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Action of DDT on two clones of a marine green alga Pyramimonas

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Action of DDT on two clones of a marine green alga Pyramimonas

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Content ACTION OF DDT ON TWO CLONES OF A MARINE GREEN ALGA PYRAMIMONAS by Nicole Marie Louise Morel - - A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY {Biological Sciences) October, 1975 UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 90007 This dissertation, written by Nicole Marie Louise Morel ·-------------------------------------------- -- ------------------------------------· under the direction of h.e."J: ... Dissertation Com mittee, and approved by all its members, has been presented to nnd accepted by The Graduate School, in partial fulfillment of requirernents of the degree of DOCTOR OF PHILOSOPHY ·----------------------------------------------------------------------------------- Dean Date ................ -~ ..... 1 •••••••••••• ~ J. _ : __ . p ACKNOWLEDGEMENTS My deepest thanks and gratitude go to my husband, Francois, for his encouragements, his help and his impatience which allowed me to complete this work. I am very grateful to Dr. B.C. Abbott, my advisor, who provided me with continued financial, moral and scientific support. I thank Dr. J.J. Morgan who provided me with space in his laboratory and kind advice. Dr. J.J. McCarthy's friendly and expert help in the tedious work of evaluating the data and preparing the manuscript is gratefully acknowledged. J.G. Rueter helped me in the laboratory, S. Demeris and J. Coleman typed the various stages of the manuscript. To them and all my other friends who supported me during this long Journey I wish to express my gratitude and I invite them to drop by sometimes for dinner. Acknowledgement is made for support for this research from a Ford Motor Company Grant. Support was also supplied in the form of an Assistantship from the U.S.C. Sea Grant Program. . . ll TABLE OF CONTENTS INTRODUCTION BACKGROUND I History and General Review II Effect of DDT on Photosynthesis OBJECTIVES OF THE THESIS MATERIALS AND METHODS I Cultures (origin-techniques-media) II Biomass Indices (cell counts, chlorophyll content, packed volume) III Physiological Indices ( 14 c-bicarbonate uptake. o 2 evolution) IV Problems with Technique V Statistical Evaluation RESULTS I Resolution of Discrepancies II Comparative Study of Pyramimonas 1 and Pyramimonas 13-10 - III Detailed Study of Pyramimonas 13-10 IV Pyramimonas 1 - Promotion of a lag phase synergistic effect of copper and DDT DISCUSSION I Lag Phase and DDT Sensitivity II Biochemical Foundations for Lack of Sensitivity of DDT. Possible Role of Photosystem I and II III Speculations on the Existence of c 3 - c 4 Metabolish in Algae. Link be tween c 3 and c 4 Metabolisms and Photosystem I and II Page 1 4 8 16 17 20 23 26 30 32 35 38 42 48 56 59 ... lll Page IV Resolution of Some of the Discrepancies 62 V Relevancy of the Study of Natural Conditions A) Problem of Estimating Amount of DDT in Natural Conditions B) Problem of Loss of DDT in Laboratory Conditions C) Problem of Batch Cultures CONCLUSION REFERENCES APPENDIX Introduction Background Results Discussicn References 64 66 71 74 108 112 113 114 120 126 138 . lV LIST OF TABLES AND FIGURES Table 1 - Comparsion of the Results of Experiments Table 2 by Wurster, Menzel and Bowes 14 - C Uptake of Pyramimonas 1 after 24 Hours of Incubation with DDT Table 3 - Comparison of Physiological and Physical Parameters of Cultures of Pyramimonas 1 with or without DDT Table 4 - 14c Uptake of Pyramimonas 1 with DDT Table 5 - 14c Uptake of Pyramimonas 1 after 24 Hours of Incubation with DDT Table 6 - 14 c Uptake of Pyramimonas 13-10 (in lag phase) after 24 Hours of Incubation with DDT Table 7 - Mean Cell Volume of Pyramimonas 1 and Pyramimonas 13-10 Table 8 - Chlorophyll Content of Pyramimonas 1 and Pyramimonas 13-10 Table 9 - Cell Densities of Pyramimonas 13-10 after 20 Hours of Incubation with DDT 14 Table 10 - C Uptake of Pyramimonas 13-10 (in exponential phase) after 24 Hours of Incubation with DDT Page 76 78 79 80 81 82 83 84 85 86 V Table 11 - Cell D nsity of Pyrarnimonas 1 after 20 Hours of Incubation with Copper and DDT Table 12 - 14 c Uptake of Pyrarnirnonas 1 after 24 Hours of Incubation with Copper and DDT Table 13 - Statistical Evaluation of the Syner gistic Action of Copper and DDT 14 on the C Uptake of Pyramimonas 1 Figure 1 - Growth of Pyramimonas 1 in Natural and Synthetic Sea Water Figure 2 - Standardization Curve for Oxygen Chamber Figure 3 - Effect of DDT on the Growth of Skeletonema Costatum Figure 4 - Effect of DDT on the Growth of Pyramimonas 1 14 Figure 5 - C Uptake of Pyramirnonas 1 in Page 87 88 89 90 91 92 93 Function of the Age of the Inoculum 95 Figure 6 - Growth Curve of Pyramimonas 13-10 96 Figure 7 - Growth Curve of Stock and Subcultures of Pyramimonas 13-10 Figure 8 - Slight Stimulatory Effect of DDT on the Growth of Pyramimonas 13-10 98 100 vi Page Figure 9 - Protection of Pyramimonas 1 by Iron against Copper 102 Figure 10 - Growth Curves of Pyramimonas 1 with Copper and DDT 104 Figure 11 - Growth Curves of Pyrarnirnonas 1 with Copper and DDT 106 Figure 12 - Loss of DDT by Evaporation 107 APPENDIX Figure 1 - Effect of Inoculum age on the growth of Pyramimonas 1 132 Figure 2 - Influence of Fe, EDTA and Age on the Inoculum on the Photosynthesis of Pyramimonas 1 133 Figure 3 - Antagonistic Effects of Iron and Copper on the growth of Pyrarnirnonas 1 134 Figure 4 - Effect of Copper Concentration on the Initial growth of Pyramimonas 1 Figure 5 - Effect of Copper on the growth of Pyr. 1 Figure 6 - Another Effect of Copper on the growth of Pyramimonas 1 135 136 137 vii INTRODUCTION In October, 1972 the Federal Insecticide, Fungucide and Rodenticide Act was signed into law. As part of this law a complete ban on the use of DDT in the United States was promulgated. This ban, based on controversial scien tific evidence has somewhat discouraged additional research efforts on the environmental impact of DDT utilization. It should not be forgotten however that DDT is still widely used around the world, that it is transported worldwide as an air pollutant and that it is quite resis tant to degradation. DDT is still very much a global pollutant, will be for some time, and more knowledge of its potential environmental impact is badly needed. Although the largest part of the observations on the deleterious effects of DDT has been made on organisms in terrestrial and freshwater ecosystems, oceanic systems have eventually received some attention and scores of re ports on pesticides concentrations in various marine organ isms are now available. However, there is still minimal knowledge of the potential metabolic consequences of such concentrations. This ignorance is particularly acute in the area of DDT effect on marine phytoplankton, especially with regard to photosynthesis. The two original short studies by Wurster (1968) and 1 by Menzel and co-workers (1970) which first demonstrated a decrease in the photosynthesis of phytoplankton exposed to high DDT concentrations also demonstrated large interspe cific variations in those effects. Many authors (Goldberg et al, 1971) have since concluded that, on a global scale, the only significant consequence of DDT effect on phyto plankton should thus be a minor alteration in the normal ecological successions of species. Subsequent work by Bowes (1971, 1972) which demon strated that the chloroplasts of all algal species - either sensitive or insensitive to DDT -- are themselves sensitive to DDT and DDE, further highlighted the need for an explanation of the variations in phytoplankton sensi tivity to the pesticide. A search for this explanation is the principal focus of the thesis. A set of preliminary experiments which duplicates some of the work of Wurster, Menzel and Bowes is first presented to resolve some fundamental discrepan cies in the results of these three authors. A systematic comparison of the growth and 14 c uptake of two clones of Pyramimonas which exhibit widely different sensitivities to DDT -- thus demonstrating intra, as well as inter specific variations -- is then used as the major metho dology to investigate the physiological and environmental conditions of the differences in DDT sensitivities. These investigations lead to studies of lag phase phenomena in 2 algal cultures and ofsynergistlc toxicity between trace metals and pesticides. The results are subsequently dis cussed from the phenomenological to the mechanistic -- bio- chemical viewpoint. Speculations on the relative im- portance of photosystems I and II in various stages of growth and how this relates to the possible coexistence of c 3 and c 4 metabolisms in algae are presented. Finally obvious environmental and ecological implications of the results are drawn. 3 BACKGROUND I History and General Review DDT, p-p' dichlorodiphenyltrichloroethane or more correctly 1,1,1 trichloro-2,2 bis (p-chlorophenyl ethane), was the first major synthetic insecticide. Its chemical formula can be represented as: IH _f ~- Cl CC 1 3 ====../ Cl After the discovery of its insecticidal properties by .. the Swiss chemist Paul Muller in 1939, it was used in massive quantities during World War II and thereafter. Al though it has undoubtedly saved large populations from death by malaria, thyphus and other insect-born diseases, and saved billions of dollars in preventing crop destruc tion by insect pests, its extensive use has come under heavy criticism in the last decade. Because of recent findings relating to the decrease of its effectiveness and to its side effects on other organ isms, scientists and legislators, particularly in the United States, have tried in recent years to weigh the advantages and the drawbacks of this chemical. Although a large array of insect species have devel oped resistance to DDT (for example the common house flies) 4 insects which have a more devastating impact, such as mosquitos, carriers of malaria or equine encephalitis, are still kept in check by this insecticide. Because in most of these "life and death" situations few if any alternate solutions have been found so far, it is obvious that any secondary ''environmental" concerns are disregarded. This is the case of many south American, African and Asiatic countries where DDT is still applied extensively. In the U.S. where such diseases are quite uncommon, voices raised by environmental conservatives have been strong enough to bring about a debate and eventually an almost complete ban on all uses of DDT. The litigation culminated in the Fed eral Insecticide, Fungicide and Rodenticide Act (FIFRA) as amended by the 1972 Federal Environmental Pesticide Control Act (FEPCA) signed into law October 21, 1972 (Butler, 1974). This law gives the EPA Administrator the power to decide on where and when there may be exemptions to the law. Because of a series of loopholes in the wording of the law, EPA issued, in March 1975, a new series of rules {40 CFR 164) {40 CFR 12261) to deal with requests to use a suspended or cancelled pesticide. These rules allow only very few exceptions. Although the reasons to go back to the use of DDT are few, there are instances where pest emergencies which could present risk to human health or to fish and wildlife require exemptions. Although EPA has been granting a few exemptions the 5 major reason to undertake an investigation on the poten tial environmental hazards of DDT is that it is a global pollutant. As long as DDT is used in large quantities in any part of the world, it will be found as a contaminant in all fresh and salt waters of the world. The reasons for such a widespread contamination are many. A National Academy of Sciences Panel headed by E.D. Goldberg gave a good summary of the dispersal of DDT (1971). DDT residues are carried to the oceans through a large array of transportation routes. Surface runoffs and 8 rivers seem to carry only 0.1% (10 g) of the annual pro- duction of DDT. DDT that has entered the atmosphere (by vaporization from water surface or vaporization from plants and soils) travels great distances and enters the sea by precipita tion or in dry fallout. This would account for one quarter of the estimated total annual production of DDT. A study of trade winds by Risebrought and co-workers (1968) showed DDT concentrations as high as 164 ppb (by weight in air borne particulate matter}. The oceans are the eventual sink for DDT. Although DDT is rather insoluble in water (1.2 ppb) enrichment occurs in the surface film of the sea and on all particles that sink throughout the water column. Sediments are also a reservoir: Burnett (1971) showed that in the extreme case of the sediments adjacent to the L.A. Country Sewer 6 Outfall, where the concentration reaches 100 ppm (SCCWRP 1973), sand crabs have been found that contain 45 times as much DDT as crabs living near major agricultural drainage. In spite of the ban on DDT and the decrease of its use during the '60s , Cox (1970) found a 3-fold . . increase in DDT concentrations in phytoplankton samples from Monterey Bay in California taken from 1955 to 1969. The concen trations increased from 200 ppb to 600 ppb (per unit weight of phytoplankton). DDT has two particular characteristics which render its potential impact on the biota more harmful: its chemical stability and its accumulation throughout the food chain. Its half-life is considered to vary between 4 and 15 years depending on the estimates. It degrades or is metabolized mostly into DDD Cl or DDE Cl C II CC1 2 DDD has been found in many samples of water, soil, plants or animal tissues from areas which were sprayed with DDT. DDD also has insecticidal properties (Finley and Fillmore, 1963). DDE is the main metabolite of DDT in insects. It has no insecticidal properties but it depresses electron transport in isolated algal chloroplasts. 7 1 1 DDT has been shown to be detrimental to insects, fish, birds and small mammals (rats) by affecting their nervous (Fumio Matsumara et al, 1966)or their reproductive systems (Sprague and Duffy, 1971). Little good evidence shows direct toxicity (acute or chronic) to humans through in halation, direct contact or food. The main concern for mankind is in the effect of DDT through its environmental impact. This study focuses on the effect of DDT on phyto plankters. Since phtyoplankton are the first link in a food chain leading to man, since most herbivorous zoo plankters have a very specific diet of algal species, and this specificity extends further up into the chain, some species of fish or birds or mammals, valuable to man as food, may be eradicated as a result of the use of DDT. A very fundamental knowledge of the action of DDT starting at the primary level of the food chain is vital. Not only is DDT going to be around for a long time, but it is not impossible that the acute toxicity of some new pesticides as well as the recurrence of various pests may demand a return of DDT use at least in local applications. The present occurrence (July, 1975) of encephalitis in Missouri is an example of such a situation. II DDT Effect on Phytoplankton Photosynthesis In 1968 Wurster reported that after 24 hours of in cubation as little as 10 ppb DDT reduced co 2 uptake of some 8 phytoplankton species by a large factor (as much as 30% for Skeletonema costatum, a dominant diatom in many coastal waters). Menzel in 1970 confirmed these results with organisms isolated from different oceanic environments. However, he also showed that some species were very in sensitive to the insecticide (for example: Dunaliella tertiolecta, a naked green flagellate typical of tidal pools and estuaries). Except for a few growth curves, most of the experiments described by Menzel were concerned with the effect of DDT on the uptake of radioactive bi carbonate ( 14 c) after 20 hours of incubation. According to Wurster a concentration of 22 ppb, found in some Cali fornia waters (Wurster, 1968), would decrease co 2 uptake by a significant amount for most species at population densities approximating those found in nature. Bowes' re sults (1971, 1972) are not totally in accord with those of Wurster and Menzel. A close examination of the data reveals some fundamental discrepancies. Wurster and Menzel used planktonic cultures from WHOI (Woods Hole Oceanographic Institution) and Bowes from the Food Chain Research Group (Scripps Institution of Oceanography). A summary, by clone, follows. Short-term experiments were designed to quantitate the effect of DDT on the up take of 14 c-bicarbonate over a 24 hour period. Long-term experiments examined the effect of DDT on cellular 9 doubling; DDT was added to the culture daily by Menzel (100 ppb), only once by Bowes (80 ppb). 1. Skeletonema costatum (Bacillariophyceae) a) Short term experiments: The reduction of 14 c uptake by 100 ppb was found to be 75% by Wurster and only 25% by Menzel. b) Long-term experiments: Menzel found that DDT produced a lower growth rate and what seemed to be a complete block on cell division once the population reached ~ 4 x 10 4 cells/ml, after about 2 to 3 cell divisions. Bowes found that starting from about 10 4 cells/ml the culture showed a 9 day lag phase, after which cell divi sion occurred at a rate similar to that of control cul tures. The final cell concentration reached that obtained with the control population. Cells growing in DDT were taken in their exponential phase and inoculated into fresh medium containing also 80 ppb DDT. The same phenomenon with a lag phase was observed demonstrating that growth after the lag phase was not due to a few DDT resistant cells propagating a resistant population. 2. Thalassiosira pseudonana (Cyclotella nana - Bacillari ophyceae) a) Short term experiment: Only Menzel performed this experiment; he found an 85% reduction of 14 co 2 uptake. 