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Effect of the interaction between two species of marine diatoms on their individual copper tolerance Metaxas, Anna 1989

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E F F E C T O F T H E I N T E R A C T I O N B E T W E E N T W O S P E C I E S O F M A R I N E D I A T O M S O N T H E I R I N D I V I D U A L C O P P E R T O L E R A N C E B y A N N A M E T A X A S B . S c , M c G i l l U n i v e r s i t y , 1986 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L 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 T H E D E G R E E O F M A S T E R O F S C I E N C E i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department o f Oceanography) W e accept this thesis as c o n f o r m i n g to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A A u g u s t 1989 © A n n a M e t a x a s , 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of OCEANOGRAPHY The University of British Columbia Vancouver, Canada Date August 10, 1989 DE-6 (2/88) - i i -A B S T R A C T T h e importance o f species interactions i n studies assessing the trace metal tolerance o f i n d i v i d u a l species, has been largely o v e r l o o k e d . M o s t tox ic i ty assessments i n v o l v e single-species tests. A l t h o u g h relevant i n some cases, this approach does not incorporate the b i o l o g i c a l enviroment o f an o r g a n i s m as a factor that m i g h t inf luence its s u r v i v a l . O n the other hand, this factor is incorporated i n studies at the c o m m u n i t y l e v e l . H o w e v e r , the latter, not e x a m i n i n g the mechanisms that dr ive the interactions i n the c o m m u n i t y , d o not a l l o w for predic t ion o f the outcome after the addi t ion o f a stress inducer , such as trace metals. T h e purpose o f this study was to examine the effect o f the interact ion o f t w o species o f marine diatoms (Skeletonema costatum (Cleve) G r e v i l l e and Nitzschia thermalis (Ehrenberg) A u e r s w a l d ) o n their i n d i v i d u a l copper tolerances. T h e t w o species were g r o w n i n unia lga l cultures i n order to determine their copper tolerance. 5 . costatum d i d not exhib i t growth above 5 x 10 _ 7 M ( p C u = 8.46) and N. thermalis above 6 x 10-7 M added total copper ( p C u = 8.36). Skeletonema exh ib i ted increased g r o w t h rate and l a g phase w i t h increas ing copper concentrat ion (and decreasing p C u ) . O n the other hand, Nitzschia demonstrated decreased growth rate. N o effect o n l a g phase was observed for this species. N o difference between the tolerances o f t w o strains o f S. costatum was observed. B o t h strains demonstrated unaffected g r o w t h at 1 x 10-7 M ( p C u = 9.16) and no growth at 1 x 10-5 M added total copper ( p C u = 7.16). Subsequently, Skeletonema costatum and Nitzschia thermalis were g r o w n together at three copper concentrations (1 x 10~9 M , 4 x 10-7 M and 5 x 10-7 M added total copper) . In the un ia lga l cultures that were used as controls , the two species grew as predic ted f r o m their tolerance tests. H o w e v e r , i n the m i x e d cultures, Nitzschia was the o n l y species that exhib i ted g r o w t h , regardless o f the copper concentration i n the m e d i u m . - i i i -T h i s i n h i b i t i o n o f Skeletonema i n the presence o f the second d i a t o m , was attributed to a t o x i c Nitzschia exudate. T h e effect o f the exudate appeared to be temporary, as demonstrated b y the extended l a g phase and subsequent satisfactory exponent ia l g r o w t h rate o f Skeletonema. It is suggested that exponent ia l growth rate was resumed because the exudate degrades w i t h i n a p e r i o d o f f i v e days (= l a g phase). It is s h o w n that the interact ion between the t w o d i a t o m species is more important i n determining the s u r v i v a l o f S. costatwn than its i n d i v i d u a l copper tolerance. T h i s is not the case for N. thermalis. S u c h interactions w o u l d be unaccounted for i n single-species tox ic i ty tests. O n the other hand, i f they are k n o w n , predic t ion o f h o w a c o m m u n i t y that includes these t w o species w o u l d respond to copper addit ions becomes possible . - i v -T A B L E O F C O N T E N T S Abstract i i T a b l e o f Contents i v L i s t o f Tables v i L i s t o f F igures v i i i A c k n o w l e d g e m e n t s x i Genera l Introduction 1 Chapter 1: C o p p e r tolerance o f t w o species o f marine diatoms 10 Introduct ion 10 Se lec t ion o f Organisms 17 M e t h o d s 19 Results 23 D i s c u s s i o n 41 Chapter 2 : E f f e c t o f the interact ion o f t w o species o f marine diatoms o n their i n d i v i d u a l copper tolerance 48 Introduct ion 48 Part A : Interaction o f Skeletonema costatum w i t h Nitzschia thermalis i n three copper concentrations 56 M e t h o d s 56 Results 59 D i s c u s s i o n 75 Part B : Poss ib le factors d r i v i n g the interact ion between Skeletonema costatum and Nitzschia thermalis 80 - v -M e t h o d s 80 Results 83 D i s c u s s i o n .87 G e n e r a l D i s c u s s i o n .92 References .97 A p p e n d i x 1: D i a t o m dis t r ibut ion i n f i v e t idepools sampled i n V a n c o u v e r , B r i t i s h C o l u m b i a , C a n a d a , o n f i v e occasions i n 1988 109 M e t h o d s 109 Results 112 T i d a l effects o n the t idepools 112 D i a t o m dis tr ibut ion 112 References 134 A p p e n d i x 2: G r o w t h curves o f Skeletonema costatum and Nitzschia thermalis d u r i n g the p r e l i m i n a r y sets o f tox ic i ty tests 135 A p p e n d i x 3 : T h e effect o f polycarbonate containers and m e d i u m c h e l e x i n g o n the g r o w t h o f mar ine diatoms 143 Introduction 143 M e t h o d s 146 Part 1: E f f e c t o f containers 146 Part 2 : E f f e c t o f che lex ing 148 Results and D i s c u s s i o n 149 References 163 - v i -L I S T O F T A B L E S T A B L E 2 .1 : Statist ical comparisons between g r o w t h rates o f Skeletonema costatum g r o w i n g i n un ia lga l cultures and i n the presence o f Nitzschia thermalis, i n three different copper concentrations (Exper iment 1). - = species g r o w n alone; + = species g r o w n i n the presence o f N. thermalis; N = sample size; S . D . = standard deviat ion ; N . D . = not determined; * = W i l c o x o n test used instead T A B L E 2.2: Statist ical comparisons between lengths o f l a g phase for Skeletonema costatum g r o w i n g i n un ia lga l cultures and i n the presence o f Nitzschia thermalis, i n three different copper concentrations (Exper iment 1). - = species g r o w n alone; + = species g r o w n i n the presence o f N. thermalis; N = sample s ize; S . D . = standard deviat ion ; N . D . = not determined T A B L E 2.3: Statist ical comparisons between g r o w t h rates f o r Nitzschia thermalis g r o w i n g i n unia lga l cultures and i n the presence o f Skeletonema costatum, i n three different copper concentrations (Exper iment 1). - = species g r o w n alone; + = species g r o w n i n the presence o f S. costatum; N = sample size; S . D . = standard d e v i a t i o n . . . T A B L E 2.4: Statist ical comparisons between lengths o f lag phase f o r Nitzschia thermalis i n un ia lga l cultures and i n the presence o f Skeletonema costatum, i n three different copper concentrations (Exper iment 1). - = species g r o w n alone; + = species g r o w n i n the presence o f 5 . costatum; N = sample size; S . D . = standard devia t ion T A B L E 2.5: Stat ist ical comparisons between g r o w t h rates for Skeletonema costatum g r o w i n g i n unia lga l cultures and i n the presence o f Nitzschia thermalis, i n three different copper concentrations (Exper iment 2). - = species g r o w n alone; + = species g r o w n i n the presence o f N. thermalis; N = sample size; S . D . = standard devia t ion ; N . D . = not determined; * = W i l c o x o n test used instead T A B L E 2.6: Statist ical comparisons between lengths o f lag phase f o r Skeletonema costatum g r o w i n g i n unia lga l cultures and i n the presence o f Nitzschia thermalis, i n three different copper concentrations (Experiment 2). - = species g r o w n alone; + = species g r o w n i n the presence o f N. thermalis; N = sample size; S . D . = standard deviat ion ; N . D . = not determined , T A B L E 2.7: Statist ical comparisons between g r o w t h rates for Nitzschia thermalis i n un ia lga l cultures and i n the presence o f Skeletonema costatum, i n three different copper concentrations (Exper iment 2). - = species g r o w n alone; + = species g r o w n i n the presence o f S. costatum; N = sample s ize; S . D . = standard devia t ion .73 T A B L E 2.8: Statistical comparisons between lengths o f lag phase for Nitzschia thermalis g r o w i n g i n un ia lga l cultures and i n the presence o f Skeletonema costatum, i n three different copper concentrations (Exper iment 2). - = species g r o w n alone; + = species g r o w n i n the presence o f S. costatum; N = sample size; S . D . = standard devia t ion - v i i -T A B L E 2.9: Statist ical comparisons for differences i n p H between Skeletonema costatum (=Sket), Nitzschia thermalis (=Nitz) and m i x e d cultures at the end o f E x p e r i m e n t 2. m = m e a n ( N = 5); S . D . = standard devia t ion ; T u k e y = c r i t i ca l range for pairs o f means ( in p H units) 84 - v i i i -L I S T O F F I G U R E S F I G U R E 1.1: G r o w t h curves o f Skeletonema costatum ( N E P C C 18c) at 5 x 1 0 - 7 , 4 x 10-7 and 3 x 10-7 M C u . ( A ) In vivo f luorescence; (B) C e l l counts. C i r c l e s , triangles and squares represent replicates 1-3 respect ively . F i l l e d symbols represent the points at w h i c h No and^Ni were estimated 26 F I G U R E 1.2: G r o w t h curves o f Skeletonema costatum ( N E P C C 18c) at 2 x 10-7 ,1 x 10-7 and 8 x 10-8 M C u . ( A ) In vivo f luorescence; (B) C e l l counts. C i r c l e s , triangles and squares represent replicates 1-3 respect ively . F i l l e d symbols represent the points at w h i c h No and N i were estimated 27 F I G U R E 1.3: G r o w t h curves o f Nitzschia thermalis ( N E P C C 608) at 1 x 1 0 - 6 , 9 x 10-7, 8 x 10-7,7 x 10-7 and 6 x 10-7 M C u . ( A ) In vivo f luorescence; (B) C e l l counts. C i r c l e s , triangles and squares represent replicates 1-3 respect ively . N o and N i were not est imated because no apparent exponent ia l g r o w t h occurred 28 F I G U R E 1.4: G r o w t h curves o f Nitzschia thermalis ( N E P C C 608) at 5 x 10-7, 3 x 10-7 and 1 x 10-7 M C u . ( A ) In vivo f luorescence; (B) C e l l counts. C i r c l e s , triangles and squares represent replicates 1-3 respect ively . F i l l e d symbols represent the points at w h i c h N o and N i were estimated 29 F I G U R E 1.5: G r o w t h curves o f Nitzschia thermalis ( N E P C C 608) at 4 x 10-7 and 3 x 10-7 M C u . ( A ) In vivo f luorescence; (B) C e l l counts. C i r c l e s , triangles and squares represent replicates 1-3 respect ively . F i l l e d symbols represent the points at w h i c h N 0 and N i were estimated 30 F I G U R E 1.6: E f f e c t o f added total copper concentration o n the growth rate o f Skeletonema costatum ( N E P C C 18c). ( A ) F l u o r = in vivo f luorescence; (B) C e l l no. = c e l l counts. E r r o r bars represent ± one standard devia t ion (N=3 except at 0.1 | i M , N = 9 and at 0.5 f i M , N=6) 31 F I G U R E 1.7: E f f e c t o f free cupr ic i o n act ivi ty o n the growth rate o f Skeletonema costatum ( N E P C C 18c). ( A ) F l u o r = in vivo f luorescence; (B) C e l l no . = c e l l counts. E r r o r bars represent ± one standard deviat ion (N=3 except at pCu=8 .56 , N = 5 and at p C u = 9 . 1 6 , N = 9 ) 32 F I G U R E 1.8: E f f e c t o f added total copper concentrat ion o n the growth rate o f Nitzschia thermalis ( N E P C C 608). ( A ) F l u o r = in vivo f luorescence; ( B ) C e l l no . = c e l l counts . E r r o r bars represent ± one standard devia t ion (N=3 except at 0.1 (J.M, N = 9 , at 0.3 | i M , N = 6 and at 0.5 u . M , N=5) 33 - i x -F I G U R E 1.9: E f f e c t o f free cupr ic i o n act ivi ty o n the g r o w t h rate o f Nitzschia thermalis ( N E P C C 608). ( A ) F l u o r = in vivo f luorescence; (B) C e l l no . = c e l l counts. E r r o r bars represent ± one standard devia t ion (N=3 except at pCu=8 .46 , N = 5 , at pCu=8 .66 , N = 6 and at pCu=9 .16 , N=9) 34 F I G U R E 1.10: E f f e c t o f added total copper concentrat ion o n the length o f l a g phase o f ( A ) Skeletonema costatum ( N E P C C 18c) and (B) Nitzschia thermalis ( N E P C C 608). E r r o r bars represent ± one standard devia t ion (N=3 except i n (B) at 0.5 uJVl, N=2) 35 F I G U R E 1.11: E f f e c t o f free cupr ic i o n act ivi ty o n the length o f l a g phase o f ( A ) Skeletonema costatum ( N E P C C 18c) and (B) Nitzschia thermalis ( N E P C C 608). E r r o r bars represent ± one standard devia t ion (N=3 except i n (B) at pCu=8 .46 , N=2) 36 F I G U R E 1.12: G r o w t h curves o f Skeletonema costatum ( N E P C C 676) at 1 x 10-5 and 1 x 10-7 M C u . ( A ) In vivo f luorescence; (B) C e l l counts. C i r c l e s , triangles and squares represent replicates 1-3 respect ively . F i l l e d symbols represent the points at w h i c h N 0 and N i were estimated 37 F I G U R E 1.13: G r o w t h curves o f Skeletonema costatum ( N E P C C 676) at 1 x 10-9 and 1 x 10-11 M C u . ( A ) In vivo f luorescence; (B) C e l l counts. C i r c l e s , triangles and squares represent replicates 1-3 respect ively . F i l l e d symbols represent the points at w h i c h No and N i were estimated 38 F I G U R E 1.14: E f f e c t o f added total copper concentration o n the growth rate o f Skeletonema costatum c lones N E P C C 18c ( = S. costatum) and N E P C C 676 ( = S. costatum*) . ( A ) In vivo f luorescence; (B) C e l l counts. E r r o r bars represent ± one standard devia t ion (N=3) 39 F I G U R E 1.15: E f f e c t o f free cupr ic i o n act ivi ty o n the g r o w t h rate o f Skeletonema costatum c lones N E P C C 18c ( = S. costatum) and N E P C C 676 ( = S. costatum*) . ( A ) In vivo f luorescence; (B) C e l l counts. E r r o r bars represent ± one standard devia t ion (N=3) 4 0 F I G U R E 2 .1 : G r o w t h curves o f Skeletonema costatum ( A , B ) and Nitzschia thermalis ( C , D ) g r o w n i n un ia lga l ( A , C ) and b i a l g a l ( B , D ) cultures, i n 5 x 10-7 M C u (Exper iment 1). C i r c l e s , triangles, squares, inver ted triangles and diamonds represent replicates 1-5 respect ively. F i l l e d symbols represent the points at w h i c h N o and N i were estimated 61 F I G U R E 2.2: G r o w t h curves o f Skeletonema costatum ( A , B ) a n d Nitzschia thermalis ( C , D ) g r o w n i n un ia lga l ( A , C ) and b i a l g a l ( B , D ) cultures, i n 4 x 10-7 M C u (Exper iment 1). C i r c l e s , triangles, squares, inver ted triangles and diamonds represent replicates 1-5 respect ively. F i l l e d symbols represent the points at w h i c h No and N i were est imated 62 F I G U R E 2.3 : G r o w t h curves o f Skeletonema costatum ( A , B ) a n d Nitzschia thermalis ( C , D ) g r o w n i n un ia lga l ( A , C ) and b ia lga l ( B , D ) cultures, i n 1 x 10-9 M C u (Exper iment 1). C i r c l e s , triangles, squares, inver ted triangles and d iamonds represent replicates 1-5 respect ively. F i l l e d symbols represent the points at w h i c h No and N i were est imated F I G U R E 2.4: G r o w t h curves o f Skeletonema costatum ( A , B ) a n d Nitzschia thermalis ( C , D ) g r o w n i n un ia lga l ( A , C ) and b i a l g a l ( B , D ) cultures, i n 5 x 10-7 M C u (Exper iment 2). C i r c l e s , triangles, squares, inverted triangles and d iamonds represent replicates 1-5 respect ively. F i l l e d symbols represent the points at w h i c h No and N i were estimated F I G U R E 2.5: G r o w t h curves o f Skeletonema costatum ( A , B ) and Nitzschia thermalis ( C , D ) g r o w n i n un ia lga l ( A , C ) and b i a l g a l ( B , D ) cultures, i n 4 x 10-7 M C u (Exper iment 2). C i r c l e s , triangles, squares, inver ted triangles and diamonds represent replicates 1-5 respect ively. F i l l e d symbols represent the points at w h i c h N 0 and N i were est imated F I G U R E 2.6: G r o w t h curves o f Skeletonema costatum ( A , B ) and Nitzschia thermalis ( C , D ) g r o w n i n un ia lga l ( A , C ) and b i a l g a l ( B , D ) cultures, i n 1 x 10-9 M C u (Exper iment 2). C i r c l e s , triangles, squares, inver ted triangles and d iamonds represent replicates 1-5 respect ively. F i l l e d symbols represent the points at w h i c h No and N i were est imated F I G U R E 2.7: G r o w t h curves o f Skeletonema costatum g r o w n i n filtrate f r o m cultures o f Nitzschia thermalis. ( A ) C o n t r o l ; (B) Fi l trate ; (C) Fi l trate enr iched w i t h macro- and micronutrients ; (D) Fi l trate enr iched w i t h metal stocks. C i r c l e s , triangles, squares, inverted triangles and d iamonds represent replicates 1-5 respect ively. F i l l e d symbols represent the points at w h i c h N 0 and N i were estimated - x i -A C K N O W L E D G E M E N T S I w o u l d l i k e to thank m y supervisor , D r . A . G . L e w i s , for his support and encouragement d u r i n g this study. I appreciate the valuable input o f the members o f m y committee , D r . R . E . D e W r e e d e and D r . W . E . N e i l l . I also thank D r . E . V . G r i l l w h o served o n m y committee d u r i n g the first t w o years. I especial ly thank B i l l N e i l l w h o was a lways enthusiastic, interested and car ing regarding not o n l y m y research spec i f i ca l ly but also m y academic w e l l - b e i n g and career i n general . A number o f other people were extremely h e l p f u l d u r i n g m y research. D r . W . G . S u n d a ( N a t i o n a l M a r i n e Fisher ies Service , Southeast F isher ies Center , Beaufort Laboratory) suggested the w a y i n w h i c h to calculate i n i t i a l p C u s . D o n W e b b shared w i t h m e his expertise i n science o n numerous occasions. E l a i n e S i m o n s a lways had cultures avai lable for m e whenever m i n e crashed, even o n Sundays . Peter T h o m p s o n was a lways w i l l i n g to show m e the ins and outs o f phytoplankton growth . Jay M c N e e a lways p r o v i d e d his c h e m i c a l w i s d o m . R o w a n H a i g h h e l d m y hand w h e n I was learning to ident i fy phytoplankton . J o d i M a c F a r l a n e and D o n W e b b came to P o i n t A t k i n s o n w i t h m e and he lped estimate the height o f those t idepools very accurately. E l a i n e S i m o n s and D r . F . J .R . T a y l o r ident i f ied Nitzschia thermalis i n t ime to inc lude i t i n the thesis. F i n a l l y , D r . P . J . H a r r i s o n , M a u r i c e Levasseur , M i k e St. J o h n , Peter T h o m p s o n , D o n W e b b and two anonymous reviewers c r i t i c a l l y evaluated parts o f this thesis. A number o f lab mates, S h i r l e y F r e n c h , Steve G o r m i c a n , J o d i M a c F a r l a n e , G r a h a m M c M i l l a n , Ian M c M i l l a n , C a r o l R u i z and L o u i s e W o o t t o n put up w i t h m y moods and p r o v i d e d an e n j o y f u l environment to w o r k i n (most o f the t ime). A special ment ion goes to those unforgettable lasagna feasts shared w i t h K a r e n P e r r y , T e r r i Suther land and D o n W e b b w h i c h were the o n l y source o f entertainment d u r i n g the last f e w months. D o n W e b b was a lways there for m y temper tantrums, not to ment ion m y j o y s . H e also made sure that I was the one that had to hear the a l a r m i n the mornings and so develop the independence necessary to succeed i n l i f e . F i n a n c i a l support for this study was p r o v i d e d b y a grant f r o m the International C o p p e r Research A s s o c i a t i o n to D r . A . G . L e w i s . I was supported b y an " A l e x a n d e r S. Onass i s " P u b l i c Benef i t F o u n d a t i o n (Athens , Greece) postgraduate scholarship, and S h e l l and C h e v r o n f e l l o w s h i p s through the Department o f Oceanography. - 1 -G E N E R A L I N T R O D U C T I O N C o p p e r is a trace metal that has been extensively studied i n natural waters. In the marine environment , studies o f this meta l have been pursued i n t w o direct ions . In the f irst , the purpose was to understand the dis tr ibut ion o f this metal i n natural seawater and i n the sediments (e.g. K l i n k h a m m e r 1980, B o y l e et al. 1981, G r a y b e a l and H e a t h 1984, Pedersen et al. 1986) as w e l l as its geochemica l c y c l e (e.g. B o y l e et al. 1977, S h o l k o v i t z 1978, W i n d o m et al. 1983, B a l l s 1988). In addi t ion , its chemistry i n both environments , w i t h a specia l emphasis o n the interaction o f copper w i t h organic c o m p o u n d s , has been e x a m i n e d (e.g. M e y e r s and Q u i n n 1974, D a v i s 1984, Forstner 1984, L a x e n 1984/1985 and 1985, C o a l e and B r u l a n d 1988, S o l i and B y r n e 1989). These studies have contr ibuted to the considerable breadth o f k n o w l e d g e that has n o w been achieved regarding the behaviour o f copper i n the w o r l d ' s oceans. T h e second di rec t ion i n w h i c h studies have been pursued i n v o l v e s the effect o f copper o n the biota. Studies o f the b i o l o g i c a l effects o f copper have centered m a i n l y around single-species tox ic i ty tests. T h e b i o l o g i c a l species that have been e x a m i n e d represent m a n y trophic levels . Studies have been carr ied out w i t h bacteria (e.g. S u n d a and G i l l e s p i e 1979, S u n d a and Ferguson 1983), phytoplankton (e.g. A n d e r s o n and M o r e l 1978, M o r e l et al. 1978, Stauber and F lorence 1985a and 1985b, P e k k a l a and K o o p m a n 1987) , macroalgae (e.g. Forsberg et al. 1988, H o 1988, S o d e r l u n d et al. 1988), zooplankton (e.g. L e w i s et al. 1971, G e r a s i m o v 1987, S u n d a et al. 1987, B l u s t et al. 1988) , benthic copepods and decapods (e.g. Depledge 1987, O ' B r i e n et al. 1988, V e r r i o p o u l o s and D i m a s 1988), gastropods (e.g. A m i a r d - T r i q u e t et al. 1987, K i t c h i n g et al. 1987, M i n n i t i 1987), b iva lves (e.g. W r i g h t and Z a m u d a 1987, H a r r i s o n et al. 1988, W a t k i n s and S i m k i s s 1988) and f i s h (e.g. K r e z o s k i et al. 1988, R e i d and M c D o n a l d 1988). -2 -M a r i n e phytoplankton have been studied i n great detai l . These studies have c lear ly indicated the large var iab i l i ty i n responses to elevated copper concentrations that exists a m o n g species that be long to the same trophic l e v e l . In order to understand this var iab i l i ty , studies have addressed the poss ib i l i ty o f t a x o n o m i c a l l y grouping i n d i v i d u a l species tolerances. B r a n d et al. (1986) e x a m i n e d the g r o w t h rate o f 38 species o f phytoplankton and discovered that three species, Skeletonema costatum, Thalassiosira pseudonana and T. oceanica, were more tolerant to copper than the other species tested. W h e n species were grouped t a x o n o m i c a l l y , coccol i thophores were f o u n d to exhib i t sensi t ivi ty to a w i d e r range o f copper concentrations than diatoms. O n e species o f dinoflagel late and a number o f cyanobacter ia l species were the most sensitive organisms. M a n d e l l i (1969) c lass i f i ed the dinoflagel lates as more copper sensitive organisms than diatoms. R i l e y and R o t h (1971) observed that, o v e r a l l , diatoms accumulated more copper than chlorophytes , however , there was large var iab i l i ty between species and w i t h i n classes. T h e y c o n c l u d e d that the copper content o f algae was not related to any taxonomic c lass i f icat ion. A different approach has been taken to group phytoplankton (and therefore generalize type-responses) w i t h respect to copper tolerance, based o n the oceanic reg ion o f o r i g i n o f each species. G a v i s et al. (1981) c o n c l u d e d that, general ly , species i solated f r o m nerit ic environments were more tolerant than oceanic species. W o o d (1983) reached the same c o n c l u s i o n i n an examinat ion o f phytoplankton assemblages ( s imi lar conclus ions have been reached for di f ferent ia l tolerance to P C B s and other exot ic c o m p o u n d s , e.g. F i s h e r et al. 1973, F i s h e r 1977). A l t h o u g h no clear c o n c l u s i o n was reached, M u r p h y et al. (1984) s h o w e d differences i n response to metal l i m i t a t i o n by Thalassiosira pseudonana (a nerit ic species) and T. oceanica (an oceanic member o f the genus). O n the other hand, M u r p h y and Belas tock (1980) d i d not succeed i n demonstrat ing a clear d is t inc t ion i n tolerance between species f r o m the t w o environments . Strains f r o m unpol lu ted and p o l l u t e d nerit ic and oceanic regions were - 3 -e x a m i n e d for their relat ive tolerance to a c o m p l e x industr ia l waste product . A l t h o u g h a pattern o f increas ing sensit ivity w i t h distance f r o m a p o l l u t e d o r i g i n was observed, the ranges o f tolerance o f the different groups were very broad. Therefore, s o l i d conclus ions c o u l d not be d r a w n due to the large over lap between groups. A l t h o u g h not direct ly relevant to copper tolerance, this study exempl i f i es the confus ing patterns that emerge d u r i n g attempts to generalize species ' responses to inhib i tory substances. It therefore becomes apparent that, at least w i t h i n the phytoplankton , it is very d i f f i c u l t to assign speci f ic relat ive tolerance w i t h respect to either taxonomic groups or environmenta l o r i g i n wi thout some reservations. A single-species tox ic i ty test m a y , therefore, be v a l i d for a g i v e n phytoplankton strain o f specif ic o r i g i n , but m a y not necessarily h o l d for that o f the same o r s i m i l a r species f r o m a different habitat. T h e di f ferent ia l response has been attributed, i n most cases, to adaptations o f the o r g a n i s m to c h e m i c a l characteristics o f the habitat (e.g. organic l o a d i n g and/or h i g h background trace metal concentrat ion, e .g. Jensen et al. 1976, F i s h e r and F r o o d 1980). A factor that has been i g n o r e d is the adaptation o f the o r g a n i s m to the b i o l o g i c a l component o f its habitat (i.e. other species i n the c o m m u n i t y and their interactions). Di f fe rent species i n a c o m m u n i t y interact w i t h each other i n a part icular fash ion . Spec i f i c interactions determine the pos i t ion o f each member i n the c o m m u n i t y as a w h o l e . These interactions have resulted f r o m a number o f adaptations that a l l o w the coexistence o f dif ferent species or contro l the poss ib i l i ty o f ext inct ion o f others. In the presence o f a stress inducer such as copper , different species m a y s h o w different types o f response (ranging f r o m no response to sublethal to lethal effects). It m a y be expected that the interact ion o f any pair o f species w i l l be affected, so le ly as a result o f their di f ferent ia l tolerance. If, for example , one species o f a p a i r o f competitors is more severely affected, then the success o f the other m i g h t increase. O n the other hand, the type o f interact ion between any pair o f species m i g h t determine the apparent tolerance o f these organisms to the stress agent. F o r example , al though a predator m a y not exhib i t -4-signs o f stress due to a p h y s i o l o g i c a l response to an inhib i tor , its prey might . In this case, an indirect effect o f reduct ion o f f o o d ava i lab i l i ty m a y result i n reduced success o f the predator. It becomes obvious , therefore, that a single-species tolerance test s i m p l y cannot describe the true eco log ica l tolerance o f i n d i v i d u a l species. A number o f studies has been carr ied out i n an attempt to determine the effect o f copper additions o n c o m m u n i t i e s . These studies have e x a m i n e d either the w h o l e c o m m u n i t y (e.g. a phytoplankton assemblage) or its components (e.g. species compos i t ion) . G o e r i n g et al. (1977) observed an increase i n s i l i ca uptake o f a phytoplankton assemblage at h i g h copper concentrations and H a r r i s o n et al. (1977) recorded an i n i t i a l reduct ion i n nitrate ass imi lat ion rate o f a phytoplankton assemblage that was , however , a l leviated w i t h i n a f e w days. Crossey and L a P o i n t (1988) observed an increase i n c h l o r o p h y l l a and respirat ion associated w i t h a decrease i n p r i m a r y product ion : c h l o r o p h y l l a and o f p r o d u c t i o n : respirat ion ratios i n a p o l l u t e d creek. These studies are quite h e