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The effects of sublethal concentrations of mercuric chloride on ammonium-limited Skeletonema costatum… Cloutier-Mantha, Louise 1978

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HE EFFECTS OF SUBLETHAL CONCENTRATIONS OF MERCURIC CHLORIDE ON AMMONIUM-LIMITED  SgELETONEMA COSTATUM  (GREV.) CLEVE.  by  LOUISE CLOUTIER-MANTHA B.Sc,  M c G i l l U n i v e r s i t y , 1976  THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  The F a c u l t y  of Graduate S t u d i e s  (Department of Botany)  We accept t h i s t h e s i s as conforming to the r e g u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA August, 1978 © L o u i s e Cloutier-Mantha, 1978  In  presenting  requirements  this for  thesis an  that  available  for  permission f o r  his  degree  that  the  reference  extensive  s c h o l a r l y purposes may or  partial  advanced  B r i t i s h Columbia, I agree freely  in  at  Library  and  copying  of  It  is  written  The U n i v e r s i t y of B r i t i s h Columbia 2075 wesbrook Place Vancouver, B.C. , Canada, V6T 1W5  make  the of it  I f u r t h e r agree this  thesis  for  Department  understood that copying or  permission.  Botany  of  University  shall  study.  p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l  Department of  the  be qranted to the Head of my  representatives.  allowed without my  fulfillment  gain  shall  not  be  ABSTRACT  The  e f f e c t s o f s u b l e t h a l a d d i t i o n s of  were s t u d i e d i n the marine diatom  mercuric  Skaletonema costatug. {Grev.)  Cleve grown i n ammonium-limited chemostats and batch In  the short-term  chemostat  effluents  5 pM NH^Cl nH HgCl^.  and  Hg  of  Hg exposure (679. 5  e f f l u e n t s were only perturbed  hours),  .  ammo-  Hg decreased the the  assimilation or the i n t e r n a l l y controlled  These e f f e c t s occurred  between  rate  uptake  When the e f f l u e n t was starved f o r 30 hours,  was reduced.  5.53  Hg exposure, when t h e e f f l u e n t from the  f o r the s u b s t r a t e ( i n c r e a s e d Ks value) and  rate, Vi  with  with 5 uM NH^Cl.  c u l t u r e was s t a r v e d f o r 1.5 hours,  ammonium  Vi  perturbed  c o n c e n t r a t i o n s ranging from 0.04 to  In the short-term chemostat  cultures.  (up to 5 h o u r s ) , unexposed  simultaneously  In the long-term  nium-starved  affinity  Hg exposure  were  chloride  only  1.84 and  max  3,68,  and at 0,18 nH H g C l  hours,  respectively.  2  i n e f f l u e n t s s t a r v e d f o r 1.5 and 30  The maximum rate of uptake, Vs, was not  depressed. In the long-term decreased  Hg exposure,  the s p e c i f i c  growth  at rate  least and  d e n s i t y , while the c h l o r o p h y l l a per c e l l of  0.37 the  Morphological recovery. In the  alterations long-term  were observed  experiment,  maximum  increased.  population d e c l i n e was followed by resumption  nH  of  HgCl^ cell  A period growth.  before and a f t e r the  s i x days  of  continual  iii  exposure  to  0.37  nM  HgCl^ g r a d u a l l y  without a f f e c t i n g Vs and V i  max  3.68  nM  since  HgCl^  both  were  the  assimilatory  ^^fnux^  were  (Ks  impaired.  a l s o reduced  and  the  In a d d i t i o n , the  after  of  growth  exposure  to  After  i n the Hg-treated c u l t u r e s , when a new  s t e a d y - s t a t e was e s t a b l i s h e d , the a f f i n i t y and  value)  nM HgCl^ f o r s i x days i n t h e long-term experiment.  resumption  to  to the short-term Hg exposure,  affinity  maximal uptake r a t e , Vs, was 3.68  The r e s u l t s from exposure  similar  substrate  rate  .  i n c r e a s e d the Ks value  for  the  substrate  a s s i m i l a t o r y r a t e s i n c r e a s e d i n phase D (day 23) compared  to phase A (day 6) . The recovery of growth and n u t r i e n t uptake r a t e s i n phase D, may  have been p a r t i a l l y  tolerance  mediated by the a c g u i s i t i o n  of  Hq  and the appearance o f c e l l s o f a d i f f e r e n t stage of  the s e x u a l l i f e c y c l e , as suggested  by  differences  in  cell  s i z e and chemical composition. An  attempt  was  made  t o determine whether a short-term  p h y s i o l o g i c a l response (Hg i n d u c t i o n o f thesis)  could  be  responsible  metallothionein  f o r the r e c o v e r y .  syn-  The 250 nm  absorbance p r o f i l e , o f n u t r i e n t - s a t u r a t e d c u l t u r e s exposed f o r 90 t o 116 hours to s u b l e t h a l c o n c e n t r a t i o n s o f mercury, showed no l a r g e absorbance peak i n the medium molecular corresponding  to  laetallothionein,  as  i t  weight  pool,  occurs i n animals  exposed t o heavy metals. The i n t r a c e l l u l a r d i s t r i b u t i o n and l e v e l s of Cu, Zn,  and  Hg i n S. costatum, grown i n n u t r i e n t - s a t u r a t e d batch c u l t u r e s , were  affected  by 0.37  nM H g C l . 0  A c o n c e n t r a t i o n equal to or  g r e a t e r than density, m.w.  possibly  pool.  addition  1,84 nM EqCl^ due  Exposure to  reduced  the growth  t o the accumulation 1.84 nM  of 5.53 nM reduced  HgCl^  prior  gradually  in  m.w.  fractions  m.w.  pool., Copper s l i g h t l y i n c r e a s e d i n  but  remained  constant  in  m.w.,  usually  to  the  high in  a  second  m.w.  pool.  and  the  high  low  medium  m.w.  t h e medium and low m.w.  pool  pools, i n  High l e v e l s o f Cu and  pool suggests t h a t a substance reported  cell  of a  for metallothionein,  lower may  be  i n v o l v e d i n the storage and d e t o x i f i c a t i o n of heavy metals  in  S,  than  the  increased  r e l a t i o n to t o t a l i n t r a c e l l u l a r l e v e l s . Zn i n the low m.w.  and  of Hg i n the high  Hg l e v e l s i n ths high  Upon Hg exposure, Zn l e v e l s decreased but  rate  costatum.  Thesis  Supervisor  V  TABLE OF CONTENTS Page CHAPTER I . INTRODUCTION S e c t i o n 1. Environmental Impacts, D i s t r i b u t i o n and C o n c e n t r a t i o n o f Mercury Section 2. F a c t o r s I n f l u e n c i n g Mercury T o x i c i t y ...... 2.1 Chemical Composition o f Medium ................ 2.2 S p e c i a t i o n of Mercury S e c t i o n 3. Accumulation of Metals .................... S e c t i o n 4. E f f e c t s o f Mercury S e c t i o n 5. Mercury R e s i s t a n c e ........................ Section 6. Assessment o f Experimental Design ......... Section 7. Purpose o f t h i s Study .....................  1 1 4 4 7 8 9 14 16 17  CHAPTER I I . EFFECTS OF SHORT AND LONG-TERM EXPOSURES TO SUBLETHAL LEVELS OF Hg ON NUTRIENT KINETICS 19 S e c t i o n 1. I n t r o d u c t i o n .............................. 19 S e c t i o n 2. M a t e r i a l s and Methods ..................... 22 2.1 Inoculum 22 2.2 Chemostat C u l t u r e s ............................ 22 2.3 Analyses ...................................... 23 2.4 Experimental Design ........................... 26 S e c t i o n 3. Results ................................... 30 3.1 R e s u l t s from some P r e l i m i n a r y S t u d i e s ......... 30 3.2 Growth Phases d u r i n g t h e Long-term Mercury Exposure ....................................... 3 0 3.3 S p e c i f i c Growth Rates and Nitrogen Quotas ..... 36 3.4 E f f e c t s of Mercury on Photosynthesis .......... 37 3.5 M o r p h o l o g i c a l Observations .................... 40 3.6 Mercury Analyses .............................. 41 3.7 Short-term N u t r i e n t K i n e t i c s 43 3.8 Long-term N u t r i e n t K i n e t i c s ................... 49 S e c t i o n 4. D i s c u s s i o n ................................ 54 4.1 E f f e c t s of Mercury on Photosynthesis .......... 54 4.2 E f f e c t s o f Mercury on Growth Parameters . . . . . . . 5 6 4.3 E f f e c t s of Mercury on N u t r i e n t Uptake K i n e t i c s 57 4.4 Recovery from I n i t i a l Mercury I n h i b i t i o n ...... 59 4.5 Mercury Losses ................................ 60 4.6 A p p l i c a t i o n s t o the N a t u r a l Environment ....... 61 4.7 E v a l u a t i o n o f Chemostat S t u d i e s ............... 62 f  CHAPTER I I I . THE EFFECT OF H<j EXPOSURE ON INTRACELLULAR DISTRIBUTION OF Hg, Cu, AND Zn ....................... S e c t i o n 1. I n t r o d u c t i o n S e c t i o n 2. M a t e r i a l s and Methods ..................... 2.1 Batch C u l t u r e s ................................ 2.2 Experimental C o n d i t i o n s ....................... 2.3 Analyses ...................................... S e c t i o n 3. Results 3.1 Growth  63 63 64 64 65 65 68 68  vi  3.2  Distribution  Section  o f Hq, Cu and Zn  68  4. D i s c u s s i o n  75  SUMMARY. REFERENCES APPENDICES Appendix  Medium  78  A. C o m p o s i t i o n o f A r t i f i c i a l  Seawater  A p p e n d i x B. Data D e r i v e d from t h e L o n q - t e r m s u r e o f Ammonium-limited S^ c g s t a t u m  and Hg  .. "f  Expo-  82 94  95 ge  vii  LIST OF TABLES Paqe  TABLE I . L i t e r a t u r e Summary o f t h e r i a l s on P h y t o p l a n k t o n  Effects of  TABLE I I . S t a n d a r d V a r i a t i o n s i n the Used i n t h e S h o r t and Lonq-term E x p e r i m e n t s TABLE I I I . C o n c e n t r a t i o n s of Exposure Experiment TABLE I V . N u t r i e n t term Hq Exposure  Uptake  Hq i n the  Mercu  Analyses  Lonq-term  Hq  K i n e t i c Response t o S h o r t -  TABLE V. E f f e c t o f D u r a t i o n o f S t a r v a t i o n o f Chemostat E f f l u e n t on the N u t r i e n t Uptake Response TABLE VI. N u t r i e n t Uptake term Hq Exposure  Kinetic  Response t o  Lonq-  27  44  4 7  50  5 1  TABLE V I I . Exposure t o Different C o n c e n t r a t i o n s of H q C l a t D i f f e r e n t Times d u r i n q a B a t c h C u l t u r e Experiment  66  TABLE V I I I . D i s t r i b u t i o n o f T o t a l Zn, I n t r a c e l l u l a r P o o l s due t o Hq Exposure  73  2  Cu, and Hq i n ..  LIST OF FIGURES  FIGURE 1. N u t r i e n t Uptake K i n e t i c Terminology FIGURE 2. Changes i n i n v i v c Fluorescence r i e n t s a t u r a t e d Batch C u l t u r e s FIGURE 3. Changes i n Biomass o f C u l t u r e s during Long-term Hq Exposure FIGURE 4. Changes i n Lonq-term Hq Exposure  Specific  in  Nut-  Ammonium-limited  Growth  FIGURE 5. Changes i n C h l o r o p h y l l a and s i s durinq Long-term Hq Exposure  Rate durinq  Photosynthe,..  FIGURE 6. V a r i a t i o n s i n Expected T o t a l Hg L e v e l s i n Chemostat I durinq LoEq-term Hg Exposure ............. FIGURE 7. Ammonium Uptake Rates as a Function t r a t e d u r i n q Short-term Hq Exposure  of Subs-  FIGURE 8. Ammonium Uptake Rates as a Function of Subst r a t e on Day 6 durinq Lonq-term Hq Exposure .......... FIGURE 9. Disappearance of Substrate Phase A of Lonq-term Hq Exposure FIGURE 10. Ammonium Substrate a f t e r the Exposure  with  Time d u r i n q  Uptake Rates as a Function of Recovery from the Lonq-term Hq  FIGURE 11. Changes i n i n v i v o C u l t u r e s A, B, C and D  Fluorescence  in  Batch  FIGURE 12. The 250 nm Absorbance P r o f i l e c f F r a c t i o n s C o l l e c t e d from Batch C u l t u r e s A, B, C and D  FIGURE 13. D i s t r i b u t i o n o f T o t a l Cu and Zn i n each F r a c t i o n and Gel E l u t i o n P r o f i l e of Batch C u l t u r e C ..  X  ACKNOWLEDGMENTS  This  research  would  not have been p o s s i b l e without the  much a p p r e c i a t e d h e l p , advice, encouragement and of  my s u p e r v i s o r . Dr. Paul J . H a r r i s o n .  thank the two other N. J . A n t i a  and  members  Dr.  of  my  I would a l s o l i k e t o  thesis  of  would l i k e t o o f f e r s p e c i a l thanks t o Mr. assistance  on  Andersen and Mr. The  research  collaboration was  M.J.  LeBlanc  i n chapter  with  instrumental  Dr.  this  thesis.  I  David H. Turpin f o r  many o c c a s i o n s during the experiments and  u s e f u l comments thoroughout t h i s study. E.  committee.  A. G. Lewis f o r t h e i r c o l l a b o r a t i o n and  h e l p f u l recommendations i n t h e r e v i s i o n  his  availability  D i s c u s s i o n s with Dr.  were h e l p f u l .  three  Mr.  David  A.  in  stimulating  was  conducted  Brown.. Dr. T.B. the  in  Parsons  research  on  metallothionein. 1  am  Parsons assisted  also  who  were  for  S.  to  originaly  i n the e a r l y  acknowledge Ms. computing  grateful  Dr. K.H. on  my  H a l l and Dr. T.B.  thesis  stages o f planning.  committee,  I would l i k e to  H a r r i s o n f o r her advice while  facilities,  h e l p i n g t o generate  and  Mr.  B.H.  using  Mantha and T.  t h i s computer-typed  and  thesis.  the  Diersch  1 CHAPTER I  INTRODUCTION  S e c t i o n 1. ... Environmental Impacts,, D i s t r i b u t j o n and"  Concentra-  t i o n of Mercury During  the  1950*s and  i n w i l d l i f e populations mercurials.  For  declined  Sweden.  in  Guatemala dressed  occurred  example,  alkyl  In  Canada  The  axial  River  and  Niigata  and  ingestion  Pakistan  are  1973).  i n the P r a i r i e s . (> 0.5  and  Disasters  amongst  the  best  poisonings. high  l e v e l s of Hg were found i n  Prairies  (Femreite,  muscles of pike from the Saskatoon R i v e r (Hobeser  commercial  of  piscivorous birds  Iraq,  (Krenkel,  (1965.)  p a r t r i d g e s i n the  network and  incinerated  the  outbreaks were caused by wheat-  (1968-1969),  up to 10 mg-kg-iHg sport  to  Yugoslavia,  mercurial fungicides  documented cases of human  pheasants and  due  sead-eating  community poisoning  a t Minimata (1953) and  In  1960's, s e v e r a l dramatic d e c l i n e s  et  al.,  1970).  1970). contained  Consequently,  f i s h e r i e s were c l o s e d i n the Saskatoon  parts o f the Great Lakes, as w e l l as More than one  m i l l i o n pounds  of  hunting  fish  were  mg'kqr Hg i n muscle t i s s u e , Dept. F i s h , and l  F o r e s t r y , 1971). Usually  the  major o f f e n d e r s o f Hg p o l l u t i o n are;  c h l o r - a l k a l i industry electrolytic  where  preparation  of  inorganic chlorine  used  1)  Hg  is  in  and  c a u s t i c soda,  the the and  2 2) metal recovery from Pb-Zn in;  deposits.  used  1) the f a b r i c a t i o n of e l e c t r i c a l and c o n t r o l i n s t r u m e n t s ,  2) a n t i s e p t i c s and p r e s e r v a t i v e s and  Mercury i s a l s o  paints,  and  in  pharmaceutical  products  3) f u n g i c i d e s and s l i m i c i d e s i n a g r i c u l t u r e  and the pulp i n d u s t r y . The d i s c l o s u r e general  decline  about  mercury  toxicity  i n the u t i l i z a t i o n  has  led  and commercial  to  a  production  of m e r c u r i a l s and a s t r e n g t h e n i n g of l e g i s l a t i o n on by-product d i s p o s a l and d i s c h a r g e . environment,  there  In s p i t e of a reduced i n p u t i n t o  are  still  important  and  inadequately  q u a n t i f i e d l o s s e s from anthropoqenic sources, such and  the  as  mining  smelting of Hg ores, combustion of f o s s i l f u e l s and  oils,  and improper waste d i s p o s a l o f s l u d g e s . In the ocean, the primary sources of Hq a r e ; deqassinq of the e a r t h ' s c r u s t , i n d u s t r i a l  pollution  by  atmospheric  jet  streams, p r e c i p i t a t i o n , volcanism, and u p w e l l i n q of deep water {Gardner, (Nelson  1975). et  Weatherinq, l e a c h i n q of Hq-containinq s o i l s  a l . , 1977),  environmental  fluxes  river  transport  influenced  by  man  to  oceans  are  less  important as the n a t u r a l atmospheric t r a n s l o c a t i o n o f Hq c o n t i n e n t s to oceans  (Rice e t a l . ,  1973; Windom e t a l . ,  and or  as from  1975).  Due t o d i f f e r e n c e s i n samplinq, a n a l y t i c a l techniques and the  short  residence  ocean, i t i s d i f f i c u l t  time  of  Hq  i n the mixed l a y e r of the  to q e n e r a l i z e on Hq l e v e l s i n d i f f e r e n t  water bodies (&ndren and H a r r i s s , 1975; Gardner, 1975; K u i p e r , 1976). in  the  The lowest Hq l e v e l s r e p o r t e d ranqed from 11.2 southern  hemisphere  to  33,5  nq«l  _ 1  nq-1  -1  i n the northern  3 hemisphere; the north anomaly  having  only  h i g h v a l u e s o f 364 (Bice  et a l . ,  1973;  from  1972;  1973).  Hg  v a l u e s ranged  unpolluted  sition et  ng-i"  125  0.2 5.3  levels  - 1  Hg  0.4  mg-l  specific  areas  Freshwater  and  plankton,  Miettenen, particulate  (dry weight)  _ 1  polluted  the  pollution  in  waters.  were n o t c o r r e l a t e d t o s p e c i e s  b u t t o t h e d i s t a n c e from  an  Unusually  1974).  mg*l  was  1975).  