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Utility of a carbon-14 bioassay for detecting selenium limitation in marine phytoplankton Clifford, Peter John 1987

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UTIL ITY OF A CARBON-14 BIO ASS A Y FOR DETECTING S E L E N I U M L IMITATION IN M A R I N E P H Y T O P L A N K T O N by P E T E R J O H N CLIFFORD B.Sc , Dalhousie University, 1984 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR T H E DEGREE OF M A S T E R OF SCIENCE in The Faculty of Graduate Studies (Oceanography) We accept this thesis as conforming to the required standard The University of British Columbia October 1987 ©Peter John Clifford, 1987 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of OCEANOGRAPHY The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date IS 0 c 3 t d < ^ / ? f < f - ~ ~ i i ABSTRACT A * 4 C primary productivity bioassay was developed to detect selenium limitation in marine phytoplankton. Addition of Na2Se0g to Se-deplete cultures of Thalassiosira pseudonana stimulated carbon uptake rates by up to 40%, when uptake was expressed on a per cell volume or relative basis. Recovery from Se-starvation was verified by changes in the growth rate and morphology of T. pseudonana. Carbon uptake rates of Katodinium rotundatum, grown in nutrient enriched artificial seawater supplemented with 1 0 " ^ M or 10'® M Se, were unaffected by IS^SeOg additions. Since Katodinium rotundatum did not exhibit growth responses to Se additions, it was concluded that 10" ^ M Se was sufficient for the growth of this alga, which has not displayed an obligate Se requirement. Natural phytoplankton assemblages in the Strait of Georgia were examined for Se limitation with this ^ C bioassay. Relative carbon uptake rates did not change following Na2SeOg addition, indicating that these assemblages were not Se-limited at the time of the study. iii TABLE OF CONTENTS ABSTRACT » LIST OF TABLES viii LIST OF FIGURES ix ACKNOWLEDGEMENTS xiii INTRODUCTION 1 Overview and objectives 1 Historical perspective 2 Selenium requirement in plants 3 Selenium speciation 4 Selenium uptake and incorporation 4 Test organisms 5 C H A P T E R 1. UTILITY OF A C BIO A S S A Y TO DETECT S E L E N I U M L IMITATION IN THE M A R I N E P H Y T O P L A N K T E R S THALASSIOSIRA PSEUDONANA A N D KATODINIUM ROTUNDATUM 7 Background 7 Materials and Methods 9 Algal cultures 9 Culture medium and flasks 9 Culture conditions and growth measurements 10 l ^C bioassays - 11 Nitrate analysis 13 Results 14 A. Thalassiosira pseudonana 14 Growth rates 14 Morphological changes 19 V Carbon uptake 19 B: Katodinium rotundatum 29 Growth rates 29 Morphological changes 32 Carbon uptake • 32 Discussion 43 Growth response 43 Morphological changes 45 1 4 C uptake 47 Utility of primary productivity bioassays 48 Test organisms 50 Summary **1 C H A P T E R 2: U S E OF A 1 4 C BIOASSAY TO E X A M I N E S E L E N I U M NUTRITION OF A N A T U R A L P H Y T O P L A N K T O N A S S E M B L A G E IN THE STRAIT OF GEORGIA 52 Background 52 Materials and Methods 53 Sample collection 53 Na2Se03 addition 53 1 4 C bioassays 57 Results and Discussion 58 Relative carbon uptake 58 Se requirements of natural assemblages 58 Dissolved selenium concentrations 61 Selenium concentrations in the Strait of Georgia 61 vii Summary 64 GENERAL CONCLUSIONS 65 LITERATURE CITED 66 APPENDIX 73 LIST OF TABLES Table I. Location of station and time of incubations ix LIST OF FIGURES Figure 1. Growth of T. pseudonana in +Se (A), or -Se (B) ESAW, following addition of 0 M (•), 1 0 " 1 0 M (A) or 10" 6 M (•) N a 2 S e 0 3 at Time = 0 h. The solid lines represent in vivo fluorescence, and the dashed lines represent cell density as cellsTnl"''". Values are the mean of duplicates; error bars indicate — 1 SD, and where absent the range was less than the width of the symbol 15 Figure 2. Early stationary phase T. pseudonana was transferred from Se-deplete E S A W into 2 culture tubes. At Time = 24 h (indicated by the arrow) an add-Q back experiment was conducted. One tube was spiked with 10 M Na2SeOg (• ) , and the other tube served as the control (#) 17 Figure 3. Average cell volume of T. pseudonana grown in +Se (A), or -Se (B) E S A W . Cells were grown in +Se or -Se E S A W for one transfer after the initial inoculum was obtained from ESNW. After a second transfer into + Se or -Se E S A W , the experiment was initiated at Time = 0 h; Na2SeOg was added at concentrations of 0 M (•), 1 0 " 1 0 M (A) 0 r 10" 6 M (•). Values are the mean of duplicates; error bars indicate — 1 SD, and where absent the range was less than the width of the symbol 20 Figure 4. Carbon uptake rates per cell for T. pseudonana grown in +Se (A) or -Se (B) E S A W for two transfers, following addition of 0 M (•) , l O " 1 0 M (•) or 10" 6 M (•) Na2SeOg at Time = 0 h. Values are the mean of duplicates; error bars indicate — 1 SD, and where absent the range was less than the width of the symbol 22 X Figure 5. Carbon uptake rates per cell volume for T. pseudonana grown in +Se (A) or -Se (B) ESAW, following addition of 0 M (•), 1 0 " 1 0 M (•) or 10" 6 M (•) Na 2 SeOg at Time = 0 h. Values are the mean of duplicates; error bars indicate + 1 SD, and where absent the range was less than the width of the symbol 25 Figure 6. Relative carbon uptake, expressed as disintegrations per minute (DPM) per ml of culture, for T. pseudonana grown in +Se (A) or -Se (B) ESAW. Selenium was added at 0 h as 0 M (•) , 1 0 ' 1 0 M (A) or 10" 6 M (•) N a 2 S e 0 3 . Values are the mean of duplicates; error bars indicate + 1 SD, and where absent the range was less than the width of the symbol 27 Figure 7. K. rotundatum was grown for 10 generations in E S A W supplemented with 1 0 - 1 0 M (A) or 10" 6 M (B) Na 2 SeOg. At Time = 0 h, 0 M (•) or 10" 6 M (•) Na 2 SeOg was added to the culture medium and growth was followed for 48 h. The solid lines represent in vivo fluorescence, and the dashed lines represent cell density as cells'ml"^. Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol 30 Figure 8. Average cell volume of K. rotundatum grown for 10 generations in ESAW supplemented with 1 0 " 1 0 M (A) or 10" 6 M (B) N a 2 S e 0 3 . 0 M (•) or 10* 6 M (•) Na 2 SeOg was added at Time = 0 h, and cell volumes changes monitored for 48 h. Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol xi Figure 9. Carbon uptake rate per cell for K. rotundatum grown for 10 generations in E S A W supplemented with 1 0 ' 1 0 M (A) or 10" 6 M (B) N a 2 S e 0 3 . At Time = 0 h, 0 M (•) or 1 0 - 6 M (•) N a 2 S e 0 3 was added to the cultures, and carbon uptake per cell monitored for 48 h. Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol 35 Figure 10. Carbon uptake rate per cell volume for K. rotundatum grown for 10 generations in ESAW supplemented with 1 0 ' 1 0 M (A) or 10" 6 M (B) N a 2 S e 0 3 . A t Time = 0 h, 0 M (•) or 10" 6 M (•) N a 2 S e 0 3 was added to the cultures, and carbon uptake per cell volume monitored for 48 h. Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol 37 Figure 11. Relative carbon uptake, expressed as disintegrations per minute (DPM) per ml of culture, for K. rotundatum grown for 10 generations in ESAW supplemented with 1 0 " 1 0 M (A) or 10" 6 M (B) IN^SeOg. Selenium was added at 0 h as 0 M (•) or 10" 6 M (•) N a 2 S e 0 3 . Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol 40 Figure 12. Location of Station 1 (•) in the Strait of Georgia, British Columbia, Canada 54 Figure 13. Relative carbon uptake, expressed as disintegrations per minute (DPM) per ml of sample, for a natural phytoplankton assemblage. The sample was collected from the 55% light depth at Station 1. Selenium was added at 0 h as 0 M (•), 1 0 " 1 0 M (A) or 10 ' 6 M (•) Na 2 SeOg. The standard deviation of the mean was less than the width of the symbols 59 Figure 14. Relative carbon uptake, expressed as disintegrations per minute (DPM) per ml of culture, for T. pseudonana grown in +Se (A) or -Se (B) E S A W . Selenium was added at 0 h as 0 M (•), 1 0 " 1 0 M (A) or 10" 6 M (M N a 2 S e 0 3 . Values are the mean of triplicates. These cultures were grown with full E S A W nutrient enrichment (550 uM N O 3 ) , and the incubations were 2 h in duration 74 ACKNOWLEDGEMENTS I am indebted to my academic supervisor, Dr. P.J. Harrison, for his patience, support and guidance during all phases of this research. Thanks are also extended to the members of my supervisory committee, Drs. E.V. Grill and F.J.R. Taylor. Dr. N.M. Price provided invaluable advice, particularly regarding laboratory analyses. The research presented in this thesis benefitted from discussions with my associates: W.P. Cochlan, G.J. Doucette, L.J. Jackson and P.A. Thompson. I am grateful to W.P. Cochlan for performing the nitrate analyses, his assistance in drafting the figures, and for his contributions to the research presented in Chapter 2. Financial support was provided by scholarships from NSERC and UBC, and by teaching assistantships. The research was supported by funds from a NSERC operating grant (58-0128) to Dr. P.J. Harrison. 1 INTRODUCTION Overview and objectives This thesis examines the utility of a C bioassay as a technique for identifying selenium limitation in phytoplankton. Selenium (Se) has been shown to be an essential nutrient for the coastal marine diatom Thalassiosira pseudonana, and shown to stimulate growth in the marine dinoflagellate Katodinium rotundatum. Chapter 1 examines the time-course of ^ ^C uptake by these two species, following a selenium addition to Se-limited cultures. It is suggested that carbon uptake rates normalized to cell volume, or, to disintegrations per minute (DPM) per ml sample, are a useful indicator of selenium limitation, and that measurement of ^ C uptake may be a feasible method for diagnosing Se-limitation in natural populations. The hypothesis that Se-limited algae show increased carbon uptake rates following Se addition was tested with a natural phytoplankton assemblage in Chapter 2. Results from this experiment are discussed, and suggestions made for further investigations. 