 20 20 1 Time = number of days a f t e r commencement of treatments. 74 Discussion Similar to the r e s u l t s of the f i r s t experiment with coho alevins, steelhead alevins were found to be s e n s i t i v e to the lanthanide elements. The steelhead alevins d i d not survive at lanthanum or samarium concentrations of 100 nq/1 i n the water supply. However, no m o r t a l i t i e s were observed i n the tanks containing 0 and 10 /xg/1 of lanthanum or samarium. The a l e v i n i n the tanks containing lanthanum at 100 nq/1 died f i r s t , i n d i c a t i n g that they were more se n s i t i v e to lanthanum than samarium. Although both lanthanum and samarium are l i g h t lanthanides, the t o x i c e f f e c t s of lanthanum was more immediate than those of samarium. Lanthanum i s a l i g h t e r element than samarium and the La 3 + ions are s i m i l a r to the Ca 2 + ions. From t h i s , i t would appear that the elements are a c t i v e l y taken up from the water supply as the Ca 2 + ions are. The increased t o x i c i t y of the l i g h t e r lanthanides was also observed i n the alevins i n Experiment 1. The causes of mortality were not investigated. The s e n s i t i v i t y of both coho and steelhead alevins to the lanthanides observed i n these two experiments suggest that a l a t e r stage (fry or smolts) might be better a l t e r n a t i v e s to pursue i n these mass marking studies. EXPERIMENT 3 - INVESTIGATION USING COHO {Oncorhynchus kisutch) FRY IN STATIC TANKS CONTAINING LANTHANUM IN A RANGE OF CONCENTRATIONS Introduction The f i r s t experiment using coho alevins (Experiment 1) showed that the al e v i n stage was unsuitable, at l e a s t at the concentrations employed, for t h i s type of mass marking study. Also, the 10-day cerium t r i a l using coho alevins and f r y showed that the f r y were less s e n s i t i v e than the alevins. Consequently, coho f r y were chosen for t h i s investigation. Because the lanthanide elements are not absorbed from the g a s t r o - i n t e s t i n a l t r a c t ( E l l i s , 1968; Luckey & Venugopal, 1977; and Kennelly et al., 1980), incorporation of the lanthanides into the water supply was tested, to explore the p o s s i b i l i t y of absorption through the g i l l s or other tis s u e s . For t h i s experiment one element, lanthanum, was introduced into the water supply with d i f f e r e n t groups of f i s h exposed to varying concentrations. The l e v e l s tested were between 2 and 1000 ng/1. A f t e r the l a b e l l i n g was complete, whole body analysis by ICP-MS was c a r r i e d out on i n d i v i d u a l f r y to determine the amount of element accumulated. There were two main objectives for t h i s experiment. The f i r s t was to determine i f the concentrations of lanthanum i n the water supply were to x i c to the f r y . The second objective was to 76 ascertain whether lanthanum was taken up and subsequently incorporated into the body of the f r y i n detectable amounts. Materials and Methods Experimental Design F i f t e e n coho (Oncorhynchus kisutch) f r y were placed i n each of 5 experimental tanks containing lanthanum concentrations of 0, 2, 10, 200, and 1000 fig/l for a 3-week period i n March, 1989. Because t h i s was a preliminary experiment, there were no r e p l i c a t e s f o r any of the treatments. For each treatment group, a measured amount of a 20 izg/ml aqueous lanthanum acetate stock solut i o n was added to each of the tanks containing 10 l i t r e s of ambient r i v e r water to provide the target concentrations. Once the lanthanum was added to the tanks, no water changes were made and no addi t i o n a l element was added. The f r y were obtained from Capilano River Hatchery brood stock and were not fed for the duration of the experiment to maintain the ammonia le v e l s i n the tank water at a minimum. The experiment was located i n a g a l l e r y under the hatchery rearing ponds which helped maintain the water temperature at approximately 10°C. At the conclusion of the 3-week chemical l a b e l l i n g period, the f r y were transferred to a flow-through system and provided with untreated r i v e r water f o r the rinse period. Two d i f f e r e n t durations were used f o r the r i n s e period: one week, and two weeks. Sampling and A n a l y t i c a l Method The coho f r y were sampled at the end of each of the two ri n s e periods. Seven f r y were randomly sampled from each tank for the f i r s t set of analyses, while the remaining f r y were maintained f o r the second set of analyses. The f r y were k i l l e d by asphyxiation and wet weights were recorded. After overnight oven-drying, they were weighed again and whole indivi d u a l s were placed i n sample v i a l s . They were then analyzed by ICP-MS for whole body lanthanum content. 78 Results M o r t a l i t i e s After two days, a l l the f i s h i n the 1000 nq/1 lanthanum treatment tank were dead. However, there was only one other mortality, t h i s occurring i n the 200 ng/1 treatment tank. A l l of the other f r y survived the 3-week treatment period and the 2-week rinse period. Although several of the f r y were very t h i n , as a r e s u l t of the feed being withheld, they survived the treatment and were used as a n a l y t i c a l samples. Water Analysis Water from each of the tanks was analyzed for lanthanum concentration at the beginning of the experiment and 9 days a f t e r the s t a r t of the experiment. The r e s u l t s obtained were consistently lower than the t h e o r e t i c a l l e v e l s (Table 13). The lanthanum concentrations of the water samples taken at the s t a r t of the experiment were greater than the concentrations 9 days l a t e r . 79 Table 13. Theoretical lanthanum concentrations, amount of lanthanum added to each tank, and the measured concentrations for the two sample dates. Tank Theoretical Concentration 20 /xg La/ml S. S.1 added I n i t i a l [La] [La] After 9 Days 1 2 /ig/1 La 1 ml 1.6 jug/i 1.3 /xg/1 2 10 /xg/1 La 5 ml 8.3 /ig/1 4.9 /ig/1 3 200 /ig/1 La 100 ml 150.0 /ig/1 116.0 /ig/1 4 1000 /ig/1 La 500 ml 816.0 M9/1 n/a 2 5 0 /ig / l La 0 ml <0.01 /xg/1 <o.o i /xg/1 1 SS = concentrated lanthanum acetate stock solution. 2 n/a = not analyzed. Whole Fry Analysis Analysis of whole f r y treated with lanthanum showed a d e f i n i t e accumulation of element, whereas undetectable amounts of element were present i n the untreated f r y (Figure 6). No f r y from the 1000 /xg/1 treatment group were analyzed as they had a l l died a f t e r only two days of treatment. There was a large increase i n the amount of lanthanum found i n the f r y treated with lanthanum at 2 00 /xg/1 compared to those treated with lanthanum at 2 or 10 /ig/1 a f t e r both the 1-week and the 2-week ri n s e periods (Figure 6) . Results for the f r y analyzed a f t e r the 2-week rinse period were s l i g h t l y lower than the values obtained f o r the 1-week rinse period. 80 _ 6 0) \ 0) 3 ~ 5 c 0 '•M (0 O 8 3 E •j c (0 2 n c _i 0 L a 2 La 10 La 200 Treatments (concentrat ion in water ug/l) 1-week rinse RINSE PERIOD 2-week r inse Figure 6. Lanthanum concentration i n the whole body of coho f r y following a 3-week exposure of lanthanum at 2, 10, and 200 /xg/1 and a 1-week or 2-week ri n s e period. Undetectable lanthanum i n control tank. Results are reported as mean ± S.E. space i n /xg of La per g of dry ti s s u e . 81 Discussion Introduction of lanthanum through the water supply appears to be a suitable method for marking coho f r y . The element i s absorbed from the water and incorporated into the body of the f i s h . Since the rinse periods were of s u f f i c i e n t length for mucus regeneration, the concentrations measured represented amounts of lanthanum accumulated i n the f r y , not the amounts adsorbed onto the f r y . Some of the anomalies found i n the whole body analysis could have resulted from the starved condition of the f r y , which could have been associated with a low mucus turnover. Only the 1000 /xg/1" concentration was f a t a l l y t o x i c . Since there was only one mortality i n the 200 Mg/1 treatment group, i t was considered that t h i s may have been near the upper l e t h a l l e v e l . As there were no m o r t a l i t i e s observed i n the tanks containing 2 and 10 /xg/1 of lanthanum, hish ammonia le v e l s did not appear to be a factor i n the m o r t a l i t i e s . The lowest concentrations, 2 and 10 Mg/1, did not r e s u l t i n a s u f f i c i e n t elemental deposition i n the whole body. However, the 2 00 l e v e l did r e s u l t i n a very high element deposition. Considering these r e s u l t s , i n combination with t o x i c i t y concerns, i t was decided that 100 Mg/1 would be used for future experiments. The analysis of water samples consistently yielded lower values than those which were expected. The water lanthanum concentrations i n the tanks also decreased with time. There are 82 at l e a s t three possible explanations f o r these low r e s u l t s . F i r s t , there may have been an adherance e f f e c t whereby the element formed insoluble hydroxides which adhered to the sides of the tanks and to the sides of the sample v i a l s (Luckey & Venugopal, 1977). The second may relate'to the s t a t i c nature of the tank set-ups which would decrease water l e v e l s i f uptake were occurring. Since the element i s not being replaced as the f r y absorb the lanthanum from the water supply, a decrease i n lanthanum concentration over time would be anticipated. F i n a l l y , the company that did the lanthanum analyses had a problem with the secondary standards which were used to c a l i b r a t e the ICP-MS. The containers that the standards were stored i n were not a i r - t i g h t and the solutions were evaporating over time, thus r e s u l t i n g i n low reported values. Since there i s no c o r r e c t i o n factor that can accurately be used for these r e s u l t s , the concentrations reported are only approximate values. The problem would have reduced the reported values for the f r y samples as well. The f r y i n t h i s experiment were kept i n s t a t i c tanks with no fresh supply of water or lanthanum. This method would have to be a l t e r e d to a flow-through system for hatchery a p p l i c a t i o n . This involves the water inflow to be set at a c e r t a i n rate with a concentrated aqueous lanthanide stock s o l u t i o n added at a constant rate. This would allow the constant replenishment of element taken up by the f i s h and would provide su i t a b l e water q u a l i t y to ensure production of high q u a l i t y juvenile salmon. 83 Although analyses of whole f r y indicated that lanthanum was incorporated into the f r y , they did not provide any information on d i s t r i b u t i o n of lanthanum into the i n d i v i d u a l tissues and organs. Several researchers have described these elements as bone seekers (Durbin et al., 1956; Jowsey et al., 1958; Kyker, 1961; Michibata, 1981; and Michibata & Hori, 1981). Consequently, the next experiment included the analysis of the v e r t e b r a l column, the o t o l i t h s and the scales to determine deposition s i t e . 84 EXPERIMENT 4 - INVESTIGATION OF COHO (Oncorhynchus kisutch) FRY I N A FLOW THROUGH TANK CONTAINING LANTHANUM Introduction The previous experiment i n which coho f r y , maintained i n tanks containing s t a t i c water, were exposed to varying concentrations of lanthanum demonstrated that i t i s f e a s i b l e to mark f r y using t h i s method. One lanthanide element, lanthanum, was shown to accumulate i n the whole body of coho f r y . This element was not f a t a l l y t o x i c at concentrations below 200 jug/1 during the 3-week period. The experiment was designed to investigate the uptake of lanthanum by coho f r y i n a flow-through system with a constant concentration of lanthanum. The d i s t r i b u t i o n of lanthanum into the vertebral column, o t o l i t h s and scales was analyzed. Also, the retention of lanthanum i n the vertebral columns was measured over a 2-month period. The following experiment was c a r r i e d out using a flow-through system to l a b e l the f r y , with ambient r i v e r water flowing into the tank at a set rate while the aqueous lanthanum acetate stock solution dripped i n at a constant rate. This method follows standard f i s h culture procedures more c l o s e l y . The water q u a l i t y could be maintained at a high l e v e l and the f i s h could be fed i n a normal manner. This procedure maintained the lanthanum concentration at a more constant rate, independent of uptake by f r y . 