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The role of colloids in providing a source of iron to phytoplankton Wells, Mark L. 1982

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THE ROLE OF COLLOIDS IN PROVIDING A SOURCE OF IRON TO PHYTOPLANKTON by MARK L. WELLS B.Sc., University Of B r i t i s h Columbia, Vancouver, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF o THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Departments of Oceanography and Zoology) o We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1982 Q Mark L. Wells, In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of "Zooio&y ** OcterNArteXAPiW The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) i i ABSTRACT I r o n - r i c h c o l l o i d a l m a t e r i a l was f o u n d t o be a s s o c i a t e d w i t h d i a t o m s i n t h e n a t u r a l e n v i r o n m e n t . To d e t e r m i n e i f t h i s a s s o c i a t i o n c o u l d be i m p o r t a n t t o t h e o r g a n i s m , t h e s u p p l y o f i r o n f r o m c o l l o i d a l forms t o p h y t o p l a n k t o n was i n v e s t i g a t e d . L a b o r a t o r y b i o a s s a y s w i t h t h e m a r i n e d i a t o m T h a l a s s i o s i r a  p s eudonana d e m o n s t r a t e d t h a t f r e s h l y p r e c i p i t a t e d c o l l o i d a l i r o n c o u l d r e a d i l y s u p p o r t d i a t o m g r o w t h . However, when t h e s e c o l l o i d s were aged or s u b j e c t e d t o s h o r t p e r i o d s of h e a t i n g , t h e i r o n a v a i l a b i l i t y was d r a s t i c a l l y r e d u c e d . The i r o n a v a i l a b i l t y was n o t i n c r e a s e d w i t h a d d i t i o n of t h e c o m p l e x i n g a g e n t EDTA. The r e d u c t i o n i n a v a i l a b i l i t y a p p e a r s t o be l i n k e d t o i n c r e a s e d thermodynamic s t a b i l i t y of t h e c o l l o i d a l h y d r o u s f e r r i c o x i d e s . The p r o b a b l e mechanism o f t h i s r e d u c t i o n i s d e c r e a s e d c o l l o i d a l d i s s o l u t i o n r a t e s . The s u p p l y of i r o n from c o l l o i d s t o p h y t o p l a n k t o n a p p e a r s t o be d e t e r m i n e d by t h e c h e m i s t r y of t h e c o l l o i d a l i r o n m a t e r i a l r a t h e r t h a n by t h e p h y s i c a l a s s o c i a t i o n of c o l l o i d a l i r o n and c e l l w a l l s . i i i TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i i Introduction 1 Materials and Methods 6 F i e l d Study 6 Laboratory Study 14 ( i_) Bioassays 14 ( i i ) Medium Preparation 15 ( i i i ) Iron Stock Preparations and Treatments 16 (iv) Physical and Chemical Analysis 18 Results 21 F i e l d Study 21 Laboratory Study 32 Bioassays .32 Analysis of the Autoclaved Iron Stock 60 (i) X-ray d i f f r a c t i o n 60 ( i i ) Mossbauer spectroscopy 60 ( i i i ) Thermal gravimetric analysis 63 (iv) Gel f i l t r a t i o n 66 (v) Dissolution rates 66 Discussion 71 F i e l d Study 71 iv Bioassays 73 Processes Occurring in F e r r i c Salt Solutions 76 Analysis of the Autoclaved and Fresh Iron Stocks .... 89 Conclusions . ..' 94 References 99 Appendix 1 102 Appendix 2 105 Appendix 3 107 Appendix 4 114 L I S T OF TABLES T a b l e 1. Mos s b a u e r r e s u l t s LIST OF FIGURES Figure 1. F i e l d sampling locations 7 Figure 2. X-ray energy spectrum 10 Figure 3. Determination of i r o n - r i c h c o l l o i d s 12 Figure 4. Iron-rich colloid:diatom association 22 Figure 5. Iron-rich colloidrdiatom association 24 Figure 6. Iron-rich colloid:diatom association 26 Figure 7. Iron-rich colloid:diatom association 28 Figure 8. Iron content along a transect 30 Figure 9. C e l l growth with fresh c o l l o i d a l and iron-EDTA stocks 33 Figure 10. C e l l growth with fresh c o l l o i d a l and iron-EDTA stocks added after medium autoclaving 35 Figure 11. The effect of autoclaving on iron stock a v a i l a b i l i t y 38 Figure 12. The effect of d i f f e r e n t periods of autoclaving . 40 Figure 13. The effect of heating of the iron stock 42 Figure 14. The effect of d i f f e r e n t periods of heating 45 Figure 15. Ionic strength e f f e c t s in the iron stock 47 Figure 16. The eff e c t of iron stock ageing on c e l l growth . 49 Figure 17. Comparison of c e l l growth with heated and aged iron stocks 51 Figure 18. C e l l growth with heated and aged iron-EDTA stocks 54 Figure 19. C e l l growth with goethite and hematite 56 v i i Figure 20. Growth in cultures with autoclaved iron and without iron addition 58 Figure 21. X-ray d i f f r a c t i o n pattern of an autoclaved (24 hrs) iron stock 61 Figure 22. Thermal gravimetric analysis 64 Figure 23. Comparison of the gel f i l t r a t i o n rates 67 Figure 24. Comparison of the dissolution rates 69 Figure 25. Polymerization of hydrated iron atoms 78 Figure 26. Lennard-Jones graph 81 Figure 27. F i l t r a t i o n sampling apparatus 103 Figure 28. Estimation of EDTA breakdown 109 Figure 29. S a l i n i t y effect on EDTA breakdown 111 v i i i ACKNOWLEDGEMENTS J w i s h t o e x p r e s s my s i n c e r e t h a n k s t o D r . A. G. L e w i s f o r h i s s u g g e s t i o n s , i n s t r u c t i o n and e ncouragement t h r o u g h o u t t h e s t u d y . My d eep a p p r e c i a t i o n i s e x t e n d e d t o t h e members of my commitee f o r t h e i r t i m e and p a t i e n t i n s t r u c t i o n . In p a r t i c u l a r , I t h a nk Dr . J . L e j a f o r h i s many h o u r s of d i s c u s s i o n s o f s u r f a c e c h e m i s t r y and D r . E. G r i l l f o r h i s s u g g e s t i o n s and p a t i e n t d e s c r i p t i o n s of m a r i n e c h e m i s t r y t o a b i o l o g i s t . The c o - o p e r a t i o n and a s s i s t a n c e e x t e n d e d t o me by t h e o f f i c e r s and crew of t h e C.S.S. V e c t o r i s g r e a t l y a p p r e c i a t e d . My t h a n k s a l s o go t o my f e l l o w g r a d u a t e s t u d e n t s f o r t h e i r s u p p o r t and a s s i s t a n c e and e s p e c i a l l y t o N i k o G. Z o r k i n f o r h i s d i s c u s s i o n s and d i r e c t a s s i t a n c e t h r o u g h o u t t h e s t u d y . F i n a l l y , I would l i k e t o thank t h e D e p a r t m e n t of Z o o l o g y , C h e v r o n Canada and D r . A. G. L e w i s f o r t h e i r f i n a n c i a l s u p p o r t g i v e n t o me d u r i n g t h e s t u d y . 1 INTRODUCTION Iron i s an essential micronutrient for v i r t u a l l y a l l forms of l i f e . Phytoplankton require more iron than any other trace metal (Anderson and Morel, 1980) and iron d e f i c i e n c i e s in phytoplankton have been found to cause decreased rates of both photosynthesis and assimilation (Glover 1977). In higher plants, S p i l l e r and Terry (1980) have shown that iron deficiency decreased chlorophyll A, chlorophyll B, carotene and xanthophyll concentrations as well as the photosynthetic electron transport capacity. Their study showed that with progressive iron deficiency, the number of grana per chloroplast and the number of thylakoids per grana both decreased. Similar responses to iron deficiency are expected in phytoplankton. The e f f e c t s of iron deficiency, however, are reversible when iron i s made available to the c e l l . With phytoplankton, this occurs through chemical interactions with various sources of iron in the surrounding environment. The present study deals with the supply of iron to. phytoplankton from c o l l o i d a l hydrous iron oxides which, for the sake of convenience, w i l l henceforth simply be termed c o l l o i d a l iron. Iron exists in sea water as a soluble hydrated ion, as dissolved complexes formed with organic and inorganic ligands, and also as a component of suspended par t i c u l a t e or c o l l o i d a l organic and inorganic material. Since iron forms highly insoluble hydrous f e r r i c oxides in oxygenated sea water, and the leve l s of organic ligands that might increase i t s s o l u b i l i t y are usually r e l a t i v e l y low, i t has been suggested that most iron is 2 p r e s e n t a s c o l l o i d a l f e r r i c h y d r o x i d e ( L e w i n and Chen, 1973). However, t h e o x i d a t i o n r a t e of f e r r o u s i r o n i s c o n s i d e r a b l y s l o w e r t h a n p r e v i o u s l y t h o u g h t ( Y u - J e a n and K e s t e r , 1978) and, s i n c e f e r r o u s i r o n i s mor,e s o l u b l e t h a n f e r r i c i r o n , t h e c a l c u l a t e d d i s s o l v e d i r o n c o n c e n t r a t i o n may u n d e r e s t i m a t e t h e t r u e c o n c e n t r a t i o n i f s u b s t a n t i a l i r o n r e d u c t i o n was o c c u r r i n g ( e . g . p h o t o o x i d a t i o n o f o r g a n i c c o m p l e x e s o r f e r r i c i o n c o m p l e x a t i o n by r e d u c i n g l i g a n d s ) . A l t h o u g h t h e b i o l o g i c a l l y a v a i l a b l e forms o f i r o n have not been s a t i s f a c t o r i l y d e t e r m i n e d , i t i s g e n e r a l l y a c c e p t e d t h a t t h e y a r e composed m a i n l y o f t h e m o n o n u c l e a r s p e c i e s formed w i t h v a r i o u s i n o r g a n i c or o r g a n i c l i g a n d s . C h e l a t i n g a g e n t s s u c h as e t h y e l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA), w h i c h i s a s t r o n g s y n t h e t i c c h e l a t o r o f f e r r i c i r o n , a r e known t o enhance p h y t o p l a n k t o n g r o w t h ( L e w i n and Chen, 1971), i n d i c a t i n g t h a t t h e c h e l a t i o n of i r o n d i r e c t l y o r i n d i r e c t l y a f f e c t s it's a v a i l a b i l i t y . S i n c e EDTA does not a p p e a r t o be t r a n s p o r t e d a c r o s s t h e c e l l membrane (Huntsman and B a r b e r , i n p r e s s ) i t s u g g e s t s t h a t EDTA a c t s o n l y t o i n c r e a s e t h e p o o l o f s o l u b l e i r o n . F u r t h e r m o r e , t h e r e i s e v i d e n c e t h a t some p h y t o p l a n k t o n , under i r o n d e f i c i e n t c o n d i t i o n s , have a p h y s i o l o g i c a l r e s p o n s e w h i c h r e s u l t s i n t h e p r o d u c t i o n and r e l e a s e o f o r g a n i c compounds i n c l u d i n g some termed s i d e r o p h o r e s t h a t a r e h i g h l y s p e c i f i c f o r f e r r i c i r o n ( A n d e r s o n and T r i c k , p e r s o n a l c o m m u n i c a t i o n ) . I t i s p o s s i b l e t h a t s i d e r o p h o r e s i n c r e a s e i r o n a v a i l a b i l i t y by e i t h e r i n c r e a s i n g t h e p o o l o f s o l u b l e i r o n o r by a c t i n g a s c a r r i e r compounds w h i c h c h e l a t e f e r r i c i r o n and t r a n s p o r t i t a c r o s s t h e 3 c e l l wall. However, Anderson and Morel (1980) have suggested that the f e r r i c iron species must f i r s t be reduced before uptake. They have suggested th i s reduction could occur either by photo-oxidation of ferric-organic complexes, by chelation by reducing siderophores, or by reduction at the c e l l membrane. The dissolved species of iron believed to be b i o l o g i c a l l y available should exist in equilibrium with hydrous c o l l o i d a l oxides that presumably are the predominant form of iron in seawater. Thus as the concentration of the available soluble species is reduced, replacement should occur through dissolution of the c o l l o i d . C o l l o i d a l hydrous oxides are therefore the ultimate source of iron for phytoplankton. The d i s t i n c t i o n between c o l l o i d a l and soluble iron species cannot be rigorously defined. In general, a c o l l o i d a l dispersion is considered one in which the discontinuous phase (in this case iron) i s subdivided into units that are large compared with simple molecules, but small enough that i n t e r f a c i a l forces as well as i n e r t i a l forces are s i g n i f i c a n t in governing the properties of the system (Sennett and O l i v i e r , 1965). P a r t i c l e s with diameters less than 1.0 Pm are usually considered to have these c h a r a t e r i s t i c e s . Because of these c h a r a t e r i s t i c e s c o l l o i d a l material may remain suspended i n d e f i n i t e l y , in contrast to part i c u l a t e matter. In the past, the term 'dissolved' has been used for metals in aqueous solutions that pass through a .45 nm pore size f i l t e r . Although th i s is a p r a c t i c a l working d e f i n i t i o n , i t i s not representitive of metals in true solution ( i . e . mononuclear species) since some c o l l o i d a l 4 metals w i l l not be removed by the f i l t r a t i o n process. For the purposes of thi s investigation, the following c l a s s i f i c a t i o n scheme was chosen: iron existing as free hydrated ions or as complexed species was considered dissolved while aggregates up to 1.0 jim in diameter were termed c o l l o i d s . Solids larger than 1.0 Mm in diameter were considered p a r t i c u l a t e s . There has been some suggestion that phytoplankton are able to d i r e c t l y u t i l i z e i r o n - r i c h c o l l o i d a l matter adsorbed on their surfaces. Harvey (1937) observed an association between c o l l o i d a l iron and marine diatoms in laboratory cultures and after estimating the iron requirements of the c e l l , the dissolved iron concentrations, and the iron d i f f u s i o n rate, he concluded that for phytoplankton to escape iron l i m i t a t i o n t h i s adsorption was necessary. This conclusion was later supported by Goldberg (1952). More recent estimates of true dissolved iron concentrations, however, are considerably higher than those used by Harvey (Bryne and Kester, 1976) which casts some doubt on his conclusions. At present, there i s s t i l l l i t t l e known about the a b i l i t y of c o l l o i d a l iron ( i . e . c o l l o i d a l hydrated f e r r i c oxides) to supply iron to phytoplankton. The purpose of t h i s study was to determine whether c o l l o i d a l iron occurred associated with diatoms in the natural environment, and to investigate how i t s manner of preparation a f f e c t s i t s a b i l i t y to supply iron to phytoplankton. Diatoms co l l e c t e d from d i f f e r e n t environmental locations were thus examined by scanning electron microscopy (S.E.M.) for the presence of surface associated c o l l o i d a l 5 material. Where this material was found, the elemental composition was estimated with energy dispersive X-ray analysis (EDAX). In addition, laboratory studies were made using a bioassay organism to test the a b i l i t y of iron c o l l o i d s prepared in d i f f e r e n t ways to provide a source of b i o l o g i c a l l y available iron. The c o l l o i d a l forms tested were: freshly precipitated f e r r i c hydroxide, freshly precipitated f e r r i c hydroxide that was treated by heating and ageing (yet remained X-ray amorphous), and c r y s t a l l i n e goethite and hematite. 