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The effect of copper on phytoplankton Leblanc, Michael Joseph 1979

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The E f f e c t Of Copper On P h y t o p l a n k t o n by MICHAEL JOSEPH XEBLANC B. Sc., U n i v e r s i t y o f Guelph, 1974 .THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Zoology He a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d s THE UNIVERSITY OF June, (c) Michael Joseph BRITISH COLUMBIA 1979 LeBlanc, 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D e p a r t m e n t o f D E - 6 B P 7 5 - 5 1 1 E ABSTRACT The s e n s i t i v i t i y of several species of marine phytoplankton to copper was investigated* No s p e c i f i c trends were found with respect to any general differences between diatoms and din o f l a g e l l a t e s or between d i f f e r e n t sized c e l l s . . Further investigation using a single bioassay species, Nitzschia lonqissima, indicate i t to be very sensitive to cupric ion a c t i v i t y . Several physiological systems appear to be affected to d i f f e r e n t degrees, with c e l l d i v i s i o n being more affected than photosynthetic pigment production or 1 4 C uptake. The a c t i v i t y of the enzyme ni t r a t e reductase i s greatly increased by the addition of low concentrations of copper. TABLE OF CONTENTS T i t l e Page i Abstract . . . . . . - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , . , , . . i i Table Of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i L i s t Of Tables ..................... ................ .v L i s t Of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Chapter 1 - A Comparison Of The Eff e c t s Of Copper On Thirteen Species Of Marine Diatoms And Dinof l a g e l l a t e s ................................. ..5 Introduction ............................... ...... 5 Method 11 Eesults ........................ .................. 17 Discussion .......................................31 Chapter 2 - The Effect Of Cupric Ion A c t i v i t y On The Growth Rate Of The Marine Diatom, Nitzschia longissima ................. ..... ......... 35 Introduction ....................................... 35 Method .................................... .... ...... 38 Results And Discussion ...........................47 Chapter 3 - C o n d i t i o n i n g Of Growth Medium By Phytoplankton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 0 I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 0 Method ............................................ 5 4 R e s u l t s ............................ . . . . . . . . . . . . . . 5 6 D i s c u s s i o n . . . , . , , , . . , . , , . , , . . , . , . . . , , . . , , . , , , , . . , 6 2 Chapter 4 - Some F a c t o r s A f f e c t e d By Copper T o x i c i t y In The Marine Diatom, M t z s e h i a l o n a j ^ s i m a 6 4 I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4 Method . 6 6 B e s u l t s . , , . . . 6 9 D i s c u s s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 0 General D i s c u s s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4 B i b l i o g r a p h y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 7 L i s t Of Tables Table 1.1: Species used i n the experiments ± . . . . . . . . . . . . . 16 Table 1.2: Nutrient enrichments for natural seawater ...,16 Table 1.3: Effect of copper on the growth rate of thirteen species of phytoplankton ................... 21 Table 2.1: Experimental medium .......................... 41 Table 2.2: Computed chemical speciation .................42 Table 2.3: Parameters and results of experiments ........ 43 Table 4.1: The ef f e c t of added copper on the a c t i v i t y of the enzyme nit r a t e reductase ..............,,...,,72 Table 4*2: The e f f e c t of added copper on the rate of photosynthesis ...,..,.......^ ..........,..,......73 v i L i s t Of F i g u r e s F i g u r e s 1.1 - 1.19: R e l a t i v e growth r a t e vs. c o n c e n t r a t i o n of added copper ....................... 23-28 Figu r e 1.20: The s u r f a c e area t o volume r a t i o vs. the amount of copper necessary t o cause a 50% r e d u c t i o n i n the growth r a t e .....,...,,,.,,...,,29 F i g u r e s 2.1 - 2.2: The c a l c u l a t e d c u p r i c i o n a c t i v i t y vs. the growth r a t e .................................44-46 F i g u r e s 3.1 - 3.2: The e f f e c t of copper on c e l l and f l u o r e s c e n c e growth r a t e s i n unconditioned and c o n d i t i o n e d media ...................................58 F i g u r e s 3. 3 - 3.4: The e f f e c t of copper on f l u o r e s c e n c e growth r a t e and the l a g time before the s t a r t of e x p o n e n t i a l growth at th r e e i n i t i a l inoculum s i z e s ..................................... ,60 F i g u r e s 4 . 1 - 4.4: Changes i n f l u o r e s c e n c e / c e l l of the marine diatom, N i t z s c h i a l g n g i s s i m a , during the f o u r day bioassay at f o u r added copper c o n c e n t r a t i o n s *.............................. 74 Fi g u r e 4.5: E f f e c t o f added copper on f l u o r e s c e n c e / c e l l and f l u o r e s e n c e / c e l l volume 76 F i g u r e 4.6: E f f e c t o f added copper on the apparent a c t i v i t y of the enzyme n i t r a t e reductase ............78 v i i Acknowledgments I would l i k e to thank Dr. A.S<._ Lewis for the encouragment and assistance he has given me during t h i s study. Drs. P.J. Harrison, K.J.F., H a l l and P. Dehnel have also assisted me i n my research and the preperation of t h i s thesis. . I also thank Dave Turpin for aid in the enzyme analysis and Rosemary Waters for aid and advice in the culturing of the phytoplankton species. 1 THE EFFECT OF COPPER ON PHYTOPLANKTON General Introduction Recently there has been increasing int e r e s t i n the ef f e c t s of trace metals on plankton species and communities in natural waters;. I t has also been increasingly recognized that i t can be those factors a f f e c t i n g trace metal a v a i l a b i l i t y which are most important, rather than the t o t a l metal concentration. Provasoli (1963) and Johnston (1964) noted that natural waters from dif f e r e n t sources had d i f f e r i n g capacities to support phytoplankton growth a-nd that these differences could not always be attributed to differences in s a l i n i t y , temperature or nutrient concentration.. Moreover, growth rate could often be increased by the addition of a r t i f i c i a l or natural chelators, which were:assumed to change the b i o l o g i c a l a v a i l a b i l i t y of trace metals* I t has been found that the poor growth of phytoplankton i n recently upwelled water could be. overcome by the addition of the a r t i f i c i a l chelator, EDTA, or a zooplankton homogenate, but not by the addition of various nutrients, trace mstals, vitamins or amino acids, either singly or i n combinations (Barber and Ryther, 1969),. Smayda (1974) found that those adverse water q u a l i t i e s overcome by the addition of EDTA were the second most important factors influencing phytoplankton growth in the surface waters of Narragansett Bay over an annual cycle.. There i s strong evidence therefore, that those water q u a l i t i e s controlled by chelators, namely trace metal a v a i l a b i l i t y , are important to phytoplankton qrowth i n many natural waters* Of those metals influencing phytoplankton growth in natural 2 waters, copper appears to be one of the most important.. Jackson and Morgan (1978) have indicated that the effect of chelators on growth can be best explained as decreasing t h e . a v a i l a b i l i t y of copper ions rather than increasing iron a v a i l a b i l i t y or decreasing n i c k e l or zinc a v a i l a b i l i t y . . Copper i s recognized as both an e s s e n t i a l micronutrient, necessary for growth (Manahan and Smith, 1973), and as a possible i n h i b i t o r of phytoplankton production (Steemann Nielsen and Wium-Andersen, 1970).. Natural copper l e v e l s i n the oceans range from < 1 i 0 x 1 0 - 8 M i n the open ocean (Boyle et a l , 1977; Windom and Smith, 1979) up to 5.0x10-7 M i n coastal areas (Abdullah et a l , 1972; Rica et a l , 1973) and additions of copper to seawater at l e v e l s as low as 1;6x10 - 8 M have been shown to be toxic to phytoplankton (Davey et a l , 1973; Steemann Nielsen and wium-Andersan, 1971)., Braek et al (1976) found copper to be an order of magnitude more toxic than zinc, as has Dvernell (1976) and Hollibaugh et al (in prep, a) . Mercury i s more toxic than copper (Dvernell, 1976; Hollibaugh et a l ^ in prep*, a) but i s considerably less common in the marine environment. In a mixture of approximately environmental l e v e l s of ten metals, Hollibaugh et a l (Ln prep* . a) found that almost a l l of the toxic e f f e c t was due to thecopper present.. It has only recently been recognized that the: t o x i c i t y of low l e v e l s of copper i s r e l a t e ! not to the: t o t a l metal concentration, but to the " b i o l o g i c a l l y a v a i l a b l e " copper concentration (Sunda and G u i l l a r d , 1976; Lewis et a l , 1972; Anderson and Morel, 1978) whirh i s affected by such factors as pH, and the complexing capacity of the seawater.. Experimental treatments which remove or destroy the natural organic compounds 3 in seawater, i . e . u l t r a f i l t r a t i o n or u l t r a - v i o l e t l i g h t treatment w i l l usually cause a large: decrease in phytoplankton growth, a decrease which can be reversed by the addition of a r t i f i c i a l chelators (Barber, 1973; Gnassia-Barelli et a l , 1978; Sunda and G u i l l a r i , 1976)., As there i s at present no physical or chemical method of accurately measuring the: available or uncomplexed copper concentration in seawater, b i o l o g i c a l assays have been increasingly used in studies of trace metal effects., When choosing a bioassay species, i t ' s s s n s i t i v i t y with respect to other species must be determined* In addition to variation due to experimental differences, there are major variations i n the response of d i f f e r e n t species or in d i f f e r e n t l i f e cycle stages of a p a r t i c u l a r species to diff e r e n t metal l e v e l s (Erickson et a l , 1970; Reeve et a l , 1977). Such diffarences could be extremely important in determining the species composition of natural communities and has been used as a possible explanation for succession i n plankton communities (Lewis , 1977),. In t h i s study an attempt has been made; 1) to compare the effect of added copper in unchelated natural seawater on a wide range of phytoplankton species to determine i f there are differences i n t h e i r s s n s i t i v i t y to copper and i f so, i f these differences follow any general patterns. 2) to select one sensitive species as a bioassay organism for further tests to determine other factors affecting copper t o x i c i t y and other physiological e f f e c t s of copper t o x i c i t y besides reduced growth rates. 4 3) t o use the b i o a s s a y s p e c i e s and copper b u f f e r e d medium (Anderson and M o r e l , 1978) t o determine at what c a l c u l a t e d c u p r i c i o a a c t i v i t i e s growth r a t e was affected.