85 Materials and Methods Experimental Design One hundred coho (Oncorhynchus kisutch) f r y were placed into each of 2 experimental tanks containing lanthanum at concentrations of 0 and 100 ng/1 for 3 weeks i n A p r i l , 1989. Ambient r i v e r water flowed into the tanks at a constant rate of one l i t r e per minute. The concentrated lanthanum acetate solu t i o n was added d i r e c t l y to the lanthanum treatment tank at a constant rate of 0.7 ml/min. The t h e o r e t i c a l lanthanum concentration to which the f r y were exposed was calculated to be approximately 100 fJ.g/1. The f r y were newly ponded with 993 accumulated thermal units (ATUs) and they were fed Oregon Moist P e l l e t s twice d a i l y for the duration of the experiment. After the 3-week l a b e l l i n g period, the f r y were provided with untreated water f o r a 2-month rinse period with sampling at 18 days and 2 months. During the rinse period, the flow rate of ambient water into the tanks was increased to approximately two l i t r e s per minute. Sampling and A n a l y t i c a l Method Forty l a b e l l e d f r y were randomly sampled 18 days a f t e r the termination of the lanthanum exposure. T h i r t y of these were analyzed f o r whole body lanthanum content. Preparation for whole body analysis f o r lanthanum content was as described i n the previous experiment. The other ten f r y were used f o r bony t i s s u e analysis. The vertebral columns, o t o l i t h s , and scales 86 were removed and analyzed f o r lanthanum content. The vertebral columns and o t o l i t h s were removed and prepared f o r analysis as described i n the Methodology section at the beginning of t h i s t h e s i s . Because the scales from such young f r y were too small to handle, the scales had to be c o l l e c t e d by taking a scrape from the area around the l a t e r a l l i n e , a f t e r removal of mucus. Two months a f t e r the termination of the lanthanum treatment, ten more f r y were sampled. The vertebral columns were removed and analyzed for lanthanum content. 87 Results M o r t a l i t i e s Throughout the 3-week treatment period, there were only two m o r t a l i t i e s ; one of these f i s h was extremely emaciated, the other had a r e l a t i v e l y normal appearance. During the two-month r i n s i n g period, there was one other mortality. The f r y appeared be healthy and were eating normally throughout the experiment. Water Analysis Lanthanum concentration, monitored weekly throughout the treatment period ranged from 46.8 to 53.2 /xg/1 with an average value of 50.9 /xg/1. Although these values were considerably lower than the t h e o r e t i c a l concentration of 100 /xg/1/ they were very consistent throughout the treatment period (Figure 7). Whole Fry Analysis The analysis of the t h i r t y whole f r y sampled 18 days a f t e r removal of treatment further demonstrated that f i s h do accumulate lanthanum i n t h e i r body. The r e s u l t s obtained showed low v a r i a t i o n between in d i v i d u a l s . Eighteen days a f t e r the 3-week l a b e l l i n g period the lanthanum concentration i n the whole body was 0.86 ± 0.02 /xg/g (mean ± S.E.). 88 Bony Tissue Analysis The analysis of d i f f e r e n t bony tissues produced some promising and i n t e r e s t i n g findings. The f r y had accumulated concentrations of 0.44 ± 0.02 ug/g i n the vertebral columns. The v a r i a t i o n between lanthanum concentrations i n the i n d i v i d u a l vertebral columns was low, while the concentrations of element i n the scales and o t o l i t h s were more variable, with concentrations of 2.08 ± 0.93 ng/g i n scales and 0.21 ± 0.07 /xg/g i n o t o l i t h s . Some of the scale scrapings had undetectable amounts of lanthanum present while others had unusually high lanthanum concentrations. The o t o l i t h samples showed markedly lower lanthanum concentrations than the vertebral columns; also the r e s u l t s were more variable. Figure 8 shows the concentration of lanthanum accumulated i n the various bony tis s u e s . Analysis of Vertebral Column 2-Months Post Treatment The vertebral columns from ten treated and f i v e untreated f r y were removed and analyzed. The lanthanum-treated f i s h had large concentrations of lanthanum i n t h e i r vertebral columns (0.13 ± 0.01 ug/g) compared with untreated f i s h (<0.01 ug/g). Although there were detectable concentrations of lanthanum i n treated f r y 2 months a f t e r the l a b e l l i n g was completed, the concentration present was markedly lower than the concentration present 18 days a f t e r l a b e l l i n g (0.44 ± 0.02 /xg/g). This lower value occurred l a r g e l y as a r e s u l t of a d d i t i o n a l calcium being 89 l a i d down i n the verte b r a l column. Because no dry weights were taken, the t o t a l amount of lanthanum i n the verte b r a l column was not calculated. 1 2 Week of Treatment Figure 7. Lanthanum concentrations i n the water that coho f r y were exposed to over the 3-week treatment period. Lanthanum concentration measured i n /xg/1. r 90 3.5 O) 0) .2 2.5 03 c O 2 c o O E 1.5 D c r: c 03 —J 1 0.5 0 • Vertebrae Otol i ths Sca les Individual Bony T issues Figure 8. Lanthanum concentrations i n the vertebral column, o t o l i t h s and scales of coho f r y following a 3-week exposure to 100 nq/1 La and an 18-day rinse period. Results reported represent the mean ± S.E. i n /ig of La per g of dry bony t i s s u e . Discussion As demonstrated i n experiment 3, the f r y exposed to the lanthanum at approximately 100 fig / l i n the water supply accumulated the element i n detectable concentrations. This was shown through a series of d i f f e r e n t types of samples taken from the l a b e l l e d f r y . There was l i t t l e v a r i a t i o n between the i n d i v i d u a l f r y i n lanthanum incorporation into the body tis s u e s . The accumulation of lanthanum i n the various bony tissues supports the findings of Durbin et al. (1956), namely, that these elements are accumulated i n bony tis s u e s . The vertebral column i s a suitable depository of lanthanum, as i t incorporated at detectable concentrations with minimal v a r i a t i o n between in d i v i d u a l s , i n the f r y stage. The o t o l i t h s also contained detectable concentrations of lanthanum. However, the amounts present were less than those found i n the vertebral column and they were more varia b l e . One of the values obtained (38.2 ug/g) was unusually high compared with the other r e s u l t s , which ranged from 0.02 to 0.62 ug/g. This sample may have been contaminated during the sample preparation or a n a l y t i c a l procedures. Therefore t h i s value was discarded from the s t a t i s t i c a l c a l c u l a t i o n s . Another possible problem with the analysis of the o t o l i t h samples could have been the r e l a t i v e l y small amount of material which was analyzed. In f i s h of t h i s s i z e , the o t o l i t h s weighed approximately 0.2 mg. The scale scrapings showed r e l a t i v e l y high concentrations of lanthanum compared with the vertebral columns and o t o l i t h s . V a r i a t i o n between i n d i v i d u a l samples was high. Since the fry-were wiped clean of mucus p r i o r to the scale scrapings, the high observed v a r i a t i o n could not have occurred as a r e s u l t of lanthanum adhering to the mucus or scales. In addition, the r i n s e period was s u f f i c i e n t l y long to allow f o r complete regeneration of the mucus. A l i k e l y explanation for the high v a r i a t i o n between samples i s the v a r i a t i o n i n sample mass. Since the scales were of such a small s i z e , they were just scraped from the l a t e r a l l i n e area. No sample weight was taken. Therefore i t was impossible to know how many scales were present i n each sample. A more r e l i a b l e method fo r c o l l e c t i n g scales would need to be used for more accurate r e s u l t s , but with f r y of t h i s s i z e no s a t i s f a c t o r y method was apparent. From the r e s u l t s of the analysis of various bony tissues, i t i s apparent that the vertebral column i s the most suitable t i s s u e f o r sampling, because of i t s larger s i z e , high concentration of lanthanum and low l i k e l i h o o d of contamination. The lanthanum was s t i l l present i n detectable concentrations i n the vertebral column a f t e r 2 months. The t o t a l amount of lanthanum i n the vertebral column i s unknown since the sample weights were not taken. The lanthanum concentration decreased over time due to the d i l u t i o n caused by a d d i t i o n a l calcium being l a i d down. A longer term study would have to be c a r r i e d out to determine how long the lanthanum would remain i n the vertebral column i n detectable concentrations. The water samples taken showed lower concentrations of lanthanum present i n the tank water than those which were expected. These r e s u l t s were consistent with the r e s u l t s of Experiment 2. As discussed previously, the p l a t i n g out theory and the problems with the secondary standards for ICP-MS are both possible explanations for these low values. Because of these a n a l y t i c a l problems, concentrations reported are only approximate values. Problems with the analysis of the bony ti s s u e also existed. Since there was no "expected" concentration, these problems were harder to detect. The problems with the secondary standards cast doubt on the values reported for the ti s s u e samples. However, as a r e s u l t of t h i s error, the marks l a i d down i n these tissues would have a c t u a l l y been higher than those reported i n t h i s t h e s i s . This experiment has again demonstrated that lanthanum can be used as a chemical marker. Lanthanum i s taken up from the rearing water and i s subsequently deposited i n the f i s h . The analysis of the vertebral column, o t o l i t h s , and scale scrapings indicated that lanthanum was deposited into these bony tissues i n varying amounts. Furthermore, the r e s u l t s obtained indicate that storage of lanthanum p e r s i s t s i n the bony tissues for at leas t 2 months. Of the three bony tissues o r i g i n a l l y analyzed, the vertebral column appeared to be the one best suited for further 94 research. However, the scales and o t o l i t h s should also be considered further. EXPERIMENT 5 - THE TREATMENT OF COHO (Oncorhynchus kisutch) FRY WITH LANTHANUM AND SAMARIUM AT THREE CONCENTRATIONS FOR 3 AND 6 WEEKS Introduction In Experiment 4, i n which coho f r y were exposed to lanthanum i n a flow-through system, lanthanum was incorporated into the vertebral column, o t o l i t h s and scales. The element was s t i l l present i n detectable concentrations i n the vertebral column 2 months a f t e r the conclusion of the treatment. Also the vertebral column appeared to be the best suited bony ti s s u e for continued research. This experiment was designed to investigate the e f f e c t of varying treatment concentrations and durations of treatment on the uptake and retention of lanthanum and samarium over a 10%-month post-treatment period. The elements were introduced into the water at three concentrations (0, 10, and 100 M9/1) for 3-week and 6-week durations i n a flow-through system. Throughout the 10%-month post-treatment growth period, the f r y were sampled and the vertebral columns analyzed for lanthanide content every 2 months. O t o l i t h samples were taken and analyzed for lanthanide content 2 weeks and 10% months a f t e r termination of l a b e l l i n g . Scale samples were taken 10% months a f t e r termination of l a b e l l i n g . Fry weights were also recorded to investigate possible e f f e c t s of the lanthanides on growth. 96 Materials and Methods Experimental Design The design involved a f a c t o r i a l arrangement of lanthanum and samarium concentrations (3) , durations of exposure (2), and time of sampling (6). One hundred coho f r y were placed i n each of 10 experimental tanks containing 0, 10 and 100 fig/I lanthanum or samarium i n June, 1989. The f r y were exposed to the lanthanides for 3-week and 6-week durations. There were 2 r e p l i c a t i o n s of each of the treatments. The concentrated lanthanum and samarium acetate stock solutions for the 10 fig/1 treatments were prepared with 0.36 g of lanthanum acetate (99.9% purity) or 0.31 g of samarium acetate (99.9% purity) dissolved i n 10 1 of ambient r i v e r water. For the 100 fig / l treatments, 3.56 g of lanthanum acetate or 3.14 g samarium acetate were used (Appendix 3). These concentrated stock solutions were metered into the corresponding treatment tanks at a rate of 0.7 ml/min and were replenished every 10 days. The rearing water flowed i n at one l i t r e per minute and the concentrated lanthanide stock solution and r i v e r water became mixed i n a funnel before being delivered to the tank. Two groups of f r y served as negative controls with no lanthanum or samarium introduced to the water supply. During the f i r s t replenishment of stock solution, two of the samarium treatments were inadvertently reversed. This resulted i n one group of f r y being exposed to samarium at 10 fig/I f o r 10 days then 100 fig / l f o r 11 days, with the opposite 97 reversal occurring with the other group of f r y . This also affected the 6-week treatment - samarium at 10 ng/1 f o r 10 days then 100 Mg/1 fo r 32 days and v i s a versa (Table 14) . Table 14. Theoretical lanthanum and samarium treatment concentrations. 