2 Historical perspective Selenium (Se) has been recognized as a potentially toxic element since the early 1930's, when it was linked to the poisoning of livestock (Robinson 1933, Franke 1934, Byers 1935). Toxicity appears to result from the indiscriminate substitution of selenium for sulfur in biological molecules, usually when the environmental concentration of selenium exceeds several micromolar (Stadtman 1974). Selenium is located between sulfur and tellurium in the periodic table, and has the same valencies as sulfur (-2, +2, +4, +6). Therefore, it is not surprising that selenium may act as an analogue for sulfur in some biological molecules. Only recently has selenium's role as an important micronutrient been appreciated (Frost 1972). In the 1960's several livestock diseases were linked to nutritional selenium deficiencies (Stadtman 1974). Selenium is now known to be a component in at least one mammalian enzyme, as well as several bacterial enzymes (Stadtman 1980). In many of these enzymes selenium is incorporated as a sulfur analogue, often selenocysteine. As Stadtman (1980) points out, these enzymes are Se-dependent. They are synthesized when the concentration of selenium is very low (10"® Q to 10 M), and in the presence of high sulfur concentrations. Hudman and Glenn (1984, 1985) found that selenite transport in three bacterial species was distinct from sulfate transport, since sulfate did not inhibit selenite uptake, nor could sulfate transport be demonstrated. This suggests that there are biochemical processes such as transport or assimilation which can specifically differentiate between Se and S, but these processes are still poorly understood. 3 Selenium requirement in plants Much controversy surrounds the role of selenium in plant nutrition. Trelease and Trelease (1938a, b, 1939) were the first to suggest that selenium stimulates growth and may be an essential nutrient. Rosenfeld and Beath (1964) found that selenium additions increased the dry weight of racemose milk vetch, Astragalus racemosus. Broyer and coworkers (Broyer et al. 1966) however, failed to observe stimulatory effects when selenium was added to alfalfa (Medicago sativa) and subterranean clover (Trifolium subterraneum). Their experiments were hampered by inadvertent Se-contamination, which appeared to be air-borne. Broyer et al. (1972) confirmed Rosenfeld and Beath's observation that selenium stimulates growth of A. racemosus and their own earlier observations (Broyer et al. 1966) that air-borne selenium contamination confounds interpretation of these data. In addition, they suggest that the stimulation of A. racemosus reported by Trelease and Trelease (1939) was due to the alleviation of phosphate toxicity by selenium addition. The essentiality of selenium for higher plants remains poorly understood, particularly regarding Se-phosphate interactions. Recent studies have focused on the importance of selenium in algal nutrition. Unlike studies with higher plants, it is apparent that Se is a true micronutrient for some species. Pintner and Provasoli (1968) were the first to report that selenium was required for reliable growth of three Chrysochromulina species. Subsequent investigations have demonstrated that selenium is required for, or stimulates growth of, representatives from six algal classes (Lindstrom and Rodhe 1978, Fries 1982, Lindstrom 1983, 1985, Wehr and Brown 1985, Price et al. 1987). Wheeler et al. (1982) caution that the effects of selenium depend upon the algal species, Se concentration and oxidation state, and the concentration of sulfate. 4 Selenium speciation Dissolved selenium in seawater occurs in three valence states: selenite ( S e 0 3 2 " , Se IV), selenate ( S e 0 4 2 _ , Se VI) and organic selenide (Se II) (Cutter 1982). The vertical distribution of selenium is similar to nutrients such as Si and P, with removal in surface waters and regeneration at depth (Measures and Burton 1980a). In the euphotic zone approximately 80% of the Se is present as organic selenide. Deep water is enriched with selenite and selenate, while organic selenide is not detectable (Cutter and Bruland 1984). These data suggest that the vertical distribution of Se is dominated by biological processes, and have led to the formation of a multistep regeneration model for Se (Cutter and Bruland 1984). Selenium uptake and incorporation Mechanisms of Se uptake and incorporation by phytoplankton remain unclear. Of the three species of dissolved Se occurring in seawater, selenite appears to be the most biologically active form. Wrench and Measures (1982) demonstrated selective uptake of selenite over selenate during a spring phytoplankton bloom. Apte et al. (1986) examined selenium uptake during a spring phytoplankton bloom in an enclosed experimental ecosystem, and confirmed that Se(IV) was preferentially taken up over Se(VI). Under relatively low Se concentrations, selenium-specific uptake and incorporation systems appear to operate; at higher Se concentrations, however, selenium incorporation utilizes the sulfur uptake and incorporation system (Bottino et al. 1984). Hudman and Glenn (1984, 1985) found that selenite uptake in the bacteria Butyriuibrio fibrisoluens, Bacteroides ruminicola, and Selenomonas ruminantium was inducible, and distinct from selenate and sulfate transport. 0 Selenium is incorporated into amino acids and proteins, some of which are analogues of sulfur proteins, such as selenocysteine and selenomethionine (Bottino et al. 1984). Wrench (1978) found that in Tetraselmis tetrathele and Dunaliella minuta, selenium did not merely bind to proteins, but that it was incorporated directly into their primary structure. These data are supported by studies of Se incorporation by macroalgae and marine animals, where Se is mainly associated with proteins, and is not found as inorganic species (Maher 1985a, b). Toxicity of selenium to phytoplankton depends upon its oxidation state. In general, selenate is at least one order of magnitude more toxic than selenite (Wheeler et al. 1982, Price et al. 1987). However, this response is species-specific and shows great variability among species. Test organisms In this thesis, the centric marine diatom Thalassiosira pseudonana (clone 3H) (Hustedt) Hasle and Heimdal and the athecate marine dinoflagellate Katodinium rotundatum (Lohmann) Loeblich III, are used to investigate carbon uptake following selenium addition to Se-replete and Se-deplete cultures. T. pseudonana has been chosen by many investigators examining nutrient uptake (Goldman and McCarthy 1978, Dortch et al. 1982, Parslow et al. 1984a, 1984b, Sharp et al. 1980, Smith and Piatt 1984, Price 1987), trace metal interactions and requirements (Sunda and Huntsman 1983), and selenium requirements (Doucette et al. 1987, Price 1987, Price et al. 1987). T. pseudonana has been extensively studied, grows rapidly and is easily maintained in culture. Morphological changes due to selenium limitation provide an easy and unequivocal means of determining Se-limitation. These criteria make it an ideal organism to use in examining physiological processes. 6 In contrast, K. rotundatum. is more fragile in culture and has not been extensively studied. However, it is an ecologically important species, which may be dominant at times in the plankton community (Jacobson 1987). Preliminary studies, by another student in our laboratory, suggested that variations in selenite concentration influence its growth rate. Therefore, K. rotundatum may also be an ideal organism to use to examine responses to selenium limitation. 7 C H A P T E R 1. UTILITY OF A 1 4 C BIOASSAY TO DETECT SELENIUM  LIMITATION" IN THE MARINE P H Y T O P L A N K T E R S THALASSIOSIRA PSEUDONANA A N D KATODIUM ROTUNDATUM B A C K G R O U N D Selenium occurs at picomolar to nanomolar concentrations in the marine environment (Measures and Burton 1980a, Cutter and Bruland 1984). Laboratory studies have found that the threshold concentrations required to stimulate growth of Se-limited algae are in the nanomolar range (Lindstrom and Rodhe 1978, Lindstrom 1983, 1985, Wehr and Brown 1985, Price 1987, Price et al. 1987). Furthermore, Wehr and Brown (1985) demonstrated that selenium limitation can occur in natural freshwater phytoplankton populations. Therefore, it appears that the concentrations of selenium in the environment are very similar to those required for phytoplankton growth. Recognizing that natural phytoplankton assemblages may potentially be selenium-limited, there exists a need for a simple method to detect Se limitation. Price (1987) and Price et al. (1987) examined the response of Se-limited Thalassiosira pseudonana to a selenium addition. They found that the use of either in vivo fluorescence or cell numbers, as physiological indicators, were not sensitive enough to accurately indicate recovery from Se starvation. Consequently, they suggested that measuring the increase in H ^ C O g " uptake after a selenium addition to Se-limited cells may increase the resolution and provide a more sensitive bioassay for selenium limitation. The research presented in this thesis was initiated in order to evaluate the * 4 C bioassay as a method of identifying Se-limited phytoplankton. Although the primary productivity incubation bioassay (Ryther and Guillard 1959) has been in use 8 nearly as long as the primary productivity technique (Steemann Nielsen 1952), its utility has often been questioned (O'Brien and deNoyelles 1976, Ignatiades 1977, Healey 1979, Lean and Pick 1981). Nevertheless, since preliminary data indicated that uptake was useful in assessing Se limitation, it was decided to further explore the applications of this technique. Existing analyses for selenium, such as gas chromatography or neutron activation analysis, usually require expensive equipment, highly trained personnel, or both (Measures and Burton 1980b, Orvini et al. 1981). The requirements for a simple field assay were incompatible with existing techniques, which was another reason for examining the potential of bioassays. M A T E R I A L S AND METHODS Algal cultures Thalassiosira pseudonana (clone 3) (Hustedt) Hasle et Heimdal and Katodinium rotundatum (Lohmann) Loeblich III were obtained from the Northeast Pacific Culture Collection (N.E.P.C.C. # 58 and 44, respectively), Department of Oceanography, University of British Columbia. These cultures were maintained on nutrient enriched natural seawater, and were unialgal but not axenic. Thalassiosira pseudonana (clone 3H) is a coastal isolate, first obtained in 1958 from Moriches Bay, N.Y, U.S.A. The Katodinium rotundatum strain used in these experiments is also a coastal clone, isolated in 1972 from Point Atkinson, B.C., Canada. Culture medium and flasks Cultures of T. pseudonana and K. rotundatum were grown in filter-sterilized (0.45 um) nutrient enriched artificial seawater based on ESAW (Harrison et al. 1980). In