„ 5 CHAPTER 1 A Comparison Of The Effects Of Copper On Thirteen Species Of Marine Diatoms And Dinoflagellates Introducton Copper has been increasingly recognized as an important factor a f f e c t i n g the growth and composition of phytoplankton populations. The addition of approximately environmental l e v e l s of copper (1.6x10 - 8 M) have been shown to be capable of s i g n i f i c a n t l y reducing growth in some species (Davey et a l r 1973; Steeraann Nielsen and Wium-Anderson, 1971),. . It has recently been demonstrated that i t i s the " b i o l o g i c a l l y available" copper concentratioa which i s toxic, and that i t i s controlled by such factors as t o t a l copper concentration^ pH and complexing capacity of theiseawater (Sunda and G u i l l a r d , 1976; Anderson and Morel, 1978; Sunda and Lewis, 1978).. F i e l d studies have indicated that the presence or absence of natural chelators in seawater can have important effects on phytoplankton growth (Barber and Ryther, 1969; Smayda, 1974). Because many of the. e a r l i e r experiments with copper t o x i c i t y to phytoplankton used standard growth media which contained varying amounts of a r t i f i c i a l and natural chelators (EDTA, T r i s and sediment extract), i t i s very d i f f i c u l t to compare the re s u l t s of these d i f f e r e n t experiments.. In many of these experiments, the chemical state of the copper in the experimental medium was unknown, as was the actual cupric ion concentration to which the phytoplankton species were being exposed. The use of media with chelators has also resulted i n erroneously high estimates of the copper tolerence of 6 phytoplankton. _ In a d d i t i o n to the l a r g e v a r i a t i o n s i n r e s u l t s due to d i f f e r e n t experimental techniques, there can be major d i f f e r e n c e s i n the response of d i f f e r e n t s p e c i e s or i n d i f f e r e n t l i f e c y c l e stages of a p a r t i c u l a r s p e c i e s to d i f f e r e n t metal l e v e l s ( E r i c k s o n et a l , 1970; Reeve: et a l , 1977).. Such d i f f e r e n c e s could, be extremely important i n determining the s p e c i e s composition of n a t u r a l communities and t h i s has been used as a p o s s i b l e e x p l a n a t i o n f o r s u c c e s s i o n i n plankton communities (Lewis, 1977; Thomas and S e i b e r t , 1977). Most experiments use only one or two species i n s t u d y i n g the e f f e c t s of copper, and the methodological d i f f e r e n c e s make i t v i r t u a l l y i m p o s s i b l e t o compare d i f f e r e n t experiments. For example, i n media c o n t a i n i n g no added a r t i f i c i a l or n a t u r a l c h e l a t o r s , v a r i o u s experimenters have found the amount of copper causing a 50% r e d u c t i o n i n the growth of T h a l a s s i o s i r a E§eudonana to be 0 . 7 9 x 1 0 - * M Cu (Davey et a l , 1973), 1.57x10-* M Cu ( E r i c k s o n , 1972), 3.93x10~ 7 M Cu (Jensen and Rystad, 1 9 7 6 ) and g r e a t e r than T i 57x10-* M Cu (Braek et a l , 1976). S i m i l a r i l y , the amount of copper necessary to i n h i b i t growth i n Skeletgnema costatum by 50% has been reported as l e s s than 1.57x10 -7 M Cu (Jensen and Rystad, 1976), 7.87x10-* M ( E r i c k s o n et a l , 1970) and over 1.57x10-* M Cu (Braek et a l , 1976). Those:studies which have compared the e f f e c t s of copper on two or more sp e c i e s of phytoplankton have had c o n f l i c t i n g r e s u l t s i n determining e i t h e r the amount of copper necessary to cause a t o x i c e f f e c t or i n f i n d i n g g e n e r a l t r e n d s i n the s e n s i t i v i t i e s of d i f f e r e n t s p e c i e s or groups. . Braek et a l 7 (1976) found only a small difference between three diatoms and one d i n o f l a g e l l a t e tested, with the: amount of added copper necessary to cause a 50% reduction i n growth rate ranging from 1.18x10-* to 3.94x10-* M Cu. . Bentley-Mowatt and Reid (1977), i n testing f i v e species of phytoplankton, found four species to be unaffected by copper concentrations less than 10-* M, but used a medium which contained 10~* M EDTA. In comparing 12 phytoplankton species, Erickson et a l (1970) found most species to be i n h i b i t e d by 1.57 to 7.87x10-* M Cu i n a medium containing 2.5 mM T r i s , an a r t i f i c i a l chelator which has been shown to detoxify copper (Anderson and Morel, 1978).. Ia further tests of six species i n a medium without any added chelators, they found the phytoplankton to be much more sensitive to the added copper, with almost complete growth i n h i b i t i o n in some species at 7.87x10 - 7 M Cu, the.lowest concentration of copper tested. The comparison was of rather limited s e n s i t i v i t y and precision because the c e l l s were not maintained in exponential phase of growth and because the tested copper concentrations were in increments of 7,.87x10-7 M Cu. Overnell (1976), using a medium containing 1^34 x 10~* M EDTA, compared the effects of several metals on the: rate of 0^  evolution of six phytoplankton species and found a 50% i n h i b i t i o n of growth at l e v e l s of 2-7x10~ 5 M Cu for four species including two centric diatoms, with the other two species, one a ce n t r i c diatom, being r e l a t i v e l y tolerant. In a comparison of three d i n o f l a g e l l a t e species, S a i f u l l a h (1978) found l i t t l e difference between species, with 1 i 6 x 1 0 ~ 8 M Cu having no e f f e c t and 3 . 1 5 x 1 0 - 7 M Cu t o t a l l y stopping growth i n a l l species. . 8 M a n d e l l i ( 1 9 6 9 ) , u s i n g a medium c o n t a i n i n g 4.68 x 1 0 - 6 M EDTA, c o m p a r e d t h e e f f e c t s o f c o p p e r on n i n e s p e c i e s o f p h y t o p l a n k t o n ( i n c l u d i n g t h r e e c e n t r i c d i a t o m s a n d one p e n n a t e d i a t o m and t h r e e d i n o f l a g e l l a t e s ) and f o u n d g r o w t h i n h i b i t i o n a t c o n c e n t r a t i o n s o f 3.9 t o 3 9 x 1 0 - 7 M C u , w i t h t h e t h r e e d i n o f l a g e l l a t e s b e i n g much more s e n s i t i v e (3.9 t o 8.7x10 - 7 M Cu) t h a n t h e d i a t o m s (2.52 t o 3 . 9 4 x 1 0 - 6 M C u ) . . Nor i s t h e r e any a g r e e m e n t on t h e r a n g e . o f s e n s i t i v i t i e s w i t h i n any p a r t i c u l a r g r o u p . M a n d e l l i (1969) f o u n d w i d e d i f f e r e n c e s i n s e n s i t i v i t i e s b e t w e e n d i n o f l a g e l l a t e s and d i a t o m s b u t c o m p a r a t i v e l y l i t t l e d i f f e r e n c e w i t h i n e a c h g r o u p . S a i f u l l a h (1978) f o u n d t h r e e d i n o f l a g e l l a t e s p e c i e s t o show v e r y s i m i l a r r e s p o n s e s t o c o p p e r t o x i c i t y , a s d i d B r a e k e t a l (1976) f o r t h r e e s p e c i e s o f d i a t o m s * . E r i c k s o n ( 1 9 7 0 ) , h o w e v e r , f o u n d s e n s i t i v i t i e s f o r s e v e r a l s p e c i e s o f d i a t o m s t o r a n g e o v e r an o r d e r o f m a g n i t u d e o r more, as d i d J e n s e n and E y s t a d ( 1 9 7 6 ) . C o m p a r i s o n o f t o x i c i t y e x p e r i m e n t s u s i n g c u p r i c i o n c o n c e n t r a t i o n s i n s t e a d o f t o t a l c o p p e r c o n c e n t r a t i o n s a l s o show w i d e d i f f e r e n c e s b e t w e e n d i f f e r e n t d i a t o m s p e c i e s , some o v e r two o r d e r s o f m a g n i t u d e ( J a c k s o n a n d M o r g a n , 1978) . F i e l d e x p e r i m e n t s h a v e a l s o i n d i c a t e d some i n t e r e s t i n g d i f f e r e n c e s i n s p e c i e s s e n s i t i v i t y t o c o p p e r * . Thomas and S e i b e r t (1977) f o l l o w e d t h e c h a n g e i n a n a t u r a l p h y t o p l a n k t o n p o p u l a t i o n a f t e r t h e a d d i t i o n o f v a r i o u s l e v e l s o f c o p p e r ( 0 -7 . 8 7 x 1 0 - 7 M Cu) t o s e v e r a l l a r g e p l a s t i c e x p e r i m e n t a l c o n t a i n e r s . They f o u n d t h a t c e n t r i c d i a t o m s and d i n o f l a g e l l a t e s ( i . e . . L§Etocy_lindricus, C h a e t o c e r o s , T h a l a s s i o s i r a , P e r i d i n i u m 9 and DiaoBhysis) were dominant i n the c o n t r o l s (no added copper) but disappeared from the copper t r e a t e d e n c l o s u r e s (0.79 , 1.6 and 7.87x10 - 7 M Cu) , where pennate diatoms ( i . e * N i t z s e h i a and I§¥i.£u.i.§) became dominant. T h i s o b s e r v a t i o n of the s e n s i t i v i t y of c e n t r i c s vs* pennate diatoms i s p a r t i a l l y supported by Er i c k s o n et a l (1970) who found 2 of 3 c e n t r i c s t e s t e d to be very s e n s i t i v e to added copper and the one pennate s p e c i e s t e s t e d to be comparatively i n s e n s i t i v e * In a s e r i e s of f i v e day experiments with n a t u r a l phytoplankton p o p u l a t i o n s , Ibragim and P a t i n (1976) found s i g n i f i c a n t changes i n the s p e c i e s composition o f the copper t r e a t e d communities, with the pennate diatom, N i t z s c h i a , becoming dominant and the c e n t r i c diatoms, C o s c i n o d i s c u s and R h i z p s o l e n i a , d i s a p p e a r i n g * In a study of two p o l l u t e d f j o r d s u s ing three bioassay s p e c i e s , Eide : and Jensen (1979) found the Phaeodactylum - trieornutum to be r e l a t i v e l y i n s e n s i t i v e t o t r a c e metal p o l l u t i o n , while: T h a l a s s i o s i r a pseudonana was moderately s e n s i t i v e : a n d Skeletonema costaturn was extremely s e n s i t i v e . The l i t e r a t u r e , t h e r e f o r e , shows somewhat c o n t r a d i c t o r y r e s u l t s . . Some authors have found d i n o f l a g e l l a t e s to be more s e n s i t i v e t o copper than diatoms ( M a n d e l l i , 1969; Thomas and S e i b e r t , 1977), while others have found l i t t l e d i f f e r e n c e (Braek et a l , 1976; E r i c k s o n e t a l , 1970). Some have: found c e n t r i c diatoms t o be more s e n s i t i v e than pennate diatoms (Thomas and S e i b e r t , 1977; Ibragim and P a t i n , 1976) while o t h e r s have found no d i f f e r e n c e ( M a n d e l l i , 1969). The purpose of t h i s study i s to compare the growth r a t e response t o added copper of a f a i r l y wide range of phytoplankton 10 s p e c i e s t o d e t e r m i n e : 1) i f t h e r e a r e s i g n i f i c a n t d i f f e r e n c e s b e t w e e n s p e c i e s , 2) i f t h e r e a r e a n y t r e n d s i n t h e s e n s i t i v i t y o f t h e s p e c i e s , i . e . d i a t o m s v s * d i n o f l a g e l l a t e s , l a r g e c e l l s v s i s m a l l c e l l s , g e n u s v s . g e n u s o r c e n t r i c v s . p e n n a t e d i a t o m s . The u s e o f c u p r i c i o n a c t i v i t i e s o r c o n c e n t r a t i o n s i n s t e a d o f t h e t o t a l c o p p e r c o n c e n t r a t i o n c o u l d e l i m i n a t e v a r i a t i o n s d u e t o t h e u s e o f d i f f e r e n t m e d i a . F o r t h e : p u r p o s e s o f a c o m p a r i s o n , h o w e v e r , i t i s o n l y n e c e s s a r y t o u s e t h e same c o n d i t i o n s f o r a l l s p e c i e s t e s t e d . 11 METHOD Eight species of diatoms and f i v e species of dino f l a g e l l a t e s were compared i n t h i s study (see Table 1.1 f o r the species used). The species were chosen on the basis of either previous use reported in the l i t e r a t u r e in copper bioassays or abundance in l o c a l waters. Unialgal cultures of the 13 species were obtained from the Northeast P a c i f i c Culture C o l l e c t i o n , University of B r i t i s h Columbia, Vancouver, B.C.. Natural seawater for the bioassays was collected i n December, 1977 and March, 1978 from a depth of 350 m at Geo 1748 (49«17.0« N, 123048. 8' W) , a station i n the Str a i t of Georgia, B r i t i s h Columbia* The seawater was collected with a 90 1 fibreglass and l e u c i t e sampler to minimize metal contamination, and was f i l t e r e d immediately on c o l l e c t i o n through a 0.45 um f i l t e r * The seawater was stored at 9°C i n a 200 1 acid washed polyethylene barrel. The December water was used over a period of eight weeks in December and January and the March water was used for two weeks i n May, to carry out a l l of the bioassays. The s a l i n i t y of both the sea water samples was 31.1 %o , the dissolved organic carbon concentration (DOC) was 0.9 mg 1 _ 1 and the dissolved copper l e v e l was 1.1x10 - 8 M i n the December and March waters. DOC and copper analysis methods were as reported by Cave (1 977) . . Bioassays at the beginning and end of the Dec.-Jan* .experimental period, with Nitzschia lonjjissima, Skeletonema costatum and Thalassipsira pseudonana (see Figs.. 1.1-1.6) , indicated that there was l i t t l e i f any change i n the growth supporting capacity of the stored seawater, or in the e f f e c t of 12 the added copper over the two month period of the Dec. -Jan* experiment; Dissoved organic" carbon measurements of the stored seawater also showed no change and dissolved copper showed a very s l i g h t decrease from 1,13 to 1.10x10 - 8 M Cu. . M l species except Chaetoceros crinitum were tested in the f i r s t experiment in Dec.-Jan., using the December water*. In the May experiment, C. crinitum was tested as well as three of the species previously tested. The May experiment, using the March water, was carried out to v e r i f y some of the Dec*-Jan* r e s u l t s and to add a t h i r d Chaetoceros species to the comparison. Before the sta r t of the bioassays, the seawater medium was enriched with n i t r a t e , phosphate, s i l i c a t e : and vitamins (see Table 1.2). These additions were designed to provide non-l i m i t i n g amounts of the major nutrients but not to greatly exceed the nutrient conditions that marine phytoplankton encounter i n the oceans. Copper was added as the appropriate volume of a 23. 