3 Week Duration 6 Week Duration # Tanks Treatment # Tanks Treatment 2 Control 2 Control 2 La 10 Mg/1 2 La 10 Mg/1 2 La 100 2 La 100 Mg/i 1 Sm 10 M9/1 1 Sm 10 Mg/1 1 Sm 100 M9/1 1 Sm 100 Mg/i 1 Sm 10/100 Mg/1* 1 Sm 10/100 Mg/1+ 1 sm 100/10 Mg/i" 1 sm 100/10 Mg/i + + * 10 Mg/1 fo r 10 days, 100 Mg/1 for 11 days. ** 100 Mg/1 fo r 10 days, 10 Mg/1 for 11 days. + 10 Mg/1 fo r 10 days, 100 Mg/1 for 32 days. ++ 100 Mg/1 fo r 10 days, 10 Mg/1 for 32 days. The Capilano brood stock (1988) coho f r y were ponded approximately 3 months p r i o r to the s t a r t of the experiment and, had accumulated 1,365 ATUs at the s t a r t of the treatments. The f r y weight ranged from 0.51 to 2.89 g with an average of 1.63 ± 0.08 g (mean ± S.E.). During the treatment period, the f r y were fed Oregon Moist P e l l e t s twice d a i l y . These d i e t s were analyzed for element content by ICP-MS and found to have undetectable amounts of lanthanides present (Appendix 4) . The ambient temperature of the water increased s t e a d i l y throughout the 98 l a b e l l i n g period from 7°C to 10°C. Water chemistry data for the ambient r i v e r water are given i n Appendix 5. After the 3-week l a b e l l i n g period, 50 f r y were removed from each tank and placed i n segregated mini-troughs for the grow-out period i n the g a l l e r y under the hatchery rearing ponds. The remaining f r y undergoing the 6-week l a b e l l i n g period i n the treatment tanks had continued exposure to the lanthanides for an additi o n a l 3 weeks, then were transferred to the mini-troughs. These troughs were divided into four separate 35 l i t r e sections with ambient untreated r i v e r water flowing through them at approximately 25 1/min. The f r y were maintained at the Capilano Hatchery for 1 year i n fresh untreated water. The f i s h were fed Oregon Moist P e l l e t s d a i l y and tanks maintained following to normal hatchery procedures. At the end of t h i s 1-year period, 10 of the remaining f i s h were randomly sampled from 8 of the treatment groups, f i n - c l i p p e d , and transferred to a fresh water tank at the Department of Fisheries and Oceans West Vancouver Laboratory i n June, 1990 (Table 15). After a 3-week acclimation period, each smolt received a 0.01 ml inter p e r i t o n e a l vaccination against v i b r i o s i s (Vijbrio anguillarum and V. ordalii), furunculosis (Aeromonas salmonicida), and ent e r i c redmouth (Yersinia ruckerii). The vaccine used was Ermogen-Furogen-Vibrogen bacterin supplied by Aquahealth Inc. Two weeks l a t e r , the f i s h were gradually transferred to sea water - 33% for 3 days, then 66% f o r 3 days, then f u l l strength sea water (29-31 99 g/1). These smolts were to have been maintained f o r 1 year i n sea water, then sampled and analyzed for lanthanide content i n July, 1991. Due to an error i n aquarium management, these f i s h were discarded without sampling 10 months a f t e r introduction to sea water. Unfortunately, interim samples were not taken because of small numbers of f i s h . Table 15. Labelled coho smolts transferred to the Department of Fisheries and Oceans West Vancouver Laboratory sea water tank and the f i n - c l i p s used to i d e n t i f y treatment groups. Treatment Duration # Fry F i n - C l i p Control 6 Weeks 10 Right Ventral La 100 /xg/1 3 Weeks 20 None La 100 /xg/1 6 Weeks 20 Adipose Sm 100 /xg/i 3 Weeks 10 Left Ventral + Adipose Sm 100 /xg/1 6 Weeks 10 L e f t Ventral Sm 10/100 /xg/i 6 Weeks 10 Right Ventral + Adipose 100 Sampling and A n a l y t i c a l Method The i n i t i a l sample set was taken 2 weeks a f t e r termination of the lanthanide exposures and subsequent samples were taken every 2 months for a t o t a l of 6 sample times throughout the 10%-raonth freshwater growth period. For each of the samples sets, 5 f r y were randomly sampled from each treatment group for a t o t a l of 30 f r y per treatment group over the 10% months. Wet weights were recorded, and bony ti s s u e samples were taken and prepared for ICP-MS analysis as described i n the Methodology section. Vertebral columns were removed from a l l of the treatment groups at each of the 6 sampling times. Otol i t h s were taken from a l l treatment groups at the i n i t i a l and f i n a l sampling times only. Scales were removed from f r y treated with lanthanum and samarium at 0 and 100 for a 6-week period at the f i n a l sample time only. A l l bony tissues were analyzed for lanthanum and samarium content by ICP-MS. Water samples were taken from each of the treatment tanks at the s t a r t of the experiment and then once a week fo r the duration of the l a b e l l i n g . For each sample, 10 ml were co l l e c t e d , s t a b i l i z e d with 3 drops of concentrated HCl and delivered f o r ICP-MS analysis on the same day. Three sets of spiked vertebrae standards and three sets of spiked water standards were prepared and analyzed as described i n the Methodology section. Vertebrae standards were analyzed at the same time as the l a s t three treated vertebrae sample sets only. Water standards were prepared fresh as needed using the 101 same reagents and d i s t i l l e d water and were analyzed concurrently with alternate weekly water samples taken from the treatment tanks. S t a t i s t i c a l Analysis A l l r e s u l t s were analyzed using analysis of variance using SYSTAT (Wilkinson, 1989), with differences between means tested at P<0.05, using Tukey's multiple range t e s t . Analysis of variance was ca r r i e d out on the data to determine i f there were any s i g n i f i c a n t tank e f f e c t s . Since none were shown, subsequent analyses were c a r r i e d out on the pooled data, using i n d i v i d u a l f i s h as the experimental units . The data from the lanthanum and samarium treatments were analyzed separately. The samarium treatments that were reversed were discarded from the s t a t i s t i c a l analysis because the concentrations and durations of exposure were not uniform. As a r e s u l t of these discarded groups, only one r e p l i c a t e of samarium treatments was used. The r e s u l t s for the untreated f i s h had undetectable l e v e l s present and no variance, therefore they were not included i n the s t a t i s t i c a l analyses. The data analyzed were: m o r t a l i t i e s ; growth (fry weight); lanthanide concentration and amount i n the vertebral column; and lanthanide concentration i n the o t o l i t h s and scales. 102 Results M o r t a l i t i e s The m o r t a l i t i e s for each treatment during the l a b e l l i n g period and during the 10%-month growth period are provided i n Table 16. There was no s i g n i f i c a n t difference i n the number of m o r t a l i t i e s between the groups treated f o r 3 weeks and those treated for 6 weeks. However, there were s i g n i f i c a n t l y more m o r t a l i t i e s i n the treatment tanks containing lanthanum at 100 Hg/1 during the treatment period than i n a l l other treatment tanks. Throughout the 10%-month growth period, there were only 25 m o r t a l i t i e s , with no clear e f f e c t of treatment i n evidence (Appendix 6). Table 16. Coho f r y m o r t a l i t i e s during the treatment period and during the growth period i n tanks containing lanthanum and samarium treatments. Treatment Treatment Period M o r t a l i t i e s Grow-out Period M o r t a l i t i e s Control 2%a l % a La 10/xg/l l % a 13%b La 100/xg/l l l % b 4%a Control 2%a l % a Sm 10/xg/l 0%a 6%a Sm 100/xg/l 0%a 0%a Error mean square for lanthanum treatment period = 0.779, t o t a l observations (n) = 14; error mean square f o r lanthanum growth period = 0.526, t o t a l observations (n) = 18. Total observations (n) for samarium treatment period = 2; t o t a l observations (n) f o r samarium growth period = 7. a, b. Within comparison group, values with unlike superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . 103 Growth Mean weights of f i s h and s i g n i f i c a n t e f f e c t s f or the 10% months a f t e r discontinuation of the 3-week and 6-week treatments are shown i n Tables 17 & 18. There was no s i g n i f i c a n t e f f e c t of treatment or duration on growth (Appendix 7). However, there were s i g n i f i c a n t increases i n weights between the i n i t i a l sample set measured 2 weeks post-treatment and the subsequent sample sets i n f i s h treated with both the lanthanum and with the samarium. In the lanthanum-treated f i s h , there was a s i g n i f i c a n t increase i n weights at the f i n a l weighing. Because the weighing of f i s h treated f o r 6 weeks was always done 3 weeks l a t e r than the weighing of the f i s h treated for 3 weeks, comparison of weights between f i s h treated for each of the two durations was not considered meaningful. The growth rate of the f i s h treated for 3 weeks was compared with the growth rate of the f i s h treated for 6 weeks using l i n e a r regression analysis. This comparison showed the growth rates between the two groups not to be s i g n i f i c a n t l y d i f f e r e n t . 104 Table 17. Mean body weights of f r y l a b e l l e d with lanthanum showing the s i g n i f i c a n t e f f e c t s of sampling time and duration of lanthanum exposures. Pur x Time  Means Dur 1 Dur 2 Sample Time 1 2 3 4 5 6 3.62a 7.27b 7.42b 8.05b 8.25b 9.56c 3.06s 7.17b 7. 94 b c d 8.59** 8.92cde 9.25de 4.198 7.38bc 6.90b 7.52bc 7.57bcd 9.87e Overall mean weight = 7.41 g. Start i n g weight of f r y = 1.63 g. Error Mean Square = 3.883; t o t a l observations (n) = 360. a, b, c, d, e. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Treatment x Duration i n t e r a c t i o n had no s i g n i f i c a n t differences between means. Duration 1 = 3 weeks; duration 2 = 6 weeks. Sample Time 1 = 2 weeks post-treatment; subsequent sample times every 2 months. 105 Table 1 8 . Mean body weights of f r y l a b e l l e d with samarium showing the s i g n i f i c a n t e f f e c t s of sampling time and duration of samarium exposures. Dur x Time Means Dur 1 Dur 2 Sample Time 1 | 3 . 4 5 a | | 2 . 9 6 a 3 . 9 3 a 2 | 7 . 4 4 b | j 6 . 7 2 b 8 . 1 6 b 3 | 7 . 8 0 b | | 8 . 2 0 b 7 . 4 1 b 4 | 8 . 1 6 b | j 8 . 3 8 b 7 . 9 4 b 5 | 7 . 5 9 b ] | 7 . 5 3 b 7 . 6 5 b 6 ! 8 . 5 3 b i ! 8 . 3 8 b 8 . 6 9 b Overall mean weight = 7 . 1 6 g. Start i n g weight of f r y = 1 .63 g. Error Mean Square = 2 . 9 0 2 ; t o t a l observations (n) = 1 8 0 . a, b. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0 .05) d i f f e r e n t according to Tukey's t e s t . Treatment x Duration i n t e r a c t i o n had no s i g n i f i c a n t differences between means. Duration 1 = 3 weeks; duration 2 = 6 weeks. Sample Time 1 = 2 weeks post-treatment; subsequent sample times every 2 months. 106 Downstream Contamination Untreated f r y placed i n tanks receiving the e f f l u e n t from l a b e l l e d f r y for 2 weeks did not accumulate detectable amounts of element i n t h e i r vertebral column. The elements do not appear to be "washed o f f " treated f r y and do not contaminate other f r y placed d i r e c t l y downstream. Water samples taken during the 6-week l a b e l l i n g period at 4 d i f f e r e n t locations along the Capilano River had n e g l i g i b l e amounts of lanthanum and samarium present (Table 19). The flow of the r i v e r and the flow of the hatchery e f f l u e n t appeared to d i l u t e the lanthanides i n the treatment water to nearly undetectable l e v e l s . Table 19. Concentrations of lanthanum and samarium present i n the Capilano River at 4 d i f f e r e n t locations downstream during the treatment period. Distance Downstream La (/xg/1) Sm (/xg/i) 10 metres 0.09 <0.03 1 kilometre <0.04 <0.03 2% kilometres 0.07 <0.04 3% kilometres 0.09 <0.02 Water Analysis The tanks containing lanthanum at the t h e o r e t i c a l concentration of 10 /ig/1 had mean concentrations of 2.6 to 9.5 Mg/1, and the tanks containing lanthanum at the t h e o r e t i c a l 107 concentration of 100 /xg/1 had mean concentrations of 36.9 to 69.7 jug/1 (Figure 9). Agreement between duplicates was high. The tanks containing samarium at the t h e o r e t i c a l concentration of 10 nq/1 had mean concentrations of 2.7 to 7.2 /xg/1, and the tanks containing samarium at the t h e o r e t i c a l concentration of 100 pq/1 had mean concentrations of 24.7 to 70.1 xxg/1. The reversal of the samarium treatments was r e f l e c t e d i n the water concentration values (Figure 10). These measured lanthanide concentrations were consistently lower than the t h e o r e t i c a l concentrations, but varied only s l i g h t l y from week to week. In the tanks containing the highest concentrations of lanthanides the values were lowest for the f i r s t two weeks and increased thereafter u n t i l the t h i r d week when they l e v e l l e d o f f . Values for the tanks containing the lowest treatment concentrations remained r e l a t i v e l y constant throughout the 6-week period. 108 = 80 0 1 2 3 4 5 6 Weeks After Commencement of Treatments Treatment Groups — La 10 (A) -+-• La 10 (B) - * - La 100 (A) -B- La 100 (B) F i g u r e 9. Lanthanum c o n c e n t r a t i o n s (/xg/1) i n the water t h a t coho f r y were exposed t o over the 6-week treatment p e r i o d . Undetectable lanthanum i n c o n t r o l tanks. 109 Figure 10. Samarium concentrations (/xg/1) i n the water that coho f r y were exposed to over the 6-week treatment period. Undetectable samarium i n control tanks. 11.0 Water Standards The spiked water standards analyzed by ICP-MS had a recovery of approximately 92% and 81% f o r lanthanum and samarium, respectively (Table 20). These recoveries seemed to be r e l a t i v e l y constant between duplicates of the same concentration, but tended to fluctuate between the sets of standards analyzed at d i f f e r e n t times. The concentration of element present i n the standard does not appear to a f f e c t the mean recovery rate. However, mean recovery rate of samarium was consistently lower than that of lanthanum. Table 20. Concentration of lanthanum and samarium i n water standards analyzed by ICP-MS. Time1 Calculated Concentration 2 Measured [La] 3 % La Recovery Measured [Sm] % Sm Recovery 2 weeks 10 9.0 90 8.1 81 50 44 89 40 80 100 85 85 79 79 Average 88 80 4 weeks 10 9.9 99 8.9 89 50 49 98 42 84 100 95 95 84 84 Average 97 86 6 weeks 10 9.7 97 8.2 82 50 45 90 38 76 100 88 88 77 77 Average 92 78 Overall Average 92 81 1 Time = number of weeks af t e r commencement of treatments. 