in coastal  - 1  zone  (Keckes  Atlantic  to  compo-  sources  (Windom  a l . , 1973). Even t h o u g h b a c k g r o u n d  tle of  et a l . ,  ng'l  In N o r t h  to  (Gardner,  - 1  Williams  from  coastal  occurred i n c e r t a i n  - 1  17 t o  areas,  V a r i a t i o n s i n Hg  14.7  ug-1  streams ranged Krenkel,  eastern A t l a n t i c  concern,  h e a l t h hazards  Hg-contaminated  food  chains  affinity  foods,  (Cook,  groups  (Seyferth,  through The  selenols,  of  a r e low  amino  magnified toxicity  a c i d s and  populations  t h r e s h o l d s were a s f o l l o w s : Hg nM;  Zn~1pM;  Ni,  (Hollibaugh et a l . , unpublished costaturn. with  comparable  5-10  (Berland  ug'l  Hg  of  i s due  amino and  various c e l l  lit-  in  to i t s  carboxyl components  - 1  Hg,  t h a n Cu  <10  nM;  Co,  25-100  performed  indicated C u ~ 100  Sb,  Se,  and  manuscript).  r e d u c t i o n of c e l l  e t a l . , 1977).  inhibitory  be  1978) .  phytoplankton  As(V)~300  may  accumulation  of  phosphates,  B i o a s s a y s of s i n g l e element t o x i c i t y natural  and  a r e a s s o c i a t e d with t h e i n g e s t i o n  1977).  for thiols,  terminal  levels  ug-1-  1  Cd  that nM;  Pb,  In  1971)  and  was  50-200  Cu  Cd,  and uM  Skeletonema  I n Ch1ore11a p y r a n o i d p s a ,  (Kamp-Nielsen,  on  toxicity  As(III) > 1  division and  j,n s j j t u  was  obtained ug'l-*  Hg  was  more  Cu  more toxic  4 than Zn i n  Amphidinium  S. costatum,  and  carterae,  Phaeodact^lum  Thalassiosjra tricornuturn  pseudonana.  (Braek  et a l . .  1976).  S e c t i o n 2.  2,1  F a c t o r s I n f l u e n c i n g Mercury T o x i c i t y  Chemical Composition o f Medium Heavy metal t o x i c i t y v a r i e s  rimental  variables  widely  and c o n d i t i o n s .  according  The i n f l u e n c e of  f a c t o r s a f f e c t i n g heavy metal t o x i c i t y on algae 1971;  to  expevarious  (Kamp-Nielsen,  Hannan and P a t o u i l l e t , 1972; Bice e t a l , , 1973), and  microorganisms and Mott, Faust,  and  1948;  1963;  i n v e r t e b r a t e s have been s t u d i e d  Corner wisely  and and  Sparrow, Blick,  1956;  1966;  on  (Pyefinch  Fitzgerald  and  Lewis et a l , , 1973;  W h i t f i e l d and Lewis, 1976; Gibson g t aJL., 1975). The s e n s i t i v i t y of a t e s t organism i n a is  affected  by:  pollution  1) p h y s i c a l f a c t o r s such as l i g h t  N i e l s e n and Wium-Andersen, 1971; Kamp-Nielsen, 1976), temperature et a l . ,  1977;  {De F i l i p p i s  and  Pallaghy,  Knowles and Zingmark,  1949), pH  (Wisely  exposure  (Erickson,  1971;  {Steemann Overnell,  1976a;  1978), s a l i n i t y  assay  Blinn  (Hunter,  and B l i c k , 1966), time and c o n c e n t r a t i o n 1972;  Zingmark  and  Miller,  of  1973);  2)  chemical f a c t o r s such as the degree of c h e l a t i o n , and n u t r i e n t and d i s s o l v e d oxygen c o n c e n t r a t i o n s F i t z g e r a l d and Faust, 1963;  (Corner and Sparrow,  McBrien and Hassal,  1967;  1956;  Erickson  5 e t a l . , 1970; Overnell,  1975a);  the inoculum 1972;  Lewis et a l . , 1973;  physiological  et a l . , 1970;  and Mayer, 1975;  state  and  Colwell,  1975;  3) b i o l o g i c a l f a c t o r s such as s i z e of  (Steemann N i e l s e n  Ben-Bassat  Miller,  and  Nelson  of  cells  Overnell, (Gibson,  Shieh and Barber, 1976),  1972;  and  the  Zingmark and  1973).  O f a l l the p o t e n t i a l f a c t o r s capable of i n f l u e n c i n g metal t o x i c i t y , the chemical composition o f the growth medium can be the most d i f f i c u l t of  an  f a c t o r to d e f i n e and c o n t r o l .  appropriate  choice  medium r e l i e s both on the s p e c i f i c  rements of the t e s t organisms conducted.  The  and  the  type  The i n h i b i t o r y l e v e l s determined  of  requi-  study  being  in chelator-rich  media cannot be compared to those found i n c h e l a t o r - f r e e (Jensen e t a l . , 1976;  Kayser, 1976;  Overnell,  media  1976).  I t i s well e s t a b l i s h e d that the a d d i t i o n of  natural  and  s y n t h e t i c c h e l a t i n g agents can complex t r a c e metals (Gardiner, 1976)  and  and Lewin, contains acids  i n f l u e n c e t h e i r uptake 1976;  (1971;  1973)  in i t s ability presumably agents.  George and Coombs, 1977).,  undefined complexing  (Singer,  (Cossa, 1976;  1973)  in  Schulz-Baldes  Natural  seawater  agents such as humic and  different  amounts.  found t h a t EDTA-enriched  Lewis  n a t u r a l seawater  et a l . , varied  to support growth of young stages of a copepod,  because  of  the  presence  of  natural  complexing  In c u l t u r e s of F r a g i l a r i a and A s t e r i o n e l l a with s o i l  e x t r a c t a d d i t i o n s , m e r c u r i a l s were  significantly  less  than i n completely d e f i n e d medium (Tompkins and B l i n n , The  fulvic  presence  of  decomposed  natural  plankton  toxic 1976).  and d e t r i t u s  6 i n c r e a s e d Cu t o l e r a n c e i n T. £Sgudpjian.a  {Erickson, 1972) .  absolute growth r a t e s of marine phytoplankton  were  lower  mid-winter seawater i n s p i t e of higher n u t r i e n t l e v e l s §± i.1 • i  1974).  diatoms (Jensen et a l . , tolerance  EDTA : Cu >1 rates  of  of  both  Faust,  inhibition  stimulatory  and  Although  (Overnell, (Steemann  poor  Chlorella  alkali  and  and  even  the  growth  EDTA  ( F i t z g e r a l d and  could  failed  by Cu  Oocystis.  effects  ions  they  molar r a t i o of  alleviate  to  N i e l s e n and Hium-Andersen,  Cu  n e u t r a l i z e Hg 1971).  was i n t i m a t e l y connected with n u t r i -  (Hannan and P a t o u i l l e t , 1972; Hannan et  and i n Daphnia  waters,  coastal  previously i n h i b i t e d  adverse  1975a),  three  the  In C h l o r e l l a v u l g a r i s  t i o n a l s t r e s s i n algae 1973)  on  when  which was  Heavy metal t o x i c i t y  al.,  (Jensen  The a d d i t i o n of EDTA i n c r e a s e d  P. t r i c o r p u t u m .  T.,pseudonana  1963).  toxicity  1976).  and  (Bentley-Howat and Beid, 1977),  ( E r i c k s o n , 1972). had  in  Chelated i r o n counteracted the e f f e c t s of Hg  on C. pyranoidosa (Kamp-Nielsen, 1971)  the  The  (winner et a l . ,  3.0 u g - l H g _ i  (Stokes et a l . ,  1973),  A  1977).  arrested toxic  In  the  Hg  nutrient growth  threshold  of (60  ( j g . l - i ) was demonstrated f o r a summer phytoplankton assemblage but not f o r the s p r i n g p o p u l a t i o n bly  due  t o the combined  (Blinn e t a l . ,  1977),  possi-  e f f e c t s of lower n u t r i e n t l e v e l s and  higher temperatures d u r i n g the summer.  Low  decreased  of d i s s o l v e d o r g a n i c s  the  normal  excretion  rate  (Betz, 1977), thus r e s u l t i n g i n an i n c r e a s e i n These  compounds  may  Bassat and Mayer, 1977;  be  involved  1978).  nutrient  Hg  levels  toxicity.  i n Hg v o l a t i l i z a t i o n  (Ben-  7  2.2 S p e c i a t i o n of Mercury In phytoplankton, inorganic  and elemental Hg., The  c a t i s s i m a was reduced acetate  organomercurials a r e  (PMA);  1 4  C  by 50% with;  0.5 ug«l—  1  1 u.g»l~  potassium  H» t e r t i o l e c t a freshwater  HgCl^, the accumulation with  HgCl^  and  the  0^ e v o l u t i o n o f  lipid  synthesis in  (Matson e t a l . , 1972) were more depressed by  methylmercury than HgCl^.  than  mercuric  ( H a r r i s s et a l , , 1970;  content  ( O v e r n e l l , 1976) and  algae  in  Even though PMA was more t o x i c  o f Hg i n the presence Chlorella  1976a), implying that t o x i c i t y s o l u b i l i t y of organomercurials.  (De  o f PMA was  Filippis  Colwell,  1975).  Hannan  t h a t HgCl^ was more t o x i c mercury.  The  to  1  2  less  i s not always due t o the l i p o i d C u l t u r e s of  and P a t o u i l l e t various  algae  aerobic  hetero(Nelson  (1972) r e p o r t e d than  dimethyl-  primary p r o d u c t i v i t y of F r a g i l a r i a c r o t o n e n s i s  and A s t e r i o n e l l a formosa was more a f f e c t e d HgCl  than  and Pallaghy,  t r o p h i c b a c t e r i a were more r e s i s t a n t t o PMA than HgCl^ and  than  methyl mercuric d i c y a n i d i a m i d e and  white,  The  phenyl  1  d i p h e n y l mercury  1970).  toxic  uptake of N i t z s c h i a d e l i -  MEMMI ; and 10 u g - l - i 1  more  by  Hg(NOj)^  than  (Blinn et a l . , 1977).  N-methylmercuric-1,2,3,6-tetrahydro-3,6-methano-3,4,5,6,7,7 hexachlbrophthaliamide.  8  S e c t i o n 3. Some  Accumulation  o f Metals  aspects o f t h e exchange k i n e t i c s between metals and  algae have been e l u c i d a t e d 1976;  Fujita  and  (Glooschenko,  Hashizume,  1969;  1975).  Davies,  Metal accumulation i s  p r i n c i p a l l y i n f l u e n c e d by t h e s p e c i e s of t h e metal, and of  i t s physiological state.  The uptake  the time and c o n c e n t r a t i o n of exposure  1972;  Davies,  1974;  Cossa,  1976).  g r e a t e r i n d i v i d i n g than n o n - d i v i d i n g Richardson e t a l . , cells  1974;  the  alga  v a r i e s as a f u n c t i o n (Shieh  and  Barber,  Mercury accumulation cells  (Burkett,  was  1975;  1975) and a l s o g r e a t e r i n dead and moribund  than l i v e c e l l s probably due to the c e s s a t i o n o f a c t i v e  e x c r e t i o n o f mercury  (Fujita  and  Hashizume,  1975;  Bentley-  Mowat and Heid, 1977). Metal accumulation The  first  one  i s u s u a l l y accomplished  consists  i n two phases.  o f a F r e u n d l i c h a d s o r p t i o n isotherm  which i s immediate, r a p i d and p a s s i v e .  The metal adsorbs  c e l l s u r f a c e s u n t i l a d e f i n i t e number of s i t e s (Davies,  1974;  1976;  Dolar  onto  are s a t u r a t e d  et a l . , 1971).  This adsorption  phase i s f o l l o w e d by an a c t i v e but slower uptake, d u r i n g which the metal i s t r a n s l o c a t e d across t h e c e l l membrane (Shieh Barber, uptake  1972;  Schulz-Baldes and Lewin, 1976).  was temperature  Pallaghy, exchange  1976c)., of  dependent i n C h l o r e l l a  During  accumulation,  be  T h i s phase of  (De F i l i p p i s and a  simultaneous  metals occurs between algae and the medium (Ben-  Bassat and Mayer, 1977; 1978; Betz, 1977). either  and  Excreted  Hg  can  i n o r g a n i c (Betz, 1977) or o r g a n i c , s i n c e Hg may be  9 r e l e a s e d as a compound complexed with capable of r e d u c i n g Hg* The  incorporation  S^nedra. ulna. Hg was transferred Sistis  2  (Ben-Bassat and  metal i n t o  i n t o the cytoplasm  and  peripheral  et a l . ,  metabolite,  flayer,  1978).  the c e l l i s r a p i d .  ( F u j i t a et a l . , 1977).  associated  with  with 60,000, 180,000, and  d e r i v e d from (Hammans  of  natural  deposited on t h y l a k o i d s u r f a c e s and  n i d u l a n s . Hg was  fraction  to Hg°  a  and  1976).  2 0 3  then In  lamellar  fractions components  H g - l a b e l l e d plant t i s s u e s ,  granules were almost e x c l u s i v e l y sequestered  within  and  (De F i l i p p i s  between  Pallaghy  Bassat  occupied  chromatin  nucleoli and  E f f e c t s o f Mercury  i n h i b i t o r y l e v e l s of m e r c u r i a l s on a l g a l growth  et a l . , 1972;  Hannan and  B l i n n , 1976), photosynthesis Miller,  by  Hg  1975).  S e c t i o n 4. The  spaces  Ana-  phycocyanin-rich  230,000 m.w.  intrinsic  In  a  In  1973;  De F i l i p p i s  P a t o u i l l e t , 1972;  and and  respiration Pallaghy,  (Ben-  Tompkins and (Zingmark  1976a)  and  have been  determined f o r a number of d e f i n e d c o n d i t i o n s . The e f f e c t s o f v a r i o u s m e r c u r i a l s summarized  in  Table I.  on  phytoplankton  are  In g e n e r a l , diatoms prove to be more  s e n s i t i v e to heavy metals than  dinoflagellates  (Kayser,  1977).  Out  of seven s p e c i e s , S, costatum was e s p e c i a l l y s e n s i t i v e  Hg  (Overnell,  1976).  s e n s i t i v e t o Hg (CH COO) and  Prorocentrum  Gymnodinjum followed micans  splendens by  (Kayser,  to  was  the most  Scrippsiella  faeroense  1976) .  Freshwater  10  TABLE I. VEST  L i t e r a t u r e summary of the e f f e c t s of m e r c u r i a l s on phytoplankton. 1 EFFECTS MERCURIALS CONCENREMARKS RCl'EKENCE  ORGANISM  TRATIONS  CEL:.ULAR  ( uM )  PROCESSES DIATOMS: Astei'ionella  g.r. •t  fomosa  Frafilaria  arotsnen-  Kg  ap. ap.  S  .',  1  "  inh.  r-  0.921 1.842  !; 2  stlm. f. i n h .  sic Chartoccroc Cyclotcllc. Phaecdact^tum r.uticn  p. i n h . f. "  HgCl  2  batch  0.184 0.368  «  0.368  batch  Hannan and P a t o u i l l e t , 1972  0.002  chemostat  Rice et al., 1973.  batch it  Borland ct al., 1977.  2.500  batch  O v e r n e l l , 1976.  0.5 yug.f  batch  H a r r i e s cl al., 1970.  0.5  24 h treatment  II  tricor-  Skeleionema  aottavmy  8-rg-r. s, P/S  Hi-zoahi.1 nirritj  dcliaatie-  inh. inh.  ratio  unchanged  P/S  501 i n h .  P/S  50Z i n h  KuC2  0.018-C.037  2  H;;.C1  2  MEM-MI  1  ir.othyl d i cy a n i d i amide PMA .ilph.Gnyl K«  Tompkins and B l i n n , 1976.  1.0 " 10.0 "  DIN0FLA0E1LATES: Axpkii-inim G.i~:>ic.i-:  ?asie?ae iTjletuicr.s  g-r. U.r. 6 P/S  p. Inh.  HgCl,  C.004  f. i n h . n. t.  " "  t. S.r.  i P/S  p. i n h . K 1 J ! I 0 0 ) 8  3  2  P.  turbidoi-tat (added once) 1.84 2-3.683 ( d a i l y a d d i t i o n ) O.lii-0.368 3atch 0. 363 0.037 0.368  faerc-  ,;.r. S  P/S  f. i n h  Hg(Cfi j0)., 3  P- " f. "  .W'.TURAL  Kayser, 1976.  0.3<>8  f. " P- " r. " i'aripslellc  Zingmark ar.d M i l l e r , 1973.  0.ISA-1.84? - urb i ci j:> t '(added on.:fc) 1.8^2 (dolly addition) 0.3fi8  '3.683  i;urbidoi:tat (added once)  1.842 latch 3.6;13 0.037- 0.184  POPULATIONS  Marino  phytoplankton  pri-ary productivity  p. i n h .  KsCl,  1.642  :;rock and Ma^cri, 1971.  P/S  507. i n h .  HsCl.,  0.C04  Zinr.i'urk ;:.-.d M i l l e r , 1473.  P/S  inh.  H C:,  0.0.18  •Cnauer sr.- - . i v t i r . , 1S72.  S  aethyl Kg North Sea c o a o t a l plankton Lake phytoplankton  S - '•  p. Inh.  KgCl  P/S  40% i n h .  HSC1  II  85% " iitirj.  0.006-0.018  2  II  2  0.221 3.68 3 0.048-0.107  if. r.i'.A. er.closjre  Cuiper, 1976.  ir.  Islir.n ct aZ.. 1977.  sit-:'.  enclosure  11  [GREEN ALGAE: Scened&smue  8j>.  S-r.  S. dlmorphu.r  g.r.  I j| ChUnrnjdar.onaa  lnh.  g.r.  rein'-  CTiZwelZa pyva>u>idoaa  unchanged f. l n h .  HgCl  f. l n h .  HgCl.,  «.r. K efflux +  p. lnh. stim.  H C1  stia.  HgCl.  S  lnh. strain)  (Emerson  g v.  unchanged  biomass  HgCl  stim. p. inh.  li8  n 2 1  t/S 3  leoahrysie  galbana  8.r.  8 « inh.  g-"  p. i n h . f. "  rthisTlT;  2  2  10 ug  K a l t l d a et al., 1971.  r  / S  d  1  batch, highly chelated median  Ben-Bassat. a t al., 1972.  0.368-1.050 batch  Karap-Nieisen, 1971.  [50.000-81.030 batch  1.000 0.1  S'nleh and Barber, 1972.  batch  De F i l i p p i s and Pallaah". 1976a.  jM  J..D0O  0.1 uM  o.i-i.o  HgCl,  0.363-3.683  HgCl,  10.000  "^ " ' ^ " l " i . cms chemical i s defined on page 7 P  Hringham and Kuhn, 1953.  0.037  0.037-0.368 (CH ) Hg  jHXPTOPHYTE  2  PMA  PMA tevtloleatai  batch  500.000  stiia.  PAR P/S respiration DMA, RNA protein Dunaliella  0.110  7.366  dark respi ration Chlorella  2  Hg", methyl Hg  semi-continuous cultures, 10-15 Kin exposure  batch  Overnell, 1975a.  Davies, 1974; 1975.  7.500 10.500  ™- inhibition; f . - f u l l ;  p..  p a r t l a l :  .  tla  •  .. .  t i a u W  12 p h y t o p l a n k t e r s are u s u a l l y more counterparts tant.  tolerant  than  their  marine  and nanoplankters are f r e q u e n t l y the most r e s i s -  When Cu was added to a large i n s i t u - e n c l o s u r e ,  large  c e n t r i c diatoms (e.g., Chaetoceros) and d i n o f l a q e l l a t e s d i s a p peared,  whereas,  microflaqellates  and  pennate  diatoms  ( »q»» N. d e l i c a t i s s i m a ) s u r v i v e d and were r e s p o n s i b l e f o r the e  recovery of p r o d u c t i v i t y  (Thomas and S e i b e r t ,  1977).  T e r a t o l o g i c a l s t u d i e s suggest t h a t developing or lically ication  active  stages  are  metabo-  more s u s c e p t i b l e t o metal i n t o x -  (Pyefinch and Mott, 1948; Wisely and B l i c k ,  1966).  In  A. n i d u l a n s . s t a t i o n a r y phase c e l l s were l e s s v u l n e r a b l e to Hg poisoning  than  a l . ,1976),  growing  During the l a g phase,  by  the  either  complex  volatilization ser,  1976;  growth  with  toxic  metallic  (Zingmark and M i l l e r ,  Ben-Bassat  and  cells.  concurrently  is  with  the  products  1973; Davies, 1974;  Mayer,  Kay-  1977;1978; Betz, 1977).  inoculation  of  log  cells  phase  than  (Zingmark and  1973).  specific.  