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Culture of the brine shrimp, Artemia salina L., utilizing Dunaliella tertiolecta grown in swine waste-seawater.. 1981

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CULTURE OF THE BRINE SHRIMP, ARTEMIA SALINA L. UTILIZING DUNALIELLA TERTIOLECTA GROWN IN SWINE WASTE-SEAWATER MIXTURES AND IN DEFINED INORGANIC MEDIUM by LEONARDO B. TIRO, JR. B. Sc., Xavier University, Cagayan de Oro City P h i l i p p i n e s , 197^ A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of A g r i c u l t u r a l Mechanics) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1980 0 Leonardo B. T i r o , J r . , I98O In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of this thes is for f inanc ia l gain sha l l not be allowed without my written permission. LEONARDO B. TIRO, JR. Department of A g r i c u l t u r a l Mechanics The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date -12-th January, 1981. ABSTRACT A co m p a r a t i v e s t u d y was conducted on the growth o f D u n a l i e l l a t e r t i o l e c t a u s i n g d e f i n e d i n o r g a n i c medium and swine waste-seawater medium as s o u r c e s o f n u t r i e n t s . The n i t r o g e n -1 c o n c e n t r a t i o n was m a i n t a i n e d a t c a 1200 + 5° ug~at N 1 f o r b o t h media. There were no s i g n i f i c a n t d i f f e r e n c e s i n the d a i l y measurements o f c e l l d e n s i t i e s and d r y w e i g h t s o f D. t e r t i o l e c t a grown i n d e f i n e d i n o r g a n i c medium and swine waste-seawater medium i n e i t h e r h a t c h o r c o n t i n u o u s c u l t u r e systems. The a l g a l biomass produced from c o n t i n u o u s c u l t u r e system was used as f o o d f o r the b r i n e s h rimp, A r t e m i a s a l i n a L. The b r i n e shrimp f e d w i t h D. t e r t i o l e c t a grown i n swine waste- seawater medium and t h o s e f e d w i t h _D. t e r t i o l e c t a c u l t u r e d i n d e f i n e d i n o r g a n i c medium showed no s i g n i f i c a n t d i f f e r e n c e s i n t h e i r d a i l y t o t a l l e n g t h and p e r c e n t a g e s u r v i v a l measurements. A 57-58% A r t e m i a biomass c o n v e r s i o n e f f i c i e n c y was c a l c u l a t e d . S t a t i s t i c a l a n a l y s i s f o r biomass c o n v e r s i o n e f f i c i e n c y showed no s i g n i f i c a n t d i f f e r e n c e s f o r A r t e m i a f e d w i t h D. t e r t i o l e c t a grown i n swine waste-seawater medium and t h a t f e d w i t h D. t e r t i o l e c t a c u l t u r e d i n d e f i n e d i n o r g a n i c medium. i i i TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENTS INTRODUCTION LITERATURE REVIEW A l g a l P r o d u c t i o n Algal-Waste R e c y c l i n g I n Aquaculture Use and P o t e n t i a l of Artemia For Aquaculture I n R e l a t i o n To I t s L i f e Cycle 8 N u t r i t i o n a l Value of Artemia N a u p l i i As Food I n Aquaculture Hatcheries 9 Present C u l t u r a l P r a c t i c e s of Artemia 11 MATERIALS AND METHODS lk >' iA 'Algae!; lk B r i n e Shrimp lk Media and Saltwater P r e p a r a t i o n 15 Swine Waste-Seawater Mixture 15 Defined Inorganic Medium 16 Seawater P r e p a r a t i o n 16 D e s c r i p t i o n of Culture U n i t s 17 A l g a l Batch C u l t u r e U n i t I 17 A l g a l Continuous C u l t u r e U n i t I I 18 A l g a l Continuous Culture U n i t I I I 20 Brine, Shrimp Culture U n i t 21 C u l t u r e Methods 21 A l g a l C u l t u r e Method 21 B r i n e Shrimp Culture Method 23 Chemical A n a l y s i s 2k K j e l d a h l N i t r o g e n 2k Ammonia / N i t r a t e + N i t r i t e 25 T o t a l Phosphate 25 Page i i i i i v v i v i i i 1 3 3 6 i v Page Dry Weight A n a l y s i s 25 T o t a l Suspended S o l i d s 26 RESULTS 27 Swine Waste A n a l y s i s 27 A l g a l B a t c h C u l t u r e System 27 A l g a l C ontinuous C u l t u r e System 31 B r i n e Shrimp F e e d i n g E x p e r i m e n t s 40 DISCUSSION L i v e s t o c k Waste As A N u t r i e n t Source I n A l g a l C u l t u r e ^7 Growth C o n s t a n t , C e l l D e n s i t y and Dry Weight o f D. t e r t i o l e c t a ^9 C o n t i n u o u s C u l t u r e System I n A l g a l P r o d u c t i o n 51 A r t e m i a F e e d i n g E x p e r i m e n t s 52 T o t a l Body L e n g t h and P e r c e n t a g e S u r v i v a l 52 Biomass C o n v e r s i o n E f f i c i e n c y 53 Comhined L i v e s t o c k W a s t e - A r t e m i a C u l t u r e System 5^ SUMMARY AND CONCLUSIONS 57 REFERENCES 59 APPENDICES 70 V LIST OF TABLES Table T i t l e Page 1 Swine waste analysis of TKN, NH^-N NO^-N + N02-N, PO^-P and Total Suspended Solids (TSS) during the course of the experimental work 28 2, Nitrogen (TKN, NH^-N, Organic-N;,'f NO^-N + NOg) measured i n the a l g a l effluent during the second continuous culture (Expt 5) i n Mg -at N 1 39 3 Adult Artemia biomass produced a f t e r the 14-day culture period i n wet and dry weight "basis 46 4 Growth Constant ( K ^ 0 ^ hr ) and Mean Generation Time (tg hrs) from present and e a r l i e r studies on D. t e r t i o l e c t a 5Q 5 Calculated Biomass Conversion E f f i c i e n c y (BCE %) of Artemia s a l i n a L. 55 v i LIST OF FIGURES F i g u r e T i t l e Page 1 G e n e r a l i z e d v i e w o f the a l g a l c o n t i n u o u s c u l t u r e system 19 2 F i v e - d a y measurement o f t o t a l K j e l d a h l n i t r o g e n (TKN) o f swine waste s l u r r y and t h r e e d i f f e r e n t r a t i o s o f swine waste-seawater m i x t u r e s ( l i 1 ; 2:1 ; 1:2) s t o r e d a t k°C 29 3 D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a grown i n swine waste-seawater and d e f i n e d i n o r g a n i c media i n b a t c h s y s t e m ( E x p t 1) 30 k D a i l y c e i l d e n s i t y o f D. t e r t i o l e c t a grown i n swine waste-seawater and d e f i n e d i n o r g a n i c media i n b a t c h c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c arboys (Expt 2) 32 5 D a i l y measurement o f d r y wt o f D. t e r t i o l e c t a grown i n swine waste-seawater and d e f i n e d i n o r g a n i c media i n b a t c h c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c c a r b o y s ( E x p t 2) 33 6 D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a grown i n swine waste-seawater and d e f i n e d i n o r g a n i c media i n c o n t i n u o u s c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 3) 3k 7 D a i l y measurement o f d r y wt o f D. t e r t i o l e c t a grown i n swine waste-seawater and d e f i n e d i n o r g a n i c media i n c o n t i n u o u s c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s (Expt 3) 35 8 D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a grown i n swine waste-seawater and d e f i n e d i n o r g a n i c media i n c o n t i n u o u s c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s (Expt 5) 36 9 D a i l y measurement o f d r y wt o f D. t e r t i o l e c t a grown i n swine waste-seawater and d e f i n e d i n o r g a n i c media i n c o n t i n u o u s c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 5) 37 10 Growth i n t o t a l l e n g t h o f A r t e m i a s a l i n a L. o v e r a 12-day c u l t u r e p e r i o d f e d w i t h D. t e r t i o l e c t a grown i n d e f i n e d i n o r g a n i c medium kl v i i F i g u r e T i t l e Page 11 Percentage s u r v i v a l of Artemia during the 12-day culture period fed with D. t e r t i o l e c t a grown i n defined inorganic medium 42 12 Growth i n t o t a l length measurement of Artemia s a l i n a L. over a l4-day culture period fed with swine waste and defined inorganic media grown D. t e r t i o l e c t a 43 13 Percentage s u r v i v a l of Artemia s a l i n a L. over a 14-day culture period fed with swine waste and defined inorganic media grown D. t e r t i o l e c t a 44 v i i i ACKNOWLEDGEMENTS My s i n c e r e s t g r a t i t u d e t o Dr. J . W. Z a h r a d n i k , my a d v i s e r , Department'.- o f B i o - R e s o u r c e E n g i n e e r i n g . I am d e e p l y i n d e b t e d t o a l l the members o f my committee: Dr. N. J . A n t i a , P a c i f i c Environment I n s t i t u t e , West Vancouver L a b o r a t o r y , f o r p r o v i d i n g the a l g a used i n the expe r i m e n t s and f o r h i s v a l u a b l e h e l p and a d v i c e s d u r i n g the i n i t i a l phase o f the r e s e a r c h ; Dr. N-. R. B u l l y , Department o f B i o - R e s o u r c e E n g i n e e r i n g , f o r the use o f some equipment and f o r h i s c o n s t r u c t i v e c r i t i c i s m s i n t h e p r e p a r a t i o n o f the m a n u s c r i p t ; Dr. T. R. P a r s o n s , f o r h i s comments. My s i n c e r e s t t h a n k s t o t h e s e s c i e n t i s t s f o r t h e i r encouragements and v a l u a b l e comments. I a l s o w i s h t o g r a t e f u l l y thank Dr. W. S. Hoar, Department o f Z o o l o g y , f o r the use o f h i s l a b o r a t o r y d u r i n g t h e f i r s t phase p f t h e r e s e a r c h ; Dr. J . M a r l i a v e , Vancouver P u b l i c Aquarium, f o r t h e A r t e m i a eggs; Dr. D. H i g g s , P a c i f i c Environment I n s t i t u t e , West Vancouver L a b o r a t o r y , f o r the use o f t h e f i b e r g l a s s c u l t u r e t a n k s . My s i n c e r e a p p r e c i a t i o n t o the f o l l o w i n g : Dr. P. L i a o , Department o f B i o - R e s o u r c e E n g i n e e r i n g , f o r c o n d u c t i n g some o f the c h e m i c a l a n a l y s i s ; J . P e h l k e and N. J a c k s o n , Department o f B i o - R e s o u r c e E n g i n e e r i n g , f o r t h e t e c h n i c a l a s s i s t a n c e . Mr. H. Gr e w e l , Graduate S t u d e n t , F o r e s t r y Department, f o r h i s a s s i s t a n c e i n t h e s t a t i s t i c a l a n a l y s i s ; Mr. R. P l a t o n , f o r the use o f h i s t y p e w r i t e r ; Mr. N. C a s t i l l o , f o r h i s h e l p i n t h e s e t t i n g - u p o f the c u l t u r e u n i t s . i x Ms. Debbie T u r n b u l l , I n t e r n a t i o n a l Development R e s e a r c h C e n t r e o f Canada (IDRC), Vancouver O f f i c e . My s i n c e r e g r a t i t u d e t o L i l l i a n , my w i f e , f o r h e r d e d i c a t i o n i n t y p i n g t h e m a n u s c r i p t and f o r h e r u n d e r s t a n d i n g (when the g o i n g g e t s r o u g h ) . F i n a n c i a l s u p p o r t from the I n t e r n a t i o n a l Development Res e a r c h C e n t r e o f Canada (IDRC) i s ivery s i n c e r e l y acknowledged. INTRODUCTION A r t e m i a s a l i n a L. a r e f i l t e r f e e d i n g h e r b i v o r e s and consume o n l y s m a l l p a r t i c u l a t e m a t t e r such as u n i c e l l u l a r a l g a e , y e a s t and b a c t e r i a . The s u i t a b i l i t y o f A r t e m i a f o r a q u a c u l t u r e i s enhanced by the f a c t t h a t the p h y s i c a l c u l t u r a l c o n d i t i o n s : t e m p e r a t u r e , s a l i n i t y , pH and 0^ t e n s i o n a r e the same f o r b o t h the l a r v a l s t a g e s and a d u l t s . There i s no s p e c i a l n u r s e r y environment- Most o f the p h y t o p l a n k t e r s used as f o o d f o r A . h - s a l i n a have been c u l t u r e d on d e f i n e d seawater media (Croghan, 1 9 5 8 ; P r o v a s o l i e t a l . , 1 9 5 9 ; Mason, 1 9 6 3 ; Reeve, 1 9 6 3 a ; and S i c k , 1 9 7 6 ) o r media e n r i c h e d w i t h commercial f e r t i l i z e r ( H e l f r i c h , 1 9 7 3 ) * A g r i c u l t u r a l wastes have been used s u c c e s s f u l l y f o r the c u l t u r e o f m i c r o o r g a n i s m s such as b a c t e r i a and p h y t o p l a n k t o n (Hephner, 1 9 6 2 * S c h r o e d e r , 1 9 7 4 ) and have i n c r e a s e d t h e p r o d u c t i o n o f c u l t u r e d a q u a t i c a n i m a l s (Hephner and S c h r o e d e r , 1 9 7 4 ) . The u t i l i z a t i o n o f l i v e s t o c k waste as a n u t r i e n t s o u r c e f o r p h y t o p l a n k t o n c u l t u r e and s u b s e q u e n t l y , as f o o d f o r A. s a l i n a has n o t been i n v e s t i g a t e d . The g e n e r a l o b j e c t i v e o f t h e p r e s e n t r e s e a r c h work was to e v a l u a t e the c u l t u r e p o t e n t i a l o f the bri n r e s h r i m p , A r t e m i a s a l i n a L. f e d w i t h D u n a l i e l l a t e r t i o l e c t a grown i n swine waste- seawater as compared to d e f i n e d i n o r g a n i c media i n a M a b o r a t o r y s c a l e c o n t i n u o u s c u l t u r e system. The experiments were conducted i n two phases: Phase I was the culture study between D. t e r t i o l e c t a grown i n swine waste-seawater mixture compared to that grown i n defined inorganic medium; and Phase I I was the comparative study on the s u r v i v a l and growth of Artemia on two feeding regimes: i . D. t e r t i o l e c t a grown i n swine waste-seawater mixture. i i . D.. t e r t i o l e c t a grown i n defined inorganic medium. 3 LITERATURE REVIEW A l g a l P r o d u c t i o n E a r l y p u b l i c a t i o n s by A l l e n and N e l s o n (1910) and Emerson and Lew i s (1939) p r o v e d t h a t a l g a l c u l t u r e was a s u b j e c t o f much i n t e r e s t i n t h e e a r l y 1900's. However, much o f the e a r l y work d e a l t p r i m a r i l y w i t h the s t u d y o f p h o t o s y n t h e t i c pathways i n green a l g a l c e l l s ( S a r g e n t , 19^0; P r a t t , 19^3)* M i l n e r ( I 9 6 I ) a n a l y s e d t h e c h e m i c a l c o m p o s i t i o n o f a number o f marine and f r e s h w a t e r s p e c i e s . He found t h a t f r e s h w a t e r s p e c i e s g e n e r a l l y r a n k e d much h i g h e r i n energy c o n t e n t t h a n t h e marine s p e c i e s . T h i s d i f f e r e n c e r e s u l t e d from the r e l a t i v e l y g r e a t e r f a t c o n t e n t o f t h e f r e s h w a t e r s p e c i e s . P a r s o n s e t a l . (1961) o b s e r v e d t h a t among marine a l g a l s p e c i e s c a r b o h y d r a t e , l l i p i d , p r o t e i n and ash c o n t e n t v a r i e d w i d e l y . D u n a l i e l l a s a l i n a was found t o c o n t a i n 57$ p r o t e i n , the h i g h e s t v a l u e among the s p e c i e s a n a l y z e d , however, t h e l i p i d c o n t e n t was q u i t e l o w , S.k%. The genus D u n a l i e l l a c o m p r i s e s a group o f a l g a e w i t h e t h i n c e l l envelope whose a b i l i t y t o w i t h s t a n d c e r t a i n extreme e n v i r o n m e n t a l changes i s w e l l known (Ben-Amotz and A v r o n , 1978). However, a c e l l c o a t o f D u n a l i e l l a t e r t i o l e c t a has been r e c e n t l y o b s e r v e d w h i c h appears ftofbe l a r g e l y composed, o f g l y c o p r o t e i n c o n t a i n i n g n e u r a m i n i c a c i d r e s i d u e s i n i t s m o l e c u l a r s t r u c t u r e ( O l i v e i r a e t a l . , I98O). F o r sometime, i t has been r e c o g n i z e d t h a t heavy m e t a l r e s i s t a n c e can dev e l o p i n p o p u l a t i o n s o f mi c r o o r g a n i s m s such as f u n g i , y e a s t s and b a c t e r i a w h i c h have 4 "been s u b j e c t e d t o p r o l o n g e d exposure t o s u b - l e t h a l m e t a l c o n c e n t r a t i o n s ( c . f . r.eviews by A s h i d a , I965 and A n t o n o v i c s e t a l . , 1971) b u t 7 t h e r e appear t o be v e r y few examples o f organisms whi:ch p o s s e s s an i n n a t e r e s i s t a n c e t o m e t a l s . T h i s i s the case w i t h D. t e r t i o l e c t a . The s p e c i f i c growth r a t e o f D. t e r t i o l e c t a was u n a f f e c t e d by mercury I I c o n c e n t r a t i o n s o f a t l e a s t 2.03 J i g - -1 -1 a t 1 . A t 10 / i g - a t 1 , the s p e c i f i c growth r a t e was e v e n t u a l l y r e d u c e d by Qk% b u t growth c o n t i n u e d , g i v i n g a f i n a l l e v e l o f c e l l m a t e r i a l o n l y 13% below t h a t i n a m e r c u r y - f r e e c o n t r o l ( D a v i e s , 1976). The a l g a has been shown t o be u n a f f e c t e d by DDT a t 1 ppm as measured by c e l l d i v i s i o n (Bowes, 1971). PCB gave an i n h i b i t i o n o f 43% a t 1 0 3 ppm ( L u a r d , 1973). In a d d i t i o n , t h i s s p e c i e s c o u l d t o l e r a t e f l u o r i d e c o n c e n t r a t i o n from 0 to 100 ppm ( O l i v e i r a e t a l . , 1978). By the mid - 1 9 6 0 's, the p r o d u c t i o n o f a l g a e on a se m i - commercial b a s i s c r e a t e d much i n t e r e s t . Gaucher e t a l . ( i 9 6 0 ) , D a v i s e t a l . (1961), Casey e t a l . (1963), L o o s a n o f f and D a v i s (1963) and U k e l e s (I965) i n v e s t i g a t e d the p o s s i b i l i t y o f c u l t u r i n g a l g a e on a mass s c a l e . U k e l e s and L o o s a n o