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The effect of manganese on the concentration of biologically available copper to the diatom, Thalassiosira… Kazumi, Junko 1985

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THE  T H E E F F E C T OF MANGANESE C O N C E N T R A T I O N OF B I O L O G I C A L L Y A V A I L A B L E TO T H E D I A T O M , T H A L A S S I O S I R A PSEUDONANA  COPPER  by JUNKO B.Sc,  The U n i v e r s i t y  A THESIS THE  KAZUMI of B r i t i s h  Columbia,  SUBMITTED IN PARTIAL FULFILMENT REQUIREMENTS MASTER  FOR OF  THE DEGREE  1982  OF  OF  SCIENCE  in T H E F A C U L T Y OF GRADUATE (Departments of Oceanography  We  accept to  THE  this  thesis  the required  UNIVERSITY  OF  October  ©  Junko  as  STUDIES and. Z o o l o g y )  conforming  standard  B R I T I S H COLUMBIA 1985  K a z u m i , 1985  In  presenting  degree  this  at the  thesis  in  partial  fulfilment  University of  British  Columbia,  freely available for reference and study. copying  of  department  this or  publication of  thesis by  for scholarly  his  this thesis  or  her  purposes  DE-6(3/81)  may  representatives.  Oceanography/Zoology  O c t o b e r , 1985  I agree  requirements  for  It  be is  an  advanced  that the Library shall make it  granted  for extensive  by the head  understood  that  for financial gain shall not be allowed without  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  rw.  the  I further agree that permission  permission.  Department of  of  of  my  copying  or  my written  ABSTRACT  Mn marine  was  found  diatom  to  Aquil  defined  verifying  the  results  modified  resin  f o r use  the  biologically  from  the  support  bottom better  shallow  was  similar  biologically in  the  organism the  ionic  Sunda  technique  Cu  of of  a  the  for  Cu  as  samples  active  Mn  water  probably of  active  Cu  and  Mn.  test  by  and  Mn,  to  were  estimate  found  rather  Cu c o n c e n t r a t i o n .  of  a l l depths.  the  technique  be  The  higher  the  bioassay  interaction  between  than  that  to from  from  indicating the  taken  samples  to  A  (1983)  resin  found  the  1979),  the  c o n c e n t r a t i o n was  to  in  samples  than  the  (1983).  concentration  collected  responding  3H)  Zorkin  samples  organism  estimated  samples,  by  to  a l . ,  Huntsman  fjord  the  Cu  et  Seawater  local  although  active  (Morel  seawater  and  of (clone  developed  in natural  growth  forms  biologically  of  waters  bottom was  medium  active  waters,  biologically  toxicity  pseudonana  well  was  the  Thalassiosira  chemically  cation-exchange  reduce  to  changes  in  the  iii  TABLE  LIST  OF  TABLES  LIST  OF  FIGURES  OF  CONTENTS  vi v i i  ACKNOWLEDGEMENTS GENERAL I.  THE  ix  INTRODUCTION EFFECT  OF Mn  1 ON  Cu TOXICITY  TO A MARINE  DIATOM  .  INTRODUCTION MATERIALS  4  AND  METHODS  1. B i o a s s a y s 2. M e d i a  using  6 a well  defined  medium  6  preparation  (a)  Standard  Ocean  (b)  Bioassay  medium  (c)  Trace  3.  metal  7  Water  stock  7 8  solutions  9  Procedure  4. G r o w t h R E S U L T S AND  9  rate  calculations  10  DISCUSSION  12  1. R e s p o n s e  of the bioassay  organism  t o Cu  2. R e s p o n s e  of the bioassay  organism  t o Cu a n d  Mn II.  TESTING ESTIMATE  RESIN  BIOLOGICALLY ACTIVE  INTRODUCTION MATERIALS  . . . 12  16  OF A C A T I O N - E X C H A N G E THE  4  AND  TECHNIQUE  C u AND  Mn  TO 22 22  METHODS  26  1. S a m p l e  preparation  26  2. C o l u m n  operation  26  (a)  Materials  26  iv  (b)  Preparation  (c)  Column  (d)  Elution  3.  AND  the  resin  29  procedure of  the  Determination  RESULTS 1.  of  29  columns  of  eluted  30 metals  with  GFAAS  .  DISCUSSION  33  Characterization  of  the  resin  Dowex  AG  50W-X12  III.  THE IN  33  (a)  Equilibration  (b)  Cu  and  Mn  adsorption  (c)  Effect  of  Cu  (d)  Effect  of  nutrients  EFFECT NATURAL  OF  of  on  AMBIENT  WATER  the  Mn  resin  to  Cu  and  Mn  ..  39  adsorption  41 44  LEVELS  OF  Mn  ON  Cu  TOXICITY  SAMPLES  52 52  AND  METHODS  1 . Characteristics 2.  Collection  3.  Analysis  5.  natural  1.  AND  Indian  natural  of  and the  Arm  55  seawater  water  55  samples  for  total  Mn  57 resin  method  for  natural  and  58 resin  experiments  using  water  58  DISCUSSION  Adaptation water  of  samples  Bioassays  RESULTS  55  natural  Cu  Adaptation water  of  of  dissolved 4.  33  curves  INTRODUCTION MATERIALS  31  of  samples  the  62 resin  method  for  natural 62  2. B i o a s s a y s  63  3. R e s i n  67  experiments  4. C o m p a r i s o n  of bioassay  and r e s i n  results  .. 71  G E N E R A L SUMMARY  76  REFERENCES CITED  78  APPENDIX I  86  vi  LIST  Table  Table  Table  Table  OF  TABLES  1. E f f e c t o f A q u i l n u t r i e n t s o n the adsorption o f C u a n d Mn t o Dowex AG-50W-X12 r e s i n  46  2. Dowex AG 50W-X12 r e s i n c o l u m n t e s t s e r i e s f o r estimating e f f e c t i v e metal concentrations  61  3. G r o w t h Indian  66  rate of Arm w a t e r  Thalassiosira  pseudonana i n  4. E f f e c t i v e Cu and Mn concentrations c o p p e r - e n r i c h e d I n d i a n Arm w a t e r  in 69  vii  LIST  Figure  Figure  Figure  Figure  1. G r o w t h without  rate EDTA  2. T h e e f f e c t time  versus  total  Cu a d d e d .  to  Aquil 13  concentration  over 15  The e f f e c t Cu  FIGURES  o f Cu on c e l l  3. T h e effect the presence 4.  OF  of Cu o f Mn  on c e l l  in 17  of v a r y i n g  toxicity  concentration  Mn  concentrations  on  i n EDTA-free A q u i l  19  Figure  5. R e s i n  Figure  6.  Eluate Mn v e r s u s equilibration  Figure  7.  E l u a t e Cu v e r s u s sample volume required for equilibration  (50-500ml)  Eluate Cu v e r s u s sample volume required for equilibration  (50-l000ml)  Figure  Figure  8.  9.  column  Eluate  28  Cu v e r s u s  required  sample  volume  required for 34  sample  35  volume  36 (50-800ml)  for equilibration  38  Figure  10. E l u a t e  Mn  versus  total  Mn  added  40  Figure  11. E l u a t e  Cu v e r s u s  total  Cu added  42  Figure  12. T h e effect resin  Figure  Figure  Figure  Figure  Figure  of  Mn  on Cu a d s o r p t i o n  43  1 3 . E l u a t e Mn v e r s u s p r e s e n c e o f Cu 14. T h e effect of r e s i n ( 1 0 - 5 0 0 nM  total  17. L o c a t i o n  of  Mn  added  in  the 45  F e on Cu a d s o r p t i o n Fe)  15. T h e e f f e c t o f F e on Cu r e s i n ( 1 0 0 - 5 0 0 0 nM F e ) 16. T h e effect resin  to the  adsorption  F e o n Mn  of sample  to the 48 to  the 49  adsorption  to the 51  collection  56  viii  Figure  Figure  Figure  18. E l u a t e C u v e r s u s s a m p l e v o l u m e r e q u i r e d f o r e q u i l i b r a t i o n f o r Dowex AG 50W-X8 r e s i n 1 9 . E l u a t e Mn v e r s u s s a m p l e e q u i l i b r a t i o n f o r Dowex  volume r e q u i r e d f o r AG 50W-X8 r e s i n  20. The e f f e c t o f v a r y i n g EMnC values t o x i c i t y i n n a t u r a l water samples  on  64  65  Cu 72  ix  ACKNOWLEDGEMENTS  I Dr.  A.G. L e w i s  these Dr.  past  N.G.  extend Drs.  would  my  for  few  Zorkin  sincerely his  preparation  support  years.  I  am  forh i s invaluable  appreciation  K.J. Hall  like  and  of t h i s  to  my  and also  thank  for  my  supervisor,  encouragement deeply  advice  research  E.V. G r i l l thesis..  to  grateful  o n my  research.  committee their  during  input  to I  members, i n the  1  GENERAL INTRODUCTION Copper for  the  (Cu) and manganese (Mn) are  required  growth of phytoplankton because they are e s s e n t i a l  components i n a v a r i e t y  of  cofactors  i n photosynthesis.  electron  transport  and  (Cheniae and M a r t i n ,  enzyme  systems  oxygen e v o l u t i o n  1970; Diner and J o l i o t ,  is  involved  contrast at  to  Mn,  Cu  relatively  low  response to e l e v a t e d to a r e d u c t i o n decrease  a protein  that  reactions  in  in c e l l  growth  photosynthetic  carbon  uptake (Goering  be  ( F i s h e r et al.,1981) and  (Sunda such  and G u i l l a r d , 1976).  as  assimilation,  nitrate  uptake,  i n h i b i t i o n of n i t r a t e acid  et a l . , 1977; Rueter e_t a l . , 1981), are a l s o  elevated  phosphorous  nutrition  Cu  levels.  of  In  phytoplankton  s i n c e a l k a l i n e phosphatase a c t i v i t y has i n h i b i t e d by Cu (Rueter, 1983). to  to Cell  (Harrison e_t a_l. , 1977) and s i l i c i c  a f f e c t e d by  suggested  detrimental  concentrations.  division  rate  processes,  reductase s y n t h e s i s  can  Cu l e v e l s i n c l u d e s enlargement of c e l l s  Physiological  tolerant  1976), while Cu  i n e l e c t r o n t r a n s f e r i n photosynthetic  phytoplankton  Chisolm  as  1976).  In  a  serve  i n photosystem II  important as a component of p l a s t o c y a n i n ,  due  and  Mn i s e s p e c i a l l y important f o r  is  (Rains,  nutrients  inhibit  the  addition,  may be i n f l u e n c e d been  storage  lipid  shown  to  be  High l e v e l s of Cu have been of l i p i d s .  (1981) observed t h a t , under n i t r o g e n s t r a i n s had higher  the net  S h i f r i n and  stress,  copper  f r a c t i o n s when compared to  2  intolerant  strains  The  detrimental  phytoplankton  have  presence  Mn  1983). as  of Sunda  little  as  estimated  to  that  a l .  1 nM  Mn  Thalassiosira of  oceanic  Cu  thought of  and  have  neritic  reduced  Sunda that  and  the  seawater  Huntsman  (1983)  Usually t o have  a  species  oceanic  open  et  thought of  have  also of  Cu  ambient  effect  a_l. , 1981),  algae  of  diatom,  ocean,  t o be  of an  rate  toxicity  deleterious  (Gavis  are  the  related i n the  the  containing  and  a  by  addition  i n growth  alleviated  to  Huntsman,  increase  and  Mn  levels  an  o f Mn  of  Cu  t o be  found  phytoplankton  concentrations  oceanic  shown  a l . , 1981;  Sunda  oceanica.  water p h y t o p l a n k t o n .  elevated  Sargasso  pseudonana  are  of  caused  addition  species  natural both  Cu  et  to  socialis. the  fresh  recently  (1981)  nM  Thalassiosira  levels  been  et  and  effects  (Sunda  3-10  Chaetocerous found  of marine  on  while  deficient  (Brand  et  to  a l . ,  1983). In total  the  dissolved  3.0  nM  been  found  to  dissolved usually  Some o f  been  from  in  the  fjords  the of  the  reported  to in  to  while  range  total  3.0  (such  enriched.  The  of  depleted  bottom  waters  British  central  nM  certain  circulation  result  occur  of  0.3  However,  i s often  the  waters  a l . , 1977),  range  water Mn  has  et  1980).  restricted  often  Cu  (Boyle  Bender,  are  surface  Pacific,  from  dissolved  1.5  to  Mn  has  (Klinkhammer coastal  as high  Mn  (Emerson can  areas  certain  oxygen  Columbia  North  and with  fjords),  concentrations levels, et a l . ,  offer  an  which 1979). ideal  3  situation  in  concentrations to  of  Mn  the can  effect  be  of  tested  on  naturally reducing  occurring  Cu  toxicity  phytoplankton. In  this  study,  Thalassiosira Cu  which  and  Mn  medium,  estimate was  was  the  Zorkin  tested and  investigate  response  to  et  a_l. ,  (1983)  the  1979).  and  Zorkin  Aquil.  bioassays  ambient  pseudonana.  the  i n the  available  collected  whether  to-T.  with  of  bioassay  various concentrations  determined  biologically  samples  toxicity  first  (Morel  by  then  technique water  pseudonana  Aquil  developed  the  from  a  levels  A et  a l .  local  of  Cu  can  and  the  with  marine Mn  technique  (in press)  both  conducted  both  seawater  resin  fractions  of  of  artificial  Finally,  were  organism  to Mn  resin natural  fjord  to  reduce  Cu  4  I.  