10 b) Long term experiment: Menzel again found a slower growth rate and a lower plateau with DDT (although he might not have waited long enough to be sure the culture was at the plateau level). Bowes on the contrary found as the only difference in growth, a short lag phase of less than one day when DDT was present. When growth commenced cell division rates and final population size were identical to control cul tures. 3. Coccolithus huxleyi (Chrysophyceae) a) Short term experiment: Wurster found that 100 ppb DDT reduced co 2 uptake by 65%. Menzel found a 20 % decrease. b) Long term experiment: In spite of the drastic reduction of co 2 uptake ob served in short term experiments, Menzel found no differ ence between control and DDT. Bowes also found no differ ence, but he was able to induce a short lag phase (2 days) by adding as much as 250 ppb DDT. 4. Peridinium trochoideum (Dinophyceae) a) Short term experiment: Wurster found a 65% decrease of 14 co uptake. 2 b) Long term experiment: No long term experiment was reported. Pyramimonas sp. 13-10 (Chlorophyceae) Short term experiment: 11 14 Wurster found an 80% decrease of co 2 uptake. b) Long term experiment: No long term experiment was reported. 6. Dunaliella tertiolecta (Chlorophyceae) a) Short term experiment: No effect at all reported by Menzel. b) Long term experiment: No effect at all reported by Menzel and Bowes. For the same algal species the amplitude of the ob served effect of DDT on 14 c-bicarbonate uptake appears to vary widely with the authors. The following reasons may be put forward to explain some of the variations found in short-terms experiments. Wurster clearly showed that the effect of DDT depended on the amount of insecticide per cell. Although both Wurster and Menzel mentioned that they worked with cell densities that were close to "natural" densities, only Wurster gives precise numbers. Menzel stated that he worked between 100 and 500 µg of carbon per liter and that "within these limits no effect of cell concentration on toxicity was noted." If we assume that the data from these two authors can be directly compared, we have to conclude that in Wurster's experiments the cells were more sensitive to DDT than in Menzel's experiments. There is mounting evidence that there exist among species "physio logical races" which are a function of the nature of the 12 isolation sites. This factor cannot be important here since the same clones were used by Menzel and Wurster. Other possible explanations for the discrepancies could be minor differences in methodologies or in experimental con ditions that were not reported. The discrepancies found in the long-term experiments are more difficult to account for as the data diverge not only quantitatively but also qualitatively. Menzel always found lower plateau levels with DDT, while in Bowes' ex periments cultures incubated with DDT reached the same plateau as the controls. One could argue that if DDT slightly reduced the growth rate (as in Menzel's experi ments) the carrying capacity of the flasks should remain the same with or without DDT. The drastic phenomenon of cell division blockage (after 3 cell divisions) that Menzel encountered with Skeletonema costatum was not found by Bowes and was never observed in any other species. One can perhaps assume that other variables may have altered the Skeletonema costatum results. One important observa tion is that DDT produced a longer lag phase (or induced one where otherwise there was none) in Bowes' experiments. This was not mentioned by Menzel. Overlooking a lag phase might result in an underestimation of the exponential growth rate in Menzel's experiments. Accurate cell counts with a haemacytometer are very tedious when working with less than or around 10 4 cells/ml and this may be the 13 source of the discrepancy. An altogether different hypo thesis would suggest that although Bowes' worked with the same species as Wurster and Menzel, differences in results may have came from variable sensitivities of different clones. Another reason may be that Menzel added pesti cide daily while Bowes spiked the cultures only once. Table 1 summarizes their results for a given DDT concentration (100 ppb for example), classifying them by algal species (that were studied by more than one author) and by the length of the experiment: bicarbonate uptake after 24 hours of DDT incubation and growth curves. Bowes' biochemical studies showed that DDT affects photosynthesis very much like DCMU (3-(3,4-dichlorophenyl) -1,1 dimethylurea) which has been widely studied and used to block electron transport in chloroplasts. He demon strated that, like DCMU, DDT affected the electron trans port of photosystem II in chloroplasts of sensitive and in sensitive algae. Although some species seemed able to slightly degrade DDT into DDE, DDE had exactly the same effect on the chloroplasts and thus this degradation is not a detoxification. Since all algal cells (sensitiv0s or insensitives to DDT) absorb this pesticide (Bowes), and since chloroplasts from any algal cell are sensitive to DDT (Bowes), it is important to establish why and how photosystem II of some algal species could be protected from the action of DDT. 14 Wurster, Menzel and Bowes have emphasized the dele terious effect of DDT on the co 2 uptake of some algae and the resulting extension of the lag phase. The ecological implication is a possible alteration of ecological suc cessions since the density of an algal species in the ocean fluctuates throughout the year with reasonably re current seasonal blooms. The dominance of a given phyto plankter depends on some combinations of factors which provides a competitive advantage relative to other species at that specific time. Such an advantage could be al tered by the presence of DDT. What these authors have failed to study is the possi bility that sensitivity to DDT varies with physiological and environmental conditions of the cells. Discrepancies and variations between the findings of the three authors invite one to speculate that there may be experimental designs which lead to different assessments of sensitivity. It is not inconceivable that under certain growth condi tions (i.e., within a given set of chemical factors and at a specific time in the growth cycle) an algal species may appear quite insensitive while under different circum stances it would exhibit sensitivity. 15 OBJECTIVES The fundamental discrepancies observed between the results of Menzel, Wurster and Bowes demand some resolu tion. The first objective of this study is to obtain such a resolution by replicating some of their experi ments. It should then be possible to determine a set of experimental conditions (environmental or physiological) which may lead to differences in algal sensitivity to DDT and to choose the organisms best suited to study these conditions. The second and principal objective is to es tablish the morphological, physiological or environmental factors that influence DDT sensitivity in marine phyto plankton. Going beyond these observations it will be useful to postulate on the possible biochemical endowment of the cells which underlies their responses. Accordingly the third objective is to gain a -- perhaps speculative understanding of the biochemistry of DDT sensitivity in algae. Finally as a fourth objective a reassessment of the potential environmental impact of DDT should become possible. 16 MATERIALS AND METHODS I Cultures Techniques - The following cultures were used - Skeletonama costatum was obtained from the Food Chain Research Group - Pyramimonas 1 was first obtained from the Food Chain Research Group, later from Dr. Guillard's collection at WHOI. Both were isolated from San Francisco Bay. - Pyramimonas 13-10 from Dr. Guillard's collection at 0 WHOI was isolated from the Sargasso Sea 33 ll'N, 65°15' w - Dunaliella salina was obtained from Indiana Univer- sity - Cells were grown in 125 ml erlenmeyer flasks con taining 50 ml of medium or in 250 ml flasks con taining 100 ml of medium. Stocks were maintained at 17°c in a culture room. They were kept under 4 2 fluorescent light of about 2.5 x 10 ergs/sec.cm. It seems that for chlorophyta species 2 x 10- 2 ly/min or 2.094 x 10 4 ergs/sec.cm 2 is an optimal light intensity above which the growth rate de creases almost linearly with increasing light in tensity (Strickland, 1958). To assure an even 17 lighting during the experiments a rotating table was built that could support 12 small or 8 large flasks. It rotated at a speed of 0.5 RPM under a bank of fluorescent lights kept on a 14 hour light and 10 hours of dark cycle. - Early experiments were done with F/2 medium (Guillard and Ryther, 1962). This recipe includes a set of supplements (NO 3 - PO 4 - Trace metals - Si Vitamins) which are added to filtered sea water. The problems of discrepancies between authors and the difficulty to replicate experiments made it a necessity to use an entirely artificial medium. Other workers in our laboratory having some exper ience with a synthetic sea water, it was used in all but the first series of experiments. This } artificial sea water is described in FWPCA Methods for Chemical analysis of Water and Wastes (1969). Its composition . the following: is Salt Concentration . g/1 in NaCl 24.53 CaC1 2 1.16 KB 2 0.10 NaF 0.003 MgC1 2 5.20 KCl 0.70 H 3 Bo 3 0.03 18 Salt Concentration in g/1 Na 2 so 4 4.09 g/1 NaHCO 3 0.20 g/1 SrC1 2 0.03 g/1 The supplements (NO 3 - Po 4 - Trace metals and vi tamins) of F/2 medium recipe were added to this sub stitute ocean water. It was established that the algae have the same growth rate and the same plateau with both media (Figure 1). The medium was sterilized by auto - claving or by filtration with .45µ Millipore fil ters. This latter method avoids precipitates that could plug the aperture of the Coulter Counter and give erroneous counting. - Sterile techniques were used throughout, but steril ity was not checked on a routine basis. Assays to identify bacterial contamination were performed particularly when replicate experiments were giving very different results. The medium used was a sol ution of 3g of yeast extract and 10g of peptone per liter of sea water. - In some instances the medium was washed from trace metal impurities by filtering it through an ion ex change resin (chelex 100) following Davey's metho dology (Davey, et al, 1973). 19 - In long term experiments with copper the loss of trace metal by adsorption on glassware demanded coating erlenmeyer flasks with a silicon film (SC.- 87 dry film - General Electric) as described by Davey et al (1970). - DDT (from Aldrich Chemical Company, Inc. 99 + % pure p-p' DDT) was added dissolved in acetone in quantity such that adding 5µ£ of this acetone solu tion into 50 ml of sea water would give the desired concentrations of DDT: 1, 10, 100, 200 or 1000 ppb. Pure acetone was added to the controls. Acetone or DDT in acetone were added at first with capillary tubes (Drummond Microcaps), then later with Eppen dorf pipettes which give a better delivery. In growth curves assays DDT was added daily or other wise: black arrows on the graphs label additions of DDT. II Indices of Biomass - Cell counts - Microscopic counting: cells were immobilized with an .. iodine solution (Utermohl's solution - Guillard, 1974). Addition of preservative of less than 1% in volume makes correction for volume increase unnecessary. I used at first a Max-Levy haemacytometer (Fuchs Rosenthal ultra plane - Hausser Scientific) which was 0.2 mm deep, had 2 chambers divided into 4 arrays of 4 squares. Each square 20 2 has an area of 1 mm and is subdivided into 16 squares. Total volume in both chambers is thus 0.0064 ml. At the 95% level of confidence the accuracy of counting is ± 10% if 400 objects are counted. 4 A cell density of 10 cells/ml would yield 64 cells per slide or 32 cells per chamber. To have a 10% accuracy on such a count one would have to clean, fill and count the slide at least 6 times. The ac quisition of a Coulter Counter made such a task much easier. - Electronic Particle Counting This counting system is based on electrical conduc tivity. Each particle displaces electrolyte in the aper ture producing a pulse essentially proportional to its volume. The size of the aperture was 100µ. Up to 10,000 counts no correction for coincidence (2 cells being counted as one) was needed. Under 10,000 counts the stan dard deviation was always less than 3% of the mean cell count. Calibration of the counter was done with Paper Miil- 3 berry grains of a volume of 11.4µ . It gave the following correspondence between size of the particles and the am plification (A) and intensity of the aperture current (I) of the counter. 21 I A VOLUME PER DIVISION OF DIAL 3 µ t t 4 4 4 2 22.4 2 2 11.2 2 1 5.6 1 1 + + To count Pyr 13-10, we used the settings A=l, I=2. For Pyr 1 we used A=2, I=4. - Packed Volume The packed volume of the cells was measured with clin ical centrifuge Hopkins tubes with stems graduated up to 1/100 ml to 5/100 ml and body at 1, 5 and 10 ml. 10 ml of cell suspension was centrifuged for 5 minutes at 5000 RPM. - Chlorophyll Chlorophyll was extracted in acetone and measured spectrophotometrically following the procedure described by Strickland and Parson (1972). The samples were grinded in a glass tissue grinder after filtration on a .45µ Millipore filter. Extraction of the pigments in 90% acetone was performed for a few hours in the dark. Ex tinction of the solution against a cell containing 90% acetone was measured at 750, 665, 645 and 630 nm. 22 Parson-Strickland equations were used to compute the con centrations of chlorophyll a and chlorophyll b. III Indices of Physiological Activity 14 c-bicarbonate uptake A known volume of cell suspension - usually 50ml - was incubated with 2 µc of 14 c bicarbonate solution for 4 hours. The cells were filtered on .8µ Millipore filters, glued onto aluminum planchettes and dried overnight on silica gel. The emission of radioactivity was recorded with a Nuclear Chicago glassflow counter equipped with a 8703 series Decade Scaler, 8431 printer and 1043 Low Back ground Planchet Sample changer. The counting efficiency of the system was computed to be 13%. Background was usually between 6 and 15 CPM. Strickland and Parson recommend recording 5000 counts. This was easily achieved with Pyramimonas 1 by recording 3 times for 2 minutes. It was more difficult to do with Pyramimonas 13-10, therefore two 20 minute counts were performed. Although in most instan ces total counts were in the right range, in the worst cases I could only record about 1500 counts. Coefficient of variation was between 3% and 8 ~ 0 • Correction for thick- ness of the algal cell material on the filter was not re quired since the densities were always less than 0.1 mg/ 2 cm. 23 o 2 Evolution As a corollary to 14 c bicarbonate uptake, oxygen pro duction was studied in early experiments. This was inten ded to check the possibility of DDT affecting only co 2 metabolism leaving oxygen production untouched. A silver-platinum electrode combination (Yellow Springs Ohio VSI 4004 Clark-oxygen probe) was used to measure oxygen production by the algae. A complete des cription of the system designed by Dr. P. Shugarman can be found in "Experimental Physiology" (Dunn and Arditti 1968) . The usual procedure involves first a conditioning of the algae to the experimental temperature and light in- tensity. In the initial phase nitrogen is bubbled to strip any carbon dioxide and oxygen. When the chamber is closed 10µ1 of bicarbonate (44 mg/ml) are injected with a microsyringe through a special opening. A 300 watt General Electric lamp (Cool Beam Par 56/2 mFL) illuminates the chamber from above. The current generated by the oxygen production of the algae is recorded on a strip chart recorder (Beckman). The recording is calibrated be tween 0% oxygen in the water (by adding sodiumdithionate) and 100% by shaking a bottle of distilled water before pouring it into the chamber. Those values could be ex pressed in volume of o 2 as the saturation volume of o 2 in 24 sea water at a certain temperature is known. Since only data relative to that of a control are being sought the results have not been translated in volume of oxygen but kept as percent of saturation. To determine gross oxygen production one needs to add the oxygen uptake to the net photosynthesis. The oxygen uptake by the algae and any other organisms present in the assay chamber is measured after turning off the light and covering the chamber with a foil-wrapped box. A typical reading is as follows. 100 A+B = Gross Photosynthesis % saturation A - Photosynthesis - Respiration B - Respiration 0 time (minutes) When cell concentrations were too low to produce a detect able amount of oxygen, the culture was concentrated by centrifugation (6mn at about 15000 RPM) and then resus pended at higher cell density. It was observed that each algal species can withstand a certain amount of centri fugation. Above a certain combination of speed and time of centrifugation the cells lose their ability to photo synthesize. For example at 15000 RPM above 15 minutes of centri fugation Pyramimonas 1 would not evolve oxygen anymore. To know the cell density that may be used without having shadowing of cells on each other, a standardization curve 25 was established with increasing cell density (Figure 2). 6 It shows that up to at least 1.5 x 10 cells/ml the net photosynthesis was proportional to cell density. There was no measurable shadowing effect. IV Problems in Techniques 1. Limit in the number of flasks that may be run at one time It was found that light varies enough throughout the length and the width of the lighting fixtures (4 fluor escent tubes) to make imperative the use of rotating tables as described in Materials and Methods. Lights also varied enough from one rotating table to another to impose that every experiment be done on a same table. This limited the total number of flasks to 12 when using 150 ml erlenmeyersand 8 when using 250 ml flasks. 