6 MATERIALS AND METHODS ; F i e l d Study Water samples were co l l e c t e d at a number of depths from stations in Indian Arm, the S t r a i t of Georgia, and Fitzhugh Sound as well as within the Fraser River s a l t wedge and plume (Fig. 1). The water was col l e c t e d in National Institute of Oceanography polypropylene samplers and then transferred to acid-cleaned 250 ml polypropylene bottles. Aliquots of the samples (50 ml) were immediately drawn into polypropylene syringes (see Appendix 1) and f i l t e r e d through 0.1 */m Nuclepore f i l t e r s (13 mm diameter). Five m i l l i t e r s of p r e f i l t e r e d (0.1 »m), g l a s s - d i s t i l l e d water was then passed through the sample f i l t e r to remove sa l t which would interfere with the S.E.M. examination of the sample. The f i l t e r s were then transferred to s t e r i l e p e t r i dishes, dried at room temperature, and frozen. For S.E.M. examination, the dried f i l t e r s were mounted d i r e c t l y on a metal stub with spectrographic graphite paint, sputtered with gold (ca. 100 angstrom coating) and examined with an Ortec scanning electron microscope. Where diatoms were found and p a r t i c u l a t e or c o l l o i d a l material was present on the frustules, the iron content of the material was estimated with energy dispersive X-ray analysis (Ortec). To l i m i t the electron beam exposure to the area of interest, the magnification was increased to maximum or u n t i l the c o l l o i d (or p a r t i c l e ) f i l l e d the f i e l d of view. The data are plotted as a spectrum of X-ray energies, and the peak height of a given energy l e v e l i s 7 Figure 1. F i e l d sampling locations. In Indian arm, f i l t e r samples were taken from water c o l l e c t e d from 5, 20, 50 and 200m at Ind. 2.0 and the depths of 5, 20, 50 and 175m from Ind. 1.5. In the S t r a i t of Georgia (Geo. 1748), water from 5, 20, 50 and 375m was sampled. At Fra. 1.5, samples were taken from 1 m while further upstream, water was sampled from the apex of the s a l t wedge. (The location of sampling varied due to movement of the wedge during the t i d a l cycle.) 9 p r o p o r t i o n a l t o t h e amount o f t h a t e l e m e n t i n t h e sample ( F i g . 2 ) . D i f f i c u l t i e s a r i s e w h i c h p r e v e n t t h e q u a n t i t a t i v e measurement of t h e e l e m e n t s p r e s e n t . These d i f f i c u l t i e s i n c l u d e i r r a d i a t i o n o f t h e s u r r o u n d i n g a r e a by e l e c t r o n beam s c a t t e r , X-r a y f l u o r e s c e n s e , d i f f e r i n g d e t e c t i o n c a p a b i l i t i e s d e p e n d i n g on t h e a t o m i c numbers, and a b s o r p t i o n of t h e r e l e a s e d X - r a y s by atoms w i t h i n t h e o b j e c t . In a d d i t i o n , b o t h t h e t h i c k n e s s and o r i e n t a t i o n of t h e o b j e c t w i t h r e s p e c t t o t h e d e t e c t o r a f f e c t s t h e X - r a y c o l l e c t i o n e f f i c i e n c y . Due t o t h e s e p r o b l e m s t h e t e c h n i q u e i s b e s t u s e d o n l y t o e s t i m a t e t h e e l e m e n t a l c o m p o s i t i o n of o b j e c t s . In view o f t h e l i m i t a t i o n s of t h e method, t h e f o l l o w i n g c r i t e r i a were u s e d t o i d e n t i f y i r o n - r i c h c o l l o i d a l m a t e r i a l : s p e c t r a o f c o l l o i d s of i n t e r e s t were compared w i t h t h e s p e c t r a o f t h e a d j a c e n t a r e a s of t h e f i l t e r o r f r u s t u l e s and when t h e FeKo peak h e i g h t s o f t h e c o l l o i d s were s u b s t a n t i a l l y h i g h e r t h a n t h a t o f t h e a d j a c e n t a r e a s , t h e c o l l o i d s were c o n s i d e r e d t o be i r o n - r i c h ( F i g . 3 ) . On two s e p a r a t e o c c a s i o n s where i r o n - r i c h c o l l o i d s were f o u n d a s s o c i a t e d w i t h f r u s t u l e s , t r a n s e c t s a c r o s s t h e f r u s t u l e s and c o l l o i d s were a n a l y z e d f o r i r o n c o n t e n t . In o r d e r t o t e s t i f t h e a s s o c i a t i o n of c o l l o i d a l m a t t e r w i t h d i a t o m f r u s t u l e s was a r e s u l t o n l y o f t h e c h e m i s t r y o f t h e s i l i c e o u s f r u s t u l e ( i . e . not c o n t r o l l e d by t h e o r g a n i s m ) , b o r o s i l i c a t e g l a s s p l a t e s were u s e d t o model t h e f r u s t u l e s u r f a c e . T h e s e g l a s s p l a t e s have a s i m i l a r s u r f a c e c h e m i s t r y t o t h a t o f t h e f r u s t u l e and p r e s e n t a smooth s u r f a c e where c o l l o i d a l m a t e r i a l c a n not be p h y s i c a l l y e n t r a p p e d . Water from 10 F i g u r e 2. X - r a y e n e r g y s p e c t r u m of a c o l l o i d a l p a r t i c l e c o l l e c t e d i n a f i e l d s ample. The h o r i z o n t a l a x i s r e p r e s e n t s t h e e n e r g y l e v e l s and t h e v e r t i c a l a x i s t h e number of X - r a y s m e a s u r e d . E a c h peak r e p r e s e n t s a s i n g l e e l e m e n t but i n some c a s e s m a s k i n g can o c c u r by o v e r l a p p i n g p e a k s . The p r e s e n c e o f two i r o n p e a k s , r e p r e s e n t i n g t h e FeKc and Kp X - r a y e n e r g i e s , i n d i c a t e a h i g h i r o n c o n t e n t . 11 Amount . C ' - N C L - A l - S i •:.::-'-ci m , - > - C d . • • * .. ..•'••••-Fe .:-:-Cu ~Zn 12 F i g u r e 3. D e t e r m i n a t i o n of i r o n - r i c h c o l l o i d s was done by c o m p a r i n g s p e c t r a o f t h e c o l l o i d ( l o w e r ) and a d j a c e n t a r e a s o f t h e f i l t e r o r f r u s t u l e ( u p p e r ) . C o l l o i d s h a v i n g d i f f e r e n c e s i n i r o n peak h e i g h t s s u c h as shown were c o n s i d e r e d t o be i r o n - r i c h . 14 the Fraser River salt wedge, which has abundant suspended solids (50 - 500 mg l " 1 ) , was pumped through a polypropylene tube with a p e r i s t a l t i c pump and passed continuously through a chamber containing v e r t i c a l l y mounted glass plates. (The plates were mounted v e r t i c a l l y to prevent p a r t i c l e s e t t l i n g . ) Both the chamber and the mounting stage were constructed of methyl methacrylate. After a period of exposure (about 2 hours) the glass plates were removed, dip-rinsed in f i l t e r e d (0.1 >*m) d i s t i l l e d water (to remove s a l t ) , placed in s t e r i l e dust-free p e t r i dishes, and allowed to dry. The plates were then analyzed in the same manner as the f i l t e r e d samples. Laboratory Study (i) Bioassays The bioassays were conducted to test the a b i l i t y of di f f e r e n t c o l l o i d a l iron stocks to support organism growth. The marine diatom T h a l l a s s i o s i r a pseudonana (Hustedt), Hasle and Heimdal (WHOI clone 3-H), from the N.E. P a c i f i c culture c o l l e c t i o n (the University of B r i t i s h Columbia) was used in a l l bioassays. Both the stock cultures and bioassays were conducted in batch using the a r t i f i c i a l sea water medium 'AQUIL' (Morel et a l . , 1979). A l l cultures were grown at 15°C and illuminated under Cool White twisted fluorescent l i g h t s on a 16:8 hour l i g h t dark cycle at a radiation l e v e l of 95 </Ein n r 2 s" 1. C e l l inocula from iron-limited, EDTA-free, stock cultures were added to the bioassay flasks to give i n i t i a l c e l l concentrations of 1000-2000 15 c e l l s ml" 1. C e l l concentrations were monitored da i l y during the bioassay with a Coulter Counter (model Z f ) . The length of the bioassays varied between 5 and 28 days depending on the nature of the experiment. Differences in populations were considered s i g n i f i c a n t i f they d i f f e r e d by two standard deviations. ( i i ) Medium Preparation The standard ocean water (SOW) used to prepare the bioassay medium was prepared in 20 l i t e r quantities by the addition of sa l t s to glass d i s t i l l e d water (see Appendix 2). The SOW, without added nutrients or metals, had undetectable concentrations of Fe, Cu, Cd, Mn, V, Ni, and Zn (measured by dir e c t i n j e c t i o n graphite furnace atomic absorption spectrophotometry). Four l i t e r s of medium were normally prepared at one time for each bioassay to ensure comparable medium chemistry throughout the fl a s k s . The AQUIL nutrient solutions (N0 3~, PO u 3" and Si(OH)„ passed through a column containing Chelex-100) and the metal mix (Cu, Mn, Zn, Co, Mo but excluding Fe and EDTA) were f i r s t added to the SOW. This mixture was then bubbled with f i l t e r e d (0.4 »m), acid-cleaned (6N H 2SO a) carbon dioxide to atta i n a pH of 5.5 (to prevent s a l t p r e c i p i t a t i o n during autoclaving). The medium was then autoclaved (121°C, 30 mins) in a 4 - l i t e r glass aspirator bottle. Upon cooling, the pH was measured and, i f necessary, the medium was bubbled with clean a i r (as above) to pH 8.0. Aliquots of the medium (250 ml) were then transferred to previously autoclaved 500 ml polycarbonate Erlynmeyer flasks and the c e l l inoculations were 16 added. Additions of the prepared iron stocks (see below) were made either before or after autoclaving of the medium depending on the experiment. In a l l experiments, iron was added to give a t o t a l concentration of 4.5 x 10~7M. A l l additions and inoculations were made in a class 100 laminar flow hood that had a l l possible metal parts replaced with polypropylene. The bioassay apparatus was soaked in 6N HCl and rinsed three times with glass d i s t i l l e d water prior to use. Three replicates of each test were run and every bioassay series included three flasks in which no iron was added. The pH of the individual cultures was measured i n i t i a l l y (7.9 - 8.1) and at the end of the bioassays (8.7 - 9.5). (The pH increase was due to b i o l o g i c a l action.) Bacterial contamination was tested with t r y p t i c soy broth and only low levels were infrequently found, presumably from the non-autoclaved iron stocks. ( i i i ) Iron Stock Preparations and Treatments Fresh c o l l o i d a l f e r r i c hydroxide stocks were prepared at a concentration of 4.5 x 10~"M by the addition of F e C l 3 to glass d i s t i l l e d water. No adjustments for pH, which f e l l to 2.5, were made. The rapid formation of polynuclear f e r r i c c o l l o i d s in such f e r r i c s a l t solutions i s well documented (Murphy, e_t a l (1976), van der Giessen (1968), Atkinson, et a l (1977)). C o l l o i d size was not determined but Murphy e_t a l (1976) have estimated the size to be 2-16 nm after 24 hrs of ageing. The c o l l o i d s were added to the culture media either d i r e c t l y after stock 1 7 preparation or after various treatments of the stock including: a) autoclaving (121°C, 15 psi) for 15 mins., 30 mins. and 1, 4, 12, and 24 hour periods, b) heating to 100°C for 5, 10 and 15 minute in t e r v a l s , c) heating at 50°C, 70°C, and 90°C for 5 minutes and d) ageing at room temperature for one week and three months. A Fe-SOW c o l l o i d a l stock was prepared as above, adjusted to the pH of the c o l l o i d stock in d i s t i l l e d water and added to cultures either d i r e c t l y or after 15 minutes of autoclaving. Fe-EDTA stock solutions were prepared by the addition of f e r r i c chloride (4.5 x lO _ aM) and EDTA (5.0 x 10"3M) to glass d i s t i l l e d water. The presence of the chelator EDTA in the iron stock prevents polynuclear c o l l o i d formation by the chelation of the free iron (Lewin and Chen, 1973). The Fe-EDTA stocks were added to cultures either 1-2 days after preparation (to allow equil i b r a t i o n ) or after the treatments of autoclaving (15 mins) or ageing (18 months at 10°C). A c o l l o i d a l goethite (oFeOOH) stock was prepared following the method of Forbes et a_l. (1974) . An a c i d i f i e d f e r r i c n i t r a t e solution (pH 1.9) was aged for 48 hours at room temperature and then heated at 60°C (pH 11.7) for three days. The prec i p i t a t e thus formed was confirmed to be goethite by X-ray d i f f r a c t i o n . The average c o l l o i d a l diameter in the polydispersed sol was determined with S.E.M. and found to be about 0.8 rm. The c o l l o i d a l goethite aggregates were rinsed with d i s t i l l e d water and stored at 10°C in a polyethylene bottle u n t i l use. (Iron loss to the container walls was negligible.) To determine the volume of the iron stock to be added to cultures, the iron 18 concentration of an a c i d i f i e d (pH 1.0), heated (70°C for 2 days) aliquot of the goethite stock was measured with flame atomic absorption (Techtron AA-4). A c o l l o i d a l hematite (aFe 20 3) stock was prepared by the method of Matijevic and Scheiner (1978). An ac i d i c f e r r i c n i t r a t e solution was heated for three days at 100°C to create a monodispersed c o l l o i d a l s o l . X-ray d i f f r a c t i o n confirmed that the p r e c i p i t a t e produced was hematite. The size of the monodispersed sol was determined to be 0.1 nm with S.E.M.. The aggregates were rinsed with g l a s s - d i s t i l l e d water and stored at 10°C in a polyethylene bottle u n t i l use. The iron concentration was determined in the same fashion as with the goethite stock. Both the goethite and hematite stocks were disaggregated in an ultrasonic bath before addition to culture media. (iv) Physical and Chemical Analysis The physical and chemical properties of the fresh and autoclaved c o l l o i d a l iron stocks were compared by a number of methods. These included X-ray d i f f r a c t i o n , thermal gravimetry, Mossbauer spectroscopy, gel f i l t r a t i o n , and by measuring the r e l a t i v e d i s s o l u t i o n rates. For these comparisons the period of autoclaving was 15 minutes unless otherwise noted. For