6x10 - 6 M Cu stock solution made up with CuCl^. and double d i s t i l l e d deionized water.. A l l of the:copper l e v e l s referred to i n the figures are added copper not t o t a l or i o n i c copper. After the addition of the copper, the medium was allowed to equilibrate for atleast two hours before the addition of the c e l l inoculum* Anderson and Morel (1978) have found that copper may take several hours to come to equilibrium with an a r t i f i c i a l medium and i t was considered best to allow an equal amount of time for the added copper to eq u i l i b r a t e with the natural seawater. Prior to the s t a r t of the experiment, the unialqal cultures were grown i n the enriched seawater (see table 1.2) for 4-12 13 days, depending on the growth rate of the p a r t i c u l a r species, to precondition the species to the seawater and also to minimize any carry over of contaminants•, such as a r t i f i c i a l chelators or heavy metals, to the experimental cultures.. Use.of maintenance cultures i n medium such as ES medium for the i n i t i a l c e l l inoculum can resu l t in carrying over up to 5 ug EDTA m l - 1 and 100 ug T r i s m l - 1 of inoculum (Provasoli, 1968). Both EDTA and T r i s are a r t i f i c i a l chelators with a high a f f i n i t y for copper. The i n i t i a l inoculum i n the bioassays was from an exponentially growing culture and gave st a r t i n g c e l l concentrations in the experimental vessels of 1000 c e l l s ml-*. For the bioassays, the algae were grown i n batch cultures at a temperature of 15±0.5°C, i n one l i t r e Pyrex Erlynmeyer flasks containing 500 mis of the enriched seawater medium plus the appropriate amount of added copper* The large medium volume, 500 mis, was chosen to minimize the surface area to volume r a t i o and so minimize metal adsorption to the glass surface, which could lower the metal ion concentration (Robertson, 1968),. Illumination was provided, on a 14:10 hour light:dark cycle, by fluorescent l i g h t s (GE Cool White) with a l i g h t intensity from the side of 80 uE m~2 sec-*,as measured by a LiCor Model LI 185A Quantum meter.. Four copper concentrations were tested i n each experiment; 0, 3.2, 7.9 and 15.7x10~8 M Cu (0, 2, 5 and 10 ug added Cu 1 _ 1 ) , with two re p l i c a t e f l a s k s at each concentration. The one l i t r e f l a s k s used for the experiments were double acid washed with 0.5 N HCl, rinsed several times with deionized d i s t i l l e d water and autoclaved before the: st a r t of the 14 experiment* The vessels were prerinsed with 200 mis of the medium to precondition the glass walls to minimize any adsorption of copper to the glass,. Metal analysis of the experimental medium by a t r i p l e : extraction into MIBK (Cave, 1977) showed a loss of 10% of the added copper over a 7 day period. Samples were taken daily f o r c e l l counts and fluorescence* The samples for c e l l counts were preserved by the addition of 0.10 mis of Lugol*s solution to 10 mis of sample.. C e l l concentrations of a l l but one species were determined by v i s u a l counts in a Paliner-Maloney counting chamber. . In most cases at least 200 c e l l s per sample were counted. Where the concentration was less than 2000 c e l l s ml -*, only 100 c e l l s were counted*. C e l l concentrations for Thalassiosira-fiseudonana-were determined using a Coulter Counter Model B with a 100 um aperture tube* Fluorescence was also measured daily with a Turner Fluorometer (Model 111). S p e c i f i c growth rates of the cultures were:determined by a le a s t squares linear regression of the log, D c e l l concentration or log, 0 fluorescence vs* time for the exponential portion of the growth curve. This value was then converted to doublings per day (Guillard, 1973).. In most cases, because of an i n i t i a l lag period, growth rates were based on the change in c e l l numbers or fluorescence between the f i r s t and fourth days.. S p e c i f i c growth rate has been shown i n at least one case (Berland et a l , 1977) to be a more sensitive indicator of the effect of copper t o x i c i t y than particulate C and N, culture biovolume and  l+C a s s i m i l a t i o n . 95% confidence l i m i t s are plotted on a l l of the 15 graphs. The r e l a t i v e standard deviation of the growth rate of cultures in 10 r e p l i c a t e control f l a s k s was 2.6%* Table 1.1: Bioassay species used in the experiments Skeletonema costatum (Sreville) Cleve Thalassiosira pseudonana (Hustedt) Hasle and Heimdal Thalassigsira nordenskioldii Cleve Chaetoceros compressum Lauder Chaetoceros laciniosum Schutt Chaetoceros crinitum Shutt Nitzsghia longissima (Brebisson) Ralfs Nitzsehia delicatissima Cleve Prorpce^trum minimum S c h i l l e r Heterocagsa t r i g u e t r a Ehrenb. Symnodigium simglgx (Lohm.) Kof,. Ex Swezy Gym nod inj. um v i t i l i g o Ballantine Scri£p_siella troehoidea (Stein) Loeblich III Table 1.2: Nutrient enrichments for natural seawater NaNOi NaH^PO^ Na aSi0 3 .9KX0 Thiamin* HCI B i o t i n 3.5x10-5 M 1.45x10-6 M 4.0x10-6 M 1.19x10-8 M 1.48x10-11 M 8.19x10-1* M 17 Results The r e l a t i v e effects of four concentrations of copper ( 0 , 3.2, 7.9 and 1 5 . 7 x 1 0 ~ 8 M) on the rate of increase of c e l l numbers and fluorescence for 13 species of phytoplankton, are shown in Figs.. 1.1-1.19. The growth rates are: given as a percent of the control to f a c i l i t a t e comparison between species. The points on the figures for fluorescence growth rates are shifted 2 mm to the right to prevent confusion with the c e l l number points*. The points are both taken from the same samples, however, and represent the same copper concentrations* The absolute growth rates are presented i n Table 1.3. Bioassays with three species at both the beginning and the end of the Dec.-Jan* test period (see. Figs..1.1-1.6) indicated that the water storage period had no s i g n i f i c a n t e f f e c t on the assay results* A second experiment was carried out i n May 1978 to check some of the res u l t s of the Dec.-Jan..experiment and to add a th i r d Chaetoceros species to the comparison* The res u l t s of the two experiments are presented together to f a c i l i t a t e comparisons, but the starting date of each experiment i s given with the figures* Nitzsghia lgnqissima was used twice in the f i r s t experiment and once in the May experiment (Fig..1.3, 1.4 and -1. 12) with no s i g n i f i c a n t differences between the re s u l t s i n any of the three bioassays, indicating no s i g n i f i c a n t difference in those factors affecting copper a v a i l a b i l i t y i n the seawater samples used i n the two experiments.. The species tested in both experiments were; 5. v i t i l i g o , S. troehoidea and N. longissima. In addition, C. crinitum was tested only i n the May experiment.. 18 I t should be emphasized that a l l bioassay r e s u l t s must be considered to be r e l a t i v e , and not an absolute response to a s p e c i f i c t o t a l copper concentration. I t i s very d i f f i c u l t to measure or calculate the cupric ion concentration i n seawater media and no attempt has been made to do so in t h i s experiment.„ The r e s u l t s are r e l a t i v e but are a l l that i s necessary for the comparison of species s e n s i t i v i t i e s within this experiment* The r e s u l t s shown in Figs..1.1-1.12 are a l l for diatoms while those i n Figs. . 1* 13-1. 1 9 are for dinoflagellates* I t i s apparent that there i s a great variation in the: s e n s i t i v i t y of the species to copper within each group, and neither group can be said to be more sensi t i v e than the other, . Skeletonema costaturn, one of the most abundant species i n the world, was found to be very i n s e n s i t i v e : t o added copper (Fig. 1.1 and 1.2) as i s Thalassiosira- nordenskioldii (Fig. 1.7)._ Both show no s i g n i f i c a n t reduction in growth at the highest concentration of added copper tested (15.7x10 - 8 M Cu). Thalassiosira gseudgnana, which has been used previously as a copper bioassay organism, was found to be only moderately sensitive i n t h i s study (Fig* 1*5 and 1*6), as was Nitzschia delicatissima (Fig..1.8) a species sometimes abundant in l o c a l waters. Nitzschia lgngissjma (Fig. 1.3/ 1-f and 1.12) and the three Chaetoceros species (Fig. 1.9, 1.10 and 1.11) were a l l extremely sensitive to added copper, with the lowest addition (3.15x10 - 8 M Cu) causing a s i g n i f i c a n t reduction i n growth i n a l l four cases. A l l four are species which can be abundant i n l o c a l B.C- waters* Of the dinof l a g e l l a t e s , only Hetergcap.§a trig u e t r a 19 (Fig. 1.18) i s in s e n s i t i v e to the added copper, while Gymnodinium v i t i l l i g p and Gymnodinium simplex (Fig* 1.13, 1.14 and 1.19) are moderately s e n s i t i v e , and ScrijpjDsieJLla trochoidea and Proroeentrum minimum (Fig. 1.15, 1.16 and 1.17) are very sensitive. S o r i g s i e l l a trochgidea i s the only species to show a s i g n i f i c a n t difference between the J a n . and May experiments. The difference i s probably due to variations i n the f i r s t experiment as indicated by the wide: confidence l i m i t s i n Fig..1.15.. A l l the experiments used two rep l i c a t e f l a s k s per tes t condition, with the single exception of H. trig u e t r a (Fig. 1.18) which due to experimental problems, only had one flask per test condition*. The lack of r e p l i c a t e s i s the reason for the wide confidence l i m i t s , not any large increase in the v a r i a b i l i t y of the : growth rates of t h i s species. The r e l a t i v e standard deviation was approximately the:same for t h i s species as for the other species tested. The length of the i n i t i a l lag period before the start of exponential growth was calculated for a l l conditions from the equations of the regression l i n e s . . In no case was there any relationship between the length of the lag phase and the concentration of added copper* In Fig. 1.20, the c e l l u l a r surface area to volume r a t i o of the 13 species i s plotted against the copper concentration which caused a 50% reduction i n growth* There i s no s i g n i f i c a n t r elationship between the two factors, surface area to volume r a t i o and copper t o x i c i t y . Nor i s there any relat i o n s h i p between c a l l volume and copper t o x i c i t y . Table 1.3: Effect of copper on the growth r a t e 1 of thirteen species of phytoplankton* . Growth Rate at each Sp.ec ies Date C e l l #2 Control Added Co£|>e r Cone* Or F l . 3 (x10~a-M) 1. 6 3,i 2 7.9 15. 7 S. c. Dec. C e l l # 1.56 1. 48 1.51 1.39 F l . 2.42 2.40 2.40 2.46 Jan. C e l l # 2.39 2.52 2. 10 1.97 F l . . 2. 17 2. 31 2.17 2. 10 N.l. Dec. C e l l # 1.78 1.22 0.35 0. 21 F l . . 1.89 1. 46 0.?0 0.50 Jan. C e l l # 1.73 1.05 0.26 0.20 F l . . 1.72 1.30 0.73 0. 47 May C e l l # 1. 83 1. 36 1. 00 0,.40 F l . . 1.78 1. 54 1.37 0.?2 T.p. Dec. C e l l # 1.69 1. 15 0.97 0 . 7 0 Jan. C e l l # 1. 52 1. 32 1.05 0.77 F l . . 1.77 1.69 0.96 0.77 T.n. Jan. _ C e l l # 0.60 0.47 0.59 0.58 F l . 0. 91 1.02 0.86 0.97 N.d. Dec. C e l l # 2. 17 2. 19 1.67 1.41 F l * 2. 13 2.04 1.58 1. 38 C. C O Jan. C e l l # 2. 19 1.58 0.08 0.20 F l . . 2. 19 1-71 0.73 0,19 C l . Jan. . C e l l # 1.67 1. 38 1.07 0.00 F l . 1. 76 1. 13 1.00 0.00 Growth Rate i at each Species Date C e l l Control Added-C Cone. Or Fl.£ (x10 z!-fi> 1.6 3; 2 2-9 15. 7 C. cr. May C e l l # 2.01 1.32 0.59 0.46 F l . 2.69 1.74 1.11 0. 95 G. y. . Dec. C e l l # 0.58 0.65 0.51 0.47 May C e l l # 0.60 0. 63 0.52 0.40 F l . 0.51 0.51 0.49 0.35 S. t. Jan. C e i l # 0. 54 0.38 0.31 0. 00 F l . . 0, 47 0. 42 0.42 0.14 May C e l l # 0.37 0. 37 0.33 0.21 F l . 0.35 0.36 0.34 0. 18 P. m. . Dec. . C e l l # 0. 55 0.44 0.26 0.08 F l . 0.40 0.28 0.18 0. 15 H. t. . Dec. C e l l # 0.66 0.69 0.70 0. 67 F l . 0. 52 0.49 0.51 0.44 G.s. Jan. C e l l # 0.73 0.68 0.66 0.44 F l . 0.70 0.49 0.47 0. 15 1 growth rate = doublings per day 2 refers to the growth rate of c e l l numbers 3 refers to the growth rate of fluorescence 23 Fig.1.1-1.19: Relative Growth rate as a percent of the control vs. concentration of added copper (x10 - 8 M). The effect of copper on c e l l number growth rate (•——•). The effect of copper on fluorescence growth rate (o o>. The fluorescence data (o) i s displaced 2 mm to the right to prevent confusion with the c e l l data (•). Error bars represent 95% confidence l i m i t s . . o 2k 1 0 0 CD -+-» a cr o O 5 0 0 3 ^ 1 0 0 S. c o s t a t u m Dec 0 5 10 1!5 A d d e d C o p p e r ( x 1 0 " 8 M ) F i g . 1-1 N. l o n g i s s i m a Dec a cr o L . 0 5 0 0 0 5 10 15 A d d e d C o p p e r F ig . 1-3 1 0 0 5 0 0 1 0 0 5 0 0 S.. c o s t a t u m J a n 6 5 1 0 15" A d d e d C o p p e r ( x 1 0 " 8 M ) F i g . 