THE E F F E C T  OF MN  ON  CU T O X I C I T Y  T O A MARINE  DIATOM  INTRODUCTION  The  biological  metals'  speciation  1980). as  (Morel  that  i t  metal  rather  affects  the  ionic  defined  by t h e  F l o r e n c e and  Batley,  artificial  led  to  total  of  will  1978).  metal  the  metal. In  addition  solution  will  Metal-metal  also  such  an u p t a k e found  the  ratios.  that  growth They  nutritional Cu:Mn  the response  can occur  l o w Cu:Mn  in  suggest sites,  ratios.  that C u may  metal  ratios  which  with  were  a  by c o m p e t i n g  which  of  such  the  as the  to  bind  t o adsorb  ratios  in  potentially  a metal  not  socialis  be t o x i c  of the  of phytoplankton.  i n o r on t h e c e l l .  of. C h a e t o c e r o u s  form  available  affect  either  conclusion  available  activities,  such  and G u i l l a r d ,  by f a c t o r s  of p a r t i c u l a t e s  a s Cu, c a n compete  site  ionic  (Sunda  ionic  interactions  metal,  (1981)  to  the  The c o n c e n t r a t i o n  be i n f l u e n c e d  and the l e v e l  media  concentration,  phytoplankton  and M o r e l ,  form  metal  and the c o n c e n t r a t i o n of l i g a n d s  the  high  well  1979) h a v e  the  growth  free  to  e t a_l. ,  than  Anderson  for  using  1982a;  are governed  i s the c o n c e n t r a t i o n of the free  1976;  nature  o f C u a n d Mn  (Florence,  Experiments  Aquil  toxic  effects  Sunda as  a s were  with  such  Mn  a s Mn  et a l .  detrimental high at  to phytoplankton  metal  the  Mn  at the  5  The  effect  species.  F o r example,  interaction these  metals  levels  acted  did  Gonyaulax  a  tamarensi s  an i n c r e a s e  in  stopped  (WHOI  3H) a n d a  clone  oceanica  (WHOI  completely  reversed  effect  bioassay  Sunda  the  by  growth  13-1),  must  to the interaction  This  discusses  chapter  pseudonana  (clone  concentrations experiments to The  was  characterize second  reduce  Cu  the  conducted the response  series  of  cupric  (1983) at  response  low  found  Mn  ion  pseudonana  Thalassiosira  the  effect  on a  the  was  of  response  of  species.  Thalassiosira o f C u a n d Mn  The f i r s t  different  particular  between  combinations  seawater. with  have  ion  ion activity.  since  series  of  Cu c o n c e n t r a t i o n s  of t h e organism  of b i o a s s a y s examined  toxicity.  i n the motility  that  can vary  3H) t o v a r i o u s  in artificial  Mn  interaction  be d e t e r m i n e d  Mn,  in  of Thalassiosira  t h e Mn  on  and  drop  increased  but  phytoplankton  Cu  a  activity  increasing  that  Thalassiosira  oceanic diatom,  of a metal-metal  organism  that  decrease  ion  found  With  and Huntsman  related  clone by  and  antagonistically  found  further  between  investigated the  on  Skel-0.  have  cupric  activities  (1980)  but  induced  However,  can vary  phytoplankton  clone  (1978)  not cause  activities.  The  e t a_l.  Hasle,  costatum,  and Morel  interactions  synergistically  (Huds.)  Skeletonema Anderson  Braek  o f Zn a n d C d o n  pseudonana  that  of metal-metal  to  Cu  the a b i l i t y  alone. o f Mn t o  6  MATERIALS  1.  AND  METHODS  Bioassays  The (WHOI is  using  marine  clone  3H)  were  be  centric  diatom  used  urn  using  artificial by  Thalassiosira  bioassay  and  has  particle Aquil  medium the  the  medium  in diameter)  electronic  estimated  as  copper  4.5  conducted  defined  defined  to  (approximately for  well  was  sensitive  suitable  a  and  shape  e_t a l _ . ,  chemical  model  because  consistent  counting.  for which  computer  organism  a  (Morel  pseudonana  MINEQL  i t  size  which  makes i t  The  bioassays  1979),  a  well  speciation  can  (Westall  et a l . ,  1976). Inocula  for  exponentially cultures taken  contained with  stock  initially  maintain  them  a l l Aquil  reduced  bioassay  growing  were  to  the  axenic, in  this  could  be  of  culture not  to  added  because Martell,  T.  lowered  limitation.  This  Mn  and  without  to  pseudonana to  Mn  1  nM  could  mask  trace  was to  test  to  capacity the  effect  any for of  solution  metals  Mn  cells  agent  the  flasks. of  the  Cu  levels  reaching  for  Mn  but  Preliminary  that  adopted  the  was  chelating  (EDTA).  s o l u t i o n s nor  complexing  and  the  The  care  culture  indicated  from  pseudonana.  The  the  without  carryover  stock  i t s strong 1964)  acid  T.  obtained  reasonable  state.  concentration  reduce  and  nutrients, vitamins  levels  with  were  c u l t u r e s of  ethylenediaminetetraacetic experiments  media  stock  EDTA test  was  media  (Sillen on  Mn  reducing  and Cu  7  toxicity. Enough stock c u l t u r e was added to the f l a s k s to an  initial  cells/ml.  c e l l c o n c e n t r a t i o n of approximately 1000 - 2000 The organisms were grown  controlled  s e c " on a 16:8  2  hour  1  concentrations  Counter  i n an  room at 16°C with an i n - f l a s k  100 uEin m" cell  obtain  (model  were  environmentally  light  i n t e n s i t y of  light:dark  measured  daily  cycle.  The  using a C o u l t e r  Zf) over a p e r i o d of 5 days.  2. Media p r e p a r a t i o n  (a) Standard Ocean Water Standard ocean water made  by  dissolving  MgCl .6H 0 i n 12 2  chloride  into  a l l major  liters  2  measured  (SOW) (Morel  a  20  of  liter  et  a_l. ,  seawater  salts  glass  distilled  glass  carboy.  (previously dried for 2  days  at  1979) was  water  used  acid-cleaned allow the  were  reagent  (1N H SO,,), f i l t e r e d 2  complete  mixing  medium was 8.0±0.05.  ion-exchange Laboratories)  grade.  column to  60°C)  was  injection  A l l the  (0.4 urn Nuclepore) a i r to  and e q u i l i b r a t i o n .  The f i n a l pH of  The SOW was then passed through an 100-200  mesh,  t r a c e metal i m p u r i t i e s .  SOW had undetectable l e v e l s of Mn and direct  then  The SOW was bubbled with  (Chelex-100®,  remove  (GDW)  The magnesium  added and the volume made up t o 20 l i t e r s with GDW. salts  except  graphite  furnace  Cu,  as  atomic  Bio-Rad Chelexed  measured  by  absorption  8  spectrophotometry technique  (b)  The  polycarbonate  medium  carboy.  which  nutrients  10 nM  was  The medium  appropriate  (previously  passed  given  i n Appendix with  I,  Table  acid-cleaned  approximately  during  and  cooled  t o room t e m p e r a t u r e .  Fe  formation the  of  Aquil.  full  (1979)  f o r Cu.  ferric  T h e pH  of the A q u i l  medium  in  that  added  as  differs  freshly  of  t h e low s o l u b i l i t y media  was  vitamins was  until  then t h e pH  The c a r b o y  Aquil  a n d Mn)  and  was  to with  15 p s i ,  the  before  increased  that  n o r Mn  was  trace freshly  allowed  to  t h e Fe  of  addition to  Morel  added,  EDTA  8.0,  to i f  filtered air. et a l .  and that  Fe h y d r o x i d e .  i n seawater,  t o keep  added.  preparation to allow the  precipitated  of Fe  and  121°C and  stock  to occur  from  were  necessary  a c i d - c l e a n e d and  n e i t h e r EDTA  was  to culture  with  EDTA  after  and  2  was  Finally,  hydroxide  by b u b b l i n g  Aquil  step  The Fe  hours  vitamins  medium C0  Nalgene of  nutrients  f o r 1 hr at  were a d d e d .  Fe  added  this  10 l i t e r s  Aquil  filtered  but without  for several  necessary, The  autoclaved  Cu,  stock  equilibrate  of  10 l i t e r  autoclaving.  was  (with  in a  The  This  medium  prepared  then  I.  and  6.5.  precipitation  mix  15 nM  Chelex-100)  salts,  the  metal  and  of  through  are  prevent  levels  c o n s i s t e d of  amounts  c o n c e n t r a t i o n of the A q u i l  reached  f o r Mn  prepared  The  bubbled  detection  medium  bioassay  to  The  are approximately  Bioassay  SOW  (GFAAS).  Because  has o f t e n  solubilized.  the  been  However,  9  this  procedure  strong  affinity  ferric Aquil  1983),  Trace  Cu 1 x  1 x  were  For  freshly  of the  precipitated  t o be a s e f f e c t i v e  was  as the  (Wells et  used.  prepared 1%  daily  reagent  prepared  i n 1%  from grade  from  a  a stock  solution Likewise,  stock  solution  3  soaked  rinsed  3 times  3 times  f o r a week,  with  GDW  with then  in  1N  again  i n 1N  HC1  for  about  12  were  reagent  with  t h e g l a s s and p l a s t i c w a r e were  soaked 3  of  3  GDW,  GDW,  Mn  HN0 .  rinsed  uses,  of  HN0 .  i n the experiments  with  GDW.  rinsed  3  h r s , and  times.  Procedure  All hood  manipulations  with  minimize  metal  Aquil  polycarbonate  screwcaps.  were  a l l possible  trace  autoclaved ml  because  solutions  in  2  2  subsequent  rinsed  found  study  g l a s s and p l a s t i c w a r e used  HC1  times  been  Since  of Fe a d d i t i o n  stock  3  grade  mode  freshly  10" M M n C l  initially  has f o r Cu.  has  were  CuCl  All  3.  this  stocks 3  in this  mix i n s u p p l y i n g F e t o p h y t o p l a n k t o n  metal  10~ M  stocks  EDTA  hydroxide Fe-EDTA  al.,  (c)  was n o t s u i t a b l e  done  parts  in a class  poured  erlenmeyer  Appropriate  flow  r e p l a c e d by p o l y p r o p y l e n e t o  contamination.  were  100 l a m i n a r  into  Aliquots  ml)  of  previously autoclaved  500  flasks  spikes  with  o f C u a n d Mn  (250  polypropylene were  added and  10  allowed T.  to equilibrate  pseudonana.  relative  Each  are often  speciation metal,  the  deviation  strong  and  the  later.  cultures, assumed  metal  influenced  t h e pH  any  the  the  which  growth  b y pH  change  produce  bioassay  chelators.  However,  available (e.g.  with gave a  rate  of  medium  as  was  metal  f r a c t i o n of the  from  eta l . ,  8.00±0.05 o n  o f 8.43±0.07 f o u r  similar  between  trace  Peterson  changed  t o a maximum  difference  not t o  to  the bioassay  of i n o c u l a t i o n Since  of  biologically  T h e pH d u r i n g  day  inoculation  was r u n i n t r i p l i c a t e  were n o t added  i s highly  1984).  before  5%.  pH b u f f e r s they  test  standard  approximately  overnight  in  control  significant  the  control  and t e s t  differences  days  pH  in  was  metal  spec i a t i o n .  4. G r o w t h The in  calculations  effect  terms  which  rate  o f C u a n d Mn  of growth  rate  was c a l c u l a t e d growth  rate  over  o n T. p s e u d o n a n a the period  as follows  (div d a y  1  day  was 1  expressed  to  day  4,  :  ) = l o g (N,/N ) x 0  3.322  t where end,  0  and  N,  respectively,  Since of  N  low  growth  the bioassay  are cell  concentrations  of a time period, rates  were o b t a i n e d  (probably  due  to  t  a t t h e s t a r t and  (Guillard, i n the f i r s t  the  bioassay  1973). 24  hours  organism  11  acclimatizing from the growth  to  growth rates  cultures.  the rate  medium),  calculations.  In  some  excluded  instances,  were expressed as a percentage of the c o n t r o l  T h i s was d e f i n e d by the  growth r a t e  day 0 v a l u e s were  (% of c o n t r o l )  =  relationship:  growth r a t e t e s t x 100 growth r a t e c o n t r o l  12  RESULTS  AND  DISCUSSION  1. R e s p o n s e  As  phytoplankton  elevated  Cu  pseudonana which  of the bioassay  estuarine  effect species  found  (Gavis  Cu  of  Mn  of even from  hardy  spikes  ranging added  concentration  o f Mn  ( 2 8 . 3 nM)  of  Cu  T. p s e u d o n a n a was  required  20  nM  were  conditions. organism  for  to  T h a l a s s i o s i r a pseudonana  levels  by  than  baseline  against  In  general,  oceanic  possibly  those  to  of T h a l a s s i o s i r a  related.  from  were  is (Fig.  for a  species.  t h e same  species  environments from  The  nM  this  1).  oceanic  have  regions  in  t o Cu. by  in  Cu  i n growth  o f T.  the  of  under  i n growth between  levels rate.  rate 40  of  Cu  of  100  nM  levels  of the  of  test  100 nM  in  a  et a l .  