2 Concentrations measured i n ng/1. 3 A l l values for duplicates were within 5%. Vertebrae Standards Lanthanide recoveries from vertebral columns with known added amounts were low and extremely variable. The lanthanum standards had approximately 77% recovery while the samarium standards had only 49% recovery (Table 21) . The lanthanum standards appeared to be more consistent than the samarium standards i n terms of percentage recovery. There were fluc t u a t i o n s between the r e s u l t s for the d i f f e r e n t sample sets, with the recovery varying with the amount of element present. The standards with less lanthanum or samarium had a greater recovery rate than the ones with more element. However, the duplicates within concentrations were reasonably consistent. 112 Table 21. Concentration of lanthanum and samarium i n the vertebrae standards analyzed by ICP-MS. Time1 Calculated Concentration 2 Measured [La] 3 % La Recovery Measured [Sm] % Sm Recovery 6% months 3 3 100 2 67 10 6 60 6 , 60 20 10 50 11.5 58 Average 70 62 months 3 3 100 2.5 83 10 7 70 5 50 20 14 70 9 45 Average 80 59 10% months 3 4 133 0 0 10 6 60 7.5 75 20 9.5 48 7.5 38 Average 80 25 Overall Average 77 . 49 1 Time = number of months a f t e r termination of treatments. 2 Concentration measured i n /ig/1. 3 A l l values f o r duplicates were within 10%. Vertebral Column Analysis Analysis of vertebral columns of f r y treated with lanthanum or samarium showed the elements to be present, whereas untreated f r y had undetectable amounts of element present. The values reported were not corrected for the recovery of the standards, because of the errors associated with the analysis of the standards (previously discussed). A considerably greater number of standards would have had to be prepared and analyzed before enough data would be available to use to correct the values reported. Since the recoveries of the standards were not s u f f i c i e n t l y r e l i a b l e to use to compensate f o r the r e s u l t s , no 113 correction was used. The groups treated with lanthanum or samarium at concentrations of 10 f o r 3 weeks had undetectable l e v e l s present 6% months a f t e r termination of the l a b e l l i n g . The data i n Tables 22 and 23 show the s i g n i f i c a n t e f f e c t s of treatment concentration, durations of exposure, and sampling time on the mean lanthanum and samarium concentrations present i n the vertebral columns. ANOVA tables for concentrations and amounts of element i n the vertebral column are presented i n Appendices 8 and 9. There were a s i g n i f i c a n t l y greater concentrations and amounts of element i n f r y treated with lanthanum or samarium at concentrations of 100 ng/1 for 6 weeks than i n a l l other groups. The concentration of element decreased as time progressed. The group exposed to lanthanum at 100 nq/1 f o r 6 weeks had 0.93 jug of La/g of dry vertebrae t i s s u e 2 weeks post-treatment, but the concentration was only 0.32 ug/g 10% months post-treatment. A l l other treatment groups showed si m i l a r , steady declines i n the concentration of element i n t h e i r vertebral columns (Tables 22 and 23). Tables 24 and 25 show the same e f f e c t s of treatment concentration, durations of exposure, and sampling time on the mean amounts of lanthanum or samarium i n the vertebral column of l a b e l l e d f r y . Results s i m i l a r to those f o r the concentrations were found i n these comparisons. The f i s h that were exposed to lanthanum at 100 fJ-g/1 for 6 weeks had s i g n i f i c a n t l y greater amounts of element accumulated i n t h e i r v e r t e b r a l columns. 114 The amount of element i n the vertebral columns remained r e l a t i v e l y constant throughout the 10%-month growth period. For the f i s h treated with lanthanum at 100 /xg/1 f o r 6 weeks, there was 12.23 ng present 2 weeks post-treatment and 11.83 ng present 10% months post-treatment. The amount of element present fluctuated only s l i g h t l y over the period and the average amount i n that group over the 10% months was 14.0 ng. Stable amounts were also observed i n a l l other groups (Tables 24 and 25). 115 Table 22. Mean lanthanum concentration (ug/g) i n vertebral columns of f r y l a b e l l e d with lanthanum showing the s i g n i f i c a n t e f f e c t s of lanthanum treatment concentration, duration of lanthanum exposures, and sampling time. Treatment x Duration x Time Trt 1 T r t 2 Dur 1 Dur 2 Dur 1 Dur 2 Sample Time 1 [ 0.09ab= 0.16 a b c d 0.47ef9~ 0.93~h ] 2 | 0.04" 0.12abc 0.22*** 0.639 | 3 | 0.05ab 0.15abc 0.22cd 0.589 | 4 | - 0.15abc 0.13abc 0.48fg | 5 ] - 0.10abc 0.15abc 0.41ef | 6 ! - 0.08abc 0.13abc 0.32de ! i J Means \ 0.06a 0.13a 0.22~b 0.56~b 1 i J T r t x Time Dur x Time Tr t 1 T r t 2 Dur 1 Dur 2 Means Sample Time 1 | 0. 1 2 abc 0. 70f ] j 0. 28 a b 0. 54c ; | 0. 41a 2 | 0. 08a 0. 43 e j | 0. 13a 0. 38 a c ! | 0. 2 5 a b 3 | 0. 1 0 abc 0. 40 d e | ! °* 14a b 0. ^ y 3C | | 0. 25 a b 4 | 0. 1 5 a b c 0. 3 1 c d e j | 0. 13 a b 0. 3 2 a b c | | 0. 25 a b 5 | 0. 1 Q abc 0. 2 8 bcde j ! °* 15a b 0. 25 a b | | 0. 22b 6 i 0. 08 a b 0. 22 a b c d ! ! 0. 1 3 a b 0. 20 a b i ! 0. 18b I 1 I J I I Means l" O . l l 8 0.39b 1 T 0.178 "o"~3Tb""1 Overall mean lanthanum concentration = 0.25 /xg La/g. Error Mean Square = 0.009; t o t a l observations (n) = 240. a, b, c, d, e, f, g, h. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Duration 1 = 3 weeks; duration 2 = 6 weeks. Treatment 1 = La at 10 /xg/1; treatment 2 = La at 100 ng/1. Sample Time 1 = 2 weeks post-treatment; subsequent sample times every 2 months. 116 Table 23. Mean samarium concentration (/xg/g) i n vertebral columns of f r y l a b e l l e d with samarium showing the s i g n i f i c a n t e f f e c t s of samarium treatment concentration, duration of samarium exposures, and sampling time. Treatment x Duration x Time  Trt 1 T r t 2  Dur 1 Dur 2 Dur 1 Dur 2 Sample Time 1 | 0.02ab 0.08abc 0.19bcd 0.49e 2 ; 0a 0.05ab 0.10abc 0.31d 3 | 0.03ab 0.02ab 0.16 a b c d 0.32^ 4 1 - 0.02ab 0.03ab 0.29d 5 ! - 0.02ab 0.08abc 0.23cd 6 ! — 0.02ab 0.08abc 0.22cd L. Means ! 0.02a 0. 04a 0.11b 0.3l"b ! Tr t x Time Tr t 1 T r t 2 Dur x Time Dur 1 Dur 2 Means 1 | 0.05abc 0.34~d | | 0.11ab 0.29~b j | 0.20a 2 j 0.02a 0.21bed | j 0.05ab 0.18ab | j 0.12a 3 | 0.03ab 0.24cd j | 0.10ab 0.17ab I | 0.13a 4 j 0.02ab 0.16abc | | 0.03a 0.16ab | | 0.12a 5 j 0.02ab 0.16abc ] j 0.08ab 0.12ab j j O . l l 8 6 i 0.02ab 0.15abc i I 0.08ab 0.12ab ! ! 0.11s Means I 0.03a L _ _ _ _ _ _ _ _ . 0.21b I i ! 0.08a i 0.17b Overall mean samarium concentration = 0.12 /xg Sm/g. Error Mean Square = 0.005; t o t a l observations (n) = 120. a, b, c, d, e. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Duration 1 = 3 weeks; duration 2 = 6 weeks. Treatment 1 = Sm at 10 /xg/1; treatment 2 = Sm at 100 /xg/1. Sample Time 1 = 2 weeks post-treatment; subsequent sample times every 2 months. 117 Table 24. Mean amount (ng) of lanthanum i n vertebral columns of f r y l a b e l l e d with lanthanum showing the s i g n i f i c a n t e f f e c t s of lanthanum treatment concentration, duration of lanthanum exposures, and sampling time. Treatment x Duration x Time  Trt 1 T r t 2 Dur 1 Dur 2 Dur 1 Dur 2 Sample Time 1 | 1.35a 2.68ab 7.81cd 12.23d* 2 i 0.90a 3.84abc 4.80abc 17.17f 3 | 2.008 4.33abc 6.81*° 17.24f 4 ! - 4.48abc 4.02abc 13.40ef 5 1 - 2.68ab 4.96abc 12.11de 6 j — 2.88ab 4.19abc 11.83de Means I 1.41a 3.48b 5.43c 14.00d T r t x Time Tr t 1 T r t 2 Dur x Time Dur 1 Dur 2 Means Sample Time Means 2.79s 9.71E ! 4.09a i 8.74b 1 | 2 .01a 10. 02 d e | | 4 .58ab 7. 45 a b ; j 6. 01a 2 j 2 • 37a 10. 99e | | 2 .85a 10. 51 b | j 6. 68a 3 j 3 .17 a b c 12. 02e j | 4 .40ab 10. 78 b | | 7. 59a 4 ! 4 < 4 8 a b c d 8. 7 1 c d e j | 4 • 02a 8. 94 a b | | 7. 30a 5 ! 2 .68° 8. 54b c d e ! | 4 .96ab 7. 39 a b ' j 6. 58a 6 i 2 • 88 a b 8. 01*** ! ! 4 . 19 a b 7. 36 a b I ! 6. 30a Overall mean lanthanum amount = 6.25 ng La. Error Mean Square = 7.369; t o t a l observations (n) = 240. a, b, c, d, e, f. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Duration 1 = 3 weeks; duration 2 = 6 weeks. Treatment 1 = La at 10 nq/lj treatment 2 = La at 100 nq/1. Sample Time 1 = 2 weeks post-treatment; subsequent sample times every 2 months. 118 Table 25. Mean amount (ng) of samarium i n vertebral columns of f r y l a b e l l e d with samarium showing the s i g n i f i c a n t e f f e c t s of samarium treatment concentration, duration of samarium exposures, and sampling time. Treatment x Duration x Time  Tr t 1 T r t 2  Dur 1 Dur 2 Dur 1 Dur 2 Sample Time 1 j 0.318 1.43ab 2.60ab 8.73cd 2 | 0.00s 1.55ab 2.00ab 11.20d 3 | 1.23ab 0.72ab 4.92bc 11.34d 4 ! - 0.76ab 1.19ab 10.41d 5 ! - 0.71ab 2.51ab 7.70cd 6 j — 0.86ab 2 . 64 a b 7.26cd Means i 0.52a 1.01a 2.64b 9.44b T r t x Time Dur x Time  Tr t 1 T r t 2 Dur 1 Dur 2 Means Sample Time 1 j 0.87ab 5.67abc ; j 1.468 5.08a | | 3.27s 2 | 0.78ab 6.60** | | 1.00a 6.38s | | 3.69s 3 j 0.98ab 8.13c | | 3.088 6.03s | j 4 .55s 4 | 0.76ab 5.80*° \ j 1.19a 5.59s | | 4.12s 5 j 0.718 5.11 a b c | | 2.518 4.21s ] ] 3.64s 6 ! 0.86ab 4.95abc ! ! 2.64a 4.06s ! ! 3.59s L I I J L I Means \ 0.84s 6.04b 1 \ 1.93s 5.22~b 1 Overall mean samarium amount = 3.44 ng Sm. Error Mean Square = 2.894; t o t a l observations (n) = 120. a, b, c, d. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Duration 1 = 3 weeks; duration 2 = 6 weeks. Treatment 1 = Sm at 10 /xg/1; treatment 2 = Sm at 100 iiq/1. Sample Time 1 = 2 weeks post-treatment; subsequent sample times every 2 months. 119 O t o l i t h Analysis The e f f e c t s of treatment concentration, duration of exposure, and sample time on the mean concentrations of lanthanum and samarium i n o t o l i t h s (sagittae) of l a b e l l e d f r y are indicated i n Tables 26 and 27. Appendix 10 contains the relevant ANOVA tables. The only f r y to show any s i g n i f i c a n t accumulation of element were those treated with lanthanum or samarium at 100 nq/1 for 6 weeks. In samples taken 10% months post-treatment, samarium was undetectable i n a l l groups, while lanthanum was detectable, but at a very low l e v e l , only i n the groups treated with lanthanum at the highest concentration. 120 Table 26. Mean lanthanum concentration i n o t o l i t h s of f r y l a b e l l e d with lanthanum showing the s i g n i f i c a n t e f f e c t s of lanthanum treatment concentration, duration of lanthanum exposures, and sampling time. Treatment x Duration x Time Trt 1 Dur 1 Dur 2 T r t 2 Dur 1 Dur 2 Sample Time 1 j"o5 "drd4~aB '57lYair~~~0~~36b"~\ 2 ! 0 a 0 .01 a 0 . 01 8 0 . 0 4 a b I i i Means H6a "b"~d3aB " o V o 6 a b ~Q~.~2& \ L I T r t x Time  Tr t 1 T r t 2 Sample Time 1 r~6~"o2SB "OTYA6 I 2 ! 0 .01 a 0 . 02 a b ! i i Means r~6~~6l~a~ ! i i Dur x Time Dur 1 Dur 2 Means ["6764"*"" ~0Tl3 i r "j ! 0 a 0 . 0 2 a b ! i i \~o~~o~2* "drdP"""! I_ I j 0 .09 a ! o . o i b I Overall mean lanthanum concentration = 0.08 /xg La/g dry tiss u e ( r e l a t i v e to calcium). Error Mean Square = 0 .005 ; t o t a l observations (n) = 30 (2 missing values). a, b. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Duration 1 = 3 weeks; duration 2 = 6 weeks. Treatment 1 = La at 10 nq/1; treatment 2 = La at 100 ng/1. Sample Time 1 = 2 weeks post-treatment; sample time 2 = 10% months post-treatment. 121 Table 2 7 . Mean samarium concentration i n o t o l i t h s of f r y lab e l l e d with samarium showing the s i g n i f i c a n t e f f e c t s of samarium treatment concentration, duration of samarium exposures, and sampling time. Treatment x Duration x Time Tr t 1 T r t 2 Dur 1 Dur 2 Dur 1 Dur 2 Sample Time 1 T65 ~o~a o"oTa~~ !T ¥ u i r " j 2 ! 0.01 s 0a 0a 0.01 a ! i i Means H55 ~0~a "o'TYP" ~6'731ir~~! L , I Sample Time Trt x Time Dur x Time Trt 1 T r t 2 Dur 1 Dur 2 Means To 5 ~o"~2~6b~"j r6"."6i"a" ~6~.~iTa | r~6~~6iTa~~~"j ! o a o a ! ! o a o a I I o b ! i i i i i J Means T o 5 ~0~~13ir~~! H65 ~0~(D8T> ! i . i i i Overall mean samarium concentration = 0 . 0 7 /xg Sm/g dry tissue ( r e l a t i v e to calcium). Error Mean Square = 0 . 0 0 0 ; t o t a l observations (n) = 1 6 . a, b. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0 .05 ) d i f f e r e n t according to Tukey's t e s t . Duration 1 = 3 weeks; duration 2 = 6 weeks. Treatment l = Sm at 10 fig/1; treatment 2 = Sm at 100 fig/1. Sample Time 1 = 2 weeks post-treatment; sample time 2 = 10% months post-treatment 122 Scale Analysis S i g n i f i c a n t concentrations of lanthanum and samarium were present 10% months af t e r the termination of l a b e l l i n g i n the scales of f r y treated with lanthanum or samarium at 100 ng/l for 6 weeks (Appendix 11) . No scale samples were taken from any other treatments. The lanthanum-treated f r y had more element accumulated i n t h e i r scales than the samarium-treated f r y or the control f r y (Table 28). Because no samples were taken d i r e c t l y a f t e r the termination of l a b e l l i n g , the i n i t i a l concentration of element present i s unknown. Table 28. Concentration of lanthanum and samarium found i n scales of coho f r y treated with lanthanum and samarium at 0 and 100 ug/1 for 6 weeks showing the s i g n i f i c a n t differences. Treatment [Lanthanide] Control 0a La at 100 1.48b Sm at 100 M5/1 0.46c Overall mean element concentration = 0.65 Mg La or Sm.g dry ti s s u e ( r e l a t i v e to calcium). Error Mean Square = 0.002; t o t a l observations (n) =6. a, b, c. Within comparison group, mean values with unlike superscript l e t t e r s were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Comparing the accumulation of lanthanum i n the three bony tis s u e s (vertebral column, o t o l i t h s and scales) , the scales had the greatest concentration of lanthanum 10% months post-treatment. The concentrations observed i n the vertebral columns 123 and o t o l i t h s decreased as time progressed and the. lanthanum i n the o t o l i t h s was almost undetectable a f t e r 10% months (Figure As found i n the lanthanum-treated f i s h , the scales of the samarium-treated f i s h had the greatest concentration of samarium 10% months post-treatment. The vertebral columns and the o t o l i t h s had approximately equal concentrations of samarium 2 weeks post-treatment, with the concentrations decreasing markedly 10% months post-treatment (Figure 12). 124 2000 1500 D) C o 03 c g 1000 c o o E 13 C CO c CO 500 0 no data Vertebrae Otoliths Scales TIME POST-TREATMENT 10 1/2 Months 2 Weeks 1 Figure 11. Concentration of lanthanum present i n the bony tissues of coho f r y 2 weeks and 10% months a f t e r termination of the treatments. Results reported as mean ± S.E. i n /ig of La/kg of vertebrae, or i n nq of La/kg dry tissue ( r e l a t i v e to calcium) i n o t o l i t h s and scales. Undetectable lanthanum i n the bony tis s u e s of untreated f r y . 125 600 3 N 500 D C 0 400 CO 1_ CO E co CO 300 c 0 0 c 0 0 200 100 no. d a t a Vertebrae Otoliths Scales TIME POST-TREATMENT 2 Weeks 10 1/2 Months Figure 12. Concentration , of samarium present i n the bony ti s s u e s of coho f r y 2 weeks and 10% months a f t e r termination of the treatments. Results reported as mean ± S.E. i n ng of Sm/kg of vertebrae, or i n ng o f Sm/kg dry ti s s u e ( r e l a t i v e to calcium) i n o t o l i t h s and scales. Undetectable samarium i n the bony ti s s u e s of untreated f r y . 126 Discussion During the exposure period there were s i g n i f i c a n t m o r t a l i t i e s i n the groups treated with lanthanum at 100 nq/1 i n contrast to the n e g l i g i b l e m o r t a l i t i e s associated with the other lanthanum and a l l of the samarium treatments. Lanthanum at t h i s concentration may have been s l i g h t l y high f o r these young f r y , but samarium was better tolerated. A possible explanation for the increased lanthanum t o x i c i t y i s that l i g h t lanthanides may have been accumulated i n the f r y at an accelerated rate and thereby resulted i n increased mortality; however there are no published r e s u l t s to support t h i s hypothesis. Throughout the 10%-month growth period r e l a t i v e l y few m o r t a l i t i e s were observed. These m o r t a l i t i e s appeared randomly over time and among treatments, i n d i c a t i n g that once the exposure to the element had been terminated, the t o x i c e f f e c t s were diminished. There was a s i g n i f i c a n t increase i n f r y weight two weeks post-treatment, a f t e r which the growth l e v e l l e d o f f . There are at l e a s t three possible explanations for the slow growth rate observed over the 10% months. F i r s t , f i s h generally tend to consume less food when they are fed i n small groups (Brett, 1979). Second, the temperature decreased s t e a d i l y 4 months post-treatmnet also r e s t r i c t i n g the growth rates. Third, subdivisions of the mini-troughs into 35 1 sections, may not have allowed s u f f i c i e n t space f o r exercise. F i n a l l y , the f r y were under stress when they were disturbed during the d a i l y 127 cleaning, as they had nowhere to hide. Due to the conditions of the experiment, none of these situations could have been avoided. As found i n the previous experiments, the lanthanide concentration i n each of the treatment tanks were consistently lower than the t h e o r e t i c a l concentration, and the values fluctuated over the 6-week treatment period. The e a r l i e s t weeks had the lowest r e l a t i v e concentration with the values l e v e l l i n g o f f a f t e r the t h i r d week. The low recovery observed i n the early weeks could have been a r e s u l t of the adsorption of the elements onto the sides of the treatment tanks (Luckey & Venugopal, 1977). Once the available adsorption s i t e s had been f i l l e d , the lanthanides introduced into the water presumably did not appear to adhere to the sides. Another p o s s i b i l i t y was v a r i a b i l i t y i n ICP-MS analysis. The a b i l i t y of ICP-MS to accurately detect the d i f f e r e n t lanthanides varies, as reported by Douglas & Houk (1985) and Houk & Thompson (1988). The r e s u l t s of the water and vertebrae standards demonstrate t h i s c h a r a c t e r i s t i c , where the recovery of lanthanum tended to be greater than the recovery of samarium. Samarium i s harder to quantify because there are 7 isotopes i n the mass range of 144 to 154, with the most abundant isotope without any interference being 147Sm at 15.0%. This means the instrument i s quantifying samarium using a low i s o t o p i c abundance and therefore needs greater amounts of samarium i n the sample fo r detection (Table 3). Lanthanum i s more r e l i a b l e to 128 analyze f o r because the isotope of i n t e r e s t ( 1 3 9La) has an abundance of 99.91% with no interference. For t h i s reason, lanthanum i s preferable to samarium for analysis when using ICP-MS (Longerich et al., 1987). In addition to v a r i a t i o n i n detection of elements, both types of standards showed differences between operating days of the instrument. There was a weekly v a r i a t i o n i n observed recovery for both lanthanum and samarium (Table 20 and 21) . This could have occurred as a r e s u l t of the plasma and mass spectrometer parameters not being adjusted to the optimum mass range, r e s u l t i n g i n some loss i n s e n s i t i v i t y for s p e c i f i c elements. However, the v a r i a t i o n observed was not very large. Published reports indicate that t h i s i s not usually a s i g n i f i c a n t problem (Taylor, 1986). The recovery rate was less for the t i s s u e standards than for the water standards. The recovery of lanthanum and samarium i n the water standards was 92% and 81%, respectively, and 77% and 49% i n the vertebrae standards, respectively. I t i s possible that, as a r e s u l t of the preparation method described i n the Methodology section, the n i t r i c acid was not able to digest the e n t i r e sample. Future vertebrae standards were prepared using a d i f f e r e n t method. Since the r e s u l t s obtained from the standards were variable and since there was an i n s u f f i c i e n t number of standards analyzed, there was no correction factor that could have been applied with any r e l i a b i l i t y to the measured concentrations i n 129 the samples. The reported values for the bony t i s s u e samples are probably low also; therefore i t would be expected that any marks l a i d down were act u a l l y greater than those reported. Previous experiments i n t h i s thesis and the r e s u l t s of Durbin et al. (1956) have demonstrated that the lanthanides are bone seeking elements. The r e s u l t s of the analysis of the vertebral columns, o t o l i t h s and scales confirm that lanthanum and samarium are taken up from the water and are deposited i n the bony tissues of f r y . Greater concentrations of elements and longer exposure periods resulted i n increased accumulation of element. The concentration of lanthanum and samarium i n the verteb r a l column and o t o l i t h s decreased over the 10% months. This was due to the continual deposition of calcium i n the bony tissues d i l u t i n g the o r i g i n a l lanthanide deposited. This d i l u t i o n e f f e c t was demonstrated throughout a l l of the treatment groups. I f the i n i t i a l amount accumulated i s not s u f f i c i e n t l y large, t h i s d i l u t i o n e f f e c t could reduce concentrations below the l i m i t s of detection of the ICP-MS (Longerich et a l . , 1987). As had been previously shown with t e t r a c y c l i n e , the marks l a i d down remain i n the bony tissues because the bony tissues grow i n a concentrical manner (Casselman, 1987). Over the 10% months, the amount of element present i n the vertebral column remained r e l a t i v e l y constant. This indicates that once the element has been deposited into the bony tissues i t remains there. Although the concentration of element declined st e a d i l y , 130 the actual amount of element present stayed approximately the same. The r e s u l t s indicate that greater amounts of lanthanum than samarium were accumulated i n the bony ti s s u e s . However, t h i s f i n d i n g may be misleading due to the problems of analyzing samarium, as discussed previously. ICP-MS has a greater s e n s i t i v i t y f o r detection of lanthanum than f o r samarium (Douglas & Houk, 1985; Longerich et a l . , 1987; and Houk & Thompson, 1988). Af t e r 10% months, the scales had markedly higher concentrations of lanthanum than either the vertebral column or the o t o l i t h s . However, amount of element accumulated i n the o t o l i t h s and scales i s not known since no weights of these bony tissues were taken. Once again, t h i s experiment proved that the lanthanide elements are accumulated i n the bony tissues and that t h i s storage p e r s i s t s . For the development of an e f f e c t i v e marking t o o l , more than one element would need to be incorporated into the t i s s u e . The following experiment investigated the e f f e c t of introducing two light-weight lanthanides (lanthanum and cerium) to the same f r y and to the same smolts. Parameters studied included element deposition i n bony tissues and t o x i c i t y . 131 EXPERIMENT 6 - THE TREATMENT OF COHO (Oncorhynchus kisutch) FRY AND SMOLTS WITH LANTHANUM AND CERIUM IN VARIOUS COMBINATIONS FOR 4 WEEKS Introduction Experiment 5 demonstrated that lanthanum and samarium are accumulated i n the vertebral columns, o t o l i t h s and scales of coho f r y , and that the elements remain i n these bony tissues for at l e a s t 10% months post-treatment. To develop an e f f e c t i v e mass marking technique, more than one element would need to be incorporated i n the bony tis s u e s . The present experiment investigated the e f f e c t s of introducing two elements to the same groups of f i s h . The two lanthanides with the lowest atomic weight, lanthanum and cerium, were used because previous studies (Experiments 1, 2, and 5) had shown the light-weight lanthanides to be more t o x i c than the heavier ones. Coho f r y and smolts were used to investigate the t o x i c e f f e c t s of lanthanide administration and the accumulation of the foregoing elements into the bony tis s u e s . Fish were exposed to various combinations of lanthanum and cerium at a concentration of 100 /xg/1 f o r 4 weeks. 132 Materials and Methods Experimental Design The design involved a f a c t o r i a l arrangement of stages of development (2) and treatment combinations of lanthanum and cerium (4), with two missing blocks. T h i r t y coho f r y (average weight of 0.52 g) were placed i n each of 8 experimental tanks and f i f t e e n coho smolts (average weight of 19.44 g) were placed i n each of 4 experimental tanks. Each tank containing various combinations of lanthanum and cerium f o r a 4 week period s t a r t i n g i n A p r i l , 1990. The treatment combinations used were: (i) lanthanum at 100 jug/l for 2 weeks then cerium at 100 ng/1 for 2 weeks - f r y and smolts; ( i i ) no element f o r 1 week then lanthanum and cerium each at 100 nq/1 for 2 weeks the no element for 1 week - f r y only; ( i i i ) lanthanum and cerium each at 50 j L i g / 1 f o r 4 weeks - f r y only; and (iv) no element f o r 4 weeks -f r y and smolts. There were 2 r e p l i c a t i o n s of each of the lanthanide treatments and of the negative controls (Table 29). The lanthanides were introduced into the flow-through system as described i n Experiment 5. The calc u l a t i o n s and concentrations of the treatments are presented i n Appendix 12. 