1-2 N. l o n g i s s i m a Jan 0 5 10 15 A d d e d C o p p e r F i g . 1 -4 2 5 o ? 1 0 0 +-> d c r o L. o 5 0 0 T. pseudonana Dec 0 5 10 15" A d d e d C o p p e r ( x 1 0 " 8 M ) F i g . 1 -5 ^ 1 0 0 -+-> d c r o (3 5 0 0 1 0 0 5 0 0 T. pseudonana Jan 0 5 10 15" A d d e d C o p p e r ( x 1 0 " 8 M ) F ig . L 6 1 0 0 5 0 T. n o r d e n s k i o l d i i Jan 0 5 10 1 5 ~ A d d e d C o p p e r F i g . 1-7 N. d e l i c a t i s s i r n a Dec O 5 10 15 A d d e d C o p p e r F ig . 1«8 2 6 o^lOO d cr 5 0 o O 0 C. pompressum Jan 5*100 -+-> d o o 5 0 0 0 5 10 15 A d d e d C o p p e r ( x 1 0 " 8 M ) F ig . 1-9 C. c r i n i t u m May 0 5 10 15 A d d e d C o p p e r F ig . 1-11 1 0 0 5 0 0 C. l a c i n i o s u m Jan 0 5 10 15 A d d e d C o p p e r ( x 1 0 * 8 M ) F ig . 1 -10 1 0 0 5 0 0 N. l o n g i s s i m a May 0 5 10 15 A d d e d C o p p e r F ig . 1-12 27 5^100 a c r o L. o 5 0 0 A, G. v i t i l l i g o , .Dec 0 5 1 0 15~ A d d e d C o p p e r ( x 1 0 " 8 M ) F ig . 1-13 S. t r o e h o i d e a Jan i 100 +-> a c r +-» o o 0 5 10 15 A d d e d C o p p e r F ig . 1 -15 1 0 0 5 0 0 G. v i t i l l i g o May 6 5 10 15~ A d d e d C o p p e r ( x 1 0 " 8 M ) F i g . 1-14 S. t r o e h o i d e a May 1 0 0 5 0 0 0 5 10 15 A d d e d C o p p e r F i g . 1 -16 28 1 0 0 +-» d o L . o 5 0 0 P. minimum Dec o <D •+-> d si o L . CD 1 0 0 5 0 0 0 5 10 15 A d d e d C o p p e r ( x 1 0 " 8 M ) F ig . 1-17 G. simplex Jan 0 5 10 15 A d d e d C o p p e r F ig . 1 - 1 9 1 0 0 5 0 0 H. t r i q u e t r a Dec 0 5 10 15" A d d e d C o p p e r ( x 1 0 " 8 M ) F i g . 1 - 1 8 29 F i g . . 1.20: The s u r f a c e area to volume r a t i o (SA:V) of each s p e c i e s vs. the amount of copper necessary to cause an 50% r e d u c t i o n i n the growth rate of each s p e c i e s (150). Below the f i g u r e are the c a l c u l a t e d SA:V and 150 val u e s f o r each s p e c i e s as w e l l as the i d e n t i f i c a t i o n number f o r each s p e c i e s used i n the f i g u r e . The f i v e s p e c i e s shown to the r i g h t of the break i n the X a x i s d i d not show a 50% r e d u c t i o n i n growth w i t h i n the range of copper c o n c e n t r a t i o n s t e s t e d i n the experiment. 5-O +-> o o < cr s g a c L_ — =J o oo > Q 1 11 8 3 , 13 # 10 1 ^ 12 »» 4 ~ 0 5 1 0 1 5 C o p p e r C o n c e n t r a t i o n C a u s i n g a 50%> R e d u c t i o n in G r o w t h (x 1 0 " 8 M ) Fig . 1«20 Species SAxVol. > 1 Skeletonema costatum 0.92 2 N i t z s c h i a longissima 1.22 3 . 0 3 T h a l a s s i o s i r a pseudonana 1 .30 8.0 4 T h a l a s s i o s i r a n o r d e n s k i o l d i i 0.28 / 5 N i t z s c h i a delicatissirna 2.7^ / 6 Chaetoceros compressum 0 . 5 9 3 . 0 7 Chaetoceros laciniosum 0 .61 2. 8 8 Chaetoceros crinitum 0.71 3 - 5 9 Gymnodinium v i t i l i g o 10 S c r i p p s i e l l a troehoidea 11 Prorocentrum minimum 12 Heterocapsa t r i q u e t r a 13 Gymnodinium simplex 0 . 5 0 0 . 2 5 1.04 0 . 3 7 1 .20 / 1 0 . 0 / 9 . 0 31 Discussion Those experiments, in the past, which have compared the effects of copper on several species of phytoplankton have had c o n f l i c t i n g r e s u l t s i n determining either the amount of copper necessary to cause a toxic effect or i n finding general trends in the s e n s i t i v i t i e s of d i f f e r e n t species or groups. Comparisons of the e f f e c t s of copper on phytoplankton species have found somewhat contradictory r e s u l t s with some experimenters finding general differences between dinoflagellates and diatoms (Mandelli, 1969; Thomas and Seibert, 1977) and within the diatoms, differences between centrics and pennates (Ibragim and Patin, 1976; Thomas and Seibert, 1977) and others finding no such general differences (Braek et a l , 1976; Erickson et a l , 1970). The present study has found both diatoms and di n o f l a g e l l a t e s to be very variable in their s e n s i t i v i t i e s to copper, ranging from complete growth i n h i b i t i o n to no growth effect within the copper concentrations tested. No general statement as to the s e n s i t i v i t y of either group can be made. Pennate and ce n t r i c diatoms also show wide ranges of s e n s i t i v i t y , and neither can be said to be more: sensi t i v e than the other* The r e s u l t s reported here do support the findings of some of the f i e l d experiments (Thomas and Seibert, 1977; Hollibaugh et a l , i n prep, a) where i t was found that pennate diatoms, l i ^ z s c h i a delicatissima i n p a r t i c u l a r , became dominant i n copper treated containers and the -haetoeeros species, which were abundant in the controls, disappeared. In the present study, 3 2 the three Chaetpcergs species tested were among the most sensitive to added copper, while Nitzschia delicatissima was only moderately sensitive. The only general trend in s e n s i t i v i t y to copper observed in th i s study was for species of the same genera to have s i m i l a r s e n s i t i v i t i e s . This was especially apparent with the three Chaetoeerps species tested, a l l of which were very sensitive. The other genera where more than one species were tested (Thalassigsira, Nitzschia and gYmngdinium) were not as close i n the s e n s i t i v i t i e s of the two species but at le a s t did not show a wide difference* as suggested i n e a r l i e r studies (Erickson et a l , 1970; Mandelli, 1969; Thomas and Seibert, 1977; Hollibaugh et a l , i n prep* a) and supported in t h i s study* phytoplankton vary i n t h e i r s u s c e p t i b i l i t y to copper. Some species show a great decrease i n growth rate at added copper l e v e l s l i t t l e greater than natural environmental l e v e l s * . Such differences in s e n s i t i v i t y could r e s u l t in drastic changes i n community structure of phytoplankton populations. This could in turn affect higher trophic levels because of such factors as food preference (Hag, 1967) and f i l t e r i n g e f f i c i e n c y of zooplankton (Frost, 1975). Thomas and Seibert (1977) have suggested that one result of increasing copper lev e l s i n the oceans would be a decrease i n phytoplankton taxonomic d i v e r s i t y , with a r e s u l t i n g dominance of re s i s t a n t species* . This, however, does not necessarily mean a decrease in productivity or biomass* Several studies have indicated a s i g n i f i c a n t c o r r e l a t i o n between added copper or cupric ion a c t i v i t y and the length of 33 the i n i t i a l lag phase in the bioassay. Barber (1973) found the lag time i n several Chaetoceros species to be decreased by the addition of chelators and increased by the addition of metals.. Morel et a l (1978) has shown that the lag phase of Skeletonema costatum increases with increasing cupric ion a c t i v i t y . Jackson and Morgan (1978), i n analyzing the data of several previous authors, have observed that there seems to be two types of responses to excessive lev e l s of copper. The f i r s t type of response i s a depression i n growth rate with increasing copper concentration which does not decrease markedly with time. The second type of response i s a lag phase in growth increasing with increasing copper concentration, followed by an exponential growth at a rate i n d i f f e r e n t to the i n i t i a l concentration of copper. The present study has found no species showing the second type of response. While many of the species showed a lag phase before exponential growth, i n no case could the lag phase be related to the concentration of added copper. It must be stated, however, that the shortness of the bioassay (four days) could allow the recovery after an extended lag phase to be missed (Stockner and Antia, 1976). This only seems possible for a few of the most sensitive species, the Chaetoceros species i n p a r t i c u l a r . In conclusion, t h i s study has found no general difference in the t o x i c i t y of copper to either diatoms or d i n o f l a g e l l a t e s , or within the diatoms, between centric or pennate species* There i s no relationship between copper t o x i c i t y and surface area to volume r a t i o or to the length of the:lag phase before 3 4 exponential growth. There i s some in d i c a t i o n that the species of a p a r t i c u l a r genera have sim i l a r s e n s i t i v i t i e s to copper and thi s i s especially apparent with the Chaetoceros species. 35 Chapter 2 The Effect Of Cupric Ion A c t i v i t y On The Growth Rate Of The Marine Diatom, Nitzschia-lgngissima Introduction There has been an increasing int e r e s t i n the effects of heavy metals, copper i n p a r t i c u l a r , on phytoplankton species and communities in natural waters. I t has been demonstrated that the addition of approximately environmental l e v e l s of copper (1.6x10-8 M) to seawater i s capable of s i g n i f i c a n t l y reducing growth in some phytoplankton species (Davey et a l , 1973; Steemann Nielsen and Wium-Andersen^ 1971) but i t has only recently been f u l l y recognized that i t i s both the:concentration and the chemical species of the metal present that are important in determining the effect of the metal on phytoplankton. . Of the trace metals naturally present i n seawater; copper appears to be most important in influencing phytoplankon growth. Hollibaugh et a l (in prep, b) compared the e f f e c t on phytoplankton growth of different-strengths of a mixture of ten metals whose r e l a t i v e proportions were those found in natural seawater* They found that almost a l l of the t o x i c i t y of the metal mixture was due to the copper present. That i s , copper at a p a r t i c u l a r concentration, either alone or with the nine other metals, would have the same eff e c t on growth rate.. In another paper by the same authors (Hollibaugh et a l , in prep, a) they determined the comparative t o x i c i t y of 10 metals i n d i v i d u a l l y and found copper to be the most toxic, with the exception of mercury, which while more toxic, i s present in natural seawater in considerably lower concentrations than copper. Also 36 supporting t h i s , Jackson and Morgan (1978) reinterpreted Davey's (et a l , 1973) and Barber's (1973) experiments and attributed the effects on growth and * * C assimilation to changes i n the cupric ion concentration rather than to changes i n i r o n , zinc or n i c k e l ion concentrations. Early works by Provasoli (1963) and Johnston (1964) demonstrated the importance of chelating agents in making seawater a suitable medium for phytoplankton growth, presumably by c o n t r o l l i n g the speciation of the metals present. I t has been found that recently upwelled waters, which were high i n dissolved metals and low in dissolved organic compounds, were unsuitable for phytoplankton growth u n t i l either a r t i f i c i a l chelators or an extract of homogenized zooplankton were added to the seawater (Barber and Ryther, 1969).. Treatment of seawater by u l t r a v i o l e t i r r a d i a t i o n , which i s known to break down organic matter which could complex and detoxify trace metals, has been found i n many cases to cause an increase in the; t o x i c i t y of trace metals (Barber, 1973; Sunda and G u i l l a r d , 1976). The fact that the t o x i c i t y change can be reversed through the addition of a r t i f i c i a l chelators such as EDTA would further indicate that the toxic action of the 0.V..irradiation was through the break down of nontoxic organic trace metal complexes to more to x i c metal species, probably the i o n i c species (Sunda and Lewis, 1978) . Several studies (Sunda and G u i l l a r d , 1976; Anderson and Morel, 1978; Sunda and Lewis, 1978) have indicated that the t o x i c i t y i s related to the cupric ion concentration (Cu 2 +) and not the t o t a l copper concentration, the inorganically complexed 37 copper species, EDTA. copper or T r i s copper complexes or copper complexed with natural organic compounds (Sunda, 1975; Sunda and Lewis, 1978). Their studies have related the cupric ion a c t i v i t y i n seawater to such factors as growth rate, »*C uptake and c e l l m o t i l i t y in various phytoplankton species. At present there i s no method of d i r e c t l y measuring cupric ion a c t i v i t y i n seawater although s p e c i f i c ion electrodes have been used with some success in fresh water (Swallow et a l , 1978; Sunda and Lewis, 1978). Lacking a method of d i r e c t measurement i t has been necessary to use computer models and thermodynamic calculations, such as those developed by Westall et a l (1976) and Sunda (1975), to calculate the speciation of copper in copper t o x i c i t y experiments with