preliminary experiments with T. pseudonana grown on full E S A W nutrient enrichment, when cultures reached stationary phase, the pH of the culture medium often measured pH 9.3 to 9.5. This suggests that the pH-tolerance of T. pseudonana may be exceeded when it is grown on full E S A W nutrient enrichment, if precautions are not taken to control the pH of the medium. Therefore, for my experiments, nutrient concentrations in the culture medium were reduced to 1/10 those suggested by Harrison et al. (1980); this succeeded in maintaining pH < 8.5 for cultures in late exponential phase. Reagent grade chemicals were used throughout, and nutrient enrichment solutions were prepared in deionized distilled water (DDW). Modifications to E S A W included replacing F e N H ^ C S O ^ ' O H ^ O by an equimolar concentration of F e C l g ^ r ^ O , and by adding all the iron to a solution with a resultant EDTA:Fe molar ratio of 1.6. The remaining N a 2 E D T A added to ESAW was included with the trace metal stock, and 0.0126 N a 2 M o 0 4 and 0.0059 g-L" 1 N i C l 2 - 6 H 2 0 were also added. Na 2 glyceroPO^ was replaced with an equimolar concentration of N a 2 H P 0 4 . N a 2 S i 0 3 - 9 H 2 0 was prepared and added following Suttle et al. (1986). Se was added to E S A W , when required, as an aqueous solution of Na 2 SeOg Glassware and polycarbonate (PC) vessels were used for culturing algae and storing ESAW. Prior to use, they were soaked for at least 24 h in 10% (v/v) HC1, thoroughly rinsed with DDW, followed by at least 24 h of soaking in DDW. Price et al. (1987) report that this procedure successfully removes adsorbed Se from glassware, preventing Se contamination of Se-deplete ESAW. Culture conditions and growth measurements Cultures were continuously illuminated from two sides by Vita-Lite® UHO and Vita-Lite® VHO daylight fluorescent tubes, filtered through a 3 mm thick sheet of blue Plexiglas® (No. 2069, Rohm and Hass). The irradiance at the surface of 2 1 + the culture vessels measured 125 uE - m s . Temperature was maintained at 18 — 1 C by a temperature-regulated water bath. Al l experiments were conducted on batch cultures, which were stirred daily when samples were taken. Cultures were unialgal but not axenic, although bacterial numbers were kept to minimal densities by employing aseptic techniques throughout. Cell growth was monitored by in vivo chlorophyll a fluorescence measured on a Turner Designs model 10 fluorometer, and cell counts on a Coulter Counter® model TA II. Average cell volumes were computed from the cell 11 distributions in the various channels. T. pseudonana was counted using a 70 um aperture sample tube, which was calibrated with 5.07 um diameter microspheres. K. rotundatum was counted using a 200 um aperture sample tube, which was calibrated with 44.2 um diameter microspheres. In experiments designed to examine the effects of N a 2 S e 0 3 on carbon uptake rates, cells were preconditioned in the medium for at least 10 doublings. Thalassiosira pseudonana was inoculated from nutrient enriched natural seawater (ESNW) into nutrient enriched artificial seawater (ESAW), containing no added Se (-Se) or E S A W containing 10 ' 8 M N a 2 S e 0 3 ( + Se). The cultures were serially diluted to maintain the cells in exponential growth. Following the second transfer into Se-replete or Se-deplete E S A W , N a 2 S e 0 3 was added to the cultures at concentrations of 0, l O ' ^ C "1 A or 10 M , and C bioassays were initiated. Katodinium rotundatum was inoculated from nutrient enriched natural seawater (ESNW) into nutrient enriched artificial seawater (ESAW), with either 1 0 " 1 0 M or 10" 6 M N a 2 S e 0 3 enrichment. The cultures were serially diluted to maintain the cells in exponential growth. After at least 10 generations in the 1 0 " 1 0 M or 10 ' 6 M Se ESAW, N a 2 S e 0 3 was added to the cultures at concentrations of 0 M or 10 -^ M Se, and bioassays were initiated. Water mounts of T. pseudonana and K. rotundatum were prepared for light microscopy and examined with a Zeiss Photomicroscope II light microscope using Nomarski interference optics and brightfield illumination. ^ ^ C bioassays N a H 1 4 C 0 3 (New England Nuclear; 4.5 mCrmmol" 1) was prepared following Strickland and Parsons (1968), filter-sterilized (0.22 um filters) and refrigerated before use. Disposable polypropylene pipette tips used for inoculations were soaked in 10% HCI and thoroughly rinsed with DDW prior to use, in order to reduce trace metal contamination (Fitzwater et al. 1982). Carbon uptake rates were measured using a fixed incubation time (4 h) and variable Se enrichment. At the beginning of each experiment (t = 0 h), 250 ml samples were removed from the experimental or control cultures and transferred to 250 ml PC screw-top bottles. For T. pseudonana cultures, Na9SeOg was added to duplicate bottles at 0 , 10 or 10 M concentrations. This range was chosen Q because 10 M was previously determined by Price et al. (1987) to be saturating for T. pseudonana. For K. rotundatum cultures, Na2SeOg was added to duplicate bottles at 0 or 10 M concentrations. At the beginning of each ^ 4 C incubation, 5 ml aliquots from each PC bottle were transferred to 15 ml sterile polystyrene tubes. Two uCi N a H ^ C O g was added to each tube, which was incubated at 18 C for 4 h. Cells were collected by filtration onto Whatman GF/F glass-fibre filters. In order to prevent cell breakage during filtration, vacuum pressure differentials were < 100 mm Hg for T. pseudonana and <25 mm Hg for K. rotundatum cultures (Goldman and Dennett 1985). Filters were placed in glass scintillation vials containing 0.2 ml of 0.5 N HCI, which removed inorganic 1 4 C (Lean and Burnison 1979, Goldman and Dennett 1985, Hitchcock 1986). After a 2 h interval, 10 ml Aquasol II scintillation fluor (New England Nuclear) was added to each vial. Zero-time blanks were used in order to correct for cell and bottle adsorption of 1 4 C . Total ^ 4 C activity of the stock solution was determined by adding 20-25 ul H ^ C O g " stock solution (nominal activity 0.4-0.5 uCi) to scintillation vials containing 0.2 ml phenethylamine (Iverson et al. 1976), an amount shown to maximize inorganic label retention but minimizing quenching. Samples were counted on an Isocap 300 liquid scintillation counter, and quench correction was by the channels-ratio method. Carbon uptake rates were determined using the formula provided by Parsons et al. (1984). Total carbon dioxide was determined using a modification of Parsons et aZ.'s (1984) method; sample volume was decreased from 100 to 20 ml, and the volume of 0.01 N HC1 added was decreased from 25 to 5 ml. Nitrate analysis At the conclusion of each ^ C bioassay experiment, samples from each PC bottle were removed with an acid-washed syringe for nitrate (NO3) analysis. Samples were gently filtered under pressure through combusted (460 C for 4 h) Whatman GF7F filters (mounted in Milipore Swinnex® filter holders) into acid-washed polyethylene bottles and stored frozen (- 20 C) until analysis. Nitrate concentrations were measured with a Technicon Autoanalyzei® II following the procedure outlined in Wood et al. (1967). R E S U L T S A: Thalassiosira pseudonana Growth rates Selenium enrichment had no effect upon the growth of the +Se culture (Fig. 1A). Exponential growth rates (k = 1.7 d"^) were equivalent to the maximum growth rates of T. pseudonana cultures grown on ESNW. Cell counts verified that fluorescence was an accurate indicator of algal growth, and have been included for comparison with the fluorescence data (Fig. 1A). In contrast, growth of the -Se cultures was positively correlated with Se addition (Fig. IB). Cultures with 0 M Se enrichment had the lowest growth rates (k = 0.89 ± 0.09 d" 1). Those cultures with 1 0 " 1 0 M Se enrichment had intermediate growth rates (k = 1.1 - 0.17 d" 1), while cultures with the highest (10" 6 M) Se enrichment exhibited the highest growth rates (k = 1.9 - 0.05 d"*), compared to the + Se cultures. Enhancement of growth was observed within 12 h of the Se addition, indicating that the -Se cultures were Se-limited. Nitrate was present in saturating concentrations (NO 3 > 10 uM) at the conclusion of theexperiment, precluding the possibility that cultures were N-limited. Further evidence that the -Se cultures were Se-limited is presented in Q Figure 2 in an add-back experiment. The addition of 10 M Na2Se03 to an aliquot of the 0 M Se culture stimulated growth; the aliquot without Se addition remained in stationary phase. Figure 1. Growth of T. pseudonana in +Se (A), or -Se (B) ESAW, following addition of 0 M (•), 1(T 1 0 M (•) or 10"6 M (•) Na 2Se0 3 at Time = 0 h. The solid lines represent in vivo fluorescence, and the dashed lines represent cell density as cells 'mr*. Values are the mean of duplicates; error bars indicate — 1 SD, and where absent the range was less than the width of the symbol. ]n vivo Fluorescence jn vivo Fluorescence |LU J 9 d s||9o | U J J a d S | p o Figure 2. Early stationary phase T. pseudonana was transferred from Se-deplete ESAW into 2 culture tubes. At Time = 24 h (indicated by the arrow) an add-Q back experiment was conducted. One tube was spiked with 10 M Na2SeOg (•), and the other tube served as the control (•). ]n vivo Fluorescence o _, i o Morphological changes T. pseudonana grown in Se-replete medium did not exhibit any morphological changes over the course of the experiment. The Coulter Counter® cell size distribution indicated that the average cell volume of the +Se cultures remained constant (Fig. 3A), and was verified by microscopic examination. Al l cells appeared healthy, with no observable chain formation or cell elongation. However, at the beginning of the bioassay experiment, the average cell volume of the Se-starved cells was two times greater than the Se-replete cells (Fig. 3B). Microscopic examinination showed that the cells were twisted and bent; some were greatly elongated, and chain formation was prevalent. Cells continued to increase in size, and were three times larger than Se-replete cells by the conclusion of the experiment (Fig. 3B). Selenium addition partially ameliorated these symptoms of Se-starvation: cell volumes decreased in comparison to the sample with 0 M Se enrichment (Fig. 3B), and microscopic examination revealed that cell distortion, cell elongation and chain formation were greatly reduced 24 h after Se addition. Carbon uptake Carbon uptake rates per cell are presented in Figure 4. Uptake rates per cell for the +Se culture were fairly constant from 0 to 18 h, but showed a sharp decline at 24 h. This decrease in carbon uptake per cell is attributable to a decrease in the growth rate of the culture, since by 24 h the -t-Se culture appears to have entered early stationary phase (cf. Fig. IA). Selenium enrichment appears to have had little effect upon C uptake in the +Se cultures, although minor stimulation did occur at 6 and 12 h for the cultures receiving 1 0 " 1 0 M and 1 0 ' 6 M N a 2 S e 0 3 enrichment (Fig. 4A). 