Mercuric  isolated  Hq (CH^COO)^ §t a l . ,  et  i o n s or s t i m u l a t e  The e f f e c t s o f m e r c u r i a l s . o n photosynthesis  in  medium  These  Mercury was l e s s t o x i c when added during the  Miller,  (Hammans  e x c r e t i o n from l i v e c e l l s and d i s s o l v e d orga-  n i c s l e a c h i n g from dead and moribund may  cells  probably due to the presence o f m e t a b o l i t e s i n the  growth medium. conditioned  logarithmically  chloride inhibited  chloroplasts degraded  1976).  (Kiminura  phycocyanin  In Chlamydamonas  in  are  species  plastocyanin  activity  and A.  Katok,  1972)  and  n i d u l a n s (Hammans  reinhardtii.  Hg  interfered  13  with PS I and PS I I r e a c t i o n s ( O v e r n e l l , 1975b); costaturn reduced  was  the  most  photosynthesis  sensitive  potassium  by only 50% ( O v e r n e l l , 1976).  i o n s as a primary  pointed  to  excretion  e f f e c t of Hg p o i s o n i n g  Sublethal  potassium  at c o n c e n t r a t i o n s s i m i l a r t o or g r e a t e r  those  reguired  for  levels  the  inhibition  P. t e r t i o l e c t a and P. t r i c o r n u t u m 1976).  In  Ankistrodesmus  syntheses  jbjEaunii  species  (Tompkins  in  and  and  ENA syntheses  p r o t e i n syntheses.  while  caused  photosynthesis 1975a;  than in  Davies,  and Eugjena g r a c i l i s . Hg  et a l . ,  galactolipid 1972).  Fraqilaria  and  In C h l o r e l l a . HgCl^  The  and same  Asterionella-  stimulated  PMA i n i t i a l l y decreased  Both m e r c u r i a l s had a  on biovolume and e x c r e t i o n In S. costatum,  metals  and B l i n n , 1976)., G a l a c t o l i p i d s a r e one o f  the major c h l o r o p l a s t l i p i d s . DNA  of  activity,  (Matson  i n h i b i t i o n appeared to occur  heavy  (Overnell,  inhibited galactosyl transferase chlorophyll  of  (Kamp-  N e i l s e n , 1971). leakage  S.  a l g a t e s t e d , 2.5 uM HgCl^  An i n v e s t i g a t i o n on C. pyranoidgsa of  Although  DNA, BNA  similar  {De F i l i p p i s and P a l l a g h y ,  effect 19 76a).  (Berland e t a l . , 1977) and n a t u r a l p o p u l a t i o n s  (Thomas e t a l . , 1977), the C:N r a t i o remained constant  despite  a r e d u c t i o n i n growth and p h o t o s y n t h e s i s . In  metal-treated  algae,  morphological  occurred i n P. t r i c o r n u t u m and C h l o r e l l a TMiassiosira  isolated  (D. S e i b e r t ,  personnal  underwent swelling  osmotic  from  Hg-treated  a l t e r a t i o n s have  (Nuzzi, 1972), and i n in  situ  enclosures  communication)., P^ty^fta-'-ferAgfrtjie^lil'-  disturbances  (Bentley-Mowat and B e i d ,  resulting 1977),  in  considerable  A f t e r 12-14 days o f  14 exposure  to  50-100  deviated  from  pg^l-^BqCl ,  their  normal  colonies  stellate  of  A.  foraosa  c o n f i g u r a t i o n t o form  l a r g e c y l i n d r i c a l s t a c k s of up t o 30 c o l o n i e s , agglomerated mucilagenous s e c r e t i o n s  (Tompkins  and  Blinn,  1976).  by  Cell  volumes of I . galbana almost doubled at 10.5 uM HgCl^ probably due  to  the  impairment  of  involved i n c e l l d i v i s i o n S.  methionine  p r o d u c t i o n , which i s  (Davies, 1 974).  f a e r o e n s e . responded to Hg(CH COO)  tbecae,  releasing  of  naked,  The  by  dinoflagellate,  the  motile c e l l s ,  bursting  of  and formation of  v e g e t a t i v e r e s t i n g stages (Kayser, 1976).  S e c t i o n 5.  Mercury R e s i s t a n c e  The t o l e r a n c e o f c e r t a i n algae to e l e v a t e d has  been assessed i n comparative s t u d i e s  and Reid, 1977).  T o l e r a n c e may  of an e x c l u s i o n mechanism  et a l . ,  levels  Bentley-Mowat development  (Davies, 1976), by v o l a t i l i z a t i o n of Mayer,  a c c l i m a t i o n response (Stokes e t a l . , Say  (e.g.,  be a c q u i r e d by the  Hg from the medium (Ben-Bassat and  1976;  metal  1977).  1973;  1975),  or  Stockner and  by  an  Antia,  T o l e r a n c e may a l s o be i n n a t e , as  shown by Pediastrum boryanum which could s u r v i v e and reproduce at of  1 mg-1 1.77  -1  HgCl^ i n s p i t e o f a c e l l u l a r c o n c e n t r a t i o n  x 10»*  (Richardson  et a l . ,  1975).  Differences i n  t h r e s h o l d l e v e l s f o r F. c r o t o n e n s i s and A. formosa with s u r f a c e t o volume r a t i o s Tolerance  to  heavy  recovery from i n i t i a l  metal  factor  correlated  (Blinn & i •_§!•, 1977),  metals  can  inhibition  be  acquired  following  (Kamp-Nielsen,  1971;  15 Ben-Bassat  et a l . ,  1972; F u j i t a  and Hashizume, 1975; Davies,  1976; De F i l i p p i s and P a l l a g h y , 1976a; 1976b; Berland  et a l . ,  1977). Besistance croorganisms. Pseudomgnas  due  to v o l a t i l i z a t i o n  of Hg i s common i n mi-  In Staphylococcus aureus aeruginosa  and  P. £uteda  recovery from the i n i t i a l i n h i b i t i o n the  phenylmercury  the  r e d u c t i o n of H g  hydrolysis  may  have  (Nelson  et a l . ,  (Clark et a l . ,  of PMA  of PMA  1977),  was  to benzene  1977),  mediated  by  and Hg*  and  2  to Hg° by mercuric r e d u c t a s e followed by  + 2  a rapid v o l a t i l i z a t i o n process  (Weiss e t a l . ,  of  Hg°  occurred 1973).  from in  the  medium.  The  H g - r e s i s t a n t marine  Volatilization  was  same  bacteria  genetically  c o n t r o l l e d i n E. £oli and Aerofcacter ae£Ojg.en.e§ (Komura and ziro,  1971;  Summers  and  Lewis, 1973).  C e l l s of Ch l o r e l l a,  which became H g - r e s i s t a n t , r e e s t a b l i s h e d normal and  were  more  efficient  (De F i l i p p i s and P a l l a g h y , 1976c).  tract  Chiorella  weight,  contained  non-enzymatic,  growth  i n Hg° v o l a t i l i z a t i o n  sitiva cells from  a  Ka-  natural,  light-induced,  rates  than Hg-senThe c e l l  ex-  low  molecular  reducing  compound  capable of d i f f u s i n g Hg° from the spent medium (Ben-Bassat and Mayer,  1977;  20 uM H g C l  2  1978).  prevented DCMU  volatilization  due  while an uncoupler mul;ated  Pretreatment  0^  Mayer, 1978).  to of  evolution  inhibition  competition  of  Chlorelia of  with  10-  light-induced  Hg°  between  PS I ,  methylamine,  and  volatilization  HgCl^ and DCMO, transiently s t i (Ben-Bassat  and  16 Section  6,  Two  Assessment of Experimental Design  basic  pollution  experimental  studies  to  approaches  determine  (sublethal)  Batch studies empt  determine,  the  (Kuiper,  ecological  repercussions  cultures  grow  usually  cultures,  substances  short,  Since  batch  The  and  t o Cu  grown i n batch  att-  natural  and  their  Gibson e t  at  added the  once and  exhaustion  of  the of  the bioassay i s  may  overestimate  be m i s l e a d i n g l y  p h y s i o l o g i c a l s t a t e of the  varied  few  are  bioassays  with the u t i l i z a t i o n o f the  example, t o l e r a n c e  an on  1977)  a  growth-promoting  duration  culture  only  essential  until  the  and  periods.  Menzel, 1971;  observed t o x i c l e v e l s may  because the d e n s i t y  palea  all  logarithmically  occurs.  toxicity.  time  systems i n  et a l . ,  in  (lethal)  pollutants  (Dunstan and  batch  toxic  of  Blinn  With  and  vary  1976;  G r i c e e t a l . , 1977).  elements  aguatic  effects  1975;  nutrients  acute  used  pollutants.  c u l t u r e s have been i n t e n s i v e l y used and  populations  al.,  to  l e v e l s over short to long  were conducted i n n a t u r a l  to  been  the e f f e c t s of  Bioassay organisms have been exposed chronic  have  high  population  most l i m i t i n g n u t r i e n t .  For  by a f a c t o r of 3 0 i n N i t z s c h i a  cultures  (Steemann  cultures  are  Nielsen  and  Wium-  Andersen, 1971). However,  batch  ranges, screening 1970;  Nuzzi,  1976), and  for different  1972)  or  useful i n estimating  mercurials  suitable  determining the a c t i o n of  test a  (Harriss  organisms toxicant  toxic  et  aJL.,  (Overnell, on  various  17 metabolic A  processes.  few  studies  have been done using c o n t i n u o u s c u l t u r e s  such as a t u r b i d o s t a t  (Kayser, 1976),  or  et a l . ,  the  algal  1973).  In  c o n t r o l l e d by the r a t e (Harrison  et a l . ,  of  chemostat, medium  1976).  a  inflow  Culture  chemostat growth or  dilution  u n l i m i t e d and are  easily  rate.  dilution  constant  is rate and  for  (e.g.,  n u t r i e n t s and  sensitive,  mode of metal  exposure)., T h i s technigue i s s u i t a b l e f o r the maintenance nutrient-limited  populations  and  may  often  months. useful  nutrient-stressed,  especially  during  In the f u t u r e , t h i s mode of c u l t u r i n g may in  assessing  and  of  be more r e a l i s t i c than  batch c u l t u r e s because phytoplankton of the upper p h o t i c are  a  Experimental time i s t h e o r e t i c a l l y  v a r i a b l e s , which make the assay more controllable  rate  volume, c e l l d e n s i t y  c e l l u l a r chemical composition are r e l a t i v e l y specific  (Rice  zone  the summer prove  more  understanding the e f f e c t s of lower  l e v e l s of metals.  S e c t i o n 7.  Purpose o f t h i s  Study  In g e n e r a l , t h e r e i s a l a c k of research effects  of environmentally  documenting  encountered mercury l e v e l s .  over, s i n c e most i n v e s t i g a t i o n s have used c o n c e n t r a t i o n s to  6  orders  terminal rather  than  term s u b t l e e f f e c t s of mercury have been examined the  Moreof  of magnitude higher than the c o n c e n t r a t i o n s  i n t h i s study, the short-term  In  the  past,  a  myriad  the  3  used long-  (Table I ) . ,  of s t u d i e s have determined  the  18 e f f e c t s of m e r c u r i a l s the  effects  of  studies reported parameters 1976;  on d i f f e r e n t c e l l u l a r  some  factors  the  processes  influencing  effects  of  heavy  and/or  toxicity.  metals  on  A few biomass  i n c o n t i n u o u s c u l t u r e s (Rice e t a.1., 1973; Kayser,  Bentley-Mowat and Reid,  tolerance  1977) and some  briefly  related  to n u t r i e n t s but under saturated c o n d i t i o n s  (Hannan  e t a l . , 1973; Morel e t a l . , 1978)., Nutrient under  these  survival will of  the  limitation  can  conditions  of  species to increase will  competition. (pollution)  primary  nutrient  production  the  populations. uptake  and  d e f i c i e n c y , chances o f  depend on the n u t r i e n t uptake k i n e t i c  nutrient-stressed  nutrient  limit  The  affinity  responses  ability  f o r the  of  a  limiting  favour the chances o f s u c c e s s f u l i n t e r s p e c i f i c However, the i m p o s i t i o n o f  on a population  may impair t h i s c o m p e t i t i v e Recovery i n algae  a  secondary  stress  t h a t i s already n u t r i e n t - s t r e s s e d ability.  p r e v i o u s l y i n h i b i t e d by  heavy  metals,  may be a t t r i b u t e d t o s e v e r a l f a c t o r s ,  one p o s s i b l e reason f o r  the  be  recovery  complexing  from  agents  i n v e s t i g a t e d with Thus,  such  as  different  may  the  production  metallothionein.  This  effects  ecological  of  of was  l e v e l s o f mercury exposure.  i t appears that t h i s work i s the f i r s t  documents the nearly  inhibition  long-term  mercury  study which  exposures,  at  l e v e l s , on n u t r i e n t - l i m i t e d diatoms, grown  i n continuous c u l t u r e s  (chemostats).  19  CHAPTER I I  EFFECTS OF SHORT AND LONG-TEEM EXPOSURES TO SUBLETHAL  IJIIiS Section  1.  In/troductiQn  Nutrient cribed  OF Hg. ON NflTRIENT KINETICS  uptake k i n e t i c s i n  phytoplankton  can  be  des-  i n terms of the f o l l o w i n g eguation which i s s i m i l a r t o  the Hichaelis-Menten eguation f o r enzyme k i n e t i c s ; (1) where V - r a t e of  uptake  V = Vmax'S (Ks «• S)-»  of n u t r i e n t uptake  (hr  - 1  ( h r ) ; Vmax = maximum  ) ; S = concentration  o f the l i m i t i n g  (uM) , and Ks = h a l f - s a t u r a t i o n constant when  V = Vmax/2.  species  affinity  limiting  membrane. and  take  up  following  by  (uH) , the value  a  has  of  the  Conway e t a l ,  a  high  substrate  Newer terminology a s s o c i a t e d  the r a t e of uptake  described  nutrient of  capacity  The  or  across the c e l l  with the disappearance  limiting  nutrient  has  been  (1976) and i t i s presented i n the  paragraphs.  In n u t r i e n t - l i m i t e d c u l t u r e s , uptake and a s s i m i l a t i o n the  S  Values o f Ks are s p e c i e s s p e c i f i c and a low  Ks value i n d i c a t e s t h a t a to  rate  - 1  limiting  nutrient  u s u a l l y occurs i n 3 phases ( F i g . 1),  f i r s t phase c o n s i s t s o f a r a p i d r a t e o r surge uptake, to  of  represent  which  appears  across  the c e l l membrane and i n t o an i n t e r n a l pool., I t occurs  over the time period Ts.  the  transport  Vs,  of t h e n u t r i e n t  During t h e second phase, the r a t e o f  20  13  s  t t t o s t K  ~ Substrate  :  s  (pM)  FIGURE 1. Nutrient uptake k i n e t i c  terminology. (A) The disappearance of the  l i m i t i n g n u t r i e n t with time. (B) The rate of uptake of the l i m i t i n g n u t r i e n t as a f u n c t i o n of the substrate. ned i n Section  2.1  (From Conway et a l . , 1976). Symbols are d e f i -  21 uptake , V i , decreases  as the i n t e r n a l pool becomes  full.  At  t h i s p o i n t , the r a t e o f a s s i m i l a t i o n of the l i m i t i n g n u t r i e n t , Vi,  (the  molecules  mobilization  from  the  internal  v i a a s s i m i l a t o r y enzymes) becomes the r a t e  step c o n t r o l l i n g the uptake r a t e . external  substrate  limiting  During the t h i r d phase, the  c o n c e n t r a t i o n i s low.  The  c o n t a i n i n g the l i m i t i n g n u t r i e n t , becomes as  pool i n t o l a r g e r  internal  gradually  pool,  depleted  the r a t e of a s s i m i l a t i o n exceeds the uptake r a t e .  In  this  phase, the uptake r a t e , ve, i s under e x t e r n a l c o n t r o l s i n c e i t i s l i m i t e d by the low nutrient  in  the  e x t e r n a l c o n c e n t r a t i o n of  medium.  the  limiting  Thus, the three phases i n v o l v e d i n  the t r a n s l o c a t i o n of the s u b s t r a t e a c r o s s  the  cell  membrane  are Vs, V i arid Ve. The two  substrate  phases of  indicates  c o n c e n t r a t i o n at the j u n c t u r e between the  uptake,  the  Vi  beginning  and of  Ve,  the next  t h i r d phase of uptake, Ve may  latter  case,  The  constant  (actual  which  and  cease when the c o n c e n t r a t i o n  of  or p a r t i a l l y d e p l e t e d .  i s d e f i n e d as So. Ks)  The  actual  half-  i s determined by adding (apparent  Ks)  and  represents  hyperbola  (see  estimates  the  only  dashed true  line  estimated u s i n g Vs.. The one in  datum  point  F i g , 1B),  value of Vmax.  The  on  the  the So 0.,  value of  the  probably  In  limiting  apparent Ks i s c a l c u l a t e d by assuming t h a t So =  In t h i s study, Vmax was Vs,  phase, Ve.  St  the  apparent h a l f - s a t u r a t i o n constant value.  as  t h e r e s i d u a l c o n c e n t r a t i o n of the  n u t r i e n t s t i l l remaining saturation  defined  During  the l i m i t i n g n u t r i e n t i s completely the  is  uptake under-  maximum a s s i m i l a t i o n  22 r a t e , Vi.  , was e s t i m a t e d by f i t t i n g an hyperbola to  the  rn&x  Ve  and V i data.  S e c t i o n 2.  M a t e r i a l s and Methods  2. 1 Inoculum Chemostat  cultures  skeletonema  were  inoculated  culture  of  cost aturn  Pacific  C u l t u r e C o l l e c t i o n , #18,  with  (Grev. )  an  cleve  unialgal (Northwest  U n i v e r s i t y o f B r i t i s h Colum-  bia) , i s o l a t e d from P a t r i c i a Bay, B r i t i s h Columbia, Canada.  2.2 Chemostat  Cultures  A l l glassware was autoclaved and c u l t u r e tered  through a 0.22  um M i l l i p o r e f i l t e r  b a c t e r i a at n e g l i g i b l e l e v e l s . nium-limited  chemostats  in  S. costatum was grown i n ammoartificial  *f» medium (Appendix A).  n u t r i e n t s i n the i n f l o w N a S i 0 , 35.3  seawater  of  were:  (f/3) ; KH P0 , 3.68  trace  metals (f/20) and vitamin  ZnS0 = 7.65; 4  2.61  medium 2  4  C o C l . 6H 0= 4.39; 2  and vitamin B ^ -  2  The  chemostat  1  system  NH^Cl,  (f/20).  constituents  10.0  (f/100) ;  C o n c e n t r a t i o n s (nM)  (f/20) were: CuS0 = 4  MnCl^ 41^0=  °*05 u g ' l - .  as f e r r i c seguestrene (1.17  with  (Appendix A)  C o n c e n t r a t i o n s (uM) of the major  3  2  fil-  i n an attempt t o keep  made from reagent grade s a l t s and enriched of  medium was  0.91;  Iron and EDTA  Na^oCy  3.93; 2^0=  were  added  described  (Davis  uM). was  previously  23 Si al.,  1973).  