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

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HE EFFECTS OF SUBLETHAL CONCENTRATIONS OF MERCURIC CHLORIDE ON AMMONIUM-LIMITED SgELETONEMA COSTATUM (GREV.) CLEVE. by LOUISE CLOUTIER-MANTHA B.Sc, McGill University, 1976 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n The Faculty of Graduate Studies (Department of Botany) We accept t h i s thesis as conforming to the reguired standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1978 ©L o u i s e Cloutier-Mantha, 1978 In presenting t h i s thesis i n p a r t i a l f u l f i l 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 f o r reference and study. I further agree that permission for extensive copying of t h i s t h e s i s for scholarly purposes may be qranted to the Head of my Department or his representatives. It 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 Botany The University of B r i t i s h Columbia 2075 wesbrook Place Vancouver, B.C. , Canada, V6T 1W5 ABSTRACT The e f f e c t s of sublethal additions of mercuric chloride were studied i n the marine diatom Skaletonema costatug. {Grev.) Cleve grown i n ammonium-limited chemostats and batch cultures. In the short-term Hg exposure (up to 5 hours), unexposed chemostat efflu e n t s were simultaneously perturbed with 5 pM NH^Cl and Hg concentrations ranging from 0.04 to 5.53 nH HgCl^. In the long-term Hg exposure (679. 5 hours), ammo-nium-starved effluents were only perturbed with 5 uM NH^Cl. In the short-term Hg exposure, when the effluent from the chemostat culture was starved for 1.5 hours, Hg decreased the a f f i n i t y for the substrate (increased Ks value) and the rate of ammonium assimilation or the i n t e r n a l l y controlled uptake rate, Vi . When the eff l u e n t was starved f o r 30 hours, only Vi was reduced. These effects occurred between 1.84 and max 3,68, and at 0,18 nH HgCl 2 i n effluents starved for 1.5 and 30 hours, respectively. The maximum rate of uptake, Vs, was not depressed. In the long-term Hg exposure, at least 0.37 nH HgCl^ decreased the s p e c i f i c growth rate and the maximum c e l l density, while the chlorophyll a per c e l l increased. A period of population decline was followed by resumption of growth. Morphological alt e r a t i o n s were observed before and a f t e r the recovery. In the long-term experiment, six days of continual i i i exposure to 0.37 nM HgCl^ gradually increased the Ks value without a f f e c t i n g Vs and Vi . The re s u l t s from exposure to max 3.68 nM HgCl^ were sim i l a r to the short-term Hg exposure, since both the substrate a f f i n i t y (Ks value) and the assimilatory rate ^^fnux^ were impaired. In addition, the maximal uptake rate, Vs, was also reduced a f t e r exposure to 3.68 nM HgCl^ for six days i n the long-term experiment. After resumption of growth i n the Hg-treated cultures, when a new steady-state was established, the a f f i n i t y for the substrate and assimilatory rates increased i n phase D (day 23) compared to phase A (day 6) . The recovery of growth and nutrient uptake rates i n phase D, may have been p a r t i a l l y mediated by the acgu i s i t i o n of Hq tolerance and the appearance of c e l l s of a d i f f e r e n t stage of the sexual l i f e c y c l e , as suggested by differences i n c e l l s i z e and chemical composition. An attempt was made to determine whether a short-term physiological response (Hg induction of metallothionein syn-thesis) could be responsible for the recovery. The 250 nm absorbance p r o f i l e , of nutrient-saturated cultures exposed for 90 to 116 hours to sublethal concentrations of mercury, showed no large absorbance peak i n the medium molecular weight pool, corresponding to laetallothionein, as i t occurs i n animals exposed to heavy metals. The i n t r a c e l l u l a r d i s t r i b u t i o n and l e v e l s of Cu, Zn, and Hg in S. costatum, grown i n nutrient-saturated batch cultures, were affected by 0.37 nM HgCl 0. A concentration equal to or greater than 1,84 nM EqCl^ reduced the growth rate and c e l l density, possibly due to the accumulation of Hg i n the high m.w. pool. Exposure to 1.84 nM HgCl^ prior to a second addition of 5.53 nM reduced Hg levels i n ths high m.w. pool. Upon Hg exposure, Zn leve l s decreased i n the high and low m.w. fractio n s but gradually increased i n the medium m.w. pool., Copper s l i g h t l y increased in the high m.w. pool but remained constant in the medium and low m.w. pools, i n r e l a t i o n to t o t a l i n t r a c e l l u l a r l e v e l s . High leve l s of Cu and Zn in the low m.w. pool suggests that a substance of a lower m.w., than usually reported for metallothionein, may be involved i n the storage and deto x i f i c a t i o n of heavy metals in S, costatum. Thesis Supervisor V TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION 1 Section 1. Environmental Impacts, D i s t r i b u t i o n and Concentration of Mercury 1 Section 2. Factors Influencing Mercury Toxicity ...... 4 2.1 Chemical Composition of Medium ................ 4 2.2 Speciation of Mercury 7 Section 3. Accumulation of Metals .................... 8 Section 4. Effects of Mercury 9 Section 5. Mercury Resistance ........................ 14 Section 6. Assessment of Experimental Design ......... 16 Section 7. Purpose of t h i s Study ..................... 17 CHAPTER I I . EFFECTS OF SHORT AND LONG-TERM EXPOSURES TO SUBLETHAL LEVELS OF Hg ON NUTRIENT KINETICS 19 Section 1. Introduction .............................. 19 Section 2. Materials and Methods ..................... 22 2.1 Inoculum 22 2.2 Chemostat Cultures ............................ 22 2.3 Analyses ...................................... 23 2.4 Experimental Design ........................... 26f Section 3. Results ................................... 30 3.1 Results from some Preliminary Studies ......... 30 3.2 Growth Phases during the Long-term Mercury Exposure ....................................... 3 0 3.3 Sp e c i f i c Growth Rates and Nitrogen Quotas ..... 36 3.4 Effects of Mercury on Photosynthesis .......... 37 3.5 Morphological Observations .................... 40 3.6 Mercury Analyses .............................. 41 3.7 Short-term Nutrient Kinetics 43 3.8 Long-term Nutrient Kinetics ................... 49 Section 4. Discussion ................................ 54 4.1 Effects of Mercury on Photosynthesis .......... 54 4.2 Effects of Mercury on Growth Parameters .......56 4.3 Effects of Mercury on Nutrient Uptake Kinetics 57 4.4 Recovery from I n i t i a l Mercury In h i b i t i o n ...... 59 4.5 Mercury Losses ................................ 60 4.6 Applications to the Natural Environment ....... 61 4.7 Evaluation of Chemostat Studies ............... 62 CHAPTER I I I . THE EFFECT OF H<j EXPOSURE ON INTRACELLULAR DISTRIBUTION OF Hg, Cu, AND Zn ....................... 63 Section 1. Introduction 63 Section 2. Materials and Methods ..................... 64 2.1 Batch Cultures ................................ 64 2.2 Experimental Conditions ....................... 65 2.3 Analyses ...................................... 65 Section 3. Results 68 3.1 Growth 68 v i 3.2 D i s t r i b u t i o n of Hq, Cu and Zn 68 S e c t i o n 4. D i s c u s s i o n 75 SUMMARY. 78 REFERENCES 82 APPENDICES . . 94 Appendix A. Composition of A r t i f i c i a l Seawater and " f Medium 95 Appendix B. Data Derived from the Lonq-term Hg E x p o -sure of Ammonium-limited S^ cgstatum ge v i i LIST OF TABLES Paqe TABLE I. L i t e r a t u r e Summary of the E f f e c t s of Mercu r i a l s on Phytoplankton TABLE I I . Standard V a r i a t i o n s i n the Analyses Used i n the Short and Lonq-term Experiments 27 TABLE I I I . Concentrations of Hq i n the Lonq-term Hq Exposure Experiment 44 TABLE IV. N u t r i e n t Uptake K i n e t i c Response to Short-term Hq Exposure 4 7 TABLE V. E f f e c t of Duration of S t a r v a t i o n of Chemostat E f f l u e n t on the N u t r i e n t Uptake Response 50 TABLE VI. Nutrient Uptake K i n e t i c Response to Lonq-term Hq Exposure 5 1 TABLE VII. Exposure to D i f f e r e n t Concentrations of HqCl 2 at D i f f e r e n t Times durinq a Batch C u l t u r e Expe-riment 66 TABLE V I I I . D i s t r i b u t i o n of T o t a l Zn, Cu, and Hq i n I n t r a c e l l u l a r Pools due to Hq Exposure .. 73 LIST OF FIGURES FIGURE 1. Nutrient Uptake Kinetic Terminology FIGURE 2. Changes i n i n vivc Fluorescence in Nut-r i e n t saturated Batch Cultures FIGURE 3. Changes i n Biomass of Ammonium-limited Cultures during Long-term Hq Exposure FIGURE 4. Changes i n Specific Growth Rate durinq Lonq-term Hq Exposure FIGURE 5. Changes i n Chlorophyll a and Photosynthe-s i s durinq Long-term Hq Exposure ,.. FIGURE 6. Variations i n Expected Total Hg Levels i n Chemostat I durinq LoEq-term Hg Exposure ............. FIGURE 7. Ammonium Uptake Rates as a Function of Subs-trate durinq Short-term Hq Exposure FIGURE 8. Ammonium Uptake Rates as a Function of Subs-trate on Day 6 durinq Lonq-term Hq Exposure .......... FIGURE 9. Disappearance of Substrate with Time durinq Phase A of Lonq-term Hq Exposure FIGURE 10. Ammonium Uptake Rates as a Function of Substrate after the Recovery from the Lonq-term Hq Exposure FIGURE 11. Changes in i n vivo Fluorescence i n Batch Cultures A, B, C and D FIGURE 12. The 250 nm Absorbance P r o f i l e cf Fractions Collected from Batch Cultures A, B, C and D FIGURE 13. Distribution of Total Cu and Zn i n each Fraction and Gel Elution P r o f i l e of Batch Culture C .. X ACKNOWLEDGMENTS This research would not have been possible without the much appreciated help, advice, encouragement and a v a i l a b i l i t y of my supervisor. Dr. Paul J. Harrison. I would also l i k e to thank the two other members of my thesis committee. Dr. N. J. Antia and Dr. A. G. Lewis for t h e i r collaboration and helpful recommendations i n the revision of t h i s thesis. I would l i k e to offer s p e c i a l thanks to Mr. David H. Turpin for his assistance on many occasions during the experiments and useful comments thoroughout t h i s study. Discussions with Dr. E. Andersen and Mr. M.J. LeBlanc were h e l p f u l . The research in chapter three was conducted in collaboration with Mr. David A. Brown.. Dr. T.B. Parsons was instrumental i n stimulating the research on metallothionein. 1 am also grateful to Dr. K.H. Hall and Dr. T.B. Parsons who were o r i g i n a l y on my thesis committee, and assisted i n the early stages of planning. I would l i k e to acknowledge Ms. S. Harrison for her advice while using the computing f a c i l i t i e s , and Mr. B.H. Mantha and T. Diersch for helping to generate t h i s computer-typed t h e s i s . 1 CHAPTER I INTRODUCTION Section 1. .. Environmental Impacts,, Distributjon and" Concentra-tion of Mercury During the 1950*s and 1960's, several dramatic declines i n w i l d l i f e populations occurred due to the ingestion of mercurials. For example, sead-eating and piscivorous birds declined i n Sweden. In Yugoslavia, Iraq, Pakistan and Guatemala community poisoning outbreaks were caused by wheat-dressed a l k y l mercurial fungicides (Krenkel, 1973). Disasters at Minimata (1953) and Niigata (1965.) are amongst the best documented cases of human poisonings. In Canada (1968-1969), high levels of Hg were found in pheasants and partridges i n the P r a i r i e s (Femreite, 1970). The a x i a l muscles of pike from the Saskatoon River contained up to 10 mg-kg-iHg (Hobeser et a l . , 1970). Consequently, sport and commercial f i s h e r i e s were closed in the Saskatoon River network and parts of the Great Lakes, as well as hunting i n the P r a i r i e s . More than one m i l l i o n pounds of f i s h were incinerated (> 0.5 mg'kqrlHg i n muscle tissue, Dept. Fish, and Forestry, 1971). Usually the major offenders of Hg p o l l u t i o n are; 1) the c h l o r - a l k a l i industry where inorganic Hg i s used i n the e l e c t r o l y t i c preparation of chlorine and caustic soda, and 2 2) metal recovery from Pb-Zn deposits. Mercury i s also used i n ; 1) the fab r i c a t i o n of e l e c t r i c a l and control instruments, 2) antiseptics and preservatives i n pharmaceutical products and paints, and 3) fungicides and s l i m i c i d e s in agriculture and the pulp industry. The disclosure about mercury t o x i c i t y has led to a general decline in the u t i l i z a t i o n and commercial production of mercurials and a strengthening of l e g i s l a t i o n on by-product disposal and discharge. In s p i t e of a reduced input into the environment, there are s t i l l important and inadequately quantified losses from anthropoqenic sources, such as mining and smelting of Hg ores, combustion of f o s s i l f u e l s and o i l s , and improper waste disposal of sludges. In the ocean, the primary sources of Hq are; deqassinq of the earth's crust, i n d u s t r i a l pollution by atmospheric j e t streams, p r e c i p i t a t i o n , volcanism, and upwellinq of deep water {Gardner, 1975). Weatherinq, leachinq of Hq-containinq s o i l s (Nelson et a l . , 1977), r i v e r transport to oceans and environmental fluxes influenced by man are l e s s or as important as the natural atmospheric translocation of Hq from continents to oceans (Rice et a l . , 1973; Windom et a l . , 1975). Due to differences in samplinq, a n a l y t i c a l techniques and the short residence time of Hq i n the mixed layer of the ocean, i t i s d i f f i c u l t to qeneralize on Hq l e v e l s i n d i f f e r e n t water bodies (&ndren and Harriss, 1975; Gardner, 1975; Kuiper, 1976). The lowest Hq l e v e l s reported ranqed from 11.2 nq-1 - 1 i n the southern hemisphere to 33,5 nq«l _ 1 i n the northern 3 hemisphere; the north eastern A t l a n t i c c o a s t a l zone was an anomaly having o n l y 14.7 n g - i " - 1 (Gardner, 1975). Unusually high values of 364 ug-1 - 1 occurred i n c e r t a i n s p e c i f i c areas (Bice e t a l . , 1973; W i l l i a m s et a l . , 1974). Freshwater streams ranged from 17 to 125 n g ' l - 1 Hg (Keckes and Miettenen, 1972; Krenkel, 1973). In North A t l a n t i c plankton, p a r t i c u l a t e Hg values ranged from 0.2 to 0.4 m g * l _ 1 (dry weight) i n u n p o l l u t e d areas, to 5.3 m g - l - 1 i n c o a s t a l p o l l u t e d waters. V a r i a t i o n s i n Hg l e v e l s were not c o r r e l a t e d t o s p e c i e s compo-s i t i o n but to the d i s t a n c e from the p o l l u t i o n sources (Windom et a l . , 1973). Even though background l e v e l s are low and may be of l i t -t l e concern, h e a l t h hazards are a s s o c i a t e d with the i n g e s t i o n of Hg-contaminated foods, through magnified accumulation i n food chains (Cook, 1977). The t o x i c i t y of Hg i s due to i t s a f f i n i t y f o r t h i o l s , s e l e n o l s , phosphates, amino and c a r b o x y l t e r m i n a l groups of amino a c i d s and v a r i o u s c e l l components ( S e y f e r t h , 1978) . Bioassays of s i n g l e element t o x i c i t y performed j,n sjjtu on n a t u r a l phytoplankton p o p u l a t i o n s i n d i c a t e d t h a t t o x i c i t y t h r e s h o l d s were as f o l l o w s : Hg <10 nM; C u ~ 100 nM; Pb, Cd, and A s ( V ) ~ 3 0 0 nM; Zn~1pM; N i , Co, Sb, Se, and As(III) > 1 uM (Hollibaugh et a l . , unpublished manuscript). In Skeletonema costaturn. comparable r e d u c t i o n of c e l l d i v i s i o n was obtained with 5-10 u g ' l - 1 Hg, 25-100 ug-1- 1 Cd and 50-200 u g ' l - * Cu (Berland et a l . , 1977). In Ch1ore11a pyranoidpsa, Hg was more i n h i b i t o r y than Cu (Kamp-Nielsen, 1971) and Cu was more t o x i c 4 than Zn in Amphidinium carterae, Thalassiosjra pseudonana. S. costatum, and Phaeodact^lum tricornuturn (Braek et a l . . 