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Culture of the freshwater cladoceran, Daphnia Pulex F. utilizing Scenedesmus obliquus grown in dairy… Castillo, Nelson M. 1981

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CULTURE OF THE FRESHWATER CLADOCERAN, DAPHNIA PULEX F\ UTILIZING SCENEDESMUS OBLIQUUS GROWN IN DAIRY WASTE MEDIUM by NELSON M. CASTILLO B. Sc., U n i v e r s i t y of the P h i l i p p i n e s , Quezon C i t y P h i l i p p i n e s , 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in 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 t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH October 1981 COLUMBIA © N e l s o n M. C a s t i l l o , 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y , s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Q ^ O ^ ! S n ^ QUx&qV\flg*> The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V ancouver, Canada V6T 1W5 Date S A W \&j R?\  J i i ABSTRACT Scenedesmus o b l i q u u s was grown in batch c u l t u r e s at v a r y i n g c o n c e n t r a t i o n s of d i g e s t e d d a i r y medium ranging from 200 to 2,000 ug-at • N - l 1 . Higher growth r a t e s were observed at low N-c o n c e n t r a t i o n s while higher c e l l y i e l d s were observed at high N-c o n c e n t r a t i o n s . A e r a t i o n enhanced both a l g a l growth r a t e s and biomass y i e l d s . R e s u l t s show an advantage i n a d j u s t i n g the n i t r o g e n to phosphorus atomic r a t i o of the medium. More biomass was produced i n c u l t u r e s with higher N:P r a t i o s . The a l g a l biomass produced was used as food f o r the freshwater c l a d o c e r a n , Daphnia pulex F. Three fe e d i n g l e v e l s -1 were used: 50,000, 100,000 and 150,000 c e l l s - m l . However, no s i g n i f i c a n t d i f f e r e n c e s were observed i n both Daphnia biomass y i e l d s and biomass c o n v e r s i o n e f f i c i e n c i e s . The tendency of Scenedesmus c e l l s to s e t t l e down in the bottom and c l i n g to the s i d e s of the tank presented a major problem i n the study.-I n t e n s i v e f e e d i n g d i d not i n c r e a s e the biomass p r o d u c t i o n of Daphnia, although l a r g e r - s i z e d a d u l t s with l a r g e r brood s i z e s were produced. Animals i n c u l t u r e reached a d e n s i t y of 1.24 animals-mf Aand obtained c o n v e r s i o n e f f i c i e n c i e s as h i g h as 40-50%. Dr. J,W. Zahradnik T h e s i s S u p e r v i s o r i i i TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS . i i i LIST OF TABLES v LIST OF FIGURES . v i ACKNOWLEDGEMENTS ix INTRODUCTION 1 LITERATURE REVIEW '.. 3 R e c y c l i n g of Wastes through Aquaculture 3 A l g a l P r o d u c t i o n from Waste 5 Daphnia as a P o t e n t i a l F i s h Food i n Aquaculture 8 Present S t a t e - o f - t h e - A r t on Daphnia C u l t u r e 12 MATERIALS AND METHODS 14 Algae 14 Daphnia pulex 14 Media P r e p a r a t i o n 15 A l g a l C u l t u r e Medium 15 Daphnia C u l t u r e Medium 15 D e s c r i p t i o n of C u l t u r e U n i t s 16 A l g a l C u l t u r e U n i t 16 Daphnia C u l t u r e Unit 18 C u l t u r e Methods 18 A l g a l - C u l t u r e Method 18 Daphnia C u l t u r e Method 19 Chemical A n a l y s i s 21 Kheldahl N i t r o g e n 21 Ammon i a / N i t r a t e / N i t r i t e 21 i v T o t a l Phosphate 22 . Ortho-Phosphate 22 A l g a l C e l l Weight 22 Growth Rate Experiments ( Daphnia ) 23 Length-Weight R e l a t i o n s h i p ( Daphnia ) 23 RESULTS 25. Dair y Waste A n a l y s i s .25 A l g a l Experiments 26 Daphnia Experiments 37 A l g a l C e l l Weight 68 Growth Rate Experiments 68 Length-Weight R e l a t i o n s h i p 71 Brood S i z e i n R e l a t i o n to Daphnia l e n g t h 71 DISCUSSION 77 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 Production 77 A l g a l Growth on Manure Medium 78 Daphnia Biomass Pro d u c t i o n 81 Pop u l a t i o n Growth and S i z e S t r u c t u r e 82 Growth Rate Experiments 84 Length-Weight R e l a t i o n s h i p 85 N u t r i t i o n a l Inadequacy of C e r t a i n Algae Daphnia C u l t u r e 85 T o x i c i t y i n Daphnia C u l t u r e 87 Scale-up C o n s i d e r a t i o n s 89 SUMMARY AND CONCLUSIONS 91 REFERENCES 94 APPENDICES 103 V LIST OF TABLES Table T i t l e Page 1 Summary of chemical composition of each media and the a l g a l experiment i n which they were used 25 2 Summary of the a l g a l experiments, the d i f f e r e n t -treatments and the r e s u l t s 27 3 Chemical composition of media before and a f t e r a l g a l growth . 3 3 4 Amount of algae consumed by Daphnia at 3-day i n t e r v a l s . 4 2 ,5 Fecundity of Daphnia c u l t u r e s i n a l l f i v e experiments 69 6 Ammonia, pH and DO l e v e l s measured d a i l y i n Daphnia c u l t u r e tanks i n D^ Expts. #4 #5 70 7 Daphnia brood s i z e i n r e l a t i o n to t o t a l l e n g t h 75 v i LIST OF FIGURES Fi g u r e T i t l e Page 1 A g e n e r a l i z e d view of the A l g a l C u l t u r e Unit 17 2 D a i l y c e l l d e n s i t y of S.obiiquus grown i n v a r y i n g N-concentrations of d a i r y waste medium ( A l g a l Expt.#1) 28 3 D a i l y c e l l d e n s i t y of S.obiiquus grown i n v a r y i n g N-concentrations o f . d a i r y waste medium ( A l g a l Expt.#2) 30 4 D a i l y c e l l d e n s i t y of S.obiiquus grown i n v a r y i n g N-concentrations of d a i r y waste medium ( A l g a l Expt.#3) 31 5 D a i l y c e l l d e n s i t y of S.obliquus grown i n v a r y i n g . N-concentrations of d a i r y waste medium ( A l g a l Expt.#4). . 3 2 6 D a i l y c e l l d e n s i t y of S.obiiquus grown i n v a r y i n g N-concentrations of d a i r y waste medium ( A l g a l Expt.#5) • 35 7 D a i l y c e l l d e n s i t y of S.obiiquus grown i n v a r y i n g N - c o n c e n t r a t i o n s of d a i r y waste medium ( A l g a l Expt.#6) 36 8 D a i l y c e l l d e n s i t y of S.obliquus grown i n d a i r y waste medium with Nitrogen to Phosphorus atomic r a t i o s of 17 and 64 ( A l g a l Expt.#7) 38 9 D a i l y c e l l d e n s i t y of S.obliquus grown i n d a i r y waste medium with Nitrogen to Phosphorus atomic r a t i o s of 4, 22 and 69 ( A l g a l Expt.#9) 39 10 D a i l y c e l l d e n s i t y of S.obiiquus grown i n d a i r y waste medium with and without a e r a t i o n ( A l g a l Expt.#8) 40 11 N-consumed vs. A l g a l y i e l d 41 12 Daphnia biomass at the three feeding l e v e l s at 3-day i n t e r v a l s ( D^ Expt.#l) 45' 13 Biomass co n v e r s i o n e f f i c i e n c y of Scenedesmus to Daphnia at the three f e e d i n g l e v e l s at 3-day i n t e r v a l s ( EL Expt.#l) 45 14 Daphnia biomass at the three feeding l e v e l s at 3-day i n t e r v a l s ( D^ Expt.#2) 46 15 Biomass c o n v e r s i o n e f f i c i e n c y of Scenedesmus to Daphnia at the three f e e d i n g l e v e l s at 3-day •VI1 i n t e r v a l s ( IK Expt'.#2) 46 16 Daphnia biomass at the three feeding levels..at 3-day i n t e r v a l s (-'IK Expt. #3)' 47 17 Biomass conversion e f f i c i e n y of Scenedesmus to Daphnia at the three f e e d i n g l e v e l s at 3-day . i n t e r v a l s (. IK" Expt. #3) 47 18 Daphnia biomass at 3-day i n t e r v a l s fed with 100,000 Scenedesmus c e l l s ml 2 to 3 times d a i l y ( IK. Expt.#4 and #5) ; 48 19 Biomass conver s i o n e f f i c i e n c y of Scenedesmus to Daphnia i n EK_ Expts.#4 and #5 a t . i n t e r v a l s 48. 20 P r o p o r t i o n of j u v e n i l e , young a d u l t and a d u l t Daphnia i n terms of number and biomass i n c u l t u r e s at the three feeding l e v e l s at 3-day i n t e r v a l s ( EL Expt.#1) 49 21 P r o p o r t i o n of j u v e n i l e , young a d u l t and a d u l t Daphnia in terms of number and biomass i n c u l t u r e s at the three feeding l e v e l s at 3-day i n t e r v a l s ( IK Expt.=2) 50 22 P r o p o r t i o n of j u v e n i l e , young a d u l t and a d u l t Daphnia i n terms of number and biomass i n c u l t u r e s at the three feeding l e v e l s at 3-day i n t e r v a l s ( IK Expt.#3) 51 23 P r o p o r t i o n of j u v e n i l e , young adult, and a d u l t Daphnia i n terms of number and biomass at 3-day i n t e r v a l s i n EK_ Expt.#4 and #5 52 24 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 50,000 c e l l s ml fee d i n g l e v e l at 3-day i n t e r v a l s ( IK Expt.#1) 53 25 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 100,000 c e l l s ml feeding l e v e l at 3-day i n t e r v a l s ( IK Expt.#1) 54 26 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 150,000 c e l l s ml fee d i n g l e v e l at 3-day i n t e r v a l s ( IK Expt.#1) 55 27 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 50,000 c e l l s ml fee d i n g l e v e l at 3-day i n t e r v a l s ( IK Expt.#2) 56 28 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 100,000 c e l l s ml feeding l e v e l at 3-day i n t e r v a l s ( IK Expt.#2) 57 29 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 150,000 V I 1 1 c e l l s ml feeding l e v e l at 3-day i n t e r v a l s ( EL Expt.#2) 58 30 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 50,000 c e l l s ml feeding l e v e l at 3-day i n t e r v a l s ( EL Expt.#3) 59 31 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 100,000 c e l l s ml feeding l e v e l at 3-day i n t e r v a l s ( EL_ Expt.#3) 60 32 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 150,000 c e l l s ml feeding l e v e l at 3-day i n t e r v a l s ( EL Expt.#3) . 6 1 33 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 3-day i n t e r v a l s fed. with 100,000 c e l l s ml 2 to 3 times d a i l y ( IK Expt.#4- Repl ."l) 62 34 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 3-day i n t e r v a l s fed with 100,000 c e l l s ml 2 to 3 times d a i l y ( IL Expt.#4- Repl.2) 63 35 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 3-day i n t e r v a l s fed with 100,000 c e l l s ml 2 to 3 times d a i l y ( IL Expt.#4- Repl.3) 64 36 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 3-day i n t e r v a l s fed with 100,000 c e l l s ml 2 to 3 times d a i l y ( Expt.#5- Repl.1) 65 37 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 3-day i n t e r v a l s fed with 100,000 c e l l s ml 2 to 3 times d a i l y ( EL Expt.#5- Repl.2) 66 38 Daphnia s i z e - f r e q u e n c y s t r u c t u r e at 3-day i n t e r v a l s fed with 100,000 c e l l s ml 2 to 3 times d a i l y ( EL Expt.#5- Repl.3) 67 39 D a i l y t o t a l l ength of D.pulex at the three food c o n c e n t r a t i o n s (Growth Rate Expt.#1) 72 40 D a i l y t o t a l l ength of D.pulex at the three food c o n c e n t r a t i o n s (Growth Rate Expt.#2) 73 41 Length-weight r e l a t i o n s h i p of D.pulex 74 ACKNOWLEDGEMENT My s i n c e r e s t g r a t i t u d e to Dr. J.W. Zahradnik, my a d v i s o r , Department of Bio-Resource E n g i n e e r i n g . I am deeply indebted to a l l members of my committee: Dr. P.J. H a r r i s o n , Department of Oceanography and Dr. W.E. N e i l l , I n s t i t u t e of Animal Resource Ecology, f o r t h e i r h e lp at the i n i t i a l stages of the experiments and t h e i r c o n s t r u c t i v e c r i t i c i s m s i n the p r e p a r a t i o n of the manuscript; Dr. V. Lo f o r h i s comments. My s i n c e r e s t a p p r e c i a t i o n to the f o l l o w i n g : Dr. P. L i a o , Department of Bio-Resource E n g i n e e r i n g , for conducting most of the chemical a n a l y s i s ; J . Pehlke and N. Jackson, Department of Bio-Resource E n g i n e e r i n g , f o r the t e c h n i c a l a s s i s t a n c e ; to the r e s t of the Bio-E s t a f f and graduate students f o r t h e i r moral support. F i n a n c i a l support from the I n t e r n a t i o n a l Development Research Centre (I.D.R.C.) i s very s i n c e r e l y acknowledged. INTRODUCTION A q u a c u l t u r i s t s world-wide have expressed the need f o r n u t r i o n a l l y adequate d i e t s in animal husbandry. The search f o r s u i t a b l e feeds i s s t i m u l a t e d by the c o s t s and u n p r e d i c t a b l e supply of Artemia. sp. Cysts and manufactured d i e t s have only been p a r t i a l l y s u c c e s s f u l due to u n c o n t r o l l e d l e a c h i n g of vitamins and minerals as soon as the food touches the water (Norman et a l . , 1979). The p o s s i b i l i t y of u s i n g cladocerans (such as Daphnia and Moina ) which can e a s i l y be grown e s p e c i a l l y i n wastewater have a l r e a d y been i n v e s t i g a t e d by a number of r e s e a r c h e r s (Bogatova and Askerov, 1958; Dewitt and Candland, 1971; Dinges, 1974, 1976; Norman et a l . , . 1979; Rees and O l d f a t h e r , 1980). Daphnia i s an u n e x p l o i t e d p o t e n t i a l source of animal p r o t e i n and can be a p r o t e i n supplement i n f i s h food p e l l e t s used by h a t c h e r i e s and commercial f i s h - f a r m e r s . Daphnia (Cladocera:Daphniidae) are f i l t e r - f e e d e r s and feed upon b a c t e r i a , c e r t a i n u n i c e l l u l a r green algae, and a mixture of v a r i o u s protozoa and protophytes from the sediments of ponds (Banta, 1921). In past s t u d i e s , the green algae such as, C h l o r e l l a , Chlamydomonas and Scenedesmus have been used as food f o r the c u l t u r e of Daphnia (Watanabe et a l . , 1955; Richman, 1958; Sasa et a l . , 1960; S c h i n d l e r , 1968; Gordon, 1975). 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 of microorganisms such as b a c t e r i a and phytoplankton (Hephner, 1972; Schroeder, 1974). The u t i l i z a t i o n of l i v e s t o c k wastes as a n u t r i e n t source f o r phytoplankton c u l t u r e and subsequently, as food f o r Daphnia has not been i n v e s t i g a t e d y e t . 2 The general objective of the present . research was to evaluate the culture potential of the freshwater cladoceran, Daphnia pulex fed with the green alga, Scenedesmus obiiquus grown in dairy manure medium. The experiments were conducted in two phases: Phase I - Culture studies of Scenedesmus obliquus grown in dairy manure medium; Phase II - Culture studies of Daphnia pulex fed with waste-grown algae. 3 LITERATURE REVIEW  R e c y c l i n g of Wastes through Aquaculture For hundreds of years people have been using wastes of household and farm animals as f e r t i l i z e r s i n fishponds to s t i m u l a t e p r o d u c t i o n of a c c e p t a b l e food organisms fo r f i s h and other a q u a t i c animals (Brown and Nash, 1979). The i n t e g r a t i o n of a g r i c u l t u r a l wastes with f i s h c u l t u r e i s being p r a c t i c e d e s p e c i a l l y i n l e s s e r developed c o u n t r i e s . On the other hand, aquaculture as a means for wastewater treatment i s being i n v e s t i g a t e d i n more developed c o u n t r i e s . Aquaculture systems have been i n t e g r a t e d with wastewater s t a b i l i z a t i o n ponds to b e n e f i t from l a r g e q u a n t i t i e s of a q u a t i c food m a t e r i a l produced as a by-product of m i c r o b i a l degradation, and to a s s i s t i n the s t a b i l i z a t i o n of the pond. In most cases, n a t u r a l foods present i n ponds and other a q u a t i c containments are not s u f f i c i e n t to support the f i s h e s p e c i a l l y when grown i n great d e n s i t i e s . Supplemental feeding with a r t i f i c i a l d i e t s should be given, which i s o f t e n the l a r g e s t s i n g l e o p e r a t i n g expense i n f i s h - f a r m i n g (Hepher and Schroeder, 1974). One way of reducing the need f o r t h i s i s by i n c r e a s i n g the amount of n a t u r a l food i n the pond. I n v e s t i g a t o r s have demonstrated many times to have i n c r e a s e d plankton p r o d u c t i o n of s m a l l ponds as much as 50 to 200% by the a d d i t i o n of commercial and n a t u r a l f e r t i l i z e r s , and i t i s w e l l known that sewage e f f l u e n t s g r e a t l y i n c r e a s e the plankton of l a k e s and r i v e r s (Pennak, 1946; Schroeder, 1974). 4 Animal and domestic wastes are i d e a l as a growth medium f o r the p r o d u c t i o n of algae s i n c e they are r i c h i n a l l macro- and m i c r o - n u t r i e n t s necessary f o r a l g a l growth; E f f l u e n t s from secondary and t e r t i a r y sewage treatments, mixed with seawater had been used as a source of n u t r i e n t s to grow s i n g l e - c e l l e d marine algae i n mass c u l t u r e s (Songer et a l . , 1974; Ryther, 1975; Schroeder and Hepher, 1976; T r i e f et a l . , 1976). Animal wastes such as swine and cow-shed manure were a l s o e f f e c t i v e i n growing algae (Schroeder, 1977; Maddox et a l . , 1978). Numerous i n v e s t i g a t o r s have a p p l i e d a g r i c u l t u r a l wastes d i r e c t l y i n fishponds and r e p o r t e d i n c r e a s e d f i s h y i e l d s and decreased supplemental fe e d i n g (Buck et a l . , 1976; Moav et a l . , 1977; Schroeder, 1977; Maddox et a l . , 1979). The e f f e c t i v e n e s s of these wastes i n f i s h c u l t u r e maybe based on a food chain that s t a r t s with b a c t e r i a and protozoa a c t i v e i n the decomposition of the organic matter i n the manure. There i s a b i g p o t e n t i a l of i n c o r p o r a t i n g wastes i n f i s h c u l t u r e . However, we are s t i l l faced with a number . of r e s t r a i n t s f o r i t s wider a p p l i c a t i o n . A l l e n and Hepher (1976) enumerates them: (1) u n s a t i s f a c t o r y d i s s o l v e d oxygen l e v e l s i n ponds (2) presence of t o x i c m a t e r i a l s i n wastewaters (3) presence of unacceptable t a s t e s and odours i n f i s h (4) presence of p a r a s i t e s and d i s e a s e s (5) v a r i o u s problems of p u b l i c h e a l t h (6) d i f f i c u l t i e s of s a t i s f y i n g pond e f f l u e n t standards (7) meeting p u b l i c acceptance of the p r a c t i c e . 5 A l g a l Product ion From Waste C h l o r e l l a , Scenedesmus and Chlamydomonas have been r e p o r t e d to be the p r i n c i p a l c o n s t i t u e n t s of sewage o x i d a t i o n ponds and s u f f i c e as a d i e t f o r Daphnia and r e l a t e d c l a doceran Crustacea (Hintz et a l . , 1966; H i n t z and Heitman, J r . , 1975; Gordon, 1975) . P r o l i f e r a t i o n of blue-green algae has a l s o been noted i n waters r e c e i v i n g domestic o u t f a l l s . However, these algae serve as i n f e r i o r food to cladocerans compared to green a l g a e . A r n o l d (1971) in h i s study on seven d i f f e r e n t s p e c i e s of blue-green algae found that i n g e s t i o n , a s s i m i l a t i o n , s u r v i v o r s h i p and r e p r o d u c t i o n of Daphnia pulex fed with blue-green algae • were lower than those fed with green a l g a e . The micro-algae C h l o r e l l a , Scenedesmus and Chlamydomonas are e x c e l l e n t feeds f o r Daphnia (Watanabe et a l . , 1955; Richman, 1958; S c h i n d l e r , 1968). L i s t e d below are the c a l o r i c values of these algae obtaned by Richman (1958): Chlamydomonas r e i n h a r d i 5,269 c a l / g C h l o r e l l a pyrenoidosa 5,444 c a l / g Scenedesmus o b l i q u u s 5,507 c a l / g . Of the three a l g a l s p e c i e s mentioned above, only Scenedesmus can be grown s u c c e s s f u l l y i n the l a b o r a t o r y using cow-shed manure. Scenedesmus i s commonly found i n freshwaters and s o i l s but i t can a l s o withstand domestic sewage p o l l u t i o n ( T r a i n o r et a l . , 1976) . Because of i t s simple n u t r i t i o n a l requirements and r a p i d growth r a t e s , i t can be e a s i l y maintained i n the l a b o r a t o r y . Members of t h i s genus are coenobic and occur i n the plankton, among benthic algae i n q u i e t bodies of water. The c y l i n d r i c a l •6. c e l l s , with rounded or p o i n t e d ends are l a t e r a l l y j o i n e d i n groups of 4, 8 or 16. The t e r m i n a l c e l l s and some of the other c e l l s , i n some s p e c i e s have s p i n e s . Reproduction i s by autocolony formation i n which each p a r e n t a l c e l l forms a m i n i a t u r e colony, that i s l i b e r a t e d through a tear i n the p a r e n t a l w a l l (Bold and Wynee, 1978). The genus Scenedesmus i s composed of two groups, the " o b l i q u u s " or non-spiny and the spiny group. The " o b l i q u u s " group i s c h a r a c t e r i z e d by the f o l l o w i n g : • (1) c e l l s f u s i f o r m (2) no ornamental p e c t i c l a y e r surrounding colony (3) u n i c e l l s form by fragmentation of c o l o n i e s as they age (4) c e l l s may be j o i n e d end.to end i n a Dactylococcus stage (5) may reproduce s e x u a l l y by f u s i o n of b i f l a g e l l a t e d segments ( T r a i n o r et a l . , 1976). Dimentman et a l . ( l 9 7 5 ) observed that Scenedesmus . o b l i q u u s was the absolute dominant sewage al g a i n the shallow treatment ponds near Jerusalem and H a i f a ( I s r a e l ) . T h i s sewage grown a l g a allowed a c o n s i d e r a b l e growth r a t e of f i v e s p e c i es of f a i r y shrimps anostracans s t u d i e d and hastened maturation and egg p r o d u c t i o n . D i f f e r e n t s p e c i e s of Scenedesmus were a l s o shown to be compatible in growing daphnids (D'Agostino and P r o v a s o l i , 1970; Lampert, 1976; Rees and O l d f a t h e r , 1980). P r o v a s o l i and P i n t n e r (19 ) l i s t the p r e - r e q u i s i t e s and v e r s a t i l e media for p h o t o - a u t o t r o p h i c a l g a e : (a) t o t a l - s o l i d s c o n c e n t r a t i o n s (b) c o n c e n t r a t i o n of major elements to s u i t the p r e v a l e n t ions r e q u i r e d 7 (c) adequate sources of N, and growth f a c t o r s (d) sources of P, and avoidance of p r e c i p i t a t e s i n a l k a l i n e pH's (e) pH b u f f e r i n g ( f ) t r a c e metal b u f f e r i n g . Phosphates o f t e n become t o x i c above 5 - 2 0 mg% except f o r organisms l i v i n g i n p o l l u t e d waters and ammonia tends to become t o x i c above 3 - 5 mg% i n a l k a l i n e media except f o r Eurobionts l i v i n g i n p o l l u t e d waters. Most algae u t i l i z e n i t r a t e s although Scenedesmus tends to . show a s,light p reference f o r NH^over NO^ (Krauss, 1958 ) . In the 1960's, r e s e a r c h e r s began e x p l o r i n g the p o s s i b i l i t y of c u l t u r i n g algae on a mass s c a l e (Davis et. a l . , 1961; Casey et a l . , 1963; Loosanoff and Davis, 1963; Ukeles, 1965). These were d i r e c t e d most e s p e c i a l l y f o r a q u a c u l t u r a l purposes. Three types of mass c u l t u r e were i d e n t i f i e d : batch, semi-continuous and continuous (Wisely and Purday, 1961; Monod, 1950; Herbert, 1958, 1961). Goldman (1978) has i d e n t i f i e d the problems r e l a t i n g s p e c i f i c a l l y to mass c u l t u r e of a l g a e : (1) c u l t u r e mixing (2) n u t r i e n t a v a i l a b i l i t y and a d d i t i o n (3) s p e c i e s c o n t r o l (4) CO^addition (5) water supply and e v a p o r a t i o n (6) a l g a l s e p a r a t i o n and h a r v e s t i n g . 