marine phytoplankton. The aim of t h i s investigation i s to determine and quantify the r e l a t i o n s h i p between calculated cupric ion a c t i v i t y and growth rate of the diatom species Nitzschia longissima and to compare t h i s r e l a t i o n s h i p with those reported for other species and calculated estimates of cupric ion a c t i v i t y in natural seawater* 38 Method Stock cultures of the pennate diatom Nitzschia lonaissima were obtained from the Northeast P a c i f i c Culture: Co l l e c t i o n at U.B.C. This species was chosen as a bioassay organism for t h i s study because of i t s s e n s i t i v i t y to copper, as determined i n previous work comparing the e f f e c t s of copper on 13 species of diatoms and dinof l a g e l l a t e s (Chapter 1). Natural seawater for the bioassays was collected i n February and March, 1978 as in Chapter 1* The seawater was stored at 9°C i n a 200 1 acid washed polyethylene barrel and was used over a period of two months, Feb. To A p r i l , to carry out several bioassays to determine the effect of calculated cupric ion a c t i v i t y on the growth rate of the phytoplankton species.. A two month storage period had previously been shown to have l i t t l e or no ef f e c t on the capacity of the seawater to support phytoplankton growth or aff e c t copper t o x i c i t y bioassays. This water apparently had a low natural complexing capacity as suggested by the fa c t that; 1) addition of small amounts of copper (1.6x10 - 8 M) were shown to have a toxic e f f e c t on the bioassay species 2) comparison of bioassays using high intensity u l t r a v i o l e t (UV) treated and non-UV treated seawater showed no s i g n i f i c a n t difference* Treatment with U.V* i r r a d i a t i o n i s known to break down the dissolved organic compounds which could complex and detoxify copper (Armstrong et a l , 1966; Barber, 1973; Sunda and Lewis, 1978) . . In any case, any naturally occurring organic compounds 39 present would have to be either extremely strong chelators and/or present i n very high concentrations to s i g n i f i c a n t l y a f f e c t the cupric ion a c t i v i t y in a medium in which the copper ion a c t i v i t y i s buffered by the addition of comparatively large concentrations of the chelators, EDTA and T r i s (Morel et a l , 1978; Sunda and G u i l l a r d , 1 976). The medium used i n the experiments consisted of f i l t e r e d seawater plus added nutrients, vitamins, a r t i f i c i a l chelators and varying amounts of copper (Table 2.1). Computation of the eguilibrium chemical speciation, especially the cupric ion concentration, was performed with the computer program MINEQL (Westall et a l , 1976). Limitations on the computations were as described i n Anderson and Morel (1978).. Table. 2.2 l i s t s the chemical constituents and concentrations used in the calculations* Concentrations of i o n i c species were corrected to a c t i v i t i e s using the Davies modification of the Debye Huckel approximation for a c t i v i t y c o e f f i c i e n t s (Stumm and Morgan, 1970).. After the: addition of the: various enrichments, the medium was allowed to equilibrate for at least two hours before the i n i t i a l c e l l inoculum was added. Anderson and Morel (1978) have found that some chelators, i . e * . EDTA, can take several hours to e q u i l i b r a t e in seawater* The i n i t i a l c e l l inoculum was from an exponentially growing culture and gave a starting c e l l concentration i n the experimental vessels of 1000 c e l l s ml-*.. For the bioassays, the algae were grown i n batch cultures at a temperature of 15±0.5<>C in 1 1 b o r o s i l i c a t e glass Erlynmeyer fl a s k s containing 500 mis of the experimental medium. Illumination was provided on a 14:10 light:dark cycle by no fluorescent l i g h t s (WH Cool White SHO) with an incident l i g h t i n tensity of 100 uE m - 2 sec -* as measured by a quantum sensor. Two re p l i c a t e f l a s k s were run at each test concentration. The 1 1 Erlynmeyer flasks used for the bioassays were double acid washed with 0.5N HCI, rinsed several times with deionized d i s t i l l e d water and autoclaved before the sta r t of the experiment. The vessels were prerinsed with 200 mis of the medium to condition the glass walls to minimize any adsorption of copper*_ Samples were taken d a i l y for c e l l counts and fluorescence* The c e l l count samples were preserved by the addition of 0.10 mis of Lugol's solution to 10 mis of sample. C e l l concentrations were determined by v i s u a l counts i n a Palmer-Maloney counting chamber.. In most cases, at least 200 c e l l s were counted.. Where the c e l l concentration was less than 2000 c e l l s ml-i, only 100 c e l l s per sample were counted. Fluorescence was measured with a Turner Fluorometer Model 111. The pH was determined at the beginning and end of the assays, using a Fisher Accutron 140. . S p e c i f i c growth rates of the cultures were used as an indication of copper t o x i c i t y and were determined as in Chapter 1. 41 Table 2.1: Experimental Medium Natural Seawater S a l i n i t y 31.1%o Dissolved Copper 1.0 X 10"« M Dissolved Zinc 7.6 X 10~ 8 M Dissolved Organic Carbon 0.9 ug l ~ i Enrichment NaN03 NaHA PO^  Na ASi0 3 . 9HA0 Thiamin* HCl Bio t i n B ex EDTA Tr i s Copper 3.50 x 10-s M 1.45 x 10-s M 4.0 x 10-6 M 1. 19X10-B M 1.48x10-11 M 8.19x10-1i M 5.0 x 10-* M 1.0 x 10-3 M 32.2 to * 010 x 10-6 H Table 2.2: Computed chemical speciation Species T o t a l 1 Ionic Cone. (M) Cone* (M) 42 Dominant Aqueous Form Bromide 7. 48x10" 4 7.48x10" * • Br~ 100 Calcium 9. 11. io- 3 7.95x10- 3 Caz* 87. 3 Carbonate 2. 07x10- 3 1.17x10- 4 HC03- 61. 1 Chloride 4. 85x10- I 4.85x10- 1 c i - 100 Cobalt 1. 09x10- 9 5.05x10- I I COEDTA2+ 92. 4 Copper 1. 00x10" 8 7.43x10" 1 3 CuTris-^OH-^ 50.4 EDTA 5. 00x10- 7 5.40x10- 1 S CaEDTA2 + 79. 7 Iron 5. 00x10- 7 1.05x10- 2 1 Fe (0H)3 99.8 Magnesium 4. 73x10" 2 4.00x10- Z Mg2+ 84.5 Manganese 3. 64x10- 8 1.18x10- 8 MnCl+ 46. 2 Nitrate 5. 50x10- 5 5.50x10- 5 NO3- 100 Phosphate 5. 00x10- 6 1.48x10- 9 HPO H 2" . 51.4 Potassium 9. 07x10- 3 8.77x10- 3 K+ 96.7 S i l i c a t e 1. 00x10- 5 6.69x10- 1 t HiSiO^ 90.4 Sodium 4. 16x10- 1 4.10x10- 1 Na 1* 98.6 Strontium 8.01. 10- S 7.86x10- S S r 2 + 98. 2 Sulfate 2. 51.10- 2 1.18x10- 2 so^.2- 46. 8 Tr i s 1. 00x10- 3 7-72.10-4 T r i s 77. 1 Zinc 7. 65x10- a 2.33x10- 9 ZnEDTA 2 + 96. 3 1 concentrations based on Riley and Chester (1972) except for EDTA and copper which were added, and manganese and zinc which were measured. Table 2.3: Parameters And Re s u l t s Of Experiments Exper- TotCu4 Growth Rate* iment* (UM) pH 2 pCu* 3 C e l l # F l u o r . 1 5. 01 8. 18±,.09 9.49±.22 0.08±.12 0.20±.34 2. 01 8.18±.08 9.95±.20 0.31±.20 0.86±.24 1.01 8.21±.09 10. 42±. 14 0.90±. 42 1.40±.30 0. 11 8.22+.07 11.55±.Q9 1.92±. 18 2. 11±. 12 0. 01 8.23±.08 12.65±.08 1.84±.14 2.04±. 10 2 32.3 8.17±.07 8.86+.17 0. 12±.41 .000 5. 01 8.24±.12 9. 6 0+. 3 0 0. 16±.27 0.28±.75 2. 01 8.18±.10 9.95±.20 0.04±.22 0.23±. 13 1. 01 8.20±.10 10.35±.20 0.06±. 20 0.50±.11 0. 11 8.30±.02 11.64t.04 1.90±.16 2. 35±.24 0. 11 8.30±.05 11.64±.06 1.97+.21 2.36±.15 0.01 8.30±.Q4 12.61±.04 1.95+,. 15 2.3U.11 3 1. 66 7.95±„08 9.49+.20 0.01±.08 0.24±. 10 0. 51 8.00+.06 10.40±.10 0.21±.04 0.42±.08 0. 21 8.17±.13 12.21±.16 1.6 5±,.08 1.57±.06 0. 11 8.23±.14 11.50±.15 1.83±.11 1. 58±.05 1 T o t a l added copper plus the 1 x 1 0 - 8 M t h a t was a l r e a d y present i n the seawater 2 Median c u l t u r e pH - l i m i t s g i v e the range i n c u l t u r e pH durin g the experiment 3 Median c u l t u r e pCu* (negative l o g of the c u p r i c i o n a c t i v i t y ) - l i m i t s g i v e the range due to the v a r i a t i o n s i n pH * Growth r a t e = d i v i s i o n s per day, the e r r o r l i m i t s i n d i c a t e the 95% confidence l i m i t s . 44 'O Figs. 2.1 and 2 . 2 : The calculated pCu* (-log of the cupric ion a c t i v i t y ) vs* the growth rate of c e l l numbers :(•) and fluorescence (o), respectively, of the bioassay species Nitzschia lonqissima. The horizontal bars on each point represent variations in the calculated pCu* due to v a r i a t i o n ^ in pH during the course of the four day experiments. The v e r t i c a l bars are the 95% confidence l i m i t s for the measured growth (jrate at each test condition* G r o w t h R a t e co O *o C e l l N u m b e r s ( d o u b l i n g s d a y ) -A ro b b 2] -o <P £ o l ro + 00 o b G r o w t h Rate - F l u o r e s e n c e ( d o u b l i n g s d a y ) b ro b CD f 31 ~o CQ' O -c O ro * ro + ro + - f -CA) Results And Discussion HI The purpose of these experiments was to evaluate the t o x i c i t y of copper for Nitzschia i2£3i§sima, by r e l a t i n g the effect on growth rate to the calculated cupric ion a c t i v i t y . I t was also to compare i t s s e n s i t i v i t y with that of other species whose s e n s i t i v i t y to cupric ion a c t i v i t y have been determined. Recent work i n both fresh and s a l t waters has indicated that copper t o x i c i t y i s related to the concentration (or activity) of certain chemical species of copper present, esp e c i a l l y the cupric ion a c t i v i t y and not to the t o t a l copper concentration present (Sunda and S u i l l a r d , 1976; Anderson and Morel, 1978; Howarth and Sprague, 1978; Andrew et a l , 1977; Jackson and Morgan, 1978; Sunda and Lewis, 1978). The cupric ion a c t i v i t y can be affected by such factors as pH and the presence of organic and inorganic complexing agents as well as by the t o t a l copper concentration* Figures 2.1 and 2*2 show the e f f e c t of the calculated pCu* (the negative log of the calculated cupric ion a c t i v i t y ) on the growth rate, as determined from c e l l numbers and fluorescence measurements, respectively, of Nitzschia lonqissima,. . There i s a very abrupt change i n the growth rate between pCu*«s 10.6 and 11.6, with maximum growth rate above pCu* 11.6, almost complete growth i n h i b i t i o n below pCu* 10.6 and approximately 50% growth i n h i b i t i o n at a pCu* of 10.9.. Copper t o x i c i t y data reported for other species can be compared with some degree of confidence when the copper concentrations are expressed as pCu* rather than as t o t a l copper added to a medium of unknown complexing capacity. Sunda and 48 G u i l l a r d (1976), using a si m i l a r method for determining pCu*, found 50% i n h i b i t i o n of the diatom Thalassiosira pseudonana and the green alga Nannoehloris atomus at a pCu* of 9.3.. Morel et a l (1978) have found no reduction i n the growth of the diatom Skeletonema costatum above a pCu* of 8.4,. Anderson and Morel (1978), using the motility and »*C uptake of Gonyaulax tamarensis c e l l s as an indicator of copper t o x i c i t y , found a 50% effect at a pCu* of 10.4.. Reuter et a l (1979) found a pCu* of 10 to cause a 50% i n h i b i t i o n of J*C f i x a t i o n i n the marine blue green algae O s c i l l a t o r i a t h e i b a u t i i . Based on a mean value of 1.4x10 - 8 M copper i n coastal seawater samples (Chester and Storey, 1974), i t i s possible to estimate the cupric ion a c t i v i t y i n seawater (assuming only inorganic complexation)* At a pH of 8*2, calculations give a pCu* of 9.6-9*7 (Anderson and Morel, 1978; Sunda and G u i l l a r d , 1976), a value considerable higher than that required to cause a major reduction i n the growth of Nitzschia longissima.. As t h i s calculation does not include any possible organic complexation, i t represents a maximum a c t i v i t y for that pH and copper concentration* At that pCu*, Gonyaulax tamarensis also would not be able to grow and Ti pseudonana, 0* Theibautii and N. atomus would have p a r t i a l growth i n h i b i t i o n while S* costatum would not be affected at a l l * I t i s obvious that there are some major variations in the s e n s i t i v i t i e s of di f f e r e n t species to copper.. It i s also i n t e r e s t i n g that the ranges of s e n s i t i v i t i e s spans the estimated levels of cupric ion a c t i v i t y i n natural seawater*. This would indicate that one of the e f f e c t s of copper on natural H9 communities could be to cause a s h i f t i n the species composition, though not necessarily a decrease in productivity. This i s what has been observed in the CEPEX experiments (Thomas et a l , 1977; Thomas and Siebert, 1977), where observations on the e f f e c t s of copper on the community structure of the phytoplankton population showed major changes i n the species composition but l i t t l e change i n primary productivity or biomass between the controls and the copper treated enclosures. Such changes could have important effects on higher levels of the food chain through such factors as food preference and d i f f e r i n g f i l t e r i n g e f f i c i e n c i e s of the zooplankton.. I t i s also in t e r e s t i n g to note that the two most sensitive species are the pennate diatom Nitzschia lonqissima and the di n o f l a g e l l a t e Gonyaulax tamarensis (Anderson and Morel, 1978). Some previous comparisons of species s e n s i t i v i t i e s to copper have suggested that dinoflagellates are a great deal more sensitive than diatoms (Mandelli, 1969; Thomas et a l , 1977). The present study does not support such a generalization. The pennate diatom species used here, Nitzschia longissima, appears to be more sensi t i v e than the d i n o f l a g e l l a t e , Gonyaulax tamarensis, or the blue green alga, O s c i l l a t o r i a - t h e i b a u t i i , and considerably more sensitive than the c e n t r i c diatoms, Thalassiosjra pseudonana and Skeletonema costatum. . 