Cu,  rapid  i n growth  previously  by R u e t e r  rate  experimental  and  resulted  Reduction  has been  pseudonana  the  Aquil  response  i n growth  rate,  nM  increasing  a concentration  i n growth  20  the  The e f f e c t decrease  range  Cu c o n c e n t r a t i o n s clone  a  change  the  in  to characterize  Although  greatest  nM,  containing  t o be d e l e t e r i o u s  increases  decrease  elevated  shown  20-120  Aquil  50% d e c r e a s e  found  occurred  20  linear  a  neritic  than  increments,  where  be  resistant  obtained  differently  e t a l . , 1981).  Copper  of  respond  forms  can  clones  t o be more  t o Cu  the response  levels  a r e more  some  phytoplankton  been  may  concentrations,  Furthermore, of  species  to various  the  organism  rates  reported (1981),  13  Figure  1.  Growth EDTA.  rate Bars  versus total represent ±2  Cu a d d e d standard  to Aquil without deviations.  14  Gavis  et  a l .  Changes days  after  (Fig.  2).  in growth  As  delay  in  the  In  and/or  caused  of  an  its ability  of  examined  the  the  marine  data  with  one  of  most  toxic  amino cell  diatom  the  Cu  to  bind  most the is  photosynthetic  more  Cu  has  and  pool  to  also  Guillard that  could  be  the  due  within  inhibit  nutrient  and  Cu  such  the as  reacts  1978).  toxicity  of  to the  uptake silicic  reactive  thought  apparatus  Once to  and  Cu  of  such  on  Jones  (1981)  (among  other  Cu,  metals,  was  found  bound  to  the  et  a l . ,  metals)  correlated  metals.  cell  groups outer  and  the  as  the  Fisher  and  are  change  functional  groups)  decouple  (Fisher  organism  ligands  with  japonica  reactivity  any  phytoplankton  sulfhydryl  diatom.  to  organic  In  (Davies,  sulfur  Cu  to  Asterionella  sulfur  to  of  nature.  relative  the  acting  essential  pseudonana,  carboxyl,  nutrient  be  became  suggest  metal  days  Morel,.1981).  functional  groups,  of  may  a  4  2  time  Sunda  (1983) of  after  rate  by  evident  exposure  to added  clone  important Cu  and  growth  for this  were  marked  Cu  response  detrimental effects by  surface  the  an  Cu  more  of  Morel-Laurens  case,  and  Thalassiosira (e.g.  shown  added  became  detrimental effect  this  (Rueter  to  in  delayed  assimilation  The  its  a  and  exhaustion  cell.  acid  Such  Morel  and  decrease  previously  (1976).  due  (1983).  concentration  the  pronounced.  the  the  Zorkin  rate  inoculation  increased  been  (1 98 1 ) , a n d  be  is the  sulfhydryl  division 1981).  these  which to  to  and  the  Overnell  15  Figure  2.  The  effect  of  Cu  on  cell  A 160 nM C u . Bars d e v i a t i o n s exceed the  concentration  are present s i z e of the  over  time  when ± 2 s t a n d a r d symbol.  16  (1975) both  has  the  found  that  and  modified  Hill  once  inside  Mehler  II  and  photosystem  2.  of  the  bioassay organism  In test was to  the  organism  to  a  final of  a  final  level  of  levels  o f Mn  (28.3  shown  as  a  without  the  a  of  delay the  to  Mn  spike  2 days,  controls  The  varied.  prepared: and  1  3  to  the  prior  3 to  in  in  each  a  growth  both  experiment, used as  was  at Aquil medium, of  Cu  to a  Cu 4  which  was  spikes.  added  to  response of  The  Mn  of is  cultures  rate  after  Mn  spike  the  rates. Cu  i n the and  (28.3  Mn of  series  Aquil  were  concentration, which  nM).  Mn  to  concentrations  in  divided Six  toxicity second  t h e Mn  control  levels  added  contained  i n growth  liters  test  Mn  was  absence  with  of  flasks  was  The  the  of  set  nM).  reducing  the  Mn  ( F i g . 3).  investigated  used  addition  rate  on  was  of  (1  of  level  sets  No  presence, and  similar  were  fixed  control  Cu  Mn  s e t , Mn  reduction  Mn  a  nM.  the c u l t u r e s  which  liters  liters the  showed  100  A  i n growth  limit-  measures  response  i n two  second  and  the  further  For  concentration  nM)  of  was  liter  nM.  showed  experiments  were  1000  whereas  effect  pseudonana  in a  Cu  difference  of  and  the  of  to Aquil  while  can  which  t o Cu  presence  added  Cu  activities.  experiments,  concentration  flasks  pseudonana  of  was  set  Aquil  T.  Cu  of  i n the  one  give  and  series  t o Cu  determined. obtain  T.  first  cell,  reactions  photosystem  Response  I  the  Mn  into  the was  the  Mn  added flasks  concentrations  17  Figure  3.  The e f f e c t o f Cu p r e s e n c e o f Mn V l O O ^ M C u  on cell Symbols: ^  concentration in © c o n t r o l (0.1 nM C  U  W  l  t  h  1  0  0  0  n  M  M  the Cu, n  ;  18  (1,  10,  50,  concentrations tested. and  Mn  level  to  dependent  higher  the  of  of  already al.,  been  1981),  believe  of  reduce  the  target  sites  sites  across  the  reversal enzymatic compete  Cu  extra  of  o f Mn  added  each  between Cu  to  seven nM)  the  added  the  Cu  toxicity  controls  growth  that  of  by  the  Mn  seems  ( F i g . 4).  The  the  bioassay  medium,  Cu  had  the  on  of  Cu  Such  and  Cu  were  ensured  to  reduced.  pseudonana  of  120  present  excess  be  (1981)  reduction  onto  within the  in  (120  an  the  growth nM),  the  effect  socialis T.  has  (Sunda  oceanica  Cu  et  (Sunda  by  the  organism  and  oxides.  by or  inhibition  for the  caused  the  uptake  due  Hill  by  Mn from  site  on  a  to  the  thought  to  potential  for  (Habermann,  chloroplasts  same  not  example,  (1983)  i s purely  protecting  For of  Mn  is  competing  chloroplasts  the  Mn  either  membrane.  be  Huntsman  toxicity  cell  to  and  Cu  manganese of  Sunda  cell  believed  Cu  and  the  Cu  in  of  of  isolated  sites  with  and  for Chaetocerous  toxicity  was  100  concentrations  to  response  reverse  reactions  seems  a l .  physiological  to  high  at  1983).  et  that  adsorption  Mn  nM)  rates  limiting  effect  at  80,  the  amount  Thalassiosira  Sunda  60,  growth  not  reported  Huntsman,  found  the  1000  alleviation  level  Mn  40,  the  was  the  the  and  without  on  However,  effect  of  The  detrimental  rate.  and  20,  tested  organism.  less  ("0",  bioassays  test be  500  Comparison  the  the  100,  transport  Mn and  has  been  Mehler  1969).  The  protecting  the  Cu.  Mn  may  the  surface  also of  19  o o  .0  48.0  72.0  TOTAL CU ADDED  Figure  4.  (NM)  The e f f e c t of varying Mn concentrations on Cu toxicity i n EDTA-free A q u i l . Symbols: A 1000 nM Mn; * 5 0 0 nM Mn; O 100 nM Mn; X 50 nM Mn; + 1 0 nM Mn; © 1 nM Mn. Vertical bars p r e s e n t when ± 2 standard deviations exceed the s i z e of t h e symbol.  20  the  cell.  found in  A  f o r Cu  the  cell Morel,  Zn,  1981).  (Foster  cell,  the  nature  or  covalent  and  Morel,  of  the  the  interaction  uptake  and  predominantly  groups  and  believe  that  Cu  phytoplankton  uptake  sites  affected  by  solely  Cu.  by  amino  per  (Rueter  and  Cd-stressed to reverse  caused  the  by  Cd  (II)  acids. ratios,  the  cell;  both  Mn  and type  the Cu of  by  plotted  and  or  (II)  form  weak  such  such  a_l.  detrimental  effect  the  presence  two  first while  the  uptake  is  thought  second site  is  as  (1981)  the  of  a  strong,  N-donors, et  as  termed  forms  Sunda  a  Mn  ( I ) was  which S-  chemical  O-donors Cu  to  according to  which  to  due  in  electrostatic  elements  Cu  either  (1980) an  metals  to  sites  metal's  against  bonds  i s caused  first  up  partially  Richardson  metal  on  The  taken  to  been  activity  found  the  Both  bonds  h i g h Cu:Mn  Fe  be  phosphodiester groups.  covalent  at  of  may  to  group  covalent  non-essential"  of  and  to  "toxic  to  and  parameter  and  and  surface,  site  carboxylate  Cu  f o r uptake  "micronutrient"  electrostatic  of  initially  metals  characteristics.  termed  zinc  pseudonana  addition  has  1982).  Nieboer  parameter  bonding  amount  rate  sites  in  w e i s f l o q i i was  cell  interaction  sulfhydryl  in the  the  growth  on  increase  Thalassiosira  of  characteristics.  were  of  c o m p e t i t i o n among  the  their  drop  an  T h a l a s s i o s i ra  inhibition  The  a  Similarly,  of  toxicity  c o m p e t i t i o n f o r uptake  i n which  caused  cultures  cultures the  and  medium in  similar  of  types to  be  affected  characterised  21  by  Sunda  et  functional  groups  bind.  At  the  which  Mn,  groups. S-  or  cell. high  elevated  at  modify  the  Cu:Mn  displacing altering  the  possibly  both of  Cu  (II)  Cu,  the  b i n d s more the  second  functional  bind;  The  may  levels  usually  N-donating  or  (1981)  to which  Similarly,  strongly block  a l .  high  groups  groups  detrimental  effects  ratios  therefore  the  Mn  at  functional  Cu  the groups  of  at  the  Mn  (II)  the  of  uptake  to  which of  can  displace functional  site Cu Cu,  may  have  (I) Cu  (I)  may  of  the  the  Cu  to phytoplankton  at  caused  Cu  site, second  surface  can  on  be  first  O-donating  ( I I ) may  to  concentrations  functional  may  and  weakly type  have  and  by by  site.  the  possibly  22  II.  TESTING  OF  ESTIMATE  A  CATION-EXCHANGE  THE  BIOLOGICALLY  RESIN  ACTIVE  TECHNIQUE  CU  AND  TO  MN  INTRODUCTION  Ion-exchange analytical (e.g.  Filby  low  et  of  of  resin, small  also  a  adsorbed  of  The  adsorption by  capacity  and  well  the  sample  and  Usually, are  more  the  as  can  of  the  the  be  those likely  size  concentration 1966).  increases,  will  the  more  available metals t o be  not  metals the  As  The  can  of  be  the  concentration tend  for adsorption  by  or the  technique  to  onto  weakly  resin  1972). metal  the  bound  several  the  is  ion-exchange  the  nature  the  minimized.  the  will  large  thus  (Dorfner,  charge  a  to  relatively of  and  metals  adsorbed  in a  ion-exchange  resin  and  resin  samples,  cross-linkage,  of  with  i n .volume  the  onto  working  through  possible.  of  waters An  When  passed  the  1976).  the  recovered  trace  of  size  by  be  of  sample.  is  therefore  metals  degree mesh  given  reductions  handling  (Inczedy,  solution less  the  influenced  as  in  and are  little  a  f o r c o n t a m i n a t i o n by  affected  also  eluate  Batley,  ability  solution  metals  magnitude  involves  chances  is  of  and  for  in natural  e s p e c i a l l y when  the in  extensively  metals  Florence  is  metals  used  trace  technique,  d i l u t e metal  volume  been  of  1974;  levels,  the  the  orders  a l ., the  metal  concentrate volume  have  measurements  advantage very  resins  of  ligands of  be  ion in  ligands cpmplexed  the  bound  It  by  ion-exchange  resin. ligands resin.  23  Consequently, determine Suter, and  resins  metal  1979)  However,  can  metal-ligand  Chelex-100  most  biologically  of  are  press).  To  species, GLU the  and  final  constants  Cu  that  of  the  the  and  Figura waters.  metal-EDTA 1984)  adsorb  Zorkin  an  i o n , which  the  test  and (e.g.  onto  et  a  a l . , in  overestimation  are  thought  Cu  and  the  to  of  be  the  samples  the  resin  was  resin  was  therefore  and  ionic  used  in  Cu  metal. or  the  increased,  decreased. concluded  The to  As  be  the  natural this  a l .  the  such and  of  of  et  only  ligands  samples  and  use  Zorkin  adsorbed  ligands  onto  the  containing  of  seawater  binding  for  organic  strongly  concentration  in a r t i f i c i a l  (1983)  a  50W-X12) was  the  Mn  resin  for levels  Cu  AG  technique  (1983) added  the  s o l u t i o n and  (Dowex  Zorkin  seawater  in  to  estimate  and  ensure  eluate  adsorbed to  theory in  to  to  1983;  lead  resin  to  given  concentration  Cu  between  available  Zorkin NTA  trace  (Sunda,  found  to  available.  