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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 C o p p e r On P h y t o p l a n k t o n  by  MICHAEL  JOSEPH XEBLANC  B. S c . , U n i v e r s i t y  o f Guelph,  1974  .THESIS SUBMITTED I N PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF  SCIENCE  in THE FACULTY OF GRADUATE STUDIES Department o f Z o o l o g y  He a c c e p t t h i s t h e s i s a s c o n f o r m i n g to the r e q u i r e d  THE UNIVERSITY  standards  OF B R I T I S H COLUMBIA  J u n e , 1979 (c) Michael Joseph LeBlanc,  1979  DE-6  In  presenting this  thesis in partial  an a d v a n c e d d e g r e e a t the L i b r a r y I further for  shall  the U n i v e r s i t y  make i t  agree that  freely  this  thesis for  It  Department  f i n a n c i a l gain shall  of  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  the requirements I agree  r e f e r e n c e and copying of  this  that  not  copying or  for  that  study. thesis  by t h e Head o f my D e p a r t m e n t  i s understood  permission.  of  B r i t i s h Columbia,  extensive  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  written  B P 75-51 1 E  of  available for  permission for  by h i s r e p r e s e n t a t i v e s . of  fulfilment  or  publication  be a l l o w e d w i t h o u t  my  ABSTRACT The s e n s i t i v i t i y to  copper was  respect  to  investigation lonqissima, activity. to than  investigated*  any  dinoflagellates  general or  using  a  No  it  to  between  different  single  sized  bioassay be  very  diatoms cells..  species,  photosynthetic  with  pigment  with and  Further Nitzschia  s e n s i t i v e to c u p r i c i o n  S e v e r a l p h y s i o l o g i c a l systems appear to degrees,  phytoplankton  s p e c i f i c trends were found  differences  between  indicate  different  activity  of s e v e r a l s p e c i e s of marine  be  affected  c e l l d i v i s i o n being more a f f e c t e d production  or  1 4  C  uptake.  The  of the enzyme n i t r a t e reductase i s g r e a t l y i n c r e a s e d by  the a d d i t i o n of low c o n c e n t r a t i o n s of  copper.  TABLE OF CONTENTS  Title  Page  Abstract  i  ......-...............................,,.,,..ii  Table Of Contents L i s t Of Tables  ....................................ii  ..................... ................ .v  L i s t Of F i g u r e s  ......................................vi  Acknowledgments  ......................................vi  General I n t r o d u c t i o n  Chapter 1 - A  .................................1  Comparison Of The E f f e c t s  Of Copper  On T h i r t e e n Species Of Marine Diatoms And Dinof l a g e l l a t e s  ................................. ..5  I n t r o d u c t i o n ............................... ...... 5 Method  11  E e s u l t s ........................ .................. 17 Discussion  .......................................31  Chapter 2 - The E f f e c t Of C u p r i c Ion A c t i v i t y On The Growth Rate Of The Marine Diatom, Nitzschia  l o n g i s s i m a ................. ..... ......... 35  I n t r o d u c t i o n ....................................... 35 Method .................................... .... ...... 38 R e s u l t s And D i s c u s s i o n  ...........................47  Chapter  3 - C o n d i t i o n i n g Of Growth  Method  By  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 0  Phytoplankton Introduction  Medium  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 0  ............................................ 54  R e s u l t s ............................ . . . . . . . . . . . . . .  . . . , . , , , . . , . , , . , , . . , . , . . . , , . . , , . , , , , . . , 6 2  Discussion  Chapter In  4 - Some F a c t o r s A f f e c t e d By The  Marine Diatom,  64  .......................................80  Discussion  Bibliography  lonaj^sima  . , , . . . 6 9  Besults  General  Toxicity  66  .  Discussion  Mtzsehia  Copper  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4  Introduction Method  56  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 7  L i s t Of Tables  Table 1.1: Species used i n the experiments Table 1.2: N u t r i e n t enrichments Table 1.3: E f f e c t  ± . . . . . . . . . . . . . 16  f o r n a t u r a l seawater ...,16  of copper on the growth r a t e o f  t h i r t e e n s p e c i e s o f phytoplankton  ................... 21  Table 2.1: Experimental medium .......................... 41 T a b l e 2.2: Computed chemical s p e c i a t i o n .................42 Table 2.3: Parameters and r e s u l t s of experiments Table 4.1: The e f f e c t of added copper of  ........ 43  on the a c t i v i t y  the enzyme n i t r a t e reductase ..............,,...,,72  Table 4*2: The e f f e c t o f added copper  of photosynthesis  on the r a t e  ...,..,.......^..........,..,......73  vi List  Figures  the  vs. Figures  1.20: The s u r f a c e  area  t o volume  amount o f c o p p e r n e c e s s a r y i n t h e growth  t o cause a  rate  .....,...,,,.,,...,,29  t h e growth r a t e .................................44-46 3.1 - 3.2: The e f f e c t  conditioned  o f c o p p e r on c e l l  growth r a t e s i n u n c o n d i t i o n e d media  3. 3 - 3.4: The e f f e c t  of e x p o n e n t i a l inoculum 4.1  o f c o p p e r on  and  growth  at three  fluorescence  the s t a r t  initial  s i z e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,60 - 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 ,  the  four  day b i o a s s a y  copper c o n c e n t r a t i o n s Figure  and  ...................................58  growth r a t e and t h e l a g t i m e b e f o r e  Figures  ratio vs.  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  fluorescence  Figures  rate vs.  o f added c o p p e r . . . . . . . . . . . . . . . . . . . . . . . 23-28  50% r e d u c t i o n Figures  Figures  1.1 - 1.19: R e l a t i v e growth  concentration Figure  Of  4.5: E f f e c t  at four  during  added  * . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74  o f added c o p p e r 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 Figure  4.6: E f f e c t  activity  o f added  76  c o p p e r on t h e a p p a r e n t  o f t h e enzyme n i t r a t e  reductase  ............78  vii Acknowledgments  I would l i k e to thank Dr. and  assistance  he  A.S<._ Lewis f o r the encouragment  has given me during t h i s study.  H a r r i s o n , K.J.F., H a l l and P.  Drs.  Dehnel have a l s o a s s i s t e d  me  P.J. in  my r e s e a r c h and the p r e p e r a t i o n o f t h i s t h e s i s . . I a l s o thank Dave Turpin f o r a i d i n the enzyme a n a l y s i s and Rosemary  Waters  for  phytoplankton s p e c i e s .  aid  and  advice  i n the c u l t u r i n g o f the  1 THE  EFFECT OF COPPER ON PHYTOPLANKTON General  Introduction  Recently there has been i n c r e a s i n g i n t e r e s t i n the of  trace  waters;.  metals on plankton s p e c i e s  effects  and communities i n n a t u r a l  I t has a l s o been i n c r e a s i n g l y r e c o g n i z e d t h a t i t can be  those f a c t o r s a f f e c t i n g t r a c e metal a v a i l a b i l i t y which are important, r a t h e r than the t o t a l metal c o n c e n t r a t i o n . (1963)  and  different  Johnston  sources  (1964) had  noted  that  differing  natural  most  Provasoli waters  capacities  to  from  support  phytoplankton growth a-nd t h a t these d i f f e r e n c e s c o u l d not always be  attributed  to  differences  nutrient concentration.. increased  by  the  metals*  salinity,  It  change  has  temperature  Moreover, growth r a t e  a d d i t i o n of a r t i f i c i a l  which were:assumed to trace  in  the  been  could  or n a t u r a l  biological  found  that  a d d i t i o n o f the a r t i f i c i a l  the  vitamins  or  amino  acids,  combinations  (Barber and Ryther,  that  those  adverse  EDTA  were  the  phytoplankton  1969),.  water  second  growth  most  important  i n the s u r f a c e  qualities  controlled  metal a v a i l a b i l i t y , a r e important many n a t u r a l  chelators, of  poor growth o f  be. overcome  by  nutrients,  either Smayda  singly (1974)  trace or i n found  water q u a l i t i e s overcome by t h e a d d i t i o n o f factors  influencing  waters of Narragansett Bay  over an annual c y c l e . . There i s strong those  be  c h e l a t o r , EDTA, or a zooplankton  homogenate, but not by the a d d i t i o n of various mstals,  often  availability  phytoplankton i n r e c e n t l y upwelled water could the  or  evidence t h e r e f o r e ,  by to  c h e l a t o r s , namely phytoplankton  that trace  qrowth  in  waters*  Of those metals i n f l u e n c i n g phytoplankton growth i n n a t u r a l  2 waters, copper and Morgan  appears to be one  of the most important..  (1978) have i n d i c a t e d  growth  can  copper  ions  that the e f f e c t of c h e l a t o r s on  be best e x p l a i n e d as d e c r e a s i n g t h e . a v a i l a b i l i t y of rather  than  increasing  iron  decreasing n i c k e l or z i n c a v a i l a b i l i t y . . both  an  availability  1973), and as a p o s s i b l e i n h i b i t o r  production  of  copper l e v e l s i n the oceans range from < 1 i 0 x 1 0  M  i n c o a s t a l areas  Windom and  phytoplankton  has  at l e v e l s as low as  Mercury al^  (1976)  and  Hollibaugh  i s more t o x i c than copper  Braek  et  ten  all  of the t o x i c e f f e c t was  and  1;6x10  than  ( D v e r n e l l , 1976; in  H o l l i b a u g h et the  metals, H o l l i b a u g h et a l (Ln prep* . a) found that  levels  of  concentration, concentration Anderson pH,  copper but (Sunda  is  to  the and  and Morel, 1978)  and  treatments  the complexing  M  zinc,  marine levels almost  due to t h e c o p p e r present..  I t has only r e c e n t l y been recognized that the: t o x i c i t y low  -8  et a l (1976)  In a mixture of approximately environmental  of  7  a l (in prep, a) .  i n prep*, a) but i s c o n s i d e r a b l y l e s s common  environment.  open  (Davey et a l , 1973;  and wium-Andersan, 1971).,  Dvernell  the  up to 5.0x10-  found copper to be an order of magnitude more t o x i c as  Natural  Rica et a l , 1973)  have been shown t o be t o x i c to phytoplankton Nielsen  M in  - 8  Smith, 1979)  (Abdullah et a l , 1972;  a d d i t i o n s of copper to seawater  Steemann  (Manahan  (Steemann N i e l s e n and Wium-Andersen, 1970)..  (Boyle et a l , 1977;  or  Copper i s recognized as  e s s e n t i a l m i c r o n u t r i e n t , necessary f o r growth  and Smith,  ocean  Jackson  relate!  not  to  "biologically Guillard,  1976;  the: t o t a l available"  of  metal copper  Lewis et a l , 1972;  whirh i s a f f e c t e d by such c a p a c i t y of the seawater..  factors  as  Experimental  which remove or destroy the n a t u r a l o r g a n i c compounds  3  in  seawater,  i.e. ultrafiltration  l i g h t treatment  will  usually  or  cause  a  l a r g e : decrease  phytoplankton growth, a decrease which can be addition  of a r t i f i c i a l c h e l a t o r s  et a l , 1978;  Sunda and G u i l l a r i ,  measuring  concentration increasingly  in used  in  the  Gnassia-Barelli  chemical  or  of  method  uncomplexed  biological  studies  by  in  1976).,  the: a v a i l a b l e  seawater,  reversed  (Barber, 1973;  As there i s at present no p h y s i c a l or accurately  ultra-violet  assays  trace  copper  have  been  metal e f f e c t s . ,  choosing a bioassay s p e c i e s , i t ' s s s n s i t i v i t y  with  of  respect  When to  other s p e c i e s must be determined* In  addition  to v a r i a t i o n due to experimental d i f f e r e n c e s ,  there are major v a r i a t i o n s i n the response of d i f f e r e n t or  in  different  life  d i f f e r e n t metal l e v e l s 1977).  Such  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 t o (Erickson  diffarences  et  could  al, be  1970;  been  used  as  plankton communities  a  possible  Reeve  extremely  determining the s p e c i e s composition of n a t u r a l has  species  explanation  et  important  communities  al, in and  f o r succession i n  (Lewis , 1977),.  In t h i s study an attempt has been made; 1) to compare the e f f e c t of added copper i n seawater if  on  unchelated  natural  a wide range of phytoplankton s p e c i e s to determine  there are d i f f e r e n c e s i n t h e i r s s n s i t i v i t y t o copper  and  if  so, i f these d i f f e r e n c e s f o l l o w any general p a t t e r n s . 2)  to  select  further  tests  toxicity  and  one s e n s i t i v e s p e c i e s as a bioassay organism f o r to  determine  other  besides reduced growth  other  physiological rates.  factors effects  affecting  copper  of copper t o x i c i t y  4  3) t o use  the  bioassay  (Anderson  and  Morel,  cupric  ioa activities  species 1978)  to  and  copper  determine  buffered a t what  g r o w t h r a t e was affected.„  medium  calculated  5 CHAPTER 1 A Comparison Of The Marine  E f f e c t s Of Copper On T h i r t e e n Species Of Diatoms And  Dinoflagellates  Introducton Copper has been i n c r e a s i n g l y factor  affecting  populations. of copper  growth  as  an  and composition of  important  phytoplankton  The a d d i t i o n of approximately environmental  (1.6x10  significantly 1973;  the  recognized  M)  -8  reducing  Steeraann  have  been  growth  in  Nielsen  and  shown some  to  capable  been  demonstrated  available"  copper  c o n c e n t r a t i o a which i s t o x i c , and that  controlled  by such f a c t o r s as t o t a l capacity  Sunda and  have i n d i c a t e d that the presence can  (Barber and  Ryther,  Because t o x i c i t y to contained  1969;  many  of  "biologically  experimental  used  phytoplankton  experiments  standard  of a r t i f i c i a l  extract),  the chemical  c o n c e n t r a t i o n t o which exposed. erroneously  The  is and  1976;  or absence of n a t u r a l c h e l a t o r s  the. e a r l i e r  amounts  medium  it  1978).. F i e l d s t u d i e s  it  with  growth  growth  was  state  unknown, as was  the  phytoplankton  which  and n a t u r a l c h e l a t o r s  is  of  copper  media  very  difficult  the r e s u l t s of these d i f f e r e n t experiments..  