50 C H A P T E R 3 Conditioning Of Growth Medium By Phytoplankton Introduction The a v a i l a b i l i t y and t o x i c i t y of trace metals, especially copper, in seawater, depends on the metal speciation. Several studies (Sunda and G u i l l a r d , 1976; Anderson and Morel, 1978; Sunda and Lewis, 1978) have indicated that the most toxic species i s the cupric ion, with the various inorganically or organically bound species being either non-toxic or considerably less toxic* Any reduction i n the cupric ion concentration, i . e . through the addition of organic compounds capable of complexing copper, w i l l result i n a reduction in the t o x i c i t y of the copper i n the medium. If organic complexation does not take place, the l e v e l s of cupric ion naturally present in seawater have been calculated to be high enough to cause a reduction in growth i n several species of phytoplankton (Sunda and G u i l l a r d , 1976; Anderson and Morel, 1978; LeBlanc, Chapter 2; Sunda, pers* comm*). The presence of natural dissolved organic compounds capable of complexing copper could be important i n c o n t r o l l i n g copper t o x i c i t y . The amount of copper present in the organically bound form varies in dif f e r e n t seawaters, but has been measured to be i n the range of 6% to 40% or more of the t o t a l copper present (Florence and Batley, 1977a; Mantoura et a l , 1978; Smith, 1976; Williams, 1969; Schmidt, 1978 a and b). Measurements of the complexing capacity of seawater as determined by s p e c i f i c ion electrodes (Williams and Baldwin, 1976) , anodic stripping voltammetry (Florence and Batley, 1977b) and b i o l o g i c a l assay 51 (Gillespie and Vaccaro, 1978) have shown that the complexing capacity i s greatly reduced when u l t r a v i o l e t i r r a d i a t i o n i s used to oxidize the dissolved organic compounds present i n the seawater. Fi e l d studies have found that i n some natural upwelling situations, where there are low l e v e l s of dissolved organic compounds in the seawater, phytoplankton growth w i l l i n i t i a l l y be depressed (Barber and Ryther, 1969). The growth rate can be greatly increased during t h i s i n i t i a l period by the addition of either EDTA, an a r t i f i c i a l chelator, or a homogenized zooplankton extract. Barber and Ryther (1969) have suggested that natural organic chelators, released by organisms as the water ages at the surface, may be partly responsible for the increased phytoplankton growth away from the upwelling region., Phytoplankton are generally considered to be the main source of the dissolved organic matter in the oceans (Anderson and Zeutschel, 1970; Thomas, 1971) and i t i s of i n t e r e s t to determine i f phytoplankton species are capable of d i r e c t l y producing compounds capable of affecting trace metal a v a i l a b i l i t y , p a r t i c u l a r i l y the copper speciation.. The possible production and release of copper complexing compounds i s also of importance:to work on t o x i c i t y bioassays designed to aid in the study of copper speciation i n the oceans., It i s necessary that any bioassay species not be capable of changing the complexing capacity of the medium* Changes which reduced the cupric ion a c t i v i t y , and therefore the copper t o x i c i t y , would invalidate the assay. I t has been suggested that there are at least two d i f f e r e n t 52 types of phyotoplankton response to copper t o x i c i t y (Jackson and Morgan, 1978)* The f i r s t type of response i s a decrease in the growth rate with increasing copper concentrations, a depression which does not change markedly with time* The second type of response i s a lag phase i n growth which increases with increasing copper concentration* followed by an exponential growth at a rate i n d i f f e r e n t to the i n i t i a l copper concentration; This type of lag phase ef f e c t has been reported for the green algae Ch l o r e l l a pyrenoidgsa (Steemann Nielsen and Wium-Andersen, 1970), the diatom Nitzschia palea (Steemann Nielsen and Wium-Andersen, 1971), several Chaetoceros species (Barber, 1973; Huntsman and Barber, 1975) and i n Skeletonema costatum (Morel et a l , 1978). I t i s believed that those phytoplankton species showing the second type of response could be excreting organic compounds which condition the medium by complexing the free cupric ions i n solution and therefore decrease the t o x i c i t y of the medium. Steemann Nielsen and Wium-Andersen (1971) have reported that both N. galea and S. costatum excrete organic matter i n the presence of copper.. Swallow et a l (1978), however, found that, of eight marine and fresh water phytoplankton species tested for the i r a b i l i t y to excrete organic substances capable of complexing i o n i c copper, only one species was capable of affecting the free ion concentration. It i s also worth noting that the eight species were selected for th e i r known a b i l i t y to release large quantities of organic materials* Further work by one of the authors (McKnight, 1978) using a more sensitive methodology, showed that of 14 species tested, 9 were capable of 53 producing copper complexing agents. Huntsman and Barber (1975) have found that a medium conditioned by the growth of phytoplankton could contain both i n h i b i t i n g compounds which affected the growth rate of the phytoplankton, and stimulating compounds which affected the length of the i n i t i a l lag phase. Gnassia-Barelli et a l (1978) have found that media conditioned by the growth of six d i f f e r e n t phytoplankton species could decrease the t o x i c i t y of copper to a Haptophycean bioassay species, Criegsehaera elonqata. This study w i l l attempt to determine i f the marine diatom, Nitzschia longissima, i s capable of producing compounds which can a f f e c t the t o x i c i t y of copper i n sea water* Possible effects on both the i n i t i a l lag phase and growth rate were investigated in t h i s study* 54 Method The bioassay procedure was the same as that in chapter 1. Nitzschia lqnqissima was chosen as a bioassay organism oa the basis of i t s s e n s i t i v i t y , consistency of response to copper and ease of handling* Seawater for the experiments was collected in Jan..and March, 1978 from Geo 1748. The water was enriched with N03, PO^, Si0 3 and vitamins as i n Chapter 1, Table 1^2. No a r t i f i c i a l or natural complexing agents were added to the medium* The s a l i n i t y of both of the seawater samples used i n the experiments was 31.1%>; the dissolved organic carbon concentration was 0.9 mg 1 _ 1 ; and the dissolved copper concentration was 1.1x10 - 8 M. Conditioning of Media To determine i f the bioassay species was i n any way conditioning i t s medium, N. Longissima was grown for three days in the standard (Chap. 1) medium + 1.6x10 - a M Cu.. The copper was added to stimulate any possible production of copper detoxifying compounds, but not to cause a major i n h i b i t i o n in growth. at the end of the three day conditioning perioa, the c e l l s were removed by f i l t r a t i o n (0.45 um c e l l u l o s e acetate f i l t e r ) and the f i l t r a t e (the 'conditioned* medium) was then used to star t another bioassay. More copper was added to give t o t a l added copper concentrations of 1,6, 3.2 and 7*9x10 - 8 M. At the same time more experimental medium was prepared using fresh or 'unconditioned* seawater, with added copper concentrations of 0, 1.6, 3.2 and 7.9x10~8 M* an inoculum of N. longissima was added and the bioassays were monitored for the normal four day period, and growth rates determined by the usual 5 5 methods (see chapter 1). Effect of C e l l Inoculum Size The bioassay procedures were as i n Chapter 1 with the exceptions that the bioassays were monitored for eight days, not four, and the i n i t i a l c e l l inoculum sizes were, approximately 1000, 100 and 10 c e l l s ml-. Because of d i f f i c u l t i e s i n obtaining accurate c e l l counts at the lower concentrations, the resu l t of the bioassay was followed using fluorescence readings rather than the c e l l numbers., Fluorescence readings were made using a Turner Model 111 Fluorometer* The s t a r t i n g fluorescence readings were 10, 1 and 0*1 units*. The l a s t figure (0.1) i s estimated rather than measured because i t was below the detection l i m i t s of the fluorometer* Growth rates were determined as in Chapter 1, from the change in l o g l 0 fluorescence with time for the exponential portion of the growth curve and the intercepts with the i n i t i a l fluorescence were determined from the eguation of the regression l i n e . The intercepts gave the length of the i n i t i a l lag period of the culture before the s t a r t of exponential growth.. 56 Results Conditioning experiment Two bioassays were carried out to determine whether the bioassay species, N. Longissima, could change i t s medium and s i g n i f i c a n t l y a f f e c t i t s response to added copper. Such changes or 'conditioning' of the medium would presumably be due to excretion of organic compounds, which through complexation could reduce the concentration of the most toxic species of copper* Bioassays using fresh medium and the conditioned medium were run together and the r e s u l t s for c e l l and fluorescence;growth rates are presented i n Figs. 3.1 and 3.2, respectively. There i s no control culture (0 added copper) for the conditioned medium because i t had already had 1.6x10 - 8 N of copper added during the conditioning period to stimulate the excretion of copper complexing organic compounds. There i s no s i g n i f i c a n t difference between either the conditioned or the unconditioned medium, in d i c a t i n g that during exponential growth Nitzschia longissima does not noticeably change or condition i t s medium to reduce copper t o x i c i t y . Lag time experiment A second experiment was carried out to determine whether N. longissima could condition i t s medium. If the c e l l s in the experimental cultures must i n some way 'condition* the medium through the excretion of organic compounds before they can begin exponential growth, the length of the i n i t i a l lag time should be proportional to the concentration of the toxic factor which must be overcome by the 'conditioning', i . e . copper.. The lag phase should also be proportional to the i n i t i a l c e l l concentration. This second experiment used v a r y i n g copper and i n i t i a l inoculum s i z e s t o determine whether N* 1on^issima-was modifying the t o x i c e f f e c t of the added copper. F i g s . . 3.3 and 3.4 show the r e s u l t s of the experiment* N e i t h e r copper c o n c e n t r a t i o n o r i n i t i a l c e l l c o n c e n t r a t i o n a f f e c t s the l e n g t h of the i n i t i a l l a g phase and th e r e i s no s i g n i f i c a n t d i f f e r e n c e i n the e f f e c t copper has on growth r a t e at the t h r e e inoculum s i z e s . _ 5 8 Figs. 3.1 and 3.2: The e f f e c t of copper on c e l l and fluorescence growth rates (in doublings per day) i n unconditioned (•) and conditioned ( o ) media._ 9 5 % confidence l i m i t s are.given for each point. A d d e d 4 8 C o p p e r ( x 1 0 ~ 8 W, F i g . 3 -1 A d d e d C o p p e r ( x 1 0 u f s / F i g . 3 - 2 60 Figs. 3.3 and 3.4: The ef f e c t of rate and the lag time before the three i n i t i a l inoculum sizes. 95 for the growth rates. copper on fluorescence growth st a r t of exponential growth at % confidence l i m i t s are given 0 4 8 A d d e d C o p p e r ( X 1 0 " 8 M ) F ig . 