1976). Section 2. Factors Influencing Mercury Toxicity 2,1 Chemical Composition of Medium Heavy metal t o x i c i t y varies widely according to expe-rimental variables and conditions. The influence of various factors affecting heavy metal t o x i c i t y on algae (Kamp-Nielsen, 1971; Hannan and P a t o u i l l e t , 1972; Bice et a l , , 1973), and on microorganisms and invertebrates have been studied (Pyefinch and Mott, 1948; Corner and Sparrow, 1956; F i t z g e r a l d and Faust, 1963; wisely and Blick, 1966; Lewis et a l , , 1973; Whitfield and Lewis, 1976; Gibson gt aJL., 1975). The s e n s i t i v i t y of a test organism in a pollution assay i s affected by: 1) physical factors such as l i g h t {Steemann Nielsen and Wium-Andersen, 1971; Kamp-Nielsen, 1971; Overnell, 1976), temperature {De F i l i p p i s and Pallaghy, 1976a; Blinn et a l . , 1977; Knowles and Zingmark, 1978), s a l i n i t y (Hunter, 1949), pH (Wisely and B l i c k , 1966), time and concentration of exposure (Erickson, 1972; Zingmark and M i l l e r , 1973); 2) chemical factors such as the degree of chelation, and nutrient and dissolved oxygen concentrations (Corner and Sparrow, 1956; Fitzgerald and Faust, 1963; McBrien and Hassal, 1967; Erickson 5 et a l . , 1970; Lewis et a l . , 1973; Nelson and Colwell, 1975; Overnell, 1975a); and 3) b i o l o g i c a l factors such as s i z e of the inoculum (Steemann Nielsen et a l . , 1970; Shieh and Barber, 1972; Ben-Bassat and Mayer, 1975; Overnell, 1976), and the physiological state of c e l l s (Gibson, 1972; Zingmark and M i l l e r , 1973). O f a l l the potential factors capable of influencing metal t o x i c i t y , the chemical composition of the growth medium can be the most d i f f i c u l t factor to define and cont r o l . The choice of an appropriate medium r e l i e s both on the s p e c i f i c requi-rements of the test organisms and the type of study being conducted. The i n h i b i t o r y l e v e l s determined in c h e l a t o r - r i c h media cannot be compared to those found in chelator-free media (Jensen et a l . , 1976; Kayser, 1976; Overnell, 1976). It i s well established that the addition of natural and synthetic chelating agents can complex trace metals (Gardiner, 1976) and influence t h e i r uptake (Cossa, 1976; Schulz-Baldes and Lewin, 1976; George and Coombs, 1977)., Natural seawater contains undefined complexing agents such as humic and f u l v i c acids (Singer, 1973) in d i f f e r e n t amounts. Lewis et a l . , (1971; 1973) found that EDTA-enriched natural seawater varied i n i t s a b i l i t y to support growth of young stages of a copepod, presumably because of the presence of natural complexing agents. In cultures of F r a g i l a r i a and Asterionella with s o i l extract additions, mercurials were s i g n i f i c a n t l y less toxic than i n completely defined medium (Tompkins and Blinn, 1976). The presence of decomposed natural plankton and detritus 6 increased Cu tolerance in T. £Sgudpjian.a {Erickson, 1972) . The absolute growth rates of marine phytoplankton were lower i n mid-winter seawater i n spite of higher nutrient l e v e l s (Jensen §± i.1 • i 1974). Chelated iron counteracted the ef f e c t s of Hg on C. pyranoidosa (Kamp-Nielsen, 1971) and on three coastal diatoms (Jensen et a l . , 1976). The addition of EDTA increased the tolerance of P. tricorputum. when the molar r a t i o of EDTA : Cu >1 (Bentley-Howat and Beid, 1977), and the growth rates of T.,pseudonana which was previously i n h i b i t e d by Cu (Erickson, 1972). In C h l o r e l l a vulgaris and Oocystis. EDTA had both stimulatory and adverse e f f e c t s (Fitzgerald and Faust, 1963). Although a l k a l i ions could a l l e v i a t e Cu t o x i c i t y (Overnell, 1975a), they f a i l e d to neutralize Hg i n h i b i t i o n (Steemann Nielsen and Hium-Andersen, 1971). Heavy metal t o x i c i t y was intimately connected with n u t r i -t i o n a l stress in algae (Hannan and P a t o u i l l e t , 1972; Hannan et a l . , 1973) and in Daphnia (winner et a l . , 1977). In nutrient poor waters, even 3.0 ug-l _ iHg arrested the growth of Ch l o r e l l a (Stokes et a l . , 1973), A toxic Hg threshold (60 (jg.l-i) was demonstrated for a summer phytoplankton assemblage but not for the spring population (Blinn e t a l . , 1977), possi-bly due to the combined effects of lower nutrient l e v e l s and higher temperatures during the summer. Low nutrient levels decreased the normal excretion rate of dissolved organics (Betz, 1977), thus r e s u l t i n g i n an increase i n Hg t o x i c i t y . These compounds may be involved in Hg v o l a t i l i z a t i o n (Ben-Bassat and Mayer, 1977; 1978). 7 2.2 Speciation of Mercury In phytoplankton, organomercurials are more toxic than inorganic and elemental Hg., The 1 4 C uptake of N i t z s c h i a d e l i -catissima was reduced by 50% with; 1 u.g»l~1 phenyl mercuric acetate (PMA); 0.5 ug«l— 1 methyl mercuric dicyanidiamide and MEMMI1; and 10 u g - l - i diphenyl mercury (Harriss et a l , , 1970; white, 1970). The potassium content and 0^  evolution of H» t e r t i o l e c t a (Overnell, 1976) and the l i p i d synthesis in freshwater algae (Matson et a l . , 1972) were more depressed by methylmercury than HgCl^. Even though PMA was more toxic than HgCl^, the accumulation of Hg in the presence of PMA was less than with HgCl^ i n C h l o r e l l a (De F i l i p p i s and Pallaghy, 1976a), implying that t o x i c i t y i s not always due to the l i p o i d s o l u b i l i t y of organomercurials. Cultures of aerobic hetero-trophic bacteria were more resistant to PMA than HgCl^ (Nelson and Colwell, 1975). Hannan and P a t o u i l l e t (1972) reported that HgCl^ was more toxic to various algae than dimethyl-mercury. The primary productivity of F r a g i l a r i a crotonensis and Asterionella formosa was more affected by Hg(NOj)^ than HgCl 2 (Blinn et a l . , 1977). 1 N-methylmercuric-1,2,3,6-tetrahydro-3,6-methano-3,4,5,6,7,7 hexachlbrophthaliamide. 8 Section 3. Accumulation of Metals Some aspects of the exchange ki n e t i c s between metals and algae have been elucidated (Glooschenko, 1969; Davies, 1974; 1976; F u j i t a and Hashizume, 1975). Metal accumulation i s p r i n c i p a l l y influenced by the species of the metal, the alga and i t s physiological state. The uptake varies as a function of the time and concentration of exposure (Shieh and Barber, 1972; Davies, 1974; Cossa, 1976). Mercury accumulation was greater i n dividing than non-dividing c e l l s (Burkett, 1975; Richardson et a l . , 1975) and also greater i n dead and moribund c e l l s than l i v e c e l l s probably due to the cessation of active excretion of mercury (Fujita and Hashizume, 1975; Bentley-Mowat and Heid, 1977). Metal accumulation i s usually accomplished i n two phases. The f i r s t one consists of a Freundlich adsorption isotherm which i s immediate, rapid and passive. The metal adsorbs onto c e l l surfaces u n t i l a de f i n i t e number of s i t e s are saturated (Davies, 1974; 1976; Dolar et a l . , 1971). This adsorption phase i s followed by an active but slower uptake, during which the metal i s translocated across the c e l l membrane (Shieh and Barber, 1972; Schulz-Baldes and Lewin, 1976). This phase of uptake was temperature dependent i n Chlorella (De F i l i p p i s and Pallaghy, 1976c)., During accumulation, a simultaneous exchange of metals occurs between algae and the medium (Ben-Bassat and Mayer, 1977; 1978; Betz, 1977). Excreted Hg can either be inorganic (Betz, 1977) or organic, since Hg may be 9 released as a compound complexed with a natural metabolite, capable of reducing Hg*2 to Hg° (Ben-Bassat and flayer, 1978). The incorporation of metal into the c e l l i s rapid. In S^nedra. ulna. Hg was deposited on thylakoid surfaces and then transferred into the cytoplasm (Fujita et a l . , 1977). In Ana-S i s t i s nidulans. Hg was associated with a phycocyanin-rich f r a c t i o n and with 60,000, 180,000, and 230,000 m.w. f r a c t i o n s derived from peripheral and i n t r i n s i c lamellar components (Hammans et a l . , 1976). In 2 0 3 H g - l a b e l l e d plant tissues, Hg granules were almost exclusively sequestered within n u c l e o l i and between spaces occupied by chromatin (De F i l i p p i s and Pallaghy 1975). Section 4. Effects of Mercury The i n h i b i t o r y l e v e l s of mercurials on a l g a l growth (Ben-Bassat et a l . , 1972; Hannan and P a t o u i l l e t , 1972; Tompkins and Blinn, 1976), photosynthesis and r e s p i r a t i o n (Zingmark and M i l l e r , 1973; De F i l i p p i s and Pallaghy, 1976a) have been determined for a number of defined conditions. The e f f e c t s of various mercurials on phytoplankton are summarized i n Table I. In general, diatoms prove to be more sensitive to heavy metals than dinoflagellates (Kayser, 1977). Out of seven species, S, costatum was es p e c i a l l y sensitive to Hg (Overnell, 1976). Gymnodinjum splendens was the most sen s i t i v e to Hg (CH COO) followed by S c r i p p s i e l l a faeroense and Prorocentrum micans (Kayser, 1976) . Freshwater 10 TABLE I. Literature summary of the effects of mercurials on phytoplankton. VEST ORGANISM 1 CEL:.ULAR PROCESSES EFFECTS MERCURIALS CONCEN-TRATIONS ( uM ) REMARKS RCl'EKENCE DIATOMS: Astei'ionella fomosa g.r. • t p. inh. f. " Kg!;12 0.921 1.842 batch Tompkins and B l i n n , 1976. Frafilaria arotsnen-sic stlm. f. inh. " 0.184 0.368 « Chartoccroc ap. Cyclotcllc. ap. Phaecdact^tum tricor-r.uticn S . ' , r - inh. HgCl 2 0.368 batch II Hannan and P a t o u i l l e t , 1972 Skeleionema aottavmy 8-r- inh. 0.002 chemostat Rice et al., 1973. g-r. s, P/S r a t i o inh. unchanged KuC2 2 0.018-C.037 batch it Borland ct al., 1977. P/S 501 inh. H;;.C12 2.500 batch Overnell, 1976. Hi-zoahi.1 dcliaatie-nirritj P/S 50Z inh MEM-MI1 ir.othyl d i -cy a n i d i -amide PMA .ilph.Gnyl K« 0.5 yug.f 0.5 1.0 " 10.0 " batch 24 h treatment Harries cl al., 1970. DIN0FLA0E1LATES: Axpkii-inim ?asie?ae G.i~:>ic.i-: iTjletuicr.s g-r. U.r. 6 P/S p. Inh. f. inh. n. " t . " t. HgCl, C.004 0.ISA-1.84? 1.8^2 0.3fi8 0.3<>8 - urb i ci j:> t '(added on.:fc) (d o l l y addition) Zingmark ar.d M i l l e r , 1973. Kayser, 1976. S.r. i P/S p. inh. f. " P- " r. " P . K 81J!I 300) 2 0.368 1.84 2-3.683 O.lii-0.368 0. 363 0.037 turbidoi-tat (added once) (daily addition) 3atch i'aripslellc faerc-,;.r. S P/S f. inh P- " f. " Hg(Cfi 3j0)., '3.683 1.842 3.6;13 0.037- 0.184 i;urbidoi:tat (added once) latch .W'.TURAL POPULATIONS Marino phytoplankton p r i - a r y productivity p. inh. KsCl, 1.642 :;rock and Ma^cri, 1971. P/S 507. inh. HsCl., 0.C04 Zinr.i'urk ;:.-.d M i l l e r , 1473. P/S inh. H SC:, aethyl Kg 0.0.18 •Cnauer sr.- - . i v t i r . , 1S72. North Sea coaotal plankton S - '• p. Inh. KgCl 2 0.006-0.018 if. r.i'.A. er.closjre Cuiper, 1976. Lake phytoplankton P/S II 40% inh. 85% " ii t i r j . HSC12 II 0.221 3.68 3 0.048-0.107 ir. sit-:'. enclosure Islir.n ct aZ.. 1977. 11 [GREEN ALGAE: Scened&smue 8j>. S. dlmorphu.r I j| ChUnrnjdar.onaa rein'-CTiZwelZa pyva>u>i-doaa Chlorella (Emerson strain) Dunaliella tevtloleatai S-r. g.r. g.r. + «.r. K efflux dark respi ration g v. biomass PAR P/S respiration DMA, RNA protein t/S lnh. unchanged f. lnh. f. lnh. p. lnh. stim. s t i a . lnh. unchanged stiia. stim. p. inh. jHXPTOPHYTE leoahrysie galbana 8.r. g-" 8« inh. p. inh. f. " HgCl 2 Hg", methyl Hg HgCl., H SC1 2 HgCl. HgCl 2 PMA l i 8n 12 PMA (CH3)2Hg HgCl, 0.110 0.037 10 ug r 1 7.366 0.368-1.050 [50.000-81.030 500.000 1.000 0.1 jM J..D0O HgCl, 0.1 uM 0.037-0.368 o.i-i.o 0.363-3.683 10.000 batch batch, highly chelated median batch batch batch semi-continuous cultures, 10-15 Kin exposure Hringham and Kuhn, 1953. Kaltlda et al., 1971. Ben-Bassat. at al., 1972. Karap-Nieisen, 1971. S'nleh and Barber, 1972. De F i l i p p i s and Pallaah". 1976a. 7.500 10.500 batch Overnell, 1975a. Davies, 1974; 1975. r t h i s T l T ; P d / S" ^ " ' ^ " l " ™- inhibition; f.