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Toxic effects of selenite and selenate on marine microalgae : a physiological and ultrastructural study Wong, Donald Chun Kit 1990

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TOXIC  E F F E C T S O F S E L E N I T E AND S E L E N A T E ON M A R I N E M I C R O A L G A E . A P H Y S I O L O G I C A L AND U L T R A S T R U C T U R A L S T U D Y . by DONALD CHUN K I T WONG  B.Sc.(Hon.),  The U n i v e r s i t y  A THESIS  SUBMITTED  of  British  Columbia,  IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS  FOR T H E DEGREE O F  MASTER OF S C I E N C E  in THE FACULTY OF GRADUATE (DEPARTMENT  We a c c e p t to  this  the  thesis  Donald  as  conforming  standard  OF B R I T I S H  July (c)  O F BOTANY)  required  THE UNIVERSITY  STUDIES  COLUMBIA  1990  Chun K i t  Wong,  1990  1986  In presenting  this  degree at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department  this or  publication of  and study.  thesis for scholarly by  this  his  or  her  Department of The University of British Columbia Vancouver, Canada  requirements that the  I further agree  purposes  representatives.  may be It  thesis for financial gain shall not  permission.  DE-6 (2/88)  the  for  an  advanced  Library shall make it  that permission for extensive granted  is  by the  understood be  that  allowed without  head  of  my  copying  or  my written  ABSTRACT  Seven  species  different  taxonomic  responses known  to  to  two  be  Selenate severe  marine  proved  different  to  toxicity  phytoplankters  divisions  prevalent  —P  (10  of  in  be  were  tested  molecular  species  seawater,  more  selenite  toxic  than  was  only  observed  at  of  both  selenate  and  for of  to  toxic selenium  and  selenate.  selenite,  although  high  concentrations  —T  and  10  M)  concentrations,  growth  was  in  tested.  In  most  viable, swimming  species both  the  speed  completely some o f  percentage  were  the  of  drastically  under  Dunaliella  tertiolecta  cells  possessed  to  the  lacked  flagella  alterations  both  carterae.  showed,  adaptation  frequently  after  to  concentrations  high of  even  that  for  while  growth  and  selenium  stimulatory meaningful  were to  their  electron  circumstances, flagella  became  non-motile  these  striking  morphology, tertiolecta  cells and  exposures,  generally  growth.  signs  on  of  Lower  non-toxic  These  of  Pavlova  concentrations.  inferences  ii  and  much s h o r t e r that  prolonged  remained  Scanning  Despite  Dunaliella  selenium  cells  these  inhibited  that  these  those  altogether.  in  Amphidinium lutheri  controls,  species  reduced.  that,  At  severely  motile  revealed  compared  selenite.  or  microscopy  suggest  assigned  and  observations  selenium  toxicity  Abstract  both  the  must  be  concentration considered  selenium  main  of  involved the  were  the  microalgae  even  in  shortage  growth. and  suggesting play  a  major  microalgae  the  with  in  to  toxic  the  to  high  selenite  or  rates.  produced  later  upon  among  suggested  that  affected  by  decreases  or  major  reductions  exposure  of  of  cell  surface  of  and  indicative  loss  concentrations  nitrogen  were  the  detoxification  well  changes  variability  which  and  as  Other  significant  in  selenate  chloroplasts  severely  products  changes  lutheri  greater  to  and  studies  Pavlova  vacuoles,  were  energy  shedding  role  with  led  occurred  coincided  that  physiological  showed  storage in  These  and  lipids,  systems  the  adaptation  The major a l t e r a t i o n s  These  of  of  consideration.  photosynthetic  these  studied.  toxicity.  for  mitochondria,  nucleus,  but  elimination  toxicant  and  transducing  severe  into  treated  the  contents,  selenium  the  in  length  tertiolecta,  coat,  respiratory  energy  of  cell  the  potential  taken  carterae  the  and  ultrastructural  observed  carbon  the  Dunaliella  Amphidinium  as  and  concentrations  The cells  range  cell  and  coat  to  the  material,  material  might  adaptation  selenium.  of  T A B L E OF C O N T E N T S Abstract  i i  List  of  Tables  List  of  Figures  Acknowledgement  . . . .v vi .  x  Introduction Materials  1  and Methods  .  8  Results I. Effects of selenite and s e l e n a t e on t h e growth m o t i l i t y of seven species of marine microalgae II. Effects of selenite and selenate toxicity on ultrastrucutre and physiology of three species marine microalgae  12 and 12 the of . 18  Discussion I. Effects of selenite and s e l e n a t e on t h e growth m o t i l i t y of seven species of marine microalgae II. Effects of selenite and selenate toxicity on ultrastructure and physiology of three species marine microalgae  25 and 25 the of 32  Conclusions  and Proposed  Future Research  References.  40 82  Appendix I. Composition of Selenium added s e p a r a t e l y at  iv  microalgae growth medium. various concentrations. ..99  LIST Table  I.  Species  of  OF T A B L E S  microalgae  used  in  the  present  study..42  Table II. Growth of microalgae in selenite. a, adaptation period (days) ; b, exponential growth rate (d ; c, m a x i m u m y i e l d (OD ggo n m ) / ' g r o w t h ; NM, v a l u e s too low or inadequate for measurement with confidence. Values in brackets represent the percentages with respect to the control 43 N  G  n  o  Table III. Growth of microalgae i n s e l e n a t e . a, adaptation period (days) ; b, exponential growth rate (d - -) ; c, m a x i m u m y i e l d (OD ggo ) ; NG, no g r o w t h ; NM, v a l u e s too low or inadequate for measurement with confidence. Values in brackets represent the percentages with respect to the control 44 -  n  1  m  Table I V . Mean (x) diameter (nm) and l e n g t h (jzm) of the f l a g e l l a of Dunaliella t e r t i o l e c t a , and P a v l o v a l u t h e r i , and o f t h e haptonema o f P a v l o v a l u t h e r i grown w i t h and without selenite, p l u s m e a n (x) diameter and l e n g t h of the flagella (/xm) of Amphidinium c a r t e r a e , grown with a n d w i t h o u t s e l e n a t e . O b s e r v a t i o n s made w i t h a T E M , SEM and/or Light microscope. Corresponding standard errors o f m e a n s (sem) shown 45 T a b l e V . M e a n (x) a n d s t a n d a r d physiological activities of w i t h and without s e l e n i t e  error (±sem) o f t h e means o f Dunaliella tertiolecta grown 46  Table VI. Mean (x) and standard error (±sem) of the absorption by different pigments from two in vivo a b s o r p t i o n s p e c t r a r e p l i c a t e measurements of Dunaliella t e r t i o l e c t a grown w i t h and w i t h o u t s e l e n i t e . 47 T a b l e V I I . M e a n (x) and s t a n d a r d e r r o r (±sem) of the of physiological activities of Pavlova lutheri w i t h and without s e l e n i t e  means grown 48  T a b l e V I I I . M e a n (x) a n d s t a n d a r d e r r o r ( ± s e m ) of the means of physiological activities of log phase Amphidinium c a r t e r a e grown w i t h and w i t h o u t s e l e n a t e 49  V  LIST Fig.  OF FIGURES  1. Growth curves of Pavlova l u t h e r i exposed to 10 M (A) a n d 1 0 ~ M (•) s e l e n i t e , and c o n t r o l w i t h o u t selenium addition (o) . V e r t i c a l b a r s i n d i c a t e t h e s t a n d a r d e r r o r o f m e a n . n=3 50 3  4  Fig.  2. Growth curves 10~ M s e l e n i t e (•) (o). V e r t i c a l bars n=3 4  Fig.  of D u n a l i e l l a t e r t i o l e c t a exposed to and c o n t r o l w i t h o u t s e l e n i u m addition indicate the standard e r r o r of mean. 52  3. Growth curve of Amphidinium carterae in 10~ M selenate without (•) and w i t h (•) previous adaptation, and in 10 M selenate with prior adaptation to 10 M selenate (A) • Corresponding control growth without selenium addition is also shown (o). Vertical abrs i n d i c a t e t h t e s t a n d a r d e r r o r o f mean 54 3  - 2  Fig.  3  4. Percentage and swimming s p e e d o f m o t i l e cells in cultures of Dunaliella tertiolecta and P a v l o v a lutheri grown w i t h 10 M selenite (A) and Amphidinium c a r t e r a e adapted to grow _ i n 1 0 M s e l e n a t e (B)Motility symbols: control (•) , 10~ M s e l e n i t e (S) , 10 _ M s e l e n a t e (S) . S w i m m i n g s p e e d s y m b o l s : c o n t r o l (H) , 10 M s e l e n i t e (0) , 10~ M selenate (0) . V e r t i c a l b a r s i n d i c a t e t h e standard e r r o r o f mean. D u n a l i e l l a t e r t i o l e c t a : 9 days, n = 644. 17 d a y s , n = 419. 30 d a y s , n = 5 5 7 . Pavlova l u t h e r i : 18 days, n = 447. 29 d a y s , n = 5 5 3 . A m p h i d i n i u m c a r t e r a e : n = 164 56 4  - 3  4  4  3  Figs. 5 a n d 6. Scanning electron micrograph of Dunaliella tertiolecta g r o w n w i t h 1 0 M s e l e n i t e f o r 17 d a y s (Fig. 5) and 30 days (Fig. 6) displaying conspicuously short flagella. F i g . 5 b a r = 1/xm ( 2 0 , 0 0 0 X ) . F i g . 6 , b a r = 1 /xm (25,000X) 58 - 4  Fig.  7. Scanning electron micrograph of tertiolecta grown w i t h o u t s e l e n i u m a d d i t i o n (similar for 30 d a y s ) . N o t e long f l a g e l l a . (11,000X)  Fig.  8. Scanning electron micrograph of Dunaliella tertiolecta grown with 10 M selenite for 17 days ( s i m i l a r f o r 30 d a y s ) . N o t e t h e a b s e n c e o f f l a g e l l a . Bar = 1 /xm ( 2 6 , 0 0 0 X ) 58 4  vi  Dunaliella f o r 17 days B a r = 2 jum 58  List  of  Figures  Figs. 9 and 10. Mitochondria of Dunaliella tertiolecta g r o w n w i t h ( F i g . 9) a n d w i t h o u t ( F i g . 10) 10 M selenite for 17 days. S e l e n i u m grown c e l l s possess rather long m i t o c h o n d r i a . The c r i s t a e d i s p l a y u n u s u a l configurations (arrows) and t h e m a t r i x i s more d e n s e . Figs. 9, bar = 0 . 5 /xm ( 4 0 , 0 0 0 X ) . F i g . 1 0 , b a r = 0 . 5 fin ( 6 6 , 0 0 0 X ) 60 4  Figs. 11 a n d 1 2 . Dunaliella tertiolecta grown w i t h (Fig. 11) and without ( F i g . 12) 1 0 ~ M s e l e n i t e f o r 17 days. N o t e t h e s m a l l e r amount o f s t a r c h ( F i g . 11a c o m p a r e w i t h Fig. 12a, Sth), and s m a l l e r p y r e n o i d (Fig. lib compare w i t h F i g . 12b, Pyr) i n s e l e n i t e grown c e l l s . These cells are also largely devoid of lipid inclusions (Fig. 11a compare with Fig. 12a, Lip) . Fig. 11a, bar = 1 /m (19,000X). F i g . l i b , b a r = 1.5 /urn ( 1 5 , 0 0 0 X ) . Fig. 12a, bar = l / i i (17,000X). Fig. 12 b, bar = 1.5 /xm (12,000X) 62 4  F i g s . 13 t o 1 5 . D u n a l i e l l a t e r t i o l e c t a grown w i t h ( F i g s . 13 and 14) and without (Fig. 15) 10~ M s e l e n i t e for 30 days. Large vacuoles (Vac), lack of lipid inclusions (Lip), smaller chloroplasts (Chi), pyrenoid (Pyr) and l e s s s t a r c h (Sth) c h a r a c t e r i z e the s e l e n i u m grown cells. Fig. 13, b a r = 1.5 /xm ( 1 5 , 0 0 0 X ) . F i g . 14, b a r = 1 . 5 /xm ( 1 3 , 0 0 0 X ) . F i g . 1 5 , b a r = 2 . 0 /nm ( 1 1 , 0 0 0 X ) 64 4  Figs. 16 a n d 1 7 . C e l l coat of D u n a l i e l l a t e r t i o l e c t a grown w i t h 1 0 M s e l e n i t e f o r 17 d a y s , r e v e a l e d b y a l c i a n b l u e staining. Note the less dense cell coat of selenium grown (Fig. 16) compared t o control cells (Fig. 17) . F i g s . 16 a n 1 7 , b a r = 0 . 3 /xm ( 7 1 , 0 0 0 X ) 66 - 4  Figs. 18 a n d 1 9 . C e l l coat of D u n a l i e l l a t e r t i o l e c t a grown w i t h 10 M s e l e n i t e f o r 30 d a y s , r e v e a l e d b y a l c i a n b l u e staining. Note the absence of cell coat in selenium grown (Fig. 18) compared t o control cells (Fig. 19) . F i g s . 18 a n d 1 9 , b a r = 0 . 3 /xm ( 7 1 , 0 0 0 X ) 66 Figs. 20 a n d 2 1 . P a v l o v a l u t h e r i grown w i t h ( F i g . 20) and without ( F i g . 21) 1 0 ~ M s e l e n i t e f o r 18 d a y s . Note the small size of both chloroplasts and mitochondria in s e l e n i u m grown compared t o c o n t r o l c e l l s . F i g . 20, b a r = 0 . 7 /xm ( 2 8 , 0 0 0 X ) . F i g . 2 1 , b a r = 0 . 7 /xm ( 3 0 , 0 0 0 X ) 68 4  Figs. 22 (Fig. days,  and 23. 22) and revealed  C e l l c o a t o f P a v l o v a l u t h e r i grown w i t h without (Fig. 23) 10 ^ M s e l e n i t e for 18 by a l c i a n b l u e s t a i n i n g . The c e l l c o a t of  List  of  Figures  s e l e n i u m grown c e l l s i s l e s s dense t h a n t h a t cells. F i g s . 22 a n d 2 3 , b a r = 0 . 3 /nm ( 6 0 , 0 0 0 X )  of  control 70  Figs. 24 a n d 2 5 . C e l l c o a t o f P a v l o v a l u t h e r i grown w i t h (Fig. 24) and w i t h o u t ( F i g . 