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Bromophenols in Rhodomela larix : a study in chemical ecology Phillips, David William 1980

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BROMOPHENOLS IN RHODOMELA LARIX: A STUDY IN CHEMICAL ECOLOGY by DAVID WILLIAM PHILLIPS B.S., Western Carolina University, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1980 0DAVID WILLIAM PHILLIPS, 1980 In presenting th is thesis in par t ia l fu l f i lment o f f the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th i s thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It i s understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my writ ten permission. Department nf BOTANY  The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date 30 JUNE 1980 ABSTRACT In t h i s study aspects of the ecology, b i o l o g y and chemistry of Rhodomela l a r i x (Turner) C. Agardh are i n v e s t i g a t e d and an e f f o r t made to r e l a t e the f i n d i n g s to a d e t e r m i n a t i o n o f the reasons f o r the pro-d u c t i o n of bromophenols by red algae. Environmental f a c t o r s o c c u r r i n g , i n l o c a l t i d e -p o o l s which might a f f e c t the p r o d u c t i o n or exudation o f bromophenols by t h i s a l g a were examined i n summer over a three month p e r i o d . Measurements of a l g a l biomass and l e v e l s of t o t a l phenols i n the pools show that R. l a r i x produces and exudes copious amounts of these compounds. The e c o l o g i c a l i m p l i c a t i o n s o f t h i s r a t e of r e l e a s e are i n t r o d u c e d . Chemotaxonomic comparison of the m o r p h o l o g i c a l l y d i f f e r e n t t i d e p o o l and low i n t e r t i d a l p o p u l a t i o n s of R. l a r i x i n d i c a t e s that the two are s u f f i c i e n t l y s i m i l a r to be c o n s i d e r e d the same s p e c i e s . S e v e r a l bromophenols p r e v i o u s l y thought to be a r t i f a c t s are now c o n s i d e r e d to be n a t u r a l c o n s t i t u e n t s of algae. A high-performance l i q u i d chromatographic method was developed f o r the s e p a r a t i o n , i d e n t i f i c a t i o n and i i i q u a n t i t a t i v e d e t e r m i n a t i o n o f r e d a l g a l bromophenols. The method i s e v a l u a t e d on the b a s i s o f s e v e r a l chromato-g r a p h i c p a r a m e t e r s , and s t e p s i n i t s development and improvement are d i s c u s s e d . P r e v i o u s s t u d i e s on r e d a l g a l p h e n o l s have gener-a l l y o v e r l o o k e d q u a n t i t a t i v e c o n s i d e r a t i o n s . T h i s s t u d y p r e s e n t s d a t a f o r v a r i o u s a s p e c t s o f the q u a n t i t a t i v e a n a l y s i s o f the major b r o m o p h e n o l i c s u b s t a n c e ( l a n o s o l , 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l a l c o h o l ) i n R. l a r i x . Temporal d e t e r m i n a t i o n o f l a n o s o l c o n c e n t r a t i o n s demon-s t r a t e s t h a t the h i g h e s t l e v e l s o c c u r i n w i n t e r months. P o p u l a t i o n a l d i f f e r e n c e s were o b s e r v e d and the h i g h e s t l e v e l s w i t h i n a s i n g l e p l a n t were found t o be i n the youngest r e g i o n s . C o n t r o l o f the e x u d a t i o n o f bromophenols by R. l a r i x was s t u d i e d u s i n g a r t i f i c i a l c o n d i t i o n s w h i c h f e l l w i t h i n t h e ranges o f e n v i r o n m e n t a l c o n d i t i o n s p r e -v i o u s l y m o n i t o r e d . E x u d a t i o n o f l a n o s o l i s enhanced i n l i g h t , a t h i g h e r t e m p e r a t u r e s and a t lower s a l i n i t i e s . pH has l i t t l e e f f e c t on e x u d a t i o n . The r a t e s o f exu-d a t i o n found i n t h e s e e x p e r i m e n t s c o r r e s p o n d t o t h o s e measured,in the t i d e p o o l . E x a m i n a t i o n o f the b r o a d spectrum a n t i b i o t i c a c t i v -i t y o f l a n o s o l p r o v e s t h i s compound t o be e f f e c t i v e even a t low l e v e l s a g a i n s t a wide v a r i e t y o f organisms. The i v e f f e c t i s enhanced a t low pH, and comparison w i t h R. l a r i x exudates s u g g e s t s l a n o s o l t o be the major a c t i v e com-ponent . A d i s c u s s i o n o f t h e e c o l o g i c a l s i g n i f i c a n c e o f bromophenol p r o d u c t i o n i s p r e s e n t e d and an argument made f o r t h e p r o d u c t i o n o f t h e s e compounds as a n t i b i o t i c s u b s t a n c e s . Comparison w i t h p r e v i o u s e x p e r i m e n t s d e s c r i b -i n g the t e m p o r a l a n t i b i o t i c a c t i v i t y o f a n o t h e r Rhodomela s p e c i e s s u p p o r t s t h i s c o n c l u s i o n . V TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x PROEM 1 CHAPTER I. Aspects of the ecology of Rhodomela l a r i x 3 CHAPTER I I . Bromophenols of. Rhodomela l a r i x : Chemotaxonomy of morphologica1 forms 23 CHAPTER II I . HPLC Separation of red al g a l bromo-phenols . . . 31 CHAPTER IV. Temporal, interpopulational and i n t r a t h a l l i a l measurement of lanosol levels i n Rhodomela  l a r i x 53 CHAPTER V. Exudation of bromophenols by Rhodomela l a r i x 67 CHAPTER VI. Broad spectrum a n t i b i o t i c a c t i v i t y of bromophenols from Rhodomela  l a r i x 83 PERSPECTIVES 101 -FOOTNOTES . 106 REFERENCES 107 v i LIST OF TABLES I. Dominance, frequency and importance values for the algae i n the two tidepools. . . . 17 2 II. X Species association of algae i n the two pools 21 III . Retention times, capacity factors and r e l a -t i v e retentions of standards of bromo-phenols separated by reverse phase HPLC . 41 IV. Resolution as a function of column e f f i -ciency, s e l e c t i v i t y and capacity using di f f e r e n t solvent systems i n the sepa-r a t i o n of 3,5-dibromo-4-hydroxybenzyl alcohol and the corresponding acid . . . 45 V. A summary of data available on quanti-tat i v e aspects of bromophenols i n red algae 55 VI. Quantitative comparison of lanosol con-centrations i n three d i s t i n c t popula-tions of Rhodomela l a r i x 63 VII. Exudation measured as t o t a l phenols over a twenty-four hour period by tidepool and subtidal forms of Rhodomela l a r i x . . 73 VIII. Quantitative determination of exuded lanosol and other observations under varying experimental conditions 76 IX. A n t i b i o t i c a c t i v i t y of lanosol against three species of marine fungi 97 v i i LIST OF FIGURES 1. Map of Bath Island, B r i t i s h Columbia, and environs, and d e t a i l showing location of the tidepools studied. . . . . . . . 7 2. Diagram of pools 1 and 2 showing ranges i n s t r a t i f i c a t i o n of s a l i n i t y and temperature and l i g h t attenuation with depth 11 3. Environmental measurements made i n pool 1 and pool 2 during three consecutive months 13 4. Total phenols i n pool 1 and pool 2 over a twenty-four hour period given as parts per m i l l i o n of phloroglucinol . . 19 5. Temperature programmed GLC analysis of Rhodomela l a r i x bromophenols 28 6. Reverse phase HPLC separation of fourteen bromophenol standards 40 7. HPLC Analysis of the petroleum ether and chloroform fractions from the large scale extraction of Rhodomela l a r i x . . 46 8. HPLC Analysis of the ethyl acetate and methanol fractions from the large scale extraction of Rhodomela l a r i x . . 48 9. Reverse phase separation of bromophenols extracted from P r i o n i t i s l y a l l i i and Ceramium washingtoniense 51 10. A t y p i c a l HPLC separation of Rhodomela l a r i x bromophenols used for the quanti-tative determination of lanosol . . . . 60 v i i i 1 1 . S e a s o n a l v a r i a t i o n o f l a n o s o l c o n t e n t i n R h o d o m e l a l a r i x a n d c h a n g e i n t h e d r y t o w e t w e i g h t r a t i o o v e r t h e p e r i o d o f one y e a r 62 1 2 . L a n o s o l c o n t e n t i n d i f f e r e n t p a r t s o f t h e R h o d o m e l a l a r i x t h a l l u s 65 1 3 . HPLC o f t h e t w e n t y - f o u r h o u r e x u d a t e o f t h e s u b t i d a l f o r m o f R h o d o m e l a l a r i x . . . . 75 1 4 . E f f e c t o f t e m p e r a t u r e a n d s a l i n i t y o n t h e p r o d u c t i o n o f c o l o r e d m a t t e r b y R h o d o m e l a  l a r i x d u r i n g a n e i g h t h o u r e x u d a t i o n e x -p e r i m e n t 78 1 5 . E f f e c t o f pH a n d l i g h t o n t h e p r o d u c t i o n o f c o l o r e d m a t t e r b y R h o d o m e l a l a r i x d u r i n g an e i g h t h o u r e x u d a t i o n e x p e r i m e n t . . . . 79 1 6 . E f f e c t o f l i g h t , s a l i n i t y , t e m p e r a t u r e a n d pH o n e x u d a t i o n o f l a n o s o l b y R h o d o m e l a l a r i x 8 1 1 7 . A n t i b i o t i c a c t i v i t y o f l a n o s o l a g a i n s t C a n d i d a a l b i c a n s , E s c h e r i c h i a c o l i , Sac c h a r o m y c e s c e r e v i s i a e a n d ~ S t a p h y 1 o c o c c u s a u r e u s 9 1 1 8 . The e f f e c t o f pH o n t h e a n t i b i o t i c a c t i v i t y o f l a n o s o l a n d l a n o s a l t a g a i n s t E s c h e r i c h i a c o l i 93 1 9 . E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f l a n o s o l i n p u r e f o r m , i n a l g a l e x t r a c t s a n d i n e x u d a t e s o n t h e g r o w t h r a t e o f E s c h e r i c h i a c o l i 96 2 0 . A t y p i c a l s n a i l r e p u l s i o n e x p e r i m e n t s h o w i n g t h e e f f e c t s o f l a n o s o l a n d l a n o s a l t o n t h e b e h a v i o r o f L i t t o r i n a s c u t u l a t a 99 2 1 . D e g r e e o f r e p u l s i o n o f L i t t o r i n a s c u t u l a t a b y v a r y i n g c o n c e n t r a t i o n s o f l a n o s o l a n d l a n o s a l t 100 i x ACKNOWLEDGEMENTS I wish to thank Drs. Raymond Andersen, Bruce Bohm, Robert De Wreede, Ronald Foreman, G i l b e r t Hughes, Robert Scagel, Tony Swain and N e i l Towers f o r t h e i r c a r e f u l r e a d i n g and c r i t i c i s m o f t h i s manuscript. S p e c i a l thanks to Dr. Robert De Wreede f o r h i s generous a s s i s t a n c e and enthusiasm d u r i n g the many f i e l d t r i p s to Bath I s l a n d , and to Dr. Ronald Foreman f o r use of the f a c i l i t i e s there. Dr. James C r a i g i e k i n d l y pro-v i d e d the bromophenol standards and Dr. Guenther E i g e n d o r f the use of the GLC-MS. Dr. Raymond Andersen deserves thanks f o r the many h e l p f u l d i s c u s s i o n s concerning spectroscopy and syntheses. Ms. J u l i e C e l e s t i n o O l i v e r i a helped w i t h the i d e n t i f i c a t i o n of the algae and Ms. Z. Abramowski and Ms. E. Graham w i t h the i n i t i a l m i c r o b i o l o g i c a l experiments. Many thanks to Dr. N e i l Towers f o r h i s continued support and encouragement throughout the past f o u r years. My w i f e , Helen, t o l e r a t e d , supported, encouraged, e d i t e d and typed the manuscript, and d i d whatever e l s e i t took to get us through t h i s experience. Needless to say, "thanks" i s i n s u f f i c i e n t g r a t i t u d e f o r her many con-t r i b u t i o n s . 1 PROEM The e c o l o g i s t ' s i n c r e a s i n g a w a r e n e s s o f t h e i n t r i c a t e a n d s u b t l e c h e m i c a l b a l a n c e i n n a t u r e a n d t h e c h e m i s t ' s a c c e s s t o m o d e r n i n s t r u m e n t a t i o n e n a b l i n g h i m t o d e t e c t s m a l l q u a n t i t i e s o f n a t u r a l p r o d u c t s h a v e l e d t o t h e e v o l u -t i o n o f a new a n d e x c i t i n g b r a n c h o f s c i e n c e c a l l e d c h e m i c a l e c o l o g y . C h e m i c a l e c o l o g y a t t e m p t s t o u n r a v e l t h e c o m p l e x i n t e r r e l a t i o n s h i p s o f o r g a n i s m s a t b o t h c h e m i c a l a n d b i o l o g -i c a l l e v e l s . One c o r n e r o f t h i s b r o a d f i e l d d e a l s w i t h t h e i n t e r a c t i o n s o f p l a n t s a n d o t h e r l i v i n g c r e a t u r e s , a n d h a s b e e n c a l l e d p h y t o c h e m i c a l e c o l o g y ( H a r b o r n e 1 9 7 2 ) . W h e t h e r t h e i n t e r a c t i o n i s p l a n t t o p l a n t , p l a n t t o a n i m a l o r p l a n t t o m i c r o b e , i t c a n be d e s c r i b e d as b e i n g a n t a g o n i s -t i c , b e n e f i c i a l o r m u t u a l l y i n d i f f e r e n t . The m a j o r i t y o f p h y t o c h e m i c a l e c o l o g i s t s f i n d a n t a g -o n i s t i c i n t e r a c t i o n s t h e m o s t i n t e r e s t i n g . The p r o d u c t i o n o f d e f e n s i v e compounds i n r e s p o n s e t o p r e d a t i o n ( h e r b i v o r y ) a n d p a r a s i t i s m i s common t h r o u g h o u t t h e p l a n t k i n g d o m . V u i l l e m i n ( 1 8 8 9 ) f i r s t u s e d t h e t e r m " a n t i b i o s i s " t o d e -s c r i b e t h e c o u n t e r a c t i o n o f o n e o r g a n i s m t o t h e l i f e o f a n o t h e r , a c o n c e p t w h i c h h a s g r o w n t o i n c l u d e t h e s t u d y o f a n t i b i o t i c s p r o d u c e d b y m a r i n e o r g a n i s m s , n o t t o m e n t i o n t h o s e t h o u s a n d s o f t e r r e s t r i a l o r i g i n . 2 This d i s s e r t a t i o n takes a step i n furthering our understanding of the role of an unusual group of "secondary" metabolites which are widespread among red algae. Aspects of the ecology, chemistry and biology of a p a r t i c u l a r red alga are approached from the standpoint of attempting to re l a t e "form to function," form being the production of the compounds and function t h e i r raison d'etre. I sincerely hope that even i f the reader remains unconvinced by the f i n a l v erdict, I w i l l have at least stimulated imaginative speculation, i f not the desire to i n t e n s i f y research into the chemical ecology of the oceans. CHAPTER I ASPECTS OF THE ECOLOGY OF RHODOMELA LARIX 3 4 INTRODUCTION T i d e p o o l s a r e s c a t t e r e d t h r o u g h o u t t h e r o c k y i n t e r -t i d a l z o n e a l o n g much o f t h e c o a s t o f B r i t i s h C o l u m b i a , C a n a d a . W i t h i n t h e s e p o o l s a v a r i e t y o f o r g a n i s m s c a n be f o u n d , i n c l u d i n g many s p e c i e s o f r e d a l g a e ( R h o d o p h y t a ) . Red a l g a e g e n e r a l l y o c c u r i n t h e l o w i n t e r t i d a l t o s u b -t i d a l r e g i o n s o f t h e s e a s h o r e ( F r i t s c h 1 9 4 5 , S c a g e l 1 9 6 7 ) , a n d p r e f e r l o w e r l i g h t i n t e n s i t i e s ( B i e b l 1 9 6 2 , J o h n s o n e t a l . 1 9 7 4 ) a n d / o r l o n g e r p e r i o d s o f i m m e r s i o n ( D o t y 1 9 4 6 ) t h a n do many o f t h e b r o w n a n d g r e e n m a c r o p h y t e s . The o c c u r r e n c e o f r e d a l g a e i n p o o l s h i g h i n t h e l i t t o r a l z o n e s h o u l d t h e r e f o r e p o s e many d i f f e r e n t a n d u n u s u a l p r o b l e m s f o r t h e s e s p e c i e s . The e n v i r o n m e n t a l c o n d i t i o n s i n t i d e -p o o l s a r e q u i t e u n s t a b l e a n d o f t e n e x t r e m e , i n d i c a t i n g t h a t a v a r i e t y o f p h y s i o l o g i c a l a d a p t a t i o n s m u s t h a v e o c -c u r r e d f o r s u c h a l g a e t o s u r v i v e i n t h e t i d e p o o l h a b i t a t . A l t h o u g h some w o r k h a s b e e n done on t h e m o n i t o r i n g o f p h y s i c a l a n d e n v i r o n m e n t a l c o n d i t i o n s i n p o o l s i n o t h e r p a r t s o f t h e w o r l d ( K l u g h 1 9 2 4 , J o h n s o n a n d S k u t c h 1 9 2 8 , P y e f i n c h 1 9 4 3 , G a n n i n g 1 9 7 1 , E d e l s t e i n a n d M c L a c h l a n 1 9 7 5 ) , l i t t l e o r n o s u c h w o r k h a s b e e n done o n t h e N o r t h P a c i f i c c o a s t ( s e e H u m p h r e y a n d Macy 1 9 3 0 ) . The a b o v e s t u d i e s 5 c o n s i d e r e d f a c t o r s s u c h as d i s s o l v e d o x y g e n , l i g h t , t e m p e r -a t u r e , s a l i n i t y a n d pH a n d how t h e s e m i g h t a f f e c t t h e d i s -t r i b u t i o n o f a l g a e i n c o a s t a l t i d e p o o l s . O n l y r e c e n t l y h a v e f a c t o r s s u c h as t h e r e l e a s e o f d i s s o l v e d o r g a n i c m a t t e r ( K h a i l o v a n d B u r l a k o v a 1 9 6 9 ) a n d o t h e r c h e m i c a l s u b s t a n c e s ( R a g a n a n d J e n s e n 1 9 7 9 ) b e e n c o n s i d e r e d i n r e l a t i o n t o t h e p r e s e n c e o r a b s e n c e o f a n a l g a l s p e c i e s i n a p a r t i c u l a r h a b i t a t . B i o l o g i c a l f a c t o r s s u c h as i n t e r - o r i n t r a - s p e c i f i c c o m p e t i t i o n b e t w e e n p l a n t s o r p l a n t s a n d a n i m a l s h a v e b e e n f r e q u e n t l y ' : o v e r l o o k e d . T h e s e t o o c o u l d p l a y a n i m p o r t a n t r o l e i n . d e t e r m i n i n g t h e d i s t r i b u t i o n o f a l g a e i n t i d e p o o l s . I n t h i s c o n t e x t , I w i s h t o r e p o r t o n some e x p e r i m e n t s p e r f o r m e d d u r i n g t h e summer o f 1978 o n t w o t i d e p o o l s i n B r i t i s h C o l u m b i a . P h y s i c a l a n d c h e m i c a l c h a n g e s i n t h e e n v i r o n m e n t a l c o n d i t i o n s i n t h e s e p o o l s w e r e m o n i t o r e d o v e r a t h r e e m o n t h p e r i o d . The a l g a l f l o r a i n t h e t w o p o o l s w e r e i d e n t i f i e d a n d t h e a b u n d a n c e ( p e r c e n t a g e c o v e r a n d s t a n d i n g c r o p ) o f e a c h s p e c i e s was d e t e r m i n e d . I n a d d i t i o n , an a t t e m p t was made t o m o n i t o r t h e e x u d a t i o n f r o m t h e a l g a e o f s e v e r a l c l a s s e s o f c h e m i c a l c o m p o u n d s . I t was h o p e d t h a t t h i s p h a s e o f t h e e x p e r i m e n t s w o u l d g i v e some i n d i c a -t i o n o f t h e p h y s i o l o g i c a l s t r e s s e s t o w h i c h t h e s e a l g a e w e r e s u b j e c t e d d u r i n g c h a n g i n g e n v i r o n m e n t a l c o n d i t i o n s . 6 MATERIALS AND METHODS The S t u d y S i t e The t w o t i d e p o o l s s t u d i e d w e r e l o c a t e d o n B a t h I s l a n d , B r i t i s h C o l u m b i a , Canada ( F i g u r e l a ) , i n t h e S t r a i t o f G e o r g i a . A r o c k l e d g e c o n t a i n i n g a s e r i e s o f t i d e p o o l s s u r r o u n d s t h e i s l a n d a t a h e i g h t o f a b o u t 4 . 5 m a b o v e z e r o t i d e l e v e l (MLLW, C a n a d i a n C h a r t D a t u m ) . The p o o l s c h o s e n w e r e a t 4 . 4 m ( p o o l 1 ) a n d 3 . 7 m ( p o o l 2 ) a b o v e MLLW ( F i g u r e l b ) a n d h a d maximum d e p t h s o f 53 a n d 4 1 cm r e s p e c -t i v e l y . L e v e l s o f t h e p o o l s a n d r e l e v a n t t i d a l c y c l e s w e r e d e t e r m i n e d u s i n g t h e C a n a d i a n T i d e a n d C u r r e n t T a b l e s ( 1 9 7 8 ) , w i t h P o i n t A t k i n s o n as a r e f e r e n c e p o r t a n d S i l v a Bay as a s e c o n d a r y p o r t . S t a n d a r d s u r v e y i n g i n s t r u m e n t s w e r e e m p l o y e d . M e a s u r e m e n t o f P h y s i c a l a n d C h e m i c a l F a c t o r s L i g h t i n t e n s i t y r e a d i n g s w e r e made u s i n g a L i - C o r M o d e l L I - 1 8 5 q u a n t u m m e t e r , w i t h t h e p r o b e ( M o d e l UWQ 2 1 9 2 ) i n a v e r t i c a l p o s i t i o n 1 . R e a d i n g s w e r e made a t e a c h p o o l ' s s u r f a c e e x c e p t i n t h e c a s e o f d e p t h v s . l i g h t i n t e n s i t y p r o f i l e s . T e m p e r a t u r e a n d s a l i n i t y ( g i v e n as p a r t s p e r t h o u s a n d s a l i n i t y , ° / o o ) p r o f i l e s , as m e a s u r e d w i t h a c a l i b r a t e d Y S I S a l i n o m e t e r , w e r e d e t e r m i n e d o v e r a 24 h o u r p e r i o d 7 F i g u r e 1. Map of Bath I s l a n d , B r i t i s h Columbia, and e n v i -rons ( l A ) y and d e t a i l (IB) showing l o c a t i o n of the t i d e -pools s t u d i e d . 