- f u l l ; p.. p a r t l a l : . t l a . . . t i a u W i . cms chemical i s defined on page 7 • 12 phytoplankters are usually more tolerant than th e i r marine counterparts and nanoplankters are frequently the most r e s i s -tant. When Cu was added to a large in s i t u - enclosure, large c e n t r i c diatoms (e.g., Chaetoceros) and din o f l a q e l l a t e s disap-peared, whereas, microflaqellates and pennate diatoms (e»q»» N. delicatissima) survived and were responsible f o r the recovery of productivity (Thomas and Seibert, 1977). Teratological studies suggest that developing or metabo-l i c a l l y active stages are more susceptible to metal intox-i c a t i o n (Pyefinch and Mott, 1948; Wisely and B l i c k , 1966). In A. nidulans. stationary phase c e l l s were less vulnerable to Hg poisoning than logarithmically growing c e l l s (Hammans et a l . ,1976), probably due to the presence of metabolites in the growth medium. During the lag phase, the growth medium i s conditioned by excretion from l i v e c e l l s and dissolved orga-nics leaching from dead and moribund c e l l s . These products may either complex with toxic metallic ions or stimulate v o l a t i l i z a t i o n (Zingmark and M i l l e r , 1973; Davies, 1974; Kay-ser, 1976; Ben-Bassat and Mayer, 1977;1978; Betz, 1977). Mercury was less t o x i c when added during the log phase than concurrently with the inoculation of c e l l s (Zingmark and M i l l e r , 1973). The effects of mercurials.on photosynthesis are species s p e c i f i c . Mercuric chloride inhibited plastocyanin a c t i v i t y i n i s o l a t e d chloroplasts (Kiminura and Katok, 1972) and Hq (CH^COO)^ degraded phycocyanin in A. nidulans (Hammans §t a l . , 1976). In Chlamydamonas r e i n h a r d t i i . Hg interfered 13 with PS I and PS I I reactions (Overnell, 1975b); Although S. costaturn was the most sensitive alga tested, 2.5 uM HgCl^ reduced photosynthesis by only 50% (Overnell, 1976). An investigation on C. pyranoidgsa pointed to excretion of potassium ions as a primary e f f e c t of Hg poisoning (Kamp-Neilsen, 1971). Sublethal l e v e l s of heavy metals caused potassium leakage at concentrations similar to or greater than those reguired for the i n h i b i t i o n of photosynthesis i n P. t e r t i o l e c t a and P. tricornutum (Overnell, 1975a; Davies, 1976). In Ankistrodesmus jbjEaunii and Eugjena g r a c i l i s . Hg in h i b i t e d galactosyl transferase a c t i v i t y , g a l a c t o l i p i d and chlorophyll syntheses (Matson et a l . , 1972). The same i n h i b i t i o n appeared to occur i n F r a q i l a r i a and Asterionella-species (Tompkins and Blinn, 1976)., Galactolipids are one of the major chloroplast l i p i d s . In Ch l o r e l l a . HgCl^ stimulated DNA and ENA syntheses while PMA i n i t i a l l y decreased DNA, BNA and protein syntheses. Both mercurials had a sim i l a r e f f e c t on biovolume and excretion {De F i l i p p i s and Pallaghy, 19 76a). In S. costatum, (Berland et a l . , 1977) and natural populations (Thomas et a l . , 1977), the C:N r a t i o remained constant despite a reduction in growth and photosynthesis. In metal-treated algae, morphological a l t e r a t i o n s have occurred i n P. tricornutum and C h l o r e l l a (Nuzzi, 1972), and i n T M i a s s i o s i r a i s o l a t e d from Hg-treated i n s i t u enclosures (D. Seibert, personnal communication)., P^ty^fta-'-ferAgfrtjie^lil'-underwent osmotic disturbances r e s u l t i n g i n considerable swelling (Bentley-Mowat and Beid, 1977), After 12-14 days of 14 exposure to 50-100 pg^l-^BqCl , colonies of A. foraosa deviated from their normal s t e l l a t e configuration to form large c y l i n d r i c a l stacks of up to 30 colonies, agglomerated by mucilagenous secretions (Tompkins and Blinn, 1976). C e l l volumes of I. galbana almost doubled at 10.5 uM HgCl^ probably due to the impairment of methionine production, which i s involved in c e l l d i v i s i o n (Davies, 1 974). The d i n o f l a g e l l a t e , S. faeroense. responded to Hg(CH COO) by the bursting of tbecae, releasing of naked, motile c e l l s , and formation of vegetative resting stages (Kayser, 1976). Section 5. Mercury Resistance The tolerance of certain algae to elevated metal levels has been assessed in comparative studies (e.g., Bentley-Mowat and Reid, 1977). Tolerance may be acquired by the development of an exclusion mechanism (Davies, 1976), by v o l a t i l i z a t i o n of Hg from the medium (Ben-Bassat and Mayer, 1975), or by an acclimation response (Stokes et a l . , 1973; Stockner and Antia, 1976; Say et a l . , 1977). Tolerance may also be innate, as shown by Pediastrum boryanum which could survive and reproduce at 1 mg-1-1 HgCl^ in sp i t e of a c e l l u l a r concentration factor of 1.77 x 10»* (Richardson et a l . , 1975). Differences i n threshold levels for F. crotonensis and A. formosa correlated with surface to volume r a t i o s (Blinn &i •_§!•, 1977), Tolerance to heavy metals can be acquired following recovery from i n i t i a l metal i n h i b i t i o n (Kamp-Nielsen, 1971; 15 Ben-Bassat et a l . , 1972; F u j i t a and Hashizume, 1975; Davies, 1976; De F i l i p p i s and Pallaghy, 1976a; 1976b; Berland et a l . , 1977). Besistance due to v o l a t i l i z a t i o n of Hg i s common in mi-croorganisms. In Staphylococcus aureus (Weiss et a l . , 1977), Pseudomgnas aeruginosa and P. £uteda (Clark et a l . , 1977), recovery from the i n i t i a l i n h i b i t i o n of PMA was mediated by the phenylmercury hydrolysis of PMA to benzene and Hg*2 and the reduction of Hg + 2 to Hg° by mercuric reductase followed by a rapid v o l a t i l i z a t i o n of Hg° from the medium. The same process may have occurred i n Hg-resistant marine bacteria (Nelson et a l . , 1973). V o l a t i l i z a t i o n was gen e t i c a l l y controlled i n E. £oli and Aerofcacter ae£Ojg.en.e§ (Komura and Ka-z i r o , 1971; Summers and Lewis, 1973). C e l l s of Ch lor e l l a, which became Hg-resistant, reestablished normal growth rates and were more e f f i c i e n t in Hg° v o l a t i l i z a t i o n than Hg-sen-s i t i v a c e l l s (De F i l i p p i s and Pallaghy, 1976c). The c e l l ex-tra c t from C h i o r e l l a contained a natural, low molecular weight, non-enzymatic, light-induced, reducing compound capable of d i f f u s i n g Hg° from the spent medium (Ben-Bassat and Mayer, 1977; 1978). Pretreatment of C h l o r e l i a with 10-20 uM HgCl 2 prevented DCMU i n h i b i t i o n of light-induced Hg° v o l a t i l i z a t i o n due to competition between HgCl^ and DCMO, while an uncoupler of PS I, methylamine, trans i e n t l y s t i -mul;ated 0^ evolution and v o l a t i l i z a t i o n (Ben-Bassat and Mayer, 1978). 16 Section 6, Assessment of Experimental Design Two basic experimental approaches have been used in pollution studies to determine the e f f e c t s of pollutants. Bioassay organisms have been exposed to acute (lethal) and chronic (sublethal) l e v e l s over short to long time periods. Batch cultures have been intensively used and only a few studies were conducted in natural aguatic systems i n an att-empt to determine, the e f f e c t s of pollutants on natural populations (Kuiper, 1976; Blinn et a l . , 1977) and t h e i r e c o l o g i c a l repercussions (Dunstan and Menzel, 1971; Gibson et a l . , 1975; Grice et a l . , 1977). With batch cultures, a l l e s s e n t i a l growth-promoting elements and toxic substances are added at once and the cultures grow logarithmically u n t i l the exhaustion of nutrients occurs. Since the duration of the bioassay i s usually short, batch culture bioassays may overestimate t o x i c i t y . The observed toxic levels may be misleadingly high because the density and physiological state of the population vary with the u t i l i z a t i o n of the most l i m i t i n g nutrient. For example, tolerance to Cu varied by a factor of 3 0 i n Nitzschia palea grown in batch cultures (Steemann Nielsen and Wium-Andersen, 1971). However, batch cultures are useful i n estimating toxic ranges, screening for different mercurials (Harriss et aJL., 1970; Nuzzi, 1972) or suitable test organisms (Overnell, 1976), and determining the action of a toxicant on various 17 metabolic processes. A few studies have been done using continuous cultures such as a turbidostat (Kayser, 1976), or a chemostat (Rice et a l . , 1973). In the chemostat, a l g a l growth rate i s controlled by the rate of medium inflow or d i l u t i o n rate (Harrison et a l . , 1976). Culture volume, c e l l density and c e l l u l a r chemical composition are r e l a t i v e l y constant for a s p e c i f i c d i l u t i o n rate. Experimental time i s t h e o r e t i c a l l y unlimited and variables, which make the assay more sensitive, are easily controllable (e.g., nutrients and mode of metal exposure)., This technigue i s suitable for the maintenance of nutrient-limited populations and may be more r e a l i s t i c than batch cultures because phytoplankton of the upper photic zone are often nutrient-stressed, especially during the summer months. In the future, t h i s mode of culturing may prove more useful i n assessing and understanding the e f f e c t s of lower level s of metals. Section 7. Purpose of t h i s Study In general, there i s a lack of research documenting the e f f e c t s of environmentally encountered mercury l e v e l s . More-over, since most investigations have used concentrations of 3 to 6 orders of magnitude higher than the concentrations used i n t h i s study, the short-term terminal rather than the long-term subtle e f f e c t s of mercury have been examined (Table I ) . , In the past, a myriad of studies have determined the 18 effects of mercurials on d i f f e r e n t c e l l u l a r processes and/or the effects of some factors influencing t o x i c i t y . A few studies reported the e f f e c t s of heavy metals on biomass parameters i n continuous cultures (Rice et a.1., 1973; Kayser, 1976; Bentley-Mowat and Reid, 1977) and some b r i e f l y related tolerance to nutrients but under saturated conditions (Hannan et a l . , 1973; Morel et a l . , 1978)., Nutrient l i m i t a t i o n can l i m i t primary production and under these conditions of nutrient deficiency, chances of su r v i v a l w i l l depend on the nutrient uptake k i n e t i c responses of the nutrient-stressed populations. The a b i l i t y of a species to increase the uptake a f f i n i t y f o r the l i m i t i n g nutrient w i l l favour the chances of successful i n t e r s p e c i f i c competition. However, the imposition of a secondary stress (pollution) on a population that i s already nutrient-stressed may impair t h i s competitive a b i l i t y . Recovery in algae previously i n h i b i t e d by heavy metals, may be attributed to several factors, one possible reason for the recovery from i n h i b i t i o n may be the production of complexing agents such as metallothionein. This was investigated with different levels of mercury exposure. Thus, i t appears that t h i s work i s the f i r s t study which documents the e f f e c t s of long-term mercury exposures, at nearly ecological l e v e l s , on nutrient-limited diatoms, grown i n continuous cultures (chemostats). 19 CHAPTER II EFFECTS OF SHORT AND LONG-TEEM EXPOSURES TO SUBLETHAL IJIIiS OF Hg. ON NflTRIENT KINETICS Section 1. In/troductiQn Nutrient uptake k i n e t i c s i n phytoplankton can be des-cribed i n terms of the following eguation which i s similar to the Hichaelis-Menten eguation for enzyme k i n e t i c s ; (1) V = Vmax'S (Ks «• S)-» where V - r a t e of nutrient uptake ( h r - 1 ) ; Vmax = maximum rate of uptake ( h r - 1 ) ; S = concentration of the l i m i t i n g nutrient (uM) , and Ks = half-saturation constant (uH) , the value of S when V = Vmax/2. Values of Ks are species s p e c i f i c and a low Ks value indicates that a species has a high capacity or a f f i n i t y to take up a l i m i t i n g substrate across the c e l l membrane. Newer terminology associated with the disappearance and the rate of uptake of the l i m i t i n g nutrient has been described by Conway e t a l , (1976) and i t i s presented i n the following paragraphs. In nutrient-limited cultures, uptake and assimilation of the l i m i t i n g nutrient usually occurs in 3 phases (Fig. 1), The f i r s t phase consists of a rapid rate or surge uptake, Vs, which appears to represent the transport of the nutrient across the c e l l membrane and into an internal pool., I t occurs over the time period Ts. During the second phase, the rate of 20 13 t t t : ~ s o K s s t Substrate ( p M ) FIGURE 1. Nutrient uptake k i n e t i c terminology. (A) The disappearance of the l i m i t i n g nutrient with time. (B) The rate of uptake of the l i m i t i n g nutrient as a function of the substrate. (From Conway et a l . , 1976). Symbols are d e f i -ned i n Section 2.1 21 uptake , V i , decreases as the i n t e r n a l pool becomes f u l l . At t h i s point, the rate of assimilation of the l i m i t i n g nutrient, V i , (the mobilization from the i n t e r n a l pool into larger molecules via assimilatory enzymes) becomes the rate l i m i t i n g step c o n t r o l l i n g the uptake rate. During the t h i r d phase, the external substrate concentration i s low. The i n t e r n a l pool, containing the l i m i t i n g nutrient, becomes gradually depleted as the rate of assimilation exceeds the uptake rate. In t h i s phase, the uptake rate, ve, i s under external control since i t i s limited by the low external concentration of the l i m i t i n g nutrient i n the medium. Thus, the three phases involved in the translocation of the substrate across the c e l l membrane are Vs, Vi arid Ve. The substrate concentration at the juncture between the two phases of uptake, Vi and Ve, i s defined as St and indicates the beginning of the next phase, Ve. During the t h i r d phase of uptake, Ve may cease when the concentration of the l i m i t i n g nutrient i s completely or p a r t i a l l y depleted. In the l a t t e r case, the r e s i d u a l concentration of the l i m i t i n g nutrient s t i l l remaining i s defined as So. The actual half-saturation constant (actual Ks) i s determined by adding the apparent half-saturation constant (apparent Ks) and the So value. The apparent Ks i s calculated by assuming that So = 0., In t h i s study, Vmax was estimated using Vs.. The value of Vs, which represents only one datum point on the uptake hyperbola (see dashed l i n e in Fig, 1B), probably under-estimates the true value of Vmax. The maximum assimilation 22 rate, Vi. , was estimated by f i t t i n g an hyperbola to the Ve rn&x and Vi data. Section 2. Materials and Methods 2. 1 Inoculum Chemostat cultures were inoculated with an unialgal culture of skeletonema cost aturn (Grev. ) cleve (Northwest P a c i f i c Culture C o l l e c t i o n , #18, University of B r i t i s h Colum-bia) , i s o l a t e d from P a t r i c i a Bay, B r i t i s h Columbia, Canada. 