f f and D a v i s were p r i m a r i l y i n t e r e s t e d i n o b t a i n i n g a r e l i a b l e f e e d s o u r c e f o r clam and o y s t e r l a r v a e . The e f f o r t s o f L o o s a n o f f and D a v i s (1963) were d i r e c t e d toward c u l t u r i n g a l g a e i n l a r g e o u t d o o r v a t s r e s e m b l i n g s m a l l ponds. A n o t h e r t y p e o f mass c u l t u r e u n i t was d e v i s e d by W i s e l y and P u r d a y ( I 9 6 I ) , t h e b a t c h c u l t u r e system. T h i s u n i t c o n s i s t e d o f l a r g e drums c o n t a i n i n g 200 l i t e r s o f I s o c h r y s i s g a l b a n a c u l t u r e s . I l l u m i n a t i o n showed l i g h t i n t e n s i t y t o be re d u c e d 5 g r e a t l y a t . t h e p e r i p h e r y o f the c u l t u r e . T h i s r e d u c t i o n i n l i g h t i n t e n s i t y may have i n f l u e n c e d the d e n s i t y o f the c u l t u r e . A primary drawback to batch c u l t u r e l i e s i n the h a n d l i n g and maintenance of such u n i t s . A l s o , steady s t a t e c o n d i t i o n i n batch c u l t u r e cannot occur without a d d i t i o n a l n u t r i e n t supplementation. Furthermore, such u n i t s must be p e r i o d i c a l l y dismantled, scrubbed down,andc.reinoculated w i t h f r e s h c u l t u r e s i n order to keep a h e a l t h y supply of a l g a e . Because of the i n h e r e n t d i f f i c u l t i e s a s s o c i a t e d with batch c u l t u r e , many experimenters turned to the continuous c u l t u r e of a l g a e . Much of the r e c e n t work done with continuous a l g a l c u l t u r e s has been based on b a c t e r i o l o g i c a l models p i o n e e r e d by Monod (1950), Novick and S z i l a r d (1950) and Herbert et a l . (1956) and Herbert (1958; 1961) a l l o f whom c o n t r i b u t e d to the use of mathematical models f o r continuous c u l t u r e s . The e s s e n t i a l f e a t u r e of the continuous c u l t u r e l i e s i n the f a c t t h a t i t i s s e l f - r e g u l a t i n g . Once the i n f l o w i n g medium has been s e t at a constant v a l u e , the system w i l l a u t o m a t i c a l l y a d j u s t i t s e l f to the steady s t a t e (Herbert, 1958). There are e s s e n t i a l l y two types of continuous c u l t u r e u n i t s , the 'chemostat' (Novick and S z i l a r d , 1950) and the ' t u r b i d o s t a t ' (Herbert, 1958). Although these u n i t s are s i m i l a r i n o p e r a t i o n , the b a s i c p r i n c i p l e by which growth r a t e (̂ 1) i s c o n t r o l l e d d i f f e r s . The t u r b i d o s t a t r e g u l a t e s growth through c o n t r o l o f c e l l d e n s i t y w h i le the chemostat r e g u l a t e s the growth r a t e of the c e l l s by v a r y i n g the amount of ' l i m i t i n g growth s u b s t r a t e ' (Monod, 1950). 6 A l g a l W a s t e - R e c y c l i n g System i n A q u a c u l t u r e C u l t u r e o f marine a l g a e a t h i g h "biomass l e v e l s i n b o t h l a b o r a t o r y and l a r g e - s c a l e o u t d o o r c u l t u r e s u s i n g m u n i c i p a l waste and seawater m i x t u r e s has been conducted (Dunstan and Me n z e l , 1971; Goldman and S t a n l e y , 197*0 • I n t e r e s t i n t h i s c u l t u r e t e c h n i q u e has been g e n e r a t e d by the d e m o n s t r a t i o n t h a t waste w a t e r n u t r i e n t s may be r e c y c l e d i n marine a q u a c u l t u r e systems d e s i g n e d f o r s i m u l t a n e o u s t e r t i a r y t r e a t m e n t o f waste and f o o d p r o d u c t i o n ( R y t h e r e t a l . , 1972; 1 9 7 5 ) . A s e r i e s o f pap e r s i n t h e l i t e r a t u r e i n d i c a t e t h e s u i t a b i l i t y o f l i q u i d o r g a n i c wastes such as sewage and s l u r r y as media f o r the c o n t r o l l e d c u l t i v a t i o n o f f r e s h w a t e r m i c r o - a l g a e ( G a r r e t and A l l e n , 1976; Dugan e t a l . , 1971; G a r r e t e t a l . , 1976). The c o m p o s i t i o n and n u t r i t i o n a l p o t e n t i a l o f a l g a e have a l s o a t t r a c t e d i n t e r e s t f o r many y e a r s (Burlew ( e d ) , 1 9 5 3 ) • Of p a r t i c u l a r i n t e r e s t a r e r e p o r t s on the n u t r i t i v e v a l u e s o f sewage grown a l g a e f o r r a t s , c h i c k , p i g s , c a t t l e and sheep (Cook e t a l , 1963; H i n t z e t a l . , 1 9 6 6 ) . Most o f the i n v e s t i g a t i o n s i n the 1960's and mid - 1 9 7 0's were concerned w i t h t h e c u l t u r e o f a l g a e on m u n i c i p a l sewage e. g., i n C a l i f o r n i a , U.SAA. (Oswald e t a l , 1 9 5 9 ) . i n T h a i l a n d (McGarry e t a l . , 1 9 7 2 ) , A u s t r a l i a (Bureau o f E n v i r o n m e n t a l S t u d i e s , 1975) and I s r a e l ( S h e l e f 5 e t a l . , 1 9 7 2 ) . I n i t i a l work w i t h a n i m a l wastes d u r i n g t h i s p e r i o d i n c l u d e d p i g wastewater s t u d i e s i n the U."*S'. A. and i n t h e P h i l i p p i n e s . The l a t t e r made use o f r o o f s o f p i g houses as s h a l l o w ponds f o r a l g a l c u l t u r e ( E u s e b i o , 1 9 7 6 ) . 7 One of the areas o f u n c e r t a i n t y i n the use of waste-grown algae f o r animal food ( e i t h e r a q u a t i c or t e r r e s t r i a l ) i s the q u e s t i o n of t o x i c a n t s such as heavy metals which may he p r e s e n t i n the s u b s t r a t e and may have p o s s i b l e c o n c e n t r a t i n g e f f e c t s i n pond organisms and c a r r y o v e r as t o x i c r e s i d u e s i n the foods d e r i v e d from the animals. Although animal wastes may c o n t a i n t o x i c a n t s from f e e d r e s i d u e s and growth s t i m u l a n t s such as copper added to the r a t i o n , these are g e n e r a l l y more p r e d i c t a b l e and c o n t r o l l a b l e than with sewage (Dodd, 1 9 7 9 ) ' An important c o n s i d e r a t i o n i n u t i l i z i n g l i v e s t o c k waste or sewage i s the use o f c e r t a i n s p e c i e s of algae t h a t c o u l d t o l e r a t e c o n c e n t r a t i o n of t o x i c compounds or c o u l d u t i l i z e c e r t a i n s o r g a n i c n i t r o g e n s o u r c e s . D. t e r t i o l e c t a have been known to u t i l i z e c e r t a i n o r g a n i c n i t r o g e n sources such as hypoxanthine, urea, ©-glucosamine ( A n t i a et a l . , 1975; 1 9 8 0 ) . The p l a n k t o n i c microalgae which predominate i n h i g h - r a t e ponds are too s m a l l to be s a t i s f a c t o r i l y f i l t e r e d or screened from the pond e f f l u e n t by c o n v e n t i o n a l equipment which employ f a b r i c m a t e r i a l such as m i c r o s t r a i n e r s . Consequently, over many years the search f o r an e f f e c t i v e , low cost method of h a r v e s t i n g which does not decrease the q u a l i t y o f a l g a l product has p a r a l l e l e d the r e s e a r c h i n pond technology. A l g a l h a r v e s t i n g and removal were s t u d i e d e x t e n s i v e l y by Golueke and Oswald ( I 9 6 5 ) and Oswald ( 1 9 6 3 ) . Dodd ( 1979) gave a review on d i f f e r e n t t e c h n o l o g i c a l and managerial advancement of a l g a l h a r v e s t i n g . But s t i l l to date no economical method of h a r v e s t i n g has been s u c c e s s f u l . 8 One h i g h l y e f f i c i e n t method o f u t i l i z i n g t h e c u l t u r e d biomass produced i s t o f e e d i t to a q u a t i c f i l t e r f e e d e r s , such as some s p e c i e s o f c a r p , z o o p l a n k t o n and A r t e m i a . Use and P o t e n t i a l o f A r t e m i a I n A q u a c u l t u r e I n R e l a t i o n To I t s L i f e C y c l e The b r i n e s h r i m p , A r t e m i a s a l i n a L. i s h i g h l y a d a p t a b l e to an e x t r e m e l y wide range o f s a l i n i t i e s and t e m p e r a t u r e s . The A r t e m i a i s a n o n - s e l e c t i v e p a r t i c l e f i l t e r - f e e d e r . C o n t r a r y t o many o t h e r c r u s t a c e a n s , i t s f o o d r e q u i r e m e n t s do n o t change d u r i n g growth (Reeve, 1 9 6 3 a ; 1 9 6 3 b ) and i t has a unique r e p r o d u c t i v e c y c l e which i s v e r y w e l l - s u i t e d t o a c u l t u r a l system even i n h i g h d e n s i t i e s ( H e l f r i c h , 1 9 7 3 ; S o r g e l o o s and P e r s o o n e , 1 9 7 5 ; T o b i a s e t a l . , 1 9 7 9 ) . A r t e m i a have a r a p i d g e n e r a t i o n time (maximum o f about 3 - 4 weeks), a h i g h f e c u n d i t y ( o v e r 1 0 0 o f f s p r i n g day ) and r e p r o d u c e c o n t i n u o u s l y t h r o u g h o u t t h e i r s ix-month to one y e a r l i f e span ( G i l c h r i s t , i 9 6 0 ; Bowen, 1 9 6 2 ; Nimura, 1967). The s u i t a b i l i t y o f A r t e m i a f o r a q u a c u l t u r e i s enhanced by the f a c t t h a t t h e p h y s i c a l c u l t u r a l c o n d i t i o n s such a s : t e m p e r a t u r e , s a l i n i t y , pH, Og t e n s i o n a r e t h e same f o r "both the l a r v a l s t a g e s and a d u l t s . That i s , t h e r e i s no s p e c i a l n u r s e r y ; environment/for a l l s t a g e s . D i f f e r e n c e s i n growth r a t e and time t o a t t a i n m a t u r i t y c i t e d i n the l i t e r a t u r e a r e p r o b a b l y a f u n c t i o n o f : ( 1 ) s p e c i e s o f a l g a l f o o d a v a i l a b l e , ( 2 ) the p r e s e n c e o r absence o f b a c t e r i a and ( 3 ) p h y s i o l o g i c a l s t a t e o f the a l g a l f o o d ( G i b o r , 1 9 5 6 a ; 9 Mason, 1963; Reeve, 1963a; Sick, 1976). In addition to the above environmental conditions, geographical strains have been included ( G i l c h r i s t , 1956; Baid, 1963; D'Agostino, 1965; Sorgeloos et a l . , 1975). However, recent studies showed that Artemia can be reared and reproduced successfully using inanimate food (Person-le Ruyet, 1975; Jacob, 1978; Sorgeloos et a l . , 1980). Technically, the advantagesof Artemia f o r aquaculture i s "'u that i t s t a r t s as dry cysts. These cysts are, i n f a c t , inactive embryos and are commercially available, can be stored f o r years and only have to be incubated for 24-48 hours i n seawater to produce free-swimming n a u p l i i . N u t r i t i o n a l Value of Artemia Nauplii As Food In Aquaculture Hatcheries The e a r l i e s t s i g n i f i c a n t r e a l i z a t i o n of Artemia as an excellent source of food f o r early stages of l a r v a l f i s h has been documented (Seale, 1933; Rollefsen, 1939). Moreover, recent studies showed that brine shrimp are very well accepted by l a r v a l fishes and crustaceans (May, 1970; 1971; Houde, 1972). I t i s not exactly known whether t h i s can be attributed i n t h e i r biochemical composition (Benijts et a l . , 1975; Watanabe et a l . , 1978b), or t h e i r t h i n carapace (1 pm) or the fa c t that they are a moving prey (Houde, 1972) or a combination of a l l these fa c t o r s . Furthermore, studies showed that a diet of l i v e Artemia gives better r e s u l t s than any preparation of dead Artemia ( S e r f l i n g et a l . , 1974; Beck, 1979; Schauer and 10 and Simpson, 1 9 7 9 ) ' Recent f i n d i n g s showed t h a t w h i t e f i s h l a r v a e metamorphosed e q u a l l y w e l l i f f e d A r t e m i a p r e v i o u s l y f r o z e n i n l i q u i d n i t r o g e n o r l i v e A r t e m i a , hut n o t when f e d s l o w - f r o z e n n a u p l i i . R e c e n t l y , emphasis on l a r v a l r e a r i n g o f f i n f i s h e s and c r u s t a c e a n s has been p l a c e d on the use o f g e o g r a p h i c a l s t r a i n s o f t h e b r i n e shrimp (Reed, 1 9 6 9 ; Provenzano and Goy, 1 9 7 6 ; O l e y n i k o v a and P l e s k a c h e u s k a y a , 1979; U c a l , 1 9 7 9 ; Watanabe, 1 9 7 9 ) . From t h e s e s t u d i e s , iifcttwas c o n c l u d e d t h a t the A r t e m i a from G r e a t S a l t Lake (USA) showed poor performance compared w i t h o t h e r s t r a i n s such as t h o s e from Buenos A i r e s ( A r g e n t i n a ) , Shark Bay ( A u s t r a l i a ) , Macau a r e a ( B r a z i l ) , C h a p l i n Lake (Canada), L a v a l d u c ( F r a n c e ) , G u j a r a t a r e a ( I n d i a ) , T i e n t s i n ( P e o p l e ' s R e p u b l i c o f C h i n a ) , B a r o t a c Nuevo ( P h i l i p p i n e s ) , C a d i z a r e a ( S p a i n ) , San F r a n c i s c o Bay (USA). V a r i o u s t h e o r i e s have been s u g g e s t e d t o e x p l a i n t h e p o o r performance o f the G r e a t S a l t Lake (GSL) s t r a i n o f A r t e m i a . One o f t h e s e p l a c e s t h e blame on a c c u m u l a t i o n o f r e s i d u a l p e s t i c i d e s i n t h e GSL A r t e m i a ( S l o d b o k i n , 1 9 6 8 ) . I t i s a l s o p o s s i b l e t h a t GSL A r t e m i a might have d e v e l o p e d immunity a g a i n s t t o x i c a l k a l o i d s e c r e t e d by a l g a l ^ b l o b m s e i n the l a k e and c o n . c o n c e n t r a t e d i n t h e A r t e m i a c y s t s ( P r o v a s o l i , I969). A n o t h e r s u g g e s t i o n blames m i n e r a l d i f i c i e n c y . .> S i n c e t h e use o f A r t e m i a i s p r e s e n t l y l i m i t e d i n most cases t o f r e s h l y h a t c h e d n a u p l i i , t h e use o f a d u l t A r t e m i a has been s u g g e s t e d f o r f u r t h e r s t u d i e s . A d u l t A r t e m i a a r e twenty t i m e s l a r g e r and weigh 5 0 ° t i m e s more t h a n f r e s h l y h a t c h e d n a u p l i i (Reeve, 1 9 6 3 c ) . T h e i r n u t r i t i o n a l v a l u e changes c o n s i d e r a b l y 11 d u r i n g growth. T h e i r f a t d e c r e a s e s from 20$ t o l e s s t h a n 10$ o f t h e d r y w e i g h t and t h e p r o t e i n c o n t e n t I n c r e a s e s from 42$ to over 60$ ( H e l f r i c h , 1973; B e n i j t s e t a l . , 1975). Whereas n a u p l i i a r e d e f i c i e n t i n h i s t i d i n e , m e t h i o n i n e , p h e n y l a l a n i n e and t h r e o n i n e , a d u l t A r t e m i a a r e r i c h i n a l l e s s e n t i a l amino a c i d s ( G a l l a g h e r and Brown, 1975; Watanabe e t a l . , 1978a; C l a u s e t a l . , 1979). P r e s e n t C u l t u r a l P r a c t i c e s o f A r t e m i a E x p o n e n t i a l l y i n c r e a s i n g demand f o r b r i n e shrimp c y s t s w o r l d - w i d e by aquarium h o b b y i s t s and a q u a c u l t u r e h a t c h e r i e s exceeded t h e y e a r l y h a r v e s t o f a p p r o x i m a t e l y 3° t o 5° m e t r i c t o n s ( S o r g e l o o s , 1976). As a r e s u l t , commercial a q u a c u l t u r e has been impeded s e r i o u s l y . Macrobrachium and P e n a e i d s a r e e n t i r e l y dependent on A r t e m i a d i e t d u r i n g J h e i r l o n g l a r v a l development ( B l e d s o e e t a l . , 1978; Glude,e 197.8; S m i t h e t a l . , 1978). I n a d d i t i o n , T h i r d World c o u n t r i e s c o u l d h a r d l y a f f o r d t o i m p o r t the v e r y e x p e n s i v e c y s t s . N a t u r a l b r i n e shrimp p o p u l a t i o n a r e s t i l l t he most i m p o r t a n t s o u r c e o f c o m m e r c i a l l y a v a i l a b l e A r t e m i a . However, they- areooialy e x p l o i t e d i n a few a r e a s i n Canada, F r a n c e and the USA w i t h t h e t o t a l y e a r l y o u t p u t from t h e s e c o u n t r i e s o f c a . 