133 Table 29. Theoretical lanthanum and cerium treatment / concentrations. Tanks With Fry Tanks With Smolts Week Treatment Week Treatment 1 0 1 0 2 0 2 0 3 0 3 0 4 0 4 0 1 La 100 Mg/1 1 La 100 Mg/1 2 La 100 /xg/i 2 La 100 Mg/i 3 Ce 100 Mg/i 3 Ce 100 Mg/1 4 Ce 100 Mg/i 4 Ce 100 Mg/i 1 0 2 La + Ce 100 Mg/1 3 La + Ce 100 Mg/1 4 0 1 La + Ce 50 Mg/1 2 La + Ce 50 Mg/1 3 La + Ce 50 Mg/1 4 La + Ce 50 Mg/1 The coho f r y used i n t h i s experiment were recently ponded with 944 ATUs; and the smolts used were Capilano brood stock (1988) ponded approximately 1 year ago (4,148 ATUs). The water temperature ranged from 4.5 to 6.5 °C throughout the l a b e l l i n g period. The f i s h were fed Oregon Moist P e l l e t s d a i l y f or the duration of the experiment. After the 4-week l a b e l l i n g period was completed, the f i s h were provided with untreated r i v e r water for a 2-week rin s e period. Sampling and A n a l y t i c a l Method Two weeks a f t e r the termination of the treatments (during which time a l l f i s h were maintained i n untreated water) a l l f r y 134 and smolts were k i l l e d , and 5 f i s h from each group were randomly selected for analysis. Vertebral columns and o t o l i t h s were removed from both f r y and smolts. Vertebrae standards with known amounts of lanthanides were prepared as described i n the Methodology section. A l l bony tissu e samples and standards were analyzed f o r lanthanum and cerium content by ICP-MS. Water samples were taken from each of the treatment tanks at the s t a r t of the l a b e l l i n g and then once a week f o r 4 weeks. Water standards with known amounts of lanthanides were prepared and analyzed at the same time as the weekly water samples taken from the treatment tanks. Preparation of both water standards and samples was the same as for Experiment 5. S t a t i s t i c a l Analysis A l l r e s u l t s were analyzed using analysis of variance using SYSTAT (Wilkinson, 1989), with differences between means tested at P<0.05, using Tukey's multiple range t e s t . The r e s u l t s for the untreated f i s h had no variance, therefore they were not included i n the s t a t i s t i c a l analyses. The data analyzed were: m o r t a l i t i e s ; lanthanide concentration and amount i n the vertebr a l columns; and lanthanide concentration i n the o t o l i t h s . Analysis of variance was c a r r i e d out on the data to determine i f there were any s i g n i f i c a n t tank e f f e c t s . Since none were shown, subsequent analyses were c a r r i e d out on the pooled data, using i n d i v i d u a l f i s h as the experimental units. 135 Results M o r t a l i t i e s M o r t a l i t i e s i n the tanks containing lanthanum at 100 /xg/1 for the f i r s t 2 weeks then cerium at 100 /xg/1 f o r the l a s t 2 weeks of the treatment were s i g n i f i c a n t l y greater than m o r t a l i t i e s i n a l l other treatments (Appendix 13). A l l other tanks had n e g l i g i b l e numbers of m o r t a l i t i e s (Table 30). Table 30. Total m o r t a l i t i e s during the treatment period i n tanks containing lanthanum and cerium treatments. Treatment Fry Smolts Control l % a 0.5%a La 100/xg/l (2 weeks) then Ce 100/xg/l (2 weeks) 0%a 0.5%a 0 (1 week) then La + Ce each at 100/xg/l (2 weeks) then 0 (1 week) 12%b -La + Ce each at 50/xg/l (4 weeks) l % a -Error mean square = 0.500, t o t a l observations (n) = 30. a, b. Values with unlike superscript l e t t e r s were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Water Analysis The water i n the tanks containing lanthanum at the t h e o r e t i c a l concentration of 50 fig/1 had mean concentrations ranging from 40 to 56 fig/1, and the water i n the tanks containing lanthanum at the t h e o r e t i c a l concentration of 100 /xg/1 had mean concentrations ranging from 94 to 107 /xg/1. The recovery for lanthanum was r e l a t i v e l y constant throughout the treatment period and, had an average value of 98% (Table 31). 136 The water i n the tanks containing cerium at the t h e o r e t i c a l concentration of 50 Mg/1 had mean concentrations ranging from 44 to 59 and the tanks containing cerium at the t h e o r e t i c a l concentration of 100 Mg/1 had mean concentrations ranging from 100 to 117 Mg/1* The recovery for cerium was r e l a t i v e l y constant throughout the treatment period and, had an average value of 107% (Table 31). Table 31. concentration of lanthanum and cerium i n water samples from tanks analyzed by ICP-MS. Time1 Calculated Concentration 2 Measured [La] 3 % La Recovery Measured [Ce] % Ce Recovery I n i t i a l 50 56 111 59 118 100 94 94 102 102 Average 103 110 Week 1 50 50 100 53 106 100 98 98 100 110 Average 99 108 Week 2 50 52 104 54 108 100 107 107 123 123 Average 106 116 Week 3 50 40 80 44 87 100 94 94 107 107 Average 87 97 Week 4 50 43 86 46 92 100 100 100 117 117 Average 93 105 Overall Average 98 107 1 Time = number of weeks a f t e r commencement of treatments. 2 Concentration measured i n nq/1. 3 A l l values for duplicates were within 5%. 137 Water Standards Analysis of water standards produced high and steady recoveries f o r both lanthanum and cerium. The average values were 105% f o r lanthanum and 117% for cerium (Table 32). Agreement between duplicates was high (within 5%). The mean recovery rate for cerium was consistently higher than that of lanthanum. Table 32. Concentration of lanthanum and cerium i n water standards analyzed by ICP-MS. Time1 Calculated Concentration 2 Measured [La] 3 % La Recovery Measured [Ce] % Ce Recovery I n i t i a l 50 46 92 55 110 100 92 92 103 103 Average 92 - 107 Week 1 50 48 96 55 110 100 97 97 100 100 Average 97 105 Week 2 50 59 118 64 128 100 120 120 120 120 Average 113 124 Week 50 49 98 60 120 3 100 100 100 115 115 Average 99 118 Week 4 50 61 122 66 132 100 115 115 135 135 Average 119 134 Overall Average 105 117 1 Time = number of weeks a f t e r commencement of treatments. 2 Concentration measured i n fig/1. 3 A l l values f o r duplicates were within 5%. 138 Vertebrae Standards Analysis of vertebrae standards spiked with known amounts of lanthanum, cerium and samarium resulted i n extremely variable values for each of the elements. The average recovery rates were 88% for lanthanum and 136% for cerium (Table 33) . The recovery d i d not vary with the amount of lanthanide spike present. There was moderately good agreement between duplicates (within 10%). Table 33. Concentraiton of lanthanum and cerium i n vertebrae standards analyzed by ICP-MS. Cone. (Mg/g) [La] 1 % La [Ce] % Ce 0.40 0.34 85 0.57 142 0.80 0.72 90 1.04 130 Average 88 136 1 A l l values for duplicates were within 10%. Vertebral Column Analysis Analysis of vertebral columns of treated f r y and smolts showed lanthanum and cerium to be present, whereas untreated f r y and smolts had undetectable l e v e l s of element present. Values were not corrected for the recovery of the vertebrae standards because of lack of s u f f i c i e n t numbers of standards analyzed and v a r i a b i l i t y of r e s u l t s . The data i n Table 34 show the e f f e c t s of lanthanum and cerium treatments on mean lanthanide concentrations i n the vertebral columns of f r y and smolts and 139 those i n Table 35 show the e f f e c t s on mean amounts of lanthanides i n the vertebral columns of f r y and smolts. ANOVA tables f o r the concentrations and amounts are presented i n Appendix 14. There were s i g n i f i c a n t l y greater concentrations of both lanthanum and cerium i n the vertebral columns of f r y (0.48 ug/g) than i n the vertebral columns of smolts (0.10 ug/g). Comparing the concentrations present i n the vertebral columns of f r y , there were no differences between treatments or elements (Table 34) . Because smolts were exposed to the f i r s t treatment only and f r y were exposed to a l l three treatments, comparisons of element present i n f r y and smolts between the treatments were not considered meaningful. Amounts of lanthanum and cerium present i n the vertebral columns of the f r y d i f f e r e d markedly from amounts i n smolts with the smolts accumulating markedly greater amounts (8.94 ng) than the f r y (1.49 ng) . There were no differences observed between lanthanum and cerium or between the three treatments (Table 35). Comparisons of amounts of elements present i n f r y and smolts between the treatments were not possible. 140 Table 34. Mean lanthanide concentrations (Mg/g). i n vertebral columns of f r y and smolts l a b e l l e d with lanthanum and cerium showing the s i g n i f i c a n t e f f e c t s of lanthanum and cerium treatments. Treatment x Stage x Lanthanide Fry Smolts La Ce La Ce Treatment 1 | 0.54bc 0.59c 0.10a 0.09s 2 | O.W1* 0.47*° - -3 ! 0.44^ 0.37b - -Means i 0.49a 0.48a 0.10b 0.09b T r t x Lanthanide La Ce Tr t x Stage Fry Smolts Means Treatment 1 i o . 32 a 0. 34 a 2 i ° - 47a 0. 47 a 3 i 0. 44 8 0. 37 a Means ! o . 39 a 0. 38 a ! 0.57a ] 0.47ab I 0.41b 0.10B 0.48a 0.10b | 0.338 ! 0.47b l : o i 41 ab Overall mean lanthanide concentration = 0.40 Mg/g-Error Mean Square = 0.016; t o t a l observations (n) = 80. a, b, c. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Treatment 1 = La at 100 /xg/1 for 2 weeks, then Ce at 100 for 2 weeks; treatment 2 = La + Ce each at 100 Mg/1 f o r 2 weeks, 0 f o r 2 weeks; treatment 3 = La + Ce each at 50 Mg/1 for 4 weeks. 141 Table 35. Mean lanthanide amounts (ng) i n vertebral columns of f r y and smolts l a b e l l e d with lanthanum and cerium showing the s i g n i f i c a n t e f f e c t s of lanthanum and cerium treatments. Treatment x Stage x Lanthanide  Fry Smolts La Ce La Ce Treatment 1 | 1. 71a 1. 81a 9.19b 8.68b i i i 2 ] 1. 33a 1. 28a - - i 1 3 I 1. 52a 1. 27a - - i i Means ! 1. 52a 1. 45a 9.19b 8.68b i T r t x Lanthanide Trt x Stage La Ce Fry Smolts Means Treatment 1 i 5 - 45a 5. 25a 2 | 1. 3 3®k 1. 28 a b 3 ! 1. L-__. 52 a b 1. 27 b Means ! 3. 44a 3. 26a I 1.49a 8.94b L Overall mean lanthanide amount = 2.68 ng. Error Mean Square = 4.219; t o t a l observations (n) =80. a, b. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Treatment 1 = La at 100 pq/1 f o r 2 weeks, then Ce at 100 /xg/1 for 2 weeks; treatment 2 = La + Ce each at 100 nq/1 f o r 2 weeks, 0 f o r 2 weeks; treatment 3 = La + Ce each at 50 nq/1 f o r 4 weeks. 142 O t o l i t h Analysis Analysis of o t o l i t h s of f r y and smolts l a b e l l e d with lanthanum and cerium showed detectable concentrations to be present, whereas untreated f i s h had undetectable concentrations of the element. Appendix 15 contains the relevant ANOVA table. There was no s i g n i f i c a n t difference i n the concentration of element between the d i f f e r e n t treatments. The smolts had s i g n i f i c a n t l y lower concentrations of element present (0.04 M<3/g) than the 0.14 /xg/g accumulated i n the f r y (Table 36). 143 Table 36. Mean lanthanide concentrations (ug/g) i n o t o l i t h s of f r y and smolts l a b e l l e d with lanthanum and cerium showing the s i g n i f i c a n t e f f e c t s of lanthanum and cerium treatments. Treatment x Stage x Lanthanide Fry Smolts La Ce La Ce Treatment 1 j 0.14b 0.22a 0.02b 0.05b | 2 j 0.10b 0.12b - i — i j 3 ! 0.10b i 0.14b - i — i Means \ 0.11ab 0.16a 0.02b 0.05b 1 Tr t x Lanthanide T r t x Stage La Ce Fry Smolts Means -Treatment 1 j 0.08a 0.14a | | 0.188 0.04a j j 0.11a 2 j 0.10a 0.128 j ] 0.11b j j O . l l 8 3 i 0.108 0.148 i I 0.12ab i — i ! 0.128 I 1 I . J I I Means T 0.098 0.148 \ !~0.14a 0.04~b 1 I I I I Overall mean lanthanide concentration = 0.11 /xg/g. Error Mean Square = 0.004; t o t a l observations (n) = 32. a, b. Within comparison groups (enclosed by a dashed l i n e ) , mean values without a common superscript l e t t e r were s i g n i f i c a n t l y (P<0.05) d i f f e r e n t according to Tukey's t e s t . Treatment 1 = La at 100 ixg/1 for 2 weeks, then Ce at 100 jug/l f o r 2 weeks; treatment 2 = La + Ce each at 100 iig/1 for 2 weeks, 0 f o r 2 weeks; treatment 3 = La + Ce each at 50 ng/1 for 4 weeks. 144 Discussion The tanks containing f r y that were treated with lanthanum at 100 /xg/1 for 2 weeks followed by cerium at 100 /xg/1 for 2 weeks had a s i g n i f i c a n t l y larger number of m o r t a l i t i e s than a l l other tanks containing f r y and smolts. As discussed previously, the l i g h t lanthanides as a group may have an increased t o x i c a f f e c t on young f r y ; however, the elements were better tolerated by smolts. The spiked water standards and the water samples taken from the treatment tanks showed that the lanthanum concentrations were close to the t h e o r e t i c a l values, and that the cerium concentrations were higher than expected. The values were reasonably consistent over the 4-week treatment period. However, these r e s u l t s were inconsistent with those obtained from previous experiments. Two possible explanations for the better recovery rates observed i n t h i s experiment are: (i) the ICP-MS instrument may have been better adjusted for the detection of the lanthanides, allowing f o r more accurate values; and ( i i ) accuracy of analysis for lanthanum and cerium content was enhanced by the high i s o t o p i c abundances of the isotopes used f o r measurement ( 1 3 9La at 99.91% and 1 4 0Ce at 88.48%) and by the lack of interference from other isotopes (Longerich et al., 1987). As was observed i n Experiment 5, analysis of vertebrae standards spiked with known amounts of lanthanides resulted i n variable recovery rates. Lanthanum had a low average recovery 145 (88%), whereas cerium had a high average recovery (136%). This v a r i a t i o n was most l i k e l y due to the d i f f e r e n t i a l a b i l i t y of ICP-MS i n the detection of d i f f e r e n t elements (Douglas & Houk, 1985; and Houk & Thompson, 1988). Results from the analysis of vertebral column samples showed the cerium l e v e l s to be consistently higher than the lanthanum l e v e l s . This could have been a r e s u l t of an a n a l y t i c a l problem rather than a difference i n the l e v e l s of accumulation between the two elements. Because the analysis of spiked standards produced variable r e s u l t s , no correction factor was