25) 10~ M s e l e n i t e for 29 days, revealed by alcian blue staining. Note the thinness of this coat in selenium compared to the control cell. Figs. 24 and 25, bar = 0.3 /xm (60,000X) 70 4  Figs. 26 a n d 2 7 . Amphidinium c a r t e r a e at log phase adapted to grow with (Fig. 26) and w i t h o u t (Fig. 27) 10 M selenate. Note the differences in the nucleus (Nuc), vacuoles (Vac) of selenium grown compared t o control cells. F i g . 2 6 , b a r = 2 . 5 /xm ( 7 , 6 0 0 X ) 72 - 3  F i g s . 28 a n d 2 9 . C h l o r o p l a s t s o f A m p h i d i n i u m c a r t e r a e a t log p h a s e a d a p t e d t o grow w i t h ( F i g . 28) and w i t h o u t (Fig. 29) 10 M selenate. Note the vesiculation of the t h y l a k o i d s i n s e l e n i u m grown compared t o c o n t r o l cells. Fig. 28, b a r = 0.2 /xm ( 8 1 , 0 0 0 X ) . F i g . 29, b a r = 0 . 5 /xm (41,000X) . .72 Fig. 30. Cell size, expressed as number o f . p o i n t s over cross section, of Dunaliella tertiolecta and Pavlova lutheri after growth f o r v a r i o u s p e r i o d s of time with 10 M selenite, and Amphidinium c a r t e r a e at log phase a d a p t e d t o grow w i t h 1 0 ~ M s e l e n a t e (0). Corresponding control growth without selenium addition is also shown (•). V e r t i c a l b a r s i n d i c a t e t h e s t a n d a r d e r r o r o f mean. Dunaliella tertiolecta: 9 d a y s , n = 5 0 . 17 d a y s , n = 7 2 . 30 d a y s , n = 49. P a v l o v a l u t h e r i : 18 d a y s , n = 51. 29 d a y s , n = 5 2 . A m p h i d i n i u m c a r t e r a e : n = 49 74 4  3  Fig. 31. Mean volume density of cell components of Dunaliella tertiolecta a f t e r growth f o r v a r i o u s periods of t i m e w i t h 10 M s e l e n i t e (Q) . C o r r e s p o n d i n g c o n t r o l growth without selenium addition is also shown (•). Vertical bars indicate the standard error of mean. Nucleus (Nuc) and cytoplasm (Cyt) volume d e n s i t i e s are expressed per cell volume. Chloroplast (Chi), Mitochondria (Mit), Golgi (Gig), vacuoles (Vac), and lipid inclusions (Lip) volume densities are expressed p e r c y t o p l a s m i c volume. P y r e n o i d (Pyr) and s t a r c h (Sth) volume d e n s i t i e s are expressed per chloroplast volume. D u n a l i e l l a t e r t i o l e c t a : 9 d a y s , n = 5 0 . 17 d a y s , n = 7 2 . 30 d a y s , n = 49 76 4  List  of  Figures  Fig.  32. Mean volume d e n s i t y o f c e l l components o f P a v l o v a lutheri after growth f o r v a r i o u s p e r i o d s of time with 10~ M s e l e n i t e , and Amphidinium c a r t e r a e , at log phase, adapted to grow w i t h 1 0 M s e l e n a t e (0) . C o r r e s p o n d i n g control growth without selenium addition is also shown (•) . V e r t i c a l b a r s i n d i c a t e t h e s t a n d a r d e r r o r o f m e a n . Nucleus (Nuc), cytoplasm (Cyt) and p e r i p h e r a l cisternae (PCs) expressed per cell volume. Chloroplast (Chi), Mitochondria (Mit), Golgi (Gig), vacuoles (Vac), storage vesicles (StV), projectile bodies (PjB) and storage products starch and lipids (StP) expressed per cytoplasmic volume. Plastoglobuli (Pig) and pyrenoid (Pyr) expressed per c h l o r o p l a s t volume. P a v l o v a l u t h e r i : 18 d a y s , n = 5 1 . 29 d a y s , n = 5 2 . A m p h i d i n i u m c a r t e r a e : n = 49 78 4  - J  Fig. 33. In vivo absorption spectrum of Dunaliella tertiolecta grown w i t h 1 0 M s e l e n i t e (a,b) or without selenium a d d i t i o n (c,d) f o r 17 d a y s . N o t e t h e difference i n t h e s h a p e o f t h e a b s o r p t i o n c u r v e s b e t w e e n 5 0 0 nm a n d 6 0 0 n m . n=4 80 - 4  ix  ACKNOWLEDGEMENT  I Dr.  would  Luis  of  willingness  of  lab  Dr.  F.  Weis  for  J .  for  Hanh,  his  R.  the  also  are  his  for  up also  his  due  marvelous for  for  grad  students  to  Brian,  physiological  lab  Laurie  technical  Mr. Peter  of  helpful  the  help,  Dr.  to  this  to -  Thana  Bisalputra and  to  Dr.  algal  the  Klut,  Lucy  and m o t i v a t i o n his  component  this  this  several P.  to  Dr.  and  Samuels,  -  for  to  finish  study.  to  Mr. M.  (former  technical  J .  physiology  investigation,  a l l  Dr.  the  of  suggestions,  Thompson f o r  x  toward  research,  and  of  supervisor,  encouragement  support,  assistance, in  ideas,  introduction  his  my  supervision  and  a component  to  guidance  the  directions  technical  and t o  but  for  ultrastructural  his  Randy,  my g r a t i t u d e  only  pick  to  Taylor  companionship,  with  me  facilities  present)  work,  to  research  Harrison and  not  Thanks  introducing  years  express  my w o r k ,  investigation. for  to  Oliveira,  completion and  like  their this  assistance  INTRODUCTION  Selenium present  in  the  (Robberecht high  is  heart  to  of  not  Polo  Early  cattle loss  disease".  to  the  be  the  present. animals  horses  was  the feeds  at  his of  plants  of  from  that  mane  other  a  similar  genus  hair  and  loss,  (Hewitt  and  poisoning  a  by  and  was  areas.  fatal  soreness t a i l ,  fatal  was  described  -  found  of  of  disease the  disease,  "blind  continent.  discovered  of  to  be  found  the  elements  administered  were  due  was  (Franke and P a i n t e r  Astragalus  feet  "alkali  the  search  by  called  Selenium  selenate  1  liver  (Giessel-Nielsen  parts  symptoms  plant  Asia  systematic or  failure,  low  to  disease  affected  at  all)  causes  at  -  another  at  toxicant  not  animals  M  time  characterized the  (if  selenium  North America  selenite  the  of  a  1983) 10  element  selenium  case  ° M to  10  trace  in  (Wilbur  both  many  journeys  alkali  from the  produced  and  of  is  essential  oldest  from  1930s,  Sodium  It  blindness  reported  toxicant,  range  respiratory  Concurrently,  staggers", In  of  settlers  hair  the  animals  and  The  one  and of  an  identified  in  metalloid  concentrations,  joints  although  or  1982).  paralysis,  1963).  1976).  and  both  At high  Nicholas  Marco  in  and Van G r i e k e n  atrophy,  erosion  The  water  concentrations  organisms.  and  minor-metal  sea  concentrations  in  a  found  to  to  lab  1935). to  be  Introduction  associated selenium  with was  also  accumulators selenium  "blind  staggers".  found  (Underwood  from  amounts.  They  livestock  especially.  staggers"  due  These  plants  which  would  available 1962)  to  were  (Wilbur  1983).  also  able  These  livestock amounts  determine  the  toxic  levels  mix  Selenium  may  chlorosis  also  in  selenium  non-accumulators  are  without show  for  with  inhibit  in  able  to  (Underwood the  form  inorganic so  economically performed  and  are  to and  selenium-  low  growth  accumulate  of  forms  organisms  from  toxic  soil,  readily  selenium and  (Hurd—Karrer  showing  chlorosis  the  cropping  plant  that  1971).  were  plants  to  (Walker  in  were  avoid  great  forms  different  either  2  and  studies  leaves  Selenium—accumulators of  them  of  to  "blind  to  usually  problems  Se-  caused  selenium  organic  as  selenium  non-accumulators,  of  large  or  retained  demonstrated  poisoning  selenium,  that  areas.  of  unavailable,  important  accumulators  amounts  convert  plants,  Farmers  and  forms  subsequently  to  be  mixture  counter-measures.  such  selenium  inorganic  a  absorbed  of  selenium-  Se-methylselenocystine,  was  acute  other  or  called  selenium-accumulators  otherwise  to  in  toxic  It  of  containing  selenate,  and  concentrations  plants  They  soil  presented  ingestion  these  1962).  the  methylselenomethionine  the  in  High  cause 1935).  large  amounts  effects  while  inhibited  in  growth  Introduction  (Hurd-Karrer such  as  193 5 ) .  vegetables  potentially  Many  groups  Selenium  selenium,  It trace fed  to  in  interactions  been  observed  (Hewitt was  not  amounts  of  as  a  liver  necrosis,  but  yeast  prevented  this  sufficient  cystine  antiliver  necrosis  Different variety  of  amounts natural  in  between  the  the  sole  human  1963,  and  and  a  Kramer before  vitamin  sulfhydryl  for  protein E or  animals.  Rats  developed  American  vitamin  for  protection.  products.  3  did  designated was  Concentrates  1988). that  latter  activity  cellular  found  The  was  uptake  Inorganic  a n d Ames was  fatal  brewer's  not  contain  This  third  "Factor  found  have  to  1962).  sulfate  condition. E  They  selenium  potent  it  of  groups.  table.  Wan 1 9 9 0 ) .  also  of  sulfhydryl  (Underwood  essential  source  3  daily  generation  permits  selenium  1950s  Factor  are  proposed,  of  periodic  This  is  are  that  been  and  with  (Mikkelsen  factor  of  have  macromolecules  cystine,  or  exceed  removal  reacting  selenium  yeast  far  processes,  selenite,  until  concentrations  sulfur,  and N i c h o l a s  non—accumulators  1989).  properties.  many  by  toxicity  sulfur  especially  Torula  for  up  and  a n d Meek  when  similar  sulfur  also  oxidant  animals  oxidative  next  many  reach  selenium  species  is  Synergistic have  in  oxygen  replace  of  taken  also  substitution  acting  exhibit  to  (Banuelous  modes  including  can  harmful  requirements  active  Selenium,  a  in  a  3". wide  garlic-like  Introduction  odour,  suggesting  selenite same  was  the  able  fashion  as  Chicks  fed  to  the  diathesis  mortality.  Symptoms  and  prevented found,  this  effective  was  and  losses  in  sheep,  (Underwood  necrosis,  failure  (World  growth  diatoms  are  amounts  of  amounts  necrosis  Torula poor  edema  on  A substance  was  the  in  and poor  in  of the  pancreatic  breast,  wings  to  to  cause  in  in  and  was  be  an that  lambs,  postnatal some  muscular  degeneration  3  found  pigs,  fertility  and also  Factor  myopathies in  high  yeast  observed  dietetrica  Deficiencies  and  investigations  and  developed  growth  similar  treating  growth  yeast  Brewer's  subsequently  widespread  areas  dystrophy, reproductive  H e a l t h O r g a n i z a t i o n 1987) .  Many p h y t o p l a n k t e r s for  of  with  hepatosis  1962).  Small  liver  hemorrhage.  effective  foals,  diet  included  More  selenium.  against  along  disease.  cure.  calves  liver  same  selenite  of  3.  subcutaneous  and  selenium  protect  Factor  exudative  neck,  presence  (Harrison elongated vesicular  have et  al.  with and  been  found t o . r e q u i r e  1988) .  altered  lipid  Selenium  membrane  inclusions  selenium deficient  systems,  (Doucette  large et  al.  1987) . The research.  role It  anti-oxidative  of is  selenium clear  that  processes  in  in it  trace is  animals.  4  amounts required Selenium  is  s t i l l  for  one  is  a  under of  the  co-factor  Introduction  of  the  enzyme  destruction plants would  glutathione  of  and  peroxidases  to  possibly  the  current  Recently, Kesterson  high  from  caused  the  area  the  dispersed  al.  from from  the  volcanos  tend  to  Sedimentary selenium. favour by  The  the  plants  Selenium  rocks  and is  Rainfall,  is  also pH  bioavailability 1987)  also  of  selenate  readily  and of  effects and  from  along  soil  to  the  (World  waters,  animals  in  1987,  sulfur, washes  is this  around  (Wilbur  1983).  concentrations  is  the  Soils  from  these  greatly  leached  composition  selenium  with  earth.  and  in  California,  on  quickly  which  coal  aging  Ohlendorf  derived  dissolved  found  irrigation  high  soils  This  and  in  selenium  contain  of  passed  and  the  in  1987),  by  Health  rocks  absorbed rainfall.  surrounding a l l  of  affect  soil. the  Organization  . The  lead,  rich  alkalinity  formation  Refuge  toxic  to  the  1988).  selenium  Rainfall  atmosphere be  of  Selenium,  al.  carcinogens  Presser  volcanos.  et  in  1983).  Life  of  1985,  1987).  selenium  some  foothills  concerns  acts  Harrison  (Wilbur  Wild  coastal  serious  et  of  (Combs  and  concentrations  (Marshall  Ohlendorf  (Price  effects  and  which  animals  theories  Reservoir  have  in  phytoplankters  counter-act  according  peroxidase  mining,  zinc,  milling,  phosphate,  smelting uranium,  5  and and  refining selenium  of  copper,  itself  a l l  Introduction  cause  emissions  Atmospheric these  activities.  such  components,  is  found  products sewage,  in  1979;  Lemly  grown  on  amounts  of  1985a,  precipitation  plants  generate  amended  with  from  the  (Cutter selenium  most  sources  contribute  oceanic  has  emission  surface  increases  and in  Church  small  control  significantly waters,  waters  (Hamilton  and  half  the  pollution  by  nickel 25%  air  mining while  (Wilbur  coal  Buhl  the  1983).  