1000 m e t r i c t o n s . The p o t e n t i a l f u t u r e h a r v e s t from n a t u r e where A r t e m i a has, t o d a t e , been r e c o r d e d from more t h a n 150 h a b i t a t s c o u l d c o n s i d e r a b l y i n c r e a s e (Persoone and S o r g e l o o s , 1980). A major i n n o v a t i o n i n t h e t e c h n o l o g y o f A r t e m i a b a t c h c u l t u r i n g i s t h e a i r - w a t e r - l i f t raceway, o r i g i n a l l y d e v e l o p e d 12 f o r t h e i n t e n s i v e c u l t u r e o f p o s t - l a r v a l P e n a e i d shrimp (Mock e t a l . , 1973) out m o d i f i e d f o r "brine shrimp ( S o r g e l o o s e t a l . , 1977; B o s s u y t and S o r g e l o o s , 1 9 8 0 ) . Monodiet u s i n g whey-powder o r r i c e b r a n p r o v e d s u f f i c i e n t . However, i n v i e w o f t h e d i f f e r e n c e s i n f a t t y a c i d c o m p o s i t i o n , i t i s s u g g e s t e d t h a t s t u d i e s a r e needed to e v a l u a t e t h e n u t r i t i o n a l v a l u e o f b r i n e shrimp r a i s e d on waste p r o d u c t s f o r m v a r l d u s s c u l t u r e d organisms ( D o b b e l e i r e t a l . , 1 9 8 0 ) . A s i d e from t h e a i r - w a t e r - l i f t raceway t e c h n o l o g y , a much more i n t e n s i f i e d mass p r o d u c t i o n can be a c h i e v e d i n f l o w - t h r o u g h systems ( T o b i a s e t a l . , 1979)* The f l o w t h r o u g h t e s t c a r r i e d out i n S t . C r o i x (US V i r g i n I s l a n d s ) were r u n s w i t h the e f f l u e n t o f t h e 2 a l g a l ponds o f t h e l o c a l A r t i f i c i a l U p w e l l i n g P r o j e c t ( R o e l s e t a l . , 1 9 7 6 ) . T o b i a s e t a l . ( 1979) c a l c u l a t e d 3 -1 t h a t i n a 1 nr tank 25 k g a d u l t biomass ( o r 12000 a n i m a l s 1 ) can be produced w i t h i n 2 weeks from an i n i t i a l 30 g- c y s t s u s i n g the above system. However, th e maximum p r o d u c t i v i t y p o t e n t i a l has n o t been a c h i e v e d due to lowowater temperature ( 2 2 - 2 5°C). R e c e n t l y , a n o t h e r i n t e r e s t i n g s o u r c e o f A r t e m i a p r o d u c t i o n has come i n t o p e r s p e c t i v e . T e r t i a r y t r e a t m e n t p l a n t s f o r i n d u s t r i a l e f f l u e n t s o f h i g h s a l i n i t y a r e c a p a b l e o f p r o d u c i n g s u b s t a n t i a l amount o f a d u l t A r t e m i a ( M i l l i g a n e t a l . , 1 9 7 9 ) . Other s i m i l a r s t u d y was conducted i n Bombay, I n d i a . The i n i t i a l s t u d y p r o v e d s u c c e s s f u l i n p r o d u c i n g g r a v i d f e m a l e s r e a r e d i n manured ponds ( p i g dung) w i t h superphosphate as a d d i t i o n a l f e r t i l i z e r . P h y t o p l a n k t o n p o p u l a t i o n c o n s i s t i n g o f N a v i c u l a sp_., and Amphiphora sp. bloomed under t h i s c o n d i t i o n and s e r v e d as f e e d f o r t h e s h r i m p . However, ground n u t o i l cake and y e a s t 13 were added as supplement (Dwivedl et a l . , 1979). Aside from an improved p e r s p e c t i v e f o r the use of Artemia i n the aquaculture h a t c h e r i e s , i t has become obvious t h a t other a p p l i c a t i o n s show very high p o t e n t i a l even i n c l u d i n g d i r e c t use i n human n u t r i t i o n . I n a t a s t e p a n e l l t e s t i n Hawaii of an experimental shrimp tempura prepared from f r o z e n b r i n e shrimp the response was q u i t e favorable•. (Davidson, 197*0 • I f not used d i r e c t l y as human food, Artemia meal can be used as a r i c h source of animal p r o t e i n i n l i v e s t o c k d i e t s (Anonymous, 1978)• I n t h i s context, b r i n e shrimp may be used as a va l u a b l e a l t e r n a t i v e to f i s h meal, e s p e c i a l l y i n co u n t r i e s that are e n t i r e l y dependent on f i s h meal imports. 14 MATERIALS AND METHODS A l g a e D u n a l i e l l a t e r t i o l e c t a , a e u r y h a l i n e a l g a l s p e c i e s o b t a i n e d 1 from Dr. N. J . A n t i a ' s c o l l e c t i o n s was used i n t h e e x p e r i m e n t s . The a l g a was m a i n t a i n e d under a x e n i c c o n d i t i o n s i n twenty f i v e ml Erl e n m e y e r f l a s k s i n a l t e r n a t i n g l i g h t and da r k n e s s a t 18°C. The a l g a was t h e n exposed t o a 12 L,: 12 D regime f o r a c o u p l e of days "before t r a n s f e r r i n g t o f i f t y ml Erlenmeyer f l a s k s and where t h e y were m a i n t a i n e d under a x e n i c c o n d i t i o n s as a " s t a r t e r " c u l t u r e . When needed, the Erlenmeyer f l a s k c u l t u r e s were i n o c u l a t e d i n t o one l i t e r o f seawater and a l l o w e d t o r e a c h a d e n s i t y s u i t a b l e f o r i n o c u l a t i n g t h e l a r g e r e x p e r i m e n t a l c u l t u r e v e s s e l s . T h i s a l g a was used f o r the co m p a r a t i v e growth s t u d y u s i n g two d i f f e r e n t media, namely, swine w a s t e - s e a w a t e r r and d e f i n e d i n o r g a n i c media. L i k e w i s e , a l g a l "biomass produced from each c u l t u r e media d u r i n g c o n t i n u o u s c u l t u r e was used f o r the f e e d i n g experiment o f t h e "brine s h rimp. B r i n e Shrimp The "brine s h r i m p , A r t e m i a s a l i n a L. eggs were o b t a i n e d 2 from Dr. J . M a r l i e v e . T h i s s t r a i n o f A r t e m i a came from San F r a n c i s c o Bay, C a l i f o r n i a (USA). R e s e a r c h S c i e n t i s t , P a c i f i c Environment I n s t i t u t e 4 l 6 0 M a r i n e D r i v e , West Vancouver, B. C. > R e s e a r c h S c i e n t i s t , Vancouver P u b l i c Aquarium i n S t a n l e y P a r k P. 0 . Box 3232 Vancouver, B. C. V6B 3X8 15 The b r i n e shrimp eggs were s t o r e d a t 4°C p r i o r t o use. The eggs were h a t c h e d i n a f u n n e l shaped bottom p l e x i g l a s s c o n t a i n e r a t a d e n s i t y o f 10 g 1 . The temp e r a t u r e and s a l i n i t y were m a i n t a i n e d a t 28 + 1°C and 31+1 p p t r e s p e c t i v e l y i n t he h a t c h i n g f u n n e l s . The temp e r a t u r e was m a i n t a i n e d i n a water b a t h h e a t e d by a g l a s s thermo-heater (Supreme Heatmaster T h e r m o s t a t i c 100 w a t t s ) . The newly h a t c h e d n a u p l i i were s e p a r a t e d by a s e p a r a t o r d e s i g n e d a f t e r S o r g e l o o s and P e r s o o n e ^ (1975) Media and S a l t w a t e r P r e p a r a t i o n Swine Waste-Seawater M i x t u r e Swine waste samples were o b t a i n e d from the f i n i s h i n g hog in - h o u s e d b a r n f a c i l i t y a t t h e UBC Farm. Wastes were t a k e n from t h e s u p e r n a t a n t p o r t i o n o f the manure p i t by u s i n g a B i l g e pump (Whale gusher 8 pump MK I I I w i t h 3-81 cm i n l e t and o u t l e t ) m a n u a l l y o p e r a t e d and the waste f i l t e r e d w i t h a n y l o n , s c r e e n mesh (0.3 - 0 . 5 cm)aat the o u t l e t . The f i l t e r e d swine wastes were t h e n s t o r e d i n a p l a s t i c c o n t a i n e r (20 1) and t r a n s p o r t e d t o t h e l a b o r a t o r y ( B i o - R e s o u r c e E n g i n e e r i n g Department). . The swine wastes were s t o r e d i n a 4°C w a l k - i n r e f r i g e r a t o r . New samples o f swine waste were o b t a i n e d from the UBC b a r n e v e r y f o u r days. The samples were a n a l y s e d f o r t o t a l K j e l d a h l n i t r o g e n , NH^-N, N0^-N + N0 2-N and t o t a l phosphate w i t h i n 24 h r s o f c o l l e c t i o n . The r a t i o between swine waste and seawater volume was v a r i e d depending on the n i t r o g e n c o n c e n t r a t i o n , b u t on the 16 average ca 5-10% wastes and 90-95% seawater. The n i t r o g e n -1 c o n c e n t r a t i o n was m a i n t a i n e d a t ca 1200 + 50 j i g - a t N 1 D e f i n e d I n o r g a n i c Medium D. t e r t i o l e c t a has a c o n s p i c u o u s r e q u i r e m e n t f o r sodium ( M c l a c h l a n , i 9 6 0 ) . The medium, . ' f / 2 * d e v e l o p e d by G u i l l a r d (1975) c o n t a i n s s u f f i c i e n t sodium, o t h e r r m a g . o r r n u t r i e n t s such as n i t r o g e n , phosphorus w i t h t r a c e m e t a l i o n s and v i t a m i n s s e r v e d as a n u t r i e n t s o u r c e . The e f f e c t o f f / 2 medium and swine waste-seawater m i x t u r e on a l g a l growth was compared (see Appendix 1 f o r f / 2 c o m p o s i t i o n ) . The s t e r i l e n u t r i e n t s were added t o n o n - s t e r i l i z e d c arboys (20 1) and f i l l e d w i t h 15 1 seawater b e f o r e a l g a l i n o c u l a t i o n . There was no s t e r i l i z a t i o n a ttempt i n any o f the c u l t u r e e x p e r i m e n t s . Seawater P r e p a r a t i o n S i n c e t h e Z o o l o g y Department has seawater f a c i l i t i e s , t he 3 e x p e r i m e n t a l work was i n i t i a l l y c onducted a t Dr. W. S. Hoar's l a b o r a t o r y ;. However, a t the o n s e t o f s p r i n g I98O, t h e seawater had a h i g h c o n c e n t r a t i o n o f mixed a l g a l p o p u l a t i o n due t o h i g h t e m p e r a t u r e . A p r e l i m i n a r y . e x p e r i m e n t showed t h a t t h e s y n t h e t i c s e a w a t e r , F o r t y Fathoms ( B i o - c r y s t a l M a r i n e M i x ; Appendix 2 ) , when supplemented w i t h n u t r i e n t s c o u l d be used t o c u l t u r e P r o f e s s o r , Department o f Z o o l o g y U n i v e r s i t y o f B r i t i s h Columbia Vancouver, B.C. V6T 1W5 17 D. t e r t i o l e c t a . T h i s a r t i f i c i a l s e a w a t e r was u s e d i n f u r t h e r e x p e r i m e n t s . The s e a w a t e r u s e d f o r A r t e m i a c u l t u r e was t r a n s p o r t e d f r o m t h e Z o o l o g y D e p a r t m e n t t o t h e B i o - R e s o u r c e E n g i n e e r i n g D e p a r t m e n t . The s e a w a t e r was a g e d f o r a week b e f o r e i t was u s e d D e s c r i p t i o n o f C u l t u r e U n i t s A l g a l B a t c h C u l t u r e U n i t A l g a l c u l t u r e u n i t s w e re c o n s t r u c t e d a t D r . W. S. H o a r ' s l a b o r a t o r y a t t h e Z o o l o g y D e p a r t m e n t a n d a t t h e a q u a c u l t u r e l a b o r a t o r y o f t h e B i o - R e s o u r c e E n g i n e e r i n g D e p a r t m e n t . B a t c h c u l t u r e e x p e r i m e n t s w e r e c o n d u c t e d b o t h i n U n i t I a n d U n i t I I . C u l t u r e c o n t a i n e r s f o r U n i t I I w e r e 20 1 b o r o s i l i c a t e c a r b o y s . F o r U n i t I ( E x p e r i m e n t I ) , , D . t e r t i o l e c t a w e r e g r o w n i n o n e - l i t e r w i d e m o u t h E r l e n m e y e r f l a s k s . P l a s t i c t u b i n g ( T y g o n R-3603 0.476 x O .635 x 0.14 cm) f i t t e d t o g l a s s t u b i n g • ( 0 . 5 cm o. d.) c o n n e c t e d t h e c u l t u r e f l a s k s w i t h t h e a e r a t i o n s y s t e m . A i r was f i l t e r e d b e f o r e i t was i n t r o d u c e d i n t o t h e c u l t u r e f l a s k s w i t h a b s o r b e n t c o t t o n c i n s i d e a N a l g e n e f i l t e r u n i t . G l a s s t u b i n g o u t l e t s w e r e a l s o p r o v i d e d f o r a i r p r e s s u r e b u i l d - u p a n d a s s a m p l i n g p o r t s . R u b b e r s t o p p e r s ( n o . 8) s u p p o r t e d t h e g l a s s t u b i n g ? w h i c h a l s o p r e v e n t e d l e a k a g e a n d e v a p o r a t i o n . The t e m p e r a t u r e i n t h e c u l t u r e v e s s e l s was m a i n t a i n e d a t 24 + l ° c b y p l a c i n g t h e v e s s e l s , i n t h e wooden w a t e r b a t h (115 cm x 49 cm x 9 cm) w i t h w a t e r f l o w i n g t h r o u g h a t 1 1 h r . 18 Four 4 0-Watt*Cool W h i t e " F l u o r e s c e n t Tubes (F 24712; H. 0. Lamps) p r o v i d e d c o n t i n u o u s i l l u m i n a t i o n . Two tubes were i n s t a l l e d h o r i z o n t a l l y on b o t h s i d e s a p p r o x i m a t e l y 15-20 cm away from t h e c u l t u r e f l a s k s . F l u o r e s e c e n t f i x t u r e s (BL-248, 98 W a t t s , 0.81 amps, 120 v o l t s ) p r o v i d e d power f o r t h e l i g h t s . These f i x t u r e s were a t t a c h e d on Handy-angle s u p p o r t frames. The frame was cov e r e d w i t h peg board (122 x 45 cm). B a t c h c u l t u r e s u s i n g 20 1 b o r o s i l i c a t e c a r b o y s was conducted i n U n i t I I w i t h a se t - u p s i m i l a r t o c o n t i n u o u s c u l t u r e e x p e r i m e n t s . A l g a l C ontinuous C u l t u r e U n i t I I U n i t I I was used f o r c o n t i n u o u s c u l t u r e e x p e r i m e n t s as w e l l as f o r b a t c h c u l t u r e experiment u s i n g 20 1 b o r o s i l i c a t e ( P y r e x ) c u l t u r e c o n t a i n e r . T h i s U n i t was housed i n a peg board-Handy a n g l e frame (230 x 131 x 220 cm). A bank o f f o u r f l u o r e s c e n t tubes (4 f t , 40 w a t t s " c o o l w h i t e " ) were a t t a c h e d on bo t h s i d e s o f t h e c u l t u r e c o n t a i n e r s . L i k e w i s e , two tubes were a t t a c h e d overhead. The c o n t i n u o u s c u l t u r e a p p a r a t u s c o n s i s t e d o f a n u t r i e n t r e s e r v o i r c onnected t o t h r e e c a r b o y s and a common y i e l d 4 - l i t e r a s p i r a t o r b o t t l e s . The a l g a l e f f l u e n t was c o l l e c t e d and s t o r e d i n 20 1 carb o y s ( F i g u r e 1 ) . The n u t r i e n t m i x t u r e s ( d e f i n e d i n o r g a n i c and swine waste- . seawater media) were s t o r e d s e p a r a t e l y i n p l a s t i c c o n t a i n e r s (20 1) on an e l e v a t e d p l a t f o r m . A m u l t i - c h a n n e l p e r i s t a l t i c c a s e t t e pump (Manostat, New Yo r k , N.Y.) was used t o c o n t r o l the AER F i g u r e ! . . G e n e r a l i z e d view o f the a l g a l c o n t i n u o u s c u l t u r e system. . A, a i r ; AE, a l g a l e f f l u e n t ; APP, a i r p r e s s u r e p o r t ; AS, a i r s t o n e ; AER, a l g a l e f f l u e n t r e s e r v o i r ; BC, b o r o s i l i c a t e c a r b o y s ; CYB, common y i e l d b o t t l e ; FL, f l u o r e s c e n t l i g h t s ; N I , n u t r i e n t i n f l o w ; NR, n u t r i e n t r e s e r v o i r ; PP, p e r i s t a l t i c pump; RS, r u b b e r s t o p p e r ; SP, s a m p l i n g p o r t . i n f l o w o f n u t r i e n t s t o a l l c u l t u r e c o n t a i n e r s . The f l o w r a t e was m a i n t a i n e d a t an average o f 2.4 1 day . Tygon t u b i n g ( I . D. 0.4-76 cm) was used t o c c o n v e y t h e n u t r i e n t s . A i r was f i l t e r e d b e f o r e i t was i n t r o d u c e d i n t o t h e c u l t u r e c o n t a i n e r t h r o u g h a Nalgene f i l t e r u n i t w i t h a b s o r b e n t c o t t o n . G l a s s t u b i n g o u t l e t s were p r o v i d e d f o r a i r - p r e s s u r e b u i l d - u p and as a s a m p l i n g p o r t . A r u b b e r s t o p p e r (no. 11) s u p p o r t e d the g l a s s t u b i n g , p r e v e n t e d l e a k a g e and e v a p o r a t i o n . There was no attempt t o s t e r i l i z e t h e c u l t u r e c a r b o y s , s t o p p e r s , t y g o n and g l a s s t u b i n g s . However, c u l t u r e v e s s e l s were acid-washed and r i n s e d w i t h d i s t i l l e d w a t e r and oven d r i e d . The t e m p e r a t u r e i n t h e c u l t u r e c o n t a i n e r was m a i n t a i n e d a t 24 + 1°C by p l a c i n g i n wooden wa t e r b a t h (244 cm x 122 cm x 15.2 cm) w i t h w a t e r f l o w i n g t h r o u g h a t 1 1 h r A l g a l C o n t i n u o u s C u l t u r e U n i t I I I The seawater a t t h e Z o o l o g y Department had a h i g h c o n c e n t r a t i o n o f c o n t a m i n a n t s which b a s i c a l l y c o n s i s t e d o f m i x t u r e s o f a l g a l f l a g e l l a t e s d u r i n g the summer t i m e . A l s o , t h e l a b o r a t o r y a t t h e Z o o l o g y Department was to be r e n o v a t e d b e f o r e the a n t i c i p a t e d c o m p l e t i o n o f the work. E x p e r i m e n t a l work was moved t o t h e a q u a c u l t u r e l a b o r a t o r y o f the B i o - R e s o u r c e ^ - i ^ i ^ e r E n g i n e e r i n g Department. U n i t I I I se,±-up was s i m i l a r t o t h a t o f U n i t I I . However, the whole s e t - u p was conducted i n a c o n t r o l l e d e n v i r o n m e n t a l chamber ( C o n v i r o n Model E8M, C o n t r o l l e d E n v i r o n m e n t a l L t d . , W i n n i p e g , M a n i t o b a , Canada. Temperature was c o n t r o l l e d and m a i n t a i n e d a t 24 C, C o n t i n u o u s i l l u m i n a t i o n was p r o v i d e d w i t h 6 f l u o r e s c e n t tubes (Powertubes 48" VHO 'Cool W h i t e 1 F48T1Z-CW, S y l v a n i a , Canada) p l a c e d overhead. I l l u m i n a t i o n was measured by Quantum/Radiometer/Photometer s e n s o r s (Lambda L I - 1 8 5 ) • -1 -2 The a v e r a g e . l i g h t i n t e n s i t y was ca 0 . 0 4 c a l min cm ( 0 . 0 4 - -1 0 . 