the X-ray d i f f r a c t i o n analysis, the autoclaved and non-autoclaved solutions were adjusted to pH 7.5 and rapidly mixed to produce aggregates by c o l l o i d a l coagulation. (This i s o e l e c t r i c pH was determined experimentally.) After the aggregates had se t t l e d they were extracted by pipet, deposited 1 9 on an X-ray s l i d e and dried at room temperature. The X-ray d i f f r a c t i o n patterns of these dried samples was scanned between 10 and 50 degrees 20 using Mn f i l t e r e d FeKo radiation. Fresh and autoclaved (15 min, 30 min, 1 hr, 4 hr, 12 hr, and 24 hr) c o l l o i d a l iron stocks were also scanned by camera techniques using 6 hour exposure periods. Thermal gravimetric analysis was performed on both fresh and autoclaved c o l l o i d a l iron stocks using subsamples of the batches previously prepared for X-ray d i f f r a c t i o n analyses. The samples were held at 115°C for 4 mins to remove moisture followed by a 40°C min - 1 temperature increase to 700°C. The percent weight change of the sample was recorded. For Mossbauer spectroscopy, the autoclaved and non-autoclaved c o l l o i d a l iron stocks were adjusted to their i s o e l e c t r i c pH (ca. 7.5) and rapidly mixed to produce aggregates of iron that were allowed to s e t t l e . The aggregates were repeatedly rinsed by centrifugation with d i s t i l l e d water to remove the chloride ion. The supernatant was discarded and the p e l l e t s transferred to Mossbauer c e l l s . These c e l l s were then sealed with- epoxy and quick frozen in l i q u i d nitrogen. The spectrum of each sample was then measured for a 24 hour period at l i q u i d nitrogen temperatures (see appendix 4). To determine the gel f i l t r a t i o n rates, a polycarbonate column (60 cm) was f i l l e d with Sephadex G-10. Because this gel readily adsorbs iron from solution, i t was f i r s t treated with a 1 M zirconium solution to block the available adsorption s i t e s of the gel and thus permit the passage of iron (Murphy et a l , 20 1975). A 1.0 ml aliquot of the iron stock was introduced at the top of the gel and eluted with a 1 M zirconium solution at a flow rate of 6.0 ml h r - 1 . Three m i l l i t e r fractions were col l e c t e d and, after a c i d i f i c a t i o n (1% HCl), the iron concentrations were determined by direct i n j e c t i o n graphite furnace atomic absorption spectrophotometry. To measure the c o l l o i d a l d i s s o l u t i o n rates, the dissolved f e r r i c iron was measured over time with the colorimetric chelating reagent s u l p h o s a l i c y l i c acid by the method of Langford et al_ (1977). As free f e r r i c ions are chelated by this reagent, the equilibrium between dissolved and c o l l o i d a l iron is disrupted, causing the s o l i d to dissolve. The rate of this solution under a given set of conditions i s in part related to the thermodynamic s t a b i l i t y of the c o l l o i d s and i s seen to decrease with increasing s t a b i l i t y . Thus, the rates of colour development in the autoclaved and non-autoclaved iron stocks indicate the r e l a t i v e s t a b i l i t i e s of the c o l l o i d a l matter. In thi s experiment, the pH of the s u l p h o s a l i c y l i c acid solution was f i r s t adjusted to that of the iron stocks (2.5) and then the solution was mixed (1:1) with the sample in a 10 cm spectrophotometer c e l l . The c e l l was immediately placed in a spectrophotometer (Bausch and Lomb 2000) and the absorbance at 500 nm measured for 30 mins. 21 RESULTS F i e l d Study . C o l l o i d a l material was found at a l l of the sampling locations (Fig. 1). At those stations where samples were taken from a series of depths (Ind. 0.0, Ind. 1.5, Ind. 2.0 and Geo 1748), the amount of c o l l o i d a l material retained on the 0.1 yin pore diameter f i l t e r was found to increase with increasing depth. One exception to th i s was Fra. 1.5 where the maximum was at the surface within the water of the Fraser River plume. Extremely high levels of c o l l o i d a l and par t i c u l a t e material were found in the Fraser River salt wedge. At each station some of the c o l l o i d a l material was found to be i r o n - r i c h . For example, about 26% of the c o l l o i d a l material examined in the bottom waters of Indian Arm (at Ind. 2.0) had substantial amounts of iron. Diatoms were found in the upper part of the water column at every station and in the bottom waters of Indian Arm and the Fraser River (in the sa l t wedge). Some diatom frustules within the Fraser River plume, the Fraser rive r s a l t wedge and in the deep waters of Indian Arm had c o l l o i d a l matter associated with their surfaces. In many cases th i s c o l l o i d a l matter contained substantial amounts of iron. Figures 4-7 show some examples of ir o n - r i c h . c o l l o i d s associated with diatoms. Quantitative estimates of th i s association were not made. The iron content along a transect crossing a diatom frustule towards an i r o n - r i c h c o l l o i d i s shown in F i g . 8. The euphotic zones within Indian 22 F i g u r e 4. I r o n - r i c h c o l l o i d : d i a t o m a s s o c i a t i o n was f o u n d on a number of o c c a s i o n s . T hese s p e c t r a were t a k e n from t h e a r e a s shown i n t h e p h o t o g r a p h . 23 -•--Ha •••-Na .V>-P - S - C I S . - • • • - c i - F e - F e •/ - G e - > : - - G e u r e 5. A n o t h e r example of i r o n - r i c h c o l l o i d : d i a t o m a s s o c i a t i o n . T h e s e s p e c t r a were t a k e n from t h e shown i n t h e p h o t o g r a p h . 25 26 F i g u r e 6. A n o t h e r example o f i r o n - r i c h c o l l o i d : d i a t o m a s s o c i a t i o n . T h e s e s p e c t r a were t a k e n from t h e a r e a s shown i n t h e p h o t o g r a p h . 28 F i g u r e 7. A n o t h e r example o f i r o n - r i c h c o l l o i d r d i a t o m a s s o c i a t i o n . T h e s e s p e c t r a were t a k e n form t h e a r e a s shown i n t h e p h o t o g r a p h . a n ? ) . 29 -Na Si */ ' . .•••-ci ••••-Na - S i "Ail • ••--ci -Ge -Ge 30 F i g u r e 8. I r o n c o n t e n t a l o n g a t r a n s e c t o f a c o l l o i d a s s o c i a t e d f r u s t u l e was m e a s u r e d t o a t t e m p t t o d e t e r m i n e w h e t h e r c o l l o i d a l d i s s o l u t i o n was o c c u r r i n g . The s p e c t r a were measured from 1 t o 3 t o w a r d s t h e c o l l o i d . T h e r e a p p e a r s t o be a s l i g h t i n c r e a s e i n t h e i r o n c o n t e n t t o w a r d s t h e c o l l o i d , however, t h e d i f f e r e n c e s a r e p r o b a b l y o n l y due t o e l e c t r o n beam s c a t t e r d u r i n g measurement. 32 Arm, the S t r a i t of Georgia, and F i t z h u g h Sound co n t a i n e d many diatoms; however, no c o l l o i d a l a s s o c i a t i o n was observed. A d d i t i o n a l l y , l i t t l e c o l l o i d a l matter was present on the f i l t e r s from these areas. As mentioned i n the m a t e r i a l s and methods s e c t i o n , b o r o s i l i c a t e g l a s s p l a t e s were exposed to sample water and then examined by S.E.M. to determine the degree of c o n t r o l the s i l i c e o u s f r u s t u l e might e x e r t over the apparent c o l l o i d a l a s s o c i a t i o n . C o l l o i d a l m a t e r i a l was found to be a s s o c i a t e d with the p l a t e s and i n some cases these c o l l o i d s were found to be i r o n - r i c h . L aboratory Study Bioassays The growth in c u l t u r e s s u p p l i e d with f r e s h c o l l o i d a l i r o n and iron-EDTA stocks was found not to be s i g n i f i c a n t l y d i f f e r e n t demonstrating that EDTA d i d not enhance the supply of i r o n from f r e s h l y p r e c i p i t a t e d c o l l o i d a l hydroxide to T. psuedonna ( F i g . 9 ) . The same s e r i e s of experiments was run using glutamic a c i d with s i m i l a r r e s u l t s . To determine i f the i r o n remained a v a i l a b l e with no c h e l a t i n g agent i n the medium duri n g a u t o c l a v i n g , f r e s h c o l l o i d a l i r o n was added to the medium before a u t o c l a v i n g . Growth i n these c u l t u r e s was found to be no d i f f e r e n t than i n c u l t u r e s with f r e s h c o l l o i d a l i r o n added a f t e r a u t o c l a v i n g ( F i g . 1 0 ) . Since EDTA was shown not to i n c r e a s e the i r o n a v a i l a b i l i t y , c u l t u r e s having f r e s h c o l l o i d a l i r o n added ure 9. C e l l growth with fresh c o l l o i d a l (•) and iron-EDTA stocks ( A ) added afte r medium autoclaving i s shown. The X-axis represents days of the bioassay and the Y axis the log of the c e l l number ml" 1. One S.D. was less than the symbol s i z e . Growth in the two tests was not s i g n i f i c a n t l y d i f f e r e n t . 35 F i g u r e 10. C e l l g r o w t h w i t h f r e s h c o l l o i d a l s t o c k a d d i t i o n b e f o r e medium a u t o c l a v i n g ( • ) was f o u n d n o t t o be s i g n i f i c a n t l y d i f f e r e n t f r o m a d d i t i o n s a f t e r medium a u t o c l a v i n g ( A ) . One S.D. was l e s s t h a n t h e s y m b o l s i z e . 37 after medium autoclaving were chosen as the control for subsequent experiments. The fresh c o l l o i d a l iron stock was then autoclaved, heated, and aged to determine what treatments of the stock might reduce the a v a i l a b i l i t y of the iron. Autoclaving of the fresh c o l l o i d a l iron stock before addition to the cultures caused a reduction in the rate of organism growth (Fig. 11). This growth reduction did not vary s i g n i f i c a n t l y with d i f f e r e n t periods of autoclaving (Fig. 12). The growth l i m i t i n g factor, as determined by the addition of both nutrients (N0 3" , P0 f t 3~ , and Si(OH) a ) and metals (Cu, Mn, Zn, Co, Mo, and Fe), was found to be iron. To determine i f the increased pressure during autoclaving (15 p s i ) , contributed to t h i s change in the iron stock, aliquots of a fresh c o l l o i d a l iron stock were heated to 100°C (at atmospheric pressure) for 5, 10 and 15 minute intervals before addition to cultures. The growth in these experiments did not d i f f e r s i g n i f i c a n t l y and was similar to that of cultures supplied with autoclaved iron stock (Fig. 13). Fresh c o l l o i d a l iron was again the only nutrient addition that increased organism growth. C e l l numbers in cultures supplied with the iron stocks heated for 5 and 10 minutes were found to increase very slowly over a period of 21 days after growth had ceased in cultures without added iron and in the control. To provide a better indication of the degree of heating required to cause th i s change in iron a v a i l a b i l i t y , aliquots of a fresh c o l l o i d a l iron stock, prepared at room temperature (ca. 21°C), were heated for 5 minute intervals at 50°C, 70°C and 90°C 38 Figure 11. Growth in cultures supplied with autoclaved c o l l o i d a l stock (•. ) was s i g n i f i c a n t l y less than that of the non-autoclaved fresh c o l l o i d a l iron ( A ). This reduction in growth was found to be due to iron l i m i t a t i o n (by nutrient addition). One S.D. was less than the symbol s i z e . C e l l Numbers/ml' 103 104 10s 10' 40 F i g u r e 12. F r e s h c o l l o i d a l s t o c k s were a u t o c l a v e d f o r 15 m i n u t e s ( A ) , 4 h o u r s ( O K and 24 h o u r s (W ) p e r i o d s b e f o r e a d d i t i o n t o c u l t u r e s . G rowth i n t h e s e c u l t u r e s was s i g n i f i c a n t l y below t h e c o n t r o l (• ) but n o t s u b s t a n t i a l l y d i f f e r e n t f r o m one a n o t h e r - Bar r e p r e s e n t s one S.D. where g r e a t e r t h a n symbol s i z e . 41 ure 13. Growth in cultures supplied with iron stocks heated (100°C) for 5 mins. (A) , 10 mins^ ( O h and 15 mins. (tf) was s i m i l a r and s i g n i f i c a n t l y less than the control ( • ) . Growth in these cultures, however, continued during a monitoring period of 28 days. _ Bar represents one S.D. where greater than symbol s i z e . 44 before being added to the cultures. Cultures supplied with the aliquot heated to 50°C had less growth than the control, but s i g n i f i c a n t l y better growth than cultures supplied with the aliquots heated to 70°C and 90°C (Fig. 14). There was no s i g n i f i c a n t difference in growth between the experiments at the two higher temperatures. Since reduced organism growth was not found when the iron was autoclaved in the culture medium (Fig. 10), the effect of ionic strength on the a v a i l a b i l i t y of the c o l l o i d a l iron was investigated. A Fe-SOW c o l l o i d a l stock was autoclaved (15 min) before being added to cultures. Growth in these cultures was found not to vary s i g n i f i c a n t l y from the control and was substantially greater than the autoclaved F e - d i s t i l l e d water stock (Fig. 15). To determine the e f f e c t of ageing on the a v a i l a b i l i t y of iron, fresh c o l l o i d a l iron stocks (in d i s t i l l e d water at room temperature and pH 2.5) were aged for one week and three months before being added to cultures. Three, months of ageing resulted in a s i g n i f i c a n t growth reduction while one week of ageing did not a f f e c t growth (Fig. 16). The growth pattern of the cultures supplied with three month old iron stock was similar to those of cultures supplied with an autoclaved iron stock (Fig. 17). Additions of EDTA to the cultures did not enhance growth. Trace metal and nutrient additions demonstrated that the l i m i t i n g factor was iron. To determine how the presence of a chelating agent in the iron stock affected the change in iron a v a i l a b i l i t y , fresh iron-ure 14. Growth in cultures supplied with an iron stock heated to 50°C for 5 minutes ( A ) was s i g n i f i c a n t l y below that of the control ( • ) but better than those cultures supplied with iron stocks heated to 70°C ( O ) and 90°C ( A ) . Bar represents one S.D.1 where greater than symbol s i z e . Cell Numbers/ml id? io4 io5 i'o 47 F i g u r e 15. Growth i n c u l t u r e s s u p p l i e d w i t h d i s t i l l e d w a t e r a u t o c l a v e d i r o n s t o c k ( • ) and a u t o c l a v e d Fe-SOW s t o c k ( A ) were compared t o t h