6  and  secondary  Furr  that  et  al.  vegetables  absorbs  selenium  large  power  or  vapours  which  1983) .  These  in  comes  content  Coal-fired  selenium  1990).  selenium  combustion  end  (Wilbur  particularly  coastal of  1986).  particles  to  incinerator  1983).  the  equipment  for has  sludge  (Wilbur  Health  Selenium  1976;  shown  sewage soil  1983).  al.  been  electronics  selenium  primary  et  may Other  (World  of  i n garbage  (Brown  It  glass,  source  by  effluents  situation  raw,  raised  selenium.  of  (Wilbur  1989),  combustion  escapes  same  Quebec  ash  b) .  with  largest  al.  fly  fuel  the  concentrations  selenium  Fossil  The  et  coal  soil  of  create  environment.  industrial  manufacturing  Noranda,  (Ericzon and  and  the  significantly  the  as  high  are  directly  1987). in  into  water  and dyes  Organization  levels  Mining  the  industries  industry  selenium  selenium  contaminate  been  of  content  estuarine In  Canada,  from  contributes  to  copper less  of and over and  than  Introduction  Short have  term  been  studies  performed  (Wheeler  et  studies  emphasizing  possibility  al.  of  the  longer  term  taxonomic  selenate, of  forms and  selenium  Dunaliella carterae,  not  marine  the  to  of  days)  handful et  al.  to  (up  to  selenium  in  tertiolecta, to  three  2  study,  months)  species  7  also  examined  water,  adaptation to the  two  most  selenite  and  taxonomic  marine f  and  of  different  physiological  lutheri  the  have  using  of  term  selenium  belonging  sea  long  of  and  toxicity, in  but  we  toxicity  microalgae  However,  concentrations  this  Pavlova  different  marine  1987).  microalgae  selenium  selenium  toxicity  high In  of of  u l t r a s t r u c t u r a l and  toxicity  assigned  15  only  missing.  divisions  prevalent  Price  effects  of  to a  adaptation  conspicuously  species  on  1982,  are  seven  (0  aspects  microalgae, Amphidinium  divisions.  Materials  Stock  cultures  Table  I  Cheng  (1975)  jLiE«m  - 1  •s  aliquots  a  (4  ml)  species  previously  I).  Growth  culture  algae  tests  were  sodium  final  by A n t i a  illumination  to  0.2  in  (95-100 Small  (Na SeC>3)  or  aliquots  of  2  ml  concentrations  in and  out  lamps.  selenite  added  listed  carried  fluorescent  were  at  of  described  continuous  medium w i t h  (Na2Se04),  stock  seven  cool-white  of  selenate  Methods  1 8 ° C under  from  —0 10  at  )  species  as  (Appendix  tubes  sodium  the  were m a i n t a i n e d  culture - 2  of  and  ranging  from  —7 to  10  M. A s e p t i c  experiment  to  monitored  ensure  three  sterility  times  each  density  at  600  periods  of  incubation  delayed  algal  nm  techniques of  week  after  the by  brief  (over  adaptation  were  as  used  throughout  cultures.  Growth  measurement  of  vortex-mixing.  60  days)  suggested  were  the was  optical Extended  allowed  for  by  Stockner  and  Antia  for  swimming  speed  (1976). Culture motility  screen  cells  were  system were  were v i d e o  determinations  Photographs on  samples  of in  one  measured  and the  from  second  subsequent on  distances  taped  Zeiss  duration  play a  a  backs.  Kontron  measured  calculated. 8  as  were The image  and  photomicroscope. taken  of  tracks  the of  analysis  /Ltm t r a v e l l e d  cells  a l l  the  computer per  second  Materials  For  scanning  concentrated 1%  (v/v)  by g e n t l e  OSO4  in  saline,  pH  series,  critical  in  a  7.4.  0.1M  (w/v)  4°C  with  OSO4  a  in  7.4.  0.1M  dehydrated  point  dried  and g o l d  embedded on  EM10A  a  (w/v)  a  OMU-3  acetate  added  to  to  stain  al.  and  the  1980).  electron lead  fixative  the  cell  to  coat  of  organelles  micrographs  enlarged  21,000X  for  Pavlova  lutheri  and  carterae,  for  following  the  9  and  were 4  h  1%  filtration and  after  the  out  Zeiss  for by  of  with blue 0.5%  (Oliveira  densities in  Dunaliella  described  a  were  Alcian  cells  volume  13,000X  finally  staining  1963).  pH for  sections on  at  (v/v)  saline,  examined  of  carried  procedure  for  concentration  the  individual  32,000X  was  fixed  Ultrathin  final  surface  Calculation  samples  series,  (Reynolds  a  ethanol  microscope.  by  and  buffered  electron  buffered  microscope  citrate  with  2 h  observation  and  methanol  microtome  for  for  glutaraldehyde  1961).  were  graded  microscopy,  concentrated  (Luft  a  coated  cacodylate  graded  P o l y b e d 812  Reichert  in  scanning  (v/v)  sodium then  transmission  uranyl was  in  in  2%  cells  cacodylate  centrifugation  of  (w/v)  250T  electron  gentle  were  dehydration  sodium  were  mixture  They  cut  by  and f i x e d  Cells  transmission  concentrated  microscopy,  centrifugation  Cambridge Stereoscan For  et  electron  and Methods  of  electron  tertiolecta. Amphidinium Steer  (1981).  Materials  The  Student  different  t  test  parameters  Cell Counter  s  1  counts Model  for  For  immersion  in  10  min,  reading  added  model  and  these  the  taken  a  will  and  O2  extraction  using  or  2 h  cultures  photosynthesis measurements. net  0  procedures  Approximately  values  in  vitro  for  be  A  few  cell  on  a  10%  a  were  grinding  of  a  for  Turner  HC1  were  (phaeopigment) be  of  are  noted  the  that  amount because  of of  sufficient  for  here.  in by  darkness  before  Klut  Photosynthesis as  et  al. was  after  10  2  the  evolved  for  (1984) . given  respiration,  and r e s p i r a t i o n a r e jumole 0  1 8 ° C as  respectively,  preconditioning  right  at  (adjusted  darkness  measuring  measured  measured  illumination  described  and expressed  Coulter  immediately  by  should  they  a  phaeophytin  (phaeopigment)  though  consumption  of  on  followed  phaeophytin  a  of  suspensions  an u n d e r e s t i m a t e  under  2  and  drops  It  means  conditions.  fluorescence  delay.  time,  the  measured  and r e s p i r a t i o n were  evolution  respiration)  were  2 ml of  phaeophytin  comparison purposes  net  growth  homogenization  without  Photosynthesis  the  of  readings  measurements  short  acetone,  Methods  compare  chlorophyll a  fluorometer.  sample  chlorophyll the  90%  10  concentration  to  Whatman G F / F f i l t e r s ,  by  Designs  volumes  determinations,  through  and  used  different  and c e l l TAIL  (phaeopigment) filtered  was  and  or  to  with  respiration reported consumed  as per  Materials  minute, nitrogen and  respectively. samples  analyzed  absorption on  a  diluted  on  DU-62  effects  samples,  positioned  a  against  collected  Carlo  spectrum  Beckman  scattering  were  Particulate  of  on  Erba  13  a the  short  by  the  sample  holding  cell  carbon  mm G e l m a n A / E analyzer.  tertiolecta  spectrophotometer. produced  Methods  organic  CHN  Dunaliella  and  An  was  path were  and  filters in  vivo  determined  To decrease microalgal  and  the  light  suspensions, a  detector  utilized.  i,  11  RESULTS  J.  EFFECTS  MOTILITY  OF SELENITE  OF.SEVEN  Selenite  SPECIES  was  concentrations concentration 10~ M.  AND SELENATE  OF MARINE MICROALGAE  used  ranging range  The  in  from  employed  for  concentration  ranges  tested  selenium  related  concentrations  and  concentrations. crystals  in  10  M.  on  growth  selenite  Initial were  or  Acrmenellum  and  at  f  for  10  - 2  M  with  was  these  from  two  selenate  the  the  10~ M 2  at  of  higher  at  lower  production  this  to the  species  activity  caused  at  on  problems  biological  tests  10  - 5  oculata. Dunaliella  at  M for  10~ M 3  minimal Growth  occurred  of  only  significant below  10  effects - 7  M  for  selenate. inhibited  partially  inhibition  was the 12  the  inhibited  Isochrvsis  upon the  where  no  concentrations  vixvisibilis.  effect  tertiolecta  that  completely  carterae,  Chaetoceros  showed  showed  quadruplicatum,  Amphidinium  lutheri  while  while  M,  - 7  discrepancies  solubility  at  observed  below  Selenite  of  Selenite  10  selenate  the  of  medium  .  at  X  lack  solution,  —i  to  growth to  3  for  are  the 10~ M  reasons  5  ON THE GROWTH AND  of  of  growth Pavlova  tertiolecta,  Nannochloropsis  particularly maximum  the  cralbana,  Dunaliella growth  growth  yield  severe  in  represented  Results  only  24%  also  of  the  control.  observed  lutheri  with  controls,  Chaetoceros  maximum  (Isochrysis  pronounced. well  exponential  growth  only  noticeable  rate  of  10  - 3  the  rate  increased  from  2  affected The growth shown on  by  in  higher  Table  was  both  the  well  below  exponential  much  of  - 4  the  to  and  the  rate  growth  marine  exponential  control in  in  (Fig.  the  tested.  with  The  was  only  the  significantly II).  microalgae inhibitory (Fig.  on is  growth  effects  1).  rate  13  the also  Growth  tertiolecta,  Nannochloropsis  only  that  with values  Inhibition  Aqmenellum q u a d r u p l i c a t u m .  of  a  oculata  selenite  2) .  The  period  (Table  Dunaliella  was  values  oculata  also  the  other  yield  lutheri  of  the  carterae)  adaptation  pronounced in  of  Nannochloropsis  M selenite,  severe  of  control.  p e r i o d was  of  57%  species  the  were  Pavlova  observed  concentrations  less  occurred  growth  of  and  maximum  concentrations  yield  those longer  10  in  Pavlova  species  At  s t i l l  maximum  no  lower  II.  the  days.  selenite  seven  were  inhibition  18  on  adaptation  of  these  growth  growth  whose  effect  of  to  the  Amphidinium  similar  of  duration  growth  and  Nannochloropsis  had  the  species  is  selenite  involved  other  most  growth  42  reduction  for  exception  M  and  of upon  reduction  with  in  vixvisibilis  effect  qalbana  exponential  reductions  values  The  The  correlates  effect  yield  respectively.  species less  in  Major  oculata  of The was  Results  actually  stimulated  18  at  days  10~ M  Instead,  to  3  inhibition  and 2  occurred in  the days  at  Pavlova  lutheri,  was  particularly  exponential  growth  172%  121%  and  at  lower  Isochrysis  and  adaptation  rate  No  4  in  was  Pavlova  controls  of  of  selenite.  vixvisibilis, observed.  were  at  from  signs  lutheri  maximum y i e l d  the  major  Chaetoceros  stimulation  and  dropped  concentrations  pronounced  of  10~ M.  galbana,  growth  period  This  where  the  increased  10~ M  to  selenite,  7  respectively. Selenate all  species  at  of the  the  were  selenate  selenite  at  10  J  28  also  to  Growth were  only  of  after  respectively.  14  these  maximum  accounted  for  44%  and  Equally after the  noticeable  a  rather  completely  than  that  that  inhibited  at  adaptation Growth  of  of and of this  quadruplicatum inhibited.  was  effect  oculata  and  55%  lengthy  inhibitory  pronounced  partially  lengthy  of  and  Agmenellum  only  Under  Nannochloropsis  severely  growth  rate  more  were  of  lutheri.  Overall, be  the  growth  occurred  M . The growth  galbana  days  and  days).  carterae  occurred  Pavlova  respectively. only  (27  inhibited  exponential  vixvisibilis  concentration.  and  completely  affected  growth  proved  Amphidinium  growth  its  values,  period  Chaetoceros  Isochrysis  M  except  severely  that  adaptation of  both  control  fact  - 2  tested,  circumstances, yield  10  and  However,  periods  of  18  Agmenellum  Results  quadruplicatum inhibition observed 10~ M  10~ M  in  with  Pavlova it  showed  growth  selenate growth that  is  is  lasts  20%  for  of  4  the  to  60  reaching  42%  of  maximal  yield  inoculum 10  M and  growth 61% with  (P<0.05  taken 10  J  of  the  into  Concentrations  to  140%  In  this  of  the  pronounced rate  was  with  157%  this  of  No  case,  major  control 10~ M  signs  an  total was  inhibited of  at  inhibition  by  a  to  in and  rate  control  15  45%  of An  ones  at  instance, 51%  yield  and  compared were  M  selenate  (Fig.  3).  and  including  10~ M,  had  - 2  this when  (Table  growth  cells  exponential  selenate  new  reached  when  is  control.  last  and  yield  the  growth,  and  it  of  This  and  the  the  3  of  rate  initiate  that  below,  of  10~ M  maximum  stagnates.  cases)  10  a  in  period  resumption  occurred  on  to  growth  delay  with  carterae  initial  leads  growth  growth  the  the  situation  severely  growth  showed  effects  the  and  culture  selenate  5  After  both  medium  of  significant  lutheri.  