study  The  been  possibly  (e.g.  natural  that  used  r e s i d u a l p o s i t i v e charge  (Zorkin,  cation-exchange  seawater.  a  free metal  Equilibration  resin  also  resin  levels  biologically  of  onto  could  present  1982b)  adsorb  with  been  Baccini  speciation  reported  the  the  metal  recently  cation-exchange  acidic  trace  have  (e.g.  been  have  This  Chelex-100  capacity  Florence,  complexes  Cu-histidine)  press).  the  1979;  i t has  chelates  as  complexation and  McDuffie,  such  as  (in ionic  EDTA,  analysed the  ligand  stability amount  of  adsorption  of  controlled  by  24  the  fraction  by (r  organic =  of  p<0.05)  concentration adsorbed  (ECC)  (an  the  was  found  charged  cupric  inorg)  ion.  and  adsorbed  onto  a  the  resin) to  and  occur  The  amount  the  rates  effective  amount of  except  ligands  completely of  ([Cu]  cancel  inorganic  )  Cu  be  Cu  of  Cu  which the  and were  charge  in solution  determined can  of  Cu  Thalassiosira  for a l l combinations  experimentally resin  of  complexed  relationship  biologically  growth  studied  d i d not  the  strong  expression  and  the  c o n c e n t r a t i o n not  Moreover,  concentrations  weakly  metal  between  onto  pseudonana  the  total  ligands.  0.92,  ligand  the  amount  expressed  of (Cu  of  Cu  by  the  equation:  [Cu  where Cu  Xinorg  derived  same  pH  and  limitation complexes  to  adsorb  value  salinity the  the  complexes such  the  (Zorkin  as  those  resin.  sewer  However,  in  most  complexes  in  natural  negative  charge  distribution  on  formed  Cu  the  and  with  coefficient seawater  and  has  may  be  (Clark  been  Batley,  were  waters,  form  of the  is  a  charged  also  weakly  found  charged  positively  important et  of  There  positively  HIS)  m a j o r i t y of  waters  (Florence  as  a c i d s ) can  outfalls  areas  artificial  natural  amino  with  Xinorg  however,  In  and  x  e_t a l . , i n p r e s s ) .  technique,  (such as  onto  of  [Cu]  measurements  ( e . g . ammonia  charged places  from  to  Cu  ligands  i s the  inorg] =  a l . ,  in  some  1972).  the m e t a l l o - o r g a n i c found  1980).  to  have  a  Consequently,  25  the  adsorption  resin  i s considered In  resin to the  this  determine  AG  to  the  the  efficiency  of  charged  complexes  be  relatively  unimportant.  the  adsorption  of  50W-X12) sample  concentration and  positively  chapter,  (Dowex  samples, the  of  of  in artificial  volume metals  effect  of  the  resin.  required to  nutrients  be  Cu  and  Mn  seawater for added  onto  the  to  the  i s examined  equilibration, to  the  (in particular  seawater Fe)  on  26  MATERIALS  AND  1.  preparation  Sample  Cu 1  x  and  Mn  10" M  CuCl  iron  stock  3  An  METHODS  dissolving left  2  stocks in of  0.135  to  1%  of  seawater  the  manner  same  appropriate  metals  always  for  2.  left the  Column  (a)  resin  and  3  1 x  10"*M,  FeCl .6H 0 3  as  2  for  samples  added.  hours  10" M  to  MnCl  3  from  in  liter  1  hours composed  equilibrate  3  made  to  The  (made  in  which  the  samples  before  by  then  use.  SOW  prepared  of  HN0 .  GDW,  before  previously) The  1%  was of  of  stocks  in  2  needed,  2-3 were  daily  when  described  were  2-4  prepared  passing  were them  columns.  operation  Materials  A AG  x  equilibrate  artificial  through  HN0 5  g  were  Dowex-50 c a t i o n - e x c h a n g e  50W-X12,  these  200-400 mesh)  experiments.  divinyl  benzene  (R-SO3-)  when  completely  are  Company,  attached. dry,  1958).  the  resin  copolymer  groups  Chemical  This  in  resin  is  hydrogen  is  5.0  to ion  Laboratories,  form  composed  lattice The  (Bio-Rad  which  of  was  used  a  styrene  sulfonic  exchange  in  acid  capacity,  millequivalents/gram  (Dow  27  The 12  apparatus  x 75 mm  polypropylene  and  both  grid  support  Both  ends  the  fitted  column  tips  (60 c c ) , u s e d  equilibration  with  polypropylene  were  delivered  (i.d.  1.19 mm)  polypropylene placed.  rinsing  was  (glass  1N  filter)  reagent  GDW.  with  into  with  of which  GDW.  2  grade  grade  from  system  and  t o the columns The  samples  microtubing ml  Nalgene  the samples  tubing  gas l i n e .  under N  The  Polypropylene  500  pressure  were  lead  from t h e  The  flow  of about  of  1  psi  gas.  i n the experiments  Subsequent  1N r e a g e n t  3 times  caps  of polypropylene  maintained  fiber  size.  (Fig. 5).  t o the Nitrogen  off  tips.  were  v i a polypropylene  the  cut  polyethylene.  support  connected  Locks  from  L a b o r a t o r i e s , Richmond,  5.0 cm  were  the resin  p l a s t i c w a r e used  with  washing  SOW,  column  c l e a n i n g of the  bottles  samples  in  x  screwcap  the  soaked  (i.d.) foracid  piece  grid  made  t h e bottoms  of f r i t t e d  (Bio-Rad  to  of the bottles  All  the  connected  Another  with  columns  polypropylene  3-way L u e r  to  caps  filtered  tubes  and  with  resin  was made  Econo-columns®  the  times  with  U . S . A . ) o f 1.0 cm  syringes  of  test  f o r the resin  disassembled Ca.,  consisted  HC1  f o r 3 days  was then  initially rinsed  cleaning  consisted  of  HC1  few h o u r s ,  prior  fora  3  acid to  28  chamber for ocid cleaning a n d equilibration of resin  sample  delivery  resin  F i g u r e 5. R e s i n column  tube  tube  29  (b)  P r e p a r a t i o n of  To  remove  methanol  any  then  slurried  with  the  organics the  rinsed 1 N  resin  into  with  GDW.  Finally, in  3 times  the  the  weight  resin was  screwcapped  Approximately  40  g  of  experiments,  the  g),  slurried  about  in  individual  columns.  (c)  procedure  about The to  air  0.5  ml  resin the  columns  followed  by  8 . 0 ± 0 . 0 5 ) was to  with  passed  through  All  the  the  the  effluent  resin  HC1  then at  then  prepared  ml  was  of  at  one  with  c l e a n e d of  trace  metals  HCl rinse  GDW.  in  (0.75  a  For or  by  5  ml  adding rod.  converted ml  of  1.0 into  glass and  a  use.  time.  columns.  Fifty  no  of  3N  This SOW  was (pH  =  through  the  columns  to  raise  allow  the  salts  to  come  into  (500  ml)  were  then  of  about  to resin.  columns  of  the  times  poured  passing  through  3  until  removed  resin  first  convert  until  and  were  then  stored  weighed  GDW,  column  by  to  100°C  with  was  rinsed  the  passed and  and  washed  in a dessicator  resin  i n the  form  ml  then  8.0±0.05  equilibrium  of  were  5  was  5  distilled a  The  dried  bottle  stirring  hydrogen  isopiestically  pH  and  form, was  dried  bubbles  GDW  first  detected,  resin  the  Any  was  w i t h GDW.  protonated  polypropylene  Column  resin  isopiestically distilled  the  change  resin  The at  a  samples flow  passing through  rate the  resin  was  6  the  ml/min.  discarded.  30 •  (d)  Elution  EDTA used  of the columns  and  to elute  grade  nitrilotriacetic the columns.  EDTA  monohydrate) concentration of  3 x  10" M 2  was  an  3N H C 1 .  1 x  10" M.  prepared  in eluting the  original T h e pH  pH  of  made  NTA  with  solutions needed,  with  that  solutions  of  the  these from NTA  11.0) u s i n g  o f t h e EDTA  the resin was  solution  at  were  left  more  low  adjusted to  was  final  Preliminary  isopiestically  solution  salt,  a  a n EDTA  manner.  were  Analar  (trisodium  i n t h e same  the metals pH  or  When  2  (NTA) s o l u t i o n s  were  t o make  indicated  Consequently, (from  salt)  t o GDW  of  experiments effective  These  (disodium added  acid  pH.  5.5-6.5  distilled  unadjusted  at  4.8.  Mn To ml  measure  aliquots  columns. determined  t h e amount I x  of The  Mn  by  most  EDTA  had passed  2  EDTA  adsorbed were  concentration Graphite  spectrophotometry that  tO" M  o f Mn  o f t h e Mn  (GFAAS). was  through  furnace  used the  after  resin.)  to  the  the  was  then  absorption  experiments first  20  elute  eluate  atomic  (Preliminary  removed the  in  to the resin,  10  showed ml  of  31  Cu Preliminary Cu  experiments  r e a d i n g s when t h e r e s i n  than  when  amount  i t was e l u t e d  of  Cu a d s o r b e d  was p a s s e d passed  through  adsorbed from  through  was  with  resin  to the resin,  The  eluate  polypropylene  Nalgene  was  1 x  10 m l o f NTA  t o remove most  effective used  were  screwcap  NTA  2  2  was f o u n d  samples  10" M  20 m l o f 1 x 1 0 " M  (The f i r s t  later  GFAAS  To measure t h e  2  resin  and  with  1 x 1 0 " M EDTA.  the resin.  the  more s e n s i t i v e  eluted  C u . ) T h e NTA w a s a l s o  the  metals.  indicated  for  stored  bottles  that of the  removing  forelution  Mn  of both  in  and  NTA  30  ml  analyzed  by  determined  by  GFAAS.  3. D e t e r m i n a t i o n o f e l u t e d The amount direct atomic  injection  furnace,  programmer.  the  Cu-  the  eluate  coupled and  as  used  s t a n d a r d s were used  GFAAS  i n the eluate  to  a  with  for eluting  regression  a P e r k i n - E l m e r 560  with  EDTA  or  t h e same  Three  be  HGA  NTA EDTA  t h e columns.  t o draw c a l i b r a t i o n  analysis.  a Perkin-Elmer  Perkin-Elmer  c o n c e n t r a t i o n s of the samples could  linear  was  into  Mn-enriched  the standard solutions,  concentration Mn  of  with  absorption spectrophotometer,  graphite  formed  of metals  metals  curves  400  solutions and  NTA  The Cu a n d from  estimated  absorbance  HGA  levels  which using were  32  obtained  for  each  deterioration the  first  third  two  was  sample  of  the  and  g r a p h i t e tube  r e a d i n g s were  taken  after  standard.  at  taken  least  during  in  one  To  detect  sample  succession,  analysis  of  any  analysis, while  the  the  samples.  Mn A  sample  pyrolytically line  of  current  a of  volume  coated  10  12  mA  was  of  1000°C  sec,  15  ul  was  graphite tube.  Varian Techtron  temperatures for  of  used.  100, then  120  The  injected 279.5  Mn  hollow  cathode  The  sample  was  and  500°C  atomized  at  nm  2700°C  lamp  run  at  dried  sec,  for 5  a  resonance  slowly  f o r 35  into  a at  charred at sec.  Cu An  injection  determine line  of  current  Cu  10  levels  of  i n the  a Varian Techtron of  temperatures for  volume  sec,  13  mA  of  110  then  was and  finally  25  u l was  samples.  hollow used.  150°C  The  cathode The  f o r 30  atomized  used  at  on  324.7 Cu  nm  lamp  sample sec,  the  GFAAS  resonance run  was  at  a  dried  at  charred at  2700°C  for 5  to  sec.  1000°C  33  RESULTS  1.  AND,DISCUSSION  Characterization  (a)  Equilibration  An the  volume  equilibrium volumes resin The  with  o f SOW  sample  300  necessary  containing  E l u a t e Mn with  resin-Mn  equilibration  sample  volume  equilibrium containing sample  50 nM  volumes  increments, increased  (Figs. been  change  in ml  were  resin  were  from  the 100  passed  the  found that  to  come  ( F i g . 6) u p  To d e t e r m i n e  the  to  come 35  In the f i r s t  50 t o 5 0 0  ml  to  into  ppt  SOW  test,  the  in  sample  increments.  resin-Cu  i n 50 m l  that  with  up  the  indicating  resin  rise  sample  equilibration.  to rise  test,  is into  through  50 t o 500 m l  t o 500 m l ,  second ml  to  for  found  conducted.  in  be d e t e r m i n e d  Different  experiments  i n c r e a s e d from  while  the  achieved.  for  two  7 & 8), indicating  achieved.  were  had been  Cu were  were  t o 1000  concentrations  levels  Cu,  must  required  increased  necessary  with  that  100 nM Mn  little  50W-X12  i n t h e sample.  t h e volume were  AG  t o C u a n d Mn  for  the metals  volumes  ml,  Dowex  characteristic  to determine  increments. to  of the resin  important  sample  of the resin  50  ml  volumes  Eluate  Cu  450-500  ml,  equilibration  had  34  Figure  6.  