2. Variability in Controls Since the number of flasks is very limited one has to run the same experiments at a few days intervals; this leads to two sorts of problems: a) the cells in the stock culture used for the in oculum are not exactly in the same physiological state - plus or minus 20 hours in the development of Pyramimonas 1 may create a difference of 1000 CPM in 14 c uptake for the same control: if one makes a study of variance within controls it can be very large. This variation can be 26 minimized when working at a more or less "long lasting" constant growth stage such as full exponential. In the case of Pyramimonas 1 particularly we were working at the edge of the lag phase (usually almost or entirely inex istent) and the onset of exponential growth. This is pro bably one of the worst points in the growth curve as over a day or two the co 2 uptake ability of the cells may in crease by at least one order of magnitude. Because of this problem the effect of any variable on the 14 co 2 up take should be tabulated relative to the control of that day. b) When introducing an inoculum of 2 x 10 3 cells/ml or less there may be as much as 10% variation between some flasks - knowing the cell density is extremely important for initiation of the exponential phase, a 10% difference in cell density will create more than a 10% difference in uptake of co 2 after a few hours of growth. It is well known (Hase, 1962; Fogg, 1965 etc.) that cellular meta bolism varies with the stage of the life cycle of a cell. The proportions of proteins, lipids, carbohydrates and nucleic acids is different in dividing cells and in the various stages of nondividing cells. In batch cultures it has been observed that the amount of chlorophyll, the size of the cells, the absorption of 14 c and many other para meters vary with the growth stage of the culture. On the first day of a long lag phase a non-dividing cell must 27 had a very different metabolism ("physiological" state) from a cell which is ripe and ready to start the first division of its exponential growth. The "adaptation" of the cells to a new environment is usually more rapid if there are more cells together (see Appendix). Therefore, even a 10% difference in inoculum size may lead to a larger difference in photosynthetic capacity of the whole culture after a few hours. It is important to stress that point particularly to explain for example the large difference in contrul uptake Of 14 co 2 · th 1 · in e ast experiment ceived an inoculum of 2 x 10 3 + of Table 13. Having re- 10% cells/ml a control has 2.1 x 10 3 cells/ml after 20 hours of incubation with copper; its 14 co 2 uptake is 1614 CPM. Another control with the same theoretical inoculum has already 2.4 cells/ml and a uptake of 4521 CPM after 20 hours. Expressed per 2 cells/ml, this uptake is 1537 CPM for the first one and 3768 CPM for the other. As mentioned in the preceding paragraph, such a difference may be accounted for by cells that are physiologically about 30 hours "older" than the others. A slightly larger inoculum - by as little as 10% - would mean slightly more active cells after 20 hours and in physiological terms, slightly "older" cells. 3. Sterility In short term experiments one has to manipulate the 28 flasks 4 times: - inoculating the cells - adding acetone or DDT and copper in some instances - taking a sample after 20 hours of incubation to count the cell density dd . 14 b' b - a ing C icar onate Each manipulation brings a chance of contamination. A few sterility tests were run to check for this possibility particularly when a cell density was obviously very differ ent from others. This was the case of the second experi ment in experiment 14 (Table 13) where a control had an uptake of only 486 CPM. Long term experiments are even more problematic. Growth curves may last for 2 to 3 weeks with daily sampling and DDT or acetone additions. At the plateau level some flasks clearly showed contamination by a sharp fall in cell concentration and general turbidity of the flasks. Plateau levels after 12 to 14 days may be altered by con tamination. 4. Problems of Keeping Cultures Alive when Using Artificial Medium which has Impurities (trace metals) in its Salts This problem is explained in more detail in the section on copper and DDT. Because some cultures were lost when starting to use a new batch of artificial sea 29 water, it became a routing test to check for growth with every new batch of artificial sea water before using it for experiments. By trial and error it was discovered that it takes between 48 to 96 hours to dissolve all the salts in artificial sea water unless they were added in small amounts with vigorous shaking between additions. The lethal effect of trace metal impurities in the salts, particularly on Pyramimonas 13-10, was overcome by running the medium through a chelating exchange resin Chelex 100 (Davey, et al, 1973) in the last growth curve experiments. V Problems with Statistical Analysis I encountered a series of problems which render classical parametric statistics unusable as one cannot make all the assumptions underlying those tests. 1) I am dealing with small samples 2) Samples may be unequal due to limitations in number of flasks that may be used simultaneously or to loss because of bacterial contamination 3) There is a large variation between controls from one day to another and even in some instances within the controls of a same day because the physiological state of the cells is very unstable and difficult to control in that area of the growth curve (edge of lag phase and exponential growth) 30 Therefore one needs to use non parametric statistical methods. For small and unequal samples the Wilcoxon test (or sometimes called the Mann Whitney U test) is relevant (Tate and Cleland, 1956). This test uses only the rank of each data point, as the two independent random samples are combined into a single ordered series, and the number of data points in each sample. The null hypothesis being tested in that there is no difference between the sampled population. If the observed T value is equal or less than a tabulated value, the hypothesis that there is no differ ence is discredited at the level a. 31 RESULTS I. Resolution of Discrepancies in Previous Experiments Replication of Menzel's and Bowes' experiments were first attempted with Skeletonema costatum since it ex hibited one of the largest discrepancies. As seen in Figure 3 cultures of Skeletonema costatum containing 10 and 100 ppb of DDT did not stop at the 3rd or 4th gener ation as reported by Menzel. They grew at the same rate 6 and reached the same plateau (2 x 10 cells/ml) as the controls. As in Bowes' results the only difference was a slightly longer lag phase of an extra 0.5 day with 10 ppb and 1.5 day with 100 ppb. Dunaliella salina like Dunaliella tertiolecta used by Menzel and Bowes exhibited a normal growth with con centrations of DDT as high as 5000 ppb. Contradictory results appeared when working with the green alga of the genus Pyramimonas. A species of that genus (clone 13-10 from WHOI) used by Wurster was shown to have a drastic reduction in net co 2 uptake with concentrations of DDT as low as 10 ppb. The species that I orginally obtained from the Food Chain Research Group was later on identified as Pyramimonas clone 1 from WHOI. Two short term experiments designed to replicate Wurster's methodology gave unexpected results (Table 2). 32 With DDT concentrations as high as 1000 ppb the net co 2 uptake was not reduced at all. Long term experiments gave contradictory results (Figure 4). In experiment I (Figure 4) even with the highest DDT concentration (1000 ppb), all cell counts re mained within the ± 15% error range that is expected with a haemacytometer. In the other experiment (experiment II, Figure 4) as little as 10 ppb of DDT resulted in 88% less cellular growth. The small difference in the inoculum size (less than a factor of two) seems an unlikely explanation for such an effect. In the case where growth was suppressed (experiment II, Figure 4) the effect was observed through out the entire exponential phase, but was very much re duced when the plateau was reached. Cell densities in DDT flasks were within 10 to 25% of the control on the plateau, but the packed volume of the cells differed only by 5% at the maximum (Table 3). The photosynthetic capacity of DDT and control algae as estimated with both the oxygen chamber and the 14 c-bicarbonate method differed by only 2% or 3%. Although there were fewer cells in flasks con taining DDT, they probably had a larger size. The compar ison of control and DDT flasks (Table 3) at the end of their growth curves shows that per cubic millimeter of culture both DDT and control cultures had the same ability 33 to fix co 2 and evolve o 2 after two weeks of incubation with DDT. All the experiments so far (CO 2 uptakes and growth curves) except one (growth curve II, Figure 4) imply that Pyramimonas 1 is insensitive to DDT. This contradictory result requires further investigation. Since Pyramimonas 1 appears mostly insensitive to DDT and Pyramimonas 13-10 has been shown to be quite sensitive to small amounts of DDT (Wurster), it can be concluded that one encounters variations in sensitivity not only between genera but also within a genus. A systematic com parison of the two species Pyramimonas 1 and Pyramimonas 13-10 is in order. As mentioned in Materials and Methods, the need for a very rigorous control of environmental variables made necessary the use of an artificial medium. This medium allowed the same growth rate, plateau level (Figure 1) and co 2 uptake (Figure 5) as the natural sea water medium. These properties will be further considered when discussing the induction of a lag phase with Pyramimonas 1. It should be noted that the oxygen production and the 14 co 2 uptake techniques demonstrated the same results (Table 3). As Bowes later showed photosystem II and not photosystem I is affected by DDT, therefore one can expect that both o 2 evolution and co 2 fixation will be simultaneously altered or unaltered. The study of either 34 co 2 or o 2 should therefore be enough to evaluate the sen sitivity of algae to DDT. II Comparative Study of Pyramimonas 1 and Pyramimonas 13-10 Table 4 resumes short term experiments conducted as follows: 2 x 10 3 cells/ml of Pyramimonas 1 were inoculated into 50 ml of fresh medium containing 200 ppb DDT. After 20 hours of growth 1 ml of 14 c bicarbonate (2µc) was intro duced into the medium. 4 hours after 14 c addition (24 hours after inoculation) the cells were filtered, dried and their radioactivity counted. In both natural sea water (12 pairs of flasks) and artificial medium (2 pairs of flasks) the net co 2 uptake in the flask containing DDT was higher than in control. The same stimulation by DDT was observed with an inoculum of 6.5 x 10 2 cells/ml in artificial medium (5 pairs of flasks - Table 5). This stimulation is statistically significant (P < 0.01 - Wilcoxon T-test). In contrast 7 experiments performed in the same manner on Pyramimonas 13-10 showed a 60% decrease in net co 2 uptake in the presence of 200 ppb DDT. This is demonstrated in Table 6. The difference is statistically significant (P < 0.5 - Wilcoxon T test). On the basis of these experiments one can conclude that co 2 uptake of Pyramimonas 1 is not inhibited by DDT while that of Pyramimonas 13-10 is greatly reduced by the same amount of DDT. 35 These results fully agree with Wurster's data on Pyramimonas 13-10. Examination of the results reveals that the average co 2 uptake for Pyramimonas 1 (3800CPM) is extremely high compared to Pyramimonas 13-10 ( ~ 100 CPM). It became important to see if indices of cellular biomass could account for such a 40 fold difference and perhaps could explain variations in DDT sensitivity. Cell size was the first parameter to be compared. The Coulter Counter data showed that Pyramimonas 1 mean cellular volume varied from 615 µ 3 to 450 µ 3 , while that of 3 3 Pyramimonas 13-10 varied from 120 µ to 196 µ depending upon the growth stage (Table 7). In the preceding set of experiments Pyramimonas 1 volume was 4 times larger than that of Pyramimonas 13-10 but this cannot explain the observed 40-fold observed difference in co 2 uptake. The chlorophyll content of the cells was then ana lyzed. In average Pyramimonas 1 contained 12 times as much chlorophyll a as did Pyramimonas 13-10 (Table 8). It appears that for most algal species, net assimila- 14 tion of C follows closely the chlorophyll a content of the cells (Eppley and Sloan, 1966). Therefore chlorophyll a content can explain a difference by a factor of 12 in co 2 uptake but not of 40. It is also interesting to note that the ratio of 36 chlorophyll a to b varies from 1.9 to 2.2 in Pyramimonas 1 and from 1.2 to 1.7 in Pyramimonas 13-10. Since the above factors can not fully account for the difference in 14 c bicarbonate uptake that was observed, the cells must have been in distinctly different metabolic states. By electronic particle counts it was established that Pyramimonas 13-10 cultures were in a lag phase at the time of 14 c-bicarbonate addition (Table 9 - left double column). Other growth curves established that Pyramimonas 13-10 almost always exhibits a 2 to 3 day lag phase (Figures 6 and 7). The precision of the haemacytometer counts performed for the Pyramimonas 1 study (before the acquisition of a Coulter Counter) did not permit direct determination of the growth stage at the time of the 14 c addition. The presence or absence of a lag phase could thus be identified by extrapolation of the exponential phase. A control culture of Pyramimonas 1 was allowed to grow beyond the 24th hour, when the other cultures were filtered and dried for 14 co 2 uptake measurement. Daily cell counts of some of these control flasks are shown in Figure 1 and clearly indicate that Pyramimonas 1 cultures 11 · 1 t· 1 h d · the 14 co 2 were a in an ear y exponen 1a p ase uring uptake experiments. The lag phases exhibited by a few of the cultures (up to a day) are probably only apparent lag phases due to the presence of a non-viable cells which are 37 to be expected with the few week old inocula used for these experiments. It should be pointed out that the re sults of experiment II (Figure 4), which are contradictory to all the other results obtained with Pyramimonas 1, re veal a lag phase when one extrapolates the exponential phase. From these experiments one can hypothesize that sensitivity to DDT varies with the different growth stages of the algae. Two lines of studies were therefore planned: one to find if the sensitivity of a "sensitive" species (Pyramimonas 13-10) varied with the successive growth stages of a laboratory batch culture, the other to es tablish if an "insensitive" species (Pyramimonas 1) which had only been studied in exponential growth, could be sensitized by promoting a lag phase. III Detailed Study of the Sensitivity of Pyramimonas 13-10 The hypothesis to be tested in this series of experi ments is that the sensitivity of Pyramimonas 13-10 to DDT depends on its physiological state, specifically that the sensitivity will decrease as the cells progress in their exponential growth. To test this hypothesis one could not simply add DDT at different stages of the growth of a culture. To do so, as the cell population increases, would require the addition of more and more DDT, so as to 38 keep constant the amount of DDT available per cell. Wurster clearly showed that the amount of pesticide per cell is crucial to establish sensitivity. One way to overcome this difficulty is to promote various stages of growth in cultures started with inocula of the same size but containing cells in different physiological conditions. It was observed that if one uses cells from an exponen tially growing culture of Pyramimonas 13-10 to inoculate fresh medium, the new culture goes directly into exponen tial growth without a lag phase (Figure 7). On the con trary, an inoculum from the stationary phase of the stock culture (or the late exponential phase) produces cultures that exhibit lag phases, the length of which depends on the exact origin of the inoculum. As previously mentioned, all the cultures were started with an inoculum of 2 x 10 3 cells/ml, but this inoculum was taken at different stages of the growth of the stock culture. Figure 7 shows the origin of the in ocula and Table 9 gives the cell density after 20 hours of incubation with or without DDT. There are the following growth stages: early exponential, full exponential and plateau (or stationary phase) in chronological order. After 20 hours of growth cells concentrations varied from 3 3 about 2 x 10 cells/ml (lag phase) to about 3.8 x 10 cells/ml (full exponential). Such difference in cell 39 counts requires a correction of 14 co 2 uptake for cell den sity. This allows a direct comparison of the different growth stages. Tables 6 and 10 show uptakes of 14 c bi carbonates as counts per minute (CPM) corrected for cell density. Statistical analysis of the data yield the following results: - in lag phase: for 7 controls and 7 DDT flasks, at the 95% level of conficence (P < 0.5) there is a difference between the two samples (Table 6). This confirms Wuster's experiments with Pyramimonas 13-10; DDT depresses co 2 up take during the lag phase. The average magnitude of the effect is 60% (Table 6). - in exponential phase. It can be concluded that within the precision of the data the observed 4% difference be tween the two samples is not significant (Table 10). At least 50% of the time random variation alone will account for the difference between the two samples (P >> .2). DDT does not inhibit co 2 uptake during the exponential growth. It is difficult to study precisely the efrect on cell numbers (i.e., cell division) since the cells are growing very slowly or not at all during the lag phase. Cell densities counted with the Coulter Counter at the time of 14 b. b dd. . . . T bl 9 C icar onate a itions are given in a e . In the late plateau they are identical in both control and DDT 40 flasks ( ± 3%). In the full exponential phase (Table 9) the DDT treated cells were less numerous (8%) but the differ ence was not significant (P > .20). Since the cell den sity after 20 hours in the exponential phase is at least 180% of that in the lag phase and the same amount of DDT was added to both cultures, it is important to consider the amount of pesticide available per cell. Since DDT re duces 14 c-bicarbonate uptake by 60% in lag phase, can a lesser effect (only 4% reduction in uptake) during the exponential phase be explained solely by a smaller amount of DDT per cell? Wurster showed that a difference by a factor of 2 in cell numbers leads to a decrease of DDT effect by only a few percent (probably ~ 5% if one extra polates from his figures). Therefore, in full exponential growth, we should still see a reduction of co 2 uptake of about 55% - we see only a 4% reduction which is insignifi cant as it is within the error of the analysis and which could even be accounted for by a slightly lower cell den sity in the DDT flasks (8 % lower) as mentioned earlier. Therefore, while in lag phase Pyramimonas 13-10 is ex tremely sensitive to DDT; it appears completely insensi tive when in full exponential growth. To make a better assessment of the sensitivity in the exponential phase, an inoculum of 1.9 x 10 3 cells/ml was taken from a stock culture in full exponential growth and 41 inoculated into fresh medium. 