8 i n e a c h o f t h r e e c o n s e c u t i v e m o n t h s ( J u l y , A u g u s t a n d S e p t e m b e r , 1 9 7 8 ) . pH was m e a s u r e d u s i n g a Beckman E x p a n d m a t e p o r t a b l e pH m e t e r e q u i p p e d w i t h a Beckman M o d e l 39502 e l e c t r o d e . S t a n d a r d b u f f e r s (pH 7 . 0 0 a n d 1 0 . 0 0 Amachem, S e a t t l e ) w e r e u s e d f o r c a l i b r a t i o n . D i s s o l v e d o x y g e n was m e a s u r e d b y t h e W i n k l e r m e t h o d ( S t r i c k l a n d a n d P a r s o n s 1 9 6 8 ) . T h r e e r e p l i c a t e t i t r a t i o n s w e r e done o n e a c h w a t e r s a m p l e , a n d t h e a v e r a g e v a l u e was c o n v e r t e d t o mg C ^ / l . Q u a d r a t S a m p l i n g M e t h o d s The s u r f a c e a r e a s o f t h e p o o l s a n d t h e p e r c e n t a g e c o v e r o f e a c h a l g a l s p e c i e s c o n t a i n e d w i t h i n t h e m w e r e o d e t e r m i n e d u s i n g q u a d r a t f r a m e s . A ( 1 . 5 m) f r a m e d i v i d e d 2 2 i n ( 1 0 cm) s q u a r e s was u s e d f o r p o o l 1 , a n d a ( 4 m) 2 f r a m e d i v i d e d i n t o (25 cm) s q u a r e s was u s e d f o r p o o l 2 . The n u m b e r o f s q u a r e s e n c o m p a s s e d b y e a c h p o o l was u s e d t o c a l c u l a t e s u r f a c e a r e a . The p e r c e n t a g e c o v e r f o r e a c h a l g a l s p e c i e s ( a l l o f w h i c h w e r e i d e n t i f i e d u s i n g W i d d o w s o n 1 9 7 4 o r A b b o t t a n d H o l l e n b u r g 1 9 7 6 ) was f i r s t e s t i m a t e d f o r e a c h s q u a r e a n d t h e n c a l c u l a t e d f o r t h e e n t i r e p o o l . F r e q u e n c y , d o m i n a n c e a n d , s u b s e q u e n t l y , i m p o r t a n c e v a l u e s w e r e d e t e r m i n e d f o r e a c h s p e c i e s a c c o r d i n g t o Cox ( 1 9 7 2 ) . 2 X s p e c i e s a s s o c i a t i o n v a l u e s w e r e a s s i g n e d t o a l l s p e c i e s r e l a t i v e t o R h o d o m e l a l a r i x ( T u r n e r ) C. A g a r d h ( K e r s h a w 9 1 9 6 4 ) . S t a n d i n g c r o p m e a s u r e m e n t s w e r e o b t a i n e d f o r R. l a r i x a n d P r i o n i t i s l y a l l i i H a r v e y b y s c r a p i n g a s e r i e s o o f ( 1 0 cm) s e c t i o n s c o n t a i n i n g t h e s e t w o s p e c i e s i n v a r -i o u s c o m b i n a t i o n s o f p e r c e n t a g e c o v e r . A l g a l d r y w e i g h t s w e r e d e t e r m i n e d a f t e r 5 d a y s a t 90°C ( o r u n t i l c o n s t a n t d r y w e i g h t was a c h i e v e d ) . C h e m i c a l M e a s u r e m e n t s F o r c h e m i c a l a n a l y s i s o f t i d e p o o l w a t e r , 100 m l w a t e r s a m p l e s w e r e t a k e n , f r o z e n o n D r y I c e a n d r e t u r n e d t o t h e l a b o r a t o r y . T o t a l p h e n o l s w e r e m e a s u r e d u s i n g t h e m e t h o d o f L a n g l o i s ( 1 9 7 5 ) o n a U n i c a m M o d e l SP 800 S c a n n i n g S p e c t r o p h o t o m e t e r s e t a t a c o n s t a n t w a v e l e n g t h ( 4 4 5 n m ) . S e a w a t e r was u s e d as a b l a n k , a n d v a r y i n g , b u t k n o w n , c o n -c e n t r a t i o n s o f p h l o r o g l u c i n o l i n s e a w a t e r w e r e u s e d f o r c a l i b r a t i o n a n d t h e e s t i m a t i o n o f t h e c o n c e n t r a t i o n o f t o t a l p h e n o l s i n t h e t i d e p o o l w a t e r . A t t e m p t s t o m e a s u r e c a r b o h y d r a t e ( D u b o i s e t a l . 1 9 5 6 ) a n d p r o t e i n ( L o w r y e_t a l . 1 9 5 1 ) p r o v e d u n s u c c e s s f u l due t o t h e l o w c o n c e n t r a t i o n s o f t h e s e c o n s t i t u e n t s i n t h e p o o l s ( e . g . t h e r e w e r e n o a p p a r e n t c h a n g e s i n c o n c e n t r a t i o n o v e r t i m e ) . RESULTS AND DISCUSSION The p r o b l e m o f m o n i t o r i n g p h y s i c a l a n d c h e m i c a l 10 c o n d i t i o n s i n s y s t e m s s u c h as t i d e p o o l s i s c o m p l i c a t e d b y t h e s t r a t i f i c a t i o n o f t h e s e f a c t o r s w h i c h may o c c u r w i t h i n an i n d i v i d u a l p o o l . F o r e x a m p l e , as shown i n F i g u r e 2 , p o o l 1 h a d s a l i n i t y r a n g e s o f 1 1 - 2 8 ° / o o v e r t i -c a l l y a n d 1 1 - 2 5 ° / o o h o r i z o n t a l l y d e p e n d i n g o n t h e a m o u n t o f o c e a n w a s h o r p r e c i p i t a t i o n e n t e r i n g t h e p o o l . P o o l 2 showed a s i m i l a r s a l i n i t y s t r a t i f i c a t i o n , b u t l e s s m i x i n g w i t h o c e a n w a t e r ; t h e r e f o r e , a l o w s a l i n i t y , l e s s d e n s e l a y e r o f w a t e r p r e d o m i n a t e d o v e r t h e p o o l s u r f a c e . The a l g a e i n t h e p o o l s a r e d i s t r i b u t e d o v e r m o s t o f t h e a v a i l -a b l e r o c k s u r f a c e , i n d i c a t i n g t h a t t h e i r t o l e r a n c e t o t h e s e v a r y i n g s a l i n i t i e s i s b r o a d . The d e g r e e o f t e m p e r a -t u r e s t r a t i f i c a t i o n i n t h e t w o p o o l s i s a l s o shown i n 2 F i g u r e 2 . G a n n i n g ( 1 9 7 1 ) h a s r e p o r t e d s i m i l a r r e s u l t s f o r t h e S w e d i s h t i d e p o o l s he s t u d i e d . He b e l i e v e s t h a t t i d e p o o l b i o t a a r e c o n f i n e d t o t h a t h a b i t a t b e c a u s e o f t h e i r l a c k o f d e s i c c a t i o n t o l e r a n c e a n d n o t b e c a u s e o f o s m o r e g u l a t o r y p r o b l e m s w h i c h t h e y m i g h t e n c o u n t e r . K l u g h ( 1 9 2 4 ) , o n t h e o t h e r h a n d , h a s s t r e s s e d t h e i m p o r t a n c e o f t e m p e r a t u r e a n d i t s f l u c t u a t i o n s as t h e m a i n l i m i t i n g p a r a m e t e r f o r t h e t i d e p o o l o r g a n i s m . The t e m p e r a t u r e s a n d s a l i n i t i e s w h i c h a p p e a r i n t h e g r a p h s i n F i g u r e 3 a r e a v e r a g e s o f r e a d i n g s t a k e n a t v a r y -i n g d e p t h s a n d l o c a t i o n s t h r o u g h o u t t h e p o o l s . Low s a l i n -i t i e s i n d i c a t e t h a t p r e c i p i t a t i o n ( a s l a n d s u r f a c e r u n o f f ) ( FRESH WATER 21. 2H TEMPERATURE <°C) STRAIT OF GEORGIA POOL 1 ( 2 3 u / o o ) -Q - "1700 10 - -1200 2 0 - - 1 1 0 0 3 0 " -1050 43 J nooo DEPTH (cm) LIGHT_2 _ x (uE m sec ) F i g u r e 2. Diagram o f pools 1 and 2 showing ranges i n s t r a t i f i c a t i o n o f s a l i n i t y (°/oo) and temperature and l i g h t a t t e n u a t i o n w i t h depth. 12 i s o c c u r r i n g w h i l e t h e p o o l i s e x p o s e d , a n d h i g h s a l i n -i t i e s i n d i c a t e t h e d e g r e e o f e v a p o r a t i o n o c c u r r i n g i n t h e p o o l , r e d u c i n g i t s v o l u m e a n d , i n t h e p r o c e s s , c o n -c e n t r a t i n g t h e s a l t s p r e s e n t . The m o r e s t a b l e r e a d i n g s , o f c o u r s e , o c c u r w h e n t h e t i d e i s i n . The r e s u l t s o f t h e pH d e t e r m i n a t i o n s as shown i n F i g u r e 3 c l e a r l y i n d i c a t e t h e r a t i o o f p h o t o s y n t h e s i s t o r e s p i r a t i o n o f a l g a e i n t h e p o o l . D u r i n g t h e d a y a c t i v e p h o t o s y n t h e s i s i s t a k i n g p l a c e ; o x y g e n i s e v o l v e d a n d s u b -s e q u e n t l y t h e pH o f t h e p o o l g o e s u p , n o t due t o t h e i n -c r e a s e d p r e s e n c e o f o x y g e n , b u t t o a r e d u c t i o n i n t h e l e v e l o f d i s s o l v e d CC^ i n t h e p o o l . C o n v e r s e l y , a t n i g h t w h e n t h e l e v e l o f r e s p i r a t i o n i s h i g h , o x y g e n i s c o n s u m e d , CG^ i s p r o d u c e d a n d a l o w e r i n g o f t h e p o o l ' s pH o c c u r s . K l u g h ( 1 9 2 4 ) a n d l a t e r J o h n s o n a n d S k u t c h ( 1 9 2 8 ) h a v e s u g g e s t e d t h a t t h e a c i d i t y o f t i d e p o o l s i s d e t e r m i n e d b y , r a t h e r t h a n d e t e r m i n e s , t h e b i o t a p r e s e n t . I f t h i s i s t r u e t h e n pH a l o n e c a n n o t be t h e l i m i t i n g f a c t o r f o r s p e c i e s o c c u r -r e n c e i n t i d e p o o l s . A l t h o u g h n o t i m m e d i a t e l y o b v i o u s , m e a s u r e m e n t s o f s u r f a c e l i g h t i n t e n s i t i e s a r e n o t a w h o l l y a c c u r a t e i n d i -c a t i o n o f t h e i r r a d i a n c e a c t u a l l y r e c e i v e d b y t h e a l g a e a t v a r y i n g d e p t h s i n t h e p o o l s . As much as 35% o f t h e l i g h t s t r i k i n g t h e p o o l ' s s u r f a c e i s r e f l e c t e d o f f o r i s f i l t e r e d o u t i n t h e t o p 20 cm o f p o o l w a t e r o n a c a l m 13 F i g u r e 3-. E n v i r o n m e n t a l m e a s u r e m e n t s made i n p o o l 1 a n d p o o l 2 d u r i n g t h r e e c o n s e c u t i v e m o n t h s . The s a m p l i n g d a t e s w e r e J u l y 1 8 - 1 9 ( ) , A u g u s t 1 4 - 1 5 ( •) a n d S e p t e m b e r 6 - 7 ( ) . T i d e l e v e l s a r e i n m e t e r s a b o v e MLLW. - 2 - 1 S u r f a c e l i g h t i n t e n s i t i e s ( I Q ) a r e i n uE m s e c , t e m p -e r a t u r e s i n ° C , s a l i n i t i e s ( ° / o o ) i n p a r t s p e r t h o u s a n d o f s a l i n i t y a n d d i s s o l v e d o x y g e n (O^ ) i n mg 1 * . The t i m e s c a l e i s d i v i d e d i n t o t w o h o u r i n t e r v a l s b e g i n n i n g a t 1200 h o u r s o n t h e d a y o f t h e s t u d y . The l e v e l o f t h e p o o l i n r e l a t i o n t o z e r o t i d e l e v e l i s i n d i c a t e d b y t h e f i n e h o r i z o n t a l l i n e t h r o u g h t h e g r a p h m a r k e d " T I D E S " . R e a d i n g s w e r e t a k e n a t a d e p t h o f 10 cm b e l o w t h e p o o l ' s s u r f a c e a n d i n t h e c e n t e r o f e a c h p o o l . ' - ' Q _ N 3 U l o o o "I L A r f i r pH / oo TEMPERATURE I o TIDES 15 day (Figure 2). This irradiance would decrease even further with turbulence (e.g. wind or waves) on the pool surface or with an increase i n dissolved organic matter i n the pools. Surface i n s o l a t i o n also controls the temperature of the water i n the pools to a great extent either by heating the water d i r e c t l y or by heating the surrounding rock. Dissolved oxygen i n the pools i s again correlated with photosynthesis and, therefore, pH. Figure 3 shows that as pH increases so does the l e v e l of dissolved oxygen. The oxygen leve l s of the pools were high even at the higher temperatures (warmer solutions hold less dissolved gas), i n d i c a t i v e of the high photosynthetic rate and, hence, productivity of the algae i n the pools. A l e v e l l i n g e f f e c t i n the oxygen content of the pools would occur when they are reimmersed and the turbulence of waves releases or dilutes the excess gases present. Pyefinch (1943) states that the d i v e r s i t y of the f l o r a i n a rock pool i s the key factor i n governing the range of pH and oxygen values. This appears to be the case i n the two pools studied here. The larger and more densely populated pool (see below) shows the greater rate of change i n the four variables so far discussed. The contribution of phytoplankton to the productivity of the pool has not been considered since i t should be minimal when compared to that of the attached f l o r a . 16 T a b u l a t i o n o f t h e q u a d r a t s a m p l i n g d a t a r e v e a l e d 2 t h a t p o o l 1 h a d a s u r f a c e a r e a o f 1 . 6 5 m ( e n c o m p a s s i n g o 165 ( 1 0 cm) q u a d r a t s ) a n d p o o l 2 a s u r f a c e a r e a o f 2 2 1 0 . 8 8 m ( e n c o m p a s s i n g 134 (25 cm) q u a d r a t s ) . V a l u e s o f p e r c e n t a g e c o v e r , d o m i n a n c e , f r e q u e n c y a n d i m p o r t a n c e f o r t h e a l g a e i n t h e t w o p o o l s a r e shown i n T a b l e I . R. l a r i x was d o m i n a n t i n b o t h p o o l s . G e r a m i u m w a s h i n g t o n i e n s e K y l i n a n d R a l f ' s i a c r u s t s w e r e m o r e i m p o r t a n t i n t h e h i g h e r p o o l t h a n i n t h e l o w e r p o o l , a n d P. l y a l l i i was m o r e i m -p o r t a n t i n t h e l o w e r p o o l t h a n i n t h e h i g h e r . S t a n d i n g c r o p m e a s u r e m e n t s ( a s g d r y w e i g h t , g d . w t . ) o f R h o d o m e l a a n d P r i o n i t i s g a v e a v e r a g e s o f 1 . 8 1 ( + 0 . 5 6 ) g 2 a n d 7 . 6 7 ( + 2 . 5 3 ) g r e s p e c t i v e l y p e r ( 1 0 cm) i n b o t h p o o l s . U s i n g t h e f o r m u l a e f o r s u r f a c e a r e a a n d v o l u m e o f a s p h e r e , one c a n e s t i m a t e t h e o v e r a l l s t a n d i n g c r o p o f R h o d o m e l a i n p o o l 1 ( w i t h i t s c l o s e l y s p h e r i c a l s h a p e ) a n d a t t e m p t t o c o r r e l a t e t h e p r o d u c t i o n o f t o t a l p h e n o l s ( a s s u m -i n g R h o d o m e l a t o be t h e s o l e s o u r c e ) w i t h p o o l v o l u m e . I f o u n d t h a t t h e r e was 0 . 3 9 g d . w t . / l o f t h i s a l g a i n p o o l 1 3 a n d t h a t t h e l e v e l o f t o t a l p h e n o l s r e a c h e d 0 . 5 m g / 1 d a y . T h e r e f o r e , e a c h g d . w t . o f R h o d o m e l a e x u d e s 1 . 4 1 mg o f t o t a l p h e n o l s p e r d a y . I f we assume h a l o p h e n o l s t o be a n t i b i o t i c , t h e n t h e y s h o u l d be e f f e c t i v e a t s u c h l e v e l s . The t w o p o o l s w e r e i n i t i a l l y c h o s e n b e c a u s e o f t h e h i g h p e r c e n t a g e o f R. l a r i x i n e a c h . I t was h o p e d t h a t Table I. Dominance, frequency and importance values for the algae i n the two tide pools. * The f i r s t figure i n each column refers to pool 1 and the second figure to pool 2. SPECIES COVER(%) DOMINANCE FREQUENCY IMPORTANCE (7„) Rhodomela l a r i x 45.9* 45.1* 0.76 4.91 90.3 96.0 39.8 38.6 Ceramium  washingtoniense P r i o n i t i s  l y a l l i i 9.3 3.6 0.8 20.3 0.15 0.39 0.01 2.21 63.6 33.3 16.5 7.4 7.9 79.9 1.9 23.5 R a l f s i a crusts 5.8 1.6 0.10 0.17 29.7 19.5 8.4 4.1 Bare rock 38.2 29.4 0.63 3.20 77.6 70.7 33.5 26.5 18 s i n c e R. l a r i x i s v e r y h i g h i n p h e n o l i c c o n t e n t , t h e e x u d a t i o n o f c h a r a c t e r i s t i c compounds f r o m t h i s p l a n t c o u l d b e m o n i t o r e d . H o w e v e r , t h e c o n c e n t r a t i o n s o f i n d i -v i d u a l p h e n o l i c c o m p o n e n t s i n t h e p o o l w a t e r p r o v e d t o be t o o l o w f o r i d e n t i f i c a t i o n p u r p o s e s . I t was o n l y p o s -s i b l e t o g e t a n i n d i c a t i o n o f t h e t o t a l p h e n o l s p r e s e n t i n t h e p o o l w a t e r a t a n y p a r t i c u l a r t i m e . An i n c r e a s e d r a t e o f e x u d a t i o n o f p h e n o l s a n d t h e i r s u b s e q u e n t a p p e a r -a n c e i n t h e p o o l w a t e r was j u d g e d a r e f l e c t i o n o f t h e p h y s i o l o g i c a l s t r e s s p l a c e d o n t h e p l a n t s i n t h e p o o l a t t h e t i m e o f t h e s a m p l i n g . The c h a n g e i n p h e n o l c o n c e n -t r a t i o n i n t h e p o o l s as m e a s u r e d o n J u l y 1 8 - 1 9 i s shown i n F i g u r e 4 . The l e v e l s i n c r e a s e t o maximum v a l u e s j u s t b e f o r e r e i m m e r s i o n o f t h e p o o l i n e a c h c a s e , i n d i c a t i n g t h e p o s s i b i l i t y t h a t t h e i n c r e a s e d p r o d u c t i o n o f e x t r a -c e l l u l a r m e t a b o l i t e s ( e . g . p h e n o l s ) b y t h e a l g a e m i g h t be a s t r e s s r e s p o n s e . T h e r e a r e n u m e r o u s e x a m p l e s o f t h i s k i n d o f r e s p o n s e i n t h e l i t e r a t u r e ( C r a i g i e a n d M c L a c h l a n 1 9 6 4 , S i e b u r t h 1 9 6 9 , S i e b u r t h a n d J e n s e n 1 9 6 9 ) . E x u d a t e s a r e p r o b a b l y i m p o r t a n t i n t h e e c o l o g y o f t i d e p o o l o r g a n i s m s ( L a n g l o i s 1 9 7 5 ) . Some r e s e a r c h e r s b e l i e v e t h a t e x u d a t i o n may be a f o r m o f w a s t e p r o d u c t d i s -p o s a l b y t h e a l g a e , o t h e r s t h a t t h e c o m p o n e n t s o f e x u d a t e s may a c t as a n t i b i o t i c s u b s t a n c e s ( L a n g l o i s 1 9 7 5 , Ragan a n d J e n s e n 1 9 7 8 ) o n c e i n s u f f i c i e n t c o n c e n t r a t i o n s i n t h e POOL 1 POOL 2 Figure 4. Total phenols i n pool 1 and pool 2 over a twenty-four hour period given as parts per m i l l i o n of phloroglucinol. The period of dark i s indicated by s t i p p l i n g . 20 t i d e p o o l . A l t h o u g h t h e a n t i b i o t i c a c t i v i t y o f a l g a l e x u d a t e s h a s y e t t o be d e m o n s t r a t e d c o n c l u s i v e l y , t h e r e i s some i n d i c a t i o n t h a t e x u d a t e s h a v e some e f f e c t s o n b a c t e r i a a n d i n v e r t e b r a t e l i f e h i s t o r y s t a g e s ( C o n o v e r a n d S i e b u r t h 1 9 6 6 , S i e b u r t h a n d J e n s e n 1 9 6 9 , L a n g l o i s 1 9 7 5 ) . o The X s p e c i e s a s s o c i a t i o n d a t a ( T a b l e I I ) i n d i c a t e t h a t o n l y R h o d o m e l a a n d C e r a m i u m a r e c l o s e l y a s s o c i a t e d i n t h e u p p e r p o o l a n d t h a t n o a s s o c i a t i o n w i t h R h o d o m e l a o c c u r s i n t h e l o w e r p o o l . C e r a m i u m i s a common e p i p h y t e o f R h o d o m e l a ( p e r s o n a l o b s e r v a t i o n 1977 . ) , h e n c e t h e c l o s e a s s o c i a t i o n . S i n c e C e r a m i u m a l s o c o n t a i n s h a l o p h e n o l s ( s e e C h a p t e r I I I ) , i t i s u n l i k e l y t h a t i f t h e s e compounds show some a n t i b i o t i c f u n c t i o n i n r e d a l g a e , t h e i r p r o d u c -t i o n by . R h o d o m e l a w o u l d show no e f f e c t a g a i n s t e p i p h y t i -z a t i o n b y C e r a m i u m . Work b y K h a i l o v a n d B u r l a k o v a ( 1 9 6 9 ) i n d i c a t e s t h a t as much as 37% o f t h e g r o s s p r o d u c t i v i t y o f a n a l g a may b e r e l e a s e d as d i s s o l v e d o r g a n i c m a t t e r (DOM) . P h e n o l s m u s t c o n s t i t u t e a s i g n i f i c a n t f r a c t i o n o f t h e s e e x u d a t e s . W h e t h e r t h i s r e l e a s e i s a c t i v e o r p a s s i v e i s u n k n o w n a t p r e s e n t ; h o w e v e r , a l a r g e p o r t i o n o f t h e c a r b o n b u d g e t o f an a l g a i s r e l e a s e d i n t o t h e e n v i r o n m e n t . One w o u l d q u e s t i o n s u c h h i g h l e v e l s o f w a s t e compound p r o d u c t i o n , e s p e c i a l l y i n t h e f o r m o f c o m p l e x m o l e c u l e s s u c h as Table I I . X s p e c i e s a s s o c i a t i o n of algae i n the two po o l s . The p r o b a b i l i t i e s g i v e n are t h a t the p l a n t i n quest i o n i s not a s s o c i a t e d w i t h Rhodomela l a r i x . POOL 1 ' POOL 2 SPECIES X 2 P(%) X 2 P(%) Ceramium ~ . 