0 5 l y min ) measured i n s i d e the c a r b o y s w i t h o u t the l i q u i d b e i n g p r e s e n t . B r i n e Shrimp C u l t u r e U n i t B r i n e shrimp were c u l t u r e d i n c y l i n d r i c a l f i b e r g l a s s t a n k s o f 40 l i t e r c a p a c i t y . A PVC (127 mm d i a m t e r ) s t a n d p i p e f i t t e d w i t h n y l o n s c r e e n mesh (0.1-0.3 mm) a t the o u t l e t m a i n t a i n e d the volume a t 20 l i t e r s . A handy a n g l e frame h e l d the f i b e r g l a s s t a n k s u p r i g h t and a wooden d r a i n a g e system was p r o v i d e d . -1 -1 The w a t e r column was c o n s t a n t l y are'rafte'd (600 -700 ml min ) i n o r d e r t o r e s u s p e n d the a l g a e t h r o u g h o u t the w a t e r column and t o p r o v i d e oxygen f o r the b r i n e s h r i m p s . A i r f l o w was measured by G i l m o n t Flowmeter (F - 1 2 0 0 ) . The temperature i n the t a n k s was m a i n t a i n e d a t 27 + 1°C by g l a s s t h e r m o - h e a t e r s (Supreme Heatmaster T h e r m o s t a t i c 100 w a t t s ) . C u l t u r e Methods A l g a l C u l t u r e Methods A l t h o u g h none o f the c u l t u r e u n i t s remained b a c t e r i a - f r e e , c o n t a m i n a t i o n by o t h e r a l g a e was a v o i d e d by i n o c u l a t i n g w i t h an a x e n i c c u l t u r e and f i l t e r i n g the seawater w i t h a h i g h p r e s s u r e f i l t e r u n i t (AMF/CUNO F i l t e r Model 1A1) a t t a c h e d t o a s e l f - p r i m i n g pump (Ja b s c o Model No. 12310-0001). The d e n s i t y o f t h e i n o c u l a n t was de t e r m i n e d b e f o r e i t was i n t r o d u c e d . t o t h e c u l t u r e u n i t s . The a l g a l c e l l s were counted d a i l y u s i n g a hemacytometer (New Improved Neubauer Chamber, Hausser S c i e n t i f i c ! ) . To i m m o b i l i z e the m o t i l e D. t e r t i o l e c t a , 2 o r 3 drops o f 5% f o r m a l i n were added to t h e a l g a l samples b e f o r e c o u n t i n g . The same f o u r squares on t h e g r i d o f each chamber were counted each time and the average count was 4 m u l t i p l i e d by 10 , Thus, g i v e n t h e average Q, the d e n s i t y e,' ( c e l l s ml ) o f t h e s u s p e n s i o n i n the hemacytometer was c a r a u l a t e d from the e x p r e s s i o n , d = 10* x Q Three r e p l i c a t e counts were made and the mean v a l u e , c a l c u l a t e d . S a l i n i t y was m a i n t a i n e d a t 32 + 1 p p t and was measured u s i n g a hydrometer ( S p e c i f i c g r a v i t y f o r heavy l i q u i d s TP 60/60°F range 1000-1220). Temperature r e a d i n g s were t a k e n d a i l y i n a l l c u l t u r e u n i t s . P e r i o d i c checks on pH change i n t h e c u l t u r e s h e l p e d to m o n i t o r a c i d i c and/or b a s i c c o n d i t i o n s w i t h i n t h e c u l t u r e . The pH was measured u s i n g a pH meter ( F i s h e r Accumet Model 420 D i g i t a l p H / i o n m e t e r ) . Once the a l g a l growth phase r e a c h e d t h e d e c r e a s i n g o r d e c l i n i n g l o g a r i t h m i c phase (Myers, 19^2) a t a d e n s i t y o f 6 -1 a p p r o x i m a t e l y 4 x 10 c e l l ml , the n u t r i e n t i n f l o w t o t h e c u l t u r e v e s s e l was i n i t i a t e d . Flow r a t e was m a i n t a i n e d a t -1 -1 a p p r o x i m a t e l y 2 .33 - 2.50 l i t e r day carboy . A t t h i s f l o w r a t e , t h e c e l l d e n s i t y r e a c h e d s t e a d y s t a t e , as d e t e r m i n e d by i d e n t i c a l c e l l c ounts o v e r a p e r i o d o f 2-3 days. The c e l l d e n s i t i e s would drop t o a l o w e r c o n c e n t r a t i o n a t a h i g h e r f l o w 23 r a t e . B r i n e Shrimp C u l t u r e Method The i n c u b a t i o n p e r i o d was about 48r.hours a t 26 + 1°C. . D e n s i t y d e t e r m i n a t i o n was conducted a f t e r the n e w l y - h a t c h e d n a u p l i i were s e p a r a t e d from unhatched eggssand empty c y s t s . Newly-hatched A r t e m i a were p l a c e d i n a 4 - l i t e r p l a s t i c c o n t a i n e r , m o d e r a t e l y a e r a t e d t o keep them e v e n l y suspended. F i v e s e p a r a t e samples o f 5 ml each were t a k e n from the s u s p e n s i o n . An 8 mm d i a m e t e r , 250 mm l o n g g l a s s t u b i n g was used as s a m p l i n g t u b e . Both ends o f t h e tube were opened and when s a m p l i n g , one end was c l o s e d by a thumb. Thus, th e e n t i r e w a t e r column was sampled. Each o f t h e 5 ml samples were p l a c e d i n s e p a r a t e c y l i n d e r s (50 ml c a p ) . Then th e volume i n t h e c y l i n d e r s was made up t o 25 ml o f t h e same s a l i n i t y . T h i s volume was a e r a t e d s l o w l y t o e v e n l y suspend t h e n a u p l i i . A g a i n , 5 ml were t a k e n from each c y l i n d e r and were p l a c e d i n s m a l l g l a s s p e t r i d i s h e s . S e v e r a l drops o f L u g o l ' s s o l u t i o n were added t o each p e t r i d i s h . Then, th e p e t r i d i s h e s were p l a c e d under a d i s s e c t i n g m i c r o s c o p e and t h e n a u p l i i c ounted. The number o f n a u p l i i i n t h e o r i g i n a l c o n t a i n e r was o b t a i n e d by t h e f o l l o w i n g e q u a t i o n : T o t a l no. o f n a u p l i i = T o t a l no. o f n a u p l i i counted volume o f * ~ -z - c o n t a i n e r . . -1 I n i t i a l c o n c e n t r a t i o n m each tank was 2-3 n a u p l i i l i t e r A l g a l c e l l c o n c e n t r a t i o n was m a i n t a i n e d d a i l y a t a p p r o x i m a t e l y < -1 5.0 x 1CK ml i n each t a n k . S u r v i v a l counts were conducted d a i l y i n each t a n k . A 5 ° n i l "beaker was used t o samp He the water column and t h e n a u p l i i were counted. The c u l t u r e was a g i t a t e d "by a r e l a t i v e l y s t r o n g a e r a t i o n f o r a minute "before s a m p l i n g . C o u n t i n g was done 3 t i m e s f o r each t a n k . To measure growth, t e n a n i m a l s were sampled d a i l y and p r e s e v e d from each t a n k . P r e s e r v e d a n i m a l s ( i n a 5$ f o r m a l i n solution)v:w.ere measured two weeks a f t e r t he exp e r i m e n t . The t o t a l body l e n g t h was measured from the a n t e r i o r m a r g i n o f the head t o the base o f t h e c a u d a l f u r c a ( G i l c h r i s t , 1 9 5 6 ) . D u r i n g measurement, t h e a n i m a l s were p l a c e d v e n t r a l s i d e down on a mjr.c r o s e ope s l i d e . The t e m p e r a t u r e i n t h e c u l t u r e t a n k s was m a i n t a i n e d a t 27 + 1°C by g l a s s t h e r m o s t a t i c h e a t e r s . Oxygen c o n c e n t r a t i o n was a t s a t u r a t i o n l e v e l s . C h e m i c a l A n a l y s i s K j e l d a h l N i t r o g e n The method f o l l o w e d was an a d a p t a t i o n o f W.all e t a l . (197*0 and T e c h n i c o n A u t o A n a l y s e r II ( 1 9 7 1 b ) . F i v e ml o f the sample (swine waste o r a l g a l suspensionH'were i n t r o d u c e d i n t o a 50 ml d i g e s t i o n t u b e . To enhance the o x i d a t i o n r e d u c t i o n , 0 . 5 g o f a d i g e s t i o n c a t a l y s t (composed of9 9 6 0 g K^SO^, 35 g CuSO^, and 5 g SeOg) were added t o each t u b e . The tu b e s were t h e n p l a c e d i n a d i g e s t o r r a c k a t 360°C f o r 6-12 h o u r s . The a d d i t i o n o f b o i l i n g c h i p s p r e v e n t e d e x c e s s i v e bumping d u r i n g d i g e s t i o n and a s m a l l g l a s s f u n n e l on t h e open end o f t h e tubes p r e v e n t e d s p i l l a g e 25 and e x c e s s i v e e v a p o r a t i o n . A f t e r d i g e s t i o n , the t ubes were ei:. removed from the d i g e s t o r and c o o l e d f o r about an hour. D i s t i l l e d w a t e r was t h e n added t o each o f the f l a s k s t o a volume o f 50 m l . F u r t h e r d i l u t i o n o f t h e sample w i t h d i s t i l l e d w a t e r was n e c e s s a r y when the a u t o - a n a l y s i s g r a p h i c a l r e c o r d e r went o f f - s c a l e . A p p r o x i m a t e l y 10 ml o f each d i l u t e d sample were p l a c e d on a r o t a t i n g sampler o f t h e a u t o - a n a l y s e r . Ammonia / N i t r a t e + N i t r i t e The automated p r o c e d u r e f o r the d e t e r m i n a t i o n o f t h e above n i t r o g e n o u s compounds was adapted t o t h a t o f T e c h n i c o n A u t o A n a l y s e r I I , 1969; 1971a. T o t a l Phosphate Samples were d i g e s t e d b e f o r e d e t e r m i n i n g t o t a l phosphate t h r o u g h t h e a u t o - a n a l y s e r . The d i g e s t i o n p r o c e d u r e was s i m i l a r to t h a t used f o r t h e d e t e r m i n a t i o n 1 o f t o t a l K j e l d a h l n i t r o g e n . The automated p r o c e d u r e f o r the d e t e r m i n a t i o n o o f t o t a l phosphate w a s r a d a p t e d t o t h a t o f T e c h n i c o n A u t o A n a l y s e r , 1971c. Dry Weight A n a l y s i s An i n c r e a s e i n d r y w e i g h t o f c e l l s i s a w i d e l y used method o f growth measurement. F i f t y ml a l i q u o t samples from each c u l t u r e c o n t a i n e r were f i l t e r e d t h r o u g h g l a s s f i b e r f i l t e r (Reeve A n g e l Grade 93^ AH, s i z e 4 .25 cm d i a m e t e r ) u s i n g vacuum s u c t i o n . The f i l t e r p a p er was p l a c e d on t h e . c r u c i b l e and oven d r i e d a t lOO^C f o r about an 26 hour and weighed b e f o r e u s i n g . A f t e r f i l t r a t i o n , c r u c i b l e and f i l t e r paper were oven d r i e d t o a c o n s t a n t w e i g h t . Three r e p l i c a t e s were r u n e f o r each o f t h e c u l t u r e v e s s e l . The f i l t e r paper and c r u c i b l e were weighed on a M e t t l e r b a l a n c e (Type H6 d i g cap 160 g, M e t t l e r I n s t r u m e n t , Highstown N. S. Canada). The w e i g h t was r e c o r d e d t o f o u r d e c i m a l p l a c e s . Dry w e i g h t biomass o f A r t e m i a was measured as d e s c r i b e d above. To d e t e r m i n e the v a r i a b i l i t y o f the s o l i d c o n c e n t r a t i o n i n the swine waste samples, t o t a l suspended s o l i d s were a n a l y s e d . U s i n g s u c t i o n , 100 ml samples were f i l t e r d and washed w i t h 2-5 ml d i s t i l l e d w a t e r . The c r u c i b l e and f i l t e r p aper (Whatman 7 .0 cm no. h a r d e n e d ? , a s h l e s s ) were oven d r i e d a t 103-105°C f o r an hour b e f o r e u s i n g . The oven d r i e d f i l t e r p a per was c o o l e d i n a d e s i c c a t o r and the w e i g h t was r e c o r d e d . A f t e r f i l t r a t i o n , the f i l t e r paper and c r u c i b l e were t h e n p l a c e d i n the oven f o r an hour, c o o l e d i n a d e s i c c a t o r and weighed ( g r o s s w e i g h t ) . D r y i n g and w e i g h i n g were c a r r i e d out u n t i l l c o n s t a n t w e i g h t was a c h i e v e d ( n o r m a l l y t h r e e t i m e s ) . F i v e r e p l i c a t e s were r u n f o r each sample. A n a l y s i s f o r t o t a l suspended s o l i d s was conducted f o r ' f r e s h ' s s a m p l e s t a k e n from t h e p i t - m a n u r e . T o t a l suspended s o l i d s was c a l c u l a t e d u s i r i g l r f c h e r s f o l l o w i n g e q u a t i o n : T o t a l Suspended S o l i d s TSS ppm (mg 1 1 ) = Gross wt - Tare wt (mg) 6 v o l o f sample (ml) x 10 27 RESULTS Swine Waste A n a l y s i s D u r i n g t h e c o u r s e o f t h e e x p e r i m e n t s , t h e t o t a l K j e l d a h l n i t r o g e n c o n c e n t r a t i o n (TKN = o r g a n i c n i t r o g e n and ammonia n i t r o g e n ) and o t h e r i n o r g a n i c n i t r o g e n (NOg-N + NO^-N) and PO^-P o f t h e s u p e r n a t a n t p a r t o f t h e swine waste v a r i e d o n l y s l i g h t l y and a l s o t h e r e was n o t much c o n s i d e r a b l e changes i n t o t a l suspended s o l i d s (TSS) as shown i n T a b l e 1. Measurement o f t o t a l K j e l d a h l n i t r o g e n o f t h e swine waste samples and t h r e e d i f f e r e n t ratio:s;:- o f swine waste-seawater m i x t u r e s ( 1 : 1 , 2:1 and 1 : 2 ) s t o r e d a t 4°C r e f r i g e r a t o r were c a r r i e d out t o d e t e r m i n e i f changes i n N d i s t r i b u t i o n m ight o c c u r d u r i n g s t o r a g e . The r e s u l t s showed no c o n s i d e r a b l e change o f the t o t a l K j e l d a h l n i t r o g e n c o n c e n t r a t i o n between day 1. and day 5 ( F i g u r e 2 ) . A l g a l B a t c h C u l t u r e System The f i r s t e x p eriment on the c o m p a r a t i v e growth s t u d y o f D. t e r t i o l e c t a grown i n d e f i n e d inorganicir,medium and swine waste- seawater medium was conducted i n o n e - l i t e r wide mouth Er l e n m e y e r f l a s k s i n b a t c h c u l t u r e system. F i g u r e 3 shows d a i l y c e l l d e n s i t y d u r i n g t h e seven day c u l t u r e p e r i o d . Growth c o n s t a n t (K^Q^hr ) were 0 . 0 2 3 and 0 . 0 2 4 and t h e mean g e n e r a t i o n time ( t g h r s ) were 13.0 and 1 2 . 5 f ° r d e f i n e d i n o r g a n i c and swine waste-seawater media, r e s p e c t i v e l y . A n o t h e r b a t c h c u l t u r e s t u d y ( E x p t 2 ) was conducted i n 2 0 l i t e r c a p a c i t y P y r e x c a r b o y s . However, the c u l t u r e volume was T a b l e 1. T o t a l K j e l d a h l n i t r o g e n (TKN), NH^-N, NO^-N^ N0 2~N f PO^-P and T o t a l Suspended S o l i d s (TSS) o f swine waste d u r i n g the course o f t h e e x p e r i m e n t a l work. T o t a l K j e l d a h l NH^-N N0~-N+N0?-N p o k ~ P T o t a l N i t r o g e n (TKN) ^ Suspended -1 -1 -1 _1 S o l i d s . , ( j i g - a t N 1 ) (ug-at N 1 1 ) ( u g - a t N 1 ) (/ig-a t E l ) (mg 1 l ) 25 Sept 1979* 1 4 2.5 x 10* 4 1.0 x 10^ 3-57 3 . 2 x 1 0 2 3-0 : 28 Oct 2 4.9 4 . 0 5.00 3.87 3.9 29 Nov 3 5.0 3 .5 6.43 5.16 3.31 07 Dec 3 4.6 3 .0 3.57 3.20 10 Dec 3 5.4 3 .6 3.57 6.12 3 . 4 13 Dec 3 5.1 3.6 2.14 5.80 4 .05 16 3 4.8 3 .4 5-0 04 Fed (Expt 4,. .was 3.9 2.8 5-71 3.20 3.09 d i s c o n t i n u e d ) 10 March 4 . 2 2.5 3.87 3-50 18 June 5 3.8 2.3 3.57 3 .20 3-7 22 5 3.7 2.1 5-0 2.9 3 . 0 5 26 5 3.8 2.2 3.57 3.54 3.12 30 5 4.1 2.5 5.0 4.83 05 J u l y 5 3.6 2.2 3.20 3.8 10 5 4.3 2.5 3.57 4.19 15 5 3.7 2.2 2.58 3.5 * T h i s p a r t i c u l a r swine waste sample was o b t a i n e d from R and H Farm L a n g l e y , B. C. The r e s t o f the samples were from U. B. C. No. 1 R e s e a r c h Farm. S a m p l i n g Date Experiment No. 800 n 29 700' 600- 500^ £ c G . C OJ o> o . c 400 o 5 300^ 200^ 10-0-1 0 Figure 2, -r 2 TIME (days) o f swme waste-seawater mixtures ( 1 i D f o means 30 TIME (days) F i g u r e 3- D a i l y c e l l d e n s i t y of'D. t e r t i o l e c t a grown i n swine waste-seawater ( O ) and d e f i n e d i n o r g a n i c . m e d i a (® ) i n b a t c h system (Expt 1 ) . 31 m a i n t a i n e d o n l y up to 15 l i t e r s . I n t h i s e xperiment and a l s o w i t h the l a t t e r ones, t h r e e r e p l i c a t e s were used f o r each t r e a t m e n t . F i g u r e s 4 and 5 show d a i l y c e l l d e n s i t y and a l g a l d r y w e i g h t , r e s p e c t i v e l y . A g a i n , t h e growth c o n s t a n t s were c a l c u l a t e d . S t a t i s t i c a l a n a l y s i s u s i n g a n a l y s i s o f v a r i a n c e (ANOVA) showed no s i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t s and r e p l i c a t e s f o r growth c o n s t a n t (Appendix 3 ) . The s t a t i s t i c a l a n a l y s i s was done t h r o u g h t h e UBG Computer System u s i n g a G e n e r a l L e a s t Squares A n a l y s i s o f V a r i a n c e Programme known as GENLIN ( G r e i g and B j e r r i n g , 1 9 7 7 ) . A l g a l C ontinuous C u l t u r e System C o n t i n u o u s c u l t u r e s t u d i e s were conducted i n 20 l i t e r c a p a c i t y P y r e x c a r b o y s . The f i r s t c o n t i n u o u s c u l t u r e experiment was t e r m i n a t e d on day 16 because t h e r e were some problems w i t h seawater s u p p l y i n the l a b o r a t o r y (Dr. W. S. Hoar's l a b o r a t o r y , Z o o l o g y D e p a r t m e n t ) . F i g u r e s 6 and 7 show d a i l y c e l l d e n s i t y and a l g a l d r y w e i g h t , r r e s p e c t i v e l y . S t a t i s t i c a l a n a l y s i s f o r t h e growth c o n s t a n t , c e l l d e n s i t y and d r y w e i g h t showed no s i g n i f i c a n t d i f f e r e n c e s ( P < 0 . 0 5 ) between treatmentsr>,arid r e p l i c a t e s (Appendices 4 a , 4 b , 4 c ) . The second c o n t i n u o u s c u l t u r e experiment was t e r m i n a t e d on day 2 7 . The a l g a e p r o d u c e d d a i l y from t h i s e x periment were used i n t h e f e e d i n g s t u d i e s o f A r t e m i a . F i g u r e s 8 and 9 show c e l l d e n s i t y and d r y w e i g h t , r e s p e c t i v e l y , measured d a i l y d u r i n g t h e c u l t u r e p e r i o d . T h i s experiment was conducted i n a c o n t r o l l e d e n v i r o n m e n t a l chamber a t t h e Department o f B i o - R e s o u r c e 32 T I M E (days) F i g u r e 4. D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a grown i n swi n e waste-seawater (o ) and d e f i n e d i n o r g a n i c media ( • ) i n b a t c h c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 2 ) , The above v a l u e s are ranges and means o f r e p l i c a t e s p e r t r e a t m e n t . 33 0.20-, i o> 0.15- t_ xi o 0.10- < 0.051 0 TlME(days) F i g u r e 5- D a i l y measurement o f d r y wt o f D. t e r t i o l e c t a grown i n swine waste-seawater ( O ) and d e f i n e d i n o r g a n i c media ( • ) i n a b a t c h c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e carboys (Expt 2 ) . The above v a l u e s are ranges and means o f r e p l i c a t e s p e r t r e a t m e n t . 