2.2 Chemostat Cultures A l l glassware was autoclaved and culture medium was f i l -tered through a 0.22 um M i l l i p o r e f i l t e r i n an attempt to keep bacteria at n e g l i g i b l e l e v e l s . S. costatum was grown i n ammo-nium-limited chemostats in a r t i f i c i a l seawater (Appendix A) made from reagent grade s a l t s and enriched with constituents of *f» medium (Appendix A). Concentrations (uM) of the major nutrients i n the inflow medium were: NH^Cl, 10.0 (f/100) ; Na 2Si0 3, 35.3 (f/3) ; KH2P04 , 3.68 (f/20). Concentrations (nM) of trace metals (f/20) and vitamin (f/20) were: CuS04= 3.93; ZnS04= 7.65; CoCl 2. 6H20= 4.39; MnCl^ 41^0= 0.91; N a ^ o C y 2^0= 2.61 and vitamin B^- °*05 u g ' l - 1 . Iron and EDTA were added as f e r r i c seguestrene (1.17 uM). The chemostat system was previously described (Davis 23 S i a l . , 1973). Continuous cultures were grown i n b o r o s i l i c -ate, 2 1 and 6 1 f l a t bottom b o i l i n g f l a s k s (Pyrex) maintained at 17 ± 0.5 0C. Flasks were not coated with s i l i c o n e ( S i l i c l a d , Clay Adams) since preliminary r e s u l t s indicated that adsorption of Hg onto walls was not decreased and no s i l i c a t e leached from the inner walls of the culture f l a s k s . The cultures" were continuously s t i r r e d with te f l o n covered magnetic s t i r r i n g bars at 120 rpm and were maintained without aeration to avoid v o l a t i l i z a t i o n . They were also under continuous il l u m i n a t i o n ( 50 uE•m - 2•s - 1). Spectral d i s t r i b u -t i o n from daylight fluorescent lamps (Sylvannia Powertube, VHO and Duro-Test, UHO) was corrected by using a 0.3 cm thick Plexiglas sheet (# 2069 Rohm and Hass), i n an attempt to simulate 5 m underwater l i g h t under sunny conditions for the Jerlov type 3 coastal water (Holmes, 1957). 2.3 Analyses Growth was monitored by following changes of in vivo fluorescence using a Turner Model 111 fluorometer and of c e l l density using an inverted l i g h t microscope. Length and width of c e l l s were measured using an ocular micrometer. Nutrient analyses were performed using a Technicon AutoAnalyzer . The ammonium method was ba s i c a l l y that of Koroleff (1970) as automated by Slawyk and Maclssac (1972). The methods for s i l i c a t e and phosphate followed the procedures of Armstrong et a l . , (1967) and Murphy and Siley (1962), respectively. Both 24 methods were automated by Haqer et a l . , (1969). The l*C technique was used for measuring the rate of pho-tosynthesis (Strickland and Parsons, 1972). Five uCi of NaH1*C05 (New Enqland Nuclear, Boston) were added to 100 ml of chemostat e f f l u e n t , mixed, separated into two, 50 ml bottles and incubated for 2 hours under o r i q i n a l conditions. No dark incubation was conducted. C e l l s were collected onto a 25 mm diameter 0.45 urn M i l l i p o r e f i l t e r . F i l t e r s were dissolved in s c i n t i l l a t i o n c o c k t a i l (Scintiverse, Fisher S c i e n t i f i c Co. Ltd.) and counted for r a d i o a c t i v i t y with a Unilux III Nuclear Liquid S c i n t i l l a t i o n System, Nuclear Chicaqo. Chlorophyll a was determined by the trichromatic method (Strickland and Parsons, 1972). Two hundred and f i f t y ml of chemostat effluent were f i l t e r e d onto a glass f i b e r f i l t e r co-vered with 1 ml of a saturated HgCO^ solution. Pigments were extracted for 20 hours i n cold, dark conditions i n 90% acet-one. C e l l s were homogenized, centrifuged and the absorbance spectrum (750, 665, 663, 645, 630 and 430 nm) of the supernatants for chlorophylls, carotenoids and phaeophytins were determined using a Perkin-Elmer Model 124D double beam spectrophotometer. Phaeophytin a was measured by adding 3 drops of 10% HCl to the above supernatants and reading the atsorbances at the same wavelenqhts. For the Hq analysis, 200 ml of effluent were f i l t e r e d onto a 47 mm diameter 0.45 urn Mi l l i p o r e f i l t e r . , F i l t e r s and f i l t r a t e s were analyzed for part i c u l a t e and soluble Hg, respectively. These Hq fractions were measured by a cold vapor method (Hatch and Ott, 1968) using a flameless atomic absorption spectrophotometer (Pharmacia U.V. Control and Optical Units, Model 100). Mercurials were oxidized via a H^SO^/KMnO^/K^S^Og digestion and reduced by the reagent (NH^OH^. H 2S0 4 • NaCl. This was followed by the addition of SnS0 4 resulting in the release of elemental Hg vapour. This a n a l y t i c a l technigue does not d i f f e r e n t i a t e between the various , species of inorganic and organic Hg. The detection l i m i t s of the method were 0.18 to 5.52 nM Hg (0.05-1.50 u g . i - i ) . A l l reported Hg concentrations are a mean of duplicate samples. The concentration of HgCl 2 during any time i n t e r v a l (eguation 2) and the average concentration during the entire experiment (eguation 3) were computed as follows: (2) C n + 1= C,, e - 0 < W t n 1 +C* (3) C = I ( C n + l - C n / D) (T-M where n = number of time i n t e r v a l s ; C n + 1 = concentration at t m i ( n M ) ; C n = concentration at t n (nM) ; Ca = added concentration (nM) ; D = d i l u t i o n rate (hr—^J; t^+i - t n = time int e r v a l s between two additions (hr); C = average concentration (nM) during the entire experiment and T = time of the entire experiment (679.5 hr). Since i n these cal c u l a t i o n s losses of Hg are only due to the d i l u t i o n rate, these calculated Hg concentrations are referred to as the •expected' Hg concentrations. The s p e c i f i c growth rate (p) was computed using the following eguation (Davis et a l . , 1973); 26 (4) p = D + (1/t) In i x ^ / x ^ ) where u = s p e c i f i c growth rate ( h r - 1 ) ; D = mean d i l u t i o n rate over the time i n t e r v a l ( h r - 1 ) ; and x h = c e l l densities at times t n + 1 and 1^, (10 7 c e l l s - l - 1 ) Variations i n the analyses used in these experiments are presented i n Table II. Subsamples of chemostat culture were used to determine the standard deviation, except for the Hg analyses. 2•4 Experimental Design Batch cultures were used to determine the range of sublethal concentrations of HgCl 2 capable of i n h i b i t i n g the growth rates of nutrient-saturated cultures. Pr i o r to inocu-l a t i o n , c e l l s were centrifuged and resuspended i n a r t i f i c i a l seawater to avoid a transfer of complexing agents into the fresh medium. The medium was enriched with *f/25* and the source of nitrogen was n i t r a t e . In the short-term Hg-exposure, a 6 1 chemostat culture was grown under ammonium l i m i t a t i o n with a d i l u t i o n rate of 0.04 h r - 1 . The d i l u t i o n rate was determined using the following eguation: (5) D = F.V-* where F = flow rate (ml'hr - 1) and V = volume of the culture (ml). Chemostat samples were analyzed d a i l y f or fluorescence, c e l l density and nutrient concentrations. A steady-state was reached when no trend i n these parameters was observed for 27 TABLE I I . Standard deviation f o r analyses used i n the short and long-term mercury exposure experiments. Parameter measured Mean s.d. Units C e l l numbers Fluorescence 1 1* Chlorophyll a 1.37 ± 0.33 45.59± 1.56 22.19 ± 2.85 107 cells-1"" 1 r e l a t i v e u n i t s ug c h l a/10 7 c e l l s 1 24 10 3 13 10' 22 5 1.8 Ammonium 5.09 ± 0.30 2.0 ± 0.15 ug C *107 a e l l s K hr 1 fM 6 7* 10 30 10 Hp analysis, particulate Hg soluh le 20.0 ±1.20 3.68 ± 0.22 -1 ng 1 nM Hg 6 6 6 6 % - standard deviations expressed as : percentage of the mean 1 ; also see . standard deviations i n Appendix B and Figure 3. ^: used n/2 sets of standards done on d i f f e r e n t days. The standard deviations were ca l c u l a t e d and expressed as a percentage of the mean. The l a t e r percentage was converted into the units used f o r eaeh type of a n a l y s i s . The highest standard deviation (6%) was used i n t h i s table, * the standard deviation i s expressed as a mean of the standard devia-tion of n/2 sets of duplicate measurements. 28 several days. Then 500 ml of chemostat effluent was collected and simultaneously perturbed with 5 uM NH^Cl and one of the following concentrations of HgCl^; 0.37, 1.84, 3.68, or 5.53 nM. In an attempt to increase the toxic e f f e c t of HgCl 2 on nutrient uptake, 1 1 of effluent was coll e c t e d for 12 hours and allowed to starve for 24 hours such that the average starvation period was 30 hours. Then i t was perturbed as described above, with 5 uM NH C l and one of the following concentrations of HgCl.,, 0. 18, 0.37, 1.84 or 3.68 nM. Details of the batch mode perturbation technigue have been described (Caperon and Meyer, 1972). B a s i c a l l y , the technique consisted of quickly injecting HgCl 2 and NH^Cl s i -multaneously into the ef f l u e n t and mixing thoroughly. Ammonium determinations were made as frequently as every 6 minutes i n order to cl o s e l y follow the disappearance of ammonium from the medium. Phosphate and s i l i c a t e were not added along with ammonium since t h e i r concentrations i n the chemostat e f f l u e n t were s u f f i c i e n t to ensure no l i m i t a t i o n during the perturbation experiment. From the experiments usinq short-term Hg exposure, i t appeared that semi-continuous additions of 3.68 nM HgCl 2 over a long period of time could a f f e c t ammonium-limited c e l l s . In designinq the long-term Hq exposure experiment, a lower concentration was also used in case the ef f e c t s of 3.68 nM HqCl were too severe. In the long-term experiment, a 6 1 chemostat culture (D= 0.039 ± 0.005 hr-*) was divided into three, 2 1 chemostat cultures operating at the following nearly i d e n t i c a l d i l u t i o n 29 rates: 1= 0.041 ± 0.002 hr-*; 11= 0.040 ± 0.002 hr~*; 111= 0.039 ± 0.001 h r - 1 . These d i l u t i o n rates were chosen, such that small variations i n the d i l u t i o n rate would not change the concentration of ammonium i n the culture medium. The o r i g i n a l steady-state was maintained for another 2 days to ensure that the cultures ware not disturbed by the s p l i t t i n g of the 6 1 chemostat culture, and then Hg additions were started. For 30 days, cultures I and III were semi-conti-nuously exposed to 0.37 and 3.68 nM HgCl^, respectively. A Hgcl^ reference solution (3.68 mM, or 1000 mg.l _ 1. Fisher S c i e n t i f i c Co. Ltd.) was diluted to form substock solutions of 0.37 and 3.68 juM which were made fresh every fourth day. A t o t a l of two ml of each HgCl^ substock was injected through the inflow port of each chemostat cork. Generally, the additions were made every 8 hours during the f i r s t 10 days and every 12 hours for the rest of the experiment and exceptions are noted on Fig, 6 i n section 2.3. The con t r o l , culture I I , received an equivalent volume of d i s t i l l e d deionized water. Chemostat e f f l u e n t was c o l l e c t e d for 12 hours, ( i . e . , starved f o r an averaqe period of 6 hours), and used for l*C uptake experiments, c e l l countinq and Hg analysis. The remaining e f f l u e n t was used for a perturbation experiment conducted 5 hours l a t e r , r e s u l t i n g i n an average c e l l starvation period of 11 hours. Five hundred ml of effluent were collected for chlorophyll a determination. , Three perturbation experiments (chemostat effluents I, I I , and III) were performed simultaneously and nutrient con-30 centrations were determined every 6 minutes during the i n i t i a l 18-30 minutes, and then every 18 minutes for the rest of the perturbation experiment. The uptake k i n e t i c parameters, apparent Ks and V i _ „ were 777ctx computed using the eguations outlined by Conway et a l . , (1976) and s t a t i s t i c a l l y evaluated using the methodology of Cleland (1967). Section 3. Results 3.1 Results from some Preliminary Studies The e f f e c t s of Hg on S. costat^m were f i r s t investigated using nutrient-saturated batch culture assays to determine a range of sublethal concentrations (Fig. 2). Additions of 0.37 to 7.37 nM HgCl 2 gradually decreased the i n vivo fluorescence while only 3.68 and 7,37 nM decreased the maximum growth rate without inducing a lag period. After 88 hours of exposure to HgCl^, f i n a l c e l l densities were gradually reduced from 2.50 to 0.30- 10 7 c e l l s ' l - 1 over a sublethal concentration range of 0.37 to 7.37 nM HgCl^, respectively. 3.2 Growth Phases during the Long-term Mercury Exposure The o r i g i n a l data including d i l u t i o n rate, i n vivo fluorescence, c e l l numbers, s p e c i f i c growth rate, NH 4 + effluent concentration, chlorophyll a, carotenoid;chlorophyll 31 I 1 1 i 1 1——>• . . i — — \ 0 24 4 8 6 4 8 8 T ime (hr ) FIGURE 2. Changes i n i n vivo fluorescence i n nutrient-saturated batch cultures exposed to the following additions of HgCl., (nM): (Q) 0.00; ( V ) 0.37; (•) 0.93; ( T ) 1.84; (•) 3.67 and ( A ) 7.37. 32 a, photosynthetic rate, and c e l l dimensions are tabulated in Appendix B. Changes in biomass (in vivo fluorescence and c e l l numbers) of the 3 chemostat cultures are shown i n Fig. 3. Although both measurements display the same trend, i n vivo fluorescence was l e s s variable than c e l l numbers., The growth of ammonium-limited S. cj>sta.tu,m, during the long-term exposure to sublethal doses of HgCl 2 was divided into four phases (Fig. 4). The duration of phases A and B varied, whereas, phases C and D were s i m i l a r in the three chemostats. During phase A, the control chemostat culture (II) remained at the steady-state previously held by the parent chemostat, except for a few days i n phase A (Fig. 3). The semi-continuous additions of HgCl 2 impaired the a b i l i t y of chemostat culture I and I I I c e l l populations to return to the o r i g i n a l steady-state. In phase B, Hg-treated culture populations d r a s t i c a l l y declined while the control suffered a milder population loss due to changes in the l i f e c y c l e ; the l i f e cycle w i l l be described l a t t e r . C e l l densities of cultures I I , I, and I I I were reduced to 17,0, 3.0 and 0.09$, respectively, of their o r i g i n a l densities. During the recovery period (phase C), c e l l densities rapidly increased. In phase D, a new steady-state was established i n a l l cultures (Fig. 3A) . A FIGURE 3. Changes i n (A) in vivo fluorescence and (B) c e l l numbers of ammonium-limited c e l l s during the long-term mercury exposures (nM HgCl ) i"o; ( O ) 0.00, ( A ) 0.37 and ( l l ) 3.68 . The arrow represents the time at which the f i r s t addition of mercury was made. The bars shew the standard deviations of each measurement. FIGURE 3B. 35 Time (Days) FIGURE k . Changes i n the sp e c i f i c growth rates of ammonium-limited c e l l s during the long-term mercury exposures (nM HgCl^) t o : ( 0 ) 0.00, ( A ) 0.37 and ( I J) 3.68. The four growth phases are: phase A: mainte-nance of the steady-state; phase B: decline i n s p e c i f i c growth rates; phase C: increase i n s p e c i f i c growth rates and ; phase D: return to a new steady-state. 