8.77 1 0.34 59.0 washingtoniense P r i o r i i t i s ~ T T T - 1.52 23.2 0.50 48.8 l y a l l i x R a l f s i a ~~ 2.51 14.1 0.80 78.6 c r u s t s 22 halophenols or polyphenols. These compounds must, i t seems, have some other f u n c t i o n and u n t i l f u r t h e r evidence accumulates to the c o n t r a r y , they can be c o n s i d e r e d a n t i -b i o t i c substances. S i l v a and B i t t n e r (1979) have r e c e n t l y reviewed the l i t e r a t u r e on the a n t i b i o t i c a c t i v i t y of a l g a l m e t a b o l i t e s . In t h i s chapter I have presented p r e l i m i n a r y e v i -dence f o r the exudation of p h e n o l i c compounds by R. l a r i x found i n t i d e p o o l s . T h i s h a b i t a t from a l l i n d i c a t i o n s i s a s t r e s s f u l one/promoting, at l e a s t i n the case of Rhodomela, i n c r e a s e d p r o d u c t i o n of DOM as p h e n o l i c substances. Whether these compounds f u n c t i o n as a n t i b i o t i c s remains undetermined. I f a n t i b i o t i c , t h e i r a c t i o n a g a i n s t t i d e -p o o l organisms c o u l d be enhanced under the i n c r e a s e d en-vironmental p r e s s u r e s i n h e r e n t to t h i s h a b i t a t . CHAPTER I I BROMOPHENOLS OF RHODOMELA L A R I X : CHEMOTAXONOMY OF MORPHOLOGICAL FORMS 23 24 INTRODUCTION A l g a e o f t h e d i v i s i o n R h o d o p h y t a a r e u n i q u e i n t h e d i v e r s i t y a n d a b u n d a n c e o f c a r b o n - h a l o g e n compounds w h i c h t h e y c o n t a i n . The R h o d o m e l a c e a e i s e s p e c i a l l y r i c h i n t h e s e compounds i n t h e f o r m o f e i t h e r h a l o g e n a t e d t e r p e n -o i d s o r p h e n o l s . The b r o m o p h e n o l s a r e w i d e l y d i s t r i b u t e d among g e n e r a i n t h i s f a m i l y ( F e n i c a l 1 9 7 5 , K u r a t a e_t a l . 1 9 7 3 , K u r a t a a n d A m i y a 1 9 7 5 , W e i n s t e i n e_t a l . 1 9 7 5 , C h e v o l o t - M a g u e u r e t a l . 1 9 7 6 , S a e n g e r e t a l . 1 9 7 6 , K u r a t a e t a l . 1 9 7 6 , Combaut e t a l . 1 9 7 8 , P e d e r s e n 1 9 7 8 , Ragan a n d C r a i g i e 1 9 7 8 , L u n d g r e n e t a l . 1 9 7 9 , P e d e r s e n e t a l . 1 9 7 9 , K u r a t a a n d A m i y a 1 9 8 0 ) . T h e i r u s e as t a x o n o m i c m a r k e r s h a s b e e n s u g g e s t e d ( P e g u y 1 9 6 4 , A u g i e r 1 9 6 5 ) a n d d e b a t e d ( F e n i c a l 1 9 7 5 ) . I t i s g e n e r a l l y a g r e e d t h a t t h e s e compounds a r e u s e f u l i n d i f f e r e n t i a t i n g c l o s e l y r e l a t e d s p e c i e s w i t h i n a s i n g l e h a b i t a t ( F e n i c a l 1 9 7 5 ) . R h o d o m e l a i n B r i t i s h C o l u m b i a o c c u r s i n s e v e r a l v a r i a b l e a n d s o m e t i m e s d i s t i n c t m o r p h o l o g i c a l f o r m s . Two o f t h e s e f o r m s - - o n e common t o t i d e p o o l s i n t h e u p p e r i n t e r t i d a l z o n e , t h e o t h e r t o t h e m i d d l e a n d l o w e r i n t e r t i d a l t o u p p e r s u b t i d a l z o n e o f t h e s e a s h o r e - - a r e 25 g r o u p e d w i t h i n a s i n g l e t a x o n , R. l a r i x ( T u r n e r ) C. A g a r d h . To d a t e , a l l c o l l e c t i o n s o f t h i s s p e c i e s e x a m i n e d f o r b r o m o p h e n o l s h a v e come f r o m t h e l o w e r i n t e r t i d a l ( W e i n s t e i n e t a l . 1 9 7 5 , K a t s u i e_t a l . 1 9 6 7 ) . D u r i n g i n -v e s t i g a t i o n s r e l a t e d t o t h e p r e s e n t s t u d y , i t became n e c -e s s a r y t o d e t e r m i n e w h e t h e r t h e t w o f o r m s ( t i d e p o o l a n d i n t e r t i d a l ) s h o u l d i n f a c t be c o n s i d e r e d t h e same s p e c i e s . S i n c e t h e t e c h n i q u e o f c o m b i n e d g a s - l i q u i d c h r o m a t o g r a p h y -mass s p e c t r o m e t r y (GLC-MS) i s c o m m o n l y u s e d f o r t h e s e p a -r a t i o n a n d i d e n t i f i c a t i o n o f a l g a l h a l o m e t a b o l i t e s , a c o m p a r i s o n o f t h e t i d e p o o l a n d i n t e r t i d a l f o r m s b y t h i s m e t h o d was c o n s i d e r e d s u i t a b l e f o r t h e i r a s s i g n m e n t a t t h e s p e c i e s l e v e l . MATERIALS AND METHODS C o l l e c t i o n o f S a m p l e s S a m p l e s o f t h e t i d e p o o l a n d i n t e r t i d a l f o r m s o f R. l a r i x w e r e c o l l e c t e d i n e a r l y S e p t e m b e r , 1 9 7 8 , o n B a t h I s l a n d . A f t e r r e m o v a l o f v i s i b l e e p i p h y t e s , p l a n t s w e r e w a s h e d i n f r e s h w a t e r a n d r e t u r n e d t o t h e l a b o r a t o r y o n D r y I c e . V o u c h e r s p e c i m e n s h a v e b e e n p l a c e d i n t h e P h y c o -l o g i c a l H e r b a r i u m a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . E x t r a c t i o n a n d P r e p a r a t i o n o f TMS D e r i v a t i v e s I n g e n e r a l t h e m e t h o d o f P e d e r s e n ( 1 9 7 8 ) was 26 f o l l o w e d . One g o f e a c h a l g a l t y p e was g r o u n d i n a V i r t i s h o m o g e n i z e r w i t h 25 m l 0 . 1 M s o d i u m p h o s p h a t e b u f f e r (pH 5 . 0 ) a n d s o n i c a t e d f o r 15 m i n ( 2 0 0 W ) . The e x t r a c t was f i l t e r e d t h r o u g h Whatman N o . 1 p a p e r a n d p a r t i t i o n e d i n t o e t h y l a c e t a t e (3 x 50 m l ) . The e t h y l a c e t a t e l a y e r s w e r e c o m b i n e d a n d t h e s o l v e n t r e m o v e d a t 38°C ( f r a c t i o n A ) . The w a t e r l a y e r was a d j u s t e d t o pH 8 w i t h 2 N NaOH a n d r e e x t r a c t e d w i t h e t h y l a c e t a t e ( f r a c t i o n B ) . F o r a f i n a l f r a c t i o n ( C ) , t h e w a t e r l a y e r was a c i d i f i e d t o pH 2 w i t h 1 N H C 1 , h e a t e d t o 60°C (15 m i n ) a n d e x t r a c t e d w i t h e t h y l a c e t a t e as b e f o r e . The d r i e d f r a c t i o n s w e r e t a k e n up i n 0 . 5 m l m e t h a n o l a n d t r a n s f e r r e d t o m i c r o s i l a t i o n v i a l s . M e t h a n o l was r e -moved u n d e r n i t r o g e n a n d t h e s a m p l e s w e r e d r i e d o v e r p h o s -p h o r o u s p e n t o x i d e i n a v a c u u m d e s i c c a t o r . F o r t h e p r e p a r a t i o n o f t r i m e t h y l s i l y l (TMS) d e r i v a t i v e s o f t h e b r o m o p h e n o l s , 25 u l e a c h o f a c e t o n i t r i l e a n d N , 0 - b i s -t r i m e t h y l s i l y l - t r i f l u o r o a c e t a m i d e c o n t a i n i n g 1% t r i m e t h y l c h l o r o s i l a n e ( P i e r c e C h e m i c a l C o . ) as c a t a l y s t w e r e a d d e d t o e a c h v i a l f o l l o w e d b y h e a t i n g t o 60°C f o r 15 m i n . A n a l y s i s S a m p l e s w e r e a n a l y z e d o n a V G - M i c r o m a s s mass s p e c t r o m e t e r i n t e r f a c e d t o a P y e - U n i c a m S e r i e s 104 g a s -l i q u i d c h r o m a t o g r a p h . A g l a s s c o l u m n o f 3% S E - 3 0 o n 27 a c i d w a s h e d 8 0 - 1 0 0 mesh C h r o m o s o r b - W ( V a r i a n A s s o c . ) was u s e d a t a He f l o w o f 30 m l / m i n . T e m p e r a t u r e p r o g r a m m i n g f r o m 1 0 0 - 2 5 0 ° C a t 5 0 ° C / m i n g a v e b e s t r e s u l t s . Mass s p e c t r a w e r e t a k e n a t 70 eV. I n e v e r y c a s e a n i o n a t m/e 73 was e x c e s s i v e ; t h e r e f o r e , o n l y i o n s o f g r e a t e r m/e w e r e r e c o r d e d . RESULTS AND DISCUSSION F i v e b r o m o p h e n o l s w e r e i d e n t i f i e d i n t h e e x t r a c t s o f t w o m o r p h o l o g i c a l f o r m s o f R h o d o m e l a l a r i x . The s t r u c -t u r e s o f t h e s e compounds a n d a t y p i c a l GLC t r a c e ( i n t e r -t i d a l f r a c t i o n A ) a r e shown i n F i g u r e 5 . F r a c t i o n A c o n t a i n e d compounds 1 ( i n t e r t i d a l f o r m o n l y ) , 2_, 3 a n d 4 ; f r a c t i o n B, 2 , 4 a n d 5 ; f r a c t i o n C, 2 ( i n t e r t i d a l o n l y ) , 3_ ( t i d e p o o l o n l y ) a n d 4 . The f o l l o w i n g mass s p e c t r a l d a t a w e r e o b t a i n e d f o r t h e f i v e c o m p o u n d s . R e l a t i v e i n t e n s i t i e s (>10% o n l y ) a r e g i v e n i n p a r e n t h e s e s f o l l o w i n g e a c h m / e . Compound 1 : m /e 74 ( 7 3 ) , 75 ( 8 2 ) , 77 ( 9 1 ) , 147 ( 2 7 ) , 205 ( 1 8 ) , 2 2 1 ( 1 0 0 ) , 222 ( 1 8 ) , 223 ( 1 8 ) , 257 ( 4 5 ) , 259 ( 3 6 ) , 2 7 1 ( 2 7 ) , 345 ( 8 2 ) , 347 ( 9 1 ) , 360 ( 3 6 ) , 362 ( 2 7 ) . Com-p o u n d 2 : m /e 74 ( 1 0 0 ) , 75 ( 7 4 ) , 77 ( 2 1 ) , 349 ( 1 2 ) , 3 5 1 ( 2 4 ) , 353 ( 1 5 ) , 423 ( 5 3 ) , 425 ( 1 0 0 ) , 427 ( 5 9 ) , 438 ( 3 2 ) , 440 ( 5 6 ) , 442 ( 3 5 ) . Compound 3 : m/e 74 ( 2 5 ) , 2 5 x 2 . 5 x 2 ' CHO R = H=1 3 0 2 8 2 6 2 4 2 2 2 0 1 8 16 14 12 1 0 8 6 4 2 0 T i m e ( m i n ) 2 5 0 2 2 5 2 0 0 V 7 5 1 5 0 1 2 5 1 0 0 T e m p e r a t u r e ( ° C ) F i g u r e 5. Temperature programmed GLC a n a l y s i s of Rhodomela l a r i x bromophenols. The r e l a t i v e p o s i t i o n of compound 5 i s shown by an i n s e r t e d dashed l i n e . 29 75 ( 3 2 ) , 147 ( 2 7 ) , 149 ( 1 0 0 ) , 257 ( 8 4 ) , 259 ( 9 3 ) , 267 ( 1 1 ) , 3 3 1 ( 1 4 ) , 345 ( 2 0 ) , 347 ( 2 0 ) , 4 3 4 ( 3 4 ) , 436 ( 4 1 ) . Compound 4 : m /e 75 ( 8 0 ) , 147 ( 4 8 ) , 149 ( 1 0 0 ) , 335 ( 3 2 ) , 337 ( 7 2 ) , 339 ( 3 2 ) , 4 1 1 ( 1 2 ) , 423 ( 1 6 ) , 425 ( 2 4 ) , 427 ( 1 6 ) , 433 ( 2 8 ) , 435 ( 3 2 ) , 512 ( 2 0 ) , 514 ( 4 4 ) , 516 ( 2 4 ) . Compound 5 : m/e 74 ( 2 8 ) , 75 ( 3 0 ) , 77 ( 1 2 ) , 105 ( 2 0 ) , 137 ( 1 3 ) , 139 ( 1 7 ) , 147 ( 1 0 0 ) , 148 ( 2 2 ) , 149 ( 1 2 ) , 198 ( 1 2 ) , 207 ( 1 3 ) , 259 ( 1 2 ) , 335 ( 2 2 ) , 337 ( 4 0 ) , 339 ( 2 3 ) , 345 ( 1 8 ) , 347 ( 2 3 ) , 409 ( 3 2 ) , 4 1 1 ( 6 5 ) , 413 ( 3 5 ) , 4 2 4 ( 1 0 ) , 426 ( 2 0 ) , 428 ( 1 2 ) . The t w o f o r m s o f R h o d o m e l a e x a m i n e d h e r e o c c u r w i t h -i n a b o u t 3 m (12 f t ) v e r t i c a l d i s t r i b u t i o n o n B r i t i s h C o l u m b i a s h o r e s . A l t h o u g h t h e i r m o r p h o l o g i e s a r e q u i t e d i s t i n c t , i t i s i n t e r e s t i n g t h a t t h e y c o n t a i n a l l b u t one o f t h e same b r o m o p h e n o l s . Compound 1_ o c c u r r e d o n l y i n e x t r a c t s o f t h e i n t e r -t i d a l f o r m . I f t h e s e a l d e h y d e s a r e t o be c o n s i d e r e d a r t i -f a c t s o f e x t r a c t i o n p r o c e d u r e s ( W e i n s t e i n e_t a l . 1 9 7 5 , S a e n g e r e t a l . 1 9 7 6 ) , t h e n t h e p r e s e n c e o f t h i s compound i n one o r t h e o t h e r o f t h e t y p e s i s i n c o n s e q u e n t i a l . I c a n n o t a r g u e a g a i n s t t h i s s u p p o s i t i o n b u t c a n o n l y q u e s t i o n t h e p r o d u c t i o n o f s u c h a r t i f a c t s u n d e r t h e m i l d e x t r a c t i o n p r o c e d u r e s u s e d h e r e . M o s t w o r k e r s who h a v e e x a m i n e d m a r i n e a l g a e f o r h a l o m e t a b o l i t e s f i n d s u f f i c i e n t e v i d e n c e t o s e p a r a t e p o p u -l a t i o n s i n t o d i s t i n c t c h e m i c a l t y p e s i f n o t i n f a c t 30 d i s t i n c t s p e c i e s ( F e n i c a l a n d N o r r i s 1 9 7 5 , F e n i c a l 1 9 7 6 , Crews et_ a l . 1 9 7 7 , Caccamese e t a l . 1 9 7 9 ) . F o r R h o d o m e l a  l a r i x , h o w e v e r , i t a p p e a r s t h a t t h e t i d e p o o l a n d i n t e r -t i d a l f o r m s s h o u l d be c l a s s e d w i t h i n a s i n g l e s p e c i e s , d i f f e r e n c e s i n b r o m o p h e n o l c h e m i s t r y n o t w a r r a n t i n g s e p a -r a t i o n a t t h i s l e v e l . S i m i l a r c h e m i c a l i n v e s t i g a t i o n o f o t h e r h e t e r o m o r p h i c s p e c i e s s h o u l d , i n t h e f u t u r e , l e n d f u r t h e r c r e d e n c e t o t h e c o n c e p t o f p l e o m o r p h i s m i n m a r i n e a l g a e . CHAPTER I I I HPLC SEPARATION OF RED ALGAL BROMOPHENOLS 31 32 INTRODUCTION S i n c e t h e i r i n i t i a l d i s c o v e r y as m a r i n e n a t u r a l p r o d u c t s ( M a s t a g l i a n d A u g i e r 1 9 4 9 ) , h a l o p h e n o l s h a v e r e -c e i v e d i n c r e a s i n g a t t e n t i o n . T h e s e compounds h a v e b e e n i s o l a t e d f r o m a n u m b e r o f a l g a e r e p r e s e n t i n g s e v e r a l o r -d e r s o f t h e R h o d o p h y t a ( F e n i c a l 1 9 7 5 ) . P a p e r c h r o m a t o g r a p h y ( P e g u y 1 9 6 4 ) , t h i n l a y e r c h r o m a -t o g r a p h y ( K u r a t a e t a l . 1 9 7 3 , W e i n s t e i n e t al. 1 9 7 5 , Ragan a n d C r a i g i e 1 9 7 8 , C r a i g i e a n d G r u e n i g 1 9 6 7 , G l o m b i t z a a n d S t o f f e l e n 1 9 7 2 , S t o f f e l e n e t a l . 1 9 7 2 , C h a n t r a i n e et a l . 1 9 7 3 , P e d e r s e n e_t a l . 1 9 7 4 ) , c o l u m n c h r o m a t o g r a p h y ( C h e v o l o t - M a g u e u r e t a l . 1 9 7 6 , K u r a t a e_t a l . 1 9 7 6 , Combaut e t a l . 1 9 7 8 , L u n d g r e n e t a l . 1 9 7 9 , K u r a t a a n d A m i y a 1 9 8 0 , G l o m b i t z a a n d S t o f f e l e n 1 9 7 2 ) , gas l i q u i d c h r o m a t o g r a p h y (GLC) ( R a g a n a n d C r a i g i e 1 9 7 8 , P e d e r s e n e t a l . 1 9 7 9 , S t o f f e l e n e_t a l . 1 9 7 2 ) a n d c o m b i n e d GLC-mass s p e c t r o m e t r y (GLC-MS) ( S a e n g e r e t a l . 1 9 7 6 , P e d e r s e n 1 9 7 8 , S t o f f e l e n e t a l . 1 9 7 2 , P e d e r s e n e t a l . 1 9 7 4 , P e d e r s e n a n d D a S i l v a 1 9 7 3 , P e d e r s e n a n d F r i e s 1 9 7 5 , P e d e r s e n a n d F r i e s 1 9 7 7 , a l s o see C h a p t e r I I ) h a v e b e e n u s e d e x t e n s i v e l y f o r t h e s e p a r a t i o n l e a d i n g t o s u b s e q u e n t s t r u c t u r a l e l u c i d a t i o n o f t h i s g r o u p o f p h e n o l s . P e r h a p s t h e m o s t u s e f u l i n f o r m a t i o n 33 r e g a r d i n g t h e a r r a y o f h a l o p h e n o l s w i t h i n e a c h a l g a so f a r e x a m i n e d h a s come f r o m GLC-MS a n a l y s e s . U n f o r t u n a t e l y , s u c h c o m p l e x i n s t r u m e n t s a r e e x p e n s i v e a n d g e n e r a l l y u n -a v a i l a b l e . H o w e v e r , r e c e n t a d v a n c e s i n t h e d e v e l o p m e n t o f h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC) s y s t e m s make t h i s c h r o m a t o g r a p h i c m e t h o d c o m p a r a t i v e l y i n e x p e n s i v e a n d t h e r e f o r e m o r e w i d e l y a v a i l a b l e f o r t h e s c r e e n i n g o f o r g a n i s m s f o r v a r i o u s g r o u p s o f c o m p o u n d s . L i q u i d c h r o m a -t o g r a p h y (LC) h a s now a d v a n c e d t o t h e p o i n t t h a t i t r i v a l s p a c k e d c o l u m n GLC i n a p p l i c a t i o n a n d p r e c i s i o n . HPLC c o l u m n s p a c k e d w i t h 5 - 1 0 um m i c r o p o r o u s b e a d s a r e c a p a b l e o f g e n e r a t i n g up t o 2 5 , 0 0 0 t h e o r e t i c a l p l a t e s p e r m e t e r . A v a r i e t y o f a c c u r a t e a n d s e n s i t i v e d e t e c t i o n s y s t e m s i s a v a i l a b l e w h i c h , w h e n c o u p l e d t o m o d e r n LC e q u i p m e n t , makes t h e s e p a r a t i o n a n d d e t e c t i o n o f m i c r o - t o n a n o g r a m q u a n -t i t i e s o f m o s t compounds p o s s i b l e . B e c a u s e o f t h e e a s e o f s a m p l e p r e p a r a t i o n ( d e r i v a -t i z a t i o n as i n GLC n o t n e c e s s a r y ) a n d t h e r e s o l u t i o n a n d s e n s i t i v i t y a c h i e v e d b y t h i s m e t h o d , I c h o s e t o a p p l y i t t o t h e d e t e r m i n a t i o n o f b r o m o p h e n o l s f r o m t h r e e common s p e c i e s o f r e d a l g a e i n B r i t i s h C o l u m b i a . R h o d o m e l a  l a r i x ( T u r n e r ) C. A g a r d h , h a v i n g r e c e i v e d t h e s c r u t i n y o f s e v e r a l p r e v i o u s c h e m i c a l i n v e s t i g a t i o n s ( W e i n s t e i n e t a l . 1 9 7 5 , K a t s u i e_t a l . 1 9 6 7 ) was c h o s e n as a m o d e l o r g a n i s m o n w h i c h t o t e s t t h e e f f e c t i v e n e s s o f t h e HPLC 34 m e t h o d . MATERIALS AND METHODS C h e m i c a l s S t a n d a r d s o f 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l a l c o h o l ( 2 ) ( l a n o s o l ) , 3 - b r o m o - 4 , 5 - d i h y d r o x y b e n z a l d e h y d e ( 3 ) , 3 , 5 - d i b r o m o - 4 - h y d r o x y b e n z y l a l c o h o l ( 5 ) , 3 , 5 - d i b r o m o -4 - h y d r o x y b e n z o i c a c i d ( 6 ) a n d 3 , 5 - d i b r o m o - 4 - h y d r o x y b e n z y l m e t h y l e t h e r ( 1 4 ) w e r e k i n d l y p r o v i d e d b y D r . J . S. C r a i g i e ( A t l a n t i c R e g i o n a l L a b o r a t o r y , H a l i f a x , Nova S c o t i a ) . T e t r a m e t h y l a m m o n i u m c h l o r i d e (TMA) a n d 3 , 4 - d i h y d r o x y -b e n z a l d e h y d e w e r e p u r c h a s e d f r o m A l d r i c h C h e m i c a l Co. a n d HPLC g r a d e a c e t o n i t r i l e f r o m F i s h e r S c i e n t i f i c . A l l o t h e r c h e m i c a l s a n d s o l v e n t s w e r e f r o m v a r i o u s s o u r c e s a n d w e r e o f t h e h i g h e s t q u a l i t y o b t a i n a b l e . S y n t h e s i s o f O t h e r B r o m o p h e n o l s The f o l l o w i n g s y n t h e s e s w e r e p e r f o r m e d o n a m i c r o -s c a l e t o p r o v i d e a d d i t i o n a l s t a n d a r d s f o r . c o m p a r i s o n w i t h c r u d e e x t r a c t s . P u r i f i c a t i o n o f t h e p r o d u c t s was n o t a t t e m p t e d due t o t h e s m a l l q u a n t i t i e s i n v o l v e d . The r e -a c t i o n p r o c e d u r e s u t i l i z e d a r e w e l l k n o w n a n d f o r t h e m o s t p a r t p r o d u c e a s i n g l e p r o d u c t . 3 - B r o m o - 4 , 5 - d i h y d r o x y b e n z y l a l c o h o l ( 1 ) . A p p r o x i m a t e l y 1 . 0 mg ( 3 0 uM) s o d i u m b o r o h y d r i d e was s l o w l y a d d e d t o a s m a l l a m o u n t o f s l i g h t l y c o o l e d m e t h a n o l c o n t a i n i n g 35 2 . 0 mg ( 1 . 0 uM) o f 3_. When t h e r e a c t i o n h a d c e a s e d , 0 . 1 m l w a t e r was a d d e d t o h y d r o l y z e t h e h y d r o b o r a n e a n d f r e e t h e a l c o h o l . 3 - B r o m o - 4 , 5 - d i h y d r o x y b e n z y l m e t h y l e t h e r ( 4 ) . To 0 . 3 m l o f t h e r e a c t i o n m i x t u r e f r o m !L a b o v e , t w o d r o p s o f 2 N h y d r o c h l o r i c a c i d (HC1) w e r e a d d e d . A f t e r h e a t i n g f o r one h r o n a s t e a m b a t h , a m i x t u r e o f u n r e a c t e d 1_ a n d i t s b e n z y l m e t h y l e t h e r w a s . f o u n d . 2 , 3 - D i b r o m o - 4 , 5 - d i h y d r o x y b e n z a l d e h y d e ( 7 ) . One mg ( 0 . 4 uM) o f 2_ was d i s s o l v e d i n 0 . 3 m l d i m e t h y l s u l f o x i d e a n d 0 . 2 m l a c e t i c a n h y d r i d e a n d h e a t e d o n a s t e a m b a t h f o r 15 m i n ( A l b r i g h t a n d G o l d m a n 1 9 6 5 ) t o y i e l d a s i n g l e p r o d u c t . 2 . 3 - D i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l m e t h y l e t h e r ( 8 ) . Two d r o p s o f 2 N HC1 w e r e a d d e d t o 1 . 