3k F i g u r e 6. D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a grown i n swine waste-seawater ( O ) and d e f i n e d i n o r g a n i c media ( #) i n c o n t i n u o u s c u l t u r e system using- 20 1 b o r o s i l i c a t e c a r b o y s ( E x p t 3 ) • The above v a l u e s are ranges and means o f r e p l i c a t e s p e r t r e a t m e n t . 35 u ' ' I 1 1 1 1 1 1 1 1 1 1 1 1 r - — i 1 5 10 1 5 TIME (days) F i g u r e 7 . D a i l y measurement o f d r y wt Of D. t e r t i o l e c t a grown i n swine waste-seawater (o ) and d e f i n e d i n o r g a n i c media (• ) i n c o n t i n u o u s c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a r b o y s (Expt 3 ) . Arrow i n d i c a t e s s t a r t o f c o n t i n u o u s c u l t u r e . The above v a l u e s are ranges and means o f r e p l i c a t e s p e r t r e a t m e n t . 36 1 5 10 15 20 25 30 TIME (days) F i g u r e 8. D a i l y c e l l d e n s i t y o f D. t e r t i o l e c t a grown i n swine waste-seawater ( O ) and d e f i n e d i n o r g a n i c media ( • ) i n c o n t i n u o u s c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c a rboys (Expt 5 ) ' Arrow i n d i c a t e s s t a r t o f c o n t i n u o u s c u l t u r e . The above v a l u e s are ranges and means o f r e p l i c a t e s p e r t r e a t m e n t . 37 0.35n 0.30H 0.05H u ' — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — | — i — i 1 5 10 15 20 25 30 TIME (days) F i g u r e 9. D a i l y measurement o f d r y wt o f D.. t e r t i o l e c t a grown i n • s w i n e - w a s t e — s e a w a t e r (o) and d e f i n e d i n o r g a n i c media ( • ) i n c o n t i n u o u s c u l t u r e system u s i n g 20 1 b o r o s i l i c a t e c arboys ( E x p t 5)• Arrow i n d i c a t e s s t a r t o f c o n t i n u o u s c u l t u r e . The above v a l u e s a r e ranges and means o f r e p l i c a t e s p e r t r e a t m e n t . 38 E n g i n e e r i n g Department A q u a c u l t u r e L a b o r a t o r y . S t a t i s t i c a l a n a l y s i s showed a h i g h l y s i g n i f i c a n t d i f f e r e n c e ( P < 0 . 0 1 ) between t r e a t m e n t s b u t i t showed no s i g n i f i c a n t d i f f e r e n c e s f o r r e p l i c a t e s , ( A p p e n d i x 5 a ) . The d a t a a n a l y s e d were from day 8 ( o n s e t o f n u t r i e n t i n f l o w ) up t o day 2 7 . However, the r e s u l t s were d i f f e r e n t when d a t a were a n a l y s e d i n segments (Appendices 5"b, 5 c , 5 d ) « Thus, t h e f o l l o w i n g were o b t a i n e d : Days F - R a t i o n 8 - 1 4 t r e a t m e n t h i g h l y s i g n i f i c a n t ( P < 0 . 0 1 ) r e p l i c a t e n o t s i g n i f i c a n t 1 5 - 2 2 t r e a t m e n t b o t h n o t s i g n i f i c a n t r e p l i c a t e 2 3 - 2 7 t r e a t m e n t b o t h n o t s i g n i f i c a n t r e p l i c a t e The v a r i a b i l i t y o f the above r e s u l t s was p r o b a b l y due to t h e v e r y dynamic system. S t a t i s t i c a l a n a l y s i s f o r growth c o n s t a n t showed no s i g n i f i c a n t d i f f e r e n c e s f o r t r e a t m e n t s and r e p l i c a t e s ( Appendix 6 ) . However, f o r a l g a l d r y w e i g h t , s i g n i f i c a n t d i f f e r e n c e s f o r t r e a t m e n t ( P < 0 . 0 1 ) and f o r r e p l i c a t e s ( P < 0 . 0 5 ) were o b s e r v e d (Appendix 7 ) . N u t r i e n t s i n t h e form o f n i t r o g e n such as t o t a l K j e l d a h l n i t r o g e n , NH^-N, 0 r g a n i c - N i a n d NO^-N + NOg-N were measured i n t h e a l g a l e f f l u e n t d u r i n g t h e second c o n t i n u o u s c u l t u r e ( E x p t 5 ) t o d etermine whether t h e s e n u t r i e n t s were f u l l y u t i l i z e d ( T a b l e 2 2 ) . 1 39 T a b l e 2". N i t r o g e n (TKN, NH^-N, ©rganic-N",- NO^-N + N 0 2 ) measured i n t h e a l g a l e f f l u e n t d u r i n g the second c o n t i n u o u s c u l t u r e (Expt 5) i n ug-at N 1 .(SW, swine waste seawater medium; DM, d e f i n e d i n o r g a n i c mediumO Date Sample TKN NH^-N O r g a n i c NO^-N+NOg-N No. N i t r o g e n 26 June SW 1 3.8 X !0 2 3.2 X 2 10^ 6.0 X IO 1 u n d e t e c t a b l e (d9) 2 3.6 3-5 1.0 I I 3 3.4 2.8 6.0 i t DM 1 2.0 1.4 X 10 1 1.9 X 2 10 1.78 x 10 2 2 1.7 1.4 1.6 1.85 3 2.1 1.8 1.9 1.28 30 June SW 1 3.2 2.1 X 2 10 l . l X 2 10 u n d e t e c t a b l e (dl3) 2 2.8 1.4 1.4 I I 3 2.9 2.1 8.0 X I O 1 i t DM 1 1.3 3.8 X 10 1 9.2 1.07 x i o 2 2 1.6 2.1 1.4 X i o 2 1.28 3 1.4 1.8 1.2 1.14 06 J u l y SW 1 2.5 1.8 X 2 10 7.0 X i o 1 u n d e t e c t a b l e (dl9) 2 2.1 1.3 8.0 i t 3 1.8 1.1 7.0 If . DM 1 7.1 X i o 1 3.6 X 10 1 3-5 1.42 x 10 2 2 1.4 X 2 10 2.6 1.1 X 2 10 1.64 3 1.4 2.1 1.2 1.14 10 J u l y SW 1 2.7 1.4 X 2 10 1.3 u n d e t e c t a b l e (d23) 2 2.3 1.4 9.0 X i o 1 i t 3 1.9 8.9 X i o 1 1.0 X 2 10^ I I DM 1 9.3 X i o 1 1.9 7.4 X I O 1 1.28 x 10 2 2 3.6 1.9 2.0 1.50 X 3 1.3 X 10 2 1.8 1.1 X i o 2 1.78 Sample nos 1, 2 and 3 were o b t a i n e d from each o f t h e c u l t u r e v e s s e l s . ^ O r g a n i c N i t r o g e n * (TKN) (NH--N) 40 Brine Shrimp Feeding Experiment s Figure 10 shows the growth i n t o t a l length of Artemia (fr e s h l y measured and preserved samples) during the 12-day culture period. In t h i s preliminary experiment the a l g a l density was maintained d a i l y at the range 3«0 to 5*0 x 10-̂  c e l l s ml . Figure 11 shows the percentage su r v i v a l for t h i s experiment. Figures 12 and 13 show the t o t a l length measurement and percentage s u r v i v a l of the shrimp,respectively (during the 14-day culture period. In th i s p a r t i c u l a r experiment four r e p l i c a t e s were used for each treatment. S t a t i s t i c a l analysis f o r t o t a l length measurement showed no s i g n i f i c a n t differences f o r treatments and r e p l i c a t e s (Appendix 8). However, s t a t i s t i c a l analysis for percentage su r v i v a l showed highly s i g n i f i c a n t differences (P<0.01) for treatments and r e p l i c a t e s . The overalllmean length of Artemia i n treatment 2 (Artemia fed with swine waste grown algae) was higher than tr.eatmenteibifcArtemia fed with defined inorganic medium grown algae). Furthermore, s t a t i s t i c a l analysis showed d i f f e r e n t r e s u l t s when percentage su r v i v a l data were analysed i n segments (days 1-7 and 8-14). The analysis f o r the f i r s t seven days showed highly s i g n i f i c a n t differences (P<0.01) i n percentage surv i v a l y f o r treatments and r e p l i c a t e s . However, during the l a s t seven days the percentage su r v i v a l showed no s i g n i f i c a n t differences f o r treatments and r e p l i c a t e s (Appendices 9a, 9t>, 9c). U " 1 1 1 1 1 1 1 1 1 1 1 1 1 ! 1 1 5 . • 10 15 TIME (days) F i g u r e . 1 0 . Growth i n t o t a l l e n g t h o f A r t e m i a s a l i n a L. over a 12-day c u l t u r e p e r i o d f e d w i t h D. t e r t i o l e c t a grown i n d e f i n e d i n o r g a n i c medium. The above v a l u e s a r e average measurements o f a n e s t h e s i z e d samples ( • ) and 2-weeks o l d p r e s e r v e d samples (o). 42 F i g u r e 11. P e r c e n t a g e s u r v i v a l o f A r t e m i a s a l i n a L. f e d w i t h D. t e r t i o l e c t a grown i n d e f i n e d i n o r g a n i c medium. The above v a l u e s are ranges and means o f r e p l i c a t e s . 43 F i g u r e 12. Growth i n t o t a l l e n g t h o f A r t e m i a s a l i n a L. o v e r a 14-day c u l t u r e p e r i o d f e d w i t h swine waste-seawater ( O ) and d e f i n e d i n o r g a n i c ( # ) media grown D. t e r t i o l e c t a . The above v a l u e s a r e ranges and means o f f o u r r e p l i c a t e s p e r t r e a t m e n t . T 5 10 TIME (days) 15 gure 1 3 . P e r c e n t a g e s u r v i v a l o f A r t e m i a s a l i n a L. ov e r a • 1'4-day c u l t u r e p e r i o d f e d w i t h swine waste- seawater (o ) and d e f i n e d i n o r g a n i c media ( • ) grown D. t e r t i o l e c t a . The above v a l u e s are ranges and means o f f o u r r e p l i c a t e s . Adult Artemia produced i n the system a f t e r the 14-day culture period were measured i n wet and dry weight basis (Table 3 ) . S t a t i s t i c a l analysis f o r wet and dry weight of Artemia showed no s i g n i f i c a n t differences between treatments and r e p l i c a t e s (Appendices 10a and 10b, r e s p e c t i v e l y ) . 46 T a b l e 3- A d u l t A r t e m i a biomass produced a f t e r t h e l 4-day c u l t u r e p e r i o d i n wet and d r y w e i g h t b a s i s . A r t e m i a Fed W i t h Wet Weight Dry Weight Swine Waste-Seawater (g) (g) Grown A l g a e — —- 1 18.82 1.82 2 19.^6 2.13 3 19.43 2.06 4 20.47 2.40 X 19.55 + 0.68 2.10 + 0.24 A r t e m i a Fed W i t h D e f i n e d I n o r g a n i c Medium Grown A l g a e 1 16.00 1.50 2....* 18.06 1.91 3 19.08 2.01 4 18.05 1.87 X 17.80 + 1.29 1/82 +0.22 47 DISCUSSION Livestock Waste As A Nutrient Source In A l g a l Culture Analysis of t o t a l Kjeldahl nitrogen (TKN), -NĤ -N, NO^-N + NOg-N PO^-P and t o t a l suspended s o l i d s (TSS) of the swine waste samples col l e c t e d during the course of the experiments showed that the nutrient content of the waste varied only s l i g h t l y . The constancy of the manure nutrients concentration found i n these experiments i s s i g n i f i c a n t (Table 1). I t indicates that f o r a p a r t i c u l a r farm i t m.aybe .unnecessary t'ooanalyse each batch of waste and that analysing the waste p e r i o d i c a l l y may be adequate to provide the necessary information f o r c a l c u l a t i n g the required nutrient loading rates. In t h i s case, the samples were taken from the same batches of f i n i s h i n g hogs,and just p r i o r to p i t fl u s h i n g which may have increased the uniformity of the manure samples. In the present study, the highest percentage of nitrogen found i n l i v e s t o c k waste was i n the form of ammonia nitrogen which i s in,;.- agreement with the r e s u l t s of Dunstan and Menzel (1971) and Thomas et a l . (1974) fo r the sewage. This form of inorganic nitrogen i s r e a d i l y u t i l i z e d by phytoplankton as long as toxic l e v e l s are not attained. Although i n the swine waste-seawater mixtures, ammonia nitrogen concentration was about 5°0 ug-at -1 N 1^ and- 5°% higher than that of defined inorganic medium, t h i s NH^-N l e v e l did not appear to exhibit toxic effects on the algae. 48 C e r t a i n s p e c i e s o f p h y t o p l a n k t o n (Hemiselmis v i r e s c e n s and D u n a l i e l l a t e r t i o l e c t a ) can a l s o f u l l y u t i l i z e o r g a n i c s o u r c e s o f n i t r o g e n ( A n t i a and Charney, 1968; A n t i a e t a l . , 1 9 7 5 ) . I f t h e s e p h y t o p l a n k t o n were grown on l i v e s t o c k waste o r sewage, th e y c o u l d have a g r e a t advantage i n terms o f n i t r o g e n u t i l i z a t i o n and c o n v e r s i o n e f f i c i e n c y s i n c e l a r g e amounts o f o r g a n i c n i t r o g e n are found i n t h e s e w a s t e s . I t s h o u l d he n o t e d , however, t h a t i n the p r e s e n t s t u d y v e r y l i t t l e o r g a n i c n i t r o g e n i s p r e s e n t i n t h e medium due'Lto p r e f i l t r a t i o n . A l t h o u g h a n i m a l wastes may c o n t a i n t o x i c a n t s from f e e d r e s i d u e s and growth s t i m u l a n t s such as copper added t o t h e r a t i o n , t h e s e a r e g e n e r a l l y more p r e d i c t a b l e and c o n t r o l l a b l e t h a n i n t h e case o f sewage (Dodd, 1 9 7 9 ) . V a r i a b i l i t y o f n u t r i e n t s found i n sewage c o u l d a l s o a f f e c t t he s p e c i e s c o m p o s i t i o n o f p h y t o p l a n k t o n i n c u l t u r e (Dunstan and Tenore, 1 9 7 2 ) grown on t h i s medium. Moreover, the problem o f t o x i c a n t s i n l i v e s t o c k waste i s c o n s i d e r a b l y l e s s i n d e v e l o p i n g c o u n t r i e s t h a n i n h i g h l y i n d u s t r i a l i z e d c o u n t r i e s . The l a t t e r uses a h i g h l y i n t e n s i v e l i v e s t o c k c u l t u r e system i n v o l v i n g g r e a t e r amounts o f s y n t h e t i c s t i m u l a n t s i n t h e i r f o o d r a t i o n . I n the p r e s e n t s t u d y , t h e p r e s e n c e o f t o x i c a n t s i n t h e swine i s p r o b a b l y i n s i g n i f i c a n t . However, f u r t h e r s t u d i e s s h o u l d be conducted on t r a c e m e t a l s a c c u m u l a t i o n n o t o n l y on the a l g a e but a l s o on the n e x t t r o p h i c l e v e l . 4 9 Growth C o n s t a n t , C e l l D e n s i t y And Dry Weight o f D. t e r t i o l e c t a I n "both h a t c h and c o n t i n u o u s a l g a l c u l t u r e systems, th e growth c o n s t a n t ( K ^ 0 ^ h r ) and mean g e n e r a t i o n t i m e ( t g h r s ) o b t a i n e d i n a l l e x p e r i m e n t s have s i m i l a r v a l u e s e i t h e r i n d e f i n e d i n o r g a n i c o r swine waste-seawater media. The v a l u e s o b t a i n e d ( T a b l e 4 ) were s i m i l a r t o e a r l i e r s t u d i e s ( A n t i a and K a l m a k o f f , 1 9 6 5 ; J i t t s . e t a l . , 1 9 6 4 ) i n which th e v a l u e s 1 , r e p o r t e d a r e c o n s i d e r e d t o be c l o s e t o o p t i m a l . These r e s u l t s i n d i c a t e t h a t the c u l t u r e c o n d i t i o n s i n t h e p r e s e n t s t u d y were o p t i m a l i n most cases and t h a t n e i t h e r t h e h i g h l e v e l s o f NH^-N n o r p o s s i b l e t o x i c a n t s i n the manure were h a v i n g a d e t r i m e n t a l e f f e c t on a l g a l growth r a t e . A l g a l c e l l d e n s i t i e s attained*..: a f t e r 7 - 8 days growth d u r i n g 6 -1 b a t c h c u l t u r e were 5 t o 7 x 10 c e l l s ml f o r b o t h media. S i m i l a r v a l u e s were a t t a i n e d d u r i n g c o n t i n u o u s c u l t u r e s and were m a i n t a i n e d f o r t h e d u r a t i o n o f the e x p e r i m e n t s . T h i s showed t h a t the swine waste-seawater medium hasv.neither i n h i b i t o r y n o r t o x i c e f f e c t on the growth o f D. t e r t i o l e c t a when compared w i t h the d e f i n e d i n o r g a n i c medium. I n a s i m i l a r , s t u d y , u s i n g 15 l i t e r volume c u l t u r e s , i t was demonstrated t h a t s econdary t r e a t e d sewage e f f l u e n t i s an e x c e l l e n t medium f o r the growth o f mixed p o p u l a t i o n s o f marine p h y t o p l a n k t o n (Dunstan and M e n z e l , 1 9 7 1 ) . The a l g a l d r y w e i g h t measured d a i l y f o r swine w a s t e - seawater and d e f i n e d i n o r g a n i c media d u r i n g c o n t i n u o u s c u l t u r e ( F i g u r e 2 ) was n o t c o n s t a n t . I t ranged from 0 . 1 2 t o 0 . 2 5 g 1 I t was o b s e r v e d t h a t a l t h o u g h the c o n c e n t r a t i o n o f c e l l s remained 50 Table 4. Growth Constant ( K ^ 1 0 ) ) h r -1) and Mean Generation Time (tg hrs) from present and e a r l i e r studies on D. t e r t i o l e c t a . Defined Inorganic Swine Waste-Seawater Medium Mixtures ( K ( i p ) h r _ 1 ) (tg hrs) ( K ( 1 0 ) h r _ 1 ) (tg hrs) Present Study Expt 1 0.023 13.0 0.024 12.5 Expt 2 0.023 + 0 . 0 1 2 . 9 2 + 0 . 0 0.023 + 0.001 12.95+0^34 Expt 3 0.023 ± 0.002 13.92 + 0.36 0.0231 + 0.001 13.02+0.05 Expt 5 0.023 + 0.002 12.85 + 0.49 0.024 + 0.004 12.71+0.67 Antia and Kalmakoff (19'';r) (1965) 0.025 12.0 J i t t s ' e t a l . (1964) ' ' O.030 51 f a i r l y constant, the algal "biomass decreased at d i f f e r e n t times (Figures 8 and 9 ) . I t was observed that the a l g a l "biomass tended to increase shortly a f t e r the nutrient media "being added to the culture was replenished from the stock solution. Moreover, the higher v a r i a b i l i t y was observed i n swine waste-seawater medium due to considerable amount of suspended s o l i d s i n the swine waste- seawater mixture as compared to defined inorganic medium. S i g n i f i c a n t difference was found i n the a l g a l dry weight produced d a i l y f o r both media. Further research should be carr i e d out to determine possible reasons why at the time of replenishing the nutrients-solution an increase i n a l g a l biomass was observed. Continuous Culture System In Alga l Production One of the major values of the present work was the demonstration that the continuous culture system can produce f a r more a l g a l biomass than the batch system. This culture system when compared to a batch system i s f a r more conducive f o r optimal productivity of the algae. Although the batch system was capable -1 -1 -1 of producing 0.015 g dry wt 1 day (0.10 dry wt 1 in. : 6 days), .thercontinuous culture system was capable of producing 0.2 to 0.25 g dry wt l - 1 d a y - 1 . •C I t should be pointed out that the nutrients i n both media were i n excess and thereby the nutrients were not f u l l y u t i l i z e d by the algae during continuous culture (Table 2). I t i s suggested that additional culture vessels should be added i n series to f u l l y u t i l i z e the nutrients, thus r e s u l t i n g to a higher biomass. 