36 3.3 S p e c i f i c Growth Rates and N itrog.en 2uotas The effect of HgCl^ on s p e c i f i c growth rates i s shown in Fig. 4. On day 1, s p e c i f i c growth rates varied between 0.035 - 0.040 h r - 1 i n a l l cultures but by day 6, a d e f i n i t e trend of declining s p e c i f i c growth rate was set i n a l l c u l -tures. In chemostat I I I , a decrement i n c e l l d i v i s i o n began as early as day 3 and s p e c i f i c growth rates remained undetectable from days 12 to 16. Cultures I I and I sustained a low s p e c i f i c growth rate during phase B. In phase C, c u l -tures I I , I, and III successively recovered with s p e c i f i c growth rates as high as 0.085, 0.077 and 0.135 h r - 1 , respectively, and d i v i s i o n rates up to 3 times per day. Although these maxima occurred only one day, r e l a t i v e l y high s p e c i f i c growth rates were maintained for 5, 7 and 11 days at exposures of 0.00, 0.37 and 3.68 nM HgCl^, respectively. New steady-state growth rates resumed after approximately 20.days. The amount of nitrogen per c e l l (pM N«10 7 c e l l s - * ) followed the opposite trend to c e l l density. As c e l l densities and ammonium assimilation decreased, the nitrogen per c e l l increased from about 1.7 in phase A, to 6.38, 25.03, and 15.87 i n cultures I I , I, and I I I , respectively, at the end of phase B. During phase C, s p e c i f i c growth rates sharply increased, and the nitrogen per c e l l concomitantly decreased as the cultures became nitrogen-limited again. In phase D, the nitrogen per c e l l for the control culture was 5055 lower than i n phase A probably due to differences i n c e l l volume and 37 the amount of nitrogen per c e l l . 3.4 Effects of Mercury on Photosynthesis Changes i n chlorophyll a and J*C uptake during phase A are presented in Fig.5. Chlorophyll a per c e l l increased with time. The largest amounts were observed on day 8 (Fig. ,5A). During phase ft, the average carotenoid : chlorophyll a r a t i o s were 2.39, 2.17 and 2.04 for cultures I I , I and I I I , respectively (Appendix A). A pigment r a t i o of 2.39 was also reported f o r ammonium-starved S. costatum (Harrison et. a l . , 1977). Since d i r e c t chlorophyll a determinations were not conducted a f t e r day 8, the chlorophyll a : fluorescence r a t i o was determined from values obtained i n phase A and was used to convert fluorescence readings to chlorophyll a. This r a t i o was 0.33, 0.4 3 and 0.45 for chemostats I I , I and I I I , respectively. After 22-29 days, chlorophyll a guotas i n chemostats I I , I, and III were 1.41 ± 0.53, 2.17 ± 0.54, and 2.33 ± 0.90 ug c h l a«10 7 c e l l s - 1 , respectively (Appendix B). Values f o r phaeophytin a were too variable to be used with confidence. The **C uptake gradually decreased with time u n t i l day 5 (Fig. 5B) . Between days 1 and 4, a 50% reduction in photosynthesis occurred in chemostats II and I I I , while no obvious trend was observed in chemostat I. On day 6, chemos-tats I I , I, and III achieved photosynthetic rates of 2.52, 5.39, and 4.54 ug C • 10 7 c e l l s - * . h r _ i , respectively. The 38 0.0 1 — i 1 1 — i 1 1 0 2 4 6 8 10 Ti mc (Days) FIGURE 5. Changes i n (A) chlorophyll a_, and (B) photosyntru- L i c rate and (C) photosynthe.tic assimilation rate during phase A of the long-term mercury exposures' (nM HgCl 2) at (Q) 0.00; ( A ) 0.37 snd ( 0 ) 3.68 . The. arrow indicates the end of phase A for each culture. T IME (Days) FIGURE 5B and 5C. 40 photosynthetic assimilation rate was reduced to about the same value on day 6 f o r a l l treatments (Fig.,5C).. 3.5 Morphological Observations During phases A and B, chains of 1 to 2, elongated, curved and highly vacuolated c e l l s , connected by short s i l i c a rods, and c e l l c l u s t e r s around lysed c e l l s , were more freguently observed i n Hg-treated chemostats than i n chemostat culture I I. S t a t i s t i c a l analyses, including the analysis of variance and three a fiosteriori range tests (Duncan's multiple range tes t , Newman-Keul's tes t and Tukey's test) were used i n an attempt to determine whether the e f f e c t s of the three treatments on c e l l dimensions were 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 (0.00, 0.37 and 3.68 nM HgCl ) . Up to day 5, the c e l l dimensions i n the three treatments were not s i g n i -f i c a n t l y d i f f e r e n t . During the l a t e r stages of phase A, c e l l s exposed to 3.68 nM HgCl^ were s i g n i f i c a n t l y longer than the two other chemostat populations. In phases C and D, c e l l s were s i g n i f i c a n t l y shorter than i n phase B in a l l chemostats. Chemostat III c e l l s were longer than c e l l s of chemostats I and II c e l l s , the l a t t e r being s t a t i s t i c a l l y indistinguishable. Scanning electron micrographs revealed most of the c h a r a c t e r i s t i c s already observed with the inverted microsco-pe, (e.g., short s i l i c a rods). Only i n the Hg-treated cultures, at the end of phase C, populations consisted of 41 post-auxospore c e l l s , (1-3%) and chains of 4 to 6 c e l l s (1-8%) connected with longer s i l i c a rods. Consequently, these populations were more heterogeneous than the control. Although no gametes and r e l a t i v e l y few post-auxospore c e l l s were observed, synchronized sexual reproduction may have occurred, due to the increased frequency of shorter c e l l s toward the end of phase C i n a l l cultures. The wide c e l l s which were formed during phase C, quickly became narrower under ammonium-limited conditions i n phase D. This may explain the absence of these wide c e l l s in chemostat culture I I since the c e l l s were always under ammonium l i m i t a t i o n . 3.6 Mercury Analyses Variations i n the expected t o t a l Hg concentrations in culture I are shown in Fig. 6. Since the same pattern of Hq fluctuations with time was observed for culture I I I , i t was not included i n Fig. 6. Expected t o t a l concentrations (calculated using equation 2) represent maximum Hq l e v e l s . The accumulation or v o l a t i l i z a t i o n of Hg by the c e l l s , the adsorption onto frustules and inner walls of culture flasks are not considered i n these calculations. Expected t o t a l concentrations take into account only the changes i n concentrations as a r e s u l t of the semi-continuous additions and losses due to the d i l u t i o n rate of the culture. The average expected t o t a l concentrations (calculated using equation 3), in cultures I and I I I , were 0.33 ± 0.07 and 42 FIGURE 6. Variations i n the expected t o t a l mercury concentrations i n chemostat I during the long-term mercury exposure to 0.37 nM HgCl^ The average expected t o t a l concentration, which only takes into account the mercury losses due to the d i l u t i o n rate of the chemostat, was 0.33t0.07 nM HgC^. During the f i r s t ten days, the concentration of the addition was 0.12 nM HgCl 2 except when ( Q ) 0.06 and (®) 0.24 were ad-ded. The expected t o t a l concentrations of mercury between each addition and the average expected concentration over the entire experiment were calculated using equations 2 and 3, respectively. 43 3.46 ± 0.78 nM HgC]^, respectively for the entire experiment. Based on studies on Cu t o x i c i t y , (Sunda and G u i l l a r d , 1976), i t i s possible that Hg t o x i c i t y may be related to mercuric ion a c t i v i t y . However, the present work deals with t o t a l Hq concentrations, because there i s no technique which d i r e c t l y measures mercuric ion a c t i v i t y . Table I I I shows that the concentration of particulate Hq increased with concentration and time of Hq exposure. Particulate Hq values in cultures exposed to 0.37 and 3.68 nM HgCl^, accounted f o r 22-58% and 15-41%, respectively, of the t o t a l expected concentrations. In cultures I and I I I , most of the soluble Hq could not be recovered, possibly due to the a n a l y t i c a l technique used. The small amounts of Hg i n the control c e l l s were probably due to contamination from reagent grade s a l t s used i n the preparation of the culture medium (Table I I I ) . 3.7 Short-term Nutrient Kinetics The nutrient uptake k i n e t i c responses of cultures exposed to sublethal doses of HgCl,,, for 5 minutes to 5 hours, are shown in Fig. 7., The values of the nutrient uptake k i n e t i c parameters are presented i n Table IV. The e f f e c t s of Hg on the uptake kinetics of NH^+-limited S. costaturn starved f o r 1.5 hours, occurred at a threshold concentration between 1.84 and 3.68 nM HgCl 2 (Table IV). The l a t t e r addition reduced V i 1 ) i ; ! r by 37% and increased Ks from 44 TABLE III. Concentrations of mercury In the long-term experiment. F i l t e r s and filtrates from 200 ml of chemostat effluent were analyzed for p a r t i c u -late and soluble mercury, respectively. Each value is the mean of duplica-tes. 'Measured' refers to values obtained from the mercury analysis. 'Expected total' refers to values obtained using equations 2 and 3, and 'expected soluble' refers to the difference between the total expected and measured particulate value. The percentage loss (% Loss) is the difference between the 'expected' and 'measured' soluble Hg, divided by the 'expected' —18 soluble Hg, and multiplied by 100 . Atg - attog =10 g. CONTROL EXPOSED TO 0. 37 nM • EXPOSED TO 3. 68 -riM DAY 2 4 6 2 4 6 2 4 6 MEASURED: Particulate Hg: (atg'.cell *) 0.91 1.61 2.45 5.16 7.56 8.32 33.46 207.93 (nM.) 0.07 0.11 0.07 0.18 0.11 0.52 1.36 1.18 Soluble Hg: (nM) N.I). N.D. 0.18 .0.33 N.D. N.D. 0.41 N.D. 0.55 EXPECTED: Total Hg : (nM) 0.00 0.00 0.00 0.33 0.32 0.40 3.46 3.32 3.24 Soluble Hg :(nM) 0.00 0.00 0.00 0.25 0.13 0.29 2.95 1.95 2.06 % LOSS: 100% 100% 86% 100% 73% A B 1 - T O U R E R . . Ammonium uptake rates as a function of the substrate, for ammonium-limited c e l l s starved for (A) 1 . 5 0 hours and (C) 3 0 . 0 hours, during the phort-term (un to 5 hours) mercury-exposure (nM HgCl ) to 0 . 0 0 (O) and 3 . 6 8 ( i \ ) . figures (B) and (!)) show the disanpearance of ammonium with time. c 0.20 f 0.15 + 0 1.0 2.0 3.0 4.0 S ( p M NHJ) D O ^ W U) W u i O u i ' o b i b b i b u i Time (hr) 47 TABLE IV. Nutrient kinetic response to short-term mercury exposure. The nutrient kinetic parameters are defined in section l?of this chapter.The Ks values are the actual Ks-*5 apparent Ks + So. Values of the standard errors are shown for V i m a x and Ks; the Vs values represent the average uptake rate during the time period, Ts, over which the surge uptake oc-curs. N.D. •= not detectable. 1.5 HOURS OP STARVATION CONCENTRATION Vs . Ts V i max Ks So (nM HgCl 2) (hr" 1) (hr) (hr ) (uM NH^) (uM NH^) 0.00 0.44 0.26 0.178 ± 0.017 0.27 ± 0.13 N.D. 0.37 0.50 0.26 0.148 ± 0.006 0.21 ± 0.05 N.D. 1.84 0.93 0.16 0.167 ± 0.011 0.01 + 0.02 N.D. 3.68 0.45 0.26 0.112 ± 0.004 1.11 ± 0.07 1.08 5.53 1.89 0.06 0.117 ± 0.005 0.85 + 0.07 0.83 30.0 HOURS OF STARVATION CONCENTRATION Vs . Ts Vi max Ks So (nM HgCl 2) (hr*-1) (hr) (hr ) (pM NHJ) (UM-NH^) 0.00 1.74 0.06 0.012 ± 0.003 0.02 ± 0.00 N.D. 0.18 0.46 0.26 0.070 ± 0.010 0.51 ± 0.29 0.32 0.37 2.18 0.06 0.060 ± 0.000 0.54 ± 0.11 0.50 1.84 0.55 0.26 0.050 ± 0.000 0.00 ± 0.03 N.D. 3.68 0.50 0.26 0.060 ± 0.000 0.20 ± 0.08 0.16 48 0.28 to 1.11 uM NH^+. The increase in Ks was primarily due to the cessation of uptake when 1.08 uM NH^Cl s t i l l remained i n the medium (Fig. 7k). The Vs values were s i m i l a r when a l l values were calculated over the same time i n t e r v a l of 0.26 hour., In N H ^ * -limited e f f l u e n t s starved for 30 hours, v i w a x was s i g n i f i c a n t l y reduced by exposure to a concentration as low as 0.18 nM HgCl_. Increased time of starvation of NH.+-2 4 l i m i t e d chemostat ef f l u e n t s (from 1. 5 to 30 hours) resulted in a decrease i n the Ks value of the control cultures from 0.27 to 0.02 uM NH^Gl (Table IV). Since t h i s l a t t e r value cf Ks for the control culture was near the l i m i t s of detection, t h i s made i t d i f f i c u l t to determine the ef f e c t s of Hg exposure on Ks. /Short-terra Hg exposure (1.50 hours of starvation) did not appear to af f e c t Vs (Table IV), since the mean uptake rate over the concentration range from 0.00 to 5.53 nM HgCl^ was 0.51 ± 0.06 h r - 1 when the uptake was calculated at a time of 0.26 hrjJVVariations i n the Vs values r e f l e c t the d i f f i c u l t y i n making t h i s measurement. However, both N H 4 + starvation and Hg treatment resulted i n a very sharp and reduced Ve region of the uptake curve (Fig, 7A and C). The So values (Table IV) represent the amount of NH + remaining at the end of the perturbation experiment when Ve = 0 for 18 to 60 minutes. However, on some occasions the uptake slowly resumed after a few hours. Of secondary i n t e r e s t , i s the eff e c t of duration of starvation of eff l u e n t from the chemostat on the nutrient 49 uptake response (Table V). When effluents were starved for 11 hours, Ks and St values decreased while Vi„ a„remained •' max constant. This indicated that ammonium-limited populations responded to 11 hours of nitrogen deficiency by increasing their a f f i n i t y for the substrate. Between 11 to 30 hours, the rate of mobilization of nitrogen from the i n t e r n a l nutrient pool into the assimilatory system was decreased. Between 30 and 72 hours, ammonium deficiency had adverse effects on a l l the phases of the nutrient uptake rates. 3 • 8 Long.