200 ppb of DDT were added daily to 4 flasks (D 1 to D 4 ) while 4 controls (c 1 to c 4 ) received only acetone (Figure 8). Flasks containing DDT grew faster than the controls for 3 days. On the third day DDT flasks contained 65% more cells than the controls. Every DDT flask had a cell density higher than any control., Even with such a small sample a Wilcoxon test shows that the stimulation is as significant as the test permits (P < 0.5). After the third day in DDT the growth rate slowed down and ventually resumed a "normal" slope like the controls. IV Pyramimonas 1: Promotion of a Lag Phase and Syne rgistic Effect of Copper and DDT The logical inquiry which followed the results ob tained with Pyramimonas 13-10 was to ask if the apparently insensitive species Pyramimonas 1 becomes sensitive when in lag phase. However, this species did not normally ex hibit a lag phase, even when started with an inoculum as 2 low as 6.5 x 10 cells/ml (Table 5). After 20 hours of these conditions the 5 DDT flasks had in average ~ 12% more cells than the 5 control flasks. Their 14 c uptake per cell was identical. As will be explained in more de tail in the Discussion section and the Appendix, the lag phase has been linked to two families of parameters: those determining the physiological (bio-chemical) state 42 of the cells and those defining the external milieu. To promote a lag phase I tried to manipulate both. Creation of a lag phase with an inoculum of 2 x 10 3 cells/ml was attempted using old stock cultures (from 1 to 5 weeks old). Figure 5 shows the dependency of the uptake of 14 c-bicarbonate (for 2 x 10 3 cells/ml) on the age of the stock culture. The older the stock culture the smaller was the uptake of 14 c-bicarbonate, going from 7000 to 3500 CPM as the age of the stock culture went from 1 to 5 weeks (experiment A figure 5), but in no case was a lag phase observed, as defined by zero increase in cell number over 24 hours or more. Even with removal of EDTA there was no evidence of a lag phase with an old inoculum. Iron was found to protect the cells in some way; an aged(over 2 -5 days) FeC1 3 solutions (stored as 1.2 x 10 M) added to medium without EDTA yielded a very high co 2 uptake without lag phase (experiment B Figure 5). The addition of "fresh" iron solution (made the day of the experiment) to medium without EDTA (experiment C figure 5) reduced the 14 c uptake to its original level of experiment A (Figure 5). Only when diminishing the total amount of iron and using freshly made solutions of iron for every experiment could the growth of a culture be slowed (experiment D, Figure 5). Even under these restrictive conditions no lag phase was obtained. Included in these curves (Figure 5) are points 43 for natural and synthetic sea water to emphasize their similarity. At this juncture it was decided to attempt a drastic modification of the trace metals chemistry of the growth medium, to the point of toxicity if necessary. A series of otherwise normal media were prepared with no EDTA, and various ferric chloride and copper sulfate additions. The stock Iron and Copper solutions were prepared fresh for each experiment to alleviate difficulties with "ageing" of the solutions and adsorption on the wall of the containers. The details of the evolution of the methodology are given in the Appendix. Figure 9 shows that a reduction in the iron concentration, as low as one tenth of the normal F/2 value, did not limit growth by itself. Copper was then added. A protective action of iron -7 from copper is clearly seen at 4.4 x 10 M Cu (Figure 9): with a normal (1.2 x 10-SM) concentration of iron abso lutely no effect on growth is observed, while with 1/10 of this iron concentration and the same amount of copper growth is delayed by at least 2 days. The concentration of iron necessary to allow a toxic effect of copper varies with different batches of artificial sea water medium. As much as 3/10 of the original F/2 concentration of iron was necessary with some batches of medium to allow for normal cell growth without any copper addition. The variability 44 of this basic amount may be linked to the uneven amount of trace metal impurities found in chemicals used to make up the medium (see Appendix). Considering that this could be used as a tool to slow the growth of Pyramimonas 1 and to promote a lag phase, the sensitivity of this alga to DDT was tested. Depending on the batch of sea water either 1/10 or 3/10 of F/2 concentration of iron and 1.4 x l0- 7 M of copper were added. A total of 10 flasks with 200 ppb DDT, 8 flasks with only copper and 7 flasks with neither copper or DDT were inoculated with 2 x 10 3 cells/ml. Table 11 and Table 12 summarize the results. The total absorption of 14 c-bi carbonate in flasks containing copper and DDT averaged 64% less than in flasks containing only copper. Flasks with copper alone absorbed only 33% less co 2 than the con trol without copper and DDT. A Wilcoxon T -test shows that the difference is significant (P < .01) (Table 13). The cell densities in all the flasks used for co 2 up take measurements have been monitored. In the control Pyramimonas 1 has a strong tendency to go directly into exponential phase; one can observe differences in cell densities after 24 hours of growth. Compared to controls with copper, DDT always reduces cell division by at least 15% (Table 11). The inoculum was always 2 x 10 3 cells/ml ( ± 10%). From the densities observed after 20 hours of 45 growth (Table 11) it appears that copper promoted a perfect lag phase even in the last set of experiments (Day 14) where the control without copper obviously grew directly in exponential phase. It seems therefore that copper promotes a lag phase and enhances sensitivity to DDT in growing cultures of Pyramimonas 1: it is observed as a significant decrease in 14 c-bicarbonate uptake. Figure 10 illustrates the effect of DDT on growth of Pyramimonas 1 in the presence of copper. An inoculum of 2 x 10 3 cells/ml, from a stock solution in its 5th day of stationary phase, is introduced into 500 ml erlenmeyer flasks containing 250 ml of medium with all the modifi cations discussed in the Appendix: No EDTA, 1/10 Fe concentration of F/2. Two concentrations of copper -8 -7 8 x 10 and 1.2 x 10 M Cu are added to the flasks which are all coated with silicone. The first observation is that neither 8 x 10- 8 or 1.2 x l0- 7 M copper are sufficient to promote a lag phase with that inoculum. Another interesting result is that -8 8 x 10 M Cu does not slow down the exponential growth -7 rate, 1.2 x 10 M Cu does. In spite of this slower rate there is no lag phase and the addition of DDT does not in troduce any significant additional decrease in growth rate. A second experiment with some modifications is des- 46 cribed on Figure 11. The inoculum is reduced to 1.2 x 10 3 -7 cells/ml and 1.6 x 10 M Cu is added. In this experiment all flasks are coated with silicone. The inoculum is taken from a culture at its 10th day of stationary phase. An inoculum of that age has some non-viable cells that may produce an apparent lag phase. This is indeed observed in control curves without copper. The addition of 1.6 x l0- 7 M Cu promotes a small lag phase of an extra half day. The controls with copper have the same exponential growth rate as controls without copper. The growth curves are simply shifted to the right. Cultures that received 200 ppb DDT -7 and 1.6 x 10 M Cu after a longer lag phase (1/2 to 3/4 extra day) exhibit an exponential growth phase for a very short time only (24 hours). It slows down quickly and sharply to an almost complete block on cell division. If after a few days of quasi stationary phase DDT is added only every third day (instead of every day) the growth rate picks up very sharply. 47 DISCUSSION I Lag Phase and DDT Sensitivity The two algal species that are contrasted for DDT sensitivity in this thesis behave quite differently when inoculated into a F/2 medium. Pyramimonas 13-10, the sensitive species, always exhibits a lag phase and has a longer doubling time than Pyramimonas 1 which does not normally show a lag phase. The inescapable conclusion from the bulk of the data is that the cells are inhibited by DDT when -- and only when in lag phase. The physiological-biochemical state that corresponds to a lag phase is very poorly understood. The mere presence or absence of a lag phase with a given species of alga in a given medium is usually an empirical observation. The length of the lag phase -- when it is observed -- depends on the growth stage of the stock cul ture used for the inoculum. It can be sometimes reduced to zero when the inoculum is taken during the exponential phase. Spencer (1954) gives a diagram describing such relationship for a marine diatom. 48 logarithm of cell numbers (stock culture) lag time (hrs) of subcultures 20 TIME (hrs) t I 140 An increase in the size of the inoculum has also been shown to diminish the duration of the lag phase. Two explanations have been put forward to explain this: - Some factor produced by the cells themselves is necessary for optimum growth. It must reach a certain level in the medium for the cells to start growing. This extracellular production may be secondary to the onset of some physio logical states of the cells and can therefore be related to the "age" of the inoculum. - Some toxic factor present in the medium is inactivated by a metabolite excreted by the cells (copper for example may be such a toxic chemical that needs to be complexed before growth can start.) This would explain how the components of the medium would influence the length of the lag phase. The Appendix describes in some detail how the chemical 49 speciation of trace metals (particularly copper) can influ ence the length of the lag phase. It also stresses the variable response of a certain inoculum size to a constant concentration of copper in function of the "physiological" age of the cells. The first of these two components of the lag phase (the nature of the chemistry of the medium) is clearly affecting Pyramimonas 13-10 in our experimental set-up; in some instances it was necessary to remove trace metal impurities from artificial sea-water to allow the growth of Pyramimonas 13-10, while Pyramimonas 1 grew well under all but the most severe increase in trace metal concentra tions. Pyramimonas 13-10 which appears a great deal more sensitive to trace metals than ~ramimonas 1 usually ex hibits a lag phase, while it took several months of re search to induce a lag phase in Pyramimonas 1. In general it appears that oceanic species (like Pyramimonas 13-10), which live in a rather constant en vironment, are more delicate and more sensitive to a large array of variables than coastal species (like Pyramimonas 1) which are subject to frequent and ample environmental fluctuations (Fisher et al, 1973). From the empirical observation that DDT affect the cells only when they are in lag phase, the question arises whether a slow carbon assimilation or a diminished rate of 50 cell division is the significant lag phase characteristics in promoting DDT sensitivity. To approach the problem one may compare the assimilation number, which is a measure of their capacity to take up co 2 , and their specific growth rate which quantitates the uptake efficiency, i.e., the conversion of the absorbed co 2 into cell biomass. As mentioned before, it has been established that 14 c-bicar bonate uptake closely follows chlorophyll a content in most instances (Eppley and Sloan, 1966). Eppley (1972) further linked the rate of photosynthetic carbon assimilation per weight of chlorophyll a to the growth rate and carbon/ chlorophyll a ratio. A study (Mullin et al, 1966) of the relationship between carbon content, cell volume and area in phytoplankton indicates that the cell carbon per unit cell volume varies inversely with cell volume. Experimen tal points were fitted by the equation log 10 c = .76 log 10 v - .29 where C is the carbon content in picograms and Vis the volume in 3 ( µm) • An increase in volume by a factor of 4 (as it is the case for Pyramimonas 1 versus Pyramimonas 13-10) would thus yield an increase of cell carbon by a factor of 3 approximately. The chlorophyll a content difference between Pyramirnonas 1 and 13-10 in lag phase and in exponential phase is by a factor of about 10 to 12. A difference in assimilation number by a factor of 3 is 51 therefore very likely; this is about what is found as well in lag phase as in exponential phase. The specific growth rate (doublings of carbon per day) is more informative. Eppley (1972) relates this rate to the total cell carbon (C), the net increase of carbon per day ( !:£.) and the chlorophyll a (Cha) content by the equa tion: l (C/Cha + 6 C/Cha ) og2 C/Cha When comparing Pyramimonas 13-10 to Pyramimonas 1 one observes a reduction of chlorophyll a by a factor of 10, a reduction of net co 2 uptake by a factor of 30 and a reduc tion of cell carbon by only a factor of 3. This yields a specific growth rate lower for Pyramimonas 13-10 than for Pyramimonas 1. Increase in cell number indeed reflects this tendency as doubling time is about 20 hrs for Pyrarnimonas 1 and 30 hours for Pyramirnonas 13-10. This result is most in- triguing a~ in generaL smaller species tend to reproduce faster unless they excrete some organics while they grow (Eppley, 1972). Thus it appears that in spite of a rate of co 2 assimi lation, that is not abnormal when compared to the other spe cies, Pyrarnimonas 13-10, the sensitive species,may exhibit a loss of carbon which results in a specific growth rate slower than expected. Excretion of organics by 52 Pyramimonas 13-10 would be quite compatible with the rou- tine appearance of a lag phase in that species. It can be postulated that Pyramimonas 13-10, an oceanic species, may exude some complexing agent before being able to pursue its growth in a standard laboratory medium which contains non -8 negligable amounts of copper (4 x 10 M). It has been proposed that phytoplankton population in their natural environment may excrete a proportion of the carbon fixed in photosynthesis in the form of dissolved organic material (DOM). Thomas (1971) observes that al though the rate of release of DOM decreases seaward (from estuaries to open sea), the percent of photoassimilated carbon released as DOM increases seaward. He found that the Georgia estuary 7% is released, in Southeastern USA surface coastal waters less than 13 % , less than 21% . in . in coastal waters below the surface and less than 44% for the westernmost Sargasso Sea. The nature of all extracellular products is still uncertain. The demonstration that 92% of the extracellular products released by a natural popu lation of Stephanodiscus hantzshii in situ was in the form of glycolate (Watt, 1965), the almost exclusive excretion of glycolate by Chlorella cultures and the measurements by Shah and Wright (1974) in the Gulf of Maine, showing levels of glycolic acid as high as 78 µg/ t , suggest that this substance is often the major component. 