in  exponential  control.  while  exponential  without  similar  Amphidinium  later  M selenate  maximum  inoculated  no  from  occurred  of  days  days  with  III).  control,  50  A  minimal  unusual.  followed  maximum  only  inhibition  rather  that  that  (Table  3  contrasts  selenite.  lutheri  10" M selenate The  selenate  3  observed  selenite,  3  with  in  except growth  effect the III).  4  for rate  was  Pavlova increased  s t i l l  exponential  more growth  Results  It that of  was  most  evident  of  the  selenium  instead  of  water/air  from  microalgae  settled  being  at  as  the  speed  flagellar  and  the  Pavlova  Amphidinium were  In  was  significantly  was  the  (P<0.05) went  (Fig.  from  days), cells  into  dropped  and  dropped  from  recovered  back  of  cells  motile  control days. days  after  Their then  percentage  to  recover  the  the  (9  (30  38.7%  with  controls  days)  days),  of  the  of  Pavlova then speed  the  of  also  selenate  motile  cells and  remained as  the  culture  percentage  of  of  to  8.5%  the to  the to  (17  motile to  6.4%  motile  47.4%  so  motile  phase  cells  but  then  percentage  21.6%  of  the  back  to  30.95%  after  29  dropped  to  72.1%  after  18  to  89.3%  of  the  motile  cells  in  Amphidinium  16  and  stationary  decreased  recover  tertiolecta  3  Similarly,  lutheri  these  swimming  10~ M  that  control  4).  the  (P<0.05),  control speed  the  (Fig.  to  of  the  selenite,  4  cells  swimming  89.8%  swimming  10~ M  vessel  near  view  cells,  percentage  those  phase  days,  In  Dunaliella tertiolecta,  80.8%  of  18  of  of  culture  Dunaliella  grow  the  than  decline from  respectively, also  In  exponential  and  cases,  speed  4).  motile  to  cultures  concentrations  the  were.  of  the  concentrated  with  adapted  lower  swimming  of  grown  all  or  of  high  of  controls  structure  lutheri  examined.  bottom  percentage  carterae  with  distributed the  observations,  inspection  grown  the  evenly  interface  and  visual  control  by  day  29.  carterae,  The after  Results  adaptation control;  to  growth  while  the  in  —3 M selenate,  10  swimming  speed  was  12.4%  of  the  to  73.9%  of  the  that  in  dropped  control. Light  and  Dunaliella major  had  tertiolecta  changes  compared  to  differences  (P<0.05)  to  the  were the  also  of  controls  after  17  to  days  diameter  and  selenite,  and  selenate  were  observed  in  major both all  the three  of in  Amphidinium the  the  changes  the  (P<0.05) length  same  as  haptonema were  flagella  of 5  10  the  and  lutheri  carterae  adapted  controls.  haptonema)  species.  17  the  flagella  cells  examined  with  dropped  No  internal and  flagella  from  10  - 4  M  4%  flagella in  grow  10~ M 4  in  10~ M  difference (Table  IV).  structure basal  in  selenite  The  grown to  7) .  statistically  lacking  lutheri  shaft  Fig.  length  8).  no  the  increased  (Fig.  Pavlova  selenite,  are  Pavlova  in  6  grown w i t h  exposure  of  M  of  Cells  numbers  the  - 4  flagellar  IV).  cells  observed (and  mean  their  in  52%  length  (Table  and  41.3%  the  in  Figs.  flagella and  revealed  diameter  However,  control  observed  grown  the  (compare  in  significant 59.4%  in  control.  flagella  microscopy  cultures  occurred  the  shorter  The  electron  body  3  was No of of  Results  II.  EFFECTS  OF SELENITE  ULTRASTRUCUTRE  AND SELENATE  TOXICITY  ON THE  AND PHYSIOLOGY OF THREE SPECIES  OF MARINE  MICROALGAE  The and  effects  Pavlova  carterae was  Dunaliella  After  17  with  10) .  Fig. and  12a)  grown  Fig.  obvious  cells  (compare  the  the  after  9  Figs.  also  with in  30  to  14  grown  day  12b, old  be  were  with  tended  and  present were Fig.  18  15).  Fig. to  These when  the  10  - 4  M  longer also  in  the  inclusions. 11a  be  with  smaller  changes the  were  selenite  mitochondria.  depleted in  of  arrows,  reduction  branched  smaller  9,  lipid  cultures  in had  Fig.  cut  Pyr) .  long  There  cristae  (compare  region  level.  cells The  clear  starch  Fig.  Amphidinium  growth  (compare  a  inclusions and  of  cytoplasmic less  vacuoles  13  days  tertiolecta  ultrastructure  matrix.  pyrenoid  tended  lipid  gross  selenite  was  on  ultrastructural  in  displayed  Large  cytoplasmic  at  of  the lib  3  configurations  There  and  10~ M  denser  possessed  Chloroplasts deposits.  selenate  a  number  Chloroplasts  more  and  days  with  unusual  even  on D u n a l i e l l a  difference  displayed  (compare  M selenite  examined  mitochondria  Fig.  - 4  tertiolecta  selenite.  size  10  lutheri,  were  little  of  of  starch  cytoplasm  conspicuously  and  absent  Results  Dunaliella possesses wall  a  to  day  old  that  17  more  fibrous  The  to  material cells  cell  seemed  (compare  stroma.  numerous  Fig.  21). less  thickness changes is  of  were  in  the  selenate.  In  osmiophilic  in  cell  of  the and  coat  no  (compare  with  the  Amphidinium nucleus,  the  in  10  coat  except  the  more  grown  M  selenite,  - 4  and  smaller  (compare  in  had  a  less  and  more  20  with  Fig.  selenite  the  grown  (compare  for  sharp  decrease  major  ultrastructural  a  other Fig.  24  with  changes carterae  Fig.  cells  cells  22  with  in  the  Fig.  25) .  This  observed  in  the  cells  chromosomal  perichromatic  19  became  19).  control  control  17).  selenite  also  cell  Fig.  from  were  the  manner  coat  mitochondria  observed  ultrastructure  Fig.  with  a  cell  smaller  days,  contrast  altogether  revealed  displayed  when  were  than 29  days)  cell using  compact  16  lutheri  Furthermore, the  After  less  Fig.  (30  a  material  morphology  growth  compared t o  dense  23).  of  a  of  that  studies  grown c e l l s  in  coat  with  Pavlova The  when  18  instead  coat  (compare  missing  days  of  cell  cultures  be  Fig.  18  chloroplasts dense  to  protoplast  Cytochemical  arranged  cell  older  cover  selenite  cultures  in  natural  cell  the of  coat  control  a  1980).  cultures  in  After  al.  visualize  difference  pronounced  was  et  blue  compared  is  glycocalyx-like  (Oliveira  Alcian  Fig.  tertiolecta  granules  grown  material more  in  10~ M  was  evident  3  less in  Results  selenium large  treated  and  Fig.  26  contained  with  producing  Fig.  large  chloroplasts used or  for  could  coat  be  of  the  30  the  control  grown  in  Cells  of  29  days  selenite Pavlova  were  also  size  .  was  with  unable  selenium  than  to a  10  no  The  no  in  instance, aging  30  (P<0.10 no  selenium  adapted  significant  changes  to in  grow cell  in size  of 17  P<0.30 when  while  4  however,  or  days  reduction  control  9  and  size  size  Cells  for  10~ M s e l e n i t e  in  cell  study.  those  (P<0.05). for  18  and  P<0.30  decrease  treated  . carterae  cells.  on t h e  after  significant  amphiesma  comparisons  (P<0.15  in  within  methodology  the  M selenite  increase  grown  observed with  - 4  under  reversed  l a r g e r than the this  selenium  controls  was  tended  lutheri  in  the  process  29).  Therefore,  of  dilated  appearance  preserve  microalgae  grown  showed  Fig.  were  (compare  grossly  grown and c o n t r o l  effect  of  became  to  vacuoles  inclusions  . .  Amphidinium showed  In  of  The  vesiculated  microalga.  shows t h e  cells  respectively). cell  this  The  28  was  species  respectively).  a  Fig.  studies  larger  cells.  amounts  with  tertiolecta  were  control  The t h y l a k o i d s  areas  of  three  Dunaliella days  27).  made b e t w e e n  Figure  in large  (compare  these  cell  than  and  in  cells.  —T 10  M  selenate  compared  to  the  control. Morphometric the  analysis,  major organelles  of  expressed  as  volume  Dunaliella tertiolecta 20  density,  was  of  conducted  Results  at  9,  31). the  17  and  Major  30  volume  (P<0.001). there  73%  (Mit)  volume  (P<0.005). (P<0.001)  less  the  The  vacuolar  and the  volume  difference control  in  and  the  the  though  there  starch  granules  volume  was  density  There lutheri  observed  in  significant. organelles  no  not  in  18  10  19%  nearly  days were  27%  organelles  further  mitochondria  than  the  control  decreased (Lip)  (Pyr)  decreased  between  the  volume the  43%  significant  selenite  many (Fig.  in  after  The  volume  (P<0.005).  proved to in  changes 32) .  the  smaller  changes  detected  also,  the  (P<0.10), density  of  chloroplast  (P<0.001).  as  was  and  Overall,  smaller the  Overall,  76%  (P<0.005),  M  - 4  in  4  other  were  in  (P<0.001). by  days,  pyrenoid  when  (P<0.005).  statistically  10~ M s e l e n i t e  that  No  of  days  P<0.001).  inclusions  decrease  decreased  after  chloroplasts  was  (Fig.  decreased  compartment  lipid  17  volume  smaller  larger  of  86%  (Sth)  were  grown  observed  a  after  (Lip)  30  (Vac)  grown  selenite  (P<0.001)  72%  volume  cells  13%  M  - 4  in  (Sth;  After  s t i l l  There  73%  90%  starch  control.  (P<0.001).  10  observed  were  became  was  to  inclusions  (Pyr)  (Chi)  to  only  increased  lipid  Pyrenoids  chloroplasts  exposure  were  (Mit)  of  was  compared  of  differences  mitochondria  The  99%  days  be  Pavlova  only  change  density  days  density of  of  Differences  statistically  volume 29  in  of  growth  not most in  Results  selenite,  although  (P<0.20), control.  and With  selenate, was  changes include  than  the  P<0.15),  increase;  p<0.20).  Table  and  V  phaeophytin  a  content  in is  was  Respiration  and was  mitochondria. was  protoplast  days,  per  also  has  is  cell,  observed per  tertiolecta  fim  dramatically for  less no  cell  the  control.  when  per  cultures  22  the  cell  of  or  V) .  grown  The with  p e r jim per  under the  expressed colour 4  3  control. per  10~ M  /*m  these  difference  were  The  pigment  or  expressed  values  with  the  70-75%  (Table  grown  chlorophyll a  significant  when  (10%  less  reduced  when  (17%  P<0.20),  protoplast  (expressed  Other  chloroplast  than  per  was  nucleus  tertiolecta.  20-30%  M  significant  the  and  10  32).  not  the  analysis  increase;  observed  accounted 43%  in  (VIn)  (Fig.  of  (19%  (phaeopigment)  However,  or  volume  Dunaliella  Photosynthesis  circumstances  Dunaliella  17  expressed  chloroplast)  /tim3  the  smaller than  grow  inclusions  (P<0.050)  P<0.55),  pigmentation  chloroplast.  respiration  that  to  statistically  in  16%  (P<0.25)  adapted  mitochondria  shows for  decrease  be  increase;  selenite  larger  with  controls to  remained  found by m o r p h o m e t r i c  vacuoles  modification  (50%  4  the  proved  pyrenoid  10~ M  of  21%  carterae  difference  volume  that  increase;  mitochondria  sole  the  greater  chloroplasts  Amphidinium  the  that  140%  the  the  of  /xm in per the  selenite  Results  changes  from  green  exponential Dunaliella change nm  phase.  in  the 33).  There  was  chlorophyll nm  enough  in  the  increased  b.  increase  (430-440  in  nm)  of  but  and  they  in  spectra  Chlorophyll  not  a  and  Pavlova  in  ratio  of  is  not  in  studies  not  the  nm the  detected  a  chlorophyll  b  at  cell  were  the  shift  ratio 680  size  of  nm  was  measured  contents  were  significant. conducted  However,  because  with  was  with  and  carterae.  taken  There  The  statistically  Amphidinium  p i g m e n t a t i o n were  at  coincides  430-440  (Table  34% i n c r e a s e  of  a.  600  peaks,  increase  ratio  a  to  carbon and n i t r o g e n  were  were  the  is  carotene  significant.  chlorophyll  physiological and  this  of  nm a n d the  of  increase  that  Changes  Counter  an  However,  (P<0.001).  lutheri  in  also  39%  absorption  40-50%  and a  at  Similar  culture  nm ( P < 0 . 0 1 0 ) ,  the  there  changed  ratio  absorption  studied  Pavlova  a  in  of  also  of  spectrum  500  some  the  was  end  that  between  pigments,  statistically  small  the  shows  between  a  Coulter  also  There  absorption  chlorophyll  by  620-630  be  time  increase  at  absorption  spectrum  ratio  32%  to  chlorophyll  this  the  chlorophyll  corresponding to  a  at  to  of  vivo  different  (P<0.050).  carotene large  a  in  at  The to  reddish/brown  The  shape  corresponding  680  a  tertiolecta  (Fig.  VI) .  to  no  selenium  in  with vivo  changes  in  treatments.  