52 M c A l l i s t e r e t a l (1964) c o n c l u d e d t h a t the maximum p h o t o s y n t h e t i c e f f i c i e n c y ( P m a x ) ? o r D. t e r t i o l e c t a was a c h i e v e d a t 0.1 l y min . I n the p r e s e n t s e t - u p , l i g h t was p r o b a b l y l i m i t i n g . S i n c e t h e l i g h t i n t e n s i t y from t h e f l u o r e s c e n t tubes was between 0.04 to 0.05 l y min , i n c r e a s i n g t h e l i g h t i n t e n s i t y w i l l i n c r e a s e t h e p h o t o s y n t h e t i c p r o d u c t i o n . F u r t h e r m o r e , u s i n g c o n t i n u o u s c u l t u r e s , i t i s p o s s i b l e t o p r o v i d e a more u n i f o r m p r o d u c t d a i l y by s e l e c t i n g growth c o n d i t i o n s t o o b t a i n the d e s i r e d c o m p o s i t i o n o f the p r o d u c t . F o r example, by s e l e c t i n g t h e f l o w r a t e , a p r o d u c t o f h i g h o r low p r o t e i n c e l l s c o u l d be produced c o n s i s t e n t l y ( B a l l a r d , 1972; Palmer e t a l . , 1975; Lampert, 1976). On the o t h e r hand, i t i s a c h a r a c t e r i s t i c o f b a t c h c u l t u r e s t h a t t h e i r c o m p o s i t i o n w i l l v a r y c o n t i n u o u s l y as t h e y get o l d e r (Taub and D o l l a r , 1965). A r t e m i a F e e d i n g E x p e r i m e n t s T o t a l Body L e n g t h and P e r c e n t a g e S u r v i v a l T o t a l body l e n g t h and p e r c e n t a g e s u r v i v a l measurements o f the b r i n e shrimp d u r i n g t h e 14-day c u l t u r e p e r i o d showed no s i g n i f i c a n t d i f f e r e n c e s when f e d swine waste grown a l g a e w i t h t h o s e f e d d e f i n e d i n o r g a n i c medium grown a l g a e . D a i l y o b s e r v a t i o n s on t h e i r swimming o r f e e d i n g b e h a v i o r gave no ap p a r e n t d i f f e r e n c e s between t h e two t r e a t m e n t s . The r a p i d growth o f A r t e m i a between 2 t o 7 mm s t a g e i n t h e p r e s e n t s t u d y was r e m a r k a b l e . Mason (1963) o b s e r v e d a s i m i l a r growth p a t t e r n . These l e n g t h s c o r r e s p o n d t o the p e r i o d s when growth r a t e i s most r a p i d and h i g h l y dependent upon f o o d l e v e l 53 and with the time when food f i r s t shows i t s e f f e c t , days 4-6 (Provasoli et a l , 1959). Although the same number of algae are being fed and those algae contain about 25$ less organic nitrogen (Table 2 ) , t h i s did not a f f e c t the subsequent growth rate of the Artemia. The algae are s t i l l able to grow and reproduce while the Artemia are feeding on them. Scott and Middleton ( 1 9 7 9 ) have shown that turbot larvae v with D. t e r t i o l e c t a given during the r o t i f e r feeding stage showed poor animal growth and s u r v i v a l compared to larvae i n tanks with either Isochrysis galbana, Phaedactylum tricornoturn or Pavlova l u t h e r i added. Since D. t e r t i o l e c t a i s known to be not toxic, Its e f f e c t on the turbot larvae Is probably due to "a dietary e f f i c i e n c y . I t has been pointed out that the n u t r i t i o n a l state of the a l g a l food i s important for the growth of herbivores (Parsons et a l . , 1 9 6 1 ) . This i s probably the cause of low su r v i v a l of Artemia i n the present study. Tobias et a l . (1979) achieved 80$ s u r v i v a l of Artemia fed with diatom Chaetoceros curvisetus (clone STX - I 6 7 ) i n a flow-through raceway system. Biomass Conversion E f f i c i e n c y The biomass conversion e f f i c i e n c y ($) i s defined here as a measure of the amount of biomass of Artemia produced by a given quantity of algae fed during the culture period. The formula i s given as: dry weigth of Artemia produced B. C. E . $ = _ _ — _ — _ _ x i n n dry weight of algae fed x 1 U U where B. C. E . i s biomass conversion e f f i c i e n c y The biomass conversion e f f i c i e n c i e s f o r Artemia fed with swine waste-seawater grown algae and defined inorganic grown algae were 57$ and 5 8 $ ,respectively (Table 5 ) - The calculated algal-Artemia conversion e f f i c i e n c i e s showed no s t a t i s t i c a l l y s i g n i f i c a n t differences between treatments and r e p l i c a t e s (Appendix 1 1 ) . F i f t y percent protein-N conversion e f f i c i e n c y from D. t e r t i o l e c t a to Artemia c u l t u r e d l i n the laboratory was reported by Reeve ( 1 9 6 3 c ) . H e l f r i c h (1963) indicated a 50$ conversion e f f i c i e n c y .(ash-free dry wt ) was attained i n a f e a s i b i l i t y study conducted on Christmas Island f o r a production scheme of Artemia eggs and adult. The present study cannot be d i r e c t l y compared with the above studies since the units used were i n g dry wt. The present r e s u l t s , however, may be said to have reached a comparable conversion e f f i c i e n c y . Reports on e f f i c i e n c y of growth of Artemia are quite varied i n terms of units used such as wet weight, dry weight, ash-free dry weight, protein-N and mg C (Gibor, 1957, Mason, 1963; H e l f r i c h , 1973; Sick, 1976 and Bossuyt and Sorgeloos, 198O). I t i s suggested that the use of such units should have common parameters such that r e s u l t s w i l l be comparable and also applicable to aquacultural studies. Combined Livestock Waste-Artemia Culture System The pres.ent study shows that the use of swine waste as a source of nutrients i n a l g a l culture i s highly comparable to defined inorganic media. The study also shows that Artemia fed 55 Table 5* Calculated Biomass Conversion E f f i c i e n c y (BCE %) of Artemia s a l i n a L. Parameters Measured Fed With Swine Waste Grown D. t e r t i o l e c t a Fed With Defined Inorganic Medium Grown D. t e r t i o l e c t a A l g a l Production (X = g dry wt day - 1) 0.17 + 0.72 0.15 ±0.61 Corresponding Al g a l C e l l Density (X = c e l l m l - 1 ) 5.78 + 1.65 x 10° 5.31 + 1.62 x ier C e l l Weight (X = g dry wt c e l l " 1 ) 2.99 + 1.01 x I O - 1 1 2.78 + 0.9 x I O - 1 1 A l g a l C e l l Censity Maintained as Feed For Artemia day ~1 (X = c e l l ml 1 ) 5.0 + 0.50 x 10 5 5.0 + 0.50 x 10 5 No. of Alg a l C e l l s Added As Feed For Artemia day-1 (X = c e l l s d a y - 1 8.67 + 1.50 x 10y 9.97 + 1.0 x 10' A l g a l Dry Weight Added As Feed In 14-Day Culture Period (X = g dry wt) 3.63 + 1.40 3.10 + 1.09 Artemia Biomass Produced After 14 Days (X = g dry wt) 2.10 + 0.42 1.82 + 0.38 Biomass Conversion E f f i c i e n c y (fo) 57-93 + 5-84 58.79 + 7.19 56 O H algae grown i n swine waste-seawater mixture grow as well as those fed with algae grown i n defined inorganic medium. This i s an important consideration, es p e c i a l l y for an agriculture-aquaculture integrated system, and i t shows,that Artemia has a high p o t e n t i a l i n converting a l g a l biomass grown i n a g r i c u l t u r a l wastewater into much needed protein, p a r t i c u l a r l y i n Third World countries. For example, Artemia can be used as a substitute f o r f i s h meal. In t h i s regard, integrated livestock waste-Artemia production system can be most economically f e a s i b l e i n Third World countries. I t should be pointed out, however, that there are s t i l l numerous areas of uncertainties i n the said system, such as b i o l o g i c a l , engineering and economic problems. I t i s suggested that future studies should be conducted on outdoors f o r algal-Artemia production scheme so that data, e s p e c i a l l y on engineering and economic aspect can be applied to aquacultural production ventures. 57 SUMMARY AND CONCLUSIONS A n a l y s i s o f t o t a l K j e l d a h l nitrogen(TKN(), NH^-N, NO^-N + Nbg-N, PO^-P and t o t a l suspended s o l i d s ( T S S ) o f t h e swine waste samples showed o n l y s l i g h t v a r i a t i o n d u r i n g the cour s e o f the e x p e r i m e n t a l work. I n b o t h "batch and c o n t i n u o u s c u l t u r e systems.,, t h e a l g a l growth c o n s t a n t s o b t a i n e d i n a l l e x p e r i m e n t s were found t o have s i m i l a r v a l u e s f o r b o t h d e f i n e d i n o r g a n i c and swine waste-seawater media. The p r o d u c t i o n parameters m a i n t a i n e d d u r i n g a l g a l c u l t u r e i n b o t h b a t c h and c o n t i n u o u s c u l t u r e systems were * as f o l l o w s : t e m p e r a t u r e , 2h + 1°C; s a l i n i t y , 32+1 p p t ; f l u o r e s c e n t l i g h t i n t e n s i t y , &a 0.04 c a l -1-2 -1 min cm ( o r 0.04 l y min" ). The a l g a l c e l l d e n s i t y a t t a i n e d a f t e r 7-8 days growth f o r b a t c h c u l t u r e was 5 t o 7 x 10 c e l l s _ i ml i n b o t h media. S i m i l a r v a l u e s were a t t a i n e d d u r i n g c o n t i n u o u s c u l t u r e s and s u c c e s s f u l l y m a i n t a i n e d f o r the d u r a t i o n o f the e x p e r i m e n t s . The r e s u l t s show t h a t t h e use o f swine waste-seawater m i x t u r e s as a n u t r i e n t s o u r c e i n a l g a l c u l t u r e i s comparable t o d e f i n e d i n o r g a n i c medium. One advantage o f u s i n g l i v e s t o c k waste as a n u t r i e n t s o u r c e f o r a l g a l c u l t u r e i s t h a t n u t r i e n t l o a d i n g such as n i t r o g e n and phosphorus can be p r e d i c t e d . I t i s a l s o cheap and a v a i l a b l e i n many a r e a s . The t o t a l body l e n g t h and p e r c e n t a g e survivalinmeasurements o f A r t e m i a d u r i n g the 14-day c u l t u r e p e r i o d showed no s i g n i f i c a n t d i f f e r e n c e s between A r t e m i a f e d swine waste-seawater grown D. t e r t i o l e e t a and thos e f e d d e f i n e d i n o r g a n i c medium grown 58 D. t e r t i o l e c t a . The calculated biomass conversion e f f i c i e n c i e s for Artemia fed with swine waste-seawater grown algae and defined inorganic medium grown algae were 57% and 58%, respectively. The algal-Artemia conversion e f f i c i e n c i e s showed no s t a t i s t i c a l l y s i g n i f i c a n t differences between treatments and r e p l i c a t e s . The study shows that Artemia fed on algae grown i n swine waste-seawater mixture grow as well as those fed with algae grown i n defined inorganic medium. 59 REFERENCES A l l e n , E. J . and E. W. Nelson. 1910. On the a r t i f i c i a l c u l t u r e of marine plankton organisms. J . Mar. B i o l . Assoc. U. K. 8: 421 -74. Anon. 1978. Aquaculture development f o r Hawaii. Aquaculture P l a n n i n g Program, Dept. of Pla n n i n g and Economic Development, State of Hawaii, USA. 222 pp. A n t i a , N. J . and V. Charney. I968. Nature of the n i t r o g e n compounds supporting phototrophic growth of the marine cryptomonad Hemiselmis v i r e s c e n s . J . P r o t o z o o l . 15(1): 198-201 A n t i a , N. J . and J . Kalmakoff. I965. Growth r a t e s and c e l l y i e l d s from axenic mass c u l t u r e s of fourteen species of marine phytoplankton. Oceanogr. and^iimnol. Manuscript Report S e r i e s No. 203 F i s h . Res. Bd.~Can. 23 pp. A n t i a , N. J . , B. R. Berland and D. J . Bonin. 1980. Proposal f o r an abridged n i t r o g e n turnover c y c l e i n c e r t a i n marine p l a n k t o n i c systems i n v o l v i n g hypoxanthine-guanine e x c r e t i o n by c i l i a t e s and t h e i r r e u t i l i z a t i o n by phytoplankton. Mar. E c o l . Prog. Ser. 2: 97-103. A n t i a , N. J . , B. R. Berland, D. J . Bonin and S. Y. M a e s t r i n i . 1975. Comparative e v a l u a t i o n of c e r t a i n organic and in o r g a n i c sources of n i t r o g e n f o r phototrophic growth of marine microalgae. J . Mar. B i o l . Assoc. U.K. 55s 519-39. Antonovics, J . , A. D. Bradshaw and R. G. Turner. 1971. Heavy metal t o l e r a n c e i n p l a n t s . Advances i n E c o l o g i c a l Research. 7: I -85 . Ashida, J . I965. Adaptation of f u n g i to metal t o x i c a n t s . Ann. Rev. Phytopathology. 3: ±53~7M^ B a i d , I . C. 1963. The e f f e c t of s a l i n i t y on growth and form of Artemia s a l i n a L. J . E x p 1 t a l . Z o o l . 153: 279-83. B a l l a r d , K. A. 1972. The production c h a r a c t e r i s t i c s of three continuous c u l t u r e s of Monochrysis l u t h e r i . M. Sc. Thesis. College of . F i s h e r i e s , U n i v e r s i t y of Washington, S e a t t l e , Washington, USA. "48 pp. Beck, A. D. 1979* Laboratory c u l t u r e and fe e d i n g of the A t l a n t i c S i l v e r s i d e s , Menidia menidia 63-85. I n " C u l t i v a t i o n of F i s h Fry and I t s L i v e Food. EMS Spec. P u b l . No. 4" (Styczynska- Jurewics, E.T. B a c k i e l , E. Jaspers, G. Persoones, eds.) I n s t i t u t e f o r Mar. S c t f c . Res., Bredone (Belgium). 534 pp. 6o Ben-Amotz, A. and M. Avron. 1 9 7 8 . On the mechanism of osmoregulation i n D u n a l i e l l a . ^ u l n " "Energetics and S t r u c t u r e of H a l o p h i l i c Micro-organisms (S. R. Caplan and M. Ginzburg, e eds.). E l s e v i e r , Amsterdam, pp 5 2 9 - 4 1 . B e n i j t s , F., E. Van Voordin and P. Sorgeloos. 1 9 7 5 - Changes i n biochemical composition of the e a r l y l a r v a l stages of Artemia s a l i n a 1. Tenth European Symp. Mar. B i o l . Ostend, Belgium, 1 7 - 2 3 Sept. Bledsoe, L. J . , R. A. Sh l e s e r , J . B. Glude. 1 9 7 8 . Development of research p r i o r i t i e s f o r aquaculture p l a n n i n g . Washington Sea Grant, WSG-MR78-2 U n i v e r s i t y of Washington, S e a t t l e , Washington, USA. 5 2 pp. Bossuyt, E. and P. Sorgeloos. I 9 8 O . Technological aspects of the batch c u l t u r i n g of Artemia i n high d e n s i t i e s . I n "The Brine Shrimp Artemia" (G. Persoone, P. Sorgeloos, 0. A. Roels, E. Jaspers, eds.) v o l . 3 . UUniversa P r e s s , Wetteren, Belgium. (In press) Bowen, S. T. 1 9 6 2 . The genetics of Artemia s a l i n a . I . The reprodu c t i v e c y c l e . B i o l . B u l l . 1 2 2 : 2 5 - 3 2 . Bowes, G. W. 1 9 7 1 . Molecular oceanography. Ph. D. D i s s e r t . U n i v e r s i t y of C a l i f o r n i a San Diego, C a l . USA. Bureau of Environmental S t u d i e s . 1 9 7 5 « Department o f Environment, Housing and Community Development. A l g a l h a r v e s t i n g from sewage. Environmental Study Report No. 1. A u s t r a l i a n Government P u b l i s h i n g S e r v i c e , Canberra. Burlew, J . S. (Ed.) 1 9 5 3 . A l g a l c u l t u r e from l a b o r a t o r y to p i l o t p l a n t . Carnegie I n s t i t u t i o n of Washington, Wash., D. C. Casey, R. P., J . A., L u b i t z , R. J . Ben o i t , R. J . Weisman and H.. Chan. 1 9 6 3 . Mass c u l t u r e of C h l o r e l l a . Food Tech. 1 7 : 8 5 - 8 9 . Claus, C., F . s B e n i j t s , G. Vandeputte and W. Gardner. 1 9 7 9 . The biochemical composition of the l a r v a e of two s t r a i n s of Artemia s a l i n a L. reared on two d i f f e r e n t a l g a l foods. J . E x p ' t a l . Mar. B i o l . E c o l . 3 6 : 1 7 1 - 8 3 . Cook, B. B., E. W. Lau and B. M. B a i l e y . I963. The q u a l i t y of waste, grown green algae I . Q u a l i t y of p r o t e i n i n mixtures of algae, non f a t powdered m i l k and c e r e a l s . J . Nut. 81: 2 3 ~ 9 « Croghan, p. C. 1 9 5 8 . The s u r v i v a l of Artemia s a l i n a L. i n various media. J . Exp. B i o l . 3 5 ( 1 ) : 2 1 3 - l F i 61 D a v i d s o n , J . R. 1974. A new i n d u s t r y - and perhaps a new p r o t e i n s o u r c e . Sea Grant T e c h n i c a l R e p o r t UNIHI - Se a g r a n t MR-74-02. 3 pp. D'Ag o s t i n o , A. S. 1965* Comparative s t u d i e s o f A r t e m i a s a l i n a (Development and P h y s i o l o g y ) . Ph.D. D i s s e r t . Dept. Of B i o l o g y New York U n i v e r s i t y , N. Y. D a v i e s , A. G. 1976. An assessment on th e "basis o f mercury t o l e r a n c e i n D u n a l i e l l a t e r t i o l e c t a . _J_. Mar. B i o l . A s s . U. K. 56: 39-57. D a v i s , E. A., J . D e d r i c k , C. S. F r e n c h , H. W. M i l n e r , J . Myers, J . H. C. Smith and H. A. Spheer. 1961. L a b o r a t o r y experiments on C h l o r e l l a c u l t u r e a t th e C a r n e g i e I n s t i t u t i o n Washington Dept. o f P l a n t B i o l o g y . I n " A l g a l C u l t u r e From L a b o r a t o r y To P i l o t P l a n t ( J . S. Bu r l e w , ed.) C a r n e g i e I n s t i t u t i o n o f Washington, P u b l . 600 Wash. D. C. D o b b e l e i r , J . , N. Adam, E. B o s s u y t , E. Bruggeman and P. S o r g e l o o s . 