Continuous c u l t u r e s were grown  a t e , 2 1 and 6 1 f l a t bottom b o i l i n g f l a s k s at  17  ±  0.5 C .  Flasks  0  ( S i l i c l a d , C l a y Adams) that  adsorption  silicate The  aeration  were  stirring to  since  Hg  leached from  cultures"  magnetic  of  were  not  onto  borosilic-  (Pyrex) maintained  coated  preliminary  in  with  results  silicone indicated  w a l l s was not decreased and no  the i n n e r w a l l s o f the c u l t u r e continuously  stirred  flasks.  with t e f l o n covered  bars at 120 rpm and were maintained  avoid  volatilization.  continuous i l l u m i n a t i o n  They  ( 50 u E • m • s ) . - 2  were  also  Spectral  - 1  without under  distribu-  t i o n from d a y l i g h t f l u o r e s c e n t lamps (Sylvannia Powertube, VHO and  Duro-Test,  UHO)  was  corrected  P l e x i g l a s sheet (# 2069 Rohm simulate  5 m  underwater  and  Hass),  fluorescence  by  to  1957).  following  cells  of  microscope.  method  vivo  was  basically  s i l i c a t e and phosphate  Maclssac  that  of  (1972).  Koroleff The  width  Nutrient  using a Technicon AutoAnalyzer .  automated by Slawyk and  (1967)  in  Length and  were measured u s i n g an o c u l a r micrometer.  analyses were performed ammonium  changes  using a Turner Model 111 f l u o r o m e t e r and o f c e l l  d e n s i t y using an i n v e r t e d l i g h t  al.,  attempt  Analyses Growth was monitored  of  i n an  l i g h t under sunny c o n d i t i o n s f o r the  J e r l o v type 3 c o a s t a l water (Holmes,  2.3  by using a 0.3 cm t h i c k  The  (1970) as  methods f o r  f o l l o w e d the procedures of Armstrong e t  and Murphy and S i l e y  (1962), r e s p e c t i v e l y .  Both  24 methods were automated by Haqer e t a l . , (1969). The *C technique was used f o r measuring  the r a t e  l  tosynthesis (Strickland NaH *C0  Parsons,  1972).  Five  uCi of  (New Enqland Nuclear, Boston) were added t o 100 ml of  1  5  chemostat and  and  of pho-  e f f l u e n t , mixed, separated i n t o two,  50 ml  bottles  incubated f o r 2 hours under o r i q i n a l c o n d i t i o n s .  i n c u b a t i o n was conducted. diameter  C e l l s were c o l l e c t e d onto  0.45 urn M i l l i p o r e f i l t e r .  scintillation  cocktail  Co. Ltd.)  counted  and  No dark a  25 mm  F i l t e r s were d i s s o l v e d i n  (Scintiverse,  Fisher  for radioactivity  Scientific  with a Unilux I I I  Nuclear L i q u i d S c i n t i l l a t i o n System, Nuclear Chicaqo. Chlorophyll (Strickland chemostat  a was determined by t h e  and  Parsons,  effluent  1972).  trichromatic  Two hundred and f i f t y  were f i l t e r e d onto a glass  one.  C e l l s were homogenized, c e n t r i f u g e d (750, 665,  663,  645,  supernatants f o r c h l o r o p h y l l s , determined  using  spectrophotometer. drops  of  onto  a  filtrates  were  10%  630  the 430  nm)  carotenoids  and  phaeophytins  measured  by  analyzed These  adding  3  wavelenqhts. 200 ml  of  effluent  were  47 mm diameter 0.45 urn M i l l i p o r e f i l t e r . ,  respectively.  of the  t o the above supernatants and r e a d i n g the  the Hq a n a l y s i s ,  were  absorbance  and  Phaeophytin a was  HCl  and  a Perkin-Elmer Model 124D double beam  atsorbances a t the same For  Pigments  f o r 20 hours i n c o l d , dark c o n d i t i o n s i n 90% a c e t -  spectrum  were  ml of  f i b e r f i l t e r co-  vered with 1 ml of a s a t u r a t e d HgCO^ s o l u t i o n . extracted  method  Hq  for particulate fractions  were  and  filtered  F i l t e r s and soluble  Hg,  measured by a c o l d  vapor method absorption  (Hatch  and O t t , 1968) u s i n g  spectrophotometer  O p t i c a l U n i t s , Model 100). H^SO^/KMnO^/K^S^Og 2  SnS0  4  analytical  and  U.V.  were  technigue  does  of  not  Control  reduced  by  were  A l l reported  Hg  to  5.52  reagent  addition  Hg vapour.  differentiate  0.18  and  via a  the  the  nM  concentrations  of This  between  i n o r g a n i c and organic Hg.  l i m i t s of the method  atomic  oxidized  i n t h e r e l e a s e o f elemental  various , species  ug.i-i).  Mercurials  flameless  T h i s was followed by  4  resulting  (Pharmacia  digestion  (NH^OH^. H S0 • NaCl.  a  the  The d e t e c t i o n Hg  (0.05-1.50  are  a  mean o f  time  interval  d u p l i c a t e samples. The  concentration  (eguation  2)  experiment  (eguation  of  HgCl  during  2  and t h e average c o n c e n t r a t i o n during t h e e n t i r e 3) were computed as f o l l o w s :  (2)  C  (3)  C  n + 1  = =  C,, I  e-0<W n t  (C  n  +  l  -  C  n  where n = number o f time i n t e r v a l s ; C t  m i  (nM);  C  concentration intervals  =  n  concentration  between  two  (nM)  the entire  calculations  1  +  C*  / D)  =  n + 1  at  (T-M  t  n  concentration (nM) ;  C  =  a  (nM) ; D = d i l u t i o n r a t e ( h r — ^ J ; t^+i - t  concentration of  any  additions  (hr); C  =  n  at  added = time  average  d u r i n g the e n t i r e experiment and T = time  experiment  losses  (679.5 h r ) .  Since  in  these  of Hg a r e only due t o the d i l u t i o n r a t e ,  these c a l c u l a t e d Hg c o n c e n t r a t i o n s  are r e f e r r e d  to  as the  •expected' Hg c o n c e n t r a t i o n s . The  specific  f o l l o w i n g eguation  growth  rate  (p)  was  (Davis et a l . , 1973);  computed u s i n g the  26 (4)  p =  D  +  where u = s p e c i f i c growth r a t e over the time i n t e r v a l times t  n  +  1  and 1^, ( 1 0  Variations  to  rate  - 1  and x  - 1  cells-l  - 1  h  = c e l l d e n s i t i e s at  )  i n the a n a l y s e s used i n these experiments are  presented i n T a b l e I I . used  )  ( h r ) ; D = mean d i l u t i o n  (hr ) ; 7  i x ^ / x ^  (1/t) In  determine  Subsamples o f chemostat  culture  were  the standard d e v i a t i o n , except f o r the Hg  analyses.  2•4 Experimental Design Batch c u l t u r e s sublethal  were  used  concentrations  of  to HgCl  2  determine  cells  seawater  to a v o i d a t r a n s f e r o f  fresh  Prior to  were c e n t r i f u g e d and resuspended  medium.  The  medium  range  of  capable of i n h i b i t i n g the  growth r a t e s o f n u t r i e n t - s a t u r a t e d c u l t u r e s . lation,  the  complexing  in artificial  agents  was enriched with  inocu-  into  the  *f/25* and the  source o f n i t r o g e n was n i t r a t e . In the short-term Hg-exposure, a was  grown  0.04 h r  - 1  under  .  ammonium  The  dilution  6 1  chemostat  culture  l i m i t a t i o n with a d i l u t i o n r a t e of rate  was  determined  using  the  f o l l o w i n g eguation: (5)  D = F.V-*  where  F = flow  rate  (ml'hr )  (ml).  Chemostat samples were analyzed d a i l y f o r f l u o r e s c e n c e ,  -1  and V = volume of the c u l t u r e  c e l l d e n s i t y and n u t r i e n t c o n c e n t r a t i o n s . reached  when  no  trend  A steady-state  i n these parameters  was  was observed f o r  27  TABLE I I . Standard d e v i a t i o n f o r a n a l y s e s used i n the s h o r t and long-term mercury exposure  Parameter measured  Cell  experiments.  Mean  numbers  Fluorescence  1 1*  Chlorophyll a  s.d.  Units  1.37 ± 0.33  10  45.59± 1.56  relative  22.19 ± 2.85  ug c h l a / 1 0 c e l l s  5.09 ± 0.30  7  cells-1""  1  units 7  ug C *10 a e l l s 7  1  K hr  1  24  10  3  22  13  5  10'  1.8  6  10 30  7* Ammonium  2.0 ± 0.15  fM  Hp analysis, p a r t i c u l a t e Hg soluh le  20.0 ±1.20 3.68 ± 0.22  ng 1  10  -1  6 6  6 6  nM Hg  % - standard d e v i a t i o n s expressed as : percentage of the mean 1 ; a l s o see . s t a n d a r d d e v i a t i o n s i n Appendix B and F i g u r e 3. ^: used n/2 s e t s of s t a n d a r d s done on d i f f e r e n t  days.  The s t a n d a r d  d e v i a t i o n s were c a l c u l a t e d and expressed as a percentage of the mean. The  later  percentage was c o n v e r t e d i n t o the u n i t s used f o r eaeh type  of a n a l y s i s . The h i g h e s t s t a n d a r d d e v i a t i o n  (6%)  was used i n t h i s  table,  * the s t a n d a r d d e v i a t i o n i s expressed as a mean of the s t a n d a r d tion  of n/2 s e t s of d u p l i c a t e measurements.  devia-  28 s e v e r a l days.  Then 500 ml o f chemostat  e f f l u e n t was c o l l e c t e d  and s i m u l t a n e o u s l y perturbed with 5 uM NH^Cl following nM.  and one  c o n c e n t r a t i o n s of HgCl^; 0.37, 1.84, 3.68, o r 5.53  In an attempt to i n c r e a s e the t o x i c e f f e c t  nutrient  o f the  uptake,  1 1  and allowed t o s t a r v e starvation  period  of  HgCl  2  on  of e f f l u e n t was c o l l e c t e d f o r 12 hours f o r 24 hours  was  such  30 hours.  that  Then  d e s c r i b e d above, with 5 uM NH C l and  the  average  i t was perturbed as  one  of  the  following  c o n c e n t r a t i o n s of HgCl.,, 0. 18, 0.37, 1.84 or 3.68 nM. Details  of  been d e s c r i b e d technique  batch  (Caperon  consisted  multaneously Ammonium  the  into  and  the e f f l u e n t  6 minutes i n order to from  Meyer,  1972).  Basically,  of quickly injecting HgCl  determinations  ammonium  mode p e r t u r b a t i o n technigue have  the  were  made  closely  medium.  and  follow  2  and NH^Cl s i -  mixing  as  thoroughly.  frequently the  the  as every  disappearance  of  Phosphate and s i l i c a t e were not  added along with ammonium s i n c e t h e i r  c o n c e n t r a t i o n s i n the  chemostat  t o ensure no l i m i t a t i o n  effluent  d u r i n g the  were  perturbation  sufficient experiment.  From  the  experiments  usinq short-term Hg exposure, i t appeared t h a t semi-continuous additions  of  3.68  nM H g C l  2  over a long period of time could  a f f e c t ammonium-limited c e l l s . exposure  experiment,  a  In d e s i g n i n q the long-term  lower c o n c e n t r a t i o n was a l s o used i n  case the e f f e c t s o f 3.68 nM HqCl In  the long-term  (D= 0.039 ± 0.005  Hq  experiment,  were too severe. a  6 1  chemostat  culture  hr-*) was d i v i d e d i n t o t h r e e , 2 1 chemostat  c u l t u r e s o p e r a t i n g at the f o l l o w i n g nearly i d e n t i c a l  dilution  29 rates:  1=  0.041 ±  0.039 ± 0.001 h r that  small  - 1  . These d i l u t i o n r a t e s  variations  the c o n c e n t r a t i o n original  0.002 h r - * ; 11= 0.040 ± 0.002 h r ~ * ; 111= were  chosen,  i n the d i l u t i o n r a t e would not change  of ammonium  steady-state  was  in  the  culture  maintained  medium.  the  6  started.  1  chemostat  culture,  For 30 days, c u l t u r e s I  nuously  exposed  Hgcl^ r e f e r e n c e  to  The  f o r another 2 days t o  ensure t h a t the c u l t u r e s ware not d i s t u r b e d by of  such  the  splitting  and then Hg a d d i t i o n s were and  I I I were  semi-conti-  0.37 and 3.68 nM HgCl^, r e s p e c t i v e l y .  s o l u t i o n (3.68  mM,  or  1000  mg.l .  A  Fisher  _ 1  S c i e n t i f i c Co. Ltd.) was d i l u t e d t o form substock s o l u t i o n s of 0.37  and  3.68  juM which were made f r e s h every f o u r t h day. A  t o t a l of two ml o f each HgCl^ substock  was  the  cork.  inflow  port  of  each  chemostat  injected  G e n e r a l l y , the  a d d i t i o n s were made every 8 hours during the f i r s t every 12 hours f o r the r e s t of the experiment are  noted on F i g , 6 i n s e c t i o n 2.3.  r e c e i v e d an e q u i v a l e n t  uptake  f o r an  volume o f d i s t i l l e d  cell  effluent  was  conducted  5  later,  starvation  hours period  f o r 12  countinq  remaining  of  used  and  deionized  averaqe p e r i o d o f 6 hours),  experiments,  exceptions  and  resulting  in  water.  hours,  (i.e.,  and used f o r *C l  Hg  analysis.  f o r a perturbation  11 hours.  10 days and  The c o n t r o l , c u l t u r e I I ,  Chemostat e f f l u e n t was c o l l e c t e d starved  through  an  F i v e hundred  The  experiment  average  cell  ml of e f f l u e n t  were c o l l e c t e d f o r c h l o r o p h y l l a determination. , Three p e r t u r b a t i o n II,  and  experiments  (chemostat  I I I ) were performed simultaneously  effluents  I,  and n u t r i e n t con-  30 c e n t r a t i o n s were determined every 6 minutes d u r i n g 18-30  the i n i t i a l  minutes, and then every 18 minutes f o r t h e r e s t  perturbation The  o f the  experiment.  uptake k i n e t i c parameters, apparent Ks and V i _ „ were 777ctx  computed using t h e e g u a t i o n s o u t l i n e d by Conway e t a l . , (1976) and  statistically  evaluated  using  the methodology of C l e l a n d  (1967). Section  3.  Results  3.1 R e s u l t s from some P r e l i m i n a r y The using  e f f e c t s of Hg on S. costat^m were f i r s t  nutrient-saturated  to 7.37 nM H g C l  2  investigated  batch c u l t u r e assays t o determine a  range o f s u b l e t h a l c o n c e n t r a t i o n s  while  Studies  gradually  ( F i g . 2 ) . A d d i t i o n s o f 0.37  decreased the i n v i v o  fluorescence  only 3.68 and 7,37 nM decreased the maximum growth r a t e  without i n d u c i n g a l a g period. HgCl^,  final  t o 0.30- 1 0  7  A f t e r 88 hours of exposure  to  c e l l d e n s i t i e s were g r a d u a l l y reduced from 2.50  cells'l-  1  over a s u b l e t h a l c o n c e n t r a t i o n  range  of  0.37 t o 7.37 nM HgCl^, r e s p e c t i v e l y .  3.2 Growth Phases during t h e Long-term Mercury Exposure The  original  fluorescence, effluent  cell  data  including  numbers,  concentration,  dilution  specific  growth  rate,  in  rate,  vivo NH  + 4  c h l o r o p h y l l a, c a r o t e n o i d ; c h l o r o p h y l l  31  I  0  1  i  1  24  1  1——>•  . .i——\  48 64 Time (hr)  88  FIGURE 2. Changes i n i n vivo fluorescence i n n u t r i e n t - s a t u r a t e d batch c u l t u r e s exposed to the f o l l o w i n g additions of HgCl., (nM): (V)  0.37;  (•)  0.93;  (T)  1.84;  (•)  3.67  and  (A)  7.37.  (Q)  0.00;  32  a, photosynthetic Appendix  r a t e , and  c e l l dimensions are  in  biomass  (in  vivo  fluorescence  numbers) of the 3 chemostat c u l t u r e s Although  both  fluorescence  measurements  was  i n t o f o u r phases  (II)  display  to  sublethal  ( F i g . 4).  whereas,  S. cj>sta.tu,m,  d u r a t i o n of  milder life  control  the  steady-state  previously  impaired  2  In  drastically  will  B,  Hg-treated  to changes i n the l i f e  described  latter.  Cell  their  original  3.0  recovery  (phase C), c e l l d e n s i t i e s r a p i d l y  3A) .  of  to r e t u r n to the  of  In phase D, a new  the  (Fig. 3).  the a b i l i t y  respectively, period  culture  culture  d e c l i n e d while the c o n t r o l s u f f e r e d a  l o s s due be  phase  B  three  held by  days i n phase A  c u l t u r e s I I , I, and I I I were reduced t o 17,0,  (Fig.  and  the  population cycle  A  chemostat  the  divided  During phase A,  steady-state.  populations  was  2  phases  c u l t u r e I and I I I c e l l p o p u l a t i o n s  original  during  D were s i m i l a r i n the  semi-continuous a d d i t i o n s of H g C l  chemostat  F i g . 3.  and  at  cell  numbers.,  doses of H g C l  parent chemostat, except f o r a few The  in  C  remained  phases  The  shown  and  the same t r e n d , i n v i v o  ammonium-limited  exposure  chemostats.  are  l e s s v a r i a b l e than c e l l  growth o f  long-term  varied,  in  B.  Changes  The  tabulated  steady-state  was  densities.  cycle;  the  d e n s i t i e s of and  0.09$,  During  the  increased.  established i n a l l cultures  A  FIGURE 3. Changes i n (A) i n v i v o fluorescence and (B) c e l l numbers of ammonium-limited c e l l s during the long-term mercury exposures (nM HgCl ) i"o; ( O ) 0.00, ( A ) 0.37 and ( l l ) 3.68 . The arrow represents the time at which the f i r s t a d d i t i o n of mercury was made. d e v i a t i o n s of each measurement.  The bars shew the s t a n d a r d  FIGURE 3B.  35  Time (Days) FIGURE k .  Changes i n the s p e c i f i c growth rates of ammonium-limited  c e l l s during the long-term mercury exposures (nM HgCl^) t o : ( 0 ) 0.00, (A)  0.37 and ( I J ) 3.68. The four growth phases are: phase A: mainte-  nance of the steady-state;  phase B: d e c l i n e i n s p e c i f i c growth r a t e s ;  phase C: increase i n s p e c i f i c growth rates and ; phase D: return to a new steady-state.  36 3.3  Specific The  Growth Rates and  effect  F i g . 4.  On  of HgCl^ on s p e c i f i c growth r a t e s i s shown i n  day  0.035 - 0.040  N itrog.en 2uotas  1,  hr  specific in  - 1  growth  rates  varied  a l l c u l t u r e s but by day  6, a  t r e n d o f d e c l i n i n g s p e c i f i c growth r a t e was s e t tures. as  In  early  as  day  3  and  specific  from days 12 to 16.  