53 The secretion of glycolate by Chlorella was discovered in 1956 (Tolbert & Zill). Fogg reports (1965) that the addition of 1 µg/t of glycolic acid abolishes the lag phase shown by a small inoculum of C. pyrenoidosa at limiting light intensity. Equivalent addition of glucose or other organic acids did not have a similar effect. Probably al so related to the lag phase phenomenon is the fact that young cultures excrete more organic than older ones (Fogg, 1965). A better understanding of the link between lag phase and organic excretion -- mostly that of glycolic acid - at a biochemical level could shed some light on how DDT may interfere with normal growth. Glycolate (COOH-CH 2 OH) is a very intriguing compound. Its synthesis is a function of light and increases with intensity up to saturation at 1200 fc (Gibbs, 1971). The use of inhibitors that block its catabolism have shown that a massive flow of carbon normally moves through this path way (Tolbert, 1971) more than what is needed for glycine or serine synthesis (its usual catabolites). Algae have brought the mystery even further: they do not use glyco late to synthesize serine. They mostly synthesize glycine out of it, if they metabolize it at all. the 1st enzyme involved in glycolate catabolism is a dehydrogenase with low activity which is extreme ly sensitive to co 2 levels contrary to the very 54 active and co 2 insensitive oxidase of higher plants. Since glycolate has two routes to follow (catabolism or excretion) and since the first enzyme involved has a low activity particularly in culture conditions where high co 2 is bubbled, a large part of the glycolate (up to 90%) can be excreted. In the dark it may be taken up by the cells. (Researchers are not unanimous on this point). A high level of CO 2 inhibits the dehydro genase and therefore glycolate is excreted. With low co 2 levels, glycolate is mostly metabolized. Saturating light intensities or heavy inocula have an effect on the lag phase similar to that of gly colic acid. The cells are probably able to estab lish the necessary concentrations of extracellular glycolic acid. This effect is not shown by all algae (Fogg, 1965). Fogg also reports that there is an equilibrium between intra and extracellular glycolate concentrations, the lag phase could therefore be a delay needed to establish that equilibrium. A possibility that has not been mentioned by these authors is that glycolic acid may play a role similar to EDTA. Chelating agents have been shown to have the same effect on the lag phase as a heavy inoculum. Although glycolic 55 acid is a small compound and therefore an unlikely chelator, the possibility of polymer formation could make it a candidate for chelation. II Biochemical Foundations for Lack of Sensitivity to DDT Possible Role of Photosystem I and II In the light of the restricted knowledge of biochem ical events related to the lag phase, it is worth examin ing the results of Bowes' biochemical experiments. He found that DDT and DDE were both toxic to all al gal chloroplasts studied, whether the algae themselves were sensitive or insensitive to DDT. He further demon strated that DDT interfers (like DCMU) with electron trans port in photosystem II and that DDT penetrates all algal cells (sensitive or insensitive). Some explanation for the apparent insensitivity of some algae is then called for: how can the cells be unaffected by an internal con centration of pesticide which blocks phytosystem II? The possibility of the presence of a detoxifying enzyme as is found in higher organisms seems remote. Although there is some conversion of DDT to DDE in some algae (Bowes), DDE is just as toxic to chloroplasts. (This was the hypothesis that I originally pursued). One is therefore compelled to assume that the basic metabolism of algae either allows sequestration of DDT away from the chloroplasts or can function in spite of the "shutting- 56 off" of phytosystem II, at least sometimes for some algae. The possibility of sequestration will not be pursued since no datum is available on the localization of DDT within the cells. To approach the other hypothesis one needs to under stand how lag phase, photosystem II, release of organics, trace metal toxicity and DDT sensitivity might be linked. If indeed DDT works like DCMU, not only should it affect the electron transport of phytosystem II but it could interfere, like DCMU, in the metabolism of glycolic acid. If one of the effects of excretion of glycolic acid or perhaps other organics is to shorten the lag phase, the inhibition of its synthesis by DDT in algae that usually secrete it (perhaps Pyramimonas 13-10, in our case) would lead to a longer lag phase, which is indeed what has been found by Bowes when working with 4 different organ isms. Compared to cells that usually do not excrete organ ics, these cells would obviously have a slower (lag-phase type) metabolism and would take longer to establish the necessary concentration of exudates and start their ex ponential growth. In exponential growth less and less excretion is seen and probably almost none of that excretion is needed. It is likely that the shutting-off of the synthesis of the exudate shows a time delay during 57 which the chemical is wasted. This again may explain why DDT stimulates rather than slows down both Pyramimonas 1 and 13-10 growth in early exponential. Indeed both Pyramimonas 1 and 13-10 appeared to grow faster in early exponential with DDT than without (Table 4-5, Figure 8). This may be due to the shutting off of that wasteful excretion. While this exudate is needed in the external medium in lag phase and its ab sence is detrimental to the cells, in early exponential the prevention of this energy "leak" may allow the cells to funnel all their co 2 uptake into cell material and therefore increase net co 2 uptake. This appears to lead to a larger cell number (Table 5). As algal metabolism changes when it enters a full exponential growth, the shut-off of the excretion is probably complete, so that DDT has no effect either positive or negative on any algal growth. In this line of argument it is interesting to note that Skeletonema costatum (a sensitive species) does pro duce exudate in variable amount and that it does produce more when copper is added to the medium (Steeman-Nielsen and Wium Andersen, 1970). Because copper does increase exudate excretion and does promote a lag phase (perhaps because it mobilizes a lot of cellular "energy" to complex copper) one can see how DDT can amplify this effect. By cutting down the synthesis of that exudate DDT will slow 58 the cell metabolism even further and as cells in some cases will not be able to complex enough the copper to come out of their lag phase this synergistic action may be lethal. III Speculation on the Possibilities of Finding Basically Different Metabolism in Algae-c 3 or c 4 Metabolism and Their Relation to Photosystem I and II Algae, like higher plants, do not fit one single metabolic "mold." There may be numerous metabolic varia tions which could account for greater sensitivity in the lag phase of a species and for the presence or absence of lag phase. At this stage of knowledge of metabolic dif ferences, the ratio of chlorophyll a to b can be hypo thesized to be a very significant criterion. In higher plants, different ratios of chlorophyll a to b have been shown to reflect differences in metabolism. In these plants there exists more than one pathway for carbon up take. The pathways do not have the same efficiency. In his 1973 review on the subject, Black states that plants using the c 4 cycle (4 carbon compounds intermediates) can have a maximum rate of net photosynthesis 5 times as large as that of the c 3 plants using only the typical Calvin cycle. The main differences between those two metabolisms are to be found in the enzyme kinetics and photorespiration. PEP carboxylase is the main carboxylating enzyme of c 4 plants, it is much more efficient at capturing co 2 than 59 RuDP carboxylase. In c 3 plants photorespiration plays a large role in loss of efficiency of co 2 uptake. This phe nomenon involves the glycolate pathway, the function of which is still unknown as mentioned earlier. This path way carries on the synthesis of glycolic acid from existing sugars in the chloroplasts. The oxydation of this compound in higher plants, or dehydrogenation in algae, is followed by its catabolism outside the chloroplasts with loss of co 2 and therefore a smaller net co 2 uptake. It seems that both types of plants (c 3 or c 4 ) have all the necessary enzymes for normal Calvin and c 4 cycles, but they may not all be active in different plants. This is linked to the fact that there is a different balance of photosystems I (PSI) and II (PSII) in c 3 and c 4 plants and that those systems are linked with co 2 uptake as well as glycolic acid synthesis (and therefore photorespiration). c 4 plants seem to have a low amount of PSII in their chloro plasts (Mayne, 1971). Different ratios of PSI to PSII result in different ratio of chlorophyll a to chlorophyll b. Indeed Mayne and co-workers (1971) and Anderson et al (1971) showed that in higher plants c 4 cells exhibited higher chlorophyll a to chlorophyll b ratios than c 3 cells. If this holds true in the case of the alga Pyramimonas 1, which has the highest chlorophyll a/chlorophyll b ratio would be more of 60 a c 4 type while Pyramimonas 13-10 could be more a c 3 type alga. Therefore Pyramimonas 1 could have less photo system II than Pyramimonas 13-10. Since DDT has been shown by Bowes to affect PSII, Pyramimonas 1, which could have little of that system, should be the least sensitive of the 2 species and its 14 co 2 absorption would not be reduced as drastically as for Pyramimonas 13-10. There may have been less of a need for exudates and therefore less of a need for PSII. This would result in a short or non existing lag phase as was observed. It is not impossible that the same reasoning could explain some of the discrepancies in results of workers using species originating from different environments. Two algal cultures having the same generic and species names but originating one from the west coast, the other from the east coast of the United States, could be en dowed with slightly different photosynthetic apparatus. Little is known of that possibility. Similar variations in the ratio of phytosystem I and II could arise throughout the growth stages of a cell and explain variations in sensitivity for a single algal species throughout its growth curve. This is pure specu lation at this stage but certainly suggests some inter esting studies. 61 IV Resolution of Some of the Discrepancies Wurster, Menzel, and Bowes have clearly shown that DDT affects certain algal species by lowering their co 2 uptake. Bowes further linked this effect to an extension of the lag phase. Cultures resumed normal growth after the onset of the exponential phase. It now appears that there is a simple reason for such a behavior; it is during the lag phase that cells are sensitive to DDT, once they evolve out of that "physiological" state they are inaffected by DDT and grow normally. Not only did Pyramimonas 13-10 (a sensitive species) show this pattern but Pyramimonas 1 (a species which appeared at first insensitive) shows a decrease in co 2 uptake if forced into the conditions of a lag phase. This result explains very well the discrepan cies of the early results with Pyramimonas l; independently of the absolute size of the inoculum, sensitivity to DDT appears directly linked to the presence and length of a lag phase. The variations in estimates for DDT sensitiv ity at equal concentrations of cells and DDT can be ex plained the same way, depending on the physiological conditions of the cells, a wide variety of responses will be observed. The induction of a lag phase with Pyramimonas 1 led to two main observations: the outcome of the sensitivity of an alga to a certain chemical depends widely on 62 a) the physiological state of the cells b) the environmental factors which influence this physiological condition and which can be modi fied by the cells themselves. The changes in the physiology and metabolism of algae as they evolve from lag phase to exponential phase and eventually to a plateau are still very poorly documented. Some data of changes in chlorophyll content, cell size, proteins and lipid composition have been collected only scarcely and recently. The biochemical events underlying the changes have not been investigated in any depth. The large differences in growth that were encountered with Pyramimonas 1 in artificial medium batches that had very minute difference in iron (such as 3/10 F/2 versus 1/10 F/2 iron) points out the difficulty in working with natural sea water for phytoplankton bioassays. The complex syner- gistic or antagonistic interactions of chemicals making up the medium (and also found in natural sea water) such as copper, iron, chelating agents and DDT make it virtually impossible to reach any conclusion without rigorous control of these variables. Complex interactions among trace chemicals may indeed be at the origin of some of the discrepancies described in "Background" section. Since there is no information available about the growth stage of the cells at the time of the experiments and since the 63 detailed composition of the seawaters used for medium preparation is not known, it can be expected that the sen sitivity of a given amount of DDT will appear to vary wide ly within the same species and even the same clone. V Relevancy of the Study to Natural Conditions Three basic difficulties are encountered in the dis cussion of the relevancy of these laboratory studies to natural conditions: - The actual concentration of DDT in natural waters available to organisms is difficult to assess. - The loss of DDT from a laboratory flask by co volatization with water and adsorption on the walls of the flask cannot yet be predicted. - No laboratory experiment - be it continuous culture or batch culture - gives a really good duplication of natural events. A) Real Concentration of DDT in Nature The concentration of DDT available in natural waters is not known with any certainty. It is well established that DDT solubility in aqueous systems is very low: 1.2 ppb (Bowman et al, 1960). Measurements of most waters will therefore yield very low quantities. Indeed most of the highest estimates are at the maximum in the tens of ppb (California waters). An interesting experi ment conducted in Florida by Crocker and Wilson (1965) 64 gives a good picture of the kinetics of "disappearance" of DDT from the main water bodies. They added DDT to a tidal marsh ditch to yield a concentration of 70 ppb. The bottom water had no detectable DDT after 1 day. The sur face water showed a decrease down to a few ppb DDT in a few days and the pesticide was undetectable after 14 days. Obviously DDT had not disappeared and could be found accumulated in sediments (760 ppb), fish (90 ppm), vegetation (9 ppm) and oily patches at the surface (133 ppb). Indeed, Seba and Corcoran (1969) found that surface slicks are effective concentrators of chlorinated hydrocarbons. They found that although the presence of pesticides was undeterminable in the surrounding water, surface slicks contained as much as 13 ppb. These observations are important since high biological activities were seen associated with the slicks. Parker and Barsom (1970) stress the chemical and biological importance of surface microlayers in aquatic ecosystems specifically as possible reservoir for hydrophobic presticides. Odum, Woodwell and Wurster (1969) mention the accumulation of DDT and its metabolites on organic plant detritus particularly in estuaries. These detritus with their associated bacteria and other microorganisms constitute a reservoir where the concentration of DDT residues can be thousand times greater than the concentration in the water. 65 The concentrations of DDT to which a phytoplankter is effectively exposed in the sea is hard to estimate: the cells will absorb DDT from the very dilute soluble pool which itself is in contact with large reservoirs at all interfaces. B) Loss of DDT in Laboratory Conditions DDT disappears from the medium via two routes, - it is lost by volatization with water and subse quent adsorption into the cotton plug or on the glassware - it is lost by absorption by the cells and by ad sorption on glassware Using both a purely aqueous and an acetone-water dispersion method Acree (Acree et al, 1962) found that DDT was lost from the placid surface of distilled water in a jar at an unexpectedly high rate. Figure 12 gives an estimate of the loss in function of the initial DDT concentration, after 24 hours at 25° C. They estimated that above 300 ppb the absolute amount lost by co-valiti zation was almost constant for increasing amount of DDT. Below 100 ppb the loss was almost linearly decreasing. They found that the method of dispersion had no great effect on co-volatization rates. To explain such large losses in spite of DDT low solubility in water, they proposed that the affinity of DDT for the upper surface allows it to volatize faster 66 than if it were uniformly distributed in the aqueous dis persion. The extremely hydrophobic nature of DDT and therefore its heterogeneity in aqueous suspension accounts for this behavior. DDT also disappears through another route; it is absorbed by the algal cells. After 7 days Scenedesmus obliquus concentrated DDT by a factor of 600 from an initial concentration of 1 ppm (Gregory et al, 1969). Sodergren (1968) found that Chlorella accumulated 52 to 77% of 14 c-DDT in 24 hours from an initial concentration of less than 0.6 ppb. He concluded that DDT was accumu lated by absorption since no DDT was released from the cells when they were resuspended in medium void of DDT. He also found that the mechanism responsible for uptake was very rapid; only a few seconds. Bowes made a budget of recovery of DDT after a few days of experiments (3 or 4) and at the end of the ex periment (1 to 2 weeks). He usually spiked his medium with DDT only once at the beginning of the experiment. Without any algal cell in the system and after 14 days of incubation with 80 ppb of DDT he found 30% of the DDT in the plug, 20% on the flask and the rest in the water. If 30% of the original DDT concentration in the medium was lost per 24 hours (as one would expect from Acree's work), one should recover almost all the DDT in the plug. How 67 much DDT is really available to cells when daily additions are used as was done in some experiments -- is hard to estimate and represents a problem when one has no direct way of measuring its concentration in the medium. To study the effect of a given concentration of DDT on the growth of an algal species, one has a few possi bilities. 1) If there is access to instrumentation to measure DDT in the medium, one ought to monitor the amount left in the medium day by day and readjust it to the original concentration. 