phaeophytin a  (phaeopigment)  decreased  by  lutheri,  photosynthesis  decreased  by  while  23  Results  30-50% /xm  3  when  expressed  chloroplast.  respiration decrease  per  T h e r e was  expressed  was  /xm  contents  of  50% 3  a  carterae  decrease  chlorophyll  a  of  when  /xm  basis  chloroplast  decreased  by  conspicously per  /xm  J  expressed  per  mitochondria. (P<0.001), decrease little  though  in  the  variation  Counter.  The  protoplast  cells  (5  adapted  to  to  on  11%)  per  a  /xm  3  there  is  no  decreased  of 18%  of the  when  carbon  and  decreased  expressed  50  carbon size nitrogen  per  24  and  4 0%  /xm  protoplast  3  photosynthetic but  this  expressed by  30  to  the  cell  per  decreased  by  cell and  rate was or  /xm  3  6%  significant  protoplast  measured  or  4 0% w h e n  or  cell  /xm3  value  per  statistically  per  in  (phaeopigment)  per  per  selenate  3  a  The  (P<0.050)  respectively.  10~ M  protoplast  carbon  amount  The  in  increased  of  cell  15-17%  when  cell,  when  amount  in  amount  also  grow  VIII) .  Respiration  of  per  However,  of  were  chloroplast,  3  amount  the  or  (Table VII) .  (Table  cell, The  order  approximately  /xm  in  protoplast.  3  expressed  smaller  protoplast.  the  phaeophytin  3 5% p e r  protoplast  3  respectively,  and  concentrations, 3  40%  protoplast  Amphidinium showed  and  the  /xm  mitochondria.  3  per  nitrogen  /xm  /xm in  or  per  reduction  27%  and  cell  or  a  smaller  per  cell  per  per  expressed  approximately  cell,  the and  13%  due  to  Coulter per  /xm  3  (P<0.050),  Discussion  I. EFFECTS MOTILITY  is  evident  microalgae  however,  emphasize  nutrient  sufficient  consistent selenite  almost  with at  the  fern  Morrison  study  or  in  and  al. et  al.  1979;  is  usually  of  of  Wheeler  observed  cells  grown  under  our  findings  1987) .  yeast  show  eliminate  This  is  in  observed  (Brown and  several as  that  as  in  (Arenholt-Bindsley  that growth  contrast at  lower  Smith  agronomic  well  are  1979),  species  of  a  number  et  al.  of  1988;  1988). growth of other  et  in  must,  not  toxicity  1989) ,  cells  total  al.  1989),  al.  several  those  et  does  in  One  3  in  several  inhibition  (10~ M).  authors  it  that  growth  Overall  other but  selenite  and Medina  observed  of  level  et  occurs  conditions.  cultured  Severe  this  (Price  of  (Carlson  mammalian  levels  3  (Elmore  plants  saturated  10~ M reduces  concentrations  results  partial  those  high  present  only  that  phytoplankters  with  the  exhibit  at  ON THE GROWTH AND  OF MARINE MICROALGAE  from  selenite  of  AND SELENATE  OF SEVEN SPECIES  It marine  OF SELENITE  al. only  the  inhibition  phytoplankters  studies 1982; at  (toxicity)  (Sielicki  Gotsis  rather  25  1982).  high  tested 1973;  is  also  in  our  Moede  However,  concentrations  et  this of  Discussion  selenite.  Despite  a  resistance  higher  types,  a  this  clear  distinction  For  luridum  (Sielicki  et  the  al.  al.  chlorophycean  1979). lower  showed  minimal  is  variety al.  of  or  Sielicki is  saturation  to  survive,  inhibition.  A l l  in  et  more  level  and  in  other  to  Nannochloropsis  on  organisms  in  (10~ M), it  showed  species  by  other  4  of  observed (Moede  (10~ M  et cell  or  lower)  phytoplankters.  observations (Gotsis  using  1982;  were  in  Pavlova  Price  and 10  - 3  26  selenate,  was  strong at  with  a et  (Price  For  to  able  high  results et be  al. more  instance,  vixvisibilis while  At  growth  this  the  proved  selenite.  Chaetoceros M  of  studies  phytoplankters  selenite.  lutheri  inhibited  agreement  than  than  signs  phytoplanktonic  oculata  with  seawater  only  2  selenate  was  1973).  soluble  is  be  selenite  5  dimorphus  contrast  other  cannot  10~ M  situation  growth  for  cyanobacterium  at  selenite  with  al.  marine  inhibited  of  effect  other This  Overall,  completely  no  . although  concentration.  sensitive  similar  cell  allows  them  inhibited  display  other  that  marine  Scenedesmus  phytoplanktonic  its  1987).  alga  consistent  Selenate  obtained  a  to  than  between  the  concentrations  also  1987;  and  Nonetheless,  types,  This  levels  severely  1973)  toxicity  threshold  instance, was  phytoplankters  selenium  toxicity  Phoridium  in  to  of  concentration  of  established.  tendency  selenite  were at  Discussion  this At  concentration  showed  10~ M selenate,  sharp  4  were  required  and  Anabaena  similar  also  (Sarma  and  and  variabilis  to  a l l  (1982).  to  be  toxic  phytoplanktonic  The  reasons  known.  However,  uptake  of  absorbed  this  the  greater  may  by  the  (Ulrich  and  Shrift  1968).  Tweedie  that  filamentous  fungi,  selenate  in  up  by  by  a  the  cells  different  mechanisms sulfate  in  conditioning phytoplankters  via  transport  were  ions  part  is  true  the  same  to  be  (Wheeler  et  al.  selenium  27  Segel  1982;  toxicity  by not  plants  (1970)  selenite  Selenium  Shrift  showed  were but  taken also  transport  affected 1954).  medium (Kumar  is  Peterson  terrestrial  differentially  sulfate-containing  against  and  transporter,  mechanism.  shown  in  and  is  selenite  (Butler  and  al.  differential  Indeed,  for  et  exhibited  to  duckweed  1989)  by W h e e l e r  sensitivity  species.  were  (Bennett  related  the  desmids  concentrations  tested  a  selenate  several  selenite  while  and  of  to  in  nidulans  regarding  compared be  amounts  to  period  1971)  studies  pyrenoidosa  higher  these  reverse  Prakash  lower  growth.  Anacystis  concentration  species  selenate  selenium in  for  on  adaptation  our  Even  Chlorella  the  in  inhibitory  1984).  to  to  This  impact  the  and  in  severely  Jayaraman  phytoplankters  1967),  (Kumar  occurred  be  in  resumption  quadruplicatum.  proved  significant  increases  growth  situation  Aqmenellum  found  for  no  by Pre-  protects and  Prakash  Discussion  1971;  Wheeler  et  seawater  medium  cells  to  the  also  tempted  to  lutheri  effects  of  in by  of  selenate  In growth react such Ray  of  activity  the  the is  1973).  and  Padmaja et  it  to  inhibitory  of  in  of the  line,  selenium  is  inhibited  the  phosphate  influence  species  is  of  the  selenium.  toxicity  causing  ions  strongly  sulfhydryl-enzymes  Krebs  cycle  (Ray  and  succinic  dehydrogenase  rate.  Lactate  dehydrogenase  to  such  a  condition  Selenite  plant  cells also  (Sielicki acts  photophosphorylation of  selenium  as  (Pick on  of  inhibits  chlorophyll concentration  Selenate  28  from  Selenium  1990).  actions  of  of  of  response  higher  far  selenium  the  One  similar  also  and i n h i b i t  decreases  of  may  activity  1989).  a  Thus,  different  al.  and  al.  inhibitor The  1979).  response  influence  Along  understood.  in  et  a direct  the  tolerance  phosphate  metabolic  (Nebbia  higher  of  of  selenium.  form  seawater  increased  the  of  not  dehydrogenase  aerobic  the  in  element  al.  not  role  cells.  mechanism  is  phytoplankters  transfer  the  et  The d i m i n i s h e d  photosynthesis  1973;  in  s u l f h y d r y l groups  succinic  hypoxia  an  phytoplankters  with  reduces  may b e  these  the  inhibition  1975).  both  table,  content  species  that  phosphorus,  general,  as  upon  sulfate  crucial  selenate  in  The  molecular  (Moede  concentration  a  speculate  to  periodic  response  play  sulfate  uptake the  may  1982) .  different  Pavlova  the  al.  an and the  et  in al.  energy Avron energy  Discussion  producing strong  mechanisms  growth  microalgae of  H2O2  and  These  higher  to  are  also  extensive membranes  (Csallany  and  cell, and  are  also  microalgae selenium damage  the  Selenium and  the  lutheri this  internal  structure  showed  change  the  producing systems  no  percentage  in  a  of  these  flagella motile  in  organelles.  length, cells  29  the  but  in  decreased  the  and  motile  of the  cells Pavlova  tertiolecta.  length  the  of  II).  tertiolecta.  in  changes  effects beyond  Dunaliella  decrease  observable  in  In  the  of  Hewitt  species  Part  number  Dunaliella  carterae.  by  of  the  of  structural  extend  (see  of  permeability  the  inhibition  accompanied without  of  growth  reduces  balance  membrane  some  to  perodixation  1990,  on  speed  flagella  al.  confirming that  and Amphidinium  is  et  study,  toxicity  swimming  redox  our  energy  1988) .  particularly  membrane  and  (eg.  concentrations  the  (Nebbia  Ames  lipid  in  reaction  species  and  cells,  the  observed the  oxygen  Higher  in  with  (Kramer  mechanisms,  detectable in  active  disturb  functions  toxicity  to  to  Along  increased  1986).  explains  inhibition  the  Organelle  are used  to  through  transport  1963).  alterations  damage  known  membrane  Nicholas  total  produced  Menken  affecting  other  and/or  certainly  concentrations.  biological  selenium  cells  s u l f h y d r y l groups,  02")  cause  plant  reduction  at  selenium  of  of  diameter  Pavlova both over  the or  lutheri  microalgae, time.  The  Discussion  decrease the  in  the  presence  that  of  selenium  effects  the  compounds  of  sulfhydryl  The  by  groups  are  activities  synthetic  rate  in  the  polymerase  activity  will  is  dynamic their  integrity  1987). it  is  balance  This being  One  ability  of  in  seems  adversely  of and  carterae to  in  point studies to  cells  occur  in  our  30  and  RNA  and  RNA  other  depend to  and  further  of  in  synthesis.  flagella  on  a  maintain Nadezhdina  attention  and  taken  into  laboratory. must  on  selenium over  This  cultures  and  protein  that  treated  selenite  A decrease  (Vorobjeo  adapt  selenium.  some  exchange  deserves  pursued  However,  continuously  functionality  future  of  for  subunit  ascertain  inhibition  affect  of  selenium-  DNA  1987).  to  that  protein  microalgae  also  due  structures  actively  concentrations  it  cell  al.  forms  to  of  inhibit  et  suggest  these  subject.  in  toxicity,  different  difficult  to  relevant  interesting  Amphidinium  known  results  for  the  observed  selenium  interaction  problem c e r t a i n l y  consideration  more  (Frenkel  particularly  cytoskeleton-based  of with  on  the  polymerase  This  are  not  our  reason(s)  studies  formed  was  Therefore,  associated  effects  lack  cells  effects  occur.  dependent  to  motile  general  also  species  of  selenate.  besides  specialized  due  number  is with  of  be  toxicity time  to  clearly 10~ M 3  is  the  higher seen  in  selenate  and  Dunaliella  tertiolecta  Discussion  and  Pavlova  lutheri  adaptation  was  the  of  cells  exposed  to  only  - 4  to  al.  at of  of  1987;  selenium  et  Price  Rotruck  bacteria  (Combs  et  the  This  1973;  al.  1987;  growth  1986;  other  no  effect  is  in  are  that  or  selenium in  may  al.  Stadtman  and  1982; and 1980).  proved  with  the  as  an  (Harrison (Combs  Morris  Stewart Foltz  be  Lower  behaves  animals  of  severe  even  microalgae  1988),  of  s t i l l  species  agreement  Levander et  speed  continuously  results  to  cases,  phytoplankters.  Schwartz  31  swimming  concentrations  that  Levander  al.  these  some  showed  both  cultures  selenium for  In  possibility  Harrison  Combs  1981; et  for  in  extended  authors  and  and  al.  be  growth.  element  Combs  to  least  to  older  Although  high  other  trace  1988;  Levander 1978;  at  selenite.  recovery  in  emphasize  stimulatory  essential  al.  need  inhibition  observations  et  in  temporary,  be  species  they  concentrations  with  by t h e  M selenite.  and  microalgae, growth  detected both  10  preliminary  treated  et  1984; et  1957)  al. and  Discussion  II.  EFFECTS  OF SELENITE  ULTRASTRUCTURE  AND SELENATE  TOXICITY  ON THE  AND PHYSIOLOGY OF THREE SPECIES  OF MARINE  MICROALGAE  The  main  observed  in  ultrastructural  cells  concentrations well  as  changes coat, showed  involve  the  observed  size,  greater  and  An  increase  in  lower the  cell  of  is  decrease  This  nucleus, and  length  of  the  coincides  17  However,  compartment  no  different This  in  The  from  adaptation  respiration  the  18%  increase  is  the  by  to of  in  these  Dunaliella in  10  - 4  by  M an  unusual  cristae  cultures  with  overall  grown  also  observed  32  old  volume  cells  volume  concentration  and  cell  tested.  