these experiments,  has  c o n c e n t r a t i o n ^ pH  Lewis,  r  Smayda, 1974).  phytoplankton varying  the  It  (Sunda and G u i l l a r d ,  have important e f f e c t s on  (EDTA, T r i s and sediment compare  is  copper  of theiseawater  Anderson and Morel, 1978;  i n seawater  it  1971),. .  recently  complexing  of  species (Davey et a l  Wium-Anderson, that  be  levels  the  to  In many o f  copper  in  the  the a c t u a l c u p r i c i o n species  were  being  use of media with c h e l a t o r s has a l s o r e s u l t e d i n  high  estimates  of  the  copper  tolerence  of  6  phytoplankton. _ In  addition  different  stages  (Erickson  differences species used  et  could,  a  communities  (Lewis,  it  effects virtually  example, chelators, causing  E§eudonana Cu and  0.79x10-*  M Cu  than  cause  M Cu  Seibert,  T i 57x10-*  by  over  7  of  M Cu Cu  has  effect  of  Rystad, M Cu  or  different  the in  of  or  make For  natural copper  Thalassiosira  and  1.57x10-* M  Rystad,  et  al,  to i n h i b i t as  1976) 1976).  growth i n less  than  (Erickson  e t a l , 1976).  the e f f e c t s have  general groups. .  of copper  had  amount o f c o p p e r  species  or  1976), 7.87x10-* M  finding  studying  amount o f  reported  (Braek  been  differences  the  (Braek  phytoplankton  either  in  artificial  necessary  the  1977).  (Jensen  been  has  experiments.  growth  which have compared  species  toxic  added  Such  i n plankton  (Davey e t a l , 1 9 7 3 ) ,  M  1.57x10-*  i n determining  sensitivities  50%  this  species  have f o u n d the  1977)..  succession  o r two  no  3.93x10~  ( J e n s e n and  and  more  a  and  methodological  t h e amount o f c o p p e r  Those:studies  results  the  t o be  a l , 1970)  or  and  containing  Skeletgnema costatum  two  for  o n l y one  in  Similarily,  et  communities  Thomas and  use  1972),  metal  in determining  reduction  greater  1.57x10-7  species to d i f f e r e n t  important  50%  (Erickson,  different  extremely  1977;  media  in  to  major  al,  various experimenters  a  species or  i m p o s s i b l e t o compare d i f f e r e n t  in  be  Reeve: e t  explanation  of copper,  can  due  1970;  of n a t u r a l  Most e x p e r i m e n t s the  a particular  be  in results  there  of d i f f e r e n t  al,  possible  variations  techniques,  of  composition  as  large  i n the response  cycle  levels  the  experimental  differences life  to  on  conflicting  necessary  to  trends in  the  Braek  et  al  7 (1976) one  found  only a small d i f f e r e n c e between t h r e e diatoms  d i n o f l a g e l l a t e tested,  necessary  to  with  cause a 50%  the: amount  reduction  of  added  copper  i n growth r a t e ranging  1.18x10-* t o 3.94x10-* M Cu. . Bentley-Mowatt and  Reid  and  from  (1977), i n  t e s t i n g f i v e s p e c i e s of phytoplankton, found f o u r s p e c i e s to unaffected  by copper c o n c e n t r a t i o n s  medium which contained In  comparing  10~*  12  l e s s than 10-*  phytoplankton  Cu i n a medium c o n t a i n i n g 2.5 has  been  shown  but used a  M EDTA. species,  (1970) found most s p e c i e s to be i n h i b i t e d by  which  M,  mM  Tris,  an  Erickson  1.57  t o 7.87x10-* M  artificial  to d e t o x i f y copper  et a l  chelator  (Anderson and  Morel,  1978).. Ia f u r t h e r t e s t s of six s p e c i e s i n a medium without added  chelators,  s e n s i t i v e t o the inhibition  in  concentration limited  they  maintained  species  and  in exponential  copper c o n c e n t r a t i o n s Overnell  copper,  (1976),  with  at  of copper t e s t e d .  sensitivity  The  precision  were i n  phytoplankton  six  dinoflagellate  between s p e c i e s , with  3.15x10  - 7  M Cu t o t a l l y  of  species 5  7,.87x10 1^34  on  and  1i6x10~  8  -7  x 10~*  the:  rate found  M Cu f o r four  the other two  Saifullah  stopping  the.lowest rather  because the t e s t e d  of  relatively tolerant. species,  growth  because the c e l l s were not  metals  c e n t r i c diatoms, with  c e n t r i c diatom, being  and  was  of growth at l e v e l s of 2-7x10~  i n c l u d i n g two  difference  comparison  u s i n g a medium c o n t a i n i n g  evolution  three  Cu,  increments  several  complete M  -7  phase of growth and  of  inhibition  almost  7.87x10  compared the e f f e c t s of  any  found the phytoplankton t o be much more  added some  be  In a (1978)  M  M EDTA, of  0^  a  50%  species  s p e c i e s , one comparison found  M Cu having  no  Cu.  a of  little effect  growth i n a l l s p e c i e s . .  8 Mandelli EDTA,  (1969),  compared  phytoplankton diatom  the  dinoflagellates  being  i s  within  any  differences  3.9  three centric  similar  to  there  39x10  group.  i n sensitivities  inhibition  with  M of  pennate  the  at  three  ( 3 . 9 t o 8.7x10  on t h e r a n g e . o f Mandelli  M Cu)  -7  sensitivities  (1969)  found  dinoflagellates  difference  within  three dinoflagellate  t o copper  a n d one  growth  Cu,  species  - 6  M Cu)..  between  little  found  responses  M  - 7  4.68 x 1 0  nine  diatoms  and found  any agreement  particular  (1978)  on  much more s e n s i t i v e  comparatively  Saifullah  toxicity,  and  wide diatoms  each  group.  s p e c i e s t o show  as d i d Braek  et a l  very (1976)  three species of diatoms*. Erickson  species as  copper  (2.52 t o 3.94x10-6  the diatoms Nor  for  of  and t h r e e d i n o f l a g e l l a t e s ) of  but  a medium c o n t a i n i n g  effects  (including  concentrations  than  using  of diatoms  did  Jensen  experiments copper  t o range  and  using  however,  (Jackson  diatom  cupric  Field  Seibert  i n  population 7.87x10  - 7  containers.  followed  after M  They  Comparison  concentrations show  wide  some  over  two  to  found  also  or  of  of  differences orders  more,  toxicity  instead  indicated  sensitivity the change  the addition Cu)  f o r several  an o r d e r o f m a g n i t u d e  also  have  species  (1977)  sensitivities  of  total between  magnitude  1978) .  experiments  differences  found  (1976).  ion  species,  and Morgan,  over  Eystad  concentrations  different  (i.e..  (1970),  large  centric  L§Etocy_lindricus, C h a e t o c e r o s ,  copper*.  i n a natural  of various  several  that  to  some  levels  Thomas  and  and  phytoplankton  of  plastic  diatoms  interesting  copper  (0-  experimental dinoflagellates  Thalassiosira,  Peridinium  9 and  DiaoBhysis)  but  disappeared  and  7.87x10  I§¥i.£.i.§) u  of  very  from  t h e copper  became d o m i n a n t .  treated  diatoms  e t a l (1970) who found to  added  Patin  with n a t u r a l  (1976)  composition diatom,  found  o f the copper  Nitzschia,  (i.e*  is  copper  centrics  and  sensitivity  supported tested  In a s e r i e s  o f f i v e day  significant  changes  communities,  dominant  be  t h e one p e n n a t e s p e c i e s  p o p u l a t i o n s , Ibragim  becoming  by  to  phytoplankton  treated  1.6  N i t z s e h i a and  partially  2 of 3  copper)  (0.79,  enclosures  t o be c o m p a r a t i v e l y i n s e n s i t i v e *  experiments  (no added  This observation of the  v s * pennate  sensitive  tested  i n the controls  M Cu) , where p e n n a t e d i a t o m s  - 7  centrics  Erickson  were dominant  and  in  the  species  with  the  pennate  and t h e 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 *  I n a study  o f two  polluted  E i d e : and  Jensen  (1979)  fjords found  insensitive pseudonana extremely The results.. sensitive Seibert, et  using three bioassay species, the  to  Phaeodactylum  trace  was m o d e r a t e l y  Thalassiosira  Skeletonema  therefore,  shows  Some a u t h o r s  have f o u n d  dinoflagellates  to  copper  1977),  while  to  Seibert,  1977; I b r a g i m  difference  response  sensitive:and  while:  literature,  a l , 1976; E r i c k s o n  The  pollution,  t o be r e l a t i v e l y  costaturn was  sensitive.  diatoms  no  metal  - trieornutum  be  than  o t h e r s have  more s e n s i t i v e  of  contradictory to  be  more  1969; Thomas and difference  have: f o u n d  pennate diatoms  (Braek centric  (Thomas and  1976) w h i l e o t h e r s have  found  1969).  this  t o added c o p p e r  little  Some  than  and P a t i n ,  (Mandelli,  found  e t a l , 1970).  (Mandelli,  purpose  diatoms  somewhat  study  i s t o compare t h e growth  of a f a i r l y  wide range  rate  of phytoplankton  10  species  to  determine:  1)  i f  there  are  significant  2)  i f  there  are  any  i.e.  diatoms  genus v s . The of to  the  vs*  trends  of  total  the  comparison, conditions  centric  cupric  copper  use  of  all  vs.  sensitivity  i t  large  ion a c t i v i t i e s  different is  species  between  pennate  concentration  however, for  i n the  dinoflagellates,  genus o r use  differences  media. only tested.  of  cells  the  species,  v s i small  cells,  diatoms.  or  could  species,  concentrations eliminate  For  necessary  the: to  instead  variations purposes use  the  due  of  a  same  11 METHOD Eight  species  of  diatoms  and  five  species  d i n o f l a g e l l a t e s were compared i n t h i s study (see Table the  species  either  previous  bioassays the  used).  or  The  use  species  reported  abundance  1.1  in  the  literature  i n l o c a l waters.  in  December, 1977 (49«17.0« British  Columbia*  was  filter*  The  was  Culture  from a depth of 350  seawater  filtered  immediately  m at Geo  i n the S t r a i t was  of  collected  for  two  weeks i n May,  contamination,  on c o l l e c t i o n through a 0.45  sea  water  samples  organic carbon c o n c e n t r a t i o n  waters.  a 90 1  acid  um  washed  water  was  to c a r r y out a l l of the b i o a s s a y s .  d i s s o l v e d copper l e v e l was  March  1748  The December water was used over a p e r i o d  The s a l i n i t y of both the dissolved  in  Georgia,  with  The seawater was stored at 9°C i n a 200 1 barrel.  B.C..  collected  eight weeks i n December and January and the March  used  the  1978  bioassays  and l e u c i t e sampler to minimize metal  polyethylene of  and March,  the  N, 123048. 8' W) , a s t a t i o n  fibreglass and  for  copper  U n i a l g a l cultures of  C o l l e c t i o n , U n i v e r s i t y of B r i t i s h Columbia, Vancouver, seawater  for  were chosen on the b a s i s o f  13 s p e c i e s were obtained from the Northeast P a c i f i c  Natural  of  1.1x10  -8  was  31.1  (DOC)  was 0.9 mg 1  M in  the  %o , the _ 1  December  and and  DOC  and copper a n a l y s i s methods were as r e p o r t e d  at  the  by Cave (1 977) . . Bioassays  beginning  and  Jan* .experimental p e r i o d , with N i t z s c h i a costatum  and  Thalassipsira  i n d i c a t e d t h a t there was supporting  pseudonana  little  if  any  end  of  lonjjissima, (see change  the  Skeletonema  Figs.. in  Dec.-  1.1-1.6) ,  the  growth  c a p a c i t y of the s t o r e d seawater, or i n the e f f e c t of  12 the added  copper  over  Jan* experiment; stored  the  Dissoved  seawater a l s o  two  month  showed  no  Dec. -  change  and  dissolved  t o 1.10x10  -8  copper  M Cu. .  using the December water*. In the  experiment, C. c r i n i t u m was  species  the  s p e c i e s except Chaetoceros c r i n i t u m were t e s t e d i n the  f i r s t experiment i n Dec.-Jan., May  of  o r g a n i c " carbon measurements of the  showed a very s l i g h t decrease from 1,13 Ml  period  previously tested.  water, was  t e s t e d as w e l l as t h r e e  The May  of  the  experiment, using the March  c a r r i e d out to v e r i f y some of  the  Dec*-Jan*  results  and to add a t h i r d Chaetoceros s p e c i e s t o the comparison. Before  the s t a r t  of the b i o a s s a y s , the seawater medium  enriched with n i t r a t e , phosphate, Table  1.2).  These  additions  s i l i c a t e : and were  l i m i t i n g amounts of the  major  exceed  conditions  the  nutrient  encounter i n the oceans. volume of a 23. 6 x 1 0 double  distilled  -6  designed  nutrients  Copper  but  that  was  vitamins  as  not  to  After  the  appropriate  M Cu stock s o l u t i o n made up with CuCl^. and  deionized  the  greatly  phytoplankton  water..  All  of the:copper l e v e l s  r e f e r r e d t o i n the f i g u r e s are added copper not t o t a l copper.  (see  t o provide non-  marine  added  was  addition  of  the  copper,  or  ionic  the medium  was  allowed to e q u i l i b r a t e f o r a t l e a s t two hours before the a d d i t i o n of the c e l l inoculum* copper may artificial  Anderson  take s e v e r a l hours to come medium  and i t was  amount of time f o r the added natural  and Morel (1978) have found t h a t to  equilibrium  an  c o n s i d e r e d best t o allow an equal copper  to  equilibrate  with  the  seawater.  P r i o r to the s t a r t of the experiment, the u n i a l q a l were  with  grown  in  the  enriched seawater  (see t a b l e  1.2)  cultures for  4-12  13 days, depending on the growth r a t e of the p a r t i c u l a r s p e c i e s , t o p r e c o n d i t i o n the s p e c i e s t o the seawater and any  also  to  minimize  c a r r y over o f contaminants•, such as a r t i f i c i a l  chelators or  heavy metals, t o the e x p e r i m e n t a l c u l t u r e s . . Use.of  maintenance  cultures  in  medium  such  as  ES  medium  f o r the i n i t i a l  inoculum can r e s u l t i n c a r r y i n g over up to 5 ug 100  ug  Tris m l  - 1  of inoculum  T r i s are a r t i f i c i a l The  EDTA  ( P r o v a s o l i , 1968).  ml  cell and  - 1  Both EDTA and  c h e l a t o r s with a high a f f i n i t y  f o r copper.  