3 - 4 62 Discussion The occurrence of copper complexing compounds has been shown to be of importance in c o n t r o l l i n g phytoplankton growth i n some areas (Barber and Ryther, 1969; Smayda, 1974). The source of those organic compounds responsible for copper complexation in seawater must be known before meaningful predictions can be made as to the expected copper complexing capacity of seawater at d i f f e r e n t times or i n d i f f e r e n t areas. A knowledge of the sources of the compounds w i l l also aid in the i d e n t i f i c a t i o n of the structures of the compounds. If a c t i v e l y growing phytoplankton c e l l s are the source, as has been suggested by Jackson and Morgan (1978), then the possible i n h i b i t o r y e f f e c t s of copper in seawater w i l l be less important because the phytoplankton species are themselves capable of overcoming i t . If the a b i l i t y to produce copper complexing agents i s species s p e c i f i c (McKnight, 1978; Swallow et a l , 1978) i t might indicate a competitive advantage f o r some species i n situations where copper might be inhibitory* If the copper complexing compounds are the result of the degradation of dead phytoplankton c e l l s , then the phytoplankton would not be able to e f f e c t i v e l y condition t h e i r medium, or at least such conditioning must occur at a much slower pace than i f the c e l l s were capable of ac t i v e l y excreting such compounds*. The fact that t h i s study has found Nitzschia longissima to be incapable of conditioning i t s medium under the conditions tested, does not indicate that phytoplankton i n general lack such a capacity. The studies of McKnight (1978), Huntsman and Barber (1975) and Gnassia-Barelli et a l (1978) indicate that some p h y t o p l a n t k o n s p e c i e s a r e c a p a b l e o f r e d u c i n g the t o x i c e f f e c t of copper through a c o n d i t i o n i n g of t h e i r medium. N i t z s c h i a - l o n g i s s i m a has been shown t o be e x c e p t i o n a l l y s e n s i t i v e t o copper (Chapters 1 and 2) which might be t a k e n as an i n d i c a t i o n of an i n a b l i l i t y t o reduce the t o x i c e f f e c t o f copper* Lack o f s e n s i t i v i t y , however, cannot be t a k e n as evi d e n c e of an a b i l i t y t o c o n d i t i o n the growth medium. Skeletonema constatum has been shown t o be very i n s e n s i t i v e t o copper (Chapter 1; Mor e l e t a l , 1978) but has been found not t o c o n d i t i o n i t s medium i n response t o copper i n h i b i t i o n , so t h a t an a b i l i t y t o c o n d i t i o n media cannot be the s o l e f a c t o r c o n t r o l l i n g p h y t o p l a n k t o n s e n s i t i v i t y t o copper. _ CHAPTER 4 Physiological Effects Of Copper Toxicity In The Marine Diatom, Nitzschia longissima Introduction The effect of heavy metals on phytoplankton growth i s not well understood. Different experiments have found widely d i f f e r i n g values for heavy metal t o x i c i t y and i t has only recently been recognized that both the t o t a l amount and the chemical speciation of the metals are important i n determining t h e i r e f f e c t on phytoplankton* Copper has been found to be one of the most important and most int e r e s t i n g of the heavy metals (Hollibaugh et a l , i n prep..b) and i s both a micronutrient necessary for phytoplankton growth (Manahan and Smith, 1973) and a toxic agent which can cause reduced phytoplankton growth at extremely low concentrations (Steeman Nielsen and Wium-Andersen, 1970; Davey et a l , 1 973),. An area of increasing i n t e r e s t i s the mechanism of copper t o x i c i t y i n marine phytoplankton* Phytoplankton species vary widely in t h e i r s e n s i t i v i t y to copper and the reasons for t h i s variation are not at present known. Aside: from species differences, i t i s also possible that physiological differences can cause variations i n the e f f e c t copper w i l l have on a particular species*. The ef f e c t of copper on c e l l growth rates has been the most often used indicator of t o x i c i t y , and has been found to be, i n at least some cases, the most sensitive indicator of copper t o x i c i t y (Berland et a l , 1977). There have been only a few studies of other physiological systems that are affected* Anderson and Morel (1978) found the motility of a 65 marine d i n o f l a g e l l a t e to be an extremely sen s i t i v e and f a s t indicator of copper t o x i c i t y * Uptake of l 4 C has been used i n several studies (Anderson and Morel, 1978; Berland et a l , 1977) as has the rate of chlorophyll 'a' (or fluorescence) increase ( S a i f a l l a h , 1977), chlorophyll »a' concentration per c e l l (Morel et a l , 1978), maximum y i e l d growth, mean c e l l volume and shape, part i c u l a t e carbon and nitrogen (Berland et a l , 1977), oxygen evolution (Overnell, 1976), ni t r a t e uptake, n i t r a t e reductase synthesis (or ac t i v i t y ) (Harrison and Davies, 1977) and copper concentration per c e l l (Sunda and G u i l l a r d , 1976). Studies of the various e f f e c t s of copper t o x i c i t y w i l l aid i n determining the mechanism (s) of the to x i c action of copper, which may lead to an explanation f o r the varying s e n s i t i v i t i e s of d i f f e r e n t species. This study w i l l look at some of the physiological e f f e c t s of copper t o x i c i t y i n the bioassay species, Nitzschia longissima. In p a r t i c u l a r , mention w i l l be made of effects on the rate of c e l l d i v i s i o n , the rate of increase of fluorescence, c e l l volume, fluorescence/cell, **C uptake and the a c t i v i t y of the enzyme ni t r a t e reductase. 66 Method The marine diatom, Nitzschia longissima, was used as a bioassay species because of i t s previously demonstrated s e n s i t i v i t y to copper (see Chapter 1).. . A unialgal culture of the species was obtained from the Northeast P a c i f i c Culture Col l e c t i o n * . Seawater for the bioassay experiments was c o l l e c t e d in December, 1977 and March, 1978, and treated as i n Chapter 1. The seawater was enriched with n i t r a t e , phosphate, s i l i c a t e and vitamins (see Chapter 1, Table 1*2) at leve l s high enough to provide non l i m i t i n g amounts of the major nutrients but not to greatly exceed the nutrient conditions that marine phytoplankton encounter i n the oceans. Copper was added from a 23.6x10~6 M copper stock solution made with CuCl^ and double d i s t i l l e d deionized water* A l l of the copper l e v e l s referred to in the figures are added copper concentrations, not t o t a l copper or i o n i c copper concentrations._ Other conditions for the medium preparation are as described in Chapter 1. The bioassays were run for 4 days with samples being taken dai l y for c e l l counts (using Palmer-Maloney counting chambers), fluorescence readings (using a Turner Model 111 Fluorometer). C e l l sizes were measured for the t h i r d day samples* As i n Chapter 1, s p e c i f i c growth rates were determined by a least squares l i n e a r regression of the log >o c e l l concentration or fluorescence vs* time for the exponential portion of the growth curve. These s p e c i f i c growth rates were changed to doubling per day by an arithmetic conversion (Guillard, 1973).. C e l l sizes were determined by visual measurement of at least 30 c e l l s at each test condition* 67 Enzyme A c t i v i t y Measurements M a t e r i a l f o r the enzyme.assays was obtained by f i l t e r i n g the bioassay c u l t u r e s a t the end of the 4 day bi o a s s a y s . N i t r a t e reductase was assayed by measuring the NADH dependent formation of n i t r i t e as d e s c r i b e d i n Eppley e t a l (1969) with the m o d i f i c a t i o n t h a t the KN0 3, MgSO^ and NADH c o n c e n t r a t i o n s were as de s c r i b e d i n H a r r i s o n (1973) and the enzyme e x t r a c t s were incubated f o r 45 minutes. Two r e p l i c a t e enzyme assays were run f o r each sample* then averaged t o give the a c t i v i t y of the sample* Two separate t e s t c u l t u r e s were run f o r each t e s t c o n d i t i o n , making at l e a s t four enzyme assays of each copper c o n c e n t r a t i o n ( 0, 1.6, 3.2 and 7.9x10 - 8 M Cu)._ In a d d i t i o n , the e n t i r e experiment was run twice to co n f i r m the r e s u l t s . In the f i r s t assay, the t e s t e d copper c o n c e n t r a t i o n s were 0, 1.6 and 3.15x10-8 M Cu, w h i l e . i n the second 0, 1.6 and 7.9x10~ 8 M Cu were t e s t e d (due t o an experimental e r r o r the 3 . 1 5 x 1 0 - 8 M CU r e s u l t s were i n v a l i d i n the: second experiment). For the 7 . 9 x 1 0 - 8 M Cu sample, the two r e p l i c a t e f l a s k s had to be pooled to give enough c e l l s f o r the assay. The a c t i v i t y measurement t h e r e f o r e i s based o n l y on 2 enzyme assay r e p l i c a t e s from one pooled sample. i* C Uptake Measurements Medium was prepared as f o r the growth experiments d e s c r i b e d i n Chapter 1. An inoculum from an e x p o n e n t i a l l y growing c u l t u r e was added to give an i n i t i a l c e l l c o n c e n t r a t i o n of 2000 c e l l s ml-*. The c u l t u r e s were incubated with the added copper f o r one hour^ then t r a n s f e r r e d t o three 125 ml BOD b o t t l e s , two l i g h t and one dark.. 0.5 mis of a **C l a b e l e d NaHCOs s o l u t i o n (1.25 68 uCi) was added and then the bottles were incubated at 15 C and a l i g h t i n t e n s i t y of 80 uE m~2 s e c - 1 for two hours; The cultures were f i l t e r e d and the f i l t e r placed in 15 mis of ScintiVerse s c i n t i l l a t i o n f l u i d i n a v i a l . A c t i v i t y i n the v i a l s was determined on a Dnilux III Liquid S c i n t i l l a t i o n Counter. Photosynthetic rates were calculated as described i n Strickland and Parsons (1972). 69 Eesults Growth Eate Growth rate i s severely affected by the addition of low concentrations of copper* i s can be seen in Chapter 1, Figs. 1.3, 1.4 and 1.12., the addition of 1.6 or 3.2x10 - 8 M Cu causes a s i g n i f i c a n t depression i n the rate of increase of both c e l l numbers and fluorescence. I t i s in t e r e s t i n g to note that fluorescence increase i s consistently less affected than the c e l l number increase. Further investigation of the relationship between c e l l numbers, fluorescence and added copper, indicate differences i n the increase of c e l l number and fluorescence, with the differences increasing with increasing copper concentration. Fluorescence per c e l l showed an increase: both with increasing copper concentration and with increasing time of exposure!. Figs. 4.1 to 4*4 show the e f f e c t of added copper (0, 1.6, 3.2 and 7.9x10 - 8 M Cu) and exposure time (0 to 4 days) on the fluorescence/cell r a t i o * The control (0 added copper) remained r e l a t i v e l y stable over the course of the four day bioassay, but the three added copper conditions showed a gradual increase to over double the control value i n the case of the fourth day 7.9x10~8 M Cu treatment. The 1.6x10~8 M Cu condition showed a decrease i n fluorescence/cell a f t e r the t h i r d day, indicating a possible adaptation on the part of the c e l l s to the added copper. The change could also be attributed to a change in the t o x i c i t y of the medium, but other experiments (see chapter 3) have found no decrease i n the t o x i c i t y of the medium after three days growth 70 of phytoplankton c e l l s in i t . The 3.15x10-8 M Cu additions shows no increase i n fluorescence/cell between the t h i r d and fourth days, also indicating a possible adaptation on the part of the c e l l s . There i s no such change at 7.9x10 _ 8 M CU, but i t i s possible such a change could have been observed i f the assay were followed f o r a longer period of time. Although i t would at f i r s t appear that fluorescence i s being greatly affected by copper, when the fluorescence/cell i s converted to fluoresence/cell volume (see Fig., 4.5), there i s no increase,. In f a c t , there i s a slight,; though not s t a t i s t i c a l l y s i g n i f i c a n t decrease with increasing copper concentration* When examined i n l i g h t of the e f f e c t of copper on c e l l numbers i t i s apparent that the toxic action of the copper i s on c e l l d i v i s i o n and not the production within the c e l l of those chlorophyll pigments responsible for fluorescence. Enzyme Assay The changes i n apparent n i t r a t e reductase a c t i v i t y with added copper concentration i s shown in Fig. 4.6 and Table 4.1. Nitrate reductase a c t i v i t y appears to be very sensitive to added copper, with the smallest copper addition, 1.6x10 - 8 M CU, causing a 500% increase i n the apparent enzyme a c t i v i t y , and the a c t i v i t y at the highest copper concentration, 7.9x10 - 8 M CU, being an order of magnitude greater than the c o n t r o l . i*C Uptake The e f f e c t of added copper on the rate of photosynthetic **C assimilation i s given i n Table 4.2. The photosynthetic rate per c e l l shows a small decrease with added copper, but the e f f e c t i s not as great as t h a t shown by growth r a t e . Table 4.1: The e f f e c t of added copper on the a c t i v i t y of the enzyme n i t r a t e reductase i n the marine diatom, N i t z s c h i a l o n g i s s i m a . Added Cu C e l l Cone. _ Enz. Act. X 1 0 - 8 M ( X 1 0 + 6 i - i ) (un i - i h - i ) 0 247 1.07 0 46.6 .246 0 41.2 .166 0 49.6 .315 avg. 1 1.6 23.2 .525 1. 