growth  I,  and  rates  as  respectively,  III  specific  high  and  as  0.085,  division  rates  steady-state  followed densities  15.87  of  phase  amount the and  and  3.68  nM  C,  with  cul-  specific  and  0.135  up  to  3 times per  day,  relatively  hr  - 1  ,  day. high  11 days at  HgCl^, r e s p e c t i v e l y .  New  of  nitrogen  opposite ammonium  trend  per to  cell cell  (pM  N«10  7  density.  1.7  i n phase A,  -  As  a s s i m i l a t i o n decreased, the  from about  cells *) cell  nitrogen  to 6.38,  25.03,  i n c u l t u r e s I I , I , and I I I , r e s p e c t i v e l y , at the B.  i n c r e a s e d , and the  0.37  I sustained  0.077  only one  began  growth r a t e s resumed a f t e r approximately 20.days.  per c e l l i n c r e a s e d and  recovered  cul-  remained  In phase  growth r a t e s were maintained f o r 5, 7 and  exposures of 0.00,  The  all  rates  C u l t u r e s I I and  successively  Although these maxima occurred  as  growth  s p e c i f i c growth r a t e during phase B.  tures I I ,  in  definite  chemostat I I I , a decrement i n c e l l d i v i s i o n  undetectable a low  between  During  phase C, s p e c i f i c growth r a t e s s h a r p l y  the n i t r o g e n  cultures  per c e l l  concomitantly  became n i t r o g e n - l i m i t e d again.  the n i t r o g e n per c e l l  end  f o r the c o n t r o l c u l t u r e  than i n phase A probably  was  decreased In phase D, 5055  lower  due t o d i f f e r e n c e s i n c e l l volume and  37  the  amount of n i t r o g e n per c e l l .  3.4 E f f e c t s of Mercury on P h o t o s y n t h e s i s Changes are  in  chlorophyll a  presented i n F i g . 5 .  time.  and *C uptake during phase A J  C h l o r o p h y l l a per c e l l i n c r e a s e d with  The l a r g e s t amounts were observed on day 8  During were  (Fig. ,5A).  phase ft, the average c a r o t e n o i d : c h l o r o p h y l l a r a t i o s 2.39,  2.17  respectively  and  2.04  (Appendix  for cultures  A).  A pigment  Since  direct  chlorophyll a  and I I I ,  r a t i o of 2.39 was a l s o  reported f o r ammonium-starved S. costatum 1977).  II, I  (Harrison  et. a l . ,  d e t e r m i n a t i o n s were not  conducted a f t e r day 8, the c h l o r o p h y l l a : f l u o r e s c e n c e  ratio  was determined from v a l u e s obtained i n phase A and was used t o convert  fluorescence  was 0.33,  0.4 3  respectively.  and  readings 0.45  After  t o c h l o r o p h y l l a.  f o r chemostats  22-29  days,  II, I  chlorophyll a  chemostats I I , I , and I I I were 1.41 ± 0.53, 2.17 2.33 ± 0.90  ug c h l a « 1 0  cells  7  - 1  ,  respectively  Values f o r phaeophytin a were too v a r i a b l e  This r a t i o  to  and I I I , guotas  ± 0.54,  in and  (Appendix B). be  used  with  decreased with time u n t i l  day 5  confidence. The (Fig.  **C uptake g r a d u a l l y  5B) .  Between  photosynthesis  days  occurred  1 in  and  4,  50%  a  chemostats  5.39, and  I,  and  4.54  III  in  I I and I I I , while no  obvious trend was observed i n chemostat I. tats I I ,  reduction  On day 6,  chemos-  achieved p h o t o s y n t h e t i c r a t e s of 2.52,  ug C • 10  7  cells-*. hr  _ i  ,  respectively.  The  38  0.0  1  — i  0  1  2  1  — i  4 6 Ti mc (Days)  1  1  8  10  FIGURE 5. Changes i n (A) c h l o r o p h y l l a_, and (B) photosyntru- L i c rate and (C) photosynthe.tic a s s i m i l a t i o n rate during phase A of the longterm mercury exposures' (nM HgCl ) at ( Q ) 0.00; ( A ) 0.37 2  3.68 .  snd ( 0 )  The. arrow i n d i c a t e s the end of phase A f o r each c u l t u r e .  T I M E (Days) FIGURE 5B and 5C.  40 p h o t o s y n t h e t i c a s s i m i l a t i o n r a t e was reduced t o about the same value on day 6 f o r a l l treatments (Fig.,5C)..  3.5 M o r p h o l o g i c a l O b s e r v a t i o n s During  phases  A  and  B,  chains  of 1 to 2, elongated,  curved and highly vacuolated c e l l s , connected by s h o r t rods,  and  cell  clusters  around  lysed  cells,  silica  were  more  f r e g u e n t l y observed i n Hg-treated chemostats than i n chemostat culture I I . S t a t i s t i c a l a n a l y s e s , i n c l u d i n g the a n a l y s i s of and  three  a  fiosteriori  t e s t , Newman-Keul's attempt  to  from  (Duncan's m u l t i p l e  t e s t and Tukey's test)  determine  treatments on c e l l  range t e s t s  whether  dimensions  the were  were  effects  used of  the c e l l dimensions i n t h e t h r e e treatments  exposed  to  3.68  Chemostat  in  an  three  different Up to day 5, not  signi-  During t h e l a t e r stages of phase A, c e l l s nM HgCl^ were s i g n i f i c a n t l y l o n g e r than the  two other chemostat p o p u l a t i o n s . were s i g n i f i c a n t l y  were  range  the  significantly  each other (0.00, 0.37 and 3.68 nM HgCl ) .  ficantly different.  variance  In phases  C  and  D,  cells  s h o r t e r than i n phase B i n a l l chemostats.  I I I c e l l s were l o n g e r than c e l l s of chemostats I and  I I c e l l s , the l a t t e r being s t a t i s t i c a l l y Scanning  electron  micrographs  indistinguishable.  revealed  most  c h a r a c t e r i s t i c s a l r e a d y observed with the  inverted  pe, (e.g.,  in  short  silica  c u l t u r e s , at t h e end o f  rods).  phase  C,  Only  populations  the  of  the  microscoHg-treated  consisted  of  41 post-auxospore connected  with  populations Although were  c e l l s , (1-3%) longer  were  observed,  o c c u r r e d , due toward  end  relatively  few  than  the  phase  ammonium-limited  of  quickly  conditions  control.  reproduction  frequency  C,  these  post-auxospore  in  phase  e x p l a i n the absence of these wide c e l l s i n  cells  may  have  shorter  phase C i n a l l c u l t u r e s .  which were formed during under  Consequently,  sexual  increased  of  rods.  heterogeneous  synchronized  t o the  the  silica  more  no gametes and  and chains of 4 t o 6 c e l l s (1-8%)  The  cells  wide c e l l s  became  narrower  D.  This  chemostat  may  culture  I I s i n c e the c e l l s were always under ammonium l i m i t a t i o n .  3.6  Mercury  Analyses  Variations  in  the  expected  c u l t u r e I are shown i n F i g . 6. fluctuations not  included  (calculated  with  time  in  F i g . 6.  using  The accumulation adsorption  observed  frustules in  take  average equation  due  expected 3),  in  total  2) represent  to  total  by  calculations.  into  account the  dilution  the  levels.  cells,  I  and  only  Expected the  semi-continuous  the  in  additions  (calculated  I I I , were 0.33  total  changes  r a t e of the c u l t u r e .  concentrations  cultures  concentrations  maximum Hq  these  the  Hq  and inner w a l l s of c u l t u r e f l a s k s  c o n c e n t r a t i o n s as a r e s u l t of losses  of  f o r c u l t u r e I I I , i t was  Expected  equation  are not considered  and  S i n c e the same p a t t e r n  o r v o l a t i l i z a t i o n o f Hg  onto  concentrations  was  t o t a l Hg c o n c e n t r a t i o n s i n  ± 0.07  The using and  42  FIGURE 6.  V a r i a t i o n s i n the expected t o t a l mercury concentrations i n  chemostat I during the long-term mercury exposure to 0.37 nM H g C l ^ The average expected t o t a l concentration, which only takes i n t o account the mercury losses due to the d i l u t i o n rate of the chemostat, was 0.33t0.07 nM HgC^. During the f i r s t ten days, the concentration of the a d d i t i o n was 0.12 nM H g C l  except when ( ) 0.06 and ( ® ) 0.24 were adQ  2  ded. The expected t o t a l concentrations of mercury between each a d d i t i o n and the average expected concentration over the e n t i r e experiment were c a l c u l a t e d using equations 2 and 3, r e s p e c t i v e l y .  43 3.46 ± 0.78 nM HgC]^, r e s p e c t i v e l y f o r the e n t i r e experiment. Based on s t u d i e s on Cu t o x i c i t y , it  (Sunda and  i s p o s s i b l e t h a t Hg t o x i c i t y may  activity.  However,  concentrations,  present  work  deals  with t o t a l Hq which  directly  ion a c t i v i t y .  I I I shows that the c o n c e n t r a t i o n of p a r t i c u l a t e Hq  increased  with  Particulate  concentration  Hq values  and  time  of  Hq  exposure.  i n c u l t u r e s exposed to 0.37 and 3.68 nM  HgCl^, accounted f o r 22-58% and 15-41%, r e s p e c t i v e l y , t o t a l expected c o n c e n t r a t i o n s . the  soluble  Hq  could  a n a l y t i c a l technique control  1976),  be r e l a t e d t o mercuric i o n  because t h e r e i s no technique  measures mercuric Table  the  Guillard,  grade s a l t s used i n the  p o s s i b l y due to the  The s m a l l amounts  c e l l s were probably  the  In c u l t u r e s I and I I I , most of  not be recovered,  used.  of  of  Hg  i n the  from  reagent  culture  medium  due t o contamination  preparation  of  the  (Table I I I ) .  3.7 Short-term The to  Nutrient Kinetics  n u t r i e n t uptake k i n e t i c responses of c u l t u r e s exposed  sublethal  doses  of  HgCl,,, f o r 5 minutes t o 5 hours, are  shown i n F i g . 7., The values o f the parameters a r e presented The  nutrient  uptake  kinetic  i n Table IV.  e f f e c t s of Hg on t h e uptake k i n e t i c s o f NH^+-limited  S. costaturn s t a r v e d f o r 1.5 hours, concentration  occurred  between 1.84 and 3.68 nM H g C l  l a t t e r a d d i t i o n reduced V i  1 ) i ; ! r  by 37%  and  at 2  a  threshold  (Table I V ) . The  increased  Ks  from  44  TABLE I I I . Concentrations of mercury In the long-term experiment.  Filters  and f i l t r a t e s from 200 ml of chemostat effluent were analyzed f o r p a r t i c u late and soluble mercury, respectively. tes.  Each value is the mean of duplica-  'Measured' refers to values obtained  from  the mercury analysis.  'Expected total' refers to values obtained using equations 2 and 3, and 'expected soluble' refers to the difference between the total expected and measured particulate value.  The percentage loss (% Loss) is the difference  between the 'expected' and 'measured' soluble Hg, divided by the 'expected' —18 soluble  Hg, and multiplied by 100 . Atg - attog =10 CONTROL  DAY  2  g.  EXPOSED TO 0.37 nM • EXPOSED TO 3.68 -riM  4  6  2  4  6  2  (atg'.cell *)  0.91  1.61  2.45  5.16  7.56  8.32  33.46  (nM.)  0.07  0.11  0.07  0.18  0.11  0.52  1.36  1.18  N.I).  N.D.  0.18  .0.33  N.D.  N.D.  0.41  N.D.  0.55  Total Hg (nM)  0.00  0.00  0.00  0.33  0.32  0.40  3.46  3.32  3.24  Soluble Hg :(nM)  0.00  0.00  0.00  0.25  0.13  0.29  2.95  1.95  2.06  100%  100%  86%  100%  73%  4  6  MEASURED: Particulate Hg:  Soluble Hg: (nM)  207.93  EXPECTED: :  % LOSS:  A  B  Ammonium uptake rates as a function of the substrate, f o r ammonium-limited c e l l s starved for (A) 1 . 5 0 hours and (C) 3 0 . 0 hours, during the phort-term (un to 5 hours) mercury-exposure (nM HgCl ) to 0 . 0 0 ( O ) and 3 . 6 8 ( i \ ) . f i g u r e s (B) and (!)) show the disanpearance of ammonium with time. 1-TOURER..  c  D  0.20 f  0.15 +  0  1.0  2.0 3.0 S ( p M NHJ)  4.0  O ^ W U) W u i O u i ' o b i b b i b u i  Time (hr)  47 TABLE IV. Nutrient k i n e t i c response to short-term mercury exposure. The nutrient k i n e t i c parameters are defined i n section l ? o f t h i s chapter.The Ks values are the actual Ks-* apparent Ks + So. Values of the standard 5  errors are shown for V i  m a x  and Ks; the Vs values represent the average  uptake rate during the time period, Ts, over which the surge uptake occurs.  N.D. •= not detectable. 1.5 HOURS OP STARVATION  CONCENTRATION (nM HgCl )  Ts (hr)  0.00  0.44  0.26  0.178 ± 0.017  0.27 ± 0.13  N.D.  0.37  0.50  0.26  0.148 ± 0.006  0.21 ± 0.05  N.D.  1.84  0.93  0.16  0.167 ± 0.011  0.01 + 0.02  N.D.  3.68  0.45  0.26  0.112 ± 0.004  1.11 ± 0.07  1.08  5.53  1.89  0.06  0.117 ± 0.005  0.85 + 0.07  0.83  Ks  So  2  1  V i max (hr )  Ks (uM NH^)  So (uM NH^)  Vs . (hr" )  30.0 HOURS OF STARVATION CONCENTRATION  Vs .  Ts  V i max  (hr)  (hr )  (pM NHJ)  (nM HgCl )  (hr* )  0.00  1.74  0.06  0.012 ± 0.003  0.02 ± 0.00  N.D.  0.18  0.46  0.26  0.070 ± 0.010  0.51 ± 0.29  0.32  0.37  2.18  0.06  0.060 ± 0.000  0.54 ± 0.11  0.50  1.84  0.55  0.26  0.050 ± 0.000  0.00 ± 0.03  N.D.  3.68  0.50  0.26  0.060 ± 0.000  0.20 ± 0.08  0.16  2  -1  (UM-NH^)  48 0.28 to 1.11 uM NH^ .  The i n c r e a s e i n Ks was p r i m a r i l y due t o  +  the  of uptake when 1.08 uM NH^Cl s t i l l  cessation  the medium ( F i g . 7k). values  were  The Vs values  calculated  over  were  similar  significantly  reduced  low as 0.18 nM HgCl_. 2  decrease  hours,  v  i  w a  x  by exposure t o a c o n c e n t r a t i o n as  Increased time o f s t a r v a t i o n  l i m i t e d chemostat e f f l u e n t s a  when a l l  the same time i n t e r v a l of 0.26  hour., In N H ^ * - l i m i t e d e f f l u e n t s starved f o r 30 was  remained i n  of  NH.+4  (from 1. 5 to 30 hours) r e s u l t e d i n  i n t h e Ks value o f t h e c o n t r o l c u l t u r e s from 0.27  t o 0.02 uM NH^Gl  (Table I V ) .  Since t h i s l a t t e r  value  cf  Ks  f o r the c o n t r o l c u l t u r e was near the l i m i t s o f d e t e c t i o n , t h i s made  i t d i f f i c u l t to determine the e f f e c t s o f Hg exposure on  Ks. /Short-terra Hg exposure  (1.50 hours  of  starvation)  did  not appear to a f f e c t Vs (Table I V ) , s i n c e the mean uptake r a t e over  the  concentration  0.51 ± 0.06 h r  - 1  range from 0.00 t o 5.53 nM HgCl^ was  when the uptake was c a l c u l a t e d at a  time  of  0.26 hrjJVVariations i n the Vs values r e f l e c t the d i f f i c u l t y i n making t h i s measurement. treatment  resulted  in  However, both N H  4  the  amount  of  NH +  values  remaining  p e r t u r b a t i o n experiment when Ve = 0 f o r 18 However,  on  s t a r v a t i o n and Hg  a very sharp and reduced Ve r e g i o n o f  the uptake curve ( F i g , 7A and C ) . The So represent  +  (Table  IV)  a t the end o f the to  60  minutes.  some o c c a s i o n s t h e uptake slowly resumed a f t e r a  few hours. Of secondary i n t e r e s t , starvation  of  effluent  i s the  from  effect  of  duration  of  the chemostat on the n u t r i e n t  49 uptake response hours, •'  Ks  constant.  (Table  and  St  This  V).  When e f f l u e n t s were starved  values  decreased  indicated  responded t o 11 hours of  of  mobilization  nitrogen  and the  72  by  increasing  from the i n t e r n a l n u t r i e n t  system was decreased.  phases of the n u t r i e n t  Nutrient  populations  Between 11 t o 30 hours, the  hours, ammonium d e f i c i e n c y  3 • 8 Long.- term N u t r i e n t  a  deficiency  of n i t r o g e n  pool i n t o the a s s i m i l a t o r y  Vi„ „remained max  t h a t ammonium-limited  t h e i r a f f i n i t y f o r the s u b s t r a t e . rate  while  f o r 11  Between  30  had adverse e f f e c t s on a l l  uptake r a t e s .  Kinetics  uptake k i n e t i c parameters measured during  phases  a and D of the long-term Bg exposure a r e presented  in  VI.  In c u l t u r e I I I , the n u t r i e n t k i n e t i c s d e v i a t e d  considera-  bly  from  culture  the regular I  ( F i g . 8).,  showed  pattern  significant  Despite  the  value  culture III contrast measured.  as day 4, whereas  of  only  ,  day  resulted  +  - 1  by  8  1.3 t o 1.5 uM NH^Cl i n  a d d i t i o n o f NH„  f o r Vs of 1.21 h r  uptake took place  early  deviations  presence  c u l t u r e I e f f l u e n t , the spike normal  as  Table  in a  a f t e r which no s i g n i f i c a n t  f o r 7.1 hr (Fig.8B,  day  8).  However, i n  (day 6), the maximum recorded Vs was 0.70 hr~*, i n  to  culture I I  (day 5) where a Vs of 1.19 h r — was 1  These comparisons were  made  at  a  standard  time  i n t e r v a l of 0.10 hour. In  general,  the Ve region  during  of the uptake  phase k i n the Hg-treated curve  gradually  became  cultures, gentler  50  TABLE V.  The e f f e c t s of duration of s t a r v a t i o n of chemostat e f f l u e n t on the  n u t r i e n t uptake k i n e t i c s . 1 of t h i s chapter.  The uptake k i n e t i c parameters are defined i n section  The Ks values are the a c t u a l Ks, where a c t u a l Ks = apparent  Ks + So; n. d. = not detectable.  Vs  Ts  Vi max  (hr)  (hr )  (hr)  (hr )  0.00  0.27  2.60  0.124  0.50  0 .20  1.50  0.A4  0.26  0.178  0.27  n . d.  Conway and Harr i s o n , 1977 Table IV  11.00  0.37  0.30  0.129  0.59  n . d.  Tavle VI  30.00  1.74  0.06  0.102  0.02  n . d.  Table IV  72.00  0.17  0.70  0.088  0.10  n . d.  