l p f u l i n assessing the o v e r a l l impact o n the c o m m u n i t y and the impact o f the c o m m u n i t y response o n their habitat. Unfor tunate ly , they are not very exp l i c i t as to the speci f ic changes expected w i t h i n the c o m m u n i t y due to di f ferent ia l response o f i n d i v i d u a l members . Changes i n species c o m p o s i t i o n , dominance and succession have been e x a m i n e d for some c o m m u n i t i e s . T h e most c o m m o n l y encountered effects o f increased copper levels i n v o l v e a shift i n species dominance and a change i n the species divers i ty . E f f l e r et al. (1980) observed an i n i t i a l increase i n cyanobacter ia i m m e d i a t e l y after l o w copper treatment o f a lake . T h i s was accompanied by a sharp decrease i n bacterial populat ions w h i c h , however , recovered very q u i c k l y . Daphnia also demonstrated an i n i t i a l decrease f o l l o w e d by large osc i l la t ions . In a s i m i l a r study, M c K n i g h t (1981) observed the disappearance o f the dominant dinof lagel late Ceratium f o l l o w e d by an increase i n cyanobacteria . Daphnia was replaced by a c y c l o p o i d c o p e p o d . Y a s u n o and F u k u s h i m a -5 -(1987) , i n an examinat ion o f p o l l u t e d r ivers , s h o w e d that the d i a t o m Acnanthes minutissima dominated a l l po l lu ted areas. S i m i l a r studies have been carr ied out i n the marine environment . T h o m a s et al. (1980b) recorded the disappearance o f Nitzschia delicatissima and Chaetoceros spp. f r o m a phytoplankton assemblage exposed to a h i g h metal m i x concentration and a shift i n dominance to Skeletonema costatum and Thalassiosira spp. H o l l i b a u g h et al. (1980) recorded S. costatum as the dominant species i n a l l meta l concentrations o f a number o f different metals. T h e presence and density o f other species depended u p o n the concentration o f the speci f ic metal . Sanders et al. (1981a) observed a shift i n dominance f r o m a Skeletonema and Chaetoceros spp. dominated assemblage to an Amphiprora paludosa var . hyalina dorriinated assemblage u p o n the addi t ion o f copper. Sanders et al. (1981b) e x a m i n e d the d i a t o m succession i n elevated copper. In the undisturbed c o m m u n i t y , a b l o o m o f Skeletonema was f o l l o w e d b y a b l o o m o f Chaetoceros. U n d e r copper addit ions, Skeletonema r emained unaffected and the Chaetoceros b l o o m was replaced b y a Navicula or Amphiprora b l o o m . Sanders and C i b i k (1988) also e x a m i n e d i n the laboratory, a shift i n dominance o f a phytoplankton c o m m u n i t y isolated f r o m Chesapeake B a y . Thalassiosira pseudonana increased at h i g h arsenic concentrat ion, compared to the contro l , and eventual ly flagellates dominated . A b l o o m o f Cerataulina pelagica present i n the controls never occurred at h i g h arsenic levels . In h i g h s i lver levels , S. costatum was more abundant and b looms were more p r o l o n g e d than i n the contro l . O n the other hand, the concentrat ion o f Rhizosolenia fragilissima was not affected at a l l . A s a rule , c o m m u n i t y d ivers i ty tends to decrease i n elevated metal concentrations. T h i s has been demonstrated i n a number o f studies. L a P o i n t et al. (1984) and R o l i n e (1988) have demonstrated this for aquatic macroinvertebrates i n p o l l u t e d streams. Y a s u n o and F u k i s h i m a (1987) and Crossey and L a P o i n t (1988) reached s i m i l a r conc lus ions for attached algae communi t ies f r o m p o l l u t e d streams. F i n a l l y , Stokes et al. -6-(1973) demonstrated increased m i c r o a l g a l b iomass and divers i ty w i t h increas ing distance f r o m a smelter o u t f l o w i n a lake near S u d b u r y , Ontar io . Harrass and T a u b (1985) attempted to examine the impact o f copper o n communi t ies i n the laboratory and compare these to c o m m u n i t y responses i n the f i e l d . T h e y used freshwater m i c r o c o s m s (Standard A q u a t i c M i c r o c o s m ) that i n c l u d e d ten a lgal and f i v e invertebrate species. A t the l o w copper concentration (0.8 x 10-5 M added copper) , Daphnia was severely affected. T h i s resulted i n a lgal b iomass accumulat ion and a roti fer increase. A t 3.2 x 10-5 M C u , a l l t rophic levels were comple te ly inh ib i ted f o r extended periods o f t ime. T h e authors c o n c l u d e d that these results were quite s i m i l a r to c o m m u n i t y responses observed i n the f i e l d . Shannon et al. (1985) c o m p a r e d the Standard A q u a t i c M i c r o c o s m ( S A M ) w i t h the M i x e d F l a s k C u l t u r e ( M F C ) . T h e latter also inc ludes a standard c o m m u n i t y . T h e dif ference is that the M F C examines the o v e r a l l c o m m u n i t y responses, such as product ion and respirat ion, wi thout cons ider ing the species-specif ic responses because i t assumes that "the system l e v e l variables are species-independent" (Shannon et al. 1985). T h e authors c o n c l u d e d that o v e r a l l M F C s demonstrated a higher sensi t ivi ty i n the c o m m u n i t y response to metal addit ions , but l o w e r r e p r o d u c i b i l i t y compared to S A M . Studies that have been p e r f o r m e d at the c o m m u n i t y l e v e l are certainly m o r e realist ic than single-species tox ic i ty tests i n that they take into considerat ion the b i o l o g i c a l c o m p l e x i t y o f a species ' habitat. T h e y d o demonstrate that the entire status o f the c o m m u n i t y changes w i t h the addi t ion o f an inh ib i tor such as copper at h i g h concentrations. F r o m these studies, it is c lear that i t is not a lways the same species that dominates the phytoplankton assemblage. F o r example , i n the studies ment ioned above, S. costatum does dominate i n some instances but is i n h i b i t e d i n others. T h e response o f species that d o not have h i g h abundances is even more unpredictable. P a r t l y , the source o f this var iab i l i ty can be attributed to the different interactions o f the species i n the different habitats. T h e studies at the c o m m u n i t y l e v e l are unfortunately very descr ipt ive . -7-C o p p e r is added and the response o f the c o m m u n i t y is recorded. In the end, c o m m u n i t y interactions appear to contro l the response o f the organisms. F o r example , i n the study b y Harrass and T a u b (1985), w h e n Daphnia decreased, phytoplankton abundance increased. T h e l imitat ions o f both approaches (i.e. s ingle species and c o m m u n i t y bioassays) , therefore, become obvious . A single-species tox ic i ty test, by d e f i n i t i o n , isolates the o r g a n i s m f r o m its interspecif ic b i o l o g i c a l environment . A study at the c o m m u n i t y l e v e l (as per formed to date) describes quite prec ise ly the effects o n the o v e r a l l c o m m u n i t y structure but fa i ls to examine the mechanisms o f change. In addi t ion , no associat ion can be made between i n d i v i d u a l species tolerances and their response at the c o m m u n i t y l e v e l . Interactions between species m a y p l a y a s ignif icant ro le i n cont ro l l ing species abundance, regardless o f their speci f ic copper stress status. T h i s effect w i l l be part icular ly pronounced i n the manifestat ion o f sublethal rather than lethal effects. F e w laboratory studies have actual ly addressed the importance o f species interactions o n the metal tolerance o f part icular species. R i c e et al. (1981) c o n c l u d e d that, for some metals (e.g. C d , Z n , N i ) , an increase i n detrital f o o d quantity resulted i n a decrease i n meta l content i n the tissue o f the polychaete Capitella capitata. T h e y c o n c l u d e d that the increase i n popula t ion density ( w h i c h resulted f r o m the higher f o o d quantity) increased compet i t ion for avai lable metals w h i c h , i n turn, decreased the average metal content per i n d i v i d u a l . T h e opposite was the case for C u . N o effect was observed w i t h F e or M n . L e B l a n c (1985) used the cladocerans Daphnia pulex and D. magna. In the absence o f copper , D. pulex over took the cultures. In single species cultures i t was s h o w n that w i t h increased exposure t ime, D. magna deve loped copper tolerance whereas D. pulex d i d not. W h e n cul tured together i n elevated copper concentrations, w i t h no p r i o r p e r i o d o f acc l imat izat ion , an i n i t i a l b l o o m o f D. magna was eventual ly replaced by D. pulex d o m i n a t i o n . T h e p e r i o d before the shift i n the species that contro l l ed the cultures was extended, i f the organisms were f irst in t roduced to a l o w e r i n i t i a l copper -8-concentrat ion ( w h i c h w i l l then inh ib i t D. pulex a n d acc l imat ize D. magna). A n examinat ion o f this study reveals that a l though D. magna was the most tolerant species, this tolerance was overr idden by the fact that D. pulex was the superior compet i tor . Sanders (1986) observed a shift i n phytoplankton dominance towards a b l o o m o f T. pseudonana i n increased arsenic levels . H e also d iscovered , however , that this part icular species o f phytoplankton was not preferential ly grazed b y zooplankton . H e c o n c l u d e d that a l though the zooplankton m i g h t not be direct ly affected at a part icular metal concentrat ion, an indirect effect w o u l d lead to a shortage o f the preferred f o o d i tem. T h e importance o f f o o d concentrat ion to copper tolerance o f D. magna was demonstrated b y G e r a s i m o v (1987). F o r a l l copper concentrations (below a certain leve l ) , increased f o o d density enhanced s u r v i v a l and increased g r o w t h o f this species. T h o m a s and R o b i n s o n (1987) ascertained that the presence o f bacteria or the filtrate f r o m a non-axenic culture o f Amphora coffeaeformis enhanced metal tolerance o f the d i a t o m . T h e y c o n c l u d e d that a soluble c o m p o u n d (< 0.22 |im) was produced b y the interact ion o f the d i a t o m and bacteria that a l leviated copper tox ic i ty . T h e importance o f the presence o f other organisms f o r the i n d i v i d u a l tolerances o f species becomes very apparent. P o p u l a t i o n responses to a toxicant m a y depend o n both the o r i g i n o f the species i n v o l v e d as w e l l as the organisms w i t h w h i c h the habitat is shared. T o date, most trace metal studies have ignored this c o m p l i c a t i n g factor a n d have concentrated o n the effect o n organisms i n single-species cultures. T h e purpose o f this study was to examine the effect o f a s imple two-species interact ion o n i n d i v i d u a l species copper tolerances. T h e group o f organisms that I . chose to study were marine diatoms (Class Bac i l l a r iophyceae ) . T h e y are p r i m a r y producers , their response to copper has been extensively studied, they are re la t ive ly easy to culture and they have short generation t imes. -9-T h e t w o species that I used were Skeletonema costatum (Grev i l l e ) C l e v e and Nitzschia thermalis (Ehrenberg) A u e r s w a l d . F i rs t , I e x a m i n e d their i n d i v i d u a l copper tolerances us ing a number o f bioassays. T w o g r o w t h curve parameters were e x a m i n e d , exponent ia l growth rate and length o f l a g phase (Chapter 1). Subsequently, I assessed the effect o f their interact ion o n their predetermined tolerance. T h e t w o species were g r o w n at a cont ro l copper concentrat ion (1 x 10 _ 9 M C u - as suggested by M o r e l et al. (1979) for the preparation o f an ar t i f i c ia l seawater m e d i u m ) . In addi t ion , they were g r o w n at part ia l ly stressful copper concentrations. I then compared their performance w h e n g r o w n alone to w h e n g r o w n i n the presence o f the second species (Chapter 2 A ) . S ince the interact ion between the t w o species overrode their copper tolerance, I exarnined the poss ible factors that c o u l d be d r i v i n g the interact ion and, therefore, c o n t r o l l i n g the species s u r v i v a l irrespective o f copper concentrat ion (Chapter 2 B ) . B o t h phytoplankton cu l tur ing and trace metal methodology i n v o l v e a number o f tradit ional , "acceptable" protocols w h i c h are not necessari ly compat ib le . These i n c l u d e the use o f containers made o f dif ferent materials and the cu l tur ing m e d i u m preparation. S ince i n this study both protocols h a d to be incorporated, I exarnined the effect o f the t w o procedures o n phytoplankton growth ( A p p e n d i x 3). -10-C H A P T E R 1 : C o p p e r Tolerance o f T w o Species o f M a r i n e D i a t o m s I N T R O D U C T I O N T h e b i o l o g i c a l effects o f copper i n both the marine and freshwater environments have been studied extensively . These studies i n v o l v e both the p h y s i o l o g i c a l responses o f microalgae to elevated (or reduced) copper concentrations as w e l l as the importance o f c h e m i c a l speciat ion to copper tox ic i ty . A copper requirement has been demonstrated for some species, Chlorella vulgaris, Oocystis marssonii ( M a n a h a n and S m i t h 1973) and Gonyaulax tamarensis (Schenck 1984). O n e o f the f irst studies to demonstrate the detr imental effect o f copper to phytoplankton was by Steeman N i e l s e n and W i u m - A n d e r s e n (1970). These authors e x a m i n e d the copper tolerance o f Chlorella pyrenoidosa and Nitzschia palea. S ince then, a large number o f studies have e x a m i n e d the p h y s i o l o g i c a l responses to increased copper concentrations. G r o w t h rate reduct ion w i t h increas ing copper levels is one o f the most prominent observations (e.g. E r i c k s o n 1972, Jensen et al. 1976, M o r e l et al. 1978, F i s h e r et al. 1981, N a k a m u r a et al. 1986). F o r some species o f algae, there have been reports o f an increase i n the length o f l a g phase i n response to copper elevations (e.g. Steeman N i e l s e n and W i u m - A n d e r s e n 1970, Bartlett et al. 191 A, B e n t l e y - M o w a t and R e i d 1977, M o r e l et al. 1978). There have also been reports o f a reduct ion i n photosynthetic rate (e.g. Steeman N i e l s e n et al. 1969, Steeman N i e l s e n and W i u m A n d e r s e n 1970, E r i c k s o n 1972, O v e r n e l l 1976, R a o and S ivasubramanian 1985) and o f an increase i n the rate o f respirat ion (Rao and S ivasubramian 1985) as symptoms o f copper tox ic i ty . T h e latter authors also descr ibed a decrease i n m a x i m u m photosynthetic rate (Pmax) and a dif ference i n the i n i t i a l slopes o f Photosynthesis vs . Irradiance curves. A number o f studies have e x a m i n e d other p h y s i o l o g i c a l responses o f phytoplankton cel ls to copper tox ic i ty . Al terat ions i n c e l l m o r p h o l o g y (e.g. E r i c k s o n -11 -1972, S u n d a and L e w i s 1978, T h o m a s et al. 1980a, F i sher et al. 1981) and reduct ion i n c e l l m o t i l i t y ( A n d e r s o n and M o r e l 1978) are t w o examples . P e k k a l a and K o o p m a n (1987) observed an increase i n s i n k i n g rate w i t h increas ing copper concentration after 30 minutes o f contact t ime. F i sher et al. (1981) e x a m i n e d a number o f p h y s i o l o g i c a l effects due to elevated copper. T h e y reported an increase i n the levels o f C • c e l l - 1 , N • c e l l - 1 , c h l a • ce l l -1 and D N A • ce l l -1 a s signs o f copper e levat ion. K a n a z a w a and K a n a z a w a (1969) and R i c e et al. (1973) suggested that copper interferes w i t h transmembrane i o n transport. Perhaps one o f the most important effects at the ce l lu lar l e v e l , espec ia l ly for diatoms, is the interference o f copper w i t h s i l i c i c a c i d uptake. Va lente et al. (1987) demonstrated an i n h i b i t i o n o f spore format ion o f Chaetoceros protuberans i n the presence o f elevated copper, w h i c h was reversed i n the presence o f h i g h s i l i c i c a c i d concentrations. Rueter (1983) s h o w e d that s i l i c i c a c i d uptake is reduced i n increas ing copper levels . U n d e r S i -s tarvat ion, both the uptake rate and the content o f the S i - p o o l s i n the cel ls were higher i n l o w e r copper concentrations. A l t h o u g h this was the case w i t h Thalassiosira weisflogii, a different response was observed i n T. pseudonana. T h e results o f Rueter et al. (1981) suggested for this species a possible increase i n uptake (manifested b y an increase i n S i - c e l l quota), as w e l l as an increase i n the s i l i con-uptake threshold, w i t h increas ing copper concentrat ion. In addi t ion , copper sensit ivity was reduced i n the presence o f higher S i ( O H ) 4 i n the m e d i u m . T h e authors c o n c l u d e d that there is compet i t ive i n h i b i t i o n between the two elements and proposed a m o d e l whereby C u and S i use the same transport site to enter the c e l l . O n the other hand, T h o m a s et al. (1980a) c o n c l u d e d that for Skeletonema costatum, copper d i d not act as a s i l i c o n compet i t ive inhib i tor . S u n d a a n d G u i l l a r d (1976) demonstrated that the copper species that is most tox ic to phytoplankton is the cupr ic i o n (Cu2+). Canter ford (1979) and Canter ford and Canter ford (1980) s h o w e d that an increase i n the concentration o f the chelat ing agent -12-E D T A ( w h i c h complexes i o n i c copper) i n the phytoplankton m e d i u m , enhanced the copper tolerance o f Ditylwn brightwellii. N a k a m u r a et al. (1986) c o n c l u d e d that g r o w t h sensit ivity o f Chattonella antiqua to copper was dependent u p o n the E D T A concentration and not the total copper concentrat ion. Sunda a n d L e w i s (1978) suggested that growth rate i n Monochrysis lutheri was negat ively associated w i t h ion ic copper concentrat ion. G a v i s et al. (1981) e x a m i n e d the effect o f cupr ic i o n concentration o n the g r o w t h o f phytoplankton quite extensively . T h e y determined three types o f responses o f g r o w t h to increase i n p C u (the negative l o g a r i t h m o f free cupr ic i o n act ivi ty) . T h e "one-step" response ( w h i c h has a single threshold o f g r o w t h i n h i b i t i o n ) , "one-step inc ip ient " ( w h i c h is a no-response curve) , and a " two-step" response ( w h i c h inc ludes a l o w e r and a higher threshold o f part ia l and complete i n h i b i t i o n respect ively) . H o w e v e r , F lorence and Stauber (1986) demonstrated that some l igands (such as phenanthroline) f o r m complexes w i t h copper that are very tox ic as w e l l . T h i s type o f c o m p l e x catalyzes the product ion o f exc i ted o x y g e n species (oxygen free radicals) f r o m m o l e c u l a r o x y g e n (such as H 2 O 2 ) ins ide the c e l l . These free radicals then react w i t h most b i o l o g i c a l substances (such as amines , enzymes etc.) and m a y , therefore, inh ib i t a number o f processes i n the c e l l (e.g. l i p i d ox idat ion) . F lorence et al. (1984) suggested that the l i p i d so lubi l i ty o f the copper complexes is what is o f p r i m a r y importance i n copper tox ic i ty and not s i m p l y whether the i o n i c copper is c o m p l e x e d . T h e c e l l membrane is a site o f copper b i n d i n g before the metal enters the c e l l . G a v i s et al. (1981) suggested that negative charges o n the membrane b i n d the pos i t ive ly charged cupr ic i o n . T h e y proposed that the cupr ic ions b i n d to c a r b o x y l and a m i n o groups that are present o n the c e l l surface. F i s h e r and Jones (1981) c o n c l u d e d that the EC50 (the concentrat ion o f metal at w h i c h growth rate is reduced to 5 0 % o f the control) o f Asterionella japonica for several metals , i n c l u d i n g copper , was correlated w i t h the so lubi l i ty o f their sulphides. A negative correlat ion was f o u n d w i t h the stabil i ty constants o f the metals for cysteine and methionine ( w h i c h are su l fhydry l - conta in ing -13-a m i n o acids) . T h e y c o n c l u d e d that s u l f h y d r y l b i n d i n g o f metals is the manner i n w h i c h i n h i b i t i o n occurs . It has been demonstrated that cel ls remove copper f r o m the m e d i u m i n proport ion to the levels present (e.g. M a n d e l l i 1969, B e n t l e y - M o w a t and R e i d 1977). In natural seawater, f r o m an area o f u p w e l l i n g , phytoplankton copper uptake was not large enough to account for any changes i n copper concentrat ion i n the water ( K n a u e r and M a r t i n 1973). In the laboratory, an i n i t i a l r a p i d uptake f o l l o w e d b y copper release has been documented for some species (e.g. M a n d e l l i 1969, B e n d e y - M o w a t and R e i d 1977, A n d e r s o n a n d M o r e l 1978). M a n d e l l i (1969) also s h o w e d an increase i n copper uptake w i t h increas ing temperature and sal ini ty . Jones et al. (1987) demonstrated reduct ion (Cu(IJ) —> C u (I)) o n the c e l l w a l l . T h e y suggested a non-renewable c e l l w a l l component as be ing responsible for the reduct ion . T h e y also suggested that the reduct ion rate o f Cu(II) at the c e l l surface is not related to the cupr ic i o n act ivi ty but rather to the total copper concentration and the C u - o r g a n i c complexes . T h e authors c o n c l u d e d that a trans-p l a s m a l e m m a N A D ( P ) H - c y t o c h r o m e c reductase-type e n z y m e was responsible f o r the reduct ion . A f e w studies have addressed the mechanisms o f microalgae reactions that prevent copper tox ic i ty . Foster (1977) suggested copper exc lus ion . A less tolerant strain o f Chiorella vulgaris showed higher copper uptake than a more tolerant one. In Amphora spp. , copper was f o u n d b o u n d o n spherical , polyphosphate bodies o r o n electron dense, i rregular-shaped bodies ( D a n i e l and C h a m b e r l a i n 1981). T h e latter had a h i g h copper : sul fur ratio and were considered bodies used for the deact ivat ion and r e m o v a l o f the meta l f r o m the c y t o p l a s m . T h e polyphosphate bodies conta ined copper inconsistent ly and were suggested to p r o v i d e inoUscriminate b i n d i n g sites. C l o u t i e r - M a n t h a and B r o w n (1980) proposed that i n Skeletonema, a m i n o acids are used for d e t o x i f y i n g metals. -14-Skeletonema costatum is a phytoplankton species that has been used extensively f o r copper tox ic i ty tests. M a n d e l l i (1969) determined the copper l e v e l o f growth i n h i b i t i o n i n a 16:8 h L : D c y c l e as 8 x 10-7 M C u and i n continuous l ight as 2.6 to 4 x 10-6 M . Jensen et al. (1976) obtained an 8 0 % reduct ion i n g r o w t h w i t h the addi t ion o f 1.6 x 10-7 M C u to natural seawater o f u n k n o w n copper background l e v e l . A c c o r d i n g to O v e r n e l l (1976), this species suffered a 5 0 % reduct ion i n photosynthetic rate i n the presence o f 5 x 10-5 M C u . B e r l a n d et al. (1977) successful ly grew this species i n concentrations up to 1.6 x 10-6 M C u added to natural seawater i n the absence o f chelators. In the same study, a decrease i n g r o w t h (no i n h i b i t i o n was ever obtained) at 3.2 x 10-6 M was observed. F i s h e r and F r o o d (1980) observed a h a l v i n g i n g r o w t h rate w i t h the addi t ion o f 8 x 10-8 M C u to natural seawater. F r o m the results o f K h a n and S a i f u l l a h (1986), 5 . costatum s h o w e d progress ive ly decreasing g r o w t h rates w i t h increasing copper levels w i t h a complete i n h i b i t i o n around 3.2 - 4.8 x 10-7 M (no added E D T A ) . F i n a l l y , perhaps the most detai led study o f the copper tolerance o f this species is b y M o r e l et al. (1978). N o apparent effect o n growth was observed i n copper concentrations up to 6 x 10-6 M C u . T h i s study was p e r f o r m e d i n A q u i l ( M o r e l et al. 1979) i n the presence o f 5 x 10-6 M E D T A ( p C u = 8.8). T h e copper i n h i b i t i o n l e v e l l ies somewhere around 1 x 10-6 M (Fi tzgera ld and Faust (1963) suggested that one part E D T A i m m o b i l i z e s one part copper ; as M o t e k a i t i s and M a r t e l l (1987) caut ioned, however , this w i l l depend o n the E D T A concentrat ion; these authors s h o w e d that at 1 x 10-9 M E D T A less than 1% o f the copper is b o u n d whereas i n 1 x 10-7 almost a l l o f the meta l is bound) . G a v i s et al. (1981) c o n c l u d e d that the g r o w t h rate o f S. costatum demonstrates a two-step response to the negative l o g o f cupr ic i o n act ivi ty . Increased copper concentrations result i n a number o f other p h y s i o l o g i c a l effects for S. costatum. M o r e l et al. (1978) f o u n d an increase i n l a g phase w i t h increas ing copper concentrat ion, however , this effect was reversed i n the presence o f h i g h s i l i c i c ac id . T h o m a s et al. (1980a) observed elongated S. costatum ce l ls i n the presence o f h i g h -15-copper levels but no c e l l aberrations were apparent. N o effect o n c e l l shape was observed b y F i sher and F r o o d (1980). T h e same authors f o u n d a decrease i n g r o w t h rate w i t h increas ing ce l lu lar copper l o a d . B e s i d e centric diatoms, pennates are also c o m m o n l y used bioassay microalgae . M a n d e l l i (1969) demonstrated g r o w t h i n h i b i t i o n i n the pennate d i a t o m Nitzschia closterium s i m i l a r to that observed for S. costatum. Steeman N i e l s e n and W i u m -A n d e r s e n (1970) observed g r o w t h i n h i b i t i o n o f Nitzschia palea i n 2 x 10-7 M C u ( in the absence o f E D T A , i r o n added as a fresh precipitate) at an i n i t i a l c e l l concentrat ion o f 10,000 cel ls • m L - 1 and i n 8 x 10-8 M C u at an i n o c u l u m size o f 100 cel ls • m L - 1 . F i sher and F r o o d (1980) e x a m i n e d the effect o f copper o n the g r o w t h o f Nitzschia closterium and Asterionella japonica. T h e g r o w t h o f these t w o species was reduced to a th i rd o f the contro l for the former and a ha l f for the latter i n the presence o f 8 x 10-8 M C u added, to natural seawater ( w h i c h averaged ~1 x 10"^ M background copper concentration). F i s h e r et al. (1981) s h o w e d a decrease i n g r o w t h rate i n the presence o f 4 x 10-7 M C u i n f/2 enr iched natural seawater. L u m d s e n and F l o r e n c e (1983) demonstrated a d e c o u p l i n g o f photosynthesis f r o m c e l l d i v i s i o n for N. closterium. A l t h o u g h their g r o w t h rate was reduced i n m e d i u m conta in ing - 4 . 