Eluate Mn versus equilibration. (100  sample volume nM Mn i n 35 p p t  required SOW.)  for  35  SV  F i g u r e 7. E l u a t eiredCu f  versus °  r  e  (  3  u  sample i  l  i  b  r  a  t  i  o  volume n  (50  (50-500 ml) nM Cu i n 35 ppt  36  Figure  8.  Eluate Cu versus sample volume required for equilibration (50 SOW).  (50-1000 ml) nM C u i n 3 5 p p t  37  The to  amount  increase  at salinities  presumably cations Since  of metal  because  for binding  natural  salinities required  below reduced  sites  on  between  50 a n d 800 m l w e r e  SOW  (35  Eluate  the  ppt  SOW  of  indicating  500  resin  can  be  fast,  as  liquid  the and  be  increased  flow  with metals  Zorkin  influenced  for  a  GDW)  small  (1983)  may  erode  the resin have  can thus  volume that  flow  was  volumes  of  25  ppt  5 8 . 3 nM C u . to  sample  i n adsorbed  sample  volume  sample  Cu, of  volumes  f o r the e q u i l i b r a t i o n flow  rate  small when the  bead.  o f t h e sample volume  the flow  rate i s  thickness This  of  would in  of the r e s i n  rates  1966).  equal  the  reduce  order  be a c c o m p l i s h e d  (Inczedy,  of the  sample  to travel  and the e q u i l i b r a t i o n  sample  Cu  experiments.  relatively  t h e sample  found  a  have  volume  sample  Consequently,  the  the molecules  the resin in  at  study  increase  change  equilibration  surrounding that  by  A  to  1983).  with  6 liters  SOW,  t h e major  sample  containing  little  required  the  using  i n a l l of the  volume  in this  found  in  (Zorkin,  equilibrium  found  (Fig. 9).  required  film  with  with  to the resin.  distance  react  used  sample  delivered  the  500 m l  500 m l w e r e  may  with  was  found  Different  resin-Cu equilibration  approximately  The  into  tested  diluted  ml,  ppt,  salinity.  Cu c o n c e n t r a t i o n s were  volumes  of  lower  resin  25  t o come  at  ppt  t o be u s e d  approximately  tested  the resin  competition with  the  samples  f o r the resin  onto  t h e 35  of  seawater  of  adsorbed  to with  quickly However,  t o o r above  2.0  38  Figure  9.  Eluate Cu versus sample required for equlibration SOW).  volume (58.3  nM  (50-800 C u i n 25  ml) ppt  39  ml/min d i d not  i n f l u e n c e the sample volume r e q u i r e d f o r  equilibration  of  Dowex  AG  t h i s study) with the metals the  flow  rates  approximately channeling  the  50W-X12 (the same r e s i n used i n in  the  sample.  i n t h i s study were kept  5 to 6 ml/min to  reduce  of the s o l u t i o n through  Nevertheless,  f a i r l y constant at  the  possibility  of  the columns at very high  flow r a t e s .  (b) Cu and Mn  adsorption  curves  In order to determine the amount of Cu and Mn onto  the  resin  r e s i n has  to  composition.  when  be  natural  calibrated  The  c a l i b r a t i o n can be reduced and  metal  levels  water samples are used, the with  number  standards  of  standards  i f metal  adsorbed  levels  onto  the  r e l a t i o n s h i p over the range of metal tested.  The  t e s t e d u s i n g Mn nM  in  35  ppt  concentration. linear,  even  linearity  of  the  The Mn  with  two  when 5000 nM Mn  S i m i l a r l y , the l i n e a r i t y of the tested  using  nM  25  in  ppt  concentration.  75,  The  with Cu  two  for  standards  to  be  relationship  was  and  500  for  found  (Figs.  each to  10 &  replicates curve  100,  250  run was  be  13).  relationship  50,  l i n e a r a t the low c o n c e n t r a t i o n s although  250 run  was  Cu-resin  adsorption  required  100,  present  Cu c o n c e n t r a t i o n s of 10, SOW,  known  concentrations  curve  was  a  r e s i n show a l i n e a r  replicates  adsorption  of  i n the  Mn-resin  c o n c e n t r a t i o n s of 50, SOW,  adsorbed  was  and  500  for  each  found  to be  the slope  of  the  40  Figure  10.  Eluate  Mn  versus  total  Mn  added  ( i n 35  ppt  SOW).  41  adsorption  curve  indicating higher curve  that  decreased  the r e s i n  concentrations. at high  importance  used  in  this  have  also  been  (c)  found  o f Cu  Since  the  i t  with  determine p p t SOW  aliquots added.  Each  eluate  Cu  Mn Mn  concentrations in  the  (Fig.  eluate  12).  significant  Cu  ±2  little  o f Cu  under  were and  to  be  curves  seemingly 1983).  always  under  water  passed  the  same  samples.  50  resin and  will  Mn  test  in  for adsorption  o f Mn nM of  on  Cu  Cu was  was  run  there  concentration this standard  was  when  Cu  onto  divided  500,  1000  the  a  5000  and  Mn  resin.  5000  of  500 nM  the range slight  nM  Mn  considered  deviations  water  ml were  duplicate.  over  was  to  5 liters  into  and  in  used  natural  adsorption,  100,  although  be  whether  d i d n o t c h a n g e much  However, as  time  to  concentration  tested  of the  of  (Zorkin,  solutions  o f Cu  another  spikes  levels  even  the  adsorption  the e f f e c t  and  slope  t o be  11),  at  of the a d s o r p t i o n  the n a t u r a l  species  containing  (Fig.  t o Cu  levels  slightly  necessary  one  expected  slopes  cation-exchange  was  sensitive  conditions  as  nM  the d i f f e r e n t  was  a t t h e same  o n Mn  the ionic  interfered  35  The  to differ  conditions  250  o f t h e low  standard  the columns  samples,  To  the  Effect  estimate  because  operating  operating  not as  However,  study.  Consequently, through  was  Cu c o n c e n t r a t i o n s  practical  identical  after  for  was  not  The of  Mn  decrease present to  be  a l lthe  data  42  Figure  11.  Eluate  Cu  versus  total  Cu  added  ( i n 25  ppt  SOW).  43  (M _  cn"  <_D  1  CJ  lil t—°P m  (M'  ~~l 0.0  Figure  100.0  1 200.0  1  —i 300.0  TOTRL MN ADDED (NM)  .400.0  (X10 ) 1  1 500.0  1 2 . T h e e f f e c t o f Mn o n C u a d s o r p t i o n to the resin (100-5000 nM Mn, 50 nM Cu i n 35 p p t SOW). V e r t i c a l b a r s p r e s e n t when ± 2 s t a n d a r d d e v i a t i o n s exceed t h e s i z e of t h e symbol.  44  points were Mn to  were  found  analysed adsorbed  be  (Fig.  samples,  (d)  onto  another  Effect  of  Since Aquil  was  the natural  nutrients  was  to  obtain  levels  follows:  nutrients except Si  into  Si  Cu  t h e amount  added  o f 50  was  nM  Mn  onto  of  metals  a n d Mn  found  Cu the  d i d not  of  had resin  in  the  interfere  to the r e s i n .  at Aquil  50  7  a n d Fe were  growth,  added  to 5 l i t e r s  nM  and  Cu  1 liter  the Aquil only  aliquots,  iron  (1.25 x  Fe  at  to the seawater  would  affected,  as both  Cu  o f C u a n d Mn  to  o f 25 Mn.  which  ppt  The were  10" M);  a n d Mn silicic  SOW  was  of a l l  I, Table of  1) only  4) a d d i t i o n  5  (6.25 x  Aquil  samples adsorbed and  of  10~ M); 5  concentration  run f o r each  acid  SOW  treated  2) a d d i t i o n  concentration  added  of  of  ( F e ) ; 3) a d d i t i o n  r e p l i c a t e s were  amount  nM  with  the influence  (see Appendix  Two  the  100  of n u t r i e n t s ;  ( S i ) and  concentration  whether  a r e t o be a m e n d e d  the adsorption  concentrations  of  10" M).  samples  on  were  five  S i at 5 times  be  water  of  for silicate  x  that  1) n o a d d i t i o n  5) a d d i t i o n (4.51  f o r the l e v e l s  tested  a n d Mn  at the Aquil  only  of  f o r phytoplankton  Cu  divided  Mn  samples  nutrients  resin.  as  adsorption  eluate  between  the presence  for adsorption  the  then  the  concluded  nutrients  added  t h e same  and the t o t a l  that  Consequently, i t  one  the resin  on  When  the r e l a t i o n s h i p  indicating  effect 13).  with  f o r Mn,  linear,  little  to overlap.  to onto  addition. determine the  resin  hydrated  Fe  Figure  13.  E l u a t e Mn o f Cu (50  v e r s u s t o t a l Mn nM Cu i n 35 p p t  added SOW).  in  the  46  (III)  oxides  ASV-labile Si  have  Cu  been  demonstrated  i n s e a w a t e r due t o a d s o r p t i o n  a n d Fe p a r t i c l e s  (Lumsden  al.,  1983).  Table  1 shows  not  differ  when  nutrients  added  t o the samples.  observed  to  when  An  that  eluate  and  onto  colloidal et  Cu c o n c e n t r a t i o n s d i d levels  i n eluate  together  the  1983; F l o r e n c e  various  increase  the nutrients  10" M) were  and F l o r e n c e ,  decrease  with  o f S i were  C u was  Aquil  however,  levels  of Fe  (4.51  x  Table  1. E f f e c t o f A q u i l n u t r i e n t s o n t h e a d s o r p t i o n of Cu a n d Mn ( 5 0 nM C u , 100 nM Mn i n 25 p p t SOW) t o Dowex AG 50W-X12 resin. See A p p e n d i x , T a b l e 1 for Aquil nutrient levels. Where appropriate, () = ±2 standard deviations.  7  added.  resin)  No  7.90  (0.49)  4.27  (0.25)  8.42  (0.25)  3.92  (0.07)  8.48  (0.33)  3.22  (0.92)  8.93  (0.16)  3.69  (0.57)  (1.94)  3.87  (0.00)  All nutrients except S i and Fe A q u i l l e v e l s of S i (1.25 x 10"  M)  5  5 times A q u i l of S i (6.25 x  levels 10" M) 5  A q u i l l e v e l s of Fe (4.51 x 1 0 "  To Cu  further  was a d d e d  11.11  M)  7  test  the effect  to 5 l i t e r s  concentration  of  50  concentrations  o f 10, 5 0 ,  of  nM.  resin)  (nM/g  Cu  nutrients  (nM/g  Mn  Test  o f F e on e l u a t e  25  ppt  SOW  In  the  first  100 a n d 5 0 0 nM  to  were  Cu  obtain  levels, a  Cu  experiment,  Fe  tested,  while  47  in  the  second  experiment,  5000  were  1000  and  each  concentration.  to  have  nM  little  while  Fe  to  increase  (Figs.  &  increases Cu  Fe  Cu  15). Fe  The  rise  onto  and  resin.  As  The high that  Benjamin  flow  rates  which  may  also  Fe  2  and  and  in  1000  to  that  for  Fe  onto  slower  oxides  water  particles  from  the  rather  et a l . ,  onto  the  that  with  metals  were  report  in the  have  the  beads Cu  when  with  indicating  trapped  resin  and  samples  possibly  in  the  (Inczedy,  adsorbed the  Huang  conditions, i f  in  colloids.  eluted Elliot  rise  expected  (Swallow  become  may  eluate  is  is  adsorption  packed  been  Cu  s o l u t i o n , more  have  in  the  levels  (1980)  found  further  contamination Fe  14),  dramatically  change  hydroxides  a  were  however,  high  found  (Fig.  nM  little  Cu  were  levels  Fe  at  nM)  levels  Leckie  resin.  3  Cu  concentrations,  tightly  surface  Al 0  Cu  ferric  may  have  the  (10-100  concentrations,  trapped  normal onto  Fe  the  Fe  to  visibly  oxides  between  through  under  adsorbed  Fe  for  adsorption  were  nM)  run  nM  available  s o l u t i o n by  r e p l i c a t e s were  Cu  due  introduced  These  passed that  well,  colloidal  them,  high  500,  indicating  eluate  colloidal  (500-5000  1966).  in  less  from  interstices  not  100,  100  1000  15),  of  eluate  resulted  made  colloids  removed  (Fig.  probably  to  1980),  levels  at  the  eluate  was  since,  of  between  Beyond  unexpected adsorb  on  concentrations  Two  levels  the  in  stock.  more  Low  effect  concentrations  eluate  tested.  concentrations  14  Fe  onto  NTA  was  (1979)  found  less NTA:Cu  Cu  (II)  was  ratio  in  48  Figure  14.  The effect of F e on C u a d s o r p t i o n t o t h e ( 1 0 - 5 0 0 nM F e , 50 nM C u i n 25 p p t SOW).  resin  49  Figure  15.  The e f f e c t o f Fe ( 1 0 0 - 5 0 0 0 nM F e ,  on 50  Cu nM  adsorption to the C u i n 25 p p t SOW).  resin  50  solution  was  concentration the  greater o f t h e NTA  Cu a d s o r b e d  adsorbed levels  onto  when  Since  the  (Table  1).  to  obtain  50,  100 a n d 500 nM found  even  in  decrease  this by  the  thought  t h e network  eluate  Mn  concentrations  reaction  between  do  levels  was  the Fe  again  resin.  One onto  Mn-NTA  Fe  (Fig.  the will  o f Mn  NTA  slower  o f 35 p p t  SOW  16).  