2) If one cannot measure DDT (as was the case in my experiments), there are two alternatives a) To spike the culture only once. This is what Bowes did. This method has the disadvantage that the initial concentration in the medium will de crease very fast (at his first check after a few days of experiments Bowes found all the DDT in the cells), and that the intracellular concentration will be drastically decreased as the cell density in creases by 3 orders of magnitude throughout the growth curve. b) To add DDT every day (or every 2nd or 3rd day as was done in some growth curves). This replenishment of DDT as it is removed by the organisms appears 68 more "natural" than the preceding solution. The lack of monitoring of the extracellular DDT raises some questions on what real quantity of DDT is responsible for an observed effect. If a culture is already in exponential phase when DDT is added and if its doubling time is close or even smaller than a day (as is the case for Pyramimonas 1), the new cells may absorb the same amount of DDT as the population of the preceding day. There may then be a con stant concentration of DDT per cell. In the experiments reported here growth curves were used as a means of checking if, for example, Pyramimonas 13-10 was really insensitive to DDT during the exponential phase (Figure 8). In spite of probable daily increase in the extracellular concentration of DDT, the cell number increase of Pyramimonas 13-10 was not slowed down at all. On the contrary a small stimulation was observed over the first few days. Growth curves with Pyramimonas 1 without copper (Figure 4) do not present any problem either. Once again even if extracellular concentration of DDT was increasing it did not have an effect on the rate of growth during the exponential. The only case where a question may arise in interpreting the data is when copper is added to Pyramimonas 1. 69 Figure 11 shows that copper and DDT produce a longer lag phase. It also shows that very suddenly there is a sharp decline in growth rate. As long as copper concentra tion is low enough to still allow a doubling time under 24 hours, the amount of DDT per cell may be constant. As the cell division rate slows down and the doubling time largely exceeds 24 hours the amount of DDT per cell very probably increases after daily addition of DDT. This could explain a lower plateau in the curve with copper and DDT. This hypothesis of increasing concentration of DDT per cell is quite in agreement with the observe "acceler ation" of growth when DDT is added only every 3rd day instead of every day (Figure 11). Indeed this explanation of increasing concentration of DDT particularly as the cell enters the stationary phase of growth may well ex plain the discrepancies in plateau levels observed be tween Bowes' data (he added DDT only once) and Menzel's data (he added DDT daily). The discrepancies observed with Skeletonema costatum may also be linked to that phenomenon. The curves drawn in Menzel's report look very much like the curve with copper and DDT on Figure 11. His medium may very well have had some trace metal con taminants which through a synergistic coupling with DDT would completely stop growth after the 4th day. 70 C) Batch Cultures Although there is an increasing trend toward per forming exclusively continuous cultures experiments in many laboratories, batch-cultures studies, such as this one, can yield many important results. As Jannash (1974) recently pointed out a chemostat is not meant to and cannot reproduce the events in a natural habitat; sus tained diversity and variability of species composition are the main reasons why it cannot be so. The limita tions introduced in a chemostat for the purpose of under standing events taking place are seldom encountered in nature (dilution rate, one substrate truly limiting over an indefinite period of time, etc.). The limitations of batch cultures as approximations of real systems are rather obvious and have been dwelled upon by many authors (Rice et al, 1973). However, bloom events observed in fresh waters as in the oceans are rather close to batch situations. For example in regions of upwellings, such as the coast of Peru, large influxes of nutrients at certain times of the year bring about an explosion in phytoplankton growth such as might be approximated by a batch culture. It is worth noting that Barber's results, which showed little growth in nutrient rich waters from the Cromwell current upwelling, have been interpreted by 71 Jackson (Jackson and Morgan, 1975), as growth repression by copper. The last series of experiments reported here might be directly applicable to the potential impact to DDT in such a situation. The experiments reported here clearly show that an oceanic andacoastal species of the same genus of Chloro phyceae both exhibit a sensitivity to DDT that varies with their metabolic state. The use of 200 ppb of DDT was selected to allow a direct comparison of these data with previous works on the subject. This concentration is about 10 times higher than the amount measured in polluted areas. Thus it would be necessary to extend this work to smaller concentrations of DDT in order to reach accep table conclusions about the real impact of DDT in natural environments. However, the results bring into light another perhaps more important problem: that of synergistic toxicity between copper and DDT and quite possibly between a large variety of other trace metals and chlorinated hydrocarbons. The amounts of copper used in these experiments are similar to concentrations found in oceanic and freshwaters. Comparing the potential synergistic toxicity of DDT and copper (and for that matter of any other pollutant and trace metals) in coastal and oceanic water is not simple. Although the concentrations of both pesticides and trace 72 metals is lower in the open ocean than in coastal waters, the results of this thesis indicate that oceanic species (like Pyramimonas 13-10) might exhibit a more drastic sensitivity than coastal species (like Pyramimonas 1) because of their greater general fragility and suscep tibility to toxicants. Fresh water bodies that are sub jected to larger variations in pollutants (pesticides and trace metals) because of direct discharges in waterways and run-offs, will probably offer the best "natural" examples of synergistic toxicity. 73 CONCLUSION This study showed that in spite of apparent original differences in behavior when incubated with DDT, two species of chlorophyceae did behave in the same manner if similar "physiological states" were imposed on them: they were inhibited by DDT when in lag phase, insensitive in exponential phase. Stimulation by DDT was demonstrated in certain physiological conditions (early exponential phase). Assessments of toxicity by bioassays are rather uninformative and misleading if the biochemical state of the cells as well as the chemistry of the medium is not precisely known. The discrepancies found in the previous works on the subject could all be explained by a lack of control of these parameters. The potential impact of DDT on algal photosynthesis has so far been considered minimal since not all species appeared sensitive and since relatively high levels of DDT were needed to see a major reduction of co 2 uptake. The discovery of a synergistic effect with relatively small quantities of copper reopens the question. More studies are obviously needed, but it appears now possible that even small concentrations of copper and probably other metals may render toxic to some important algae the low levels of DDT already present in natural waters. Because continuous culture systems are normally 74 operated to maintain a steady state they may overlook some transient aspects of toxicity of a chemical. It appears therefore important to run batch cultures as well. That algal cells are DDT sensitive in their lag phase and that a lag phase can be promoted through trace metal toxicity, underscore the relevance of algal physiological questions to ecological considerations. By piecing together the evidence gathered in this as well as in other works a circumstancial case can be made for the coexistence of c 3 and c 4 metabolisms in algae. This opens avenues of research where different ratios of photosystems I and II would be evaluated and linked to specific physiological responses. The poten tial existence of pure c 4 or c 3 types of metabolisms in unicellular algae would make them extremely good simple models to study problems in plant physiology. In addition, such studies would have important ecological relevance. 75 Table 1 Comparison of the results of experiments performed by Wurster, Menzel and Bowes. (a) Short term experiments: Reduction of 14 c uptake after 24 hours of incubation with 100 ppb DDT, expressed as percent of control. from figures. Data are extrapolations (b) Long term experiments: growth curves parameters (exponential growth rate and plateau level) of cultures which lasted 8 days and were inoculated daily with 100 ppb in Menzel's experiments, which lasted 14 days and were spiked with DDT only once in Bowes' experiment. 76 (a) ' Wurster Menz 1 Skeletonema costatum 75% 25% Coccoli thus Luxleyi 65% 20% (b) MENZEL BOWES I Exponential Plateau Exponential Plateau Level Growth Rate L v 1 Growth Rate Skeletonema I costatum slower compl te cell long lag same as d·v·s·on block phase after control after 3 which normal divisions growth rate Cyclotella nana slower low r plateau short lag same as phase normal control growth rate Coccoli thus Luxleyi same as same as same as same as control control control control ,'c -le (extend lag * phase with 250 ppb) -le *No effect on growth curve was 1 ~bserved by either author in spite of a drastic reduction of C bicarbonate uptake in 24 hours experiments. Table 1 77 (a) CPM % of Control Control 4750 DDT (1000 ppb) 5553 116% (b) CPM/100 ml % of Control of culture - Control 8212 DDT (100 ppb) 8112 99% Table 2. 14 C Uptake of Pyramimonas 1 after 24 Hours of Incubation with DDT with Two Different Inocula (a) 2 x 10 3 cells/ml (b) 1.5 x 10 3 cells/ml CPM = counts/min 78 '1 \.D Assay for Oxygen Evolution CO 2 Uptake Chlorophyll Packed cell/ml 50 ml of A+ B CPM mg/m 3 Volume Culture % of ? 2 Saturation 3 (counts/min) µ per nunute CONTROL 2.96 141844 107.8 .002 3.4 X 10 5 DDT 5 (1000 ppb) 2.89 1379 31 92.8 .0019 2.8 X 10 as % of Control Table 3. 98% 97% 86% 95 % 83% Comparison of Some Physiological Parameters (Oxygen Evolution, 14 c Uptake, Chloroplyll Content) and Physical Parameters (Cell Number and Packed Volume) of Cultures of Pyramimonas 1 at the End of their Growth Curve with or without 1000 ppb DDT (Growth Curves Described in Figure 4, Experiment #II). Day of Control Flasks DDT Flasks Experiment CPM/50 ml of culture CPM/50 ml of culture 1 2 3 4 5 6 7 Total % of C Table 4. 2476 3791 2911 3916 4264 4218 4850 4358 3449 4155 2540 4158 7016 7434 7496 7306 3176 4598 4639 4562 3761 3714 3580 3410 1264 1582 2097 2342 53519 59624 112% 14 C Uptake of Pyramimonas 1 in Normal F/2 Medium after 24 Hours of Incubation with 200 ppb DDT. Experiments 1-6 Done in Natural Seawater, 7 in Artificial Water. 2 Replicates on each Day. Inocula of 2 x 10 3 cells/ml from Stocks at the Palteau Phase 80 CONTROL DDT % OF CONTROL Cells/ml 6.5 X 10 2 8.9 X 10 2 after 24 hrs. X 10 2 X 10 2 6.7 7.5 6.0 X 10 2 6.7 X 10 2 7.0 X 10 2 7.2 X 10 2 6.0 X 10 2 7.8 X 10 2 Total 32.2 35.7 118% CPM/50 ml of 569 712 culture 565 688 625 671 594 719 670 782 Total 3023 3579 118% CPM/2 x 10 3 1751 1600 cells/ml 1687 1835 2083 2065 1786 1820 2233 2172 Total 9540 9492 99.5% Table 5. 14 C Uptake of Pyramimonas 1 after 24 Hrs. of Incubation with 200 ppb DDT. Same Experiments as in Table 4 but all the Replicates Were Done on the Same Day with Inocula from the Same Stock Culture. Inoculum Size= 6.5 x 10 2 cells/ml. CPM = counts/min. 81 Table 6. CONTROL DDT 5 CPM/10 cells 5 CPM/10 cells 29 22 54 24 65 34 87 44 172 54 204 65 205 80 Total 816 Total 323 40% of Control 14 C Uptake of Pyramimonas 13-10 (in lag phase) after 24 Hours of Incubation with 200 ppb DDT. The Cell Densities of the Flasks are Given in Table 9. (CPM = counts/min) 82 LAG EXPONENTIAL PLATEAU Pyramimonas 1 600 450 615 Pyramimonas 13-10 140 120 196 Table 7. 3 Mean Cell Volume (µm) of Pyramimonas 1 and Pyramimonas 13-10 at Different Stages of their Growth Curve 83 Species Cells/ml Chlorophyll a Chlorophyll b Chlorophyll Chlorophyll Chlorophyll Chlorophyll + 3 3 a+b 3 a+b/cell a/cell a/chlorophyll mg/m mg/m Medium mg/m picogram picogram B Pyr. 1 6.3 X 10 5 41.04 18.72 59.76 9.49 6.5 2.19 synthetic Pyr. 1 7.2 X 10 5 41.28 21.64 62.92 8.74 5.73 1.91 1 natural , seawater ' 6 Pyr. 13-10 1.3 X 10 7.6 4.5 12.08 0.93 0.58 1.68 synthetic Pyr. 13-10 2.3 X 10 6 10.8 9.3 20.1 0.87 0.47 1.16 synthetic Pyr. 13-10 3.3 X 10 6 17.72 11 28.72 0.87 0.54 1.61 natural seawater Pyr. 13-10 2.5 X 10 6 13.44 10.56 24.00 0.95 0.54 1.27 natural seawater Table 8. Chlorophyll Content of Pyramimonas 1 and RYramimonas 13-10 in Natural Seawater and Artificial Medium 00 ~ a) LAG PHASE b) EXPONENTIAL PHASE CONTROL DDT CONTROL DDT 2 2.1 3.8 3.3 2 2.3 3.5 3.3 3.1 3.0 1.8 1.9 1.9 1.9 2.4 2.4 1.8 1.7 2.7 2.5 2.3 2.0 l* 1.1* 2.8 2.3 .9* .7* average 1.63 average 1.67 average 2.94 average 2.69 98% of control 94% of control - * old stock - 1/2 non viable cells from inoculum of 2 x 103cells/ml Table 9. Cell Densities of Pyramimonas 13-10 after 20 Hours of Incubation with 200 ppb DDT at Two Stages of its Growth Curve: a) Lag Phase b) Exponential Phase. Inoculum in all Cases is 2 x 10 3 cells/ml. Cell Density= 3 Xx 10 cells/ml 85 CONTROL 5 CPM/10 cells 415 365 401 300 408 285 175 Total 2349 DDT 5 CPM/10 cells 375 353 228 383 360 233 332 Total 2264 = 96% of control Table 10. 14 c Uptake of Pyramimonas 13-10 (in Exponential Phase) after 24 Hours of Incubation with 200 ppb DDT. The Cell Densities of the Flasks are given in Table 9 86 OCu +Cu Day of CONTROL CONTROL DDT. ,- Cu DDT. Experiment C DDT Cu ODDT 2 1.9 1.6 11 2.0 1.8 2.8 2.6* 2.4 12 2.4 2.8 2.8 2.5 2.2 2.4 2.0 13 2.2 2.2 1.6 5.3 2.1 1.5 14 1.9* 1.4 5.5 2.4 1.5 1 * average 2.9 average 2.3 average 1.7 79% of C 74% of Cu Data in last column are only as a check that DDT by itself does not inhibit cell growth. Already proven by data in Table 4 and Table 10 * Bacterial contamination found at time of cell count 2.1 2.2 OCu Table 11. Cell Density of Pyramimonas 1 after 20 Hours of Incubation -7 with 1.4 x 10 M Copper and 200 ppb DDT. Inoculum 2 x 10 3 cells/ml. Cell Density= X x 10 3 cells/ml Medium= F/2 without EDTA with Reduced Iron Iron Solution Made the Day of the Experiment 87 OCu +Cu Day of CONTROL CONTROL DDT. + Cu DDT. OCu Experiment C ODDT Cu ODDT 1264 1654 393 1582 11 1307 742 2591 * 1015 12 1440 1783 2281 850 2338 1258 755 2342 13 1855 1578 517 5804 1614 324 14 * 292 3896 4521 572 * Average 2790 1837 690 % of 67% of C 36% of Cu Control * discarded data: bacterial contamination Table 12. 14 C Uptake of Pyramimonas 1 after 24 Hours of Incubation with 1.4 x 10- 7 M Copper and 200 ppb DDT. Inoculum 2 x 10 3 cells/ml. CPM = counts/min (correspond to cell densities in Table 11). CPM for 50 ml of culture 88 CONTROL Cu DDT + Cu CPM/2 x 10 3 cells/ml Rank CPM/2 x 3 10 cells/ml Rank 1741 16 491 3 1307 12 824 8 1629 15 846 9 1048 10 1200 11 1435 13 680 5 1537 14 755 6 3768 17 646 4 432 2 417 1 763 7 Nl = 7 Nl N = 10 N2 2 I R=97 I R=56 T.01(7,10)=37 Wilcoxon T. test. the difference between the two samples is significant with a 99% level of confidence (P < .01) Table 13. Statistical Evaluation of the Synergistic Inhibition of 14 c Uptake of Pyramimonas 1 by Copper and DDT. Compare Cu (Control with Copper) and DDT with Copper Data from Table 12. All Data are Normalized, i.e., Expressed as CPM/10 5 cells 89 Q) u 0 ® Natura I Sea Water + • Synthetic Sea Water 5 7 9 I I 13 Time 1n Days F i g u re I Comp a r i s o n o f t he g row t h o f Py r a m i m o no s in natural and synthetic sea water 15 90 \.0 r-' • 12 I I I ~· 10 CD + C: 9 ·- 5 Q) C: 4 Q) Cl ~ 3 >< 0 2 I • 0 25 50 75 100 200 300 400 Cell density ( X x 104 cells/m-t) Figure 2 Standardization curve for oxygen evolution assay in oxygen chamber. Net photosynthesis (A) plus respiration ( B) in function of time (minutes) versus eel I density - Assay done with Pyramimonas I. Q) u 10 4 inoculum.,...._ _ _.._ ___ 0 ! DDT additions • Control + DDT 10 3 ---------------------~___,, _______ _ 0 2 4 6 8 10 Time in Days Figure 3 Effect of I ppb and 100 ppb DDT on the growth of Skeletonema costatum 92 f--:+~ ± D • • • I 10 5 • ~ I E 10 4 I ' (/) I • I (1) I u I I I • I I I I 10 3 I 0 I I I (.) + Contra I :;:: 0 --- I I Experiment (1) - el0ppbDDT ~ ::J 0 (.) / I I ® 1000 ppb DDT (1) 0 -------- ..c C: f- ·- Experiment [ o Contra I ]] • 10 ppb DDT I O 2 '------'--..___---'--'--__.___..___-'---A----'-------___,----__.______. 0 2 4 6 8 10 12 Time in Days F i g u r e 4 E ff e c t of IO pp b and I 000 pp b DDT on t he g row t h of Py ram i mo n as I. Pre I i m in a r y res u I t s . Experiment I and II performed with inocula of different size and different origin 14 93 Figure 5. 14 Variations of C uptake of Pyramimonas 1, after 4 h f . b . . h 14 b. b . f ours o incu ation wit C icar onate, in unc- tion of the age of the inoculum (2xl0 3 cells/m) in the presence or absence of EDTA, with freshly made or old solutions of iron. Curve A represents experiments done with standard F/2 medium. Curve B represents experiments without EDTA and with iron solution not made on the day of the experiment. Curve C represents experiments without EDTA and with iron solution made on the day of the experiment. Curve D represents experiment without EDTA, with freshly made iron in reduced concentration. Either synthetic ( ~) or natural (S.W.) sea water was used in the experiments. 94 \..0 V1 V, >,.. 0 0 I E ::, 50 ; 30 u 0 C: "+- 0 (1) ~ 1 0 AO Standard F/2 medium B D No E OTA - aged iron CA No EDTA - fresh iron D V No EDTA - fresh iron in reduced concentration L 6 C D L ~ 2,000 4 000 os.w. ~ A I 14 co 2 Uptake (CPM) Figure 5 L □ B S. W · O OL 6,000 Q) u 3 5 7 9 I I 13 15 Time 1n Days Figure 6 Standard growth curve of Pyramimonas 13-10 (4 replicates) 96 Figure 7. Growth Curve of a stock culture and subcultures of Pyraminonas 13-10 A - typical stock culture growth curve B - subculture with an inoculum taken during the exponential growth phase of the stock and demonstrating no lag phase C - subculture with an inoculum taken at the stationary phase of the stock and showing a significant lag phase. 