growth  increased per  but  accompanied  day  since  strategy  per  is  as  Other  vacuoles,  in  of  matrix,  in  rates.  organisms  days  selenate  chloroplasts  lipids,  affected  30  in  and  changes  and  carbon content,  most  r e s p i r a t i o n expressed  selenium  lutheri.  the  and  density  mitochondrial  addition.  high  17  respiratory rate.  amount  selenite  photosynthetic  nitrogen  increase  the  arrangement.  high  mitochondria and  were  after  selenite.  in  physiological  v a r i a b i l i t y among t h e  Mitochondria tertiolecta  with  the  respiratory  were  cell  treated  and  73%,  volume the  protoplast without  compensate  of  total or  per  selenium for  mitochondria observed  a  in  mitochondrial  due  the to  Pavlova volume  Discussion  lessened as  the  15% w h e n  contrast, 10  - 3  M  respiratory  per  cell  or  changes  per  carterae  respiration  observable  rate  f r o m 27% t o  volume was  in  the  low  protoplast.  adapted  increased  as  to  In  grow  approximately  ultrastructure  or  in 35%  volume  mitochondria. mitochondria  Prorocentrum micans  (10~  to  3  10~ M) and  selenite,  yeast  mitochondria  with  J  in  M)  in  the  the  The  caused  swelling  of  the  1980).  was  inhibited  and  swelling  the  After  When  albidus  cristae  6  appeared was  recorded  hours  incubation  with  10~ M 2  enlarged  electron  opaque  et  cells  growth  in  and  5  (10~ M)  ppm as  as  cells cell  al.  selenite.  1988) .  —  loss  (Browne DNA  No  may b e  1  cristae  a  mitochondria This  also  selenite  6  well  to  2mg-l - -  the  mitochondrial  isolated  33  ppm  cohnii  in  and n e r v e  muscle  selenite,  in  and  epithelial  (Morrison  grown  swell,  mitochondria  10~ M  50-100  exhibited  after  tadpoles,  and  the  observed  U l t r a s t r u c t u r a l damage  Daphnia  mammary  respiration after  1986).  mitochondria In  at  were  Crypthecodinium  epithelial  epithelial  by  in  1979).  muscles,  disorganized.  Dumont  Smith  invertebrate  selenite.  in  not  shortened  become  cristae  but  cristae  selenite  Cryptococcus  (Brown and  mitochondria occurs  dilated  Soyer-Gobillard  the  inclusions  with  grown w i t h  range,  4  (Prevot  (10  the  Amphidinium  selenate,  Giant in  in  expressed  after  without of  drop  of and  synthesis inclusions change up due  to to  in 10 the  Discussion  short  incubation  observed  in  (Ignesti  pig  et  the  with  selenium,  of  but  organelle  selenium and  not  it  Nonetheless,  it  inhibition  in  the  mitochondrial in  signs likely  the  of to  require  inhibition suggested  inhibitory  that  selenium  is  on  It  (1966, a  the  many  major  material considers  states,  fact,  to  34  be  due  rather  than  type  of  transitions  has  by  also  groups  the  without more  selenium  to  of  observed  this  dehydrogenating  1985).  cause  paralleled  sulfhydryl  al.  are  of  It  et  synthesis  alterations  new  observed  al.  increases  phosphorylation.  proposed  et  mitochondria  these  density  synthesis  protein is  of  documented  protein  the  In  the  well  manifestation  removes  in  (Smith  is  the  1968)  range  chloramphenicol  Lewko  here  of  phosphorylation.  effect  This  that  respiratory  oxidative  with  was  selenite  previously  that  of  synthesis  of  oxidative  volume  changes  between  1979;  fxK  the  synthesis  Indeed,  suggest  Hackenbrock  ultrastructural occurring  and  been  1972).  tested  alterations. size  in  occurs  unlikely  species  swelling  inhibition.  urease.  is  10-100  cytoplasmic  Cruickshank  respiration  The i n c r e a s e  protein  inhibits  (Gruenwedel  is  our work.  of  with  This  Smith-Johannsen and Gibbs  that  for  in  inhibition  mitochondria  1984) .  used  in  inhibition  both  since  m i t o c h o n d r i a l m a t r i x has  growth  1970;  heart  al.  concentrations of  time,  the been  necessary exerts  enzymes removal  an and of  Discussion  sulfhydryl  groups  (Underwood rates  1977).  This  exhibited  selenium.  after  by  These  increase  in  to  when  (Klut  by  showed  association  with  phaeophytin  a  in  selenite.  10  - 4  M  extraction the  actual  grown w i t h exhibited in  both  will  the  authors  This  is  showing  a  respiratory presence  conflict  interesting  to  an  of  with  Amphidinium  higher  the  carterae note  that  increase  in  concentrations  other  in  min  in  the and  will  of  organelle  Dunaliella  reduction  of  amounts  the  pyrenoid  and  volume  the  amount with  to  an  and  volume  chlorophyll when  and  underestimate this  of the  starch.  rate energy  a,  grown  should the  of  of  cultures  The  also  decrease  photosynthesis  available  observations is  a  be  Dunaliella tertiolecta less  in  chlorophyll  between  photophosphorylation  35  of  short  purposes  selenium.  tertiolecta  photosynthesis  lead  severely  chloroplast  c h l o r o p h y l l a,  consistent that  the  displayed  to  Both  chloroplast reduce  in  in  is  the  comparison  smaller  certainly  cells.  10  and without a  It  Although  amount for  be  also  decreases  of  to  observed  (phaeopigment)  time  sufficient  seem  is  selenium.  lutheri  in  lower  processes  1984).  chloroplast  affected  microalgae  cells  al.  oxidative the  exposed  et  to  explain  selenate.  carterae  respiration  Pavlova  the  respiration  Amphidinium  The  could  findings  adaptation  fluoride  essential  of  to  the other  inhibited  by  Discussion  selenium that  (Pick  selenium  products, This and  is  of  such  as  further  the  et  al.  Prevot  in  starch  the  selenite. inhibits  selenium  on  When grown the  vulgaris,  in  these  grown  which  is  the  also  uptake it  in  phosphate  aggravate  amount  the  presence  an  inhibitor an  observed  note  more  of  exogeneous  disappearance  to  of  observed  of  with  selenate  (Moede  general  the  "changes"  grown  that  in microalgae the  nitrogen  also  dinoflagellates  interesting  of  can  two  the  in  circumstances,  prevented  the  in  were  storage  inclusions.  of  Reductions content  of  lipid  analysis  Chlorella  Atrazine,  observations  et  effects  al. of  photophosphorylation.  with  Burnham  blue-green  10~ M to 2  of  there  was  luridum a  c h l o r o p h y l l a and p h o t o s y n t h e s i s  were  affected  biosynthesis  Phormidinium  5  The  enzyme  alga,  10~ M s e l e n i t e ,  1973).  chlorophyll an  (1966)  vesicles  the  amount  cells.  own  depletion  a n d S o y e r - G o b i l l a r d (1986)  is  hence  the  our  cytoplasmic  chlorophyll  glucose,  It the  to  and  the  Under  supply,  1980);  of  herbicide  starch.  lead  starch  and  photosynthesis. energy  and w i t h  c o r r o b o r a t e d by t h e  deposits  Shoton  1973)  treatments  carbon content  starch by  and Avron  was  found  seedlings  to  proposed (Padmija  by be to  of  Mung  10~ M selenium. 5  paralleled be  et  involved al.  36  by  1989).  decrease  (Sielicki  Bean, The the in In  was in and  Phaseolus  reduction  in  inhibition  of  chlorophyll Dunaliella  Discussion  tertiolecta  and  phaeophytin  a  same  likely  This  a  occurring, the  with  is  indicative  breakdown  the  inhibition  of  of  the  However,  with  then  or  breakdown.  that  a  in  specific  chlorophyll  Amphidinium  of  decrease  an in  of  a  synthesis  phaeophytin  a  thesis  a to was  This  chlorophyll  a  (phaeopigment)  reinforce  showing  to  is  adapted  (phaeopigment).  further  of not  chlorophyll a  in  responses  the  is  carterae in  by  chlorophyll  increase  this  toxicity  in  and  breakdown  (phaeopigment)  observations I  massive  phaeophytin  reduction  Part  a  chlorophyll a  approximately  reduction  the  of  either  These  discussed exhibit  a  3  greater  that  phaeophytin  10~ M selenate,  the  decreased  suggests  cause  10%  both  to  but  concentration. grow  lutheri.  (phaeopigment)  amount.  chlorophyll  Pavlova  those  that  different  cells  forms  of  selenium. In shows  a  vivo  decrease  relative  to  chlorophyll 440  nm.  longer  is  unclear.  in  a  at  680  thylakoids  are  a  there  to  shorter  given  adapted  dilated  is  seems to  and  of of  Dunaliella  tertiolecta  chlorophyll a  absorption  decrease  nm t h a t  However, was  amount  and  wavelengths  that  spectra  the  carotene,  Overall,  at  carterae  absorption  the  to  further be  a  fact  grow  the  in is  absorbance  accentuated  shift  ones.  selenium  37  in  The  from  at  reason  for  in  10  selenate,  M  known  to  430-  absorption  that J  of  this  Amphidinium  react  many with  Discussion  sulfhydryl cause  groups  lipid  The  that  were of  osmiophilic than  treated  the  tips  Garberg  in  the  et  polymerase  Ames to  which  can  1988);  it  is  shifts  in  the  chromosomes The  control  cells.  observed  Selenium (breaks  while  activities  to  repair  gaps)  successfully  since  genetic  chromosomal are  perichromatic 1984).  granules The  it  al.  since  10~ M in  1987).  significance  38  DNA s t r a n d  (Snyder  However, observed the  of  their  the is  in  The  1988,  RNA a n d DNA  it  cells  of  sativum  The l a s t  dinoflagellates  speculation.  Allium  decrease  selenate.  3  amount  and caused  would  alterations  to  chromosomes  i n h i b i t i n g both  material.  deleterious adapt  also  densely  seedlings  the  in  were  organization  meiotic  hepactocytes  (Frenkel et  hazardous  damaged  and  the  increased  and  more  barley  in  in  altered  chromosomes  When  alterations  were  1967) .  1988),  were  perichromatic granules  fibroblasts  al.  especially  (Spector  species  lead  3  aberrations  is  carterae,  can  10~ M selenate.  selenate,  Ting  in  the  damage  and  ( M u k h e r j e e and Sharma 1 9 8 8 ) ,  breakage  that  with  and the  with  and  chromosomal root  (Kramer  carterae  chromatin fibers  (Walker  oxygen  spectra. Amphidinium  stained  active  thylakoid  ultrastructurally less  form  peroxidation  conceivable absorption  to  effect ability  unlikely  Amphidinium  were  able  to  function  of  is  change  unknown is  beyond  Discussion  A  cell  present  i n most,  Dunaliella al.  structure  of  in  fixation  used  the  the  exchangeable (1980)  protective  role,  surface.  It  is  utilized  to  bind  coat  the very  lutheri  can  it  was  of  bind  of  cell  study.  A  cell  coat  Amphidinium by  carterae  the  When g r o w n become  It  has  1977).  them  cell  coat  in  theorized  that  this  might  upon  surface.  This  thin.  39  the  shedding, mechanism  M  and  in et  an al.  Dunaliella  from cell  - 4  suggested  Oliveira  contaminants  10  thinner  been  retain  method  in  the  remove  et  coat-  and  that  wall.  cell  species  al.  cell  (Oliveira  preserved  cations et  rigid  is  a  in  disappeared.  material  exhibits  studies.  conceivable  and  coat  a  this  both  removing  then  such  not  present  (Leppard  it  a  occur was  surface  without  also  in  to  dispersal  and  cell  coat  coat  observed  from  the  eventually  tertiolecta  ions  but  in  form  cells  possesses  known  cell  cell  all  surface  1985),  Dunaliella  that  not  its  also  al.  selenite,  glycocalyx-like  Pavlova  on is  et  if  and  layer  (Klut  or  tertiolecta  1980),  like  coat  play  the coat  excess would  a  cell may  be  selenium make  the  C O N C L U S I O N S  In that  conclusion,  high  growth  selenium  and  by  1983) . fish  High  levels  reproductive Indeed,  a  et  effluent  arms  increase  in  to  other  of  the  as  a  the  by fish  (Marcogliese  by  only  but  predation  et  disturbances  of  in  the  40  lower at  has  been  selenium  from  selenium  from  Freshwater  times,  in  water  freshwater  times  decrease  the  damage  the  (Wilbur mass  of  liver,  (Sorensen  1988).  species  diversity  in  been  by  the  attributed expansion  the  selenium 1989).  high  tissue  after  al.  It  selenium  4000  also  N . C . has  populations result  of  to  toxicity  1973).  1000  connective  Lake,  al.  amount  not  their  cause  at  to  their  et  zooplankton  Belews  fish  of  concentrate  1986),  size-selective  planktivorus  reservoir  in  most  selenium  and  lead  fact  to  adaptation  bioaccumulation.  