Conway and Harr i s o n , 1977  STARVATION TIME  _ 1  _ 1  Ks  So  Source  (jM NH}") (pM NH^)  51  TABLE V I . ure.  Nutrient  The n u t r i e n t uptake k i n e t i c parameters a r e d e f i n e d  t h i s chapter. Ks + So. Ks.  uptake k i n e t i c response t o the l o n g - t e r m mercury i n section  expos1 of  The Ks v a l u e s a r e the a c t u a l Ks where a c t u a l Ks *• apparent  V a l u e s of  standard  errors  a r e shown f o r V i ( V i * V i ) and max  N.D. = n o t d e t e c t a b l e .  The u n i t s of t h e n u t r i e n t uptake k i n e t i c , parameters are; Ts= h r , V i ( V i V i max ' K s » u M N H , So = uM NH^ . ) ?  h  r  +  Vs « h r  -1  +  4  CHEMOSTAT CULTURE I I (CONTROL) DAYS  2  4  5  Vs  0.37  0.62  Ts  0.30  Vi  6  19  26  1.19  0.94  0.38  0.20  0.10  0.10  0.30  0.129±.Q06  0.080+.003  0.1451.009  0.129±.011  0.145± .006  0.129 + .011  Ks  0.59  0.25  0.44 ±.11  0.49  0.06  0.12 ± .08  So  0.26  N.D.  N.D.  +.14  ±.09  0.20  ±.16  ± .02  N.D.  N. D.  CHEMOSTAT CULTURE I (EXPOSURE TO 0.37 nM HgCl DAYS  2  4  5  Vs  0.28  0.31  0.28  Ts  0.50  0.30  Vi  6.104±.005  Ks So  2  )  19  23  0.99  1.62  0.48  0.60  0.10  0.10  0.20  0.110±.017  0.186±.078  0.152±.024  0.122+,.004  0.183+ -009  0.18 ±.10  1.22 ±.57  0.03 ±1.67  1.34 +.71  0.01 ±..02  0.01 ± .02  .0.04  0.25  N.D.  N.D.  N.D.  N.D.  CHEMOSTAT CULTURE I I I (EXPOSURE TO 3.68 nM HgCl ) DAYS  2  4  5  6  23  26  Vs  0.37  0.54  0.63  0.70  0.58  0.30  Ts  0.40  0.20  0.20  0.10  0.20  0.20  Vi  0.134±.007  0.123±.013  0.121±.020  0.06U.008  0.170±. 007  0.105±,.003  Ks  0.35 ±.07  1.36 ±.36  1.80 ±.52  2.83 ±.23  0.15  0.05 ±,.02  So  0.04  0.63  0.85  2.43  N.D.  +. 03  N.D.  52  2.0  FIGURE 8.  3.0 Time (hr)  4.0  The disappearance of the substrate with time during a perturb-  ation experiment conducted on d i f f e r e n t days during phase A of the longterm mercury exposure (nM HgCl-) to (A) 3.68  ( • ) , and (B) 0 . 3 7 ( Z \ ) .  53  A  <ao  n  3.0  FIGURE 9.  4.0 5.0 Tims (hr)  7.0  80  9.0  (A) Disappearance of ammonium with time, a f t e r the a d d i t i o n of  5 fM NH^ • on day 6 (phase A) of the long-term mercury exposure (nM HgCl^) to ( O ) 0.00; ( A ) 0.37 and (•) 3.68 . These data were used to c a l c u l ate  the  ammonium uptake rates as a f u n c t i o n of the substrate i n f i g u r e (B).  54 with  duration  exposure. values  ( F i g . 8)  and  concentration  T h i s change was r e f l e c t e d by  the  (Fig.  9) of Hg  increase  i n Ks  or a l o s s of a f f i n i t y f o r the s u b s t r a t e i n c u l t u r e I I I  (Table VI).  I t appeared t h a t vs was a l s o reduced i n chemostat  c u l t u r e I I I compared t o the c o n t r o l c u l t u r e I I . In phase D, Hg-treated lower Vi_  Ks (Fig.,10)  c u l t u r e s had a  higher  ^i^ax  a  n  d  than i n phase A ( F i g . 9). I n the c o n t r o l ,  values were s i m i l a r t o values obtained  i n phase A but Ks  values were reduced i n phase D (Table V I ) .  S e c t i o n 4.  Discussion  ^ • 1 E f f e c t s of Mercury on Under  ammonium  Photosynthesis  limitation  (e.g., i n the c o n t r o l c u l t u r e  II) , the r e d u c t i o n i n p h o t o s y n t h e t i c t o a decrease i n chlorophyll a  photosynthesis  per  cell.  1975b)  and  and  Transient  t h e s i s have a l s o been observed 1975a;  a s s i m i l a t i o n r a t e was due  in  a  increase  in  r e d u c t i o n s i n photosyn-  nutrient-limited  nutritionally  1975).  The  simultaneous  e f f e c t s of mercury and ammonium l i m i t a t i o n  (days  enhancement limitation assimilation  of  in  phase  chlorophyll a  alone. rate  The in  small  D)  resulted  synthesis increase  mercury-treated  Stone,  natural  communities  and  and  perturbed  (Thomas,  phytoplankton  1 - 5 i n phase A  (Falkowski  slight  in a  qreater  than under ammonium in  photosynthetic  cultures  (day 6) was  55  0.195  r  S (|-'M FIGURE 10.  NH ) 4  Ammonium uptake r a t e s as a f u n c t i o n of the substrate a f t e r  the recovery (phase D) from the long-term mercury exposure (llM HgC^) ( O ) 0.00;  ( A ) 0.37 and (•)  3.68 .  to  probably  due to the c e s s a t i o n o f  the amount of n i t r o g e n spite  of  an  atg»cell-i  ammonium  limitation,  and c h l o r o p h y l l a per c e l l  increase  in  particulate  (attog = 10~*8 g) .  treatments.  comparable  in  a l l  I n the c o n t r o l and mere u r y - t r e a t e d c u l t u r e s , c e l l  doubled  and  c e l l volume s t i l l  returned  i n c r e a s e d upon  to their o r i g i n a l levels mercury  n i t r o g e n and c h l o r o p h y l l a per c e l l cell  increased i n  mercury up t o 207.93  In phase D, i n v i v o f l u o r e s c e n c e was  numbers  since  volume,  the values  exposure.  while  I f the  were expressed per u n i t o f  would appear to be s i m i l a r among a l l  cultures. The  i n c r e a s e i n c h l o r o p h y l l a content  stimulation  of  synthesis,  may  o f a new f r u s t u l e .  elongated  g r e a t e r c e l l u l a r content  and  proteins).  Navicula  of  T h i s has  pelliculosa  been  (Coombs  observed et  This  in  al.,1967).  would  to  a  produce  (e.g., DNA, BNA silicate-starved Thus, mercury  might a r r e s t c e l l d i v i s i o n by i n t e r f e r i n g with the of the s i l i c a t e  due  o r to a d u p l i c a t i o n o f o r g a n e l l e s  without the formation cells  be  regulation  metabolism.  4.2 E f f e c t s o f Mercury on Growth Parameters The  effect  of  mercury on biomass and n u t r i e n t k i n e t i c s  were p r o p o r t i o n a l to t h e c o n c e n t r a t i o n c u l t u r e I I I was t h e f i r s t c u l t u r e to steady-state). values  In  culture  o f the a d d i t i o n s be  I I I , high  disturbed  (e.g.,  from i t s  particulate  mercury  were reached sooner than i n c u l t u r e I (Table I I I ) .  57  Periods o f c e l l been  reported  in a l l  loss i n  (Davis e t a l , , 1973).  nutrient-limited  increased  likelihood  reproduction. a decrease limiting  nutrient-limited  chemostat for  cultures  As c e l l s became narower cultures,  the  there  recurrence  of  For a s i l i c a t e - l i m i t e d c u l t u r e s o f S.  per c e l l and the occurrence  was  an  sexual costaturn  i n c e l l d e n s i t y , an i n c r e a s e i n t h e amount nutrient  have  o f the  of wide, s h o r t  c e l l s i n d i c a t e d t h a t synchronized sexual r e p r o d u c t i o n occurred (Davis e t a l . , Hg-treated  1973).  cultures,  S i m i l a r o b s e r v a t i o n s were made f o r t h e and  to  a  very l i m i t e d e x t e n t , i n t h e  control culture.  A d d i t i o n s of mercury, which r e s u l t e d i n the  earlier  i n c u l t u r e s I and I I I populations  did  decline  not appear to i n t e r f e r e with presumed s e x u a l  processes  (phase  C)  since  resumption  of  (phase A),  reproduction  growth  occurred  simultaneously i n a l l c u l t u r e s .  U.3 E f f e c t s of Mercury on N u t r i e n t Dotake K i n e t i c s I n the short-term mercury exposure and i n c u l t u r e I , the maximum  rate  of  uptake, Vs, was not a l t e r e d .  and I I I (days 2 t o 4 ) , the a c t u a l Ks i n c r e a s e d , loss  i n the  affinity  apparent  Ks  (apparent  remained unchanged.  Ks + So)  Ks values were reduced.  Ks values were a l s o reduced r a t e . Vs.  indicating  a  f o r the s u b s t r a t e , while the maximum  assimilatory rate, ' i ^ ^ * actual  In c u l t u r e s I  The enzymatic  as  increased  Although,  the  i n c u l t u r e I , the  I n c u l t u r e I I I , the apparent well  inhibition  as  the  maximum  by heavy metals  uptake  i s usually  58 classified  as  noncompetitive (Lehninger, 1975) .  uncompetitive and noncompetitive i n h i b i t i o n can decrease  in  Vi  „ or  Vs,  the  additional  Even  though  result  in  decrease  a  i n the  wax  apparent Ks inhibited  values the  indicated  uptake  long-term, low l e v e l term higher l e v e l at  low l e v e l s  in  mercury  uncompetitively cultures.  Under  (0.37 nM HgCl^) exposure and under  short-  ammonium-limited  (>1.84 nM HgCl^) exposure, the  uptake  rate  (estimated by Ks) appeared to be more s e n s i t i v e  than the i n i t i a l uptake were  that  reguired  to  (Vs) .  Much higher l e v e l s  decrease Vs or V i  .  of  mercury  The reasons f o r the  max greater  sensitivity  of  the  c o n c e n t r a t i o n s are not c l e a r .  rate  of  uptake  at  low  The r e d u c t i o n i n Vs or V i ^ ^ i n  the  long-term high l e v e l  (3.68 nM HgCl^) exposure could be due  to  the  of  high  affinity  r e s u l t i n g i n the b i n d i n g  of  m e r c u r i a l s f o r s u l f h y d r y l groups, some  mercuric  ions  onto  cell  membrane enzymes ( e i t h e r enzymes i n v o l v e d i n the h y d r o l y s i s of ATP the  or  carrier  enzymes).  short-term experiment  recovery cell  (1.5 hours of s t a r v a t i o n ) , a f t e r the  (phase D) i n the long-term experiment, and changes i n  morphology  concentration between 1.84 where  Changes i n n u t r i e n t k i n e t i c s from  the  i n culture I I I ,  suggest  f o r r e c o v e r y from mercury and 3.68  first  nM HgCl^., The  e f f e c t o f mercury  that  the  threshold  inhibition  , occurred  threshold  i n h i b i t i o n was observed on  n u t r i e n t uptake k i n e t i c s was reduced t o 0.18 increased  period  concentration  nM  HgCl^  (30 hours) of ammonium s t a r v a t i o n .  by  an  59 Mercury I n h i b i t i o n  4.4 Recovery from I n i t i a l Recovery and  from  Hashizume,  (De F i l i p p i s  mercury  1975;  and  i n h i b i t i o n i n continuous  Kayser,  Pallaghy,  1976)  and  batch  (Fujita cultures  1976c; Berland e t a l . , 1977) are  known.  They have been a t t r i b u t e d t o mercury l o s s e s  from  medium  (Davies,  when c e l l  d e n s i t i e s are through  1974)  high  either  or  a  by  volatilization  decrease  in  particulate  the  mercury  an uptake e x c l u s i o n mechanism(Ben-Bassat e t a l . , 1972;  Ben-Bassat  and  Mayer,  1975; 1977; De F i l i p p i s and P a l l a g h y ,  1S76b). In phase C of resumed  when  cell  mercury  particulate  the  long-term  densities values  accumulation  occurred  F i l i p p i s and  Pallaghy,  were  in  highest.  1976c), after  divisions  (Davies, 1974).  The  mercury-treated  in the  content).  galbana  (De and  i n i t i a t i o n o f a few c e l l  was  in  specific  probably  appearance of c e l l s of a new l i f e c y c l e stage  chlorophyll a  metal  Chlorella  IsochrjrsJLs  increase  cultures  c e l l u l a r chemical composition  growth  Greatest  mercury-resistant  tertiolecta,  in  exposure,  were minimum and p o s s i b l y when  Qunaliella  rates  mercury  growth  due to the  with a d i f f e r e n t  (as seen from t h e  nitrogen  and  I n n u t r i e n t - l i m i t e d diatoms, during  v e g e t a t i v e r e p r o d u c t i o n , changes i n the c e l l u l a r chemical comp o s i t i o n , r e d u c t i o n i n width and e l o n g a t i o n o f c e l l s  possibly  produced  stages,  populations  of  found  different  Werner (1971)  also  that  Coscinodiscus  asteromphalus,  physiological  during  cell  diminution  changes i n chemical  in  composition  60 occurred and the s e n s i t i v i t y  to  metabolic  with d i f f e r e n t l i f e c y c l e stages or c e l l After  the  been  may  be due t o  acguired  several  from  genetic  conseguence of sexual r e p r o d u c t i o n . all  cells  i n phase C may  r e s i s t a n c e upon operating of a few  on  mercury  capable  of  phase  would  et a l , , (1977) suggested to  the  initial  be  The a c g u i s i t i o n of factors.  The  type may  or  pressure  high  survival  external  or  and t h e r e f o r e growth d u r i n g the  t h a t recovery may  physiological  state  given.  of  Berland  be due t o a the  cells  return but no  The r e s u l t s from the l o n g change  be as important as a pure p h y s i o l o g i c a l  in  adapta-  metabolism.  Mercury Losses Losses were independent  losses  from  volatilization the  a  a physiological  selective  with  as  some  have r e s u l t e d i n the  coping  could  recombination,  t i o n mediated by a b i o c h e m i c a l change i n c e l l  4.5  It  mercury exposure i n t h i s study suggest t h a t a  cell  uptake)  i n i t i a t e d by these c e l l s .  s u g g e s t i o n of mechanism was term  of  Alternatively,  exposure.  phase C c e l l s may  cells  rates  a l s o have developed  i n t e r n a l c o n c e n t r a t i o n s of Hg, recovery  widths.,  i n the mercury-treated c u l t u r e s .  mercury-tolerance have  varied  r e c o v e r y , improvement of n u t r i e n t uptake ( i n c r e a s e  i n the a f f i n i t y f o r the s u b s t r a t e and occurred  inhibitors  the  used  of  medium  the  initial  were  w i t h i n the c u l t u r e s .  In  probably  doses. not  continuous  These due  to  cultures,  r e g u l a t i o n of a constant volume i n s i d e the c u l t u r e  flasks  61 depends on a constant Consequently,  partial  pressure  the  a i r space. the  s u r f a c e of the medium, and the amount o f Hq adsorbed onto  the  walls  The  mercury  i n the s o l u t i o n were probably at e q u i l i b r i u m .  a c t u a l low recovery o f s o l u b l e presence  of  dead  above  and  the  a i r pressure i n  of  a  heat-stable  mercury  may  be  photosynthetic  due  t o the  metabolite  i n the  used medium (Ben-Bassat and Mayer, 1977), r e s p o n s i b l e f o r the r e d u c t i o n of Hq* the  analytical  2  to Hq° . procedures  Thus, t h e major l o s s may come from f o r mercury d e t e r m i n a t i o n s ,  r e q u i r e heatinq of aqueous samples a t 95°C Therefore,  the  heat-stable  durinq  photosynthetic  2  which  hours.  metabolite  enhance the r a t e o f v o l a t i l i z a t i o n d u r i n q the h e a t i n q  would  process.  4.6 A p p l i c a t i o n s t o t h e N a t u r a l Environment In n a t u r a l waters, mercuric  ion a c t i v i t y  would  probably  be l e s s than i n the a r t i f i c i a l seawater due t o the presence o f unknown  quantities  detritus,  dissolved  concentrations  of  natural  complexinq m a t e r i a l s such as  orqanics.  equivalent  to  Therefore,  total  mercury  those used i n c u l t u r e s may not  produce the same e f f e c t s i n n a t u r a l seawater. During p e r i o d s of seasonal n u t r i e n t  limitation,  mercury  p o l l u t i o n may s e r i o u s l y i n t e r f e r e with the c o m p e t i t i v e of  a  diatom.  Under n u t r i e n t - l i m i t e d c o n d i t i o n s , the a b i l i t y  to take up the l i m i t i n g n u t r i e n t i s s e v e r e l y reduced i s increased) mercury.  ability  by  exposure  to  a  secondary  stress  (Ks value such  as  T h i s may r e s u l t i n a change i n the dominant s p e c i e s  62 or replacement by d i f f e r e n t a l g a l groups such  as  flagellates  {Thomas and S e i b e r t , 1977),  4,7 E v a l u a t i o n of Chemostat S t u d i e s In  this  study,  the use of chemostats i n determining the  e f f e c t s of n e a r l y e c o l o g i c a l t o t a l c o n c e n t r a t i o n s on  an  unialgal  population,  already  situation  than  Long-term, low l e v e l level  The  rather  than  into  the  natural  short-term,  high  experiments.  continuous  s t r o n g l y urged.  reproduction, bioassays.  cultures  tend  and  especially  morphology and during  sexual  t o make diatoms u n s u i t a b l e t e s t organisms  From these experiments, the use bioassay  of  are i l l - d e f i n e d ,  chemostat i n p o l l u t i o n s t u d i e s  chemostat  i s c o n s i d e r e d as a r e f i n e d  Since c o n d i t i o n s which induce c e r t a i n  i n algae  compared  I t i s f o r t h i s reason t h a t the  The n o t i c e a b l e changes i n c e l l  for pollution  technigue.  long-term  c u l t u r e s t o determine t h r e s h o l d e f f e c t s i s  physiology, during vegetative  stages  This  t h r e s h o l d c o n c e n t r a t i o n where the e f f e c t s occur, are  the short-term of  environment.  using continuous c u l t u r e s .  almost an order of magnitude lower i n the  for  of an  e f f e c t s may simulate more c l o s e l y t h e d i s p e r s a l mode of  s i m u l a t i o n i s best achieved  use  imitation  stress  i f batch c u l t u r e s had been used.  effects  industrial pollutants  to  mercury  under a primary  ( n u t r i e n t l i m i t a t i o n ) , has allowed a c l o s e r ecological  of  could  the be  life  suitability improved  s p e c i e s which are l e s s v a r i a b l e during t h e i r l i f e  by  cycle.  