20 Figure 3. Average cell volume of T. pseudonana grown in +Se (A), or -Se (B) ESAW. Cells were grown in +Se or -Se ESAW for one transfer after the initial inoculum was obtained from ESNW. After a second transfer into +Se or -Se ESAW, the experiment was initiated at Time = 0 h; lS^SeOg was added at concentrations of 0 M (•), 10"10 M (A) or 10"6 M (•). Values are the mean of duplicates; error bars indicate — 1 SD, and where absent the range was less than the width of the symbol. Figure 4. Carbon uptake rates per cell for T. pseudonana grown in +Se (A) or -Se (B) ESAW for two transfers, following addition of 0 M (•), 10"10 M (•) or 10"6 M (•) Na2SeOg at Time = 0 h. Values are the mean of duplicates; error bars indicate — 1 SD, and where absent the range was less than the width of the symbol. Carbon uptake rates per cell for the -Se culture increased slightly over the 24 h experimental period, but there was no consistent response to Na9SeOg enrichment (Fig. 4B). There was a small increase in C uptake per cell at 6 and 12 h for the -Se cultures receiving 10 M and 10 M Na 9 Se03 enrichment; recall that this was also observed for the -l-Se cultures. When carbon uptake is expressed on a per cell volume basis, differences following Se enrichment are readily apparent between the +Se and -Se cultures (Fig. 5). In the +Se culture, Se enrichment had little effect upon C uptake per cell volume, although uptake at 6 and 12 h was slightly enhanced with increasing Se addition (Fig. 5A). However, Se enrichment had dramatic effects upon C uptake per cell volume in the -Se culture. Whereas C uptake per cell volume was constant for samples receiving 1 n (\ 0 M or 10 M Se enrichment, the sample with 10 M Se-enrichment showed a ca. 40% increase in carbon uptake (Fig. 5B). Relative carbon uptake data, expressed as disintegrations per minute (DPM) per ml of culture, corroborates these observations that 10"^ M Se enrichment caused enhanced C uptake in Se-starved T. pseudonana (Fig. 6B), but had no effect upon uptake rates of the control ( + Se) culture (Fig. 6A). 25 Figure 5. Carbon uptake rates per cell volume for T. pseudonana grown in +Se (A) or -Se (B) ESAW, following addition of 0 M (•), 10'10 M (A) or 10-6 M (•) Na2SeOg at Time = 0 h. Values are the mean of duplicates; error bars indicate — 1 SD, and where absent the range was less than the width of the symbol. Figure 6. Relative carbon uptake, expressed as disintegrations per minute (DPM) per ml of culture, for T. pseudonana grown in +Se (A) or -Se (B) ESAW. Selenium was added at 0 h as 0 M (•), 10"10 M (A) or 10"6 M (•) Na 2Se0 3. Values are the mean of duplicates; error bars indicate — 1 SD, and where absent the range was less than the width of the symbol. 28 B: Katodinium rotundatum Growth rates When Katodinium rotundatum was grown on 1 0 " 1 0 M Se and given no additional selenium supplement, it displayed growth rates (k = 0.63 i 0.06 d"^) that were not significantly different from cultures with 10" M Se additions (k = 0.71 — 0.20 d"^) (Fig. 7A). Fluorescence was less sensitive as an indicator of algal growth, compared to growth as measured by cell counts (Fig. 7A). Typical growth rates, determined from preliminary investigations with K. rotundatum grown in ESAW containing 10" 6 to 10" 8 M additions of Se, are slightly higher (k = 0.80 — 0.08 d"1) than the rates measured in this experiment. This suggests that the calculated experimental values for growth of the 10" M culture are too low. The similarity of growth rates between the cultures receiving 0 M and 10 M Se additions implies that 10 M Se concentrations satisfy the growth requirements of this alga. However, since an obligate Se requirement by K. rotundatum has not yet been demonstrated , there is no a priori reason to assume that this alga requires Se for growth. The high variability between replicates makes it difficult, if not impossible, to reach a firm conclusion regarding the Se requirements of K. rotundatum. Similarly, selenium enrichment had no effect upon the growth of the 10* 6 M Se cultures (Fig. 7B). Cultures with 0 M Se enrichment and those with 10" 6 M Se enrichment had growth rates (k = 0.88 — 0.11 d " 1 , 0.81 — 0.12 d " 1 ; respectively) comparable to the mean growth rate (k = 0.80 — 0.08 d"^) realized when K. A rotundatum is cultured in ESAW. over a range of Na2SeOg concentrations (10 to 10" ^ M). As previously observed with K. rotundatum grown on 10"^^ M. Se. cell counts Figure 7. K. rotundatum was grown for 10 generations in ESAW supplemented with 10"10 M (A) or 10"6 M (B) Na 2Se0 3. At Time = 0 h, 0 M (•) or 10"6 M (•) Na2Se03 was added to the culture medium and growth was followed for 48 h. The solid lines represent in vivo fluorescence, and the dashed lines represent cell density as cells'ml"^. Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol. 33U33S3JOn|J OA IA Ul displayed unusually high variability between duplicates. Although the mean growth rates for K. rotundatum grown on 10 M Se are slightly higher than for K. rotundatum grown on 1 0 " ^ M Se, the large variations in the data prevent conclusive statements regarding the effects of selenium enrichment upon growth. Morphological changes A preliminary study that I conducted indicated that K. rotundatum appeared to increase in cell volume, following Na 2 SeOg addition. However, this observation could not be confirmed in this subsequent investigation. Selenium enrichment had no effect upon the cell volume of K. rotundatum grown on 1 0 " 1 0 M Se (Fig. 8A), nor could selenium enrichment be shown to effect a change in cell size of the 10"® M culture (Fig. 8B). It is puzzling why the 10" ^  M Se cultures showed a temporary increase in mean cell volume at 24 and 36 h (Fig. 8A), while the 10"® M Se cultures showed a temporary decrease in mean cell volume (Fig. 8B) over the same time period. Due to the large variability, however, it is difficult to draw concrete conclusions from these data. Carbon uptake Carbon uptake rates indicated that K. rotundatum was not Se-limited. Katodinium rotundatum grown on 10" * u M Se exhibited slightly reduced carbon uptake rates, both on a per cell and a per cell volume basis (Fig. 9A and 10A, respectively), fi fi following addition of 10" M Na 9 SeOg. Cultures of K. rotundatum grown on 10 M Se showed the opposite result following Na2SeOg enrichment. Carbon uptake per cell (Fig. 9B) and carbon uptake per cell volume (Fig. 10B) were slightly higher in the cultures receiving Se enrichment, compared to the cultures with no Se enrichment. Figure 8. Average cell volume of K. rotundatum grown for 10 generations in ESAW supplemented with 10"10 M (A) or 10"6 M (B) Na 2Se0 3. 0 M (•) or 10"6 M (•) Na2Se03 was added at Time = 0 h, and cell volumes changes monitored for 48 h. Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol, Figure 9. Carbon uptake rate per cell for K. rotundatum grown for 10 generations in ESAW supplemented with 10" 1 0 M (A) or 10"6 M (B) N a 2 S e 0 3 . At Time = 0 h, 0 M (•) or 10"6 M (•) Na 2 SeOg was added to the cultures, and carbon uptake per cell monitored for 48 h. Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol. 37 Figure 10. Carbon uptake rate per cell volume for K. rotundatum grown for 10 generations in ESAW supplemented with 10"10 M (A) or 10'6 M (B) Na2SeOg. At Time = 0 h, 0 M (•) or 10"6 M (•) Na 2Se0 3 was added to the cultures, and carbon uptake per cell volume monitored for 48 h. Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol. However, it is difficult to discern trends in these data, particularly since variability was high. It is possible that when K. rotundatum is cultured in ESAW supplemented with 10"^ M NaQSeOg, selenium is present in quantities that are saturating for growth. If this were the case, then enhancement of carbon uptake would be unlikely to occur following addition of selenium to the medium. This was likely true for these experiments, since Se additions to K. rotundatum grown on 10*^ M Se did not affect carbon uptake rates. The growth rate data support this hypothesis that 10"^ M Se is sufficient for maximum growth of K. rotundatum, since selenite additions failed to elicit an increase in growth rate. Indeed, it is also possible that K. rotundatum does not require any Se for growth. Interpretation of these data is confounded by the large variability between duplicate cultures. Much of this variation may be due to imprecise cell counts, and subsequent cell volume determinations, that occurs when counting K. rotundatum with a Coulter Counter®. Moreover, even the relative carbon uptake measurements, expressed as DPM per ml of culture, do not show significant trends in carbon uptake following selenium enrichment of the cultures (Fig. 11). Relative carbon uptake measurements for K. rotundatum grown on 10" 1 0 M Se (Fig. 11A) display high variability, even though this parameter is independent of cell density or cell volume measurements, and therefore does not require use of the Coulter Counter®. Variability in relative carbon uptake for the 10"® M Se cultures (Fig. 11B) is minimal, yet there is still no distinct pattern for carbon uptake, following Se enrichment. It appears that selenium additions to K. rotundatum cultures have no effect upon carbon uptake rates. Furthermore, contrary to earlier observations in our laboratory, it is not clear that selenium additions between 10"® M and 10* ^  M IS^SeOg elicit a growth 40 Figure 11. Relative carbon uptake, expressed as disintegrations per minute (DPM) per ml of culture, for K. rotundatum grown for 10 generations in ESAW supplemented with 10" ^  M (A) or 10"6 M (B) Na2Se03. Selenium was added at 0 h as 0 M (•) or 10"6 M (•) Na 2Se0 3. Values are the mean of duplicates; error bars indicate the range of duplicates, and where absent the range was less than the width of the symbol. 