0 mg ( 0 . 4 uM) 2 i n 0 . 5 m l m e t h a n o l . H e a t i n g o n a s t e a m b a t h f o r one h r p r o d u c e d 8 . 3 , 6 - D i b r o m o - 4 , 5 - d i h y d r o x y b e n z a l d e h y d e ( 9 ) . The d e t a i l e d p r o c e d u r e f o r t h e p r e p a r a t i o n o f t h i s compound h a s b e e n g i v e n b y L u n d g r e n e t a l . 1 9 7 9 . F r e s h l y s u b l i m e d 3 . 4 - d i h y d r o x y b e n z a l d e h y d e ( 1 . 8 g , 0 . 1 3 M) was u s e d as s t a r t i n g m a t e r i a l . C o n c e n t r a t i o n o f t h e r e a c t i o n s o l v e n t p r o d u c e d w h i t e c r y s t a l s w h i c h w e r e r e c r y s t a l l i z e d f r o m e t h a n o l ( y i e l d = 0 . 3 8 g , 2 1 % ) , mp 1 7 8 - 1 8 0 ° C . I n f r a r e d a n d p r o t o n m a g n e t i c r e s o n a n c e s p e c t r a a g r e e d w i t h t h o s e r e p o r t e d i n t h e a b o v e r e f e r e n c e s . 3 . 5 - D i b r o m o - 4 - h y d r o x y b e r i z a l d e h y d e ( 1 0 ) . One mg ( 0 . 5 uM) 36 o f 5_ was w o r k e d up as i n 7_ a b o v e . H o w e v e r , t h i s r e a c t i o n p r o d u c e d t h r e e p r o d u c t s as m o n i t o r e d b y HPLC. I n a d d i t i o n t o 10 t h e o t h e r p r o d u c t s a r e t h o u g h t t o b e t h e h e m i -a c e t a l (12_) a n d a c e t a l ( 1 3 ) o f t h e s t a r t i n g m a t e r i a l . A q u e o u s a c i d a d d i t i o n a n d c o n t i n u e d h e a t i n g c a u s e d t o t a l c o n v e r s i o n t o t h e a l d e h y d e . A n a l t e r n a t i v e o x i d a t i o n p r o c e d u r e w h i c h y i e l d e d t h e a l d e h y d e as s o l e p r o d u c t c o n -s i s t e d o f h e a t i n g f o r 15 m i n 1 . 0 mg o f t h e a l c o h o l ( 5 ) a n d 2 mg m a n g a n e s e d i o x i d e i n 0 . 5 m l m e t h a n o l . 2 , 3 - D i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l e t h y l e t h e r ( 1 1 ) . The p r o c e d u r e was t h e same as i n t h e p r e p a r a t i o n o f 8_ e x c e p t t h a t m e t h a n o l was r e p l a c e d b y e t h a n o l . C h r o m a t o g r a p h y HPLC was p e r f o r m e d o n a V a r i a n M o d e l 5 0 0 0 LC l i n k e d t o a V a r i s c a n 634 S S p e c t r o p h o t o m e t e r s e t a t a c o n s t a n t w a v e l e n g t h o f 280 nm. The e l u t i o n s o l v e n t c o n -s i s t e d o f 40% a c e t o n i t r i l e ( i n g l a s s d i s t i l l e d w a t e r ) c o n t a i n i n g 10 mM e a c h o f TMA a n d d i b a s i c s o d i u m p h o s p h a t e ( b u f f e r ) . The pH o f t h e s o l v e n t was a d j u s t e d t o 3 . 2 - 3 . 5 w i t h c o n c e n t r a t e d H C 1 . I n i t i a l l y , g r a d i e n t e l u t i o n w i t h 80%, a c e t o n i t r i l e a n d w a t e r ( b o t h c o n t a i n i n g a d d i t i v e s ) was u s e d t o d e t e r m i n e o p t i m a l s o l v e n t s t r e n g t h f o r b e s t r e s o l u t i o n o f t h e s t a n -d a r d s i n t h e s h o r t e s t t i m e . Once e s t a b l i s h e d , i s o c r a t i c e l u t i o n w i t h t h e 40% a c e t o n i t r i l e s o l v e n t p r o v e d t h e m o s t u s e f u l a n d e c o n o m i c a l . O c t a d e c y l s i l a n e c o l u m n s o f M i c r o p a k MCH-10 ( V a r i a n ) w e r e u s e d f o r s e p a r a t i o n s i n t h e r e v e r s e p h a s e mode . A l l compounds a n a l y z e d as s t a n d a r d s w e r e u s e d i n c o n c e n t r a t i o n s o f a b o u t 1 mg p e r m l i n m e t h a n o l t o g i v e r o u g h l y e q u i v a l e n t d e t e c t o r r e s p o n s e s . I n j e c t i o n v o l -umes w e r e t y p i c a l l y 1 - 1 0 u l . E v a l u a t i o n o f t h e C h r o m a t o g r a p h i c M e t h o d T h r e e m o d i f i e d s o l v e n t s y s t e m s a n d a m i x t u r e o f compounds 5_ a n d 6_ w e r e u s e d ( i n a d d i t i o n t o t h e s o l v e n t s y s t e m d e s c r i b e d a b o v e ) f o r t h e e v a l u a t i o n o f c o l u m n s e l e c t i v i t y a n d e f f i c i e n c y : A , a c e t o n i t r i l e a n d w a t e r ( 4 : 6 ) o n l y ; B, A p l u s 10 mM b u f f e r (pH 3 . 2 ) ; C, A p l u s 10 mM TMA; D, 40% a c e t o n i t r i l e s o l v e n t c o n t a i n i n g b o t h a d d i t i v e s . E a c h s y s t e m u s e d i n d e p e n d e n t l y g a v e a n i n d i -c a t i o n o f t h e e f f e c t i v e n e s s o f an i n d i v i d u a l c o m p o n e n t t o t h e m e t h o d as a w h o l e . F r o m t h e s e m e a s u r e m e n t s f o u r c h r o m a t o g r a p h i c p a r a m e t e r s w e r e c a l c u l a t e d : c a p a c i t y f a c t o r ( k 1 ) , r e l a t i v e r e t e n t i o n («.) , n u m b e r o f t h e o -r e t i c a l p l a t e s ( n ) a n d r e s o l u t i o n ( R ) . F o r m u l a e u s e d i n t h e s e c a l c u l a t i o n s a r e as f o l l o w s ( S n y d e r a n d K i r k -l a n d 1 9 7 4 ) : 38 n = 5 . 5 5 4 / t \2 R = s/n r-oj- l W k ' \ 4 V « / I k ' + 1 J ( a ) ( b ) ( c ) w h e r e t = r e t e n t i o n t i m e i n m i n , w , = p e a k w i d t h a t h a l f h e i g h t , t = r e t e n t i o n t i m e o f a n o n r e t a i n e d s o l u t e a n d ° ' o k'-^ a n d k * 2 = c a p a c i t y f a c t o r s f o r t w o compounds s e p a r a t e d ( 1 e l u t i n g b e f o r e 2 ) . The t e r m s i n t h e r e s o l u t i o n e q u a -t i o n i n d i c a t e d b y t h e b r a c k e t e d l e t t e r s ( a ) , ( b ) a n d ( c ) r e p r e s e n t c o l u m n e f f i c i e n c y , s e l e c t i v i t y a n d c a p a c i t y r e s p e c t i v e l y . C o l l e c t i o n a n d E x t r a c t i o n o f A l g a e R h o d o m e l a l a r i x . A p p r o x i m a t e l y 2 k g f r e s h w e i g h t o f a l g a w e r e c o l l e c t e d i n t h e s u b t i d a l z o n e o f f B a t h I s l a n d . V i s i b l e e p i p h y t e s h a v i n g b e e n r e m o v e d , t h e p l a n t s w e r e w a s h e d i n f r e s h w a t e r a n d i m m e d i a t e l y f r o z e n a t - 8 0 ° C . F o l l o w i n g l y o p h i l i z a t i o n , t h e a l g a e w e r e g r o u n d i n a W i l e y m i l l t o p a s s t h r o u g h a 2 mm mesh s c r e e n ( y i e l d = 447 g d . w t . ) . S o x h l e t e x t r a c t i o n o f t h e d r i e d 39 m a t e r i a l u s i n g a s e r i e s o f s o l v e n t s ( l i g h t p e t r o l e u m e t h e r , c h l o r o f o r m , e t h y l a c e t a t e a n d m e t h a n o l ) g a v e f o u r f r a c t i o n s . E a c h f r a c t i o n was r e d u c e d i n v o l u m e i n v a c u o ( £ 4 0 ° C ) t o a b o u t 50 m l . The l a r g e a m o u n t o f s o l i d ( 4 2 . 9 g ) d e p o s i t e d o n c o n c e n t r a t i o n o f t h e m e t h a n o l e x -t r a c t was r e m o v e d b y f i l t r a t i o n a n d i d e n t i f i e d as t h e d i p o t a s s i u m s u l f a t e s a l t o f l a n o s o l ( W e i n s t e i n e t a l . 1 9 7 5 ) . One m l o f e a c h o f t h e f o u r f r a c t i o n s was t a k e n t o d r y n e s s , r e d i s s o l v e d i n 1 . 0 m l m e t h a n o l a n d a n a l y z e d b y HPLC f o r b r o m o p h e n o l s . C e r a m i u m w a s h i n g t o n i e n s e K y l i n a n d P r i o n i t i s l y a l l i i H a r v e y . T e n g o f e a c h a l g a w e r e g r o u n d i n a W a r i n g b l e n d e r i n b o i l i n g 80% m e t h a n o l a n d r e f l u x e d f o r one h r on a s t e a m b a t h . The e x t r a c t s w e r e a c i d i f i e d t o pH 2 w i t h 1 N H C 1 , w a r m e d t o 60°C f o r 15 m i n a n d f i l t e r e d . The m e t h a n o l was r e m o v e d i n v a c u o a n d t h e r e m a i n i n g w a t e r e x t r a c t e d t h r e e t i m e s w i t h e t h y l a c e t a t e . The e t h y l a c e t a t e l a y e r s w e r e c o m b i n e d a n d t h e s o l v e n t r e m o v e d i n v a c u o . The r e s i d u e s w e r e t a k e n up i n 5 . 0 m l m e t h a n o l a n d u s e d f o r HPLC a n a l y s e s . RESULTS AND DISCUSSION S e p a r a t i o n o f S t a n d a r d Compounds F i g u r e 6 i l l u s t r a t e s t h e s e p a r a t i o n o f 14 b r o m o p h e n o l 40 i ' i " i 1 1 1 1 1 1 1 1 1 1 r 0 2 4 6 8 1 0 12 1 4 T i m e (min) F i g u r e 6. Reverse phase HPLC s e p a r a t i o n o f f o u r -teen bromophenol standards. The r e l a t i v e p o s i t i o n of compound 11 i s shown by a dashed l i n e . 4 1 Table I I I . R e t e n t i o n times, c a p a c i t y f a c t o r s and r e l a t i v e r e t e n t i o n s of standards of bromophenols separated by r e v e r s e phase HPLC. t = 1 . 8 min. r o COMPOUND t R k' a (min) 1) 3-bromo - 4 , 5 -dihydroxybenzy1 a l c o h o l 3 . 1 0 . 72 1 . 82 2 ) 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y -b e n z y l a l c o h o l 4 . 15 1 . 13 1 . 17 3) 3-bromo - 4 , 5-dihydroxybenz-aldehyde 4 : 55 1 . 53 1 . 12 4 ) 3-bromo - 4 , 5-dihydroxybenzyl methyl ether 4 . 9 1 . 72 1 . 16 5 ) 3 , 5-dibromo - 4-hydroxybenzyl a l c o h o l 5. ,4 2 . 0 1 . 1 1 6 ) 3 , 5-dibromo - 4-hydroxybenzoic a c i d 5. ,8 2 . ,22 1 . ,13 7) 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y -benzaldehyde 6. ,3 2 . ,5 1 . , 3 1 8 ) 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y -b e n z y l methyl ether 7. . 7 3. ,28 1 . .15 9 ) 2 , 5 - d i b r o m o - 3 , 4 - d i h y d r o x y -benzaldehyde 8. .6 3. ,78 1 , , 10 1 0 ) 3 , 5-dibromo - 4-hydroxybenz-aldehyde 9. ,25 4 . , 1 4 1 , ,13 I D 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y -b e n z y l e t h y l ether 10 . .0 4 , .67 1 , .02 1 2 ) Hemiacetal of 7 above 10. . 4 4 , .78 1 . .27 13 ) A c e t a l of 7_ above 12 .7 6, . 06 1 , . 0 2 14 ) 3 , 5-dibromo - 4-hydroxybenzyl 12, .9 6, . 17 methyl ether 42 s t a n d a r d s a t a f l o w r a t e o f 1 m l / m i n ( p r e s s u r e 1120 p s i ) . R e t e n t i o n t i m e s , c a p a c i t y f a c t o r s a n d r e l a t i v e r e t e n t i o n d a t a f o r t h e s e compounds a r e g i v e n i n T a b l e I I I . The e l u t i o n o r d e r o f t h e compounds i s , as e x p e c t e d , t h e r e v e r s e o f t h a t o f a n o r m a l p h a s e s e p a r a t i o n . The m o r e p o l a r a l c o h o l s a n d a c i d s e l u t e f i r s t , f o l l o w e d b y a l d e h y d e s a n d e t h e r s . o - D i h y d r o x y f u n c t i o n s i n c r e a s e p o l a r i t y w h i l e a n o - d i b r o m o f u n c t i o n r e d u c e s p o l a r i t y . M e t h y l a n d e t h y l e t h e r s a r e r e n d e r e d q u i t e n o n p o l a r c o m -p a r e d t o t h e i r f r e e h y d r o x y l c o u n t e r p a r t s . The e t h y l e t h e r o f l a n o s o l ( 1 1 ) i s i n c l u d e d t o show t h e e f f e c t o f e x t e n d e d c h a i n l e n g t h o n r e t e n t i o n t i m e . A l s o o f i n t e r e s t i s t h e d e c r e a s e d p o l a r i t y o f t h e p - ( 9 ) v e r s u s t h e o -d i b r o m o a l d e h y d e ( 7 ) . E v a l u a t i o n o f t h e C h r o m a t o g r a p h i c M e t h o d I n i t i a l a t t e m p t s a t t h e s e p a r a t i o n o f b r o m o p h e n o l s b y r e v e r s e p h a s e LC m e t w i t h l i t t l e s u c c e s s : l a n o s o l was i r r e v e r s i b l y r e t a i n e d o n t h e c o l u m n , a c i d s s h o w e d n o r e t e n t i o n a n d a l d e h y d e s g a v e l o n g r e t e n t i o n t i m e s w i t h v e r y b r o a d a n d t a i l i n g p e a k s . Some a u t h o r s h a v e n o t e d t h a t r e p e a t e d i n j e c t i o n s o f a n i r r e v e r s i b l y r e t a i n e d c o m -p o u n d e v e n t u a l l y g i v e r e s u l t s ( v a n Sumere e_t a l . 1 9 7 9 ) ; h o w e v e r , t h e l e v e l o f t h e compound r e t a i n e d i s p r o b a b l y i n c r e a s e d t o t h e p o i n t t h a t a l l c o l u m n r e a c t i v e s i t e s a r e f i l l e d b e f o r e e l u t i o n o c c u r s . Such g r o s s c o n t a m i n a t i o n 43 m u s t be c o n s i d e r e d u n d e s i r a b l e s i n c e i t i n e v i t a b l y s h o r t -ens c o l u m n l i f e , n o t t o m e n t i o n i t s e f f e c t o n e f f i c i e n c y . I n an e f f o r t t o r e d u c e t h e s e p r o b l e m s , s u i t a b l e s o l v e n t m o d i f i e r s w e r e s o u g h t w h i c h w o u l d i m p r o v e s e l e c -t i v i t y a n d e f f i c i e n c y w h i l e m a i n t a i n i n g c a p a c i t y . S i n c e m o s t p h e n o l s a r e q u i t e a c i d i c ( k = 10 a n d a r e e a s i l y c o n v e r t e d t o p h e n o x i d e a n i o n s i n s o l u t i o n , t h e f i r s t m e t h o d c h o s e n t o i m p r o v e r e t e n t i o n c a p a b i l i t y was i o n s u p p r e s s i o n . T h i s t e c h n i q u e i s c o m m o n l y u s e d f o r t h e LC o f p h e n o l s ( v a n Sumere e_t a_. 1 9 7 9 ) as i t g e n e r a l l y i m p r o v e s p e a k s y m m e t r y b y s u p p r e s s i n g t a i l i n g . F o r many c o m p o u n d s , h o w e v e r , t h e r e i s s t i l l t h e p r o b l e m o f i r r e v e r s i b l e r e t e n t i o n , w h i c h c a n be o v e r c o m e s i m p l y a n d e f f e c t i v e l y b y a d d i n g a p h a s e - a l t e r i n g r e -a g e n t t o t h e s o l v e n t . TMA h a s b e e n u s e d f o r t h i s p u r p o s e ( B u r c e , p e r s o n a l c o m m u n i c a t i o n ) . T h i s m o d i f i e r s e l e c -t i v e l y b o n d s t o u n b l o c k e d s i l a n o l g r o u p s i n t h e c o l u m n p a c k i n g m a t e r i a l . Compounds i n t e r a c t i n g w i t h t h e s t a t i o n -a r y p h a s e o n a r e v e r s e p h a s e c o l u m n a r e t h e n e x p o s e d t o a c o m p l e t e l y n o n p o l a r e n v i r o n m e n t . The p o s s i b i l i t y o f h y d r o g e n b o n d i n g t o s i l a n o l f u n c t i o n s i n t h e p a c k i n g , as I b e l i e v e o c c u r s i n t h e c a s e o f l a n o s o l , i s t h e r e f o r e . n e g a t e d . The c o m b i n a t i o n o f i o n s u p p r e s s i o n a n d TMA a d d i t i o n i m p r o v e s s e p a r a t i o n c a p a b i l i t y c o n s i d e r a b l y . The r e s u l t s 44 o f t h e e v a l u a t i o n o f e a c h c o m p o n e n t t a k e n s e p a r a t e l y a n d t h e n i n c o m b i n a t i o n a r e shown i n T a b l e I V . As n o t e d f r o m t h i s t a b l e , TMA t r i p l e s c o l u m n s e l e c t i v i t y a n d a l s o i n -c r e a s e s c o l u m n e f f i c i e n c y i n t h e s e p a r a t i o n o f compounds 5 a n d 6 , a c l o s e l y r e l a t e d a l c o h o l a n d a c i d . S e l e c t i v i t y i s r e d u c e d b y a d d i t i o n o f t h e b u f f e r , a l t h o u g h i n t h e d u a l c o m p o n e n t s y s t e m e f f i c i e n c y i s s t i l l m a i n t a i n e d a t a h i g h l e v e l . S e l e c t i v i t y c a n e a s i l y be i n c r e a s e d b y r e d u c i n g s o l v e n t s t r e n g t h o r f l o w r a t e . A n a l y s i s o f R h o d o m e l a E x t r a c t s The t e s t o f a c h r o m a t o g r a p h i c s y s t e m comes when i t i s a p p l i e d t o t h e s e p a r a t i o n o f a c o m p l e x m i x t u r e s u c h as a c r u d e p l a n t e x t r a c t . The s e p a r a t i o n s a c h i e v e d f o r t h e f o u r f r a c t i o n s o f t h e S o x h l e t e x t r a c t o f R. l a r i x a r e shown i n F i g u r e s 7 a n d 8 . M o s t o f t h e s t a n d a r d compounds a p p e a r i n t r a c e a m o u n t s i n t h e p e t r o l e u m e t h e r f r a c t i o n . L a n o s o l i s t h e m a j o r c o m p o n e n t i n t h e c h l o r o f o r m f r a c t i o n , a n d t h e a l d e h y d e ( 7 ) i n t h e e t h y l a c e t a t e f r a c t i o n . M e t h y l e t h e r s ( 4 , 8 , 1 4 ) a p p e a r o n l y i n t h e m e t h a n o l e x t r a c t . I n p r e v i o u s s t u d i e s t h e a l d e h y d e s a n d m e t h y l e t h e r s o f l a n o s o l a n d r e l a t e d compounds h a v e b e e n c o n s i d e r e d t o b e a r t i f a c t s o f i s o l a t i o n p r o c e d u r e s ( W e i n s t e i n e t a l . 1 9 7 5 , S a e n g e r e t a l . 1 9 7 6 ) . T h e r e i s l i t t l e d o u b t t h a t e t h y l a n d m e t h y l e t h e r s f a l l i n t o t h i s c a t e g o r y ; h o w e v e r , T a b l e I V . R e s o l u t i o n as a f u n c t i o n o f c o l u m n e f f i c e n c y , s e l e c t i v -i t y a n d c a p a c i t y u s i n g d i f f e r e n t s o l v e n t s y s t e m s i n t h e s e p a r a t i o n o f 3 , 5 - d i b r o m o - 4 - h y d r o x y b e n z y l a l c o h o l a n d t h e c o r r e s p o n d i n g a c i d . A b b r e v i a t i o n s u s e d a r e g i v e n i n t h e t e x t . SOLVENT SYSTEM EFFICIENCY SELECTIVITY CAPACITY R A) A c e t o n i t r i l e : w a t e r 1 1 8 0 - 0 B) A p l u s lOmM b u f f e r 8 . 7 2 0 . 2 8 0 . 4 6 1 .12 C) A p l u s lOmM TMA 9 . 2 2 0 . 8 8 0 . 4 4 3 . 6 1 D) B p l u s lOmM TMA 8 . 9 6 0 . 2 6 0 . 4 5 1 .05 46 F i g u r e 7. HPLC a n a l y s i s of the petroleum ether and c h l o r o f o r m f r a c t i o n s from the l a r g e s c a l e (Soxhlet) e x t r a c t i o n of Rhodomela l a r i x . The assignment of peaks i n p a r e n t h e s i s i s u n c e r t a i n . 47 F i g u r e 7 48 F i g u r e 8. HPLC a n a l y s i s of the e t h y l a c e t a t e and methanol f r a c t i o n s from the l a r g e s c a l e (Soxhlet) e x t r a c t i o n of Rhodomela l a r i x . 49 7 50 t h e p r e s e n c e o f a l d e h y d e 7_ t h r o u g h o u t a l l f o u r f r a c t i o n s c a u s e s me t o b e l i e v e t h a t s u c h compounds a r e n a t u r a l c o n -s t i t u e n t s o f t h e a l g a e . E v e n t h e m o s t m i l d e x t r a c t i o n p r o c e d u r e s ( P e d e r s e n 1 9 7 8 , s e e a l s o C h a p t e r I I ) show t h e p r e s e n c e o f t h e s e c o m p o u n d s . The p o s s i b i l i t y o f t h e i r e x i s t e n c e i n o t h e r s p e c i e s e x a m i n e d so f a r s h o u l d , t h e r e -f o r e , be r e e v a l u a t e d . C h r o m a t o g r a p h i c A n a l y s i s o f O t h e r Red A l g a e I n t h i s s t u d y t w o o t h e r a l g a l s p e c i e s , C e r a m i u m  w a s h i n g t o n i e n s e a n d P r i o n i t i s l y a l l i i , w e r e e x a m i n e d f o r t h e p r e s e n c e o f b r o m o p h e n o l s . The LC t r a c e s f o r t h e e x -t r a c t s o f t h e s e t w o s p e c i e s a p p e a r i n F i g u r e 9 . B o t h a l g a e c o n t a i n b r o m o p h e n o l s , C e r a m i u m c o n t a i n i n g o n l y one m a j o r compound ( 2 ) . The i d e n t i t y o f l a n o s o l i n t h i s s p e c i e s was v e r i f i e d b y c o c h r o m a t o g r a p h y w i t h t h e a u t h e n t i c s t a n d a r d a t d i f f e r e n t s o l v e n t s t r e n g t h s a n d f l o w r a t e s . P r i o n i t i s , l i k e R h o d o m e l a , c o n t a i n s a v a r i e t y o f b r o m o p h e n o l s , t h e m o s t a b u n d a n t b e i n g l a n o s o l a n d t h e m - d i b r o m o a c i d ( 6 ) a n d a l c o h o l (5_). A m o r e c a r e f u l e x -t r a c t i o n o f b o t h P r i o n i t i s a n d C e r a m i u m may l e a d t o t h e i d e n t i f i c a t i o n o f a n e v e n w i d e r a r r a y o f p h e n o l i c com-p o u n d s . I n c o n c l u s i o n , HPLC i s a r a p i d a n d s e n s i t i v e m e t h o d f o r t h e d e t e r m i n a t i o n o f b r o m o p h e n o l i c compounds i n a l g a e . C o l u m n s e l e c t i v i t y a n d e f f i c i e n c y i n r e v e r s e p h a s e 51 T - 1 i 1 1 1 1 1 1 1 1 1 1 1 1 1— 0 2 4 6 8 1 0 12 1 4 T i m e (min) 6 1 i — i — i — i — i — i — i — i — i — i — i — i — i — i — 1 0 2 4 6 8 1 0 12 1 4 T i m e (min) F i g u r e 9. Reverse phase s e p a r a t i o n o f bromophenols e x t r a c t e d from P r i o n i t i s l y a l l i i (bottom) and Ceramium washingtoniense ( t o p ) . Compounds i n p a r e n t h e s i s are t e n t a t i v e l y i d e n t i f i e d . 