8 x 10"7 M C u , their photosynthetic rate was not affected. N o such effect was observed for Asterionella glacialis. F lorence and Stauber (1986) and Stauber and F lorence (1987) suggested a m e c h a n i s m for such d e c o u p l i n g i n N. closterium. It i n v o l v e d the depression o f the reduced g luta th ione :ox id ized glutathione ratio and the subsequent suppression o f mi to t i c d i v i s i o n (for w h i c h a h igh ratio is required) . T h e o x i d a t i o n o f the reduced glutathione is ca ta lyzed by cupr ic ions . T h i s react ion is cy toso l i c . In addi t ion , i o n i c copper inhibi ts glutathione reductase, the e n z y m e that c o u l d reverse the react ion (Florence and Stauber 1986). In terms o f other p h y s i o l o g i c a l responses, T h o m a s et al. (1980a) observed very f e w c e l l aberrations i n pennate diatoms g r o w i n g i n elevated copper. Nitzschia delicatissima was sometimes present i n the t w i n n e d state. N. closterium was sometimes -16-d iscovered w i t h abnormal ly s w o l l e n ce l ls . N. pungens never s h o w e d any m o r p h o l o g i c a l alterations. F i sher et al. (1981) observed an increase i n c e l l v o l u m e for A. japonica i n the presence o f elevated copper. T h e same result was obtained by F i s h e r and F r o o d (1980), a l though no effect o n c e l l m o r p h o l o g y was observed for N. closterium. Stauber and F lorence (1987) observed no copper effect o n the ultrastructure o f the chloroplasts , nucleus , m i t o c h o n d r i a or the c e l l membrane o f the same species. T h e s imi lar i ty i n meta l tolerance o f different strains o f the same species has been the topic o f o n l y f e w studies. M o s t o f them i n v o l v e d two strains o f S. costatum (Ske l -0 and Skel -5) w h i c h were isolated f r o m t w o different f jords i n N o r w a y . Jensen et al. (1976) obta ined very s i m i l a r g r o w t h responses to copper addit ions for these two strains. B o t h were reduced to 2 0 % g r o w t h o f the contro l i n 1.6 x 10-7 M added C u (to f i l tered seawater w i t h u n k n o w n copper content). F o r z i n c , different types o f g r o w t h responses were obtained for the t w o strains (Jensen et al. 191 A). S k e l - 0 demonstrated reduced but uninterrupted g r o w t h i n elevated z i n c concentrations. S k e l - 5 , o n the other h a n d , at the same z inc levels , s h o w e d n o r m a l g r o w t h for three days f o l l o w e d by a r a p i d dec l ine i n c e l l numbers . B o t h strains demonstrated equal tolerance to z inc i n terms o f photosynthetic rate ( O v e r n e l l 1976). B r a e k et al. (1976) e x a m i n e d the c o m b i n e d effect o f copper and z i n c o n the same t w o strains o f 5 . costatum. S y n e r g i s m between the t w o metals was observed f o r both strains. In this study however , Ske l -5 was proven s l ight ly more sensitive to copper addi t ion ( in the absence o f z inc) than Ske l -0 . T h e c o m b i n e d effect o f c a d m i u m and z inc was not quite so s i m i l a r for the two strains (Braek et al. 1980). S k e l - 0 was more sensitive to c a d m i u m whereas Ske l -5 was more sensitive to z i n c . In c o m b i n a t i o n , the t w o metals acted synergis t ica l ly i n S k e l - 5 , whereas increased z inc concentrat ion a l lev ia ted c a d m i u m tox ic i ty i n S k e l - 0 . A l t h o u g h both strains accumulated the same amount o f z i n c i n the course o f the experiments, S k e l - 0 accumulated more c a d m i u m than S k e l - 5 (Braek et al. 1980). -17-T h i s chapter examines the i n d i v i d u a l copper tolerances o f Skeletonema costatum and the pennate Nitzschia thermalis. T h e parameters o f growth that I chose to observe were exponent ia l g r o w t h rate and length o f l a g phase. I sha l l discuss the change i n these g r o w t h parameters w i t h increas ing copper concentrat ion. In addi t ion , I w i l l examine the s imi lar i ty i n copper tolerance between t w o strains o f S. costatum. SELECTION OF ORGANISMS T h e d i a t o m species that I chose to examine are species that are present i n h i g h abundances i n t idepools . T i d e p o o l s can be considered, to a certain extent, s m a l l , par t ia l ly enclosed ecosystems d u r i n g at least the p e r i o d that they r e m a i n isolated f r o m the ocean (during l o w tide). D u r i n g the p e r i o d o f complete i so la t ion ( w h i c h c o u l d vary f r o m hours to days) , the interact ion o f diatoms that are present there w i l l be mainta ined rather than become d i l u t e d by water m o v e m e n t (the latter might be the case i n a large, d y n a m i c b o d y o f water). D i a t o m species that are f o u n d together i n t idepools might interact w i t h each other i n a part icular manner. A n y effect that such an interact ion m i g h t have o n their copper tolerances w i l l p l a y a ro le i n determining the structure o f the c o m m u n i t y under copper stress. F i v e t idepools as w e l l as the sea-surface were sampled o n f i v e occasions d u r i n g M a y - J u n e , 1988, at P o i n t A t k i n s o n , W e s t V a n c o u v e r , B r i t i s h C o l u m b i a . T h e s a m p l i n g strategy, the enumerat ion procedure and a s u m m a r y o f the results are presented i n A p p e n d i x 1. A number o f patterns regarding species ' abundance emerged f r o m this study: (1) Skeletonema costatum was m o s t l y abundant at the sea-surface and i n l o w e r t idepools . (2) Pennates, as a group, were not very abundant at the sea-surface, but were the group w i t h the highest abundance i n a l l o f the t idepools o n a l l f i v e occasions. (3) Melosira demonstrated l o w densities at the sea-surface. T h i s genus reached large concentrations i n most t idepools . -18-(4) G e n e r a o f benthic diatoms such as Odontella, Surirella a n d Gyrosigma were almost non-detectable at the sea-surface, yet consistently appeared i n the t idepools i n moderate abundance. O f the descr ibed groups, I chose S. costatum and the pennates to use i n this study. T h i s was due to the h i g h abundances that both these taxa attained i n most t idepools . T h e actual species that were used i n this thesis were not i so lated f r o m t idepool samples. T h e y were both obtained f r o m the N o r t h East P a c i f i c Cul ture C o l l e c t i o n , U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r . T h e y were isolated f r o m l o c a l waters, S. costatum f r o m the Strait o f G e o r g i a and Nitzschia thermalis f r o m Jer icho B e a c h , V a n c o u v e r . Unfor tunate ly , I a m not able to determine whether the latter was i n c l u d e d i n the group o f pennates that were present i n the t idepool samples. H o w e v e r , this species has been observed i n several different areas: i n the marine environment i t has been f o u n d i n the B a y o f F u n d y , N e w B r u n s w i c k (Linklet ter et al. 1977) and i n a marine mudf la t o f the E e m s - D o l l a r d estuary, i n the Netherlands ( A d m i r a a l and Pelet ier 1979). In addi t ion , i t has been c lass i f i ed as a marine l i t toral d i a t o m ( L e w i n 1963), a l though the locat ion f r o m w h i c h i t was isolated is not ment ioned i n that study. In freshwater, i t has been col lec ted f r o m lakes i n the S w i s s A l p s ( B r u n 1880). It has also been f o u n d i n h i g h abundances o n the w a l l s o f water reservoirs , and i n swamps and sloughs i n South A f r i c a ( C h o l n o k y 1968). In east A f r i c a , i t was f o u n d as a m e m b e r o f the lake per iphyton and i n r ivers and ponds , w i t h the highest relat ive abundance reached i n a s m a l l "sal ted" p o n d (Gasse 1986). F o g e d (1978), i n a c o m p i l a t i o n o f data o n the diatoms o f eastern A u s t r a l i a , c lass i f i ed this species as cosmopol i tan . Unfor tunate ly , this Nitzschia species has been extensively m i s i d e n t i f i e d (e.g. see V a n L a n d i n g h a m 1978) and no consistent habitat pattern exists. S ince this species has been f o u n d i n temperate estuaries as w e l l as i n freshwater, its presence was quite l i k e l y i n the brack ish t idepools o f P o i n t A t k i n s o n (wi th respect to its tolerance to p h y s i c a l factors such as sal ini ty and temperature). I therefore felt that i t was a reasonable representative o f the pennate group that was present i n the t idepool samples. -19-M E T H O D S Stock cultures o f t w o strains o f Skeletonema costatum ( N E P C C 18c and N E P C C 676) and one strain o f Nitzschia thermalis ( N E P C C 608) were obtained f r o m the N o r t h East P a c i f i c Cul ture C o l l e c t i o n , Department o f Oceanography, U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r . O n c e obtained, the cultures were mainta ined i n polycarbonate 250 m L E r l e n m e y e r f lasks , u s i n g ar t i f i c ia l seawater ( A q u i l ) prepared as i n M o r e l et al. (1979) except for t w o modi f i ca t ions . I d i scovered that under the laboratory condit ions used, trace meta l contaminat ion was in t roduced into the A q u i l w h e n I bubbled CO2 to drop the p H before autoc laving. F o r this reason, I o n l y raised the p H o f both the seawater and the C h e l e x - 1 0 0 res in to 7.5 instead o f 8.0 ( w h i c h is r e c o m m e n d e d i n M o r e l et al. 1979). In this manner , after autoc laving a n d a l l o w i n g the m e d i u m to stand f o r at least 48 hrs. , the p H was raised o n l y up to - 8 . 4 and no noticeable precipitate was observed. T h e second alteration i n the procedure was i n the preparation o f the trace metal m i x . I ron ch lor ide was added as fresh Fe-precipitate ( iron was a l l o w e d to precipitate i n d i s t i l l e d d e i o n i z e d water - N a n o p u r e - for 24 hrs.) rather than a F e - E D T A m i x . T h e reason for this m o d i f i c a t i o n was the very h i g h af f in i ty that E D T A has for copper (99.5%, Jackson and M o r g a n 1978), w h i c h m i g h t inf luence the copper tox ic i ty results. T h e metal stock solut ion was prepared b y the addi t ion o f the r e m a i n i n g trace metals in to the F e -precipitate stock, a l l o w i n g at least 2-3 hrs. for equi l ibra t ion o f the solut ion. B e f o r e use, the f lasks were washed w i t h Sparkleen Detergent, soaked i n I N H C L for at least 24 hrs. , r i n s e d three t imes w i t h N a n o p u r e water, and autoclaved i n a Cast le standard laboratory autoclave, at 21 p s i for 20 minutes . T h e m i c r o a l g a l cultures were kept i n a culture chamber at - 1 6 ° C , under a 16:8 h L : D c y c l e , at an irradiance o f - 9 0 \\E • m"2 • s ' l . A l l cultures were u n i a l g a l but not axenic (although sterile technique was used throughout) . G e n e r a l l y , the same procedure was f o l l o w e d w i t h the replicate d i a t o m cultures d u r i n g the experiments . B o t h the f lasks and the m e d i u m were processed i n a s imi lar -20-manner as for the stocks. A f t e r the m e d i u m was autoc laved and a l l o w e d to stand for at least 24 hrs. , 200 m L were added to the w a s h e d f lasks . Subsequently, the fresh metal a n d v i t a m i n stocks were added to the appropriate concentrations. F i n a l l y , the freshly prepared copper stocks (prepared b y the d i sso lut ion o f C u S 0 4 i n D D W ) were i n c l u d e d i n v o l u m e s necessary to attain the desired concentrations. T h e f lasks were a l l o w e d to equil ibrate w i t h the m e d i u m for 24 hrs. after w h i c h they were inoculated w i t h al iquots f r o m exponent ia l ly g r o w i n g d i a t o m stock cultures. A l l transfers were p e r f o r m e d i n a class 100 laminar f l o w h o o d w i t h a l l poss ib le meta l parts replaced by p o l y p r o p y l e n e . E v e r y 24 hrs. , 10 m L aliquots were r e m o v e d f r o m the exper imental f lasks after gentle shaking to ensure homogeneous d i a t o m dis t r ibut ion , and used for c e l l counts and f luorescence measurements. In vivo f luorescence was measured us ing a Turner Des igns M o d e l 10 f luorometer . C e l l counts were p e r f o r m e d us ing a P a l m e r M a l o n e y count ing sl ide under a Z e i s s Standard 14 c o m p o u n d microscope . T h e lowest c e l l density that I c o u l d detect us ing the c e l l counts was 200 cel ls • m L - 1 . F o r each replicate, three subsamples were counted us ing f i v e f ie lds per subsample. T h e f ie lds represented a r a n d o m c e l l d is t r ibut ion as r e c o m m e n d e d by L u n d et al. (1958). Three sets o f tox ic i ty tests were r u n for both S. costatum (strain N E P C C 18c) and N. thermalis. E a c h set was des igned to examine a narrower range o f copper concentrations than the preceding set. In the f irst set, the concentrations o f added total copper were 1 x 10-5 M , 1 x 10-7 M , 1 x 1 0 - 1 1 M (treatments) and 1 x 10-9 M . F o r each o f the subsequent sets, the concentration that was used as the " c o n t r o l " was the highest f r o m the preceding set at w h i c h no s igni f i cant ly different g r o w t h f r o m the next l o w e r concentrat ion was observed (as demonstrated by a Student 's t- or a W i l c o x o n test) . T h e f i n a l set o f tox ic i ty tests was p e r f o r m e d at the 1 x 10-7 M l e v e l ( 0 . 1 - 1 | i M added C u ) for both species. In order to compare copper tolerance between strains o f the same species, I attempted the same procedure for the second stock o f S. costatum ( N E P C C 676). T h i s -21-strain was p r o v e n very sensitive and therefore I o n l y managed to carry out tests us ing o n l y the widest range o f copper concentrations (first set). A s a result the compar i son was a c c o m p l i s h e d at a very general l e v e l . Three replicates were used f o r each copper concentrat ion, for a l l tests that were carr ied out w i t h a l l three strains. Repl icates were arranged i n a systematic des ign o n shelves, ensuring equal l ight intensity i n the interior o f a l l the containers. F o r each culture, t w o parameters o f g r o w t h were e x a m i n e d , exponent ia l g r o w t h rate and length o f l a g phase. E x p o n e n t i a l g r o w t h rate was determined u s i n g K e and k as i n G u i l l a r d (1973) (where K e = [In ( N i / N 0 ) ] / [ti - to] and k (div • d a y 1) = K e / 0.6931). T h e exponent ia l por t ion o f the growth curve was determined as the part w i t h the steepest o v e r a l l s lope. N o and N i fo r each replicate were chosen as the points beneath o r b e y o n d w h i c h there was an apparent devia t ion f r o m this slope. T h e length o f the l a g phase was determined as the t ime between inoc u la t ion and the time at w h i c h N o was estimated. T h e effect o f both added total copper concentrat ion and i n i t i a l p C u o n exponent ia l g r o w t h rate and l a g phase was explored us ing l inear regressions. In i t ia l p C u i n the m e d i u m at p H 8.4 was ca lculated assuming o n l y inorganic c o m p l e x a t i o n o f copper , p r i m a r i l y b y carbonate ions (as suggested by B y r n e and M i l l e r (1985)). It was ca lculated f r o m the f o l l o w i n g equations: p[Cu2+] = - l o g [total copper] + l o g a p C u = p [ C u 2 + ] + 0.68 where p[Cu2+] is the negative l o g o f the free cupr ic i o n concentrat ion, [total copper] is the added total copper concentrat ion, a is the rat io o f total copper concentration to free cupr ic i o n concentrat ion at the p H o f the m e d i u m , and 0.68 is the negative l o g o f the act iv i ty coef f ic ient (0.21) i n seawater. T h e value for the act ivi ty coeff ic ient was est imated by B y r n e and M i l l e r (1985). B y r n e et al. (1988) est imated l o g a for copper i n seawater at 2 5 ° C a n d p H 7.6 and 8.2 (both these factors affect the ion ic concentration o f copper i n seawater). F r o m extrapolat ion and due to the l inear i ty o f the relat ionship -22-between l o g a and p H w i t h i n this narrow range o f p H values , l o g a at p H 8.4 was estimated to equal 1.48. B y us ing the t w o equations, I was able to convert the total copper concentration to p C u . F o r the regressions o f growth rate against copper concentration and p C u , I used a l l the values that I obtained d u r i n g any set o f tox ic i ty tests that i n c l u d e d the specif ic concentrat ion. F o r the regressions o f l a g phase, o n l y the values f r o m the last set o f tox ic i ty tests were i n c l u d e d . T h e regressions were est imated us ing the M G L H m o d u l e o f S Y S T A T ( W i l k i n s o n 1985) o n a C o m m o d o r e P C - 1 0 LT. A s ignif icance l e v e l o f 0.05 was used throughout. -23-R E S U L T S T h e f i n a l sets o f tox ic i ty tests f o r S. costatum (18c) and for N. thermalis are presented i n F i g s . 1.1-1.5. T h e results f o r the p r e l i m i n a r y sets are i n c l u d e d i n A p p e n d i x 2. T h e copper concentrations that I used for S. costatum i n the f i n a l set were 0.08 j i M and 0.1 to 0.5 | i M C u i n 0.1 LIM intervals (F igs . 1.1,1.2). F o r N. thermalis, the f i n a l copper concentrations were 0 .1 , 0 .3 ,0 .5 and 0.6 to 1.0 \iM C u i n 0.1 LLM intervals (F igs . 1.3,1.4). In addi t ion , I ran an extra experiment at 0.3 and 0.4 \iM C u ( F i g . 1.5). O n e o f the reasons for this was to determine the react ion o f this o r g a n i s m at a copper concentration c loser to the lethal one. S. costatum grew very w e l l i n concentrations o f 0.08-0.4 | i M C u a n d d i d not g r o w at a l l i n 0.5 | i M added C u (Figs . 1.1 and 1.2). O n e o f the replicates o f the 0.5 |J.M C u treatment d i d , however , demonstrate signs o f potential increase. S ince no growth was ever observed i n a l l the other replicates that I ever ran at this concentrat ion, I d i d not use that replicate i n m y g r o w t h rate estimations. In addi t ion to the i n h i b i t i o n o f g r o w t h at 0.5 u . M added total C u , there was an increase i n the length o f l a g phase as the latter concentrat ion was approached. F i g . 1.6 demonstrates the change i n g r o w t h rate w i t h increas ing copper concentrat ion for both types o f biomass measurements and F i g . 1.7 the change i n g r o w t h w i t h p C u . L i n e a r regressions demonstrated a s ignif icant increase i n g r o w t h rate w i t h total copper concentrat ion up to but e x c l u d i n g the lethal l e v e l o f 0.5 | i M (reg. coeff . = 2.13; 1.56, r2 = 0 .681; 0 .333, N = 24, p <0.001; <0.01 for the in vivo f luorescence and the c e l l counts est imations, respect ively) . A n inverse relat ionship was observed between growth rate a n d p C u (reg. coeff . = -0.842; -0.582, r2 = 0.666; 0 .291, N = 24, p <0.001; <0.01 for in vivo f luorescence and the c e l l counts estimations, respect ively) . L a g phase demonstrated a s i m i l a r increase w i t h added total copper (reg. coeff . = 10.10; 10.47, r2 = 0 .451; 0.422, N = 24, p < 0.01 f o r f luorescence and c e l l count measurements, respect ively) and a decrease w i t h p C u (reg coeff . = -4.27; -4 .53, r2 = -24-0.409; 0 .403, N = 24, p < 0.05 f o r f luorescence and c e l l counts, respect ively) (Figs . l . l O A a n d l . H A ) . N. thermalis was s l ight ly more tolerant than S. costatum. T h i s species d i d g r o w i n copper concentrations o f up to 0.5 p :M i n c l u s i v e (Figs . 1.3-1.5). In these results, I have i n c l u d e d the f i n a l set o f tox ic i ty tests as w e l l as a last experiment that I conducted i n order to add the results concerning g r o w t h at a concentration o f 0.4 added total C u ( F i g . 1.5). U n l i k e Skeletonema, this species s h o w e d a different type o f g r o w t h response to increas ing added total copper concentrations and p C u ( F i g . 1.8). F o r the f luorescence measurements, a s ignif icant decrease was observed between 0.1 and 0.5 p :M added total C u (reg. coeff . = -0.710, r2 = 0 .211, N = 23 , p < 0.05). T h e relat ionship was even stronger w h e n the growth rates were e x a m i n e d between 0.3 and 0.5 p :M (reg. coeff . = -2.25, r2 = 0.598, N = 14, p = 0.001). T h e relat ionship o f g r o w t h rate w i t h p C u was o n l y present between 8.66 and 8.16 w h i c h corresponds to 0.3 and 0.5 nM total copper (reg. coeff . = 2 .25, r2 = 0.598, N = 14, p = 0.001) ( F i g . 1.9). N o s ignif icant pattern was observed for the c e l l counts , probably because o f the larger error associated w i t h the g r o w t h rate at 0.5 p :M added total C u and p C u = 8.56. T h e length o f the lag phase was not affected b y increas ing total copper concentration or decreasing p C u (Figs . 1.9B, 1.10B). A s ment ioned i n the methods section, the second strain o f S. costatum ( N E P C C 676) d i d not g r o w after the first set o f tolerance tests (1 x 10 -5 ,1 x 10-7 ,1 x 10-9 and 1 x 1 0 - H M added total copper) . A s a result , I o n l y present the results f r o m that set (F igs . 1.12-1.13). T h i s strain ( N E P C C 676) grew adequately up to 1 x 10-7 M C u and d i d not g r o w at a l l at 1 x 10-5 M C u . T h i s is i n agreement w i t h the results for the first strain (18c), as demonstrated i n F i g s . 1.14-1.15. Therefore , i n general , the t w o strains showed s i m i l a r copper tolerance, at least w i t h i n the same t w o orders o f magnitude. T h e y both grew at copper concentrations up to 1 x 10-7 M and neither grew at 1 x 10"^ M added total copper. -25-F r o m the f luorescence measurements, the second strain (676) demonstrated a m u c h l o w e r g r o w t h rate (Figs . 1 .14A, 1 .15A) . T h i s was suspected to be p r i m a r i l y due to a p o o r p h y s i o l o g i c a l state o f this strain d u r i n g the experiments . In addi t ion , a s ignif icant increase i n g r o w t h rate w i t h both total copper concentration and p C u was observed i n the c e l l count estimates (Figs . 1 .14B, 1.15B) (reg. coeff . = 0.223; -0.223, r2 = 0.630; 0.630, N = 8, p < 0.05 for total copper and p C u respect ively) . 26 CU u c CD O (f) CD ^_ O J D Li_ 1 0 . 0 -1 . 0 0 -0 . 1 0 0 -0 . 0 1 0 0 .001 o o o J/5 "CD o 1 0 0 - -F I G U R E 1.1: G r o w t h curves o f Skeletonema costatum ( N E P C C 18c) at 5 x 1 0 " 7 , 4 x 1 0 " 7 a n d 3 x l O 7 M C u . ( A ) In vivo f luorescence ; ( B ) C e l l counts . C i r c l e s , triangles a n d squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h No and N i were est imated. 27 1 0 . 0 - -. . . o -c CD O CO CD v_ O J D 1 . 0 0 - . . o 0 . 1 0 0 -0 . 0 1 0 -• o . A - J : o — o o o o — o 2 X 1 0 " 7 M C u 1 X 1 0 - 7 M Cu 8 X 1 0 ~ 8 M Cu 0 .001 0 8 10 12 14 0.1 -I 1 1 1 1 1 1 1 0 2 4 6 8 10 12 14 D a y s F I G U R E 1.2: G r o w t h curves o f Skeletonema costatum ( N E P C C 18c) at 2 x 1 0 " 7 , 1 x 1 0 - 7 a n d 8 x 10-8 M C u . ( A ) In vivo f luorescence ; (B) C e l l counts . C i r c l e s , triangles a n d squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 a n d N i were estimated. 28 F I G U R E 1.3: G r o w t h curves o f Nitzschia thermalis ( N E P C C 608) at 1 x 1 0 ' ° , 9 x 1 0 " 7 , 8 x 10-7, 7 x 10-7 a n d 6 x 10-7 M C u . ( A ) In vivo f luorescence ; ( B ) C e l l counts . C i r c l e s , tr iangles a n d squares represent replicates 1-3 respect ive ly . N 0 a n d N i were not est imated because n o apparent exponent ia l g r o w t h o c c u r r e d . 29 1.000-O <5 0.100 4-o CO O [Z — 0.01 Oo 0.001 O O = 5 X 10 ' M Cu O O = 3 X 10_Z M Cu O ----- 0 = 1 X 1 0 M Cu 0 cr: 'A' O .A. " " A - - A 8 o o O ~ci5 O 1 0 -1.0o [] 0.1 4^ o o o o o - - - - - o 5 X 10_Z M Cu 3 X 10 M Cu 1 X 1 0 - 7 M Cu / / A \ .O' 0 • A r . •O'v-Days ,. _ o -8 • o 8 B F I G U R E 1.4: G r o w t h curves o f Nitzschia thermalis ( N E P C C 608) at 5 x l O ' ' , 3 x 1 0 ' ' and 1 x 10-7 M C u . ( A ) In vivo f luorescence ; (B) C e l l counts . C i r c l e s , tr iangles a n d squares represent repl icates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h NQ a n d N i were est imated. 30 A Days F I G U R E 1.5: G r o w t h curves oi Nitzschia thermalis ( N E P C C 608) at 4 x 1 0 ' 7 and 3 x 1 0 " 7 M C u . ( A ) In vivo f luorescence; (B) C e l l counts . C i r c l e s , triangles and squares represent replicates 1-3 respect ively . F i l l e d s y m b o l s represent the points at w h i c h N 0 and N i were est imated. 31 o > II CD D _C o 1 . 5 -1.0- • 0 . 5 -O 0 .0 Fluor 0.7 o > II CD -t—1 O o O 0 .0 Cell no. B 0.1 0.2 0 .3 0 .4 0 . 5 0 .6 C o p p e r C o n c e n t r a t i o n ( / x M o l a r ) 0.7 F I G U R E 1.6: E f f e c t o f a d d e d total copper concentrat ion o n the g r o w t h rate o f Skeletonema costatum ( N E P C C 18c). ( A ) F l u o r = in vivo f luorescence ; (B) C e l l no . = c e l l counts . E r r o r bars represent ± one standard d e v i a t i o n (N=3 except at 0.1 | i . M , N = 9 and at 0.5 p : M , N = 6 ) . 32 >N o > '"O o •4—1 O _^ O A Fluor 9.4 o > II CU o o o 1.5 1.0 0 . 5 -0 .0 8 .4 8 .6 B Cell no. 8.8 9 .0 pCu 9.2 9 .4 F I G U R E 1.7: E f f e c t o f free c u p r i c i o n ac t iv i ty o n the g r o w t h rate o f Skeletonema costatum ( N E P C C 18c). ( A ) F l u o r = in vivo f luorescence ; (B) C e l l no . = c e l l counts . E r r o r bars represent ± one standard d e v i a t i o n (N=3 except at p C u = 8 . 5 6 , N = 5 and at p C u = 9 . 1 6 , N = 9 ) . 33 A Fluor Cell no. 0.2 0 .3 0 .4 0 .5 0 .6 0 .7 0 .8 0.9 C o p p e r C o n c e n t r a t i o n ( / x M o l a r ) F I G U R E 1.8: E f f e c t o f added total copper concentrat ion o n the g r o w t h rate o f Nitzschia thermalis ( N E P C C 608) . ( A ) F l u o r = in vivo f luorescence ; (B) C e l l no . = c e l l counts . E r r o r bars represent ± one standard d e v i a t i o n (N=3 except at 0.1 p . M , N = 9 , at 0.3 p . M , N = 6 and at 0.5 \iM, N = 5 ) . 34 0 .5 0 .0 8.1 8.2 8 . 3 8 .4 8 .5 8.6 8 .7 8 .8 8 .9 9 .0 9.1 9.2 1.5 1 . 0 -0 . 5 -0 .0 B T ' 1 Cell no. A It A H h 8.1 8 .2 8 .3 8 .4 8 .5 8 .6 8.7 8 .8 8 .9 9 .0 9.1 9 .2 pCu F I G U R E 1.9: E f f e c t o f free c u p r i c i o n act iv i ty o n the g r o w t h rate o f Nitzschia thermalis ( N E P C C 608) . ( A ) F l u o r = in vivo f luorescence ; ( B ) C e l l no . = c e l l counts. E r r o r bars represent ± one standard dev ia t ion (N=3 except at p C u = 8 . 4 6 , N = 5 , at pCu=8 .66 , N = 6 a n d at p C u = 9 . 1 6 , N = 9 ) . 35 10 8 6 -4 -2 -<p-o-1 A i T : = • • • = fluorescence o o = cell counts 0 0.1 0.2 0 .3 0 .4 0 .5 6 T 4 -2 - I o-1 - O " 1 B • = fluorescence o = cell counts 0 0.1 0.2 0 .3 0 .4 0 . 5 Copper Concentration ( /xMolar ) F I G U R E 1.10: E f f e c t o f added total copper concentrat ion o n the length o f l a g phase o f ( A ) Skeletonema costatum ( N E P C C 18c) a n d (B) Nitzschia thermalis ( N E P C C 608) . E r r o r bars represent ± one standard devia t ion (N=3 except i n ( B ) at 0.5 p : M , N=2) . 36 00 o CD 00 O _ C C L CT> D 1 0 T 8 -6 -4 -• -T •o-1 •cp O _c c CD 0 8 .5 • • = fluorescence o o = cell counts 8.6 8.8 9 .0 9 .2 6 T D CD 00 O _c Q_ D o c CD 2 -0 B • • = fluorescence o• o = cell counts • • ? I i 8 . 3 8 .5 8.7 8 .9 9.1 pCu F I G U R E 1.11: E f f e c t o f free c u p r i c i o n a c t i v i t y o n the length o f l a g phase o f ( A ) Skeletonema costatum ( N E P C C 18c) a n d ( B ) Nitzschia thermalis ( N E P C C 608) . E r r o r bars represent + one standard d e v i a t i o n (N=3 except i n (B) at pCu=8 .46 , N = 2 ) . 37 o — 0 = 1 0 ^ M C u , . A - - : : : : : t i A o o = 1 0 M C u ,.-"';.«-"....-o 1 3 5 7 9 1 3 5 7 9 Days F I G U R E 1.12: G r o w t h curves o f Skeletonema costatum ( N E P C C 676) at 1 x 10~ 5 and 1 x 10-7 M C u . ( A ) In vivo f luorescence ; ( B ) C e l l counts . C i r c l e s , triangles and squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 and N i w e r e est imated. 38 Days F I G U R E 1.13: G r o w t h curves o f Skeletonema costatum ( N E P C C 676) at 1 x 1 0 ' 9 a n d 1 x 10-11 M C u . ( A ) In vivo f luorescence ; (B) C e l l counts . C i r c l e s , triangles a n d squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 a n d N i were est imated. 39 D T 3 > 1 . 0 - T •o-1 o o • • -o S. costatum S. costatum* 0) o 0 . 5 " T 1 T 1' o o 0 .0 10 - 1 1 10 - 9 10" •7 10 - 5 t 2 .0 > o B 1 . 5 -i . o « -0 . 5 -•o-i I T •o r ° o = s . costatum • • = S. costatum* O O 0 .0 1 0 ' •11 » - 9 ^ - 7 10 a 1 0 _ / 10 C o p p e r C o n c e n t r a t i o n ( Molar ) - 5 F I G U R E 1.14: E f f e c t o f a d d e d total c o p p e r concentrat ion o n the g r o w t h rate o f Skeletonema costatum c lones N E P C C 18c ( = S . costatum) a n d N E P C C 676 ( = S. cos ta tum*) . ( A ) In vivo f luorescence ; ( B ) C e l l counts . E r r o r bars represent ± one standard d e v i a t i o n (N=3) . 4 0 L O -CI o • • •° I o - " i S. costatum S. costatum* .-•6 0 . 5 -T 'i 0 . 0 ^ 7 1 1 13 B 2 . 0 - 1 o o = S. costatum • • = S. costatum* 1 . 5 -1.0 T o -•'1 •o— ± i 0 . 