i n i t i a l inoculum i n the b i o a s s a y s was from an e x p o n e n t i a l l y  growing c u l t u r e and gave s t a r t i n g  cell  c o n c e n t r a t i o n s i n the  experimental v e s s e l s o f 1000 c e l l s ml-*. For at  the  b i o a s s a y s , the algae were grown i n batch c u l t u r e s  a temperature o f 15±0.5°C,  flasks the  containing  appropriate  volume,  500  amount  mis,  which  one  litre  Pyrex  was  of  added  chosen  minimize  could  copper*  The  large  metal  lower  adsorption  the  metal  to  ion  I l l u m i n a t i o n was p r o v i d e d , on a  light:dark  by f l u o r e s c e n t l i g h t s  cycle,  2  (0,  tested  medium  the  glass  concentration 14:10  hour  (GE Cool White) with a  i n t e n s i t y from the s i d e o f 80 uE m~  a L i C o r Model LI 185A Quantum meter.. were  plus  t o minimize the s u r f a c e area t o  (Robertson, 1968),.  light  Erlynmeyer  500 mis of the enriched seawater medium  volume r a t i o and so surface,  in  sec-*,as measured  Four copper c o n c e n t r a t i o n s  i n each experiment; 0, 3.2, 7.9 and 15.7x10~  8  2, 5 and 10 ug added Cu 1  _ 1  by  ) , with two r e p l i c a t e  flasks  M Cu at  each c o n c e n t r a t i o n . The  one  litre  f l a s k s used f o r the experiments were double  a c i d washed with 0.5 N HCl, r i n s e d s e v e r a l times with distilled  water  and  autoclaved  before  the: s t a r t  deionized of  the  14 experiment* medium  The v e s s e l s were p r e r i n s e d  to  precondition  the  a d s o r p t i o n of copper  to  the  experimental  by  a  1977)  medium  glass glass,.  triple:  with  200  mis  of  the  walls  to  minimize  any  Metal  analysis  of  e x t r a c t i o n i n t o MIBK  showed a l o s s of 10% of the added  copper  over  the  (Cave,  a  7  day  period. Samples The  were taken d a i l y f o r c e l l counts and f l u o r e s c e n c e *  samples f o r c e l l counts were preserved by  0.10  mis  of  Lugol*s  solution  to  10  the  mis  of  addition sample..  c o n c e n t r a t i o n s of a l l but one s p e c i e s were determined counts  in  least  200  by  of Cell  visual  a Paliner-Maloney counting chamber. . In most cases a t cells  per  c o n c e n t r a t i o n was  sample  were  counted.  l e s s than 2000 c e l l s m l * ,  Where  the  only 100 c e l l s were  -  counted*.  C e l l c o n c e n t r a t i o n s f o r Thalassiosira-fiseudonana-were  determined  using a  aperture  tube*  Coulter  Counter  Fluorescence  Turner Fluorometer  (Model  Model  was  also  B  with  a  100  um  measured d a i l y with a  111).  S p e c i f i c growth r a t e s of the c u l t u r e s were:determined by least or  squares  log,  0  c e l l concentration  D  f l u o r e s c e n c e vs* time f o r the e x p o n e n t i a l p o r t i o n of the  growth curve. day  l i n e a r r e g r e s s i o n of the log,  (Guillard,  T h i s value was  then converted  to  r a t e has been shown i n at l e a s t one a  more  sensitive  95%  case  indicator  t o x i c i t y than p a r t i c u l a t e C and assimilation.  per  on the change i n c e l l numbers or  f l u o r e s c e n c e between the f i r s t and f o u r t h days..  be  doublings  1973).. In most cases, because of an i n i t i a l l a g  p e r i o d , growth r a t e s were based  to  a  N,  S p e c i f i c growth  (Berland e t  of  culture  the  al,  1977)  e f f e c t of  copper  biovolume  confidence l i m i t s are p l o t t e d  and  +C  l  on a l l of the  15 graphs.  The  r e l a t i v e standard d e v i a t i o n of the  c u l t u r e s i n 10 r e p l i c a t e c o n t r o l f l a s k s was  growth  2.6%*  rate  of  Table 1.1: Skeletonema  Bioassay s p e c i e s used costatum  (Sreville)  T h a l a s s i o s i r a pseudonana  i n the  experiments  Cleve  (Hustedt) Hasle and  Heimdal  T h a l a s s i g s i r a n o r d e n s k i o l d i i Cleve Chaetoceros compressum  Lauder  Chaetoceros l a c i n i o s u m Schutt Chaetoceros c r i n i t u m Shutt Nitzsghia  l o n g i s s i m a (Brebisson) R a l f s  N i t z s e h i a d e l i c a t i s s i m a Cleve  Prorpce^trum  minimum  Schiller  Heterocagsa t r i g u e t r a Symnodigium simglgx Gym nod inj. um  vitiligo  Ehrenb.  (Lohm.) Kof,.  Ex Swezy  Ballantine  Scri£p_siella t r o e h o i d e a (Stein) L o e b l i c h I I I  Table 1.2:  N u t r i e n t enrichments  for natural  NaNOi  3.5x10-5 M  NaH^PO^  1.45x10-6 M  Na Si0 a  3  .9K 0 X  4.0x10-6 M  Thiamin* HCI  1.19x10-8 M  Biotin  1.48x10-11 M 8.19x10-1* M  seawater  17 Results The  relative  e f f e c t s of four c o n c e n t r a t i o n s  7.9 and 1 5 . 7 x 1 0 ~  3.2,  numbers  and  8  M)  on  fluorescence  shown i n F i g s . . 1.1-1.19.  the  points  shifted  on  the  of  increase  of  cell  f o r 13 s p e c i e s of phytoplankton, are The  growth  percent of the c o n t r o l to f a c i l i t a t e The  rate  of copper ( 0 ,  figures  rates  a r e : given  as  a  comparison between s p e c i e s .  f o r f l u o r e s c e n c e growth r a t e s a r e  2 mm to the r i g h t t o prevent  confusion  with  the  cell  number p o i n t s * . The p o i n t s are both taken from the same samples, however,  and  represent  the  same  absolute growth r a t e s are presented with  three  species  at  Dec.-Jan* t e s t p e r i o d water  storage  both  copper c o n c e n t r a t i o n s * in  Table  the beginning  (see. Figs..1.1-1.6)  period  had  no  1.3.  The  Bioassays  and the end o f t h e  indicated  that  the  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 c a r r i e d out i n May some  of  the  results  experiments  comparisons,  but  are  to  check  of the Dec.-Jan..experiment and t o add a  t h i r d Chaetoceros s p e c i e s t o the comparison* two  1978  presented  the s t a r t i n g  The r e s u l t s o f the  together  to  facilitate  date of each experiment i s given  with the f i g u r e s *  N i t z s g h i a l g n q i s s i m a was used  twice  first  and once i n the May experiment  (Fig..1.3,  experiment  and -1. 12) any  i n the 1.4  with no s i g n i f i c a n t d i f f e r e n c e s between the r e s u l t s i n  of the t h r e e b i o a s s a y s , i n d i c a t i n g no s i g n i f i c a n t d i f f e r e n c e  i n those f a c t o r s a f f e c t i n g copper a v a i l a b i l i t y  in  the  seawater  samples used i n the two experiments.. The s p e c i e s t e s t e d i n both experiments  were; 5. v i t i l i g o ,  S. t r o e h o i d e a and N. l o n g i s s i m a .  In a d d i t i o n , C. c r i n i t u m was t e s t e d only i n the May experiment..  18 I t should considered  be emphasized t h a t a l l bioassay  to  be  r e l a t i v e , and  not an absolute  s p e c i f i c t o t a l copper c o n c e n t r a t i o n . measure  or  calculate  results  It i s  results  be  response t o a  very  difficult  to  the c u p r i c i o n c o n c e n t r a t i o n i n seawater  media and no attempt has been made to do so i n t h i s The  must  experiment.„  are r e l a t i v e but are a l l t h a t i s necessary  f o r the  comparison of s p e c i e s s e n s i t i v i t i e s w i t h i n t h i s experiment* The while  r e s u l t s shown i n Figs..1.1-1.12  those  are  all  for  diatoms  i n F i g s . . 1* 13-1. 1 9 are f o r d i n o f l a g e l l a t e s *  It i s  apparent t h a t t h e r e i s a great v a r i a t i o n i n the: s e n s i t i v i t y the  species  t o copper w i t h i n each group, and n e i t h e r group  of can  be s a i d to be more s e n s i t i v e than the other, . Skeletonema costaturn, one the  world,  ( F i g . 1.1  was  found  and  1.2)  to  of the most abundant  species  be very i n s e n s i t i v e : t o added copper  as  is  Thalassiosira- nordenskioldii  ( F i g . 1.7)._ Both show no s i g n i f i c a n t r e d u c t i o n i n growth at highest  concentration  of added copper t e s t e d  T h a l a s s i o s i r a gseudgnana, which has copper  bioassay  organism,  s e n s i t i v e i n t h i s study delicatissima waters. three  was  (Fig*  (Fig..1.8)  species  1*5  been used  found and  to  all  -8  M  four cases.  l o c a l B.COf  Cu)  - 8  M Cu).  previously be  1*6),  ( F i g . 1.3/ ( F i g . 1.9,  extremely s e n s i t i v e to added copper, with (3.15x10  (15.7x10  the  as  a  o n l y moderately  as  was  Nitzschia  a s p e c i e s sometimes abundant i n l o c a l  N i t z s c h i a lgngissjma Chaetoceros  in  1-f and 1.10 the  1.12)  and  and  the  1.11)  were a l l  lowest  addition  causing a s i g n i f i c a n t r e d u c t i o n i n growth i n  A l l four are s p e c i e s which can be  abundant  in  waters* the  dinof l a g e l l a t e s ,  only  Hetergcap.§a  triguetra  19 (Fig.  1.18)  i s insensitive  Gymnodinium and  vitilligp  to  the  added  copper,  and Gymnodinium simplex  (Fig*  1.13, 1.14  1.19) are moderately s e n s i t i v e , and ScrijpjDsieJLla  and  Proroeentrum  minimum  ( F i g . 1.15,  while  trochoidea  1.16 and 1.17) are very  sensitive. S o r i g s i e l l a trochgidea significant The  difference  difference i s  experiment  as  is  the  between  probably  indicated  only  the  due  to  species  to  show  a  J a n . and May experiments. variations  in  by  the  wide: confidence  A l l t h e experiments used  two  replicate  the  first  limits  in  Fig..1.15..  c o n d i t i o n , with the s i n g l e exception which  due t o experimental  flasks  of H. t r i g u e t r a  test  ( F i g . 1.18)  problems, only had one f l a s k per t e s t  c o n d i t i o n * . The l a c k of r e p l i c a t e s i s the reason confidence  per  f o r the  wide  l i m i t s , not any l a r g e i n c r e a s e i n the v a r i a b i l i t y o f  the : growth  rates  of  this  d e v i a t i o n was approximately  species.  The  relative  standard  the:same f o r t h i s s p e c i e s as f o r the  other s p e c i e s t e s t e d . The  length  exponential equations  of  the i n i t i a l  l a g p e r i o d before  growth was c a l c u l a t e d f o r a l l of  the  regression  r e l a t i o n s h i p between  the  lines..  length  of  the s t a r t o f  conditions  from  the  In no case was there any the  lag  phase  and  the  c o n c e n t r a t i o n o f added copper* In the  F i g . 1.20, the c e l l u l a r s u r f a c e area t o volume r a t i o of  13 s p e c i e s i s p l o t t e d a g a i n s t the copper c o n c e n t r a t i o n  caused a 50% r e d u c t i o n relationship r a t i o and  between  copper  in the  toxicity.  growth* two  There  is  no  which  significant  f a c t o r s , s u r f a c e area t o volume Nor  i s there  any  relationship  between c a l l volume and copper t o x i c i t y .  Table 1.3: E f f e c t of copper on the growth r a t e  1  of  t h i r t e e n s p e c i e s of phytoplankton* . Growth Rate at each Sp.ec i e s  Date  C e l l #2 C o n t r o l  Added Co£|>e r Cone*  Or F l . 3  (x10~ -M) a  1. 6 S. c.  Dec.  1. 48  1.51  1.39  Fl.  2.42  2.40  2.40  2.46  Cell #  2.39  2.52  2. 10  1.97  Fl. .  2. 17  2. 31  2.17  2. 10  Cell #  1.78  1.22  0.35  0. 21  Fl. .  1.89  1. 46  0.?0  0.50  Cell #  1.73  1.05  0.26  0.20  Fl. .  1.72  1.30  0.73  0. 47  Cell #  1. 83  1. 36  1. 00  0,.40  Fl. .  1.78  1. 54  1.37  0.?2  Dec.  Cell #  1.69  1. 15  0.97  0.70  Jan.  Cell #  1. 52  1. 32  1.05  0.77  Fl. .  1.77  1.69  0.96  0.77  Cell #  0.60  0.47  0.59  0.58  Fl.  0. 91  1.02  0.86  0.97  Cell #  2. 17  2. 19  1.67  1.41  Fl*  2. 13  2.04  1.58  1. 38  Cell #  2. 19  1.58  0.08  0.20  Fl. .  2. 19  1-71  0.73  0,19  Cell #  1.67  1. 38  1.07  0.00  Fl.  1. 76  1. 13  1.00  0.00  Dec.  May  T.n.  N.d.  C.  CO  Cl.  15. 7  1.56  Jan.  T.p.  7.9  Cell #  Jan.  N.l.  3,i 2  Jan. _  Dec.  Jan.  Jan. .  Growth Rate iat each Species Date  Cell  Control  Or Fl.£  (x10z!-fi> 1.6  C. c r .  G. y. .  S. t .  H. t . .  G.s.  3; 2  2-9  15. 7  Cell #  2.01  1.32  0.59  0.46  Fl.  2.69  1.74  1.11  0. 95  Dec.  Cell #  0.58  0.65  0.51  0.47  May  Cell #  0.60  0. 63  0.52  0.40  Fl.  0.51  0.51  0.49  0.35  Ceil #  0. 54  0.38  0.31  0. 00  Fl. .  0, 47  0. 42  0.42  0.14  Cell #  0.37  0. 37  0.33  0.21  Fl.  0.35  0.36  0.34  0. 18  Cell #  0. 55  0.44  0.26  0.08  Fl.  0.40  0.28  0.18  0. 15  Cell #  0.66  0.69  0.70  0. 67  Fl.  0. 52  0.49  0.51  0.44  Cell #  0.73  0.68  0.66  0.44  Fl.  0.70  0.49  0.47  0. 15  May  Jan.  May  P. m. .  Cone.  Added-C  Dec. .  Dec.  Jan.  1  growth r a t e = d o u b l i n g s per day  2  r e f e r s to the growth r a t e of c e l l  3  r e f e r s t o the growth r a t e of  numbers  fluorescence  23  Fig.1.1-1.19:  Relative  Growth r a t e as a percent  vs. c o n c e n t r a t i o n of added copper  (x10  - 8  copper on c e l l number growth r a t e ( • — — • ) . on  f l u o r e s c e n c e growth r a t e (o  i s d i s p l a c e d 2 mm cell  data  (•).  to the r i g h t to  o>.  The  prevent  M). The  of the c o n t r o l The  effect  effect  of copper  f l u o r e s c e n c e data confusion  E r r o r bars represent 95% confidence  of  with  limits..  o  (o) the  2k  100  100  50  50  CD -+-»  a cr o O  S.  0  costatum  0 5 Added (x  10 1!5 Copper  6  S.. c o s t a t u m  5  Jan  10  Added  15"  Copper  (x10" M)  8  8  1-1  Fig.  N. l o n g i s s i m a  3^100  0  10" M) Fig.  a cr  Dec  Dec  N. l o n g i s s i m a J a n  100  50  50  0  0  1-2  o L.  0  0  5  Added Fig.  10 Copper 1-3  15  0  5  Added Fig.  10 Copper 1-4  15  25  100  o?100 +->  d cr  50  50  o  L.  o  T. pseudonana Dec  0  0  5  10  Added  15"  T. pseudonana J a n  0  Copper  0  5  10  Added  (x10" M)  15"  Copper  (x10" M)  8  8  Fig. 1-5  Fig.  ^100  L6  100  -+->  d cr  50  50  o  (3  T. n o r d e n s k i o l d i i J a n  0  0  5 Added Fig.  10 Copper 1-7  15~  N. d e l i c a t i s s i r n a Dec  O  5  Added Fig.  10 Copper 1«8  15  26  C. pompressum Jan  o^lOO  C. l a c i n i o s u m J a n  100  d  cr  50  50  0  0  o O  0  5  10  Added  15  Copper  0 5 Added  (x10" M)  (x10* M)  8  Fig.  8  1-9  Fig.  C. c r i n i t u m  5*100  10 15 Copper  May  1-10  N. l o n g i s s i m a  100  May  -+-> d  50  50  o o  0  0  5  Added  10 Copper  F i g . 1-11  15  0  0  5  Added  10 Copper  F i g . 