e c o n t r o l ( <0>) . The i n c r e a s e d i o n i c s t r e n g t h o f t h e Fe-SOW s t o c k a p p e a r e d t o p r e v e n t t h e change i n a v a i l a b i l i t y . Bar r e p r e s e n t s one S.D. where g r e a t e r t h a n symbol s i z e . C e l l Numbers/ml' IO3 IO4 10s io6 u r e 16. The e f f e c t o f i r o n s t o c k a g e i n g on c e l l g r o w t h was f o u n d t o v a r y w i t h t h e p e r i o d o f a g e i n g . One week o f a g e i n g a t 21°C ( A ) was not s i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l ( • ) w h i l e 3 months o f a g e i n g ( O ) p r o d u c e d a much l o w e r r a t e of g r o w t h . The r e d u c e d g r o w t h was a g a i n f o u n d t o be due t o i r o n l i m i t a t i o n . One S.D. was l e s s t h a n t h e symbol s i z e . ure 17. The growth patterns of cultures supplied with iron stocks heated at 100°C for 5 minutes ( A ) and aged at 2l°Cfor 3 months ( • ) , were found to be s i m i l a r . The growth in these tests are expressed as a percent of the control growth over time. ' P e r c e n t g r o w t h 0 20 40 60 BO 100 53 EDTA stocks (Fe: 4.5 x lO-ftM, EDTA: 5.0 x 10-3M) were autoclaved (15 mins.) or aged (14 months at 10°C) before addition to the cultures. (Because of a report by Lockhart and Blakely (1975) on the degradation of EDTA, the percent loss of EDTA during the autoclaving and the bioassays was estimated (appendix 3) and found not to be s i g n i f i c a n t . ) Growth in cultures supplied with these iron stocks was not s i g n i f i c a n t l y d i f f e r e n t from each other or from that of cultures supplied with fresh c o l l o i d a l iron (Fig. 18). C o l l o i d a l goethite and hematite were tested to determine their a b i l i t y to supply iron to the organism. The growth in cultures supplied with these two forms of iron was similar but s i g n i f i c a n t l y below that of cultures supplied with freshly prepared c o l l o i d a l iron (Fig. 19). The growth patterns were also similar to those of cultures with no added iron. Additions of EDTA to the cultures did not enhance growth. Growth l i m i t a t i o n was again found to be due to iron l i m i t a t i o n . Using a l i g h t microscope, both c o l l o i d a l goethite and hematite were observed to be attached to the l i v i n g diatom c e l l s in the test cultures. In each bioassay series there was always substantial growth for the f i r s t 2-3 days in cultures that had no added iron. However, cultures supplied with autoclaved, heated and three month old iron stocks had s i g n i f i c a n t l y lower growth when compared with those cultures without added iron (Fig. 20). It therefore appears that the addition of these stocks actually reduced the available concentration of iron. 54 Figure 18. When cultures were supplied with Fe-EDTA stocks that were autoclaved ( A ) or. aged for 18 months at 21°C < C\) , the growth was not s i g n i f i c a n t l y d i f f e r e n t from the control ( • ) . One S.D. was less than the symbol s i z e . 56 F i g u r e 19. G r o w t h i n c u l t u r e s s u p p l i e d w i t h c o l l o i d a l g o e t h i t e ( O ) and h e m a t i t e ( A ) was f o u n d t o be s i g n i f i c a n t l y below t h a t o f t h e c o n t r o l ( • ) . Reduced g r o w t h was f o u n d t o be due t o i r o n l i m i t a t i o n . Bar r e p r e s e n t s one S.D. where g r e a t e r t h a n symbol s i z e . 58 Figure 20. Growth in cultures without iron addition (O) occurred for the f i r s t 2-3 days of the bioassays but was s i g n i f i c a n t l y less than the control ( O ) . Cultures supplied with autoclaved c o l l o i d a l stock ( A ) had s i g n i f i c a n t l y lower growth than the cultures without added iron. Bar represents one S.D. where greater than symbol s i z e . 60 A n a l y s i s of t h e A u t o c l a v e d I r o n S t o c k To d e t e r m i n e how a u t o c l a v i n g c h a n g e d t h e c o l l o i d a l i r o n s t o c k , t h e a u t o c l a v e d and f r e s h c o l l o i d a l i r o n s t o c k s were examined u s i n g X - r a y d i f f r a c t i o n , M o ssbauer s p e c t r o s c o p y , t h e r m a l g r a v i m e t r y , g e l f i l t r a t i o n and d i s s o l u t i o n r a t e measurements. No a n a l y s i s was p e r f o r m e d on t h e t h r e e month o l d i r o n s t o c k b e c a u s e o f i n s u f f i c i e n t sample q u a n t i t i e s . (i_) X - r a y d i f f r a c t i o n No c r y s t a l l i n i t y was d e t e c t a b l e i n t h e f r e s h l y p r e p a r e d o r a u t o c l a v e d (30 mins.) i r o n s t o c k s . M i c r o c r y s t a l l i t e s , however, were o b s e r v e d i n t h e a u t o c l a v e d s t o c k u s i n g a p e t r o g r a p h i c m i c r o s c o p e w i t h c r o s s e d N i c h o l s . A l t h o u g h c r y s t a l l i n i t y was n o t d e t e c t e d by X - r a y d i f f r a c t i o n , t h e i r o n i n t h e sample a p p e a r e d t o be b i r e f r i n g e n t . ( M i c r o c r y s t a l l i t e s c o u l d r e m a i n u n d e t e c t e d w i t h t h e X - r a y t e c h n i q u e s used.) When i r o n s t o c k s were a u t o c l a v e d f o r d i f f e r e n t l e n g t h s o f t i m e (15 min, 30 min, 1 h r , 4 h r , 12 h r , and 24 h r ) , a d i f f r a c t i o n p a t t e r n was d e t e c t e d u s i n g a camera t e c h n i q u e i n t h e i r o n s t o c k a u t o c l a v e d f o r 24 h r ( F i g . 2 1 ) . ( i i ) M o s s b a u e r s p e c t r o s c o p y M o s sbauer s p e c t r o s c o p i c a n a l y s i s d i d not d e m o n s t r a t e any s i g n i f i c a n t d i f f e r e n c e between t h e a u t o c l a v e d and n o n - a u t o c l a v e d s t o c k s . The h y p e r f i n e s p l i t t i n g and t h e c h e m i c a l i s o m e r s h i f t o f t h e two s t o c k s were i d e n t i c a l w i t h i n t h e e r r o r o f t h e t e c h n i q u e 61 F i g u r e 21. The X - r a y d i f f r a c t i o n p a t t e r n o f t h e i r o n s t o c k a f t e r a 24 hour p e r i o d o f a u t o c l a v i n g . The bands on t h e f i l m r e p r e s e n t t h e d i f f r a c t i o n p a t t e r n o f a c r y s t a l s t r u c t u r e . No bands were d e t e c t e d a f t e r s h o r t e r p e r i o d s of a u t o c l a v i n g . 62 o 63 ( t a b l e 1 ) . ( F o r a b r i e f d e s c r i p t i o n o f M o s s b a u e r s p e c t r o s c o p y see a p p e n d i x 4.) T h e r e was, however, a s l i g h t i n c r e a s e i n t h e peak w i d t h ( r ) o f t h e a u t o c l a v e d s t o c k . T a b l e 1. M o s s b a u e r a n a l y s i s of t h e a u t o c l a v e d and f r e s h i r o n s t o c k . The e r r o r o f t h e a n a l y s i s i s a b o u t .02. I r o n T ype S p l i t t i n g ( a ) S h i f t ( A ) Peak W i d t h ( V ) ( r,) ( r a ) F r e s h c o l l o i d a l .665 .428 .634 .525 i r o n A u t o c l a v e d .673 .441 .775 .648 c o l l o i d a l i r o n ( i i i ) T h e r m a l q r a v i m e t i c a n a l y s i s The p e r c e n t w e i g h t change o f t h e a u t o c l a v e d and non-a u t o c l a v e d c o l l o i d a l i r o n s t o c k s upon h e a t i n g i s shown i n F i g . 22. The n o n - a u t o c l a v e d c o l l o i d a l i r o n was f o u n d t o have a h i g h e r p e r c e n t w e i g h t l o s s t h a n t h e a u t o c l a v e d m a t e r i a l . The non-a u t o c l a v e d sample had a s t e a d i l y d e c r e a s i n g p e r c e n t w e i g h t l o s s w i t h i n c r e a s i n g t e m p e r a t u r e , w h i l e t h e a u t o c l a v e d sample had a r a p i d w e i g h t l o s s between 450°C and 5 0 0 ° C . B o t h samples had no f u r t h e r w e i g h t change above 500°C. 64 F i g u r e 22. T h e r m a l g r a v i m e t r i c a n a l y s i s . The p e r c e n t w e i g h t l o s s , t h r o u g h t h e t e m p e r a t u r e range t e s t e d , i s shown f o r t h e f r e s h ( o ) and a u t o c l a v e d ( • ) i r o n s t o c k s . The r e s u l t s show t h a t t h e a u t o c l a v e d s t o c k had a l o w e r d e g r e e of p e r c e n t w e i g h t l o s s w h i c h s u g g e s t s a l o w e r d e g r e e of h y d r a t i o n . In a d d i t i o n , t h e a u t o c l a v e d i r o n s t o c k had a r a p i d w e i g h t l o s s between 450°C and 500°C. 66 ( i v ) Gel f i l t r a t i o n The r e s u l t s of t h i s a n a l y s i s show t h a t , with a u t o c l a v i n g , the c o l l o i d a l s i z e (or molecular weight) of the p a r t i c l e s was i n c r e a s e d ( F i g . 23). T h i s i n c r e a s e c o u l d be due to e i t h e r c o a g u l a t i o n of the e x i s t i n g p o l y c a t i o n i c c o l l o i d s or a d i s s o l u t i o n - r e p r e c i p i t a t i o n p r o c e s s . (v) D i s s o l u t i o n r a t e s The d i s s o l u t i o n rates, of the c o l l o i d s i n the two stocks were compared by c o l o r i m e t r i c measurement of the d i s s o l v e d f e r r i c i r o n c o n c e n t r a t i o n s over time. The absorbance change of the two samples over a 30 minute i n t e r v a l i s shown in F i g . 24. The non-autoclaved i r o n stock had a higher i n i t i a l absorbance and a g r e a t e r r a t e of i n c r e a s e than that i n the autoclaved stock. Absolute c o n c e n t r a t i o n s were not c a l c u l a t e d s i n c e the experiment was used only to i l l u s t r a t e a d i f f e r e n c e between the i r o n s t o c k s . g u r e 23. C o m p a r i s o n o f t h e g e l f i l t r a t i o n r a t e s . The g e l f i l t r a t i o n r a t e o f t h e f r e s h c o l l o i d a l s t o c k ( A ) was m e a s u r e d a n d compared t o t h a t o f t h e a u t o c l a v e d c o l l o i d s t o c k ( • ) ' . The r e s u l t s a r e p l o t t e d as i r o n c o n c e n t r a t i o n v s volume e l u t e d f r o m t h e c o l u m n . The g e l bed volume was d e t e r m i n e d t o be 15 m i l l i t e r s . The a u t o c l a v e d s t o c k was f o u n d t o p a s s d i r e c t l y t h r o u g h t h e column i n d i c a t i n g t h a t t h e c o l l o i d s were t o o l a r g e t o e n t e r t h e g e l b e a d s . However, t h e n o n - a u t o c l a v e d s t o c k p a s s e d s l o w e r t h r o u g h t h e column p r e s u m a b l y b e c a u s e t h e c o l l o i d s p a s s e d t h r o u g h t h e g e l b e a d s . T h e s e r e s u l t s s u g g e s t t h a t t h e c o l l o i d a l s i z e o f t h e f r e s h i r o n s t o c k i n c r e a s e d upon a u t o c l a v i n g . concentration (ppm) 0 1 2 3 4 5 6 7 ure 24. Comparison of the d i s s o l u t i o n rates . The d i s s o l u t i o n rates of autoclaved (•) and fresh c o l l o i d a l stocks ( A ) were measured c o l o r i m e t r i c a l l y over 30 mins and are plotted as absorbance over time. The results show that the d i s s o l u t i o n rate of the fresh c o l l o i d a l stock i s much more rapid than that of the autoclaved stock. 70 Time (mins) 71 DISCUSSION F i e l d Study Colloids, were found to be associated with diatom frustules in three l o c a l i t i e s sampled: the bottom waters of Indian Arm (Ind 2.0), the surface water in the Fraser River plume (Fra 1.5) and the Fraser River s a l t wedge. The water in these l o c a l i t i e s was similar in that each contained abundant c o l l o i d a l material. In some cases the associated c o l l o i d s were i r o n - r i c h . Although th i s association was not found to be ubiquitous among the l o c a l i t i e s sampled, the results of the glass plate experiments suggest that i t i s possible for any frustule surface to become coated by c o l l o i d a l material. Factors that could have contributed to the absence of observed c o l l o i d a l association with diatom frustules in the surface waters of Indian Arm, the St r a i t of Georgia and Fitzhugh Sound include the lower colloid:diatom ratios in these areas and the limited resolution of the S.E.M.. (Although the S.E.M. is capable of very high resolution, i t i s greatly decreased with the lower beam current used in this case to reduce the damage to the b i o l o g i c a l material.) It i s possible that the observed colloid-diatom association, in areas with abundant c o l l o i d a l material, may have been an a r t i f a c t of the f i l t r a t i o n process (by s e t t l i n g of c o l l o i d a l material onto the f r u s t u l e s ) . In these l o c a l i t i e s , however, c o l l o i d a l material was found associated with areas of diatom frustules not exposed to p a r t i c l e s e t t l i n g from above 72 ( i . e . underneath frustule overhangs). In addition, exposed surfaces of frustules were in some cases found to be 'material free', yet the surrounding area of the f i l t e r contained large amounts of c o l l o i d a l material. These observations suggest that at least some of the associated c o l l o i d s are not a r t i f a c t s and that c o l l o i d a l material i s in fact associated with diatoms in the natural environment. This i s supported by observations of surface associated c o l l o i d a l material in laboratory cultures (Harvey 1937, Wells, Honours thesis and present study). Results from the glass plate experiments suggest that the association of c o l l o i d a l material with diatoms need not be controlled by the diatom and may be the result of a passive process. However, organisms without s i l i c e o u s frustules may behave d i f f e r e n t l y . Massalski and Leppard (1979) observed the association of clay p a r t i c l e s with freshwater algae and bacteria and found that f i b r i l l a r c o l l o i d s , produced within the c e l l , acted to bridge the clays to the c e l l surface. Such an association between the c o l l o i d and the c e l l would appear to be a c t i v e l y controlled by the c e l l . At present, the role of the c o l l o i d a l association with organisms is unknown but i t has been suggested that i t is of n u t r i t i o n a l value (Harvey 1937, Goldberg 1952). However, i t remains to be shown that metals within c o l l o i d s are available to organisms. The bulk of the laboratory work in this study was directed towards answering t h i s question. If the i r o n - r i c h c o l l o i d a l material associated with diatoms was being u t i l i z e d , i t i s prerequisite that d i s s o l u t i o n must occur before uptake. Thus, a r e - d i s