5 -0 .0 7 1 1 13 pCu F I G U R E 1.15: E f f e c t o f free cupr ic i o n act iv i ty o n the g r o w t h rate o f Skeletonema costatum c lones N E P C C 18c ( = S. costatum) a n d N E P C C 676 ( = S. c o s t a t u m * ) . ( A ) In vivo f luorescence ; (B) C e l l counts . E r r o r bars represent ± one standard dev ia t ion (N=3). -41 -D I S C U S S I O N T h e results that I obtained general ly agree w i t h those o f other researchers. T h e tolerance o f both species to added total copper is w i t h i n the 0.1-1.0 p i M range. M o s t o f the studies that i n v o l v e d either S. costatum o r pennate diatoms obtained reduct ion i n g r o w t h w i t h i n the same range o f copper concentrations. F o r Skeletonema, the copper l e v e l o f g r o w t h i n h i b i t i o n that I obta ined (5 x 10-7 M C u ) is quite s i m i l a r to the one obtained b y M a n d e l l i (1969) and very s i m i l a r to that o f K h a n and S a i f u l l a h (1986). T h e s imi lar i ty w i t h the latter study is o f great importance because the authors d i d not i n c l u d e E D T A i n the m e d i u m preparation. T h e results o f Jensen et al. (1976) demonstrate a l o w e r copper tolerance l e v e l for this species (1.6 x 10-7 M ) . These authors however , used copper additions to natural seawater w h i l e the background concentrat ion remained u n k n o w n . S ince neither total copper nor cupr ic i o n act ivi ty is i n c l u d e d i n their study, I cannot compare their results to m i n e . O n the other hand, B e r l a n d et al. (1977) obtained h igher copper tolerance for the same species ( inhib i t ion at 1.6 x 10-6 M ) . A g a i n these authors added copper to natural seawater. T h e background concentrat ion is not k n o w n and neither is the degree o f complexa t ion . O n c e again, no c o n c l u s i o n c a n be made b y c o m p a r i s o n o f the two studies. M o r e l et al. (1978) c o n c l u d e d that the l e v e l o f i n h i b i t i o n f o r Skeletonema was 6 x 10-6 M C u i n the presence o f 5 x 10-6 M E D T A . T h i s corresponded to p C u = 8.8 w h i c h is s l ight ly higher than 8.46, the value o f i n h i b i t i o n i n m y study. In this study, Nitzschia thermalis demonstrated s l ight ly higher tox ic i ty tolerance ( inhib i t ion at 6 x 10-7 M ) than Skeletonema costatum. O n c e again, the copper levels that i n h i b i t e d N. thermalis i n this study are s i m i l a r to those that have been s h o w n to inh ib i t other species o f pennates (e.g. Steeman N i e l s e n and W i u m - A n d e r s e n 1970, F i s h e r et al. 1981, L u m d s e n and F l o r e n c e 1983). -42-There are problems associated w i t h determining the copper concentrat ion at w h i c h i n h i b i t i o n o f growth occurs f o r a part icular species. O n e o f the major di f f i cul t ies relates to the c o m p o s i t i o n o f the m e d i u m used. H i g h organic c o m p l e x a t i o n decreases copper b ioava i lab i l i ty (e.g. A n d e r s o n and M o r e l 1978, Jackson a n d M o r g a n 1978, Sunda and L e w i s 1978). I f the m e d i u m that is used for cu l tur ing microa lgae is natural seawater (unfi l tered or not U V - t r e a t e d to remove the organics) then the amount o f copper that has to be added to the m e d i u m i n order to make i n h i b i t i o n apparent ought to be quite large. O n the other hand, i f natural seawater is used, w i t h undetermined copper concentration, one w i l l o n l y obta in relat ive values o f copper that m i g h t induce tox ic i ty . F i n a l l y , the presence o f an ar t i f i c ia l chelator w i l l compl ica te measurement o f the amount o f copper present i n a f o r m that w i l l affect the organism. A s ment ioned earl ier , E D T A (as w e l l as N T A ) has a very h i g h af f in i ty for copper and w i l l c o m p l e x i t i n the m e d i u m . A number o f studies (e.g. S teeman N i e l s e n and W i u m - A n d e r s e n 1970, K h a n and S a i f u l l a h 1986, K a z u m i et al. 1987), i n c l u d i n g this one, a v o i d the addi t ion o f E D T A and i n c l u d e the i r o n as a fresh precipitate. A l t h o u g h this procedure avoids the very strong b i n d i n g o f copper to E D T A , i t introduces a n e w site f o r copper adsorption w h i c h also binds copper quite strongly (suggested by Jackson and M o r g a n 1978, L a x e n 1984/1985, Stauber and F lorence 1985a). In addi t ion , as an experiment progresses, organics that are released f r o m the organisms (e.g. organic exudates b y algae and lys i s o f dead cel ls) w i l l c o m p l e x copper and thus render i t unavai lable to the organisms (e.g. Steeman N i e l s e n and W i u m -A n d e r s e n 1971, M c K n i g h t and M o r e l 1979, F i sher and Fabr is 1982). S o , over the course o f a one-week a lga l bioassay, the c h e m i c a l speciat ion i n the m e d i u m w i l l change i n such a w a y that i t makes i t i m p o s s i b l e to determine prec ise ly h o w m u c h o f the copper is s t i l l present i n a f o r m that is avai lable to the organism. A number o f methods have been deve loped to establish either the degree o f copper c o m p l e x a t i o n or the i o n i c copper concentrat ion i n the m e d i u m . These i n v o l v e m o d e l l i n g o f the c h e m i c a l species present i n seawater (e.g. Z i r i n o and Y a m a m o t o 1972, -43-W e s t a l l et al. 1976, B o r g m a n n 1981, S u n d a and H u n t s m a n 1983, Z u e h l k e and Kester 1983, B r a n d et al. 1986), equi l ibrat ion w i t h M n 0 2 ( V a n den B e r g 1982), var ious types o f ion-exchange resins (e.g. M i l l s et al. 1982, M a c k e y 1983, Z o r k i n et al. 1986, S u n d a and H a n s o n 1987), determination o f the lab i le f ract ion by A n o d i c S t r i p p i n g V o l t a m m e t r y (e.g. F lorence et al. 1982), bacterial bioassays (e.g G i l l e s p i e and V a c c a r o 1978, Sunda and F e r g u s o n 1983, S u n d a et al. 1984, M o r e l et al. 1988), copper ion-spec i f i c electrodes ( D a v e y et al. 1973) or a c o m b i n a t i o n o f the above (e.g. B a t l e y and F lorence 1976, F i g u r a and M c D u f f i e 1980, B u c k l e y and V a n den B e r g 1986, H e r i n g et al. 1987). A l l o f these methods have their l imi ta t ions . S o m e are based o n assumptions that are too s impl i s t i c , others d o not have the sensit ivity required to determine the species o f copper that are present i n very l o w concentrations. W i t h others, the species that are measured are not exact ly k n o w n . F o r a l l these reasons, comparisons between studies for speci f ic levels o f copper that cause growth i n h i b i t i o n are very d i f f i cu l t . T h e fact that i n most cases the results o f different studies, us ing different m e d i a and general ly s l i g h d y different c u l t u r i n g condi t ions , are w i t h i n the p M o l a r l e v e l o f each other is very encouraging. T h e increase i n l a g phase o f S. costatum that was observed d u r i n g this study w i t h increase i n copper concentrat ion agrees w i t h the results obtained b y M o r e l et al. (1978). These authors also c o n c l u d e d that there was an increase i n length o f l a g phase at copper concentrations w i t h i n the same order o f magnitude o f the l e v e l that caused complete g r o w t h i n h i b i t i o n (at the 1 p , M l e v e l f o r their study, compared to the 0.1 p . M for m y study or at p C u values between 9.5 and 8.5 for both studies). M o r e l et al. (1978) suggested " c o n d i t i o n i n g " o f the m e d i u m and/or adaptation o f the cel ls to the h i g h copper concentrations as possible mechanisms for the effect o n lag phase. A number o f studies have demonstrated the excret ion o f organic exudates b y microalgae (e.g. M c K n i g h t and M o r e l 1979, F i s h e r and Fabr is 1982). A l t h o u g h these substances m a y not be excreted spec i f i ca l ly i n response to metals they nevertheless have an af f in i ty for some metals , -44-i n c l u d i n g copper. T h e organics w i l l alter the copper speciat ion i n the m e d i u m b y b i n d i n g the metal and m a y render the metal less b ioavai lable . O n e can, therefore, suggest that the m e d i u m becomes " c o n d i t i o n e d " b y the cel ls themselves and that the c o n d i t i o n i n g occurs d u r i n g the extended l a g phase. A n o t h e r p o s s i b i l i t y is that the s u r v i v i n g cel ls m a y become "adapted" to the h i g h copper concentration d u r i n g the extended l a g phase. T h i s adaptation m a y impl ica te an intracel lular p h y s i o l o g i c a l response, for example the act ivat ion o f de tox i f i ca t ion processes. O n e o f the most interesting results i n this study is the increase o f g r o w t h rate o f Skeletonema w i t h increas ing copper concentrat ion, i n a manner s i m i l a r to the l a g phase. A l t h o u g h I cannot suggest a probable m e c h a n i s m for this, i t is poss ib le that d u r i n g the extended l a g phase the p h y s i o l o g y o f the o r g a n i s m changes i n a w a y that enhances its g r o w t h rate. It is also poss ible that a selection m e c h a n i s m for more tolerant cel ls takes place . A s the i n h i b i t o r y copper l e v e l is approached, i t m a y be expected that a larger port ion o f the popula t ion w i l l be r e m o v e d due to copper stress. I f this is the case, the popula t ion that does remain is g o i n g to be smal ler but able to withstand h igher copper concentrations. It is also poss ible that the more tolerant cel ls also demonstrate h igher g r o w t h rates. If this is the case and as the copper tolerance threshold is b e i n g approached, the proport ion o f more " f i t " cel ls ( w i t h higher copper tolerance and d i v i s i o n rates) w i l l increase i n re la t ion to the cel ls that are "unf i t " and so the o v e r a l l growth rate o f the popula t ion w i l l increase. N. thermalis d i d not demonstrate an increase i n l a g phase. There are no k n o w n studies that suggest an increase i n l a g phase for any pennate diatoms. O n the other hand, this species s h o w e d a decrease i n g r o w t h rate w i t h increas ing copper concentrat ion. T h i s also is consistent w i t h most tox ic i ty studies. F r o m these results, i t is suggested that copper tox ic i ty is i n v o k e d i n different manners i n the t w o species. L u m d s e n a n d F lorence (1983) demonstrated a d e c o u p l i n g o f photosynthesis f r o m c e l l d i v i s i o n under copper stress for N. closterium. D u r i n g such -45-d e c o u p l i n g , photosynthetic rate is not affected whereas c e l l d i v i s i o n is inhib i ted . N o such effect has been demonstrated for S. costatum. T h e proposed m e c h a n i s m that caused tox ic i ty to Nitzschia was attributed to cytoso l i c reactions. A l t h o u g h such a m e c h a n i s m has never been suggested for Skeletonema, i t has not been tested either. It is k n o w n , however , that no d e c o u p l i n g occurs (e.g. O v e r n e l l 1976). In this study, no difference was observed between the g r o w t h measurements carr ied out us ing in vivo f luorescence or c e l l counts. A l t h o u g h not tested direc t ly , no d e c o u p l i n g o f the t w o processes is suggested for either o f the species used. T h e m e c h a n i s m o f copper tox ic i ty for S. costatum has been expla ined as compet i t ive i n h i b i t i o n o f sil icate uptake ( M o r e l et al. 1978). F i sher et al. (1981) demonstrated copper interference w i t h s i l i c i c a c i d uptake i n the pennate d i a t o m Asterionella japonica. A l s o , a number o f studies have demonstrated interactions between copper and other metals. Manganese has been s h o w n to reduce copper tox ic i ty ; increased copper concentrations have been s h o w n to result i n l o w e r ce l lu lar manganese content (e.g. Sunda et al. 1981, Sunda and H u n t s m a n 1983, Stauber and F l o r e n c e 1985b, K a z u m i et al. 1987). Rueter and M o r e l (1981) c o n c l u d e d that increased z i n c concentrat ion i n the m e d i u m decreased copper i n h i b i t i o n o f s i l i c i c a c i d uptake. These studies have been carr ied out w i t h both centric and pennate diatoms and there is no reason to bel ieve that any dis t inc t ion should be made based o n groups o f diatoms. H o w e v e r , i t is poss ible that i n one o f the t w o species used i n this study, the amount o f a certain meta l required to outcompete copper successful ly f o r the b i n d i n g sites was higher than i n the other, therefore caus ing a different p h y s i o l o g i c a l response. M u r p h y et al. (1984) demonstrated that v a r i e d i r o n , manganese and copper concentrations i n v o k e d different responses i n Thalassiosira pseudonana and T. oceanica. E a c h o f these species demonstrated manganese l i m i t a t i o n i n different copper concentrations. T h e former required manganese i n l o w copper and h i g h i r o n leve ls , w h i l e the latter d i d not. O n the other hand, T. oceanica required higher manganese concentrations i n elevated copper than T. pseudonana. -46-A n y one or a c o m b i n a t i o n o f these toxic i ty mechanisms m a y be operating i n each o f the t w o species that I e x a m i n e d i n this chapter. S ince I d i d not evaluate the means by w h i c h growth i n h i b i t i o n was i n v o k e d I cannot determine the operating factors. T h e o n l y c o n c l u s i o n that I can reach is that the results strongly suggest that these are different for the t w o species. T h e results o f the compar i son between the copper tolerances o f the two strains o f S. costatum are very general . T h e second strain ( N E P C C 676) appeared to be i n very p o o r p h y s i o l o g i c a l state, and d i d not survive for a l o n g enough p e r i o d . A s a result I was not able to carry out very detai led bioassays and I can o n l y conc lude that the tolerance l e v e l o f this strain is between 1 x 10-7 M C u and 1 x 10-5 M C u . T h i s is the same range that I obtained f r o m the first set o f tox ic i ty tests for strain N E P C C 18c ( A p p e n d i x 2). T h e s imi lar i ty i n copper tolerance between strains is i n agreement w i t h the results o f Jensen et al. (1976). T h e t w o strains used i n m y study were isolated f r o m adjacent bodies o f water, B u r r a r d Inlet and the Strait o f G e o r g i a . It is quite l i k e l y that these t w o strains be long to the same popula t ion (i.e. S. costatum i n the Strait o f Georg ia ) and, therefore, there is no reason to bel ieve that they should have different p h y s i o l o g i c a l responses to copper stress. T h e o n l y dif ference perhaps arises f r o m the fact that one o f the strains (676) was isolated f r o m more coastal waters w h i c h m i g h t demonstrate elevated copper levels compared to the more open waters o f the Strait o f G e o r g i a (such as f o u n d for the N o r t h P a c i f i c O c e a n - B r u l a n d 1980, b y other authors for the East Coast o f N . A m e r i c a - B o y l e et al. 1981, and for the Northwestern A d a n t i c - H u i z e n g a and K e s t e r 1983). M u r p h y and Be las tock (1980), i n their examinat ion o f tolerance to c h e m i c a l stress o f 11 strains o f T. pseudonana and 6 o f S. costatum, c o n c l u d e d that strains i solated f r o m coastal p o l l u t e d waters were more tolerant than strains isolated f r o m open waters. O n the other hand, i t has been demonstrated that strains o f species (such as Chaetoceros compressum, S. costatum, N. closterium mdA.japonica) i solated f r o m open ocean water were m o r e tolerant to metal stress than strains f r o m coastal waters w i t h -47-elevated meta l levels (Fisher and F r o o d 1980). T h i s was attributed to the adaptation o f the open ocean species to the l o w e r c o m p l e x a t i o n capaci ty o f the water (and therefore to higher metal b ioava i lab i l i ty ) . It can be c o n c l u d e d f r o m this chapter that a l though the tolerances o f the t w o species, S. costatum and N. thermalis, are w i t h i n 0.1 p : M C u , Nitzschia is s l ight ly more tolerant than Skeletonema. In addi t ion , i t was observed that the t w o species demonstrated different responses i n g r o w t h rate and l a g phase to elevated copper concentrations. It i s , therefore, suggested that the m e c h a n i s m o f tox ic i ty and/or the detoxi f i ca t ion process is different i n the t w o species. I suggest that the copper concentrations that w o u l d be o f p r i m a r y interest i n e x a m i n i n g the tolerance o f these t w o species w h e n g r o w n together are 0.4 \iM and 0.5 p : M C u . A t the first copper l e v e l , Skeletonema has demonstrated increased growth rate and l a g phase (both o f w h i c h I consider as signs o f copper stress) and at the second l e v e l , Skeletonema demonstrates no g r o w t h and Nitzschia shows signs o f copper stress b y a reduct ion i n g r o w t h rate. In the next chapter, I sha l l examine the effect o f the interact ion w i t h a second species that demonstrates different stress responses, o n the i n d i v i d u a l tolerance o f each species at these "sensi t ive" copper concentrations. -48-C H A P T E R 2: E f fec t o f the interact ion o f t w o species o f marine diatoms  o n their i n d i v i d u a l species copper tolerance I N T R O D U C T I O N M o s t o f the laboratory studies that have been carr ied out to assess the p h y s i o l o g i c a l and e c o l o g i c a l effects o f tox ic i ty o n microalgae have used unia lga l cultures. T h e f e w studies that have i n v o l v e d more than one species d i d not i n a l l cases demonstrate s i m i l a r responses for a species g r o w i n g alone and i n the presence o f a second one. A number o f general p h y s i o l o g i c a l studies have e x a m i n e d the interact ion o f t w o species that were co-cul t ivated i n the same cultures. O n e o f the f irst was by R i c e (1954) w h o e x a m i n e d the interaction between Chlorella vulgaris and Nitzschia frustulum. A s imi lar study was p e r f o r m e d by Pratt (1966) o n the interact ion o f Skeletonema costatum and Olisthodiscus luteus. O t h e r studies i n v o l v e the interact ion o f Chlamydomonas globosa and Chlorococcum ellipsoideum (Kroes 1971), o f S. costatum a n d Thalassionema nitzschioides (Fedorov and K u s t e n k o 1972), a n d o f Thalassiosira pseudonana and Dunalliela tertiolecta ( M o s s e r et al. 1972). L a n g e (1974) e x a m i n e d the interact ion o f three species o f cyanobacter ia , Microcystis aeruginosa, Nostoc muscorum and Phormidium foveolarum, Sze and K i n g s b u r y (1974) that o f Chlamydomonas spp. and Staurastrum paradoxum and Elbrachter (1977) that a m o n g Biddulphia ( = Odontella) regia, Coscinodiscus concinnus, Ceratium horridum and Prorocentrum micans. A d d i t i o n a l studies i n c l u d e the interactions o f P. micans, S. costatum and Chaetoceros didymus ( U c h i d a 1977), o f Thalassiosira pseudonana and Pheaodactylum tricornutum (Sharp et al. 1979) and the compet i t ive interact ion a m o n g Scrippsiella faeroense, Prorocentrum micans a n d Gymnodinium splendens ( K a y s e r 1979). In addi t ion , D e J o n g and A d m i r a a l (1984) e x a m i n e d the interactive g r o w t h o f Navicula salinarwn, Nitzschia -49-closterium and Amphiprora cf. paludosa and R i j s t e n b i l (1989) that o f S. costatum and Ditylum brightwellii. In most o f the studies ment ioned, the outcome o f the interact ion is i n h i b i t o r y for at least one o f the species i n v o l v e d . Nitzschia inhibi ts the g r o w t h o f Chlorella whereas the opposite does not occur (R ice 1954). Olisthodicus over took Skeletonema i n most cases (Pratt 1966) and so d i d Ditylum (Ri j s tenbi l 1989). F i n a l l y , Prorocentrum inh ib i ted both Skeletonema and Chaetoceros ( U c h i d a 1977). A number o f observers have c o n c l u d e d that the outcome o f the interact ion depends u p o n the i n i t i a l c e l l number rat io o f the species i n v o l v e d . In a l l species pairs e x a m i n e d by K a y s e r (1979) i n batch cultures, the species w i t h the highest i n i t i a l c e l l density was the one that dominated the culture at the end o f the exper imental p e r i o d . T h i s was also the case i n studies between A . cf . paludosa and N. closterium (De J o n g and A d m i r a a l 1984). T h e species w i t h the higher i n o c u l u m size was the one that overtook the cultures. F e d o r o v a n d K u s t e n k o (1972) d iscovered that Thalassionema overtook the b i a l g a l cultures, regardless o f nutrient concentrat ion, w h e n its i n o c u l u m size was larger than Skeletonema. T h e reverse was not achieved. W h e n the phosphate : nitrate ratio was too l o w for Skeletonema, this species d i d not overtake the cultures regardless o f the respective i n o c u l a . In the study by R i c e (1954), the i n i t i a l c e l l concentrat ion was important as w e l l . H o w e v e r , a m u c h larger concentrat ion o f Chlorella was necessary to inh ib i t Nitzschia g r o w t h than o f Nitzschia to inh ib i t Chlorella. In the studies by L a n g e (1974) and Sze and K i n g s b u r y (1974), the outcome o f species interactions was not altered w i t h different i n o c u l u m sizes. T h e c o n c l u s i o n o f a number o f the studies that e x a m i n e d the interact ion o f species i n culture is one o f "a l le lopathy" (as i n Sharp et al. 1979). T h i s invo lves the negative effect o f compounds excreted into the m e d i u m b y one m i c r o a l g a l species o n another species present. T h e effect is usual ly determined b y observ ing the g r o w t h o f an organism i n culture m e d i u m that contains filtrate (to remove the cells) f r o m a culture o f the -50-inhib i tory species. R i c e (1954) observed a decrease i n both growth rate and m a x i m a l y i e l d for both species w h e n g r o w n i n each other's fi ltrates. A l t h o u g h some i n h i b i t i o n d i d occur by their o w n fi ltrate, this was not as pronounced as the effect b y the other species. Olisthodiscus demonstrated satisfactory g r o w t h i n Skeletonema f i ltrate whereas the opposite was not achieved (Pratt 1966). T h e growth o f Skeletonema was least inh ib i ted i n the highest d i l u t i o n o f the O. luteus f i l trate. Chlamydomonas was inh ib i ted b y the addi t ion o f Chlorococcum f i ltrate into its o w n m e d i u m (Kroes 1971). O n the other hand, Chlamydomonas has been s h o w n to induce s i m i l a r i n h i b i t i o n to Staurastrum (Sze and K i n g s b u r y 1974). B o t h S. costatum and C. didymus were i n h i b i t e d i n P. micans filtrate ( U c h i d a 1977) and T. pseudonana demonstrated increased l a g phase a n d reduced m a x i m a l y i e l d i n the presence o f Pheaodactylum f i ltrate (Sharp et al. 1979). S i m i l a r conc lus ions were reached by K a y s e r (1979) for the filtrates o f P. micans and G. splendens w h i c h increased the length o f l a g phase and b y R i j s t e n b i l (1989) for both species used i n his study. In an attempt to characterize the i n h i b i t o r y phytoplankton exudates, R i c e (1954) and Sharp et al. (1979) c o n c l u d e d that the detr imental effect is r e m o v e d b y autoc lav ing the filtrate. In addi t ion , U c h i d a (1977) suggested that the size o f the exudates o f the species that he used must have been quite large because their effect was r e m o v e d b y f i l t e r ing the m e d i u m through a ce l lu lose f i l ter . Pratt (1966) indicated that the effects are m a x i m a l at the beg inning o f a filtrate experiment because o f subsequent degradation o f the exudate. In some instances, the effect o f exudates appears to depend u p o n the phase o f g r o w t h d u r i n g w h i c h i t was excreted. Sharp et al. (1979) f o u n d that the i n h i b i t o r y substance was excreted d u r i n g the stationary phase o f P. tricornutum. S i m i l a r results were obtained by K a y s e r (1979). F e d o r o v and K u s t e n k o (1972) f o u n d an inhib i tory effect o f the senescence exudates o f Thalassionema o n Skeletonema. H o w e v e r , fo r Thalassionema, the i n h i b i t o r y effect was pronounced for Skeletonema exudates o f -51 -exponent ia l ly g r o w i n g ce l l s . N o difference between exudates o f these t w o phases was recorded f o r Ditylum o n Skeletonema (R i j s tenbi l 1989). In some o f the aforementioned studies, other factors that m i g h t inf luence the outcome o f b i a l g a l culture growth were examined . Nutr ient exhaust ion b y one o f the species i n a m u l t i a l g a l culture was suggested as an alternate explanat ion. F o r example , R i c e (1954) added nutrients to the cultures to reverse the outcome but was unsuccessful . T h i s was also the case i n the studies by L a n g e (1974) and b y Sze a n d K i n g s b u r y (1974). U c h i d a (1977) added nutrients to the filtrates but the i n h i b i t o r y effect persisted. A s ment ioned earl ier , i n the study by F e d o r o v and K u s t e n k o (1972), nutrient levels contro l led the outcome i n the cases o f S. costatum dominance but not w i t h T. nitzschioides. These t w o species demonstrated growth enhancement i n the presence o f different nutrients (nitrate for Skeletonema and phosphate f o r Thalassionema). In the presence o f a l o w nitrate : phosphate rat io i n the m e d i u m , Skeletonema c o u l d not overtake the cultures (Fedorov and K u s t e n k o 1972). In a l l other cases, i t d i d . E lbrachter (1977) c o n c l u d e d that nutrient l i m i t a t i o n was responsible for a l l the species inh ib i t ions that he observed (Biddulphia b y Coscinodiscus, Ceratium b y Biddulphia and Prorocentrum by Biddulphia and Coscinodiscus). K a y s e r (1979), us ing mar ine dinoflagel lates , observed very l i t t le i n h i b i t i o n i n filtrate cultures and no effect at a l l i n continuous cultures and c o n c l u d e d that the effect that was observed i n batch cultures was due to nutrient exhaust ion. It has thus been demonstrated that a l though species exhibi t part icular g r o w t h characteristics w h e n g r o w n alone, they m a y be severely affected (to the point o f complete g r o w t h inhib i t ion) i n the presence o f a second species. It m a y , therefore, be suggested that g r o w t h under metal stress m a y be i n f l u e n c e d b y the presence o f a second species. O n e important c o m m o n aspect between the results obtained f r o m studies o f species interactions i n m u l t i a l g a l cultures and i n d i v i d u a l species ' metal tolerances is the importance o f organic exudates. S o far , I have discussed their importance i n species -52-interactions. H o w e v e r , their importance i n phytoplankton metal tolerance requires a l i t t le more attention. S to lzberg and R o s i n (1977) c o n c l u d e d that extracel lular , m e t a l - b i n d i n g , organic matter was produced by Skeletonema costatum. These exudates had a c o m p l e x i n g capaci ty o f 3 to 4 x 10-7 M . S w a l l o w et al. (1978) e x a m i n e d the effect o f extracel lular organic exudates o f nine species o f phytoplankton ( k n o w n to produce organic exudates) o n copper c o m p l e x a t i o n i n the m e d i u m . O f these, o n l y the freshwater species Gloeocystis gigas was p r o v e n to produce organic compounds that d o actual ly c o m p l e x copper. T h e authors suggested that the sensi t ivi ty o f the l i g a n d measurement technique was not l o w enough to detect potent ial l igands i n the other species. M c K n i g h t and M o r e l (1979) e x a m i n e d the product ion o f copper c o m p l e x i n g organic l igands i n a number o f freshwater phytoplankton . T h e results were quite var iable . D i a t o m s d i d not produce any l igands , f i v e chlorophytes and t w o chrysophytes p r o d u c e d measureable concentrations (a weak organic ac id-type l igand) and four cyanophytes produced very strong l igands (probably more than one l igand) . M c K n i g h t and M o r e l (1980) subsequently determined copper c o m p l e x a t i o n b y hydroxymate siderophores p r o d u c e d by certain species o f cyanobacteria . In cultures o f Anabaena cylindrica, increased cupr ic i o n act ivi ty was observed i n cultures o f h i g h i r o n content. T h e authors c o n c l u d e d that w h e n i r o n is i n l o w concentrat ion i n the m e d i u m , copper binds onto the siderophore. W h e n the i r o n concentration increases, this metal displaces copper o n the c o m p o u n d . T h e y detected t w o types o f l igands , w e a k ones i n l o w e r i r o n concentrations and stronger ones i n higher i r o n levels . Cul tures o f Synecchoccus leopoliensus, o n the other hand, d i d not demonstrate increased cupr ic i o n concentrat ion i n elevated i r o n . F i sher and Fabr is (1982) e x a m i n e d the copper c o m p l e x i n g abi l i ty o f exudates produced by Skeletonema costatum, Asterionella japonica and Nitzschia closterium. C o p p e r was the metal that was preferably b o u n d by the exudates produced b y a l l o f these species d u r i n g exponent ia l g r o w t h , and z i n c d u r i n g senescence. T h e y d i d not f i n d any differences i n c o m p l e x i n g capaci ty between the different d i a t o m species exudates and suggested that these l igands -53-are stronger than refractory d i s s o l v e d organic carbon f o u n d i n natural seawater (e.g. h u m i c acids) . S. costatum was f o u n d to produce t w o l igands that b i n d z i n c , a weaker and a stronger one (Imber et al. 1985). T h e condi t iona l stabil i ty constants o f the l igands excreted by the cyanobacter ium, Microcystis aeruginosa d i f fered, depending o n the p e r i o d o f g r o w t h d u r i n g w h i c h they were produced. T h e l i g a n d concentration (complexat ion capacity) and the stabi l i ty constants increased w i t h aging o f the cultures ( O g i w a r a and K o d a i r a 1989). A l t h o u g h studies have been p e r f o r m e d o n the c o m p l e x i n g abi l i ty o f organic exudates for copper , i t s t i l l remains unclear as to whether their product ion is s t imulated b y increased copper levels i n the surrounding m e d i u m . Steeman N i e l s e n and W i u m -A n d e r s e n (1971) a n d Florence et al. (1982) have suggested s t imulat ion b y ambient concentrations for Nitzschia palea and S. costatum, and Nitzschia closterium respect ively. L a n g e (1974) attributed the i n h i b i t i o n by Phormidium foveolarum o f Nostoc muscorum and Microcystis aeruginosa, to exudates that remove