6 26.9 .512 1.6 19.3 .501 1.6 18.9 .505 1.6 20.5 .499 avg* 3.2 8.33 .289 3.2 7.94 .185 avg. 7. 9 2.34 *094 Act. per C e l l (uM h - 1 10« c e l l s ) 4.33 5. 28 4 . 0 4 6.35 22.7 19.5 2 5.9 26.6 24.3 23.80±2;8 4 34. 2 24.6 29.4±6.79 40.3 1 ± one standard d e v i a t i o n T a b l e 4.2: The e f f e c t o f ad d e d c o p p e r on t h e r a t e o f p h o t o s y n t h e s i s Added Cu (X10 - 8 M) 0 1.6 3. 2 7.9 P h o t o s y n t h e s i s (mgC m - 3 h _ 1 ) 3. 103 2.520 2.223 1.917 % o f C o n t r o l 100 81.2 71,. 6 61,. 8 74 F i g s , 4.1 to 4.4: Changes i n f l u o r e s c e n c e / c e l l of the diatom, N i t z s c h i a l o n g i s s i m a , during the f o u r day bioassay at four added copper c o n c e n t r a t i o n s * . E r r o r bars represent ± one standard d e v i a t i o n * _ 10 | I to u Ll 5 l 1 0 O C u 1.6 C u 2 D a y s F i g . 4 - 2 7.9 Cu 2 D a y s F i g . 4 - 4 76 Fig., 4 . 5 : E f f e c t of added copper on fluorescence/cell (o) and fluorescence/cell volume (•) on the t h i r d day of bioassay. Error bars represent ± one standard deviation. 78 F i g 4.6: E f f e c t of added copper on the apparent a c t i v i t y of the enzyme n i t r a t e r eductase. Each p o i n t i s the average of two r e p l i c a t e s . 4 0 30 20 10 Ol 0 8 r-8 Added C o p p e r (x10 ° M ) Fig. 4 - 6 80 Discussion Studies of the d i f f e r i n g i n h i b i t o r y e f f e c t s of copper on phytoplankton species can help i n determining both the reasons for varying phytoplankton s e n s i t i v i t i e s to copper i n h i b i t i o n and the actual mechanism of the toxic action of copper on phytoplankton i n general. The rate of c e l l d i v i s i o n i s one of the most often used indicators of the e f f e c t of copper on phytoplankton species. I t has been found i n at least some cases (Berland et a l , 1977) to be the.most sensitive indicator of copper t o x i c i t y and in the present study was found to be more sensitive to added copper (ie; to be affected at lower copper concentrations) than the rate of increase of fluorescence or the rate of **C uptake. Several other observations provide evidence that the i n h i b i t i o n of c e l l d i v i s i o n i s one of the primary effects of excess copper levels in t h i s species; Fluorescence per c e l l , an indicator of the concentration of photosynthetic pigments in the c e l l , was observed to increase with increasing copper concentration, as did c e l l volume. A comparison of the fluorescence per c e l l volume showed l i t t l e difference between the d i f f e r e n t copper concentrations, i n d i c a t i n g l i t t l e e f f e c t by the copper on the production within the c e l l of those photosynthetic compounds responsible for fluorescence. Rosko and Rachlin (1977) have observed in copper stressed C h l p r e l l a , that c e l l d i v i s i o n did not keep pace with chlorophyll 'a 1 production, i n d i c a t i n g that copper exserted i t s primary ef f e c t on c e l l d i v i s i o n and a much less harmful e f f e c t on chlorophyll 'a' synthesis. An increase in c e l l volume with increasing copper concentration as found i n 81 t h i s study has a l s o been found i n a number of other s t u d i e s (E r i c k s o n et a l , 1970; Steemann N i e l s e n and Ramp-Nielsen* 1970; Eosko and R a c h l i n , 1977; H o r e l et a l , 1978) and other s p e c i e s ( T h a l a s s i o s i r a pseudonana, Chlore11a E i r e n o i d o s a , C h l o r e l l a v u l g a r i s and Skeletonema costatum, r e s p e c t i v e l y ) , Saboski (1977) found t h a t low l e v e l s of the t r a c e metals mercury or t i n c o u l d cause abnormally l a r g e c e l l s i n the diatom N i t z s c h i a l i e b e t h r u t t i . Morel e t a l (1978), however, found t h a t although copper d i d cause an i n c r e a s e i n c e l l volume, i t a l s o caused a systematic decrease i n the c h l o r o p h y l l • a 1 c o n c e n t r a t i o n per c e l l i n Skeletonema costatum and Gross e t a l (1970) found copper to cause a r e d u c t i o n i n the p h o t o s y n t h e t i c pigment c o n c e n t r a t i o n i n n o n - d i v i d i n g Chlore11a c e l l s . A change i n c e l l volume i s not n e c e s s a r i l y an i n d i c a t i o n of an i n h i b i t i o n of c e l l d i v i s i o n . R i i s g a r d (1979) has found that the e u r y h a l i n e marine f l a g e l l a t e , D u n a l i e l l a marina, i s unable to r e g u l a t e volume at e l e v a t e d copper c o n c e n t r a t i o n s , probably due to changes i n c e l l membrane p e r m e a b i l i t y * The i n c r e a s e i n f l u o r e s c e n c e / c e l l along with the i n c r e a s e i n c e l l volume observed i n t h i s study would i n d i c a t e , however, t h a t the c e l l volume i n c r e a s e was not due to a d i l u t i o n of the c e l l contents but t o an i n h i b i t i o n of c e l l d i v i s i o n * N i t r a t e r eductase, an important enzyme i n t h e . c o n v e r s i o n o f N03" to NH^1" (Falkowski, 1975), was more s t r o n g l y a f f e c t e d by copper than growth r a t e , but the e f f e c t was an i n c r e a s e i n a c t i v i t y , not an i n h i b i t i o n * T h i s i n c r e a s e cannot be e x p l a i n e d s o l e l y by the i n c r e a s e i n c e l l volume. The mechanism of copper's i n f l u e n c e on the enzyme a c t i v i t y i s not c l e a r but r a p i d and l a r g e i n c r e a s e s i n n i t r a t e reductase a c t i v i t y i n 82 phytoplankton have been reported i n response to increasing NO^  li m i t a t i o n (Eppley and Renger, 1974; Harrison, 1973)* It i s possible copper could be aff e c t i n g the uptake of N03~ into the c e l l , thereby decreasing the apparent concentration of N03~ to the c e l l , which could lead to an increase in n i t r a t e reductase a c t i v i t y . Harrison et a l (1977) have observed a large decrease in NO^ " assimilation on copper treated phytoplankton populations. Several f i e l d and lab studies, however, have found n i t r a t e reductase a c t i v i t y to vary d i r e c t l y with the N03~" concentration in the media (Eppley et a l , 1969; Eppley et a l , 1970).. Although no conclusions can be drawn as to the cause of copper's stimulation of nitrat e reductase a c t i v i t y , the magnitude of the effect makes i t an interesting area for further study. In a study of the effect of heavy metals on enzyme a c t i v i t y i n Selene cucubalus (Mathys, 1975), i t was found that n i t r a t e reductase was an order of magnitude more sensative to copper than glucose 6 phosphate dehydrogenase, and several orders of magnitude more sensative than malate dehydrogenase and i s o c i t r a t e dehydrogenase. It should be noted that the e f f e c t observed by Mathys (1975) was a decrease i n a c t i v i t y , not an increase as found here* No attempt has been made here to determine the exact mechanism of copper t o x i c i t y on marine phytoplankton.. Some observations have been made on which systems are most l i k e l y d i r e c t l y affected by the toxic action, namely c e l l d i v i s i o n and nit r a t e reducatase a c t i v i t y or N03~ uptake, and i n d i c a t i n g possible areas for further physiological studies* A further point might be mentioned concerning e c o l o g i c a l 83 and physiological studies of trace metal e f f e c t s . To avoid either underestimating or overestimating the possible effect of copper on natural systems, i t would appear best not to u t i l i z e only one parameter of e f f e c t , but determine the effects on several physiological systems. 84 General Discussion Studies of the e f f e c t of trace metals on phytoplankton can be an important aid i n determining the importance of natural levels of trace metals i n the c o n t r o l l i n g of phytoplankton growth and community structure, as well as helping i n the prediction of the possible effects of man-made pollution i n the marine environment; The studies in Chapter 1 have indicated that phytoplankton species can vary widely in t h e i r response to low levels of copper, in d i c a t i n g that the presence of i n h i b i t i n g l e v e l s of copper, either at natural levels or at elevated l e v e l s , through p o l l u t i o n , could be an important factor i n c o n t r o l l i n g the species composition of the. phytoplankton community. This supports the findings of several f i e l d studies which have reported the addition of copper to natural phytoplankton to cause large changes in the species composition of the population (Thomas et a l , 1977; Ibragim and Patin, 1976). Equilibrium calculations, bioassays and f i e l d studies have indicated that the levels of copper naturally present i n seawater could be i n h i b i t i n g in some situations (Sunda and Gu i l l a r d , 1976; Anderson and Morel, 1978; Barber and Ryther, 1969; Jackson and Morgan, 1978). Measurement of the amount of •available' copper i n seawater in not possible, at present, with chemical or physical methods, but through the use of sensitive bioassay species, an in d i c a t i o n of the state of copper a v a i l a b i l i t y can be found. This study has developed a sensitive and r e l a t i v e l y rapid b i o l o g i c a l assay for copper a c t i v i t y i n seawater using the marine diatom, Nitzschia longissima. This species was found to be stable i n i t s response to copper and 85 amenable to handling i n laboratory conditions._ The s e n s i t i v i t y of the species was compared with 12 other marine diatom and din o f l a g e l l a t e species, and Nitzschia longissima was found to be r e l a t i v e l y sensitive to copper but was by no means unusual in i t s response (Chapter 1), with several of the tested species, the Chaetoceros species i n p a r t i c u l a r , being affected more. In a further study using copper buffered media and a computer equilibrium model (MINEQL), t h i s species was compared with four other species tested under si m i l a r conditions (Sunda and G u i l l a r d , 1976; Anderson and Morel, 1978; Morel et a l , 1978; Reuter et a l , 1979) and was found to be affected at a much lower calculated cupric ion concentration. Another area of i n t e r e s t , both from an environmental standpoint and i n terms of using t h i s species as a bioassay species, was whether Nitzschia longissima was capable of al t e r i n g i t s medium to reduce the toxic e f f e c t of copper. The source . and concentration of the organic compounds i n the ocean which could complex and detoxify trace metals i s unknown, but could be important i t determining the concentration of •available 1 or toxic copper species. Nitzschia longissima was found not to a l t e r i t s medium, ind i c a t i n g that atleast some species are incapable of releasing organic chelators to reduce copper t o x i c i t y . Observation of the physiological parameters affected by copper (Chapter 4) indicate that c e l l d i v i s i o n i s affected before or more strongly than carbon uptake or the production of photosynthetic pigments* The a c t i v i t y of the enzyme n i t r a t e reductase i s strongly affected by copper, but the mechanism of 86 t h i s action i s unclear and represents an int e r e s t i n g area for further research. Nitzschia lpngissima has been shown to be an e f f e c t i v e bioassay species* I t i s very sensitive to copper but i s not overly sensative to laboratory handling conditions. Within the time period and the copper concentrations tested, N. !ong_issima does not change i t s * medium to reduce copper t o x i c i t y as some species have been found to do (Gnassio-Barelli et a l , 1979). 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