applied to the r e s u l t s of the analysis of unknown samples. Reported values for the marks l a i d down i n the bony tissues were therefore only approximate values. A l l f r y and smolts exposed to lanthanum and cerium received approximately equal doses of each element over the treatment period. The r e s u l t s of the analysis of the vertebral columns showed the f r y to contain markedly higher concentrations of lanthanum and cerium than the smolts. However, the smolts had greater amounts of element i n t h e i r vertebral columns than the f r y . The smolts had accumulated more element i n t h e i r vertebral columns than the f r y , but because of the increased s i z e of t h e i r bony ti s s u e s , the smolts had lower concentrations of element present. The larger f i s h have a greater d a i l y growth increment, therefore accumulated higher l e v e l s of both calcium and the lanthanide elements i n t h e i r vertebral columns than the f r y . Weiss (1974) and Das et al. (1988) have suggested that La 3* ions and Ca2* ions enter the f i s h by the same route and that La 3* 146 a c t i v e l y competes with Ca2*. This i s a l i k e l y explanation for the elevated lanthanum and cerium l e v e l s found i n the f r y and smolts. There was no s i g n i f i c a n t difference i n the lanthanum or cerium concentrations found i n the vertebral columns of the f r y treated with 50 or 100 jug/1 of element. The lanthanides seem to be a c t i v e l y taken up from the water supply at the same rate regardless of the exposure concentration. The o t o l i t h s of both f r y and smolts were shown to have lanthanum and cerium deposited i n them. The o t o l i t h s of the f r y had markedly greater concentrations of both elements i n contrast to the concentrations i n the o t o l i t h s of the smolts. The r e l a t i v e amount of element present i s not known since the o t o l i t h s were not weighed. This experiment confirms the findings of the previous work reported i n t h i s t h e s i s that the lanthanide elements are taken up from the water supply and deposited into bony ti s s u e s . 147 CONCLUSIONS AND RECOMMENDATIONS The seri e s of experiments c a r r i e d out for t h i s thesis indicate that the lanthanides are absorbed from the water supply and subsequently incorporated into the bony tissues of coho f r y and coho smolts. Longer exposure times and greater treatment concentrations r e s u l t i n increased element accumulation i n the bony tis s u e s . Coho f r y exposed to lanthanum or samarium at 100 /xg/1 f o r 6 weeks had detectable l e v e l s of element i n t h e i r v e r t e b r a l columns, o t o l i t h s , and scales 10% months post-treatment . When lanthanum and cerium were introduced into the water supply of coho f r y and smolts, both elements were accumulated i n the bony tissues i n approximately equal concentrations f o r a l l methods of applicat i o n . When the elements were added to the water supply at the same concentration and for the same duration, higher concentrations of lanthanum and cerium were accumulated i n the f r y than i n the smolts; but, the smolts had greater amounts of element present. Since larger f i s h have a greater d a i l y growth increment, they accumulate more element i n t h e i r bony t i s s u e s . Thus, i t may be more e f f e c t i v e to expose larger f i s h to the elements. Not only would t h i s r e s u l t i n increased element deposition, but i t would also r e s u l t i n decreased t o x i c e f f e c t s . 148 From analysis of untreated f i s h i t was shown that the lanthanides are not present i n the bony tissues i n detectable l e v e l s and should not i n t e r f e r e i n the detection of marked f i s h . At concentrations of 100 i n the water supply, the lanthanides were severely t o x i c to coho and steelhead alevins, but were only s l i g h t l y t o x i c to coho f r y . The alevins may accumulate the lanthanides at a rate which i s f a s t e r than t h e i r development rate or t h e i r excretion processes, thereby r e s u l t i n g i n an overdose and death. Coho smolts d i d not seem to be adversely affected by lanthanum or cerium at 100 ng/1. The light-weight lanthanides appeared to be more tox i c than the heavier ones. The rate of uptake may vary for the i n d i v i d u a l lanthanides, therefore r e s u l t i n g i n d i f f e r e n t i a l t o x i c e f f e c t s . Because the mechanism for the t o x i c actions of these elements i n f i s h i s not f u l l y understood, a marking strategy to follow may include exposing larger f i s h to lower concentrations of the elements for longer durations. Future developments of ICP-MS technology are of great importance to t h i s type of mass marking. Innovations i n electrothermal vaporization or laser ablation may lead to greater improvements i n s e n s i t i v i t y of detection. Because of the natural d i l u t i o n of the l a b e l by increased calcium deposition as the f i s h grows, the concentration present i n mature f i s h w i l l be markedly reduced. I f s u f f i c i e n t quantities are deposited i n bony tissues, and detection l i m i t s are 149 improved, i t should be possible to i d e n t i f y marked f i s h throughout t h e i r l i f e cycle. Although there are 15 lanthanide elements and yttrium (not a lanthanide, but has s i m i l a r properties), not a l l of them may be s u i t a b l e or f e a s i b l e to use for marking f i s h . There i s a well documented odd/even r e l a t i o n s h i p between the elements i n the lanthanide s e r i e s ; the elements with even atomic numbers are more abundant and easier to p u r i f y than the elements with odd atomic numbers (Taylor, 1964; Topp, 1965; and Kilbourn, 1988). The implication of t h i s r e l a t i o n s h i p i s that the even numbered lanthanides are more commercially av a i l a b l e . Also, the l i g h t -weight lanthanides tend to be more abundant than the heavier elements; therefore, these would tend to be less expensive. Since these elements are available i n large quantities at r e l a t i v e l y low cost, i t would be very f e a s i b l e to mark entire hatchery populations. Along with the natural elemental abundances, the a n a l y t i c a l properties need to be considered. The lanthanide elements with high abundance of a single isotope with l i t t l e or no interference from other isotopes, w i l l be preferable for analysis by ICP-MS. Lanthanides that appear to be f e a s i b l e to obtain on a commercial basis and to analyze include: yttrium, lanthanum, cerium, praseodymium, neodymium, samarium, gadolinium, terbium, dysprosium, holmium, and ytterbium. Before t h i s marking technique can be f u l l y u t i l i z e d on a large-scale, several areas need to be explored. These include: 150 (i) s u i t a b i l i t y of a l l f e a s i b l e lanthanides. for marking, e s p e c i a l l y the less expensive and more abundant ones; ( i i ) species differences i n t o x i c i t y , uptake and retention of the elements; ( i i i ) s u i t a b i l i t y of chlorides as well as acetates because of r e l a t i v e l y lower costs, greater a v a i l a b i l i t y and ease of handling of chlorides; (iv) determine the amount of element that would need to be incorporated into the bony tissues of the juvenile salmon to be detectable i n the mature adult; (v) marking of large production groups of both f r y and smolts; (vi) environmental impact study with p a r t i c u l a r attention to the t o x i c e f f e c t s on juvenile salmon and invertebrates exposed to the e f f l u e n t ; also the p o t e n t i a l problem of l a b e l l i n g f i s h downstream; ( v i i ) e f f e c t s of lanthanide administration on histopathology and immunocompetence of l a b e l l e d f i s h ; and ( v i i i ) lanthanide accumulation and retention i n the opercula because of the perceived sampling benefits of centre punching the lanthanide-enriched portion. 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Acute l e t h a l doses of lanthanide elements i n rats and mice. Lanthanide Compound Animal Route Mg metal/kg Ref Yttrium Oxide Rat IP 395 1 Yttrium Chloride Rat IP 132 1 Mouse IP 88 2 Yttrium N i t r a t e Rat IP 117 1 Lanthanum Oxide Rat PO >8500 1 Lanthanum Chloride Rat PO 2370 1 Rat IP 197 1 Mouse IP 211 2 Mouse SC >500 2 Lanthanum Sulfate Rat PO >2450 1 Rat IP 134 1 Lanthanum N i t r a t e Rat PO 1450 1 Rat IP 145 1 Mouse IP 131 3 Lanthanum Acetate Rat PO 4400 1 Rat IP 209 1 Lanthanum C i t r a t e Chloride Mouse IP 44 4 Cerium Chloride Rat IV 50 2 Mouse IP 201 2 Cerium Nit r a t e Rat PO 1355 3 Rat IP 93 3 Rat IV 2-16 3 Mouse IP 151 3 Praseodymium Chloride Mouse SC 944 2 Praseodymium N i t r a t e Rat PO 1134 3 Rat IP 79 3 Rat IV 2-25 3 Mouse IP 94 3 Neodymium Chloride Mouse IP 200 2 Neodymium N i t r a t e Rat PO 905 3 Rat IP 89 3 Rat IV 2-22 3 Mouse IP 89 3 Samarium Chloride Mouse PO >840 5 Mouse IP 241 5 164 Lanthanide Compound Animal Route Mg metal/kg Ref Samarium Nitrate Rat PO 901 3 Rat IP 96 3 Rat IV 3-20 3 Mouse IP 106 3 Europium Chloride Mouse PO 2075 6 Mouse IP 228 6 Europium Nitrate Rat PO >1704 3 Rat IP 72 3 Mouse IP 109 3 Gadolinium Chloride Mouse PO >850 5 Mouse IP 232 5 Gadolinium Nitrate Rat PO >1743 3 Rat IP 80 3 Mouse IP 105 3 Terbium Chloride Mouse PO 2175 7 Mouse IP 234 7 Terbium Nitrate Rat PO >1753 3 Rat IP 91 3 Mouse IP 168 3 Dysprosium Chloride Mouse PO 3290 6 Mouse IP 251 6 Dysprosium Nitrate Rat PO 1103 3 Rat IP 105 3 Mouse IP 110 3 Holmium Chloride Mouse PO 3140 6 Mouse IP 243 6 Holmium Nitrate Rat PO 1078 3 Rat IP 97 3 Mouse IP 115 3 Erbium Chloride Mouse PO 2700 6 Mouse IP 233 6 Erbium Nitrate Rat IP 83 3 Rat IV 13-19 3 Mouse IP 81 3 Thulium Chloride Mouse PO 2635 7 Mouse IP 204 7 Thulium Nit r a t e Rat IP 104 3 Mouse IP 93 3 165 Lanthanide Compound Animal Route Mg metal/kg Ref Ytterbium Chloride Mouse PO 2995 7 Mouse IP 186 7 Ytterbium Nitrate Rat PO 1148 3 Rat IP 94 3 Mouse PO 93 3 Lutetium Chloride Mouse PO 4400 6 Mouse IP 195 6 Lutetium Nitrate Rat IP 125 3 Mouse IP 108-130 3 (1) (2) (3) (4) (5) (6) (7) Cochran Kyker & et al, Cress, Bruce et al, Graca et al, Haley et al, Haley, 1965 Haley et al. 1950 1957 1963 , 1962 , 1961 , 1963 166 Appendix 2. ICP-MS Analysis for lanthanide content of 6% sodium hypochlorite bleaching solution used to digest traces of f l e s h o f f bony t i s s u e s . Lanthanide Concentration (Mg/1) Yttrium <0.37 Lanthanum <0.10 Cerium 0.36 Praseodymium 0.40 Neodymium 2.04 Samarium <0. 98 Europium <0.27 Gadolinium 0.88 Terbium 0.19 Dysprosium <0.50 Holmium 0. 09 Erbium 0.03 Thulium <0.10 Ytterbium 0.46 Lutecium 0.07 167 Appendix 3. Information used and re s u l t s of computer d r i p c a l c program used for c a l c u l a t i o n of lanthanide treatments used i n Experiment 5. Lanthanum at 10 mg/1 A. Data Required Element Name: Lanthanum Compound Name: Lanthanum acetate Atomic Weight of Element: 138.91 (La) Gram Formula Weight of Compound: 343.07 (La acetate) Increase i n Element Required: 10 /xg/1 = 0.01 mg/1 S o l u b i l i t y of Compound: 168.8 g/1 Stock Solution Container Size: 10 1 Flow Rate: 1 1/min Number of Units to Treat: 1 Number of Days Stock Solution to Last: 10 days B. Calculation Results To Raise La by 0.01 mg/1: La acetate stock solution: 0.04 g/1 - 10 1 requires 0.3 6 g Drip rate for stock: 0.7 ml/min - Unit flow @ 1 1/min - Stock l a s t s 10 days for 1 unit Lanthanum at 100 ug/l A. Data Required Element Name: Lanthanum Compound Name: Lanthanum acetate Atomic Weight of Element: 138.91 (La) Gram Formula Weight of Compound: 343.07 (La acetate) Increase i n Element Required: 100 /xg/1 = 0.1 mg/1 S o l u b i l i t y of Compound: 168.8 g/1 Stock Solution Container Size: 10 1 Flow Rate: 1 1/min Number of Units to Treat: 1 Number of Days Stock Solution to Last: 10 days B. Calculation Results To Raise La by 0.1 mg/1: La acetate stock solution: 0.3 6 g/1 - 10 1 requires 3.56 g Drip rate for stock: 0.7 ml/min - Unit flow § 1 1/min^ - Stock l a s t s 10 days f o r 1 unit 168 Samarium at 10 tm/1 A. Data Required Element Name: Samarium Compound Name: Samarium acetate Atomic Weight of Element: 150.4 (Sm) Gram Formula Weight of Compound: 381.53 (Sm acetate) Increase i n Element Required: 10 fig/l = 0.01 mg/1 S o l u b i l i t y of Compound: 150 g/1 Stock Solution Container Size: 10 1 Flow Rate: 1 1/min Number of Units to Treat: 1 Number of Days Stock Solution to Last: 10 days B. Calculation Results To Raise Sm by 0.01 mg/1: Sm acetate stock solution: 0.03 g/1 - 10 1 requires 0.31 g Drip rate for stock: 0.7 ml/min - Unit flow @ 1 1/min - Stock l a s t s 10 days for 1 unit Samarium at 100 mg/1 A. Data Required Element Name: Samarium Compound Name: Samarium acetate Atomic Weight of Element: 150.4 (Sm) Gram Formula Weight of Compound: 381.53 (Sm acetate) Increase i n Element Required: 100 M9/.1 = 0- 1 ^ 9/1 S o l u b i l i t y of Compound: 150 g/1 Stock Solution Container Size: 10 1 Flow Rate: 1 1/min Number of Units to Treat: 1 Number of Days Stock Solution to Last: 10 days B. Calculation Results To Raise Sm by 0.1 mg/1: Sm acetate stock solution: 0.31 g/1 - 10 1 requires 3.14 g Drip rate for stock: 0.7 ml/min - Unit flow § 1 1/min - Stock l a s t s 10 days for 1 unit 169 Appendix 4. ICP-MS analysis of Oregon Moist P e l l e t s fed to coho f r y during l a b e l l i n g period i n Experiment 5. To t a l Element Concentrations (ug/g) Element Mass Cone. Element Mass Cone. Lithium 7 0.103 Beryllium 9 <0.007 Boron 11 <1.43 Sodium 23 773 * Magnesium 24 9586 * Aluminium 27 14.2 S i l i c o n 28 178 Phosphorus 31 8740 Potassium 39 3460 Calcium 44 15700 Scandium 45 0.167 Titanium 49 0.93 Vanadium 51 0.547 Chromium 52 0.269 Manganese 55 24.8 Iron 56 58.5 * Cobalt 59 0.056 Nickel 62 0. 