metals f r o m the m e d i u m . M u r p h y et al. (1976) demonstrated that a hydroxamate chelator produced b y the cyanobacter ium Anabaena flos-aquae (or its associated bacteria) r e m o v e d i r o n f r o m the m e d i u m d u r i n g a speci f ic p e r i o d i n ni trogen f i x a t i o n . T h i s i n turn i n h i b i t e d Scenedesmus due to i r o n l i m i t a t i o n . V a n D e n B e r g et al. (1979) determined that Anabaena cylindrica, Navicula pelliculosa and Scenedesmus quadricauda produce copper l igands o f different strengths and concentrations. H o w e v e r , the f i n a l i o n i c copper concentration i n the m e d i u m d i d not d i f f e r between species. T h e y subsequently d iscovered that i n the absence o f copper, Chlorella vulgaris grew better i n the Anabaena f i ltrate and worse i n the Scenedesmus f i l trate. In the presence o f elevated total copper , the filtrates w i t h the stronger l igands were more benef ic ia l . B r o w n et al. (1988) demonstrated that the copper tolerance o f Thalassiosira profunda was s l ight ly enhanced i n the presence o f Amphora coffeaeformis exudate. T h e authors suggested that copper is b o u n d onto the muci lage -54-that is excreted by the pennate. These are the f e w studies that attempted to l i n k the excret ion o f an exudate b y a phytoplankter and the different manners i n w h i c h i t m a y affect the s u r v i v a l o f a second species i n different copper concentrations. A second outcome o f m u l t i a l g a l cultures that m a y affect the apparent tolerance o f an o r g a n i s m to a meta l is nutrient l i m i t a t i o n . In the interactions that are e x a m i n e d i n the laboratory this factor w i l l come into p lay o n l y i n batch cultures. A s s h o w n i n a number o f studies, nutrient exhaust ion b y one species was the factor that l i m i t e d the g r o w t h o f other species. M e t a l s have been s h o w n to increase the length o f l a g phase i n some species (see Chapter 1). If one o f the species demonstrates an increase i n l a g phase then, by the t ime it starts g r o w i n g , the m e d i u m m i g h t be nutrient-deplete. In this case, a l though exponent ia l g r o w t h w i l l be observed w h e n the organism is g r o w n alone, no such g r o w t h w i l l be observed i n the b i a l g a l cultures. T h i s type o f outcome w i l l depend u p o n the i n d i v i d u a l nutrient requirements a n d nutrient uptake rates o f a l l species i n v o l v e d . It becomes apparent, therefore, that organisms m a y m o d i f y the m e d i u m as w e l l as alter its c h e m i c a l speciat ion. T h i s i n turn w i l l affect their meta l tolerance d irec t ly . It has also been demonstrated that the c o m p l e x i n g capacity o f excreted organics varies between species. S o m e species produce very strong l igands , some produce weak ones, some d o not produce any at a l l . In addi t ion , i t has been s h o w n that the interact ion between species that are cul tured together affects their i n d i v i d u a l growth . In a number o f studies, the inh ib i tory effect was attributed to exudates o f the species that dominated. In others, it has been attributed to nutrient deplet ion w h i c h w i l l become more l i k e l y i n the case o f extended lag phase o f a species u p o n copper addi t ion . It can , therefore, be expected that the apparent meta l tolerance o f a species m a y be altered i n the presence o f a second species. T h i s m a y be achieved either b y i n h i b i t i o n , as demonstrated by the b i a l g a l experiments , or b y a m e d i u m m o d i f i c a t i o n , as demonstrated b y the different c o m p l e x i n g capabil i t ies o f organic exudates o f different species and by nutrient deplet ion. -55-V e r y f e w studies o f this type have been carr ied out. M o s s e r et al. (1972) e x a m i n e d the interact ion between Thalassiosira pseudonana (a sensitive species) and Dunaliella tertiolecta (a tolerant species) i n the presence o f P C B s and D D T . Thalassiosira was the dominant species i n the contro l m i x e d cultures, demonstrat ing very h i g h g r o w t h rates compared to Dunaliella. In h i g h toxicant levels , this species was more inh ib i ted i n the presence o f Dunaliella than i n un ia lga l cultures. T h e result was attributed to compet i t ion for nutrients. G o l d m a n and Stanley (1974) e x a m i n e d the relat ive growth o f Phaeodactylum tricornutum, Thalassiosira pseudonana and Dunaliella tertiolecta i n wastewater-seawater mixtures . In this study, the authors demonstrated that the single species bioassays ( w h i c h determined Phaeodactylum as the dominant species) d i d predict the outcome o f the m i x e d cultures ( in w h i c h Phaeodactylum dominated w i t h i n a f e w days) . A study carr ied out by D a s h o r a and G u p t a (1978) suggested an effect o f chlor ine and copper o n species combinat ions (of Scenedesmus obliquous, Selenastrum minutum and Phormidium luridum). H o w e v e r , i t is d i f f i c u l t to assess the value o f this study to m y research, since no examinat ion o f the i n d i v i d u a l species tolerances was i n c l u d e d . In the previous chapter o f this thesis, I discussed the speci f ic copper tolerances o f Skeletonema costatum and Nitzschia thermalis. It was demonstrated that Nitzschia is s l ight ly more tolerant than Skeletonema and the copper effect ("stress") was manifested di f ferent ly i n the t w o species. F o r this part o f the study, the t w o species were cul tured together i n order to examine the effect o f the presence o f a second species (that m i g h t be responding di f ferent ly to copper stress, as w e l l as m o d i f y i n g the m e d i u m ) o n their apparent i n d i v i d u a l copper tolerances. T h e chapter is c o m p o s e d o f two parts. In the f irst part, I discuss the effect o f the second species o n the expected g r o w t h and s u r v i v a l at three copper concentrations for each o f the t w o organisms. In the second section, I discuss poss ib le factors that m a y p l a y a ro le i n the interaction o f the t w o species and m a y , therefore, determine the apparent copper tolerance o f i n d i v i d u a l species. -56-P A R T A : Interaction of Skeletonema costatum with Nitzschia thermalis in three copper concentrations. M E T H O D S T h e same general exper imental procedure was f o l l o w e d d u r i n g these experiments as i n Chapter 1. T h e o n l y difference w i t h the experiments descr ibed i n this section is that the stock cultures f r o m w h i c h the i n o c u l a were r e m o v e d were i n late exponent ia l phase to the beg inning o f senescence. T h e large number o f replicates used d u r i n g the experiments required a large number o f cel ls for inocu la t ion that c o u l d o n l y be obtained f r o m dense cultures. E n o u g h ce l l s were avai lable by inocula t ing f r o m stocks that were late i n the exponent ia l phase or early senescence. A l i q u o t s were r e m o v e d w h e n the stock cultures reached an appropriate concentrat ion. I subsequently determined the n u m b e r o f cel ls • m L - 1 i n the stock cultures us ing a P a l m e r M a l o n e y count ing s l ide under a Z e i s s Standard 14 c o m p o u n d microscope . T h e n , the v o l u m e o f the i n o c u l u m necessary to obtain a density o f ~ 5 0 0 cel ls • m L - 1 i n the first experiment and - 1 , 0 0 0 cel ls • m L ' l i n the second one was calculated. T h e actual popula t ion sizes that I d i d obtain us ing this method were 400 ± 60 and 800 ± 80 ce l ls • m L - 1 for the f irst and second experiments respect ively (mean ± S . E . , N = 45) . Three treatments were used w i t h respect to the b i o l o g i c a l species. O n e u n i a l g a l treatment for each o f the t w o species was i n c l u d e d . These served as controls f o r the g r o w t h o f Skeletonema and Nitzschia i n the absence o f a second species. I n addi t ion , a th i rd treatment was i n c l u d e d i n w h i c h S. costatum and N. thermalis were inoculated s imultaneously . T h i s treatment was used to determine the effect that the second species had o n the g r o w t h and s u r v i v a l o f the other species. T h e i n o c u l u m size was the same for both species and a l l treatments. A s a result , the m i x e d cultures rece ived twice the number o f cel ls c o m p a r e d to the unia lga l f lasks . -57-E a c h o f these treatments was e x a m i n e d at three copper concentrations. T h e first , 1 x 10-9 M C u , served as a contro l . It has been demonstrated (Chapter 1) that, i n this concentrat ion, both species show satisfactory g r o w t h . Therefore , no effect o n their g r o w t h should be observed as a result o f copper . T h e second copper concentration that was used was 4 x 10-7 M (= 0.4 \iM) C u . F r o m the previous chapter, i t was expected that Nitzschia should g r o w equal ly w e l l w i t h the contro l treatment at this l eve l . O n the other hand, Skeletonema should demonstrate a p r o l o n g e d l a g phase and an increase i n growth rate due to the copper stress. T h e last copper l e v e l that was used was 5 x 10-7 M (= 0.5 LIM). I n this concentrat ion, Skeletonema should not g r o w at a l l . Nitzschia should either g r o w equal ly w e l l w i t h the contro l (1 x 10-9 M) or show a sl ight decrease i n g r o w t h rate c o m p a r e d to the contro l . A n y deviat ions f r o m these expectations s h o u l d be attributed to the interaction w i t h the second species present i n the same batch culture. F i v e replicates were used for each o f the above nine treatments and a l l treatments were r u n s imultaneously . A c o m b i n a t i o n o f r a n d o m and systematic designs was used i n order to arrange the posi t ions o f the f lasks o n the culture chamber shelves. Three shelves w i t h a total o f four l ight sources were avai lable for use. I ass igned a replicate per l ight source for each o f the nine treatments. I then r a n d o m l y assigned the f i f t h replicate to one o f the l ight sources. O n c e a l l f lasks were associated w i t h a l ight source, they were r a n d o m l y pos i t ioned w i t h i n each source u s i n g a r a n d o m number table. T h e experiment was r u n twice i n order to v e r i f y the consistency o f the results. A s ment ioned earlier, d u r i n g the f irst r u n the i n o c u l u m p r o v i d e d a c e l l concentration o f - 5 0 0 cel ls • m L - 1 for each species. In the course o f the experiment , I observed s l ight ly extended l a g phases w h i c h I attributed to the unusual ly l o w i n o c u l u m size. D u r i n g inocu la t ion , a number o f cel ls m i g h t die due to the mechanica l process o f transferring them, as w e l l as due to the change i n m e d i u m c o m p o s i t i o n . I f the i n o c u l u m size is not large enough, the number o f cel ls that d o survive m i g h t be too s m a l l to reflect an increase i n popula t ion s ize (g iven the accuracy o f m y count ing procedure w h i c h cannot detect -58-densities b e l o w 200 cel ls • mL-1). j f the popula t ion increase is not detectable, i t w i l l be perce ived as an extended l a g phase. Therefore , to overcome such problems i n the second experiment , the i n o c u l u m was increased to p r o v i d e an i n i t i a l concentration o f -1,000 cel ls • mL -1 . W i t h i n each copper concentrat ion, the g r o w t h rate and l a g phase were compared between t w o treatments (species g r o w i n g alone a n d i n the presence o f the second species). A l l comparisons were per formed us ing a Student 's t-test. In cases where no g r o w t h was obtained i n one o f the treatments, a W i l c o x o n rank s u m test was used instead. In cases where no g r o w t h was observed i n either o f the t w o treatments, no statistical c o m p a r i s o n was carr ied out. T h e Student 's t- and the W i l c o x o n rank s u m tests were carr ied out u s i n g E P I S T A T (Gustafson 1984) o n a C o m m o d o r e PC-10 n. A s igni f icance l e v e l o f 0.05 was used throughout. -59-R E S U L T S T h e results are s h o w n i n F i g s . 2.1-2.3 for experiment 1 and F i g s . 2.4-2.6 for experiment 2. Skeletonema d i d g r o w satisfactori ly i n 1 x 10-9 M C u , exhib i ted longer lag phase and g o o d g r o w t h rate i n 4 x 10-7 M and d i d not g r o w at a l l i n 5 x 10-7 M C u . O n the other hand, this species d i d not show any g r o w t h i n the presence o f Nitzschia (wi th the except ion o f one replicate i n 1 x 10-9 M C u i n the first r u n o f the experiment) . Nitzschia grew i n a l l copper concentrations whether i n u n i - or b i a l g a l exper imental f lasks . T h e statistical comparisons between the t w o treatments (single species versus double species addit ions) , fo r each copper concentrat ion and both experiments , are s h o w n i n Tables 2.1-2.8. 5 . costatum demonstrated s igni f icant ly higher g r o w t h i n a l l copper concentrations w h e n g r o w n alone than w h e n g r o w n i n the presence o f Nitzschia. T h e o n l y except ion was i n 4 x 10-7 M C U d u r i n g the second run o f the experiment (Table 2.5). In this case, there was no s ignif icant di f ference between the t w o treatments. N o statistical comparisons to determine differences i n l a g phase were poss ible since no g r o w t h at a l l was observed i n the treatments that i n c l u d e d Nitzschia. F o r N. thermalis, there was no s ignif icant dif ference i n growth rate between the t w o treatments i n any copper concentrat ion, i n both runs o f the experiment (Tables 2.3 and 2.7). T h e same was true f o r the l a g phase (Table 2.4 and 2.8), w i t h one except ion. T h e treatment that i n c l u d e d Skeletonema i n 1 x 10-9 M added C u demonstrated an extended lag phase c o m p a r e d to the single species addi t ion (Table 2.8). A s m a y be noted f r o m the tables, a number o f replicates were not incorporated i n the growth rate and l a g phase estimations o n a number o f occasions . In experiment 1, at the 4 x 10-7 M C u l e v e l , replicate 4 o f Skeletonema g r o w i n g alone d i d not c o m e out o f l a g phase before the terminat ion o f the experiment (Table 2.2). In the same experiment , i n 1 x 10-9 M C u , replicate 3 o f N. thermalis d i d not demonstrate a pronounced -60-exponent ia l g r o w t h rate pattern d u r i n g any stage o f the exper imental p e r i o d (Tables 2.3 and 2.4). D u r i n g exper iment 2 , one o f the replicates o f S. costatum, g r o w i n g alone i n 4 x 10 _ 7 M C u , became contaminated w i t h Nitzschia o n day 12 and was complete ly e x c l u d e d f r o m the calculat ions (Tables 2.5 and 2.7). In the same treatment, a second replicate d i d not show exponent ia l g r o w t h (although it demonstrated signs o f increase) before the e n d o f the experiment. 61 100 f 1000 10--_l E \ 1.0--in Cel > 0.1 > o \ / \ / \ 10 12 Doys 14 16 18 20 a 100 T 10 1.0 • 0.1 ; o / o B 10 12 Days 14 16 18 20 22 FIGURE 2.1: Growth curves of Skeletonema costatum (A, B) and Nitzschia thermalis (C, D) grown in unialgal (A, C) and bialgal (B, D) cultures, in 5 x 10-7 M Cu (Experiment 1). Circles, triangles, squares, inverted triangles and diamonds represent replicates 1-5 respectively. Filled symbols represent the points at which No and Ni were estimated. 62 F I G U R E 2.2: G r o w t h curves o f Skeletonema costatum ( A , B ) and Nitzschia thermalis ( C , D ) g r o w n i n u n i a l g a l ( A , C ) and b i a l g a l ( B , D ) cultures , i n 4 x 10-7 M C u ( E x p e r i m e n t 1). C i r c l e s , tr iangles , squares, i n v e r t e d triangles and d i a m o n d s represent replicates 1-5 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N o and N i w e r e est imated. 63 100T 10--1.0-v 100 T 10 o-" Q 8 10 12 14 16 18 20 22 Days 1.0-*?;?' % o °-. ,<? 3-S.S. A. 'O-^T \ 0.1 E d i Cb B aai' 1—3i-afe-e--a B 0 2 4 6 8 10 12 14 16 18 20 22 Days F I G U R E 2 .3 : G r o w t h curves o f Skeletonema costatum ( A , B ) a n d Nitzschia thermalis ( C , D ) g r o w n i n u n i a l g a l ( A , C ) and b i a l g a l ( B , D ) cultures , i n 1 x 10-9 M C u ( E x p e r i m e n t 1). C i r c l e s , t r iangles , squares, inver ted tr iangles a n d d i a m o n d s represent replicates 1-5 respec t ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 and N i were es t imated. 64 F I G U R E 2.4 : G r o w t h curves o f Skeletonema costatum ( A , B ) a n d Nitzschia thermalis ( C , D ) g r o w n i n u n i a l g a l ( A , C ) a n d b i a l g a l ( B , D ) cultures , i n 5 x 10-7 M C u (Exper iment 2) . C i r c l e s , t r iangles , squares, inver ted triangles and d i a m o n d s represent replicates 1-5 respec t ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 a n d N i were est imated. 65 F I G U R E 2 .5 : G r o w t h curves o f Skeletonema costatum ( A , B ) a n d Nitzschia thermalis ( C , D ) g r o w n i n u n i a l g a l ( A , C ) and b i a l g a l ( B , D ) cultures , i n 4 x 10-7 M C u ( E x p e r i m e n t 2) . C i r c l e s , t r iangles , squares, inver ted triangles a n d d i a m o n d s represent repl icates 1-5 respec t ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 and N i were est imated. 66 Days D a y s F I G U R E 2.6: G r o w t h curves o f Skeletonema costatum ( A , B ) a n d Nitzschia thermalis ( C , D ) g r o w n i n u n i a l g a l ( A , C ) and b i a l g a l ( B , D ) cul tures , i n 1 x 10-9 M C u (Exper iment 2) . C i r c l e s , triangles, ' squares, inver ted tr iangles a n d d i a m o n d s represent replicates 1-5 respec t ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 and N i were est imated. -67-T A B L E 2 .1 : Stat ist ical comparisons between growth rates o f Skeletonema costatum g r o w i n g i n un ia lga l cultures and i n the presence o f Nitzschia thermalis, i n three different copper concentrations (Exper iment 1). - = species g r o w n alone; + = species g r o w n i n the presence o f N. thermalis; N = sample size; S . D . = standard deviat ion ; N . D . = not determined; * = W i l c o x o n test used instead. C o p p e r Treatment M e a n G r o w t h S . D . t-value Concentrat ion Rate ( M added C u ) (N) 5 x 1 0 " 7 4 x 10-7 1 x 10-9 + + 0 (5) 0 (5) 0.97 (4) 0 (5) 1.37 (5) 0.22 (5) 0 N . D . N . D . 0 0.18 * <0.050 0 0.34 4.31 <0.005 0.49 -68-T A B L E 2.2: Statist ical comparisons between lengths o f l a g phase for Skeletonema costatum g r o w i n g i n un ia lga l cultures and i n the presence o f Nitzschia thermalis, i n three different copper concentrations (Exper iment 1). - = species g r o w n alone; + = species g r o w n i n the presence o f N. thermalis; N = sample s ize; S . D . = standard deviat ion ; N . D . = not determined. C o p p e r Treatment M e a n L a g S . D . t-value Concentrat ion Phase ( M added C u ) (N) 5 x 1 0 " 7 4 x 1 0 " 7 1 x 10-9 + N . D . (5) N . D . (5) 14.4 (4) N . D . (5) 8.6 (5) N . D . (5) N . D N . D . 3.9 N . D . 4.7 N . D . N . D . N . D . N . D . N . D . N . D . N . D . -69-T A B L E 2 .3 : Statist ical comparisons between g r o w t h rates for Nitzschia thermalis g r o w i n g i n u n i a l g a l cultures and i n the presence o f Skeletonema costatum, i n three different copper concentrations (Exper iment 1). - = species g r o w n alone; + = species g r o w n i n the presence o f S. costatum; N = sample size; S . D . = standard deviat ion . C o p p e r Treatment M e a n G r o w t h S . D . t-value Concentrat ion Rate ( M added C u ) (N) 5 x 1 0 " 7 4 x 10-7 1 x 10-9 + 1.21 (5) 1.02 (5) 1.22 (5) 1.21 (5) 1.07 (4) 1.13 (5) 0.32 0.36 0.42 0.31 0.41 0.68 0.882 >0.100 0.050 >0.100 0.155 >0.100 -70-T A B L E 2.4: Statistical comparisons between lengths o f l a g phase for Nitzschia thermalis i n unia lga l cultures and i n the presence o f Skeletonema costatum, i n three different copper concentrations (Exper iment 1). - = species g r o w n alone; + = species g r o w n i n the presence o f 5 . costatum; N = sample size; S . D . = standard devia t ion . C o p p e r Treatment M e a n L a g S . D . t-value Concentrat ion Phase ( M added C u ) (N) 5 x 1 0 " 7 4 x 1 0 " 7 1 x 10-9 + + + 2.5 (5) 3.3 (5) 1.7 (5) 2.3 (5) 0.83 (4) 1.13 (5) 1.2 1.2 1.5 0.9 1.0 0.54 1.08 >0.100 0.741 >0.100 0.922 >0.100 -71 -T A B L E 2 .5 : Statist ical comparisons between growth rates for Skeletonema costatum g r o w i n g i n unia lga l cultures and i n the presence o f Nitzschia thermalis, i n three different copper concentrations (Exper iment 2). - = species g r o w n alone; + = species g r o w n i n the presence o f N. thermalis; N = sample size; S . D . = standard deviat ion ; N . D . = not determined; * = W i l c o x o n test used instead. C o p p e r Treatment M e a n G r o w t h S . D . t-value Concentrat ion Rate ( M added C u ) (N) 5 x l ( r 7 4 x 10-7 1 x 10-9 + 0 (5) 0 (5) 1.34 (3) 0 (5) 1.26 (5) 0 (5) 0 0 0.77 0 0.13 0 N . D . N . D . =0.100 <0.010 -72-T A B L E 2.6: Stat ist ical comparisons between lengths o f l a g phase for Skeletonema costatum g r o w i n g i n un ia lga l cultures and i n the presence o f Nitzschia thermalis, i n three different copper concentrations (Exper iment 2). - = species g r o w n alone; + = species g r o w n i n the presence o f N. thermalis; N = sample size; S . D . = standard devia t ion ; N . D . = not determined. C o p p e r Treatment M e a n L a g S . D . t-value Concentrat ion Phase ( M added C u ) (N) 5 x l 0 - 7 - N . D . N . D (5) + N . D . N . D . (5) 4 x 1 0 - 7 - 7.1 0.9 (3) + N . D . N . D . (5) 1 x 1 0 - 9 - 4.1 1.6 (5) + N . D . N . D . (5) N . D . N . D . N . D . N . D . N . D . N . D . -73-T A B L E 2.7: Statist ical comparisons between growth rates for Nitzschia thermalis i n unia lga l cultures and i n the presence o f Skeletonema costatum, i n three different copper concentrations (Exper iment 2) . - = species g r o w n alone; + = species g r o w n i n the presence o f S. costatum; N = sample s ize; S . D . = standard deviat ion . C o p p e r Treatment M e a n G r o w t h S . D . t-value Concentrat ion Rate ( M added C u ) (N) 5 x l 0 " 7 - 1.18 0.31 (5) + 1.72 0.55 (5) 4 x 1 0 - 7 _ 1.07 0.48 (5) + 1.24 0.31 (4) 1 x 1 0 - 9 - 1.57 0.39 (5) + 2.09 0.87 (5) 1.94 >0.050 0.611 >0.100 1.23 >0.100 -74-T A B L E 2.8: Statist ical comparisons between lengths o f l a g phase for Nitzschia thermalis g r o w i n g i n unia lga l cultures and i n the presence o f Skeletonema costatum, i n three different copper concentrations (Exper iment 2). - = species g r o w n alone; + = species g r o w n i n the presence o f S. costatum; N = sample s ize; S . D . = standard devia t ion . C o p p e r Treatment M e a n L a g S . D . t-value Concentrat ion Phase ( M added C u ) (N) 5 x 1 0 " 7 4 x 10-7 1 x 10-9 + + 0.92 (5) 1.5 (5) 1.5 (5) 1.6 (4) 0.58 (5) 1.48 (5) 0.95 0.50 0.9 0.6 0.53 0.57 1.32 >0.100 0.050 >0.100 2.58 <0.050 -75-D I S C U S S I O N O n e c o m m o n pattern arose f r o m both experiments . Regardless o f the copper concentration i n w h i c h these t w o species were g r o w i n g , Nitzschia thermalis had an inhib i tory effect o n Skeletonema costatum. T h e opposite interact ion does not appear to occur . I f i t d i d occur , the effect was not strong enough to reverse the outcome o f the experiments. T h e t w o species, w h e n i n un ia lga l cultures, behaved as expected. A f e w points o n the behaviour o f i n d i v i d u a l replicates, however , require c lar i f i ca t ion . A s noted earlier i n this chapter, extended l a g phases were observed d u r i n g the first r u n o f this experiment . T h i s was corrected b y increasing the i n o c u l u m size d u r i n g the second r u n . If the l a g phases f r o m both species are c o m p a r e d between experiments , a decrease i n length o f l a g phase can be seen (Table 2.2 w i t h 2.6 and T a b l e 2.4 w i t h 2.8 for 5 . costatum and N. thermalis respect ively) . In addi t ion , several replicates were not i n c l u d e d i n the g r o w t h calculat ions because o f the lack o f demonstrat ion o f exponent ia l g r o w t h d u r i n g the exper imenta l p e r i o d . F o r Skeletonema, there were t w o such occasions. B o t h o f these were g r o w i n g i n 4 x 10-7 M C u . In this concentrat ion, this species demonstrated extended l a g phase as a s ign o f copper stress. B o t h the experiments were terminated w h e n there were 50 m L o f m e d i u m r e m a i n i n g i n the exper imental f lasks (this d e c i s i o n was reached i n order to a v o i d any artifacts due to the increased surface : v o l u m e rat io o f the m e d i u m w h e n such a s m a l l v o l u m e remained) . A s a result, i n both these instances, the experiment was terminated before the f lasks demonstrated exponent ia l increase. O n one occas ion , one Nitzschia replicate that was g r o w i n g i n 1 x 10-9 M C u d i d not show strong exponent ia l increase at any point o n its g r o w t h curve ( F i g . 2 . 