52 se p a r a t i o n s can be improved by the a d d i t i o n o f m o d i f i e r s which a l t e r the nature o f the s t a t i o n a r y phase or sup-press i o n i z a t i o n when, f o r example, the s e p a r a t i o n o f a c i d i c m e t a b o l i t e s i s necessary. I s o c r a t i c e l u t i o n w i t h s o l v e n t s c o n t a i n i n g m o d i f i e r s produces adequate s e p a r a t i o n o f a wide v a r i e t y o f bromophenols c o n t a i n i n g many types of f u n c t i o n a l groups. Under these c o n d i t i o n s the need f o r chromatographs equipped w i t h complex g r a d i e n t forming devices i s e l i m i n a t e d . \ CHAPTER IV TEMPORAL, INTERPOPULATIONAL AND INTRATHALLIAL MEASUREMENT OF LANOSOL LEVELS IN RHODOMELA LARIX 53 54 INTRODUCTION F e n i c a l ( 1 9 7 5 ) h a s s u g g e s t e d t h a t r e d a l g a l h a l o -m e t a b o l i t e s , r a t h e r t h a n b e i n g i n v o l v e d i n p r i m a r y m e t a -b o l i c p a t h w a y s , f u n c t i o n i n an e x o c r i n e s y s t e m p r o v i d i n g t h e a l g a c o n t a i n i n g t h e m w i t h a s e l e c t i v e e n v i r o n m e n t a l a d v a n t a g e . The n a t u r e o f t h i s a d v a n t a g e , i f i t e x i s t s , i s u n c l e a r ; h o w e v e r , e x c r e t i o n o f d i s s o l v e d o r g a n i c m a t -t e r (DOM) b y a l g a e i s w e l l d o c u m e n t e d ( H e l l e b u s t 1 9 7 4 , W a n g e r s k y 1 9 7 8 , Ragan a n d J e n s e n 1 9 7 9 ) . Such m a t e r i a l s may o n l y be w a s t e p r o d u c t s o f m e t a b o l i s m , b u t t h e y may a l s o be a c t i v e l y p r o d u c e d as a n t i b i o t i c s u b s t a n c e s ( L a n g l o i s 1 9 7 5 , Ragan a n d J e n s e n 1 9 7 8 ) . The s t u d y o f v a r i a t i o n s i n t h e t e m p o r a l a b u n d a n c e o f b r o m o p h e n o l s i n r e d a l g a e h a s r e c e i v e d l i t t l e a t t e n t i o n . Our p r e s e n t k n o w l e d g e c o n s i s t s o n l y o f q u a n t i t a t i v e d a t a d e r i v e d f r o m a l g a l e x t r a c t i o n s w h i c h , u n l e s s v e r y c a r e -f u l l y d o n e , m u s t i n v a r i a b l y l e a d t o u n d e r e s t i m a t i o n o f compounds i n t h e p l a n t s . T a b l e V s u m m a r i z e s t h e a v a i l a b l e l i t e r a t u r e i n t h i s a r e a a n d i l l u s t r a t e s t h e g r e a t v a r i a -b i l i t y i n t h e d a t a e v e n f o r m u l t i p l e e x t r a c t s o f a s i n g l e s p e c i e s . As a r e s u l t , t h e p h y s i o l o g i c a l a n d e c o l o g i c a l T a b l e V . A summary o f d a t a a v a i l a b l e o n q u a n t i t a t i v e a s p e c t s o f b r o m o p h e n o l s i n r e d a l g a e . A l l e s t i m a t e s a r e b a s e d o n t h e a b u n d a n c e o f t h e n a t u r a l l y o c c u r r i n g p h e n o l , n o t o n d e r i v a t i v e s w h i c h may h a v e b e e n p r e p a r e d i n i s o l a t i o n p r o c e d u r e s . The compounds l i s t e d a r e as f o l l o w s : 1 ) 3 , 5 - d i b r o m o - 4 - h y d r o x y p h e n y l a c e t i c a c i d , 2 ) 3 , 5 - d i b r o m o - 4 -h y d r o x y p h e n y l p y r u v i c a c i d , 3 ) p e r d e s m e t h y l c y c l o t r i b r o m o v e r a t r y l e n e , 4 ) 2 , 3 - d i b r o m o -4 , 5 - d i h y d r o x y b e n z y l m e t h y l e t h e r , 5 ) 2 , 3 , 2 ' , 3 ' - t e t r a b r o m o - 4 , 5 , 4 ' , 5 1 - t e t r a h y d r o x y d i -p h e n y l m e t h a n e , 6 ) 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l a l c o h o l , 7 ) 2 , 3 - d i b r o m o - 5 - h y d r o x y -b e n z y l - 1 ' , 4 - d i s u l f a t e ( p o t a s s i u m ) , 8 ) 3 , 5 - d i b r o m o - 4 - h y d r o x y b e n z y l a l c o h o l , 9 ) 2 , 3 -d i b r o m o - 4 , 5 - d i h y d r o x y b e n z a l d e h y d e , 1 0 ) 2 , 3 - d i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l e t h y l e t h e r , 1 1 ) 3 - b r o m o - 4 , 5 - d i h y d r o x y b e n z a l d e h y d e , 12 ) 3 , 3 ' - d i b r o m o - 4 , 4 ' , 5 , 5 1 - t e t r a h y d r o x y b i b e n z y l , 1 3 ) 3 - b r o m o - 4 , 5 - d i h y d r o x y b e n z y l m e t h y l e t h e r , 14 ) 3 , 5 - d i b r o m o - 4 - h y d r o x y b e n z y l m e t h y l e t h e r , 15 ) 2 , 4 - d i b r o m o - l , 3 , 5 - t r i h y d r o x y b e n z e n e , 16 ) 5 , 6 , 3 ' , 5 ' - t e t r a b r o m o - 3 , 4 , 2 ' , 4 ' , 6 ' -p e n t a h y d r o x y d i p h e n y l m e t h a n e , 17) b i s ( 2 , 3 , 6 - t r i b r o m o - 4 , 5 - d i h y d r o x y b e n z y l ) e t h e r . * = e s t i m a t e d a b u n d a n c e . ! = t e n t a t i v e i d e n t i f i c a t i o n . ALGA COMPOUND % d . w t . . (70 w . w t . ) REFERENCE H a l o p y t i s i n c u r v u s 1 2 ( 0 . 0 0 3 ) ( 0 . 0 0 2 ) C h a n t r a i n e e t a l . 1973 H. p i n a s t r o i d e s 1 2 3 ( 0 . 0 0 3 ) ( 0 . 0 0 3 ) ( 0 . 0 0 3 ) Combaut e t a l . 1978 O d o n t h a l i a c o r y m b i f e r a 4 5 ! 6 7 ( 0 . 0 1 ) ( 0 . 0 0 4 ) ( 0 . 0 0 8 ) ( 2 . 0 ) K u r a t a e t a l . 1973 0 . d e n t a t a 6 0 . 5 - 2 . 0 * C r a i g i e a n d G r u e n i g 1967 8 0 . 1 , ( 0 . 0 2 4 ) i b i d . , G l o m b i t z a a n d S t o f f e l e n 1972 P o l y s i p h o n i a b r o d i a e i 8 0 . 5 , 0 . 0 0 5 P e d e r s e n e t a l . 1 9 7 4 , L u n d g r e n e t a l . 1979 9 0 . 0 8 L u n d g r e n e t ' a l . 1979 T a b l e V . c o n t i n u e d . 10 0 . 0 0 6 5 0 . 0 0 5 P. l a n o s a 6 1 - 5 , 2 - 3 H o d g k i n e t a l . 1966 Ragan a n d C r a i g i e 1978 7 1 . 0 G l o m b i t z a a n d S t o f f e l e n 1972 P. m o r r o w i i 1 1 0 . 0 7 S a i t o a n d Ando 1955 P. u r c e o l a t a 1 1 0 . 0 8 K u r a t a e t a l . 1976 12 0 . 0 0 5 13 0 . 0 2 14 0 . 0 3 R h o d o m e l a c o n f e r v o i d e s .6 0.02-0.06-*, C r a i g i e a n d G r u e n i g 1 9 6 7 , ( 0 . 0 0 3 ) G l o m b i t z a a n d S t o f f e l e n 1972 8 0 . 0 0 3 C r a i g i e a n d G r u e n i g 1967 R. l a r i x 4 0 . 0 0 5 , K a t s u i e t a l . 1 9 6 7 , ( 0 . 0 0 9 ) W e i n s t e i n e_t a l . 1975 9 0 . 0 3 K a t s u i e t a l . 1967 7 ( 0 . 0 4 ) W e i n s t e i n e t a l . 1975 R. s u b f u s c a 4 0 . 0 6 K u r a t a a n d A m i y a 1975 7 9 . 2 9 0 . 0 2 R y t i p h l e a t i n c t o r i a 6 ( 0 . 0 0 9 ) C h e v o l o t - M a g u e r e t a l . 1976 10 ( 0 . 0 0 9 ) 15 ( 0 . 0 0 9 ) 16 ( 0 . 0 0 4 ) Symphyo c 1 a d i a l a t i s ' c ' u ' l a 17 K u r a t a a n d A m i y a 1980 57 s i g n i f i c a n c e o f b r o m o p h e n o l s i n r e d a l g a e i s u n k n o w n . B r o m o p h e n o l s h a v e b e e n shown t o be e f f e c t i v e a n t i b i o t i c s ( S i l v a a n d B i t t n e r 1 9 7 9 ) , b u t n o i n f o r m a t i o n i s a v a i l a b l e o n q u a n t i t a t i v e a s p e c t s o f t h e i r o c c u r r e n c e , w h i c h m i g h t be h e l p f u l i n a s s e s s i n g t h e i r i m p o r t a n c e t o t h e p l a n t . Ragan a n d J e n s e n ( 1 9 7 8 ) h a v e shown t h a t a k n o w l e d g e o f s e a s o n a l v a r i a t i o n i n c o n c e n t r a t i o n s o f p o l y p h e n o l s i n b r o w n a l g a e c a n p r o v i d e u s e f u l i n f o r m a t i o n r e g a r d i n g t h e i r s i g n i f i c a n c e . F o r t h i s r e a s o n I c h o s e t o e x a m i n e t h e r e d a l g a R h o d o m e l a l a r i x ( T u r n e r ) C. A g a r d h f o r c h a n g e s i n t h e l e v e l s o f l a n o s o l , t h e m a j o r b r o m o p h e n o l i n t h i s s p e c i e s ( s e e C h a p t e r I I I ) , o v e r a one y e a r p e r i o d . A l s o e x a m i n e d w e r e d i f f e r e n c e s i n b r o m o p h e n o l c o n t e n t among t h r e e d i s t i n c t p o p u l a t i o n s a n d w i t h i n a s i n g l e p l a n t ( i n t r a t h a l l i a l ) . I t was h o p e d t h a t an e x t e n s i v e q u a n t i -t a t i v e c h e m i c a l e x a m i n a t i o n o f t h i s a l g a w o u l d l e a d t o a c l e a r e r u n d e r s t a n d i n g o f t h e r a i s o n d ' e t r e o f b r o m o -p h e n o l s i n r e d a l g a e . MATERIALS AND METHODS C o l l e c t i o n o f A l g a l S a m p l e s U n l e s s o t h e r w i s e i n d i c a t e d , a l l a l g a l s a m p l e s w e r e c o l l e c t e d i n t h e l o w e r i n t e r t i d a l t o u p p e r s u b t i d a l z o n e o f f B a t h I s l a n d d u r i n g t h e h i g h e s t t i d e o f t h e m o n t h . 58 The p l a n t s f o r e a c h s a m p l e w e r e r a n d o m l y s e l e c t e d o v e r a l a r g e a r e a , c l e a n e d o f e p i p h y t e s a n d r e t u r n e d t o t h e l a b i n p l a s t i c b a g s o v e r i c e . A l g a e w e r e t h e n u s e d i m m e d i a t e l y o r f r o z e n ( - 2 0 ° C ) . O t h e r s a m p l e s came e i t h e r f r o m t i d e -p o o l s o n B a t h I s l a n d o r f r o m t h e l o w i n t e r t i d a l z o n e n e a r B a m f i e l d , B r i t i s h C o l u m b i a . E x t r a c t i o n P r o c e d u r e s A l l e x t r a c t i o n s w e r e done i n t r i p l i c a t e ; d r y w e i g h t s w e r e t a k e n i n d u p l i c a t e , t h e s a m p l e s h a v i n g b e e n d r i e d f o r one w e e k a t 9 0 ° C . One g o f e a c h a l g a l s a m p l e was g r o u n d i n a V i r t i s m i c r o h o m o g e n i z e r i n b o i l i n g 807 o m e t h a n o l . F o l l o w i n g one h o u r r e f l u x o n a s t e a m b a t h , p a r t i c u l a t e m a t t e r was f i l t e r e d o f f a n d t h e m e t h a n o l r e -m o v e d i n v a c u o . The r e s i d u a l w a t e r l a y e r was a c i d i f i e d t o pH 2 . 0 w i t h 1 . 0 N H C 1 , h e a t e d t o 60°C f o r 15 m i n ( t o h y d r o l y z e e s t e r s u l p h a t e s ) a n d t h e n c o n t i n u o u s l y e x t r a c t e d w i t h e t h y l a c e t a t e ( 4 h r s ) . The e t h y l a c e t a t e was r e -moved a n d t h e r e s i d u e t a k e n up i n 5 . 0 m l m e t h a n o l f o r s u b s e q u e n t c h r o m a t o g r a p h i c a n a l y s i s . C o l l e c t i o n s f r o m t h e o t h e r t w o l o c a t i o n s ( s e e a b o v e ) w e r e w o r k e d up i n t h e same way f o r r e l a t i v e c o m -p a r i s o n o f p h e n o l c o n t e n t among p o p u l a t i o n s . F o r m e a s u r e m e n t o f l a n o s o l c o n c e n t r a t i o n i n d i f -f e r e n t p a r t s o f t h e p l a n t , 1 . 0 g e a c h o f g r o w i n g t i p s , b r a n c h e s w i t h many l a t e r a l s , b r a n c h e s w i t h f e w l a t e r a l s 59 a n d h o l d f a s t s was e x c i s e d a n d e x t r a c t e d as b e f o r e . C h r o m a t o g r a p h i c M e t h o d s Q u a n t i t a t i v e d e t e r m i n a t i o n o f l a n o s o l was p e r f o r m e d b y HPLC u s i n g t h e c h r o m a t o g r a p h i c s y s t e m a n d e l u a n t s d e -s c r i b e d i n C h a p t e r I I I . I n t h i s c a s e , h o w e v e r , a w a v e -l e n g t h o f 292 nm was u s e d s i n c e t h i s i s t h e max imum a b -s o r p t i o n o f l a n o s o l ( W e i n s t e i n e t a l . 1 9 7 5 ) . The c h r o m a t o g r a p h was f i t t e d w i t h a 10 u l c a l i b r a t e d l o o p . I n j e c t i o n s o f t h i s v o l u m e w e r e a l w a y s u s e d t o m i n i m i z e p o t e n t i a l e r r o r i n t h i s p a r t o f t h e q u a n t i f i c a t i o n p r o -c e d u r e . The f l o w r a t e was 1 m l / m i n . L a n o s o l was i d e n t i f i e d b y c o m p a r i s o n w i t h t h e a u t h e n t i c s t a n d a r d . C a l i b r a t i o n c u r v e s f o r l a n o s o l i n m e t h a n o l w e r e made b y m e a s u r i n g p e a k a r e a s b y t h e w i d t h a t h a l f h e i g h t m e t h o d a n d c o n v e r t i n g t o m g / m l o f l a n o s o l a n d s u b s e q u e n t l y t o m g / g d r y w e i g h t as q u a n t i t i e s i n t h e a l g a . The l i f e h i s t o r y d a t a f o r a l g a e i n t h e s e s a m p l e s w e r e p r o v i d e d b y U l l a V i s s c h e r ( p e r s o n a l c o m m u n i c a t i o n ) . RESULTS AND DISCUSSION F i g u r e 10 shows a t y p i c a l HPLC s e p a r a t i o n o f t h e b r o m o p h e n o l s f r o m R. l a r i x . L a n o s o l was c h o s e n as an i n d i c a t o r o f t h e l e v e l o f t o t a l b r o m o p h e n o l s i n t h e p l a n t . 60 T 0.02AU 1 i — i — i — i — i — i — i — i — i — i — r 0 2 4 6 8 .10 TIME (min) F i g u r e 10. A t y p i c a l HPLC s e p a r a t i o n o f Rhodomela l a r i x bromophenols used f o r the quant-i t a t i v e d e t e r m i n a t i o n of l a n o s o l ( i n d i c a t e d by the arrow). The chromatographic c o n d i t i o n s are l i s t e d i n the t e x t . 6 1 The p e a k c o r r e s p o n d i n g t o l a n o s o l i s w e l l s e p a r a t e d a n d c a n e a s i l y a n d a c c u r a t e l y be q u a n t i f i e d b y t h i s m e t h o d . The y e a r l y r a n g e o f l a n o s o l l e v e l s i n R. l a r i x i s 1 .2 t o 3 .8% o n a d r y w e i g h t b a s i s ( F i g u r e 1 1 ) . The l e v e l s i n w i n t e r a r e e s s e n t i a l l y t h r e e t i m e s t h o s e o f t h e summer m o n t h s . Ragan a n d J e n s e n ( 1 9 7 8 ) f o u n d s i m i l a r m a x i m a a n d m i n i m a i n t h e b r o w n a l g a e w h i c h t h e y e x a m i n e d f o r p o l y p h e n o l s . The r e s u l t s o f t h e i n t e r p o p u l a t i o n a l c o m p a r i s o n s a r e shown i n T a b l e V I . The w i d e r a n g e o f l a n o s o l l e v e l s among t h e t h r e e p o p u l a t i o n s e x a m i n e d h e r e i s t y p i c a l o f t h e v a r i a t i o n o b s e r v e d f o r t h i s s p e c i e s ( T a b l e V ) . The t i d e p o o l f o r m h a d h i g h e r l a n o s o l l e v e l s i n t h e summer m o n t h s t h a n d i d e i t h e r t h e s u b t i d a l B a t h I s l a n d o r Bam-f i e l d p o p u l a t i o n s . Ragan a n d J e n s e n ( 1 9 7 8 ) h a v e p o i n t e d o u t t h a t t h e h i g h e r l e v e l s o f p o l y p h e n o l s i n b r o w n a l g a e o c c u r n o t d u r i n g t h e p e r i o d o f max imum p o t e n t i a l e p i p h y t i z a t i o n ( s p r i n g a n d summer) b u t d u r i n g t h e f a l l a n d w i n t e r m o n t h s when c o l o n i z a t i o n b y t h e s m a l l e r m a r i n e a l g a e i s a t a m i n i m u m . F o r R h o d o m e l a t h e same p a t t e r n was o b s e r v e d . B o t h t i d e p o o l a n d s u b t i d a l p l a n t s a r e t y p i c a l l y e p i p h y -t i z e d i n t h e e a r l y s p r i n g i n c r e a s i n g t o a maximum e p i p h y t e c o v e r b y e a r l y t o m i d summer . T h e r e a f t e r t h e d i v e r s i t y o f R h o d o m e l a e p i f l o r a d e c r e a s e s u n t i l i n w i n t e r n o n e i s 50 40 r 1 > a o w o 30 /"-\ 3 UP 0Q rt 20 10 -veg-REPRODUCTIVE STATUS -HI ® - II- veg-1 40 rt 30 3 ft) rt S3 rt 20 £ 10 J M M J J T IME ( m o n t h s ) A 0 N F i g u r e 1 1 . S e a s o n a l v a r i a t i o n o f l a n o s o l c o n t e n t i n R h o d o m e l a l a r i x ( s o l i d l i n e ) a n d c h a n g e i n t h e d r y t o w e t w e i g h t r a t i o o v e r t h e p e r i o d o f one y e a r ( d a s h e d l i n e ) . The d e v i a t i o n f r o m t h e mean f o r a l l s a m p l e s i s i n d i c a t e d b y v e r t i c a l b a r s a n d t h e r e p r o d u c t i v e s t a t e o f t h e p l a n t s b y h o r i z o n t a l b a r s (® = t e t r a s p o r i c p l a n t s , o_ = c a r p o s p o r i c p l a n t s , v e g = v e g e t a t i v e p l a n t s ) . Table VI. Q u a n t i t a t i v e comparison of l a n o s o l c o n c e n t r a t i o n s i n three d i s t i n c t p o p u l a t i o n s o f Rhodomela l a r i x ( c o l l e c t e d i n August 1978) . POPULATION LANOSOL REPRODUCTIVE (mg/g d.wt.) CONDITION Bamf i e l d (low -,-, Q /. n Q A \ „ i n t e r t i d a l ) 11.8 (+0.30) t e t r a s p o n c Bath I s . (low i n t e r - ± g ( o.17) v e g e t a t i v e and to h i g h s u b t i d a l ) ^- t e t r a s p o r i c T i d e p o o l (Bath Is.) 21.7 (+0.55) v e g e t a t i v e 64 o b s e r v e d . The d a t a f o r i n t r a t h a l l i a l v a r i a t i o n i n l a n o s o l c o n t e n t ( F i g u r e 1 2 ) show m a x i m a l l e v e l s o f t h e compound i n t h e y o u n g e s t a n d m o s t r a p i d l y g r o w i n g p o r t i o n s o f t h e a l g a l t h a l l u s . R h o d o m e l a i s a l m o s t i n v a r i a b l y e p i -p h y t i z e d i n t h e o l d e r r e g i o n s o f t h e t h a l l u s as a r e many o t h e r s p e c i e s o f m a r i n e a l g a e ( B a l l a n t i n e 1 9 7 9 ) . T h i s c o u l d be a r e s u l t o f t h e l o w e r c o n c e n t r a t i o n s o f l a n o s o l i n t h e s e p o r t i o n s , as c o n t r a s t e d t o t h e g r o w i n g t i p s . L e a c h i n g o u t o r e x u d a t i o n o f t h e compounds d u r i n g a p e -r i o d o f l e s s a c t i v e g r o w t h ( summer ) w o u l d l e a d t o l o w e r l e v e l s o f t h e compounds f o r t h e e n t i r e p l a n t . T h i s c o u l d e x p l a i n t h e r a p i d d r o p i n l a n o s o l c o n t e n t as summer p r o -c e e d s . H i g h e r l e v e l s o f i n s o l a t i o n a n d h i g h e r w a t e r t e m p e r a t u r e s , o r a c o m b i n a t i o n o f f a c t o r s m i g h t a f f e c t t h e l e v e l s o r r a t e o f c h a n g e i n t h e l e v e l s o f l a n o s o l i n t h e p l a n t s . F u r t h e r s t u d y c o r r e l a t i n g t h e p e r i o d o f m o s t a c t i v e g r o w t h w i t h c h a n g e s i n l a n o s o l l e v e l s s h o u l d p r o -v i d e a b e t t e r u n d e r s t a n d i n g o f t h e r o l e o f l a n o s o l i n e p i p h y t e c o n t r o l . L a n o s o l h a s b e e n shown t o b e a n e f f e c t i v e a n t i -b a c t e r i a l a n d a n t i f u n g a l a g e n t ( S i l v a a n d B i t t n e r 1 9 7 9 ) . A g a i n , as w i t h b r o w n a l g a l p h e n o l s ( R a g a n a n d J e n s e n 1 9 7 8 ) , t h e e c o l o g i c a l s i g n i f i c a n c e o f b r o m o p h e n o l s t o r e d a l g a e may be i n t h e c o n t r o l o f p a t h o g e n s r a t h e r t h a n e p i p h y t e s . 65 LANOSOL ( m g / g d . w t . ) 4 5 . 0 T I P S 4 2 . 1 REGION OF DENSE LATERALS 2 1 . 3 8 . 6 REGION OF FEW LATERALS HOLDFAST F i g u r e 1 2 . L a n o s o l c o n t e n t i n d i f f e r e n t p a r t s o f t h e R h o d o m e l a l a r i x t h a l l u s , ( c o l l e c t e d f r o m t i d e p o o l s o n B a t h I s . , B . C . i n S e p t e m b e r , 1 9 7 9 ) . 66 The c o n t r o l o f h e r b i v o r y by these compounds i s a l s o not excluded. The author has shown (Chapter VI) t h a t l a n o s o l and, at much h i g h e r c o n c e n t r a t i o n s , i t s dipot a s s i u m s u l f a t e s a l t are e f f e c t i v e as r e p e l l a n t s a g a i n s t t i d e p o o l s n a i l s . T h i s e f f e c t c o u l d extend to other p o t e n t i a l pred-a t o r s as w e l l . The p o s s i b i l i t y t h a t these compounds are waste products o f metabolism (Pedersen e_t a l . 