083 Copper 63 4.15 Zinc 66 89.3 Gallium 71 <0.007 Germanium 72 <0.004 Arsenic 75 2.16 Selenium 78 1.87 Bromine 79 26.9 Rubidium 85 1.50 Strontium 86 21.7 Yttrium 89 0.008 Zirconium 91 <0.115 Niobium 93 0. 006 Molybdenum 100 0.099 Ruthenium 102 <0.002 Rhodium 103 <0.002 Palladium 105 <0.004 S i l v e r 107 <0.002 Cadmium 112 0.118 Indium 115 i n t . s t d . T in 120 0.133 Antimony 123 <0.006 Tellurium 126 0.012 Iodine 127 8.56 Caesium 133 0. 012 Barium 138 2.23 Lanthanum 139 0.010 Cerium 140 0.018 Pra s eodym ium 141 0.004 Neodymium 145 0.014 Samarium 147 0.003 Europium 151 <0.002 Gadolinium 157 <0.003 Terbium 159 <0.002 Dysprosium 161 <0.003 Holmium 165 <0.002 Erbium 166 <0.002 Thulium 169 <0.002 Ytterbium 172 <0.002 Lutetium 175 <0.003 Hafnium 178 <0.002 Tantalum 181 <0.002 Rhenium 185 <0.002 Tungsten 186 0.089 Osmium 190 <0.002 Iridium 193 <0.002 Platinum 194 <0.005 Gold 197 <0.003 Mercury 200 0.020 Thallium 205 0.003 Lead 208 0.237 Bismuth 209 <0.002 Thorium 232 <0.003 Uranium 238 0. 035 * High values f o r these elements could be a r e s u l t of digestion problems and matrix interferences i n ICP-MS analysis. V 170 Appendix 5 . Water chemistry data f o r the ambient C a p i l a n o R i v e r water. I. Parameters and Metals - Cantest L a b o r a t o r y A. Parameters pH (pH u n i t s ) 6 . 3 3 C o n d u c t i v i t y (us/cm) 1 9 . 0 Hardness CaC0 3 4 . 0 A l k a l i n i t y HC0 3 4 . 1 7 Ammonia N <0 .01 C h l o r i d e CI 1 . 3 0 D i s s o l v e d Oxygen 7 . 9 5 N i t r i t e N <0 .05 N i t r a t e N 0 . 1 5 R e s i d u a l - f i l t e r a b l e 1 2 . 0 R e s i d u a l N o n - f i l t e r <1 .0 S i l i c a S i 0 2 3 . 1 2 Sulphate s o 4 1 .08 T u r b i d i t y (NTU) 0 . 5 5 F l u o r i d e F < 0 . 0 5 B. Metals (mcr/1) Aluminum A l < 0 . 1 5 A r s e n i c As <0 .030 Barium Ba <0 .001 Calcium Ca 1 .23 Cadmium Cd <0.02 C o b a l t Co <0.02 Chromium Cr <0 .03 Copper Cu < 0 . 0 1 5 I r o n Fe 0 . 0 9 7 Mercury Hg < 0 . 0 0 0 0 5 Potassium K <1 .0 Magnesium Mg 0 . 1 7 Manganese Mn 0 . 0 0 5 Molybdenum Mo <0.04 Sodium Na 0 . 5 3 N i c k e l N i < 0 . 0 2 5 Phosphorus P < 0 . 1 5 Lead Pb <0 .08 Antimony Sb < 0 . 1 5 T i n Sn <0 .03 Strontium Sr 0 . 0 0 6 T i t a n i u m T i <0 .006 Vanadium V <0 .01 Z i n c Zn < 0 . 0 1 5 C o n c e n t r a t i o n s r e p o r t e d as mg /1 . 171 I I . ICP-MS Analysis for Total Elemental Content - ERI Element Mass Cone. Element Mass Cone. Lithium 7 <0.03 Beryllium 9 <0.03 Boron 11 <16.3 Sodium 23 140 Magnesium 24 38.6 Aluminium 27 9.83 Calcium 44 181 Scandium 45 <0.17 Titanium 47 <0.32 Vadium 51 0.13 Chromium 52 <0.25 Manganese 55 0.37 Iron 56 9.83 Cobalt 59 0.03 Nickel 60 0.59 Copper 63 <0.05 Zinc 66 <0.04 Gallium 69 <0.14 Germanium 74 <0.05 Arsenic 75 3.04 Selenium 78 0.27 Bromine 79 <0.63 Rubidium 85 0.21 Strontium 88 3.20 Yttrium 89 <0.03 Zirconium 90 <0.05 Niobium 93 <0.02 Molybdenum 100 0.08 Ruthenium 101 <0.03 Rhodium 103 <0.02 S i l v e r 107 <0.02 Palladium 105 <0.03 Cadmium 111 1.64 Indium 115 i n t . s t d . Tin 120 0.04 Antimony 121 <0.03 Iodine 127 0.29 Tellurium 128 <0.08 Caesium 133 <0.02 Barium 138 2 .12 Lanthanum 138 0.05 Cerium 140 0.04 Praseodymium 141 <0.02 Neodymium 146 0. 03 Samarium 149 <0. 03 Europium 153 <0.02 Gadolinium 157 <0. 04 Terbium 159 <0. 02 Dysprosium 163 0.07 Holmium 165 <0. 02 Erbium 166 <0.02 Thulium 169 <0.02 Ytterbium 172 <0. 02 Lutetium 175 <0.02 Hafnium 178 <0.02 Tantalum 181 <0.02 Tungsten 184 <0.03 Rhenium 185 <0.02 Osmium 190 <0.02 Iridium 193 <0.02 Platinum 194 <0.03 Gold 197 <0.02 Mercury 202 <0.17 Thallium 205 <0.02 Lead 208 0.55 Bismuth 209 <0.02 Thorium 232 0.02 Uranium 238 0.03 Concentrations reported as /xg/l« 172 A p p e n d i x 6. ANOVA t a b l e s f o r m o r t a l i t i e s o b s e r v e d i n Experiment 5 i n lanthanum t r e a t m e n t t a n k s . 1. ANOVA t a b l e o f m o r t a l i t i e s o b s e r v e d d u r i n g 6-week t r e a t m e n t p e r i o d o n l y . R 2 = 0.779 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 16.667 2 8.333 9.091 0.015 DUR 2.083 1 2.083 2.273 0.182 TRTxDUR 0.667 2 0.333 0.364 0.709 ERROR 5.500 6 0.917 2. ANOVA t a b l e o f m o r t a l i t i e s o b s e r v e d d u r i n g grow-out p e r i o d o n l y . R 2 = 0.526 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 19.500 2 9.750 2.167 0 .196 DUR 0.333 1 0. 333 0.074 0 .795 TRTxDUR 10.167 2 5. 083 1.130 0 .383 ERROR 27.000 6 4.500 173 Appendix 7. ANOVA tables for growth (fry weight) observed i n Experiment 5 i n lanthanum and samarium treatment tanks. 1. ANOVA table of f r y weights observed during 10%-month grow-out period i n lanthanum treatment tanks. R2 = 0.536 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 7.597 2 3.799 0.978 0.377 DUR 5.649 1 5.649 1.455 0.229 TIME 1205.043 5 241.009 62.072 0.000 TRTxDUR 46.308 2 23.154 5.963 0.003 TRTxTIME 50.950 10 5.095 1.312 0.222 DURxTIME 80.843 5 16.169 4.164 0.001 TRTxDURxTIME 54.670 10 5.467 1.408 0.175 ERROR 1257.999 324 3.883 2. ANOVA table of f r y weights observed during 10%-month grow-out period i n samarium treatment tanks. R2 = 0.592 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 4.286 2 2.143 0.739 0.480 DUR 3.210 1 3.210 1.106 0.295 TIME 520.552 5 104.110 35.881 0.000 TRTxDUR 18.938 2 9.469 3.263 0. 041 TRTxTIME 14.314 10 1.431 0.493 0.892 DURxTIME 26.515 5 5.303 1.828 0.111 TRTxDURxTIME 18.143 10 1.814 0. 625 0.790 ERROR 417.824 144 2.902 174 Appendix 8. ANOVA tables for lanthanum and samarium concentration i n the vertebral columns of f r y la b e l l e d with lanthanum and samarium i n Experiment 5. 1. ANOVA table of lanthanum concentrations i n the vertebral columns during 10%-month grow-out period i n lanthanum treatment tanks. R2 = 0.866 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 5.744 1 5.744 631.489 0.000 DUR 2.845 1 2.845 312.752 0.000 TIME 1.982 5 0.396 43.589 0.000 TRTxDUR 0.885 1 0.885 97.239 0.000 TRTxTIME 1.013 5 0.203 22.281 0.000 DURxTIME 0.129 5 0. 026 2.828 0.017 TRTxDURxTIME 0.135 5 0.027 2.979 0.013 ERROR 1.965 216 0.009 2. ANOVA table of samarium concentrations i n the vertebral columns during 10%-month grow-out period i n samarium treatment tanks. R2 = 0.809 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 1.055 1 1.055 212.979 0.000 DUR 0.391 1 0.391 78.961 0.000 TIME 0.196 5 0. 039 7.898 0.000 TRTxDUR 0.224 1 0. 224 45.328 0.000 TRTxTIME 0.087 5 0. 017 3.508 0.006 DURxTIME 0.046 5 0.009 1.847 0.111 TRTxDURxTIME 0.017 5 0.003 0.702 0.623 ERROR 0.475 96 0.005 175 Appendix 9. ANOVA tables for lanthanum and samarium amounts i n the vertebral columns of f r y l a b e l l e d with lanthanum and samarium i n Experiment 5. 1. ANOVA table of lanthanum amounts i n the vertebral columns during 10%-month grow-out period i n lanthanum treatment tanks. R2 = 0.801 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 3483.696 1 3483.696 472.736 0.000 DUR 1928.231 1 1928.231 261.660 0.000 TIME 238.865 5 47.773 6.483 0.000 TRTxDUR 502.578 1 502.578 68.200 0.000 TRTxTIME 52.635 5 10.527 1.429 0.215 DURxTIME 145.757 5 29.151 3.956 0.002 TRTxDURxTIME 73.338 5 14.668 1.990 0.081 ERROR 1591.752 216 7.369 2. ANOVA table of samarium amounts i n the vertebral columns during 10%-month grow-out period i n samarium treatment tanks. R2 = 0.860 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 878.038 1 878.038 303. 385 0.000 DUR 426.851 1 426.851 147. 488 0.000 TIME 49.081 5 9.816 3. 392 0.007 TRTxDUR 274.286 1 274.286 94. 773 0.000 TRTxTIME 24.115 5 4.823 1. 666 0.150 DURxTIME 32.593 5 6.519 2. 252 0.055 TRTxDURxTIME 22.497 5 4.499 1. 555 0.180 ERROR 277.837 96 2.894 176 Appendix 10. ANOVA tables for lanthanum and samarium concentrations i n the o t o l i t h s of f r y l a b e l l e d with lanthanum and samarium i n Experiment 5. 1. ANOVA table of lanthanum concentrations i n the o t o l i t h s 2 weeks and 10% months post-treatment i n lanthanum treatment tanks. R2 = 0.794 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 0.102 1 0.102 20.486 0.000 DUR 0.049 1 0.049 9.842 0.005 TIME 0.092 1 0.092 18.566 0. 000 TRTxDUR 0. 023 1 0.023 4.601 0.043 TRTxTIME 0.071 1 0. 071 14.358 0. 001 DURxTIME 0.025 1 0.025 5.058 0.035 TRTxDURxTIME 0.015 1 0.015 2.990 0.098 ERROR 0.110 22 0.005 2. ANOVA table of samarium concentrations i n the o t o l i t h s weeks and 10% months post-treatment i n samarium treatment tanks. R2 = 0.995 SOURCE SS DF MEAN-SQUARE F-•RATIO P TRT 0.066 1 0.066 245. 649 0. 000 DUR 0.056 1 0.056 208. 904 0.000 TIME 0.064 1 0.064 237. 122 0. 000 TRTxDUR 0. 059 1 0.059 218. 712 0.000 TRTxTIME 0. 067 1 0.067 247. 565 0. 000 DURxTIME 0.057 1 0.057 210. 670 0.000 TRTxDURxTIME 0.054 1 0.054 201. 046 0.000 ERROR 0.002 8 0.000 177 Appendix 11. ANOVA tables for lanthanum and samarium concentrations i n the scales, 10% months post-treatment, of f r y la b e l l e d with lanthanum and samarium i n Experiment 5. R2 = 0.997 SOURCE TRT ERROR SS DF MEAN-SQUARE F-RATIO P 2.278 2 1.139 546.792 0.000 0.006 3 0.002 178 Appendix 12. Information used and r e s u l t s of computer d r i p c a l c program used for c a l c u l a t i o n of lanthanide treatments used i n Experiment 6. Lanthanum at 50 uq/l A. Data Required Element Name: Lanthanum Compound Name: Lanthanum acetate Atomic Weight of Element: 138.91 (La) Gram Formula Weight of Compound: 343.07 (La acetate) Increase i n Element Required: 50 = 0.05 mg/1 S o l u b i l i t y of Compound: 168.8 g/1 Stock Solution Container Size: 10 1 Flow Rate: 1 1/min Number of Units to Treat: 1 Number of Days Stock Solution to Last: 10 days B. Calculation Results To Raise La by 0.05 mg/1: La acetate stock solution: 0.18 g/1 - 10 1 requires 1.77 g Drip rate for stock: 0.7 ml/min - Unit flow @ 1 1/min - Stock l a s t s 10 days for 1 unit Lanthanum at 100 uq/1 A. Data Required Element Name: Lanthanum Compound Name: Lanthanum acetate Atomic Weight of Element: 138.91 (La) Gram Formula Weight of Compound: 343.07 (La acetate) Increase i n Element Required: 100 /xg/1 = 0.1 mg/1 S o l u b i l i t y of Compound: 168.8 g/1 Stock Solution Container Size: 10 1 Flow Rate: 1 1/min Number of Units to Treat: 1 Number of Days Stock Solution to Last: 10 days B. Calculation Results To Raise La by 0.1 mg/1: La acetate stock solution: 0.36 g/1 - 10 1 requires 3.56 g Drip rate f o r stock: 0.7 ml/min - Unit flow @ 1 1/min - Stock l a s t s 10 days f o r 1 unit 179 Cerium at 50 ttq/1 A. Data Required Element Name: Cerium Compound Name: Cerium acetate Atomic Weight of Element: 140.12 (Ce) Gram Formula Weight of Compound: 317.2 6 Increase i n Element Required: 50 /xg/1 = S o l u b i l i t y of Compound: 2 00 g/1 Stock Solution Container Size: 10 1 Flow Rate: 1 1/min Number of Units to Treat: 1 Number of Days Stock Solution to Last: (Ce acetate) 0.05 mg/1 10 days B. Calculation Results To Raise Ce by 0.05 mg/1: Ce acetate stock solution: - 10 1 requires 1.62 g 0.16 g/1 Drip rate for stock: 0.7 ml/min - Unit flow @ 1 1/min - Stock l a s t s 10 days for 1 unit Cerium at 100 UQ/1 A. Data Required Element Name: Cerium Compound Name: Cerium acetate Atomic Weight of Element: 140.12 (Ce) Gram Formula Weight of Compound: 317.26 (Ce acetate) Increase i n Element Required: 100 fig/1 = 0.1 mg/1 S o l u b i l i t y of Compound: 200 g/1 Stock Solution Container Size: 10 1 Flow Rate: 1 1/min Number of Units to Treat: 1 Number of Days Stock Solution to Last: 10 days B. Calcula t i o n Results To Raise Ce by 0.1 mg/1: Ce acetate stock solution: - 10 1 requires 3.24 g 0.32 g/1 Drip rate for stock: 0.7 ml/min - Unit flow @ 1 1/min - Stock l a s t s 10 days for 1 unit 180 Appendix 13. ANOVA t a b l e f o r m o r t a l i t i e s observed i n Experiment 6 d u r i n g 4-week l a b e l l i n g p e r i o d . R2 = 0.722 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 435.000 3 145..000 290.000 0.003 ERROR 1.000 2 0.500 181 Appendix 14. ANOVA tables for lanthanum and cerium concentrations and amounts i n the vertebral columns of f r y and smolts l a b e l l e d with lanthanum and cerium i n Experiment 6. 1. ANOVA table of lanthanum vertebral columns of f r y SOURCE SS DF TRT 0. 258 2 STAGE 2. 190 1 LN 0. 005 1 STAGExLN 0. 010 1 TRTxSTAGExLN 0. 035 2 ERROR 1. 158 72 and cerium concentrations i n the and smolts. R2 = 0.683 MEAN-SQUARE F-RATIO P 0.129 8.009 ^ 0.001 2.190 136.131 0.000 0.005 0.301 0.585 0.010 0.610 0.437 0.018 1.100 0.338 0.016 2. ANOVA table of lanthanum and cerium amounts i n the vertebral columns of f r y and smolts. R2 = 0.734 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 2.350 2 1.175 0.278 0.758 STAGE 514.735 1 514.735 121.993 0.000 LN 0.414 1 0. 414 0. 098 0.755 STAGExLN 0.196 1 0.196 0. 047 0.830 TRTxSTAGExLN 0.321 2 0.161 0.038 0.963 ERROR 303.796 72 4.219 Note: LN = lanthanide 182 Appendix 15. ANOVA tables for lanthanum and cerium concentration i n the o t o l i t h s of f r y and smolts l a b e l l e d with lanthanum and cerium i n Experiment 6. R2 = 0.53 3 SOURCE SS DF MEAN-SQUARE F-RATIO P TRT 0.021 2 0.010 2.768 0.083 STAGE 0.081 1 0.081 21.373 0.000 LN 0.014 1 0.014 3.730 0.065 STAGExLN 0.000 1 0.000 0.093 0.763 TRTxSTAGExLN 0.005 2 0.002 0.598 0.558 ERROR 0.091 24 0.004 

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