1-12  15  27  A,  5^100  100  a cr  50  50 o  G. v i t i l l i g o , .Dec  L.  o  0  0  5  10  15~  0  Added Copper (x10" M) Fig. 1-13  6  G. v i t i l l i g o  5  May  10  15~  Added Copper (x10" M) Fig. 1-14  8  8  S. t r o e h o i d e a J a n  S. t r o e h o i d e a  May  100  i100 +->  a  cr  50  +-»  o o  0  5  Added Fig.  10 Copper 1-15  15  0  0  5  Added Fig.  10 Copper 1-16  15  28  P. minimum Dec  100  100  +-» d  50  50  o  H. t r i q u e t r a  L.  o  0  0  5  10  Added  15  Copper  (x10" M) 8  Fig.  o  100  1-17  G. s i m p l e x J a n  <D •+-> d  si  50  o L.  CD  0  0 5 Added Fig.  10 15 Copper 1-19  0  0  5  10  Added  Copper  (x10" M) 8  Fig.  Dec  1-18  15"  29  Fig..  1.20:  species  surface  v s . t h e amount o f  reduction figure  The  in  t h e growth  area copper  well  as  the  figure.  the  X axis  and  the i d e n t i f i c a t i o n The  five  necessary  150  values  range o f copper c o n c e n t r a t i o n s  for  to the r i g h t  d i d n o t show a 50% r e d u c t i o n tested  in  i n the  cause  (150).  number f o r each  s p e c i e s shown  (SA:V) o f  to  r a t e o f each s p e c i e s  a r e t h e c a l c u l a t e d SA:V  as  t o volume r a t i o  an Below  each  species  each 50% the  species used i n  of the break i n  growth  within  experiment.  the  5-  O  +->  o  o  < cr 1  11  #  1  8  s g a  3, 13  c  =J o 5 oo > Q0 Copper  Causing  12 »»  10  L_ —  10  ^  4~  15  Concentration a 50%> R e d u c t i o n  in G r o w t h ( x 1 0 "  8  M)  F i g . 1«20 Species  SAxVol.  >  1 Skeletonema costatum  0.92  2 Nitzschia longissima  1.22  3 Thalassiosira  pseudonana  1.30  8.0  4 Thalassiosira  nordenskioldii  0.28 2.7^  6 Chaetoceros compressum  / /  0.59  3.0  7 Chaetoceros l a c i n i o s u m  0.61  2. 8  8 Chaetoceros c r i n i t u m  0.71  3-5  9 Gymnodinium  0.50  /  0.25  10.0  5 Nitzschia  delicatissirna  10 S c r i p p s i e l l a  vitiligo troehoidea  3.0  11 Prorocentrum minimum  1.04  12 Heterocapsa  0.37  /  1.20  9.0  triquetra  13 Gymnodinium simplex  31 Discussion Those  experiments,  in  the  past, which have compared the  e f f e c t s of copper on s e v e r a l s p e c i e s of phytoplankton conflicting  results  have  had  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  general  trends  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. Comparisons  of  s p e c i e s have found experimenters  the  effects  somewhat finding  copper  contradictory general  d i n o f l a g e l l a t e s and diatoms 1977)  of  on phytoplankton  results  differences  ( M a n d e l l i , 1969;  others  (Ibragim and P a t i n , 1976; finding  some  between  Thomas and  and w i t h i n the diatoms, d i f f e r e n c e s between  pennates  with  Seibert,  centrics  and  Thomas and S e i b e r t , 1977)  no such g e n e r a l d i f f e r e n c e s  and  (Braek et a l , 1976;  E r i c k s o n e t a l , 1970). The  present  dinoflagellates  study  to  be  has  found  within  the  copper  and  centric  inhibition  concentrations  statement as to the s e n s i t i v i t y Pennate  diatoms  and  very v a r i a b l e i n t h e i r s e n s i t i v i t i e s to  copper, ranging from complete growth effect  both  of e i t h e r  diatoms  also  to  tested.  group show  no  No g e n e r a l  can wide  growth  be  made.  ranges  s e n s i t i v i t y , and n e i t h e r can be s a i d to be more: s e n s i t i v e  of than  the other* The  results  r e p o r t e d here do support the f i n d i n g s of some  of the f i e l d experiments et  al,  in  prep, a)  (Thomas and S e i b e r t ,  where  i t was  1977;  Hollibaugh  found that pennate  diatoms,  l i ^ z s c h i a d e l i c a t i s s i m a i n p a r t i c u l a r , became dominant i n copper treated containers abundant  in  the  and  the  -haetoeeros  c o n t r o l s , disappeared.  species,  which  were  In the present study,  32  the  three  Chaetpcergs  sensitive  to  added  only moderately  species  copper,  tested  were  among  the  most  while N i t z s c h i a d e l i c a t i s s i m a  was  sensitive.  The only g e n e r a l trend i n s e n s i t i v i t y to copper observed i n t h i s study was  f o r s p e c i e s of the same genera  sensitivities.  This  was  especially  apparent  Chaetoeerps  s p e c i e s t e s t e d , a l l of which  The  genera  other  where  more  to  than  were  one  have  similar  with the t h r e e very  species  sensitive. were t e s t e d  ( T h a l a s s i g s i r a , N i t z s c h i a and gYmngdinium) were not as c l o s e  in  the s e n s i t i v i t i e s of the two s p e c i e s but at l e a s t d i d not show a wide d i f f e r e n c e * as  suggested  Mandelli,  1969;  prep* a)  and  in  earlier  Thomas and S e i b e r t , supported  their s u s c e p t i b i l i t y to decrease than  in  growth  natural  sensitivity  in  higher  preference  result  (Frost,  trophic  (Hag,  1975).  dominance  Some  1967)  in  species  because  and f i l t e r i n g and S e i b e r t  resistant  This  studies  in  a  great  in  in  community  could  in  turn  efficiency  of  zooplankton  (1977) have suggested  that  l e v e l s i n the oceans would be  species* .  have  differences  of such f a c t o r s as food  a  d i v e r s i t y , with a r e s u l t i n g This,  however,  n e c e s s a r i l y mean a decrease i n p r o d u c t i v i t y or Several  show  changes  populations.  levels  Thomas  Such  drastic  i n phytoplankton taxonomic of  al,  study* phytoplankton vary i n  levels*.  one r e s u l t of i n c r e a s i n g copper decrease  H o l l i b a u g h et  rate a t added copper l e v e l s l i t t l e g r e a t e r  environmental could  (Erickson e t a l , 1970;  1977;  this  copper.  s t r u c t u r e of phytoplankton affect  studies  not  biomass*  indicated a significant  between added copper or c u p r i c ion a c t i v i t y and  does  the  correlation length  of  33 the  initial  lag  l a g phase i n the bioassay.  Barber  (1973) found  time i n s e v e r a l Chaetoceros s p e c i e s t o be decreased  addition  of  c h e l a t o r s and  Morel et a l (1978) has  by  i n c r e a s e d by the a d d i t i o n of  shown t h a t the l a g phase  of  Morgan  authors,  (1978),  in  Skeletonema  responses  to  excessive  response i s a d e p r e s s i o n concentration  which  Jackson  a n a l y z i n g the data of s e v e r a l  have observed t h a t there  seems  levels  of  to  be  copper.  two  The  i n growth r a t e with  the  metals..  costatum i n c r e a s e s with i n c r e a s i n g c u p r i c i o n a c t i v i t y . and  the  previous types  first  increasing  type  of of  copper  does not decrease markedly with time.  The  second type of response i s a l a g phase i n growth i n c r e a s i n g with i n c r e a s i n g copper growth  at  a  concentration,  rate  followed  by  i n d i f f e r e n t to the i n i t i a l  an  exponential  concentration  of  copper. The type  present  study  of response.  has  found no  s p e c i e s showing  to  the  concentration  of  added  the  lag  copper.  s t a t e d , however, t h a t the shortness  of the bioassay  could  allow  an  missed  (Stockner  a few  the  second  While many of the s p e c i e s showed a l a g phase  before e x p o n e n t i a l growth, i n no case could related  the  recovery and  after  A n t i a , 1976).  phase  be  I t must be (four  days)  extended l a g phase to  be  This only seems p o s s i b l e f o r  of the most s e n s i t i v e s p e c i e s , the Chaetoceros s p e c i e s  in  particular. In in  conclusion,  the t o x i c i t y  or  within  the  t h i s study  to  volume  found no g e n e r a l d i f f e r e n c e  of copper to e i t h e r diatoms or diatoms,  between  There i s no r e l a t i o n s h i p between area  has  centric copper  dinoflagellates,  or pennate s p e c i e s *  toxicity  and  r a t i o or to the l e n g t h of t h e : l a g phase  surface before  34  e x p o n e n t i a l growth. of  There i s some i n d i c a t i o n t h a t  the  species  a p a r t i c u l a r genera have s i m i l a r s e n s i t i v i t i e s t o copper and  this i s especially  apparent with the Chaetoceros s p e c i e s .  35 Chapter 2 The E f f e c t Of C u p r i c Ion A c t i v i t y  On The Growth Rate Of  The  Marine Diatom, N i t z s c h i a - l g n g i s s i m a Introduction There  has been an i n c r e a s i n g i n t e r e s t  in  the  effects  of  heavy metals, copper i n p a r t i c u l a r , on phytoplankton s p e c i e s and communities  in  natural  waters.  the a d d i t i o n of approximately (1.6x10growth  M)  8  in  Steemann  to  some  seawater  environmental  and  levels  of  that  copper  i s capable of s i g n i f i c a n t l y r e d u c i n g  phytoplankton  Nielsen  I t has been demonstrated  species  Wium-Andersen^  (Davey 1971)  et  but  al, it  1973;  has only  r e c e n t l y been f u l l y r e c o g n i z e d t h a t i t i s both t h e : c o n c e n t r a t i o n and the chemical s p e c i e s of the metal present t h a t are important i n determining the e f f e c t of the metal on phytoplankton. . Of the t r a c e metals n a t u r a l l y present i n  seawater;  copper  appears to be most important i n i n f l u e n c i n g phytoplankon  growth.  Hollibaugh  et  al  (in  prep, b)  compared  the  effect  phytoplankton growth of d i f f e r e n t - s t r e n g t h s of a mixture of metals  whose  seawater*  relative  a  the  due t o the copper p r e s e n t .  toxicity  the  p a r t i c u l a r c o n c e n t r a t i o n , e i t h e r alone or with the nine o t h e r  paper  by  the  determined found  mercury, in  of  That i s , copper at  metals, would have the same e f f e c t on growth r a t e . .  and  ten  p r o p o r t i o n s were those found i n n a t u r a l  They found t h a t almost a l l of  metal mixture was  on  In  another  same authors (Hollibaugh et a l , i n prep, a) they  the comparative t o x i c i t y of copper  to  10  metals  individually  be the most t o x i c , with the e x c e p t i o n of  which while more t o x i c , i s present i n n a t u r a l  considerably  lower  concentrations  than  seawater  copper.  Also  36 supporting (et  t h i s , Jackson and Morgan  a l , 1973)  and Barber's  ion  concentration  Davey's  (1973) experiments and a t t r i b u t e d the  * * C a s s i m i l a t i o n to changes i n the  e f f e c t s on growth and ion  (1978) r e i n t e r p r e t e d  cupric  r a t h e r than to changes i n i r o n , z i n c or n i c k e l  concentrations. Early  works  demonstrated seawater  the  by  Provasoli  importance  (1963)  of  chelating  Johnston agents  found  dissolved  that  recently  metals  for  making  presumably  present.  It  has  upwelled waters, which were high i n  metals and low i n d i s s o l v e d  unsuitable  (1964)  in  a s u i t a b l e medium f o r phytoplankton growth,  by c o n t r o l l i n g the s p e c i a t i o n of the been  and  phytoplankton  organic  growth  until  compounds, either  were  artificial  c h e l a t o r s or an e x t r a c t of homogenized zooplankton were added t o the seawater  (Barber and Ryther, 1969)..  by u l t r a v i o l e t i r r a d i a t i o n , matter  which  could  (Barber, 1973;  increase  chelators  in  of  nontoxic  metal s p e c i e s ,  organic  the; t o x i c i t y  Sunda and G u i l l a r d , 1976).  such  organic  probably the  of  The f a c t  through the a d d i t i o n o f  as EDTA would f u r t h e r i n d i c a t e  the t o x i c a c t i o n of the 0 . V . . i r r a d i a t i o n was through down  seawater  complex and d e t o x i f y t r a c e metals, has been  that the t o x i c i t y change can be reversed artificial  of  which i s known t o break down  found i n many cases to cause an t r a c e metals  Treatment  the  that break  t r a c e metal complexes to more t o x i c ionic  species  (Sunda  and  Lewis,  1978) . Several Morel, 1978; toxicity not  is  studies  (Sunda  Sunda and Lewis,  and 1978)  G u i l l a r d , 1976; have  Anderson  indicated  r e l a t e d to the c u p r i c i o n c o n c e n t r a t i o n  the t o t a l copper c o n c e n t r a t i o n ,  the i n o r g a n i c a l l y  and  that  the  (Cu )  and  2+  complexed  37 copper  species,  complexed Lewis,  EDTA. copper or T r i s copper complexes  or copper  with n a t u r a l organic compounds (Sunda, 1975;  Sunda and  1978).  activity  Their  have  related  the  i n seawater t o such f a c t o r s as growth  and c e l l m o t i l i t y there  studies  Lewis, 1978). been  in  fresh  water  to  use  been  direct  Sunda  (1975),  copper t o x i c i t y  to  present  computer  models  and  calculate  the  et  al  between  calculated  cupric  quantify  ion a c t i v i t y  longissima  and  and to  t h i s r e l a t i o n s h i p with those reported f o r other s p e c i e s  and c a l c u l a t e d estimates seawater*  (1976)  s p e c i a t i o n of copper i n  r a t e o f the diatom s p e c i e s N i t z s c h i a  compare  has  experiments with marine phytoplankton.  relationship  growth  it  thermodynamic  The aim o f t h i s i n v e s t i g a t i o n i s to determine and the  with  Sunda and  measurement  c a l c u l a t i o n s , such as those developed by W e s t a l l and  uptake  used  (Swallow et a l , 1978;  Lacking a method of  necessary  At  ion  measuring c u p r i c i o n a c t i v i t y i n  seawater although s p e c i f i c i o n e l e c t r o d e s have success  r a t e , »*C  i n v a r i o u s phytoplankton s p e c i e s .  i s no method of d i r e c t l y  some  cupric  of  cupric  ion  activity  in  natural  38  Method Stock  cultures  of the pennate diatom N i t z s c h i a  lonaissima  were o b t a i n e d from the Northeast P a c i f i c C u l t u r e : C o l l e c t i o n U.B.C.  at  T h i s s p e c i e s was chosen as a bioassay organism f o r t h i s  study because of i t s s e n s i t i v i t y to previous  work  as  determined  in  comparing the e f f e c t s of copper on 13 s p e c i e s of  diatoms and d i n o f l a g e l l a t e s Natural  copper,  collected  in  The seawater  was  stored at 9°C i n a 200 1 a c i d washed p o l y e t h y l e n e b a r r e l and  was  February  seawater  (Chapter 1).  