3B) . T h e c e l l counts largely osc i l la ted around 1,000 cel ls • m L - 1 a n d it was not poss ible to decide o n a part icular point o f in f lec t ion o f the growth curve . -76-Skeletonema d i d not demonstrate a s ignif icant difference i n g r o w t h between treatments i n one o f the replicates g r o w i n g i n 4 x 10-7 M added copper (Table 2.5). F r o m the actual growth rates, however , the unia lga l cultures d i d show g r o w t h (1 .34 div i s ions • day-1) whereas i n the presence o f Nitzschia no replicates grew at a l l . I bel ieve that the lack o f s igni f icance i n v o l v e s the large variance associated w i t h the m e a n growth rate. I consider this l a c k o f s igni f icance a statistical artifact and I conc lude that i n a l l cases Skeletonema g rew better i n the absence o f the pennate. N. thermalis demonstrated a pecul iar pattern o f g r o w t h i n both u n i - and b i a l g a l cultures. T h i s species had very short l a g phases (0.58-1.48 days , Tables 2 .4 and 2.8). O n c e out o f l a g phase, it grew at a very r a p i d rate for 1-2 days , reached a plateau o f c e l l numbers and then mainta ined its popula t ion s ize for the remainder o f the exper imental p e r i o d . T h i s becomes apparent f r o m the h igh g r o w t h rates that were obtained for this species d u r i n g the second experiment , especia l ly i n 1 x 10-9 M added copper (Table 2.7). M o s t o f these values were obtained f r o m 1-2 day exponent ia l g r o w t h , as ca lculated f r o m the growth curves . Nitzschia thermalis overtook a l l the b i a l g a l cultures. S ince the cont ro l , u n i a l g a l cultures grew as expected, one m i g h t expect that Nitzschia has an advantage over Skeletonema. T h i s c o u l d be a compet i t ive advantage i n nutrient uptake. Skeletonema, as a ru le , demonstrates longer l a g phase than Nitzschia. It is therefore possible that b y the time the l a g phase o f S. costatum is over , Nitzschia, w h i c h has reached stationary phase, has c o n s u m e d most o f the nutrients i n the m e d i u m . T h i s w i l l prevent Skeletonema f r o m attaining any s ignif icant g r o w t h rate. T h e poss ib i l i ty o f nutrient deplet ion by one o f the species g r o w n i n m u l t i a l g a l cultures was c o n c l u d e d by K a y s e r (1979) o n the interact ion o f marine dinoflagel lates a n d by Elbrachter (1977) for marine diatoms. Nitzschia m a y m o d i f y the m e d i u m by affect ing the carbon-source for Skeletonema spec i f i ca l ly . D e J o n g and A d m i r a a l (1984) suggested that the deplet ion o f H C O 3 - , w h i c h w i l l subsequently result i n h i g h O2 and h i g h p H , m a y act as a growth -77-l i m i t i n g factor i n the f i e l d . It has been demonstrated i n the laboratory that increased carbonate addi t ion i n the m e d i u m enhances photosynthetic rate ( A d m i r a a l et al. 1982). T h i s , i n turn, m i g h t determine the dominant species i n an interact ion depending u p o n the rate o f carbon-uptake and the g r o w t h - l i m i t i n g carbon concentrations for the different species i n v o l v e d . O n e m a y then suggest that Nitzschia can remove the source o f carbon d i o x i d e f r o m the m e d i u m d u r i n g its g r o w t h before the l a g phase o f Skeletonema is over . In this fash ion , suff ic ient carbon for exponent ia l growth m a y never become avai lable to Skeletonema. A decrease i n the carbonate concentration i n the m e d i u m s h o u l d be manifested by an increase i n p H (as i n D e J o n g and A d r n i r a a l 1984). A n alternative explanat ion for the results obta ined i n this series o f experiments is the product ion o f exudates by Nitzschia that are inh ib i tory to Skeletonema. A s descr ibed earlier, such a type o f interact ion has been c o m m o n l y f o u n d a m o n g species o f microalgae (e.g. R i c e 1954, Pratt 1966, F e d o r o v and K u s t e n k o 1972, Sharp et al. 1979, R i j s t e n b i l 1989). A th i rd explanat ion invo lves the product ion o f an organic exudate b y Nitzschia that complexes metals i n the m e d i u m . T h i s substance m i g h t be a siderophore-type, h i g h molecular weight ( M . W . ~ 1,000), organic c o m p o u n d , secreted to c o m p l e x metals i n the m e d i u m . T h e existence o f such c o m p o u n d s is not k n o w n for marine diatoms. I f i t does exist , i t m i g h t be secreted i n order to remove i r o n f r o m the culture m e d i u m . H o w e v e r , organic c o m p o u n d s o f l o w e r m o l e c u l a r weight have also been demonstrated to c o m p l e x metals . F o r example , F i s h e r and Fabr is (1982) have documented the excret ion o f such compounds by Skeletonema, Asterionella and Nitzschia. A s a result , the presence o f such Nitzschia exudates i n the culture m e d i u m m a y lead to a speci f ic meta l l i m i t a t i o n i n Skeletonema w h i c h w i l l result i n g r o w t h i n h i b i t i o n . F r o m this experiment , Nitzschia was the o r g a n i s m that grew successful ly i n a l l the b ia lga l cultures whereas Skeletonema d i d not. I c o n c l u d e d that Nitzschia has an inhib i tory effect u p o n Skeletonema. T h i s , however , does not preclude the reverse f r o m -78-occurr ing . It is poss ib le that Skeletonema m a y be i n h i b i t i n g Nitzschia i n a s i m i l a r fash ion , but that the effect is not strong enough to overr ide its o w n i n h i b i t i o n . T h i s m a y be observed i n two instances. T h e first was d u r i n g exper iment 1, i n replicate 1 o f the cultures g r o w i n g i n 1 x 10-9 M C u . T h i s Skeletonema replicate d i d g r o w at a satisfactory g r o w t h rate (1.10 d i v • day-1) . Nitzschia grew i n the same f lasks as w e l l , a l though at a re la t ive ly l o w g r o w t h rate (0.51 d i v • day-1) . T h i s was the o n l y occas ion d u r i n g w h i c h Skeletonema grew at a l l i n the m i x e d cultures. Its lag phase i n that replicate is m u c h shorter than the mean l a g phase demonstrated b y the unia lga l cultures i n the same copper concentration (1.2 versus 8.6 days respect ively) . Therefore, it appears that the inh ib i tory effect might depend o n the species that exits l a g phase early enough that, g i v e n its g r o w t h rate, i t can dominate the culture. R i c e (1954) d i scovered a s i m i l a r s i tuation between Chlorella and Nitzschia frustulum. In this si tuation, Chlorella was never observed to dominate a m i x e d culture. H o w e v e r , w h e n this species was a l l o w e d to c o n d i t i o n the m e d i u m before Nitzschia was added to inh ib i t i t , Chlorella was able to cause a reduct ion i n the Nitzschia g r o w t h . In addi t ion , its o w n g r o w t h was not reduced at a l l , even after the addi t ion o f the second species. T h e second occas ion d u r i n g w h i c h the negative effect o f Skeletonema became apparent was d u r i n g the second experiment . A l t h o u g h there was n o s ign o f increase f o r Skeletonema, Nitzschia demonstrated increased l a g phase i n the presence o f the second species c o m p a r e d to w h e n g r o w n alone i n 1 x 10-9 M added C u . R i j s tenb i l (1989) observed a negative effect o f both Ditylum and Skeletonema exudates o n each other. H o w e v e r , the i n h i b i t i o n o n Ditylum b y exponent ia l exudates o f Skeletonema was stronger than the i n h i b i t i o n o f Skeletonema b y any type o f Ditylum exudates. It i s , therefore, quite poss ible that both species affect each other but that, o v e r a l l , Nitzschia is the least sensitive one. In the next section o f this chapter, I shal l attempt to determine whether any o f the above factors e x p l a i n the i n h i b i t o r y effect that Nitzschia imposes u p o n Skeletonema. I -79-shal l examine the poss ib i l i ty o f alterations i n the p H o f the cultures due to carbonate deplet ion, the poss ib i l i ty o f nutrient (other than carbon) deplet ion, inh ib i tory exudates i n Nitzschia cultures, and f i n a l l y the poss ib i l i ty o f organic exudates act ing as compounds that c o m p l e x and remove metals f r o m the m e d i u m , leading to trace meta l l i m i t a t i o n . -80-P A R T B : P o s s i b l e f a c t o r s d r i v i n g the i n t e r a c t i o n be tween Skeletonema costatum a n d Nitzschia thermalis M E T H O D S T h e f irst factor e x a m i n e d was a di f ferent ia l change i n p H , measured at the end o f g r o w t h , between the unia lga l and the m i x e d cultures w i t h i n each copper concentrat ion. A di f ferent ia l increase i n p H was used as an indicator o f di f ferent ia l carbonate deplet ion b y the end o f the exper imental p e r i o d . A t the end o f experiment 2 , (see Part A ) I determined the p H i n a l l the exper imental f lasks u s i n g a F i s h e r A c c u m e t ® p H M o d e l 140 meter. Three treatments were used, S. costatum cultures, N. thermalis cultures and m i x e d cultures w i t h i n each o f the copper concentrations. Subsequently , I c o m p a r e d the p H values between the three treatments u s i n g a one-way analysis o f variance. In cases where a s ignif icant dif ference was obtained, a T u k e y "honestly s ignif icant di f ference" test was used to determine w h i c h pairs o f means were different. Other factors that were e x a m i n e d were the poss ible excret ion o f an i n h i b i t o r y c o m p o u n d b y Nitzschia, nutrient deplet ion o f the m e d i u m b y Nitzschia, and f i n a l l y trace meta l Umitat ion i n d u c e d through meta l b i n d i n g b y a Nitzschia exudate. F i v e treatments were used i n this experiment , a l l o f w h i c h were r u n at 1 x 10 _ 9 M C u m e d i u m : (a) Skeletonema g r o w n i n filtrate f r o m Nitzschia cultures; (b) Skeletonema g r o w n i n Nitzschia f i l trate, enr iched w i t h a l l nutrient stocks (nitrate, phosphate, s i l icate, trace metals and v i tamins) ; (c) Skeletonema i n fi ltrate enr iched w i t h trace meta l stock o n l y ; (d) S. costatum i n fresh m e d i u m , serving as a contro l . A l l exper imenta l procedures i n this section were s imi lar to those descr ibed i n Part A . A stock culture o f N. thermalis that was i n late exponent ia l to early senescent phase was used to inoculate 15 exper imental f lasks at an i n i t i a l concentration o f ~1000 cel ls • m L - l . T h e inocula ted f lasks , as w e l l as f i v e f lasks conta in ing A q u i l that had not been -81-inoculated, were then incubated as i n Part A . T w o l ight sources were used for this experiment . T w o replicates o f each treatment were assigned to each l ight source. T h e f i f t h replicate o f a l l treatments was assigned to l ights r a n d o m l y . W h e n a l l cultures were associated w i t h a l ight source, their arrangement w i t h i n sources was r a n d o m i z e d . W h e n Nitzschia reached stationary phase, I gently f i l tered the cel ls out o f each f lask. F o r f i l t e r ing , I used 0.45 p.m M i l l i p o r e type H A fi l ters , mounted o n 25 m m S w i n n e x disc f i l ter holders . A l i q u o t s o f the culture m e d i u m were d r a w n into a 60 m L po lypropylene sterile syringe and gently pushed through the filters in to c lean f lasks . T h e f i l ters, f i l ter holders and syringes were ac id-washed pr ior to use. T h e rubber plunger-t ips o f the syringes were w r a p p e d i n tef lon tape, i n order to a v o i d introduct ion o f tox ic substances that m i g h t leach f r o m the rubber. T h e f i l t e r ing was per formed under class 100 air condi t ions . E a c h o f the treatments (a) to (c) were then r a n d o m l y assigned to f i v e o f the f i f teen f lasks that contained the Nitzschia f i ltrates. F o r treatment (a), the filtrate was not m o d i f i e d . F o r treatment (b), a l l the A q u i l nutrients were added to the filtrate. F i n a l l y , for treatment (c), o n l y the metal stock was added to the filtrate. T h e f i v e replicates that just contained A q u i l m e d i u m underwent the same f i l t rat ion procedure and no subsequent addit ions. A l l the f lasks were a l l o w e d to equil ibrate for 24 hrs. after the appropriate additions were completed . Subsequent ly , they were a l l inoculated f r o m an exponent ia l ly g r o w i n g Skeletonema stock culture to an i n i t i a l concentration o f - 1 0 0 0 cel ls • m L - 1 . T h e exper imental cultures were incubated under the same condit ions as i n Part A . T h e y were arranged i n a f a s h i o n s imi lar to the Nitzschia cultures ment ioned above. O n l y d a i l y in vivo f luorescence measurements were used to moni tor growth . These were carr ied out u s i n g a Turner M o d e l 111 f luorometer . -82-I used M a n n - W h i t n e y U-tests to compare g r o w t h rates and length o f lag phase between treatments because I c o u l d not al leviate heteroscedacity by transformation. A s igni f icance l e v e l o f 0.05 was used throughout. T h e one-way analyses o f variance and the M a n n - W h i t n e y U - and T u k e y tests were carr ied out us ing the N P A R and S T A T S modules o f S Y S T A T ( W i l k i n s o n 1985) o n a C o m m o d o r e P C - 1 0 I I . -83-R E S U L T S T h e p H was not s igni f i cant ly different between the t w o unia lga l and the m i x e d cultures at 4 x 10-7 and 1 x 10-9 M C u (Table 2.9). A t 5 x 10-7 M C u , the unia lga l Skeletonema costatum cultures exhib i ted a l o w e r f i n a l p H value than the m i x e d ones. N o other differences were detected. T h e results f r o m the experiment that e x a m i n e d the effect o f m e d i u m m o d i f i c a t i o n by Nitzschia o n Skeletonema g rowth are presented i n F i g . 2.7. T h e latter species demonstrated satisfactory g r o w t h i n the contro l m e d i u m ( F i g . 2 . 7 A ) . G r o w t h rate was 1.28 ± 0.05 d i v • d a y 1 and a l a g phase o f 1 ± 0 day (mean ± standard devia t ion , N = 5) was observed. S. costatum also exhib i ted g r o w t h i n the Nitzschia f i ltrate that was enr iched w i t h nutrients ( F i g . 2 . 7 C ) . T h e g r o w t h rate i n this treatment was 1.03 ± 0.19 d i v • day- i and the length o f l a g phase was 5.4 ± 0.89 days (mean ± standard devia t ion , N = 5). T h e cultures that were g r o w i n g i n nutrient-enriched filtrate exhib i ted s igni f i cant ly l o w e r g r o w t h rate a n d longer l a g phase than the contro l ( M a n n - W h i t n e y U-test , p < 0.05 and <0.01 respect ively) . N o growth was observed i n the Skeletonema cultures that were g r o w i n g either i n unenr iched Nitzschia f i ltrate or i n filtrate that was enr iched w i t h the metal stocks o n l y (Figs . 2 . 7 B and 2 . 7 D ) . It should be noted that replicate 5 o f the unenr iched Nitzschia f i ltrate was contaminated w i t h Nitzschia ce l ls . T h i s can be observed i n the abnormal ly h i g h (compared to the rest o f the replicates) f luorescence throughout the exper imental p e r i o d ( F i g . 2 .7B) . -84-T A B L E 2.9: Statist ical comparisons costatum (=Sket), Nitzschia thermalis E x p e r i m e n t 2. m = m e a n ( N = 5); S . D . pairs o f means ( in p H units) . for differences i n p H between Skeletonema (=Nitz) and m i x e d cultures at the end o f = standard devia t ion ; T u k e y = cr i t i ca l range for C o p p e r Treatment p H A N O V A T u k e y Concentrat ion m S . D . ( in M o l a r C u ) 5 x 1 0 ' 7 Skel Nitz M i x e d 4 x 1 0 " 7 Skel Nitz M i x e d 1 x 10-9 Skel Nitz M i x e d 7.88 0.10 P<0.05 8.31 0.34 8.53 0.38 8.02 0.12 P>0.05 8.13 0.15 8.36 0.34 8.32 0.45 P>0.10 8.08 0.18 8.22 0.37 85 CD O C CD CJ CO CD i_ O D 1 0 0 CD O C CD U CO CD o B 0 0 o O 1 0 •o o o o- •o :;,c^:::::::5-..._ v-O' : -B : : : -•V. •o. v v O o 0 6 Days 8 10 F I G U R E 2.7 : G r o w t h curves o f Skeletonema costatum g r o w n i n filtrate f r o m cultures o f Nitzschia thermalis. ( A ) C o n t r o l ; ( B ) Fi l t rate ; ( C ) F i l t ra te enr i ched w i t h m a c r o - a n d micronutr ien ts ; (D) Fi l t ra te enr i ched w i t h meta l s tocks. C i r c l e s , tr iangles, squares, i n v e r t e d tr iangles and d iamonds represent repl icates 1-5 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 and N i were est imated. 86 F I G U R E 2.7 (continued) -87-D I S C U S S I O N F r o m the f irst part o f this study, one m a y conclude that there is no difference between the three treatments i n f i n a l p H . T h e o n l y difference observed was between the S. costatum un ia lga l and the m i x e d cultures g r o w n at 5 x 10-7 M C u . T h e l o w e r p H i n the Skeletonema cultures can be e x p l a i n e d easi ly . T h e starting p H o f A q u i l before inocu la t ion was 8.4. In addi t ion , a l though axenic technique was used throughout this study, the cultures were not bacteria-free. A s a result , and since Skeletonema d i d not g r o w at 5 x 10-7 M C u , one m a y expect an actual drop i n p H i n these cultures. T h i s m a y occur since the phytoplankton is not r e m o v i n g carbonate by photosynthesis and bacterial respirat ion is adding C O 2 to the m e d i u m . A t the r e m a i n i n g t w o copper concentrations, there was no difference i n p H between any o f the cultures. T h i s indicates that the degree o f carbonate deplet ion was s imi lar i n the three treatments. T h i s result does not necessari ly exc lude the potential o f carbonate l imi ta t ion o f Skeletonema b y Nitzschia uptake i n the m i x e d cultures. S ince the p H measurements were made at the end o f the exper imental p e r i o d for a l l cultures, i t is possible that carbonate l i m i t a t i o n occurred i n a l l treatments. H o w e v e r , I d o not bel ieve that this is the case, for t w o reasons. F i r s t , p H d i d not increase d u r i n g the experiment . In most cases, the mean f i n a l p H was l o w e r than, or s i m i l a r to, the i n i t i a l va lue . T h i s suggests that the carbonate l e v e l d i d not change d u r i n g the exper imental p e r i o d . E v e n i f it d i d , but the p H meter was not accurate enough to reflect the increase or other factors such as metal speciat ion were also act ing and m a s k i n g the increase, the changes certainly d o not appear to be large enough to i n h i b i t g r o w t h . M o s t o f the p H values were - 8 . 2 w h i c h is very c lose to the l e v e l r e c o m m e n d e d by M o r e l et al. (1979) for A q u i l . T h e second factor that suggests the absence o f carbonate l imi ta t ion is that the culture vessels were not sealed. T h e caps were screwed o n the f lasks very loose ly i n order to a l l o w f o r gas exchange i n the cultures d u r i n g the experiments . In addi t ion , the f lasks were shaken -88-and opened d a i l y w h i c h w o u l d a l l o w for a r a p i d d i f f u s i o n o f C O 2 into the culture m e d i u m . T h e type o f i n h i b i t i o n that Nitzschia induces u p o n Skeletonema becomes clearer w h e n one considers the filtrate experiments . F r o m the four treatments that I used, it becomes apparent that Nitzschia m o d i f i e d the culture m e d i u m i n a manner inh ib i tory to Skeletonema. T h e m e d i u m was f i l tered w h e n the Nitzschia cultures were reaching senescence. Therefore the concentrations o f one or more nutrients were c lose to deplet ion i n the i n i t i a l m e d i u m . In this case, i f a l l the nutrients were added back to the culture m e d i u m , and assuming no other m o d i f i c a t i o n had occurred , Skeletonema s h o u l d have g r o w n i n this treatment. A s expected, no g r o w t h was observed i n the unenr iched fi l trate. T h i s treatment was stripped o f nutrients b y Nitzschia and was not, therefore, expected to support g r o w t h , regardless o f other modi f i ca t ions b y the pennate. In the filtrate w i t h a l l the m a c r o - and micronutrients added, g r o w t h was indeed observed but was negat ively affected. T h e extended lag phase and the reduced g r o w t h rate, compared to the contro l , suggest the poss ib i l i ty that some factor (other than nutrient hmitat ion) was retarding g r o w t h . T h i s factor was most ly active i n the first f i v e days o f the experiment d u r i n g the extended l a g phase o f Skeletonema. T h e subsequent exponent ia l g r o w t h that S. costatum exh ib i ted suggests that this factor d i d not persist for longer than the p e r i o d o f l a g phase. I suggest that the observed i n h i b i t i o n can be attributed to a c o m p o u n d that is excreted b y Nitzschia d u r i n g some stage o f g r o w t h and is toxic to Skeletonema. S u c h i n h i b i t o r y compounds have been reported b y a number o f authors (e.g. R i c e 1954, Pratt 1966, F e d o r o v and K u s t e n k o 1972, U c h i d a 1977, Sharp et al. 1979, R i j s t e n b i l 1989). In a l l these studies, i n h i b i t i o n o f one species b y another was attributed to a toxic exudate. A point o f interest is the extended l a g phase that Skeletonema exhib i ted i n this treatment. T h i s type o f response has also been recorded i n other studies (e.g. K a y s e r 1979, Sharp et al. 1979, R i j s t e n b i l 1989). It has been suggested by Pratt (1966) that an -89-inh ib i tory effect should be more prominent at the beg inning o f a "f i l trate" experiment. T h e same author suggested that the magnitude o f the i n h i b i t i o n should decrease w i t h t ime as a result o f degradation o f the exudate due to o x i d a t i o n , p o l y m e r i z a t i o n or bacterial decompos i t ion . In m y experiments , i t is poss ible that after ~ 5 days (Skeletonema l ag phase) most o f the exudate was degraded thus a l l o w i n g Skeletonema to attain exponent ia l g r o w t h . E x p o n e n t i a l g r o w t h rate was s igni f i cant ly l o w e r i n the nutr ient-enriched filtrate m e d i u m c o m p a r e d to the contro l . O n e poss ible explanat ion is that the c o m p o u n d was not complete ly degraded b y the end o f the f i v e day p e r i o d , thus af fect ing the g r o w t h rate. A l t e r n a t i v e l y , the role o f bacterial abundance i n the t w o treatments must be considered. O n e m a y assume that, at the t ime o f inocu la t ion , the density o f bacteria was higher i n the filtrate o f an a lga l culture that had reached senescence compared to autoc laved m e d i u m (control) . T h e m e d i u m i n w h i c h Nitzschia had been g r o w i n g was passed through a 0.45 ( i m f i l ter before it was inoculated w i t h Skeletonema. T h i s procedure p r o b a b l y r e m o v e d a por t ion o f the bacteria but p r o b a b l y a l l o w e d a large number o f t h e m to pass through the fi l ter . O n the other hand, i n the cont ro l , w h i c h was probably not complete ly sterile, the bacterial numbers were expected to have been l o w e r . It i s , therefore, poss ible that the larger bacterial numbers were somehow responsible f o r the reduct ion o f the g r o w t h rate o f S. costatum. T h i s c o u l d have been achieved i n one o f t w o w a y s . It is poss ible that the bacteria themselves are excret ing a substance that is toxic to Skeletonema. S ince i n Part A o f this chapter, no such i n h i b i t i o n was observed i n u n i a l g a l Skeletonema cultures, one must assume that such species o f bacteria have a specif ic associat ion w i t h Nitzschia. A second p o s s i b i l i t y is compet i t ion between bacteria and the d i a t o m for a nutrient. T h e higher bacterial abundance i n the nutrient-enriched filtrate m e d i u m m a y account f o r a more r a p i d r e m o v a l o f a nutrient w h i c h i n turn m a y result i n g r o w t h rate reduct ion for Skeletonema. -90-A l l studies that have e x a m i n e d the effect o f a lgal exudates have not characterized the compounds except at a very superf ic ia l l e v e l (i.e. they are removable by autoc laving or they are large because they d o not pass through a cel lulose f i l ter) . Pratt (1966) suggested that the substance that Olisthodiscus excreted w h i c h inh ib i ted Skeletonema was a tannin- l ike c o m p o u n d . H o w e v e r , no evidence for this was p r o v i d e d . T h u s , i t becomes very d i f f i c u l t to assess what component o f the organic molecule causes the i n h i b i t i o n or even whether a l l microa lgae excrete s imi lar substances. F r o m this study, I cannot conc lude whether the c o m p o u n d that is excreted by Nitzschia acts as a m e t a l - c o m p l e x i n g agent. G r o w t h was not observed i n the filtrate that was enr iched o n l y w i t h the metal stock. H o w e v e r , this suggests that the m e d i u m was either stripped o f nutrients other than metals and/or i n c l u d e d a toxic substance. T h e lack o f g r o w t h i n this treatment indicates that regardless o f whether or not the excreted c o m p o u n d b o u n d metals , this is not what l i m i t e d the g r o w t h o f Skeletonema. N. thermalis inhibi ts the g r o w t h o f S. costatum, at least temporar i ly . It was demonstrated that such an i n h i b i t i o n is probably the result o f an exudate w h i c h is tox ic to a certain extent to Skeletonema. T h e tox ic i ty appears to be part ia l ly a l leviated i n the absence o f the o r g a n i s m that produces it after a number o f days , presumably by the degradation o f the c o m p o u n d . H o w e v e r , i n the presence o f Nitzschia this c o m p o u n d w i l l be cont inuous ly produced , thus i n h i b i t i n g Skeletonema, unless i t becomes d i l u t e d (for example , i n a chemostat) . D i l u t i o n o f the filtrate o f cultures o f algae that excrete i n h i b i t o r y compounds has been s h o w n to enhance g r o w t h (Pratt 1966, Sharp et al. 1979). O n the other hand, i f this c o m p o u n d is produced d u r i n g a specif ic stage o f Nitzschia g r o w t h , Skeletonema m a y enter exponent ia l growth before Nitzschia produces the exudate. H o w e v e r , i t w i l l s t i l l become inh ib i ted as soon as the concentration o f the substance increases. If, however , Skeletonema does not enter exponent ia l growth i n t ime, it w i l l be i n h i b i t e d w h i l e Nitzschia ut i l izes the nutrients i n the culture. B y the t ime that Nitzschia ceases p r o d u c i n g it and/or the toxic substance degrades, the m e d i u m w i l l be -91-depleted o f nutrients and Skeletonema w i l l not be able to exhibi t exponent ia l growth at a l l . It, therefore, becomes clear that the interact ion between these t w o species is quite c o m p l e x . It has been demonstrated that such an interact ion w i l l overr ide the speci f ic copper tolerance o f Skeletonema. In this study, I demonstrated that regardless o f the copper concentration at w h i c h Skeletonema exhib i ted satisfactory g r o w t h , this species was i n h i b i t e d s i m p l y by the presence o f Nitzschia. O n the other hand, the single-species tolerance tests for Nitzschia quite adequately predicted the tolerance o f this species i n the presence o f Skeletonema (except for a s l ight i n h i b i t i o n at 1 x 10-9 M C u ) . A l t h o u g h not e x a m i n e d i n deta i l , a s l ight retardation o f growth o f Nitzschia is suggested by the longer l a g phase exhib i ted at that copper l e v e l . H o w e v e r , i n a l l cases Nitzschia d i d overtake the batch cultures regardless o f the copper concentrat ion i n the m e d i u m and o f the copper tolerance o f the i n d i v i d u a l species i n v o l v e d . -92-G E N E R A L D I S C U S S I O N In this s t u d y , . t h e copper tolerances o f Skeletonema costatum and Nitzschia thermalis were examined . S. costatum was i n h i b i t e d at 5 x 10-7 M and N. thermalis at 6 x 10-7 M added total copper. Skeletonema exh ib i ted an increase i n both g r o w t h rate and l a g phase w i t h increas ing copper concentrat ion. O n the other hand, Nitzschia exh ib i ted a decrease i n g r o w t h rate. N o effect o n the l a g phase o f this species was observed. W h e n these t w o species were g r o w n together, their growth response (as predicted g i v e n the copper concentration i n the m e d i u m ) was altered. F o r Nitzschia, the alteration was not very pronounced. T h i s species showed an increase i n length o f l a g phase at 1 x 10-9 M C u . T h e g r o w t h response o f Skeletonema, o n the other hand, was severely altered. T h i s species d i d not show any g r o w t h i n the presence o f Nitzschia. In an attempt to e x p l a i n this i n h i b i t i o n , i t was c o n c l u d e d that i t c o u l d be attributed to a c o m p o u n d excreted b y Nitzschia that is tox ic to Skeletonema. It was also suggested that the effect o f such a c o m p o u n d was temporary, as indica ted b y extended l a g phase f o l l o w e d by exponent ia l g r o w t h rate o f Skeletonema i n nutrient-enriched filtrate f r o m senescent Nitzschia cultures. It was , therefore, demonstrated that the interaction between these t w o species was too strong to a l l o w for the copper tolerance o f S. costatum to dictate its s u r v i v a l i n m i x e d cultures, regardless o f the copper concentration i n the m e d i u m . , Interactions such as these are expected to o c c u r i n the natural environment as w e l l . S. costatum is a d i a t o m that is c o m m o n l y col lec ted i n the p lankton o f m a n y different bodies o f water (e.g. H a r r i s o n et al. 1983, Sakshaug and A n d r e s e n 1986, Sakshaug and O l s e n 1986, T o n t 1987, M o r t a i n - B e r t r a n d 1989). It has also been co l lec ted i n the estuarine benthos (see A d m i r a a l 1984 for r e v i e w ) . O n the other hand, Nitzschia thermalis (although not c o m m o n l y ment ioned i n the literature) has been descr ibed as one o f the dominant pennates i n late A p r i l i n the E e m s - D o l l a r d estuary, i n the Netherlands ( A d m i r a a l et al. 1982). It has also been co l lec ted i n the p lankton i n the B a y o f F u n d y , -93-N e w B r u n s w i c k (Linklet ter et al. 1977) and near V a n c o u v e r , B r i t i s h C o l u m b i a (isolate 608 o f the N o r t h Eas t P a c i f i c Cul ture C o l l e c t i o n ) . It is therefore very l i k e l y that these t w o species share the same habitat o n certain occasions. I f this is true, i t can be expected that interactions, such as the one observed i n this study, m i g h t affect their coexistence i n the f i e l d . T h e magnitude o f the effect o f the interaction w i l l depend u p o n the type o f habitat that these two species share. If, for instance, they share a body o f water w i t h very strong c i r cu la t ion patterns, one m i g h t expect that the effect o n each other w i l l be a l leviated by the d i l u t i o n o f inh ib i tory c o m p o u n d s that they m a y produce . O n the other hand, i n areas o f l i t t le water exchange such d i l u t i o n w i l l not occur and an inhib i tory interact ion m a y arise. T w o examples o f such areas come to m i n d . O n e m a y be the benthos o f coastal waters. A s ment ioned, N. thermalis is one o f the dominant benthic species a n d S. costatum has been f o u n d i n the benthos (presumably after s i n k i n g out o f the plankton) . It i s , therefore, quite l i k e l y that these species w i l l share a por t ion o f the same habitat. In addi t ion , since the benthic pennates m i g h t f o r m a thick mat at the bot tom, Skeletonema ce l l s that s ink out o f the p lankton m a y encounter a h i g h concentrat ion o f tox ic exudates o v e r l y i n g the mat. A second example o f a habitat where such an interact ion m a y become very important is tidepools. In this case, water exchange w i l l depend u p o n the frequency o f f l u s h i n g o f the pools . T i d e p o o l s that are higher i n the intert idal w i l l be f l u s h e d less frequently than l o w e r - l e v e l t idepools . In such p o o l s , any exudates that are p r o d u c e d w i l l accumulate u n t i l the water is replaced b y t ida l f l u s h i n g . In addi t ion , Skeletonema w i l l tend to s ink towards the bot tom o f the t idepool after the nutrients close to the surface are depleted. T h i s species m a y then encounter increased levels o f tox ic exudates. It is quite l i k e l y that N. thermalis is present i n the t idepools that I sampled. H o w e v e r , since the pennate diatoms were not ident i f i ed , no conclusions can be d r a w n regarding the poss ib i l i ty o f these t w o species interact ing. O n the other hand, other pennate diatoms have been observed to produce filtrates that are inh ib i tory to other algae. F o r example , -94-Thalassionema nitzschioides inh ib i ted 5 . costatum (Fedorov and K u s t e n k o 1972) and Nitzschia frustulum inh ib i