8 Daphnia as a P o t e n t i a l F i s h - f o o d in Aquaculture Daphnia pulex has a c a l o r i c content of 5,350 c a l gm dry weight (Richman, 1958). Of the t o t a l c a l o r i e s , about 21 - 27% i s carbohydrate, 4 - 20% i s fat. and 47% i s p r o t e i n . A comparison'is made between the composition of a t y p i c a l dry t r o u t r a t i o n and Daphnia: Proximate Composition Composition (%Dry Matter) of Daphnia pulex (Yurokowski and Tobachek, 1978) Dry Trout Ration (Cho et a l . , 1 974) P r o t e i n Crude Fat F i b e r Gross Energy ( U t i l i z a b l e energy) 47.7 10.3 2.3 5.0 49.7 16.3 6.9 3.6 The f i g u r e s above c l e a r l y suggest that a Daphnia d i e t can meet the requirements of a f i s h r a t i o n . Daphnia i n h a b i t temporary p o o l s , small ponds and l a k e s . They are common aq u a t i c c r u s t a c e a n s which feed upon b a c t e r i a , c e r t a i n u n i c e l l u l a r green a l g a e , and a mixture of v a r i o u s protozoa and protophytes from the sediments of ponds (Banta, 1921). In some major l a k e s , the major p o r t i o n of t h e i r food c o n s i s t s of d e t r i t u s and b a c t e r i a , r a t h e r than l i v i n g algae (Pennak, 1946). 9 Daphnia e x h i b i t both sexual, and parthenogenic ( d i p l o i d ) r e p r o d u c t i o n . During f a v o u r a b l e c o n d i t i o n s Daphnia reproduces by parthenogenesis. The eggs are l i b e r a t e d a few hours before the mother moults. The young produced i n t h i s way are a l l females which mature and reproduce in the same manner. The. number of young produced i n c r e a s e s to a peak, t h e r e a f t e r d e c l i n e s with age (Anderson et a l . , 1937; Anderson and Jenkins, 1942; Frank et a l . , 1957; Richman, 1958). A parthenogenic female produce as high as 30 - 40 new Daphnia every two days when c o n d i t i o n s are optimum (Dinges, 1974). A maximum of 70 parthenogenic eggs per brood was even r e p o r t e d by Daborn et a l . ( l 9 7 8 ) of very l a r g e female Daphnia (4.4mm TL) i n aerated, sewage treatment ponds (Nova S c o t i a , Canada). However, when c o n d i t i o n s become unfavourable males appear among the o f f s p r i n g and sexual eggs are produced which r e q u i r e f e r t i l i z a t i o n . Two eggs are enclosed i n a dense, durable s t r u c t u r e c a l l e d the ephippium (Green, 1955). Females which produce e p h i p p i a may r e t u r n to parthenogenetic r e p r o d u c t i o n when c o n d i t i o n s r e t u r n to normal. The environmental s t i m u l i a s s o c i a t e d with the r e v e r s a l of parthenogenesis to sexual r e p r o d u c t i o n are the f o l l o w i n g : d e n s i t y of c u l t u r e , e v a p o r a t i o n of h a b i t a t , s t a r v a t i o n , high and low temperatures, d i e t , metabolic depresants and photoperiodism ( S t r o s s , 1965). A newly-born Daphnia i s about 0.667 mm i n t o t a l l e n g t h . The number of p r e - a d u l t i n s t a r s , the number of i n s t a r s e l a p s i n g between the time of r e l e a s e of the i n d i v i d u a l female from the brood chamber of her mother and the appearance of eggs i n i t s own brood chamber, i s u s u a l l y from 4 - 6 depending on 10 environmental c o n d i t i o n s . Adult females range from 1.5 - 3.5 mm in t o t a l l e n g t h . The r a t e of growth of Daphnia, the age at sexual m a t u r i t y , the s i z e of the broods, the i n t e r v a l between broods and the age at death are i n f l u e n c e d by food c o n c e n t r a t i o n , temperature, crowding and other e x t e r n a l c o n d i t i o n s . Food i s necessary f o r the growth of Daphnia. Semi-starved animals do not grow as q u i c k l y as w e l l - f e d animals. S t a r v a t i o n decreases growth i n two ways: i t i n c r e a s e s the d u r a t i o n of the i n s t a r s and reduces the increment at each moult (Ingle et a l . , 1937; Dunham, 1938). Daphnia are f i l t e r - f e e d e r s . Food p a r t i c l e s c o l l e c t e d on the f i l t e r i n g setae are swept i n t o the food groove by t u f t s of l a t e r a l l y i n c l i n e d s e t u l e s l o c a t e d on the second, t h i r d , and f o u r t h t h o r a c i c appendages. In the food groove i t moves forward to the m a x i l l u l e s and mandibles which pass i t i n t o the o r a l c a v i t y where i t c o l l e c t s u n t i l a p e r i s t a l t i c wave of the oesophagus c a r r i e s i t i n t o the midgut (McMahon and R i g l e r , 1963). Temperature a f f e c t s the frequency of moulting and hence the frequency with which young are produced. Reproduction occurs every 2.0 days at 25°C; every 2.6 days at 20°C; and every 8.0 days at 11°C ( H a l l , 1964). An i n c r e a s e i n temperature, up to a s u b - l e t h a l l e v e l , i n c r e a s e s the i n i t i a l growth r a t e by sho r t e n i n g the d u r a t i o n of i n s t a r s (MacArthur and B a i l l i e , 1929; Green, 1955). The l o n g e v i t y i n Daphnia magna v a r i e d as an i n v e r s e f u n c t i o n of temperature between 8°C and 28°C (MacArthur and B a i l l i e , 1929). H a l l (1964) r e p o r t e d that the median l i f e s p a n of D.galeata mendotae i s 30 days at 25°C; 60 - 80 days 11 at 20 C; and about 150 days at 11°C. Temperature a l s o a f f e c t s the f i l t e r i n g and metabolic r a t e s of Daphnia. F i l t e r i n g r a t e and maximum feeding r a t e of D.magna both continued to i n c r e a s e as temperature i n c r e a s e d to 24°C, but above t h i s temperature, the rate r a p i d l y decreased (McMahon, 1965). Increase i n temperature i n c r e a s e d metabolic r a t e s such as heartbeat rate and C 0 2 p r o d u c t i o n (MacArthur and B a i l l i e , 1929). Brown and C r o z i e r (1927) r e p o r t e d b e t t e r Daphnia c u l t u r e s i n terms of s u r v i v a l r a t e at room temperature (21°C) than i n any other temperatures. A l l of these f i n d i n g s suggest that the best o p e r a t i n g c o n d i t i o n s for the c u l t u r e of D.pulex i s i n the range 20 - 24°C. St u d i e s show that photoperiodism a f f e c t s the growth of Daphnia. S t r o s s (1965) found that short day photoperiods (12:12, 13:11, 14:10 vs. 16:8LD) induced sexual r e p r o d u c t i o n . On the other hand, Gordon (1975) using two photoperiods (18:6 and 16:8LD) found photoperiod to have a l i t t l e e f f e c t on Daphnia biomass p r o d u c t i o n with 16:8LD a c h i e v i n g s l i g h t l y more biomass. There i s a l i m i t to how many Daphnia a given volume of water can h o l d depending on the environmental c o n d i t i o n s . Increased d e n s i t y i s accompanied, over a wide range, by decreased b i r t h r a t e and lowered growth r a t e (Frank et a l . , 1957; Frank, 1960; Smith, 1963; Mace, 1975). Increased crowding of young lengthens the immature p e r i o d , whereas decreased crowding of a d u l t s i n c r e a s e s the brood s i z e . The amount of 0^present i n the water i s a l s o important i n the c u l t u r e of Daphnia. The r a t e of O gconsumption per animal i n c r e a s e s with body s i z e but on a u n i t weight b a s i s , the rate of 0^uptake i s higher i n the smaller animals. Animals l a r g e r than 12 1.00 mm show a r e l a t i v e l y constant r a t e of consumption per u n i t weight, the mean being 7.21 ul«mg hr (Richman, 1958). 0 -1 l e v e l s of 3 mg-1 i s c o n s i d e r e d to be the lower l i m i t i n D.pulex c u l t u r e s (Richman, 1 9 5 8 ; I v l e v a , 1973; K r i n g and O'Brien, 1976). Below these l e v e l s the f i l t e r i n g r a t e s of Daphnia are Og _ dependent. D.pulex o f t e n possesses c o n s i d e r a b l e haemoglobin which i s an adaption to enable i t to withstand low Ogconcentration (Fox, 1948, 1951; Kri n g and O'Brien, 1976). Animals that have prolonged, exposure to low 0^ c o n c e n t r a t i o n s respond by f a c u l t a t i v e l y i n c r e a s i n g t h e i r haemoglobin l e v e l s . T h i s enhances such f u n c t i o n s as f i l t e r i n g r a t e s , egg p r o d u c t i o n , g e n e r a l a c t i v i t y and s u r v i v o r s h i p of Daphnia at low Og c o n c e n t r a t i o n . D.magna can s u r v i v e at a pH range of 5.4 to 9.5 (Klugh and M i l l e r , 1926). However, most i n v e s t i g a t o r s working on Daphnia c u l t u r e found that these animals g e n e r a l l y do very w e l l i n a s l i g h t l y a l k a l i n e medium with a pH of somewhere between 7.6 to 8.6 (Klugh and M i l l e r , 1926; Viehoever, 1935; I v l e v a , 1973; C o n k l i n and P r o v a s o l i , 1978). Present S t a t e - o f - t h e - A r t on Daphnia C u l t u r e For y e a r s , c l adocerans have shown c o n s i d e r a b l e merit as t e s t animals f o r s t u d i e s i n p o p u l a t i o n behaviour ( P r a t t , 1943; Frank, 1952, 1957; Slobodkin, 1954). They are easy to c u l t u r e , t h e i r l i f e c y c l e i s s h o r t , they do not have f r e e egg or l a r v a l stages and, they produce homogeneous p o p u l a t i o n s through 13 parthenogenic r e p r o d u c t i o n . General l a b o r a t o r y methods f o r c u l t i v a t i n g daphnids have evolved from the " s t a b l e t e a " of Banta (1939) to the d e f i n e d media of Murphy (1970) and D'Agostino and P r o v a s o l i (1970). In the l a b o r a t o r y (Bio-Resource E n g i n e e r i n g Department, U.B.C.), stock c u l t u r e s of Daphnia have been maintained using r e c o n d i t i o n e d pond and d i s t i l l e d water c o n t a i n i n g d a i r y manure/ and some green a l g a l s p e c i e s as food. Most of the s t u d i e s done so f a r on Daphnia were b a s i c experiments. To date, only a few s t u d i e s have been focussed on the mass c u l t u r e of Daphnia f o r i t s p o s s i b l e a p p l i c a t i o n i n aquaculture (Bogatova and Askerov, 1958; Dewitt and Candland, 1971; Dinges, 1974, 1976; Rees and O l d f a t h e r , 1980). A l l l a r g e s c a l e Daphnia s t u d i e s were based on the u t i l i z a t i o n of Daphnia f o r the improvement of wastewater e f f l u e n t . Dewitt and Candland (1971) r e p o r t e d a commercial Daphnia harvest of 40 tons i n C a l i f o r n i a s t a b i l i z a t i o n ponds, with one pond y i e l d i n g 25 tons -z -1 at a rate of 1.5 tons per acre per day(0.0406 kg. m day ). Bogatova and Askerov (1958), mass c u l t u r i n g Daphnia i n concrete tanks on media i n c l u d i n g yeast and f e r t i l i z e r , achieved a s u b s t a n t i a l y i e l d of 76.7 l b s per acre foot per•day(0.0854 kg-m" day"1) . Ik MATERIALS AND METHODS Algae Scenedesmus o b i i q u u s , a major contaminant of a l g a l c u l t u r e s in the l a b o r a t o r y (Bio-Resource Eng. Dept., U.B.C), was i s o l a t e d i n June, 1980. The a l g a was grown and maintained under noh-axenic c o n d i t i o n s i n 25-ml Erlenmeyer 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 darkness (16:8LD) at 20° C. The a l g a was then t r a n s f e r r e d to 20.0-ml Erlenmeyer f l a s k s where they were maintained as " s t a r t e r " c u l t u r e s . These were used f o r i n o c u l a t i n g ' c u l t u r e s i n the experiments. Daphnia pulex The freshwater c l a d o c e r a n , Daphnia pulex F., was obtained from the C i v i l E n g i n e e r i n g Laboratory ( U . B . C ) , and was o r i g i n a l l y c o l l e c t e d from Deer Lake (Burnaby, B.C.) i n J u l y , 1978. Stock c u l t u r e s of Daphnia pulex were maintained i n 20-1 a q u a r i a . The c u l t u r e medium was r e c o n d i t i o n e d pond water mixed with d i s t i l l e d water. Digested d a i r y manure and some green a l g a l s p e c i e s were added as food from time to time. These c u l t u r e s were kept at l a b o r a t o r y c o n d i t i o n s at 2 0 i 2 ° C and a photoperiod of 16:8LD. S l i g h t a e r a t i o n was p r o v i d e d to keep the -X d i s s o l v e d oxygen above the minimum l i m i t ( 3.0 mg-O^l ). 15 Media P r e p a r a t i o n A l g a l C u l t u r e Medium: Fresh d a i r y manure was c o l l e c t e d from the U n i v e r s i t y of B r i t i s h Columbia Dairy Barn. Raw wastes were c o l l e c t e d i n 25-1 p l a s t i c p a i l s and immediately taken to the l a b o r a t o r y . Two kilograms of manure was p l a c e d i n each of three 50-1 p l a s t i c garbage buckets c o n t a i n i n g 20 l i t e r s of d e c h l o r i n a t e d water. They were then aerated v i g o r o u s l y with the use of a i r s t o n e s to promote a e r o b i c d i g e s t i o n of the wastes. L i d s were provided to prevent any growth of photo-autotrophic organisms. A f t e r 10 days, the supernatant was f i l t e r e d through 9-cm g l a s s f i b e r f i l t e r s (Reeve A n g e l ) . The l i q u i d was then p l a c e d i n p l a s t i c bags and s t o r e d i n the f r e e z e r at -10°C. The samples were analyzed f o r t o t a l K j e l d a h l n i t r o g e n (TKN), ammonia-nitrogen, n i t r a t e - n i t r o g e n , n i t r i t e - n i t r o g e n , ortho-phosphate and t o t a l phosphates by c o l o r i m e t r i c methods a f t e r two days of f r e e z i n g . The r a t i o between d i g e s t e d d a i r y waste and d i s t i l l e d water volume in the medium 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 , but on the average the r a t i o was ca 10 - 15% wastes and 85 - 90% d i s t i l l e d water. The -1 n i t r o g e n c o n c e n t r a t i o n used v a r i e d from 200 to 2,000 ug-at-N«l . Daphnia C u l t u r e Medium: Water was c o l l e c t e d from a U.B.C pond l o c a t e d i n f r o n t of the P h y s i c s Department. T h i s was f i l t e r e d using 9-cm g l a s s f i b e r f i l t e r s . T h i s was then mixed with d i s t i l l e d water and used f o r growing Daphnia. In the Daphnia experiments, r e c o n d i t i o n e d water from the 16 stock cultures.was used. T h i s was a l s o f i l t e r e d using g l a s s f i b e r f i l t e r s . The green a l g a , Scenedesmus o b i i q u u s , was su p p l i e d as food for Daphnia. . D e s c r i p t i o n of C u l t u r e U n i t s  A l g a l C u l t u r e U n i t : To p r o v i d e a uniform temperature to the a l g a l c u l t u r e s , a b i g water bath c o n t a i n e r (102cm x 64cm x 10cm) was c o n s t r u c t e d out of P l e x i g l a s . A Temporite Package water c h i l l e r (TR4-20 3/4HP 20GPH) maintained the water temperature at 20°C. A l i q u i d c i r c u l a t i n g pump (Model D-6 E a s t e r n , 3 1/2GPM at 0 pressure) r e c i r c u l a t e d the water from the c u l t u r e s to the c h i l l e r and back to the c u l t u r e s . A g e n e r a l i z e d view of the whole system i s shown i n F i g u r e 1. A l l c u l t u r e s were arranged i n a blocked randomized design f a s h i o n . The a l g a l experiments were conducted using 1 - l i t e r f l a t bottom b o i l i n g f l a s k s (Experiments #1 - #6, #9) and 1-gal j a r s (Experiments #7 and #8). Six VHO (Daylight 48" Sylvannia) f l u o r e s c e n t lamps provided i l l u m i n a t i o n f o r the growth of algae. -1 -2 -1 An i n i t i a l l i g h t i n t e n s i t y of ca 0.03 ly-min (113 uE.m sec ) was used. On the s i x t h day of growth when the c u l t u r e s got t h i c k e r , - 1 the l i g h t i n t e n s i t y was i n c r e a s e d to ca 0.05 ly^min (161 uE-m sec~ L) by lowering the l i g h t source. I l l u m i n a t i o n was measured by Quantum/Radiometer/Photometer Sensors (Lambda LI-185). A timer r e g u l a t e d the day and night c y c l e (16:8LD) of the c u l t u r e s . No a e r a t i o n was used i n Experiments #1 t h r u #3. The f l a s k s were f i t t e d with Neoprene rubber stoppers (No.8). A e r a t i o n was 18 i n c o r p o r a t e d beginning with Experiment #4. A i r was f i r s t passed through 1N H ^ SO^ . s o l u t i o n to dry i t and to get r i d of contaminants {e.g.NHo, i n a i r ) and washed with d i s t i l l e d water before being d i s t r i b u t e d i n t o the c u l t u r e s . P l a s t i c tubing (Tygon R-3'603 0.476cm x 0.635cm x 0.140cm) f i t t e d to g l a s s tubing (0.5cm OD) connected the c u l t u r e f l a s k s , to the a e r a t i o n system. Glass tubing o u t l e t s were a l s o provided to prevent a i r pressure b u i l d - u p . Neoprene rubber stoppers (No.8) supported the g l a s s t u b i n g . Daphnia C u l t u r e U n i t : Daphnia pulex was c u l t u r e d i n 50cm x 30cm x 15cm aquaria of 20-1 c a p a c i t y . The c o n t a i n e r s were made shallow because shallow c u l t u r e s tend to favour Daphnia growth (Viehover, 1935). T h i s tendency was a l s o observed i n the l a b o r a t o r y . The experiments were conducted i n s i d e a C o n t r o l l e d Environment (CONVIRON) chamber. Temperature was made constant at 20° C and a photoperiod of 16:8LD was maintained throughout the experiments. L i g h t i n t e n s i t y was ca 0.01 ly-miri" i(50 uE-m^sec" 1). No a e r a t i o n was p r o v i d e d . However, the d i s s o l v e d oxygen and pH were checked r e g u l a r l y using an Oxygen meter (Model 54 YSI) and a pH/Ion meter ( F i s h e r Model 420 D i g i t a l ) . C u l t u r e Methods A l g a l C u l t u r e Methods: None of 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 , however, there was no contamination by other a l g a e . The d e n s i t y of the inoculum was determined before i t was 19 i n t r o d u c e d to the c u l t u r e u n i t s . I n i t i a l i n o c u l a t i o n s averaged ca 5,000 - 10,000 cells«ml . Two to three g e n e r a t i o n s were allowed f o r the algae to a c c l i m a t i z e to the new c u l t u r e c o n d i t i o n s . The a l g a l c e l l s were then counted d a i l y using a haemacytometer (American O p t i c a l C o r p o r a t i o n ) . F i v e squares on the g r i d of each chamber were counted and the average count, Q, 4 . was m u l t i p l i e d by 10 . Thus, given the average Q, t h e . d e n s i t y ( c e l l s ml..