However,  with  eluate  previously, trapped  Cu  levels,  as only  from  of i s  the kinetics  n o t be a f f e c t e d  the Fe  for  high that  f o rthe Cu-NTA.  the resin, a  a  samples  explanation  than  o f 10,  tested,  becoming  through  be e l u t e d  of  levels  i s passed  will  d i d not  i n the presence  Fe o x i d e s ,  even  amounts  and, as noted  Unlike  tested  Cu  concentrations  observed  possible  is  Mn  Cu  eluate  and Fe s p i k e s  The e l u a t e  Cu  eluate  the resin  to 5 liters  o f 500 nM was  on  and v a r i o u s  over  the  added.  effect  100 nM,  high  removed  as  i n the high  onto  not increase  adsorbs  levels  of  added.  rate  the  when  amount  added  unchanged  of Fe.  Mn  small  was  the  have  well  t o be due t o t h e F e o x i d e s  of  although  Mn  adsorbed  Fe c o n c e n t r a t i o n s  levels  Therefore,  Mn  presence  high  Mn  of n u t r i e n t s  were  i n the flow  was  eluate  Mn  may  o f Fe were  dramatic  concentration  t o remain  containing  resulting  a  of  i n the presence  were  as  o f F e on e l u a t e  level  a  oxides,  concentrations  such  though  a n d Fe  Fe  resin,  the e f f e c t  Si  study  the  Fe has  Therefore,  in this  the  elevated  1.0.  used  onto  levels,  change  than  the  relatively  oxides.  51  o co"  CD <\i"  CD  -  —  ^  —o  UJ —  cr-' ZD  , 0 0  Figure  16.  10.0  —I 20.0  1 30.0  TOTAL FE ADDED (NM)  1  I 5  (X10  1  0  0  )  The effect of F e o n Mn a d s o r p t i o n t o t h e ( 1 0 - 5 0 0 nM F e , 100 nM C u i n 35 p p t SOW).  resin  52  III.  THE E F F E C T  OF AMBIENT NATURAL  LEVELS WATER  OF MN  ON  CU T O X I C I T Y  IN  SAMPLES  INTRODUCTION  The well  established  (e.g. The  biological  Sunda  of  a  analysis  models  estimate for  i s  of  occurring  between  specified  conditions  strength  thermodynamic determined  be a d j u s t e d  and  errors  various  of  i n order  in calculating  (Stumm a n d M o r g a n ,  (Westall  et  adsorption solids  such  silicic  acid  as  ions,  unknown  (Rueter  et  however,  equilibrium  estimated  through  MINEQL,  thermodynamic  the major  1980).  under  and i o n i c  However,  constants)  than  reactions  species  temperature  complex  MINTEQ) o r  seawater  t o be a p p l i e d  the  are often and  have  t o seawater,  the chemical  speciation  may  1981).  models  a s MINEQL  also  Most  and  hydrated  the relevant a l . , 1981). models  such  do n o t i n c l u d e  ligands  colloidal  because  1978).  chemical  f o r them  a _ l . , 1976)  of free  and Morel,  using  equilibrium  less  speciation  T h e 'computer  and  Batley,  has been  samples. by  pressure,  (e.g.  in solutions  water  elements  and  data  to  result  the  (Florence  (e.g.  speciation  the  i t s chemical  usually  models  natural  chemical  by  i n seawater  1976; A n d e r s o n  metal  of  most  of a metal  influenced  t h e o r e t i c a l computer  direct  data  t o be  and G u i l l a r d ,  speciation  either  effect  to  or  on  ( I I I ) oxides and  equilibrium Despite  are useful  the e f f e c t of  complexes Fe  thus  for  constants these  are  drawbacks  estimating  the  53  chemical have of  speciation  often  a  metal  Anderson  been with  and  The  its  Morel,  bioavailability 1978;  analysis  ultrafiltration, combinations  of  these 1981;  by  consequently  it  may  speciation  give  a  and  and  (e.g.  compare the  to  The  a  normally  determination, these  results  analysis  to of  equilibrium  estimate of  1976;  chemical  are  direct  bioavailability  various  Batley,  theoretical  realistic  (e.g.  or  1983).  of  by  stripping  methods  method  However,  more  sample  dialysis),  to  estimated  Anodic  methods  the  be  water  e_t a _ l . ,  compared  the  also  ( F l o r e n c e and  difficult  when  can  separation  such  on  predictions.  samples  models,  is  and  speciation  organisms  (usually  Florence  dependent  the  to  the  techniques  operationally  water  of  chemical  determined  equilibrium  metals  ion-exchange  Davies,  species  of  techniques or  system,  1982).  direct  voltammetry)  defined  in studies associating  speciation  electrochemical  and  used  chemically well  chemical  through  Hart  in a  of  chemical  metal  in  the  use  natural  waters. This  chapter  cation-exchange and  described  available modified  on  the  resin  in  in this  same  with  2,  natural study  results  technique,  in chapter  Cu  Bioassays  discusses  to water  from  developed estimate  the  samples.  The  f o r a s s e s s i n g Mn  T h a l a s s i o s i ra pseudonana  water  samples,  and  by  the  Zorkin  results  a  (1983)  biologically technique  in natural were  of  also  compared  was  waters. conducted to  the  54  amount  of  Cu  whether  ambient  biologically Cu  to  and  from  (Whitfield,  the  Indian of  inlet  and  bottom  Arm,  thus  local  Mn  (IV)  a_l. ,  are  occasionally in  the  high  the  Since  to  high  i n deep  water  1983),  of  Mn  oxygen  in  concentrations,  undergo  Arm  were  dissolved  soluble  Indian  (Burling,  deep  of  of  with  depletion  oxides  toxicity  fjord  oxygen  of  samples  especially  converted  1979).  marine  determine  concentration  water  concentration  insoluble  to  i n f l u e n c e the  Mn,  p r i m a r i l y to  and  resin  the  natural  low  e_t  extremely  high  the  affect  At  (Emerson  accumulates  a  onto  waters.  state  only  The  The  i s due  oxidation  the  could  dissolved  1976).  particulate,  over  levels  a v a i l a b l e Cu  concentrations  the  adsorbed  phytoplankton.  collected  in  Mn  Mn  a  Mn  change (II)  completely the  waters  of  the  fjord,  concentrations  of  dissolved  in  ions turns  soluble  Mn  resulting  in  Mn.  55  MATERIALS  1.  AND  METHODS  Characteristics  The  seawater  shallow-silled (Fig.  17).  deep,  samples  fjord  The s i l l  although  1976).  occurs  Indian  brackish  water  A  above  influenced  by t h e v o l u m e  only Arm  have  been  Gilmartin  2.  from with The  documented  of n a t u r a l  Seawater  from  collected  at station  located  five  at the deepest  each  depth  the e l a s t i c samples  filters  200 m  estuarine  circulation  outflowing layer  denser,  saltier  of the f j o r d ,  water  runoff,  of Georgia  which i s  water,  Davidson  of  layer  tidal  characteristics by  m  mixing occurs  of Indian  (1976)  and  (1962).  Collection  were  well  i s over  mixing  The p h y s i c a l  B.C.  of the basin  thin  Strait  a  26  a  of fresh  of the incoming  Arm,  i s only  inflowing  Complete  occasionally.  depth  with  an  Indian  of the i n l e t  multi-layer  Arm,  from  northeast of Vancouver,  a t t h e mouth  1983).  density  taken  located  (Burling,  and  were  t h e maximum  (Davidson, in  o f I n d i a n Arm  were  within  were  seawater  depths IND-2  point  ( 1 0 , 5 0 , 1 0 0 , 150 a n d 2 0 0 ( 4 9 ° 2 3 . 5 ' N,  of  the  gathered  using  replaced  b y o n e made  filtered  through  24  hours  after  inlet. 8 liter from  Gelman  1 2 2 ° 5 2 . 5 ' W) , Forty  liters  Niskin  bottles  silicone 196  collection.  m)  mm,  rubber. 0.45  The i n i t i a l  urn 2  56  Figure  17.  Location  of  sample  collection.  57  liters  of  before  filtered  each  sample  carboy  was  was  used  filled  as  with  liter  polyethylene  were  used  within  collection.  The  Niskin  carboys  initially  stored  dark,  for  3.  and  a  were  day,  then  Analysis and  five  of  3  rinsed  natural  times  water  d i s s o l v e d Mn  depths  was  HC1.  samples The  from  a  Mn  ppt  samples  was  stripping  voltammetry  2.5  SOW.  for  determined  1N  with  (ASV)  3  total  in  months  the after  holders  and  1 N  HC1  dissolved  Cu  soaked  plated  using  deposition  a  in  in  a  placed metal  2  pH  collected  injection  GFAAS  curve  by  1  with  N  in  the  derived  from  differential  distilled  374  60  a  Princeton  polarographic  the  Teflon  between  -0.3  sec.  Ten  GFAAS  ml  of  i n the pulse  the  samples  to  The  ASV  graphite  the  to  walls)  and  the  Research A  seawater  total  mercury  electrode  -0.15  chlorosilane-coated borosilicate  adsorption  water anodic  HC1.  and  were  analysis  Applied  V  after  samples  analyser.  rotating  from  isopiestically  d i s s o l v e d Cu  isopiestically  potential of  direct  samples  acidifying  onto  time  water  after  model  was  the  Total  Corporation  reduce  samples  samples  16°C  filter  HC1,  concentrations  w e r e made w i t h  was  6N  discarded  The  at  to  filters,  GDW.  to  measurements  by  carboys  with  by  standard  25  Mn-enriched  in  determined  the  estimated  film  sample.  weeks  r i n s e d with  Total  distilled  pH  bottles,  and.  Mn  acidifying  of  2  rinse  the  i n 20  were  a  V  with  a  sample cell  (to  Cu  was  58  determined  by  through  the  samples  were  4.  passing a  electrodes. determined  A d a p t a t i o n of  The  Dowex  200-400  mesh)  samples  was  previously 10" M the  resin.  all  prepared  5.  was of  AG  50W-X8  EDTA  in the  each  water  bubbling  for  acid-cleaned air.  A l l  acid  cleaned The  ensure  that  organism.  AG  50W-X12.  6-12 (1  N  i n the  and they  15  20  H SO„)  plasticware  trace would  Freshly  adsorbed  Cu  the  liter  (depending  on  filtered  (0.4  as  samples  not  prepared  water  mix  become Fe  seawater described  aliquot and Mn  Mn  were (but  were  water  one  depth The  sample)  urn  pH by  with  Nucleopore)  and  carboys)  was  previously.  amended without  deficient  x  from  8.00±0.05  the  described  3  2.  from  to  bottles  of  stocks  natural  adjusted  (including  metal  and  as  in chapter  of  samples  polyethylene carboy.  and  and  Cu  using  liters  same m a n n e r  water  ml  and  method.  natural  manner A  the  Laboratories,  20  experiments  hours 2  same  discussed  measured  natural  nutrients  the  with  V  in  water  (Bio-Rad  in  experiment,  was  for natural  the  -0.15  and  concentrations  experiments  manner  V  standard addition  solution  to a  Cu  resin  remove  resin  transferred the  to  -0.9  between  method  i n the  used  The  the  resin  Dowex  B i o a s s a y s and  For  by  prepared  was  The  the  used  for  EDTA  2  potential  to  stock, allowed  with  EDTA the to  or  Aquil Mn)  to  bioassay  equilibrate  59  for  several  the  Aquil  hours,  experiments  had  eluate  Figs.  14 &  Cu  was  15).  numbers  or  Mueller,  pers.comm.).  water  was  spikes  of  samples  were  in  left  40,  the bioassay A portion  used  in  manner were  the  80,  and  sample,  Counter.  were  precipitation may  be  counted  (B.  nutrients, and  then  added.  Cu The  t o use  was  i n t h e same  the water  with  to  samples  A  used  Cu  of  250  ml  between  1000-2000  15 of  concentrations.  concentration.  pseudonana  of  total  any  f o r the c o n t r o l s  t h e Cu  T.  a period  preserve  containing  were  run f o r each  over  cell  samples  conducted  and  each  for testing  inoculated  water  present.  flasks,  used  were  the  to inoculation with T h a l a s s i o s i r a  were  concentrations  numbers  to influence  nM  However,  3 flasks  r e p l i c a t e s were  cell  were  prepared:  Three  were  160  natural  which  prior  the r e s t  (e.g.  to equilibrate prior  previously.  that  have  experiments.  of the prepared  while  samples  and  overnight  erlenmeyer  were  not expected  120  resin  F e may  in  of the bioassays,  hours,  reduce  polycarbonate  term  used  experiments  t o e q u i l i b r a t e f o r 2-3  left  organics  spike  the influence resin  (10% of  8  of T h a l a s s i o s i r a pseudonana  After  bioassays,  to  10' M  of the  not autoclaved  natural  was  Fe  x  the a d d i t i o n  as d e s c r i b e d  pseudonana  the  rates  60,  then  Fe  a t 4.51  smaller  the short  of added growth  The  in  During  level  added  to minimize  levels  reduced  the  also  Fe c o n c e n t r a t i o n ) .  