- term Nutrient Kinetics Nutrient uptake k i n e t i c parameters measured during phases a and D of the long-term Bg exposure are presented i n Table VI. In culture I I I , the nutrient k i n e t i c s deviated considera-bly from the regular pattern as early as day 4, whereas culture I showed s i g n i f i c a n t deviations only by day 8 (Fig. 8)., Despite the presence of 1.3 to 1.5 uM NH^Cl i n culture I ef f l u e n t , the spike addition of NH„ + resulted i n a normal value for Vs of 1.21 h r - 1 , after which no s i g n i f i c a n t uptake took place for 7.1 hr (Fig.8B, day 8). However, i n culture III (day 6), the maximum recorded Vs was 0.70 hr~*, i n contrast to culture II (day 5) where a Vs of 1.19 h r — 1 was measured. These comparisons were made at a standard time i n t e r v a l of 0.10 hour. In general, during phase k i n the Hg-treated cultures, the Ve region of the uptake curve gradually became gentler 50 TABLE V. The effects of duration of starvation of chemostat effluent on the nutrient uptake k i n e t i c s . The uptake k i n e t i c parameters are defined i n section 1 of this chapter. The Ks values are the actual Ks, where actual Ks = apparent Ks + So; n. d. = not detectable. Vs Ts V i Ks So Source max ( h r _ 1 ) (hr) ( h r _ 1 ) (jM NH}") (pM NH^) 0.00 0.27 2.60 0.124 0.50 0 .20 Conway and Har-ri s o n , 1977 1.50 0.A4 0.26 0.178 0.27 n . d. Table IV 11.00 0.37 0.30 0.129 0.59 n . d. Tavle VI 30.00 1.74 0.06 0.102 0.02 n . d. Table IV 72.00 0.17 0.70 0.088 0.10 n . d. Conway and Har-r i s o n , 1977 STARVATION TIME (hr) 51 TABLE VI. Nutrient uptake k i n e t i c response to the long-term mercury expos-ure. The nu t r i e n t uptake k i n e t i c parameters are defined i n section 1 of t h i s chapter. The Ks values are the ac t u a l Ks where a c t u a l Ks *• apparent Ks + So. Values of standard er r o r s are shown f o r V i (Vi * V i ) and max Ks. N.D. = not detectable. The units of the nutrie n t uptake kinetic, parameters Ts= hr, V i (Vi Vi max ) ? h r ' K s » u M NH 4 +, So = uM NH^  + . CHEMOSTAT CULTURE II (CONTROL) are; Vs « hr -1 DAYS 2 4 5 6 19 26 Vs 0.37 0.62 1.19 0.94 0.38 Ts 0.30 0.20 0.10 0.10 0.30 V i 0.129±.Q06 0.080+.003 0.1451.009 0.129±.011 0.145± .006 0.129 + .011 Ks 0.59 +.14 0.25 ±.09 0.44 ±.11 0.49 ±.16 0.06 ± .02 0.12 ± .08 So 0.26 0.20 N.D. N.D. N.D. N. D. CHEMOSTAT CULTURE I (EXPOSURE TO 0.37 nM HgCl 2) DAYS 2 4 5 19 23 Vs 0.28 0.31 0.28 0.99 1.62 0.48 Ts 0.50 0.30 0.60 0.10 0.10 0.20 Vi 6.104±.005 0.110±.017 0.186±.078 0.152±.024 0.122+, .004 0.183+ -009 Ks 0.18 ±.10 1.22 ±.57 0.03 ±1.67 1.34 +.71 0.01 ±. .02 0.01 ± .02 So .0.04 0.25 N.D. N.D. N.D. N.D. CHEMOSTAT CULTURE I I I (EXPOSURE TO 3.68 nM HgCl ) DAYS 2 4 5 6 23 26 Vs 0.37 0.54 0.63 0.70 0.58 0.30 Ts 0.40 0.20 0.20 0.10 0.20 0.20 V i 0.134±.007 0.123±.013 0.121±.020 0.06U.008 0.170±. 007 0.105±, .003 Ks 0.35 ±.07 1.36 ±.36 1.80 ±.52 2.83 ±.23 0.15 +. 03 0.05 ±, .02 So 0.04 0.63 0.85 2.43 N.D. N.D. 52 2.0 3.0 4.0 Time (hr) FIGURE 8. The disappearance of the substrate with time during a perturb-ation experiment conducted on different days during phase A of the long-term mercury exposure (nM HgCl-) to (A) 3 .68 (•), and (B) 0 .37 (Z\). 53 3.0 4.0 5.0 Tims (hr) A <aon 7.0 80 9.0 FIGURE 9. (A) Disappearance of ammonium with time, after the addition of 5 fM NH^ • on day 6 (phase A) of the long-term mercury exposure (nM HgCl^) to (O) 0.00; ( A ) 0.37 and (•) 3.68 . These data were used to c a l c u l -ate the ammonium uptake rates as a function of the substrate i n figure (B). 54 with duration (Fig. 8) and concentration (Fig. 9) of Hg exposure. This change was r e f l e c t e d by the increase i n Ks values or a loss of a f f i n i t y for the substrate i n culture III (Table VI). I t appeared that vs was also reduced i n chemostat culture III compared to the control culture I I . In phase D, Hg-treated cultures had a higher ^ i ^ a x a n d lower Ks (Fig.,10) than in phase A (Fig. 9). In the con t r o l , V i _ values were s i m i l a r to values obtained i n phase A but Ks values were reduced in phase D (Table VI). Section 4. Discussion ^ • 1 Effects of Mercury on Photosynthesis Under ammonium l i m i t a t i o n (e.g., in the control culture II) , the reduction i n photosynthetic assimilation rate was due to a decrease in photosynthesis and a s l i g h t increase in chlorophyll a per c e l l . Transient reductions i n photosyn-t h e s i s have also been observed in nutrient-limited (Thomas, 1975a; 1975b) and n u t r i t i o n a l l y perturbed natural phytoplankton communities (Falkowski and Stone, 1975). The simultaneous effects of mercury and ammonium limit a t i o n (days 1 - 5 i n phase A and in phase D) resulted in a qreater enhancement of chlorophyll a synthesis than under ammonium li m i t a t i o n alone. The small increase in photosynthetic assimilation rate i n mercury-treated cultures (day 6) was 55 0.195 r S (|-'M NH4) FIGURE 10. Ammonium uptake rates as a function of the substrate after the recovery (phase D) from the long-term mercury exposure (llM HgC^) to (O) 0.00; ( A ) 0.37 and (•) 3.68 . probably due to the cessation of ammonium l i m i t a t i o n , since the amount of nitrogen and chlorophyll a per c e l l increased in spite of an increase i n particulate mercury up to 207.93 atg»cell-i (attog = 10~*8 g) . In phase D, i n vivo fluorescence was comparable i n a l l treatments. In the control and mere ury-treated cultures, c e l l numbers doubled and returned to the i r o r i g i n a l l e v e l s while c e l l volume s t i l l increased upon mercury exposure. I f the nitrogen and chlorophyll a per c e l l were expressed per unit of c e l l volume, the values would appear to be si m i l a r among a l l cultures. The increase in chlorophyll a content may be due to a stimulation of synthesis, or to a duplication of organelles without the formation of a new frustule. This would produce elongated c e l l s of greater c e l l u l a r content (e.g., DNA, BNA and proteins). This has been observed in s i l i c a t e - s t a r v e d Navicula p e l l i c u l o s a (Coombs et al.,1967). Thus, mercury might arrest c e l l d i v i s i o n by i n t e r f e r i n g with the regulation of the s i l i c a t e metabolism. 4.2 Eff e c t s of Mercury on Growth Parameters The e f f e c t of mercury on biomass and nutrient k i n e t i c s were proportional to the concentration of the additions (e.g., culture III was the f i r s t culture to be disturbed from i t s steady-state). In culture III, high particulate mercury values were reached sooner than i n culture I (Table I I I ) . 57 Periods of c e l l loss i n nutrient-limited cultures have been reported (Davis et a l , , 1973). As c e l l s became narower in a l l nutrient-limited chemostat cultures, there was an increased li k e l i h o o d f o r the recurrence of sexual reproduction. For a s i l i c a t e - l i m i t e d cultures of S. costaturn a decrease i n c e l l density, an increase in the amount of the l i m i t i n g nutrient per c e l l and the occurrence of wide, short c e l l s indicated that synchronized sexual reproduction occurred (Davis et a l . , 1973). Similar observations were made for the Hg-treated cultures, and to a very limited extent, in the control culture. Additions of mercury, which resulted i n the e a r l i e r decline in cultures I and III populations (phase A), did not appear to i n t e r f e r e with presumed sexual reproduction processes (phase C) since resumption of growth occurred simultaneously i n a l l cultures. U.3 Effects of Mercury on Nutrient Dotake Kinetics In the short-term mercury exposure and i n culture I, the maximum rate of uptake, Vs, was not altered. In cultures I and I I I (days 2 to 4), the actual Ks increased, indicating a loss in the a f f i n i t y for the substrate, while the maximum assimilatory rate, ' i ^ ^ * remained unchanged. Although, the actual Ks (apparent Ks + So) increased i n culture I, the apparent Ks values were reduced. In culture I I I , the apparent Ks values were also reduced as well as the maximum uptake rate. Vs. The enzymatic i n h i b i t i o n by heavy metals i s usually 58 c l a s s i f i e d as noncompetitive (Lehninger, 1975) . Even though uncompetitive and noncompetitive i n h i b i t i o n can r e s u l t in a decrease i n Vi „ or Vs, the additional decrease i n the wax apparent Ks values indicated that mercury uncompetitively inhibited the uptake i n ammonium-limited cultures. Under long-term, low l e v e l (0.37 nM HgCl^) exposure and under short-term higher l e v e l (>1.84 nM HgCl^) exposure, the uptake rate at low l e v e l s (estimated by Ks) appeared to be more sen s i t i v e than the i n i t i a l uptake (Vs) . Much higher l e v e l s of mercury were reguired to decrease Vs or Vi . The reasons for the max greater s e n s i t i v i t y of the rate of uptake at low concentrations are not clear. The reduction i n Vs or V i ^ ^ in the long-term high l e v e l (3.68 nM HgCl^) exposure could be due to the high a f f i n i t y of mercurials for s u l f h y d r y l groups, re s u l t i n g i n the binding of some mercuric ions onto c e l l membrane enzymes (either enzymes involved in the hydrolysis of ATP or c a r r i e r enzymes). Changes i n nutrient k i n e t i c s from the short-term experiment (1.5 hours of starvation), a f t e r the recovery (phase D) in the long-term experiment, and changes i n c e l l morphology i n culture I I I , suggest that the threshold concentration for recovery from mercury i n h i b i t i o n , occurred between 1.84 and 3.68 nM HgCl^., The threshold concentration where the f i r s t e f f e c t of mercury i n h i b i t i o n was observed on nutrient uptake ki n e t i c s was reduced to 0.18 nM HgCl^ by an increased period (30 hours) of ammonium starvation. 59 4.4 Recovery from I n i t i a l Mercury I n h i b i t i o n Recovery from mercury i n h i b i t i o n i n continuous (F u j i t a and Hashizume, 1975; Kayser, 1976) and batch cultures (De F i l i p p i s and Pallaghy, 1976c; Berland et a l . , 1977) are known. They have been attributed to mercury losses from the medium (Davies, 1974) either by v o l a t i l i z a t i o n when c e l l densities are high or a decrease in particulate mercury through an uptake exclusion mechanism(Ben-Bassat et a l . , 1972; Ben-Bassat and Mayer, 1975; 1977; De F i l i p p i s and Pallaghy, 1S76b). In phase C of the long-term mercury exposure, growth resumed when c e l l densities were minimum and possibly when mercury particulate values were highest. Greatest metal accumulation occurred i n mercury-resistant C h l o r e l l a (De F i l i p p i s and Pallaghy, 1976c), in IsochrjrsJLs galbana and Qunaliella t e r t i o l e c t a , after the i n i t i a t i o n of a few c e l l d i v i s i o n s (Davies, 1974). The increase i n s p e c i f i c growth rates i n mercury-treated cultures was probably due to the appearance of c e l l s of a new l i f e cycle stage with a d i f f e r e n t c e l l u l a r chemical composition (as seen from the nitrogen and chlorophyll a content). In nutrient-limited diatoms, during vegetative reproduction, changes in the c e l l u l a r chemical com-position, reduction i n width and elongation of c e l l s possibly produced populations of di f f e r e n t physiological stages, Werner (1971) also found that during c e l l diminution i n Coscinodiscus asteromphalus, changes i n chemical composition 60 occurred and the s e n s i t i v i t y to metabolic i n h i b i t o r s varied with d i f f e r e n t l i f e cycle stages or c e l l widths., After recovery, improvement of nutrient uptake (increase i n the a f f i n i t y for the substrate and the rates of uptake) occurred i n the mercury-treated cultures. The a c g u i s i t i o n of mercury-tolerance may be due to several factors. It could have been acguired from genetic recombination, as a conseguence of sexual reproduction. A l t e r n a t i v e l y , some or a l l c e l l s i n phase C may also have developed a physiological resistance upon mercury exposure. The s e l e c t i v e pressure operating on phase C c e l l s may have resulted i n the survival of a few c e l l s capable of coping with high external or i n t e r n a l concentrations of Hg, and therefore growth during the recovery phase would be i n i t i a t e d by these c e l l s . Berland et a l , , (1977) suggested that recovery may be due to a return to the i n i t i a l physiological state of the c e l l s but no suggestion of mechanism was given. The results from the long-term mercury exposure i n t h i s study suggest that a change i n c e l l type may be as important as a pure physiological adapta-ti o n mediated by a biochemical change i n c e l l metabolism. 4.5 Mercury Losses Losses were independent of the i n i t i a l doses. These losses from the used medium were probably not due to v o l a t i l i z a t i o n within the cultures. In continuous cultures, the regulation of a constant volume inside the culture flasks 61 depends on a constant a i r pressure i n the dead a i r space. Consequently, the p a r t i a l pressure of mercury above the surface of the medium, and the amount of Hq adsorbed onto the walls and i n the solution were probably at equilibrium. The actual low recovery of soluble mercury may be due to the presence of a heat-stable photosynthetic metabolite i n the used medium (Ben-Bassat and Mayer, 1977), responsible f o r the reduction of Hq*2 to Hq° . Thus, the major l o s s may come from the a n a l y t i c a l procedures f o r mercury determinations, which require heatinq of aqueous samples at 95°C durinq 2 hours. Therefore, the heat-stable photosynthetic metabolite would enhance the rate of v o l a t i l i z a t i o n durinq the heatinq process. 