1980. New a s p e c t s o f th e use o f i n n e r d i e t s f o r h i g h d e n s i t y c u l t u r i n g o f b r i n e s h r i m p . I n "The b r i n e s h r i m p , A r t e m i a , E c d l o g y i , \ : C u l t u r i n g , Use i n a q u a c u l t u r e " (G. Per s o o n e , P. S o r g e l o o s , 0. A. R o e l s and E. J a s p e r s , eds.) v o l . 3- U n i v e r s a P r e s s . W e t t e r e n , B e l g i u m . ( I n p r e s s ) Dodd, J . c. 1979- A l g a e p r o d u c t i o n and h a r v e s t i n g from a n i m a l wastewater. A g r i c u l t u r a l Wastes. 1: 23~7- Dugan, G. L., C. G. Golueke and W. J . Oswald. 1971. A b s t r a c t s . E x c e r p t s and Reviews o f S o l i d Waste L i t e r a t u r e (C. G. Golueke, e d . ) . S a n t . Eng. Res. Lab. U n i v e r s i t y o f C a l i f o r n i a , B e r k e l e y , T T O n Dunstan, W. N. and D. W. M e n z e l . 1971. C o n t i n u o u s c u l t u r e o f n a t u r a l p o p u l a t i o n o f p h y t o p l a n k t o n i n d i l u t e , t r e a t e d sewage e f f l u e n t . L i m n o l . Oceanogr. 16(4): 623-32. Dunstan, W. M. and K. R. Tenore. 1972. I n t e n s i v e o u t d o o r c u l t u r e o f marine p h y t o p l a n k t o n e n r i c h e d w i t h tr^eatod sewage e f f l u e n t . A q u a c u l t u r e . 1: 181 -92. D w i v e d i , S. N., S. R. Ansan and M. Q. Ahmed. 1979. Mass c u l t u r e o f b r i n e shrimp under c o n t r o l l e d c o n d i t i o n s i n cement ponds a t Bombay, I n d i a . P r e s e n t e d a t I n t ' 1 . Sympo. o f B r i n e Shrimp, A r t e m i a s a l i n a . Corpus C h r i s t i , Texas, USA. 20-23 A u g u s t / Emerson, R. and 0. M. L e w i s . 1939. F a c t o r s i n f l u e n c i n g t he e f f i c i e n c y o f p h o t o s y n t h e s i s . Amer. J . B o t . 26: 808-22. E u s e b i o , J . A. 1976. R e c y c l i n g system i n t e g r a t e d p l a n t and a n i m a l f a r m i n g . U n i v e r s i t y o f the P h i l i p p i n e s , Los Banos Tech. B u l l . 1(1). 15 pp. 6 2 G a l l a g h e r , M. and W. D. Brown. 1975• C o m p o s i t i o n o f San F r a n c i s c o Bay b r i n e shrimp ( A r t e m i a s a l i n a ) . J . A g r i c . Food Chem. 2 4 ( 4 ) : 6 3 0 - 3 2 . G a r r e t , M. K. and M. D. B. A l l e n . 1976. P h o t o s y n t h e t i c p u r i f i c a t i o n o f the l i q u i d phase o f a n i m a l s l u r r y . E n v i r o n . P o l l u t . 1 0 : 1 2 7 - 3 9 . G a r r e t , M. K., J . J . S t r a i n and M. D. B. A l l e n . 1 9 7 6 . C o m p o s i t i o n o f the p r o d u c t o f a l g a l c u l t u r e i n t h e phase o f a n i m a l s l u r r y . J . Fd. Sc.. A g r i c . 2 7 : 6 0 3 - 1 1 . G i b o r , A. 1 9 5 6 a . Some e c o l o g i c a l r e l a t i o n s h i p s between p h y t o p l a n k t o n and z o o p l a n k t o n . B i o l . B u l l . 111: : 2 3 0 - 3 4 . G i l c h r i s t , B. M. i 9 6 0 . Growth and form o f t h e b r i n e shrimp A r t e m i a s a l i n a L. P r o c . Z o o l . Soc. London. 1 3 4 ( 2 ) ; 2 2 1 - 3 4 . G i l c h r i s t , B. M. 1 9 5 6 . The oxygen consumption o f A r t e m i a s a l i n a L. i n d i f f e r e n t s a l i n i t i e s . H y d r o b i o l o g i a . 8 : 5 4 - 6 5 • G l u d e , J . B. 1 9 7 8 . The f r e s h w a t e r prawn, Macrobrachium r o s e n b e r g i i (de Man). A l i t e r a t u r e r e v i e w and a n a l y s i s o f t h e use o f t h e r m a l e f f l u e n t s i n the c u l t u r e o f t h e f r e s h w a t e r prawn. A q u a c u l t u r e C o n s u l t a n t , S e a t t l e , Wash., USA. 59 PP• Goldman, J . C. and H. I . S t a n l e y . 1 9 7 4 . R e l a t i v e growth o f d i f f e r e n t s p e c i e s o f marine a l g a e i n was t e w a t e r - s e a w a t e r m i x t u r e s . Mar. B i o l . 2 8 : 1 7 - 2 5 . Golueke, C. G. and W. J . Oswald. 1 9 6 5 - H a r v e s t i n g and p r o c e s s i n g sewage-grown p l a n k t o n ! c a l g a e . J o u r n a l o f the Water P o l l u t i o n C o n t r o l F e d e r a t i o n , USA. 3 7 ( 4 ) : 4 7 1 - 9 8 . G r e i g , M. and J . B j e r r i n g . 1 9 7 7 - A G e n e r a l L e a s t Squares A n a l y s i s o f V a r i a n c e Programme. U. B. C. G e n l i n Computing C e n t r e U n i v . o f B r i t i s h Columbia, Vancouver-, B. C. r e v i s e d , Sept.. 1 9 7 8 . G u i i l a r d , P. R. L. 1 9 7 5 . C u l t u r e o f p h y t o p l a n k t o n f o r f e e d i n g marine i n v e r t e b r a t e s . I n " C u l t u r e o f M a r i n e I n v e r t e b r a t e ; . A n i m a l s " (W. L. Smit h and M. H. Chanley, eds.) Plenum P r e s s . New York.. 2 9 - 6 0 . H e l f r i c h , P. 1973« The f e a s i b i l i t y o f b r i n e shrimp p r o d u c t i o n on C h r i s t m a s I s l a n d . Sea Grant Tech. Rep. UNIHI-Sea G r a n t - T R - 7 3 - 0 2 . Hephner, B. 1 9 6 2 . P r i m a r y p r o d u c t i o n i n f i s h p o n d s and i t s a p p l i c a t i o n t o f e r t i l i z a t i o n e x p e r i m e n t s . L i m n o l . Ocean. 7: 1 3 1 - 3 6 . 63 Hephner, B. and G. L. S c h r o e d e r . 1974. Wastewater u t i l i z a t i o n i n t e g r a t e d a q u a c u l t u r e and a g r i c u l t u r e systems. I n "Wastewater Use i n t h e P r o d u c t i o n o f Food and F i b e r - P r o c e e d i n g s " . EPA-660/2-7404l. H e r b e r t , D. 1958. Some p r i n c i p l e s o f c o n t i n u o u s c u l t u r e . I n "Recent p r o g r e s s i n M i c r o b i o l o g y " (G. T. T o n e v a l l , ed.) A l m q u i s t and W i k e s e l l , S t o c k h o l m . 381-96. H e r b e r t , D. 1961. A t h e o r e t i c a l a n a l y s i s o f c o n t i n u o u s c u l t u r e system. I n "Continuous c u l t u r e o f m i c r o o r g a n i s m . Soc. Chem. I n d . Monograph. 12: 21-53. H e r b e r t , D., R. E l s w o r t h and R. C. T e l l i n g . 1956. Continuous c u l t u r e o f b a c t e r i a , t h e o r y and expe r i m e n t . J . Gen.." M i c r o b i o l . H i n t z , H. F., Heitman, W. C. We i r , D. T. T o r e l l and 'J. H. Meyer. 1966. N u t r i t i v e v a l u e s o f a l g a e grown on sewage. J . An i m a l Sc. 25(3) : 675-81. Houde, E. D. 1972. Some r e c e n t advances and u n s o l v e d problems i n t he c u l t u r e o f marine f i s h l a r v a e . P r o c . I l l Ann. Wkshp. Wor l d M a r i c u l t u r e Soc. 3: 83-112. J a c o b , P. G. 1978. C o n t i n u o u s c u l t u r e o f the b r i n e s h r i m p , A r t e m i a s a l i n a . I n d i a n J . Mar. Sc. 7s 3 ° 6 - 7 . J i t t s , H. R., C. D. M c A l l i s t e r , K. Stephens and J . D. H. S t r i c k l a n d . 1964. The c e l l d i v i s i o n r a t e s o f some marine p h y t o p l a n k t e r s as f u n c t i o n o f l i g h t and t e m p e r a t u r e . J . F i s h . Res. Bd. Can. 21: 139-57- Lampert, W. 1976. A d i r e c t l y c o u p l e d a r t i f i c i a l t w o-step c h a i n f o r l o n g term e x p e r i m e n t s w i t h f i l t e r - f e e d e r s a t c o n s t a n t f o o d c o n c e n t r a t i o n s . Mar. B i o l . 37: 349~55« L o o s a n o f f , V. L. and H. C. D a v i s . 1963- R e a r i n g o f b i v a l v e m o l l u s c . I n "Advances i n M a r i n e B i o l o g y " ( F . S. R u s s e l , ed.) v o l . 1. Academic P r e s s , N. Y. 1-36. L u a r d , E. J . 1973- S e n s i t i v i t y o f D u n a l r e l l a and Scenedesmus (C h l o r o p h y c e a e ) t o c h l o r i n a t e d h y d r o c a r b o n s . P h y c o l o g i a . 12(1/2): 29-33 . Mason, D. T. 1963. The growth r e s p o n s e o f A r t e m i a s a l i n a L. t o v a r i o u s f e e d i n g r e g i m e s . C r u s t a c e a n a . 5- 138-50. May, R. C. 1970. F e e d i n g l a r v a l marine f i s h e s i n t h e l a b o r a t o r y , A r e v i e w . C a l i f . C o o p e r a t i v e Oceanic F i s h e r i e s I n v e s t i g a t i o n s R e p o r t . S e p t . 14: 76-83. 6 4 May, R. C. 1971. An a n n o t a t e d b i b l i o g r a p h y o f a t t e m p t s t o r e a r the l a r v a e o f marine, f i s h e s i n the l a b o r a t o r y . NOAATechnical R e p o r t NMFS SSRF - 6 3 2 . M c A l l i s t e r , C. D., N. Shah and J . D. H . S t r i c k l a n d . 1964. M a r i n e p h y t o p l a n k t o n p h o t o s y n t h e s i s as a f u n c t i o n o f l i g h t i n t e n s i t y : a comparison o f methods. J . F i s h . Res. Bd. Can. 21: 159-81. McGarry, M. G., C. D. L i n and J . L. M e t r o . 1972. P h o t o s y n t h e t i c y i e l d s and by p r o d u c t r e c o v e r y from sewage o x i d a t i o n ponds. I n "Advances i n Water P o l l u t i o n R e s e a r c h " ( S . H. J e n k i n s , ed.) U. K. 521-35. M c L a c h l a n , J . i 9 6 0 . The c u l t u r e o f D u n a l i e l l a t e r t i o l e c t a B u t c h e r - a e u r y h a l i n e organism. Can. J . M i c r o b i o l . 6: 367-79- M i l l i g a n , D. J . , J . A. Q u i c k , S. E. H i l l , J . A.?i':Morris and R. S. Hover. 1979- S e q u e n t i a l use o f b a c t e r i a , a l g a e and b r i n e shrimp t o t r e a t i n d u s t r i a l wastewater a t p i l o t p l a n t s c a l e . I n t e r n a t i o n a l Symposium on t h e B r i n e Shrimp, A r t e m i a s a l i n a . Corpus C h r i s t i , Texas, USA. 2 0 - 2 3.August. M i l n e r , H. W. 1961. The c h e m i c a l c o m p o s i t i o n o f a l g a e . I n " A l g a l C u l t u r e From L a b o r a t o r y To P i l o t P l a n t " ( J . B u r l e w , e d . ) . C a r n e g i e I n s t i t u t i o n o f Washington, P u b l i c a t i o n 600. Washington, D. C. Mock, C. R., R. A. N e a l and B. R. S a l s e r , 1973- A c l o s e d raceway f o r t h e c u l t u r e o f the s h r i m p . P r o c e e d i n g s 4 t h Annual Meeting«\'WMS ( J . W. A v a u l t , J r . , e d . ) . L o u i s i a n S t a t e U n i v e r s i t y . B a t o n Rouge Los Angeles,' USA. 431 pp. Monod, J . 1950. Technique de c u l t u r e c o n t i n u e t h e o r i e e t a p p l i c a t i o n e s . Ann. I n s t . P a s t e u r . 79: 3 9 ° - 4 l 0 . Myers, J . 1962. L a b o r a t o r y c u l t u r e s . I n " P h y s i o l o g y and B i o c h e m i s t r y o f A l g a e " (R. A. L e w i n , e d . ) . New York and London.. 603-15. N o v i c k , A. and L. S z i l a r d . 1950- E x p e r i m e n t s w i t h the chemostat on spontaneous m u t a t i o n s o f b a c t e r i a . P r o c . N a t ' l Acad. S c i . u. s. 36: 708-19. Nimura, Y. 1967. B i o l o g y o f the b r i n e s h rimp. B u l l . J a p . Soc. S c t f c . F i s h . 3 3 ( 7 ) . O l i v e i r a , L., N. J . A n t i a and T. B .# B i s a l p u t r a . 1978. C u l t u r e s t u d i e s on the e f f e c t from f l u o r i d e p o l l u t i o n on the growth o f marine p h y t o p l a n k t e r s . J . F i s h . Res. Bd. Can. 35(11): 1500-04, 65 O l i v e i r a , L., T. B i s a l p u t r a and N. J . A n t i a . I98O. U l t r a s t r u c t u r a l o b s e r v a t i o n o f the s u r f a c e c o a t o f D u n a l i e l l a t e r t i o l e c t a from s t a i n i n g w i t h c a t i o n i c dyes and enzyme t r e a t m e n t s . New P h y t o l . 85V 385-92. O l e y n i k o v a , F. A. and T. G. P l e s k a c h e u s k a y a . 1979• A r t e m i a s a l i n a as f o o d i n m a r i c u l t u r e . P r o c . 7th J a p a n - S o v i e t J o i n t Symp. A q u a c u l t u r e . S e p t . 1978. Tokyo, Japan. Oswald, W. J . 1963. The h i g h - r a t e pond i n waste d i s p o s a l . Developments i n I n d u s t r i a l M i c r o b i o l o g y , American I n s t i t u t e o f B i o l o g i c a l S c i e n c e s . USA. 4: 112-119. P a l m e r , F. E., B a l l a r d , K. A. and F. B. Taub. 1975* A c o n t i n u o u s c u l t u r e a p p a r a t u s f o r t h e mass p r o d u c t i o n o f a l g a e . A q u a c u l t u r e . 6: 319-31. P a r s o n s , T. R., K. Stephens and J . D. H. Strickland. : ; . 1 9 6 l . On t h e c h e m i c a l c o m p o s i t i o n o f e l e v e n s p e c i e s o f marine p h y t o p l a n k t e r s . J . F i s h . Res. Bd. Can. 18: 1001-16. P e r s o o n e , G. and P. S o r g e l o o s . 1980. G e n e r a l a s p e c t s o f th e e c o l o g y and bio g e o g r a p h y o f A r t e m i a . I n "The b r i n e shrimp, Artemia/' v o l . 3« E c o l o g y , C u l t u r i n g , Use I n aquaculture:" (G. P e r s o o n e , P. S o r g e l o o s , D. A. R o e l s and E. J a s p e r s , e d s . ) . U n i v e r s a P r e s s , W e t t e r e n , B e l g i u m , t i n p r e s s ) . P e rson-Le Ruyet, J . 1976. R e a r i n g o f A r t e m i a ( B r a n c h i o p o d a ) l a r v a e on i n e r t f o o d , S p i r u l i n a maxima (Cyanophyceae). " A q u a c u l t u r e . ,8: 157-67. ( I n F r e n c h ) P r a t t , R. 1943. S t u d i e s on C h l o r e l l a v u l g a r i s . V I I . I n f l u e n c e o f age o f the c u l t u r e on r a t e s o f p h o t o s y n t h e s i s and r e s p i r a t i o n Amer. J . B o t . 30: 404-08. P r o v a s o l i , L. 1969. L a b o r a t o r y methods i n c u l t i v a t i o n . I n "Marine B i o l o g y , P r o c . 5th I n t e r d i s c i p l i n a r y C onference on Mar i n e B i o l o g y , P r o c . 5th I n t e r d i s c i p l i n a r y C onference on Mar i n e B i o l o g y " ( J . D. L. Co s t l o w , e d . ) . Gordon and B r e a c h , New York. 606 pp.' P r o v a s o l i , L., K. S h i r a i s h i , a n d J . R. Lance. 1959. N u t r i t i o n a l i d i o s y n c r a s i e s o f A r t e m i a and T r i g i o p u s i n monoaxenic c u l t u r e . Ann. New York Acad. Sc. 77: 250-60. Provenzano, A. L., J r . and J . W. Goy. 1976. E v a l u a t i o n o f a s u l p h a t e l a k e s t r a i n o f A r t e m i a as ,a f o o d f o r l a r v a e o f the gr a s s shrimp, Palemonetes p u g i o . A q u a c u l t u r e . 9- 343-5°• Reed, P. H. 1969- S t u d i e s o f d i e t and d i e t c o n c e n t r a t i o n e f f e c t s on Dunganese Crab zoea. P r o c . N a t ' l . S h e l l f i s h Assoc. 59-12. Reeve, M. R. 1963a. The f i l t e r f e e d i n g o f A r t e m i a . I . I n pure c u l t u r e s o f p l a n t c e l l s . J . Exp_. B i o l . 40: 195-205. 66 Reeve, M. R. 1963b. The f i l t e r feeding of Artemia. II... In suspensions of various p a r t i c l e s . J . Exp. B i o l . 40: 207-14. Reeve, M. R. 1963c. Growth e f f i c i e n c y i n Artemia under laboratory conditions. B i o l . B u l l . 125(1): 133-45. Roels, 0. A., K. C. Haines and J. B. Sonderlin. 1976. The p o t e n t i a l y i e l d of a r t i f i c i a l upwelling mariculture. I n "Proc. 10th European Symposium on Marine Biology" Ostend, Belgium. 17-23 Sept. Research i n Mariculture at Laboratory and p i l o t - s c a l e . Universa Presee, Wetteren, Belgium. 620 pp. Rollefsen, G. 1939- A r t i f i c i a l rearing of f r y of seawater f i s h . Preliminary communication. Rapp. Proc-Verb. Reun. Cons, perm. Explor. Mer. 109: 133. Ryther, J . H., W. M. Dunstan, K. R. Tenore and J . E. Huguenin. 1972. Controlled eutrophication increasing food production from the sea by r e c y c l i n g human wastes. Bio Science 22: 144-52 Ryther, J . H., J . C. Goldman, C. E. Gif f o r d , J . E. Huguenin, A. S. Wing, J . P. Clarner, L.&0. Williams and B. E. Lapointe. 1975. Physical models of integrated waste r e c y c l i n g marine polyculture systems. Aquaculture. 5 : 163-77. Sargent, M. C. 1940. E f f e c t of l i g h t i n t e n s i t y on the development of the photosynthetic mechanism. Plant P h y s i o l . 15: 275_90. Schauer, P. S. and K. L. Simpson. 1979. Fatty acid as biochemical indicators of dietary a s s i m i l a t i o n i n the marine f i s h , A t l a n t i c s i l v e r s i d e s . In " F i n f i s h N u t r i t i o n and Fishfeed Tech. Proc. World Sympo . " v o l . 2. 20--23:,:June 1978 Hamburg, Germany. Heeneman, Verlagsgessellschaf B e r l i n . 565-79• Schroeder, G. 1974. Use of f l u i d cowshed manure i n f i s h ponds. Bamidgeh. 25: -104-13. Scott, A. P. and C. Middleton. 1979 • U n i c e l l u l a r algae as a food f o r Ti$rbot (Scaphthalmus maximus L.) larvae- the importance of dietary long-chain polyunsaturated f a t t y acids. Aquaculture. 18: 227-40. Seale, A. 1933- Brine shrimp (Artemia) as a s a t i s f a c t o r y l i v e food f o r f i s h e s . Trans. Amer. Fi s h . Soc. 63: 129-30. S e r f l i n g , S. A., J . C. Van 01st and R. F. Ford. 1974. An automatic feeding device and use of live-' andif ro z en Artemia - f o r c u l t u r i n g l a r v a l stages of the American lobster, Homarus americanus. Aquaculture. 3(3): 311-14. 67 S h e l e f , G., M. Schwarz, H. S c h e c h t e r . 1972. P r e d i c t i o n o f p h p t o s y n t h e t i c biomass p r o d u c t i o n i n a c c e l e r a t e d a l g a l - b a c t e r i a l wastewater t r e a t m e n t system. P r o c e e d . S i x t h I n t ' l Water P o l l u t i . Res. Conf. I s r a e l No. 9 A/5/91-19/10 18-23 June. S i c k , L. V. 1976. N u t r i t i o n a l e f f e c t o f f i v e s p e c i e s o f marine a l g a e on t h e growth development and s u r v i v a l o f t h e b r i n e shrimp, A r t e m i a s a l i n a . Mar. B i o l . 