97 A • 10 6 /l /. ~ E '- 10 5 I C/) I - - Q) u • I I I I 10 4 I • I I • C • I t ./ ♦ • ·--✓ 10 3 0 2 4 6 8 10 12 14 Time In Days Figure 7 98 Figure 8. Slight stimulatory effect of 200 ppb DDT on the growth of Pyramimonas 13-10 when the inoculum is taken in the exponential phase of the stock culture. Each point is the mean of four data points. 99 Q) u • Cont r o I (average of 4 Flasks) x 200ppb DDT (average of 4 Flasks) 4replica foreach data point I O 3 L.....----'--"---~-'---~-.___,___._~-'----~-._~_____, 0 2 4 6 Time ,n Days Figure 8 8 10 12 14 100 Figure 9. Protection of Pyramimonas 1 by iron against copper. The experiments are performed in medium without EDTA, with either l.2xl0-SM Fe or l.2xl0- 6 M -7 -7 Fe (freshly made), l.2xl0 Mor 4.4xl0 M Cu are added. 101 E ..... "' 1 0 4 C1) u 1 0 3 1 \ •---- 5 1 0 ~ 1.2 x 1 o- 5 M -6 1.2 xl O M u Days Control 4 x 1 o- 8 M 0 0 /:,. -7 1. 2 x 10 M • • 4.4 x 1 o- 7 M • 'Y Expt. -#-1 Expt.#2 Expt.-#3 -Experiments .:J/=1, 2, 3 done with inocul a from different cultures -No EDTA Figure 9 15 102 Figure 10. Growth curves of Pyramimonas 1 with l.2xl0 7 M, or -7 l.6xl0 Cu and 200 ppb DDT. The experiments are performed in silicone coated flasks. The inoculum is 2xl0 3 cells/ml. 103 Cl) Q) u DDT additions • 4 x IO - 8 M Cu ( F / 2 con cent rat i on ) 0 DDT o 1.2 x I0- 8 M Cu - 200 ppb DDT + 1.6 x I0- 7 M Cu - 0 DDT O l.6x 10 M Cu -200ppb DDT 10 2 .__~_.,_-L-_'--_._-'--_._-'--___ .__--'-_.__--'-___, 0 2 4 6 8 Time in Days Figure 10 10 12 14 104 Figure 11. -7 Growth curves of Pyramimonas 1 with l.6xl0 M Cu and 200 ppb DDT. The experiments are performed in silicone coasted flasks. The inoculum is 3 l.2xl0 cells/ml. 105 10 6 10 5 ~ E ' 10 4 (/J - Q) u ♦ ♦ 0 2 ♦ • + 0 t ♦ ♦ ! ♦ ! -----· • /. - -+ + +-- 11/ I/ :f 4 x I 0- 8 M C u ( F / 2 Con c en t rat i on ) 0 D D T -7 l.6xl0 MCu-ODDT 1.6 x 10-? M Cu -200ppb DDT additions of DDT each data point average of two replicates 4 6 8 Time 1n Days Figure II 10 12 14 106 I-' 0 -...J C: 0 +- 70 0 ~ \+ + Radiometric analysis +- C: o Ultraviolet analysis (1) (.) C: 60 0 (.) - 0 ·- +- 50 ·- C: ·- '+- 0 ~ 40 0 +- "' 0 30 ~ 0 0 20 10'-----'----'---__. __ _._ ______ ~--~--~-=-.._=-____,J 100 200 300 400 500 600 1000 DDT pp b Figure 12 Loss of DDT by evaporation from water as a function of the initial con c e n t r a t i o n of DD T. P lo t fr o m data of Acree et a I ( l 9 6 2 ) References Acree, F., M. Beroza and M.C. Bowman (1962) Agricultural and Food Chemistry 11, 278 Anderson, J.M., K.C. Woo and N.K. Board~an (1971) Biochimica and Biophysica Acta 245, 398 Black, C.C. (1973) Ann. Rev. Plant Physiol. 14, 253 Bowes, G.M. and R.W. Gee (1971) J. of Bioenergetics 2, 47 Bowes, G.M. (1972) Plant Physiology 49, 172 Bowman, M.C., F. Acree and M.K. Corbett (1960) Pesticide Monitoring J. 8, 406 Burnett, R. (1971) Science 174, 606 Butler, W.A. (1974) in BNA publication Environment Reporter Current Developments Cox, J .L. (1970) Science 170, 71 Crocker, R. and A. Wilson (1965) Trans. Amer. Fish Soc. 94, 152 Davey, E.W., J.H. Gentile, S.J. Erickson and P. Betzer (1970) Limnol. and Ocean. 15, 486 Davey E.W., M.J. Morgan and S.J. Erickson (1973) Lirnnol. and Ocean. 18, 993 Dunn, A. and J. Arditti (1968) Experimental Physiology Holt, Rinehart and Winston Inc. Eppley, R.W. and P.R. Sloan (1966) Physiologia Plantarum 19, 47. Eppley, R.W. (1972) Fishery Bulletin 70, 1063 Finley, F.B. and R.E. Fillmore (1963) Am. Inst. Biol. 108 Sci. P bl. 13, 41 Fisher, N., L.B. Graham, E.J. Carpenter and C.F. Wurster (1973) Nature 241, 548 Fogg, G.E. (1965) Algal Cultures and Phytoplankton Ecology, the University of Wisconsin Press, Madison, Wisconsin Furnia Matsumara and R.D. O'Brien (1966) J. Agr. Food Chem. 14:39 Gibbs, M. (1971) in Photosynthesis and Photorespiration M.D. Harch, C.B. Osmond and R.O. Slatyer eds., Wiley Interscience, N.Y. Goldberg, E.D., P.Butler, P. Meier, D. Menzel, R.W. Risebrough and L.F. Stickel (1971) in Chlorinated Hydrocarbons in the Marine Environment. NAS Report Gregory, W.W., J.K. Reed and L.E. Priester (1969) J. Protozool 16, 69 Guillard, R.R.L. and J.H. Ryther (1962) Can. J. Microbial. 8, 229 Guillard R.R.L. (1973) in Handbook of Phycological Methods. Culture Methods and Growth Measurements, J.R. Stein ed. Cambridge University Press Hase, E. (1962) in Physiology and Biochemistry of Algae, R.A. Lewin ed., Academic Press, N.Y. Jackson, G.A. and J.J. Morgan (1974) Presented at the 37th Annual Meeting of the American Society of Limn. and Ocean. in Seattle, Washington Jannasch, H.W. (1974) Limn. and Ocean. 19, 716 109 M nahan, S.E. and M.J. Smith (1973) Envir. Scien . and Tech. 7, 829 Mayne, B.C. (1971) Plant Phys. 47, 600 Menzel, D.W., J. Anderson and A. Randtke 167, 1724 (1970) Science Mullin, M.M., P.R. Sloan and RW. Eppley (1966) Limn. and Ocean. 11, 307 Odum, W.E., G.M. Woodwell and C.F. Wurster (1969) Science 164, 576 Parker, B. and G. Barsom (1970) Bioscience 20, 87 Rice, H.V., D.A. Leighty and G.C. McLeod (1973) Critical Reviews in Microbiology 3, 27 Risebrought, R.W., J.J. Griffin, R.J. Huggett and E.D. Goldberg (1968) Science 159, 1233 SCCWRP (Southern California Coastal Water Research Projeot) (1973) Report TR 104 Seba, D.B. and E.F. Corcoran (1969) Pesticides Aonitoring Journal 3, 190 Shah, N.M. and R.T. Wright (1974) Marine Biology 24, 121 Sodergren, A. (1968) Oikos 19, 126 Spencer, C.P. (1954) J. Mar. Biol. Assoc. W.K. 33, 285 Sprague, J.B. and J.R. Duffy (1971) J. Fish. Res. Bd. Canada 28, 59 Steeman Nielsen E. and S. Wium Andersen (1970) Marine Biology §_, 93 Strickland, J.D.H. (1958) J. Fish. Res. Bd. Canada 15, 453 Strickland, J.D.H. and T.R. Parsons (1972) A Practical 110 Handbook of Seawater Analysis Fish. Res. Bd. Canada Bull. 167. Ottawa Tate, M.W. and R.C. Cleland (1957) Non Parametric and Shortcut Statistics Interstate Printers and Publishers, Inc. Danville, Illinois Thomas, J.P. (1971) Marine Biology 11, 311 Tolbert, N.E. and L.P. Zill (1956) J. Biol. Chem. 222, 895 Tolbert, N.E. (1971) in Photosynthesis and Photorespira- tion Hatch et al eds, Wiley Interscience Watt, W.D. (1966) Proc. Roy. Soc. London B. 164, 521 Wurster, C. (1968) Science 159, 1474 111 APPENDIX LAG PHASE PROMOTION IN THE GROWTH OF PYRAMIMONAS l BY MANIPULATION OF THE TRACE METAL CHEMISTRY OF THE MEDIUM Published as Technical Note No. 17 BY Nicole M.L. Morel and Francois M.M. Morel Water Quality Laboratory Ralph M. Parsons Laboratory For Water Resources and Environmental Engineering Department of Civil Engineering Massachusetts Institute of Technology 112 Introduction The initiation of the exponential growth in a batch culture is usually unpredictable. It 1s common, although not universal, to observe a period of time during which cell density remains constant or even decreases before it increases in a geometric fashion. This period is commonly referred to as the lag phase of the culture. Two general hypotheses -- not mutually exclusive have been put forward to explain the variations in the dur ation of the lag phase: 1) the cells have to "adjust" to the new environment or 2) the medium has to be "con ditioned" to allow normal growth. It is relatively well documented that the cells go through different physio logical states in their life cycles and that some of these states predominate in the various stages of growth of a culture. That time be needed for the transition from one physiological state to another seems rather probable. Experiments with laboratory batch cultures have yielded some evidence that the duration of the lag phase in some phytoplankton species is dependent upon the trace metal chemistry of the growth medium (Steeman Nielsen, and Wium-Andersen, 1970). The dual role that trace metals play, as necessary nutrients (growth factors) and toxic agents for phytoplankton growth is extensively documented in the literature. However, a clear quantitative picture has yet to emerge from the mass of confusing and sometimes 113 contradictory data. The role of chelating agents seems to be predominant in relating the trace metal chemistry of the medium to the lag phase phenomenon. An appealing and commonly accepted view is that the cells themselves con dition the medium by exudating chelating agents. The time necessary for this conditioning would correspond to the lag phase. As mentioned earlier, the lag phase is not a univer sal phenomenon and large variations are found among vari ous clones of the same species. For example, of two clones of the marine green alga Pyramimonas that are com monly cultured, one normally exhibits a lag phase (Pyramimonas 13-10) and one does not (Pyramimonas 1). In relation with studies of pesticide effects on Pyramimonas, it became important to induce a lag phase in the growth of Pyramimonas 1. This turned out to require extensive modi fications of the normal culturing procedures. The report describes these modifications which consist mostly in manipulations of the trace metal chemistry of the growth medium. Background Spencer (1954) made a detailed study of the lag phase of Nitzchia closterium and observed that the length of that phase depended on the age of the inoculurn: - With an inoculurn from a stock culture in exponen tial growth the lag period is reduced to zero 114 - If the stock culture is at the plateau phase a lag period is observed; the longer the stock culture has re mained at the plateau level the longer the lag period. He also remarked that even cells taken in full exponential growth exhibit a lag phase if the inoculum is small. Riley (1943) had already observed this inoculum size dependency. Working with the same alga he had found that the presence or absence of an initial lag is dependent on the cell concentration of the inoculum, all other physical and chemical conditions being identical. Tamiya (1963-1966) performed a very thorough study of the different stages of the life cycles of Chlorella grown in synchronized culture. He identified two major cell types: D and L. Exponentially growing non-synchronized populations consist largely of the D-type cells (Fogg 1959). The cells have a very large concentration of pro tein (as much as 70% of the dry weight Fogg 1959), high levels of chlorophyll and nucleic acid. They synthesize very small quantities of fats or carbohydrates, have a high photosynthetic activity and a photosynthetic quotient (0 2 /co 2 ) of about 1. As the culture grows older the dominant cell type be comes L. The protein content of these cells is less than 10%. They synthesize large quantities of storage mate rials (polysaccharides) and fats. Their photosynthetic activity is low and their photosynthetic quotient increases 115 as they synthesize more reduced substances. Tamiya (1966) mentions that quotients as high as 10 have been reported. Although there are large variations in the proportions of proteins, carbohydrates, fats and nucleic acids within the life cycle of a single species, different species of algae have a tendency to have the same proportions of those metabolites when grown under approximately similar con ditions (Collyer and Fogg 1955). It can therefore be expected that upon transfer in fresh medium cells may have to adjust their metabolic machinery from fat and carbohydrate formation to protein and nucleic acid synthesis before they can divide. Cells that are already in exponential growth before transfer to fresh medium are already geared to produce mostly protein and other protoplasmic constituents (Fogg 1965). They may therefore start dividing without any adaptation time and do not exhibit a lag period. A second explanation for the lag phase phenomenon proposes that the growth medium itself has to be condi tioned before division and exponential growth can be ini tiated. This hypothesis does not exclude the first one since the conditioning of the medium by the cells may first require that certain needed enzymes be synthesized. Early observations clearly indicated that cells inoculated into re-enriched medium from an old culture show less lag than similar cells inoculated into fresh medium (Fogg, 116 1959), suggesting that some factor produced by the cells themselves is necessary for optimum growth. Fogg and his co-workers (1955, 1958, 1963a, 1963b) who did pioneering work in this area were the first to hypothesize the biological and ecological importance of cellular exudates at low concentrations. Fogg (1963) suggested that a certain equilibrium of a given chemical is required before growth can resume. Nalewajko et al (1963) demonstrated for example that 1mg of glycolic acid which is excreted by Chlorella and many other phytoplankton abolishes the lag phase of Chlorella while addition of pyruvic acid or glucose does not. The release of organic material by natural phyto plankton populations has been reported by many authors, (Watt, 1966; Hellebust, 1965-67; Thomas, 1971). Glycolic acid, polysaccharides and amino acids -- free or combined -- have been reported to represent the major fractions of the released organics. The report of large releases of photo-assimilated carbon have recently been questioned due to experimental procedures (Berman and Holm Hanson 1974). Most of the evidence described thus far implies little about the role the released organics might actually play. It has been suggested that at least some of them may be important in complex formation (Johnston, 1964). They may be able to modify the chemistry of the medium through their chelating capacities. 117 From empirical evidence early workers had recognized the importance of complexing agents in the formulation of growth media. Relatively large concentrations of strong chelating agents (usually EDTA, NTA or citrate) are in cluded in common recipes for growth medium as partial re placements for "soil extracts" (Provasoli et al., 1957; Droop, 1961). Johnston (1964) who systematically studied the influence of the medium composition on the growth of various phytoplankters demonstrated the importance of che lating agents. He postulated that trace metals (especially iron) were made available for growth through chelation. Barber (Barber and Ryther, 1969; Barber et al., 1971; Barber, 1973) attempted to demonstrate this effect through a series of laboratory and field experiments. He related the great variations found in the productivity of up welling waters to the presence or absence of natural organic ligands. He also demonstrated that the deleteri ous effect of U.V. photo-oxidation of such waters could be reversed by additions of iron, EDTA, or both. In batch cultures of Chaetroceros socialis, he demonstrated that EDTA addition considerably shortened the duration of the lag phase (without changing the exponential growth rate) in the same fashion that increase in cell density short ened the lag phase. He observed that at certain high cell density the lag period with and without EDTA were similar. He proposed that each phytoplankton cell has a certain 118 capacity to synthesize and excrete compounds that make the growth medium more favorable in the same fashion as EDTA. The larger the cell density, the faster this condition is achieved. It was originally thought {Johnston 1964) that the role of chelating agents was to make iron available for cell growth. This view seemed confirmed by the observation that iron additions had the same beneficial effect on cell growth as addition of chelators. Steeman Nielsen (1970) was the first to propose that the role of chelating agents could be to protect the cells against toxicity of trace metals such as copper. He and co-workers (1969-1970) con firmed earlier observations by Fitzgerald and Faust (1963) that the effect of copper toxicity depends on the concen tration of iron as well as that of chelating agents in the medium. In the absence of chelator, increased concentra tions of iron decrease the toxicity of copper. Davey (1973) and Barber (1973) reached the conclu sions that availability of iron and protection from poisonous trace metals are both important, that chelating agents can play this dual role, and that it is experimen tally difficult to distinguish between the two effects. Following Stumm (1969), Barber noted that addition of ferric chloride can both enhance the availability of iron and reduce the availability of toxic metals (copper) by adsorption on precipitated iron hydroxide colloid. 119 Recently Sunda (1975) confirmed the protective role of copper chelating agents on phytoplankton growth with experiments on the diatom Thalassiosira pseudonana and to a lesser extent, with the green alga Nannochloris atomus. He demonstrated that copper uptake and toxicity were uniquely determined by the cupric ion activity in the medium. Whether or not it has to do with exudation of che lating agents and detoxification of the medium, the con ditioning of the growth medium during the lag phase seems clearly dependent upon cellular activity. Nonetheless the changes that can take place in the medium due to simple "ageing" should not be overlooked. For example the adsorption and precipitation of some heavy metals are likely to take place on a scale of a few days (Stumm and Morgan, 1970). The surface chemistry and hence the ad sorption properties of iron colloids change in that time period (Gadde et al 1974). Finally a number of photo chemical reactions could drastically modify the trace metal chemistry of the medium (Stolzberg and Hume, 1975; Haines and Blakeley 1975). Results As experience had suggested, Pyramimonas 1 never exhibits a measurable lag phase under standard culture conditions no matter how old the cells of the inoculum are. This is seen in Figure 1 where the same growth rate is 120 observed in cultures inoculated with cells from an expo nentially growing culture or from a culture in its third week of stationary phase. It should be noted however that increasing the age of the inoculum depresses the carbon uptake measured after 24 hours. An illustration of this can be found in the points of experiment A in figure 2 which correspond to unmodified F/2 medium. In order to induce a measurable lag phase (defined here as no increase in cell number over 24 hours), systematic variations in the trace metal chemistry of the medium are attempted. Figure 2 summarizes several series of experiments performed sequentially in an attempt to reduce the co 2 up take and thus create a lag phase. 