-), d, of the suspension i n the haemacytometer was c a l c u l a t e d from the e x p r e s s i o n , d = I0 4x Q. Four r e p l i c a t e counts were made and the mean value , c a l c u l a t e d . When the c u l t u r e s reached t h e i r peak c o n c e n t r a t i o n and s t a r t e d to l e v e l - o f f , the experiment . was terminated. For Experiment #5, #6 and #8, the c u l t u r e medium was f i l t e r e d twice using g l a s s f i b e r f i l t e r s and then a n l a l y z e d f o r n u t r i e n t s remaining or n u t r i e n t s not consumed by the a l g a e . Daphnia C u l t u r e Method: Before the experiments were conducted, stock c u l t u r e s of Daphnia were maintained f o r s e v e r a l g enerations at the experimental c o n d i t i o n s to account f o r e f f e c t s of a c c l i m a t i o n . In Experiment #1, 1,000 Daphnia pulex were randomly taken from a homogeneously mixed stock c u l t u r e and p l a c e d i n each of three a q u a r i a c o n t a i n i n g 10 l i t e r s of r e c o n d i t i o n e d pond water. The i n i t i a l d e n s i t y was one Daphnia per ml of water. The green a l g a , Scenedesmus o b l i q u u s , grown from d a i r y manure medium, was used f o r f e e d i n g the Daphnia i n the experiments. C e l l -1 c o n c e n t r a t i o n s of 50,000, 100,000 and 150,000 cells«ml were 20 maintained once a day i n each aquarium, r e s p e c t i v e l y . T h i s was done by f i r s t making c e l l counts of the a l g a l feed and the. c u l t u r e tanks before f e e d i n g , and then c a l c u l a t i n g the volume of feed needed. Only a l g a l c e l l s in t h e i r , l o g a r i t h m i c phase of growth were used as was suggested by Ryther (1954). B a c t e r i a and d e t r i t u s a l s o developed in the tanks and undoubtedly c o n t r i b u t e d to the food supply, but at a l l times the algae represented the major food source. The i n i t i a l Daphnia biomass was determined and the increase in biomass and t o t a l number of Daphnia were noted every three days u n t i l peak l e v e l s were reached. F i f t y Daphn i a were randomly c o l l e c t e d from each aquarium. Each animal was placed on a s l i d e and i t s t o t a l l e n g t h measured with an o c u l a r micrometer. They were then d i v i d e d i n t o three s i z e c l a s s e s : 0.60 - 1.70 mm, 1.71 - 2.30 mm and>2.30 mm. They were placed on weighed 4-cm aluminum f o i l s t r i p s , d r i e d f o r 24 hours at 60 C, c o o l e d i n a d e s i c c a t o r and weighed immediately with a CAHN-21 Automatic E l e c t r o b a l a n c e . G r a v i d females were noted r e c o r d i n g the number of eggs and embryos. The procedure above was repeated f o r Experiments #2 and #3. However, i n Experiments #4 and #5, only one feeding l e v e l was -1 used, 100,000 c e l l s * ml . T h i s time food was monitored two to three times a day. More a t t e n t i o n was given to the p h y s i c o -chemical c o n d i t i o n s of the water. D i s s o l v e d oxygen and pH l e v e l s were monitored d a i l y . Accumulation of ammonia was a l s o determmined d a i l y with the use of a Technicon Auto-Analyzer I I . 21 Chemical 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 followed was an adaption of Wall et a l . (1974) and that i n the Technicon Auto-Analyzer Manual (1971b). F i v e mis of the sample ( d a i r y manure medium) were introduced i n t o a 50 ml d i g e s t i o n tube. To enhance the o x i d a t i o n - r e d u c t i o n , 0.5 g of a d i g e s t i o n c a t a l y s t (composed of 960g K^SO^, 35g CuSO^and 5g SeOg) were added to each tube. The tubes were then placed i n a d i g e s t o r rack at 3 6 0 0 C f o r 6 - 12 hours. B o i l i n g c hips were added to prevent e x c e s s i v e bumping during d i g e s t i o n and a small g l a s s funnel was p l a c e d on the open end of the tubes to prevent s p i l l a g e and exc 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 tubes were removed from the d i g e s t o r and allowed to c o o l f o r one to two hours. Each tube was d i l u t e d to 50 ml/' with d i s t i l l e d water. Approximately 10 ml of 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 of the Auto-Analyzer f o r T o t a l K j e l d a h l N i t r o g e n d e t e r m i n a t i o n . Whenever the a u t o - a n a l y s i s g r a p h i c a l r e corder went o f f - s c a l e , the sample was r e d i l u t e d and again run on the Auto-Analyzer. A m m o n i a / N i t r a t e / N i t r i t e : The automated procedure f o r the de t e r m i n a t i o n of the above nitrogeneous compounds was done through c o l o r i m e t r i c methods and adapted to that of Technicon Auto-Analyzer II Manual (1969, 1971a). 22 T o t a l Phosphate: Samples were d i g e s t e d f o l l o w i n g the same procedure as with the d e t e r m i n a t i o n of T o t a l K j e l d a h l N i t r o g e n . The automated procedure f o r the dete r m i n a t i o n of t o t a l phosphate was done through c o l o r i m e t r i c methods and adapted to that of Technicon Auto-Analyzer II Manual (1972). Ortho-Phospahte: The automated procedure f o r the dete r m i n a t i o n of o r t h o -phosphate was done through c o l o r i m e t r i c methods and adapted to that of Technicon Auto-Analyzer II Manual (1973)., A l g a l C e l l Weight The weight per a l g a l c e l l was determined by d r y i n g and weighing a known volume of a l g a l suspension of known c e l l c o n c e n t r a t i o n . Two hundred ml of a l g a l suspension was f i l t e r e d on a pre-weighed g l a s s f i b e r f i l t e r which was then p l a c e d i n a c r u i c i b l e , d r i e d at 60°C f o r 30 minutes and weighed on a M e t t l e r H6 D i g i t a l Balance. T h i s was done i n t r i p l i c a t e s . The weight per a l g a l c e l l was c a l c u l a t e d from the f o l l o w i n g equation: . , (Dry wt. F i l t e r + a l g a e ) - ( D r y wt. F i l t e r ) ( m g ) a l g a l c e l l weight = — ( m g . c e l l - : r ) (200 ml) • ( C e l l cone, c e l l s . m l ) Four a d d i t i o n a l t r i a l s were conducted using d i f f e r e n t a l g a l c u l t u r e s . 23 Growth Rate Experiments (Daphnia) The growth rate of Daphnia was determined at three d i f f e r e n t food l e v e l s : 50,000, 100,000 and 150,000 Scenedesmus -1 c e l l s - ml . G r a v i d female Daphnia were p l a c e d i n a 2-1 Erlenmeyer f l a s k and s u p p l i e d with excess food. The f o l l o w i n g day, the newly-born young were c o l l e c t e d and used i n the experiment. Two h u n d r e d - f i f t y Daphnia each were placed i n three 1-1 Erlenmeyer f l a s k s c o n t a i n i n g 500 mlf1 of r e c o n d i t i o n e d pond water from p r e v i o u s Daphnia c u l t u r e s . Food was adjusted to the d e s i r e d c o n c e n t r a t i o n and monitored once a day. The experiment was conducted i n s i d e the CONVIRON at 20°C, a photoperiod of -1 2 - 1 16:8LD, and a l i g h t i n t e n s i t y of ca 0.01 ly.min (50 uE«m sec ). The i n i t i a l l e n g t h (TL) was measured using an o c u l a r micrometer. Ten Daphnia were sampled from each f l a s k and measured d a i l y u n t i l Day 10. The appearance of the f i r s t brood and the number of progeny were noted down. A second t r i a l was repeated. T h i s time, food was monitored twice a day. Length-Weight R e l a t i o n s h i p A length-weight r e l a t i o n s h i p on Daphnia was performed. L i v e animals were removed from the experimental f l a s k s by means of a 10-ml p i p e t t e , p l a c e d on a s l i d e and measured with an o c u l a r micrometer. They were then p l a c e d on weighed 4-cm' aluminum f o i l s t r i p s , d r i e d f o r 24 hours at 60°C, c o o l e d i n a d e s i c c a t o r and weighed immediately. Newly-borns, j u v e n i l e s and a d u l t s with and without eggs and embryos were measured. 25 RESULTS Dairy Waste A n a l y s i s Six d a i r y manure media were used d u r i n g the course of the. experiments. Table 1 below summarizes the v a r i o u s chemical composition (TKN, NH^ -N, NOs-N, NOg-N, t o t a l PCfy-and PO^-P) of each medium and the a l g a l experiments i n which they were used. Table 1. Summary of chemical composition of each medium and the a l g a l experiment i n which they used. Media* TKN NH^-N N0 3-N N0 2-N PO^-P T o t a l PO^ A l g a l ug-at.N-l" 1 Expt.# I 9. 5x1 03 1 .7x10 2. 1 4x 1 0 1 . 9x 1 0 1 1 3 . 6x1 0 - 1 II 2. 6x1 1 .2x10* 3. 4x1 0* 2 .7xl0 a 5 1 .1x10 - 2 III 3. 1x1 0 3 1 .3x1 0 2. 1 X 1 0 3 1 .5x1 0^  3 % .6x10 - 3, 4 IV 4 . 8x1 0* 1 .2X10 3 3. 4x1 0Z 4 .3x1 0Z 5 Z . 0x1 0 1 . 2x1 0 2 5, 6 V 6. 1x10 2 . 6x 1 0^  3. 3x1 0 1 1 . 3x1 02 1 z .7x1 0 2 .2x10 8 7 VI 7. 0x1 Q 3 . 1 x 1 0 3 4. 2x1 0 1 1 .4x1 0* 1 .5x1 0Z 1 .6X10 2 8, 9 Note that the chemical composition v a r i e d from one medium to the oth e r . Although the d a i r y wastes were c o l l e c t e d from the same source, the r a t e of manure d i g e s t i o n c o u l d have been d i f f e r e n t each time. 26 A l g a l Experiments A summary of a l l the a l g a l experiments, the d i f f e r e n t treatments and the r e s u l t s i s shown i n Table 2. A l g a l Experiment #1 was conducted to determine the growth of Scenedesmus ob i i q u u s with i n c r e a s i n g n i t r o g e n c o n c e n t r a t i o n s . S t a t i s t i c a l a n a l y s i s using a n a l y s i s of v a r i a n c e (ANOVA) showed s i g n i f i c a n t d i f f e r e n c e s between treatments (Appendix 11a). The s t a t i s t i c a l a n a l y s i s was done through the U.B.C. Computer System using a General Least Squares A n a l y s i s of Variance Programme known as GENLIN (Greig and B j e r r i n g , 1977). Maximum c e l l y i e l d s i n c r e a s e d as n i t r o g e n c o n c e n t r a t i o n i n the medium in c r e a s e d . S t a t i s t i c a l a n a l y s i s showed s i g n i f i c a n t d i f f e r e n c e s between treatments (Appendix 11b). D a i l y c e l l d e n s i t i e s are p l o t t e d i n F i g u r e 2. In A l g a l Experiments- #2, #3 and #4, the growth of Scenedesmus was 'again determined at f i v e d i f f e r e n t n i t r o g e n c o n c e n t r a t i o n s (400, 800, 1,200., 1,600 and 2,000 ug-at • N- l " 1 ) . S t a t i s t i c a l a n a l y s i s showed s i g n i f i c a n t d i f f e r e n c e s between treatments f o r both growth r a t e s and c e l l y i e l d s i n Experiment #2 (Appendices 12a and 12b). Experiment #3 showed s i m i l a r r e s u l t s . S t a t i s t i c a l a n a l y s i s showed s i g n i f i c a n t d i f f e r e n c e s between treatments f o r both growth r a t e s and c e l l y i e l d s (Appendices 13a and 13b). A e r a t i o n enhanced both growth r a t e s and c e l l y i e l d s i n Experiment #4. S t a t i s t i c a l a n a l y s i s showed s i g n i f i c a n t d i f f e r e n c e s between treatments f o r both growth r a t e s and c e l l y i e l d s (Appendices 14a and 14b). I Table 2 . Summary of the a l g a l experiments, the d i f f e r e n t treatments and the r e s u l t s . Algal TKN NH4-N N03-N N02-N , P04-P Total P04 Aeration pH K C e l l Y i e l d Expt. ug-at'W" 1 ug-at'W ug-at'Ml uj-at'W" ug-at-P-l ug-at-P-r-1 (dlv.day") (maximum) 2.0XI0 2 3.5x10 S.OxlO"1 3 . 6 X 1 0 " 1 3.5 - 7.72 0.88±0.99 0.99x10* 4.0 7.0 , 1.0x10° 7.2 7.0 - 8.35 1.18+0.03 1.42 #1 8.0 ^ 1.4x10* 2.0 1.4x10° 1.4x10 - without 8.51 1.31+0.14 1.44 1.6X10 2.8 4.0 2.9 2.8 - 8.95 0.94+0.01 1.72 3.2 5.6 8.0 5.8 5.6 - 9.20 0.61+0.01 2.07 4.0x1O& 1.8x10 5.2x10 4.1x10 7.7 - 8.65 1.21+0.03 0.82X10 6 8.0 , 3.6 I.OxlO 2 8.2 1.5x10 - 8.71 1.10+0.03 1.36 #2 1.2x10* 5.4 1.6 1.2x10 2.3 - without 8.69 1.00+0.05 1.59 t.6 7.2 2.1 1.6 3.1 - 8.65 0.95+0.03 2.61 2.0 9.0 2.6 2.0 3.9 - 8.63 0.8810.06(1.35+0.07) 2.75 4.0x10* 1.6x10 2.6x10 Z 1.9x10 2.1x10 - 8.84 1.22*0.04 1 . 3 8 X 1 0 6 8.0 3.2 5.2 3.8 4.2 - 8.83 1.13+0.04 1.33 #3 1.2x10* 4.8 7.8 5.7 6.3 - without 8.84 0.9910.07 1.13 1.6 6.4 I.OxlO 3 7.6 8.4 4 - 8.79 0.9710.04 1.23 2 . 0 8.0 1.3 9.5 1.1x10 - 8.84 0.8310.04(1.18+0.03) 2.00 4.0x10* 1.6x10 2.6xl0 2 1.9x10 2.1x10 - 9.12 1.90+0.09 S.01X10 6 8 . 0 3.2 5.2 3.8 4.2 - 9.16 1.27+0.03 7.91 #4 1.2x10* 4.8 7.8 5.7 6.3 - with 9.10 0.98+0.02(1.14+0.03 11.11 1.6 6.4 1.0x10' 7.6 8.4 , - 9.04 0.84+0.02(0.93+0.04) 12.79 2.0 8.0 1.3 9.5 1.1x10 - 9.O0 0.75+0.03(0.92+0.03) 15.28 2 . 0 X 1 0 2 4.9x10 I.SxIO 2 1.8x10 1.9 5.2 7.66 0.98+0.09 1.80x106 *S 4.0 9.8 „ 2.6 3.6 3.8 1.0x10 with 8.27 0.98+0.04(1.24+0.04) 4.42 8.0 2.0x10 5.2 , 7.2 7.6 2.1 8.81 0.9810.04(1.15+0.09) 7.15 1.6x10* 3.9 1.0x10* l^xlO"* 1.5X1CT 4.2 8.87 1.01*0.04 9.69 2.0x10* 3.8x10^ 1.3x102 1.8x10 2.9 5.2 7.55 1.75+0.07 2.30X106 *6 4.0 7.6 2.6 3.6 5.8 1.0x10 with 8.32 1.7410.07 4.89 8.0 „ 1.5x10° 5.2 7.2 , 1.2x10 2.1 8.86 1.7410.02 7.47 1.6x10 3.0 1.0x10' 1.4x10 2.3 4.2 9.00 1.30+0.06 11.68 #7 8 0 x t 0 2 3.5xlO Z 4.3 . 1.7x10 2 3x10 2.8x10 with 8.80 1.3410.02 8.01x10fe 8.0 3.5 1.1x10* 1.7 2.3 2.8 8.80 1.30+0.00 9.17 *8 8.0x10 2 3.5x10 Z 4.8 1.7x10 1.7x10 1.8x10 with 8.83 1.90+0.15 3.98x10* 8.0 3.5 4.8 1.7 1.7 1.8 without 8.83 1.60+0.12 1.38 8.0x10 Z 3.Sx10 Z 4.8 1.6x10 9.3x10 9 5x10 8.92 1.4610.04 4.50x1O6 *9 8.0 3.5 4.B 1.6 1.7 1.8 with 8.92 1.4910.06 4.59 8.0 3.5 8.0x10* 1.6 1.7 1.8 8.92 1.41+0.09 6.41 28 TIME ( days) F i g u r e 2. D a i l y c e l l , d e n s i t y o f S_. o b l i q u u s grown i n v a r y i n g N - c o n c e n t r a t i o n s of d a i r y manure medium ( A l g a l E x p t . #1). The v a l u e s - a r e means and ranges o f r e p l i c a t e s p e r t r e a t m e n t (n=3K (200 ug-at N l " 1 * ; H-OOo • 800 * ; 1,600* ; 3,200 •) 29 When the l i g h t i n t e n s i t y was in c r e a s e d from 0.03 to 0.05 ly. min"-4-, growth r a t e s d r a m a t i c a l l y i n c r e a s e d i n c u l t u r e s with high n i t r o g e n c o n c e n t r a t i o n s . In Experiments #2 and #3, growth r a t e s -1 ~ i at 2,000 ug-at-N-1 i n c r e a s e d from 0.88 to 1.35 div.day and from 0.83 to 1.18 div.day"'*". In Experiment #4, growth r a t e s at 1,200, 1,600 and 2,000 ug-at•N•l^were i n c r e a s e d to 1.14, 0.93 and 0.92 -1 div.day , r e s p e c t i v e l y . D a i l y c e l l d e n s i t i e s f o r Experiments #2, #3 and #4 are p l o t t e d i n F i g u r e s 3, 4 and 5. Experiment #5 was conducted mainly to determine the amount of n u t r i e n t s consumed by the a l g a e . Nitrogen c o n c e n t r a t i o n s of 200, 400, 800 and 1,600 ug-at-N • l^were used. No s i g n i f i c a n t d i f f e r e n c e s were found between growth r a t e s (Appendix 15a). However, s i g n i f i c a n t d i f f e r e n c e s were found between c e l l y i e l d s (Appendix 15b). When the l i g h t i n t e n s i t y was i n c r e a s e d to 0.05 -1 ly.min , higher growth r a t e s were a t t a i n e d at c o n c e n t r a t i o n s 400 - l and 800 ug-at-N-1 . Some d i s c r e p a n c i e s were obtained during the dete r m i n a t i o n of TKN at the end of the experiment since the samples were not 100% f r e e of a l g a e . Thus, higher TKN values were obtained.at the end than i n i t i a l l y . Experiment #6 was conducted with improved means of s e p a r a t i n g the algae from the c u l t u r e medium. Although the same n i t r o g e n c o n c e n t r a t i o n s were used as i n Experiment #5, higher growth r a t e s were achieved. S i g n i f i c a n t d i f f e r e n c e s were found-between treatments (Appendix 16a). Maximum c e l l y i e l d s were a l s o found to be s i g n i f i c a n t l y d i f f e r e n t (Appendix 16b). Table 3 shows the amount of n u t r i e n t s consumed by the algae dur i n g the course of the experiment. A l l of the NO3-N, N0 2 -N and PO^-P was 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 o f S_. o b l i q u u s grown i n v a r y i n g N - c o n c e n t r a t i o n s o f d a i r y waste medium ( A l g a l E x p t . #2). The v a l u e s a r e means and ranges o f r e p l i c a t e s p e r t r e a t m e n t (n=3). (^00 ug-at N l - 1 * ; 800 o; 1,200A ; 1,600 A; 2,000B) 31 TIME (days) Figure Daily c e l l density of S. obliquus grown i n varying N-concentrations of dairy waste medium (Algal Expt. #3). The values are means and ranges of re p l i c a t e s per treatment (n=3). (^00 ug-atN 1 _ 1 • ; 8 0 0 o ; 1,200Aj 1.600A; 2 , 0 0 0 a ) 32 TIME ( d a y s ) F i g u r e 5 » D a i l y c e l l d e n s i t y o f S. o b l i q u u s g r o w u i n v a r y i n g N - c o n c e n t r a t i o n s o f d a i r y waste medium ( A l g a l E x p t . #k) \ w i t h a e r a t i o n . The v a l u e s a re means and ranges o f r e p l i c a t e s p e r t r e a t m e n t (n=3). (^00 u g - a t N ; 800 o; 1,200 A; 1,600 A; 2,000 •) T a b l e 3 . C h e m i c a l ' c o m p o s i t i o n o f m e d i a b e f o r e and a f t e r a l g a l g r o w t h . TKN NH4-N N03-N N02-N. P04-P ^ T o t a l Pfl4 u g - a t - N - l " 1 u g - a t - N - l " u g - a t - N - l " 1 - ug-at«N-l ug-at-P. f ug-at.P-1 i n out In out . 1n out In out i n out i n out 2.0x10 1.7x10 3.BX10 -?*0 1.3x10 0 1.8x10 O 3.0 0 5.0 A l g a l 4.0 2.1 7.6 4.0 2.G O 3.6 O 6.0 O 1.0x10 - * Ex p t . + /C6 8.0 2.3 1.5x10 1.1x10 5.2 0 7.2 O 1.2x10 0 2.0 -1.6x10 3 2.5 3.0 1.8 1.0x10* O 1.4x10 8 O 2.4 O 4.0 a 2 2 * 8.0x10 4.4x10 3.5x10 4.0 5.0 O 1.6x10 O 1.7x10 O 1.8x10 - T A l g a l ( w / a l r ) Expt. ^ HS 8.0 5.1 3.5 7.6x10 5.0 3.0 1.6 2.7 1.7 1.0x10 1.8 (w/o a i r ) 'HfHad d i f f i c u l t y measuring v e r y d i l u t e c o n c e n t r a t i o n s In -AutoAnalyzer. 3^ a s s i m i l a t e d by a l l c u l t u r e s . Traces of NH^-N were found i n the c u l t u r e s c o n t a i n i n g the lowest n i t r o g e n c o n c e n t r a t i o n (200 ug-at-N • 1 ) and about 5 - 7% of i t were l e f t i n the r e s t of the c u l t u r e s . T o t a l K j e l d a h l Nitrogen (organic + ammonia) was p a r t l y consumed by the a l g a e . A p p r e c i a b l e amounts of TKN -1 ranging from 170 to 250 ug-at'N-1 were l e f t in the c u l t u r e s . P l o t s of d a i l y c e l l d e n s i t i e s f o r Experiments #5 and #6 are shown in F i g u r e s 6 and 7. In Experiment #7, the n i t r o g e n to phosphorus atomic r a t i o of the medium was adjusted to determine any e f f e c t s on the c e l l y i e l d s of the a l g a e . KNO^was added to make a N:P r a t i o of 64. T h i s was t e s t e d a g a i n s t the o r i g i n a l medium (N:P r a t i o of 17). As expected, 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 on the growth r a t e s (Appendix 17a). A s l i g h t l y higher maximum c e l l y i e l d was achieved with N:P r a t i o of 64. However, s t a t i s t i c a l a n a l y s i s 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 the two values (Appendix 17b). Note that when the experiment was terminated, the a l g a l c u l t u r e s with N:P r a t i o of 64 have not reached t h e i r peak c o n c e n t r a t i o n s yet. Had they been given more time to grow, t h e i r y i e l d c o u l d have been s i g n i f i c a n t l y d i f f e r e n t from those with N:P r a t i o of 17. Experiment #9 was conducted to check again, the e f f e c t of v a r y i n g N:P r a t i o s on the c e l l y i e l d s of the a l g a e . The r a t i o s i n v e s t i g a t e d were 4, 22 ( o r i g i n a l medium) and 69. As expected, growth r a t e s were not a f f e c t e d by v a r y i n g the N:P r a t i o (Appendix 18a). T h i s time, s i g n i f i c a n t d i f f e r e n c e s were obtained between c e l l y i e l d s (Appendix 18b). P l o t s of the d a i l y c e l l d e n s i t i e s f o r Experiments #7 and #9 35 T I M E (days) 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 S. o b l i q u u s grown i n v a r y i n g N - c o n c e n t r a t i o n s o f d a i r y waste medium ( A l g a l E x p t . #5){ w i t h a e r a t i o n . The v a l u e s a re means and ranges o f r e p l i c a t e s p e r t r e a t m e n t (n=3). (200 ug-at N l --*- • ; 400 o ; 800 A ; 1,600 A) 36 TIME (days) F i g u r e • ? • D a i l y c e l l d e n s i t y o f S. o b l i q u u s grown i n v a r y i n g N - c o n c e n t r a t i o n s o f d a i r y waste medium ( A l g a l E x p t . #6); w i t h a e r a t i o n . The v a l u e s a r e means and ranges o f r e p l i c a t e s p e r t r e a t m e n t (n=3). (200 ug-at N 1 ; 400 o ; 800 A; 1,600 A) 37 are shown i n F i g u r e s 8 and 9. A e r a t i o n had a pronounced e f f e c t on a l g a l growth as shown by the r e s u l t s of Experiment #8. Both higher growth r a t e s and c e l l y i e l d s were obtained by aerated c u l t u r e s . S t a t i s t i c a l a n a l y s i s showed s i g n i f i c a n t d i f f e r e n c e s between these values (Appendices 19a and 19b). A l l of the NO3-N, NOg-N and PO^-P, most of the NH^-N and about h a l f of the TKN were consumed by aera t e d c u l t u r e s (See Table 3). On the other hand, only p a r t i a l amounts of n u t r i e n t s were consumed by non-aerated c u l t u r e s . D a i l y c e l l d e n s i t i e s f o r Experiment #8 are p l o t t e d i n F i g u r e 1.0. Scenedesmus seemed to have u t i l i z e d some of the ino r g a n i c n i t r o g e n present i n the medium. There was a high c o r r e l a t i o n (r* =0.9729) found between the N consumed and the a l g a l y i e l d . F i g u r e 11 shows t h i s r e l a t i o n s h i p . Daphnia Experiments Table 4 shows the amount of algae consumed by Daphnia i n a l l f i v e experiments. As expected, more algae were consumed at higher f e e d i n g l e v e l s . In g e n e r a l , Daphnia biomass i n c r e a s e d from Day 0 to a peak l e v e l ( u s u a l l y at Day 9) a f t e r which i t s t a r t e d to drop o f f . In both Experiments #1 and #2, f e e d i n g l e v e l s of 100,000 and -1 150,000 c e l l S ' m l achieved the hi g h e s t biomass v a l u e s . A maximum - l y i e l d of 126.4 mg f o r 100,000 c e l l s - m l and 108.8 mg f o r 150,000 - i c e l l s , ml were a t t a i n e d i n Experiment #1. Higher y i e l d s were reached i n Experiment #2: 264.2 and 228.7 mg. At the lowest 38 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 S. o b l i q u u s grown i n d a i r y waste medium w i t h n i t r o g e n t o phosphorus atomic r a t i o s o f 17 and 6k ( A l g a l E x p t . #7); w i t h a e r a t i o n . The v a l u e s are. means and ranges o f r e p l i c a t e s p e r t r e a t m e n t (n=3). (NiP o f 17 o j N:P o f 6k • ) 39 TIME (days) F i g u r e 9 ' D a i l y c e l l d e n s i t y o f S. o b l i q u u s grown i n d a i r y waste medium w i t h n i t r o g e n t o phosphorus atomic r a t i o s o f 4 , 22 and 69"' ( A l g a l E x p t . # 9 ) ; w i t h a e r a t i o n . The v a l u e s a r e means and ranges o f r e p l i c a t e s p e r t r e a t m e n t ( n = 3 ) . (N:P o f 4»;N:P o f 2 2 o;N:P o f 69 *•) 40 TIME (days ) F i g u r e 10. D a i l y c e l l d e n s i t y o f S. o o l i q u u s grown i n d a i r y waste medium w i t h and w i t h o u t a e r a t i o n ( A l g a l E x p t . # 8 ) . The v a l u e s a re means and ranges o f r e p l i c a t e s p e r t r e a t m e n t (n=3)• ( w / a i r • ; w/o a i r o) F i g u r e 11. N-consumed vs . a l g a l y i e l d . k2 Table 4.. Amount of algae consumed by Daphnia at 3-day i n t e r v a l s . ( i n mg.) Expt. c e l l s - m l 1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 50,000 - 56.2 114.5. 