and  for  March,  1978  the  bioassays  as  i n Chapter 1*  used over a period of two months, Feb. several  bioassays  was  To A p r i l , t o  carry  to determine the e f f e c t of c a l c u l a t e d  out  cupric  ion a c t i v i t y on the growth r a t e of the phytoplankton s p e c i e s . . A two  month storage p e r i o d  little  or  had  previously  been  shown  to  no e f f e c t on the c a p a c i t y of the seawater to support  phytoplankton growth or a f f e c t copper t o x i c i t y b i o a s s a y s . water  have  apparently  had  suggested by the f a c t  a  low  natural  complexing  This  c a p a c i t y as  that;  1) a d d i t i o n of s m a l l amounts of copper  (1.6x10  -8  M)  were  shown  to have a t o x i c e f f e c t on the bioassay s p e c i e s 2) comparison  of b i o a s s a y s using high i n t e n s i t y  treated  non-UV  and  difference*  treated  Treatment  seawater  with U.V*  copper  no  (UV)  significant  i r r a d i a t i o n i s known to break  down the d i s s o l v e d organic compounds detoxify  showed  ultraviolet  which  (Armstrong et a l , 1966;  could  complex  and  Barber, 1973; Sunda and  Lewis, 1978) . . In any case,  any  naturally  occurring  organic  compounds  39 present  would  have  and/or present affect ion  the  in  to  be  very  either  high  concentrations  concentrations  of  1978;  G u i l l a r d , 1 976).  Sunda and  and  plus  varying  the  chelators,  EDTA and  added  chemical  concentration,  was  speciation,  et a l , 1976).  described  i n Anderson and  chemical  constituents  calculations*  2.1).  have  found  and  concentrations  activity  inoculum was  that  some  from an e x p o n e n t i a l l y concentration  at  a  Computation  (1978)..  the  the  Table.  of  coefficients of  ion  2.2  MINEQL  lists  used  the  in  the  Debye  (Stumm  and  the: v a r i o u s  added.  the to  Huckel Morgan,  enrichments,  Anderson and  The  hours Morel  initial cell  growing c u l t u r e and  the algae  15±0.5<>C  in  Erlynmeyer f l a s k s c o n t a i n i n g 500  mis  Illumination  a  was  cupric  the  before (1978)  c h e l a t o r s , i . e * . EDTA, can take s e v e r a l  bioassays, of  the  program  gave a  i n the experimental v e s s e l s of 1000  temperature  of  of i o n i c s p e c i e s were c o r r e c t e d  hours to e q u i l i b r a t e i n seawater*  For  chelators  allowed to e q u i l i b r a t e f o r at l e a s t two  the i n i t i a l c e l l  et a l ,  artificial  Morel  1970).. A f t e r the: a d d i t i o n was  (Morel  L i m i t a t i o n s on the computations were as  Concentrations  for  large  filtered  computer  a c t i v i t i e s using the Davies m o d i f i c a t i o n approximation  copper  of  especially  performed with the  (Westall  Tris  consisted  nutrients, vitamins,  amounts of copper (Table  eguilibrium  medium  significantly  a d d i t i o n of comparatively  medium used i n the experiments  seawater  to  chelators  c u p r i c ion a c t i v i t y i n a medium i n which the  a c t i v i t y i s b u f f e r e d by the  The  extremely strong  provided  on  inoculum  starting  was cell  c e l l s ml-*..  were grown i n batch c u l t u r e s 1  1  borosilicate  glass  of the experimental medium. 14:10  light:dark  cycle  by  no fluorescent lights intensity Two  of  (WH  100 uE m  with  an  incident  light  s e c * as measured by a quantum sensor.  -2  -  r e p l i c a t e f l a s k s were run a t each t e s t c o n c e n t r a t i o n . The  double  1 1 Erlynmeyer acid  washed  deionized d i s t i l l e d experiment. medium of  Cool White SHO)  with  0.5N  used  for  HCI,  to  the  bioassays  water and a u t o c l a v e d before the s t a r t of the with  200  Samples The  a d d i t i o n of 0.10  were cell  taken count  daily  for  samples  cell  of  the  were  determined  by  mis  visual  Palmer-Maloney counting chamber.. In most cases, were counted.. Where the c e l l  2000 c e l l s  ml-i,  Fluorescence  was  only  100  cells  counts  and  were preserved by the  mis o f Lugol's s o l u t i o n to 10  concentrations  The pH was  mis  c o n d i t i o n the g l a s s w a l l s to minimize any a d s o r p t i o n  fluorescence*  cells  were  r i n s e d s e v e r a l times with  The v e s s e l s were p r e r i n s e d  copper*_  Cell  flasks  of  sample.  counts at  least  in a 200  c o n c e n t r a t i o n was l e s s than per  sample  were  counted.  measured with a Turner Fluorometer Model  determined a t the beginning and end  of  the  111.  assays,  using a F i s h e r Accutron 140. . Specific indication 1.  growth  rates  of  the  cultures  were used as an  of copper t o x i c i t y and were determined as i n  Chapter  41 Table 2.1: Experimental Medium  Natural Seawater Salinity  31.1%o  Dissolved  Copper  1.0 X 10"« M  Dissolved  Zinc  7.6 X 10~ M  D i s s o l v e d Organic Carbon  8  0.9 ug l ~ i  Enrichment  NaN0  3.50 x 10-s M  3  1.45 x 10-s  NaH PO^ A  N a S i 0 . 9H 0  4.0 x 10-6  Thiamin* HCl  1. 19X10-B M  Biotin  1.48x10-11 M  B  8.19x10-1i M  A  ex  3  A  M  M  EDTA  5.0 x 10-*  Tris  1.0 x 10-3  Copper  32.2 t o * 010 x 10-6  M  M H  42 Table 2.2: Computed chemical s p e c i a t i o n Species  Total  1  Cone. (M)  1  Ionic Cone*  Dominant (M)  Bromide  7. 48x10" 4  7.48x10" *  Calcium  9. 11. i o - 3  7.95x10-  Carbonate  2. 07x10- 3  Chloride  Aqueous Form  •  Br~  100  Caz*  87. 3  1.17x10- 4  HC0 -  61. 1  4. 85x10- I  4.85x10- 1  ci-  Cobalt  1. 09x10- 9  5.05x10-  COEDTA2+  92. 4  Copper  1. 00x10" 8  7.43x10" 1  3  CuTris-^OH-^  50.4  EDTA  5. 00x10- 7  5.40x10- 1  S  CaEDTA +  79. 7  Iron  5. 00x10- 7  1.05x10- 2  1  Fe (0H)  99.8  Magnesium  4. 73x10" 2  4.00x10- Z  Mg +  84.5  Manganese  3. 64x10- 8  1.18x10-  MnCl+  46. 2  Nitrate  5. 50x10- 5  5.50x10- 5  NO3-  Phosphate  5. 00x10- 6  1.48x10- 9  HPO " .  51.4  Potassium  9. 07x10- 3  8.77x10-  3  K+  96.7  Silicate  1. 00x10- 5  6.69x10-  1 t  HiSiO^  90.4  Sodium  4. 16x10- 1  4.10x10- 1  Na *  98.6  Strontium  8.01. 10- S  7.86x10-  Sr  98. 2  Sulfate  2. 51.10- 2  1.18x10- 2  so^. -  46. 8  Tris  1. 00x10- 3  7-72.10- 4  Tris  77. 1  Zinc  7. 65x10- a  2.33x10- 9  ZnEDTA  concentrations  based  3  3  I I  8  S  100  2  2  100 2  H  1  2 +  2  on R i l e y and Chester  EDTA and copper which were added, and manganese were measured.  3  2+  96. 3  (1972) except f o r and  zinc  which  Table  2.3: P a r a m e t e r s And R e s u l t s  Exper-  TotCu4  iment*  (UM)  1  2  Total  1  present 2  Growth R a t e * pH  Cell  3  #  Fluor.  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  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  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  added  copper  plus  the  1  x 10  - 8  M that  was  already  i n the seawater  Median c u l t u r e  the  pCu*  2  5. 01  32.3  3  Of E x p e r i m e n t s  pH - l i m i t s  give  the range i n c u l t u r e  pH  during  experiment  3  Median c u l t u r e  -  limits  give  * Growth r a t e 95% c o n f i d e n c e  pCu*  (negative  l o g of the cupric  ion  activity)  t h e r a n g e due t o t h e v a r i a t i o n s i n pH = d i v i s i o n s per day, t h e e r r o r limits.  limits  indicate the  44  'O  Figs.  2.1  activity)  and vs*  fluorescence Nitzschia  2.2: the  The  calculated  growth  (o),  lonqissima.  during the  of  respectively, The  represent v a r i a t i o n s in the pH  rate  pCu* (-log  course of the  cell of  horizontal calculated four  day  of the c u p r i c  numbers the bars  pCu* due  bioassay on  95% c o n f i d e n c e l i m i t s f o r the  at each t e s t  condition*  each  and  species point  to v a r i a t i o n ^ in  experiments.  bars are the  :(•)  ion  The  vertical  measured growth (jrate  Growth  Rate  Cell  Numbers  O  *o  co  2] <P  ro  -o £  ol  +  -A  (doublings  day  ) ro  b  b  Growth  00  o b  CD  31 CQ'  ro ro  ~o O c  *  Rate - F l u o r e s e n c e  (doublings  b  f + + -f-  CA)  )  ro b  O  ro  day  HI  R e s u l t s And The  purpose  toxicity  of  these  Discussion  experiments  of copper f o r N i t z s c h i a  was  to  i2£3i§sima,  by  e v a l u a t e the relating  the  e f f e c t on growth r a t e t o 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 . was  also  to compare i t s s e n s i t i v i t y with that of other s p e c i e s  whose s e n s i t i v i t y to c u p r i c ion a c t i v i t y have Recent  work  in  copper t o x i c i t y  both  species  activity  of  ion  present  (Sunda and S u i l l a r d , Sprague,  Morgan, 1978; can  be  copper  1976;  by  present,  1978;  Anderson  Andrew  such  factors  et  especially  and  of the  concentration Morel,  a l , 1977;  The c u p r i c as  that  (or a c t i v i t y )  and not to the t o t a l copper  Sunda and Lewis, 1978).  affected  determined.  f r e s h and s a l t waters has i n d i c a t e d  cupric  and  been  i s r e l a t e d t o the c o n c e n t r a t i o n  c e r t a i n chemical  Howarth  It  1978;  Jackson and  ion  activity  pH and the presence o f  o r g a n i c and i n o r g a n i c complexing agents as well as by the  total  copper c o n c e n t r a t i o n * Figures (the  2.1  and 2*2  show the e f f e c t o f the c a l c u l a t e d  n e g a t i v e l o g of 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 )  growth  rate,  as  determined from c e l l numbers and  on  pCu* the  fluorescence  measurements, r e s p e c t i v e l y , of N i t z s c h i a lonqissima,. . There i s a very abrupt change i n the growth  r a t e between  pCu*«s  10.6  11.6,  with maximum growth r a t e above pCu* 11.6, almost  growth  inhibition  inhibition  and approximately  complete  50%  toxicity  with  some  data degree  reported of  for  other s p e c i e s can be  confidence  when  the  copper  c o n c e n t r a t i o n s are expressed as pCu* r a t h e r than as t o t a l added  to  growth  at a pCu* of 10.9..  Copper compared  below pCu* 10.6  and  a  medium  of unknown complexing c a p a c i t y .  copper  Sunda and  48 Guillard found  (1976), u s i n g a s i m i l a r method  f o r determining  50% i n h i b i t i o n of the diatom T h a l a s s i o s i r a pseudonana and  the green a l g a Nannoehloris atomus a t a pCu* of 9.3.. al  pCu*,  (1978)  have  Skeletonema (1978),  Morel  et  found no r e d u c t i o n i n the growth of the diatom  costatum above a pCu* of 8.4,.  using  the  motility  and  Anderson  »*C  uptake  and  of  Morel  Gonyaulax  tamarensis c e l l s as an i n d i c a t o r of copper t o x i c i t y , found a 50% e f f e c t at a pCu* o f 10.4.. Reuter et a l (1979) found a 10  pCu*  to cause a 50% i n h i b i t i o n of *C f i x a t i o n i n t h e marine J  green algae O s c i l l a t o r i a Based seawater  of  1.4x10  M  -8  copper  in  coastal  (Chester and Storey, 1974), i t i s p o s s i b l e t o  estimate the c u p r i c i o n a c t i v i t y inorganic  blue  theibautii.  on a mean value samples  of  complexation)*  At  in  seawater  (assuming  only  a pH of 8*2, c a l c u l a t i o n s g i v e a  pCu* o f 9.6-9*7 (Anderson and Morel, 1978; Sunda  and  Guillard,  1976), a value c o n s i d e r a b l e higher than t h a t r e q u i r e d to cause a major  r e d u c t i o n i n the growth of N i t z s c h i a l o n g i s s i m a . . As t h i s  c a l c u l a t i o n does not i n c l u d e any p o s s i b l e o r g a n i c it  represents  concentration* not  be  able  a  maximum  activity  f o r that  At t h a t pCu*, Gonyaulax to  grow  and  complexation,  pH  tamarensis  T i pseudonana,  0*  and  copper  also  would  T h e i b a u t i i 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 a f f e c t e d  at a l l *  I t i s obvious t h a t t h e r e a r e some major v a r i a t i o n s sensitivities  of  different  species  to  copper..  It  i n the i s also  i n t e r e s t i n g t h a t the ranges of s e n s i t i v i t i e s spans the estimated l e v e l s o f c u p r i c i o n a c t i v i t y i n n a t u r a l seawater*.  This  indicate  on  that  one  of  the  effects  of  copper  would natural  H9  communities  could  composition,  be  to  cause  a  shift  in  the  species  though not n e c e s s a r i l y a decrease i n p r o d u c t i v i t y .  This i s what has been observed i n the CEPEX experiments et the  al,  1977;  effects  Thomas and S i e b e r t , of  phytoplankton  copper  on  population  composition but l i t t l e  the  1977), where o b s e r v a t i o n s on community  showed  major  could  have  structure  of  important  the  changes i n the s p e c i e s  change i n primary p r o d u c t i v i t y or  between the c o n t r o l s and the copper changes  (Thomas  treated  biomass  enclosures.  Such  e f f e c t s on higher l e v e l s of the  food chain through such f a c t o r s as food preference and  differing  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 a l s o i n t e r e s t i n g to note that the two most species  are  the  pennate  d i n o f l a g e l l a t e Gonyaulax Some  previous  have suggested sensitive  N i t z s c h i a l o n q i s s i m a and the  tamarensis (Anderson and Morel,  comparisons that  than  diatom  of  are  (Mandelli,  to  be  a  1969;  The present study does not support such a pennate  great  deal  sensitive  than  the  generalization.  dinoflagellate,  more  sensitive  than  T h a l a s s i o s j r a pseudonana and Skeletonema  the  centric  costatum. .  The  appears  Gonyaulax  tamarensis, or the blue green a l g a , O s c i l l a t o r i a - t h e i b a u t i i , considerably  more  Thomas et a l , 1977).  diatom s p e c i e s used here, N i t z s c h i a l o n g i s s i m a , more  1978).  s p e c i e s s e n s i t i v i t i e s to copper  dinoflagellates  diatoms  sensitive  and  diatoms,  50 CHAPTER  3  C o n d i t i o n i n g 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 t r a c e copper, studies Sunda  in  metals,  especially  seawater, depends on the metal s p e c i a t i o n .  (Sunda and G u i l l a r d , and  Lewis,  1978)  1976;  have  s p e c i e s i s the c u p r i c i o n , with  Anderson  indicated the  and  Morel,  that  various  Several 1978;  the most t o x i c  inorganically  or  o r g a n i c a l l y bound s p e c i e s being e i t h e r non-toxic or c o n s i d e r a b l y less  toxic*  i . e . through  Any the  reduction addition  in of  the c u p r i c i o n c o n c e n t r a t i o n ,  organic  compounds  capable  of  complexing copper, w i l l r e s u l t i n a r e d u c t i o n i n the t o x i c i t y  of  the copper i n the medium. If  o r g a n i c complexation does not take place, the l e v e l s of  c u p r i c ion n a t u r a l l y present i n seawater have been c a l c u l a t e d to be high enough t o cause a r e d u c t i o n  i n growth i n s e v e r a l  of phytoplankton (Sunda and G u i l l a r d , 1976; 1978;  LeBlanc,  species  Anderson and  Chapter 2; Sunda, pers* comm*).  Morel,  The presence  n a t u r a l d i s s o l v e d o r g a n i c compounds capable of complexing could be important i n c o n t r o l l i n g copper  copper  toxicity.  The amount of copper present i n the o r g a n i c a l l y bound varies  in  different  Schmidt,  complexing  capacity  electrodes  (Williams  voltammetry  more  of  the  and B a t l e y , 1977a; Mantoura  W i l l i a m s , 1969;  (Florence  form  seawaters, but has been measured to be i n  the range of 6% to 40% or (Florence  of  1978 a and  of  total  copper  e t a l , 1978;  b).  present  Smith,  Measurements  of  1976; the  seawater as determined by s p e c i f i c i o n  and and  Baldwin, Batley,  1976) ,  anodic  stripping  1977b) and b i o l o g i c a l assay  51 ( G i l l e s p i e and Vaccaro, 1978) capacity  is  greatly  have  reduced  shown  that  when u l t r a  the  complexing  violet irradiation i s  used to o x i d i z e the d i s s o l v e d o r g a n i c compounds present  in  the  seawater. Field  studies  have  found  that i n some n a t u r a l u p w e l l i n g  s i t u a t i o n s , where t h e r e are  low  compounds  phytoplankton growth w i l l  in  the seawater,  be depressed greatly  levels  (Barber and Ryther,  of  1969).  organic initially  The growth r a t e can  be  i n c r e a s e d d u r i n g t h i s i n i t i a l p e r i o d by the a d d i t i o n of  either  EDTA,  zooplankton  an  artificial  extract.  chelator,  Barber  or  and Ryther  that n a t u r a l o r g a n i c c h e l a t o r s , r e l e a s e d water  dissolved  ages  at  the  s u r f a c e , may  a  homogenized  (1969) have suggested by  organisms  as  the  be p a r t l y r e s p o n s i b l e f o r the  i n c r e a s e d phytoplankton growth away from the u p w e l l i n g r e g i o n . , Phytoplankton are source  of  considered  if  producing  Thomas, 1971)  phytoplankton  compounds  and  it  species  capable  are  of  be  possible  production  is  of  the  (Anderson  capable  of d i r e c t l y trace  and r e l e a s e of copper  necessary  that  changing the complexing reduced  the  cupric  any  on  toxicity  bioassays oceans.,  bioassay s p e c i e s not be capable o f  c a p a c i t y of the medium*  ion  metal  complexing  designed t o a i d i n the study of copper s p e c i a t i o n i n the is  to  speciation..  compounds i s a l s o of importance:to work  It  main  interest  affecting  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 The  to  the d i s s o l v e d o r g a n i c matter i n the oceans  and Z e u t s c h e l , 1970; determine  generally  activity,  and  Changes  therefore  the  which copper  t o x i c i t y , would i n v a l i d a t e the assay. It  has been suggested that there are a t l e a s t two  different  52 types of phyotoplankton response to copper t o x i c i t y Morgan, 1978)* growth  The f i r s t  type of response  i s a decrease i n  the  r a t e with i n c r e a s i n g copper c o n c e n t r a t i o n s , a d e p r e s s i o n  which does not change markedly response  is  a  lag  i n c r e a s i n g copper growth  at  a  phase  with time* in  The  growth  concentration* rate  concentration; for  (Jackson and  which  followed  indifferent  to  costatum  1970),  the  diatom  Wium-Andersen,  (Barber, 1973;  by  an  Huntsman and Barber,  with  initial  copper reported  (Steemann N i e l s e n  Nitzschia  1971),  of  exponential  This type of l a g phase e f f e c t has been  Wium-Andersen, and  type  increases  the  the green algae C h l o r e l l a pyrenoidgsa  Nielsen  second  palea  and  (Steemann  s e v e r a l Chaetoceros s p e c i e s 1975)  and  in  Skeletonema  (Morel et a l , 1978).  I t i s b e l i e v e d t h a t those phytoplankton s p e c i e s showing the second  type  of  response  could be e x c r e t i n g o r g a n i c compounds  which c o n d i t i o n the medium by complexing s o l u t i o n and t h e r e f o r e decrease Steemann  Nielsen  and  of  of copper..  toxicity  Wium-Andersen  both N. g a l e a and S. costatum presence  the  the f r e e c u p r i c i o n s i n the  medium.  (1971) have r e p o r t e d t h a t  excrete  organic  matter  in  Swallow et a l (1978), however, found  the that,  e i g h t marine and f r e s h water phytoplankton s p e c i e s t e s t e d f o r  their  ability  complexing  to  ionic  excrete copper,  organic only  one  a f f e c t i n g the f r e e i o n c o n c e n t r a t i o n . that  substances species  was  It i s also  capable capable worth  of  the  authors  (McKnight,  1978)  of of  noting  the e i g h t s p e c i e s were s e l e c t e d f o r t h e i r known a b i l i t y  r e l e a s e l a r g e q u a n t i t i e s of o r g a n i c m a t e r i a l s * one  of  F u r t h e r work  to by  using a more s e n s i t i v e  methodology, showed t h a t of 14 s p e c i e s t e s t e d , 9 were capable of  53 producing copper complexing Huntsman conditioned  and by  Barber  the  and  l e n g t h of the i n i t i a l  (1975)  have  found  that  a  medium  growth of phytoplankton could c o n t a i n  i n h i b i t i n g compounds which phytoplankton,  agents.  affected  stimulating  growth  compounds  l a g phase.  rate  which  of  the  a f f e c t e d the  G n a s s i a - B a r e l l i et  have found that media c o n d i t i o n e d phytoplankton s p e c i e s could  the  both  al  (1978)  by the growth of s i x d i f f e r e n t  decrease the t o x i c i t y of copper to a  Haptophycean bioassay s p e c i e s , C r i e g s e h a e r a e l o n q a t a . This  study w i l l attempt to determine  Nitzschia longissima, can  affect  the  i s capable of  toxicity  e f f e c t s on both the i n i t i a l investigated  i n t h i s study*  of  producing  copper  lag  i f the marine  phase  in and  diatom,  compounds  sea water* growth  which  Possible rate  were  54 Method The  bioassay  procedure was the same as t h a t i n chapter 1.  N i t z s c h i a l q n q i s s i m a was chosen as a b i o a s s a y basis  of i t s s e n s i t i v i t y ,  ease o f handling*  3  Seawater  artificial medium* the  or  natural  The water was enriched with Chapter  complexing  1,  agents  concentration  was  was  31.1%>;  0.9  mg  c o n c e n t r a t i o n was 1.1x10  1  the  _ 1  ;  Table  were  The s a l i n i t y of both of the seawater  experiments  the  f o r the experiments was c o l l e c t e d i n  and v i t a m i n s as i n  3  oa  c o n s i s t e n c y o f response t o copper and  Jan..and March, 1978 from Geo 1748. N0 , PO^, S i 0  organism  added  the  No  to the  samples  dissolved  and  1^2.  used  organic  in  carbon  dissolved  copper  M.  -8  C o n d i t i o n i n g of Media To  determine  i f the  c o n d i t i o n i n g i t s medium, N. i n the standard (Chap. was  added  to  bioassay  species  any  possible  at  the  and  the  filtration  filtrate  another bioassay.  total  copper  (0.45  or  The  copper  of  copper  inhibition  um  cellulose  in  acetate  medium) was then  More copper was added  to  give  c o n c e n t r a t i o n s of 1,6, 3.2 and 7*9x10  At the same time more experimental fresh  major  (the 'conditioned*  used t o s t a r t added  any way  end of the t h r e e day c o n d i t i o n i n g p e r i o a , the  c e l l s were removed by filter)  M Cu..  -a  production  d e t o x i f y i n g compounds, but not t o cause a growth.  in  Longissima was grown f o r t h r e e days  1) medium + 1.6x10  stimulate  was  'unconditioned*  medium  seawater,  c o n c e n t r a t i o n s of 0, 1.6, 3.2 and 7.9x10~  was  prepared  with 8  M*  added an  -8  M.  using copper  inoculum  of  N. l o n g i s s i m a was added and the b i o a s s a y s were monitored f o r the normal f o u r day p e r i o d , and growth r a t e s determined by the u s u a l  55  methods (see chapter  1).  E f f e c t of C e l l Inoculum S i z e The  bioassay  1 with  the  exceptions t h a t the bioassays were monitored  f o r e i g h t days,  not  f o u r , and the i n i t i a l c e l l  inoculum  were,  1000,  ml-.  100  and  procedures  10  cells  were  as  in  sizes  Because  Chapter  of  approximately  difficulties  o b t a i n i n g accurate c e l l counts at the lower c o n c e n t r a t i o n s , result  of the bioassay was  using a Turner Model 111 Fluorometer*  estimated  were  10,  rather  1  and 0*1  than  detection  limits  determined  as  of  in  the  followed u s i n g f l u o r e s c e n c e r e a d i n g s  r a t h e r than the c e l l numbers., F l u o r e s c e n c e readings  readings  in  u n i t s * . The  measured the  Chapter  The  because  fluorometer* 1,  from  the  were  made  starting fluorescence last figure it  (0.1) i s  was  below  the  Growth  rates  were  change  in  log  l 0  f l u o r e s c e n c e with time f o r the e x p o n e n t i a l p o r t i o n of the growth curve  and  the  determined  from  intercepts  gave  intercepts the the  with  eguation length  of of  the i n i t i a l f l u o r e s c e n c e were the  regression  the i n i t i a l  line.  The  l a g p e r i o d of the  c u l t u r e before the s t a r t of e x p o n e n t i a l growth..  56  Results C o n d i t i o n i n g experiment Two  bioassays  bioassay  species,  were c a r r i e d N.  out  to  Longissima,  determine  whether  could change i t s medium  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. or ' c o n d i t i o n i n g ' of the  medium  would  the  Bioassays together are  concentration  presumably  be  due  in Figs.  control culture  3.1  (0 added  and  and 3.2,  copper)  fluorescence;growth respectively.  for  because i t had  already  had  1.6x10  conditioning  period  to  stimulate  the  the  compounds.  difference  between  e i t h e r the c o n d i t i o n e d  longissima  during  There i s no  conditioned  excretion  organic  that  rates  medium  N of copper added during  -8  complexing  medium, i n d i c a t i n g  could  the c o n d i t i o n e d medium were run  the r e s u l t s f o r c e l l  presented  to  of the most t o x i c s p e c i e s of copper*  using f r e s h medium and and  and  Such changes  e x c r e t i o n of organic compounds, which through complexation reduce  the  There  is  of  the  copper  no  significant  or the  unconditioned  exponential  growth  Nitzschia  does not n o t i c e a b l y change or c o n d i t i o n i t s medium to  reduce copper t o x i c i t y . Lag time experiment A  second  experiment  was  c a r r i e d out to determine whether  N. l o n g i s s i m a c o u l d c o n d i t i o n i t s medium. experimental  cultures  must  i n some way  I f the c e l l s  in  the  ' c o n d i t i o n * the medium  through the e x c r e t i o n of organic compounds before they can  begin  e x p o n e n t i a l growth, the length of the i n i t i a l l a g time should  be  p r o p o r t i o n a l to the c o n c e n t r a t i o n of the t o x i c f a c t o r which must be overcome by the should  also  'conditioning', i . e .  copper.. The  be p r o p o r t i o n a l t o the i n i t i a l c e l l  lag  phase  concentration.  This  second  experiment  s i z e s t o determine effect  o f t h e added  of the experiment* concentration there growth  i s no  used  varying  whether N* copper. Neither  affects  significant  r a t e at the three  the  c o p p e r and  initial  inoculum  1on^issima-was modifying the t o x i c F i g s . . 3.3  and  3.4  copper c o n c e n t r a t i o n length  show t h e  or i n i t i a l  of the i n i t i a l  d i f f e r e n c e i n the e f f e c t inoculum s i z e s . _  results  l a g phase  copper  has  cell and on  58  Figs.  3.1  fluorescence unconditioned  and  3.2:  growth (•)  The  rates and  effect (in  conditioned  l i m i t s a r e . g i v e n f o r each p o i n t .  of  copper  on  doublings  per  (o)  95 %  media._  cell  and  day)  in  confidence  4 Added Fig.  Added Fig.  Copper  8 (x10~  8  W,  3-1  Copper 3-2  ( x 1 0  u  f s /  60  Figs. rate  3.3 and 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  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 a t  three i n i t i a l inoculum s i z e s . f o r the growth  rates.  9 5 % confidence  l i m i t s are  given  0  4 Added  8  Copper (X10" M) 8  Fig.  3-4  62  Discussion The  occurrence  of  copper  shown to be of importance  complexing  compounds has been  i n c o n t r o l l i n g phytoplankton growth i n  some areas  (Barber and Ryther, 1969;  of  o r g a n i c compounds r e s p o n s i b l e f o r copper  those  i n seawater made  as  Smayda, 1974).  the  to the expected copper complexing  structures  of  copper  the  cells  Jackson and Morgan in  compounds.  