ted Chlorella vulgaris (R ice 1954). It i s , therefore, quite l i k e l y that other pennates that are present i n the t idepools m i g h t exude substances that inh ib i t Skeletonema. Pennate diatoms are not the o n l y group o f microalgae that have been shown to produce i n h i b i t o r y exudates. S i m i l a r i n h i b i t i o n c a n be i n d u c e d b y groups o f microa lgae such as green algae, dinoflagel lates and centric diatoms (e.g. K r o e s 1971, Pratt 1966, U c h i d a 1977). R i j s t e n b i l (1989) demonstrated that compounds produced by Skeletonema were inh ib i tory to Ditylum brightwellii. It becomes quite c lear that such interactions are quite c o m m o n a m o n g microalgae . It has been proposed that interactions o f this type m i g h t become espec ia l ly important d u r i n g phytoplankton b looms and m a y determine the dominant species (Pratt 1966, R i j s t e n b i l 1989). I n this study, i t has been demonstrated that the interaction between S. costatum and N. thermalis was the determining factor i n the s u r v i v a l o f Skeletonema, i rrespect ive o f the copper concentrat ion i n the m e d i u m . A l t h o u g h Skeletonema should have exhib i ted g r o w t h i n t w o o f the concentrations used, g i v e n its predeterrnined copper tolerance, i t d i d not, i n the presence o f the second species. T h i s type o f study demonstrates the l imitat ions o f single-species tox ic i ty tests, w h i c h consider the speci f ic tolerances o f an o r g a n i s m to an inhib i tor , i n most cases, however , ignore the b i o l o g i c a l interactions o f the species. T h e chemistry o f the m e d i u m and the p h y s i o l o g i c a l response o f a part icular species p l a y a very important ro le i n the determination o f its tolerance. H o w e v e r , i t has been demonstrated that the apparent tolerance o f a species might also depend o n the other organisms that share its habitat. Studies o n the impact o f a stress inducer at the c o m m u n i t y l e v e l , have attempted to incorporate the b i o l o g i c a l environment i n the responses o f each species-member o f that c o m m u n i t y . These studies, however , are descript ive and, thus, d o not a l l o w for predic t ion o f the outcome o f stress i n d u c t i o n . S u c h predic t ion m a y o n l y be possible i f -95-the mechanisms that cont ro l c o m m u n i t y structure are k n o w n . I f the mechanisms are understood, one m a y be able to determine the types o f interactions that d o occur and their potential impact o n the popula t ion abundance o f different species. It becomes obvious that, i f one attempts to describe popula t ion responses o f a part icular species, an understanding o f the ways i n w h i c h i t interacts w i t h other species is absolutely essential. Futhermore , i f one attempts to understand the popula t ion response o f the same species to a stress inducer , one must first ensure an understanding o f the popula t ion dynamics i n the absence o f the " d i s t u r b i n g " factor. A single-species tox ic i ty test w i l l determine the response o f each species to the stress-inducer. It w i l l not, h o w e v e r , show whether the response w i l l be manifested as such i n the c o m m u n i t y . F o r example , i n m y study, Skeletonema showed increased lag phase a n d increased g r o w t h rate at 4 x 10-7 M added C u . I f o n l y this in format ion is used, one w o u l d assume that i n the natural environment at that copper concentrat ion, Skeletonema w o u l d show s i m i l a r responses. H o w e v e r , f r o m this study, this w i l l depend o n whether N. thermalis is also present. A c c o r d i n g to the results o f R i j s tenb i l (1989), the apparent response w i l l also depend o n whether Ditylum brightwellii is present i n the same habitat. If, at any point , Nitzschia is present then the copper tolerance o f Skeletonema, as demonstrated by the tox ic i ty test, w i l l be irrelevant. O n the other hand, i f the habitat is shared w i t h Ditylum, Skeletonema w i l l not be i n h i b i t e d b y the other species and its response to copper m i g h t be manifested as i n the single-species test. In addi t ion , the i n h i b i t i o n o f Skeletonema b y Nitzschia (and probably that o f Ditylum b y Skeletonema) w i l l depend o n the nature o f the habitat ( w h i c h m a y or m a y not a l l o w for d i l u t i o n o f the inh ib i tory compound) . S u c h predict ions are o n l y poss ible because o f an understanding o f species interactions and their mechanisms . 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C o m b i n e d tox ic i ty o f copper , c a d m i u m , z i n c , l ead , n i c k e l , and chrome to the copepod Tisbe holothuriae. Bull. Environ. Contam. Toxicol. 4 1 : 378-84. W a t k i n s , B . & S i m k i s s , K . 1988. T h e effect o f osc i l la t ing temperatures o n the metal i o n metabo l i sm o f Mytilus edulis. J. Mar. Biol. Ass. UK. 68: 93-100. W e s t a l l , J . C , Z a c h a r y , J . L . & M o r e l , F . M . M . 1976. M I N E Q L , a computer p r o g r a m for the ca lcula t ion o f c h e m i c a l e q u i l i b r i u m c o m p o s i t i o n o f aqueous systems. T e c h n i c a l N o t e #18, R . M . Parsons Labora tory , Department o f C i v i l E n g i n e e r i n g , Massachussetts Institute o f T e c h n o l o g y , C a m b r i d g e , Massachussetts . 91pp. M i m e o g r a p h e d . W i l k i n s o n , L . 1985. S Y S T A T . T h e system f o r statistics. V e r s i o n 2.1 . Systat Inc. , 1800 Sherman A v e . , E v a s t o n , I L 60201 , U . S . A . . 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Oceanogr. 17: 661-71. Z u e h l k e , R . W . & Kester , D . R . 1983. C o p p e r speciat ion i n marine waters. In W o n g , C . S . , B o y l e , E . , B r u l a n d , K . W . , B u r t o n , J . W . & G o l d b e r g , E . D . [Eds.] Trace Metals in Sea-water. P l e n u m Press , N e w Y o r k , N Y , U . S . A . p p . 773-88. Z o r k i n , N . G . , G r i l l , E . V . & L e w i s , A . G . 1986. A n ion-exchange procedure for quant i fy ing b i o l o g i c a l l y act ive copper i n sea water. Anal. Chim. Acta 183: 163-77 . -109-APPENDIX 1: Diatom distribution in five tidepools sampled in Vancouver.  British Columbia. Canada, on five occasions in 1988. M E T H O D S T h e sea-surface and f i v e t idepools were sampled at P o i n t A t k i n s o n ( 4 9 ° 20' N , 123° 15' W ) , V a n c o u v e r , B r i t i s h C o l u m b i a , C a n a d a o n f i v e occasions. T h e sampl ing dates were M a y 15, June 3, June 13, June 28 and J u l y 12 ,1988 . S a l i n i t y was recorded us ing an E n d e c o T y p e 102 refractometer and temperature measurements were taken us ing a m e r c u r y thermometer, for both the sea-surface and each o f the t idepools o n each date. N o temperature measurements were taken o n 3-6-88 because the thermometer broke d u r i n g transport. T h e depth o f each tidepool was recorded a n d the height above sea-level was estimated by eye. S a m p l i n g o f microalgae i n c l u d e d t w o water c o l u m n samples f r o m the sea-surface, as w e l l as t w o water c o l u m n and t w o bot tom samples f r o m each o f the tidepools. O n l y one sample was taken f r o m the sea-surface o n 15-5-1988. In addi t ion , the bot tom samples o f t idepool 2 for 3-6-1988 went anoxic due to inadequate preservation and are, therefore, not i n c l u d e d . T h e water f r o m t idepool 1 had complete ly evaporated o n 12-7-1988, so no samples were co l lec ted. W a t e r c o l u m n samples were co l lec ted u s i n g a 60 m L p o l y p r o p y l e n e syringe at m i d - d e p t h . A f t e r each 60 m L sample was p laced into a bottle, the syringe was r insed into the sample bottle us ing 30 m L o f d i s t i l l e d water. F o r the bo t tom samples, a bottomless cup was inver ted and p laced t ight ly o n the f l o o r o f each t idepool . T h e f l o o r was then gently scraped u s i n g a d u l l k n i f e i n order to remove the epibenthic microalgae . Subsequently, a 60 m L po lypropylene syringe was used to remove a l l the resuspended algae f r o m ins ide the c u p . T h e v o l u m e o f sample that was r e m o v e d was determined i n either o f t w o w a y s . W h e n the t idepool h a d a sha l low por t ion , the cup was p laced o n the f l o o r i n a manner that its top was exposed. In such -110-cases, I r e m o v e d a l l the water f r o m ins ide the c u p . W h e n the tidepool was deep (and the cup was therefore submerged), I r e m o v e d water u n t i l an o b v i o u s l y large d i l u t i o n h a d occurred to the water ins ide the c u p . S ince the layer o f epibenthic algae i n a l l deep tidepools was thick , the amount o f resuspended algae ins ide the cup was very large c o m p a r e d to the surrounding water. It was , therefore, very apparent w h e n most o f the sample was r e m o v e d f r o m inside the cup by the c lar i ty o f the water rep lac ing the sample. A f t e r the transfer o f the sample into the sample bottle, the syringe was thoroughly r insed into the same bottles us ing d i s t i l l e d water. A l l samples were preserved u s i n g L u g o l ' s solut ion and stored i n the dark u n t i l they were counted. A f t e r intense shaking o f the sample bottles (100 invers ions) a subsample was r e m o v e d and a l l o w e d to settle overnight i n 50 m L settl ing chambers . T h e v o l u m e o f the subsample that was p l a c e d i n the chamber depended o n the density o f the sample and v a r i e d f r o m 0.5 to 50 m L . T h e settl ing chambers were subsequently f i l l e d w i t h d i s t i l l e d water to 50 m L , thus d i l u t i n g the subsample. T h e settled subsamples were subsequently used f o r d i a t o m ident i f i ca t ion and enumerat ion. D i a t o m ident i f icat ion was per formed according to C u p p (1943). A l l centric diatoms were ident i f i ed to genus and the major i ty to species. D u e to the poor resolut ion o f the microscope that was avai lable , however , i t was very d i f f i c u l t to separate the dif ferent pennate genera and species w h i c h were consequently l u m p e d into one group. S o m e exceptions w i t h dist inct frustule shape were ident i f i ed to genus and i n some occasions to species. F o r each subsample, I counted a l l the diatoms i n f i v e f ie lds or 400 i n d i v i d u a l s . A l l counts were p e r f o r m e d us ing a W i l d M 4 0 inverted microscope . T h e d i a t o m abundances i n the tidepools were transformed i n one o f t w o w a y s . F o r the water c o l u m n samples, after a l l the d i lut ions were taken into account, the abundance ( in cel ls • cm-3) was d i v i d e d by the depth o f the tidepool to obtain number o f cel ls per unit surface area o f the water c o l u m n . T h e c e l l concentration i n the bot tom samples was d i v i d e d b y the surface area covered by the bot tom o f the s a m p l i n g c u p , i n order to y i e l d - u l -ceus per unit bot tom surface area. A l l concentrations at the sea-surface are presented as cel ls • m L - l . T h e t ida l height at the t ime o f s a m p l i n g was ca lculated as r e c o m m e n d e d i n the C a n a d i a n T i d e and Current Tables (1988). B y adding the former to the height above sea-l e v e l , as assessed for each o f the t idepools , I estimated the height above chart da tum for each o f the t idepools o n each s a m p l i n g date. T h e values o f the f i v e dates were then averaged and the m e a n height was used for the r e m a i n i n g calculat ions. I counted the number o f l o w and the number o f h i g h tides w i t h suff ic ient height to cover the i n d i v i d u a l t idepools d u r i n g the p e r i o d M a y 14-July 15th 1988. Subsequently, I expressed this as a percentage o f the total number o f l o w and h i g h tides d u r i n g the same per iod . In this manner , I obtained a rate o f f l u s h i n g for each o f the pools d u r i n g the two m o n t h s a m p l i n g p e r i o d . L a s t l y , I determined the length o f t ime d u r i n g w h i c h the t idepools had been i so la ted f r o m the sea-surface pr ior to s a m p l i n g . I est imated the t ime at w h i c h the t ide h a d last attained the height o f the t idepool before the sampl ing t ime. I then determined the e lapsed t ime as the i so la t ion p e r i o d o f the tidepools. -112-RESULTS Tidal effect on the tidepools T h e average height o f each t idepool above chart da tum is g i v e n i n Table 1. T i d e p o o l s 1 and 2 are o f s imi lar height (g iven the accuracy o f the height est imation) , whereas the r e m a i n i n g are o f very different heights. T h i s is also apparent i n the f l u s h i n g frequency o f the t idepools (Table 2) . W h i l e t idepools 1 and 2 are a lways f lushed at h i g h t ide, t idepool 3 is f lushed 8 0 % o f the t ime. O n the other hand, w h i l e t idepools 1 and 2 are f lushed 6 0 % o f the t ime at l o w tide, t idepool 3 is never f lushed. T i d e p o o l s 4 and 5 are never f lushed at either l o w o r h i g h tide and m a y , therefore, be considered splashpools . T h e temperature and sal ini ty gradients up the intert idal are presented i n F i g u r e 1. Temperature d i d not appear to vary w i t h the f l u s h i n g rate o f the tidepool. T h i s is not surpr is ing at the air temperatures that s a m p l i n g occurred. These v a r i e d between 16° C and 2 7 ° C d u r i n g the study. G i v e n the s m a l l depth o f the tidepools (4 to 34.5 c m over a l l s a m p l i n g dates) and the i so la t ion p e r i o d before s a m p l i n g (Table 3), the water temperature c o u l d have increased to reach s i m i l a r levels i n a l l tidepools s i m p l y b y h i g h heat transfer f r o m the air. S a l i n i t y , however , appeared to a lways drop to very l o w levels for tidepool 5 (<5 %c). T h e o n l y except ion to this was o n 28-6-1988 ( F i g . I D ) . T h i s c o u l d have been the result o f a recent s torm d u r i n g w h i c h sea-water was splashed into the t idepool . In a l l other cases, however , t idepool 5 appeared to receive most ly freshwater input . Diatom distribution T h e d i a t o m distr ibutions for a l l s a m p l i n g dates are s h o w n i n F i g s . 2-6. A t the sea-surface, for the f irst three dates, Skeletonema costatum was the most abundant species. Pennates, Thalassiosira nordenskioldii and Chaetoceros compressus were also present i n -113-T A B L E 1: H e i g h t above chart d a t u m o f f i v e t idepools located at P o i n t A t k i n s o n , V a n c o u v e r , B r i t i s h C o l u m b i a , C a n a d a , sampled o n f ive occasions i n 1988 (c .d . = chart datum). T i d e p o o l Date T i m e T i d a l height T i d e p o o l height T i d e p o o l height above c . d . above sea-level above c .d . (m) (m) (m) 1 15-5 14:35 1.71 0.0 1.70 3-6 14:45 0.49 1.5 2.00 13-6 13:35 1.10 0.1 1.20 28-6 13:20 1.04 0.0 1.00 M e a n height: 1.50 2 15-5 14:05 1.31 0.2 1.50 3-6 14:25 0.49 3.0 3.50 13-6 13:20 0.85 0.4 1.30 28-6 13:10 0.91 0.4 1.30 12-7 13:10 1.07 0.5 1.60 M e a n height: 1.80 3 15-5 14:20 1.52 3.0 4 .50 3-6 14:15 0.46 4.0 4 .50 13-6 13:05 0.85 1.5 2.40 28-6 12:55 0.70 4.0 4.70 12-7 13:55 1.49 1.5 3.00 M e a n height: 3.80 4 15-5 14:45 1.92 4.0 5.90 3-6 14:05 0.46 5.0 5.50 13-6 12:55 0.76 4.5 5.30 28-6 12:40 0.70 5.0 5.70 12-7 14:10 1.80 3.0 4.80 M e a n height: 5.40 T A B L E 1 (continued) -114-15-5 15:00 2.01 5.0 7.00 3-6 13:50 0.46 6.0 6.50 13-6 12:35 0.64 6.0 6.60 28-6 12:25 0.49 6.0 6.50 12-7 14:25 1.92 4.0 5.90 M e a n height: 6.5 -115-T A B L E 2 : F requency o f f l u s h i n g for the p e r i o d between M a y 14 and J u l y 1 5 , 1 9 8 8 f o r f i v e t idepools located at P o i n t A t k i n s o n , V a n c o u v e r , B r i t i s h C o l u m b i a , C a n a d a . T i d e p o o l T o t a l number N u m b e r o f tides F l u s h i n g 1 o f tides w i t h height above frequency t idepool height (%) L O W T I D E S 1 121 2 3 4 5 H I G H T I D E S 1 122 122 100 2 122 100 3 97 80 4 0 0 5 0 0 1 F l u s h i n g Frequency = ( N u m b e r o f tides w i t h height above t idepool height) / (total number o f tides) x 100 75 68 1 0 62 56 1 0 -116-T A B L E 3 : P e r i o d o f i so la t ion f r o m the ocean pr ior to s a m p l i n g for f ive t idepools located at P o i n t A t k i n s o n , V a n c o u v e r , B r i t i s h C o l u m b i a , C a n a d a , sampled o n f i v e occasions i n 1988. T i d e p o o l Date T i m e w h e n tidepool S a m p l i n g t ime Isolat ion last f lushed p e r i o d (hr :min) (hr :min) (hr :min) 15-5 09 :30 14:35 05:05 3-6 11:40 14:45 02:55 13-6 09 :00 13:35 04:35 28-6 08 :10 13:20 05 :10 15-5 09:05 14:05 05 :00 3-6 11:10 14:25 03:15 13-6 08:25 13:20 04:55 28-6 07 :35 13:10 05:35 12-7 07 :50 13:10 05 :20 15-5 05 :30 14:20 08 :50 3-6 07 :40 14:15 06 :35 13-6 05:25 13:05 07 :40 28-6 04 :40 12:55 08:15 12-7 04 :40 13:55 09:15 4 N E V E R F L U S H E D 5 N E V E R F L U S H E D -117-h i g h numbers . F o r the last t w o dates, there was a shift i n dominance (in terms o f c e l l numbers) . S. costatum, T. nordenskidldii and C. compressus decreased i n abundance. T h e pennates, o n the other hand, were the most dominant group w i t h Melosira moniliformis and Cerataulina pelagica (on 12-7-1988) present i n h i g h concentrations. T h e pattern o f dominance observed at the sea-surface was not f o l l o w e d tightly i n the t idepools . O n the three dates that i t was dominant at the sea-surface, 5 . costatum was present i n concentrations greater, equal o r l o w e r than the pennates i n tidepools 1 and 2. In t idepool 3, its concentration was l o w e r than the pennates and i n t idepools 4 and 5, i t was either l o w e r or non-existent. O n the t w o dates that i t was not very abundant at the sea-surface, this species was present i n very l o w concentrations i n t idepools 1, 2 and 3 and absent f r o m tidepools 4 and 5. A s imi lar pattern o f disappearance w i t h increasing height up the intert idal was observed w i t h the other species that attained the highest abundance at the sea-surface, o n later dates. C. pelagica, a l though i n s i m i l a r concentrations to the pennates at the sea-surface, h a d m u c h l o w e r concentrations i n t idepools 2 and 3 and disappeared i n tidepools 4 and 5 ( F i g . 6). T h e o n l y except ion appears to be M. moniliformis. T h i s species h a d a dis tr ibut ion s i m i l a r to the pennates both at the sea-surface and i n the tidepools, b e i n g present even i n t idepool 5 o n 12-7-1988. O n the other h a n d , the pennates presented a different d is t r ibut ion relat ive to the other groups. Regardless o f their abundance at the sea-surface relative to the other diatoms, they were a lways one o f the most or the most abundant group i n the tidepools. T h e y were a lways i n concentrations greater than 100,000 cel ls • cm-2 both i n the water-c o l u m n and the bot tom samples i n tidepools 1-5. T h e concentrations were m u c h higher i n some cases, for e x a m p l e i n the bot tom samples and d u r i n g the dates o f their highest abundance i n the sea-surface samples. In addi t ion , they were a lways present i n t idepool 5. In most instances, they were present i n concentrations greater than 100,000 cel ls • cm-2, w h e n the f e w other species present were at densities b e l o w 10,000 cel ls • cm-2. -118-A number o f species not abundant at the sea-surface, were also present i n the t idepools . E x a m p l e s o f these are Odontella (most iy aurita), Climacosphenia moniligera, Surirella fastuosa var . recedens and Gyrosigma spencerii. Odontella was f o u n d almost e x c l u s i v e l y i n the bot tom samples. E v e n w h e n present i n the w a t e r - c o l u m n , it was i n m u c h l o w e r concentrations. It was a lways present i n t idepool 1, its abundance increased i n tidepool 2 to decrease o r disappear above t idepool 3. It was almost never present i n t idepool 5 (wi th the except ion o f very l o w abundance o n 13-6-1988). C. moniligera and S. fastuosa var . recedens f o l l o w e d a s imi lar pattern o f dis tr ibut ion except that they demonstrated a higher upper l i m i t . O n most s a m p l i n g dates, their d is t r ibut ion extended to tidepool 4 and, i n some cases, tidepool 5 i n l o w abundances. Gyrosigma spencerii was present at its highest concentrations m o s t l y i n t idepool 3 and, i n some cases, i n t idepools 2 and 4. It was usua l ly abundant i n the bot tom samples. O n a l l s a m p l i n g dates, t idepool 4 had a l o w e r number o f "groups" than t idepools 1, 2 and 3 and t idepool 5 had the lowest " g r o u p " divers i ty o f a l l ( F i g . 7) (S ince not a l l groups o f diatoms were ident i f i ed to species, I have used the term " g r o u p " to s i g n i f y ident i f iable groups. F o r example , both the species Skeletonema costatum and the group pennates were used as "groups" . ) . -119-Explanation of abbreviations used in the Figures A . l o n = Acnanthes longipes C . c o = Chaetoceros compressus C e r = Cerataulina pelagica C h a = Chaetoceros spp. C l i = Climacosphenia moniligera C o s = Coscinodiscus spp. D i t = Ditylum brightwellii G y r = Gyrosigma spencerii L . b o r = Lauderia annulata L i e = Licmophora abbreviata M e l = Melosira moniliformis and M. nummuloides N i t = a l l pennates that c o u l d not be assigned easi ly to genera or species (most ly N a v i c u l o i d e a e and N i t z s c h i o i d e a e - see text) O d o = Odontella (most ly aurita) S E A = sea-surface samples S k e l = Skeletonema costatum S u r = Surirellafastuosa var . recedens T . gra = Thalassiosira gravida T . n o r = Thalassiosira nordenskidldii T P 1 - T P 5 = t idepool 1 - t idepool 5 respect ively 120 SEA TP1 TP2 TP3 TP4 TP5 F I G U R E 1: Temperature a n d sa l in i ty measurements at the ocean surface and i n f i v e t idepools o f i n c r e a s i n g height i n the inter t idal at P o i n t A t k i n s o n , B r i t i s h C o l u m b i a o n five occas ions : ( A ) M a y 15, ( B ) June 6, ( C ) June 13, (D) June 28 a n d (E) J u l y 12, 1988. 121 F I G U R E 1 (continued) 122 100000• • 10000--1000• • 100-Skel Nit T.gr Cha Oit Odo Cli Sur Mel Cos Gyr Cer ^ 10000-o o o — 1000-CM £ u o 100--10--B ^3 " surface m « bottom -+-Skel Nit Mel Odo Cos Clim Sur AJon Uc L.bor 10000 -ESS = surface EZi = bottom — 1000• • 100-10--+- -+-Skel Nit Mel Odo Cos Clim Sur AJon Uc L.bor F I G U R E 2 : D i a t o m abundance at the ocean surface waters a n d i n f i v e t idepools o f i n c r e a s i n g height i n the intert idal at P o i n t A t k i n s o n , B r i t i s h C o l u m b i a o n M a y 15, 1988. ( A ) O c e a n surface and (B)- (F) T i d e p o o l s 1-5 respec t ive ly . E r r o r s bars represent standard error o f the mean (N=2). 123 10000+ - 1000^ 100 + 10 + D ~ surface ^ = bottom -+-Skel Nit Mel Odo Cos Clim Sur AJon Lie L.bor ^ 10000 + o o o — 1000 + CM £ o 100 + 10 ^3 — surface 555 - bottom Skel Nit Mel Odo Cos Clim Sur AJon Lie Lbor = surface 653 = bottom Skel Nit Mel Odo Cos Clim Sur AJon Lie L.bor F I G U R E 2 (continued) 124 1000--100-JL J L I J L J L Skel Nit T.noC.co Dit Odo Cli Sur Mel Cos Gyr Cer Skel Nit T.nor Mel Odo Cos Cli Sur Gyro § 1000--O ES = surface £22 = bottom -1*-Skel Nit T.nor Mel Odo Cos Cli Sur Gyro F I G U R E 3: D i a t o m abundance at the ocean surface waters and i n f i v e t idepools o f increas ing height i n the in ter t ida l at P o i n t A t k i n s o n , B r i t i s h C o l u m b i a o n June 3 r d , 1988. ( A ) O c e a n surface a n d (B) - (F) T i d e p o o l s 1-5 respect ive ly . E r r o r bars represent standard error o f the m e a n (N=2). 125 Skel Nit T.nor Mel Odo Cos Cli Sur Gyro § 1000J o E u 0) o 100-10-k\N = surface E29 = bottom Skel Nit T.nor Mel Odo Cos Cli Sur Gyro § 1000 + O ESI = surface ESS = bottom 1 Skel Nit T.nor Mel Odo Cos Cli Sur Gyro F I G U R E 3 (continued) 126 100000 £ 10000-<P 1000 o 100 1 Skel Nit T.noC.co Oit Odo Cli Sur Mel Cos Gyr Cer 100000 •• o o 10000-1000--CM E u 100-\ tn 10-"55 O 1 •- i l E 3 = surfoce ZZ2 - bottom Ske Nit T.no C.co Mel Odo Cos Cli Sur Gyro F I G U R E 4 : D i a t o m abundance at the ocean surface waters a n d i n f i v e t idepools o f i n c r e a s i n g he ight i n the inter t idal at P o i n t A t k i n s o n , B r i t i s h C o l u m b i a o n June 13, 1988. ( A ) O c e a n surface a n d (B)- (F) T i d e p o o l s 1-5 respect ive ly . E r r o r bars represent s tandard error o f the mean (N=2). 127 Ske Nit T.no C.co Mel Odo Cos Cli Sur Gyro 100000• - £ o o o 10000 -E S — surfoce 653 = bottom CSI E u 0) 000-100-10-1 • Ske Nit T.no C.co Mel Odo Cos Cli Sur Gyn >+ F o O 10000--100--G S = surface ZS = bottom i -+- -+-Ske T.no C.co Mel Odo Cos Sur Gyro F I G U R E 4 (continued) 128 100000 •• £ 10000 0> 1000 o 100 J_ 1 JL Skel Nit T.noC.co Dit Odo Cli Sur Mel Cos Gyr Cer O o o CM E o 0) 100000 •• 10000 1000 100 10 1 B E9 « surface ESS = bottom Skel Nit T.no Mel Odo Cos Cli Sur Gyr 100000 I c o o o CM E o a> o 10000--1000--100 10 1 M ^3 = surface ESS = bottom Skel Nit T.no Mel Odo Cos Cli Sur Gyr F I G U R E 5 : D i a t o m abundance at the ocean surface waters and i n five t idepools o f increas ing height i n the inter t idal at P o i n t A t k i n s o n , B r i t i s h C o l u m b i a o n June 28 , 1988. ( A ) O c e a n surface a n d (B) - (F) T i d e p o o l s 1-5 respect ive ly . E r r o r bars represent standard error o f the m e a n (N=2) . 129 CSS = surface 553 = bottom O O 10000•• o Skel Nit T.no Mel Odo Cos Cli Sur Gyr Skel Nit T.no Mel Odo Cos Cli Sur Gyr 100000 •• o O 10000•• o ES = surface ESS - bottom Skel Nit T.no Mel Odo Cos Cli Sur Gyr F I G U R E 5 (continued) 130 100000 £ 10000•• <u 1000 o 100 X X r j i l J , U • rfi Skel Nit T.noC.co Dit Odo Cli Sur Mel Cos Gyr Cer Ske Nit T.no Cer Mel Odo Cos Cli Sur Gyr F I G U R E 6: D i a t o m abundance at the ocean surface waters a n d i n f o u r t idepools o f increas ing he ight i n the inter t idal at P o i n t A t k i n s o n , B r i t i s h C o l u m b i a o n J u l y 12, 1988. ( A ) O c e a n surface a n d (B) - (E) T i d e p o o l s 2-5 respec t ive ly . E r r o r bars represent standard error o f the mean (N=2). 131 100000 o O 10000 o 1000 100 10 CM £ o ESS = surface ESS = bottom Ske Nit T.no Cer Mel Odo Cos Cli Sur Gyr 00000 •- £ o o o 10000-\ 1000->— CM E 100 -o \ jn 10--"o O 1 -• ESS = surface ESS = bottom Ske Nit T.no Cer Mel Odo Cos Cli Sur Gyr F I G U R E 6 (continued) 132 F I G U R E 7: D i a t o m d i v e r s i t y at the ocean surface a n d i n f i v e t idepools o f increas ing height i n the in ter t ida l at P o i n t A t k i n s o n , B r i t i s h C o l u m b i a o n f i v e occas ions : ( A ) M a y 15, ( B ) June 6, ( C ) June 13, (D) June 28 and (E) J u l y 12, 1988. 133 F I G U R E 7 (continued) -134-R E F E R E N C E S C a n a d i a n T i d e and Current Tables . 1988. V o l u m e 5. Juan D e F u c a Strait and Strait o f G e o r g i a . F isher ies and Oceans , C o m m u n i c a t i o n s Directorate , Informat ion and Publ i ca t ions B r a n c h , O t t a w a , Ontar io , Canada . 85 p p . C u p p , E . E . 1943. M a r i n e p lankton diatoms o f the west coast o f N o r t h A m e r i c a . U n i v e r s i t y o f C a l i f o r n i a Press , B e r k e l e y and L o s A n g e l e s , U . S . A . Repr in t 1977, Ot to K o e l t z Sc ience Publ i shers , K o e n i g s t e i n , W . G e r m a n y . 237 p p . -135-A P P E N D I X 2: G r o w t h c u r v e s o f Skeletonema costatum a n d Nitzschia thermalis  d u r i n g the p r e l i m i n a r y sets o f t o x i c i t y tests 136 0 2 4 6 8 10 12 Days F I G U R E 1: G r o w t h curves o f Skeletonema costatum at 1 x 1 0 - 5 a n d 1 x 1 0 " 7 M C u (first set o f t o x i c i t y tests). ( A ) In vivo f luorescence; (B) C e l l counts . C i r c l e s , tr iangles a n d squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 a n d N i were est imated. 137 CD U c CD U CO CD o O O O _co O 1 0 . 0 0 1 . 0 0 0 -0 . 1 0 0 -0 . 0 1 0 -0 .001 : :• O . - • ^ 2 : : > , : 5 — o o 10 9 M o - — o 1 0 ~ 1 1 M 0 1 0 0 . 0 -8 10 12 10 .0 1.0 0.1 I , a ^ f f : ' o is '° B o o-o 1 0 - 9 M O 10 1 1 M 0 8 10 12 Days F I G U R E 2 : G r o w t h curves o f Skeletonema costatum at 1 x 1 0 ' 9 a n d 1 x 1 0 ' 1 1 M C u (first set o f t o x i c i t y tests). ( A ) In vivo f luorescence; (B) C e l l counts . C i r c l e s , triangles a n d squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N o a n d N i w e r e est imated. 138 0 2 4 6 8 Days F I G U R E 3 : G r o w t h curves o f Skeletonema costatum at 5 x 10~ 7 a n d 1 x 10~ 7 M C u (second set o f t o x i c i t y tests). ( A ) In vivo f luorescence; ( B ) C e l l counts . C i r c l e s , triangles a n d squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 a n d N i were estimated. 139 [Z 0 . 