41 response in K. rotundatum. These data indicate that growth measurements are highly variable, and hence it is difficult to accurately determine K. rotundatum's growth rate. DISCUSSION Growth response The addition of selenite stimulated the growth of Se-deplete T. pseudonana, but had no effect on the growth of K. rotundatum preconditioned in ESAW supplemented with 10" ^  M or 10"® M Na2Se03. Growth stimulation due to Se addition is not surprising, since recent studies have demonstrated a requirement for selenium by marine algae. T. pseudonana has been shown to have an obligate requirement for selenium (Price et al. 1987), and preliminary investigations in our laboratory suggested that K. rotundatum exhibited changes in growth rate in response to selenium addition. However, based upon growth rate data in this thesis, K. rotundatum displayed no evidence that it requires selenium for growth. In their pioneering study, Pintner and Provasoli (1968) reported that selenium was essential for reliable growth of 3 Chrysochromulina spp., and determined that 10" M H 2 SeOg additions provided optimal growth for these species. They noted that the selenium requirement was particularly evident when chemical cleanliness was increased, presumably since trace metal contamination of the culture medium was reduced. Fries (1982) examinined the selenium nutrition of two marine macrophytes in axenic culture, the brown alga Fucus spiralis and the red alga Goniotrichum alsidii, and concluded that optimal growth was obtained with additions of 10* 8 and 10" ^  M Na2SeOg. In their investigation with six marine phytoplankters, Wheeler et al. (1982) demonstrated that selenite additions may stimulate growth when sulfate concentrations are very low, but they caution that responses to Se addition depend upon algal species, concentration and oxidation state of selenium, and sulfate concentrations. Marine ultraplankton clones from the Sargasso Sea have been shown to require Se for adequate growth in culture: surprisingly, selenium was essential for oceanic, but not coastal clones of Micromonas spp. (Keller et al. 1984). Thalassiosira pseudonana has an obligate requirement for Se, and optimal growth was observed over a wide range of selenite concentrations (10"^ to 10"* M) (Price et al. 1987). Tetraselmis tetrathele and Dunaliella minuta have the ability to take up selenite (Wrench 1978), but growth responses or obligate requirements have not yet been demonstrated for these species. Freshwater phytoplankton also demonstrate growth requirements for selenium. The dinoflagellate Peridinium cinctum and the diatom Stephanodiscus hantzschii show growth responses to selenium (Lindstrom 1983), and obligate selenium requirements have been demonstrated by the dinoflagellates Peridinium cinctum (Lindstrom and Rodhe 1978) and Peridiniopsis borgei (Lindstrom 1985). Recently, Wehr and Brown (1985) presented evidence that Chrysochromulina breviturrita requires selenium, and suggest that episodic inputs of atmospheric Se may trigger blooms of this alga in lakes in Ontario. Furthermore, C. breviturrita was able to utilize selenite, selenate, dimethylselenide (DMSe) and selenomethionine as Se sources. Typically, phytoplankton grow best with selenite as a Se source. High selenate concentrations are often toxic (Wheeler et al. 1982, Lindstrom 1983, 1985, Price et al. 1987). An exception to these general results is reported by Sielecki and Burnham (1973). They found that when the blue-green alga Phormidium luridum was exposed to 10"° M selenite, growth was inhibited and the cells lost essentially all photosynthetic capabilities. Preferential uptake of selenite, shown in laboratory experiments, is supported by studies of natural phytoplankton assemblages. During a bloom in Bedford Basin, N.S., dominated by Thalassiosira nordenskioldii, Chaetoceros septentrionale and Gymnodinium spp., Se(IV) was selectively assimilated over Se(VT) (Wrench and Measures 1982). Apte et al. (1986) measured selenium concentrations in an enclosed experimental ecosystem which was moored in a Scottish loch, and observed that SetTV) showed preferential bio-utilization compared to Se(VT). The physiological and biochemical bases for this apparent preference for selenite is not clear. Broyer et al. (1972) suggested that selenium reduced phosphate-toxicity in the terrestrial plant Astragalus, whereas Wheeler and coworkers propose that Se acts to reduce harmful effects of sulfur-deficiency in marine phytoplankton. Animal experiments have indicated that selenium possesses anticarcinogenic properties, possibly by alleviating metal toxicity (Garberg and Hogberg 1986). This hypothesis is supported by a study on the green flagellate Dunaliella minuta (Gotsis 1982). Gotsis exposed D. minuta to selenium, mercury and copper at various selenium:mercury and selenium:copper ratios. Selenium, mercury and copper inhibited growth of D. minuta when they were added separately to the culture. However, when selenium/mercury and selenium/copper were added together, they had antagonistic effects towards each other. Price (1987) discovered that T. pseudonana manufactured the selenoenzyme glutathione peroxidase, which had previously been found only in mammals and birds (Stadtman 1980). Therefore, it is possible that selenium is a constituent of Se-specific enzymes in phytoplankton, which may be synthesized in the presence of amounts of sulfur that are orders of magnitude higher. Morphological changes Selenium-replete cultures of T. pseudonana showed no morphological changes in response to Se addition. Cultures of T. pseudonana starved for selenium contained chains of cells, and solitary cells were often elongated along the pervalvar axis. These changes are symptomatic of Se-starvation (Price et al. 1987), indicating that Se concentrations of the -Se culture medium were < 10 M. and that trace metal contamination was minimized. These morphological changes by T. pseudonana in response to Se-stress have been termed primary elongation and secondary elongation by Doucette et al. (1987). Primary elongation is the initial response to Se-limitation, in which T. pseudonana forms short chains of cells. This is thought to be due to a failure of the sibling cells to separate. If the cells become Se-starved, individual cells become greatly elongated, due to the blockage of mitotic and cytokinetic functions of mitosis (Doucette et al. 1987). Other investigators have also observed that cell size may change in relation to the selenium nutrition of an organism. Selenite addition to cultures of the bacterium Cryptococcus albidus caused a significant increase in cell diameter, often near 20% (Brown and Smith 1979). In a spherical organism, such as Cryptococcus, this is equivalent to cell volume increases of approximately 70%. In experiments with Chlorella vulgaris, Shrift et al. (1961) noted that giant cells were often formed in response to selenomethionine addition. Lindstrom (1983) reports that in the dinoflagellate P. cinctum, Se-starved cells increased in diameter by up to 17%. An interesting finding of my research is the decrease in cell size of Se-starved T. pseudonana, following 10 M Na2SeOg addition. This is cicumstantial evidence that the cells possessed the ability to take up Se, since by the end of the experiment, cells of the 10"® M add-back culture were nearly normal in size, while those in the 0 M and 10"*^ M Se add-back cultures continued to increase in size. Although K. rotundatum did not exhibit any gross morphological changes in response to Se enrichment, it is possible that ultrastructural changes may have occurred that were not apparent by light microscopy. In conjunction with gross morphological changes, Se-starved T. pseudonana cells exhibit drastic changes at the ultrastructural level (Doucette et al. 1987). Therefore, it is not unreasonable to propose that these may also occur with K. rotundatum, in response to different amounts of Se-enrichment. ^^C uptake Addition of Nc^SeOg to Se-replete cultures of T. pseudonana resulted in enhanced carbon uptake rates, when uptake was expressed on a per cell volume or per cell basis. This illustrates the importance that uptake rates should not be calculated solely on a per cell basis; this may bias interpretation of the data, if cells exhibit morphological changes during the course of an experiment. Twenty-four hours after selenium was added back to Se-deplete T. pseudonana cultures, carbon uptake rates per cell volume in the cultures receiving 10"® M Se enrichment were approximately 50% higher than the rates in the cultures with 0 M enrichment. Cultures with 1 0 " ^ M enrichment showed a more modest enhancement, with uptake rates per cell volume approximately 15% higher than the control cultures. These trends are also evident when C uptake is expressed as D P M per ml, with the 10" 6 M and 1 0 " 1 0 M Se cultures exhibiting 60% and 25% increases in uptake (respectively) compared to the control. For Se-deplete T. pseudonana, * 4 C primary productivity bioassays accurately indicated recovery from Se-limitation, which was verified by the concommitant changes in growth rate and cell volume. Since the calculation of relative carbon uptake rates does not require that cell densities or cell volumes be determined a priori, relative carbon uptake shows merit as a parameter useful for field studies. It is insensitive to variations in cell size, which is an asset if phytoplankton cells sizes are changing. In these experiments, carbon uptake rates per cell underestimated total carbon uptake as the cell volume decreased in the 10 M add-back culture. Alternatively, carbon uptake could be standardized on a per volume basis, but this is impractical for any conditions other than unialgal samples. Carbon uptake rates for K. rotundatum, grown at different selenium concentrations, did not change following the addition of 0 M or 1 0 " ^ M Se. A lack of 1 A enhancement in C uptake, following addition of Se, is interpreted as evidence that K. rotundatum was not Se-limited. If K. rotundatum was not Se-limited at the time of the ^ 4 C bioassay, this would explain the reason for the negative response to the ^^C bioassay. Since K. rotundatum did not display growth responses to Se additions, this is further confirmation that 1 0 " ^ M Se is saturating for growth of this dinoflagellate. Therefore, it appears that this ^^C bioassay provided a credible method of estimating whether or not K. rotundatum was Se-limited. Utility of primary productivity bioassays The usefulness of primary productivity bioassays in detecting nutrient limitation of phytoplankton has been hotly debated in the