t r i b u t i o n of iron across the 73 frustule surface from the s o l u b i l i z a t i o n of the c o l l o i d might be expected. In fact, there does appear to be a s l i g h t gradational increase in iron content across the frustule towards the c o l l o i d (Fig 8), however the difference i s notygreat and could be an a r t i f a c t of the electron beam scatter. It i s apparent that EDAX does not have the resolution necessary to define these small differences i f they e x i s t . Bioassays Previously published reports have suggested that metal chelating agents such as EDTA are required in phytoplankton cultures to achieve optimal growth (Provasoli, 1963, Canterford, 1979). Chelating agents are believed to enhance growth by preventing p r e c i p i t a t i o n and maintaining the iron in a soluble state. As a re s u l t , i t was desirable to exclude chelators when testing the a b i l i t y of c o l l o i d s to supply iron to the organism. Thus the purpose of the i n i t i a l experiments was to develop a bioassay technique that achieved optimum growth of the organism without the addition of a chelating agent. The growth in cultures supplied with fresh c o l l o i d a l iron was not s i g n i f i c a n t l y d i f f e r e n t from those cultures supplied with iron complexed by EDTA (Fig. 9). From these results i t is apparent that optimal growth of T. psuedonana in Aquil can be obtained without a chelating agent i f the iron i s supplied in the form of a freshly prepared f e r r i c hydroxide c o l l o i d a l suspension. Experiments were then conducted to determine what treatments of the c o l l o i d a l iron stock, prior to i t s addition to 74 cultures, would reduce the iron a v a i l a b i l i t y . These treatments included autoclaving (121°C, 15 p s i ) , heating, and ageing. In a l l cases the treated iron stocks were added to previously autoclaved culture media. Growth in cultures supplied with treated iron stocks was compared to that of the control (fresh untreated c o l l o i d a l iron stocks added after autoclaving of medium) to determine i f these treatments were responsible for a change in iron a v a i l a b i l i t y . The results showed that autoclaving (15 mins, 15 p s i , 121°C) of the iron stock caused a reduction in organism growth (Fig. 11) which was found to be due to iron l i m i t a t i o n . Longer periods of autoclaving did not cause further substantial growth reduction (Fig. 12). Growth reduction also occurred when iron stocks were heated (100°C, 1 atm pressure), suggesting that pressure variations (during autoclaving) did not contribute to the change in iron a v a i l a b i l i t y . Although growth in cultures supplied with heated iron stocks was reduced i t continued for a period of 21 days of monitoring after growth in the control had ceased (Fig 13). Even after 28 days, growth was continuing indicating the iron was s t i l l being made available to the organism but at a very slow rate. It i s possible that c e l l numbers could have eventually reached the f i n a l l e v e l seen in the control cultures. The decreased growth rate in cultures supplied with the iron stocks heated to 50°C and 70°C (5 mins.) (Fig. 14) demonstrates that while the decrease in a v a i l a b i l i t y was gradual, i t occurred within the narrow temperature range between 75 21°C and 70°C. Again, the growth in the cultures supplied with the heated iron stock had not ceased at the end of the experiment. The iron stock ageing experiments showed that while one week of ageing did not reduce growth, patterns of reduced growth did occur after three months of iron stock ageing (at 21°C) . EDTA additions to the cultures supplied with heated or aged iron stocks was found not to enhance growth. However, addition of EDTA to the iron stocks before autoclaving or extended ageing prevented the reduction in iron a v a i l a b i l i t y (Fig. 19). Since EDTA prevents the formation of polynuclear f e r r i c c o l l o i d s , by chelation of the aquo-ions, these results suggest that the formation of iron c o l l o i d s i s a necessary precursor to the process that reduces the iron a v a i l a b i l i t y . Thus the chemical and/or physical changes occurring in the c o l l o i d a l iron as a result of heating and extended ageing would appear to cause the reduction in iron a v a i l a b i l i t y . These changes are well documented and w i l l now be b r i e f l y discussed. 76 Processes Occurring in F e r r i c Salt Solutions The transformations that occur in c o l l o i d a l iron stocks during heating and ageing are presumably similar and comparible to those reported to occur during hydrolysis of f e r r i c ' s a l t s (Murphy e_t a l 1976, van der Giessen 1968, Dousma and de Bruyn 1976). After addition to d i s t i l l e d water, f e r r i c chloride w i l l dissociate and the F e + 3 ions liberated are rapidly hydrated, the coordination sphere containing six H 20 molecules forming an octahedron. This complex then rapidly releases a hydrogen ion to form [Fe(H 20) 5(OH)] 2 + (van der Giessen 1968). The e l e c t r o s t a t i c repulsion of the hydrated ions can be overcome through a combination of van der Waals and hydrogen bonding, which causes the hydrated ions to associate with one another resulting in the formation of c o l l o i d a l sized polynuclear complexes. The rate of th i s aggregation i s largely controlled by the pH, temperature, the concentration of the aquo-ions, and the type of anions present. In the c o l l o i d a l iron stocks prepared for these experiments, aggregation occurred within minutes, as was shown by a change in color from a translucent yellow to a deep amber. Murphy et aJL (1976) found that these c o l l o i d s were spheres 1.5 -3.0 nm in diameter which later linked to form larger rod-like structures. Adjacent hydrated ions within the polynuclear complex may undergo olation to form an OH (or ol) bridge between the ions resulting, in the loss of water (van der Giessen, 1968, Dousma and deBruyen, 1976). A proton may then be s p l i t off from the o l bridge to form an oxo bridge. This l a t t e r process is c a l l e d oxolation. The olation and oxolation processes are 77 i l l u s t r a t e d in F i g . 25. While the i n i t i a l loss of protons from the aquo-ions occurs very rapidly, the olation and oxolation processes proceed slowly at room temperature and may take hundreds of hours for completion (van der Giessen, 1968). The rate of these processes, however, greatly increases at higher temperatures. While the forces causing aggregation of the aquo-ions and ordering within the polynuclear complexes are chemical in nature, they are similar to those i n t e r f a c i a l forces c o n t r o l l i n g ion adsorption to surfaces. Since i n t e r f a c i a l forces are important in the supply of iron from the c o l l o i d to the organism, I have chosen to i l l u s t r a t e the factors c o n t r o l l i n g the aggregation and subsequent ordering processes with a general description of the energies of adsorption. While perhaps not s t r i c t l y v a l i d in a chemical sense, i t w i l l provide a basic understanding of i n t e r f a c i a l forces that w i l l be necessary to assess later conclusions. The relationship between energies of adsorption and distance from a surface are best described using the Lennard-Jones graph (Fig. 26). Both the energy input and release are represented by the v e r t i c a l axis while the horizontal axis depicts distance from the surface. With adsorption, an approaching ion w i l l s l i p within a defined distance (A) from the surface to form a non electron sharing bond (eg. e l e c t r o s t a t i c or van der Waals), thereby releasing the adsorption energy (Ea). Both the distance (A) and the Ea w i l l vary not only with d i f f e r e n t ions and adsorbates, but also with d i f f e r e n t locations 78 F i g u r e 25. The h y d r o l y s i s , o l a t i o n and o x o l a t i o n p r o c e s s e s i r o n atoms u n d e r g o upon a d d i t i o n t o an a queous s o l u t i o n a r e shown. I n i t i a l l y , t h e h y d r a t e d f e r r i c i o n r a p i d l y l o o s e s a p r o t o n . The r e s u l t a n t h y d r a t e d i o n may t h e n u n d e r g o o l a t i o n (3) and s u b s e q u e n t l y o x o l a t i o n ( 4 ) . T h e s e b r i d g i n g r e a c t i o n s p r o c e e d v e r y s l o w l y a t room t e m p e r a t u r e . W i t h t i m e , however, randomly o r i e n t a t e d o l and oxo b r i d g e s w i l l f o r m w i t h i n t h e c o l l o i d . 1. Fe++ 6H 2 0<=Fe(H 2 0)g + 2. Fe(H 20) 6^H 20^[Fe(H 20) 5(OH)] + Hp 2[Fe(Hp) (OH)]^ 2+ H ( H p F < o / F e ( H p ) H 4+ + 2H2Q 4+ (hip) Fe H Q > e ( H 2 0 ) H + H 2Q 3+ ( H O ) F e < > F e ( H O ) H * H3Q 80 on h e t e r o g e n o u s s u r f a c e s . D e s o r p t i o n ( o r i o n r e l e a s e ) may o c c u r w i t h an e n e r g y i n p u t e q u a l t o E a . B e c a u s e t h i s i s u s u a l l y s m a l l t h e a d s o r p t i o n i s r e l a t i v e l y weak; t h i s f o r m of a d s o r p t i o n i s te r m e d p h y s i c a l a d s o r p t i o n . S i n c e e n e r g y i s r e q u i r e d t o move t h e a d s o r b e d i o n c l o s e r t o t h e s u r f a c e , t h e d i s t a n c e (A) w i l l f l u c t u a t e w i t h t h e e n e r g y i m p a r t e d t o t h e i o n by m o l e c u l a r c o l l i s i o n s . T h e r e e x i s t s a c r i t i c a l d i s t a n c e f r o m t h e s u r f a c e w i t h i n w h i c h t h e r e w i l l be a s p o n t a n e o u s s h a r i n g of e l e c t r o n s between t h e i o n and t h e s u r f a c e , f o r m i n g a more s t a b l e bond and r e d u c i n g t h e a d s o r p t i o n d i s t a n c e ( A ) . T h i s r e a c t i o n i s much more e x o t h e r m i c (Ead) t h a n p h y s i c a l a d s o r p t i o n and i s t e r m e d c h e m i c a l a d s o r p t i o n ( o r c h e m i s o r p t i o n ) . The e n e r g y i n p u t n e c e s s a r y t o c a u s e t h i s change i s known as t h e e n e r g y o f a c t i v a t i o n ( E a c t ) . I t w i l l v a r y w i t h d i f f e r e n t i o n s and l o c a t i o n s on t h e h e t e r o g e n o u s s u r f a c e . S i n c e r e l e a s e o f t h e c h e m i s o r b e d i o n r e q u i r e s an i n p u t e q u a l t o b o t h t h e E a c t a n d E a d , c h e m i s o r p t i o n i s l e s s r e a d i l y r e v e r s e d . As a r e s u l t , t h e c h e m i s o r b e d i o n i s i n c o r p o r a t e d i n t o t h e s t r u c t u r e o f t h e s o l i d and d e s o r p t i o n i s t h e n c o n t r o l l e d m a i n l y by t h e bond s t a b i l i t y . I f we now c o n s i d e r t h e change t h a t o c c u r s i n t h e c o l l o i d a l i r o n s t o c k s u s e d i n t h i s s t u d y , t h e same p r i n c i p a l s a p p l y . The h y d r a t e d f e r r i c i o n s i n t h e i n i t i a l c o l l o i d a l s i z e d p o l y n u c l e a r c o m p l e x e s a r e l i n k e d by r e l a t i v e l y weak b o n d i n g (van d e r Waals and h y d r o g e n ) s u c h as i n p h y s i c a l a d s o r p t i o n . I f t h e E a c t i s s u p p l i e d , a d j a c e n t h y d r a t e d i o n s w i l l move t o w i t h i n t h e c r i t i c a l d i s t a n c e and p a r t o f t h e w a t e r - h y d r o x i d e s h e a t h 81 F i g u r e 26. L e n n a r d - J o n e s g r a p h . The e n e r g y of i o n s a p p r o a c h i n g a s u r f a c e i s d e p i c t e d i n t h i s g r a p h . Ea i s t h e e n e r g y r e l e a s e d upon p h y s i c a l a d s o r p t i o n and E a c t i s t h e a c t i v a t i o n e n e r g y r e q u i r e d f o r t h e i o n - t o s h i f t t o c h e m o s o r p t i o n ; a much more e x o t h e r m i c r e a c t i o n ( E a d ) . The e n e r g i e s shown w i l l v a r y w i t h d i f f e r e n t i o n s and s u r f a c e s . 82 83 s u r r o u n d i n g e a c h i r o n atom w i l l be l o s t as H 2 0 ( F i g . 2 5 ) , e s t a b l i s h i n g o l b r i d g e s between t h e f e r r i c i o n s (much t h e same as t h e c h a n g e f r o m p h y s i c a l t o c h e m i c a l a d s o r p t i o n ) . T h i s r e a c t i o n r e s u l t s i n c o n d e n s a t i o n ( i . e . d e h y d r a t i o n ) o f t h e p o l y n u c l e a r c o m p l e x e s . Once t h e i n i t i a l o l a t i o n o c c u r s , f u r t h e r c o n d e n s a t i o n i s c a t a l y z e d by t h e p r e s e n c e o f t h e o l b r i d g e s and o v e r t i m e a network of b r i d g e s w i t h i n t h e p o l y n u c l e a r complex would be f o r m e d . ( C a t a l y s i s o c c u r s by a r e d u c t i o n ' i n t h e a c t i v a t i o n e n e r g y . ) Thus b e c a u s e o f t h e i n c r e a s e i n t h e s t r e n g t h of b o n d i n g between t h e f e r r i c i o n s of t h e c o l l o i d , t h e thermodynamic s t a b i l i t y o f t h e o x i d e phase i s i n c r e a s e d . The o v e r a l l r a t e o f t h i s change i n t h e c o l l o i d a l i r o n s t o c k would be d e t e r m i n e d by b o t h t h e r a t e of h y d r a t e d i o n a g g r e g a t i o n and by t h e r a t e o f t h e r e a c t i o n w h i c h s u b s e q u e n t l y t r a n s f o r m s t h e d i s o r g a n i z e d , amorphous s o l i d t h a t i n i t i a l l y forms i n t o one h a v i n g a more o r d e r e d s t r u c t u r e and g r e a t e r thermodynamic s t a b i l i t y . A l t h o u g h t h e p r o c e s s e s of a g g r e g a t i o n and s u b s e q u e n t d e h y d r a t i o n have been d e s c r i b e d s e p a r a t e l y , one must u n d e r s t a n d t h a t b o t h may o c c u r s i m u l t a n e o u s l y . As p o l y n u c l e a r c o m p l e x e s form and grow by a g g r e g a t i o n o f a q u o - i o n s from s o l u t i o n , o l a t i o n and o x o l a t i o n w i l l o c c u r w i t h i n t h e c o l l o i d s . The e x t e n t t o w h i c h t h e s e r e a c t i o n s o c c u r d u r i n g a g g r e g a t i o n i s d e p e n d e n t on t h e t e m p e r a t u r e o f t h e s o l u t i o n . W i t h i n c r e a s i n g t e m p e r a t u r e s , t h e e n e r g y o f t h e i n t e r a c t i n g m o l e c u l a r u n i t s i n c r e a s e s p e r m i t t i n g a g r e a t e r r a t e o f r e a c t i o n . Thus t h e r e l a t i o n s h i p between a g g r e g a t i o n , o