are  the  the  A  will  to produce  (McKnight,  1978;  a c o m p e t i t i v e advantage copper might be  knowledge  the  If  of  actively  be  less  important  capable of  the  not  growing  copper complexing  Swallow et a l , 1978)  f o r some  species  in  by  effects  because  overcoming  the it.  agents i s s p e c i e s i t might  indicate  situations  where  inhibitory*  copper  complexing  compounds are the r e s u l t of the  degradation of dead phytoplankton c e l l s , then the would  be  seawater  (1978), then the p o s s i b l e i n h i b i t o r y  seawater  ability  specific  If  c a p a c i t y of  can  source, as has been suggested  phytoplankton s p e c i e s are themselves If  complexation  of the compounds w i l l a l s o a i d i n the i d e n t i f i c a t i o n of  phytoplankton  of  source  must be known before meaningful p r e d i c t i o n s  at d i f f e r e n t times or i n d i f f e r e n t areas. sources  The  phytoplankton  be able to e f f e c t i v e l y c o n d i t i o n t h e i r medium, or a t  l e a s t such c o n d i t i o n i n g must occur at a much slower pace than i f the c e l l s were capable of a c t i v e l y e x c r e t i n g such compounds*. The f a c t t h a t t h i s study has found N i t z s c h i a l o n g i s s i m a be  incapable  of  conditioning  t e s t e d , does not i n d i c a t e that such Barber  a  capacity.  to  i t s medium under the c o n d i t i o n s phytoplankton  The s t u d i e s of McKnight  (1975) and G n a s s i a - B a r e l l i et  al  in  general  lack  (1978), Huntsman and  (1978)  indicate  that  some  phytoplantkon  effect  of  copper  Nitzschiasensitive an  indication Lack  evidence  of an  has  are a  capable of reducing the t o x i c  conditioning been  shown  of  to  their  be  exceptionally  ( C h a p t e r s 1 and 2) w h i c h m i g h t an  be  taken  as  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  sensitivity, ability  constatum  medium.  to  however,  cannot  be  condition  the  growth  has been shown t o be v e r y  taken  as  medium.  insensitive  to  ( C h a p t e r 1; M o r e l e t a l , 1978) b u t has been f o u n d n o t t o  condition an  of  of  Skeletonema copper  through  longissima t o copper  copper*  species  i t s medium i n r e s p o n s e t o c o p p e r  ability  controlling  to  condition  media  cannot  inhibition, be  the  phytoplankton s e n s i t i v i t y t o copper. _  sole  so  that factor  CHAPTER 4 P h y s i o l o g i c a l E f f e c t s Of Copper T o x i c i t y In The  Marine Diatom,  Nitzschia longissima Introduction The e f f e c t well  of heavy metals on phytoplankton growth  understood.  Different  d i f f e r i n g values f o r recently  been  heavy  experiments  metal  recognized  that  have  toxicity both  e f f e c t on phytoplankton*  found  and  it  in  et  al,  in  widely  has  only  determining  Copper has been found to be  of the most important and most i n t e r e s t i n g of the (Hollibaugh  not  the t o t a l amount and the  chemical s p e c i a t i o n of the metals are important their  is  prep..b)  necessary f o r phytoplankton growth  and  heavy  metals  i s both a m i c r o n u t r i e n t  (Manahan and Smith,  a t o x i c agent which can cause reduced  one  phytoplankton  1973)  and  growth  at  extremely low c o n c e n t r a t i o n s (Steeman N i e l s e n and Wium-Andersen, 1970;  Davey et a l , 1 973),. An  area  of i n c r e a s i n g 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* widely  in  variation  species  are  not  at  present  known.  Aside: from  i t i s also possible that p h y s i o l o g i c a l  can cause v a r i a t i o n s species*.  in  the  effect  copper  indicator  be,  at  have  on  a  The e f f e c t of copper on c e l l growth r a t e s  least  of copper t o x i c i t y  been only a few affected*  in  species  differences  will  has been the most o f t e n used i n d i c a t o r of t o x i c i t y , found to  vary  t h e i r s e n s i t i v i t y t o copper and the reasons f o r t h i s  differences,  particular  Phytoplankton  some  cases,  the  and has been  most  There  have  s t u d i e s of other p h y s i o l o g i c a l systems t h a t  are  Anderson  and  (Berland et a l , 1977).  sensitive  Morel  (1978) found the m o t i l i t y of a  65 marine d i n o f l a g e l l a t e t o be indicator  of  copper  several studies as  has  the  (Saifallah,  an  extremely  toxicity*  sensitive  Uptake of  (Anderson and Morel,  l 4  and  fast  C has been used i n  1978; Berland  et a l ,  r a t e of c h l o r o p h y l l 'a' (or f l u o r e s c e n c e )  1977)  increase  1977), c h l o r o p h y l l »a' c o n c e n t r a t i o n 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,  particulate evolution  carbon  and  ( O v e r n e l l , 1976), n i t r a t e  synthesis  (or a c t i v i t y )  c o n c e n t r a t i o n per c e l l the  nitrogen  various  (Berland e t a l , 1977), oxygen uptake,  nitrate  (Harrison and Davies,  (Sunda and G u i l l a r d ,  e f f e c t s o f copper t o x i c i t y  1977) and copper  1976).  an  explanation  Studies  of  w i l l a i d i n determining  the mechanism (s) of the t o x i c a c t i o n of copper, which to  reductase  may  lead  f o r the v a r y i n g 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 of  copper  longissima.  w i l l look a t some of the  toxicity  in  the  physiological  bioassay  species,  In p a r t i c u l a r , mention w i l l be made o f  effects  Nitzschia effects  on  the r a t e o f c e l l d i v i s i o n , the r a t e of i n c r e a s e of f l u o r e s c e n c e , cell  volume,  f l u o r e s c e n c e / c e l l , **C uptake and t h e a c t i v i t y o f  the enzyme n i t r a t e  reductase.  66  Method The marine diatom, N i t z s c h i a bioassay  species  because  s e n s i t i v i t y to copper the  species  was  longissima,  of  i t s previously  (see Chapter 1).. . A  obtained  C o l l e c t i o n * . Seawater  from  seawater was  unialgal  used  as  a  demonstrated culture  the Northeast P a c i f i c  f o r the bioassay experiments was  i n December, 1977 and March, 1978, The  was  of  Culture  collected  and t r e a t e d as i n Chapter  1.  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)  provide  non  at  levels  high  enough  to  l i m i t i n g amounts of the major n u t r i e n t s but not t o  g r e a t l y exceed the n u t r i e n t c o n d i t i o n s t h a t marine phytoplankton encounter i n the oceans. copper  stock  solution  d e i o n i z e d water* figures  are  Copper made  was  with  added  from  CuCl^  a  and double  A l l of the copper l e v e l s r e f e r r e d  added  copper  23.6x10~  6  M  distilled  to  in  the  c o n c e n t r a t i o n s , not t o t a l copper or  i o n i c copper c o n c e n t r a t i o n s . _  Other c o n d i t i o n s  for  the  medium  p r e p a r a t i o n are as d e s c r i b e d i n Chapter 1. The  b i o a s s a y s were run f o r 4 days with samples being taken  daily for cell fluorescence  counts readings  (using Palmer-Maloney (using  C e l l s i z e s were measured f o r Chapter  1,  specific  growth  curve.  the  third  rates  day  l o g >o  cell  determined  by  condition*  As  concentration  vs* time f o r the e x p o n e n t i a l p o r t i o n of the  These s p e c i f i c  each t e s t  samples*  in  were determined by a l e a s t  growth r a t e s were changed  day by an a r i t h m e t i c conversion ( G u i l l a r d , were  chambers),  a Turner Model 111 F l u o r o m e t e r ) .  squares l i n e a r r e g r e s s i o n of the fluorescence  counting  or  growth  t o d o u b l i n g per  1973)..  Cell  sizes  v i s u a l measurement of at l e a s t 30 c e l l s at  67 Enzyme  Activity  Material the  Measurements  f o r t h e enzyme.assays  bioassay  cultures  at  the  was  end  obtained  of  the  N i t r a t e r e d u c t a s e was a s s a y e d by m e a s u r i n g formation the  of  nitrite  modification  were  as  that  described  as d e s c r i b e d t h e KN0 , 3  in  were i n c u b a t e d f o r 45 m i n u t e s . run  f o r each  sample*  sample*  Two  condition,  making a t l e a s t  concentration the e n t i r e the and  (  0,  first  assay,  four  the t e s t e d  were  7.9x10-8  M Cu s a m p l e ,  therefore pooled  Two r e p l i c a t e t o give  invalid  enough  in  cultures  were  enzyme  assays - 8  i s based  only  t h e second  Chapter  ml-*. hour^ and  extracts  enzyme a s s a y s were of the  r u n f o r each of  each  M Cu)._  test  copper  In a d d i t i o n ,  the r e s u l t s .  0, 1.6 and 7 . 9 x 1 0 ~  the: second  f o r the assay. on 2 enzyme  the  In  The a c t i v i t y  M Cu M  CU  For the  f l a s k s had t o be  assay  8  3.15x10-8  experiment).  pooled  measurement  replicates  from  one  experiments  described  Measurements  Medium was p r e p a r e d a s f o r t h e g r o w t h  was  concentrations  sample.  i * C Uptake  in  with  c o p p e r c o n c e n t r a t i o n s were 0, 1.6  t h e two r e p l i c a t e  cells  dependent  the a c t i v i t y  (due t o an e x p e r i m e n t a l e r r o r  results  NADH  NADH  was r u n t w i c e t o c o n f i r m  3.15x10-8 M Cu, w h i l e . i n  give  the  and  1.6, 3.2 and 7 . 9 x 1 0  experiment  were t e s t e d  to  test  4 day b i o a s s a y s .  (1973) and t h e enzyme  then averaged  separate  filtering  i n E p p l e y e t a l (1969)  MgSO^  Harrison  by  added  1.  An i n o c u l u m  to  give  The c u l t u r e s  an i n i t i a l  dark..  an e x p o n e n t i a l l y cell  growing  concentration  were i n c u b a t e d w i t h t h e added  then t r a n s f e r r e d one  from  t o three  125 ml BOD  0.5 mis o f a **C l a b e l e d  o f 2000 copper  bottles, NaHCO  s  culture cells  f o r one  two  light  s o l u t i o n (1.25  68 uCi) was added and then the b o t t l e s were incubated at 15 C and a light  i n t e n s i t y of 80 uE m~  were  filtered  2  scintillation determined  and  fluid in on  a  Photosynthetic rates and Parsons  the  (1972).  a  Dnilux  sec  filter vial. III  - 1  f o r two hours;  The  cultures  placed i n 15 mis of S c i n t i V e r s e Activity Liquid  were c a l c u l a t e d  in  the  Scintillation  as described  in  vials  was  Counter. Strickland  69 Eesults Growth Eate Growth  rate  concentrations 1.3,  is  severely  of copper*  a f f e c t e d by the a d d i t i o n of low  i s can be seen i n  Chapter  1.4 and 1.12., the a d d i t i o n of 1.6 or 3.2x10  a s i g n i f i c a n t depression numbers  and  fluorescence  fluorescence. increase  cell  It  Further  numbers,  the  differences  less  M Cu causes  -8  of  interesting  Figs.  both  to  affected  cell  note  that  than  the  i n v e s t i g a t i o n of the r e l a t i o n s h i p  fluorescence  d i f f e r e n c e s i n the i n c r e a s e with  is  i s consistently  c e l l number i n c r e a s e . between  i n the rate of i n c r e a s e  1,  of  cell  and added copper, i n d i c a t e number  increasing  and  with  fluorescence,  increasing  copper  concentration. Fluorescence increasing exposure!.  per  copper Figs.  remained r e l a t i v e l y  -8  over  f o u r t h day 7.9x10~ The  1.6x10~  fluorescence/cell adaptation change the  increase: with  ratio*  s t a b l e over  8  control  the  course  (0 t o 4 (0 of  added copper c o n d i t i o n s  double 8  The  days)  in  on  added copper) the  four  day  showed a g r a d u a l  M Cu treatment. M  Cu  after  condition the  third  showed  a  decrease  in  day, i n d i c a t i n g a p o s s i b l e added  copper.  The  a l s o be a t t r i b u t e d to a change i n the t o x i c i t y of  medium, but other experiments (see chapter 3) have found  decrease  with  the c o n t r o l value i n the case o f the  on the part of the c e l l s t o the  could  both  i n c r e a s i n g time o f  M Cu) and exposure time  bioassay, but the three to  and  an  4.1 t o 4*4 show the e f f e c t of added copper (0,  fluorescence/cell  increase  showed  concentration  1.6, 3.2 and 7.9x10 the  cell  the t o x i c i t y o f the medium a f t e r t h r e e  no  days growth  70 of phytoplankton The  c e l l s in i t .  3.15x10-  M  8  fluorescence/cell  Cu  additions  between  the  third  i n d i c a t i n g a p o s s i b l e adaptation There  is  no  such  such a change  shows  on  could  have  been  and the  change at 7.9x10  no  _8  increase  fourth part  in  days,  of  also  the  cells.  M C U , but i t i s p o s s i b l e  observed  if  the  assay  were  followed f o r a longer period of time. Although  it  would  at  first  appear that f l u o r e s c e n c e i s  being g r e a t l y a f f e c t e d by copper, when the f l u o r e s c e n c e / c e l l converted no  to  f l u o r e s e n c e / c e l l volume (see F i g . , 4.5),  increase,.  In  fact,  there  is  a  significant  concentration*  When examined i n l i g h t of the e f f e c t  cell  numbers  it  is  of those  increasing  not copper  of  copper  apparent that the t o x i c a c t i o n of  copper i s on c e l l d i v i s i o n and cell  with  there i s  though  statistically  on  decrease  slight,;  not  the  production  is  within  the the  c h l o r o p h y l l pigments r e s p o n s i b l e f o r f l u o r e s c e n c e .  Enzyme Assay The  changes  in  apparent  n i t r a t e reductase  added copper c o n c e n t r a t i o n i s shown i n F i g . N i t r a t e reductase copper,  with  causing a 500%  activity  the  4.6  activity  and  with  Table  4.1.  appears t o be very s e n s i t i v e to added  smallest  copper  addition,  1.6x10  M CU,  -8  i n c r e a s e i n the apparent enzyme a c t i v i t y , and  a c t i v i t y a t the highest copper  concentration,  7.9x10  -8  M  the CU,  being an order of magnitude g r e a t e r than the c o n t r o l . i*C The **C  Uptake effect  of  added copper on the rate of  a s s i m i l a t i o n i s given i n Table 4.2.  per c e l l shows a s m a l l  decrease  with  The added  photosynthetic  photosynthetic copper,  but  rate the  effect  i s not  as  great  as  that  shown by  growth  rate.  T a b l e 4.1:  The e f f e c t  enzyme n i t r a t e  o f added  reductase  c o p p e r on t h e a c t i v i t y  i n the marine diatom,  of the  Nitzschia  longissima.  Added Cu C e l l X10-8  M  Cone. _  (X10+6  i - i )  Enz. (un  Act. i - i h-i)  per  (uM h -  1  0  247  1.07  4.33  0  46.6  .246  5. 28  0  41.2  .166  4.04  0  49.6  .315  6.35  1.6  23.2  .525  22.7  1. 