177.6 £1 100,000. - 124.5 253.2 386.4 .' 150,000 - 195.1 398.2 599.8 50,000 - 60.9 109.6 173.5 242.3 #2 100,000 - 123.5 244.4 366.7 492.6 150,000 - .175.6 361.3 546.4 737.7 50,000 - 57.7 116.3 175.9 244.7 #3 100,000 - 117.6 246.7 370.9 494.7 150.000 - 173.8 364.3 532.2 #4 100,000 - 216.4+ 437.5± 746.3± 1118.5+ 11.9 27.9 26.4 29.9 #5 100,000 - 191.7± 377.8+ 715.0* 1081.8+ 14.7 41.4 41.4 41.4 43 feeding l e v e l (50,000 cells«ml ), minimum biomass values were recorded: 69.0 mg i n Experiment #1 and 102.1 mg i n Experiment #2. However, these f i n d i n g s were reversed i n Experiment #3. The highest biomass was a t t a i n e d at the lowest feeding l e v e l . Biomass val u e s were 138.7, 99.1 and 76.2mg at 50,000, 100,000 and 150,000 c e l l S ' i n l * feeding l e v e l s , 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 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 the three treatments due to the wide range of values obtained i n the three experiments (Appendix 20a). The maximum number of Daphnia i n the c u l t u r e s was i n the range 7,000 and 8,000, a t t a i n i n g d e n s i t i e s of 0.7 - 0.8 Daphnia' m l \ These numbers were obtained at a l l three feeding l e v e l s . Maximum biomass conve r s i o n e f f i c i e n c i e s were i n the range 40 --1 50%. At 50,000 c e l l s • ml feeding l e v e l , biomass conve r s i o n e f f i c i e n i e s of 45.3, 47.2 and 49.3% were o b t a i n e d . At 100,000 • - A ' . c e l l s . m l f e e d i n g l e v e l , v a l u e s were 43.2, 49.5 and 21.8%. At -1 . 150,000 c e l l s • ml f e e d i n g l e v e l , values were 36.2, 38.2 and 15.9%. However, s t a t i s t i c a l a n a l y s i s 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 treatments due to the wide range of valu e s obtained (Appendix 20b). I n t e n s i v e feeding d i d not i n c r e a s e the amount of Daphnia produced i n Experiment #4 amd #5. Maximum y i e l d s of only 143.4 and 234.6 mg were obt a i n e d . S t a t i s t i c a l a n a l y s i s showed s i g n i f i c a n t d i f f e r e n c e s at 5% l e v e l of s i g n i f i c a n c e between the two t r i a l s conducted (Appendix 21a). A much higher frequency of 12,400 Daphnia or a d e n s i t y of 1.24 Daphnia .ml*' was a t t a i n e d by c u l t u r e s i n Experiment #5. T h i s was due to the i n c r e a s e d r e p r o d u c t i o n r a t e s with more food a v a i l a b l e . As expected, biomass conver s i o n e f f i c i e n c i e s were much lower due i n t e n s i v e f e e d i n g . Maximum e f f i c i e n c i e s recorded were 17.7 and 21.0%. S t a t i s t i c a l a n a l y s i s 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 the two values (Appendix 21b). To get a c l e a r e r view of the biomass and biomass conver s i o n e f f i c i e n c i e s obtained at each feeding treatment, data were p l o t t e d i n graphs (Raw<data i s t a b u l a t e d i n Appendices 1 t h r u 3). Increase i n Daphnia:biomass f o r Experiments #1. thru #5 are shown in F i g u r e s 12, 14, 16 and 18, r e s p e c t i v e l y . Conversion e f f i c i e n c i e s on the other hand, are shown in F i g u r e s 13, 15, 17 and 19. The p r o p o r t i o n of j u v e n i l e s (0.60 - 1.70 mm), young a d u l t s (1.71 - 2.30 mm) and a d u l t s ( >2.30 mm) i n the c u l t u r e s are d e p i c t e d i n F i g u r e s 20 t h r u 23. The p r o p o r t i o n in terms of biomass i s a l s o i n c l u d e d . The i n c r e a s e i n Daphnia frequency c o i n c i d e d with the i n c r e a s e i n biomass except fo r Experiment #2 -1 (100,000 cells«ml feeding l e v e l ) and Experiment #5, where at Day 12, the number of a d u l t s s t i l l i n c r e a s e d d e s p i t e the decrease in the t o t a l number of animals in the c u l t u r e . G e n e r a l l y , most of the biomass was accounted f o r by the a d u l t animals which weigh so many times more than the j u v e n i l e s . A more d e t a i l e d s i z e - f r e q u e n c y s t r u c t u r e of Daphnia i n the c u l t u r e s was i n v e s t i g a t e d . The number of Daphnia was noted i n increments of 0.20 mm at a l l f e e d i n g l e v e l s . F i g u r e s 24 t h r u 38 d e p i c t these r e s u l t s (Raw data i s t a b u l a t e d i n Appendices 4 t h r u 8). G e n e r a l l y , the Daphnia p o p u l a t i o n i n c r e a s e d from Day 0 to a peak l e v e l a f t e r which huge m o r t a l i t i e s i n young occu r r e d r e s u l t i n g to a d e c l i n e i n p o p u l a t i o n . There was a b i g i n c r e a s e 4 5 150 0 3 6 9 T I M E ( days) F i g u r e 12. Daphnia biomass a t t h e t h r e e f e e d i n g l e v e l s a t 3-day i n t e r v a l s (D. E x p t . #1). (50,000 c e l l s m l - 1 * 100 ,0000. 150,0004-) T I M E (days) F i g u r e 13• Biomass c o n v e r s i o n e f f i c i e n c y o f Scenedesmus to Daphnia a t t h e t h r e e f e e d i n g l e v e l s a t 3-day--, i n t e r v a l s (D. E x p t . #1). (50,000 c e l l s m l - l # ) 100 ,0000; 150,000,4.) 6 9 12 T I M E days F i g u r e 14. Daphnia biomass a t t h e t h r e e f e e d i n g - l e v e l s a t 3-day i n t e r v a l s (D. E x p t . # 2 ) . (50,000 c e l l s m l - 1 ^ ! ioo,ooo05 150,0004) T I M E (days) F i g u r e 15. Biomass c o n v e r s i o n e f f i c i e n c y o f Scenedesmus t o Daphnia a t t h e t h r e e f e e d i n g l e v e l s a t 3-day i n t e r v a l s (D. E x p t . # 2 ) . (50,000 c e l l s m l - 1 ^ ; ioo,oooO; 150,0004) 47 TIME (days) F i g u r e 16. D a p t o l a biomass a t t h e t h r e e f e e d i n g l e v e l s a t 3-day i n t e r v a l s (D. E x p t . #3). (50,000 c e l l s m l " 1 ^ ; I O O . O O O O ; 1 5 0 , O O O A ) TIME (days) F i g u r e 17. Biomass c o n v e r s i o n e f f i c i e n c y o f Scenedesmus t o Daphnia a t t h e t h r e e f e e d i n g l e v e l s a t 3-day i n t e r v a l s (D. E x p t . #3). (50,000 c e l l s m l _ 1 0 i 100,0000; 150,000,4) 200H o E < 2 O CO iooh TIME Figure 18. Daphnia hiomass at 3-day i n t e r v a l s f ed w i t h 100,000 Scenedesmus c e l l s ml :2 to 3 times d a i l y (D. Expts. #4 and #5)• The values are means and ranges of r e p l i c a t e s v(n=3). (Expt.#4g; Expt.#5D) ;25 O ffl "9" T 1 TIME 6 ( d a y s ) Figure 19 12 Biomass conversion e f f i c i e n c y of Scenedesmus to Daphnia i n D. Expts. #4 and #5 at 3-day i n t e r v a l s . The values are means and ranges'of r e p l i c a t e s (n=3)« (Expt.#4>; Expt.#5d) 200t E CO «21001 o CD 3 6 TIME (days) 10000fr cc UJ co 3 5000 4 9 D 0.61-1.70 mm Hi.71-2.30 mm • > 2.30 mm 3 6 TIME (days) ( A ) 10000I cc HI CQ 2 z> z 5000h 3 6 TIME (days) ( C ) 3 6 TIME (days) F i g u r e 20. The p r o p o r t i o n o f j u v e n i l e , young a d u l t and a d u l t Daphnia i n terms o f number and biomass i n c u l t u r e s a t 3-day i n t e r v a l s (D. E x p t . #1). (A-50,000 c e l l s m l " 1 B- 100,000 c e l l s ml-1, C- 150,000 c e l l s ml-1) lOOOOf DC UJ m 3 5000 D 0.61-1.70 mm I 1.71-2.30 mm • >2.30 mm 50 3 6 TIME (days) 3 6 9 TIME (days) 6 TIME (days) TIME TIME (days) The p r o p o r t i o n o f j u v e n i l e ; , y o u n g a d u l t and a d u l t Daphnia i n terms o f number and biomass i n c u l t u r e s a t t h e t h r e e f e e d i n g l e v e l s a t 3-day i n t e r v a l s (D. E x p t . #2). (A- 50,000, B- 100,000, C- 150,000 c e l l l s ml-1) F i g u r e 22. The p r o p o r t i o n o f j u v e n i l e , young a d u l t and a d u l t Daphnia i n terms o f number and "biomass i n c u l t u r e s a t the t h r e e f e e d i n g l e v e l s a t 3-day i n t e r v a l s (D. E x p t . #3). (Avo50,000f B- 100,000, C- 150,000 c e l l s ml-1) 52 300h —200 E CO CO < 5 100 03 10000H DC 111 m F i g u r e 23. TIME The p r o p o r t i o n o f j u v e n i l e , young a d u l t and a d u l t Daphnia i n terms of number and "biomass a t 3-day i n t e r v a l s i n D. E x p t s . #k (B)'-arid #5 ( A ) . F i g u r e 2k. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 5°» 0 0 0 c e l l s ml f e e d i n g l e v e l a t 3-day i n t e r v a l s (D. E x p t . # i ) . 0 . 6 0 0 L 8 0 1 J 0 O 1 . 2 0 1 . 4 0 1 . 6 0 1 . 8 0 2 X > 0 2 . 2 0 2 . 4 0 2 . 6 0 2 . 8 0 3 . 0 0 3 . 2 0 3 . 4 0 TOTAL LENGTH (mm) F i g u r e 25. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 100,000 c e l l s f e e d i n g l e v e l a t 3-day i n t e r v a l s (D. E x p t . #1). 55 1,0001 D A Y 0 2,000|« 1,000 D A Y 9 060 0 80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.8 3.00 3.20 3.40 3£0 T O T A L L E N G T H (mm) F i g u r e 26. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 150,000 c e l l s ml"" 1 f e e d i n g l e v e l a t 3-day i n t e r v a l s (D. E x p t . #1). 1,000 D A Y O 3,0O01-2000 1,0001-DAY 12 1,000h 0.60 0 80 1.00 1.20 1.40 1.60 1.80 2X)0 2.20 2.40 2.60 2.80 3 00 3 20 T O T A L L E N G T H ( m m ) F i g u r e 27. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 5 ° » 0 0 0 c e l l s f e e d i n g l e v e l a t 3-day i n t e r v a l s (D. E x p t . #2). i.oooh DAY O 57 1,000 DAY 3 DAY 6 2,000 2,000K 1,000 DAY 12 a60 0 80 1.00 1.20 140 1.60 180 2.00 2.20 2.40 2.60 2.80 3.00 T O T A L L E N G T H ( m m ) _ -j F i g u r e 28. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 100,000 c e l l s ml f e e d i n g l e v e l at 3-day i n t e r v a l s (D. Expt. #2). 58 1,000* DAY O DAY 6 i,oooK > o z LU o LU cc 2,000 1,000 DAY 9 0.60 0-80 1.00 1.20 1.40 1.60 180 2.00 2.20 2.40 2.60 2.80 3.00 3.20 T O T A L L E N G T H ( m m ) F i g u r e 29. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 150,000 c e l l s ml f e e d i n g l e v e l a t 3-day i n t e r v a l s (D. E x p t . #2). -1 1,000 DAY O 59 F i g u r e 30. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 5 ° » 0 0 0 c e l l s m l " f e e d i n g l e v e l a t 3-day i n t e r v a l s (D. E x p t . #3). 60 1,<XX* DAY O 1,000* DAY 3 DAY 6 1,0001- DAY 9 1,0004" DAY 12 0.60 0.80 tOO 1.20 140 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 T O T A L L E N G T H (mm) F i g u r e 31. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 100,000 c e l l s m l - 1 f e e d i n g l e v e l a t 3-day i n t e r v a l s (D. E x p t . #3). LOOCM- DAY O T O T A L L E N G T H ( m m ) F i g u r e 32. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 150,000 c e l l s ml f e e d i n g l e v e l a t 3-day i n t e r v a l s (D. E x p t . #3). 62 D A Y O 1,00(* 1,0001- D A Y 3 D A Y 6 1,000f 5,000+ >-o z 4,000 3,000}-2£00t 1,000-1,0001 0.60 0.80 1.00 1.20 140 160 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3A0 3.60 3.8—40o" T O T A L L E N G T H { m m ) F i g u r e 33- Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 3-day i n t e r v a l s f e d w i t h 100,000 c e l l s m l - 1 2 t o 3 t i m e s d a i l y (D. E x p t . #4- R e p l . 1). 63 i^ oor D A Y O 0 . 6 0 0 . 8 0 1 . 0 0 W O 1 4 0 1 .60 1 , 8 0 2 J O O 2 . 2 0 2 . 4 0 2 . 6 0 2 . 8 0 3 . 0 0 3 . 2 3 . 4 0 3 . 6 0 3 . 8 0 T O T A L L E N G T H (mm) F i g u r e 34. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 3-day i n t e r v a l s f e d w i t h 100,000 c e l l s m l - 1 2 t o 3 t i m e s d a i l y ( D . E x p t . #4- R e p l . 2). 6 4 1,000* DAY 0 O 1,OOOf DAY 3 2,000 > 1,000 o z L U 3 o L U O DAY 6 ipooj-DAY 9 i poo f DAY 12 0.60 0.80 1.00 1.20 1A0 1.60 1.80 2X>0 2.20 2 AO 2.60 2.80 3.00 3.20 3.40 3.60 3.80 T O T A L L E N G T H (mm) F i g u r e 35• Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 3-day i n t e r v a l s f e d w i t h 100,000 c e l l s m l " 1 2 t o 3 t i m e s d a i l y (D. E x p t . # 4 - R e p l . 3). T O T A L L E N G T H [mm) F i g u r e 36. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 3-day i n t e r v a l s f e d w i t h 100,000 c e l l s m l - 1 2 t o 3 "times d a i l y (D. E x p t . #5- R e p l . 1). 6 6 F i g u r e 37 . Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 3-day i n t e r v a l s fe'd w i t h 100,000 c e l l s m l - 1 2 t o 3 t i m e s d a i l y (D. E x p t . #5r R e p l . 2 ) . 67 1,000 L D A Y 0 1,000 k D A Y 3 1,000 D A Y 6 D A Y 9 2,000 1,000 D A Y 12 1,000 0.60 0.80 1.00 1.20 140 1.60 1.80 2.00 2.20 240 2.60 2.80 3.00 3.20 3.40 3.60 3.80 T O T A L L E N G T H (mm) F i g u r e 38. Daphnia s i z e - f r e q u e n c y s t r u c t u r e a t 3-day i n t e r v a l s f e d w i t h 100,000 c e l l s ml - 1 2 t o 3 t i m e s d a i l y (D. E x p t . #5- R e p l . 3). 6 8 in the number of j u v e n i l e Daphnia due to the high r e p r o d u c t i v e rate of the few a d u l t s . There was a l s o a steady increase i n the number of a d u l t s up to the peak l e v e l u n t i l food became l i m i t i n g and g r e a t l y reduced t h e i r r e p r o d u c t i v e rate and s u r v i v a l . The t o t a l number of eggs and embryos at each sampling day was recorded (Table 5). E p p h i p i a l eggs were a l s o . noted when c u l t u r e s aged. In Experiments #4 and #5, water q u a l i t y was monitored d a i l y . Table 6 shows the f l u c t u a t i o n s i n pH and Og l e v e l s and the accumulation of NH^-N as c u l t u r e s aged. A l g a l C e l l Weight The weight of Scenedesmus was determined on c e l l c o n c e n t r a t i o n s ranging from 4.14 to 9.20 x 10 per 200 ml of sample. The mean weight was 4.5846 x 10 mg per Scenedesmus c e l l ± a standard d e v i a t i o n of 1.0818 x 10 . The c o e f f i c i e n t of v a r i a t i o n was 23.6%. Growth Rate Experiments Growth r a t e s were e s s e n t i a l l y the same at the three feeding l e v e l s up to the t h i r d or f o u r t h day a f t e r which growth r a p i d l y l e v e l l e d - o f f with the average maximum l e n g t h at the end of the experimental p e r i o d being dependent on the food l e v e l . S t a t i s t i c a l a n a l y s i s shows s i g n i f i c a n t d i f f e r e n c e s between the lengths of Daphnia i n both experiments (Appendices 22a and 22b). 6 9 T a b l e 5. F e c u n d i t y of Daphnia c u l t u r e s i n a l l f i v e e x periments T o t a l # of eggs and embryos Expt. c e l l s - m l " 1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 # 1 5 0 , 0 0 0 1 0 0 , 0 0 0 1 5 0 , 0 0 0 9 6 0 1 , 4 5 6 4 , 1 3 0 5 , 3 0 2 2 , 180 ( 1 4 5 ) 1 , 7 8 4 1 , 8 9 9 6 3 6 * ( 7 0 6 ) 7 9 3 % ( 6 1 4 ) 1 , 1 1 0 ( 3 9 2 ) * -#2 5 0 , 0 0 0 1 0 0 , 0 0 0 1 5 0 , 0 0 0 5 0 0 3 , 168 2 , 7 2 0 3 , 0 0 9 3 , 2 8 0 ( 2 1 9 ) 4 , 8 6 4 ( 6 0 8 ) 1 , 5 6 0 ( 2 6 0 ) 1 , 7 6 8 ( 2 9 5 ) * 3 , 0 8 0 (308)* 3 , 3 2 9 ( 3 0 3 ) 1 , 1 7 6 ( 3 9 2 ) 1 , 2 5 4 ( 2 7 8 7 ) * (460)* #3 . 5 0 , 0 0 0 1 0 0 , 0 0 0 1 5 0 , 0 0 0 8 2 9 2 , 1 4 6 2 , 7 3 6 2 , 4 0 7 1 , 0 6 4 3 , 4 7 9 1 , 4 4 4 2 , 1 9 8 ( 4 1 9 ) * 8 0 7 ( 1 6 1 r 0 * ( 61 r 1 , 8 5 5 j. ( 1 4 2 7 * 2 1 8 M ( 8 5 1 V 2 , 8 3 0 8 , 7 2 2 5 , 1 3 0 1 , 6 2 8 -( 8 8 ) * #4 1 0 0 , 0 0 0 180 2 , 8 3 6 1 1 , 3 4 4 3 , 5 0 2 9 , 3 8 4 ( 1 0 9 ) 2 , 5 2 0 9 , 7 9 2 1 2 , 7 9 9 7 , 5 4 3 2 , 1 2 7 7 , 1 9 1 1 1 , 7 0 4 7 , 4 0 0 #5 1 0 0 , 0 0 0 2 8 0 2 , 2 1 0 6 , 0 4 8 8 , 9 7 6 6 , 5 3 3 1 , 6 3 2 5 , 2 7 4 1 3 , 6 9 6 1 0 , 1 2 2 iff ( ) e p p h i p i a l eggs T a b l e 6. Ammonia, pH and DO l e v e l s measured i n Daphnia c u l t u r e t a n k s i n Expe r i m e n t #4 and #5. NH4-N (ug-at' N< 1 ) Expt DAY 0 DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 DAY 1 1 DAY 12 DAY 13 MA #5 14 . 3 28 . 6 14.3 18.6 15.0 2 1.4 15.7 10.7 16.4 17.9 19.3 19.3 24 . 3 17.9 26 . 4 19.3 28 . 6 23 . 6 32 .9 45.0 38.6 60. 7 38 . 6 63.6 43.6 75 . 7 67.9 101.4 pH Expt DAY 0 DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 DAY 1 1 DAY 12 DAY 13 #4 8 . 38 8 . 06 7 . 83 8 .09 8.11 8.61 8.47 8 . 80 8 . 49 8 .92 8 . 30 8 . 87 8.19 8 .60 8.11 8 . 28 7 . 88 7 . 98 7 .65 7 .93 7 . 48 7.65 7 . 36 7 . 53 7 . 60 7 . 42 7 . 74 7.71 DO (ug-a t' Expt DAY 0 DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 DAY 1 1 DAY 12 DAY 13 #4 #5 240.6 231 . 3 206 . 3 265 .6 231 . 3 265.6 262 . 5 268.8 259.4 268 . 8 237 . 2 268 . 8 253 . 1 268 .8 243.8 268.8 265.6 253 . 1 200.0 22 1 . 9 206 . 3 209.4 23 1 . 3 212.5 240.6 178 . 1 200.0 187.5 o 71 When fed twice a day, Daphnia a t t a i n e d l a r g e r s i z e s . At Day 10, the average t o t a l lengths of Daphnia at 50,000, 100,000 -1 and 150,000 c e l l s . m l . feeding l e v e l were 2 . 9 1 , 2.96 and 3.01 mm, r e s p e c t i v e l y . In c o n t r a s t , t o t a l l e ngths of 2.36, 2.44 and 2.52 mm were a t t a i n e d by Daphnia when fed only once a day. F i g u r e s 3 9 and 40 d e p i c t these r e s u l t s (Raw data i s t a b u l a t e d i n Appendix 9) . Length-Weight R e l a t i o n s h i p The r e l a t i o n between the l e n g t h and dry weight of parthenogenic female Daphnia with and without eggs and embryos in the brood chamber i s shown in F i g u r e 41 (Raw data i s , t a b u l a t e d i n Appendix 10). These valu e s f i t two l i n e s , one d u r i n g j u v e n i l e stage and the other one d u r i n g a d u l t stage. The r e g r e s s i o n equation was Y = 14.88X - 10.57 (r = 0.7268) with animals between 0.80 and 1.82 mm i n t o t a l l e n g t h and Y = 80.28X 146.18 ( r 3 = 0.9355) with animals l a r g e r than 1.8.2 mm. The length-weight r e l a t i o n was the same r e g a r d l e s s of f e c u n d i t y . Brood S i z e i n R e l a t i o n to Length Daphnia brood s i z e was i n v e s t i g a t e d d u r i n g the course of the growth and feeding r a t e experiments. Table 7 shows the number of eggs and embryos i n r e l a t i o n to Daphnia s i z e . The average number of young per brood i n c r e a s e d f o r the f i r s t few broods f o l l o w e d by an i r r e g u l a r but r e l a t i v e l y h i g h average 72 TIME (days) Figure 3 9 . Daily t o t a l length of D. pulex at the three food con-centrations (Growth Rate Expt. # 1 ) . The values are means and ranges of r e p l i c a t e s per treatment (n= 1 0 ) . ( 5 0 , 0 0 0 c e l l s ml" 1*; lOO.OOOB; 1 5 0 , 0 0 0 0 ) 73 0.8h TIME (days) F i g u r e 40. D a i l y t o t a l l e n g t h o f D. p u l e x a t t h e t h r e e f o o d con-c e n t r a t i o n s (Growth Rate E x p t . #2). The v a l u e s a re means and ranges o f r e p l i c a t e s p e r t r e a t m e n t (n=10). (50,000 c e l l s m l - l # ; 100,000 150,000Q) 74 0.8 1.0 2.0 3.0 T O T A L L E N G T H (mm) F i g u r e 41. L e n g t h - w e i g h t r e l a t i o n s h i p o f D. p u l e x . (w/eggs and embryostf-'jv.w/o eggs and embryos#) Table 7. Daphnia brood s i z e i n r e l a t i o n to t o t a l l e n g t h . (number of eggs and embryos) Length(mm.) Fed 50,000 c e l l s «ml 1x a day Fed 100,000 c e l l s - ml" 1x a day Fed 1 50 , 000 _± c e l l s - m l 1x a day Fed 100,000 ^ c e l l s . m l 2-3x a day 1.80-2.00 2-4 - '- . 2.01-2.20 3-5* 3-4 , 3-4 , -2.21-2.40 3-6 3-6 3-6 3-6 2.41-2.60 3-9 4-9 5-9 4-9 2.61-2.80 4-9 4-9 4-13 6-16 2.81-3.00 6-12 6-12 6-10 6-17 3.01-3.20 6-12 6-7 8-12 9-15 3.21-3.40 6- 10-14 1 3- • ; 8-21 3.41-3.60 12-33 3.61-3.80 20-35 3.81-4.00 21-76 p r o d u c t i o n of young. A maximum of 35 recorded. young per brood was 77 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 Although the d a i r y manure was p r e - d i g e s t e d f o r 10 days p r i o r to i t s use, organic and ammonia-nitrogen s t i l l comprised most of the n i t r o g e n content except f o r Medium III i n which l a r g e amounts of n i t r a t e - and n i t r i t e - n i t r o g e n were observed. The composition of each medium v a r i e d from one batch to another and t h i s might be a t t r i b u t e d to the d i f f e r e n c e i n the d i g e s t i o n r a t e s of the manure. Nitrogen appears to be the major n u t r i e n t l i m i t i n g primary p r o d u c t i o n i n world's, oceans as w e l l as i n c e r t a i n freshwater systems (Owens and Osaias, 1976). Among the n i t r o g e n forms, ammonium appears to be the primary i n o r g a n i c N form which enters i n t o s y n t h e t i c r e a c t i o n s ; both N03and NO^must f i r s t be reduced to NH^prior to a s s i m i l a t i o n i n t o p r o t e i n s . Ammonium i s the most e n e r g e t i c a l l y favourable source of i n o r g a n i c N because of i t s reduced s t a t e , and both l a b o r a t o r y and f i e l d s t u d i e s demonstrate that NH4" i s taken up i n p r e f e r e n c e to NO^and N0 2when a l l forms are present (Owens and Osaias, 1976). The green a l g a , Scenedesmus o b i i q u u s , i s thought to have a s l i g h t p r e f e r e n c e f o r NH4 over NOj and N02~ (Krauss, 1958). However, i n the present study, no p r e f e r e n c e was found. Both NO3" and NOg were always completely a s s i m i l a t e d by the algae but s u b s t a n t i a l amounts of NH^were always l e f t i n the medium a f t e r growth. Animal wastes may c o n t a i n t o x i c substances from r e s i d u e s and growth s t i m u l a n t s added to the food r a t i o n of the animals. 