l a t i o n and o x o l a t i o n w i l l change w i t h 84 d i f f e r e n t conditions. While the i n i t i a l bridging of the iron atoms w i l l be randomly orientated, over time the structure w i l l change to a c r y s t a l l i n e one. C h a r a t e r i s t i c a l l y , the structure within the hydrous f e r r i c oxides w i l l undergo constant alt e r a t i o n s over time progressing through two or more c r y s t a l l i n e forms before the most thermodynamically stable structure is achieved (Langmuir and Whittlemore, 1971). (The most stable structure varies under d i f f e r e n t conditions.) Thus one must not consider hydrous f e r r i c oxides as s t a t i c polynuclear complexes but as constantly changing units. To determine whether c r y s t a l l i z a t i o n of the c o l l o i d a l iron would reduce the growth of the organism, the a b i l i t y of c o l l o i d a l goethite and hematite to supply iron to the organism was tested. Goethite and hematite are probably the most thermodynamically stable f e r r i c oxyhydroxide and oxide under earth surface conditions (Langmuir and Whittlemore, 1971). The growth in cultures supplied with these forms of iron was s i g n i f i c a n t l y below that of cultures supplied with fresh c o l l o i d a l iron (Fig. 20). This reduction was found to be due to iron l i m i t a t i o n . Although association of c o l l o i d a l goethite and hematite with the l i v i n g diatoms of the cultures was observed, the growth patterns were similar to those of cultures with no added iron indicating that there w a s ' l i t t l e or no iron supplied to the organism. These results suggest that the a v a i l a b i l i t y of , c o l l o i d a l iron changes in p a r a l l e l with the thermodynamic s t a b i l i t y of the structure within the c o l l o i d . It i s also 85 apparent that iron a v a i l a b i l i t y i s not d i r e c t l y related to the proximity of the c o l l o i d to the organism as suggested by Harvey (1937) and Goldberg (1952). If variation in the thermodynamic s t a b i l i t y of the structure within the c o l l o i d does affect the a v a i l a b i l i t y of iron, i t must be related to i t s affect on the mononuclear iron species, one or more of which are believed to be available. For the moment, l e t us assume that one or a number of dissolved f e r r i c species are available to the organism. Upon uptake of the dissolved f e r r i c species by the organism, their replacement in the medium would occur through a re- e q u i l i b r a t i o n of the solution with the c o l l o i d a l iron. In a given solution, the rate of such replacement from a c o l l o i d is controlled by both the rate of monomer removal from the c o l l o i d , which i s dependent on the active surface area, and the magnitude of the activation energy of the dissocia t i o n reaction. This energy of activation increases with increasing s t a b i l i t y of the f e r r i c ion bonding configuration. Thus, the thermodynamic s t a b i l i t y of the polynuclear complexes may be related to iron a v a i l a b i l i t y through changes in the c o l l o i d a l dissolution rate. However, another effect of increasing thermodynamic s t a b i l i t y i s a reduction in the mononuclear iron species concentration. While these reduced concentrations may not be of importance i f the iron species released from the c o l l o i d i s in a b i o l o g i c a l l y available form, a more s i g n i f i c a n t effect might occur i f reduction or conversion to some other s p e c i f i c species is necessary before uptake is possible. I f , for the moment, i t 86 i s assummed t h a t r e d u c t i o n o f t h e f e r r i c i o n i s n e c e s s a r y b e f o r e u p t a k e by t h e o r g a n i s m c a n o c c u r ( A n d e r s o n a n d M o r e l , 1980), t h e n a ^ p o s s i b l e p a t h w a y o f i r o n s u p p l y t o t h e o r g a n i s m m i g h t be a s shown b e l o w . A d e c r e a s e i n t h e d i s s o l v e d f e r r i c i r o n c o n c e n t r a t i o n may d e c r e a s e t h e r a t e o f r e d u c t i o n ( s t e p 2) a n d t h u s t h e i r o n s u p p l y t o t h e o r g a n i s m . I f t h e r a t e o f r e d u c t i o n ( o r a ny o t h e r f e r r i c i o n c o n v e r s i o n ) i s ' t h e r a t e d e t e r m i n i n g s t e p , a d d i t i o n o f EDTA t o t h e medium c o n t a i n i n g h e a t e d o r a g e d i r o n w o u l d i n c r e a s e t h i s r a t e , a n d t h u s o r g a n i s m g r o w t h , by i n c r e a s i n g t h e c o n c e n t r a t i o n o f t h e m o n o n u c l e a r i r o n s p e c i e s . H o w e v e r , a d d i t i o n o f EDTA t o c u l t u r e s s u p p l i e d w i t h h e a t e d o r A P o s s i b l e P a t h w a y o f I r o n S u p p l y a g e d i r o n s t o c k s d i d n o t e n h a n c e g r o w t h s u g g e s t i n g t h a t low m o n o n u c l e a r i r o n c o n c e n t r a t i o n s was n o t t h e m a j o r c a u s e o f g r o w t h r e d u c t i o n b u t r a t h e r t h a t t h e r e d u c e d d i s s o l u t i o n r a t e o f 87 the c o l l o i d s controlled organism growth. Growth in cultures supplied with heated, autoclaved, and three month old iron stocks was not only less than the control, but was s i g n i f i c a n t l y lower than that of cultures with no added iron. A possible explanation for this phenomenon i s the adsorption of residual available iron (present by contamination) to the added c o l l o i d s of the stock. The metal scavenging a b i l i t i e s of c o l l o i d a l hydrous oxides are well documented and adsorption of the residual available iron in the cultures may occur within hours (Kim and Z e i t l i n , 1971). The i n i t i a l adsorption may be physical, however, i f the s t a b i l i t y of the c o l l o i d a l oxide of these stocks was higher, the structuring present would catalyze chemisorption of the iron (Leja, personal comm.). The ions would then be incorporated into the c o l l o i d structure and release of these adsorbed ions would then be determined by the s t a b i l i t y of 'the c o l l o i d . Although the residual iron would also be scavenged by c o l l o i d s in the control cultures (fresh c o l l o i d a l iron addition), the growth demonstrates that these c o l l o i d s provide a good supply of iron to the organism. While autoclaving of the fresh c o l l o i d a l iron stock before addition to the cultures was shown to reduce organism growth, the i n i t i a l laboratory experiments showed that autoclaving of the Aquil medium with iron (Fe: 4.5 x 10"7M) had no deleterious ef f e c t on growth (Fig. 10). It was found that the higher ionic strength of the solution was, in part, responsible for the lack of reduction in iron a v a i l a b i l i t y . This s a l t e f f e c t might result 88 because of the occlusion of foreign ions by the precipitate which then interferes with the formation of oxide bonds between the iron atoms (Langmuir and Whittlemore, 1971). Thus, a hydrous oxide phase of lower thermodynamic s t a b i l i t y might be expected in the higher ionic strength solution after the same period of heating suggesting more rapid dissolution rates and higher mononuclear iron concentrations. This in turn would be expected to increase the iron a v a i l a b i l i t y . In addition to higher ionic strength, the lower iron concentration in the medium during autoclaving (Fe: 4.5 x 10~7M) would also decrease the rate of hydrated ion aggregation and thus the rate of restructuring. In summary, i t i s suggested that autoclaving, heating and extended ageing a l l cause the c o l l o i d a l phase to undergo similar st r u c t u r a l changes that presumably increase their thermodynamic s t a b i l i t y . These structural modifications, moreover, appear to decrease the b i o l o g i c a l a v a i l a b i l i t y of the c o l l o i d a l iron by reducing the c o l l o i d d i s s o l u t i o n rate. To investigate this hypothesis further, the autoclaved and fresh c o l l o i d a l iron stocks were a n a l y t i c a l l y compared. 89 A n a l y s i s of the Autoclaved and Fresh Iron Stocks To determine whether the s t r u c t u r e of the c o l l o i d a l i r o n p r e p a r a t i o n s was measurably a l t e r e d by short p e r i o d s of a u t o c l a v i n g , v a r i o u s p r o p e r t i e s of the a u t o c l a v e d and f r e s h c o l l o i d a l i r o n stocks (both i n d i s t i l l e d water) were compared. The thermodynamic s t a b i l i t y of the c o l l o i d a l phase i s determined by i t s s u r f a c e area and s t r u c t u r e , the l a t t e r being i n turn determined by f a c t o r s such as mineralogy, degree of o r d e r i n g degree of h y d r a t i o n and by the presence of i m p u r i t i e s . The e f f e c t of i m p u r i t i e s can be d i s r e g a r d e d i n the present case because of the method of stock p r e p a r a t i o n . Thus the r e l a t i v e thermodynamic s t a b i l i t i e s of the c o l l o i d a l i r o n stocks should i n c r e a s e as the degree of o r d e r i n g i n c r e a s e s and the degree of h y d r a t i o n and the s u r f a c e area decrease. The techniques used to estimate these c h a r a c t e r i s t i c s i n c l u d e d X-ray d i f f r a c t i o n , Mossbauer spectroscopy, thermal g r a v i m e t r i c a n a l y s i s , and g e l f i l t r a t i o n . No o r d e r i n g was d e t e c t e d i n the a u t o c l a v e d (15 min) or f r e s h i r o n stock by X-ray d i f f r a c t i o n u s ing e i t h e r wavelength scanning or camera techniques. However, a f t e r 24 hrs of a u t o c l a v i n g , evidence of c r y s t a l l i z a t i o n was detected by the camera technique. T h i s suggests that e a r l y stages of o r d e r i n g might be present a f t e r 15 minutes of a u t o c l a v i n g . The i n a b i l i t y to d e t e c t o r d e r i n g i n the l a t t e r stock i s l i k e l y due to the l i m i t a t i o n s of the d i f f r a c t i o n technique. Mossbauer spectroscopy, which was a l s o used to compare the degree of o r d e r i n g i n the two i r o n stocks showed a s l i g h t 90 increase in peak width of the autoclaved c o l l o i d a l stock that could possibly be due to a l t e r a t i o n in the electron spin orientations of some iron atoms ( i . e . altered bond formation). These observations, however, could be equally well explained by differences in sample thickness or by alt e r a t i o n s in the orientation of the atomic ferromagnetic f i e l d during freezing of the sample. The i n a b i l i t y of Mossbauer spectroscopy to measure a s i g n i f i c a n t difference between the stocks demonstrates that reduction in iron a v a i l a b i l i t y requires very l i t t l e change in the measurable chemical environment of the atom. However, the ferromagnetic properties of iron interfere to some extent in the analysis and i t may be possible to achieve a better . comparison of the stocks with more extensive Mossbauer studies. The degree of hydration of the autoclaved and fresh c o l l o i d a l stocks was compared with thermal gravimetric analysis. The samples were f i r s t heated to 115°C for 4 minutes (to drive off the water molecules associated with pores and surfaces) and then to 700°C. Because the i n i t i a l heating (4 mins, 115°C) would also cause some loss of chemically bound water (through o l a t i o n ) , the technique was used only to compare the stocks rather than to measure their actual degrees of hydration. The autoclaved stock was found to have a lower degree of hydration than that of the fresh stock (Fig. 22) suggesting that olation or oxolation between iron atoms had occurred in the autoclaved c o l l o i d s . Additionaly,' the autoclaved stock had a rapid water loss between 450°C and 500°C. One possible explanation for thi s phenomenon may be the release of trapped or chemically bound 91 water molecules. During the ordering process some water molecules can be incorporated within the structure and at higher temperatures s u f f i c i e n t energy may be provided to release t h i s water. The temperature at which th i s release occurs w i l l vary depending on the strength and configuration of the bonds incorporating the water molecules into the structure. The narrow temperature range of release in t h i s case would suggest that these molecules must have had similar bonding configurations. Another p o s s i b i l i t y for t h i s sudden water release might be a simultaneous change in atomic bonding configurations ( i . e . structure) within the s o l i d that resulted in the loss of hydroxide ions (through further olation) as water. In either case, the sudden weight change between 450°C and 500°C further suggests that ordering occurred within the c o l l o i d s during autoclaving. In an attempt to compare the surface areas of the c o l l o i d a l iron stocks, their gel f i l t r a t i o n rates were measured. The results showed that the c o l l o i d a l ' size increased upon autoclaving of the iron stock (Fig. 23). Although the size of well c r y s t a l l i z e d c o l l o i d s can be used to estimate their surface area, the surface areas of amorphous f e r r i c hydroxides cannot be estimated in t h i s fashion as they have a porous, g e l - l i k e structure through which ions can migrate. Thus, measurement of the exterior surface area greatly underestimates the actual surface area. If ,the c o l l o i d size increase in the autoclaved iron stock was due only to coagulation of existing polynuclear complexes, i t would not appreciably reduce the actual surface 92 area of the c o l l o i d a l stock (although exchange between the internal surface and exterior solution would be slowed). However, Murphy et a l (1976) suggested that during heating of such polymeric c o l l o i d a l suspensions, a d i s s o l u t i o n -rep r e c i p i t a t i o n reaction occurs that results in increased st r u c t u r a l order. If t h i s did occur in the heated iron stocks, the porosity of the c o l l o i d s would be reduced (and thus the surface area), which in turn would reduce the number of s i t e s available to react with the solution. However, th i s r e p r e c i p i t a t i o n process was seen by Murphy et a_l after a period of weeks in solutions that were four orders of magnitude more concentrated than the stocks used in t h i s study and did not result in an appreciable change in c o l l o i d a l size (although aggregates were later seen to form). Thus the increase in c o l l o i d size i s probably due to just coagulation of existing c o l l o i d s , a phenomenon that should not appreciably a l t e r the thermodynamic s t a b i l i t y of the hydrous oxide phase. This suggests that the increased s t a b i l i t y was mainly due to increases in the degree of condensation and, hence, of ordering. Since the rate of dissolution is expected to covary with changes in thermodynamic s t a b i l i t y , the dissolution rates of the c o l l o i d a l phase in the two stocks were compared. The results indicate that the d issoluti on rate of the fresh c o l l o i d a l iron stock was more rapid than that of the autoclaved iron stock (Fig. 24). This provides evidence supporting the hypothesis that decreased growth in cultures supplied with the heated iron stocks was due to reduced rates of c o l l o i d a l d i s s o l u t i o n . The 93 very low r a t e of d i s s o l u t i o n i n t h i s stock c o u l d be the reason f o r the continued low r a t e of growth seen i n c u l t u r e s s u p p l i e d with heated ( 100°C) i r o n s t o c k s . While no s i n g l e technique p r o v i d e d unequivocal evidence of a change i n the thermodynamic s t a b i l i t y of the c o l l o i d a l i r o n , the combined evidence suggests that the s t a b i l i t y i n c r e a s e d d u r i n g a u t o c l a v i n g . Thus while the changes which made the c o l l o i d a l i r o n n o n - a v a i l a b l e c o u l d not be p r e c i s l y documented, i t i s evident that the decrease i n a v a i l a b i l i t y o c c u r r e d f o l l o w i n g only minor changes i n the thermodynamic s t a b i l i t y . 