literature. In their seminal paper, Ryther and Guillard (1959) propose nutrient enrichment experiments as a method of determining which nutrients limit phytoplankton production. They found that Sargasso Sea samples that were not enriched with nutrients, had ^ 4 C uptake rates that were only 25% as much as samples enriched with nutrients prior to the * 4 C incubations. By omitting various nutrients from their enrichment mixture, they were able to assess which nutrients were limiting production. The ^ C primary productivity bioassay also proved useful to Glooschenko and Curl (1971), when they studied the nutrient status of phytoplankton populations off the Oregon coast and in the mid-North Pacific Ocean. They showed that when strong upwelling was present, nutrient enrichment of samples had no effect upon H ^ C O g uptake. When upwelling was not present, additions of N, P or Fe significantly increased carbon uptake rates. Glooschenko and Curl (1971) concluded that N, P, and Fe each were limiting to primary production, depending on the time of year. In their study on the selenium requirement of P. cinctum, Lindstrom and Rodhe (1978) noted that when other nutrients were in excess, photosynthesis, which was measured by oxygen evolution, was regulated exclusively be selenium concentration. It is unfortunate that they did not also determine photosynthetic rates with the ^ 4 C technique, since this would have provided valuable information on the relative worth of the ^ 4 C primary productivity bioassay in assessing Se limitation. Numerous studies exist, however, which dispute the ability of ^ 4 C nutrient enrichment bioassays to properly assess nutrient limitation in phytoplankton. Using 4 h incubation times, Steemann Nielsen and A l Kholy (1956) did not find increases in photosynthetic rates of N-deficient or P-deficient Chlorella, following addition of NO3 or PG* 4 . O'Brien and deNoyelles (1976) examined the use of batch bioassays, chemostat assays and primary productivity incubations for measuring nutrient limitation. Although both the batch bioassay and chemostat assays clearly indicated N and P limitation, the primary productivity bioassay showed no increase in ^ 4 C uptake with addition of the limiting nutrients. In her investigations in an oligotrophic area of the Aegean Sea, Ignatiades (1977) concluded that nutrient additions did not affect the photosynthetic rates of phytoplankton over 21 h incubation periods. An excellent review of the short-term responses of nutrient-deficient algae to nutrient additions was provided by Healey (1979). He concluded that photosynthetic responses could not reliably be used in short-term enrichments for detecting nutrient limitation. Furthermore, he argued that nutrient-deficient algae might be expected to show decreases in C uptake following nutrient enrichments. This was confirmed by Lean and Pick (1981), who examined nutrient-deficient freshwater phytoplankton populations, and discovered that uptake was depressed following P O ^ additions. It is apparent that primary productivity bioassays do not always provide accurate estimates of nutrient limitation. Elapsed time, between the addition of the limiting nutrient and the measurement of uptake, appears to determine whether or not a bioassay is successful. Studies in which bioassays do not reliably indicate nutrient limitation, typically, are studies with very short time periods (i.e. several hours) between addition of the nutrient and initiation of the incubations. If I had measured carbon uptake by T. pseudonana at only 4 h following the addition of 10 M Na2SeOg, I would have been unable to detect the subsequent enhancement of uptake. Thus, it is important that test organisms be allowed sufficient time to respond to additions of limiting nutrients, before incubations are initiated. For a fast-growing diatom such as T. pseudonana, 24 h appears to suffice. For K. rotundatum, which grows only 50% as fast as T. pseudonana, a 48 h period is more suitable. The length of the incubation period may also affect the reliability of this ^• 4C assay. In preliminary experiments, I incubated cells for 2 h following addition, before I collected them for filtration. I achieved more reliable estimates of the degree of Se-limitation when the incubation period was increased to 4 h (see Appendix). Test organisms Thalassiosira pseudonana proved to be an ideal bioassay organism to detect Se-limitation. Se-deplete cells displayed unequivocal enhancement of uptake, following the addition of Na 2 SeOg. Morphological changes in response to Se-starvation also provide further evidence for the degree of Se limitation. T. pseudonana 51 grows very quickly and is hardy, characteristics which are desirable in a test organism. In contrast, Katodinium rotundatum did not show changes in carbon uptake following selenite additions. Since selenium addition failed to elicit a growth response, I concluded that 10'^ M Na2SeG"3 was sufficient for maximum growth of this alga. K. rotundatum is a fragile, athecate dinoflagellate, which grows slowly. It does not appear that K. rotundatum is suitable for use as a bioassay organism to detect possible Se-limitation in natural phytoplankton populations. S U M M A R Y Carbon-14 bioassays reliably and sensitively indicated the nutritional status of T. pseudonana. Addition of selenite to Se-deplete cultures stimulated carbon uptake rates, when expressed on a per cell volume or a relative basis. Recovery from Se-starvation was confirmed by changes in the growth rate and morphology of T. pseudonana. Carbon uptake rates by K. rotundatum grown at different selenium concentrations did not change following the addition of 0 M or 10"^ M Se. Contradictory to preliminary findings by members of our research group, these data indicate that K. rotundatum does not display growth responses to Se additions, and were confirmed by cell counts and in vivo fluorescence. C H A P T E R 2. USE OF A i 4 C BIOASSAY TO E X A M I N E S E L E N I U M NUTRITION  OF A N A T U R A L P H Y T O P L A N K T O N A S S E M B L E IN THE STRAIT OF GEORGIA B A C K G R O U N D In Chapter 1, Se-limited T. pseudonana cultures exhibited an increase in l ^ C uptake rates following selenium addition. The magnitude of the increase was positively correlated with the amount of selenite added. Initial indications were that * ^ C primary productivity bioassays provided a viable technique for determining the degree of Se limitation in marine phytoplankton. However, this hypothesis had been tested only upon unialgal laboratory cultures. In order to determine if this procedure was applicable to the field, it was necessary to test it with natural phytoplankton assemblages. The opportunity to examine the selenium nutrition of natural phytoplankton populations arose during a cruise off the mouth of the Fraser River in July 1987. Inorganic nitrogen (NOg and N H ^ + ) concentrations were < 1 uM, suggesting N-limitation. M A T E R I A L S AND METHODS Sample collection One 24 h time course experiment was conducted in the Strait of Georgia, B.C., Canada aboard the C.S.S. Vector (July 1987). The station location is shown in Figure 12, and described in Table 1. The photosynthetically active radiation (P.A.R.) was monitored continuously with a Lambda Instruments LI-185 light meter equipped with a LI-190S Surface Quantum Sensor, and subsurface light levels measured with a LI-192S Underwater Quantum Sensor. At approximately 1200 h PDT on 29 July, 1987, water was collected with a 5 L PVC Niskin bottle from the depth corresponding to 55% of the sea surface irradiance. The amber rubber spring closure of the Niskin bottle had been replaced by silicone tubing in order to reduce toxicity to phytoplankton (Price et al. 1986). Aft^SeOg addition Duplicate 250 ml samples were placed in 250 ml PC screw-cap bottles, and Na2SeOg was added at concentrations of 0 , 10" ^ u or 10"® M Se. Bottles were incubated under natural light in clear Plexiglas® deck incubators, cooled with surface seawater and covered with neutral density screening to simulate the irradiance at the 55% light depth. gure 12. Location of Station 1 (•) in the Strait of Georgia, British Columbia, Canada. 55 Table I Location of station and time of incubations Station Description Date Incubation P.A.R. and Time (PDT) (uE-m"2^"1) Location 1: 48 55.8 N 123 17.9 W Estuarine 29VII87 1215-1645 30VII87 1215-1630 >1000-1500 >1000-1500 C bioassays Bioassays were initiated at 1215 h PDT on 29-30 July, 1987. Aliquots (50 ml) were removed from each PC bottle and transferred to 60 ml Wheaton glass bottles. A 2.0 uCi H ^ C O g " aliquot was added to each sample. The sample was incubated for approximately 4.5 h under the conditions described above, and the cells collected by gentle filtration (< 50 mm Hg) onto Whatman GF/F filters. Zero-time blanks were determined by adding 2.0 uCi H ^ C O g " to duplicate 50 ml samples and filtering them immediately. Filters were placed into glass scintillation vials containing 0.2 ml of 0.5 N HC1, and 10 ml Aquasol II fluor added after 2 h. Internal standards were determined by adding 25 ul * ^ C stock solution to 0.2 ml phenethylamine, followed by the addition of fluor. Samples were counted on an Isocap 300 liquid scintillation counter, and quench correction was by the channels-ratio method. RESULTS AND DISCUSSION Relative carbon uptake Relative carbon uptake of a natural phytoplankton assemblage, expressed as D P M per ml of sample, is presented in Fig. 13. Carbon uptake was constant over the period measured, and selenium addition had no effect upon relative carbon uptake. It appears that phytoplankton at this station, at this time, are not Se-limited. Low ambient concentrations of inorganic nitrogen suggest that they are N-limited, instead. Se requirements of natural assemblages The impetus behind testing for Se limitation in marine coastal waters were the observations of several other workers, and their findings of Se limitation in lakes and the open ocean. In their study of P. cinctum, Lindstrom and Rodhe (1978) were the first to suggest that selenium may be a limiting nutrient in the natural environment. Their speculations were supported by the findings of Wrench and Measures (1982), who measured the concentrations of dissolved selenium during a phytoplankton bloom. Selenium IV was selectively assimilated by the phytoplankton, over Se (VI); following the decay of the bloom there was a rapid regeneration of Se (IV), indicating that Se (IV) is cycled