cycle o f the using  63  CHAPTER I I I  TH E EFFECT OF Hg  EXPOSUSE ON  INTRACELLULAR DISTBIBOTION M AND  Cuj,  Section  1.  Mi^  Zn  Introduction  Repeated  exposure of phytoplankton to t r a c e elements  increase tolerance  {De  is  whether  not  known  F i l i p p i s and a  Pallaghy,  complexing  1976c) , agent  may  but  it  such  as  m e t a l l c t h i o n e i n p l a y s a r o l e i n t h i s process,  Metallothionein  is a  (10,000)  low  molecular  synthesis  is  p r o t e i n can Hg,  I t can  weight  stimulated  bind and  (m.w.)  by  protein  exposure to heavy metals.  a l s o s t o r e Cu  ; Brown and  This  d e t o x i f y heavy metals such as Ag, Cd  and  and Zn when they occur i n excess of  the l e v e l s r e g u i r e d f o r metalloenzymes 1975  whose  Chatel,1978).  (Bremmer  and  Davies,  D e l e t e r i o u s e f f e c t s may  occur  when the r a t e of heavy metal accumulation exceeds the r a t e metallothionein  synthesis  et a l . , 1973;  Brown and  such  and  as  Cd  metalloenzymes  (Brown,  its  binding  Parsons, 1978).  Hg may  exert t o x i c e f f e c t s by;  or  capacity  Hence,  heavy  accumulate i n the high  1)  substituting  1977;  Brown  and  for  m.w.  Cu  and  guarternary  or t e r t i a r y enzyme s t r u c t u r e ,  binding  active  other  or  (Winge metals pool  and  Zn  in  Parsons, 1978), 2)  a l t e r i n g the to  of  s i t e s l e a d i n g to  or  3)  conformational  changes. M e t a l l o t h i o n e i n has been u b i g u i t o u s l y found i n  land  and  marine  animals ( P i s c a t o r ,  1964;  Buhler and K a g i , 1974;  Howard  and N icicles s, 1977a; 1977b), but i t s presence i n phytoplankton remains to be c l a r i f i e d .  McLean et a l . ,  (1972) may  a m e t a l l o t h i o n e i n - l i k e f r a c t i o n binding Cd green a l g a e , exposed The  Zn  in  blue-  Cd.,  i n i t i a t i o n of l o g a r i t h m i c growth i n algae p r e v i o u s l y  inhibited  by  factors. may  to r a d i o a c t i v e  and  have found  One  be  heavy  metals  may  be  attributed  to  p o s s i b l e reason f o r the recovery from  the  production  of  complexing  several  inhibition  agents  such  as  metallothionein. The aim o f t h i s study was of  a  short-term  metal  exposure  metallothionein  possibility  b i o c h e m i c a l a d a p t a t i o n i n response to heavy  in  S. cost,atum  would  be  metals and the subseguent the  to i n v e s t i g a t e the  intracellular  and  to  determine  whether  r e s p o n s i b l e f o r s e g u e s t e r i n g heavy recovery.  levels  and  The e f f e c t s of  HgCl^  on  d i s t r i b u t i o n of Cu and Zn are  a l s o examined.„  S e c t i o n 2.  2.1  M a t e r i a l s and Methods  Batch C u l t u r e s C u l t u r e s of Skeletonema  seawater  (Davis et a l . ,  medium  (appendix  borosilicate, 6  A).  1 flat  costatum were grown i n a r t i f i c i a l  1973)  enriched  Batch bottom  with  cultures boiling  modified were  flasks  ,  f/2  grown  l  in  continuously  65  stirred  at  120  rpm.  Growth was  monitored  by changes  v i v o f l u o r e s c e n c e with a Turner Model 111 f l u o r o m e t e r c e l l d e n s i t y using an i n v e r t e d  in i n  and  in  microscope.  2.2 Jxjaer imenta 1 C o n d i t i o n s  log  During the f i r s t  70 hours of the experiment, two  phase  were  cultures  grown  with  no pre-exposure to Hg  { c u l t u r e s A and B) while two other c u l t u r e s exposed subset  t o 1.84  VII).  {C  and  D)  were  nM H g C l ; a f t e r 70 hours, one c u l t u r e o f each 2  ( c u l t u r e s B and D)  (Table  unialgal  Culture  p e r i o d of 116 hours  was E was  perturbed  with  exposed t o 0.37  5.53  nM  nM H g C l  HgCl  2  over a  2  (Table V I I ) .  2.3 Analyses Early  stationary  centrifugation  for  6  phase minutes  polycarbonate t e s t tubes. were  harvested  after  70  cells at  were  650«g  at  harvested 6°C  in  The c e l l s from the c o n t r o l  by 50 ml  culture  hours while the c e l l s from the Hg-  exposed c u l t u r e s were harvested between 90 and  116 hours.  The  c e l l s remaining i n the supernatants were c o l l e c t e d onto a 0 . 4 5 uM M i l l i p o r e f i l t e r , resuspended i n  artificial  seawater  and  r e c e n t r i f u g e d as above. One 0.9%  gram of c e l l s  NaCl f o r 5  minutes  (wet weight) was homogenized i n 3ml of using  v a r i a b l e speed l a b homogenizer homogenate  a  TBI-B  STIB-R  Model  at a speed s e t t i n g of 4.5.  563C The  was c e n t r i f u g e d at 27,000-g f o r 10 minutes using a  66  TABLE VII. Exposure to different concentrations of HgCl at different times during a batch culture experiment.  2  TREATMENT Culture A Culture B Culture C Culture D Culture E  CONCENTRATION (n^ HgCl ) ADDED AT 0.00 hr 70.00 hr 0.00 0.00 1.84 1.84 0.37  0.00 5.53 0.00 5.53 0.00  67 S o r v a l Super/speed EC2-B The  supernatant  (Pharmacia) buffer.  column  automatic  was  fractionated  (9 X 60  cm)  with  on  a  centrifuge. Sephadex  a NH.HCO,  0.01  elution  was  determined with a Perkin-Elmer  124D double beam spectrophotometer.  Absorbances  and 280 nm determined the r e l a t i v e amount of metal-bound tances and aromatic amino identified  as  positions  in  acids,  respectively.  intracellular  pool  by  r e l a t i o n to t h e medium m.w.  from n a t u r a l l y  occurring  identification  of  duck  liver  metallothionein  comparing  Perkin-Elmer  spectrophotometer  Hodel  306  their  f r a c t i o n s obtained  (1964) and Leber Copper and Zn  were determined i n each f r a c t i o n using d i r e c t flame  with deuterium background  was measured by a c o l d vapor method of  were  metallothionein.  as d e s c r i b e d i n Brown and C h a t e l (1978).  fractions  subs-  The  was done by comparing i t s  e l u t i o n p r o f i l e with those o f P i s c a t o r  mercury  Peaks  at 250  being i n the p o s i t i o n o f the high (enzyme-con-  t a i n i n g ) and low m.w.  a  G-75  F r a c t i o n s o f 2 ml were c o l l e c t e d .  U l t r a v i o l e t absorbance Model  refrigerated  aspiration  atomic  each peak, u t i l i z i n g a 30 cm c e l l  C o n t r o l and O p t i c a l U n i t s Model 100).  levels with  absorption  correction. on  (1974)  the  Total  combined  (Pharmacia  UV  68 S e c t i o n 3.  3.1  Results  Growth At the beginning of the  exponentially  (Fig.11).  HgCl^  concentrations  HgCl had  while  Growth r a t e was  a growth r a t e o f 1.92 cultures  cultures  unaffected  equal to or g r e a t e r  ± 0.27  (C  and  d i v i s i o n s per day.  The  (C and  by 55%  3.2  D i s t r i b u t i o n of Hcu  shows  nM  than 1,84  nM  nm  c o n s i s t e d of two m.w.  pool  m.w.  pool  (fractions  absorbance 2  t o heavy metals, there  The  7)  peaks, and  a f t e r 24 nM  pre-  hours,  HgCl.,.  cultures  (Fig.12)  one  in  the  a major peak i n the  It high low  In c o n t r a s t to the absorbance  f r a c t i o n s derived was  Hg-  no l a r g e  from animals exposed  absorbance  peak  in  the  pool. typical  c u l t u r e exposed to 13.  to  ( f r a c t i o n s 12 to 20).  p r o f i l e s of c y t o p l a s m i c  medium m.w.  the  abundance of metal-bound substances.  major  B)  Zn  absorbance p r o f i l e f o r a l l  relative  and  c e l l d e n s i t i e s i n the  D) were reduced by 25%  Cu and  {A and  reduced growth r a t e of  a f t e r 46 hours of pre-exposure to 1.84  250  the  a  grew  by 0.37  d i v i s i o n s per day had  and  The  ± 0.37 D)  exposed c u l t u r e s  Fig.  all  reduced i t . , A f t e r 70 hours, c o n t r o l c u l t u r e s  2  exposed 1.31  experiment,  g e l e l u t i o n p r o f i l e of a n u t r i e n t - s a t u r a t e d 1.84  nM  T h i s p r o f i l e was  HgCl  2  (culture  c h a r a c t e r i z e d by;  C)  is  shown  in  1) high l e v e l s of  69  200  r  10  5ioo u  %  80 6  0  E  o 40 i_  o D LL  20  :  ~T  2  Time  1  3  4  (Days)  FIGURE 11. Changes i n the i n v i v o fluorescence  5  i n the f o l l o w i n g batch  c u l t u r e s : ( O ) c u l t u r e A; (@) c u l t u r e B; ( A ) c u l t u r e C and ( A ) c u l ture D.  The time and concentration  Table V I I .  of mercury exposure are given i n  The arrow i n d i c a t e s the time (70 hours) at which the addi-  t i o n of 5.53 nM HgCl^ was made to cultures B and D.  70  Fraction  Number  FIGURE 12. The 250ranabsorbance p r o f i l e of f r a c t i o n s c o l l e c t e d from the followoing c u l t u r e s : (O) C and (A)  c u l t u r e D.  c u l t u r e A; (^)  c u l t u r e B; (A)  culture  The time and concentration of mercury exposure  are given i n Table V I I .  7-1  1.0 D)  0.8  £  0.6  D  0.4  u  0.2  ~  005  •  r  JR  ^0.04  £ 0.03  5  10  Fraction  15  20  Number  FIGURE 13. The g e l e l u t i o n p r o f i l e of the 280 nm absorbance and of the amounts of Cu and Zn i n i n t r a c e l l u l a r pools of batch c u l t u r e C, i n i t i a l l y exposed to 1.84 nM HgCl . 2  I = high molecular weight pool;  I I = medium molecular weight p o o l , and I I I = low molecular weight pool.  72  Cu  and  Zn i n  and  lesser  smaller  the  low  amounts  amounts  of  m.w, Cu i n  of  Cu  correspond  to  the  determined  in  previous  Gel  profiles  elution  shape  but  the  pool the  and  position  m. w.  Zn i n  those  of  other  of  2)  high  large  amounts  pool  (I),  fractions  (Bouguegneau  Hg e x p o s u r e s  metals  in  each  et  Zn 3)  w h i c h may (II)  a l . ,  were peak  of  and  metallothionein  studies  from  level  (III),  as  1S75),  similar varied  in  (Table  VIII). In  all  decreased  cultures, and  increased,  concentrations by  38  the  to  high  20%  in  due t o  and  low m.w.  i n the  cultures  additions m.w.  (Table  501  occurred  of  intracellular  Zn  respectively,  as  VIII).  Total  Hg e x p o s u r e . fractions  m e d i u m m.w, B and  HgCl  fractions,  fractions  total  (5.53  2  whereas  remained  and  Cu  constant  a  Cu  function Zn  with  in  12.5%  the of  of  a  in Zn by  highest  i n the  medium  total  Hg  decreased  increase  Copper increased  (ca.  of  T o t a l Cu i n c r e a s e d  perturbed  levels  levels  i n Zn o c c u r r e d  gradual  fractions.,  nM).  a  intracellular  A decrease  D w h i c h were  and  high m.w.  intracellular  Cu). Total and  D which were  (5.53 m.w.  intracellular  nM). pool.  (culture culture  perturbed culture  D)  reduced  values  for  Hg  was  85% of  total  B which had no  fl,  detectable  highest  of S.  the  Hg l e v e l s  in  Hg a p p e a r e d to  b y 75% In  over  the  high  C and  E  were  cultures  additions  costatum  pre-exposure.  found  cultures  only  by t h e  B,  Pre-exposure  accumulated Hg  In  Hg was  of in  1.84  HgCl^  the  high  nM H g C l  when c o m p a r e d  culture m.w., lower  D,  pool. than  B  a l l  2  to the  Although the  Hg  TABLE VIII.  Distribution  of t o t a l Zn, Cu and Hg i n the i n t r a c e l l u l a r  pools  of nutrient-saturated c e l l s  with (cultures C, D and E). and without (cultures A and B) previous exposure to HgCl . Time and concentration  of HgCl  2  exposure f o r the different  metal levels fcrnole g - c e l l s "  Zn  TOTAL TREATMENT  1  cultures are given in Table V I I .  Data are the compilation o f  (wet weight)) from p r o f i l e s such as i n Fig. 13.  (p mole • q cells"')  Cu  (?] c 1e • a c 5lis"')  HI GH  DI'J E LOW  HIGH  MEDIUM LOW  aw  KM  HW  HW  POOL  POOL  POOL  PGCL  POOL  . 27 0  .0 32  .0 35  .275  .0 44  .034  .253  .041  TOTAL  Ha (;i:r:ol e •<-[ c e l l  TO? A  T  HIG H  REDIUM LOM  aw  KM  P 00 L  PCOL  POOL  POOL  ND  KB  ND  K0  . 197  ND  ND  ND  ND  . 032  .ISO  ND  ND  ND  MD  CULTURE  k  . 0231  .0083  ND  CCLTORE  £  . C136  .0065  ND  CULTURE  Q  .0115  . 0035 . 0009  CULTURE 3  .0144  .0045 .001 1  .0087  . 228  .042  . G4C  . 246  .013  CULTURE D  .0130  .GC35 .0017  . CC73  .326  .062  . 040  . 223  . 0034  . 0071  ND = not detectable.  <->  .0117 . 0034  . 0021 ND  ND ND  LEAF 74 OMITTED IN PAGE NUMBERING.  75 standards, that  a q u a l i t a t i v e examination o f the Hg a n a l y s i s showed  t o t a l i n t r a c e l l u l a r Hg i n c r e a s e d with Hg c o n c e n t r a t i o n s ,  and the highest l e v e l s of Hg were found i n the high m.w. followed by the medium m.w.  S e c t i o n 4.  pool  fractions.  Discussion  Even though a  concentration  as  low  as  0.37  nM HgCl  2  f a i l e d t o reduce growth r a t e s , i t was s u f f i c i e n t t o change the levels  and  d i s t r i b u t i o n o f i n t r a c e l l u l a r Cu and zn i n S, c o s -  taturn.  The displacement  o f Zn by Hg  on  metallothionein  a l s o been observed by Kagi and V a l l e e (1960). Zn  and  Cu  by  other  heavy  metals  has  Displacement of  appears t o be a g e n e r a l  c h a r a c t e r i s t i c of metal-induced bindinq p r o t e i n s . In S. m.w.  costaturn, more Cu than Zn occurred i n t h e medium  fractions,  which  i s not t y p i c a l o f m e t a l l o t h i o n e i n s .  T h i s i n d i c a t e s t h a t t h i s p r o t e i n may not play a major r o l e the  detoxification  m.w.  {enzyme-containing) pool  excess  Cu  and  C h a t e l , 1978). Zn §i Cu  appeared a  2«*  Zn  of heavy metals. was  occurred  I n ducks, when t h e high  apparently  saturated, (Brown and  In Cd and Hq-induced t h i o n e i n s i n r a t h a l f the bindinq s i t e s  1975), o r i n r e s i d u a l amounts with  i n animals  Zn  i n metallothionein  i n approximately  in  livers, (Hinge  s m a l l e r amounts o f  (Kagi and V a l l e e , 1960).  In S. cgstatum, t h e decrease o f t o t a l i n t r a c e l l u l a r Zn a t any  Hg  concentration  r e g u l a t e d by an  and  exclusion  of  Hq upon Hg-preexposure may be  mechanism.  When  the  freshwater  76 green  alga  Chlorella  was  exposed  s e n s i t i v e component of the Zn uptake number  of  chum  salmon  greater  pool.  and a summer  failure  than  1.84  4  nS  also  z o o p l a n k t o n * assemblage effects  o f these  heavy  metals  simultaneously Hi*r  then  were  occurring  1975;  in  the high  pathological  observed.  Yoshikawa,  1976;  Brown  D e l e t e r i o u s e f f e c t s can be caused by the and  Zn  from  metalloproteins  proteins non-functional 1975). have  Since a  high  elements  of  pools,  medium m.w, This  affinity  pool  observation  •zooplankton . 4  by  The excess pool  with  (Bouguegneau  and Parsons, 1978). displacement  1974; Bremmer and  at  the  sulfhydryl  tertiary  of  Cu  Davies,  and  groups,  guarternary  occur.  a  and low  also  molecular  weight  simultaneous i n c r e a s e of Zn i n the  was found i n S,, costatum has  of  heavy metals r e n d e r i n g the  for  decrease of Zn i n t h e high accompanied  rate  of subgroups l i b (eg. Zn, Cd and Hg)  protein  s t r u c t u r a l l e v e l s could The  (Friedberg,  binding  denaturation  by  In  as t h e r a t e  m.w,  effects  in  (Brown and  heavy metals exceeds the  appears  HgCl^,  found  m e t a l l o t h i o n e i n s y n t h e s i s or i t s b i n d i n g c a p a c i t y . of  the  the d e t e c t i o n of Hg  t o d e t o x i f y heavy metals occurs  of bioaccumulation  and  w a l l was reduced (De  A s i m i l a r p a t t e r n was  Parsons, 1978) when d e l e t e r i o u s animals,  temperature  inhibited  growth r a t e s c o i n c i d e d with  i n the high m.w.  a  1976c).  At exposures egual t o or of  Zn,  was  Zn exchange s i t e s on the c e l l  F i l i p p i s and P a l l a g h y ,  reduction  to  been  The medium m.w.  reported  in  this  f o r the  study. summer  p o o l i n S. costatum does  not  77 appear  to  be  the  main  .costaturn approximately respectively  were  storage  70%  and  found  s i t e f o r Cu and 60%  in  the  of  total  low  m. w.  Zn. , In Cu  S.  and  pool  Zn,  like  in  'zooplankton* and  u n l i k e higher organisms.  T h i s pool may  as  of Zn and  f o r the enzymes i n  a  reservoir  the high m.w. metabolism present  pool. and  such  may  play  detoxification  in excessive  organics  It  p o s s i b l y of Cu, a  dominant  role  act  in  the  of t r a c e metals when they are  amounts.  The  low  m.w.  pool  contains  as amino a c i d s , n u c l e i c a c i d s , e t c . , which are  known as metal complexing  agents.  