these on  was  to obtain  5 days  cells/ml. using  a  The initial Cell  Coulter  60  The used 500  remainder  of the prepared  in the resin ml  experiments.  aliquots  bottles.  The  calibrate  and  Cu  the resin  columns  the  natural  SOW  with  t h e a p p r o p r i a t e Cu  500  ml  bottles. and  Cu-enriched  column  as  each  were  eluted  with  eluate  f o r Cu  with  i n Table sample  20 m l  3 times  a n d Mn  with  divided  into  polypropylene  at the  were  used  same  time  (made  from  also  500 m l  to  divided  polypropylene  for  each  standard  the  same m a n n e r  sample. run  in  prepared  standard  was  a t one  passed  as  time.  through  a  2.  and  of 3 x  into  12 c o l u m n s or a  was  standards  prepared  were  sample  run  were  ml  spikes)  placed  was  sample  (pH = 8 . 0 0 ± 0 . 0 5 )  The  o r Mn  seawater  columns  of a  shown  Since columns  natural  previously,  replicate  and  500  prepared  samples.  replicates  resin  described One  aliquots  Three  The  seawater  were  water  sample  into  standards  as  into  The  placed  a n d Mn  natural  standard  had  3  in succession.  10" M 2  EDTA  GFAAS.  prior  replicates, The  columns  to analysis  the were  of the  61  Table  2. Dowex AG 50W-X12 r e s i n c o l u m n t e s t . s e r i e s f o r estimating e f f e c t i v e metal c o n c e n t r a t i o n s .  Column 1 2 3 4 5 6 7 8 9 10 1 1 12  No.  Metal  Concentration  None ( b l a n k ) 40 nM C u 80 nM C u 120 nM C u No a d d e d C u 40 nM C u 60 nM C u 80 nM C u 120 nM C u 160 nM C u 100 nM Mn 300 nM Mn  Solut ion D i l u t e d SOW Cu s t a n d a r d s  (SOW)  IT ff  Natural  seawater  ii  »i  Mn  standards  (SOW)  62  RESULTS  1.  AND  DISCUSSION  Adaptation  of  the  Preliminary indicated  that  effective 50W-X12 their  experiments  neither  resin.  1 x  Other  eluents  hot  (80°C)  included  for natural  with  10" M  natural NTA  2  the  eluents  effectiveness  pH  method  i n e l u t i n g a l l of  The  low  resin  Cu  EDTA:NTA  2  x  warm  (40°C)  (5.0)  with  3 x  10" M 2  3  x  10" M  10" M NTA;  2  salt);  3 N  20  3 N, ml  5 N  from  cations  of  the  were  seawater  also  cations  GFAAS  atomization  EDTA but more  or not  the  a  a l l of  high  (Kingston  (which the  et  salt  for eluting  the  to  1 x  3  ammonium acids,  acid.  The  are  the to  trace  columns.  ratio  during  of is the  Consequently,  to  chelators  as  metals  were  of  high  sensitivity  volatilized  a l . , 1978).  seawater  in  signal  A the  presence  especially reduce  and  acid.  major  cations)  the  10" M,  adsorbed  background  the  and  i n removing  interference,  remove  seawater  (as  (9.5)  hydrochloric  eluate,  cations  background  NTA  suitable  as  1 x  for  solution;  pH  ft  AG  resin.  effective  the  found  Dowex  the  (50:50) high  were  tested  acetic, sulfuric  was  by  the been  the  step  HC1  the  from  10" M,  acid  a l t h o u g h any  in has  when  N  1 x  distilled  eluted  readings,  produced  minimize  3  resin  concentrations, the  nitric,  isopiestically  aliquot  metals  grade  metals  samples EDTA  2  from  EDTA;  2  10" M  therefore  a  1-Pyrrolidinecarbodithioic  1 N,  Mn  the  samples  seawater 1 x  i n removing  and  reagent  and  were  10' M 2  nor  water  from  such  the  considered  resin to  be  63  Since v a r i o u s c o n c e n t r a t i o n s and eluent volumes of EDTA and  NTA  d i d not remove the metals from the r e s i n Dowex AG  50W-X12, a d i f f e r e n t  r e s i n with a lower c r o s s - l i n k a g e (Dowex  AG 50W-X8) was t e s t e d .  With the Dowex AG 50W-X8, 20 ml of 3  x 10" M EDTA was found to be e f f e c t i v e  in eluting  2  adsorbed onto the r e s i n . with the  I t was  presumed  the lower c r o s s - l i n k a g e has l a r g e r EDTA molecules  complex  could  could  diffuse  diffuse out  containing  50  through the columns. from  nM  resin  pores through  which  more e a s i l y .  Cu  The sample  and  with  relatively  that the r e s i n had (Figs.  18  &  little  attained  to  achieve  100 nM Mn were passed tested  100 to 1000 ml i n 100 ml increments.  ml,  To determine the  sample volumes of 25  volumes  c o n c e n t r a t i o n s were found to r i s e up  the metal-EDTA  50W-X8  e q u i l i b r a t i o n with Cu and Mn, d i f f e r e n t SOW  the  i n and  sample volume r e q u i r e d f o r the Dowex AG  ppt  that  the metals  to  increased  E l u a t e Cu and Mn approximately  400  change to 1000 ml, i n d i c a t i n g equilibrium  with  Cu  and  Mn  19). Consequently, sample volumes of 500 ml  were used f o r these r e s i n  experiments.  2. Bioassays Cu was added to 6 l i t e r s of n a t u r a l seawater at  each  and  160 nM.  rates  depth  of  (Table 3 ) .  collected  t o obtain c o n c e n t r a t i o n s of 40, 60, 80, 120  With more Cu added t o the medium,  T h a l a s s i o s i ra However,  pseudonana  the  growth  were found t o decrease  the d e t r i m e n t a l  effect  of  Cu  at  64  Figure  18. E l u a t e . Cu versus sample volume required for e q u i l i b r a t i o n f o r Dowex AG 50W-X8 r e s i n (50 nM Cu i n 35 ppt SOW).  65  Figure  19. E l u a t e Mn versus sample e q u i l i b r a t i o n f o r Dowex AG Mn i n 2 5 p p t SOW).  volume required for 50W-X8 r e s i n (100 nM  66  Table  3. Growth r a t e Arm w a t e r . deviations.  of T h a l a s s i o s i r a pseudonana i n Indian Where a p p r o p r i a t e , 0 = ±2 standard  Depth (m)  Copper added (nM)  10  0 40 60 80 1 20 160  1 .89 1 .52 1 .29 1 .24 1.17 0.89  (0.02) (0.02) < 0.03) ( 0.01) ( 0.02) ( 0.02)  81 68 66 62 47  (1 ) (2) (1 ) (2) (2)  0 40 60 80 1 20 160  1 .80 1 .50 1 .30 1.32 1.15 0.89  ( 0. 1 4 ) ( 0.04) < 0.01 ) ( 0.05) I 0.05) < 0.02)  83 72 73 64 49  (3) ( 1 ) (4) (4) (2)  0 40 60 80 120 160  2.20 0.03) 2.05 ,0.02) 2.01 < 0 . 0 2 ) 1 .89 10.01) 1 .53 [0.02) [0.02) 1.31  93 91 86 70 60  (1 ) (1 ) (1) (1 ) (2)  0 40 60 80 120 160  2.16 2.16 1 .95 1 .96 1 .76 1 .37  [0.04) [0.02) [0.11) [0.06) [0.10) (0.06)  100 90 91 81 63  (1 ) (6) (3) (6) (4)  0 40 60 80 120 160  1 1 1 1 1 1  (0.04) (0.05) (0.04) (0.02) (0.02) (0.01)  98 100 96 85 65  (3) (2) (1 ) (1) (1)  50  1 00  150  200  elevated test  deepest rate  depths  (as compared  was  .88 .84 .88 .80 .60 .22  found  grown i n w a t e r (150  %  R  concentrat ions  o r g a n i s m was  Growth r a t e (divisions/day)  and  200  to  samples m).  to the control)  was  For  Control  b e r e d u c e d when t h e collected example,  not affected  from the  the  growth  when  a Cu  67  spike the  of  60  nM  same C u  growth  was  added  spike  rate  by  added 32%.  contain  acids,  fulvic  and  humic  concentration  of  such  the  Ralph, rates  3.  1983) of  T.  Resin  of  of  sample,  from  each  were  prepared  for  the  metal the  reduced  been  shown  deep  (e.g.  amino A  high  previously  (e.g.  water  the  i s that  ligands  explain  Borgmann  the  better  to and  growth  samples.  full  had  onto to  SOW  strength  nM  Cu,  and  at  the  same  100  and  time  samples.  The  sample related,  and  300  nM  and  a  In  order  resin  same  ppt).  GDW. Mn  under of  were  with  the Cu  same or  of  adsorbed  passed  relationship  can  the  seawater  s t a n d a r d s of from as  a  Cu  or  Mn  that  of  the  SOW  40,  was 80  the  made  and  onto  the  the  120  columns  conditions  through be  2.  described  the  through  operating  Mn  in Table  diluted  passed  of  to determine  salinity The  liters  as  from  Standards  standard are this  manner  calibrated  the  9  indicated  same  the  be  of  amount  and  as  using  s t a n d a r d s w e r e made  SOW  and  depth  i n the  These  to  conducted  bioassays.  adsorbed  resin  were  ( a p p r o x i m a t e l y 25-27  be  sample  while  shallow waters.  Cu  deep  sample,  explanation  has  may  i n the  samples  can  water  than  ligands  added  a  m  water  occurring  acids)  experiments  composition.  when  m  possible  spikes  the  10  therefore  seawater  previously  from  200  bioavailable  pseudonana  samples  known  the  naturally  and  resin  natural  amount  the  experiments  The  The  amount  to One  waters  reduce  more  to  as  resin  columns  expressed  as  68  the  effective  in  press).  Cu  a n d Mn  are  T h e EMC  by  various  the  total  Cu  sample  exactly  the  standards.  hydroxide  extent and  samples  of metal  hydroxide  ions  ions  used  the  thought  EMC  i n t h e sample  present  in  standards  considered  and  to  Mn  be  by l i g a n d s  f o r Cu and by l i g a n d s  are  than  the  ions f o r  for  other  the  Cu a n d  natural  a measure  other  will  natural  to influence  the  Cu  metals  a  f o r Cu and c h l o r i d e  for  total  these  for  present  ligands  for  ions  complexation  same  Cu  the bioassays  more  Cu  added  concentrations indicating However,  that not  spikes were to  the  (ECC)  of the  carbonate  than  chloride  a l l of  sample  produced  sample  without  contained  Cu  found was  the  addition  onto  C u was Cu  spike  4 3 . 8 nM,  (Table the  suggests  some  naturally occurring  ligands,  4),  adsorbed  onto the  to a  m  while  the seawater  Cu  resin.  50  water  t h e same  o f Cu had a b a c k g r o u n d  This  With  the e f f e c t i v e  increase  adsorbed  o f a 40 nM  that  to  tested  experiments.  samples,  added  a n ECC o f o n l y the  120 a n d 160 nM)  i n the resin  seawater  were  more  as the a d d i t i o n  nM.  (40, 60, 80,  tested  resin,  17.3  those  the  f o r Mn. The  in  as  et. a l . ,  f o r C u o r Mn  concentration  same  are  ions  T h e EMC  i f the ligands  major  on b o t h  t o which  only  Consequently,  seawater  Mn  carbonate  (see Zorkin  a r e dependent  ligands.  i n t h e SOW  and  (EMC)  and the e x t e n t  or  The only  speciation  Mn.  values  concentration  seawater  Mn  concentration  bound  equal  are  metal  samples which  water ECC o f  probably would  tend  69  Table  Depth (m)  10  4. E f f e c t i v e Cu a n d Mn concentrations in coppere n r i c h e d I n d i a n Arm w a t e r . Where a p p r o p r i a t e , () = ±2 s t a n d a r d d e v i a t i o n s . [] = Copper present in unenriched w a t e r . ECC = e f f e c t i v e copper concent r a t i o n , EMnC = e f f e c t i v e m a n g a n e s e c o n c e n t r a t i o n . Manganese p r e s e n t = t o t a l d i s s o l v e d manganese.  Copper Added (nM) [14.2  (1 . 5 ) ]  0 40 60 80 1 20 1 60 50  [16.5  [54.5  150  [13.2  17.29 43.81 67.40 79.70 126.30 152.97  (0.24) (2.53) (0.24) (4.62) (5.39) (4.62)  14.83 43.45 67.27 78.35 129.74 159.70  (0.85)  (1.81)  (1.49) (0.65) (2.49) (3.42) (3.43)  (3 . 9 ) ] 12.85 40.62 59.35 77.26 120.61 153.43  (0.45) (0.89) (2.53) (1.56) (3.97) (3.57)  (1 . 3 ) ] 16.96 0 43.62 40 6 2 . 18 60 80 78.26 130.72 120 160 154.49  (0.63) (1.08) (1.02) (1.18) (2.95) (5.27)  0 40 60 80 1 20 160 200  (0.23) (1.79) (0.62) (0.71 ) (1.18)  (5 . 3 ) ]  0 40 60 80 120 160  Manganese Present (nM) <  16.52 43.33 62.61 77.68 120.14 154.98  (2 . 4 ) ]  0 40 60 80 1 20 160 100  ECC Concentration (nM)  [17.9  < 1 Tf fl f! ff ft  10 II II II  IT  II  60.4 n n »i ft  EMnC Concentration (nM)  (0.6)  29.4 ti ti n  II  ii  II  II  633.5 H n H n ft  (9.4)  n ti ft  1443.5 n »i ii ft n n  (20.1 )  2504.1  (26.7)  It fl fl fl ft  (6.4)  II  TI  »!  369.9 n  (0.72)  1281.7  (23.6)  ii II II  ft 1671.7 «  ft tt it  (18.1)  70  to  reduce  the  amount  the  resin.  The  vertical  Cu  addition  also  found  to  occurring twice  ECC  differ,  level  of  although  (Table  a  of  and  added  Cu  available  profile  of  ECC  total  For  the  example, dissolved  corresponding This  ligands high  for  dissolved  suggesting  total  4).  concentration sample  the  ligands.  