3 5 : 69-78. S l o b o d k i n , L. B. 1968. A s p e c t s o f the f u t u r e e c o l o g y . B i o l . . S c. Tokyo. 18: 16-23. S m i t h , T. I . J . , J . S. Hopkins*and P. A. S a n d i f e r . 1978. Development o f a l a r g e s c a l e A r t e m i a h a t c h i n g systems u t i l i z i n g r e c i r c u l a t e d w a t e r . I n "Proceed. 9th Ann. Meet. WMS" ( J . V. A v a u l t , e d . ) . L o u i s i a n a S t a t e U n i v e r s i t y , B a t o n Rouge, L. A. U. SA A. 807 pp. S p e c t r o v a , L. V. 1979. R e a r i n g l i v e f e e d i n the USSR. I n " C u l t i v a t i o n o f f i s h f r y and i t s l i v e f o o d " (E. S t y c z y n s k a - J u r e w i c s , T. B a k i e l , E. J a s p e r s and G. Pers o o n e , eds.) Spec. P u b l . No. 4 . I n s t . Mar. S c t f c . Res. Bredene, B e l g i u m . 534pp. S o r g e l o o s , P. 1976. The b r i n e s h r i m p , A r t e m i a s a l i n a : A b o t t l e n e c k i n m a r i c u l t u r e . FAO Tech. Conf. A q u a c u l t u r e . K y o t o , Japan. S o r g e l o o s , P. and G. Pe r s o o n e , 1975- T e c h n o l o g i c a l improvements f o r t he c u l t i v a t i o n o f i n v e r t e b r a t e s as f o o d f o r f i s h e s and c r u s t a c e a n s . I I . H a t c h i n g and c u l t u r i n g o f the b r i n e shrimp, A r t e m i a s a l i n a L. A q u a c u l t u r e . 6: 303-17. S o r g e l o o s , P., M. Baeza-Mesa, F. B e n i j t s and G. Pe r s o o n e . 1975- R e s e a r c h on the c u l t u r i n g o f the b r i n e s h r i m p , . A r t e m i a s a l i n a L. a t t h e S t a t e U n i v e r s i t y o f Ghent. 10th European Sympo. Mar..:,. B i o l . 17-23 S e p t . B e l g i u m . 1: 473-95. S o r g e l o o s , P., M. Baeza-Mesa, E. B o s s u y t , E* Bruggeman, J . D o b b e l i e r , D. V e r s i c m e l e , E. L a v i n a and A. B e r n a r d i n o . 1980. The c u l t u r e of. A r t e m i a on r i c e b r a n : the c o n v e r s i o n o f a '-•"•at waste p r o d u c t i n t o h i g h l y n u t r i t i v e a n i m a l p r o t e i n . A q u a c u l t u r e . ( I n p r e s s ) S o r g e l o o s , P., E. B o s s u y t , E. L a v i n a , M. Baeza-Mesa and G. Per s o o n e . 1977. D e c a p s u l a t i o n o f A r t e m i a c y s t s : a s i m p l e t e c h n i q u e f o r t h e improvement o f t h e use o f t h e b r i n e shrimp i n a q u a c u l t u r e . A q u a c u l t u r e . 12(4) : 311-16. S t e w a r t , J . 1977. R e l a t i v e s e n s i t i v i t y t o l e a d o f a naked green f l a g e l l a t e , D u n a l i e l l a t e r t i o l e c t a , Water, A i r and S o i l P o l l u t i o n . 8: 243-47. Taub, F. B. and A. M. D o l l a r . 1965. ..Control o f p r o e t i n l e v e l o f a l g a e , C h l o r e l l a . J . Fd. Sc. 30: 359-64. 68. Technicon AutoAnalyser I I . I n d u s t r i a l Method AA II 33-369 W. 1 9 6 9 . AutoAnalysis f o r Nitrate + N i t r i t e i n Water (Range: 0 T 2 „ 0 ppm). Technicon Instrument Corp., Tarrytown, New York 1 0 5 9 1 . Technicon AutoAnalyser I I . I n d u s t r i a l Method AAII 9 8 - 7 0 W. 1 9 7 1 a . AutoAnalysis f o r Ammonia i n Water and Waste Water (Range 0 - 1 0 ppm). Technicon Instrument Corp., Tarrytown, New York 1 0 5 9 1 . Technicon AutoAnalyser I I . I n d u s t r i a l Method AAII 1 0 3 - 7 0 A. 1 9 7 1 h . AutoAnalysis f o r Total Kjeldahl Nitrogen (Range: 0 - 1 0 0 0 ppm). Technicon Instrument Corp., Tarrytown, New York I O 5 9 1 . Technicon AutoAnalyser I I . I n d u s t r i a l MethodlAAII 9 4 - 7 0 W. 1 9 7 1 c . AutoAnalysis f o r Phosphate (Ortho). i n Water and Waste Water (Range 0 - 1 0 ppm). Technicon instrument Corp., Tarrytown New York 1 0 5 9 1 . Thomas, W. H., D. L. R. Seibert and A. N. Dodson. 1 9 7 4 . Phytoplankton enrichment experiments and bioassays i n natural coastal seawater and i n sewage o u t f a l l r e ceiving waters o f f Southern C a l i f o r n i a . Estuarine and Coastal Mar. S c i . 2 : 1 9 1 - 2 0 6 . Tobias,vw.J., P. Sorgeloos, E. Bossuyt and 0 . A. Roels. 1 9 7 9 . The technical f e a s i b i l i t y of mass-culturing Artemia s a l i n a i n the St. Croix " A r t i f i c i a l Upwelling" mariculture system. Proceed. World Maricult. Soc. 1 0 : 203-14. Ucal, 0 . 1 9 7 9 . Artemia s a l i n a (L.) collected from Carnalti Saltern (Izmir) and i t s use as a nutrient material i n f i s h culture. Rap_p_. Comm. Int. Mer. Medit. 25/26 8: 127-28. Ukeles, R. 1 9 6 5 . A simple method fo r the mass culture of marine algae. Limnol. Oceanogr. 1 0 : 4 9 2 - 9 5 . Wall, L. L. Sr., C. W. Gehrke, T. E. Neuner, R . 0 . Cathey and P. R. Rexroad. 1974. Total protein nitrogen-evaluation and comparison fo four d i f f e r e n t methods. Presented at the 88th Ann. Meet. Ass. O f f i c i a l A n a l y t i c a l Chemists. Watanabe, T. 1979- N u t r i t i o n a l q u a l i t y of l i v i n g feeds i n seed production of f i s h . In "Proceed. 7 th Japan-Soviet Joint Symp. Aquaculture, Sept. 1978 Tokyo, Japan,-.' Watanabe, T., T. Arakawa, C. Kitg.ima, K. Fukusho and. S. F u j i t a . 1978a. N u t r i t i o n a l evaluation of proteins of l i v i n g feeds .used i n seed production of f i s h . B u l l . Jap. Soc. S c t f c . F i s h . 44(10) : 985-88. 69 VJatanabe, T., T. Arakawa, C. Kitjima, K. Fukosho and S. F u j i t a . 1978b. Proximate and mineral composition of l a v i n g feeds used i n seed production of f i s h . B u l l . Jap. Soc. 'Sctf c. F i s h . 44(9): 979-84. Wickins, S. F. 1972. The food values of the brine shrimp, Artemia s a l i n a L. to larvae of the prawn, Palaemon serratus Pennat. J . Exp. Mar. B i o l . Zool. 10: 151-70. Wisely, B. and C. Purday. 1961. An a l g a l mass culture unit f o r feeding marine invertebrate larvae. 1-B Div. Fish Ocenogr. Tech. Paper No. 12 C. S. I. RQ 0. Melbourne, A u s t r a l i a . APPENDICES 71 Appendix 1 Composition of Major Nutrients NaNO^ NaH 2 P0^'H 2 0 Trace Metals Na 2EDTA + F e C l y 6 H 2 0 + CuSO^•5H2o Z n S 0^'7H 2 0 CoCl 2*6H 2 0 MnCl 2«4H 2 0 Na2MoO^«2H 20 Vitamins Thiamin-HCl B i o t i n B 1 2 Seawater Enrichment " f / 2 " 7 5 mg ( 8 8 3 uM) 5 mg ( 3 6 . 3 uM) 4 . 3 6 mg ( ca 1 1 . 7 uM) 3 . 1 5 mg (O . 6 5 mg Fe or ca 1 1 . 7 uM 0 . 0 1 mg ( 2 . 5 ug Cu or ca 0 . 0 4 uM) 0 . 0 2 2 mg ( 5 ug Zn or ca 0 . 0 8 uM) 0 . 0 1 mg ( 2 . 5 ug Co or ca 0 . 0 5 uM) 0.18 mg ( 0 . 0 5 mg Mn or ca 0 . 9 uM) 0 . 0 0 6 mg ( 2 . 5 ug Mo or ca 0 . 0 3 uM 0 . 1 mg 0 . 5 ug 0 . 5 ug to one l i t e r Appendix 2 Composition of A r t i f i c i a l Seawater FORTY FATHOMS BIO-CRYSTALS MARINEMIX Assay Chart Average solution of Forty Fathoms Marinemix hydrated to a density of 1.025 using d i s t i l l e d water.. Figures c i t e d from actual independent laboratory analysis. Concentration (ppm) Aluminum 0.06 Antimony 0.0005 Argon trace Arsenum 0.01 Barium 0.12 Bicarbonate 174.0 Beryllium 0.0002 Bismuth trace Boron 2.1 Bromide 62.0 Cadmium 0.009 Calcium 410.0 Carbonate 10.0 Cerium 0.0007 Cesium trace Chromium 0.02 Chloride 18600.0 Copper 0.007 Cobalt 0.0025 Dysprosium trace Erbium trace Europium trace Fluoride 1.9 Gadolinium trace Gallium 0.0004 Germanium 0.00005 Gold trace Hafnium trace Helium trace Holmium trace Indium trace Iodine 0.03 Iron 0.03 Krypton trace Lanthanum trace Lead trace Lithium 0.24 Lutetium trace Magnesium 1290.0 Manganese 0.008 Composition (ppm) Mercury Molybdenum Neodynium Neon N i c k e l Niobium N i t r o g e n Palladium Phosphorus Potassium Praeseodymium P r o t a c t i n i u m Radium Radon Rubidium Ruthenium Samarium Scandium Selenium S i l i c o n Sodium Strontium S u l f u r (as SO^) Tantalium T e l l e r i u m Terbium Thalium Thulium T i n Titanium Tungsten Uranium Vanadium Xenon Ytterbium Y t t r i u m Zinc Zirconium 0.0007 0.005 t r a c e t r a c e 0.009 t r a c e 0.85 t r a c e 0.04 380.- t r a c e t r a c e t r a c e t r a c e 0.06 t r a c e t r a c e t r a c e t r a c e 4.5 110400.0 12.4 2600.0 t r a c e t r a c e t r a c e 0.00007 t r a c e 0.006 0.004 0.004 0.00005 0.0009 t r a c e t r a c e t r a c e 0.24 t r a c e Appendix 3 ANALYSIS OF VARIANCE TABLE FOR GROWTH CONSTANT (EXPT 2A) SOURCE SUM OF SQUARES PROBABILITY TEST TERM- TREATMENT 0.16666E-08 RESIDUAL RFP 0.43333E-07 RESIDUAL RESIDUAL 0 . 4 3 3 3 3 E - 0 7 TOTAL 0.88333E-07 OVERALL MEAN GROWTH 0.23283E-01 DF MEAN SQUARE F-RATIO 1. 0.16666E-08 0.76920E-01 0 . 8 0 7 5 5 2. 0.21667E-07 1.0000 0 . 5 0 0 0 0 2. 0.21667E-07 5- OVERALL STANDARD DEVIATION 0.13292E-03 Appendix 4a ANALYSIS OF VARIANCE TABLE FOR GROWTH CONSTANT (EXPT 3A) SOURCE TREAT REP RESIDUAL TOTAL GROWTH SUM OF SQUARES 0.20167E-06 0.13334E-07 0.53334E-07 0.26833E-06 OVERALL MEAN 0.22917E-01 DF 1. 2. 2. 5- MEAN SQUARE 0.20167E-06 0.66668E-08 0.26667E-07 F-RATIO PROBABILITY TEST TERM 7.5624 O.IIO7O RESIDUAL 0.25000 0.80000 RESIDUAL OVERALL STANDARD DEVIATION 0.23166E-03 -N3 ANALYSIS OF VARIANCE Appendix 4b TABLE FOR CELL DENSITY SOURCE SUM OF PROBABILITY TEST TERM SQUARES TREAT RESIDUAL REP RESIDUAL DAY RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL DENSITY 0.76163 4.2740 202.71 3.9576 5.6726 2.1127 3.4500 222.94 OVERALL MEAN 3-5733 DF 1. 2. 9. 2. 18. 9. 18. 59. MEAN SQUARE O.76163 2.1370 22.524 1.9788 0.31514 0 .23475 0.19167 F-RATIO 3.9737 11.149 117.51 10.324 1.6442 1.2248 0.06160 0.00071 0.00000 0.00103 0.15031 O.33986 OVERALL STANDARD DEVIATION 1.9439 Appendix 4c ANALYSIS OF VARIANCE TABLE FOR ALGAL DRY WEIGHT (EXPT 3C) SOURCE SUM OF DF MEAN SQUARE F-RATIO PROBABILITY TEST TERM SQUARES TREAT 0.47538E-03 1. 0147538E -03 0.79947E -01 0.78060 RESIDUAL REP 0.20040E-01 2. 0.10020E -01 1.6851 0.21339 RESIDUAL DAY 0.59008 9. O.65565E -01 11.026 0.00001 RESIDUAL TREAT*REP 0.16076E-01 2. 0.80382E -02 1.3518 0.28381 RESIDUAL REP*DAY 0.10690 18. 0.59387E -02 0.99874 0.50105 RESIDUAL TREAT*DAY RESIDUAL O.63669E-OI 9. 0.70743E -02 1.1897 O.35845 RESIDUAL 0.10703 18. 0.59462E' -02 TOTAL 0.90240 59. OVERALL MEAN OVERALL STANDARD DEVIATION DRY WEIGHT 0.18375 0.12367 Appendix 5a ANALYSIS OF VARIANCE TABLE FOR CELL DENSITY (EXPT 5 B ) SOURCE SUM OF PROBABILITY TEST TERM SQUARES TREAT RESIDUAL REP RESIDUAL DAY RESIDUAL TREAT*REP RESIDUAL REP*DAY>I RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL- DENSITY 6.6082 0.93253 294.11 O.62585 5-5737 8.1307 5.5260 321.51 OVERALL MEAN 5.5438 DF 1. 2. 19. 2. 38. 19. 38. 119. MEAN SQUARE 6.6082 0.46627 15.479 0.31293 0.14668 0.42793 0.14542 F-RATIO 45.442 3.2063 106.45 2.1519 1.0086 2.9427 0.00000 0.05167 0.00000 0.13023 0.48950 0.00227 OVERALL STANDARD DEVIATION 1.6437 Appendix $b ANALYSIS OF VARIANCE TABLE FOR CELL DENSITY (EXPT 5B1) SOURCE PROBABILITY TEST TERM TREAT RESIDUAL REP RESIDUAL DAY RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL SUM OF SQUARES DENSITY DF 8.1420 1. 0.63622 2. 31.404 6. 0.62014 2. 2.3135 12. 2.2230 6. 1.4467 11. 47.840 40. OVERALL MEAN 4.9885 MEAN SQUARE 8.1420 0.31811 5.2340 0.31007 0.19279 0.37050 0.13152 61.907 2.4187 39.796 2.3576 1.4658 2.8170 F-RATIO 0.00001 0.13471 0 .00000 0.14057 0.26708 0.06512 OVERALL STANDARD DEVIATION 1.0936 Appendix 5c ANALYSIS OF VARIANCE TABLE FOR CELL DENSITY (EXPT 5B2) SOURCE SUM OF DF MEAN SQUARE PROBABILITY TEST TERM SQUARES TREAT 1.1959 1. 1.1959 4.4383 RESIDUAL REP 0.32929 2 . 0.16464 0.61102 RESIDUAL DAY 5.0913 6. 0.848 54 3.1491 RESIDUAL TREAT*REP 0.76389 2 . 0.38194 1.4175 RESIDUAL REP*DAY 1.3014 11. 0.11831 0 . 4 3 9 0 8 RESIDUAL TREAT*DAY 1.4670 6. 0.24450 0.90740 RESIDUAL RESIDUAL 3.5029 13. 0 . 2 6 9 4 6 TOTAL 12.791 41 OVERALL MEAN OVERALL STANDARD DEVIATION DENSITY 6.6921 0-55855 F-RATIO 0.05513 0.55767 0.03924 0.27741 0.91017 0.51899 00 o Appendix 5d ANALYSIS OF VARIANCE FOR CELL DENSITY (EXPT 5B3) SOURCE SUM OF DF MEAN SQUARE F-RATIO PROBABILITY TEST TERM SQUARES TREAT 0.14963E-01 1 . 0.14963E-01 0.15520 0.70391 RESIDUAL REP 0.16247E - 0 1 2. 0.81233E - 0 2 0.84255E - 0 1 0.92000 RESIDUAL DAY 0.15365 4. 0 . 3 8 4 1 2 E - 0 1 0.39841 0 . 8 0 4 8 2 RESIDUAL TREAT*REP 0.40613 2 . 0.20306 2.1062 0.18415 RESIDUAL . REP*DAY 0.94345 8 . 0.11793 1.2232 0.39130 RESIDUAL TREAT*DAY 0 . 2 2 8 9 5 4. 0 . 5 7 2 3 8 E - 0 1 0.59368 0.67726 ^.£I>!RESIDUAL RESIDUAL 0.77131 8 . 0.96413E-01 TOTAL 2.5347 29. OVERALL MEAN OVERALL STANDARD DEVIATION DENSITY 5-8537 0.29564 Appendix 6 ANALYSIS OF VARIANCE TABLE FOR GROWTH CONSTANT (EXPT 5A) SOURCE PROBABILITY TEST TERM TREAT. RESIDUAL REP RESIDUAL RESIDUAL TOTAL GROWTH SUM OF DF SQUARES 0.10667E-06 1. 0 . 3 2 3 3 3 E - 0 6 2 . 0 . 8 0 3 3 3 E - 0 6 2 . 0 . 1 2 3 3 3 E - 0 5 5. OVERALL MEAN 0 . 2 3 5 3 3 E-01 MEAN SQUARE 0.10667E -06 0.16167E-06 0 . 4 0 1 6 7 E - 0 6 F-RATIO 0 . 2 6 5 5 6 0.40249 OVERALL STANDARD DEVIATION 0 . 4 9 6 6 6 E - 0 3 O.65763 0.71302 co ro Appendix 7 ANALYSIS OF VARIANCE TABLE FOR ALGAL DRY WEIGHT ( EXPT 5C) SOURCE PROBABILITY TEST TERM SUM OF SQUARES DF MEAN SQUARE TREAT 0.18800E-01 1. 0.18800E-01 RESIDUAL REP 0.84485E-03 2. 0.42243E-03 RESIDUAL DAY 0.46410 19. 0.24426E-01 RESIDUAL TREAT*REP 0.22687E-02 2. 0.11344E-02 RESIDUAL REP*DAY 0.11038E-01 38. 0.29048E-03 RESIDUAL TREATEDAY 0 . 2 4 5 9 4 E-01 19. 0 . 1 2 9 4 4 E-02 RESIDUAL RESIDUAL 0 . 1 9 7 1 2 E-01 38. 0.51874E-03 TOTAL 0.54135 119. F-RATIO 36.241 0.00000 0.81432 0.45052 47.087 0.00000 2.1867 0.12621 0.55996 0.96109 2.4953 0.00808 DRY WEIGHT OVERALL MEAN 0.16005 OVERALL STANDARD DEVIATION 0.67448E-01 Appendix 8 ANALYSIS OF VARIANCE TABLE FOR TOTAL LENGTH OF ARTEMIA SOURCE SUM OF PROBABILITY TEST TERM SQUARES TREAT RESIDUAL REP RESIDUAL DAY RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL GROWTH 0.36363E-03 0.74867E-01 .2.3 • 0 . 1 2 4 5 2 1 .3383 0 .68787 0 . 9 2 4 0 5 8 3 6 . 7 5 OVERALL MEAN 2.8957 DF 1. 3. 13. 3. 39. 13. 38. 110. MEAN SQUARE F-RATIO 0 .36363E-03 0 .14954E -01 0.249£6E-01 1.0263 64 .094 2635.8 0141508E-01 1.7070 0 . 3 4 3 1 5 E - 0 1 0.52913E-01 0.24317E-01 1.4111 2.1760 OVERALL STANDARD DEVIATION 2 . 7 5 8 0 0 . 9 0 3 3 2 0 . 3 9 1 8 3 0 . 0 0 0 0 0 0.18189 0 . 1 4 5 3 2 0 .03137 Appendix 9 a ANALYSIS OF VARIANCE TABLE FOR PERCENTAGE SURVIVAL OF ARTEMIA SOURCE PROBABILITY TEST TERM TREAT RESIDUAL REP RESIDUAL DAY RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL SURVIVAL SUM OF DF SQUARES 331.95 1. 242.75 3. 41120. 13. 105.10 3. 395.58 39. 591.09 13. 328.87 39. 43369. 111. OVERALL MEAN 61.393 MEAN SQUARE 331.95 80.917 3163.1 35.035 10.143 45.468 8.4326 F-RATIO 39.365 9-5958 375.10 4.1547 1.2028 5.3920 0.00000 0.00007 0.00000 0.01200 0.28346 0.00002 OVERALL STANDARD DEVIATION 19.766 Appendix 9 b ANALYSIS OF VARIANCE TABLE FOR PERCENTAGE SURVIVAL OR ARTEMIA SOURCE SUM OF PROBABILITY TEST TERM SQUARES TREAT RESIDUAL REP RESIDUAL DAY RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL SURVIVAL DF 631.86 1. 134.34 3- 10628. 6. 115.01 3. 229.41 18. 330.71 6. 160.51 20. 12311. 57- OVERALL MEAN 77.862 MEAN SQUARE 631.86 44.782 1771.4 38.338 12.745 55.119 8.0257 78.730 F-RATIO 0.00000 5.5798 0.00600 220.72 0.00000 4.7769 0.01141 1.5880 0.15837 6.8678 0.00045 OVERALL STANDARD DEVIATION 14.696 Appendix 9c ANALYSIS OF VARIANCE TABLE FOR PERCENTAGE SURVIVAL OF ARTEMIA SOURCE PROBABILITY TEST TERM TREAT'I' RESIDUAL REP RESIDUAL DAY RESIDUAL TREAT*REP RESIDUAL REP*DAY RESIDUAL TREAT*DAY RESIDUAL RESIDUAL TOTAL SURVIVAL SUM OF DF SQUARES 12.532 1. 129.96 3. 1267.5 6. 66.274 3. 74.818 18. 20.097 6. 81.204 19. 1653.3 56. OVERALL MEAN 45.368 MEAN SQUARE 12.532 43.320 211.246 22.091 4.1566 3.3496 4.2739 F-RATIO 2.9323 10.136 49.426 5.1689 0.97255 0.78373 0.10310 0.00033 0.00000 0.00883 0.52185 0.59317 OVERALL STANDARD DEVIATION 5.4335 Appendix 10a ANALYSIS OF VARIANCE TABLE FOR ARTEMIA WET WEIGHT SOURCE PROBABILITY TEST TERM TREAT RESIDUAL REP RESIDUAL TREAT*REP RESIDUAL RESIDUAL TOTAL WET WEIGHT SUM OF DF SQUARES 6.1075 1. 4.5720 3- 1.8382 3. 0.0 - 0 . 12.518 7. OVERALL MEAN 18.671 MEAN SQUARE F-RATIO 6.1075 0.0 1.00000 1.5240 0.0 1.00000 0.61272 0.0 1.00000 0.0 OVERALL STANDARD DEVIATION 1.3373 Appendix 10b SOURCE PROBABILITY TEST TERM TREAT RESIDUAL REP RESIDUAL TREAT*REP RESIDUAL RESIDUAL TOTAL DRY WEIGHT ANALYSIS OF VARIANCE TABLE FOR ARTEMIA DRY WEIGHT DF SUM OF SQUARES 0.15680 1,.' 0.25965 3. 0.60300E-01 3. 0.0 -0. 0.47675 7. OVERALL MEAN 1.9625 MEAN SQUARE F-RATIO 0.15680 0.0 1.00000 0.86550E-01 0.0 1.00000 0.20100E-01 0.0 1.00000 0.0 OVERALL STANDARD DEVIATION 0.26097 Appendix 11 ANALYSIS OF VARIANCE TABLE FOR ARTEMIA BIOMASS CONVERSION EFFICIENCY (BCE) SOURCE PROBABILITY TEST TERM TREAT RESIDUAL REP RESIDUAL TREAT*REP RESIDUAL RESIDUAL TOTAL BCE SUM OF SQUARES 1.5051 230.91 53.863 0.0 286.28 OVERALL MEAN 58.356 DF 1. 3. 3. -0. 7. MEAN SQUARE 1.5051 76.969 17.954 0.0 F-RATIO 0.0 0.0 0.0 OVERALL STANDARD DEVIATION 6.3950 1.00000 1.00000 1.00000

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