0 1 0 -0.001 -I 1 1 — i h 0 2 4 6 8 O 0.1 -I 1 1 1 H 0 2 4 6 8 Days F I G U R E 4 : G r o w t h curves o f Skeletonema costatum at 5 x 10"^ a n d 1 x 10~% M C u (second set o f t o x i c i t y tests). ( A ) In vivo f luorescence ; ( B ) C e l l counts . C i r c l e s , triangles a n d squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N 0 and N i were estimated. 140 0 2 4 6 8 1 0 0 2 4 6 8 1 0 D a y s F I G U R E 5 : G r o w t h curves o f Nitzschia thermalis at 1 x 1 0 " 5 , 1 x 1 0 - 7 , 1 x 1 0 " 9 and 1 x 10" ^ M C u (first set o f tox ic i ty tests). ( A ) In vivo f luorescence ; ( B ) C e l l counts . C i r c l e s , triangles a n d squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N o and N i were est imated. 141 E 1.0 1 X 1 0 _ 5 M Cu 5 X 1 0 6 M Cu F I G U R E 6: G r o w t h curves o f Nitzschia thermalis at 1 x 1 0 " 5 , 5 x 1 0 " 6 a n d 1 x 1 0 " 6 M C u (second set o f t o x i c i t y tests). ( A ) In vivo f luorescence ; (B) C e l l counts . C i r c l e s , tr iangles and squares represent replicates 1-3 respect ive ly . F i l l e d s y m b o l s represent the points at w h i c h N o and N i were est imated. 142 CD O C CD O CO CD o Z5 1 . 0 0 0 -0 . 1 0 0 0 . 0 1 0 -0 .001 0 o — o = 5 X 1 0 _ 7 M Cu o O = 1 X 1 0 M Cu 8 O O O in ID O — O = 5 x 1 0 7 M Cu o O = 1 X 1 0 7 W Cu 7 8 Days F I G U R E 7: G r o w t h curves o f Nitzschia thermalis at 5 x 1 0 ' 7 and 1 x 1 0 ' 7 M C u (second set o f t o x i c i t y tests). ( A ) In vivo f luorescence; (B) C e l l counts . C i r c l e s , triangles and squares represent replicates 1-3 respect ively . F i l l e d s y m b o l s represent the points at w h i c h N o a n d N i were est imated. -143-APPENDIX 3 : The effect of polycarbonate containers and medium chelexing on the growth of marine diatoms. I N T R O D U C T I O N T h e suitabi l i ty o f var ious materials used i n the cu l tur ing o f marine organisms has l o n g been o f concern (e.g. B e r n h a r d et a l . 1966, B e r n h a r d and Zattera 1970, Justice et a l . 1972, P r i c e et a l . 1986). G l a s s has been the tradit ional container f o r phytoplankton studies (except studies i n v o l v i n g s i l i con) . H o w e v e r , glass is not suitable for trace metal studies because o f a cont inuous interaction o f its i o n i c surface w i t h cations i n the m e d i u m (see B r o o k e s 1969). T h i s interact ion disrupts the meta l e q u i l i b r i u m o f the aqueous solut ion. S u c h disruptions have been demonstrated b y Robertson (1968) a n d F i t z w a t e r et a l . (1982). A s a result , trace meta l studies are n o w be ing conducted i n either s i lan ized glass (e.g. Schenck 1984) or plast ic containers. O f the plast ics , te f lon is the most favored mater ia l ; however , due to its h i g h cost, polycarbonate is more c o m m o n l y used. B e r n h a r d and Zattera (1970) tested the effect o f polycarbonate containers after autoc laving o n the g r o w t h o f three species o f marine phytoplankton and f o u n d no effect w i t h the except ion o f the c o c c o l i t h Emiliana hwcleyi (Lohmann) H a y and Sandberg (as Coccolithus huxleyi). H o w e v e r , o n l y exponent ia l growth rate was e x a m i n e d . In addi t ion , the l e v e l o f the g r o w t h effect was presented i n a qual i tat ive manner. F i t z w a t e r et a l . (1982) and W o n g et a l . (1986) demonstrated increased p r i m a r y product iv i ty i n polycarbonate f lasks . In contrast, M a r r a and H e i n e m a n n (1984) f o u n d no s ignif icant difference i n p r i m a r y product iv i ty between glass and polycarbonate containers w h e n "noncontaminat ing" procedures were used for both treatments. D u e to the var iab i l i ty i n sensi t ivi ty to culture materials reported for different diatoms (e.g. P r i c e et a l . 1986), the effect o f glass and polycarbonate o n the g r o w t h o f Nitzschia thermalis (Ehrenberg) A u e r s w a l d , Skeletonema costatum (Grev i l l e ) C l e v e and -144-Thalassiosira pseudonana (Hustedt) H a s l e and H e i m d a l was tested. O n e purpose o f this study was to test the hypothesis that there is no s ignif icant dif ference i n d i a t o m growth between the t w o types o f containers. C h e l a t i n g resins are used quite extensively to quant i fy trace metal content i n seawater. T h e y are used for one o f t w o purposes. O n e purpose is to concentrate the metal and alter the matr ix i n w h i c h the meta l is embedded. T h e other is to measure a speci f ic f ract ion o f the meta l . A t o m i c absorption spectrophotometry ( A A S ) is one o f the most c o m m o n techniques used for the determination o f trace metals. H o w e v e r , its sensit ivity and reproduc ib i l i ty m a y be quite var iable w i t h seawater samples. T h i s is attributed to interference w i t h the s o d i u m chlor ide matr ix . In addi t ion , due to the l o w concentrat ion o f trace metals i n seawater, the sensit ivity o f the A A S is sometimes not adequate for metal detection, even i n the absence o f salt-matrix problems. A s a result , i n a number o f studies the seawater is passed through a C h e l e x - 1 0 0 c o l u m n i n order to r e m o v e the salts and concentrate the trace metals (e.g. K i n g s t o n et al. 1978, B r u l a n d and F r a n k s 1979, Sturgeon et al. 1980). R e s i n chelat ion has also been used to determine speci f ic fractions o f the meta l i n seawater. U s u a l l y , i t is used i n c o m b i n a t i o n w i t h other techniques i n order to determine metal speciat ion, for example photoox idat ion , anodic s tr ipping vol tammetry , a c i d digest ion, organic extract ion etc. (e.g. F i g u r a and M c D u f f i e 1980, M a c k e y 1983). In addi t ion , i t has been used to measure seawater c o m p l e x i n g abi l i ty b y str ipping metals released f r o m organics after seawater treatment w i t h ultraviolet i rradiat ion (e.g. B a d e y and F lorence 1976). Because o f the a f f in i ty that C h e l e x res in has for trace metals , i t has been used for r e m o v i n g metal impuri t ies f r o m m e d i a used for marine cultures. D a v e y et al. (1970) deve loped the technique. T h e y obtained excel lent meta l removals w i t h the s o d i u m f o r m o f C h e l e x - 1 0 0 f r o m both ar t i f i c ia l and natural seawater; 9 9 % o f 6 5 z n , H 5 m c d , 5 4 M n -145-and 64cu; 9 5 % o f 2 1 0 p D ; 9 2 % o f 6 3 N i and 59Fe ; but o n l y 32 .6% o f H O m A g . In addi t ion , they c o n c l u d e d that no tox ic components o f the res in leached out d u r i n g the pass ing through the C h e l e x c o l u m n . T h e y observed satisfactory phytoplankton g r o w t h i n the eluted m e d i u m for a number o f species. These results were v e r i f i e d by F lorence and B a t l e y (1976). T h e determined 9 9 . 5 % retention o f C u , Z n , P b and C d by a C h e l e x c o l u m n . T h e y also observed that C u - o r g a n i c complexes and labi le C u and Z n are quanti tat ively r e m o v e d by the c o l u m n after 1 L o f eff luent natural seawater. In addi t ion , meta l impuri t ies were complete ly r e m o v e d b y C h e l e x - 1 0 0 f r o m ar t i f i c ia l seawater. A q u i l ( M o r e l et al. 1979) is an ar t i f i c ia l c h e m i c a l l y def ined m e d i u m that is used f o r phytoplankton cu l tur ing . It was developed spec i f i ca l ly for trace metal studies due to its k n o w n c o m p o s i t i o n . It is prepared i n a metal -c lean fashion. Part o f the procedure i n v o l v e s the pass ing o f the ar t i f i c ia l seawater and its enrichment stocks through a s o d i u m f o r m C h e l e x - 1 0 0 c o l u m n to r e m o v e trace metal impur i t ies . S ince this m e d i u m was used d u r i n g m y study, I determined the impact that c h e l e x i n g the m e d i u m has o n the growth o f Nitzschia thermalis and Skeletonema costatum. T h e second purpose o f this study was to test the hypothesis that there is no difference i n g r o w t h these t w o species cul tured i n che lexed c o m p a r e d to unchelexed A q u i l . -146-M E T H O D S Parti: Effect of containers Stock cultures o f Nitzschia thermalis ( N E P C C 608), Skeletonema costatum ( N E P C C 18c) and Thalassiosira pseudonana, c lone 3 H ( N E P C C B 5 8 ) were obtained f r o m the N o r t h East P a c i f i c Cul ture C o l l e c t i o n , Department o f Oceanography, U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r . G l a s s and polycarbonate 250 m L E r l e n m e y e r f lasks were ' a g e d ' p r i o r to use for this, experiment . T h e y were washed w i t h F i s h e r Sparkleen detergent, rinsed three times w i t h d i s t i l l e d water, soaked i n I N H C 1 for 48 h , rinsed three times w i t h d i s t i l l e d d e i o n i z e d water and autoclaved i n a Cast le standard laboratory autoclave. T h e procedure was repeated f i v e times before us ing the f lasks for the study. In addi t ion , this c l ean ing process was repeated (but o n l y r u n once) between experiments . A r t i f i c i a l seawater ( A q u i l ) was prepared as descr ibed i n M o r e l et al. (1979). It was autoclaved i n a polycarbonate carboy, and 200 m L aliquots o f the same batch o f m e d i u m were added to a l l f lasks . T h e stock culture o f each species was transferred to both glass and polycarbonate f lasks and incubated under the exper imental condit ions for t w o weeks . Subsequendy, the stocks were transferred to c lean f lasks o f both materials conta in ing fresh m e d i u m . W h e n they reached exponent ia l g r o w t h , aliquots were r e m o v e d to inoculate the exper imental f lasks that were made o f the same mater ia l . There was no s ignif icant difference i n the i n i t i a l i n o c u l u m size between the t w o treatments (Student's t-or W i l c o x o n rank s u m test, P>0.05). F i v e replicate f lasks o f each treatment were used. A l l transfers were p e r f o r m e d i n a class 100 laminar f l o w h o o d w i t h a l l metal parts replaced by p o l y p r o p y l e n e . T h e batch cultures were then p laced i n racks that ensured equidistance f r o m the l ight source for a l l replicates o f both treatments. F l a s k s were r a n d o m i z e d w i t h i n each set o f racks to a v o i d any systematic pattern (Runs test for randomness , P>0.05 for a l l experiments) . T h e experiment was conducted at ~ 16° C , under a 16:8h L : D c y c l e and an irradiance o f ~ 9 0 |J.E . m ' ^ . s"*. Irradiance was measured ins ide the culture vessels w i t h a L i - C o r M o d e l L I - 1 8 5 meter (2TC col lector) ; no -147-s ignif icant difference was detected between irradiances ins ide the t w o types o f f lask ( W i l c o x o n rank s u m test, P=0.310). E v e r y 24 h , 10 m L aliquots were r e m o v e d f r o m the cultures after gentle shaking to ensure homogeneous d i a t o m dis t r ibut ion , and used for c e l l counts and f luorescence measurements. In vivo f luorescence was measured us ing a Turner Des igns M o d e l 10 f luorometer . C e l l counts were per formed us ing a P a l m e r M a l o n e y count ing sl ide under a Zeiss Standard 14 c o m p o u n d microscope for N. thermalis and S. costatum, and an O l y m p u s m o d e l B H A phase contrast microscope for T. pseudonana. F o r each replicate, three subsamples were counted us ing f i v e f ie lds per subsample. T h e f ie lds represented a r a n d o m c e l l d is t r ibut ion as r e c o m m e n d e d by L u n d et a l . (1958). F o r each culture, three parameters o f the g r o w t h curve were e x a m i n e d , exponent ia l growth rate, length o f l a g phase and m a x i m a l y i e l d . E x p o n e n t i a l growth rate was calculated u s i n g K e and k as descr ibed i n G u i l l a r d (1973). T h e exponent ia l por t ion o f the g r o w t h curve was determined to be the part w i t h the steepest o v e r a l l slope. N o and N i for each replicate were chosen as the points beneath or b e y o n d w h i c h there was an apparent devia t ion f r o m this s lope. T h e length o f the l a g phase was determined as the t ime between inoc u la t ion and the t ime at w h i c h N o was estimated. T h e m a x i m a l y i e l d was determined as the highest value o f c e l l density or f luorescence that each o f the replicates o f each species attained d u r i n g the experiment . A l l comparisons between treatments were done u s i n g a Student 's t-test. In cases where heteroscedasity was observed and c o u l d not be a l leviated by transformation, a non-parametric W i l c o x o n rank s u m test was used to compare the two treatments. A s igni f icance l e v e l o f 0.05 was used throughout. -148-Part2: Effect of chelexing T h e general exper imental procedure was s imi lar between the two experiments . F o r this part, o n l y polycarbonate flasks were used for both the stock and the exper imental cultures. T h e f lasks were c leaned i n the fashion descr ibed above. T w e n t y litres o f standard ocean water ( S O W ) was prepared as i n M o r e l et al. (1979). T h e y were d i v i d e d i n t w o equal port ions. O n e por t ion was passed through a C h e l e x - 1 0 0 c o l u m n (as descr ibed i n M o r e l et al. 1979). In addi t ion , nitrate, phosphate and sil icate stocks were also prepared and d i v i d e d i n t w o port ions. O n e o f them was also passed through the C h e l e x . T h e che lexed nutrient stocks were subsequently added to the che lexed seawater ( C h e l e x e d treatment). T h e unchelexed stocks were added to the unchelexed S O W ( U n c h e l e x e d treatment). T h e two types o f m e d i a were then processed ident ica l ly (as descr ibed i n Part 1). T h e remainder o f the exper imental protoco l was s imi lar to that o f Part 1. O n l y t w o parameters o f the g r o w t h curve were e x a m i n e d , exponent ia l g r o w t h rate and length o f l a g phase. -149-R E S U L T S A N D D I S C U S S I O N Nitzschia thermalis d i d not demonstrate any difference i n exponent ia l growth rate between the t w o types o f container. T h e l a g phase o f this species was s igni f i cant ly higher i n polycarbonate according to the c e l l counts but not according to the f luorescence estimates ( F i g . l , T a b l e 1). T h e m a x i m a l y i e l d f o r this species was not incorporated i n the comparisons between treatments, due to a s ignif icant difference i n the i n i t i a l popula t ion size. In polycarbonate , Skeletonema costatum exhib i ted a s igni f icant ly l o w e r g r o w t h rate (established u s i n g the c e l l counts) and a longer l a g phase than i n glass, as s h o w n by both the f luorescence measurements and the c e l l counts ( F i g . 2 , Table 1). F o r this species, m a x i m a l y i e l d also s h o w e d a s ignif icant decrease. Thalassiosira pseudonana demonstrated a reduced g r o w t h rate, as s h o w n b y f luorescence, and a decreased m a x i m a l y i e l d , s h o w n both b y f luorescence and c e l l counts , w h e n g r o w i n g i n polycarbonate f lasks ( F i g . 3 , Table 1). T h i s study was preceded by a number o f p r e l i m i n a r y experiments w h i c h demonstrated s imi lar results. P r o b l e m s encountered d u r i n g these experiments and corrected for i n the present study were the i n o c u l u m size (a h i g h i n o c u l u m size subsequently resulted i n a short exponent ia l growth o f two days) , adequate shaking before d a i l y r e m o v a l o f a l iquots , and the i n o c u l u m state. F o r example , i n o c u l a o f S. costatum that were i n stationary phase resulted i n l o w growth rate and extended l a g phase o f up to 10 days that c o u l d not necessari ly be attributed to the container mater ia l . H o w e v e r , i n those cases the effect o f the container was s t i l l present, i f not more pronounced. T h e m e a n growth rates were 0.93-0.95 d i v • day-1 f n glass and 0-0.50 d i v • day-1 i n polycarbonate ( N = 5 , Student 's t-test, P<0.05). In this study, I ensured that the i n o c u l u m size was appropriate, the dis tr ibut ion ins ide the f lasks was homogeneous, and the i n o c u l u m was a lways i n exponent ia l g r o w t h . In addi t ion , the f i v e replicates o f both treatments were processed ident ica l ly throughout the experiment . I, therefore, conc lude that the observed effect was the result o f the container mater ia l . -150-Polycarbonate has been considered a plast ic that has l i t t le or no effect o n phytoplankton g r o w t h c o m p a r e d to glass (Bernhard and Zattera 1970). F r o m m y experiments , it appears to be a mater ia l that can affect growth rate, length o f l a g phase and m a x i m a l y i e l d . In the present study, statistically s ignif icant (rather than quali tat ive) differences for each speci f ic g r o w t h parameter were observed. G r o w t h i n polycarbonate containers appears to be associated w i t h a larger degree o f var iab i l i ty between replicate f lasks than g r o w t h i n glass ( F i g . 2 , T a b l e 1). Therefore , studies w i t h l o w repl ica t ion o f treatments m a y not detect a s igni f icant difference i n growth . T h e observed l o w e r growth i n polycarbonate containers c o m p a r e d to glass c o u l d be due to a number o f factors. I f o u n d n o dif ference i n l ight intensity between the t w o types o f f lasks. H o w e v e r , W o n g et a l . (1986) observed a 15 .1% reduct ion i n photosynthet ical ly avai lable energy ins ide polycarbonate f lasks . T h i s c o u l d potent ia l ly result i n di f ferent ia l growth . A second factor c o u l d be the plastic itself. Despi te extensive soaking and r i n s i n g o f the f lasks before use, some tox ic agent, such as the plast ic iser , m a y have been present i n h i g h enough concentrations to induce an i n h i b i t o r y effect. D y e r and R i c h a r d s o n (1962) demonstrated this for type II p o l y v i n y l ch lor ide rods . L a s t l y , the observed effect m a y be a g r o w t h enhancement i n glass containers rather than a g r o w t h i n h i b i t i o n i n polycarbonate . T h e continuous interact ion o f the glass surface w i t h the m e d i u m c o u l d enhance g r o w t h through leach ing o f a nutrient or buf fer ing o f a meta l . F o r example , P r i c e et a l . (1987) demonstrated a se len ium requirement for T. pseudonana. T h i s nutrient is absent f r o m A q u i l and c o u l d have poss ib ly been introduced to the m e d i u m i n the glass f lasks through leaching o f the glass surface. H o w e v e r , i t w i l l r e m a i n absent f r o m the polycarbonate containers. O n the other hand, the m e d i u m was autoclaved i n a standard laboratory autoclave w h i c h c o u l d p r o v i d e a source o f metal contaminat ion. T h e effect w i l l be reduced i n the glass containers since a port ion o f the contaminant w i l l be r e m o v e d through adsorption o n the surface w a l l s . -151-T h e results o f the effect o f c h e l e x i n g the m e d i u m o n phytoplankton l e d to quite different conclus ions . T h e g r o w t h rate o f Nitzschia thermalis was higher i n chelexed m e d i u m according to the f luorescence measurements but not according to c e l l counts ( F i g . 4 , Table 2). T h i s species demonstrated no effect o n the length o f l a g phase according to the c e l l counts. It d i d however s h o w an increased l a g phase i n che lexed m e d i u m accord ing to the f luorescence estimates ( F i g . 4 , Table 3). It can be observed i n F i g . 4 that, a l though a l l f i v e che lexed replicates exhib i ted h i g h growth d u r i n g the second day , there was a sudden cessation o n the third day f o r three o f the replicates. N o such pattern was apparent for the unchelexed replicates. Skeletonema costatum demonstrated higher g r o w t h rate i n the che lexed m e d i u m according to the c e l l counts (but not according to f luorescence) ( F i g . 5 , T a b l e 2). T h i s species also demonstrated longer l a g phase i n the unchelexed m e d i u m ( F i g . 5 , T a b l e 3). G r o w t h m e d i u m is che lexed i n order to r i d i t o f trace metal impuri t ies and i n order to prevent contaminat ion f r o m u n k n o w n sources ( D a v e y et al. 1970, M o r e l et al. 1979). Its effect o n phytoplankton growth was tested and f o u n d satisfactory ( D a v e y et al. 1970). In m y experiment, growth was enhanced i n most cases. T h i s can be attributed to a number o f factors. It c o u l d be the result o f h i g h metal impuri t ies i n the salts, chemica ls and/or d i s t i l l e d water used for the preparat ion o f the m e d i u m a l l o f w h i c h w i l l be r e m o v e d b y the C h e l e x c o l u m n . A second poss ible source o f contaminat ion is the glass carboy that was used for the m i x i n g o f the salts. T h e preparation procedure o f the m e d i u m is not carr ied out under class 100 condit ions but rather o n a laboratory bench. A e r o s o l s f r o m the laboratory c o u l d be contr ibut ing to the metal contaminat ion as w e l l . In a l l these cases, metals w i l l be added to the seawater and, unless they are r e m o v e d by che lex ing the m e d i u m , m a y become inh ib i tory . N. thermalis demonstrated increased l a g phase i n the che lexed m e d i u m . T h i s c o u l d be attributed to the absence o f a required metal due to r e m o v a l by the res in . A q u i l has been tested as a g r o w t h m e d i u m for a number o f species and has been considered -152-adequate. H o w e v e r , i t is possible that this is the case for most species but not Nitzschia thermalis. Perhaps this species requires one o f the metals i n higher concentrations than those added to the che lexed S O W and m a y therefore become l i m i t e d . A dif ferent poss ib i l i ty is that this species m a y have a requirement for a metal that is never added to A q u i l , such as se len ium (see P r i c e et al. 1987). L o w e r g r o w t h i n polycarbonate containers or unchelexed m e d i u m was not consistently demonstrated for both biomass measurements. In some cases, i t was exhib i ted w h e n u s i n g c e l l counts as a b iomass index but not w h e n u s i n g f luorescence. T h i s c o u l d be exp la ined i f an inh ib i tory agent affects some aspect o f a lgal p h y s i o l o g y (e.g. p igment product ion) but not another (e.g. c e l l d i v i s i o n ; as for copper i n S. costatum, see M o r e l et al. 1978). H o w e v e r , the discrepancy between the t w o biomass indices is probably the result o f different w i t h i n treatment variance for each index . In most o f the cases where there was a discrepancy, no treatment effect was observed for the i n d e x w i t h the highest var iab i l i ty . W i t h Nitzschia i n the container experiment, the discrepancy arose as a result o f the pecul iar shapes o f the growth curves . N o t m u c h g r o w t h was observed w i t h the f luorescence measurements ( F i g . 1). In glass, the f luorescence o f this species increased exponent ia l ly for 1-2 days , very late i n the experiment . T h e i n i t i a l increase i n c e l l number is not ref lected b y the f luorescence estimates. D u e to the pecul iar shape o f these growth curves , I can o n l y accept the result obtained b y the increase i n c e l l concentration. T A B L E 1. C o m p a r i s o n o f g r o w t h parameters o f species g r o w n i n glass and polycarbonate containers ( m = m e a n ( N = 5); sd = 1 standard devia t ion ; c e l l # = cel ls . m L - i m e d i u m ; f luor = f luorescence; P C = polycarbonate ; * = W i l c o x o n test) G r o w t h Species B i o m a s s index Conta iner m sd P parameter (units) E x p o n e n t i a l G r o w t h rate (k i n d i v / day) N. thermalis C e l l # G l a s s 0 .536 0.199 P C 0 .440 0.380 F l u o r G l a s s 1.55 0.430 P C 1.17 0 .680 S. costatum C e l l # G l a s s 1.08 0.09 P C 0 .889 0.098 F l u o r G l a s s 1.02 0.06 P C 0 .894 0.133 T. pseudonana C e l l # G l a s s 1.42 0.100 P C 1.24 0.180 F l u o r G l a s s 1.24 0.070 P C 1.05 0.050 >0.05 >0.05 <0.05 >0.05 >0.05 <0.05 T A B L E 1 (continued) L a g Phase length N. thermalis C e l l # G l a s s 1.2 0.00 <0.05 * (days) P C 8.28 1.95 F l u o r G l a s s 11.3 0 .00 > 0 . 0 5 * P C 9 .66 1.84 S. costatum C e l l # G l a s s 2 .20 0.450 <0.05 P C 6.4 1.95 F l u o r G l a s s 3.0 0 .000 <0.05 * P C 7.0 1.220 T. pseudonana C e l l # G l a s s 0 .970 0.380 >0.05 P C 0 .970 0.380 F l u o r G l a s s 0 .160 0.360 > 0 . 0 5 * P C 0 .000 0.000 M a x i m a l y i e l d S. costatum C e l l # G l a s s 201 ,300 23 ,100 <0.05 ( c e l l s . m L - 1 ) P C 114,900 33,600 or (relative F l u o r G l a s s 8.14 0.70 fluorescence) >0.05 P C 8.99 1.50 T A B L E 1 (continued) T. pseudonana C e l l # F l u o r G l a s s 99 ,400 27,321 P C 30 ,900 2 ,500 G l a s s 7.82 1.26 P C 3.50 0 .520 <0.05 <0.05 -156-T A B L E 2 : E f f e c t o f che lex ing ar t i f i c ia l seawater m e d i u m o n the g r o w t h rate ( in d i v • day-1) o f Skeletonema costatum (= Skel) and Nitzschia thermalis (= Nitz). (ce l l # = cel ls • m L - i m e d i u m ; f l u o r = f luorescence; C h e l e x = che lexed m e d i u m ; U n c h e l = unchelexed m e d i u m ; N = sample s ize; S . D . = standard deviat ion.) Species B i o m a s s Treatment M e a n G r o w t h S . D . t-value Index Rate ( N ) Nitz C e l l # C h e l e x U n c h e l F l u o r C h e l e x U n c h e l Skel C e l l # C h e l e x U n c h e l F l u o r C h e l e x U n c h e l 1.42 (5) 1.20 (5) 1.35 (5) 0.89 (5) 1.71 (5) 1.05 (4) 0.96 (5) 0.86 (4) 0.43 0.20 0.09 0.02 0.18 0.21 0.10 0.12 1.06 >0.100 12.7 <0.001 4.98 <0.010 1.48 >0.100 -157-T A B L E 3 : E f f e c t o f c h e l e x i n g ar t i f i c ia l seawater m e d i u m o n the length o f l a g phase ( in days) o f Skeletonema costatum (= Skel) and Nitzschia thermalis (= Nitz). (ce l l # = cel ls • m L - i m e d i u m ; f l u o r = f luorescence; C h e l e x = che lexed m e d i u m ; U n c h e l = unchelexed m e d i u m ; N = sample s ize; S . D . = standard devia t ion ; * = W i l c o x o n test used instead.) Species B i o m a s s Treatment M e a n L a g S . D . t-value Index Phase (N) Nitz C e l l # C h e l e x U n c h e l F l u o r C h e l e x U n c h e l Skel C e l l # C h e l e x U n c h e l F l u o r C h e l e x U n c h e l 1.2 (5) 1.0 (5) 3.0 (5) 1.0 (5) 0.8 (5) 6.3 (4) 0.8 (5) 0.86 (4) 1.1 1.0 0.0 0.0 0.5 3.5 0.4 0.1 0.32 >0.100 <0.010 <0.050 <0.050 158 1 0 0 T Days F I G U R E 1: G r o w t h curves o f Nitzschia thermalis. A = in vivo f luorescence ; B = c e l l counts . S o l i d l ines = glass containers; dashed l ines = po lycarbonate containers ; c i rc les , tr iangles , squares, inverse triangles and d i a m o n d s = replicates 1-5 respect ive ly ; f i l l e d s y m b o l s represent the points at w h i c h N 0 a n d N i were est imated. 159 2 4 6 8 10 12 14 D a y s F I G U R E 2 : G r o w t h curves o f Skeletonema costatum. A = in vivo f luorescence ; B = c e l l counts . S o l i d l ines = glass containers; dashed l ines = polycarbonate containers ; c i rc les , t r iangles , squares, inverse triangles and d i a m o n d s = replicates 1-5 respect ive ly ; f i l l e d s y m b o l s represent the points at w h i c h N 0 a n d N i were est imated. 160 CD o 0.1 -I 1 1 1 1 0 2 4 6 8 Days F I G U R E 3 : G r o w t h curves o f Thalassiosira pseudonana. A = in vivo f luorescence ; B = ce l l counts . S o l i d l ines = glass containers; dashed l ines = polycarbonate containers; c i rc les , t r iangles , squares, inverse triangles a n d d i a m o n d s = replicates 1-5 respec t ive ly ; f i l l e d s y m b o l s represent the points at w h i c h N 0 a n d N i were est imated. 161 0.01 -I 1 1 1 1 1 1 0 2 4 6 8 10 12 1 0 0 T 0.1 -I 1 1 1 1 1 1 0 2 4 6 8 10 12 Days F I G U R E 4 : G r o w t h curves o f Nitzschia thermalis. A = in vivo f luorescence ; B = c e l l counts . S o l i d l ines = c h e l e x e d m e d i u m ; dashed l ines = u n c h e l e x e d m e d i u m ; c i rc les , t r iangles , squares, inverse triangles and d i a m o n d s = repl icates 1-5 respect ive ly ; f i l l e d s y m b o l s represent the points at w h i c h No and N i were est imated. 162 F I G U R E 5 : G r o w t h curves o f Skeletonema costatum. A = in vivo f luorescence; B = c e l l counts . S o l i d l ines = c h e l e x e d m e d i u m ; dashed l ines = u n c h e l e x e d m e d i u m ; c i r c les , t r iangles , squares, inverse triangles and d i a m o n d s = replicates 1-5 respect ive ly ; f i l l e d s y m b o l s represent the points at w h i c h N 0 a n d N i were est imated. -163-R E F E R E N C E S B a t l e y , G . E . & F lorence , T . M . 1976. Determinat ion o f the c h e m i c a l forms o f d i s s o l v e d c a d m i u m , lead and copper i n seawater. Mar. Chem. 4 : 347-63. B e r n h a r d , M . & Zattera, A . 1970. 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