78 However, in the present study, the manure medium d i d not show any t o x i c e f f e c t s to the algae even at higher c o n c e n t r a t i o n s . The t o x i c a n t s may have been reduced to i n s i g n i f i c a n t l e v e l s through the d i g e s t i o n p r o c e s s . A l g a l growth was mainly a f f e c t e d by the l i g h t i n t e n s i t y which was much reduced at higher manure c o n c e n t r a t i o n s . A l g a l Growth on Manure Medium Most of the Scenedesmus o b i i q u u s grown i n manure medium were composed of u n i c e l l s . The media co n t a i n e d a high mineral content (e.g.Na + , K + , Ca* ) i n c l u d i n g a high phosphate c o n c e n t r a t i o n f a v o u r i n g u n i c e l l formation over coenobia. T h i s agrees with the o b s e r v a t i o n of Shubert and T a y l o r (1974) that Scenedesmus may be found only i n areas where there i s s u f f i c i e n t phosphate, e.g.near sewage o u t f a l l s or r u n - o f f s from a g r i c u l t u r a l areas. Among the n i t r o g e n c o n c e n t r a t i o n s used, the growth constant k was highest at lower c o n c e n t r a t i o n s while c e l l y i e l d s was h i g h e s t at higher c o n c e n t r a t i o n s . C e l l d i v i s i o n was a f f e c t e d by the l i g h t i n t e n s i t y r a t h e r than by the amount of n i t r o g e n present i n the medium. The l i g h t p e n e t r a t i n g the c u l t u r e s decreased as N-concentration i n c r e a s e d . T h i s accounted f o r the low c e l l d i v i s i o n r a t e s i n N-concentrated c u l t u r e s . However, when the l i g h t i n t e n s i t y was i n c r e a s e d from 0.03 to 0.05 l y . m i n ^ c e l l d i v i s i o n in N-concentrated c u l t u r e s a l s o i n c r e a s e d to normal r a t e s . High l i g h t i n t e n s i t y was not used i n i t i a l l y s i n c e p h o t o - i n h i b i t i o n might occur i n l e s s N-concentrated c u l t u r e s . 7 9 Only when the. algae had become dense was the l i g h t i n c r e a s e d . As the a l g a l c o n c e n t r a t i o n i n c r e a s e s , p e n e t r a t i o n of l i g h t i n the water decreases. A l i m i t i s reached at which f u r t h e r p r o d u c t i o n of algae ceases even though an excess of chemical n u t r i e n t s may be present (Hepher, 1962). Sorokin and Krauss (1958) observed that the l i g h t s a t u r a t i o n f o r Scenedesmus o b i i q u u s was 0.02 ly-min . At t h i s i n t e n s i t y , the growth constant k was 2.2 div.day while a t . h a l f -s a t u r a t i o n 1 . i n t e n s i t y i t was 1.5 div.day . The growth r a t e s obtained i n the present study were lower than these values and t h i s might be because the a c t u a l l i g h t i n t e n s i t i e s p e n e t r a t i n g the c u l t u r e s were very much reduced below s a t u r a t i o n l e v e l s by the medium used. However, the Scenedesmus used here might be of a d i f f e r e n t c l o n e , a slower grower, than that used by Sorokin and Krauss. The environmental c o n d i t i o n s might have been d i f f e r e n t , too. The f i r s t three a l g a l experiments were conducted without a e r a t i o n . Carbon l i m i t a t i o n might have caused the low c e l l y i e l d s achieved by the c u l t u r e s . These y i e l d s were i n c r e a s e d f i v e - f o l d or more when a e r a t i o n was introduced i n t o the system. A g i t a t i o n might have reduced the l i g h t l i m i t a t i o n to the c u l t u r e s . C e l l d i v i s i o n r a t e s a l s o i n c r e a s e d s i g n i f i c a n t l y . V a r i o u s s t u d i e s show that the a d d i t i o n of CO^through a e r a t i o n and a g i t a t i o n enhances a l g a l growth (Gates and Borchardt, 1963; T r a i n o r , 1964; T r a i n o r and Shubert, 1973; Perez et a l . , 1977). I t p r o v i d e s b e t t e r l i g h t u t i l i z a t i o n , prevents algae from s e t t l i n g or accumulating at the s u r f a c e where the l i g h t i n t e n s i t y i s s u f f i c i e n t to d e a c t i v a t e the c e l l s , and 80 p r o v i d e s a more homogeneous environment fo r the a l g a e . In the experiments, a e r a t i o n may.have a l s o enhanced b a c t e r i a l a c t i v i t y t r a n s f o r m i n g some of the organic n i t r o g e n i n t o more usable forms of nitrogen.and producing v i t a m i n s f o r a l g a l growth. I n v e s t i g a t o r s i n the past have a l s o showed that the t o t a l a l g a l biomass produced on l i q u i d wastes was. determined by the l i m i t i n g n u t r i e n t , u s u a l l y carbon . (Goldman et a l . , 1971; Foree et al.-., 1973; Dor et a l . , 1974; Oswald and Beneham, 1977). The amount of algae may be i n c r e a s e d by i n j e c t i n g COginto the ponds to give added carbon. Inorganic n i t r o g e n may be a l i m i t i n g f a c t o r with regards to the amount of *growth which can be produced (Mead et a l . , 1945). The r a t i o of n i t r o g e n to phosphorus (N:P) must t h e r e f o r e be given due c o n s i d e r a t i o n . In the experiments conducted, the r a t i o s used showed s i g n i f i c a n t d i f f e r e n c e i n a l g a l growth. Growth constants were the same as expected. A higher biomass was produced at N:P of 69 than at 4 or 2.2. Using much higher r a t i o s might r e s u l t to more c o n v i n c i n g r e s u l t s . Rhee (1974, 1978) showed that the optimal y i e l d of Scenedesmus occurred at N:P of 30. Below t h i s optimal r a t i o , y i e l d was determined s o l e l y by N - l i m i t a t i o n and above i t , by P-l i m i t a t i o n . C e l l - N remained constant up to the optimal r a t i o and i n c r e a s e d l i n e a r l y with N:P above i t . The l e v e l of c e l l - P was high at low N:P ( N - l i m i t e d s t a t e ) but decreased r a p i d l y u n t i l the r a t i o approached the optimal and remained constant at a low l e v e l at high N:P ( P - l i m i t e d s t a t e ) . No r e s i d u a l i n o r g a n i c or d i s s o l v e d N or P was d e t e c t e d i n the medium at any N:P examined, i n d i c a t i n g excess accumulation of both n u t r i e n t s . 81 T h i s excess accumulation of N and P was a l s o observed in the present study. C e l l d i v i s i o n r a t e s v a r i e d from time to time d e s p i t e u n i f o r m i t y in medium and c u l t u r e c o n d i t i o n s . T h i s o b s e r v a t i o n i s a l s o true for other a l g a l s p e c i es ( H a r r i s o n , p e r s o n a l communication). Daphnia Biomass Production S t a t i s t i c a l a n a l y s i s 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 the three feeding l e v e l s .used in terms of biomass pr o d u c t i o n and biomass conver s i o n e f f i c i e n c i e s . T h i s was due to the wide range in r e p l i c a t e s obtained i n each treatment. However, s l i g h t l y bigger biomass values were obtained at the higher f e e d i n g l e v e l s . The amount of food made a v a i l a b l e to Daphnia may have been the same d e s p i t e the f e e d i n g l e v e l . Scenedesmus c e l l s always s e t t l e d i n the bottom and d i n g e d to the s i d e s of the tanks and t h e r e f o r e , were never made very a v a i l a b l e to Daphnia. In the higher feeding l e v e l s , a l g a l c e l l s s e t t l e d f a s t e r and so the a c t u a l c o n c e n t r a t i o n of suspended food c o u l d have been the same i n each treatment. No a e r a t i o n nor mixing i n tanks was employed s i n c e Daphnia was very much a f f e c t e d by even s l i g h t water a g i t a t i o n . I n t e n s i v e feeding d i d not seem to i n c r e a s e the biomass pr o d u c t i o n of Daphnia. When fe e d i n g was conducted two to three -1 times d a i l y at 1 0 0 , 0 0 0 cells«ml , low biomass valu e s were s t i l l o b t a ined. Again, there was the problem of algae s e t t l i n g to the bottom of the tanks. The i n c r e a s e d a l g a l feed might have a l s o 82 i n c r e a s e d the t o x i c e f f e c t s of the r e s i d u e s found in the manure medium. Although low biomass val u e s were produced, Daphnia showed high biomass conve r s i o n e f f i c i e n c i e s . Maximum e f f i c i e n c i e s were in the range 40 - 50%. These values are comparable to the c o n v e r s i o n e f f i c i e n c y of 38 - 42% that Sasa et a l . (i960) obtained when C h l o r e l l a was fed to Daphnia. P o p u l a t i o n Growth and S i z e S t r u c t u r e G e n e r a l l y , p o p u l a t i o n s f o l l o w a sigmoid p o p u l a t i o n curve. A small p o p u l a t i o n of organisms introduced i n t o a s u i t a b l e c o n t a i n e r w i l l i n c r e a s e slowly at f i r s t , then i t s i n c r e a s e w i l l a c c e l e r a t e and f i n a l l y the i n c r e a s e i n p o p u l a t i o n w i l l become very slow and approach a l e v e l of upper asymptote (Smith, 1952). T h i s assumes that organisms in the p o p u l a t i o n can be c o n s i d e r e d i d e n t i c a l with each other. T h i s assumption has been demonstrated to be f a l s e to Daphnia (Slobodkin,. 1954). Daphnia p o p u l a t i o n s are c h a r a c t e r i z e d by i n t r i n s i c o s c i l l a t i o n s due to p h y s i o l o g i c a l d i f f e r e n c e s between i n d i v i d u a l Daphnia and do not f o l l o w a sigmoid p o p u l a t i o n curve. T h i s seems to be the case i n the present study. Knowledge of the age and s t r u c t u r e of metazoan p o p u l a t i o n s i s necessary to analyze growth p r o p e r l y (Slobodkin, 1954). Daphnia e x h i b i t s no s p e c i f i c c h a r a c t e r s , making i t d i f f i c u l t to d e s c r i b e the age s t r u c t u r e of the p o p u l a t i o n . Therefore, the p o p u l a t i o n s i z e s t r u c t u r e was c o n s i d e r e d i n t h i s study, i n s t e a d . In g e n e r a l , the peaks i n p o p u l a t i o n number c o i n c i d e d with 83 the maximum p r o p o r t i o n of small animals due to the high b i r t h r a t e s . On the other hand, the troughs c o i n c i d e d with the maximum p r o p o r t i o n of l a r g e animals due to the high j u v e n i l e death rate a f t e r peak l e v e l was reached. S i m i l a r r e s u l t s were observed f o r D.obtusa (Slobodkin, 1954), D.magna ( P r a t t , 1943) and D . p u l i c a r i a (Frank, 1952). In the e a r l y stages of p o p u l a t i o n growth the few a d u l t s had a high r e p r o d u c t i v e r a t e due to the abundance of food r e s u l t i n g in a s i z e - f r e q u e n c y d i s t r i b u t i o n that was skewed towards the small end. The p o p u l a t i o n i n c r e a s e d u n t i l i t was at i t s numerical peak. At t h i s p o i n t , the food supply became l i m i t i n g . In the competition f o r food, the small animals do not exert a strong a c o m p e t i t i v e pressure as l a r g e animals and so huge m o r t a l i t i e s i n young o c c u r r e d . The r e p r o d u c t i o n r a t e s of the a d u l t s was a l s o g r e a t l y .reduced. The s i z e frequency d i s t r i b u t i o n of the p o p u l a t i o n s h i f t e d towards,the l a r g e animals with low r e p r o d u c t i o n . The i n c r e a s e d s e v e r i t y of competition f o r food i n c r e a s e d the r a t i o of deaths to b i r t h s so that the t o t a l number of animals i n the p o p u l a t i o n curve grew smaller while the s i z e frequency d i s t r i b u t i o n moved towards a l a r g e r mean s i z e . Adult animals s t a r t e d to produce e p p h i p i a l eggs. The same o b s e r v a t i o n was found by Slodbokin (1954) on D.obtusa. When i n t e n s i v e f e e d i n g was conducted, Daphnia a t t a i n e d great s i z e s and high f e c u n d i t i e s . More young was produced due to the high r e p r o d u c t i o n r a t e s . As a r e s u l t , c u l t u r e s a t t a i n e d - 1 higher f r e q u e n c i e s . A d e n s i t y of 1.24 Daphnia.ml was reached, -1 much higher than the usual 0.7 to 0.8 Daphnia » ml i n previous experiments. The p r o d u c t i o n of e p p h i p i a l eggs was n e g l i g i b l e or 84 very much reduced. Maximum l e n g t h and brood s i z e of Daphnia i s appar e n t l y determined by the abundance of food ( H a l l , 1964; Daborn et a l . , 1978). Growth Rate Experiments Daphnia e x h i b i t e d d i f f e r e n t growth r a t e s at the three feeding l e v e l s when fed once a day with Scenedesmus o b l i q u u s . Growth r a t e s were e s s e n t i a l l y the same up to the t h i r d or f o u r t h day a f t e r which growth l e v e l l e d - o f f with the average maximum le n g t h at the end of the experimental p e r i o d being dependent on the food l e v e l . Adult stages are a f f e c t e d more by d i f f e r e n t food l e v e l s than are j u v e n i l e stages s p e c i f i c a l l y the growth per i n s t a r , the d u r a t i o n of i n s t a r and the maximum carapace l e n g t h (Ingle et a l . , 1937; H a l l , 1964; Richman, 1958). Higher growth r a t e s were observed at the higher food c o n c e n t r a t i o n s . Furthermore, the d u r a t i o n of the p r e - a d u l t i n s t a r s was one day sh o r t e r at the highest food c o n c e n t r a t i o n . S i m i l a r r e s u l t s were obtained when Daphnia was fed twice a day at the same food c o n c e n t r a t i o n s . However, l a r g e r - s i z e d Daphnia were produced. T h i s again shows the r e l a t i o n s h i p between the food l e v e l and the maximum s i z e a t t a i n e d by Daphnia. 8 5 Length-Weight R e l a t i o n s h i p A strong c o r r e l a t i o n was found between the length.and weight of Daphnia. The values f i t . two l i n e s , one d u r i n g j u v e n i l e stage and the other d u r i n g a d u l t stage. The same t r e n d was observed i n e a r l i e r s t u d i e s (Edmonson, 1956; Richman, 1958; S c h i n d l e r , 1 9 6 8 ; Burns, 1969; K r i n g and O'Brien, 1976). Daphnia with and without eggs and embryos i n the brood chamber showed the same r e l a t i o n s h i p . . T h i s f i n d i n g does not conform with Richman's o b s e r v a t i o n that Daphnia with eggs and embryos weighed twice as much as animals of the same length with empty brood chambers. N u t r i t i o n a l Inadequacy of C e r t a i n Algae to Daphnia C u l t u r e I n v e s t i g a t o r s show c o n t r a d i c t o r y r e s u l t s on the adequacy of c e r t a i n green algae as food f o r Daphnia. Taub and D o l l a r (1969) i n t h e i r s t u d i e s found that Daphnia f a i l e d to reproduce normally when fed with e i t h e r C h l o r e l l a pyrenoidosa or Chlamydomonas  r e i n h a r d i c u l t u r e d and suspended i n d e f i n e d medium. L i f e span was re p o r t e d s h o r t e r and o v u l a t i o n reduced, and a l a r g e percentage of o v u l a t e d eggs f a i l e d to complete embryonic development. The same a l g a l c u l t u r e suspended i n " b i o l o g i c a l l y c o n d i t i o n e d water" supported a normal l i f e span and ample but not maximal r e p r o d u c t i o n . Taub and D o l l a r suspected the algae to be d e f i c i e n t i n meeting the n u t r i t i o n a l requirements of D.pulex, e s p e c i a l l y with respect to r e p r o d u c t i o n . They f u r t h e r 86 suggested that proper u t i l i z a t i o n of the algae seemed to depend on f a c t o r s s u p p l i e d by other organisms, probably b a c t e r i a , and probably p r o v i d e d i n the water from n a t u r a l sources used i n most experiments. On the other hand, Watanabe et a l . ( l 9 5 5 ) r e p o r t e d that the micro-algae C h l o r e l l a , Chlamydomonas and Scenedesmus were e x c e l l e n t feeds fo r Daphnia. Furthermore, Sasa et a l . (1960) i n t h e i r s t u d i e s were s u c c e s s f u l i n feeding C h l o r e l l a to Daphnia o b t a i n i n g 38 - 42% c o n v e r s i o n e f f i c i e n c i e s . Gordon (1975) a l s o reported that Daphnia tend to p r e f e r mixed d i e t s . More biomass was achieved when Daphnia was fed with both C h l o r e l l a and Scenedesmus than with e i t h e r algae alone. I t must be p o i n t e d out that the a l g a l c u l t u r e s used in the above s t u d i e s were non-ax e n i c . In the present study, success was achieved i n growing Daphnia on Scenedesmus c e l l s . Growth was normal and high r e p r o d u c t i o n r a t e s were achieved e x p e c i a l l y when more food was given. Biomass conversion e f f i c i e n c i e s were a l s o h i g h . I t has to be p o i n t e d out that the c u l t u r e s were grown in r e - c o n d i t i o n e d pond water. D e t r i t u s and b a c t e r i a growing in i t may have c o n t r i b u t e d to the d i e t of the Daphnia. Ryther (1954) r e p o r t e d that the f i l t e r i n g r a t e of Daphnia was i n h i b i t e d by substances produced by the three a l g a l s p e c i e s ( C h l o r e l l a , Scenedesmus and N a v i c u l a ) he t e s t e d . The i n h i b i t i o n was mediated p a r t i a l l y by substances which d i f f u s e d from the c e l l s and accumulated in the water. A much more pronounced e f f e c t appeared to be produced by the r e l e a s e of i n h i b i t o r y products i n g e s t e d w i t h i n the animal. Minimum 87 i n h i b i t i o n was produced by a c t i v e l y growing algae from c u l t u r e s which were i n t h i e r l o g phase of growth. Maximum i n h i b i t i o n was produced by scenescent, n o n - d i v i d i n g algae. Ryther f u r t h e r noted that the f i l t r a t i o n rate of D.magna decreased as the c o n c e n t r a t i o n of food p a r t i c l e s i n c r e a s e d which he i n t e r p r e t e d as an i n c r e a s e i n i n h i b i t o r y f a c t o r as food c o n c e n t r a t i o n i n c r e a s e d . T h i s was contested by O'Brien and de N o y e l l e s (1972) who have shown that the m o r t a l i t y f a c t o r which Ryther observed was probably high pH. Other i n v e s t i g a t o r s have s i n c e demonstrated that the r e d u c t i o n i n feed i n g r a t e i s an accommodation of the animals to in c r e a s e d food c o n c e n t r a t i o n ( R i g l e r , 1961; McMahon and R i g l e r , 1963). T o x i c i t y i n Daphnia C u l t u r e Rees and O l d f a t h e r (1980) suggest that there i s a d i r e c t r e l a t i o n s h i p between pH and the number of Daphnia u l t i m a t e l y appearing i n the c u l t u r e s . Rise i n pH i n n a t u r a l and l a b o r a t o r y c o n d i t i o n s c o r r e l a t e with p h o t o s y n t h e t i c a c t i v i t y as removal of CO^by phytoplankton r e s u l t s i n decreased b u f f e r i n g a c t i v i t y of the system. T h i s r e s u l t s to decreased feeding of Daphnia and e v e n t u a l l y death. Although c l a d o c e r a n s have an upper l i m i t of pH t o l e r a n c e between 10.5 and 11.0 (Bogatova, 1952), the gr e a t e s t f i l t r a t i o n r a t e of D.pulex occurs at pH range 6.0 - 8.0 (Ivanova, 1969). Furthermore, Davis and Ozburn (1969) suggest that D.pulex w i l l not t h r i v e i n a pH o u t s i d e the range 7.0 -8.7. In the present study, pH range was w i t h i n safe l i m i t s ( 5 . 4 -9.5). A s l i g h t i n c r e a s e i n pH was observed when the c u l t u r e s 8 8 reached t h e i r peak numbers and s t a r t e d d e c l i n i n g . The t o x i c i t y of ammonia to a q u a t i c b i o t a i s g r e a t l y a f f e c t e d by the chemistry of the water in which i t i s d i s s o l v e d . Although ammonia t o x i c i t y i s i n f l u e n c e d by a l k a l i n i t y , temperature, free-Co^ and d i s s o l v e d (Brown, 1968), the f a c t o r of primary importance i s pH (McKee and Wolf,- 1968) because i t -c o n t r o l s the d i s s o c i a t i o n of ammonia in s o l u t i o n . Ammonia i s not t o x i c i n i t s u n d i s s o c i a t e d form. The higher the pH, the g r e a t e r the p r o p o r t i o n of ammonia that w i l l be u n d i s s o c i a t e d . Ammonia was a l r e a d y shown to be t o x i c to Daphnia at e l e v a t e d pH (Dinges, 1974). In one t o x i c i t y study, Parkhurst et a l . (1979) found that ammonia was t o x i c to D.magna at a very -1 . high c o n c e n t r a t i o n of 25 mg»l . D.magna t h r i v e i n lakes where high ammonia c o n c e n t r a t i o n s are found and t h i s e x p l a i n s t h e i r very high t h r e s h o l d l i m i t . On the other hand, D.pulex i s a pond s p e c i e s and i s not o r d i n a r i l l y exposed to high ammonia c o n c e n t r a t i o n s . Only D.magna was r e p o r t e d to grow i n s t a b i l i z a t i o n and e f f l u e n t ponds. In the present study, ammonia l e v e l s were very low. There was an accumulation of NH^from the f i r s t day of c u l t u r e u n t i l Daphnia reached peak l e v e l s and s t a r t e d to drop. T h i s does not n e c e s s a r i l y mean that NH^caused the m o r t a l i t i e s . At present, the t o x i c i t y of ammonia to D.pulex has not been i n v e s t i g a t e d yet, so no c o n c l u s i o n s can be made. In a d d i t i o n , the 0 ^ l e v e l s were very high, near s a t u r a t i o n l e v e l . S u s c e p t i b i l i t y of animals to NHj, p o i s o n i n g i s reduced when 0 S l e v e l s are maintained near s a t u r a t i o n l e v e l s (Kinne, 1976). Other t o x i c i t i e s to Daphnia have been i n v e s t i g a t e d . Taub and D o l l a r (1964) r e p o r t e d the t o x i c i t y of s a l t s o l u t i o n s l i k e 89 KNG^and NaNO^to D.pulex. They a l s o reported the p o s s i b i l i t y of v o l a t i l e contaminants in d i s t i l l e d water and to other t o x i c i t i e s that may be introduced through non-inert c o n t a i n e r s . Hydrogen s u l f i d e was a l s o r e p o r t e d to be t o x i c to Daphnia in s t a b i l i z a t i o n ponds (Dinges, 1974). Toxic m a t e r i a l s might have been present i n the d a i r y manure medium used which c o u l d have caused the m o r t a l i t i e s i n the present study. Scale-up C o n s i d e r a t i o n s The study was conducted with the i n t e n s i o n of a p p l y i n g i t in the P h i l i p p i n e s at SEAFDEC (Southeast Asian F i s h e r i e s Development Center) Aquaculture Department. Success was achieved in growing Scenedesmus obiiquus on d a i r y waste medium and t h i s c o u l d p o s s i b l y be expanded i n t o l a r g e - s c a l e o p e r a t i o n s f o r a q u a c u l t u r a l purposes. A g r i c u l t u r a l wastes are r e a d i l y a v a i l a b l e i n the P h i l i p p i n e s and the presence of s o l a r energy e l i m i n a t e s the problem of p r o v i d i n g a r t i f i c i a l l i g h t to the c u l t u r e s . The algae can be e i t h e r grown in b i g outdoor tanks or earthen ponds. However, there are c e r t a i n problems that must be overcome. Proper mixing should be employed to reduce a l g a l s e t t l i n g . Another problem i s h a r v e s t i n g the a l g a e . In tanks, t h i s i s no problem as water can be pumped d i r e c t l y i n t o the zooplankton c u l t u r e s . With -earthen ponds, t h i s can not be done as water becomes muddy when d i s t u r b e d . One s o l u t i o n to t h i s problem i s to have the bottom l i n e d with c o n c r e t e . Another s o l u t i o n i s to grow Daphnia with Scenedesmus sim u l t a n e o u s l y , but t h i s has to be i n v e s t i g a t e d f i r s t i f i t i s f e a s i b l e . 