1979) seems h i g h l y u n l i k e l y . Biochemical e v o l u t i o n a r y trends do not gener-a l l y l e a d to the formation o f by-products more complex and p o t e n t i a l l y more t o x i c than the s t a r t i n g m a t e r i a l s from which they are s y n t h e s i z e d (Swain 1977). N e i t h e r should bromophenols be c o n s i d e r e d storage products s i n c e t h e i r minimum accumulations occur d u r i n g the p e r i o d when environmental c o n d i t i o n s would f a v o r i n c r e a s e d photo-s y n t h e s i s and, subsequently, i n c r e a s e d food storage. From the r e s u l t s o f these experiments one can conclude that the scope of p o s s i b l e f u n c t i o n s f o r bromo-phenols has been c o n s i d e r a b l y narrowed. U n t i l a more c a r e f u l assessment of the e f f e c t i v e n e s s of l a n o s o l as a n t i b i o t i c , a n t i e p i p h y t e or a n t i h e r b i v o r e agent i s made, the s i g n i f i c a n c e of bromophenols i n the ecology o f red algae w i l l remain u n c l e a r . Work done along these l i n e s w i l l be presented i n Chapter VI. CHAPTER V EXUDATION OF BROMOPHENOLS BY RHODOMELA LARIX 67 68 INTRODUCTION The e x u d a t i o n o f t a n n i n s , p o l y p h e n o l s a n d o t h e r r e d u c i n g s u b s t a n c e s ( " G e l b s t o f f " ) b y b r o w n a l g a e h a s l o n g b e e n r e c o g n i z e d ( W a n g e r s k y 1 9 7 8 ) . Ragan a n d J e n s e n ( 1 9 7 9 ) h a v e r e c e n t l y e x a m i n e d t h e c o n t r o l o f e x u d a t i o n i n A s c o p h y l l u m n o d o s u m a n d shown t h a t t h i s a l g a p r o d u c e s p o l y p h e n o l s w h o s e r a t e o f r e l e a s e i s e n h a n c e d i n l i g h t . When s u c h a l g a e o c c u r i n t i d e p o o l s , t h e a c c u m u l a t i o n o f t h e i r y e l l o w b r o w n e x u d a t e s i s k n o w n t o be e f f e c t i v e i n a n t i b i o s i s a g a i n s t many g r o u p s o f o r g a n i s m s , r a n g i n g f r o m b a c t e r i a t o p l a n k t o n i c a n d s e s s i l e m a r i n e a n i m a l s ( C o n o v e r a n d S i e b u r t h 1 9 6 6 , S i e b u r t h a n d J e n s e n 1 9 6 9 , L a n g l o i s 1 9 7 5 ) . L e s s s t u d i e d a r e t h e e x u d a t e s o f m a r i n e r e d a l g a e , w h i c h a r e k n o w n ( K h a i l o v a n d B u r l a k o v a 1 9 6 9 , L a n g l o i s 1 9 7 5 ) t o r e l e a s e c o p i o u s a m o u n t s o f d i s s o l v e d o r g a n i c m a t t e r (DOM). T h e s e e x u d a t e s h a v e n o t b e e n c h e m i c a l l y c h a r a c t e r -i z e d , b u t o n l y e s t i m a t e s o f t o t a l p h e n o l s o r c a r b o h y d r a t e s ( o r o f b o t h as DOM) h a v e b e e n made ( K h a i l o v a n d B u r l a k o v a 1 9 6 9 , L a n g l o i s 1 9 7 5 ) . Red a l g a e c o n t a i n h i g h l e v e l s o f c a r b o n - h a l o g e n compounds ( F e n i c a l 1 9 7 5 ) , many o f w h i c h a r e a n t i b i o t i c ( S i l v a a n d B i t t n e r 1 9 7 9 ) . I f i t i s t h e s e c o m -p o u n d s t h a t a r e e x u d e d , t h e n t h e i r p r e s e n c e i n o c e a n w a t e r s 69 i n s i g n i f i c a n t c o n c e n t r a t i o n s r a i s e s i n t e r e s t i n g eco-l o g i c a l questions as to the advantage a f f o r d e d the a l g a producing them (Pedersen e_t aL. 1974, F e n i c a l 1975). Such compounds may f u n c t i o n i n c o n t r o l l i n g e p i p h y t i z a t i o n , h e r b i v o r y or f o u l i n g of marine algae by other algae, animals or microorganisms. In the marine environment, p h y s i c a l and chemical f a c t o r s such as pH, s a l i n i t y and temperature p l a y an im-p o r t a n t p a r t i n the p h y s i o l o g y of macrophytes ( B i e b l 1962, Soeder and Stengel 1974) and should a l s o a f f e c t exudation processes such as the r a t e of r e l e a s e of DOM as polyphenols and as other substances. In t h i s study I examined the e f f e c t s of s e l e c t e d environmental m o d i f i c a t i o n s on the r a t e and q u a l i t y of exudation by Rhodomela l a r i x (Turner) C. Agardh, obtained from h i g h i n t e r t i d a l t i d e p o o l s i n B r i t i s h Columbia. During the day an i n c r e a s i n g amount of y e l l o w to r e d d i s h brown d i s c o l o r a t i o n becomes apparent i n c o a s t a l p o ols c o n t a i n i n g t h i s s p e c i e s . Much o f t h i s c o l o r a t i o n i s a t t r i b u t a b l e to p h e n o l i c compounds (see Chapter I ) ; however, e a r l i e r attempts at the i d e n t i f i c a t i o n of the i n d i v i d u a l components of the exudates were un-s u c c e s s f u l . I have now reexamined the exudates of R. l a r i x i n c o n t r o l l e d l a b o r a t o r y experiments and am able not o n l y to c o n t r o l exudation w i t h i n normal ranges of environmental 70 f a c t o r s , b u t a l s o t o i d e n t i f y a n d q u a n t i f y s e p a r a t e l y t h e m a j o r p h e n o l i c c o m p o n e n t o f e x u d a t e s . MATERIALS AND METHODS C o l l e c t i o n a n d M a i n t e n a n c e o f A l g a l S p e c i m e n s A l g a e w e r e c o l l e c t e d f r o m t i d e p o o l s i n t h e h i g h i n t e r t i d a l o r f r o m t h e u p p e r s u b t i d a l z o n e o f B a t h I s l a n d . P l a n t s w e r e c l e a n e d o f e p i p h y t e s , w a s h e d i n f r e s h s e a w a t e r a n d i m m e d i a t e l y r e t u r n e d t o t h e l a b o r a t o r y w r a p p e d i n n e w s p a p e r o v e r i c e . I n t h e l a b t h e p l a n t s w e r e w a s h e d i n f i l t e r e d s e a w a t e r ( 0 . 4 5 u r n ) , r e c h e c k e d f o r e p i p h y t e s a n d m a i n t a i n e d i n 2 . 5 1 " l o w f o r m " c u l t u r e f l a s k s on g y r o -r o t a r y s h a k e r s ( 6 0 r p m ) f o r 48 h r s u n d e r m e d i u m l i g h t ( 3 3 0 u E . m ~ 2 s e c _ 1 , l i g h t c y c l e = 1 2 : 1 2 ) a t 1 5 ° C . A f t e r t h i s " a d j u s t m e n t " p e r i o d t h e a l g a e w e r e u s e d i n e x u d a t i o n e x p e r i m e n t s . E x p e r i m e n t s I n v o l v i n g M e a s u r e m e n t o f T o t a l P h e n o l s T e n g r a m s o f e a c h a l g a l t y p e ( s u b t i d a l a n d t i d e p o o l c o l l e c t i o n s ) w e r e w a s h e d i n 0 . 2 2 urn f i l t e r e d s e a w a t e r a n d t r a n s f e r r e d ( i n d u p l i c a t e ) t o 100 m l o f t h e same i n 250 m l E r l e n m e y e r f l a s k s . An a d d i t i o n a l p a i r o f f l a s k s was s e t up as a d a r k c o n t r o l b y w r a p p i n g t h e m i n s e v e r a l l a y e r s o f a l u m i n u m f o i l t o e x c l u d e l i g h t . The f l a s k s w e r e r e t u r n e d t o t h e s h a k e r a n d t h e t e m p e r a t u r e r a i s e d t o 2 0 ° C . T o t a l 7 1 p h e n o l s w e r e m e a s u r e d a t 0 , 4 , 8 a n d 24 h r s a c c o r d i n g t o t h e m e t h o d o f L a n g l o i s ( 1 9 7 5 ) . O p t i c a l d e n s i t y was r e a d o n a Pye U n i c a m M o d e l S P - 8 5 0 0 U V - V I S S p e c t r o p h o t o m e t e r a n d c o n v e r t e d t o p h e n o l c o n c e n t r a t i o n s as ppm o f p h l o r o -g l u c i n o l . E x u d a t i o n u n d e r D i f f e r e n t E n v i r o n m e n t a l C o n d i t i o n s T h r e e C o n t r o l l e d E n v i r o n m e n t s ( M o d e l E 1 5 ) g r o w t h c h a m b e r s w e r e u s e d a t t e m p e r a t u r e s o f 1 0 , 20 a n d 3 0 ° C . L i g h t c o n d i t i o n s w e r e s e t so t h a t a r a n g e o f f r o m 0 - 1 0 0 0 uE m~ s e c " c o u l d be a c h i e v e d i n t h e 2 0 ° C c h a m b e r . F o r a t t e n u a t i o n t o t h e l o w e r l i g h t i n t e n s i t i e s , m u l t i p l e l a y e r s o f c h e e s e c l o t h w e r e p l a c e d o v e r f o i l - l i n e d b o x e s . L i g h t was m e a s u r e d w i t h a L i - C o r L I - 185 Q u a n t u m m e t e r f i t t e d w i t h a M o d e l UWQ 2192 p r o b e . The l i g h t s o u r c e i n e a c h c h a m b e r was a b a n k o f 24 D u r o - t e s t V i t a l i g h t s ( 7 2 T 1 2 ) w h i c h w e r e u s e d a t f u l l o u t p u t f o r t h e h i g h e r l i g h t i n t e n s i t y . Two h u n d r e d a n d f i f t y m l e x u d a t i o n f l a s k s c o n t a i n i n g 100 m l f i l t e r e d s e a w a t e r w e r e s e t up as b e f o r e . F o r a s e r i e s o f i n c r e a s i n g s a l i n i t i e s ( 5 , 1 5 , 25 a n d 35 ° / o o , o r p a r t s p e r t h o u s a n d o f . s a l i n i t y ) a n d pHs (6, 7 , 8 a n d 9), f i l t e r e d s e a w a t e r was e i t h e r d i l u t e d w i t h g l a s s d i s t i l l e d w a t e r ( o r p a r t i a l l y e v a p o r a t e d f o r 35 ° / o o ) o r t h e pH was a d j u s t e d w i t h d i l u t e HC1 o r NaOH. I n t o e a c h f l a s k ( i n d u p l i c a t e ) f o r e a c h s e t o f c o n d i t i o n s t o . b e t e s t e d , 1 0 . 0 g o f f r e s h l y w a s h e d a n d g e n t l y b l o t t e d t i d e p o o l R. l a r i x w e r e a d d e d a n d t h e f l a s k s p l a c e d i n t h e g r o w t h c h a m b e r s u n d e r t h e a p p r o p r i a t e c o n d i t i o n s . D i s s o l v e d o x y g e n l e v e l s w e r e m o n i t o r e d i n i t i a l l y a n d a t t h e e n d o f t h e e x p e r i m e n t a l p e r i o d (8 h r s ) as an i n d i -c a t i o n o f a l g a l v i a b i l i t y . F i v e r e p l i c a t e d r y w e i g h t ( a f t e r d r y i n g f o r 1 wk a t 9 0 ° C ) d e t e r m i n a t i o n s w e r e made f o r 1 0 . 0 g s a m p l e s w a s h e d a n d b l o t t e d as a b o v e a n d t h e v a l u e s o b t a i n e d a v e r a g e d f o r t h e e s t i m a t i o n o f t h e i n i t i a l d r y w e i g h t s o f e x p e r i m e n t a l s a m p l e s . E x t r a c t i o n a n d C h r o m a t o g r a p h y o f E x u d a t e s A f t e r 8 h r s e a c h f l a s k was r e m o v e d f r o m t h e g r o w t h c h a m b e r , t h e a l g a e f i l t e r e d o f f a n d t h e m e d i u m a c i d i f i e d t o pH 2 w i t h 1 N HC1 a n d w a r m e d 15 m i n o n a s t e a m b a t h . The w a t e r was t h e n e x t r a c t e d w i t h e t h y l a c e t a t e ( 3 x 1 0 0 m l ) , a n d t h e e t h y l a c e t a t e l a y e r s w e r e c o m b i n e d a n d d r i e d o v e r a n h y d r o u s s o d i u m s u l f a t e . A f t e r t h e s o l v e n t was r e m o v e d m v a c u o ( 3 8 ° C ) , t h e r e s i d u e was t a k e n up i n 0 . 5 m l m e t h a n o l a n d u s e d f o r c h r o m a t o g r a p h i c a n a l y s i s . RESULTS AND DISCUSSION A d e f i n i t e i n c r e a s e i n B r e n t a m i n e r e a c t i v e s u b -s t a n c e s i s o b s e r v e d w h e n R h o d o m e l a l a r i x i s e x p o s e d t o m o d e r a t e i r r a d i a t i o n a n d t e m p e r a t u r e s i n a c o n t r o l l e d l a b o r a t o r y e n v i r o n m e n t ( T a b l e V I I ) . T h i s r e l e a s e o c c u r s T a b l e V I I . E x u d a t i o n m e a s u r e d as t o t a l p h e n o l s o v e r a t w e n t y -f o u r h o u r p e r i o d b y t i d e p o o l a n d s u b t i d a l ( b o t h i n t h e l i g h t a n d d a r k ) f o r m s o f R h o d o m e l a l a r i x . Q u a n t i t i e s g i v e n a r e as p a r t s p e r m i l l i o n o f p h l o r o g l u c i n o l . COLLECTION TIME ( h o u r s ) 4 8 24 T i d e p o o l f o r m 5 . 7 6 . 4 4 6 . 5 S u b t i d a l f o r m o i c n/ n ^ o i /. -i . i J \ 2 1 . 5 2 4 . 0 6 3 . 1 ( m l i g h t ) S u b t i d a l f o r m ( i n d a r k ) 6 . 3 6 . 5 4 7 . 3 74 f r o m a l g a l t h a l l i w h i c h h a v e b e e n g i v e n t i m e t o a d j u s t t o t h e " s h o c k " o f b e i n g t r a n s f e r r e d f r o m t h e i r n a t u r a l h a b i t a t t o t h e c u l t u r e c h a m b e r . The r a t e o f r e l e a s e i s r a t h e r c o n s t a n t w h e t h e r t h e a l g a e a r e k e p t i n t h e l i g h t o r d a r k . S i n c e t h e t i d e p o o l f o r m o f R. l a r i x r e l e a s e s l e s s t o t a l p h e n o l s , i t seems b e t t e r a d a p t e d t o l o n g e r a n d m o r e i n t e n s e p e r i o d s o f i n s o l a t i o n t h a n does t h e s u b t i d a l f o r m . T h i s w o u l d a p p e a r t o b e t y p i c a l o f many p h y s i o -l o g i c a l r e s p o n s e s e x a m i n e d i n m a r i n e m i c r o - a n d m a c r o -p h y t e s ( B i e b l 1 9 6 2 , S o e d e r a n d S t e n g e l 1 9 7 4 ) . I n c r e a s e d " s t r e s s " p r o m o t e s a g r e a t e r r e s p o n s e a n d i n t h e c a s e o f t h e s u b t i d a l f o r m , w h i c h i s e x p o s e d t o l o w e r l i g h t i n t e n -s i t i e s a n d c o o l e r w a t e r t e m p e r a t u r e s t h a n i t s h i g h i n t e r -t i d a l c o u n t e r p a r t f o r m o s t o f t h e y e a r , s u c h s t r e s s . i n c r e a s e s t h e r a t e o f e x u d a t i o n . HPLC a n a l y s i s o f t h e i n d i v i d u a l p h e n o l s i n t h e 24 h r e x u d a t e shows l a n o s o l t o be t h e m a j o r c o m p o n e n t ( F i g u r e 1 3 ) . P e d e r s e n e t a l . ( 1 9 7 4 ) h a v e a l s o f o u n d t h i s compound i n s e a w a t e r o f t h e P o l y s i p h o n i a b r o d i a e i z o n e on S w e d i s h s h o r e s . T h e r e f o r e , i t a p p e a r s l i k e l y t h a t e x u d a t i o n o f b r o m o p h e n o l s i s a w i d e s p r e a d p h e n o m e n o n , e v e n f o r a l g a e s t i l l i n t h e i r n a t u r a l h a b i t a t s . The e x p e r i m e n t a l c o n d i t i o n s a n d r e s u l t s g i v e n as m g / m l o f l a n o s o l e x u d e d a r e g i v e n i n T a b l e V I I I . As t e m -p e r a t u r e i n c r e a s e s so does l a n o s o l e x u d a t i o n a n d 75 F i g u r e 13. HPLC of the twenty-four hour exudate of the s u b t i d a l form of Rhodomela l a r i x kept i n the l i g h t f o r 24 hrs (see Table VII and p. 70). 76 T a b l e V I I I . Q u a n t i t a t i v e d e t e r m i n a t i o n o f e x u d e d l a n o s o l a n d o t h e r o b s e r v a t i o n s u n d e r v a r y i n g e x p e r i m e n t a l c o n d i t i o n s . U n l e s s s p e c i f i e d , t e m p e r a t u r e = 20°C, ° / o o = 2 5 , l i g h t i n t e n s i t y = 330 uE n T ^ s e c - - ' - a n d pH = 8 . The i n i t i a l o x y g e n c o n c e n t r a t i o n was t a k e n as 0 ppm a n d t h e mean i n i t i a l d r y w e i g h t was 1 . 8 5 g . ( * ) = r e p l i c a t e a t 1000 uE m ' ^ s e c " t o d e t e r m i n e r a t i o o f l a n o s o l t o l a n o s a l t e x u d e d o v e r t h e e x p e r i m e n t a l p e r i o d . CONDITION FINAL FINAL OXYGEN LANOSOL pH d r y w t . (ppm) ( m g / m l ) T 10°C 8 . 0 1 . 7 8 7 . 0 2 0 . 0 2 E M 20°C 8 . 0 1 . 9 0 6 . 5 1 0 . 0 5 P. 30°C 4 . 5 1 . 4 1 1 . 5 9 0 . 5 6 S A 5 / o o 5 . 4 1 . 3 3 5 . 2 8 0 . 6 1 L I 1 5 ° / o o 8 . 6 1 . 8 3 5 . 9 8 0 . 0 4 N I 2 5 ° / o o 8 . 6 1 . 8 0 7 . 1 3 0 . 0 4 T Y 3 5 ° / o o 8 . 5 1 . 8 5 6 . 9 5 0 . 0 3 0 6 . 7 1 . 6 6 1 . 6 6 0 . 0 4 L I 100 7 . 8 1 . 9 3 6 . 3 5 0 . 0 3 G H 330 8 . 0 1 . 9 0 6 . 5 1 0 . 0 5 T 1000 4 . 9 1 . 5 1 5 . 2 5 3 . 0 1 0 0 0 ( - H + ) 4 . 6 1 . 3 5 5 . 1 5 3 . 3 ( * ) 1 0 0 0 ( + H + ) - - - 0 . 1 7 6 7 . 9 1 . 7 9 5 . 1 9 0 . 0 3 P 7 7 . 4 1 . 7 7 6 . 2 5 0 . 0 5 H 8 8 . 9 1 . 7 8 6 . 5 1 0 . 0 4 9 8 . 9 1 . 9 8 6 . 8 6 0 . 0 4 d i s c o l o r a t i o n o f t h e m e d i u m ( F i g u r e 1 4 ) ^ An o p p o s i t e e f f e c t i s s e e n w i t h r e g a r d t o i n c r e a s i n g s a l i n i t y . L o w e r s a l i n i t i e s p r o d u c e d t h e h i g h e s t l e v e l s o f p i g m e n t a t i o n a n d l a n o s o l i n t h e m e d i u m ( F i g u r e 1 4 ) . S a e n g e r ( 1 9 7 0 ) h a s p r e v i o u s l y e x t r a c t e d t h e r e d p i g m e n t f l o r i d o r u b i n f r o m s e v e r a l s p e c i e s o f r e d a l g a e b y p l a c i n g t h e m i n d i s t i l l e d w a t e r f o r e x t e n d e d p e r i o d s . T h i s p i g m e n t u p o n h y d r o l y s i s y i e l d s a v a r i e t y o f h a l o p h e n o l s i n c l u d i n g l a n o s o l ( S a e n g e r e t a l . 1 9 7 6 ) . V a r y i n g pH h a d l i t t l e e f f e c t o n e x u d a t i o n , w h i l e h i g h e r l i g h t i n t e n s i t i e s i n -c r e a s e d e x u d a t i o n ( F i g u r e 1 5 ) w i t h o u t a p p a r e n t damage t o t h e a l g a e ( s e e o x y g e n e v o l u t i o n d a t a ) . The h i g h e s t t e m p e r a t u r e s , o n t h e o t h e r h a n d , w e r e d a m a g i n g ( r e s p i r a -t i o n e x c e e d i n g p h o t o s y n t h e s i s ) . The g r e a t e r r a t e s o f r e l e a s e f o r a l g a e u n d e r t h e s e c o n d i t i o n s w e r e p r o b a b l y due t o t h e l e a c h i n g o u t o f l a n o s o l f r o m d e a d o r d y i n g c e l l s . The i n i t i a l pH o f a l l m e d i a , e x c e p t t h o s e w h e r e t h i s f a c t o r was a l t e r e d , was 8 . 0 . I n e v e r y c a s e w h e r e t h e r e was a l a r g e r e l e a s e o f l a n o s o l i n t o t h e w a t e r s u r -r o u n d i n g t h e a l g a e , t h e pH d r o p p e d w e l l b e l o w 5 . 0 due t o an a c c u m u l a t i o n o f t h e a c i d i c p h e n o l i c s ( k p h e n o l = 10 ^ ) i n t h e m e d i u m . H o w e v e r , h a d t h e cl p h e n o l s b e e n e x u d e d as s u l f a t e . e s t e r s , t h e o b s e r v e d pH c h a n g e w o u l d n o t h a v e b e e n as g r e a t . As a means o f 78 F i g u r e 14. E f f e c t of temperature and s a l i n i t y on the p r o d u c t i o n of c o l o r e d matter by Rhodomela l a r i x during an e i g h t hour exudation experiment" TEe algae have been removed from the media to improve c l a r i t y o f the c o l o r s . (see Table V I I I f o r f u r t h e r d e t a i l s of c o n d i t i o n s ) 79 F i g u r e 15. E f f e c t of pH and l i g h t on the p r o d u c t i o n of c o l o r e d matter by Rhodomela  l a r i x d u r i n g an e i g h t hour exudation experiment. The algae have been removed from the media to improve c l a r i t y of the c o l o r s , (see Table V I I I f o r f u r t h e r d e t a i l s of conditions) 80 d e t e r m i n i n g t h e c h e m i c a l n a t u r e o f e x u d e d l a n o s o l , a - 2 - 1 r e p l i c a t e , i l l u m i n a t e d a t 1000 uE m s e c , was u s e d a n d e x t r a c t e d w i t h o u t b e i n g p r e v i o u s l y a c i d i f i e d . The m e d i u m was t h e n r e e x t r a c t e d a f t e r a c i d i f i c a t i o n . The a d d i t i o n a l l a n o s o l r e m o v e d f r o m t h e a c i d i f i e d f r a c t i o n c o n s t i t u t e d o n l y 5 . 2 % o f t h a t o b t a i n e d u p o n u n a c i d i f i e d e x t r a c t i o n ( T a b l e V I I I ) . As a r e s u l t , I c o n c l u d e t h a t l a n o s o l i s e x u d e d as t h e f r e e p h e n o l . A n o t h e r q u e s t i o n w h i c h a r i s e s i s w h e t h e r s u l f a t e e s t e r i f i e d l a n o s o l i s b r o k e n down b y b a c t e r i a o r o t h e r m i c r o o r g a n i s m s a f t e r e x u d a t i o n i n t o t h e m e d i u m . I n o r d e r t o a n s w e r t h i s q u e s t i o n , 1 mg o f t h e d i p o t a s s i u m s u l f a t e s a l t o f l a n o s o l ( s e e C h a p t e r I I I ) was a d d e d t o 10 m l " d i r t y " s e a w a t e r o b t a i n e d d i r e c t l y f r o m t h e t i d e p o o l f r o m w h i c h R. l a r i x was o r i g i n a l l y c o l l e c t e d . A f t e r 24 h r s a t 20°C u n d e r h i g h l i g h t , n o l a n o s o l c o u l d be d e t e c t e d e v e n a t . t h e h i g h e s t d e t e c t o r s e n s i t i v i t i e s . C o n v e r s i o n o f t h e d a t a i n T a b l e V I I I i n t o mg l a n o s o l e x u d e d / g d r y w e i g h t h o u r g i v e s t h e c o m p a r a t i v e r a t e s o f e x u d a t i o n i l l u s t r a t e d i n F i g u r e 1 6 . A l l o f t h e p h y s i c a l a n d c h e m i c a l m o d i f i c a t i o n s u s e d i n t h e s e e x p e r i m e n t s f a l l w i t h i n r a n g e s n o r m a l l y o c c u r r i n g i n t h e t i d e p o o l s i n summer m o n t h s ( s e e C h a p t e r I ) , a n d w i t h i n t h i s s e t o f c o n d i t i o n s t h e e x u d a t i o n r a t e s f o r t h i s s p e c i e s r a n g e f r o m a b o u t 1 - 1 5 0 u g / g d . w t . h r . o.oooi 1— 1 1 1 1 1 1 1 I 1 ' 1 1 ! 