through the biota. Similarly, Apte et al. (1986) established that Se(IV) was preferentially utilized during a spring diatom bloom in an enclosed experimental ecosystem. Circumstantial evidence suggests that Sargasso Sea phytoplankton are potentially Se-limited (N. Price, unpubl. data); this contention is 59 Figure 13. Relative carbon uptake, expressed as disintegrations per minute (DPM) per ml of sample, for a natural phytoplankton assemblage. The sample was collected from the 55% light depth at Station 1. Selenium was added at 0 h as 0 M (•), 10" 1 0 M (A) or 10"6 M (•) Na 2Se0 3. The standard deviation of the mean was less than the width of the symbols. b o m + o o Relative Carbon Uptake (DPM per ml) K> O m + o o fo O m + o o k) o m + o o t I I I I I I mf I ' » I i i i i i i i i i I i i i i i i i i OH 3 ro" con ro 4>" o supported by Keller et al. (1984), who were unable to culture certain Sargasso Sea ultraplankton clones unless Se was added to the culture medium. Chrysochromulina breviturrita was shown to have an obligate selenium requirement by Wehr and Brown (1985). A simulated Se spike to samples of lake water resulted in increases in algal growth up to 70% higher than the control. Wehr and Brown (1984) suggest that episodic blooms of C. breviturrita may be induced by atmospheric deposition of Se. Clearly, our knowledge of selenium and its significance for phytoplankton primary production has increased dramatically during the last decade. It is no longer inconceivable to suggest that, under certain conditions, selenium concentrations may limit productivity of natural phytoplankton assemblages. Dissolved selenium concentrations Analytical procedures for measuring dissolved selenium have not kept pace with the recent advances in our knowledge of selenium's importance to phytoplankters. In the benchmark study by Measures et al. (1980), selenium concentrations in seawater range from 50-800 pM for Se (IV) and 500-1500 pM for Se (VI). Using different analytical techniques, Cutter and Bruland's (1984) measurements agree with those of Measures et al. (1980). However, Cutter and Bruland (1984) propose that the majority of dissolved selenium in surface waters is present as dissolved selenide (Se II), but Measures et al. (1983) argue that the dominant species is selenate (Se VI). Selenium concentrations in the Strait of Georgia Measurements of dissolved selenium in coastal waters are scarce. Measures and Burton (1978) report that the mean concentration of Se (IV), in rivers emptying into estuaries, was approximately 250 pM. Cutter (1982) measured selenium in Saanich Inlet, a fjord connected to the Strait of Georgia. Surface concentrations of both Se (IV) and Se (VI) were in the picomolar range. Thus, it is reasonable to assume that surface concentrations of Se (IV), in the Strait of Georgia region examined in this thesis, are in the picomolar range. In addition to in situ amounts of selenium, Se may enter coastal waters through atmospheric deposition and weathering of terrestrial minerals. Coastal regions are also likely to have higher inputs of selenium from anthropogenic sources, compared to oceanic regions. Thus, it is unlikely that phytoplankton communities in a eutrophic environment, such as the Strait of Georgia, experience Se limitation. The possibility that Se limitation occurs in neritic regions cannot be overlooked, though, since previous studies have discovered Se-limited phytoplankton in situations where Se was thought to be in abundant supply (eg. Lindstrom and Rodhe 1978, Lindstrom 1983, 1985, Wehr and Brown 1985). The ^ 4 C primary productivity bioassay conducted with a natural phytoplankton assemblage from the Strait of Georgia supports the assertion that their growth is not Se-limited . Instead, nitrogen is most likely to limit primary production, particularly during the summer, the time that this experiment was conducted. This does not invalidate the use of this technique, however, but illustrates the need for refinement of this method by further testing. A note of caution It is possible to drive a phytoplankton assemblage into Se limitation. This was intentionally demonstrated in Chapter 1 of this thesis, when T. pseudonana was transferred from natural seawater into Se-deplete E S A W . However, caution must be excercised to avoid unintentionally driving phytoplankton into Se limitation. This is a particularly relevant problem for aquaculturists, who often maintain phytoplankton cultures to feed molluscs, such as mussels and oysters. Typically, phytoplankton are cultured in large volumes, and high nutrient enrichment concentrations are utilized to ensure high phytoplankton biomass. Many aquaculture managers are unaware that selenium may be required to support growth of phytoplankton, and unfortunately omit Se from their nutrient enrichment. Due to the high nutrient enrichments utilized in aquaculture operations, the phytoplankton cultures may subsequently become Se-limited, with resultant changes in their physiology. Since the physiological health and nutritional quality of the phytoplankton feed is of paramount importance for the survival of the molluscs, failure to add selenium to these large volume, high biomass phytoplankton cultures may have serious economic consequences for an aquaculture operation. SUMMARY Potential selenium limitation of a natural phytoplankton assemblage from the Strait of Georgia was examined with a ^ ^C bioassay. Relative carbon uptake rates were unaffected, following the addition of 10 M or 10 M N^SeOg to samples. It is concluded that the natural phytoplankton assemblage tested was not Se-limited, nor would Se limitation be expected in a eutrophic coastal environment. However, Se limitation may be induced by nutrient additions in aquaculture situations; Se should be added as part of the nutrient enrichment to prevent this from occurring. G E N E R A L CONCLUSIONS Carbon-14 bioassays reliably and sensitively indicated the nutritional status of T. pseudonana. Addition of selenite to Se-deplete cultures stimulated carbon uptake rates, when expressed on a per cell volume or a relative basis. Recovery from Se-starvation was confirmed by changes in the growth rate and morphology of T. pseudonana. Carbon uptake rates of K. rotundatum grown at different selenium concentrations did not change following the addition of 0 M or 1 0 " 1 0 M Se. Since K. rotundatum did not display growth responses to Se additions, it may not have been Se-limited, thus explaining the reason for the negative response to the ^ 4 C bioassay. Natural phytoplankton populations in the Strait of Georgia were examined for selenium limitation, with the ^"4C bioassay developed in Chapter 1. Relative carbon uptake rates were unaffected by the addition of 10" ^ M or 10"® M Na2SeOg, and I concluded that phytoplankton at this station in the Strait of Georgia were not Se-limited at the time of sampling. Literature Cited Apte, S.C., A .G . Howard, R.J. Morris and M.J. McCartney. 1986. Arsenic. antimony and selenium speciation during a spring phytoplankton bloom in a closed experimental ecosystem. Mar. Chem. 20: 119-130. Bottino, N.R., C H . Banks. K . J . Irgolic, P. Micks, A .E . Wheeler and R.A. Zingaro. 1984. Selenium containing amino acids and proteins in marine algae. Phytochem. 23: 2445-2452. Brown, T.A., and D.G. Smith. Effects of inorganic selenium compounds on growth, cell size, and ultrastructure of Cryptococcus albidus. Microbios Lett. 10: 55-61. Broyer, T.C., D.C. Lee and C .J . Asher. 1966. Selenium nutrition of green plants. Effect of selenite supply on growth and selenium content of alfalfa and subterranean clover. Physiol. Plant. 41: 1425-1428. Broyer, T.C., C M . Johnson and R.P. Huston. 1972. Selenium and nutrition of Astragalus: I. Effects of selenite or selenate supply on growth and selenium content. Plant Soil 36: 635-649. Byers, H.G. 1935. Selenium occurrence in certain soils in the United States, with a discussion of related topics. U.S. Dept. Agric. Tech. Bull. 482: 1-47. Cutter, G.A. 1982. Selenium in reducing waters. Science (Wash., D.C.) 217: 829-831. Cutter, G.A., and K.W. Bruland. 1984. The marine biogeochemisty of selenium: a re-evaluation. Limnol. Oceanogr. 29: 1179-1192. Dortch, Q., J.R. Clayton, Jr . , S.S. Thoresen, S.S. Bressler and S.I. Ahmed. 1982. Response of marine phytoplankton to nitrogen deficiency: decreased nitrate uptake vs enhanced ammonium uptake. Mar. Biol. (Berl.) 70: 13-19. Doucette, G.J . , N.M. Price and P.J. Harrison. 1987. Effects of selenium deficiency on the morphology and ultrastructure of the coastal marine diatom Thalassiosira pseudonana (Bacillariophyceae). J . Phycol. 23:9-17. Fitzwater. S.E.. G.A. Knauer and J . H . Martin. 1982. Metal contamination and its effect on primary production measurements. Limnol. Oceanogr. 27: 544-551. Franke, K.W. 1934. A new toxicant occurring naturally in certain samples of plant foodstuffs. I. Results obtained in preliminary feeding trials. J . Nutr. 8: 597-608. Fries, L. 1982. Selenium stimulates growth of marine macroalgae in axenic culture. J . Phycol. 18: 328-331. Frost, D.V. 1972. The two faces of selenium-can selenophobia be cured. CRC Crit. Rev. Toxicol. 1: 467-514. Garberg, P., and J . Hogberg. 1986. The role of selenium-oxygen interactions in selenium metabolism. Ambio 1986: 354-355. Glooschenko, W.A., and H. Curl, Jr . 1971. Influence of nutrient enrichment on photosynthesis and assimilation ratios in natural North Pacific phytoplankton communities. J . Fish. Res. Bd. Canada 28: 790-793. Goldman, J . C , and J . J . McCarthy. 1978. Steady state growth rate and ammonium uptake of a fast-growing marine diatom. Limnol. Oceanogr. 23: 695-703. Goldman, J . C , and M.R. Dennett. 1985. Susceptibility of some marine phytoplankton species to cell breakage during filtration and post-filtration rinsing. J . Exp. Mar. Biol. Ecol. 86: 47- 58. Gotsis, O. 1982. Combined effects of selenium/mercury and selenium/copper on the cell population of the alga Dunaiiella minuta. Mar. Biol. (Berl.) 71:217-222. Harrison, P.J. , R.E. Waters and F.J.R. Taylor. 1980. A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton. J . Phycol. 16: 28-35. Healey, F.P. 1979. Short-term responses of nutrient-deficient algae to nutrient addition. J . Phycol. 15: 289-299. Hitchcock. G.L. 1986. Methodological aspects of time-course measurements of fixation in marine phytoplankton. J . Exp. Mar. Biol. Ecol. 95: 233-243. Hudman, J .F . , and A.R. Glenn. 