90 C u l t u r i n g Daphnia under the present method.can not s t i l l be a p p l i e d to l a r g e - s c a l e o p e r a t i o n s . Other s t r a i n s of Daphnia as we l l as some other cladocerans (e.g. Moina ) should be t r i e d and h o p e f u l l y , a much more robust species can be c u l t u r e d to withstand water a g i t a t i o n . 91 SUMMARY AND CONCLUSIONS The green alga Scenedesmus obli q u u s was s u c c e s s f u l l y grown in a medium using d i g e s t e d d a i r y manure as the n u t r i e n t source. Best growth r a t e s were achieved by a l g a l c u l t u r e s c o n t a i n i n g -1 n i t r o g e n c o n c e n t r a t i o n s of 400 and 800 ug-at'N-1 . Decreasing l i g h t i n t e n s i t i e s caused by high biomass at i n c r e a s i n g n i t r o g e n c o n c e n t r a t i o n s accounted fo r the lower growth.rates a t t a i n e d by a l g a l c u l t u r e s with high N - c o n c e n t r a t i o n s . On the other hand, c u l t u r e s with high N-concentrations produced more biomass than those with low N - c o n c e n t r a t i o n s . There was a high c o r r e l a t i o n found between the N-concentration consumed and the a l g a l biomass produced. Evidence showed that Scenedesmus can a l s o u t i l i z e o rganic n i t r o g e n aside from the i n o r g a n i c n i t r o g e n forms. A e r a t i o n enhanced both a l g a l growth r a t e s and biomass y i e l d s . There was evidence of COg - l i m i t a t i o n i n c u l t u r e s without a e r a t i o n . N u t r i e n t s were r e a d i l y made a v a i l a b l e to the algae due to bubbling. The tendency of Scenedesmus c e l l s to s e t t l e down was a l s o reduced by a e r a t i o n . N i t r o g e n to phosphorus atomic r a t i o i n d i g e s t e d d a i r y manure was observed to f a l l i n the range 15 to 25. A l t e r i n g t h i s r a t i o showed s i g n i f i c a n t d i f f e r e n c e on a l g a l growth. The medium with a N:P r a t i o of 69 produced a higher biomass than the two other media (N:P's of 4 and 22). The r e s u l t s show that there i s an advantage of a d j u s t i n g the N:P r a t i o . The three feeding l e v e l s used d i d not show any s i g n i f i c a n t d i f f e r e n c e s i n both Daphnia biomass y i e l d s and biomass c o n v e r s i o n e f f i c i e n c i e s . The algae always s e t t l e d down in the 9 2 tanks before being consumed s i n c e no a e r a t i o n was pro v i d e d . The amount of food a v a i l a b l e to Daphnia may have been the same d e s p i t e the feeding l e v e l with f a s t e r a l g a l s e t t l i n g r a t e observed i n higher feeding l e v e l s . I n t e n s i v e feeding d i d not in c r e a s e the biomass production of Daphnia. However, l a r g e r - s i z e d Daphnia were produced. Brood s i z e was i n c r e a s e d to a maximal of 30 - 40 young per animal. A . - 1 . higher d e n s i t y of 1.24 Daphnia-ml was obtained as a r e s u l t of the i n c r e a s e d production of young. Daphnia seem to reach a peak d e n s i t y i n c u l t u r e and d e c l i n e a f t e r w a r d s . The drop i s suspected to be e i t h e r caused by a shortage of • food supply as c u l t u r e s get denser or by the degeneration of the water q u a l i t y . There i s the problem of algae s e t t l i n g down even with i n t e n s i v e f e e d i n g . D i s s o l v e d oxygen and pH l e v e l s were always w i t h i n safe l i m i t s . Only the ammonia l e v e l s tended to accumulate as c u l t u r e s aged but i n very small amounts. No c o n c l u s i o n s can be made reg a r d i n g N H ^ t o x i c i t y s i n c e the t o x i c i t y of NH3 to IK Pulex i s not known at the present time. T h i s merits i n v e s t i g a t i o n . The batch and semi-continuous c u l t u r e systems appear to be the best methods of growing Dahpnia e s p e c i a l l y i n l a r g e - s c a l e o p e r a t i o n s . C u l t u r e s should be t o t a l l y harvested at t h e i r peak l e v e l or p a r t i a l l y harvested with a s u f f i c i e n t p o r t i o n allowed to regenerate and so f o r t h . Scenedesmus obliquus though grown s u c c e s s f u l l y i n d a i r y manure does not appear to be a very good food f o r Daphnia. The c e l l s may be too small and i t s tendency to s e t t l e down i s a major problem. 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An a l g a l mass c u l t u r e u n i t for feeding marine i n v e r t e b r a t e l a r v a e . 1-B D i v . F i s h . Oceanogr. Tech. Paper No.12. C.S.I.R.O. Melbourne, A u s t r a l i a . , Yurkowski, M. And S.L. Tabachek. 1978. Proximate and amino-acid composition of some n a t u r a l f i s h - f o o d s . Presented at the Symposium of F i n f i s h and Feed Tech. Sponsored by European Inland F i s h . A dvisory Comm., FAO. 103 Appendix 1 Daphnia biomass i n c u l t u r e s at 3-day i n t e r v a l s . ( i n mg.) Expt. c e l l s - m l 1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 50,000 36.2 69.0 61.5 — #1 100,000 17.0 67.9 126.4 78.5 -150,000 87.6 • 108.8 65.6 -50,000 36.7 60.2 1 02. 1 72.0 #2 100,000 20.2 39.0 112.9 191.9 264.2 150,000 43.9 8.1.6 228.7 100.5 50,000 31.7 38.6 77.3 138.7 #3 100,000 18.1 38.8 66. 1 99. 1 66.6 150,000 31.3 76.2 42.7 -#4 100,000 11.4 52.8+ 88.31 143.4+ 76.11 4.5 13.9 35.3 25.4 #5 100,000 20.5 53.81 4.8 80.01 170.2+ 234.6+ 14.7 4.4 46.6 Appendix 2 Daphnia frequency i n c u l t u r e s at 3-day. i n t e r v a l s . Expt. c e l l s - m l 1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 50,000 2,800 3,633 3,533 #1 100,000 1,000 3,500 7,433 3,966 150,000 3,083 8,633 3,266 50,000 1,800 5,466 7,366 3,266 #2 100,000 1,000 2,566 7,600 7,700 6,966 150,000 2,033 6,500 7,566 2,300 50,000 2,333 2,533 5,233 7,133 #3 100,000 1,000 2,533 3,866 4,033 1,933 150,000 2,866 3,800. 1,533 5,233+ 7,655+ 3,178± 869 1918 1260 #5 100,000 1,000 1,233+ 3,511+ 12,400± 8,822± 88 407 1473 1248 #4 100,000 1,000 1,100+ 1 27 105 Appendix 3 Biomass conversion e f f i c i e n c i e s of Daphnia c u l t u r e s at 3-day i n t e r v a l s . ( i n %) Expt. c e l l s - m l 1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 50,000 - 34.2 45.3 . 25.0 # 1 100,000 40.9 43.2 15.9 — 150,000 36.2 23. 1 8.1 50,000 27. 1 36.5 47.2 21.4 #2 100,000 15.2 38.0 46.8 49.5 150,000 13.5 17.0 38.2 10.9 50,000 #3 100,000 150,000 23.6 17.6 7.5 17.6 19.5. 15.9 33.7 21.8 4.6 49.3 9.8 #4 100,000 19.2±2.6 17.5±2.4 17.7+4.9 5.7+2.1 #5 100,000 17.311.2 15.6 + 2.3 21 . 0±1.8 19.9 + 5.0 106 Appendix 4 S 1 z e - f r e q u e n c y d i s t r i b u t i o n o f Dnphnia i n c u l t u r e s at 3-day I n t e r v a l s (Expt.#1). 50,000 c e l l s - m l " 100,000 c e l l s - m l " - 1 -150.000 c e l l s •ml" C l a s s I n t e r v a l , DAY 0 DAY 3 DAY 6 DAY 9 DAY 0 DAY 3 DAY 6 DAY 9 DAY 0 DAY 3 DAY 6 DAY 9 0 .60-0 .80 120 112 73 0 120 140 149 0 120 185 345 131 0 .81-1 .00 220 728 436 1 , 201 220 840 1 , 487 1 , 348 220 493 2 , 763 1 , 764 1 .01-1 . 20 100 280 944 7 1 100 140 1 ,932 79 100 431 1 ,381 131 1 .21-1 . 40 160 728 872 2 12 160 630 743 0 160 678 1 ,036 0 1. ,41-1 . 60 40 336 73 495 40 630 595 397 40 431 690 0 1 .61-1 . 80 40 56 73 7 1 40 70 297 159 40 0 5 18 65 1 .81-2 .00 80 1 12 145 424 80 140 595 476 80 62 863 196 2 .01-2 . 20 140 26 2 18 14 1 140 4 90 149 7 14 140 62 173 326 2 .2 1-2 . 40 20 224 363 353 20 2 10 1 ,040 397 20 432 518 196 2 . ,41-2 . 60 • 20 168 0 353 20 0 297 159 20 0 173 131 2 6 1-2 .80 0 0 2 18 353 0 70 0 79 0 62 173 196 2 . 8 1-3 .00 60 0 2 18 0 60 70 0 79 GO 123 0 85 3 01-3 . 20 0 0 0 0 0 0 149 0 0 62 0 0 3 . 2 1-3 . 40 0 0 O 0 0 0 0 79 0 0 0 65 3 . 41-3 . 60 0 0 0 0 0 0 0 0 0 62 0 0 3 . 61-3 . 80 0 0 0 0 0 0 0 0 0 0 0 0 3 . 8 1-4 .00 0 0 0 0 O 0 0 0 0 0 0 0 T o t a 1 1 ,000 2.800 3, ,633 3 , 533 1 ,000 3,500 7 ,433 3,966 1 I .000 3 ,083 8.633 3.266 S i z e - f r e q u e n c y d i s t r i b u t i o n of Daphnia In c u l t u r e s at 3-day 1nterva1s(Expt.H7 ) 50.000 e e l IS' ml" 1 1OO,000 e e l 1 s • m 1 " A 150,OOO e e l 1s.ml C l a s s I n t e r v a 1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 0 . 6 1 -0 . 80 40 36 109 0 0 40 5 1 152 0 75 40 0 0 0 0 0 . 8 1 - 1 .00 100 432 1 . 858 3, 388 1 , 306 100 975 1 , , 368 770 139 100 69 1 1 ,040 15 1 0 1 .01 - 1 . 20 120 180 1,093 1, .473 587 120 257 2, , 280 308 557 120 285 1 ,430 303 0 1 .21 - 1 . 40 120 144 438 442 0 1.20 308 456 1 , 232 4 18 120 203 650 908 - 0 1 . 4 1 - 1 .60 80 180 656 590 O 80 103 912 914 557 80 122 1 , 170 908 138 1 .61 - 1 . 80 80 144 2 19 295 131 80 205 456 1 , 848 557 80 122 260 1 ,815 230 1 .81 -2 .00 80 144 109 442 131 80 0 304 154 697 80 81 650 454 46 2 . 01 -2 . 20 120 72 219 0 131 120 205 304 462 557 120 4 1 520 303 4 14 2 . 2 1 -2 . 40 140 252 109 147 131 140 154 456 924 697 140 122 520 2 ,118 460 2 . 4 1 -2 .60 40 72 438 44 2 26 1 40 308 456 462 1,811 40 244 0 303 644 2 . 61 -2 .80 60 36 109 0 457 60 0 304 462 697 60 81 0 303 368 2 . 8 1 -3 .00 20 72 109 147 131 20 0 152 154 279 20 4 1 130 O 0 3 . 01 -3 . 20 0 36 0 0 0 0 0 0 0 0 0 0 130 0 0 3 . 2 1 -3 . 40 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 . 4 1 -3 . 60 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 . 61 -3 . 80 0 0 0 0 0 0 0 0 0 O 0 0 0 0 0 3 . 8 1 -4 .00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 T o t a l 1 ,000 1 ,800 5,466 7. 36G 3,266 1 ,ooo 2 , 566 7 , 600 7 , 600 6.966 1 .000 2, ,033 6,500 7 , 566 2 , 300 > CD Ch H' X I-1 O -v3 S i z e - f r e q u e n c y d i s t r i b u t i o n of Daphnia 1n c u l t u r e s at 3-day 1 n t e r v a 1 s ( E xpt .//3 ) 50,000 c e l l s - m r 1 100.000 c e l l s - m l " - 1 150,OOO c e l l s - m l * - 1 C l a s s I n t e r v a l DAY 0 DAY 3 DAY G DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 0 .61-0.80 20 0 0 105 0 20 101 0 0 0 20 0 0 0 0 . 81 - 1.00 340 980 405 2 , 302 2 ,568 340 507 773 887 0 340 688 304 61 1 .01- 1 .20 80 140 203 1 . 150 856 80 405 155 8 1 39 80 745 228 61 1 .2 1 - 1 .40 80 280 557 209 428 80 304 6 18 823 155 80 516 532 0 1 .4 1 - 1 . 60 60 93 658 105 142 60 253 464 0 155 60 0 836 123 1 .61-1 .80 40 140 152 105 428 40 _152 464 7 16 348 40 57 380 307 1 .81 -2.00 60 47 203 105 7 14 60 203 - 309 403 385 60 1 15 684 276 1 .01-2.20 40 233 101 3 14 0 40 O 232 484 232 40 286 304 582 2 . 21-2.40 120 93 5 1 105 7 14 120 101 309 484 309 120 1 15 228 92 2 . 4 1 -2 .60 140 47 101 3 14 570 140 203 155 403 155 140 1 15 152 31 2 61-2.80 20 186 51 105 428 20 152 155 81 155 20 229 76 0 2 . 81 - 3.00 0 47 5 1 209 285 0 101 232 161 0 O 0 76 0 3 . 01-3.20 0 0 0 105 0 0 51 0 0 0 O 0 0 3 . 2 1 -3.40 0 47 0 0 0 0 0 0 0 0 O 0 0 O 3 . 41-3.60 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 . 61-3.80 0 0 0 0 0 0 0 0 0 0 0 0 0 . 0 3 . 81 - 4.00 0 \ 0 0 0 0 0 0 0 0 0 O 0 0 • 0 Tota 1 1.000 2, ,333 2, ,533 5. 233 7. 133 1 .000 2. ,533 3. ,866 4 .033 1 .933 1.000 2 ,866 3 .800 1 . 533 S i z e - f r e q u e n c y d i s t r i b u t i o n of Oaphnla In c u l t u r e s at 3-day 1nterva1s(Expt . #4 ) R e p l i c a t e 1 R e p l i c a t e 2 R e p l i c a t e 3 C l a s s I n t e r v a l DAY O DAY 3 DAY 6 DAY 9 DAY 12 DAY O DAY 3 DAY 6 DAY 9 DAY 12 DAY O DAY 3 DAY 6 DAY 9 DAY 12 0. 6 1-0. 80 o 21 187 592 44 0 0. 81-1 . 00 320 248 1 ,213 5 . 130 880 320 1. 01-1 . 20 320 83 840 395 0 320 1. .21-1. . 40 160 21 654 395 0 160 1. .41-1. .60 20 0 373 1 , 579 44 20 1. .61-1 . 80 40 21 747 197 132 40 1 .81-2 .00 40 41 0 987 176 40 2 .01-2 . 20 0 4 1 93 197 220 0 2 .21-2 . 40 O 4 1 93 0 440 O 2 .41-2 .60 40 83 93 197 132 40 2 .61-2 .80 0 103 93 197 44 0 2 .81-3 .00 60 83 0 0 0 60 3 .01-3 . 20 0 123 0 0 44 0 3 .21-3 .40 0 103 0 0 0 0 3 .41-3 .60 0 2 1 93 0 0 0 3 .61-3 .80 0 0 187 0 0 0 3 .81-4 ,oo 0 0 0 0 44 0 0 249 0 368 0 0 192 133 55 354 1,994 2 ,959 2 , 300 320 240 2 , 208 1,466 1 ,476 101 1 ,496 257 276 320 100 672 267 109 51 748 257 92 160 0 576 800 109 76 623 257 - 92 20 40 192 667 0 101 125 643 O 40 40 384 400 55 127 249 386 0 40 140 0 1 ,066 0 76 125 386 184 O 20 96 267 109 0 0 901 368 0 60 0 800 164 0 125 129 552 40 0 96 133 273 25 0 129 92 0 0 0 267 109 51 125 0 92 60 180 0 0 55 51 0 0 0 0 60 0 0 55 177 0 0 0 0 120 96 0 0 76 125 0 92 0 0 192 133 164 0 249 129 92 0 0 96 267 0 0 O O 0 0 0 0 0 0 T o t a l 1.000 1.033 4.666 9.866 2,200 1,000 1,266 6,233 6,433 4,600 1.000 1.000 4.800 6,666 2,733 S i z e - f r e q u e n c y d i s t r i b u t i o n of Daphnia In c u l t u r e s at 3-day 1nterva1s(Expt.US ) . R e p l i c a t e 1 R e p l i c a t e 2 R e p l i c a t e 3 Z1 ass Interva1 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 DAY 0 DAY 3 DAY 6 DAY 9 DAY 12 0 .61 -0 . 80 0 0 310 0 0 0 26 2 16 528 0 0 23 184 O 0 0 . 8 1 - 1 .00 160 76 2 , 165 3. , 458 1 , 294 160 78 2 ,088 4 . 224 2 ,053 160 204 1 .657 2 , 354 1 . 363 1 .01 - 1 . 20 140 5 1 155 2 , 926 296 140 52 72 2 .640 747 140 O 61 2 , 140 779 1 . 2 1 - 1 . 40 40 101 465 3 , .458 148 40 26 288 1 . 848 560 40 0 368 1 . 926 389 1 . 4 1 - 1 .60 260 101 77 1 , . 330 296 260 104 0 2 .112 747 260 45 184 1 , 926 1 , 168 1 .61 - 1 . 80 60 76 77 0 888 60 78 72 0 933 60 0 0 856 973 1 ,81 -2 .00 60 152 0 798 592 60 130 0 264 560 60 63 61 0 1 , 363 2 .01 -2 . 20 60 177 77 532 1 .036 60 182 72 264 560 60 , 226 184 2 14 779 2 . 2 1 -2 . 40 20 177 77 0 1 , 332 20 260 144 264 1 , 680 20 204 61 214 1 , 168 2 . 4 1 -2 .60 100 51 0 0 , 148 100 130 288 0 933 100 136 61 0 584 2 61 -2 . 80 40 152 77 266 148 40 52 72 792 0 40 91 0 642 194 2 . 81 -3 . 00 20 127 77 0 148 20 104 144 0 0 20 136 184 214 0 3 . 01 -3 . 20 0 0 155 266 296 0 0 72 0 373 0 0 0 0 584 3 . 21 -3 . 40 20 0 77 0 148 20 52 72 264 187 20 0 0 0 389 3 . 41 -2 . 60 20 25 77 266 0 20 26 0 0 0 20 0 0 2 14 0 3 . 61 -3 . BO 0 0 0 0 0 0 0 0 0 0 0 0 6 1 0 0 3 . 81 -4 . OO 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 T o t a l 1.00O 1.266 3.866 13000 7.400 1.000 1.300 3,600 13200 9.333 1.OOO 1,133 3.066 10700 9,733 D a l l y measurements of Daphnia t o t a l l e n g t h and s i z e range In Growth Rate Experiment #1. T o t a l Length (mm.) F e e d i n g l e v e l DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 50.000 c e l l s - m f * 0.85+0.06 1.08+0.07 1.42+0.07 1.53*0.15 1.66*0.08 1.91*0.09 1.96*0.05 2*22*0.16 2.16+0.03 2.36+0.0 100.000 e e l Is-ml"^ 0.85*0.06 1.12+0.07 1.44*0.08 1.76*0.08 1.82+0.21 2.68+0.09 2.42*0.08 2*4*5+0.12 2.43+0.06 2.44+0.0 150.OOO c e l l s . m l ' 0.85+0,06 1.12+0.05 1.45+0.08 1.74+0.09 1?88+0.20 2.15+0.13 2*^20+0.08 2.57+0.16 2.47+0.12 2.52+0.0 * f 1 r s t a d u l t i n s t a r o b s e r v e d • • • f i r s t youngs o b s e r v e d S i z e Range (mm.) F e e d i n g l e v e l DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 " DAY 7 DAY 8~ DAY 9 DAY 10 50.OOO e e l Is-ml"^ 0.83/0.89 0.99/1. 19 1.29/1.52 1.35/1.78 1.45/1.75 1.75/2.08 1.91/2.01 2.11/2.31 2.14/2.21 2.24/2.4 100.000 e e l Is. ml'+ 0.83/0.89 1 .06/1 .19 1 . 29/1 . 55 1 .62/1 .88 1 .58/2. 15 '1.91/2.18 2.31/2.54 2.28/2.61 2.34/2.51 2 . 38/2 . 6 150.OOO c e l l s - m l " 1 O.83/0,89 1.06/1.19 1.39/1.52 1.58/1.88 1.72/2.21 1.88/2.28 2.18/2.31 2.41/2.84 2.31/2.64 2.38/2.6 D a l l y measurement of Daphnia t o t a l l e n g t h and s i z e range i n Growth Rate Experiment til . -y T o t a l Length (mm.) Fe e d i n g l e v e l DAY 1 DAY 2 DAY 3 DAY 4 DAY 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 50.000 c e l l s - m f * 0.92+0.11 1.14*0.09 1.42*0.15 1.77+0.132.06+0.21 2*34*0.21 2.52+0.102*59+0.192.92*0.072.91*0.0 100.000 e e l Is.ml"* 0.92*0. 1 1 1.14+0.11 1.45+0.16 1.78+0.15 2 . 15+0 . 09 2 ?*44+0. 17 2.62t0.22 2**33+0.13 2.94+0.04 2.96+0.0 150.000 c e l l s - m l " 1 0.92+0.11 1.13+0.07 1.49±0.14 1.81+0.16 2.*32+0.10 2.51+0.14 2**61+0.16 2.71+0.10 3.00+0.11 3.01+0.0 * f t r s t a d u l t i n s t a r o b s e r v e d * * f i r s t youngs o b s e r v e d S1 ze Range (mm. ) F e e d i n g l e v e l DAY 1 DAY 2 DAY 3 DAY 4 DAY* 5 DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 50.OOO e e l Is.mf* 0.76/1 . 12 1.02/1.32 1.16/1.55 1.58/1.95 1.75/2.31 2.1 1/2.67 2.31/2.64 2.08/2.74 2.81/2.97 2.87/2.9 100.OOO e e l 1s-ml 0.76/1.12 1.02/1.09 1.19/1.78 1.55/2.01 2.05/2.28 2.24/2.67 2.28/2.94 2.44/2.81 2.87/2.97 2.90/3.0 150.OOO c e l l s - m l 0.76/1.12 1.06/1.25 1.19/1.68 1.62/1.98 2.18/2.44 2.34/2.77 2.31/2.81 2.57/2.94 2.94/3.14 2.94/3.1 112 Appendix 10 Length-weight r e l a t i o n s h i p of Daphnia with and without eggs and embryos. Daphnia w/o eggs & embryos Daphnia with eggs & embryos T.Length(mm.) Weight(l0 mg.) T.Length(mm.) WeightOO mg.) 0.84 2.1 1.93 20.5 0.92 5.3 1 .96 26.4 1.08 4.7 2.15 28.0 1.13 7.8 2.16 35.8 1.14 6.6 2.17 28.7 1 .32 7.5 2.20 20.8 1 .35 9.6 2.22 31.4 1 .42 7.5 2.22 33.2 1 .45 10.0 2.36 33.0 1 .49 10.8 2.41 37.3 1 .53 9.1 2.42 46.6 1 .66 17.3 2.44 36.0 1 .74 12.5 2.45 48.8 1 .75 23.1 2.51 51 .2 1 .76 11.7 2.51 57.0 1 .77 19.3 2.54 55.5 1 .78 17.0 2.56 56.9 1 .80 12.5 2.56 61.5 1.81 14.7 2.61 62.3 1 .82 19.2 2.63 69.6 2.06 27.0 2.64 78.0 2.07 31.2 2.66 56.7 2.08 25.0 2.66 63.5 2.15 29. 0 2.69 67. 1 2.19 38.2 2.86 69.0 2.27 24.0 2.91 82.2 2.31 44.0 2.92 89.4 2.31 50.0 2.94 76.6 2.33 31.5 3.02 84.4 2.38 45.5 3.06 114.8 2.43 32.0 3.07 96.2 2.43 47.0 3.08 97.8 2.45 59.0 3.14 108.0 2.54 66.0 3.27 124.0 2.61 57.0 3.37 144.0 2.67 68.0 3.50 146.0 2.74 55.0 3.63 150.0 2.77 90.0 3.70 141.0 2.84 78.0 3.86 173.0 2.97 91 .0 3.10 76.0 3.17 125.0 3.54 143.0 1 Appendix 11a ALG EXPT #1 Analysis for K Analysis of variance table Sum of Source squares DF TREAT 0.S9S76 4 . REP 0.1681OE-01 1 . Residual 0.12840E-01 4. Total 0.62641 9. Overal1 mean O.9830O Mean square 0.14919 0.16810E-01 0.3210OE-O2 Overal1 standard deviation O.26382 F-rat1o P r o b a b i l i t y Test term 46.477 5.2368 0.00131 0.08401 RESIDUAL RESIDUAL Frequencies, means, standard deviations for TREAT 1. 4. 2 0.87500 0.87500 2 1 . 18O0 1 . 1800 2 1 .3100 1 .3100 2 0.940O0 0.94000 0.61000 0.61000 O MEAN OSTDV o1l9^E-01 OMSEE-01 O'MM o! 14,4^ -01 0.14142E-01 S ERR M OAwlil-Ol 0.4O062E-01 0.40O62E-01 0.40062E-01 0.40062E-01 Homogeneity of variance test B a r t l e t t Degrees Layard Size Factors Chl-square P r o b a b i l i t y of freedom Ch1-square P r o b a b i l i t y warn TREAT 5.1516 0.27209 4 9.8718 0.04264 < 10 Time for homogeneity of variance test was 0.18229E-02 seconds. M u l t i p l e range tes t s Duncan test at 1% p r o b a b i l i t y level There are 4 homogeneous subsets which are l i s t e d as follows: ) . 4. ) . 2. ) , 3. ) Duncan test at 5% p r o b a b i l i t y l e v e l There are 3 homogeneous subsets which are 1tsted as follows: ( 5. ) ( 1.. 4. ) ( 2., 3. ) Time f o r mu l t i p l e range test was 0.58724E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 5 0 MEAN 0.94200 P MEAN 0.94200 0 STDV 0.25193 5 1.0240 1.0240 0.29821 S ERR M 0.25338E-01 O.25338E-01 Homogeneity of variance test B a r t l e t t Degrees Layard Factors Chl-square P r o b a b i l i t y of freedom Chi-square REP 0.10064 0.75106 1 0.20054 Time f o r homogeneity of variance test was 0.16667E-02 seconds. M u l t i p l e range t e s t s F - r a t l o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.08401 STOP Size P r o b a b i l i t y warn 0.65428 < 10 114 Appendix l i b -ALG EXPT #1 Analysis f o r CELLY Analysis of variance table Source TREAT REP Residual Total Sum of squares 0.12786E+13 0.84100E+10 O.68440E+1 1 0.135S5E+13 DF 4. 1 . 4. 