94 CONCLUSIONS C o l l o i d a l m a t e r i a l was found a s s o c i a t e d with diatom f r u s t u l e s i n three of the f i e l d l o c a l i t i e s sampled: the bottom waters of Indian Arm (Ind 2.0), the F r a s e r R i v e r plume (Fra 1.5) and w i t h i n the Fr a s e r R i v e r s a l t wedge. In many cases the a s s o c i a t e d c o l l o i d a l matter was i r o n - r i c h . I t does not appear that t h i s a s s o c i a t i o n was an a r t i f a c t of the sampling p r o c e s s . F a c t o r s that c o u l d have c o n t r i b u t e d to i t s absence i n the other areas sampled are the l a r g e number of c e l l s found r e l a t i v e to the small amount of c o l l o i d a l m a t e r i a l and the l i m i t e d r e s o l u t i o n of the scanning e l e c t r o n microscope. R e s u l t s from the g l a s s p l a t e experiments suggest that the a s s o c i a t i o n of c o l l o i d a l m a t e r i a l with diatoms may be due to the sur f a c e p r o p e r t i e s of the s i l i c e o u s f r u s t u l e and need not be b i o l o g i c a l l y c o n t r o l l e d by the diatom i t s e l f . The bioassay r e s u l t s showed that while f r e s h l y p r e c i p i t a t e d f e r r i c hydroxide was r e a d i l y a v a i l a b l e , a u t o c l a v i n g , h e a t i n g , or ageing of that same stock reduced the a v a i l a b i l i t y of the i r o n . The r e d u c t i o n i n i r o n a v a i l a b i l i t y was shown to be a gradual process o c c u r r i n g over time that was g r e a t l y a c c e l e r a t e d at higher temperatures. Although the r a t e of growth was reduced in c u l t u r e s s u p p l i e d with heated c o l l o i d a l i r o n s t o c k s , c e l l numbers i n these c u l t u r e s continued i n c r e a s i n g f o r at l e a s t 21 days a f t e r growth had ceased i n the c o n t r o l . T h i s i n d i c a t e s that i r o n was being made a v a i l a b l e but that the rate of supply was very low. Although a d d i t i o n of EDTA to the c u l t u r e s s u p p l i e d with 95 a u t o c l a v e d and aged c o l l o i d a l i r o n stocks d i d not i n c r e a s e i r o n a v a i l a b i l i t y , growth was not reduced when EDTA was added to the i r o n stock before these treatments. Since the presence of EDTA would prevent the aggregation of i r o n i n such stocks (by c h e l a t i o n of the f e r r i c i o n s ) , these r e s u l t s i n d i c a t e that the formation of c o l l o i d a l f e r r i c hydroxide i s a necessary p r e c u r s o r to the process reducing the a v a i l a b i l i t y of the i r o n stock. T h i s i n turn suggests that the process i s r e l a t e d to changes in c o l l o i d a l i r o n . Changes that occur i n f e r r i c s a l t s o l u t i o n s , such as those i n i r o n stocks used i n these b i o a s s a y s , are w e l l docummented. I n i t i a l l y , p o l y n u c l e a r c o l l o i d s form by aggregation of the hydrated f e r r i c ions and over time these c o l l o i d s become more s t a b l e through i n t e r n a l rearrangement and condensation r e a c t i o n s . Higher temperatures a c c e l e r a t e t h i s o r d e r i n g p r o c e s s . Such o r d e r i n g changes must, moreover, r e s u l t i n an i n c r e a s e i n the thermodynamic s t a b i l i t y of the c o l l o i d a l i r o n . To t e s t whether the s t a b i l i t y a f f e c t e d the supply of i r o n to the organism, g o e t h i t e and hematite were t e s t e d as sources of i r o n . These w e l l - c r y s t a l l i z e d minerals are probably the most s t a b l e f e r r i c oxyhydroxide and oxide under e a r t h s u r f a c e c o n d i t i o n s . Both were found to be poor sources of i r o n to T. psuedonana even though a s s o c i a t i o n of the c o l l o i d s with l i v i n g diatoms was observed. These r e s u l t s support the hypothesis that organism growth decreases as the thermodynamic s t a b i l i t y of the c o l l o i d a l i r o n i n c r e a s e s . To determine whether reduced i r o n a v a i l a b l i t y i n the 9 6 treated c o l l o i d a l iron stocks can be related to measurable changes in their chemical and physical properties, the autoclaved and fresh iron stocks were compared using techniques that would provide information with respect to their degree of polymerization, hydration and c r y s t a l l i n i t y . These techniques were X-ray d i f f r a c t i o n , Mossbauer spectroscopy, gel f i l t r a t i o n and thermal gravimetric analysis. No evidence for a regular c r y s t a l structure was detected in * the autoclaved and fresh c o l l o i d a l stocks by either X-ray d i f f r a c t i o n or Mossbauer spectroscopy. A longer period of autoclaving (24 hrs), however, produced detectable c r y s t a l l i n i t y , indicating that early stages of ordering could be present in the c o l l o i d a l stocks autoclaved for 15 minutes. Thermal gravimetric analysis demonstrated that the o v e r a l l degree of hydration was lower in the autoclaved stock. Further, the rapid loss of water between 450°C and 500°C indicates that some form of structure was present in the autoclaved c o l l o i d a l iron. These results suggest that the thermodynamic s t a b i l i t y of the c o l l o i d s increased during the process of autoclaving. Although c o l l o i d a l size was found to increase with autoclaving, th i s was most l i k e l y due to c o l l o i d a l coagulation which in i t s e l f would not a l t e r appreciably the thermodynamic s t a b i l i t y of the c o l l o i d s . The results of the a n a l y t i c a l comparison were unable f u l l y to characterize the non-available form of c o l l o i d a l i rpn. The change in iron a v a i l a b i l i t y of the c o l l o i d a l stocks appears to p a r a l l e l increases in thermodynamic s t a b i l i t y and 97 c o u l d be due to e i t h e r reduced d i s s o l u t i o n r a t e s or reduced mononuclear i r o n s p e c i e s c o n c e n t r a t i o n s . D i s s o l u t i o n r a t e s of the a u t o c l a v e d and f r e s h c o l l o i d a l stocks were compared and the r a t e i n the a u t o c l a v e d stock was found to be s u b s t a n t i a l l y lower. Reduced d i s s o l v e d i r o n c o n c e n t r a t i o n s c o u l d a f f e c t organism growth i f c o n v e r s i o n of the i r o n form r e l e a s e d by c o l l o i d d i s s o l u t i o n i s necessary before uptake can occur. However, s i n c e a d d i t i o n of EDTA to i r o n stocks that were aged or a u t o c l a v e d d i d not enhance growth, i t appears that c o l l o i d d i s s o l u t i o n r a t e s may be the c o n t r o l l i n g f a c t o r determining the i r o n supply to the organism. T h i s i s a very important c o n s i d e r a t i o n when examining the e f f e c t of n a t u r a l c h e l a t i n g agents (e.g. siderophores and humic m a t e r i a l s ) on organism growth i n e s t u a r i n e and oceanic environments. If changes in the thermodynamic s t a b i l i t y of the i r o n i s r e l a t e d to the reduced growth seen in the experiments, the s h i f t between a v a i l a b l e and n o n - a v a i l a b l e i r o n must be viewed as a c o n t i n u i n g process s i n c e the o r d e r i n g of f e r r i c (oxy)hydroxides c o n t i n u e s over time. The p o i n t at which the i r o n w i t h i n c o l l o i d s becomes ' n o n - a v a i l a b l e ' w i l l depend on the degree and form of o r d e r i n g and the i r o n demand of the p o p u l a t i o n . As the experiments have shown, the t r u e b i o l o g i c a l a v a i l a b i l i t y of a c o l l o i d a l metal cannot be.determined with short term b i o a s s a y s . Even in the case of c o l l o i d a l g o e t h i t e and hematite, an e q u i l i b r i u m e x i s t s between the c o l l o i d a l and mononuclear s p e c i e s of i r o n such that replacement w i l l occur as a mononuclear s p e c i e s i s removed b i o l o g i c a l l y . The i r o n w i t h i n the c o l l o i d s i s 98 then a c t u a l l y ' a v a i l a b l e ' although the r a t e of supply i s very slow. Thus, when I use the term ' n o n - a v a i l a b l e ' , i t i s with res p e c t to the i r o n demand that allows optimal growth of the p o p u l a t i o n . The r e s u l t s of t h i s i n v e s t i g a t i o n suggest that the method of i r o n stock p r e p a r a t i o n and the treatment of that i r o n stock before a d d i t i o n to c u l t u r e s d i r e c t l y c o n t r o l s the b i o l o g i c a l a v a i l a b i l i t y of the i r o n . Attempts to f u l l y c h a r a c t e r i z e the change in the c o l l o i d a l stocks that had reduced the i r o n a v a i l a b i l i t y were u n s u c c e s s f u l . The change, however, appears to be r e l a t e d to i n c r e a s e s i n the degree of p o l y m e r i z a t i o n and decreases in the degree of h y d r a t i o n that n e c e s s a r i l y i n c r e a s e s the thermodynamic s t a b i l i t y of the c o l l o i d a l hydrous oxide. While techniques such as scanning e l e c t r o n microscopy and energy d i s p e r s i v e X-ray a n a l y s i s show the a s s o c i a t i o n of c o l l o i d a l metals to organisms both in the l a b o r a t o r y and in the n a t u r a l environment, the a v a i l a b i l i t y of t h i s metal i s probably determined by the thermodynamic s t a b i l i t y of the c o l l o i d r a t h e r than the p r o x i m i t y of the c o l l o i d to the c e l l . REFERENCES Anderson, M.A. and Morel, F.M.M.. 1980. Uptake of F e ( l l ) by a Diatom in Oxic Culture Medium. Mar. B i o l . Lett. 1:263-268. Atkinson, R.J., Posner, A.M. and Quirk, J.P.. 1977. Crystal Nucleation and Growth in Hydrolysing Iron (III) Chloride Solutions. Clays and Clay Min. 25:41-56. Bryne, R.H. and Kester, D.A.. 1976. S o l u b i l i t y of Hydrous Ferric Oxide and Iron Speciation in Sea Water. Mar. Chem. 4:255-274. Canterford, G.S.. 1979. Effect of EDTA on Growth of the Marine Diatom DITYLUM BRIGHTWELLI. Aust. J. Mar. Freshwater Res. 30:765-772. Dousma, J. and de Bruyn, P.L.. 1976. Hydrolysis-Precipitation Studies of Iron Solutions 1. Model for Hydrolysis and P r e c i p i t a t i o n from Fe(IIl) Nitrate Solutions. J. C o l l o i d and Interface S c i . 56(3):527-539. Forbes, E.A., Posner, A.M. and Quirk, J.P.. 1974. The Specific Adsorption of Inorganic Hg(II)'Species and Co(lII) Complex Ions on Goethite. J. C o l l o i d and Interface S c i . 49(3):403-409. Gibb, T.G. P r i n c i p a l s of Mossbauer Spectroscopy. Studies in Chemical Physics, Chapman and H a l l , 19-76 Glover, H.. 1977. E f f e c t s of Iron Deficiency on ISOCHRYSIS GABLINANA (Chrysophyceae) and PHAEODACTYLUM TRICORNUTUM (Bacillariophyceae). J. Phycol. 13:208-212. Goldberg, E.D.. 1952. Iron Assimilation by Marine Diatoms. B i o l . B u l l . 102:243-248. Harvey, H.W.. 1937. The Supply of Iron to Diatoms. Mar. B i o l . Assn. 22:205-219. 100 Kim, Y.S. and Z e i t l i n , H.. 1971. The Role of iron(III) Hydroxide as a Collecter of Molybdenum From Seawater. Anal. Chim. Acta 46(1):1-8. Langford, C.H., Kay, R. Quance, G.W. and Khan, T.R.. 1977. Kinetic Analysis Applied to Iron in a Natural Water Model Containing Ions, Organic Complexes, Colloids and P a r t i c l e s . Analy. Lett. J_0 (J_4) : 1 249-1 260 . Langmuir, D. and Whittlemore, D.O.. Variations in S t a b i l i t y of Freshly Precipitated F e r r i c Oxyhydroxides. in Advances in Chemistry, Society of American Chemistry, 50, 1971 Lewin, J. and Chen, C . 1971. Available Iron: A Limiting Factor for Marine Phytoplankton. Limn, and Ocean. 16(4):670-675. Lewin, J. and Chen, C . 1973. Changes in the Concentration of Soluble and Particulate Iron in Sea Water Enclosed in Containers. Limn, and Ocean. 18(4):590-596. Lockhart, H.B. and Blakely, R.V.. 1975. Aerobic Photo-degradation of X(n) Chelates af ( E t h y l d i a m i n i t r i l l o ) Tetra Acetic Acid [EDTA]: Implications for Natural Waters. Environ. Lett. 9 (J_) : 1 9-3 1 . Massalshi, A. and Leppard, G.G.. 1979. Morphological Examination of F i b r i l l a r Colloids Associated with Algae and Bacteria in Lakes. J. Fish. Res. Board Can. 36:906-921. Matjevic, E. and Scheiner, P.. 1978. F e r r i c Hydroxide Sols. I I I . Preparation of Uniform P a r t i c l e s by Hydrolysis of F e ( l l l ) Chloride, Nitrate and Perchlorate Solutions. J. C o l l o i d and Interface S c i . 63(3):509-523. Morel, F.M.M., Rueter, J.G., Anderson, D.M. and G u i l l a r d , R.R.L.. 1979. AQUIL: A Chemically Defined Phytoplankton Culture Medium for Trace Metal Studies. J. Phycol. 