1 0 100 330 1000 5 15 25 35 ( 6 ) 10 ( 7 ) 20 ( 8 ) 30 ( 9 ) L IGHT S A L I N I T Y TEMPERATURE (pH) . F i g u r e 1 6 . E f f e c t o f l i g h t , s a l i n i t y , t e m p -e r a t u r e a n d pH (" ) on e x u d a t i o n o f l a n o s o l b y R h o d o m e l a l a r i x . 82 R. l a r i x c o n t a i n s f r o m 1-4% o f l a n o s o l o n a d r y w e i g h t b a s i s ( s e e C h a p t e r I V ) . A t t h e r a t e s o f r e l e a s e f o u n d h e r e , t h i s a l g a i s e x u d i n g 0 . 3 - 1 . 6 % o f i t s t o t a l l a n o s o l c o n t e n t p e r h o u r , i n d i c a t i n g an a c t i v e t u r n o v e r a n d , h e n c e , s y n t h e s i s o f t h i s compound w i t h i n t h e p l a n t . The e c o l o g i c a l a n d p h y s i o l o g i c a l i m p l i c a t i o n s o f t h e s e f i n d -i n g s a r e t h u s m o s t i n t r i g u i n g . As n o t e d i n Ragan a n d J e n s e n ( 1 9 7 9 ) , i t h a s b e e n s u g g e s t e d t h a t i n c r e a s e d r a t e s o f e x u d a t i o n may b e m o r e s i g n i f i c a n t i n t h e s p r i n g a n d summer m o n t h s . I n C h a p -t e r I V I e x a m i n e d t h e l e v e l s o f l a n o s o l i n R. l a r i x o v e r t h e p e r i o d o f a y e a r a n d f o u n d t h a t c o n c e n t r a t i o n s i n t h e p l a n t s a r e l o w e s t d u r i n g t h e summer m o n t h s , p r e -c i s e l y when t h e e n v i r o n m e n t a l c o n d i t i o n s a r e m o s t c o n -d u c i v e t o h i g h e x u d a t i o n r a t e s . The l o w e r l e v e l s o f l a n o s o l i n t h e p l a n t s f r o m May t h r o u g h A u g u s t may i n f a c t be due t o i n c r e a s e d e x u d a t i o n a n d n o t t o s l o w e d b i o -s y n t h e s i s o f b r o m o p h e n o l s . The c o r r e s p o n d i n g l y l o w l e v e l s o f b o t h p o l y p h e n o l s ( R a g a n a n d J e n s e n 1 9 7 8 ) a n d b r o m o p h e n o l s ( a s s u m i n g l a n o s o l t o be an i n d i c a t o r o f t o t a l p h e n o l s ) d u r i n g t h i s p e r i o d o f max imum p o t e n -t i a l e p i p h y t i z a t i o n o f t h e a l g a e ( s p r i n g t o m i d - summer) may be a r e f l e c t i o n o f i n c r e a s e d e x u d a t i o n as a r e s p o n s e t o h i g h e r l e v e l s o f a t t e m p t e d e p i p h y t i z a t i o n . CHAPTER VI BROAD SPECTRUM ANTIBIOTIC ACTIVITY OF BROMOPHENOLS FROM RHODOMELA LARIX 83 84 INTRODUCTION The s i g n i f i c a n c e o f a n t i b i o t i c p r o d u c t i o n t o t h e e c o l o g y o f l a n d p l a n t p o p u l a t i o n s h a s l o n g b e e n r e c o g -n i z e d ( W h i t t a k e r 1 9 7 0 ) . O n l y r e c e n t l y h a s t h e r e b e e n a n e f f o r t t o d e t e r m i n e a s i m i l a r s i g n i f i c a n c e i n t h e m a r i n e e n v i r o n m e n t ( B u r k h o l d e r 1973 a n d r e f e r e n c e s t h e r e i n ) . M a -r i n e a l g a e p r o d u c e a v a r i e t y o f " s e c o n d a r y " m e t a b o l i t e s , many o f w h i c h a r e k n o w n t o b e b i o l o g i c a l l y a c t i v e ( S i l v a a n d B i t t n e r 1 9 7 9 ) . The d i v e r s i t y o f t h e s e compounds e n -c o m p a s s e s v i r t u a l l y e v e r y c h e m i c a l c l a s s , a n d t h e y h a v e b e e n f o u n d i n a l l t h e m a j o r d i v i s i o n s o f a l g a e ( S c h e u e r 1 9 7 3 , F a u l k n e r a n d F e n i c a l 1 9 7 7 ) . M o s t o f t h e a v a i l a b l e i n f o r m a t i o n r e g a r d i n g a l g a l a n t i b i o s i s comes f r o m t e s t i n g w i t h c r u d e a l g a l e x t r a c t s . H o w e v e r , t h e n u m b e r o f compounds i s o l a t e d a n d i d e n t i f i e d h a s i n c r e a s e d s u b s t a n t i a l l y a n d , s u b s e q u e n t l y , s e v e r a l r e v i e w s h a v e a p p e a r e d o n t h e s u b j e c t ( L e w i n 1 9 6 2 , W o l t e r s 1 9 6 4 , . S i e b u r t h 1 9 6 4 , B a s l o w 1 9 6 9 , B h a k u n i a n d S i l v a 1 9 7 4 ) . U n f o r t u n a t e l y , f e w o f t h e s e compounds h a v e b e e n e x a m i n e d f o r b r o a d s p e c t r u m a n t i b i o t i c a c t i v i t y . H e n c e , we h a v e l i t t l e k n o w l e d g e o f t h e b e n e f i t a f f o r d e d a n a l g a b y t h e compound o r g r o u p o f compounds w h i c h i t p r o d u c e s . 85 A n t i m i c r o b i a l t e s t s , a l t h o u g h r a p i d , s i m p l e a n d w i d e l y u s e d , o f t e n p r o v i d e l i t t l e i n f o r m a t i o n r e g a r d i n g t h e r a n g e o f b i o l o g i c a l e f f e c t s a p a r t i c u l a r c o m p o u n d may p o s s e s s . W i t h t h i s i n m i n d , I c h o s e t o e x a m i n e t h e a c t i v -i t y o f a b r o m o p h e n o l i c s u b s t a n c e , l a n o s o l , a n d i t s n a t u -r a l l y o c c u r r i n g s a l t ( h e r e c a l l e d " l a n o s a l t , " 2 , 3 - d i b r o m o -5 - h y d r o x y - b e n z y l - l ' , 4 - d i s u l f a t e ) o n a v a r i e t y o f o r g a n i s m s , many o f t h e m m a r i n e . L a n o s a l t i s t h e m a j o r p h e n o l i c c o n -s t i t u e n t o f t h e m a r i n e a l g a R h o d o m e l a l a r i x ( T u r n e r ) C. A g a r d h ( W e i n s t e i n §_t a_l. 1 9 7 5 ) , w h i c h i s a b u n d a n t a l l a l o n g t h e P a c i f i c c o a s t o f N o r t h A m e r i c a ( A b b o t t a n d H o l l e n b u r g 1 9 7 6 ) . L i t t l e i s k n o w n o f t h e e c o l o g i c a l o r p h y s i o l o g i c a l s i g n i f i c a n c e o f b r o m o p h e n o l s t o t h i s r e d a l g a ; h o w e v e r , o n n u m e r o u s o c c a s i o n s m e n t i o n o f t h e a n t i -b i o t i c a c t i v i t y o f b r o m o p h e n o l s h a s b e e n made ( M a u t n e r e t a l . 1 9 5 3 , S a i t o a n d Samesh ima 1 9 5 5 , A l l e n a n d Dawson 1 9 6 0 , W e i n s t e i n e_t a l . 1 9 7 5 ) . The o n l y t r u l y q u a n t i t a -t i v e s t u d y o f b r o m o p h e n o l a n t i b i o t i c a c t i v i t y was done b y M c L a c h l a n a n d C r a i g i e ( 1 9 6 6 ) , i n w h i c h t h e y e x a m i n e d t h e e f f e c t o f l a n o s o l a n d t w o o t h e r p h e n o l i c s o n a n u m -b e r o f p l a n k t o n i c a l g a e . I n t h i s s t u d y I h a v e made an e f f o r t t o i n c l u d e t h e q u a n t i t a t i v e a s p e c t s o f b r o m o p h e n o l a n t i b i o s i s , b o t h f r o m e x t r a c t s a n d e x u d a t e s as w e l l as f r o m p u r i f i e d c o m p o u n d s . 86 MATERIALS AND METHODS A l g a l E x t r a c t i o n a n d I s o l a t i o n o f Compounds R. l a r i x ( a p p r o x . 2 . 5 k g , 500 g d r y w e i g h t ) was c o l l e c t e d i n t h e u p p e r s u b t i d a l z o n e o f f B a t h I s l a n d i n J a n u a r y , 1 9 8 0 . The a l g a was l y o p h i l i z e d , g r o u n d i n a W i l e y m i l l (2 mm m e s h ) a n d e x t r a c t e d i n a S o x h l e t a p p a -r a t u s w i t h a s e r i e s o f s o l v e n t s ( W e i n s t e i n e t al_. 1 9 7 5 ) . E v a p o r a t i o n o f t h e m e t h a n o l e x t r a c t p r o d u c e d a l a r g e a m o u n t o f s o l i d ( 4 4 . 7 g ) w h i c h was c o l l e c t e d , w a s h e d w i t h a n d r e c r y s t a l l i z e d f r o m m e t h a n o l . P h y s i c a l d a t a f o r t h i s compound w e r e i d e n t i c a l t o t h o s e r e p o r t e d f o r t h e d i p o t a s s i u m s u l f a t e s a l t o f l a n o s o l ( W e i n s t e i n e t a l . 1 9 7 5 ) . L a n o s o l was p r e p a r e d b y t h e m e t h o d o f H o d g k i n e t a l . ( 1 9 6 6 ) . One g o f l a n o s a l t u p o n h y d r o l y s i s p r o -d u c e d 123 mg o f t h e f r e e a l c o h o l . T h i s compound was i d e n t i c a l t o t h e a u t h e n t i c s t a n d a r d . The p u r i t y o f t h e i s o l a t e d compounds was c h e c k e d b y HPLC ( s e e C h a p t e r I I I f o r d e t a i l s ) . O t h e r e x t r a c t s w e r e p r e p a r e d b y g r i n d i n g 1 g o f R. l a r i x i n h o t 80% m e t h a n o l . The m e t h a n o l was r e m o v e d i n v a c u o a n d t h e w a t e r a c i d i f i e d t o pH 2 w i t h d i l u t e 87 HC1 a n d h e a t e d f o r 15 m i n a t 6 0 ° C . E x t r a c t i o n w i t h e t h y l a c e t a t e a n d s u b s e q u e n t r e m o v a l o f t h e s o l v e n t l e f t a d a r k g r e e n r e s i d u e w h i c h was t a k e n up i n 1 . 0 m l o f e t h a n o l (50%) a n d u s e d f o r t h e a n t i b i o t i c e x p e r i m e n t s . The q u a n t i t y o f l a n o s o l i n t h e e x t r a c t s was a s c e r t a i n e d b y HPLC a n d c o m p a r i s o n t o a c a l i b r a t i o n c u r v e p r e p a r e d u s i n g t h e s t a n d a r d . P r e p a r a t i o n o f A l g a l E x u d a t e s T e n g o f t h e a l g a e w e r e p l a c e d i n 100 m l o f 0 . 2 2 um f i l t e r e d s e a w a t e r a n d k e p t a t 20°C w h i l e b e i n g e x p o s e d t o b r i g h t l i g h t ( 1 0 0 0 uE m ~ 2 s e c _ 1 ) f o r 8 h r s . The a l g a e w e r e r e m o v e d a n d t h e w a t e r e x t r a c t e d w i t h e t h y l a c e t a t e a n d w o r k e d up as i n t h e 1 g e x t r a c t a l r e a d y d e s c r i b e d . The q u a n t i t a t i v e d e t e r m i n a t i o n o f l a n o s o l i n t h i s e x u d a t e was a g a i n a c h i e v e d b y HPLC. S c r e e n i n g f o r A c t i v i t y w i t h S t a n d a r d T e s t O r g a n i s m s I n i t i a l s c r e e n i n g u s i n g E s c h e r i c h i a c o l i , S t a p h y l o c o c c u s a u r e u s , S a c c h a r o m y c e s c e r e v i s i a e a n d C a n d i d a a l b i c a n s as t e s t o r g a n i s m s was done o n e i t h e r n u t r i e n t a g a r ( D i f c o ) f o r b a c t e r i a o r S a b o u r a u d d e x t r o s e a g a r ( D i f c o ) f o r f u n g i . A g a r p l a t e s w e r e s e e d e d w i t h t h e m i c r o o r g a n i s m t o be t e s t e d a n d s m a l l f i l t e r p a p e r d i s c s ( d i a m e t e r , 6mm) p r e v i o u s l y l o a d e d w i t h a k n o w n c o n c e n t r a t i o n o f l a n o s o l o r l a n o s a l t w e r e p l a c e d o n t h e s u r f a c e o f t h e p l a t e s . The d i a m e t e r o f t h e z o n e o f g r o w t h 88 i n h i b i t i o n was d e t e r m i n e d a f t e r 24 h r s a t 3 5 ° C . S u b s e q u e n t E x p e r i m e n t s w i t h E.: c o l i L i q u i d m e d i a f o r b a c t e r i a l g r o w t h r a t e e x p e r i m e n t s w e r e p r e p a r e d b y t h e a d d i t i o n o f 1 . 0 g g l u c o s e a n d 5 . 0 g b a c t o p e p t o n e ( D i f c o ) t o one l i t e r d i s t i l l e d w a t e r . I n one s e t o f e x p e r i m e n t s m e d i a w e r e made t o pH 6 - 1 0 w i t h e i t h e r d i l u t e HC1 o r NaOH. To 2 . 0 m l o f e a c h m e d i u m i n s m a l l t e s t t u b e s was a d d e d 0 . 5 m l o f a b a c t e r i a l s u s -p e n s i o n g r o w n up t o s t a t i o n a r y p h a s e t o g i v e an o p t i c a l d e n s i t y o f 0 . 1 . F i f t y u l o f 50% e t h a n o l c o n t a i n i n g 0 . 5 mg o f l a n o s o l o r 5 mg o f l a n o s a l t w e r e a d d e d t o e a c h t u b e a n d t h e g r o w t h r a t e ( c h a n g e i n o p t i c a l d e n s i t y ) m o n i t o r e d o v e r a 10 h r p e r i o d ( i n c u b a t i o n a t 3 5 ° C ) . O p t i c a l d e n -s i t y was r e a d o n a Pye U n i c a m SP 6 - 5 0 0 s p e c t r o p h o t o m e t e r s e t a t 520 nm. I n o t h e r e x p e r i m e n t s m e d i a w e r e p r e p a r e d a t pH 8 a n d b a c t e r i a a d d e d as b e f o r e . V a r y i n g c o n c e n t r a t i o n s o f l a n o s o l , a l g a l e x t r a c t s o r e x u d a t e s i n s m a l l a m o u n t s o f 50% e t h a n o l w e r e a d d e d a n d g r o w t h r a t e s m o n i t o r e d o v e r a 24 h r p e r i o d . E x p e r i m e n t s w i t h M a r i n e F u n g i T h r e e m a r i n e f u n g a l i s o l a t e s w e r e k i n d l y p r o v i d e d b y T . T h o m p s o n , D e p a r t m e n t o f B o t a n y , U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r , B. C. T h e s e w e r e S i g m o i d e a s p . C r a n e , D e n d r y p h i e l l a s a l i n a ( S u t h . ) P u g h e t N i c o t a n d 89 Z a l a r i o n maritimum (Linder) A n a s t a s i o u . A l l experiments w i t h these f u n g i were performed on D i f c o corn meal agar u s i n g sea water (pH 7.0). Stock c u l t u r e s of each s p e c i e s were grown up on the above agar and small agar b l o c k s 2 (0.5 cm ) were e x c i s e d and p l a c e d i n the c e n t e r of t e s t p l a t e s . Around the inoculum at a d i s t a n c e of about 2 cm was p l a c e d a s e r i e s of f i l t e r paper d i s c s c o n t a i n i n g spe-2 c i f i c l a n o s o l c o n c e n t r a t i o n s (3.0-0.03 mg/cm ). A con-t r o l d i s c c o n t a i n e d no l a n o s o l . Growth was slow f o r two o f the three s p e c i e s , and the zone of i n h i b i t i o n was ex-amined a f t e r 1-2 weeks at room temperature. For D e n d r y p h i e l l a an a l t e r n a t e method was used. Stock c u l -t u re p l a t e s of s p o r u l a t i n g m y c e l i a were i n v e r t e d and tapped over c l e a n t e s t p l a t e s to seed the e n t i r e agar sur-face w i t h c o n i d i a . L a n o s o l - c o n t a i n i n g f i l t e r paper d i s c s were p l a c e d on the s u r f a c e as b e f o r e . Germination of the c o n i d i a and m y c e l i a l growth c o u l d be observed w i t h i n two days and the zone of i n h i b i t i o n measured. Tests w i t h T i d e p o o l S n a i l s L i t t o r i n a s c u t u l a t a Gould, was c o l l e c t e d on Bath I s l a n d and maintained i n sea water at 10°C. A n t i b i o t i c t e s t i n g w i t h t h i s organism was done by a m o d i f i c a t i o n o f the method of Ohta (1979). Nine cm f i l t e r paper d i s c s (Whatman No. 1) were p l a c e d i n the bottom of s l i g h t l y l a r g e r g l a s s p e t r i d i s h e s , and 3 ml of sea water were 90 a d d e d t o e a c h . A s e r i e s o f t e s t s o l u t i o n s ( 1 0 0 u l e a c h ) 2 c o n t a i n i n g l a n o s o l ( 0 . 0 0 1 - 0 . 1 m g / c m ) i n e t h a n o l o r 2 l a n o s a l t ( 0 . 1 - 1 0 . 0 m g / c m ) i n w a t e r w e r e e v e n l y a p p l i e d o v e r t h e s u r f a c e o f f i l t e r p a p e r r i n g s ( o u t e r d i a m e t e r , 5 . 5 cm; i n n e r d i a m e t e r , 4 . 0 cm; a r e a , 1 1 . 2 cm ) a n d t h e r i n g s h u n g up a n d a l l o w e d t o a i r d r y . O n l y s o l v e n t s w e r e a p p l i e d t o c o n t r o l r i n g s . Once d r i e d , t h e r i n g s w e r e p o s i t i o n e d i n t h e c e n t e r o f t h e p e t r i d i s h e s a n d f i v e s n a i l s ( d i a m e t e r , 4 - 6 mm) w e r e p l a c e d i n s i d e e a c h r i n g . The d i s p e r s a l o f t h e s n a i l s was n o t e d a f t e r 10 m i n . T h i s e x p e r i m e n t was r e p e a t e d o n t h r e e s e p a r a t e o c c a s i o n s w i t h t w o r e p l i c a t e s p e r c o n c e n t r a t i o n i n e a c h c a s e . The r e s u l t s w e r e v e r y c o n s i s t e n t . RESULTS AND DISCUSSION Of t h e o r g a n i s m s u s e d i n t h e i n i t i a l s c r e e n i n g o f l a n o s o l a n d l a n o s a l t a c t i v i t y , E. c o l i p r o v e d t h e m o s t s u s c e p t i b l e ( F i g u r e 1 7 ) . As a r e s u l t , t h i s b a c t e r i u m was u s e d i n f u r t h e r a n t i b i o s i s e x p e r i m e n t s . L a n o s a l t 2 s h o w e d n o e f f e c t a t c o n c e n t r a t i o n s as h i g h as 1 m g / c m , w h i l e t h e l a n o s o l e f f e c t s o n E. c o l i i n p a r t i c u l a r 2 r a n g e d f r o m v e r y e f f e c t i v e a t 1 m g / c m t o b a r e l y d e t e c t -2 a b l e a t 0 . 0 1 m g / c m . F p r l i q u i d c u l t u r e e x p e r i m e n t s a l a n o s o l 91 ZONE OF INHIBITION (diameter i n mm) F i g u r e 17. A n t i b i o t i c a c t i v i t y of l a n o s o l a g a i n s t Candida a l b i c a n s ( ), E s c h e r i c h i a c o l i ( ), Saccharomyces c e r e v i s i a e (—-——) and S t a p h y l o c o c c u s - aureus (- — — -) . 92 c o n c e n t r a t i o n of 0.2 ppt was found to be e f f e c t i v e but not to stop completely the growth of E. c o l i . T h i s con-c e n t r a t i o n corresponds to the midrange dose response of 2 the i n i t i a l p l a t i n g experiments (e.g. 0.1 mg/cm ). L a n o s a l t was again i n e f f e c t i v e , even at c o n c e n t r a t i o n s ten times those of l a n o s o l (see F i g u r e 18). Conover and S i e b u r t h (1966) have suggested t h a t the a l k a -l i n e pH of sea water promotes the a c t i v i t y of a l g a l tan-n i n s (e.g. r e d u c i n g substances, polyphenols, e t c . ) as t o x i c substances to p l a n k t o n i c animals. T h i s i n c r e a s e d t o x i c i t y p robably i n c l u d e s e f f e c t s on other p l a n t s and herbivorous animals as w e l l as on microorganisms. As a means of examining t h i s e f f e c t u s i n g bromophenols, the t o x i c i t y l e v e l s of l a n o s o l and l a n o s a l t a g a i n s t E. c o l i were t e s t e d at d i f f e r e n t pHs ( F i g u r e 18). The r e s u l t s not o n l y i l l u s t r a t e the d i f f e r e n c e s i n the e f f e c t s of the two compounds, but they a l s o g i v e some i n d i c a t i o n of the e f f e c t of pH on the chemistry of l a n o s o l a n t i b i o s i s . Comparing the l a n o s o l curve to the c o n t r o l at pH 10, one notes that the maximal l e v e l s of b a c t e r i a l growth are roughly e q u i v a l e n t . The h i g h e r pH not o n l y slows the growth r a t e of E. c o l i ( c o n t r o l ) , but i t seems i n some way to d e t o x i f y l a n o s o l . The mechanism of t h i s d e t o x i f i c a t i o n procedure i s unknown. A p o s s i b l e explana-t i o n i s the p r o d u c t i o n of phenolate anions from the F i g u r e 1 8 . The e f f e c t of pH on the a n t i b i o t i c a c t i v i t y o f l a n o s o l and l a n o s a l t a g a i n s t E s c h e r i c h i a c o l i (pH 6 , 7— — — , 8 - — • • • , 9 — — — , 10 • — • — • ) . 