4.6 Applications to the Natural Environment In natural waters, mercuric ion a c t i v i t y would probably be less than i n the a r t i f i c i a l seawater due to the presence of unknown quantities of natural complexinq materials such as de t r i t u s , dissolved orqanics. Therefore, t o t a l mercury concentrations equivalent to those used i n cultures may not produce the same e f f e c t s in natural seawater. During periods of seasonal nutrient l i m i t a t i o n , mercury pollution may seriously i n t e r f e r e with the competitive a b i l i t y of a diatom. Under nutrient-limited conditions, the a b i l i t y to take up the l i m i t i n g nutrient i s severely reduced (Ks value i s increased) by exposure to a secondary stress such as mercury. This may result i n a change i n the dominant species 62 or replacement by d i f f e r e n t a l g a l groups such as f l a g e l l a t e s {Thomas and Seibert, 1977), 4,7 Evaluation of Chemostat Studies In t h i s study, the use of chemostats i n determining the eff e c t s of nearly ecological t o t a l concentrations of mercury on an unialgal population, already under a primary stress (nutrient l i m i t a t i o n ) , has allowed a cl o s e r imitation of an ecological situation than i f batch cultures had been used. Long-term, low l e v e l e f f e c t s rather than short-term, high l e v e l e f f e c t s may simulate more closely the dispersal mode of i n d u s t r i a l pollutants into the natural environment. This simulation i s best achieved using continuous cultures. The threshold concentration where the e f f e c t s occur, are almost an order of magnitude lower i n the long-term compared to the short-term experiments. I t i s for t h i s reason that the use of continuous cultures to determine threshold e f f e c t s i s strongly urged. The noticeable changes i n c e l l morphology and physiology, during vegetative and especially during sexual reproduction, tend to make diatoms unsuitable t e s t organisms for bioassays. From these experiments, the use of chemostat cultures for poll u t i o n bioassay is considered as a refined technigue. Since conditions which induce certain l i f e cycle stages i n algae are i l l - d e f i n e d , the s u i t a b i l i t y of the chemostat i n poll u t i o n studies could be improved by using species which are less variable during their l i f e c y cle. 63 CHAPTER III TH E EFFECT OF Hg EXPOSUSE ON INTRACELLULAR DISTBIBOTION M Mi^ Cuj, AND Zn Section 1. Introduction Repeated exposure of phytoplankton to trace elements may increase tolerance {De F i l i p p i s and Pallaghy, 1976c) , but i t i s not known whether a complexing agent such as metallcthionein plays a role i n t h i s process, Metallothionein i s a low molecular weight (m.w.) protein (10,000) whose synthesis i s stimulated by exposure to heavy metals. This protein can bind and detoxify heavy metals such as Ag, Cd and Hg, I t can also store Cu and Zn when they occur i n excess of the l e v e l s reguired f o r metalloenzymes (Bremmer and Davies, 1975 ; Brown and Chatel,1978). Deleterious effects may occur when the rate of heavy metal accumulation exceeds the rate of metallothionein synthesis or i t s binding capacity (Winge et a l . , 1973; Brown and Parsons, 1978). Hence, heavy metals such as Cd and Hg may accumulate in the high m.w. pool and exert t o x i c e f f e c t s by; 1) substituting for Cu and Zn i n metalloenzymes (Brown, 1977; Brown and Parsons, 1978), 2) a l t e r i n g the guarternary or t e r t i a r y enzyme structure, or 3) binding to active or other s i t e s leading to conformational changes. Metallothionein has been ubiguitously found i n land and marine animals (Piscator, 1964; Buhler and Kagi, 1974; Howard and N icicles s, 1977a; 1977b), but i t s presence i n phytoplankton remains to be c l a r i f i e d . McLean et a l . , (1972) may have found a metallothionein-like f r a c t i o n binding Cd and Zn i n blue-green algae, exposed to radioactive Cd., The i n i t i a t i o n of logarithmic growth i n algae previously i n h i b i t e d by heavy metals may be attributed to several factors. One possible reason for the recovery from i n h i b i t i o n may be the production of complexing agents such as metallothionein. The aim of t h i s study was to investigate the p o s s i b i l i t y of a short-term biochemical adaptation in response to heavy metal exposure i n S. cost,atum and to determine whether metallothionein would be responsible for seguestering heavy metals and the subseguent recovery. The e f f e c t s of HgCl^ on the i n t r a c e l l u l a r l e v e l s and d i s t r i b u t i o n of Cu and Zn are also examined.„ Section 2. Materials and Methods 2.1 Batch Cultures Cultures of Skeletonema costatum were grown i n a r t i f i c i a l seawater (Davis et a l . , 1973) enriched with modified , f / 2 l medium (appendix A). Batch cultures were grown in b o r o s i l i c a t e , 6 1 f l a t bottom b o i l i n g f l a s k s continuously 6 5 s t i r r e d at 120 rpm. Growth was monitored by changes in i n vivo fluorescence with a Turner Model 111 fluorometer and i n c e l l density using an inverted microscope. 2.2 Jxjaer imenta 1 Conditions During the f i r s t 70 hours of the experiment, two unialgal log phase cultures were grown with no pre-exposure to Hg {cultures A and B) while two other cultures {C and D) were exposed to 1.84 nM HgCl 2; a f t e r 70 hours, one culture of each subset (cultures B and D) was perturbed with 5.53 nM HgCl 2 (Table VII). Culture E was exposed to 0.37 nM HgCl 2 over a period of 116 hours (Table VII). 2.3 Analyses Early stationary phase c e l l s were harvested by centrifugation f o r 6 minutes at 650«g at 6°C i n 50 ml polycarbonate test tubes. The c e l l s from the control culture were harvested after 70 hours while the c e l l s from the Hg-exposed cultures were harvested between 90 and 116 hours. The c e l l s remaining i n the supernatants were collected onto a 0.45 uM Millipore f i l t e r , resuspended i n a r t i f i c i a l seawater and recentrifuged as above. One gram of c e l l s (wet weight) was homogenized i n 3ml of 0.9% NaCl for 5 minutes using a TBI-B STIB-R Model 563C variable speed lab homogenizer at a speed setting of 4.5. The homogenate was centrifuged at 27,000-g for 10 minutes using a 66 TABLE VII. Exposure to different concentrations of HgCl at different times during a batch culture experiment. 2 CONCENTRATION (n^ HgCl ) ADDED AT TREATMENT 0.00 hr 70.00 hr Culture A 0.00 0.00 Culture B 0.00 5.53 Culture C 1.84 0.00 Culture D 1.84 5.53 Culture E 0.37 0.00 67 Sorval Super/speed EC2-B automatic refrigerated centrifuge. The supernatant was fractionated on a Sephadex G-75 (Pharmacia) column (9 X 60 cm) with 0.01 a NH.HCO, elution buffer. Fractions of 2 ml were collected. U l t r a v i o l e t absorbance was determined with a Perkin-Elmer Model 124D double beam spectrophotometer. Absorbances at 250 and 280 nm determined the r e l a t i v e amount of metal-bound subs-tances and aromatic amino acids, respectively. Peaks were i d e n t i f i e d as being in the position of the high (enzyme-con-taining) and low m.w. i n t r a c e l l u l a r pool by comparing their positions i n r e l a t i o n to the medium m.w. f r a c t i o n s obtained from naturally occurring duck l i v e r metallothionein. The i d e n t i f i c a t i o n of metallothionein was done by comparing i t s el u t i o n p r o f i l e with those of Piscator (1964) and Leber (1974) as described in Brown and Chatel (1978). Copper and Zn lev e l s were determined i n each f r a c t i o n using dir e c t aspiration with a Perkin-Elmer Hodel 306 flame atomic absorption spectrophotometer with deuterium background correction. Total mercury was measured by a cold vapor method on the combined fr a c t i o n s of each peak, u t i l i z i n g a 30 cm c e l l (Pharmacia UV Control and Optical Units Model 100). 68 Section 3. Results 3.1 Growth At the beginning of the experiment, a l l cultures grew exponentially (Fig.11). Growth rate was unaffected by 0.37 nM HgCl^ while concentrations equal to or greater than 1,84 nM HgCl 2 reduced i t . , After 70 hours, control cultures {A and B) had a growth rate of 1.92 ± 0.37 d i v i s i o n s per day and the Hg-exposed cultures (C and D) had a reduced growth rate of 1.31 ± 0.27 d i v i s i o n s per day. The c e l l densities i n the pre-exposed cultures (C and D) were reduced by 25% a f t e r 24 hours, and by 55% after 46 hours of pre-exposure to 1.84 nM HgCl.,. 3.2 D i s t r i b u t i o n of Hcu Cu and Zn The 250 nm absorbance p r o f i l e for a l l cultures (Fig.12) shows the r e l a t i v e abundance of metal-bound substances. It consisted of two major absorbance peaks, one i n the high m.w. pool (fractions 2 to 7) and a major peak i n the low m.w. pool (fractions 12 to 20). In contrast to the absorbance p r o f i l e s of cytoplasmic fractions derived from animals exposed to heavy metals, there was no large absorbance peak i n the medium m.w. pool. The t y p i c a l gel elution p r o f i l e of a nutrient-saturated culture exposed to 1.84 nM HgCl 2 (culture C) i s shown in Fig. 13. This p r o f i l e was characterized by; 1) high l e v e l s of 69 200 r 10 5ioo u 80 % 6 0 E o 40 i _ o D LL 20 ~T : 1 2 3 Time ( D a y s ) 4 5 FIGURE 11. Changes i n the i n vivo fluorescence i n the following batch cultures: (O) culture A; (@) culture B; ( A ) culture C and (A) c u l -ture D. The time and concentration of mercury exposure are given i n Table VII. The arrow indicates the time (70 hours) at which the addi-tion of 5.53 nM HgCl^ was made to cultures B and D. 70 Frac t ion Number FIGURE 12. The 250 ran absorbance p r o f i l e of fractions collected from the followoing cultures: (O) culture A; (^) culture B; (A) culture C and (A) culture D. The time and concentration of mercury exposure are given i n Table VII. 7-1 D) £ D u 1.0 0.8 0.6 0.4 0.2 ~ 0 0 5 ^ 0 . 0 4 £ 0 .03 • r JR 5 10 15 2 0 F r a c t i o n N u m b e r FIGURE 13. The gel elution p r o f i l e of the 280 nm absorbance and of the amounts of Cu and Zn i n i n t r a c e l l u l a r pools of batch culture C, i n i t i a l l y exposed to 1.84 nM HgCl 2. I = high molecular weight pool; II = medium molecular weight pool, and I I I = low molecular weight pool. 72 C u a n d Z n i n t h e l o w m.w, p o o l ( I I I ) , 2) l a r g e a m o u n t s o f Z n a n d l e s s e r a m o u n t s o f C u i n t h e h i g h m. w. p o o l ( I ) , a n d 3) s m a l l e r a m o u n t s o f Cu a n d Zn i n t h o s e f r a c t i o n s w h i c h may c o r r e s p o n d t o t h e p o s i t i o n o f m e t a l l o t h i o n e i n ( I I ) a s d e t e r m i n e d i n p r e v i o u s s t u d i e s ( B o u g u e g n e a u e t a l . , 1 S 7 5 ) , G e l e l u t i o n p r o f i l e s f r o m o t h e r Hg e x p o s u r e s were s i m i l a r i n s h a p e b u t t h e l e v e l o f m e t a l s i n e a c h p e a k v a r i e d ( T a b l e V I I I ) . I n a l l c u l t u r e s , t o t a l i n t r a c e l l u l a r Z n a n d Cu l e v e l s d e c r e a s e d a n d i n c r e a s e d , r e s p e c t i v e l y , a s a f u n c t i o n o f Hg c o n c e n t r a t i o n s ( T a b l e V I I I ) . T o t a l i n t r a c e l l u l a r Zn d e c r e a s e d b y 38 t o 501 due t o Hg e x p o s u r e . A d e c r e a s e i n Z n o c c u r r e d i n t h e h i g h a n d low m.w. f r a c t i o n s a n d a g r a d u a l i n c r e a s e o f Z n o c c u r r e d i n t h e medium m.w, f r a c t i o n s . , T o t a l C u i n c r e a s e d by 20% i n c u l t u r e s B and D w h i c h w e r e p e r t u r b e d w i t h a h i g h e s t a d d i t i o n s o f H g C l 2 ( 5 . 5 3 n M ) . C o p p e r i n c r e a s e d i n t h e h i g h m.w. f r a c t i o n s , w h e r e a s Cu l e v e l s i n t h e medium m.w. f r a c t i o n s r e m a i n e d c o n s t a n t ( c a . 12.5% o f t o t a l i n t r a c e l l u l a r C u ) . T o t a l i n t r a c e l l u l a r Hg was o n l y d e t e c t a b l e i n c u l t u r e s B a n d D w h i c h w e r e p e r t u r b e d b y t h e h i g h e s t a d d i t i o n s o f H g C l ^ ( 5 . 5 3 n M ) . I n c u l t u r e B , 85% o f t h e Hg a p p e a r e d i n t h e h i g h m.w. p o o l . P r e - e x p o s u r e o f S . c o s t a t u m t o 1 .84 nM H g C l 2 ( c u l t u r e D) r e d u c e d t o t a l Hg l e v e l s by 75% when c o m p a r e d t o c u l t u r e B w h i c h h a d n o p r e - e x p o s u r e . I n c u l t u r e D , a l l t h e a c c u m u l a t e d Hg was f o u n d o v e r t h e h i g h m . w . , p o o l . A l t h o u g h Hg v a l u e s f o r c u l t u r e s fl, C a n d E were l o w e r t h a n t h e Hg TABLE VIII. Distribution of total Zn, Cu and Hg in the intracellular p o o l s of nutrient-saturated cells with (cultures C, D and E). and without (cultures A and B) previous exposure to HgCl . Time and concentra-tion of HgCl 2 exposure for the different cultures are given in Table V I I . Data are the compilation of metal levels fcrnole g-cells" 1 (wet weight)) from profiles such as in Fig. 13. ND = not detectable. Zn (p mole • q cells"') Cu (?] c 1 e • a c 5 lis"') Ha (;i:r:ol e •<-[ c e l l TOTAL HIGH MEDIUM LOW TOTAL HI GH DI'J E LOW TO? A T HIG H REDIUM LOM TREATMENT aw KM HW HW aw KM POOL POOL POOL PGCL POOL P 00 L PCOL POOL POOL CULTURE k . 0231 .0083 ND . 27 0 .0 32 .0 35 ND KB ND K 0 CCLTORE £ . C136 .0065 ND . 0071 .275 .0 44 .034 . 197 ND ND ND ND CULTURE Q .0115 . 0035 . 0009 .253 .041 . 032 .ISO ND ND ND MD CULTURE 3 .0144 .0045 .001 1 .0087 . 228 .042 . G4C . 246 .013 <-> .0117 . 0021 ND CULTURE D .0130 .GC35 .0017 . CC73 .326 .062 . 040 . 223 . 003 4 . 0034 ND ND LEAF 74 OMITTED IN PAGE NUMBERING. 