marine  al.  organs,  change  piscivores  of  of  the  needed  selenium  (Sandholm  and  despite  are  s t i l l  invertebrates  times  (Bennett  to  turn  concentrate  marine  167  may  R E S E A R C H  that  occurs,  concentrate  phytoplankters  times,  said  possibility  due  in  F U T U R E  selenium  water  levels  which  invertebrates  fish  be  of  the  the  fish  zooplankton  167  and  in  trophic that  water  can  concentrations  concentrations  found  it  concentrations  reduction  higher  A N D P R O P O S E D  This  ecosystem,  to  an  of  the  elimination  of  pollution can  the  in  the  certainly  lead  particularly  Conclusions  considering coastal  the  water  The  amount  energy In  severe  run,  is  the  of  the  metal  role(s) selenium  in  and  altered to  in  this  the  and  could  in the  seems with form  of  and to  the of  amount  of  shortages  in  despite  able  to  material play of  after  removal  of  coat  was  a  (Leppard  the  toxicity.  41  with  adaptation  exposure  material  from  al.  requires studies response  other  in  (Oliveira  to  This  in  role  et  broader  to  mechanisms  covering.  major  hypothesis  adapt  observed  microalgae  these  microorganisms,  occur  cell  that  toxicity.  the  severe  other  in  suggest  in  were  the  reduction  selenium  that  of  with  Although adaptation  conjunction scope  of  tested  microorganisms This  by  however,  and a d a p t a t i o n  toxicity.  and  decreases  these  volume  together  photosynthesis  indicative  surface  other  and  are  microalgae  that  cell  investigation  is  and c o i n c i d e s  detoxification and  estauries  ultrastructure, rate,  and  leads  understood  circumstances  1980)  to  chloroplasts  interesting,  surface  shedding  the  systems this  meaningful  cell  Research  damage  the  concentrations.  from  toxicant  in  which  is  effects,  far  on  chlorophyll a  selenium  are  the  of  It  of  respiration  observed  products  energy.  it  and  long  storage  high  observed  transducing  the  Future  habitats.  mitochondria  the  possibility  effects  alterations  and P r o p o s e d  the  et  al.  1977)  to  further on to  the high  Table I. Species of microalgae used i n the present study.  Strain/clone Spec i es  Cyanophyceae Agmenellum quadruplicatum Dinophyceae Amphidinium carterae  ( i sol ator/suppli er/source)  Van Ballen, ATCC No.27264 J . Mclachlan, G u i l l a r d , clone "Amphi 1"  Eustigmatophyceae Nannochloropsis oculata  Lefttey, SMBA No.66  Pyrmnes i ophyceae Isochrysis galbana Pavlova l u t h e r i  L e f t l e y , SMBA No.58 Left ley, SMBA No.261  BaciIlariophyceae Chaetoceros v i x v i s i b i I i s  Chlorophyceae Dunaliella tertiolecta  North East P a c i f i c Culture C o l l e c t i o n (Dept. of Oceanography, UBC)  Woods Hole Oceanographic  42  Institute  Table I I . Growth of microalgae i n s e l e n i t e . a, adaptation period (days); b, exponential growth rate ( d " ) ; c, maximum y i e l d (OD 600 nm)»" NG, no growth; NM, values too low or inadequate f o r measurement with confidence. Values i n brackets represent the percentages with respect to the control. 1  Selenite concentration (M) added to growth medium 10 -4  10"  10 -5  10"  10"  Alga  AgmenelI urn  quadruplicatum  Amphidinium carterae  NG  7  NM  0.028 0.48  Nannochloropsis  oculata  Isochrysis galbana  Pavlova  lutheri  9  0.56  (90)  (98)  7  7  (82)  (95)  0.048 (102)  0.57 (95)  11  0.044 0.53 (121)  (94)  0.048 0.56  0.54  (71)  (91)  0.045 1.30 (94) (93)  2 0.056 (115)  1.33 (95)  0.50 1.47 (103) (105)  0.050 1.40 (103) (100)  2  0.051 1.22 (104) (87)  4  0.026 0.50 (58) (66)  4  0.033 0.52 (72) (69)  0.055 0.86 (120) (115)  0.051 0.82 (111) (109)  4  0.047 0.79 (104) (106)  25  0.023 0.58 (53) (57)  10  0.049 0.83 (114) (81)  0.069 1.06 (160) (104)  9  0.074 1.23 (172) (121)  0.030 (70)  0.80 (79)  11  9  11  0.030 0.54  (104)  18  0.034 0.53  0.024  0.049  (80)  7  0.029 0.49 (80) (86)  (89)  (60)  u>  NM  (102)  (94)  Chaetoceros v i x v i s i b i l i s  4  0.012 0.044 (76) (42)  4  0.015 0.082 (94) (79)  4 0.017 0.097 (109) (94)  4  0.019 0.111 (120) (106)  4  0.017 0.110 (110) (104)  Dunaliella tertiolecta  2  0.047 0.33  2  0.060 0.53  2  2  0.073  1.30  2  0.078 1.33  (84)  (95)  (54)  (24)  (69)  (38)  0.078  1.30  (90)  (95)  (90)  (98)  Table I I I . Growth of microalgae i n selenate. a, adaptation period (days); b, exponential growth rate (d~^); c, maximum y i e l d (00 600 nm)<" NG-> no growth; NM, values too low or inadequate f o r measurement with confidence. Values i n brackets represent the percentages with respect to the control.  Selenate concentration (M) i n growth medium 10" Alga  10"  2  a  b  Agmenellum quadruplicatum  NG  NM  NM  18  Amphidinium carterae  NG  NM  NM  4  0.014 (29)  Nannochloropsis  NG  NM  NM  NG  NM  Isochrysis galbana  NG  NM  NM  28  Pavlova lutheri  27  9  Chaetoceros v i x v i s i b i l i s  NG  NM  NM  NG  Dunaliella tertiolecta  NG  NM  NM  2  oculata  e  2  0.019 0.56 (44) (55)  10"  3  b  c  0.023 0.39 (64) (69)  §  10"  4  b  £  a  11  0.031 0.55 (85) (96)  4  0.028 0.55 (78) (97)  4  0.040 0.51 (83) (85)  4  0.044 0.58 (94) (98)  2  0.045 1.50 (93) (107)  2  0.052 1.40 (107) (100)  0.030 0.61 (66) (82)  4  0.053 0.81 (117) (108)  4  0.052 0.82 (114) (109)  0.042 0.89 (99) (87)  9  0.060 1.07 (140) (105)  9  0.067 1.20 (157) (118)  4  0.016 0.107 (100) (103)  2  0.076 1.40 (87) (102)  0.30 (50)' NM  NM  NM  4  0.012 (75)  0.079 (76)  0.079 (91)  1.20 (88)  2  0.072 1.37 (83) (100)  Table IV. Mean (x) diameter (/un) and length (/im) of the flagella of Dunaliella tertiolecta. and Pavlova lutheri. and of the haptonema of Pavlova lutheri grown with and without selenite, plus mean (x) diameter and length of the flagella (<im) of Amphidinium carterae, grown with and without selenate. Observations made with a TEM, SEM and/or Light microscope. Corresponding standard errors of means (sem) shown. *The top figure for Pavlova lutheri flagella length refers to the long anterior flagellum and the bottom figure refers to the short posterior flagelIurn.  Diameter No Se added Species  Parameter  Pavlova lutheri  Pavlova lutheri  Flagella  Flagella  Haptonema  10'" M Se03 4  No Se added  sem  x  sem  x  sem  X  sem  0.67 1.14 1.77  21.56 19.85 23.59  0.94 0.68 2.79  15.724 15.744 15.417  0.260 0.395 0.230  16.438 9.355 10.625  0.382 0.626 0.429  18  21.06  1.22  19.28  0.91  * 9.635 3.516  0.462 0.651  0.608 0.268  29  20.35  0.72  19.40  1.36  11.293 4.358  0.326 0.584  10.286 4.323 10.041 3.795  0.641  1.823 1.963  0.000 0.000  2.083  0.000 0.000  18  12.92  29  10.80  2.02 1.10  1.24  12.06 12.73  1.98  1.998  0.468  Length 10" M Se04 3  No Se added  Days  10"*M Se03  :  x  Flagella  4  22.05 21.73 24.35  No Se added  Amphidinium carterae  -  9 17 30  Diameter  Species  10 " M Se03  Days x  Dunaliella tertiolecta  Length  Log phase 22.30  sem  x  0.77  12.6  sem  1.12  x  10.163  sem  1.244  x  9.080  sem  2.101  Table V. Mean (x) and standard error (±sem) of the means of p h y s i o l o g i c a l a c t i v i t i e s of D u n a l i e l l a t e r t i o l e c t a grown with and without s e l e n i t e .  No Se added  10" M Se03 4  Parameters  C e l l s i z e (/un ) 3  Chlorophyll per  83.239  1.497  1.119  a (/ig)  cell  8.913x10"  7  0.786x10"'  6.140x10"'  0.735x10"  8  0.127x10"  8  7.181x10"  9  1.016x10"  9  0.249x10"  8  1.621x10"  8  0.229x10"  8  3  3.175x10"  5  per /im protoplast  1.075x10"  per p n  2.097x10"  3  3  85.776  chloroplast  8  7  Phaeophytin a (fig) per  cell  4.657x10"  0.438x10"  5.617x10" 1.096x10-4  0.698x10"  per c e l l - m i n  3.026x10 •11  0.626x10 11  8.308x10"  12  per /un  3.663x10" 7.147x10 13  0.866x10"  9.689x10"  14  per /xm  protoplast  3  per /im c h l o r o p l a s t 3  0.136x10"  4  3  0.331x10"  3.712x10"  5  0.468x10"  8.378x10"  5  1.057x10"  Photosynthesis (jtmole 02 evolved) 3  protoplast-min  per /un chloroplast-min 3  13  13  1.689x10 -13  2.187x10"  13  0.040x10 12 0.168x10 -14 0.038x10-13  Respi r a t i o n (/unole 02 consumed) per c e l l - m i n per A"!  3  per jim  3  protoplast-min mitochondria-min  6.725x10 12 8.074x10 -14  0.342x10"  12  6.813x10"  12  0.163x10"  14  7.938x10"  14  3.666x10"  0.074x10"  2.090x10"  12  12  12  0.342x10 12 0.223x10 14 0.059x10"  12  Carbon (mg) per  cell  per /un protoplast 3  2.323x10"  0.106X10"  2.789x10 10  0.041x10"  2.213x10"  8  10  0.193x10"  8  2.586x10"  10  8  0.282x10"  10  Nitrogen (mg) per  cell  per /un protoplast 3  3.885x10"  9  4.697x10"  11  0.704x10"  9  0.990x10"  46  11  4.407x10"  9  5.156x10"  11  0.616x10"  9  0.832x10"  11  Table VI. Mean (x) and standard error (±sem) of the absorption by d i f f e r e n t vivo  absorption spectra r e p l i c a t e  measurements  of D u n a l i e l l a  tertiolecta  selenite.  17 day o l d c e l l s 10" M Se03  No Se added  4  Ratio of  Chi a (430-440 nm) Carotene (480 nm)  1.065  0.052  1.083  0.083  1.277  0.088  1.715  0.151  1.304  0.040  1.474  0.051  1.094  0.001  1.526  0.001  1.198  0.024  1.582  0.018  1.230  0.098  1.373  0.152  1.030  0.050  1.381  0.072  Chi b (650 nm) Chi a (620-630 nm)  1.029  0.102  0.869  0.106  Chi a (680 nm)  0.861  0.059  0.873  0.055  0.840  0.026  1.013  0.060  Chi a (430-440 nm) Chi a (620-630 nm) Chi a (430-440 nm) Chi b (650 nm) Chi a (430-440 nm) Chi a (680 nm) Carotene (480 nm) Chi a (620-630 nm) Carotene (480 nm) Chi b (650 nm) Carotene (480 nm) Chi a (680 nm) Chi a (620-630 nm)  Chi b (630 nm) Chi a (680 nm)  47  pigments from two i n  grown with  and without  Table V I I . Mean (x) and standard error (±sem) of the means of p h y s i o l o g i c a l a c t i v i t i e s of Pavl l u t h e r i grown with and without s e l e n i t e .  No Se added  10" M Se03 4  Parameters  C e l l s i z e (/un ) 3  Chlorophyll a (/ig) per c e l l  0.495  38.423  per /un protoplast per /un c h l o r o p l a s t  1.001x10" 2.601x10" 5.075x10"  Phaeophytin a (/ig) per eel I per /un protoplast per /on c h l o r o p l a s t  4.437x10" 1.153x10" 2.250x10"  Photosynthesis (/unole 02 evolved) per eel I-min per /un protoplast-min per /on chloroplast-min  4.068x10" 1.055x10" 3.275x10"  Respiration (/unole 02 consumed) per eel I-min per /un protoplast-min per /un mitochondria-min  2.135X10" 5.531x10" 7.938x10"  3  3  3  3  3  3  3  3  Carbon (mg) per c e l l  6  8  8  3  4  4  11  per /un protoplast Nitrogen (mg) per c e l l per /un protoplast  4.306x10" 1.119x10"  3  12  11  2.307x10" 5.997x10"  3  14  13  12  8  39.374  0.492  5.015x10-7 1.273x10-8 2.873x10"  0.225x10" 0.032x10" 0.072x10"  0.968x10-3 0.243x10-4 0.473x10-4  2.268x10"^ 5.757x10" 1.299x10"  0.105x10" 0.152x10" 0.034x10"  1.846x10" 0.472x10" 1.465x10"  2.195x10" 5.535x10" 2.302x10"  0.837x10" 2.016x10" 0.839x10"  0.222x10" 0.555x10" 1.084x10"  6  8  8  11  14  12  9  11  13  12  1.816x10 •11  3  5  4  4.591x10" 5.786x10"  0.397x10" 0.985x10"  3.476x10 •8 8.829x10"  0.061x10" 0.022x10"  5.969x10" 1.517x10-10  0.194x10" 0.080x10"  11  1.039x10" 0.261x10"  10  v  10  4  8  1.212x10" 3.108x10 -13 4.461x10 12  8  10  5  48  12  10  9  10  13  7  8  11  0.458x10" 1.072x10" 1.588x10"  13  12  11  13  12  8  10  9  10  Table V I I I . Mean (x) and standard error (±sem) of the means of p h y s i o l o g i c a l a c t i v i t i e s of log phase Amphidinium carterae grown with and without selenate.  No Se added  10" M Se04 3  Parameters  C e l l s i z e (/un ) 3  643.263  606.501  5.247  1.982  Chlorophyll a (/ig) 7  0.309x10"  5.999x10"  8  0.474x10"  per /un c h l o r o p l a s t  1.283x10" 1.993x10" 7.872x10"  8  1.873x10"  9.991x10" 3.677x10 •8  2.265x10" 3.788 10" 1.405x10"  Phaeophytin a (/ig) per c e l l per /un protoplast per /un c h l o r o p l a s t  2.872x10" 4.462x10" 1.763x10"  2  0.498x10" 0.760x10" 0.300x10-4  1.787x10-2 2.947x10 -5 1.093x10"  0.061x10" 0.116x10" 0.043x10"  Photosynthesis C/unole 02 evolved) per c e l l - m i n per /un protoplast-min per /un chloroplast-min  2.131x10 10 3.311x10 13 1.786x10"  0.133x10" 0.197x10" 0.400x10"  1.905x10" 3.141x10 13 1.166x10"  0.074x10" 0.138x10 -13 0.051x10"  Respiration (/tmole 02 consumed) per c e l l - m i n per /un protoplast-min  1.276x10' 10 1.984x10"^  0.061x10" 0.089x10" 0.176x10"  1.613x10" 2.658x10" 5.383x10"  0.218x10" 0.345x10" 0.699x10"  per c e l l per /un protoplast 3  3  3  3  3  3  3  per /un mitochondria-min 3  12  3  3.905x10"^  Nitrogen (mg) per c e l l per /un protoplast  2.904x10" 4.514x10"  3  5  4  1.398x10" 2.174x10"  3  2  5  Carbon (mg) per c e l l per /un protoplast  6  9  2  4  10  13  12  0.002x10" 0.003x10"  10  13  12  7  7  10  8  11  0.077x10" 0.105x10"  10  8  49  11  10  12  10  13  12  1.310x10"  7  2.161x10"  2.377x10" 3.919x10"  10  8  11  6  9  8  2  5  4  10  12  10  13  12  0.002x10"' 0.015x10"  10  0.022x10" 0.015x10 11 8  Fig.  1.  Growth (A)  curves  and  10  - 4  M  of (•)  selenium  addition  standard  error  of  Pavlova  lutheri  selenite, (o). mean.  50  and  Vertical n=3.  