15:135-141 . Murphy, P.J., Posner, A.M. and Quirk, J.P.. 1976. Characterization of P a r t i a l l y Neutralized F e r r i c Chloride Solutions. J . C o l l o i d and Interface S c i . 56(2):284-297. 101 Murphy, P.J., Posner, A.M. and Quirk. J.P.. 1975. Gel F i l t r a t i o n Chromatography of P a r t i a l l y Neutralized F e r r i c Solutions. J. C o l l o i d and Interface S c i . 52(2):229-238. Provasoli, I.. Organic Regulation of Phytoplankton f e r t i l i t y , in 'The Sea', vol 2. ed. M.N. H i l l , 1963. S p i l l e r , S. and Terry, N.. 1980. Limiting Factors in Photosynthesis. Plant Physiol. 65:121-125. Terry, N.. 1980. Limiting Factors in Photosynthesis. Plant Physiol. 65:114-120. van der Giessen, A.A.. 1968. Chemical and Physical Properties of Iron (Ill)-Oxide Hydrate. P h i l l i p s Res. Rept. suppl. Yu-Jean, L. and Kester, D.R.. 'Kinetices of Ferrous Oxidation in Aqueous Media'. Presented at the American Geophysical Meeting 1978 1 02 APPENDIX 1 The f i l t r a t i o n apparatus used f o r the c o l l e c t i o n of the f i e l d samples was designed to reduce sample contamination ( F i g . 27). Polypropylene s y r i n g e s were capped with s i l i c o n e rubber stoppers that were f i t t e d with two polypropylene n o z z l e s . One noz z l e was connected, through a two way v a l v e , to e i t h e r vacumm or n i t r o g e n pressure while the other nozzle was connected to the sample intake tube. A l l t u b i n g was c o n s t r u c t e d of p o l y v i n y l c h l o r i d e and the s y r i n g e stand of methyl me t h a c r y l a t e . Before sample f i l t r a t i o n , 1.0 N HCl was drawn through the sample intake tube i n t o the s y r i n g e (by vacumm) to c l e a n the apparatus. The a c i d was then e x p e l l e d from the s y r i n g e by n i t r o g e n pressure with adjustment of the two way v a l v e . The s y r i n g e and intake tube were then r i n s e d three times with d i s t i l l e d water by the same method. The s y r i n g e was f i t t e d with a nuclepore 'pop-top' 13 mm diameter f i l t e r holder ( c o n t a i n i n g a 0.1 nm polycarbonate f i l t e r ) and the sample was drawn i n t o the s y r i n g e . Nitrogen pressure was a p p l i e d and the d e s i r e d volume of sample f i l t e r e d . The pressure was then r e l e a s e d , the f i l t e r holder removed and the remaining sample d i s c a r d e d . The f i l t e r holder was r e a t t a c h e d to the s y r i n g e and 5.0 m i l l i t e r s of d i s t i l l e d water was drawn i n t o the s y r i n g e . Pressure was a p p l i e d u n t i l a l l of the r i n s e water had passed through the f i l t e r . 1 03 F i g u r e 27. F i l t r a t i o n s a m p l i n g a p p a r a t u s . 1 04 v a c u u m sample intake I 1 TJ pressure va l ve s t o p p e r nozz le s y r i n g e f i l te r holder 105 APPENDIX 2 For the bioassay study, standard ocean water (SOW) was prepared in 20 l i t e r quantities by the method of Morel et a l (1979). The following s a l t s were added to 18 l i t e r s of d i s t i l l e d water in the indicated quantities: NaCl 490.6 g NaSOa 81 .9 g CaCl 2 2H20 30.8 g KCl 14.0 g NaHC03 4.0 g KBr 2.0 g H 3 B O 3 0.6 g S r C l 2 6H20 0.34 g NaF 0.06 g The s a l t solution was then bubbled with acid cleaned (6N H 2SO„), f i l t e r e d (0.4 jim) a i r overnight to bring the pH into equilibrium. MgCl 3 6H20 was dried (at 70°C) and 222.0 g was added to the equilibrated s a l t solution. The volume was made up to 20 l i t e r s and the solution bubbled (as above) for 6 hours for r e - e q u i l i b r a t i o n . The SOW was then passed through a ion 106 e xchange r e s i n ( C h e l e x , Na f o r m , 100 mesh) t o remove t r a n s i s t i o n m e t a l c o n t a m i n a t i o n and s t o r e d i n a c i d - c l e a n e d 10 L g l a s s f l a s k s u n t i l u s e . 107 APPENDIX 3 Because of a recent report suggesting that photodegradation of EDTA occurs under high l i g h t i n t e n s i t i e s (Lockhart and Blakely, 1975), the ef f e c t of iron stock preparation techniques and coldroom bioassay conditions on EDTA was investigated. Iron, EDTA, and iron-EDTA solutions were placed under both l i g h t (95 »iEin M" 1 S" 1 ) and dark conditions in^the coldroom (15°C) for the period of one week. Lockhart and Blakely (1975) suggested that under high l i g h t i n t e n s i t i e s the following reaction occurred o X N - C H r C H - N / O CH a -C -OH HO-C-CH 2 I I O C H - C - O H * I I O hv © O H O - 6 - C r V ^ N - C H - C H - N o C H ^ - O H H O O C H a 2 O \ H hv Further loss of acet ic acid groups and suggested that the rate of '1' was much more rapid than '2'. Because increased temperatures may also cause t h i s degradation, the e f f e c t s of extended autoclaving on these solution was also tested. If these conditions did cause EDTA breakdown, then C0 2 production would be expected to occur. The C0 2 produced by the treatments of one week of coldroom l i g h t and dark conditions and 108 4 h o u r s of a u t o c l a v i n g was measured ( F i g . 2 8 ) . The r e s u l t s show t h a t t h e Fe-EDTA s o l u t i o n under c o l d r o o m l i g h t and a u t o c l a v e d c o n d i t i o n s p r o d u c e d more C 0 2 t h a n b o t h t h e Fe and EDTA s o l u t i o n s c ombined. S i m i l a r r e s u l t s were seen when a Cu~, EDTA s o l u t i o n was a u t o c l a v e d . I n c r e a s i n g s a l i n i t y was a l s o f o u n d t o i n c r e a s e t h e p r o d u c t i o n o f C 0 2 i n t h e e x p e r i m e n t s ( F i g . 2 9 ) . The r e s u l t s d e m o n s t r a t e t h a t b o t h h i g h l i g h t i n t e n s i t i e s and h e a t i n g c a t a l y z e t h e breakdown of EDTA and t h a t t h e r a t e o f breakdown i n c r e a s e s when t h e EDTA i s i n t h e c h e l a t e d f o r m . I t was t h e n n e c e s s a r y t o d e t e r m i n e i f t h i s EDTA breakdown was i m p o r t a n t w i t h r e s p e c t t o l o s s o f t h e c h e l a t i o n a b i l i t y o f t h e s o l u t i o n . I f EDTA breakdown o c c u r s as L o c k h a r t and B l a k e l y s u g g e s t e d , t h e n t h e l o s s of one a c e t i c a c i d g r o u p would g r e a t l y r e d u c e t h e s t a b i l i t y of t h e c h e l a t e - m e t a l c o m p l e x . Assumming t h a t o n l y r e a c t i o n '1' o c c u r r e d d u r i n g t h e t r e a t m e n t s , t h e upper l i m i t o f t h e p e r c e n t EDTA ' l o s t ' c a n be e s t i m a t e d . In r e a c t i o n '1', e a c h EDTA m o l e c u l e would s u p p l y 2 c a r b o n atoms o r : 2 x 12.01 g m o l e - 1 = 24.02 gC m o l e - 1 S i n c e t h e EDTA c o n c e n t r a t i o n was 5.0 x 10~ 2 M, t h e n : 24.02 gC m o l e - 1 x 5.0 x 10" 2 moles 1 _ 1 = 1200 mgC l " 1 The h i g h e s t C 0 2 l e v e l measured i n t h e t e s t s was t h e a u t o c l a v e d (4 h r s ) i r o n - E D T A s o l u t i o n a t 41.12 mgC l " 1 . The upper l i m i t o f EDTA ' l o s t ' w i t h t h i s t r e a t m e n t c a n be c a l c u l a t e d a s : 109 F i g u r e 28. The C 0 2 p r o d u c e d from F e , EDTA, and Fe-EDTA s t o c k s w i t h a u t o c l a v i n g (4 h r s ) and under c o l d r o o m l i g h t o r d a r k c o n d i t i o n s ( f o r 7 d a y s ) was measured. The r e s u l t s a r e shown as t h e peak a r e a o f t h e C 0 2 c u r v e ( i n i n t e r g r a t e r u n i t s ) . The Fe-EDTA s t o c k , w i t h a u t o c l a v i n g and under c o l d r o o m l i g h t c o n d i t i o n s , had s i g n i f i c a n t l y g r e a t e r C 0 2 p r o d u c t i o n t h e n e i t h e r t h e Fe o r EDTA s t o c k s . 1 10 40-•sr 3 5 O in sz -If _J L. <1> 30-2 5 a L - 2 0 M I R . d a 10 o <D LL 0 Fe edta F e edta Co ldroom dark Fe edta Coldroom l ight ! Fe ! Fe edta ! edta Fe edta Au toc laved 111 F i g u r e 29. An i n c r e a s e i n s a l i n i t y was f o u n d t o i n c r e a s e t h e p r o d u c t i o n o f C 0 2 i n t h e Fe-EDTA s t o c k . ( s e e f i g u r e 28 f o r l a b e l d e t a i l s . ) 14 1 2 -O x o d 4-c M 6H Q_ 2 0 -J—« 0 1.0 3 . 5 5 . 0 1 0 . 0 2 0 . 0 3 5 . 0 S a l in i t y 113 4 1 .J 2 mgC 1' 1 = 3.4% 1200 mgC l " 1 By t h i s method, t h e upper e s t i m a t e of t h e p e r c e n t EDTA l o s t u n d e r c o l d r o o m l i g h t c o n d i t i o n s i s 1.3%. I f r e a c t i o n '1' and '2' o c c u r r e d d u r i n g EDTA breakdown t h e s e e s t i m a t e s would d e c r e a s e . B e c a u s e o n l y 5.9 % o f t h e t o t a l i r o n i s c o m p l e x e d by EDTA i n A q u i l ( f r o m t h e s p e c i a t i o n p r o g r a m ' M i n e q u i l ' ) , t h e s e e s t i m a t e s s u g g e s t t h a t t h e EDTA l o s s i n t h e t r e a t m e n t s u s e d i n t h i s s t u d y was n o t s u f f i c i e n t t o a f f e c t t h e b i o a s s a y s . 1 14 APPENDIX 4 M o s s b a u e r s p e c t r o s c o p y i s t h e measurement o f r e s o n n a n t gamma-rays t r a n s m i t t e d t h r o u g h a sample as a f u n c t i o n o f t h e i r D o p p l e r v e l o c i t y w i t h r e s p e c t t o t h e s o u r c e . The D o p p l e r v e l o c i t y i s d i r e c t l y c o r r e l a t e d w i t h t h e e n e r g y o f t h e gamma-ray so t h e s p e c t r u m i s a c t u a l l y a r e c o r d o f t r a n s m i s s i o n ( o r a b s o r b a n c e ) as a f u n c t i o n o f e n e r g y of t h e i n c i d e n t r a d i a t i o n . The m a j o r d i f f e r e n c e f r o m o t h e r forms o f t r a n s m i s s i o n s p e c t r o s c o p y i s t h a t l o n g p e r i o d s of c o u n t i n g ( h o u r s r a t h e r t h a n m i n u t e s ) a r e r e q u i r e d b e c a u s e of t h e v e r y s m a l l e n e r g y band s c a n n e d and t h e low p h o t o n f l u x d e n s i t i e s n e c e s s a r y . The u n d e r l y i n g p r i n c i p l e o f t h e M o s s b a u e r e f f e c t i s t h e a b s o r b a n c e o f a u n i q u e e n e r g y ( i . e . , D o p p l e r v e l o c i t y ) gamma-ray by t h e e l e c t r o n f i e l d o f an atom r e s u l t i n g i n i t s t r a n s f o r m a t i o n i n t o t h e e x c i t e d s t a t e . Atoms i n t h e e x c i t e d s t a t e may t h e n r e l e a s e t h e a b s o r b e d e n e r g y ( e m i s s i o n ) . The key t o t h e M ossbauer e f f e c t i s t h e a b s o r b a n c e and emmision of t h e low e n e r g y gamma-r a y s w i t h o u t e n e r g y l o s s by e i t h e r r e c o i l or t h e r m a l b r o a d e n i n g . (Thus t h e a b s o r b a n c e and e m i s s i o n e n e r g i e s a r e i d e n t i c a l . ) The r e c o i l l e s s a b s o r b a n c e and e m i s s i o n i s o p t i m i z e d a t low t e m p e r a t u r e s . B e c a u s e of t h i s t h e a u t o c l a v e d and non-a u t o c l a v e d c o l l o i d a l samples were a n a l y s e d a t l i q u i d n i t r o g e n t e m p e r a t u r e s ( 7 8 ° K ) . To i n t e r p e r e t M o ssbauer s p e c t r a an u n d e r s t a n d i n g of what a f f e c t s t h e a b s o r b a n c e o f gamma-rays i s r e q u i r e d . T h e r e a r e t h r e e p r i n c i p a l h y p e r f i n e i n t e r a c t i o n s t o c o n s i d e r ; c h e m i c a l i s o m e r s h i f t , m a g n e t i c d i p o l e s , and e l e c t r i c q u a d r u p o l e s . The 115 c h e m i c a l i s o m e r s h i f t i s a change i n t h e e l e c t r i c monopole o r C o u l o m b i c i n t e r a c t i o n between t h e e l e c t r i c and n u c l e a r c h a r g e c a u s e d by a d i f f e r e n c e i n t h e s i z e of t h e n u c l e u s . I t i s seen as a s h i f t o f t h e a b s o r b a n c e l i n e w i t h r e s p e c t t o t h a t o f t h e e m i s s i o n e n e r g y o f t h e s o u r c e w i t h o u t c h a n g i n g t h e shape o f t h e s p e c t r a . B o t h t h e m a g n e t i c d i p o l e and q u a d r u p o l e i n t e r a c t i o n s , on t h e o t h e r hand, g e n e r a t e m u l t i p l e l i n e s p e c t r a and can c o n s e q u e n t l y g i v e a g r e a t d e a l o f i n f o r m a t i o n . M a g n e t i c h y p e r f i n e i n t e r a c t i o n w i l l o c c u r i f t h e atom i s s u b j e c t e d t o a m a g n e t i c f i e l d . The m a g n e t i c f i e l d w i l l s p l i t t h e e n e r g y l e v e l o f t h e n u c l e u s i n t o n o n - d e g e n e r a t e e q u a l - s p a c e d s u b l e v e l s . T h i s may be i l l u s t r a t e d i n t h e s p e c t r u m as a s p l i t t i n g o f t h e peak. ( F o r a more d e t a i l e d e x p l a n a t i o n o f t h i s phenomenon see G i b b ( 1 9 7 6 ) . ) S i n c e i r o n i s h i g h l y f e r r o m a g n e t i c , t h i s h y p e r f i n e i n t e r a c t i o n i s e x p e c t e d t o a f f e c t t h e s p e c t r a i n some way. The e l e c t r i c q u a d r u p o l e i n t e r a c t i o n i s d i r e c t l y r e l a t e d t o t h e e l e c t r o n s p i n of t h e n u c l e u s . Any n u c l e u s w i t h s u f f i c i e n t l y h i g h s p i n quantum number (I .= 0.5) has a n o n - s p h e r i c a l c h a r g e d i s t r i b u t i o n . The q u a d r u p o l e s p l i t t i n g i s s e n s i t i v e t o t h e symmetry of t h e e n v i r o n m e n t of t h e atom. The a b s e n c e of q u a d r u p o l e s p l i t t i n g i s i n d i c a t i v e o f c u b i c o r n e a r - c u b i c s i t e symmetry. In c o n t r a s t , t h e p r e s e n c e of q u a d r u p o l e s p l i t t i n g i n d i c a t e s s i g n i f i c a n t d i s t o r t i o n . W h i l e t h e c h e m i c a l i s o m e r s h i f t m e r e l y c a u s e s a u n i f o r m s h i f t of t h e a b s o r b a n c e l i n e s ( w i t h o u t a l t e r i n g t h e i r s e p a r a t i o n ) , b o t h t h e m a g n e t i c and q u a d r u p o l e i n t e r a c t i o n s a r e d i r e c t i o n a l d e p e n d a n t e f f e c t s . As a r e s u l t , when b o t h a r e 1 16 p r e s e n t t h e g e n e r a l i n t e r p r e t a t i o n of t h e s p e c t r u m c a n be q u i t e c o m p l e x . The Mossbauer a n a l y s i s i n t h i s s t u d y , however, was u s e d o n l y t o compare s p e c t r a o f t h e a u t o c l a v e d and rion-a u t o c l a v e d c o l l o i d a l s t o c k s a n d t o i n t e r p r e t p o s s i b l e d i f f e r e n c e s . 

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