the  depths,  and  of  is  the  was  present  that  a  degree  of  the  five  Cu  the  ECC  was  100  of  of  m  without  a  in  were  naturally  water  not  that  onto  concentration  compared  peak  adsorb  samples  presence  Cu  suggests  Cu  to  to  had  over  the  other  evident  in  relatively the  100  m  of  Cu  complexation  the high  water may  be  occurring. For water depth  each  samples, from  which  contrast, to  the  increase  overall are  vertical  similar,  somewhat  nM  greater. of  be  200  the  the  in m  indicates  Mn  may  be  occurring  in  dissolved  natural  (Table  the  10  sample  m  In found  sample  (Table  the  the  4).  to  4).  The  dissolved  values  were  found  that  like  Cu,  by  of  (EMnC) w e r e  EMnC a n d  latter  the  independent  collected  levels  the  of  This  to  to  concentrations  in  profiles  although  complexation  were  undetectable  1650  added  found  samples  e f f e e t i v e Mn  from  approximately  the  spikes  naturally  Mn  to  be  some  occurring  ligands. The contrast the slow  increase to  the  surface. oxidation  open  This  oceans  enrichment  k i n e t i c s of  Mn  levels with  w h e r e Mn  is usually  is partly  soluble  Mn  depth  due  (II)  to to  is  enriched  the  in at  extremely  insoluble  Mn  71  (IV)  (Ahrland,  manganese organic such  oxides  Indian due  previously  station  4.  Comparison  The to  growth  decrease  function 20).  organism the  resin,  et  a l .  as  and  water  samples  possible  be  the  ECC  the  indicated  and  water  increased  responsible organism  in  to  for the  The  of  with  reducing water  fjords with  reduction  of  dissolved Arm  at  the  Mn same  results  by  the  plotted  resin  analysis  of  Cu  amount  of  by  Zorkin  (1983)  pronounced deep  Cu  of  Cu  ECC In  the  in  contrast,  depth.  This  Cu  samples.  low  the  Zorkin  the  bioassay appear resin  the  shallow  EMnC  values  suggests  toxicity  in  the  both  onto  growth  ECC)  However,  values  the  Zorkin  the  the  a  (Fig.  and  From  (i.e.  samples.  as  adsorbed  however,  waters.  found  a v a i l a b l e to  the  as  were  when  r e l a t i o n s h i p between  similar  samples.  deep  the  Indian  values  shown  water  dramatically  for  amount  complexation  analysis deep  the  not  deep  and  profile  resin  ECC  related  was  to  T h a l a s s i o s i r a pseudonana  c o l l e c t e d from  more in  and  that  press).  found  some  (1974).  estimated  previously  (in  rates  described  ECC  indicated could  vertical  higher  dissolved  increase  oxygen  of  of In  of  rates  insoluble  a l . , 1983).  depletion a  of  presence  is  bioassay  the  et  Whitfield  with  of  This  results,  of  the  Mn  Such  by  in  photoreduction  dissolved  been  (IND-2)  the  (Sunda  the  oxides.  and light  Arm,  to  manganese has  by  substances  as  depth  1975)  to  that  Mn  is  the  test  (1983)  also  72  ©  o i  0.0  1 40.0  1 80.0  1 120.0  1 160.0  1 200.0  ECC (NM)  F i g u r e 20. The e f f e c t of v a r y i n g EMnC v a l u e s on Cu t o x i c i t y in n a t u r a l water samples. Symbols: X 200 m water (EMnC = 1671.7 nM); <J> 150 m water (EMnC = 1281.7 nM); * 100 m water (EMnC = 369.9 nM); A 50 m water (EMnC = 29.4 nM); © 10m water (EMnC = < 1 nM).  73  found  that  samples that  from  growth  concentrations  measured. deep  the  In t h i s water  toxicity onto  of  samples  does  i n Indian  Mn,  which  would  Stumm  have  that  The  effect  organism  of  seawater  Mn  removal  in  the  through  physiological  the  on r e d u c i n g  Cu  biologically  adsorption  to  kinetics  o f Mn slow.  to the bioassay  due  not  to  a c t i v e Cu c o n c e n t r a t i o n  processes,  of the organism  to  and Diem and  a r e extremely  be  o f Cu  available  (1981)  Cu t o x i c i t y  in  in  the oxidation  conditions  with  was  not  make C u l e s s  considered  response  and  o f Mn  not  obtained  o f t h e Mn  form  e t a_l.  on r e d u c i n g  i s therefore  reduction  Cu  verified  natural  was  t o be d u e t o t h e a d s o r p t i o n  Sunda  under  water  suggested  EMnC  values  most  the effect  Furthermore,  (II)  and  the  EMnC  ionic  organisms. (1984)  t h e deep  Arm,  although  indicate that  not appear  in  be d u e t o t h e p r e s e n c e o f  the high  If true,  oxides  better  might  available  form.  manganese  rates  study,  biologically  particulate  grew  t h e same s t a t i o n  the higher  high  the  T. p s e u d o n a n a  but  rather  to the ionic  the by the  forms of  a n d Mn. Sunda  and Huntsman  mechanism  of  the  increase  in  Cu-Mn  cupric (1 x  Thalassiosira  pseudonana  activity  of was  Cu  was  10"  1  0  -  5  to  However, be  reduced  authors  then  biochemical  found  at  stopped  (3H).  The  and  activity  M)  shown  increased.  investigated the  interaction ion  activities  effect  (1983)  that  low  the the  an  Mn  ion  growth  of  detrimental  when added  t h e Mn i o n 5  *Mn  to  74  determine that  the  the c e l l u l a r  activity also in  was  after  with  a n d Cu  by  their  be  to the free  related Mn  negative  feedback  Mn  i n Mn  cupric  thought  Mn  Mn  Mn  additions  to affect The  control,  been  found than  to the  1985).  t o be r e g u l a t e d the exact  rate  manganese  Huntsman,  although  of  Thus,  growth  ion concentration rather  i s thought  was  which,  rates.  levels.  and  rate  levels  has subsequently  (Sunda  ion  ion activity.  nor t h e growth  were  found  In  through  mechanism i s  uncertain. When  a  bioassay  biological assumption metal and  Mn  and  i n growth  in cellular  a decrease  the c e l l  the c e l l u l a r  the  concentration, further  concentration  addition,  still  into  when  decrease  on c e l l u l a r  rate  a  with  ion a c t i v i t i e s  transport  total  The  cellular  influence  of the c e l l s  decreased  a decrease  a certain  d i d not a f f e c t  content  content  was a s s o c i a t e d  However,  Mn  Mn  Mn  increased.  associated  turn,  Mn  cellular  availablity i s that  being  study  the  toxicity  verifies  this  such  toxicity (1984)  of  a  Sunda  and  from  a natural  a s Zn a n d F e a r e t h o u g h t  an  only  e_t a l .  indicates  unchelated  waters.  system  to  to  (1981), that  Sunda can  The p r e s e n t  that  i t  important Murphy study  the  Mn  environment.  t o be  the  as Cu, t h e main  demonstrated  i s considered in natural used  such  estimate  Cu t o p h y t o p l a n k t o n .  finding water  to  i s responding  have  of  used  metal  However,  (1983;1985)  characteristic metals  of  i s  the organism  tested.  Huntsman  reduce  organism  is  a  Other when  Cu  et a l .  t h e combined  75  effects under  of  presence  of  requirement absence  nutritional be  natural  deficiencies  conditions)  sensitive The  must  Fe  s p e c i e s of  species.  the  and  natural  neritic more  Mn  requirement  Cu  was  or  found  of metal  the to  Fe.  was  of  when  The  to  of  in the  oceanic  species  than  the  increase Mn,  while  presence  becoming  apparent  phytoplankton metal  combination  of  oceanic  levels  found  absence  is  likely  sensitivity  in metal  increase It  status  considered systems.  Cu  Thalassiosira.  to changes  Fe  on  (a  toxicity  is  of  and were  neritic in  the  the  Mn  Cu  or  that  the  complex  and  is considered in  76  GENERAL Cu  and  Mn  are  essential  physiological  processes  concentrations,  Mn  adverse species  has  presence  recently  of  Mn  198.3; 1 9 8 5 ) . manner  the  bioassay  study.  The  seawater  T.  the  test  of  in  absence  was  added  found  Sunda  are  affected  et  to  to  a_l.  and  1978),  the be  can  have  Mn  in  on  Cu  (1981)  and  in  this  chelating Cu  toxicity  verifying  Sunda  to  well-defined  media,  reduced,  was  toxicity  artificial test  same  it  used  a  the  Huntsman,  thus  in Aquil, of  high  i n the  pseudonana  grown  At  reduced  Sunda  of  for  phytoplankton  1981;  effect  was  Cu  some  be  Morel,  organism the  amounts  while  to  to  Thalassiosira  m o r e Mn  pseudonana  Cu  a l l algae  determine organism,  of  al. ,  and  With  findings  e_t  not  toxic,  shown  Anderson  medium,  agents. to  However,  to  toxicity been  trace  phytoplankton.  becomes  (Sunda  (e.g.  necessary  The  in  in  rarely  effects.  SUMMARY  and  the  Huntsman  (1983;1985). To  estimate  the  cation-exchange Zorkin  et  volume  a_l.  of  effect  Aquil  tested.  technique (in press)  required  linearity of  biologically  for  adsorption  active  Cu  by  Zorkin  developed was  tested  in Aquil.  equilibration of  nutrients  metals on  the  to  of the  efficiency  the  and  a  (1983)  and  The  sample  resin,  resin, of  Mn,  the  the  and  the  resin  was  77  Modifications could  be  could  not  resin.  applied be  The  Dowex  AG  allowing easily  use  the  resin  of  Thalassiosira and  the  high  that active  not  ionic  The  affected was  therefore  be  such  as  also  considered.  must  Mn  the  as  Cu.  presence  of  to  metals  to  on  found  and  the  local used  the with water  results  from  grow  which by  of  to  same  better contain  the  estimated  resin by  the  Mn,  indicating  in  biologically  Cu  effects  c o n s i d e r e d when  to  affect  to  waters,  as  a  was  the  the  changes  such  more  Bioassays  estimated  responding  complex, be  deep  Arm,  could  conducted  from  is by  applied  resin  c o n c e n t r a t i o n s as  by  toxicity  can  Mn  Cu  The  was  as  out  then  Indian  compared  50W-X12  resin.  levels  pseudonana  collected  organism  Mn.  were  metals  presumably  i n and  was  available  the  cross-linkage,  the  Mn.  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An method f o r m e a s u r i n g t h e c o n c e n t r a t i o n of a c t i v e Cu i n s e a w a t e r . A n a l . C h i m . A c t a  86  APPENDIX  I  87  Table I. Composition of Aquil medium modified from Morel et a l . (1979).  Substance Aquil s a l t s (SOW)  NaCl Na S0 2  4  20  4,.20  81. e  20  2..88 X  X  30. e  20  1..05 X  20  9,.39  X  NaHCO  4.0  20  2.,38  X  KBr  2.0  20  8..40  X  0. 6  20  1*. ,85 X  0. 314  20  6.,38  X  -4 10' -3 10' •4 10" •4 10' 10"•5  0.06  20  7.,14  X  20  5.46  X  B 0  2  2  NaF MgCl .6H 0 2  222. 0  2  X  6.3e  X  10'•5  7.14  X  10"-5  •2 10"  5.46  X  •2 10'  X  10"  S.97  X  1. 50  X  CuSO^.5H 0  0. 249  1  9.97  X  •4 10" •3 10" 10"•3  (NH ) Mo 0 .4H 0  0. 265  1  1. 50  X  CoCl .6H 0  0. 59 5  1  2. 50  X  MnCl .4H 0  0.199  0.1  1. 00  X  ZnSO^.VH 0  0. 115  0.1  4.00  X  •3 10" 10"•3  FeCl .6K 0  0. 122  1  14.  51  X  .4 10"  0.011  0. 01  1. 10  X  10  0. 01  0. 1  1.00  X  10"  0. 020  0.1  2.00  X  10"  3  2  12 Biotin B  Thiamine HC1  X  4.85  X  1. 25  2  X  8.40  1.25  1. 00  1  2  2. 36  -4 10" •3 10" -4 10" -4 10" 10"•5  1. 00  1  z 55  2  X  •2 10"  8.50  Na„£iO_.9K.0  2u  X  9.39  X  NaNO  2  1. 05  X  X  7  X  -5 10" •4 10'  1. 00  6  2. 88  -1 10' -2 10" ic"-2  X  1  2  X  1.00  1. 38  4  4.20  •2 10" 10"•1  NaHjPC^.HjO  ?  Final concentration (K)  -1 10" •2 10' 10"-2  14. 0  z  3 3 SrCl .6H 0  Vitamins  490. 6  Stock concentration (M)  KC1  H  Trace Metals  volume (liters)  CaCl .2H 0 2  Nutrients  Initial weight . (g)  0 1 1  •10 10" •9 ic" 10"•9  2. 50  X  1. 00  X  4.  00  X  •9 10" 10"•9  4.51  X  ic"  •7  g/l  5 . 5 X 10"  g/l  5.0  X  10"  g/l  1 .0  X  10"  7 7 4  g/l g/l g/l  88  Table  Depth (m) 1 0 50 100 1 50 200  I I . H y d r o g r a p h i c d a t a f o r I n d i a n Arm. C r u i s e d a t e = A u g u s t 24, 1984. Temperature (°C) 1 1 .40 1 1 .34 8.35 8.35 8.35  Salinity (ppt)  Oxygen (ml/1)  pH  24.783 26.044 26.873 26.970 26.983  4.17 3.87 1 .68 1 .47 1 .38  7.63 7.62 7.39 7.29 7.32  

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