exposed control bars  to  10  M  without  indicate  the  Fig.  2.  Growth  curves  10~ M  selenite  4  addition error  of  (o). mean.  of  Dunaliella tertiolecta («)and  Vertical n=3.  52  control bars  exposed  without  indicate  the  to  selenium standard  e on  Fig.  3  Growth  curve  selenate  adaptation  shown error  and to  growth (o) .  of  Amphidinium  without  adaptation,  control  of  10  (•) in - 3  M  n=3.  54  and - 2  M  selenium  bars  with  selenate  selenate  without  Vertical  mean.  10  carterae  (A) .  in  J  (•)previous with  prior  Corresponding  addition  indicate  10~ M  the  is  also  standard  Fig.  4.  Percentage  and  cultures  of  lutheri  grown  Motility 10~ M  with  control  of  motile  tertiolecta 10~ M  adapted  to  cells  and  selenite  4  carterae  in  Pavlova (A)  grow  in  and 10  J  M  (B). symbols:  control  selenate  3  speed  Dunaliella  Amphidinium selenate  swimming  (Q) •  (§3) /  10~ M  bars  indicate  (•) ,  (|§3) ,  4  Swimming  selenite  4  10~ M s e l e n i t e speed  (0) ,  symbols:  10~ M 3  selenate  (0) • Vertical  Dunaliella n  =  days,  tertiolecta:  419. n  carterae:  the  30 =  days, 447.  n =  n  29  164.  56  9 =  standard days,  557.  days,  n  n  error  of  mean.  644.  17  days,  =  Pavlova =  553.  lutheri:  18  Amphidinium  Percentage of motile cells ot  w o  '////////////////////////.  3  * o  o  w o  o» o  >i o  SAyyv  O  Is* o  xxxxxxxxxxx  "O 1 00  TT5T  ZXX)  p  3.  1  O  (X O  O  1  <D O  o  •• 1— —1  —4  Ot  o  o  o  8  2  ( s'uur/) poods  Percentage of motile cells  S o U3  o W  8  »  55  •  S  »  8  i  i  i  i  x%$yyyyyyyyyyyyyyyyyyyyyyyMH  \\\\\v\\\v\\\\v\v\v\\\\v I 1 1 1 1 O  O  O  O  O  -  t  f  e  1 l  o  (  s'wrt)  pddds  1 C  1 f  o  t  o  Figs.  5  and  6.  Scanning  tertiolecta  grown  (Fig.  and  5)  conspicuously  Fig.  7.  30  Scanning  electron  Bar  2  tertiolecta  jL«n  1  izm  30  days  displaying  Fig.  5  bar  =  ltxm  (25,O0OX).  micrograph  electron grown for  deflagellated. 1  flagella.  6)  17  selenium  days) .  Note  of  Dunaliella  addition long  for  17  flagella.  /xm ( 1 1 , 0 0 0 X ) .  Scanning  (similar  (Fig.  Dunaliella for  4  bar =  for  of  10~ M s e l e n i t e  grown w i t h o u t  (similar =  6,  micrograph  days  short  Fig.  days  8.  with  (20,000X).  tertiolecta  Fig.  electron  micrograph  with  30  The f l a g e l l a  58  Dunaliella  10~ M s e l e n i t e  days) .  ( 2 6 , 000X) .  of  4  Note is  lying  the  for  17 cell  nearby.  days is Bar =  Figs.  9  and  10  Mitochondria  grown  with  selenite  (Fig.  for  17  9)  days.  rather  long  unusual  configurations  more 10,  dense. bar  of  and  Dunaliella without  Selenium  mitochondria.  Figs.  = 0.5  /am  9,  bar  =  (66,000X).  60  (Fig.  grown  The  (arrows) 0.5  tertiolecta  cells  cristae and  /xm  10)  the  10~ M 4  possess display  matrix  (40,000X).  is  Fig.  61  Figs.  11  and  12  11)  Dunaliella  and  days.  without  Note  compare  the  with  smaller  with  Fig.  These  cells  inclusions  12a,  Lip).  Fig.  lib,  bar  1.5  (17,000X).  amount  Fig.  11a,  12b,  are  (Fig. bar  1  62  bar  =  1.5  Fig.  for  (Fig.  17 11a  pyrenoid  Pyr)  selenite  in  largely  compare fxm  (Fig.  smaller  also  11a =  starch  and  jum ( 1 5 , 0 0 0 X ) . 12b,  of  with  selenite  4  compare  cells.  grown  10" M  Sth),  grown  =  12)  12a,  lib  lipid  (Fig.  Fig.  (Fig.  of  tertiolecta  devoid  with  Fig.  (19,000X).  Fig.  12a,  1  bar  fxra ( 1 2 , 0 0 0 X ) .  =  [im  Figs.  13  to  15  13 for  Dunaliella tertiolecta  and 30  14)  and  days.  inclusions pyrenoid the  15,  Large  (Pyr)  bar = 2.0  smaller  and  less  grown  Fig.  (Fig.  vacuoles  (Lip),  selenium  (15,000X).  without  14,  bar  =  Aim ( 1 1 , 0 0 0 X ) .  64  15)  10" M  (Vac) ,  lack  (Figs.  selenite  4  of  lipid  chloroplasts  starch  cells.  grown w i t h  Fig. 1.5  (Sth) 13,  (Chi),  characterize bar  =  1.5  /Ltm ( 1 3 , 0 0 0 X ) .  /zm Fig.  Figs.  16  and  17  Cell  grown w i t h alcian coat  Mm  Figs.  18  and  4  staining.  selenium  control  of  10~ M s e l e n i t e  blue of  coat  cells  for  Note  grown  (Fig.  Dunaliella  17).  tertiolecta  17  days,  the  less  (Fig. Figs.  16) 16  revealed dense  cell  compared  an  17,  by  to  bar =  0.3  (71,000X).  19  Cell  grown w i t h alcian coat  control  of  10~ M s e l e n i t e 4  blue in  coat  staining.  selenium cells  Dunaliella for  Note  30 the  grown  (Fig.  19) .  Figs.  (Fig.  0 . 3 /am (71,00000 .  66  tertiolecta  days,  revealed  absence 18) 18  of  cell  compared and  19,  by  bar  to =  Figs.  20  and  21  Pavlova  without the  (Fig.  small  mitochondria cells. bar  =  Fig. 0.7  /im  21)  lutheri  20,  with  10~ M s e l e n i t e 4  size in  grown  of  both  selenium bar  =  (30,000X).  68  grown  0.7  iim  for  (Fig. 18  20)  days.  chloroplasts compared (28,000X).  to  and Note and  control Fig.  21,  Figs.  22  and  23  Cell  (Fig. 18  22)  days,  cell  coat  than 0.3  Figs.  24  and  that nm  29  of  and w i t h o u t revealed of of  Pavlova (Fig.  by  control  23)  alcian  selenium  lutheri  grown  cells.  grown  with  10~ M s e l e n i t e  for  4  blue  staining.  cells  Figs.  is  22  less  The dense  and 23,  bar  =  (60,000X).  25  (Fig.  coat  Cell 24)  days,  coat  of  and w i t h o u t revealed  the  thinness  the  control  of  by  this  cell.  Pavlova (Fig.  alcian coat  Figs.  (60,000X).  70  25)  in 24  lutheri  with  10~ M selenite  for  4  blue  staining.  selenium and  grown  25,  Note  compared  bar  =  0.3  to  Figs.  26  and  27  Amphidinium  adapted 27)  to  10~ M  28  nucleus  (Nuc),  compared  to  and log  (Fig. Note  27,  phase  without  vesiculation compared (81,000X).  to  of  bar = 2.5  /nm  to  29)  of  cells.  Fig.  bar = 0.5  72  26,  bar  /nm  in  =  the  grown 2.5  /nm  carterae  at  (7,500X).  (Fig.  selenate.  thylakoids  (Fig.  selenium  with  3  the  29,  grow  phase  without  Amphidinium  10~ M  control Fig.  (Vac)  of  log  differences  Fig.  adapted (Fig.  and  cells.  Chloroplasts  at  26) the  vacuoles  control  Fig.  29  with  selenate.  3  (7,600X).  Figs.  grow  carterae  in 28,  28)  and  Note  the  selenium bar  =  (41,000X).  grown 0.2  /nm  Fig.  30  Cell  size,  cross  section,  Pavlova of  expressed  as  of  Dunaliella  lutheri  after  with  10~ M  time  at  selenate  (Q).  selenium  addition  indicate  log  points  over  tertiolecta  and  for  selenite,  phase  of  adapted  various and  to  is  also  standard 9  days,  n  shown  (Q) .  mean.  =  17  days,  n = 49.  Pavlova  days,  n =  Amphidinium c a r t e r a e :  52.  74  lutheri:  18  10~ M 3  without  Vertical  of  days,  with  growth  error 50.  periods  Amphidinium  grow  Corresponding control  the  tertiolecta:  growth  4  carterae  number  days, n =  bars  Dunaliella n  =  72.  30  n =  51.  29  49.  150 -i  150 -i  130 -  130  I i  1  •§ 110 © O  90  I  © N  «  I  110 H  1  © O  1  90 -  1  1  Is  i 70  17  30  Dunaliella tertiolecta  18  29  Pavlova lutheri  Growth Period (days)  70  Log phase Amphidinium carterae  Fig.  31  Mean  volume  Dunaliella periods  density  tertiolecta of  time  Corresponding addition the  is  cell  (Cyt)  (Gig),  (Lip)  volume  chloroplast days,  n =  50.  of  volume.  Pyrenoid  days,  76  are  Dunaliella n = 72.  30  (Q) .  indicate (Nuc)  and  expressed  per  Mitochondria  (Vac),  densities  densities  17  are  (Chi),  vacuoles  various  selenium  bars  Nucleus  densities  volume  volume.  without  Vertical  mean.  for  of  selenite  4  (Q).  volume  cytoplasmic  (Sth)  10~ M  growth  shown  components  growth  Chloroplast  Golgi  inclusions  with  error  volume.  (Mit),  per  also  cell  after  control  standard  cytoplasm  of  are  (Pyr)  and  lipid  expressed and  starch  expressed  per  tertiolecta: days,  n =  49.  9  Dunaliella tertiolecta - 0.9  0.4 -  30 days  - 0.6  0.2 -  (0  0.0  CD  0.4  c  ~  0.3 0.9 17 days  o  E o > c  I  0.2 -  DP  0.0  r-,1  D  0.4  0.6  n s fti f l f  u i .  0.3 - 0.9  Nuc  MH  Gig  Vac  Up  Pyr  Sth  0.3 Chi  Cellular component 77  Cyt  3  CL CD  - 0.6  0.0  < o_ c CD  9 days  0.2 -  CD Q D  D  Fig.  32  Mean  volume  lutheri with  density  after  of  growth  10~ M s e l e n i t e , 4  log  phase,  (0) .  adapted  cell  for  various  and to  grow  the  is  also  standard (Cyt)  expressed  per  storage and  expressed (Pig)  cell (Mit),  -  and p y r e n o i d  (Pyr)  n  Amphidinium c a r t e r a e :  78  18  indicate  Nucleus  (Nuc),  cisternae  (PCs)  Chloroplast  days, n =  (Chi),  vacuoles  and  expressed  Pavlova  52.  lutheri:  bars  (Vac),  bodies lipids  volume.  volume. =  selenium  projectile  cytoplasmic  at  without  (Gig),  starch  time  selenate  peripheral  (StV),  of  carterae, 3  Vertical  volume.  Pavlova  10~ M  mean.  Golgi  products  per  of  and  vesicles  storage  (•) .  error  cytoplasm  Mitochondria  shown  with  growth  of  periods  Amphidinium  Corresponding control  addition  components  (PjB) (StP)  -  Plastoglobuli per  n = 49.  chloroplast 51.  29  days,  A  Pavlova lutheri 29 days  0.24-  CO  c  - 0.8  CD  0.12 -  - 0.5  TD  E  0.00  O >  0.24 -  <  lfl_J  Q>  O D  0.2  c 3  CD  18 days  - 0.8  c  cl  CD D  (/>  D  0.12 -  0.5  AT  JjJ  0.00 Nuc  MR  Gig Vac PCs StV  Pig  Chi  Cyt  0.2  Cellular component  £  0  1  8  Amphidinium carterae  c  "O  Log phase  B  , 1.0  i  - 0.8  0.12 -  <D  - 0.6  E  J3  O >  0.06 -  - 0.4  C  o 0)  0.00 Nuc Mit  Gig Vac Vln PJB StP Pyr  Cellular component 79  1 _ 0.2  Chi Cyt  CD  a < O  c 3 CD CL CD D  (A  Fig.  33  In  vivo  tertiolecta without the  absorption grown  selenium  difference  curves  between  spectrum  with  10  addition in  500  the  - 4  (c,d)  shape  nm a n d 6 0 0  80  M  of  Dunaliella  selenite *for of  nm.  17  the n=4.  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Biophys.  of  molybdenum Acta  95-106.  J . M . , and  excised  Underwood,  N.J.  processes  by  Br.  Structure.  C D .  Quantitative  and  adaptation  126  Griffiths,  Robinson. New  Cell  A.S.  Shrift.  Astragalus  E . J .  nutrition.  1977.  1968.  roots.  Trace  Academic  Plant  Selenium Physiol.  elements  Press,  97  N.Y.  302  in  absorption 43:  human p.  by  14-20.  and  animal  References  Vorobjev,  I.A.,  and  its  Rev.  Walker,  role  Cytol.  D.R.  Wheeler,  Wilbur,  World  in  Biol.  C.G.  1983.  and  Thomas  Pub.,  a  of  six  Ecol.  poison  Health  Soil  in  Sci.  Zingaro,  of  of  57:  forage 51:  K.  The  centromere  microtubules.  Int.  in  central  506-508.  and  selenate,  selenite  unicellular  marine  N.R. and  Bottino.  sulfate  algae.  J .  on  Exp.  181-194.  Selenium:  a food  Springfield,  II.  Organization. 58.  species  Irgolic,  necessary  Criteria  1987.  organization  Selenium  effect  growth  Nadezhdina.  227-293.  Can. J .  The  Health  the  106:  A . E . , R.A.  1982.  Mar.  E.S.  1971.  Alberta.  the  and  potential constituent. 126  1987.  Geneva.  98  environmental  3 60  G.  p.  Selenium. p.  Charles  Environmental  APPENDIX  I.  Selenium  Composition  added s e p a r a t e l y  of at  microalgae  various  KN0 NaH P0 -H 0 Na Si0 •9H 0 3  2  4  2  2  3  2  Vitamins: Thiamine-HC1 Biotin B12 Trace-metal ions (chelated) Na -EDTA-2H 0 FeCl -6H 0 MnS0 •4H 0 ZnS0 -7H 0 Na Mo0 •2H 0 CuS0 •5H 0 C0SO4•7H 0 2  2  4  2  4  2  2  4  2  4  2  2  Buffer: Tris-HCl, Sea  water, Pacific  concentrations.  2 50.0 34.5 84.0  mg mg mg  (2500 / L t m o l e ) ( 2 5 0 /xmole) ( 3 0 0 /xmole)  500.0 1.0 2.0  Mg Mg Mg  ( 1 . 4 8 /xmole) ( 0 . 0 0 4 1 /xmole) ( 0 . 0 0 1 3 4 /xmole)  200.0  41.3iru>T (  Ocean,  medium.  c  a  a  8 . 1 mg ( 2 1 . 8 /xmole) 2 . 7 mg ( 1 0 . 0 /xmole) 1 . 1 2 5 mg ( 5 . 0 /xmole) 0 . 5 7 5 mg ( 2 . 0 /xmole) 0 . 2 4 3 mg ( 1 . 0 /xmole) 0 . 0 2 5 mg ( 0 . 1 /xmole) 0 . 0 1 4 mg ( 0 . 0 5 /xmole)  2  3  growth  salinity  ml To  33%o  (1 1  g  or  8.3  mmol  liter  C  p H o f s t o c k s o l u t i o n s o f t h e s e c o m p o n e n t s was a d j u s t e d to 7.6-7.8 w i t h a q u e o u s H C l o r NaOH b e f o r e incorporation w i t h t h e r e s t o f t h e medium. a  8.0  kpH 6 . 8 - 6 . 9 b e f o r e i n t h e medium a f t e r  a u t o c l a v i n g . T h i s g i v e s a pH o f a u t o c l a v i n g (15 m i n a t 12 0 ° C ) .  7.6-  The seawater was diluted with equal volumes of d i s t i l l e d d e i o n i z e d w a t e r t g i v e a f i n a l s a l i n i t y o f 13.2%o before u s i n g f o r D u n a l i e l l a t e r t i o l e c t a . Undiluated seawater was u s e d f o r a l l o t h e r m i c r o a l g a e . c  99  Tris)  


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