94 bromophenols. These as s a l t s would approximate the i n a c t i v i t y o f the l a n o s a l t as shown i n F i g u r e 18. In Chapter V I have examined the exudation of bromophenols from t i d e p o o l R. l a r i x and found that d u r i n g p e r i o d s o f most copious phenol r e l e a s e there i s a d e f i -n i t e and r a p i d change i n pH o f the water surrounding the algae. As a r e s u l t the s u r f a c e of the p l a n t may be q u i t e a c i d i c , which i n c o n t r a s t to the above argument promotes a n t i b i o t i c a c t i v i t y a g a i n s t , f o r example, E. c o l i ( F i g u r e 18, pH 6). When d e a l i n g w i t h an a n t i b i o t i c substance i n pure form, one i s never sure i f t h i s i s the o n l y source or at l e a s t the major source o f a c t i v i t y seen when whole p l a n t s or crude e x t r a c t s are used. I attempted to ex-amine t h i s q u e s t i o n by making e x t r a c t s and producing exudates of R. l a r i x , q u a n t i f y i n g the l a n o s o l i n these, d u p l i c a t i n g these q u a n t i t i e s w i t h pure compound ( c o n t r o l s ) and t e s t i n g a l l three i n another s e r i e s of b a c t e r i a l growth experiments. T h i s type of experiment was thought to be an " a c t i o n spectrum" o f b i o l o g i c a l a c t i v i t y f o r R. l a r i x . T e c h n i c a l problems, e s p e c i a l l y i n t e s t s w i t h e x t r a c t s , minimized the p o t e n t i a l of the method. For example, the h i g h c h l o r o p h y l l c o n c e n t r a t i o n of the e x t r a c t s and i t s p r o g r e s s i v e degradation masked the growth of the b a c t e r i a as monitored by v i s i b l e spectrophotometry 95 ( F i g u r e 19). T h e r e f o r e , one can say n o t h i n g about the q u a n t i t a t i v e aspects of l a n o s o l a c t i v i t y i n e x t r a c t s except to r e f e r the reader to the l i t e r a t u r e , which g i v e s numerous examples of a c t i v i t y of suspected bromophenols i n e x t r a c t s of r e d algae. The experiments w i t h exudates, however, showed a c l o s e c o r r e l a t i o n w i t h the l a n o s o l con-t r o l s ( F i gure 19). T h i s i n f o r m a t i o n and the r e s u l t s of the b a c t e r i a l experiments g i v e n here l e a d to the c o n c l u -s i o n that l a n o s o l i s produced as an a n t i b i o t i c and that i t i s the primary substance of t h i s k i n d found i n R. l a r i x . The r e s u l t s of the assays w i t h marine f u n g i are g i v e n i n Table IX. As f a r as I am aware, no other work has been done u t i l i z i n g a n t i b i o t i c s d e r i v e d from marine algae and t e s t i n g t h e i r e f f e c t s on marine f u n g i . The procedure i n which D. s a l i n a c o n i d i a were peppered onto the agar s u r f a c e proved the most r a p i d and i n f o r m a t i v e i n t h a t no long experimental p e r i o d s were nec e s s a r y i n which l a n o s o l degradation c o u l d occur. Using t h i s pro-cedure l a n o s o l was shown to i n h i b i t spore germination and not j u s t m y c e l i a l growth alone. The s n a i l L. s c u t u l a t a i s v e r y common i n l o c a l t i d e p o o l s and e s s e n t i a l l y throughout the i n t e r t i d a l zone. I t i s o f t e n found on and around R. l a r i x , but there i s no evidence that i t ever consumes the a l g a . The reason f o r t h i s may be the h i g h c o n c e n t r a t i o n s of bromophenols 0.6 0.5 0.4 0.3 0.2 0.1 LANOSOL (and control) EXTRACT EXUDATE J L 8 24 8 24 TIME (hours) 8 24 F i g u r e 19. E f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f l a n o s o l i n pure form, i n a l g a l e x t r a c t s and i n exudates on the growth r a t e o f E s c h e r i c h i a c o l i . The i n i t i a l c o n c e n t r a t i o n was 0.25 ppt (———) and the d i l u t i o n s are 0.13 (• — — -), 0.06 (• ), 0.03 ( ) and 0 ( — — ) ppt. T a b l e I X . A n t i b i o t i c a c t i v i t y o f l a n o s o l a g a i n s t t h r e e s p e c i e s o f m a r i n e f u n g i ( g i v e n as t h e d i a m e t e r o f t h e z o n e o f g r o w t h i n h i b i t -i o n i n mm) . * A c t i v i t y a g a i n s t D. s a l i n a c o n i d i a l g e r m i n a t i o n , n o t t h e a d u l t m y c e l i u m as i n t h e o t h e r t h r e e e x a m p l e s . * * LANOSOL ( m g / c m 2 ) D e n d r y p h i e l l a s a l i n a SPECIES D e n d r y p h i e l l a s a l i n a * S i g m o i d e a s p . Z a l e r i o n m a r i t i m u m 3 . 0 10 18 9 20 0 . 3 7 12 7 16 0 . 0 3 0 7 0 10 0 0 0 0 0 * * - T h r e e - r e p l i c a t e s w e r e u s e d f o r e a c h , s p e c i e s t e s t e d a n d t h e a b o v e d a t a a r e a v e r a g e s o b t a i n e d f r o m e a c h s e t o f e x p e r i m e n t s . 98 i n t h e a l g a . R e s u l t s o f a t y p i c a l t e s t w i t h b o t h l a n o s o l a n d t h e s a l t a r e p i c t u r e d i n F i g u r e 20 a n d t h e c o m b i n e d r e s u l t s o f a l l e x p e r i m e n t s a r e shown i n F i g u r e 2 1 . L a n o s o l p r o d u c e d r e p u l s i o n o f t h e s n a i l s a t l e v e l s o f 0 . 0 0 1 t o 0 . 1 m g / c m w h i l e l a n o s a l t was o n l y e f f e c t i v e a t c o n c e n t r a t i o n s 100 t i m e s t h o s e o f t h e f r e e p h e n o l . T h i s m e t h o d o f t e s t i n g ( O h t a 1 9 7 9 ) was m o d i f i e d so t h a t a b e t t e r c o n t r o l o f t h e q u a n t i t i e s a p p l i e d t o t h e s a m p l e a r e a c o u l d be o b t a i n e d . A l s o , p r o b l e m s o f s n a i l r e p u l s i o n b y r e s i d u a l s o l v e n t w e r e o v e r c o m e b y t h o r o u g h l y d r y i n g t h e f i l t e r p a p e r r i n g s b e f o r e s u r r o u n d i n g t h e s n a i l s w i t h t h e m . E v i d e n c e h a s b e e n p r e s e n t e d h e r e f o r t h e l a r g e s c a l e p r o d u c t i o n o r a t l e a s t u t i l i z a t i o n o f l a n o s o l as an a n t i b i o t i c s u b s t a n c e b y R. l a r i x . .The a s s i g n m e n t o f t h i s compound t o a p a r t i c u l a r r o l e i n t h e e c o l o g y o f r e d a l g a e w o u l d a t t h i s s t a g e be m e r e l y c o n j e c t u r e . Dem-o n s t r a t i o n o f t h e b r o a d s p e c t r u m a n t i b i o t i c a c t i v i t y o f l a n o s o l i s e n o u g h t o e n c o u r a g e f u r t h e r i n v e s t i g a t i o n i n t o t h e i m p l i c a t i o n s o f s u c h phenomena i n t h e m a r i n e e n v i r o n -m e n t . M o r e d e t a i l e d m i c r o e c o l o g i c a l w o r k c o u p l e d w i t h q u a n t i t a t i v e c h e m i c a l a n a l y s i s w i l l i n v a r i a b l y h e l p t o d e t e r m i n e t h e r e a s o n s f o r t h e p r o d u c t i o n o f b r o m o p h e n o l s b y r e d a l g a e . 99 F i g u r e 2 0 . A t y p i c a l s n a i l r e p u l s i o n e x p e r i m e n t s h o w i n g t h e e f f e c t s o f l a n o s o l a n d l a n o s a l t o n t h e b e h a v i o r o f L i t t o r i n a s c u t u l a t a . T h i s p h o t o g r a p h was t a k e n 10 m i n a f t e r f i v e s n a i l s h a d b e e n p l a c e d i n t h e c e n t e r o f e a c h d i s h . 100 NUMBER OF SNAILS ESCAPING SAMPLE RING F i g u r e 2 1 . D e g r e e o f r e p u l s i o n o f L i t t o r i n a s c u t u l a t a b y v a r y i n g c o n c e n t r a t i o n s o f l a n o s o l ( ) and" l a n o s a l t (• • ) . L a n o s a l t c o n c e n t r a t i o n s a r e 100 t i m e s t h o s e l i s t e d o n t h e v e r t i c a l a x i s . 1 0 1 PERSPECTIVES T h i s d i s s e r t a t i o n h a s b e e n c o n c e r n e d w i t h a s s i g n -i n g a r o l e t o t h e p r o d u c t i o n o f b r o m o p h e n o l s b y r e d a l g a e . E a c h c h a p t e r r e p r e s e n t s a s t e p t a k e n t o w a r d s t h e f o r m u l a t i o n o f a c o n c l u s i o n r e g a r d i n g t h i s r o l e . B e -c a u s e o f t h e d i v e r s i t y i n t h e s u b j e c t m a t t e r o f t h e c h a p -t e r s , t h e c o n t r i b u t i o n o f e a c h t o t h e d i s s e r t a t i o n as a w h o l e may n e e d m i n o r c l a r i f i c a t i o n . I w i l l t h e r e f o r e a t t e m p t t o e x p l a i n t h e i m p e t u s f o r a n d t h e s i g n i f i c a n c e o f e a c h s e p a r a t e s t e p . The i m p o r t a n c e o f t h e f i r s t c h a p t e r l a y i n t h e d i s c o v e r y t h a t t h e l e v e l o f t o t a l p h e n o l s n o t i c e a b l y i n -c r e a s e s i n t h e t i d e p o o l s o v e r t h e c o u r s e o f a d a y . I c h o s e t o e x a m i n e t h e t i d e p o o l h a b i t a t f o r t w o r e a s o n s : f i r s t , t h e t i d e p o o l i s a " c l o s e d " s y s t e m - - e s s e n t i a l l y a l a r g e c u l t u r e f l a s k i n n a t u r e - - a n d s e c o n d l y , t h e r a p i d i n c r e a s e i n y e l l o w b r o w n c o l o r a t i o n ( p h e n o l i c s u b s t a n c e s ) p r o d u c e d b y t i d e p o o l a l g a e m i g h t v e r y w e l l h a v e some e f f e c t o n o t h e r o r g a n i s m s w i t h i n t h e p o o l s . M e a s u r e m e n t s o f t h e r a n g e o f e n v i r o n m e n t a l f a c t o r s o c c u r r i n g i n t h e p o o l s l a t e r e n a b l e d me t o s e t up e x u d a t i o n e x p e r i m e n t s w h i c h , b e c a u s e t h e c o n d i t i o n s u s e d f e l l w i t h i n 102 e n v i r o n m e n t a l r a n g e s , w e r e a s s u m e d t o p l a c e n o u n n a t u r a l p h y s i o l o g i c a l s t r e s s e s o n t h e a l g a e b e i n g t e s t e d . F o r some r e d a l g a e t h e r e i s s t i l l c o n f u s i o n as t o t h e i r a s s i g n m e n t a t t h e s p e c i e s l e v e l . P o p u l a t i o n s o c c u r r i n g i n d i f f e r e n t h a b i t a t s , b e i n g e x p o s e d t o d i f -f e r e n t d e g r e e s o f n a t u r a l s t r e s s , o f t e n c h a n g e i n g r o s s m o r p h o l o g y , c o n c e i v a b l y as an a d a p t i v e r e s p o n s e t o t h e i r e n v i r o n m e n t . The t i d e p o o l f o r m o f R. l a r i x h a s a q u i t e d i f f e r e n t a p p e a r a n c e f r o m t h a t g r o w i n g i n t h e u p p e r s u b t i d a l z o n e . The c h e m i c a l c o m p a r i s o n o f t h e s e t w o f o r m s g a v e me some b a s i s , a t l e a s t , f o r c a l l i n g t h e t i d e p o o l f o r m R. l a r i x . The c h e m i s t r y o f t h i s s p e c i e s was f u r t h e r e x a m i n e d b y HPLC. The m e t h o d w h i c h I d e v e l o p e d p r o v e d t o be r a p i d a n d i n f o r m a t i v e f o r t h e s c r e e n i n g o f a l g a l e x t r a c t s a n d e x u d a t e s f o r b r o m o p h e n o l s . Q u a n t i f i c a t i o n o f l a n o s o l was made e a s y a n d a c c u r a t e . The e f f i c i e n c y o f t h e m e t h o d was f u r t h e r d e m o n s t r a t e d i n t h e a n a l y s i s f o r b r o m o p h e n o l s o f t w o a d d i t i o n a l s p e c i e s , P. l y a l l i i a n d C. w a s h i n g t o n i e n s e . D e m o n s t r a t i o n o f t h e v a r i a t i o n i n b r o m o p h e n o l c o n t e n t , b o t h t e m p o r a l a n d i n t r a t h a l l i a l , p r o v i d e d t h e f i r s t r e a l c l u e i n t o t h e e c o l o g i c a l s i g n i f i c a n c e o f t h e s e c o m p o u n d s . S i e b u r t h e_t a l . ( 1 9 7 4 ) h a v e shown t h a t P o l y s i p h o n i a l a n o s a , w h i c h a l s o c o n t a i n s l a n o s o l i n 103 c o n c e n t r a t i o n s o f up t o 5°L o f i t s d r y w e i g h t ( T a b l e V ) , s u p p o r t s a d i v e r s e a n d s e a s o n a l l y c h a n g i n g e p i f l o r a . I n summer t h e P. l a n o s a e p i f l o r a i s c o m p o s e d m a i n l y o f y e a s t s a n d i n w i n t e r , o f d i a t o m s a n d f i l a m e n t o u s f u n g i . I f t h e l e v e l s o f l a n o s o l i n t h i s s p e c i e s v a r y t e m p o r a l l y as do t h o s e o f R. l a r i x , a n i n t r i g u i n g c o r r e l a t i o n a r i s e s . A l t h o u g h t h e e p i f l o r a o f P. l a n o s a was shown t o be a b u n -d a n t , t h e a l g a was i n n o way t h r e a t e n e d ( f o u l e d ) b y t h e n u m b e r o f o r g a n i s m s p r e s e n t . V a r i a t i o n s i n t h e a b u n d a n c e o f t h e e p i f l o r a c o u l d be due e i t h e r t o s e a s o n a l c h a n g e s i n t h e n u m b e r s o f a p a r t i c u l a r s p e c i e s p r e s e n t o r t o c h a n g i n g l e v e l s o f t o l e r a n c e t o a l g a l a n t i b i o t i c s ( e . g . b r o m o p h e n o l s ) b y t h a t same s p e c i e s . I n t e r e s t i n g l y , t h e l e v e l s o f " c o n t a m i n a t i o n " o f t h e P. l a n o s a t h a l l u s a p p e a r e d t o be m i n i m a l t o w a r d t h e g r o w i n g t i p s . The s e c o n d p h a s e o f t h i s s t u d y l e d t o t h e d i s -c o v e r y t h a t l a r g e q u a n t i t i e s o f b r o m o p h e n o l s ( l a n o s o l i n p a r t i c u l a r ) w e r e p r e s e n t i n t h e e x u d a t e s o f R. l a r i x . B r o w n a l g a e h a v e l o n g b e e n k n o w n t o c o n t r o l s u r f a c e f o u l i n g b y t h e p r o d u c t i o n o f p o l y p h e n o l i c s u b s t a n c e s ( C o n o v e r a n d S i e b u r t h 1 9 6 4 , S i e b u r t h a n d C o n o v e r 1 9 6 5 , R y l a n d 1 9 7 4 , a l s o see S i e b u r t h 1 9 6 8 ) . The p r e s e n c e o f b r o m o p h e n o l s - - p a r t i c u l a r l y l a n o s o l i n t h e f r e e f o r m - - i n r e d a l g a l e x u d a t e s f u r t h e r s t h e n o t i o n t h a t t h e s e c o m -p o u n d s h a v e a d e f i n i t e e c o l o g i c a l f u n c t i o n . The r a t e s 104 o f e x u d a t i o n o f l a n o s o l w e r e as h i g h as 0 . 1 5 p p t / g d . w t . h r . The e f f e c t i v e a n t i b i o t i c d o s e o f t h i s compound a g a i n s t 2 E. c o l i was l e s s t h a n 0 . 3 m g / c m , w h i c h i s a p p r o x i m a t e l y 0 . 1 - 0 . 2 p p t . T h u s t h e r a t e o f e x u d a t i o n o f b r o m o p h e n o l s seems t o be o n t h e o r d e r o f t h a t n e c e s s a r y t o c o n t r o l s u r f a c e f o u l i n g , a s s u m i n g t h a t o t h e r m i c r o o r g a n i s m s show t h e same r e s p o n s e t o l a n o s o l as d i d E. c o l i . T e s t i n g o f t h e a n t i b i o t i c a c t i v i t y o f l a n o s o l on a v a r i e t y o f o r g a n i s m s p r o v e d t h a t t h e y d i d i n f a c t show s u c h a r e s p o n s e : a l l w e r e a f f e c t e d a t some c o n c e n t r a t i o n 9 b e t w e e n 0 . 0 3 a n d 0 . 3 m g / c m . The e f f e c t on E. c o l i was g r e a t e s t a t l o w e r p H s , a n d t h e a c t i v i t y o f R. l a r i x e x u d a t e s c l o s e l y r e s e m b l e d t h a t s e e n f o r p u r e l a n o s o l . R e s u l t s o f t h e e x p e r i m e n t s w i t h t i d e p o o l s n a i l s o p e n e d t h e d o o r t o a n o t h e r a r r a y o f p o s s i b l e f u n c t i o n s f o r b r o m o p h e n o l s i n t h a t t h e y may a l s o be i n v o l v e d i n t h e r e p u l s i o n o f p o t e n t i a l h e r b i v o r e s . The l e v e l a t w h i c h l a n o s o l was e f f e c t i v e as a s n a i l r e p e l l e n t was much l o w e r t h a n t h a t n e c e s s a r y f o r t h e compound t o b e a n t i b i o t i c . F u r t h e r t e s t i n g w i t h a v a r i e t y o f m a r i n e a n i m a l s c o u l d d e m o n s t r a t e t h e f u l l s p e c t r u m o f t h i s t y p e o f e f f e c t . A f i n a l a n d p o s s i b l y m o s t c o n v i n c i n g a r g u m e n t f o r t h e e c o l o g i c a l s i g n i f i c a n c e o f b r o m o p h e n o l s comes f r o m t h e w o r k o f Roos ( 1 9 5 7 ) . He e x a m i n e d t e m p o r a l v a r i a t i o n i n t h e a n t i b i o t i c a c t i v i t y o f e x t r a c t s o f R. s u b f u s c a 105 o n s e v e r a l s p e c i e s o f b a c t e r i a . K u r a t a a n d A m i y a ( 1 9 7 5 ) h a v e shown t h i s a l g a t o c o n t a i n 9.2%> o f l a n o s a l t on a d r y w e i g h t b a s i s ( o r as l a n o s o l , 5 . 1 % ) . Roos o b s e r v e d t h a t t h e a n t i b i o t i c a c t i v i t y o f R. s u b f u s c a was l o w e s t i n t h e summer m o n t h s a n d e s s e n t i a l l y t w i c e as g r e a t i n t h e w i n t e r . R h o d o m e l a l a r i x c o n t a i n s l a n o s o l i n a p p r o x i m a t e l y t h e same c o n c e n t r a t i o n s , a n d t h e s e a s o n a l m a x i m a a n d m i n i m a f o r t h i s compound c o r r e s p o n d t o t h e s e a s o n a l m a x i m a a n d m i n i m a o f a n t i b i o t i c a c t i v i t y f o r R. s u b f u s c a . Hence t h e r e e x i s t s an i n d i r e c t c o r r e l a t i o n o f l a n o s o l c o n t e n t w i t h t e m p o r a l a n t i b i o t i c a c t i v i t y f o u n d f o r e x t r a c t s o f r e d a l g a e . M o s t c e r t a i n l y , I h a v e made b u t t h e b a r e s t b e g i n -n i n g i n a p p l y i n g t h e c o n c e p t s o f c h e m i c a l e c o l o g y , as we u n d e r s t a n d t h e m i n t h e s e n s e o f t h e i r t e r r e s t r i a l o r i g i n s , t o t h e v a s t a n d c o m p l e x m a r i n e e n v i r o n m e n t . The p o t e n t i a l f o r f u r t h e r a n a l y s e s o f t h e i n t e r a c t i o n s among m a r i n e o r g a n i s m s i s s t e a d i l y i n c r e a s i n g a n d I h o p e t h a t t h e r e o c c u r s , n o t t o o f a r i n t o t h e f u t u r e , much m o r e a n d d e -t a i l e d r e s e a r c h i n t h e c h e m i c a l e c o l o g y o f s u c h c o m p l e x e c o s y s t e m s . 106 FOOTNOTES ( 1 ) T h i s l i g h t m e t e r a n d p r o b e c o m b i n a t i o n e f f e c t i v e l y m e a s u r e s w a v e l e n g t h s b e t w e e n 400 a n d 800 nm, t h e r e g i o n o f t h e s p e c t r u m u t i l i z e d f o r p h o t o s y n t h e s i s . ( 2 ) T h i s f i g u r e r e p r e s e n t s a summary o f t h e d a t a p r e s e n t e d i n F i g u r e 3 a n d i n c l u d e s some o f t h e f a c t o r s d i s c u s s e d i n t h e t e x t w h i c h m i g h t e f f e c t c h a n g e s i n e n v i r o n m e n t a l c o n d i t i o n s o c c u r r i n g i n e a c h o f t h e p o o l s . ( 3 ) T h i s f i g u r e i s b a s e d o n t h e maximum l e v e l o f t o t a l p h e n o l s r e a c h e d i n t h e p o o l o v e r t h e p e r i o d o f a s i n g l e d a y ( a b o u t 10 h r s ) . H e n c e , o n an h o u r l y b a s i s , 0 . 0 5 mg o f t o t a l p h e n o l s w o u l d be p r o d u c e d f o r e a c h l i t e r o f t i d e p o o l w a t e r ( o r as a m o u n t p r o d u c e d p e r g r a m d r y w e i g h t o f a l g a , 0 . 1 4 m g / h r ) . ( 4 ) No c o r r e l a t i o n b e t w e e n m e d i u m d i s c o l o r a t i o n a n d l a n o s o l c o n t e n t i s i n t e n d e d . The t w o may be m u t u a l l y e x c l u s i v e o r some r e l a t i o n s h i p may e x i s t . I n a d d i t i o n , u s e o f t h e w o r d " c o r r e l a t i o n " i m p l i e s n o m a t h e m a t i c a l s i m i l a r i t y i n a n y o f i t s many u s e s i n t h i s t h e s i s ; r a t h e r i t s i m p l y i m p l i e s t h a t a m u t u a l r e l a t i o n s h i p e x i s t s b e t w e e n t w o e v e n t s , t h e b a s i s o f w h i c h h a s n o t n e c e s s a r i l y b e e n e s t a b l i s h e d . 107 REFERENCES A b b o t t , I . A . a n d G. J . H o l l e n b u r g . 1 9 7 6 . M a r i n e a l g a e o f C a l i f o r n i a . S t a n f o r d U n i v . P r e s s , S t a n f o r d ? BT7 p p . A l b r i g h t , J . D. a n d L. G o l d m a n . 1 9 6 5 . D i m e t h y l s u l f o x i d e -a c i d a n h y d r i d e m i x t u r e s . New r e a g e n t s f o r o x i d a t i o n o f a l c o h o l s . J . A m e r . Chem. S o c . 8 7 , 4 2 1 4 - 4 2 1 6 . A l l e n , M. 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