Zinc-taurine,  Cu-taurine  and  complexes were separated  from heavy  Cu-betaine  homarine  metal exposed o y s t e r s , Ostrea which no evidence of (Howard and In  animal-like  metallothionein  aigos. i n was  summary,  three  observations  costatum may  indicate  not play as major  the d e t o x i f i c a t i o n of Hg as i n v e r t e b r a t e s ;  1) the  to detect a major absorbance peak i n the medium m.w. the greater|amount of Cu than Zn medium  m.w.  failure p o o l , 2)  high  higher l e v e l s o f Hg o c c u r r i n g i n the  high  p o o l , i t i s p o s s i b l e t h a t the amino may  role  3) the i n c r e a s e of Hg i n the  m.w.  pool  Despite  acids  fold)  a  the  pool.  three  that  in  f r a c t i o n s and,  (about  m.w.  m.w.  found  N i c k l e s s , 1977a; 1977b).  m e t a l l o t h i o n e i n i n S. in  e d u l i s and C r a s s o s t r e a  of  the  be i n v o l v e d i n the d e t o x i f i c a t i o n c f lower  l e v e l s or the a c g u i s i t i o n of  tolerance.  low Hg  78 SUMMARY  The aim o f t h i s study nutrient diatom  uptake during  kinetic short  concentrations  was  of  diatom Skeletonema  or  to  examine  the  responses  of  long-term  exposures  HgCl .  The  2  an  growth  ammonium-limited to  sublethal  cosmopolitan n e r i t i c  costatum, which i s a  and  centric  predominant  species  d u r i n g t h e v e r n a l bloom, was used i n these experiments. To  achieve t h i s g o a l , two major s t e p s were f o l l o w e d .  first,  a  gross  determined  range  of  sublethal  concentrations ,  effects  ammonium-limited  of  cells  short-term  grown  in  mercury  chemostat  exposures cultures  determined by s i m u l t a n e o u s l y adding ammonium and sublethal  c o n c e n t r a t i o n s of HgCl^.  or g r e a t e r than 1.84 nM  rate  (Vi^ax^  a n (  *  effluents  from  starvation  2  the  one  on were  o f the  Only c o n c e n t r a t i o n s equal  decreased f o r the  sensitivity  average period of 30 hours. ammomium  HgCl  affinity  t i i e  attempt t o i n c r e a s e t h e (Hg) ,  was  using batch c u l t u r e s e n r i c h e d with f / 2 5 * medium.  Secondly, the  to  At  to  chemostats  the  assimilation  substrate.  a were  In an  secondary  stress  starved  f o r an  These experiments i n d i c a t e d  than  lowered t h e t h r e s h o l d e f f e c t of HgCl^ t o  0.18 nM. The r e s u l t s o f these  first  two  steps  provided  i n f o r m a t i o n i n the d e s i q n i n q o f the long-term mercury experiment.  In  the  useful exposure  l a t t e r experiment, two ammonium-limited  chemostat c u l t u r e s were exposed t o 0.37 and  3.68  nM  HgCl.,.  79 Mercury  was semi-continuously added at r e g u l a r time i n t e r v a l s  during a period of 30 days. this  experiment  nor  cultures  (cultures  did i t affect  grown  In t h i s experiment, the i m p o s i t i o n on  top  of  populations.  during  of  uptake  Decimation  of  mercury-treated c u l t u r e s r e s u l t e d i n saturation,  in  •f/2*  and  mercury exposure. long-term  mercury  a primary s t r e s s ( n u t r i e n t l i m i t a t i o n ) ,  a f f e c t e d t h e d i f f e r e n t phases o f exposed  in  the n u t r i e n t uptake k i n e t i c s o f  ammonium-limited c u l t u r e s i n t h e short-term  exposure  used  d i d not decrease the maximum growth r a t e s o f  nutrient-saturated •f/25*)  The lowest c o n c e n t r a t i o n  and  the  growth  the p o p u l a t i o n s  conditions  of  of  i n the  nutrient  which minimum c e l l d e n s i t i e s o c c u r r e d .  A  minor growth r a t e d e c l i n e was observed i n the c o n t r o l  culture  but c o n d i t i o n s of n u t r i e n t l i m i t a t i o n were maintained.  Growth  rates  simultaneously  resumed  in  a l l c u l t u r e s implying  mercury a d d i t i o n s d i d not i n t e r f e r e sexual  reproduction  with  the  occurrence  i n mercury-treated c u l t u r e s .  of growth d e c l i n e was f o l l o w e d  by a r e t u r n to  that  a  This new  of  period steady-  state. In the long-term exposure to 0.37 nM HgCl^* a decrease i n the a f f i n i t y maximum were and term  f o r the s u b s t r a t e  (increase i n Ks) occurred.  (Vs) and i n t e r n a l l y c o n t r o l l e d  The  ( V i ^ ^ ) r a t e s o f uptake  not a f f e c t e d i n the long-term exposures to 0.37 nM HgCl^ i n t h e short-term exposure  affinity  to  exposures up t o 1.84 3.68  nM H g C l  2  nM  decreased;  HgC^.  Long-  1) the s u b s t r a t e  (increased Ks) , 2) t h e i n i t i a l r a p i d t r a n s p o r t of the  s u b s t r a t e across  the  cell  membrane  at  high  (5  uM  NH C1) A  80 nutrient  levels,  Vs,  and  3)  the  internally  controlled  assimilatory rate, V i ^ ^ . In g e n e r a l , mercury i n h i b i t i o n on the of ammonium-limited c e l l s appeared t o be During  the  new  steady-state  i n Hg-treated c u l t u r e s , the  f o r the s u b s t r a t e  (Vi  of uptake were i n c r e a s e d i n phase D  to phase A (day 6). chemostat  exposed  k i n e t i c s and probably  changes  reflected  in  of  agents.  The  in  medium  made  of  p r o t e i n may  heavy  Hg  partial  in  the  in  all  cultures  mercury t o l e r a n c e .  to  the  appearance  of  stage.  to determine whether a  metals  by  short-term  s y n t h e s i s of  metal-  through  intracellular  the  complexing  weight pool, where m e t a l l o t h i o n e i n  i n animals exposed in  in  the a  high major  short-term  to  m.w.  heavy  which  recovered therefore  from the  role  in  the  Hg exposure. the  mercury role  d e t o x i f i c a t i o n p r o t e i n could not be  metal  and  high  pool, suggested t h a t t h i s  mercury exposure, the absorbance and  measured and  compared  of  (mercury induced  molecular  not play  detoxification  cells  rate  f a i l u r e t o detect an u l t r a - v i o l e t absorbance peak  u s u a l l y occurs levels  (day 23)  could be r e s p o n s i b l e f o r the recovery  sequestration  the  related  l i f e cycle  was  morphology  acquisition  p h y s i o l o g i c a l adaptation lothionein)  assimilatory  Improvement i n n u t r i e n t  2  cell  the  c e l l s of a d i f f e r e n t attempt  the  nM H g C l .  These changes were p a r t i a l l y  An  and  Recovery appeared t o be t o 3.68  kinetics  uncompetitive.  affinity )  (Ks),  nutrient  of  metabolism  and  In the long-term  metal  profiles  inhibition  were  metallothionein  ascertained.  of not  as  a  81  Concomitantly, HgCl  (0.37  2  to 5.53  d i s t r i b u t i o n of Hg, cultures.  nM  on the i n t r a c e l l u l a r l e v e l s  Zn  was  also  examined  using  as  well  concentrations in  the  Zn  growth  egual  accumulation  rates.  concentration concentration, was  possibly  In  to of  or  higher  Hg  cells  due  a d a p t a t i o n , e.g.,  intracellular to  and  than  i n the high  the  the  1. 84  Cu, Hg. nM  molecular decrease  pre-exposed t o a lower mercury  p r i o r t o the a d d i t i o n of the  and  batch  as the i n c r e a s e of i n t r a c e l l u l a r  weight f r a c t i o n s which could be r e s p o n s i b l e f o r in  of  mercury c o n c e n t r a t i o n s r e s u l t e d i n the  i n c r e a s e of i n t r a c e l l u l a r l e v e l s of  respectively,  resulted  HgC^)  Cu and  Increasing  decrease and  Mercury  the e f f e c t s of s u b l e t h a l c o n c e n t r a t i o n s  a  second  and  Hg l e v e l s decreased.  development  an e x c l u s i o n mechanism.  of  a  higher This  physiological  82 REFERENCES  Andren, A.S. and R.C. 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Weiss: Mercury i n the South p o l a r seas and i n the Northeast P a c i f i c ocean. Mar. Chem. 2, 287-299 (1974) Windom, H.L., F.E. T a y l o r and R. S t i c k n e y : Mercury i n North A t l a n t i c plankton. J . Cons. perm. i n t . E x p i o r . Mer. 35, 18-21 (1973) —,  — and E.M. Waiters: P o s s i b l e i n f l u e n c e of atmospheric t r a n s p o r t on the t o t a l mercury content of south e a s t e r n A t l a n t i c c o n t i n e n t a l s h e l f s u r f a c e waters. Deep-Sea Res. 22, 629-633 (1975)  Winge, D.R., J. Krasno and A.V. Colucci: Cadmium accumulation in rat liver: correlation between bound metal and pathology, 300-301, In: Trace element metabolism i n animals , 2. Ed. W.G. H o e s k s t r a , J.W. S u t t i e , H.E. Ganther and H. Mertz, U n i v e r s i t y Park P r e s s , Baltimore (1973) —  , R. Premakumar and K.7. Bajagolapan: Me t a l - i n d u c e d f o r m a t i o n of m e t a l l o t h i o n e i n i n r a t l i v e r . Arch. Biochem. Biophys. 170, 242-252 (1975)  93 Winner, R.W., T. K e e l i n g , R. Yeager and M.P. F a r r e l l ; E f f e c t of food type on the acute and c h r o n i c t o x i c i t y of copper to Dajghnia majna. Freshwater B i o l . 7, 343-349 (1977) Wisely, B. and R.A.P. B l i c k : M o r t a l i t y of marine invertebrates l a r v a e i n mercury and z i n c s o l u t i o n s . Aust. J . Mar. Freshwater Res. 18, 63-72 (1966) wobeser, G. , N. O. N i e l s e n and R.M. Dunlop: Mercury concentration i n t i s s u e s of f i s h from the Saskatchewan R i v e r . . J . Fish..Res. Bd. Can. 27, 830-834 (1970) Yoshikawa, H.: Comparison of cadmium treatment and phencbarb i t o l treatment f o r the m i t i g a t i o n of acute cadmium t o x i c i t y . Ind. Health 14, 45-46 (1976) Zingmark, R.G. and T .G, M i l l e r : The e f f e c t s of mercury on the photosynthesis and growth o f e s t u a r i n e and oceanic phytoplankton, 45-57. In: P h y s i o l o g i c a l ecology of e s t u a r i n e organisms, Ed. F.T. Vernberg, U n i v e r s i t y of South C a r o l i n a Press, Columbia, S.C., (1973)  94  APPENDIX A.  A r t i f i c i a l Seawater Recipe  APPENDIX B.  Data derived from the long-term mercury exposure of  ammonium-limited j>. costatum t o 0.00, 0.37 and 3.68 nM HgCl . D = d i l u t i o n r a t e (10 • hr using door 3; lenght (um) ;  i  _  "__2"  ); Fluor = i n v i v o fluorescence,  C e l l no = c e l l numbers (10 ''cells • 1  Cell  u = s p e c i f i c growth rate (hr ) ; E f f l . = e f f l u e n t 1  concentration of ammonium (uM); Q = the amount of nitrogen per c e l l (/JM N-10^  cells  ); Chi a_ = the amount of c h l o r o p h y l l a  per c e l l (ug c h l a^«10  7  cells  1  );  '• °665  absorbance r a t i o  =  of carotenold : c h l o r o p h y l l <a; Chla a.* = estimated c h l o r o p h y l l a_ using the c h l o r o p h y l l a : fluoresence r a t i o obtained during phase A;  P./S.«" photosynthetic rate (pg C-10" c e l l s * . h r ~ * ) . 7  2  fluorescence values obtained using door 30. J  II  II  II  II  II  -  ARTIFICIAL SEAWATER RECIFE  SALTS  m.w.  g-94.4 1-1  CONCENTRATION (M)  NaF  0.25  .00265  41.99  6.30 X 10-  H3BO3  2.20  .23305  61.83  3.77 X 10-  KBr  8.20  .08686  119.01  7.30 X ID"*  NaHCOj  16.40  .17373  84.01  2.07 X 10-  KC1  56.70  .60063  74.56  8.06 X 10-  335.60  3.555  142.04  2.50 X I D '  2003.0  21.218  58.44  3.63 X ID"  SrCl -6H 0  2.00  .02118  266.62  7.95 X 10-  CaCl -2H 0  127.20  1.347  147.02  9.17 X 10-  MgCl -6H 0  906.90  9.607  203.31  4.73 X  NaCl 2  2  2  2  2  2  io"  5  3  3  3  2  1  4  3  2  Ref: Keater et a l . , 1967  COMPOSITION OF V  SALTS  NaN0  STOCK SOLUTIONS (g-1-1)  3  Na S10 2  KH P0 2  3  4  T.M. MIX (mg.1-1)  MEDIUM  F  MIX (rag-1-1)  F MEDIUM (M)  150.0  150.0  1.77 X ID"  3  30.0  30.0  1.06 X 10-  4  10.0  10.0  7.35 X  lo'  5  &1SO4  19.6  19.6  0.0196  7.85 X 10-  8  ZnSO^  44.0  44.0  0.044  1.53 X 10-  7  CoCI -6H 0  20.9  20.9  0.029  8.78 X.  lo"  8  MnCl -4Il 0  3.6  3.6  0.0036  1.82 X  lo"  8  Na Mo04-2H 0 2  12.6  12.6  0.012  5.2 X 1 0 "  Ferric Sequestrene  10.0  10,000  10.0  1.0  1.0  .001  1.30  1,300  1.3  2.33 X 10-  5  8.72  8,720  8.72  2.34 X 10-  5  2  2  2  2  B  2  2  12  Fe  2  EDTA  8  2  Ref: G u l l l a r d  and Ryther, 1962; 2: McLachlan, 1973  96  Chemostat Culture I I (Control) DAY  D  FLUOR.  CELL NO.  CELL LENGTH  0  3.60  23.910.9  6.3710.42  15.9019.63  1  4.08  18.3±2.4  5.9010.43  16.2817.74  .035  2  4.09  15.5±0.1  5.8810.10  16.7415.24  .048  0.00  1.70  0.B3  p  EFFL.  Q  CHL.a  D430:D665  CHL. a *  P./S.  1.24  4.58  3. 18  1.02  3.37  3  4.00  17.8±0.3  9.32+0.63  21.45+6.30  .060  0.05  1.07  0.918  2.,14  0.55  2.77  4  4.00  27.010.7  5.2010.59  19.8018.46  .016  0.16  1.89  1.095  2.,56  1.71  2.26  5  4.10  24.3±0.4  5.6110.27  23.3819.39  .044  0.00  1.78  1.459  2,.25  1.43  6  4.36  27.5±1.4  7.2210.45  18.40+6.94  .053  0.00  1.39  1.214  2,.47  1.26  7  4.00  35.3±2.5  8.3910.04  19.99+8.58  .048  1.249  1.98  1.39  e  3.90  28.1±2.5  7.07+1.63  17.13+8.97  .032  1.600  2,.14  1.31 -  9  4.10  24.6±2.4  5.50+0.01  .030  1.48  10  3.97  29.710.6  3.0010.69  .015  3.27  21.8±0.3  3.98+0.54  .052  1.81  12  3.96  21.811.5  3.1410.22  .030  2.29  13  3.96  18.511.9  1.69+0.15  .014  3.61  14  3.80  17.010.7  1.48+0.43  .034  14.111.7  1.26+0.03  11  0.22  0.53  1.36  6.38  3.79  15 16  3.69  17  3.96  20.7+0.8  2.5110.40  16.37+5.51  .069  0.74  3.69  18  3.88  28.811.3  7.6111.05  16.7117.00  .085  0.13  1.30  19  3.92  32.2+0.8  8.6212.01  .044  20  3.92  32.5  9.58+0.30  .044  0.09  1.03  1.12  21  3.83  33.2+0.8  9.9910.11  .041  0.17  0.98  1. 10  22  4.05  38.911.4  14.4410.63  .055  0.06  0.69  0.89  38.010.5  12.36+0.87  .034  0.00  0.61  1.01  40.311.3  12.49+0.00  .040  40.011.3  6.5311.33  39.610.5  23 24 25  3.70  26  12.97+4.78  14.8116.67  2.72 .  1.25 1 .23  1.06  .035  0.00  0.87  2.02  0.43  1.00  1.31  10.0^0.86  .034  27  3.94  37.011.8  8.9311.66  .035  1.37  28  3.90  39 .0  10.7112.05  .047  1 .20  37.311.0  5.1610.97  .009  2.39  29  2'.52  Chemostat Culture I (Exposure to 0.37 nM HgCl ) 7  DAY  D  FLOOR.  CELL NO.  CELL LENGTH  EFFL.  Q  CHL.a  D430:D665  CHL. a*  0  3.70  23.8±0.4  5.90±0.36  15.90+9.63  1  4.06  15.5±1.0  5.80+0.41  16.2817.74  .038  2  4.38  14.0  2.99+0.08  18.53+8.71  .017  0.00  3  4.20  16.3+0.7  5.26+0.30  20.315.66  .066  4  4.10  10.110.9  3.90+0.35  25.38+10.70  .029  5  4.40  15.8+0.5  3.0310.00  18.7+8.51  .032  6  4.09  J9.4+0.9  2.08+0.24  26.07111.96  .027  7  4.15  14.5±1.5  2.6510.05  25.04112.60  .051  8  3.90  11.2+1.6  2.86  32.85113.84  .043  9  4.20  8.1+1.1  1.1310.02  .001  4.30  10  4.29  5.3+0.6  0.71+0.13  .023  4.95  11  4.30  1.810.8  0.81+0.07  .049  2.84  12  4.30  3.3±1.0  0.5610.22  .028  1.40  13  4.30  1.9+0.7  0.2510.14  .062  14  4.20  18.3  0.15  .021  6.27  25.C3  5.50  1.5  2  2.51 0.94  2.41  1.75  5,.17  1.72  1.29  2.55  1.16  4, .19  0.05  3.33  1.34  1.55  2.03  5. 09  0.16  1.87  1.49  2.10  1.81  2. 99  3.60  1.53  1.45  2.38  2.32  3.30  2.03  2.53  3.18  3.34  2 .04  3.20  0.13  4.74  0.93  3.17  5.73  15 16  42.716.8  0.84+0.02  11.37+6.77  10.1±2.5  1.4+0.21  13.3815.08  .062  2.26  5.53  3.13  28.0+0.4  3.29+0.51  18.9219.65  .077  0.15  3.00  3.69  2  17  4.00  18  4.18  19  4.15  29.8±0.8  4.8011.09  .057  20  4.01  32.5+1.8  8.4610.46  .064  0.10  1.17  1.67  21  4.04  32.1+3.0  5.67+0.19  .024  0.15  1.74  2.46  22  3.90  34.8+2.1  8.55+1.03  .057  0.06  1.16  1.77  23  39.1+1.6  8.8710.67  .041  0.00  1.13  1.91  24  41.2+2.2  9.4310.52  .043  40.1+1.1  9.2810.46  37.711.6  8.16+0.79  25  3.60  26  16.47+6.31  2.69  1.90  .038  0.00  0.72  1.88  .033  0.09  1.21  2.01  27  4.13  36.5±2.1  6.0810.34  .026  2.61  28  4.10  34.8±0.3  7.7910.18  .054  1.94  38.5±0.5  4.99+0.59  .040  3.35  29  P ./S.  5. 39  Chemostat Culture'III (Exposure to 3.68 nM HgCl ) 9  DAY  D  0  3.90  1  3.95  2  4.04  3 4  FLUOR.  CELL NO.  CELL LENGTH  23.3  5.5610.44  15.9019.63  5.68  16.28±7.74  .040  22.5+0.3  6.0810.6  16.8017.41  4.00  15.7*0.5  5.5711.19  3.94  11.9+0.3  5  3.90  6  4.09  7  EFFL.  Q  CHL. a_  .043  0.00  1.76  0.71  3. 20  21.3316.60  .037  0.06  1.64  1.75  2. 31  1.65  3..67  2.97+0.27  34.76+10.54  .014  0.16  1.77  1.72  2. 49  1.26  2..02  15.8+10.3  2.4510.12  29.51111.22  .031  4.43  1. 85  1.79  12.7±0.5  1.3210.23  28.2319.70  .014  4.00  6.610.5  1.06+0.46  35.37122.79  .031  8  3.80  4.310.5  0.433  23.0815.60  .003  9  3.90  3.5  0.18  8.0516.12  .001  10  4.06  2.5  11  3.90  15.510.4  12  3.96  3.312.1  13  3.94  17.214.72  14  3.88  7.710.3  D430:D665 CHL. a*  1.87  0.04  7.54  2.35  2. 26  2.88  3.96  1. 88  4.29  3.13  15.87 '  4.92  1. 70  2.78  0. 63  4.43  0.0362 0.00252  2  7.17  2  15 5.7+1.5  16  2  17  3.80  2.811.5  2  18  3.90  5.011.4  2  19  3.92  11.311.2  20  4.02  11.8+0.5  21  3.81  53.310.4  22  3.90  39.5+1.4  23 24 25  3.60  26  0.0167 0.04610.011  .081  2  0.07610.005  .060  3  0.7510.16  9.46  11.84  .135  4.99  6. 70  .103  1.42  3.49  9.23*0.34  .080  0.06  1.C8  1.91  41.511.0  6.00+0.26  .021  0.08  1.65  3.08  39.6+1.4  11.3910.37  .066  39.610.5  4.3010.83  33.411.0  3  3.45+0.59  9.1614.63  22.019.28  1.55  .000  0.00  6.9610.25  .058  0.06  .024  '  2.33  4.11  1.43  2.14  27  3.81  29.510.6  9.7712.89  28  3.90  30.212.5  6.8110.21  1.98  28.310.6  5.0710.67  2.49  29  8,. 10 4,.51  8.67 2  P. /S.  1.35  4. 54  

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