75 standards, a q u a l i t a t i v e examination of the Hg analysis showed that t o t a l i n t r a c e l l u l a r Hg increased with Hg concentrations, and the highest l e v e l s of Hg were found in the high m.w. pool followed by the medium m.w. fr a c t i o n s . Section 4. Discussion Even though a concentration as low as 0.37 nM HgCl 2 f a i l e d to reduce growth rates, i t was s u f f i c i e n t to change the le v e l s and d i s t r i b u t i o n of i n t r a c e l l u l a r Cu and zn i n S, cos-taturn. The displacement of Zn by Hg on metallothionein has also been observed by Kagi and Vallee (1960). Displacement of Zn and Cu by other heavy metals appears to be a general c h a r a c t e r i s t i c of metal-induced bindinq proteins. In S. costaturn, more Cu than Zn occurred i n the medium m.w. f r a c t i o n s , which i s not t y p i c a l of metallothioneins. This indicates that t h i s protein may not play a major r o l e i n the d e t o x i f i c a t i o n of heavy metals. In ducks, when the high m.w. {enzyme-containing) pool was apparently Zn saturated, excess Cu and Zn occurred i n metallothionein (Brown and Chatel, 1978). In Cd and Hq-induced thioneins i n rat l i v e r s , Zn appeared i n approximately half the bindinq s i t e s (Hinge §i a2«* 1975), or i n residual amounts with smaller amounts of Cu i n animals (Kagi and Vallee, 1960). In S. cgstatum, the decrease of t o t a l i n t r a c e l l u l a r Zn at any Hg concentration and of Hq upon Hg-preexposure may be regulated by an exclusion mechanism. When the freshwater 76 green alga C h l o r e l l a was exposed to Zn, a temperature s e n s i t i v e component of the Zn uptake was i n h i b i t e d and the number of Zn exchange s i t e s on the c e l l wall was reduced (De F i l i p p i s and Pallaghy, 1976c). At exposures egual to or greater than 1.84 nS HgCl^, reduction of growth rates coincided with the detection of Hg i n the high m.w. pool. A si m i l a r pattern was also found i n chum salmon and a summer 4zooplankton* assemblage (Brown and Parsons, 1978) when deleterious e f f e c t s were observed. In animals, f a i l u r e to detoxify heavy metals occurs as the rate of bioaccumulation of these heavy metals exceeds the rate of metallothionein synthesis or i t s binding capacity. The excess of heavy metals then appears in the high m.w, pool with simultaneously occurring pathological effects (Bouguegneau Hi*r 1975; Yoshikawa, 1976; Brown and Parsons, 1978). Deleterious e f f e c t s can be caused by the displacement of Cu and Zn from metalloproteins by heavy metals rendering the proteins non-functional (Friedberg, 1974; Bremmer and Davies, 1975). Since elements of subgroups l i b (eg. Zn, Cd and Hg) have a high binding a f f i n i t y for sulfhydryl groups, denaturation of protein at the t e r t i a r y and guarternary s t r u c t u r a l l e v e l s could occur. The decrease of Zn in the high and low molecular weight pools, accompanied by a simultaneous increase of Zn i n the medium m.w, pool was found in S,, costatum in t h i s study. This observation has also been reported for the summer •zooplankton 4. The medium m.w. pool in S. costatum does not 77 appear to be the main storage s i t e for Cu and Zn. , In S. .costaturn approximately 70% and 60% of t o t a l Cu and Zn, respectively were found i n the low m. w. pool l i k e i n 'zooplankton* and unlike higher organisms. This pool may act as a reservoir of Zn and possibly of Cu, f o r the enzymes in the high m.w. pool. I t may play a dominant r o l e i n the metabolism and d e t o x i f i c a t i o n of trace metals when they are present in excessive amounts. The low m.w. pool contains organics such as amino acids, nucleic acids, etc., which are known as metal complexing agents. Zinc-taurine, Cu-taurine and Cu-betaine homarine complexes were separated from heavy metal exposed oysters, Ostrea edulis and Crassostrea aigos. i n which no evidence of animal-like metallothionein was found (Howard and Nickless, 1977a; 1977b). In summary, three observations indicate that metallothionein in S. costatum may not play as major a role i n the d e t o x i f i c a t i o n of Hg as in vertebrates; 1) the f a i l u r e to detect a major absorbance peak i n the medium m.w. pool, 2) the greater|amount of Cu than Zn (about three fold) i n the medium m.w. fractions and, 3) the increase of Hg i n the high m.w. pool. Despite higher l e v e l s of Hg occurring i n the high m.w. pool, i t i s possible that the amino acids of the low m.w. pool may be involved in the d e t o x i f i c a t i o n cf lower Hg levels or the a c g u i s i t i o n of tolerance. 78 SUMMARY The aim of t h i s study was to examine the growth and nutrient uptake kinetic responses of an ammonium-limited diatom during short or long-term exposures to sublethal concentrations of HgCl 2. The cosmopolitan n e r i t i c c e n t r i c diatom Skeletonema costatum, which i s a predominant species during the vernal bloom, was used i n these experiments. To achieve t h i s goal, two major steps were followed. At f i r s t , a gross range of sublethal concentrations was determined using batch cultures enriched with ,f/25* medium. Secondly, the e f f e c t s of short-term mercury exposures on ammonium-limited c e l l s grown i n chemostat cultures were determined by simultaneously adding ammonium and one of the sublethal concentrations of HgCl^. Only concentrations equal to or greater than 1.84 nM HgCl 2 decreased the assimilation rate (Vi^ax^ a n ( * t i i e a f f i n i t y for the substrate. In an attempt to increase the s e n s i t i v i t y to a secondary stress (Hg) , e f f l u e n t s from the chemostats were starved for an average period of 30 hours. These experiments indicated than ammomium starvation lowered the threshold e f f e c t of HgCl^ to 0.18 nM. The results of these f i r s t two steps provided useful information i n the desiqninq of the long-term mercury exposure experiment. In the l a t t e r experiment, two ammonium-limited chemostat cultures were exposed to 0.37 and 3.68 nM HgCl.,. 79 Mercury was semi-continuously added at regular time i n t e r v a l s during a period of 30 days. The lowest concentration used i n t h i s experiment did not decrease the maximum growth rates of nutrient-saturated cultures (cultures grown in •f/2* and •f/25*) nor did i t a f f e c t the nutrient uptake ki n e t i c s of ammonium-limited cultures i n the short-term mercury exposure. In t h i s experiment, the imposition of long-term mercury exposure on top of a primary stress (nutrient l i m i t a t i o n ) , affected the d i f f e r e n t phases of uptake and the growth of exposed populations. Decimation of the populations i n the mercury-treated cultures resulted in conditions of nutrient saturation, during which minimum c e l l densities occurred. A minor growth rate decline was observed in the control culture but conditions of nutrient l i m i t a t i o n were maintained. Growth rates simultaneously resumed i n a l l cultures implying that mercury additions did not i n t e r f e r e with the occurrence of sexual reproduction i n mercury-treated cultures. This period of growth decline was followed by a return to a new steady-state. In the long-term exposure to 0.37 nM HgCl^* a decrease in the a f f i n i t y for the substrate (increase i n Ks) occurred. The maximum (Vs) and i n t e r n a l l y controlled ( V i ^ ^ ) rates of uptake were not affected in the long-term exposures to 0.37 nM HgCl^ and i n the short-term exposures up to 1.84 nM HgC^. Long-term exposure to 3.68 nM HgCl 2 decreased; 1) the substrate a f f i n i t y (increased Ks) , 2) the i n i t i a l rapid transport of the substrate across the c e l l membrane at high (5 uM NHAC1) 80 nutrient l e v e l s , Vs, and 3) the i n t e r n a l l y controlled assimilatory rate, V i ^ ^ . In general, mercury i n h i b i t i o n on the nutrient k i n e t i c s of ammonium-limited c e l l s appeared to be uncompetitive. During the new steady-state i n Hg-treated cultures, the a f f i n i t y for the substrate (Ks), and the assimilatory rate (Vi ) of uptake were increased in phase D (day 23) compared to phase A (day 6). Recovery appeared to be p a r t i a l i n the chemostat exposed to 3.68 nM HgCl 2 . Improvement i n nutrient k i n e t i c s and changes i n c e l l morphology i n a l l cultures probably r e f l e c t e d the a c q u i s i t i o n of mercury tolerance. These changes were p a r t i a l l y related to the appearance of c e l l s of a different l i f e cycle stage. An attempt was made to determine whether a short-term physiological adaptation (mercury induced synthesis of metal-lothionein) could be responsible for the recovery through the sequestration of heavy metals by i n t r a c e l l u l a r complexing agents. The f a i l u r e to detect an u l t r a - v i o l e t absorbance peak i n the medium molecular weight pool, where metallothionein usually occurs in animals exposed to heavy metal and high levels of Hg in the high m.w. pool, suggested that t h i s protein may not play a major role i n the metabolism and d e t o x i f i c a t i o n i n short-term Hg exposure. In the long-term mercury exposure, the absorbance and the metal p r o f i l e s of c e l l s which recovered from mercury i n h i b i t i o n were not measured and therefore the role of metallothionein as a d e t o x i f i c a t i o n protein could not be ascertained. 81 Concomitantly, the e f f e c t s of sublethal concentrations of HgCl 2 (0.37 to 5.53 nM HgC^) on the i n t r a c e l l u l a r levels and d i s t r i b u t i o n of Hg, Cu and Zn was also examined using batch cultures. 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"__2" _ i D = d i l u t i o n rate (10 • hr ); Fluor = i n vivo fluorescence, using door 3; C e l l no = c e l l numbers (10 ''cells • 1 C e l l lenght (um) ; u = s p e c i f i c growth rate (hr 1 ) ; E f f l . = effluent concentration of ammonium (uM); Q = the amount of nitrogen per c e l l (/JM N-10^ c e l l s ); Chi a_ = the amount of chlorophyll a per c e l l (ug c h l a^«107 c e l l s 1 ) ; '• °665 = absorbance r a t i o of carotenold : chlorophyll <a; Chla a.* = estimated chlorophyll a_ using the chlorophyll a : fluoresence r a t i o obtained during phase A; P./S.«" photosynthetic rate (pg C-10"7 c e l l s - * . hr~*). 2 fluorescence values obtained using door 30. J II II II II II ARTIFICIAL SEAWATER RECIFE SALTS g-94.4 1-1 m.w. CONCENTRATION (M) NaF 0.25 .00265 41.99 6.30 X 10- 5 H3BO3 2.20 .23305 61.83 3.77 X 10- 3 KBr 8.20 .08686 119.01 7.30 X ID"* NaHCOj 16.40 .17373 84.01 2.07 X 10- 3 KC1 56.70 .60063 74.56 8.06 X 10- 3 335.60 3.555 142.04 2.50 X ID' 2 NaCl 2003.0 21.218 58.44 3.63 X ID" 1 SrCl 2-6H 20 2.00 .02118 266.62 7.95 X 10- 4 CaCl 2-2H 20 127.20 1.347 147.02 9.17 X 10- 3 MgCl 2-6H 20 906.90 9.607 203.31 4.73 X io" 2 Ref: Keater et a l . , 1967 COMPOSITION OF V MEDIUM SALTS STOCK SOLUTIONS (g-1-1) T.M. MIX (mg.1-1) F MIX (rag-1-1) F MEDIUM (M) NaN03 150.0 150.0 1.77 X ID" 3 Na 2S10 3 30.0 30.0 1.06 X 10- 4 KH 2P0 4 10.0 10.0 7.35 X l o ' 5 &1SO4 19.6 19.6 0.0196 7.85 X 10- 8 ZnSO^ 44.0 44.0 0.044 1.53 X 10- 7 CoCI 2-6H 20 20.9 20.9 0.029 8.78 X. lo" 8 MnCl 2-4Il 20 3.6 3.6 0.0036 1.82 X lo" 8 Na2Mo04-2H20 12.6 12.6 0.012 5.2 X 10" 8 F e r r i c 2 Sequestrene 10.0 10,000 10.0 B12 1.0 1.0 .001 F e 2 1.30 1,300 1.3 2.33 X 10- 5 EDTA2 8.72 8,720 8.72 2.34 X 10- 5 Ref: G u l l l a r d and Ryther, 1962; 2: McLachlan, 1973 9 6 Chemostat Culture II (Control) DAY D FLUOR. CELL NO. CELL LENGTH p EFFL. Q CHL.a D430:D665 CHL. a* P./S. 0 3.60 23.910.9 6.3710.42 15.9019.63 1 4.08 18.3±2.4 5.9010.43 16.2817.74 .035 1.24 4.58 2 4.09 15.5±0.1 5.8810.10 16.7415.24 .048 0.00 1.70 0 .B3 3. 18 1.02 3 .37 3 4 . 0 0 17.8±0.3 9.32+0.63 21.45+6.30 .060 0.05 1.07 0.918 2. ,14 0.55 2.77 4 4.00 27.010.7 5 . 2 0 1 0 . 5 9 19.8018.46 .016 0.16 1.89 1.095 2. ,56 1.71 2.26 5 4.10 24.3±0.4 5.6110.27 23.3819.39 .044 0.00 1.78 1.459 2, .25 1.43 6 4.36 27.5±1.4 7 .2210.45 18.40+6.94 .053 0.00 1.39 1.214 2, .47 1.26 2'.52 7 4.00 35.3±2.5 8.3910.04 19.99+8.58 .048 1.249 1.98 1.39 e 3.90 28.1±2.5 7.07+1.63 17.13+8.97 .032 0.22 1.36 1.600 2, .14 1.31 -9 4.10 24.6±2.4 5.50+0.01 .030 1.48 10 3 . 9 7 29.710.6 3.0010.69 .015 3.27 11 21.8±0.3 3.98+0.54 .052 1.81 12 3.96 21.811.5 3.1410.22 .030 2.29 13 3.96 18.511.9 1.69+0.15 .014 3.61 14 3.80 17.010.7 1.48+0.43 .034 0 .53 6.38 3 .79 15 16 14.111.7 1.26+0.03 3.69 17 3.96 20.7+0.8 2.5110.40 16.37+5.51 .069 0.74 3.69 2.72 18 3.88 28.811.3 7.6111.05 16.7117.00 .085 0.13 1.30 . 1.25 19 3.92 32.2+0.8 8.6212.01 .044 1 .23 20 3.92 32.5 9.58+0.30 .044 0.09 1.03 1.12 21 3.83 33.2+0.8 9.9910.11 12.97+4.78 .041 0.17 0.98 1. 10 22 4.05 38.911.4 14.4410.63 .055 0.06 0.69 0.89 23 38.010.5 12.36+0.87 .034 0.00 0.61 1.01 24 40.311.3 12.49+0.00 .040 1.06 25 3.70 40.011.3 6.5311.33 14.8116.67 .035 0.00 0.87 2 .02 26 39.610.5 10.0^0.86 .034 0.43 1.00 1.31 27 3.94 37.011 .8 8.9311.66 .035 1.37 28 3.90 39 .0 10.7112.05 .047 1 .20 29 37.311.0 5.1610.97 .009 2 .39 Chemostat Culture I (Exposure to 0.37 nM HgCl7) DAY D FLOOR. CELL NO. CELL LENGTH EFFL. Q CHL.a D430:D665 CHL. a* P ./S. 0 3.70 23.8±0.4 5.90±0.36 15.90+9.63 2.51 1 4.06 15.5±1.0 5.80+0.41 16.2817.74 .038 0.94 2.41 1.75 5, .17 2 4.38 14.0 2.99+0.08 18.53+8.71 .017 0.00 1.72 1.29 2.55 1.16 4, .19 3 4.20 16.3+0.7 5.26+0.30 20.315.66 .066 0.05 3.33 1.34 1.55 2.03 5. 09 4 4.10 10.110.9 3.90+0.35 25.38+10.70 .029 0.16 1.87 1.49 2.10 1.81 2. 99 5 4.40 15.8+0.5 3.0310.00 18.7+8.51 .032 3.60 1.53 1.45 6 4.09 J9.4+0.9 2.08+0.24 26.07111.96 .027 0.13 4.74 2.38 2.32 3.30 5. 39 7 4.15 14.5±1.5 2.6510.05 25.04112.60 .051 2.03 2.53 3.18 8 3.90 11.2+1.6 2.86 32.85113.84 .043 0.93 3.17 3.34 2 .04 3.20 9 4.20 8.1+1.1 1.1310.02 .001 4.30 10 4.29 5.3+0.6 0.71+0.13 .023 4.95 11 4.30 1.810.8 0.81+0.07 .049 2.84 12 4.30 3.3±1.0 0.5610.22 .028 1.40 13 4.30 1.9+0.7 0.2510.14 .062 5.73 14 4.20 18.3 1.52 0.15 .021 6.27 25.C3 5.50 15 16 42.716.82 0.84+0.02 11.37+6.77 17 4.00 10.1±2.5 1.4+0.21 13.3815.08 .062 2.26 5.53 3.13 18 4.18 28.0+0.4 3.29+0.51 18.9219.65 .077 0.15 3.00 3.69 19 4.15 29.8±0.8 4.8011.09 .057 2.69 20 4.01 32.5+1.8 8.4610.46 .064 0.10 1.17 1.67 21 4.04 32.1+3.0 5.67+0.19 .024 0.15 1.74 2.46 22 3.90 34.8+2.1 8.55+1.03 .057 0.06 1.16 1.77 23 39.1+1.6 8.8710.67 .041 0.00 1.13 1.91 24 41.2+2.2 9.4310.52 .043 1.90 25 3.60 40.1+1.1 9.2810.46 16.47+6.31 .038 0.00 0.72 1.88 26 37.711.6 8.16+0.79 .033 0.09 1.21 2.01 27 4.13 36.5±2.1 6.0810.34 .026 2.61 28 4.10 34.8±0.3 7.7910.18 .054 1.94 29 38.5±0.5 4.99+0.59 .040 3.35 Chemostat Culture'III (Exposure to 3.68 nM HgCl 9) DAY D FLUOR. CELL NO. CELL LENGTH EFFL. Q CHL. a_ D430:D665 CHL. a* P. /S. 0 3.90 23.3 5.5610.44 15.9019.63 1 3.95 5.68 16.28±7.74 .040 1.87 8, . 10 2 4.04 22.5+0.3 6.0810.6 16.8017.41 .043 0.00 1.76 0.71 3. 20 4, .51 3 4.00 15.7*0.5 5.5711.19 21.3316.60 .037 0.06 1.64 1.75 2. 31 1.65 3. .67 4 3.94 11.9+0.3 2.97+0.27 34.76+10.54 .014 0.16 1.77 1.72 2. 49 1.26 2. .02 5 3.90 15.8+10.3 2.4510.12 29.51111.22 .031 4.43 1. 85 1.79 6 4.09 12.7±0.5 1.3210.23 28.2319.70 .014 0.04 7.54 2.35 2. 26 2.88 4. 54 7 4.00 6 .610.5 1.06+0.46 35.37122.79 .031 3.96 1. 88 4.29 8 3.80 4.310.5 0.433 23.0815.60 .003 3.13 15.87 ' 4.92 1. 70 2.78 9 3.90 3.5 0.18 8.0516.12 .001 0. 63 4.43 10 4.06 2.5 8.67 11 3.90 15.510.42 0.0362 12 3.96 3.312.12 0.00252 13 3.94 17.214.72 14 3.88 7.710.32 7.17 15 16 5.7+1.52 17 3.80 2.811.52 0.0167 18 3.90 5.011.42 0.04610.011 .081 9.46 11.84 19 3.92 11.311.22 0.07610.005 .060 20 4.02 11.8+0.53 0.7510.16 .135 4.99 6. 70 21 3.81 53.310.43 3.45+0.59 9.1614.63 .103 1.42 3.49 22 3.90 39.5+1.4 9.23*0.34 .080 0.06 1.C8 1.91 23 41.511.0 6.00+0.26 .021 0.08 1.65 3.08 24 39.6+1.4 11.3910.37 .066 1.55 25 3.60 39.610.5 4.3010.83 22.019.28 .000 0.00 ' 2.33 4.11 26 33.411.0 6.9610.25 .058 0.06 1.43 2.14 27 3.81 29.510.6 9.7712.89 .024 1.35 28 3.90 30.212.5 6.8110.21 1.98 29 28.310.6 5.0710.67 2.49 

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