9. Mean square F-rat lo 0.31966E+12 18.S82 0.8410OE+10 0.49153 0.17110E+11 Overal1 Overall mean standard deviation 0.15286E+07 0.38808E+06 P r o b a b i l i t y Test term 0.00748 0.52189 RESIDUAL RESIDUAL Frequencies, means, standard deviations for TREAT 1 . 2. 3. 4. 5. 2 2 2 2 2 0 MEAN 0.99350E+06 0.14155E+07 0.14400E+07 0.17225E+07 0.20715E+07 P MEAN 0.993S0E+0S O.14155E+07 0.14400E+07 0.17225E+07 0.20715E+07 0 STDV 2121.3 31820. 53740. 19092. 0.26941E+06 S ERR M 92493. 92493. 92493. 92493. 92493. Homogeneity of variance test B a r t l e t t Degrees Layard Size Factors Chl-square P r o b a b i l i t y of freedom Cht-square P r o b a b i l i t y warn TREAT 10.512 0.03263 4 18.098 0.00118 < 10 Time for homogeneity of variance test was 0.16797E-02 seconds. M u l t i p l e range te s t s Duncan test at 1% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as follows: ( 1.. 2., 3. ) ( 2.. 3.. 4. ) ( 4.. 5. ) Duncan t e s t at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as follows: ( 1. ) ( 2.. 3., 4. ) ( 4 . 5 . ) Time f o r m u l t i p l e range test was 0.56380E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 5 S 0 MEAN 0.15576E+07 0. 14996E+07 P MEAN 0.15576E+07 0.14996E+07 0 STOV 0.47324E+OG 0. 33587E»06 S ERR M 58498. 58498. Homogeneity of variance test B a r t l e t t Degrees Layard Factors Chi-square P r o b a b i l i t y of freedom Chi-square REP 0.41008 0.52193 1 0.61398 Time f o r homogeneity of'variance test was 0.17579E-02 seconds. M u l t i p l e range t e s t s F-rat1o Is not s i g n i f i c a n t at p r o b a b i l i t y 0.52189 STOP Size P r o b a b i l i t y warn 0.43329 < 10 Appendix 1-2 a ALG EXPT #2 Analysis for K Source TREAT REP Residual Total Analysis of variance tablt Sum of Mean squares DF square F-rat 1o P r o b a b i l i t y Test term 0.201 16 0.39996E-04 0.19360E-01 0.22056 20.781 0.00028 RESIDUAL 0.19998E-04 0.82636E-02 0.99178 RESIDUAL 0.24200E-02 4. 0.50290E-01 2 . 8. 14 . Overal1 Overall mean standard deviation 1.0260 O.12552 Frequencies, means, standard deviations for TREAT t. 2. 3. 4. 5. 3 3 3 3 3 O MEAN 1.2067 1.1000 0.99667 0.95000 0.87667 P MEAN 1.2067 I.10OO 0.99667 0.95000 0.87667 0 STDV 0.30551E-01 0.34641E-01 0.50332E-01 0.300OOE-01 0.64291E-01 S ERR M 0.28402E-01 0.28402E-01 0.28402E-01 0.28402E-01 0.28402E-01 Homogeneity of variance test B a r t l e t t Degrees Layard Size Factors Ch1-square P r o b a b i l i t y of freedom Chl-square P r o b a b i l i t y warn TREAT 1.5947 0.80974 4 2.5675 0.63259 < 10 Time for homogeneity of variance test was 0.17188E-02 seconds. M u l t i p l e range tests Duncan test at 1% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as follows: ( 5.. 4.. 3. ) ( 3 . 2 . ) ( 2 . 1 . ) Duncan test at 5% p r o b a b i l i t y level There are 4 homogeneous subsets which are l i s t e d as follows: ( 5.. 4. ) ( 4 . 3 . ) ( 1. ) Time f o r mul t i p l e range test was 0.61719E-02 seconds. Frequencies, means, standard deviations for REP 5 5 0 MEAN 1.0240 1.0260 P MEAN 1.0240 1.0260 0 STDV 0.14011 0.13278 S ERR M 0.22OO0E-01 0.22OO0E-01 Homogeneity of variance test 5 1 .0280 1.0280 0. 13368 0.220OOE-01 B a r t l e t t Degrees Layard Factors Chi-square P r o b a b i l i t y of freedom Chi-square REP 0.12459E-01 0.99379 2 0.2348IE-01 for homogeneity of variance test was 0.16146E-02 seconds. M u l t i p l e range t e s t s F - r a t l o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.99178 Size P r o b a b i l i t y warn 0.98833 < 10 STOP Appendix 12b lie-' A n a l y s i s f o r CELLY A n a l y s i s of v a r i a n c e t a b l e S o u rce TREAT REP ' R e s i d u a l T o t a l Sum of squares 0.82405E+13 0.34830E+11 0.17950E+12 0.84548E+13 DF Mean square F - r a t 1o 4. 0.20601E+13 91.81.5 2. 0. 174 15E+ 1 1 0.77614 8. 0.22438E+11 14 . P r o b a b i l i t y T e s t term 0.00000 0.49196 CELLY Overa11 mean 0. 18270E+07 Overa11 s t a n d a r d d e v i a t i o n 0. 77712E+06 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2 . 3 . 4 . ALG EXPT HI 0 MEAN P MEAN 0 STDV S ERR M 3 0.82300E+06 0.82300E+06 22000. 86483. 3 0. 13583E+07 0. 13583E+07 51326 . 86483. 3 0.15943E+07 0.15943E+07 0.14889E+06 86483. 3 0.26057E+07 0.26057E+07 88008. 86483. 5 . 0.27537E+07 0.27537E+07 0.27227E+06 86483. RESIDUAL RESIDUAL Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees La y a r d S i z e F a c t o r s C h t - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn TREAT 9.3835 0.05220 4 11.382 0.02259 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.16667E-02 seconds. M u l t i p l e range t e s t s Duncan t e s t at 1% p r o b a b i l i t y l e v e l T h e r e a r e 3 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 1. ) ( 2., 3 . ) ( 4.. 5. ) Duncan t e s t a t 5% p r o b a b i l i t y l e v e l T h e r e a r e 3 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 1. ) ( 2.. 3 . ) ( 4.. 5. ) Time f o r m u l t i p l e range t e s t was 0.56511E-02 seconds. F r e q u e h c i es, . 1 means, s t a n d a r d d e v i a t i o n s f o r REP .2 .3 0 MEAN P MEAN 0 STDV S ERR M 5 0.18180E+07 0. 18180E+07 0.87 18 1E+06 66989. 0.18900E+07 0.18900E+07 0.90660E+06 66989. 0.17730E+07 0.17730E+07 0.72321E+06 66989. Homogeneity of v a r i a n c e t e s t F a c t o r s REP B a r t l e t t Ch i - s q u a r e 0. 20056 Degrees L a y a r d S i z e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn 0.90458 2 0.58680 0.74572 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.15755E-02 seconds. STOP Appendix 13a ALG EXPT #3 Analysis f o r K Source TREAT REP Residual Total Analysis of variance table Sum of squares 0.27183 0.17334E-03 0.22093E-01 0.29409 DF 4 . 2. 8. 14 . Mean square F - r a t l o P r o b a b i l i t y Test term 0 G7957E-01 24.607 0.00015 RESIDUAL 0.86670E-04 0.31383E-01 0.96922 RESIDUAL 0.27617E-02 Overa11 Overall mean standard deviation 1.0273 0.14494 Frequencies, means, standard deviations for TREAT 1. 2. 3. 4. 5. 3 3 3 3 3 0 MEAN 1.2200 1.1267 0.99000 O.97000 0.83000 P MEAN 1.2200 1.1267 0.99000 0.970O0 O.8300O 0 STDV 0.40O00E-01 0.41633E-01 0.65574E-01 0.43589E-01 0.40O00E-01 S ERR M 0.30341E-01 0.3O341E-01 0.30341E-01 0.30341E-01 0.30341E-01 Homogeneity of variance test B a r t l e t t Degrees Layard Size Factors Chi-square P r o b a b i l i t y of freedom Chi-square P r o b a b i l i t y warn TREAT 0.68668 0.95296 4 1.1573 0.88507 < 10 Time for homogeneity of variance test was 0.17058E-02 seconds. M u l t i p l e range te s t s Duncan test at 1% p r o b a b i l i t y level There are 4 homogeneous subsets which are l i s t e d as follows: ( 5., 4. ) ( 4.. 3. ) ( 3 . 2 . ) ( 2 . 1 . ) Duncan test at 5% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as follows: ( 5. ) ( 4.. 3. ) ( 2 . 1 . ) Time f o r m u l t i p l e range test was 0.66536E-02 seconds. Frequencies, means, standard deviations for REP .1 .2 .3 5 5 0 MEAN 1.0260 1.0240 P MEAN 1.0260 1.0240 0 STDV 0.13957 0.14223 S ERR M 0.23502E-01 0.23502E-01 5 1.0320 1.0320 O. 18377 O.23502E-01 Homogeneity of variance test B a r t l e t t Degrees Layard Factors Chi-square P r o b a b i l i t y of freedom Chi-square REP 0.35667 0.83666 2 O.67542 Time for homogeneity of variance test was 0. 16016E-02 seconds. M u l t i p l e range t e s t s P r o b a b i I i t y 0.71340 Size warn < 10 F - r a t i o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.96922 STOP 118 Appendix 13b ALG EXPT #3 A n a l y s i s f o r CELLY A n a l y s i s of v a r i a n c e t a b l e S o u rce TREAT REP Res1dual T o t a l CELLY Sum of squares 0. 13804E+13 0.31712E+11 0. 17855E+12 0. 15906E+13 O v e r a l l mean 0. 14130E+07 DF 4 . 2 . 8 . 14 . Mean square 0.34509E+12 0. 1585GE+1 1 0. 22319E+11 F - r a t 1 o 15.462 0.71042 Overal1 s t a n d a r d d e v i a t i o n 0.33707E+06 P r o b a b i l i t y T e s t term 0.00078 0. 52000 RESIDUAL RESIDUAL F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 2 . 5. 3 3 3 3 3 0 MEAN 0.13757E+07 0.13278E+07 0.11325E+07 0.12327E+07 0.19963E+07 P MEAN 0.13757E+07 0.13278E+07 0.11325E+07 0.12327E+07 0.19963E+07 0.24254E+06 0.17719E+06 0.11504E+06 30587. 27186.. 86253. 86253. 86253. 0 STDV S ERR M 86253. 86253. Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s Ch1-square P r o b a b i l i t y of freedom Ch1-square P r o b a b i l i t y warn TREAT 9.1561 0.05731 4 15.583 0.00363 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.16667E-02 seconds. M u l t i p l e range t e s t s Duncan t e s t at 1% p r o b a b i l i t y l e v e l T h ere a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 3 . . 4 . . 2 . . 1 . ) / ( 5. ), Duncan t e s t at 5% p r o b a b i l i t y l e v e l T h e r e a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 3 . . 4 . . 2 . . 1 . ) ( 5. ) Time f o r m u l t i p l e range t e s t was 0.51302E-02 seconds. F r e q u e n c i e s , m e a n s , s t a n d a r d d e v i a t i o n s f o r REP 0 MEAN P MEAN 0 STDV S ERR M 0. 14700E+07 0. 14700E+07 0.37611E+06 66812. 0.13574E+07 0.13574E+07 0.34411E+06 66812. 0.14 116E+07 0. 14116E+07 0.36036E+06 66812. Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees Layard' F a c t o r s Ch1-square P r o b a b i l i t y of freedom C h l - s q u a r e P r o b a b i l i t y REP 0.28412E-01 0.98589 2 0.40041E-01 0.98018 Time f o r homogeneity of v a r i a n c e t e s t was 0.16016E-02 seconds. M u l t i p l e r ange t e s t s F - r a t i o i s not s i g n i f i c a n t a t p r o b a b i l i t y 0.52000 STOP S i z e warn < 10 11 Appendix ika. ALG EXPT #4 An a l y s i s f o r K Analysis of variance table Sum of Mean Source squares DF square F - r a t i o P r o b a b i l i t y Test term TREAT 2.5603 4. 0.64007 241.23 O.OOOOO RESIDUAL REP 0.37334E-03 2. 0.18667E-03 0.70352E-01 0.93264 RESIDUAL Residual 0.21227E-01 8. 0.26533E-02 Total 2.5819 14. Overal1 Overall mean standard deviation 1.1473 0.42944 Frequencies^'means. standard deviations for TREAT 3 3 3 3 .J — 0 MEAN 1.8967 1.2667 0.98000 0.84000 0.75333 P MEAN 1.8967 1.2667 O.98000 0.84000 0.75333 0 STDV 0.92916E-01 0.25166E-01 0.17320E-01 0.20O00E-01 0.28867E-01 S ERR M 0.29740E-01 0.29740E-01 0.2974OE-01 0.29740E-01 0.29740E-01 Homogeneity of variance test B a r t l e t t Degrees Layard Size Factors Ch1-square P r o b a b i l i t y of freedom Chl-square P r o b a b i l i t y warn TREAT 7.4238 0.11512 4 4.3685 0.35842 < 10 Time f o r homogeneity of variance test was 0.16927E-02 seconds. M u l t i p l e range tests Duncan test at 1% p r o b a b i l i t y level There are 4 homogeneous subsets which are l i s t e d as follows: ( 5.. 4. ) ( 4 . 3 . ) ( 2. ) ( 1. ) Duncan test at 5% p r o b a b i l i t y level There are 4 homogeneous subsets which are l i s t e d as follows: ( 5 . 4 . ) ( 3. ) ( 2. ) ( 1. ) Time f o r m u l t i p l e range test was 0.66667E-02 seconds. Frequencies~~means. standard deviations for REP . 1 5 5 S n u c A M 1 1420 1•15 4° 1 1 4 6 0 2 M1£N 1420 L1S40 1-1460 0 STDV O 4 22° 0.48066 0.49435 S ERR M 0.23O36E-01 O.23036E-O1 O.23036E-01 Homogeneity of variance test B a r t l e t t .D«?'1!HL Chl-square P r o b a b i l i t y warn Factors Chl-square P r o b a b i l i t y of freedom Ch square Q 9 0 B 0 2 < , 0 REP 0.13243 0.93593 •< Time for homogeneity of variance test -as 0. 16406E-02 seconds. M u l t i p l e range t e s t s F - r a t i o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.93264 120 Appendix 14b ALG EXPT #4 Analysis for CELLY Analysis of variance table Source TREAT REP Residual Total Sum of squares O.19568E+15 O.47982E+12 O.10422E+13 O.19720E+15 DF 4. 2 . 8. 14. Mean square 0.48920E+14 O.23991E+12 O.13028E+12 375.50 1 .8415 P r o b a b i l i t y Test term 0.00000 O.21985 RESIDUAL RESIDUAL Overa11 Overall mean standard deviation 0.10418E+08 0.37531E+07 Frequencies, 1 . standard deviations for TREAT 2. 3. 4. 0 MEAN P MEAN 0 STDV S ERR M O. 50108E+07 0.50108E+07 O.46429E+06 0.20839E+06 0.79088E+07 0.79088E+07 0.29175E+06 0.20839E+0G 0.11 108E+08 0.11 108E+08 0.5G896E+06 0.20839E+06 12790E+08 12790E+08 1450OE+0G 20839E+06 O.15275E+OB 0. 15275E+08 0.340OOE+06 0.20839E+06 Homogeneity of variance test B a r t l e t t Degrees Layard Size Factors Chl-square P r o b a b i l i t y of freedom Ch1-square P r o b a b i l i t y warn TREAT 2.8885 0.576G5 4 5.9449 0.20330 < 10 Time f o r homogeneity of variance test was 0. 16927E-02 seconds. M u l t i p l e range t e s t s Duncan test at 1% p r o b a b i l i t y level There are 5 homogeneous subsets which are l i s t e d as follows: ( 1. ) ( 2. ) ( 3. ) ( 4. ) ( 5. ) Duncan test at 5% p r o b a b i l i t y level There are 5 homogeneous subsets which are l i s t e d as follows: ( 1. ) ( 2. ) ( 3. ) ( 4. ) ( S. ) Time f o r m u l t i p l e range test was 0.71094E-02 seconds. Frequencies, means, standard deviations for REP . 1 5 5 0 MEAN 0.10355E+08 O.10238E+08 P MEAN 0.10355E+08 O. 10238E+08 0 STDV 0.43557E+07 O. 39274E*07 S ERR M O. 16142E+06 0. 16142E+06 Homogeneity of variance test O.10662E+0B 0. 10662E*08 0.38449E*07 0.16142E+OG B a r t l e t t Degrees Layard Factors Chl-square Probabl11ty of freedom Chi-square REP 0.G5492E-01 0.9G778 2 0.14154 for homogeneity of variance test was 0.1G01GE-02 seconds. M u l t i p l e range t e s t s F-rat1o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.21985 Probabl1ity 0.93168 Size warn < 10 Appendix 1 5 a 121 ALG EXPT 05 A n a l y s i s f o r K A n a l y s i s of v a r i a n c e t a b l e Sum of Mean Source squares DF square F - r a t 1 o TREAT 0.23583E-02 3. 0.78611E-03 0. 39915 REP 0.11667E-03 2. 0.58334E-04 0.29619E Res i d u a l 0.11817E-01 s 6. 0.19695E-02 T o t a l 0.14292E-01 11. Overa11 Overa11 mean -standard d e v i a t i o n K 0.98917 0.3G045E-01 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2 . 3. 4. 3 3 3 3 0 MEAN 0. 98000 0.98333 0.98000 1 .0133 P MEAN 0. 98000 0.98333 0.98000 „,,<•- 1 .0133 0 STDV 0. 36056E -01 0.40415E -01 0.36056E-01 0. 41633E-01 S ERR M 0. 25622E -01 0.25622E -01 0.25622E-01 0. 25622E-01 P r o b a b i l i t y T e s t term 0.75886 RESIDUAL 0.97096 RESIDUAL Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees Layard S i z e F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn TREAT 0.56569E-01 0.99648 3 0.13433 0.98742 < 10 Time f o r homogeneity of v a r i a n c e test-was 0.17058E-02 seconds. M u l t i p l e range t e s t s F - r a t 1 o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.75886 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP .1 .2 .3 4 4 4 0 MEAN 0.990*00 0.98500 0 99250 P MEAN 0.990O0 0.98500 0^99250 0 STDV 0.21603E-01 0.54467E-01 0.35940E-01 S 'ERR M 0.22189E-01 0.22189E-01 0.22189E-01 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees Layard F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y REP 2.0421 0.36022 2 2.8100 0.24536 Time f o r homogeneity of v a r i a n c e t e s t was 0.15625E-02 seconds. M u l t i p l e range t e s t s F - r a t i o Is not s i g n i f i c a n t a t p r o b a b i l i t y 0.97096 . - -STOP S i z e warn < 10 122 Appendix .15b ALG EXPT #5 Anal y s i s f o r CELLY Analysis of variance table Source Sum of squares Mean DF square F - r a t l o Probabl11ty Test term TREAT REP Residual Total 0.10474E+15 0.25474E+13 0.63371E+13 0.11362E+15 3. 0.34913E+14 2. 0.12737E+13 S. 0. 10562E-M3 1 1 . 33.055 1 .2059 0.00040 0.36289 RESIDUAL RESIDUAL CELLY Overall mean 0.57648E+07 Overal1 standard deviation 0.32139E+07 Frequencies, means, standard 1. 2. deviations for TREAT 3. 4. • MEAN P MEAN 0 STDV S ERR M 3 3 3 0.17985E+07 0.44150E+07 0.71525E+07 0. 0.179B5E+07 0.44150E+07 0.71525E+07 0. 0 15242E+06 0.46808E+06 O.60486E+06 0. O.S933SE+06 0.59335E+06 0.59335E+06 0. 3 96933E+07 9G933E+07 19581E+07 59335E+06 Homogeneity of variance test B a r t l e t t Degrees Layard Factors Chl-square P r o b a b i l i t y of freedom Ch1-square TREAT 8.8741 0.03101 3 8.7317 Time f o r homogeneity of variance test was 0.18229E-02 seconds. M u l t i p l e range te s t s Duncan test at 1% p r o b a b i l i t y level There are 3 homogeneous subsets which are l i s t e d as follows: ( 1.. 2. ) ( 2-. 3. ) ( 3.. 4. ) Duncan t e s t at 5% p r o b a b i l i t y level There are 4 homogeneous subsets which are l i s t e d as follows: ( 1. ) < 2. ) ( 3. ) ( 4. ) Time f o r mu l t i p l e range t e s t was 0.55469E-02 seconds. Frequencies, means, standard deviations f o r REP .1 .2 .3 4 4 4 0 MEAN 0.52563E*07 0.566S4E+07 0.63719E+07 P MEAN 0.525S3E*07 0.566G4E+07 0.S3719E+07 0 STDV 0.26736E*07 0.34297E+07 0.42560E+07 S ERR M 0.51385E+O6 0.51385E+O6 0.51385E+OG Homogeneity of variance t e s t B a r t l e t t Degrees Layard Factors Chl-square P r o b a b i l i t y of freedom Chl-square REP 0.34990 0.75961 2 1.1474 Time f o r homogeneity of variance test was 0.15755E-02 seconds. M u l t i p l e range t e s t s F - r a t l o 1s not s i g n i f i c a n t at p r o b a b i l i t y 0.36289 STOP Probabl1ity 0.03308 Size warn < 10 Size P r o b a b i l i t y warn 0.56345 < 10 ALG EXPT #5 Ana'lysls f o r K Source TREAT REP Res 1dua1 T o t a l Appendix 1 5 c A n a l y s i s of v a r i a n c e t a b l e Sum of squ a r e s 0.82755E-01 0. 18289E-01 0.56445E-02 0. 10G69 DF 2 . 2 . 4 . 8 . Mean square 0.41378E-01 0.91445E-02 0.14 111E-02 F - r a t 1o 29.323 6.4803 Probab111ty T e s t term 0.00408 0.05562 RESIDUAL RESIDUAL Overal1 mean 1.1311 Overal1 s t a n d a r d d e v i a t i o n 0.11548 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT . 1 . 2 . 3. 3 3 3 0 MEAN. 1.2400 1.1467 1.0067 P MEAN 1.2400 1.1467 1:0067 0 STDV 0.40000E-01 0.94517E-01 0.37859E-01 S ERR M. 0.21688E-01 0.21688E-01 0.21688E-01 Homogeneity of, v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom Ch1-square P r o b a b i l i t y warn TREAT 1.8503 0.39647 2 2.3816 0.30398 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.15885E-02 seconds. M u l t i p l e range t e s t s Duncan t e s t a t 1% p r o b a b i l i t y l e v e l T h e r e a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : (. 3.. 2. ) ( 2. , 1 . ) Duncan t e s t a t 5% p r o b a b i l i t y l e v e l T h e r e a r e 3 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 3. ) ( 2. ) ( 1. ) Time f o r m u l t i p l e range t e s t was 0.42708E-02 seconds. F r e q u e n c i e s , . 1 means, s t a n d a r d d e v i a t i o n s f o r REP .2 .3 0 MEAN P MEAN 0 STDV S ERR M 3 1 .0733 1 .0733 0. 1 1372 0.21688E-01 3 1 . 1367 1 . 1367 0. 13051 0.21688E-01 3 1 . 1833 1.1833 0. 1 1930 0. 21688E-01 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h l - s q u a r e P r o b a b i l i t y of freedom C M - s q u a r e P r o b a b i l i t y warn REP 0.32352E-01 0.98395 2 0.77128E-01 0.96217 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.15755E-02 seconds. M u l t i p l e range t e s t s F - r a t 1 o Is not s i g n i f i c a n t a t p r o b a b i l i t y 0.05562 STOP Appendix 16a ALG EXPT 06 A n a l y s i s f o r K Source TREAT REP Res 1dua1 T o t a l A n a l y s i s of v a r i a n c e t a b l e Sum of squares 0.44242 O.G1G66E-03 O.24050E-01 0. 46709 DF Mean square 12k F - r a t 1 o P r o b a b i l i t y T e s t term 6 1 1 3. 0.14747 36.792 0.00029 RESIDUAL 2. 0.30833E-03 0.76922E-01 0.92686 RESIDUAL O.40083E-02 Overa11 O v e r a l l mean s t a n d a r d d e v i a t i o n 1.6358 0.20607 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 2 . 3 1.7533 1 .7533 3 1 . 7433 1 . 7433 3 1 . 7433 1 . 7433 0 MEAN P MEAN 0 STDV 0.6G583E-01 0.66583E-01 0.15276E-01 0.56863E-01 S ERR M 0.36553E-01 0.36553E-01 0.36553E-01 0.36553E-01 3 1 .3033 1 .3033 Homogeneity of v a r i a n c e t e s t B a r t l e t t >. Degrees L a y a r d S i z e F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn TREAT 2.9917 0.39291 3 . 9.3312 0.02520 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.17447E-02 seconds. M u l t i p l e range t e s t s ' Duncan t e s t at 1% p r o b a b i l i t y l e v e l There a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 4. ) ( 2. . 3. , 1 . ) Duncan t e s t a t 5% p r o b a b i l i t y l e v e l There a r e 2 homogeneous s u b s e t s which a re l i s t e d as f o l l o w s : ( 4. ) ( 2. . 3. . 1 . ) Time f o r m u l t i p l e range t e s t was 0.45182E-02 seconds. F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP .1 .2 .3 4 4 4 0 MEAN 1.6450 1.6350 1.6275 P MEAN 1.6450 1.6350 1.6275 0 STDV 0.22189 0.19122 0.26399 S ERR M 0.31656E-01 0.31656E-01 0.31656E-01 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn REP 0.27214 0.87278 2 0.40349 0.81730 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.17839E-02 seconds. M u l t i p l e range t e s t s F - r a t 1 o Is not s i g n i f i c a n t a t p r o b a b i l i t y 0.92686 STOP 1 Appendix 16b ALG EXPT #6 Analysis for CELLY Analysis of variance table Source Sum of squares Mean DF square F - r a t i o Probabt11ty Test term TREAT REP Residual Total 0.14388E+15 0.68007E+12 0.20000E+13 0.14656E+15 3. 0.479G1E+14 2. 0.34003E+12 6. 0.33333E+12 11. Overal1 143.88 1.0201 0.00001 0.41557 RESIDUAL RESIDUAL CELLY Overal1 mean 0.65858E+07 standard deviation 0.36502E+07 Frequencies, 1 . means, standard 2. deviations for TREAT 3. 4. 3 3 3 3 0 MEAN 0.23008E+07 0.48925E+07 0.74700E+07 0.11680E+08 P MEAN O.23OO8E*07 O.48925E+07 0. 74700E+07 0.11680E+08 0 STDV 0.22500E+06 0.410O2E+06 0.10252E+07 0.26520E+06 S ERR M 0.33333E+06. 