6  26.9  .512  19.5  1.6  19.3  .501  2 5.9  1.6  18.9  .505  26.6  1.6  20.5  .499  24.3  avg.  Cell 10«  1  avg*  23.80±2;8 4  3.2  8.33  .289  34. 2  3.2  7.94  .185  24.6  avg. 7. 9  1  Act.  29.4±6.79 2.34  ± one s t a n d a r d  *094  deviation  40.3  cells)  Table  4.2: T h e e f f e c t  o f added copper  on t h e r a t e o f  photosynthesis  Added Cu  Photosynthesis  (X10-8  (mgC  M)  m  - 3  h  _ 1  % of Control  )  0  3. 103  1.6  2.520  81.2  3. 2  2.223  71,. 6  7.9  1.917  61,. 8  100  74  Figs,  4.1  Nitzschia  to  4 . 4 : Changes i n f l u o r e s c e n c e / c e l l of t h e  longissima,  during  copper c o n c e n t r a t i o n s * . deviation*  Error  the  f o u r day  bars  bioassay  represent  ±  diatom,  at four one  added  standard  _  10  10|  I  to  u  Ll  5l OCu  1.6  Cu 2  Days Fig. 4 - 2  7.9  Cu 2 Days  Fig.  4-4  76  Fig.,  4.5:  E f f e c t of added copper on f l u o r e s c e n c e / c e l l  fluorescence/cell  volume (•)  E r r o r bars represent ± one  on  the  standard  third  deviation.  day  of  (o)  and  bioassay.  78  Fig  4.6: E f f e c t  enzyme n i t r a t e replicates.  o f added  reductase.  c o p p e r on Each  the a p p a r e n t a c t i v i t y  point  is  the  average  of the of  two  40  30  20  10  Ol 0 Added  8 r-8 C o p p e r (x10 ° M ) Fig.  4-6  80  Discussion Studies  of  phytoplankton for the  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  s p e c i e s can help i n determining  v a r y i n g phytoplankton actual  The  the  of  the  toxic  action  of  r a t e of c e l l d i v i s i o n i s one  been  of  the  (ie; rate  found i n at l e a s t some cases  study  was  found  increase  of  c e l l d i v i s i o n i s one  levels in t h i s species; the  concentration  of  copper  often  It  (Berland e t a l , 1977)  to  cell  volume.  A  volume showed l i t t l e concentrations, production  provide evidence  Fluorescence  keep  comparison  less  harmful  the  uptake.  inhibition  difference  copper  between  little of  fluorescence.  an i n d i c a t o r  concentration,  of the f l u o r e s c e n c e per the  different  e f f e c t by the  those  and  1  Rachlin cell  production,  i t s primary e f f e c t on c e l l  of was as cell  copper  copper on  photosynthetic  Rosko  pace with c h l o r o p h y l l 'a  copper exserted  the  than  pigments i n the c e l l ,  observed i n copper s t r e s s e d C h l p r e l l a , t h a t not  that the  per c e l l ,  photosynthetic  indicating  for  in  of the primary e f f e c t s of excess copper  w i t h i n the c e l l  responsible  and  or the r a t e of **C  observed t o i n c r e a s e with i n c r e a s i n g did  used  species.  concentrations)  fluorescence  S e v e r a l other o b s e r v a t i o n s of  on  to be more s e n s i t i v e to added copper  t o be a f f e c t e d at lower of  copper  most  be the.most s e n s i t i v e i n d i c a t o r of copper t o x i c i t y present  and  i n general.  i n d i c a t o r s of the e f f e c t of copper on phytoplankton has  reasons  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  mechanism  phytoplankton  both  the  compounds (1977) have  division  did  indicating  that  d i v i s i o n and  e f f e c t on c h l o r o p h y l l 'a' s y n t h e s i s .  An  a  much  increase  i n c e l l volume with i n c r e a s i n g copper c o n c e n t r a t i o n as found  in  81 this  study  (Erickson Eosko  has  also  been  e t a l , 1970;  and  Rachlin,  found  i n a number  Steemann N i e l s e n 1977;  Horel  and  of  other  Ramp-Nielsen*  e t a l , 1978)  and  other  (Thalassiosira  pseudonana,  Chlore11a  Eirenoidosa,  vulgaris  Skeletonema  costatum,  respectively),  and  (1977) f o u n d could  t h a t low  cause  abnormally  liebethrutti. copper  did  systematic cell  levels  an  decrease  in  non-dividing  necessarily Riisgard  Dunaliella copper  increase  in  The cell  however, t h a t t h e of the  cell  1  3  copper  contents  solely  by  growth  not  an  the  to  t o an  rate,  increases  the in  pigment  regulate  found  of c e l l marine  but  in  was  inhibition  was  study not  the  division.  flagellate, at  due  to a  elevated membrane with  the  dilution  division*  strongly  effect  volume.  enzyme a c t i v i t y  was  The  reductase  affected an  cannot  i s not  not  would i n d i c a t e ,  of c e l l  This increase  nitrate  copper  enzyme i n t h e . c o n v e r s i o n more  cell  per  volume i s  volume  in this  a  concentration  to changes i n c e l l  important  1975),  increase  (1970)  euryhaline  due  although  concentration  1  inhibition  volume i n c r e a s e  an  •a  in f l u o r e s c e n c e / c e l l along  inhibition*  c o p p e r ' s i n f l u e n c e on large  the  probably  but  (Falkowski,  than  activity,  that  an  tin  diatom N i t z s c h i a  A change i n c e l l  volume o b s e r v e d  N i t r a t e reductase, N0 " t o NH^ "  of  increase  cell  Saboski  volume, i t a l s o c a u s e d  photosynthetic  marina, i s unable  permeability*  the  Gross et a l  cells.  found  concentrations,  in c e l l  and  indication  (1979) has  Chlorella  metals mercury or  in  chlorophyll  i n the  Chlore11a  an  cells  increase  i n Skeletonema costatum  cause a r e d u c t i o n  trace  1970; species  (1978), however, f o u n d t h a t  i n the  to  and  large  Morel e t a l cause  of the  studies  increase  be  of by in  explained  mechanism  c l e a r but activity  of  rapid in  82  phytoplankton limitation possible cell, the  been r e p o r t e d i n response t o i n c r e a s i n g NO^  (Eppley and Renger, 1974; copper  could  Harrison,  1973)*  cell,  It i s  be a f f e c t i n g the uptake o f N0 ~ i n t o t h e 3  thereby d e c r e a s i n g the apparent c o n c e n t r a t i o n  activity. in  have  of  N0 ~ to 3  which c o u l d l e a d t o an i n c r e a s e i n n i t r a t e  reductase  H a r r i s o n e t a l (1977) have observed a l a r g e  decrease  NO^" a s s i m i l a t i o n on copper t r e a t e d phytoplankton p o p u l a t i o n s .  Several  field  and  l a b studies,  however,  have found  nitrate  reductase a c t i v i t y t o vary d i r e c t l y with the N0~" c o n c e n t r a t i o n 3  i n the media no  (Eppley e t a l , 1969; Eppley e t a l , 1970)..  conclusions  can  be  drawn  as  to  the cause  Although  of copper's  s t i m u l a t i o n of n i t r a t e reductase a c t i v i t y , the magnitude of the effect  makes i t an i n t e r e s t i n g area f o r f u r t h e r study.  In i  n  a study of the e f f e c t of heavy metals on enzyme a c t i v i t y  Selene  cucubalus  (Mathys,  1975),  reductase was an order o f magnitude than  glucose  magnitude  6  more  isocitrate  phosphate  i t was found that  more  sensative  dehydrogenase,  sensative  dehydrogenase.  than It  nitrate  to  copper  and s e v e r a l orders of  malate  dehydrogenase  and  should be noted t h a t the e f f e c t  observed by Mathys (1975) was a decrease  in activity,  not an  i n c r e a s e as found here* No  attempt  has  mechanism of copper observations  have  been  made  toxicity been  made  on  here marine  to  determine the exact  phytoplankton..  on which systems a r e most l i k e l y  d i r e c t l y a f f e c t e d by t h e t o x i c a c t i o n , namely c e l l nitrate  reducatase  activity  or  N0 ~ uptake, 3  p o s s i b l e areas f o r f u r t h e r p h y s i o l o g i c a l A f u r t h e r point might be  Some  mentioned  division  and  and  indicating  studies* concerning  ecological  83  and  physiological  studies  of  trace  metal e f f e c t s .  To  avoid  e i t h e r underestimating or o v e r e s t i m a t i n g the p o s s i b l e e f f e c t copper  on  natural  of  systems, i t would appear best not to u t i l i z e  only one parameter o f  effect,  s e v e r a l p h y s i o l o g i c a l systems.  but  determine  the  effects  on  84 General D i s c u s s i o n Studies  of the e f f e c t of t r a c e metals on phytoplankton can  be an important a i d i n determining  the  levels  c o n t r o l l i n g of phytoplankton  of  trace  metals  growth and community prediction  the  structure,  as  well  as  of  natural  helping  in  the  of the p o s s i b l e e f f e c t s of man-made p o l l u t i o n i n the  marine environment; that  in  importance  The s t u d i e s i n  Chapter  1  have  indicated  phytoplankton s p e c i e s can vary widely i n t h e i r response t o  low l e v e l s of copper, i n d i c a t i n g t h a t the presence of i n h i b i t i n g l e v e l s of copper, levels,  either  through  at  pollution,  could  levels  be  an  at  elevated  important f a c t o r i n  the  community.  T h i s supports the f i n d i n g s of s e v e r a l f i e l d  studies  which  reported  natural  phytoplankton of  composition  or  controlling  have  species  natural  the  addition  of  the.  of  copper  Ibragim and P a t i n ,  E q u i l i b r i u m c a l c u l a t i o n s , bioassays and f i e l d that  the  seawater c o u l d be Guillard, 1969;  levels  of  inhibiting  1976;  Anderson  in  and  chemical  or  bioassay  species,  relatively  an  some  situations  studies present (Sunda  Barber and  Measurement of the  have in and  Ryther,  amount  of  i n not p o s s i b l e , at present, with  indication  rapid  found  to  of  the  state  of  copper  T h i s study has developed a s e n s i t i v e  biological  seawater u s i n g the marine was  naturally  1976).  p h y s i c a l methods, but through the use of s e n s i t i v e  a v a i l a b i l i t y can be found.  species  copper  Morel, 1978;  Jackson and Morgan, 1978).  • a v a i l a b l e ' copper i n seawater  and  to  to cause l a r g e changes i n the s p e c i e s composition  the p o p u l a t i o n (Thomas et a l , 1977;  indicated  phytoplankton  diatom,  assay f o r copper a c t i v i t y i n Nitzschia  longissima.  be s t a b l e i n i t s response t o copper  This and  85 amenable t o handling of  the  species  i n laboratory  was  compared  conditions._  with 12 other  The  marine diatom and  d i n o f l a g e l l a t e s p e c i e s , and N i t z s c h i a l o n g i s s i m a relatively its  s e n s i t i v e t o copper but was by no  response  (Chapter  computer  with f o u r other and  study  equilibrium  was found to be  means  unusual  1), with s e v e r a l of the t e s t e d  the Chaetoceros s p e c i e s i n p a r t i c u l a r , being In a f u r t h e r  sensitivity  using  copper  in  species,  a f f e c t e d more.  buffered  media  and  a  model (MINEQL), t h i s s p e c i e s was compared  s p e c i e s t e s t e d under s i m i l a r  conditions  (Sunda  G u i l l a r d , 1976; Anderson and Morel, 1978; Morel e t a l , 1978;  Reuter et a l , 1979) and was found t o be a f f e c t e d a t a much lower calculated cupric ion concentration. Another  area  of  interest,  standpoint  and i n terms of using  species,  was  whether  both this  Nitzschia  from  an  species  environmental  as  longissima  was  a l t e r i n g i t s medium to reduce the t o x i c e f f e c t o f source . and  concentration  o f the organic  a  bioassay  capable copper.  of The  compounds i n the ocean  which could complex and d e t o x i f y t r a c e metals  is  unknown,  but  could  concentration  of  be  •available found  important 1  not  i t determining  or t o x i c copper s p e c i e s . to  alter  s p e c i e s are i n c a p a b l e  the  Nitzschia  longissima  was  i t s medium, i n d i c a t i n g t h a t a t l e a s t some o f r e l e a s i n g organic  chelators  to  reduce  copper t o x i c i t y . Observation copper (Chapter 4) before  of  the  physiological  indicate  that  cell  parameters a f f e c t e d by division  i s affected  o r more s t r o n g l y than carbon uptake or the production  photosynthetic  pigments*  reductase  s t r o n g l y a f f e c t e d by copper, but the mechanism of  is  The a c t i v i t y  of  the  enzyme  of  nitrate  86  t h i s a c t i o n i s unclear further  an  interesting  bioassay  lpngissima  species* sensative  has  been  shown  I t i s very s e n s i t i v e t o to l a b o r a t o r y  handling  not  change  its*  N,. . l o n g i s s i m a providing  has  a  copper  but  tested,  i s not  Within the  N. !ong_issima  medium t o reduce copper t o x i c i t y as some  s p e c i e s have been found t o do  in  for  t o be an e f f e c t i v e  conditions.  time p e r i o d and the copper c o n c e n t r a t i o n s does  area  research.  Nitzschia  overly  and r e p r e s e n t s  rapid  (Gnassio-Barelli growth  rate  and  et is  a r a p i d and s e n s i t i v e assay f o r copper  seawater, and could be a v a l u a b l e  a l , 1979). capable  of  availiabiltiy  t o o l i n f u r t h e r studies of  the e f f e c t of environmental and p o l l u t i o n a l l e v e l s of copper  in  different  be  seawater  c a r r i e d out to  determine  p o s s i b l y be i n c r e a s e d conditions  conditions.  Further  i f t h e . speed  research of  the; assay  or at higher  exposure  inhibiting low  light  to  temperatures.  Also, l i t t l e  several  trace  metals  or n u t r i e n t l e v e l s . . reseaurch*  i s known of  stesses  such  a t once o r exposure t o  l e v e l s of a t r a c e metal while a l s o being  areas f o r f u r t h e r  could  by growing c u l t u r e s under continuous l i g h t  the response of phytoplankton s p e c i e s t o m u l t i p l e as  might  limited  A l l of these represent  by  possible  87  BIBLIOGRAPHY Abdullah, M.I., Royle, L.G. and M o r r i s , A*W*. 1972.. Heavy metal c o n c e n t r a t i o n i n c o a s t a l waters. Nature 235: 158-160. Anderson, D.M. Gpnyaulax 295.  And Morel, F.M.M. 1978*. Copper s e n s i t i v i t y tamarensis. Limnol* Oceanogr* 28: 283-  of  Anderson, G.C. And Z e u t s c h e l , R.P.. 1970.. Release of d i s s o l v e d o r g a n i c matter by marine phytoplankton i n c o a s t a l and offshore areas of the northeast P a c i f i c Ocean. Limnol. Oceanogr.. 15: 402-407. 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