1984. Selenite uptake and incorporation by Selenomonas ruminantium. Arch. Microbiol. 140: 252-256. Hudman, J . F . , and A.R. Glenn. 1985. Selenium uptake by Butyrivibrio fibrisolvens and Bacteroides ruminicola. FEMS Microbiol. Lett. 27: 215 220. Ignatiades, L. 1977. In situ short term enrichment experiments and evaluation of the ^" 4C method for testing oligotrophy in the sea. Hydrobiol. 56: 247-252. Iverson, R.L., H.F. Bittaker and V .B . Myers. 1976. Loss of radiocarbon in direct use of Aquasol for liquid scintillation counting of solutions containing 1 4 C - N a H C 0 3 . Limnol. Oceanogr. 21: 756-758. Jacobson, D.M. 1987. The Ecology and Feeding Biology of Thecate Heterotrophic Dinoflagellates.Ph.D. Thesis. Massachusetts Institute of Technology/Woods Hole Oceanographic Institution, Woods Hole, MA. 210 pp. Keller, M.D., R.R.L. Guillard, L. Provasoli and I.J. Pintner. 1984. Nutrition of some marine ultraplankton clones from the Sargasso Sea. Eos 65: 898. Lean, D.R.S., and B.K. Burnison. 1979. An evaluation of errors in the C method of primary production measurement. Limnol. Oceanogr. 24: 917-928. Lean, D.R.S., and F.R. Pick. 1981. Photosynthetic response of lake plankton to nutrient enrichment: a test for nutrient limitation. Limnol. Oceanogr. 26: 1001-1019. Lindstrom, K., and W. Rodhe. 1978. Selenium as a micronutrient for the dinoflagellate Peridinium cinctum fa. westii. Mitt. Internat. Verein. Limnol. 21: 168-173. Lindstrom. K. 1983. Selenium as a growth factor for plankton algae in laboratory experiments and in some Swedish lakes. Hydrobiol. 101: 35-48. Lindstrom, K. 1985. Selenium requirement of the dinoflagellate Peridinopsis borgei (Lemm). Int. Revue ges. Hydrobiol. 70: 77- 85. Maher, W.A. 1985a. Selenium in macroalgae. Bot. Marina 28: 269-273. Maher, W.A. 1985b. Characteristics of selenium in marine animals. Mar. Poll. Bull. 16: 33-34. Measures, C L , and J.D. Burton. 1978. Behaviour and speciation of dissolved selenium in estuarine waters. Nature (Lond.) 273: 293-295. Measures, C L , and J.D. Burton. 1980a. The vertical distribution and oxidation states of dissolved selenium in the northeast Atlantic Ocean and their relationship to biological processes. Earth Planet. Sci. Lett. 46: 385-396. Measures, C L , and J.D. Burton. 1980b. Gas chromatographic method for the determination of selenite and total selenium in sea water. Anal . Chim. Acta 120: 177-186. Measures, C L , R.E. McDuff and J . M . Edmond. 1980. Selenium redox chemistry at GEOSECS I re-occupation. Earth Planet. Sci. Lett. 49: 102-108. Measures, C L , B.C. Grant, B.J . Magnum and J . M . Edmond. 1983. The relationship of the distribution of dissolved selenium IV and VI in three oceans to physical and biological processes. In: Trace Metals in Seawater. Ed. C S . Wong, E. Boyle, K.W. Bruland, J .D. Burton and E.D. Goldberg. Plenum Press, N.Y. pp. 73-83. O'Brien, W.J. , and F. deNoyelles, Jr . 1976. Response of three phytoplankton bioassay techniques in experimental ponds of known limiting nutrient. Hydrobiol. 49: 65-76. Orvini, E., L. Lodola, M. Gallorini and T. Zerlia. 1981. Determination of selenium (IV) and selenium (VI) traces in natural waters by neutron activation analysis. In: Heavy Metals in the Environment, 3rd Int. Conf. 1981. 70 Parslow. J .S . , P.J. Harrison and P.A. Thompson. 1984a. Development of rapid ammonium uptake during starvation of batch and chemostat cultures of the marine diatom Thalassiosira pseudonana. Mar. Biol. (Berl.) 83: 43-50. Parslow, J .S . , P .J . Harrison and P.A. Thompson. 1984b. Saturated uptake kinetics: transient response of the marine diatom Thalassiosira pseudonana to ammonium, nitrate, silicate or phosphate starvation. Mar. Biol. (Berl.) 83: 51-59. Parsons, T.R., Y . Maita and C M . Lalli. 1984. A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press, Oxford. 173 pp. Pintner, I.J., and L. Provasoli. 1968. Heterotrophy in subdued light of 3 Chrysochromulina species. Bull. Misaki Mar. Biol. Inst. Kyoto Univ. 12: 25-31. Price, N.M. , P .J . Harrison, M.R. Landry, F. Azam and K.J .F . Hall. 1986. Toxic effects of latex and tygon tubing on marine phytoplankton, zooplankton and bacteria. Mar. Ecol. Prog. Ser. 34: 41-49. Price, N .M. 1987. Urea and Selenium Nutrition of Marine Phytoplankton: a Physiological and Biochemical Study. Ph.D. Thesis. University of British Columbia, Vancouver. 321 pp. Price, N.M. , P.A. Thompson and P.J. Harrison. 1987. Selenium: an essential element for growth of the coastal marine diatom Thalassiosira pseudonana (Bacillariophyceae). J . Phycol. 23: 1-9. Robinson, W.O. 1933. Determination of selenium in wheat and soils. J . Assoc. Offic. Agr. Chem. 16: 423-424. Rosenfeld, I., and O.A. Beath. 1964. Selenium Geobotany, Biochemistry, Toxicology, and Nutrition. Academic Press, N.Y. 412 p. Ryther, J . H . and R.R.L. Guillard. 1959. Enrichment experiments as a means of studying nutrients limiting to phytoplankton production. Deep-Sea Res. 6: 65-69. Sharp, J .H . , P.A. Underhill and A.C. Frake. 1980. Carbon budgets in batch and continuous cultures: how can we understand natural physiology of marine phytoplankton? J . Plankton Res. 2: 213-222. Shrift, A., J . Nevyas and S. Turndorf. 1961. Mass adaptation to selenomethionine in populations of Chlorella uulgaris. Plant. Physiol. 36: 502-509. Sielecki, M.. and J .C. Burnham. 1973. The effect of selenite on the physiological and morphological properties of the blue-green alga Phormidium luridum var. olivacea. J . Phycol. 9: 509-514. Smith, R.E.H. , and T. Piatt. 1984. Carbon exchange and * 4 C tracer methods in a nitrogen-limited diatom, Thalassiosira pseudonana. Mar. Ecol. Prog. Ser. 16: 75-87. Stadtman, T.C. 1974. Selenium biochemistry. Science (Wash., D.C.) 183: 915-922. Stadtman, T.C. 1980. Biological functions of selenium. TIBS 1980: 203-206. Steemann Nielsen, E. 1952. The use of radio-active carbon (C^ 4 ) for measuring organic production in the sea. J . Cons., Int. Explor. Mer 18: 117-140. Steemann Nielsen, E., and A .A . A l Kholy. 1956. Use of i 4C-technique in measuring photosynthesis of phosphorus or nitrogen deficient algae. Physiol. Plant. 9: 144-153. Strickland, J .D .H. , and T.R. Parsons. 1968. A Practical Handbook of Seawater Analysis. Bull. Fish. Res. Bd. Canada No. 167. Sunda, W.G., and S.A. Huntsman. 1983. Effect of competitive interactions between manganese and copper on cellular manganese and growth in estuarine and oceanic species of the diatom Thalassiosira. Limnol. Oceanogr. 28: 924-934. Suttle, C.A., N .M. Price, P.J . Harrison and P.A. Thompson. 1986. Polymerization of silica in acidic solutions: a note of caution to phycologists. J . Phycol. 22: 234-237. Trelease, S.F. , and H.M. Trelease. 1938a. Selenium as a stimulating and possibly essential element for indicator plants. Am. J . Bot. 25: 372-380. Trelease, S.F. , and H.M. Trelease. 1938b. Selenium as a stimulating and possibly essential element for certain plants. Science (Wash., D.C.) 87: 70-71. ' Trelease, S.F. , and H.M. Trelease. 1939. Physiological differentiation in Astralagus with reference to selenium. Am. J . Bot. 26: 530-535. Wehr, J .D . , and L .M. Brown. 1985. Selenium requirement of a bloom-forming planktonic alga from softwater and acidified lakes. Can. J . Fish. Aquat. Sci. 42: 1783-1788. Wheeler, A . E . , R.A. Zingaro and K. Irgolic. 1982. The effect of selenate, selenite, and sulfate on the growth of six unicellular marine algae. J . Exp. Mar. Biol. Ecol. 57: 181-194. Wood, E.D., F .A . J . Armstrong and F.A. Richards. 1967. Determination of nitrate in seawater by cadmium-copper reduction to nitrite. J . Mar. Biol. Assoc. U.K. 47: 23-31. Wrench, J . J . 1978. Selenium in the marine phytoplankters Tetraselmis tetrathele and Dunaliella minuta. Mar. Biol. (Berl.) 49: 231-236. Wrench, J . J . , and C.I. Measures. 1982. Temporal variations in dissolved selenium in a coastal ecosystem. Nature 299: 431-433. A P P E N D I X In experiments conducted prior to the research presented in this thesis, I examined the time course of uptake by T. pseudonana. Thalassiosira pseudonana cultures were grown in ESAW with full nutrient enrichment (i.e. 550 um NOg), and Q containing selenite additions of 10 M ( + Se) or 0 M (-Se). At Time = 0 h, selenite was added back to the cultures at 0 M, 1 0 " ^ M or 10"® M concentrations. Following the selenium add-backs, the time course of carbon uptake was followed, using 2 h incubation periods. The results from one of these preliminary studies are presented in Figure 14. Note how relative carbon uptake decreases with time in the +Se culture (Fig. 14A). This is unusual, since relative carbon uptake should increase with time, if the cells are physiologically healthy and growing properly. Also note how there is a drastic decrease in relative carbon uptake for the -Se culture (Fig. 14B). Once again, this is an anamoly, and is not expected to occur with healthy phytoplankton cells. The pH of the culture medium often attained levels as high as pH = 9.5, when T. pseudonana was grown on fully-enriched E S A W . When I lowered the nutrient concentrations in my artificial seawater medium to 10% of the full enrichment, pH rarely rose higher than pH = 8.4, for cultures entering stationary phase. Also, growth rates and carbon uptake rates behaved as expected for healthy cells (see Figs. 4A-6A). I concluded, therefore, that the pH-tolerance of T. pseudonana was probably exceeded when it was grown with full E S A W nutrient enrichment. Upon lowering nutrient concentrations in ESAW to 10% of those recommended by Harrison et al. (1980), I was able to lower the pH of the medium. This appeared to result in much more physiologically healthy cultures. I also found that 4 h incubations were Figure 14. Relative carbon uptake, expressed as disintegrations per minute (DPM) per ml of culture, for T. pseudonana grown in +Se (A) or -Se (B) ESAW. Selenium was added at 0 h as 0 M (•), 10"10 M (A) or 10"6 M (•) Na 2Se0 3. Values are the mean of triplicates. These cultures were grown with full ESAW nutrient enrichment (550 uM NOg), and the ^ 4C incubations were 2 h in duration. Relative Carbon Uptake (DPM per ml) Relative Carbon Uptake (DPM per ml) M rn + o o I I I I I I I I I I I I I • I I I I I I I I I GO — OO b o m m m + f- +• o o o O o o 4> CO o b m m •I- + O o o o o" Ul O" i i i i i i i i I i i i i i • i I i i i i i i i tO b m + o ' ' ' ' I I I I I I best for bioassay experiments with this organism, rather then the 2 h incubations which I had initially used. 

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