0.33333E+06 0.33333E+06 0.33333E+06 Homogeneity of variance test B a r t l e t t Degrees Layard Factors Chl-square P r o b a b i l i t y of freedom Chl-square TREAT 4.9G02 0.17474 3 4.3658 Time f o r homogeneity of variance test was 0.18359E-02 seconds. M u l t i p l e range t e s t s Duncan t e s t at 1% p r o b a b i l i t y level There are 4 homogeneous subsets which are l i s t e d as follows: Size P r o b a b i l i t y warn 0.22458 < 10 ( 3. ) ( 4. ) Duncan test at 5% p r o b a b i l i t y l e v e l There are 4 homogeneous subsets which are l i s t e d as follows: ( 1 ) ( 2. ) ( 3. ) ( 4. ) Time f o r m u l t i p l e range test was 0.56641E-02 seconds. Frequencies, means, standard deviations f o r REP .1 .2 .3 4 4 - 4 0 MEAN 0.65856E+O7 0.62944E+07 0.68775E+07 P MEAN 0.65B56E+07 0.62944E+07 0.68775E+07 0 STDV 0.40507E+07 0.41259E+07 0.38983E+07 S ERR M 0.28867E+06 0.28867E+O6 0. 2B867E+06 Homogeneity of variance t e s t B a r t l e t t Degrees Layard Size Factors Chi-square P r o b a b i l i t y of freedom Chl-square P r o b a b i l i t y warn REP 0.87032E-02 0.99566 2 0.18446E-01 0.99082 < 10 Time f o r homogeneity of variance test was 0.15755E-02 seconds. M u l t i p l e range te s t s F - r a t i o Is not s i g n i f i c a n t at p r o b a b i l i t y 0.415S7 STOP Appendix 17a ALG EXPT tn A n a l y s i s f o r K Source TREAT REP R e s i d u a l T o t a l A n a l y s i s of v a r i a n c e t a b l e Sum of squ a r e s 0. 12250E-02 0.22501E-03 0.22501E-03 0.16750E-02 Overal1 mean 1 . 3175 DF 1 . 1 . 1 . 3. Mean square 0.12250E-02 0.22501E-03 0.22501E-03 Overal1 s t a n d a r d d e v i a t i o n 0.23629E-01 F- r a t 1 o 5.4441 1.0000 P r o b a b i l i t y T e s t term 0. 25777 0. 50000 RESIDUAL RESIDUAL F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2 . 2 0 MEAN 1.3000 P MEAN 1.3000 0 STDV 0.0 2 1 .3350 1 .3350 0.21214E-01 N S ERR M 0.10607E-01 0.10607E-01 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees La y a r d S i z e F a c t o r s Ch1-square P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn TREAT U n c a l c u l a b l e due t o s t a n d a r d d e v i a t i o n of z e r o . Time f o r homogeneity of v a r i a n c e t e s t was 0.13281E-02 seconds. M u l t i p l e range t e s t s F - r a t 1 o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.25777 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP .1 .2 2 2 0 MEAN 1.3100 1 :3250 P MEAN 1 .3 100 1 .3250 0 STDV 0.14142E-01 0.35355E-01 S ERR M 0.10607E-01 0.10607E-O1 Homogeneity o f v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s Ch1-square P r o b a b i l i t y of freedom C h l - s q u a r e P r o b a b i l i t y warn REP 0.49545 0.48151 1 1.3304 0.24873 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.1G927E-02 seconds. M u l t i p l e range t e s t s F - r a t l o Is not s i g n i f i c a n t a t p r o b a b i l i t y 0.50000 STOP 127 Appendix 17b ALG EXPT HI A n a l y s i s f o r CELLY A n a l y s i s of v a r i a n c e t a b l e S o u rce TREAT REP Res 1dua1 T o t a l Sum of squares 0. 13340E-M3 0.53592E+13 O. 12656E+13 0.79589E+ 13 DF 1 . 1 . 1 . 3 . Mean square 0.13340E+13 0.53592E+13 0. 12656E+13 F - r a t 1o 1.0540 4.2344 P r o b a b i l i t y T e s t term 0. 49162 0. 28798 RESIDUAL RESIDUAL CELLY Overal1 mean 0. 85875E+07 Overa11 s t a n d a r d d e v i a t i o n 0. 16288E+07 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2 . 2 2 o 0 MEAN 0.91650E+07 0.80100E+07 P MEAN 0.91650E+07 0.80100E+07 0 STDV 0.24324E+07 0.84146E+06 S ERR M 0.79550E+06 0.79550E+06 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d F a c t o r s Ch1-square P r o b a b i l i t y of freedom Ch1-square TREAT 0.64187 0.42303 1 1.7215 Time f o r homogeneity of v a r i a n c e t e s t was 0.16276E-02 seconds. M u l t i p l e range t e s t s F - r a t i o 1s not s i g n i f i c a n t at p r o b a b i l i t y 0.49162 S i z e P r o b a b l 1 1 t y warn 0. 18950 < 10 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP .1 .2 2 2 0 MEAN 0.97450E+07 O.74300E+07 P MEAN 0.97450E+07 0.74300E+07 0 STDV 0.16122E+07 21213. S ERR M 0.79550E+06 0.79550E+06 Homogeneity of v a r i a n c e t e s t B a r t l e t t D e g r e e s A L a y a r d F a c t o r s C h l - s q u a r e P r o b a b i l i t y of freedom" C h l - s q u a r e REP 4.8503 0.02764 1 25.013 Time f o r homogeneity o f v a r i a n c e t e s t was 0.17188E-02 seconds. M u l t i p l e r a n g e t e s t s F - r a t l o Is not s i g n i f i c a n t a t p r o b a b i l i t y 0.28798 STOP S i z e P r o b a b i l i t y warn O.O0000 < 10 1(28 Appendix 18a ALG EXPT #9 A n a l y s i s f o r K Source TREAT REP Res 1dua t T o t a l A n a l y s i s of v a r i a n c e t a b l e Sum of squares 0.10689E-01 0.15089E-01 0.96445E-02 0.35422E-01 DF 2 . 2 . 4 . 8 . Mean square 0.53444E-02 0.75445E-02 ,0.24111E-02 F - r a t 1 o 2 . 2 166 3. 1290 P r o b a b i l i t y Test term 0. 22498 0. 15205 RESIDUAL RESIDUAL Overal1 mean 1 . 4556 Overa11 s t a n d a r d d e v i a t i o n 0.G6542E-01 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2. 3. 3 3 3 0 MEAN 1.4633 1.4933 . 1.4100 P MEAN 1.4633 1.4933 1.4100 0 STDV 0.37859E-01 0.55076E-01 0.88882E-01 S ERR M 0.28350E-01 0.28350E-01 0.28350E-01 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s Ch1-square P r o b a b i l i t y of freedom C h l - s q u a r e P r o b a b i l i t y warn TREAT 1.1662 0.55817 2 2.0261 0.36311 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.16016E-02 seconds. M u l t i p l e range t e s t s F - r a t l o 1s not s i g n i f i c a n t at p r o b a b i l i t y 0.22498 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP .1 .2 .3 3 1 .4233 1 .4233 3 1.4 300 1 .4 300 0 MEAN P MEAN 0 STDV 0.76376E-01 0.55677E-01 0.35119E-01 S ERR M 0.28350E-01 0.28350E-01' 0.28350E-01 3 1.5133 1.5133 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom REP 0.91109 0.63410 2 La y a r d C h i - s q u a r e 1 . 8634 S i z e Probab111ty warn 0.39389 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.16146E-02 seconds. M u l t i p l e range t e s t s F - r a t l o 1s not s i g n i f i c a n t at p r o b a b i l i t y 0.15205 STOP 129 ALG EXPT #9 A n a l y s i s f o r CELLY Appendix 18b Source TREAT REP Res 1dua1 T o t a l CELLY A n a l y s i s of v a r i a n c e t a b l e Sum of squares 0.69562E+13 0. 14574E+12 0.63468E+12 0.773G6E+13 Overa11 mean " 0.51661E+07 DF 2 . 2 . 4 . 8 . Mean square 0.34781E+13 0.728G9E+11 0. 158G7E+ 12 F- r a t 1 o 21 .920 0.45925 P r o b a b i l i t y T e s t term 0.00G99 0.66138 RESIDUAL RESIDUAL Overal1 s t a n d a r d d e v i a t i o n 0. 98340E+06 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2. 3 3 3 0 MEAN 0.45000E+07 0.45900E+07 0.64083E+07 P MEAN 0.45000E+07 0.45900E+07 0.64083E+07 0 STDV • 0.39752E+06 0.34868E+06 0.33258E+06 S ERR M 0.22998E+06 0.22998E+06 0.22998E+06 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom Ch1-square P r o b a b i l i t y warn TREAT 0.57151E-01 0.97183 2 0.13341 0.93547 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.16146E-02 seconds. M u l t i p l e range t e s t s Duncan t e s t at 1% p r o b a b i l i t y l e v e l There a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 1 . . 2. ) Duncan t e s t at 5% p r o b a b i l i t y l e v e l T h ere a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 1 . , 2. ) ( 3. ) Time f o r m u l t i p l e range t e s t was 0.38672E-02 seconds. Frequenc i e s , . 1 means, s t a n d a r d d e v i a t i o n s f o r REP .2 " .3 0 MEAN P MEAN 0 STDV S ERR M . 53233E+07 . 53233E+07 . 12359E+07 22998E+06 0.50117E+07 0.50117E+07 0 . 99925E + 06 0.22998E+06 Homogeneity of v a r i a n c e t e s t F a c t o r s REP B a r t l e t t C h i - s q u a r e 0.73380E-01 0. 5 1633E+07 0.51633E+07 0.11267E+07 0. 22998E+06 P r o b a b l 1 1 t y 0.96398 Degrees of freedom 2 Lay a r d Ch t-square 0. 17655 Probab11 1 ty 0.91551 S i z e warn < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.16276E-02 seconds M u l t i p l e range t e s t s F - r a t 1 o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.66138 STOP 100 Appendix 19a ALG E X P T . u s A n a l y s i s f o r K A n a l y s i s of v a r i a n c e t a b l e Mean DF square F - r a t i o P r o b a b i l i t y T e s t term 1. 0.13500 385.70 0.00258 RESIDUAL 2. 0.37050E-01 105.85 0.00936 RESIDUAL 2. 0.35002E-03 5 . Overal1 s t a n d a r d d e v i a t i o n 0.20484 So u r c e TREAT REP Res i dua1 T o t a l Sum of squares 0. 13500 0.74100E-01 0.70003E-03 0. 20980 Overa11 mean 1 . 7500 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2 . 0 MEAN P MEAN 0 STDV S ERR M 3 1.9000 1.9000 0. 14933 0.10801E-01 3 1.6000 1.6000 0.12288 0.10801E-01 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h l - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn TREAT 0.60428E-01 0.80582 1 0.14659 0.70182 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.17058E-02 seconds. M u l t i p l e range t e s t s Duncan t e s t at 1% p r o b a b i l i t y l e v e l T here a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 2. ) ( -1 . ) Duncan t e s t at 5% p r o b a b i l i t y l e v e l T here a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 2. ) ( 1 . ) Time f o r m u l t i p l e range t e s t was 0.35287E-02 seconds. F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP .1 .2 .3 2 2 2 0 MEAN 1.7 100 1.8050 1.7350 P MEAN 1.7 100 1.8050 1.7350 0 STDV 0.'28284E-01 0.21920 0.38891 S ERR M O.12757 • 0.12757 0.12757 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d F a c t o r s Ch1-square P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y REP 2.7226 O.25633 2 10.522 0.00519 Time f o r homogeneity of v a r i a n c e t e s t was 0.18880E-02 seconds. M u l t i p l e range t e s t s F - r a t i o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.87032 STOP . . •131 Appendix 19b ALG EXPT #8 A n a l y s i s f o r CELLY A n a l y s t s of v a r i a n c e t a b l e S o u rce TREAT REP R e s i d u a l T o t a l Sum of squares 0.10101E+14 0. 1232GE+12 0.11 132E+12 0. 10336E+14 DF 1 . 2 . 2 . 5 . Mean square 0.10101E+14 0.61G29E+11 0.55G62E+11 F- r a t 1 o 181 .47 1 . 1072 P r o b a b i l i t y T e s t term 0.00547 0.47456 RESIDUAL RESIDUAL CELLY Overa11 mean 0.26808E+07 Overa11 s t a n d a r d d e v i a t i o n . 0.14377E+07 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 • 2 • 3 3 • 0 MEAN 0.39783E+07 0.13833E+07 P MEAN 0.39783E+07 0.13833E+07 , 0 STDV . 0.31262E+06 0.13985E+06 S ERR M 0.13621E+06 0.13621E+06 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s Ch1-square P r o b a b i l i t y of freedom Ch1-square P r o b a b i l i t y warn TREAT 0.93982 0.33232 1 1.7922 0.18066 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.16276E-02 seconds. . M u l t i p l e range t e s t s Duncan t e s t a t 1% p r o b a b i l i t y l e v e l T h ere a r e 2 homogeneous s u b s e t s which a re l i s t e d as f o l l o w s : ( 2. ) ( 1. ) Duncan t e s t at 5% p r o b a b i l i t y l e v e l T h ere a r e 2 homogeneous subsets, which a r e l i s t e d as f o l l o w s : ( 2. ) ( 1. ) Time f o r m u l t i p l e range t e s t was 0.33594E-02 seconds. F r e q u e n c i e s , means, s t a n d a r d dev1 a t i o n s . f o r REP .1 . 2 . 3 2 2 2 0 MEAN 0.28775E+07 0.2G250E+07 0.25400E+07 P MEAN 0.28775E+07 0.26250E+07 0.25400E+07 0 STDV 0.19622E+07 0.19799E+07 0.15G27E+07 S ERR M 0.16683E+06 0.16683E+06 0.16683E+06 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom Ch1-square P r o b a b i l i t y warn REP 0.46991E-01 0.97678 2 0.14105 0.93191 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.15885E-02 seconds. M u l t i p l e range t e s t s F - r a t l o Is not s i g n i f i c a n t a t p r o b a b i l i t y 0.47456 STOP Appendix 2 0 a . 1-32 DAP EXPT/M .2,3 A n a l y s i s for- BIOM A n a l y s i s of v a r i a n c e t a b l e S o u rce TREAT REP Res1dual T o t a l Sum of squares 5437.2 18180. 12813. 36431. DF 2 . 2 . 4 . 8 . Mean square 2718.6 9090.1 3203.3 F - r a t 1o 0.84869 2.8377 P r o b a b i l i t y T e s t term 0.49291 0. 17092 RESIDUAL RESIDUAL BIOM Overa11 mean 134.80 Overal1 s t a n d a r d d e v i a t i o n 67.482 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2. 3. 0 MEAN P MEAN 0 STDV S ERR -'M 3 103.27 103.27 34.865 32.677 3 163 . 23 163 . 23 88.499 32 .677 3 137.90 137.90 80.307 32.677 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e TREAT 1.3233 0.51600 2 3.2167 Time f o r homogeneity of v a r i a n c e t e s t was 0.17968E-02 seconds. M u l t i p l e range t e s t s F - r a t i o 1s not s i g n i f i c a n t a t p r o b a b i l i t y 0.49291 P r o b a b l 1 1 t y 0. 20022 S i z e warn < 10 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP .1 .2 .3 3 3 3 0 MEAN 101.40 198.33 104.67 P MEAN , 101.40 198.33 104.67 0 STDV 29.407 85.210 31.620 S ERR M 32.677 32.677 32.677 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h l - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn REP 2.4551 0.29301 2 2.8809 0.23682 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.15885E-02 seconds. M u l t i p l e range t e s t s F - r a t i o i s not s i g n i f i c a n t a t p r o b a b i l i t y 0.17092 STOP Appendix 20b DAP EXPT#1,2,3 A n a l y s i s f o r BCEFF A n a l y s i s of v a r i a n c e t a b l e S o u rce TREAT REP R e s i d u a l T o t a l BCEFF Sum of squares 442.58 424.42 309.70 1 176 . 7 Overal1 mean 38.51 1 DF 2 . 2 . 4 . 8 . Mean square 22 1 . 29 212.21 77.424 Overa11 s t a n d a r d d e v i a t i o n 12. 128 F- r a t 1 o 2.8581 2.7408 P r o b a b i l i t y T e s t term 0. 16948 0. 17797 RESIDUAL RESIDUAL F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 0 MEAN P MEAN 0 STDV S ERR M 1 . 3 47.267 47 . 267 2.0008 5.0802 2 . 3 38 . 167 38. 167 14 .520 5.0802 3 30.100 30.100 12.338 5.0802 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom Ch1-square P r o b a b i l i t y TREAT 4.3481 0.11372 2 12.862 0.00161 Time f o r homogeneity of v a r i a n c e t e s t was 0.16016E-02 seconds. M u l t i p l e range t e s t s F - r a t i o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.16948 S i z e warn < 10 F r p a u e n d e s , means, s t a n d a r d d e v i a t i o n s f o r REP .1 -2 -3 3 3 3 0 MEAN 4 1.567 44.967 29.000 P MEAN 4 1.567 44.967 29.000 0 STDV 4.7648 5.9719 17.826 J S ERR M 5.0802 5.0802 5.0802 Homogeneity of v a r i a n c e t e s t B a r t l e t t Oegrees L a y a r d S i z e F a c t o r s C h l - s q u a r e P r o b a b i l i t y of freedom Ch1-square • P r o b a b i l i t y warn REP 3.3317 0.18903 2 3.6563 0.16071 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.15885E-02 seconds. M u l t i p l e range t e s t s F - r a t i o Is not s i g n i f i c a n t a t probab111ty 0.17797 STOP v 134-Appendix 2 1 a DAP EXPT#4,5 A n a l y s i s f o r BIOM A n a l y s i s of v a r i a n c e t a b l e S o u rce TREAT REP Res 1dua1 T o t a l BIOM Sum of squares 12485. 5698 . 1 1143.9 19327. Overal1 mean 189.02 DF 1 . 2 . 2 . 5 . Mean square 12485. 2849 . 1 571.96 Overa11 s t a n d a r d d e v i a t i o n 62 . 173 F - r a t 1 o 21 .829 4.9812 P r o b a b i l i t y Test term 0.04289 0. 16719 RESIDUAL RESIDUAL F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2. 0 MEAN ' P MEAN 0 STDV S ERR M 3 143.40 143.40 35.286 13.808 3 234.63 234.63 46.647 13.808 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d F a c t o r s Ch1-square P r o b a b i l i t y of freedom, Ch1-square TREAT O.12305 0.72575 1 0.29012 Time f o r homogeneity of v a r i a n c e t e s t was 0.16406E-02 seconds. M u l t i p l e range t e s t s Duncan t e s t a t 5% probab111ty l e v e l T h e r e a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 1 . ) ( 2. ) Time f o r m u l t i p l e range t e s t was 0.20703E-02 seconds. F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP S i z e P r o b a b i l i t y warn 0.59015 < 10 1 0 MEAN P MEAN 0 STDV S ERR M 2 158.30 158.30 39. 174 16.911 2 177.60 177.60 86.691 16.911 2 231 . 15 231 . 15 67 .670 16.911 Homogeneity of v a r i a n c e t e s t B a r t l e t t S i z e warn 0.56137 < 10 Degrees Layard F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom C h l - s q u a r e P r o b a b i l i t y REP 0.39743 0.81978 2 1.1548 Time f o r homogeneity of v a r i a n c e t e s t was 0.16927E-02 seconds. M u l t i p l e range t e s t s F - r a t i o 1s not s i g n i f i c a n t at p r o b a b i l * t y O.J.6J7J9 — STOP Appendix 21b 1-3^ DAP EXP#4,5 A n a l y s i s f o r BCEFF A n a l y s i s of v a r i a n c e t a b l e Source TREAT REP Res1dual T o t a l Sum of squares 16.007 43 . 773 10.253 70.033 DF 1 . 2 . 2. 5. Mean square 16.007 21 .887 5. 1267 F- r a t 1 o 3. 1222 4 . 2692 P r o b a b i l i t y T e s t term 0.21927 0. 18978 RESIDUAL RESIDUAL BCEFF Overal1 mean 19.367 Overa11 s t a n d a r d d e v i a t i o n 3 . 7426 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 . 2 . 3 3 0 MEAN 21.000 17.733 P MEAN 21.000 17.733 0 STDV 1.8193 4.8686 S ERR M 1.3072 1.3072 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom Ch1-square P r o b a b i l i t y warn TREAT 1.3501 0.24526 1 2.4687 0.11614 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.17318E-02 seconds. M u l t i p l e range t e s t s \ F - r a t 1 o Is not s i g n i f i c a n t at p r o b a b i l i t y O.21927 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP 1 .2 .3 0 MEAN P MEAN 0 STDV S ERR M 2 18.200 18.200 2.4042 1.6010 16.800 16.800 4.5255 1 .6010 2 23.100 23.100 0.0 1 .6010 Homogeneity o f v a r i a n c e t e s t B a r t l e t t Degrees ' L a y a r d S i z e F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom C h l - s q u a r e P r o b a b i l i t y warn REP U n c a l c u l a b l e due to s t a n d a r d d e v i a t i o n of z e r o . Time f o r .homogenelty of v a r i a n c e t e s t was 0.13412E-02 seconds. M u l t i p l e r ange t e s t s F - r a t 1 o 1s not s i g n i f i c a n t a t p r o b a b i l i t y 0.18978 STOP --• 136 Appendix 22a GROWTH R/M A n a l y s i s f o r TL Source TREAT REP Res 1dua1 T o t a l A n a l y s i s of v a r i a n c e t a b l e Sum of squares 0.64013E-01 0. 51693E-01 0.37387E-01 0. 15309 DF Mean square 2. 0.32007E-01 4. 0.12923E-01 8. 0.4G733E-02 14 . F - r a t lo 6.8488 2 . 7653 P r o b a b i l i t y Test term 0.01848 0. 10305 RESIDUAL RESIDUAL TL Overal1 mean 2 . 4427 Overal1 s t a n d a r d d e v i a t i o n 0.10457 F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 3. 5 2 . 5220 2.5220 0. 1 1862 0.30572E-01 1 . 2. 5 , 5 0 MEAN . 2.3620 2.4440 P MEAN 2.3620 2.4440 0 STOV 0.74632E-01 0.51283E-01 S ERR M 0.30572E-01 0.30572E-01 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h l - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn TREAT 2.4677. 0.29117 2 3.4273 0.18021 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.16276E-02 seconds. M u l t i p l e range t e s t s , Duncan t e s t at 5% p r o b a b i l i t y l e v e l T h ere a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 1., 2. ) . ( 2. . 3. ) Time f o r m u l t i p l e range t e s t was 0.26302E-02 seconds. F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP • 1 -2 .3 .4 3 0 MEAN 2.4567 P MEAN 2.4567 0 STDV 0.4X3414E-01 S ERR M 0.39469E-01 3 2.4333 2 .4333 0. 80829E-01 0. 39469E-01 3 2.3400 2 .3400 0.88882E-01 0. 39469E-01 3 2 . 4633 2 .4633 0. 12858 0. 39469E-01 . 5 2 . 5200 2 . 5200 0. 13454 O. 39469E;-01 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h i - s q u a r e P r o b a b l 1 1 t y of freedom C h i - s q u a r e P r o b a b i l i t y warn R E P 2.4113 0.66059 4 5.4338 0.24561 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.17448E-02 seconds. M u l t i p l e range t e s t s F - r a t i o i s not s i g n i f i c a n t at p r o b a b i l i t y 0.10305 STOP Appendix 22b 137 GROWTH R#2 A n a l y s i s f o r TL Source TREAT REP R e s i d u a l T o t a l TL A n a l y s i s of v a r i a n c e t a b l e Sum of squares 0. 24040E-01 0.93067E-02 0. 15093E-01 0.48440E-01 Overa11 mean 2.9580 DF Mean square 2 . 0. 12020E-01 4. 0.23267E-02 8. 0.18867E-02 14 . Overal1 s t a n d a r d d e v i a t i o n 0.58822E-01 F - r a t 1o 6 . 37 10 1 . 2332 P r o b a b i l i t y T e s t term 0.02213 0. 3G980 RESIDUAL RESIDUAL F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r TREAT 1 3. 5 3 .0080 3 .0080 5 5 0 MEAN . 2.9100 2.95G0 P MEAN 2.9100 2.9560 0 STDV 0.30000E-01 0.43359E-01 0.57620E-01 S ERR M 0.19425E-01 0.19425E-01 0.19425E-01 Homogeneity of v a r i a n c e t e s t B a r t l e t t Degrees , L a y a r d S i z e F a c t o r s C h i - s q u a r e P r o b a b i l i t y of freedom C h i - s q u a r e P r o b a b i l i t y warn TREAT 1.4514 0.48399 2 1.7924 0.40812 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.166G7E-02 seconds. M u l t i p l e range t e s t s Duncan t e s t at 5% p r o b a b i l i t y l e v e l T h ere a r e 2 homogeneous s u b s e t s which a r e l i s t e d as f o l l o w s : ( 1 . . 2. ) ( 2.. 3. ) Time f o r m u l t i p l e range t e s t was 0.26433E-02 seconds. F r e q u e n c i e s , means, s t a n d a r d d e v i a t i o n s f o r REP • 1 -2 .3 , a 0 MEAN 2.9600 P MEAN 2.9600 0 STDV 0.34641E-01 S ERR M 0.25078E-01 3 2 .9467 2 . 9467 0. 50332E-01 0.25078E-01 3.0000 3.OOOO 0. lOOOO i 0.25078E-01 3 2 .9233 2.9233 0.68069E-01 0.25078E-01 Homogeneity of v a r i a n c e t e s t 3 2.9600 2 .9600 0. 346.4 IE-01 0. 25078E-01 B a r t l e t t Degrees L a y a r d S i z e F a c t o r s C h l - s q u a r e Probab111ty of freedom Ch1-square P r o b a b i l i t y warn REP 2.8198 0.58841 4 3.8880 0.42138 < 10 Time f o r homogeneity of v a r i a n c e t e s t was 0.17578E-02 seconds. M u l t i p l e range t e s t s F - r a t i o i s not s i g n i f 1 c a n t . a t p r o b a b i l i t y 0.36980 

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