1. Removal of EDTA (A B figure 2). Contrary to expectations the complete removal of EDTA from an otherwise normal F/2 medium seems to increase the photosynthesis of Pyramimonas 1 as measured by 14 co 2 up take. In both EDTA free media (synthetic and natural sea water) the uptake of 14 co 2 is higher than in the controls containing EDTA (compare experiments B with the controls A in figure 2). Since it seems unlikely that the absence of EDTA per se should promote a higher photosynthetic activity, it is probably the indirect effect on the trace metal chemistry which is responsible for the observed effect. The most obvious metal to investigate is iron. 121 2. Use of freshly made solutions of iron (B ➔ C figure 2) . F/2 medium is made routinely with 'artificial seawater which is stored in Nalgene 5 gallon bottles for a few weeks. The nutrients Phosphate and Nitrate and the vitamins are in stock solutions kept in the refrigerator. The stock of trace metals including iron and EDTA, is kept in a dark refrigerated bottle. For the purpose of removing EDTA and changing the iron concentration it is necessary to make a stock solution of trace metals without EDTA or iron. Ferric chloride solutions are therefore made separately. A first step was to keep those stocks for a few weeks. Experiments Bin figure 2 correspond to this medium con taining "aged" ferric chloride solutions. By using fresh iron solutions (prepared the day of the inoculation) the photosynthetic activity is reduced to its original level in standard F/2 medium (experiment C figure 2). It is thus the presence of unchelated iron originating from an aged ferric chloride stock which promotes the fast carbon up take in experiment B, not the absence of EDTA. This effect is probably linked to variations in the size and surface chemistry of the iron colloids in function of time. For the purpose of inducing a lag phase the most obvious next attempt is to reduce the amount of "fresh" iron added to the growth medium following Steeman Nielsen's obser vation (1970) that, in the absence of EDTA, iron may 122 "protect" the cells from toxic trace metals. As the lag phase may be related to trace metal toxicity, a reduction of iron may conceivably render the medium more "toxic" and therefore promote a lag phase. 3. Reduction of the Iron Concentration (C ➔ D figure 2) • By reducing the amount of iron to 1.2 x l0- 6 M Fe (1/10 of its original concentration) one observes a decrease in co 2 uptake (experiment D figure 2). A larger reduction of the iron concentration creates an iron deficient condition in which the cells grow poorly. This minimum amount of iron was found to vary by a factor of 2 or 3 with different batches of artificial seawater. This variation will be seen to be linked to copper toxicity (see discussion). Suffice it to observe now that when reducing iron concen tration the 14 co 2 uptake is reduced but cell division is not. Adding copper is the next step in the attempt to promote a lag phase. 4. Additions of Increasing Amounts of Copper Stocks solutions of Cuso 4 also present a problem of ageing. -7 In dilute concentrations, such as 10 M, copper is rapidly adsorbed by the walls of the glass containers. The pro blem is overcome by maintaining a concentrated stock of -2 2 x 10 M Cu so 4 solution and preparing fresh solutions diluted daily. The loss of copper due to adsorption onto the walls of the containers presents a particularly diffi- 123 cult problem in long-term experiments. To minimize this loss the glassware used for the experiments is coated with a silicone film as described in Material and Methods. Un coated flasks are mentioned on the figures and in the text. As mentioned earlier the concentration of iron limits the effect of copper. Figure 3 clearly shows that the growth of Pyramimonas 1 is protected from the toxicity of copper by large amounts of iron. The growth of control cultures of Pyramimonas 1 is the same with 1.2 x 10-SM Fe or 1.2 x l0- 6 M Fe. Addition of 4.4 x l0- 7 M Cu does not inhibit the growth rate of the culture containing an iron concentration of 1.2 x 10-SM Fe; a similar culture does not grow at all with the same concentration of copper and -6 only 1/10 of the iron (1.2 x 10 M Fe). The same phenom- enon, although less drastic, is observed with 1.2 x l0- 7 M Cu (Figure 3). Having resolved the problem of antagonism between iron and copper in the culture medium, another compli cation has to be resolved before a lag phase can be pre dictably induced in the cultures. The sensitivity of the cells to copper vary with their physiological state. For example, 2 x 10 3 cells/ml taken in exponential or even early in the stationary phase are not affected at all by the addition of 1.6 x l0- 7 M Cu while 2 x 10 3 cells/ml from the same stock studied 5 days later do not grow at all with the same concentration of copper. 124 For a given physiological state of the cells there is a narrow range of copper concentrations in which sublethal effects can be observed. Under it there is no effect of copper, above it increase in cell density and co 2 uptake per cell is reduced to negligible values. This range was established for 1.5 x 10 3 cells/ml taken at about their 3rd day of stationary phase. After 24 hours of incubation a plot of the cell densities relative to the control shows a two step "S" curve (Figure 4). The first "titration" -7 -7 step is located between 1.6 x 10 Mand 2 x 10 M copper. Not only is this a narrow window but as stated earlier it may shift left or right depending on the age of the cells and the cell density. Figure 4 clearly shows that concen trations between 2 and 3.2 x l0- 7 M Cu added to this par ticular inoculum promote a perfect lag phase with no cell growth over 24 hours. Having selected a range of sublethal copper concen trations, one can observe the effects on the cell growth over a few days. In the experiment described in Figure 5, (which is excerpted from figure 3) the flasks are not coated with silicone. The amount of copper in the medium may thus be decreasing with time due to adsorption on the walls and absorption by the cells. -7 1.2 x 10 M copper . in that case promotes a lag phase and appears to slow down the exponential growth. Concentrations of 2.0 x l0- 7 M and -7 4.4 x 10 M completely destroy the culture. 125 Figure 6 describes a very different pattern of copper toxicity. After a delay of about three days there is a sharp decline of the exponential growth rate in the flasks containing copper. A similar pattern was observed system atically by Sunda (1975) working with Thalassiosira pseudonana. This type of effect might only be observed at copper concentration (or activity) below that which promotes a lag phase or, more likely, when the inoculum is taken from an exponentially growing culture rather than one in the stationary phase as was customarily done in this study. Discussion The basic goal stated in the introduction is achieved: a lag phase has been induced in batch cultures of Pyramimonas 1. In the process some insights have been gained regarding two important questions about the lag phase phenomenon: How do the chemical parameters, in the medium, inter act in controlling the lag phase, and more speculatively, what is the biochemical nature of the lag phase? The results demonstrate that the effect of copper on cellular metabolism is dependent upon the concentrations of chelator and iron in the culture medium. The presence of EDTA per se does not appear necessary for normal cell growth: an exponential growth is observed immediately upon innoculation, with or without EDTA. 126 Barber (1973) established that above a certain size inoculum, addition of EDTA does not shorten the lag phase. The reverse reasoning probably holds true: above a certain inoculum size, (which is clearly a function of the species) removal of EDTA does not lengthen the lag phase. Despite the removal of EDTA, which should markedly increase the cupric ion activity, no toxic effect of the normal F/2 copper concentration is observed. This is due either to a cupric ion activity lower than expected or to a relative insensitivity of Pyramimonas 1 compared for example to Thalassiosira pseudonana (Sunda 1975). The higher con centration of iron in F/2 medium appear to be the cause of this apparent insensitivity since copper toxicity is dem onstrated in Figure 9 to depend markedly on the iron con centration. The most logical explanation is that the copper is effectively scavenged out of solution by ad sorption on the ferric hydroxide colloid, resulting in a decrease of the cupric iron activity. The surface chem istry of iron colloids depends on many variables such as the pH of precipitation and the age of the precipitate. This is very probably the origin of the protective effect of "aged" ferric chloride solutions: whether a precipitate forms in the stock solution or in the medium itself must change drastically the adsorption characteristics. The great interdependency of the critical chemical variables, their dependency upon subtle changes in exper1- 127 mental conditions and the variability of the trace metal impurities in reagent grade chemicals can easily account for the variations observed from one batch to another and some erratic results found, for example, in establishing a minimum concentration of iron to allow "normal" growth. In addition to being a function of the various chemi cal variables in the medium, copper toxicity is observed to be also a function of the age of the inoculum. Are then two separate, additive effects being observed -- one de pendent on the medium chemistry, the other on the cell physiological state -- or are these effects related to a common biochemical mechanism? Are there several kinds of lag phases or is it a unique physiological state? Is the lag phase induced here in the growth of Pyramimonas 1 similar to that commonly observed in the growth of other algal species? Although these questions cannot be answered by the results presented in this report, it is worth speculating that there exist a unique physiological state corres ponding to the lag phase. A possible mechanism linking copper toxicity and physiological state of the cells with the lag phase phenomenon would be as follows: the cells need to change their metabolic machinery before they can divide and grow exponentially. If copper has the possi bility to block this change, it will prolong a lag phase which might be otherwise so short as to be unobservable. 128 It is known that cells at the plateau phase and at the lag phase (for those that have one) usually release relatively large quantities of organics (Fogg 1965, Ignatiades and Fogg 1973). This can be compared with the observation that the addition of copper also induces re lease of organics (Steeman Nielsen et al 1970). It should be borne in mind that phytoplankton demonstrate wide differences in sensitivity to copper toxicity (Erickson et al 1970) and that Pyramimonas 1 appear to be one of the more insensitive species. An essential similarity between the lag phase induced here in the growth of Pyramimonas 1 and that normally exhibited by other cells is the depen dence upon the growth stage of the inoculum: Pyramimonas 1 cells in exponential phase require more copper to exhibit a lag phase than cells originating from the plateau phase. If we assume that there is but one kind of lag phase which is equivalent to a specific metabolic state the question that follows is: what enables a cell like Pyramimonas 1 to change more rapidly to a new physiologi cal state than other species and grow directly in a geo metric fashion? A discussion of metabolic differences is an extremely speculative exercise at this point as little is known be yond the identification of various physiological stages from D to L types cells described for Chlorella (Tamiya 1963). 129 Some information comes from the literature on higher plants. It appears that the synthesis and release of organics such as glycolic acid (on which considerable work has been done) is directly coupled with specific photo synthetic activity of what is called the c 3 cycle. Plants that use a c 4 cycle to synthesize sugars do not produce glycolic acid and are overall more efficient. Interme diates between pure c 3 and c 4 type have been found. Using the conclusions of work done on DDT sensitivity (Morel 1976) and on direct enzyme studies (Morris 1975) it might be reasonable to postulate that algal species exhibit one type of metabolism or another at a given stage of their growth. A c 4 type metabolism is much more efficient than a c 3 and does not waste energy through organic secretion. These cells may divide without delay. A c 3 system may be too inefficient for exponential growth and may demand a switch to c 4 metabolism before exponential growth can resume. Like in higher plants both systems may be present in any cell but in differnet pro portions. Additions of copper could interfere more markedly with cells that have predominantly a c 3 cycle: not only could it block directly a specific enzyme system, but it could slow down (or even completely inhibit) the synthesis of the enzymes involved in the c 4 cycle at the time a switch from c 3 to c 4 is needed. An algal species that 130 maintains a high population of c 4 cells, even at the plateau level, may exhibit no lag phase and a lower sen sitivity to copper than a species that has mostly c 3 type cells. These speculations which deal with the enzymatic machinery of the cells extend beyond the mere comparison of D and L type of cells. They would be better investi gated with the use of synchronized cultures and specific assays for the main parameters that characterize c 3 and c 4 cycles. 131 ~ E ' "' Q) u ~ ______ .--0- 10 5 10 4 lnocu I um taken from: o 3rd week of stationary phase • E xponen tia l phase ( Na tu r a I Se aw a te r Med i u m ) 10 3 ____ _,_____,_ _ __.___.'---___.__ _ _._____...., _ _._____. ________ ___ 2 4 6 8 Days 10 12 Figure I Effect of lnoculum Age on the Growth of Pyramimonas I 14 132 I-' w w 1/) sor ~ 0 0 I E :J - :J 30 u 0 C: - '+- 0 (l) 0\ 10 <( A o S tan da rd F / 2 me d i um 8 o No EDTA - aged iron C 6 No EDTA - fresh iron D '7 No EDTA - fresh iron in reduced ooncentration 6c 2,000 4,000 14 c O 2 Uptake ( CPM) 6,000 Figure 2 Influence of Fe, EDTA and Age of the lnoculum on the Photosynthesis of .El.!· l Q) u ~ 5 10 ~ 1.2xl0- 5 M l.2x I o-5M u Days Control 0 0 6 4 x 10-BM 1.2 x ,o- 7 M • .. 4.4 x 10- 7 M • • Expt. #I Expt.#2 Expt.#3 - Ex pe r i men ts # I , 2 , 3 d one w it h i n o cu I a from different cultures -No EDTA Figure 3 Antagonistic Effects of Iron and Copper on the Grow t h of Py r a m i m on a s I 15 134 ,-.., w lJ1 ~ E '- Q) 0. 3 (/) 2 Q) u X 103 ◄ Cell density of I nocu lum 2 3 4 x,o-7 Concentration of Copper Figure 4 Effect of Copper Concentration on the Initial Growth of Pyramimonas I D 7 l.2xl0- M Cu 2 x 10- 7 M Cu 5 10 15 Days Figure 5 E f f e ct of Copper on t he Grow t h of Py r. I 136 <l) u :ij: 1.2 x 10-? M Cu 10 3 ..___,__---'---....L..---1-----'----'-----'------'-----'-----'---.L.----L---'----' I 5 10 15 Days Figure 6 Another Effect of Copper on the Growth of Pyr. I 137 References Barber R.T. and J.H. Ryther (1969) J. Exp. Mar. Biol. Ecol. 3: 191 Barber R.T., R.C. Dugdale, J.J. Macisaac and R.L. Smith ( 19 71) Inv. Pesq. 3 5: 1 71 Barber R.T. (1973) in Trace Metals and Metal-Organic Inter- actions in Natural Waters, P.C. Singer ed., Ann Arbor Science, Ann Arbor, Michigan Berman T. and D. Holm-Hansen (1974) Marine Biology 28:305 Collyer D.M. and G.E. Fogg (1955) J. Exp. Bot. 6:256 Davey E.W., M.J. Morgan and S.J. Erickson (1973) Limn. and Ocean. 18:993 Droop M.R. (1961) Botanica Marina 2:231 Fitzgerald G.P. and S.L. Faust (1963) Appl. Microbial. 11:345 Fogg G.E. and D.F. Westlake (1955) Verh. Intern. Ver. Limnol. 12:219 Fogg G.E. and J.D.A. Miller (1958) Verh. Intern. Ver. Limnol. 13:892 Fogg G.E. (1959) Symposium of the Society of Experimental Biology 13:106 Fogg G.E. (1963a) in Physiology and Biochemistry of Algae, R.A. Lewin ed., Academic Press, N.Y. Fogg G.E. (1963b) British Phycol. Bull. 2:195 138 Fogg G.E. (1965} Algal Cultures and Phytoplankton Ecology The University of Wisconsin Press. Madison, Wisconsin Gadde R.R. and H.A. Laitinen (1974} Analytical Chemistry 46:2022 Hellebust J.A. (1965} Limnol. and Ocean. 10:192 Hellebust J.A. (1967) in Estuaries, AAAS publ. #83, G.H. Lauff ed., Washington, D.C. Ignatiades L. and G.E. Fogg (1973) J. Mar. Biol. Ass. U.K. 53:937 Johnston R. (1964} J. Mar. Biol. Ass. U.K. 44:87 Lockhart H.B. and R.V. Blakeley (1975) Envir. Science and Techn. 9:1035 Morel N.M.L. (1976} PhD Thesis, University of Southern California, Los Angeles, Calif. Morris I. (1975} Personal Communication Nalewajko C., V. Chowdhuri and G.E. Fogg (1963} in Studies on Microalgae and Photosynthetic Bacteria, Japanese Society of Plant Physiologists, Tokyo. Provasoli L., J.J.A. McLaughin and M.R. Droop (1957) Arch. Mickrobiol. 25:392 Riley G.A. (1943} Bull. Bingham Ocean. Collection 8:1 Spencer C.P. (1954} J. Mar. Biol. Ass. U.K. 33:265 Steemann Nielsen E., L. Kamp Nielsen and S. Wium-Anderson (1969} Phys. Plant. 22:1121 Steemann Nielsen E. ands. Wiurn-Andersen (1970) Marine Biology 6:93 139 Stumm W. (1969) personal communication to R.T. Barber Stumm W. and J.J. Morgan (1970) Aquatic Chemistry, Wiley Interscience, N.Y. Stolzberg R.J. and D .. Hume (1975) Envir. Science and Techn. 9:648 Sunda W.G. (1975) PhD Thesis, Woodshole Oceanographic Institution Tamiya H. (1963), Symposium of the Society for Experimental Biology 17:188 Tamiya H. (1966) Annual Review of Plant Phys. 17:1 Thomas J.P. (1971) Marine Biology 11:311 Watt W.D. (1966) Proc. Roy. Soc. Land. B. 164:521 140 NI LE MARIE L )UI M REL 1948 Born in Montpclli r, ran e 1965 Gracluat d from Ly Jc des au. -Claircs, ;r noble, Franc 1966 ertificat Preparatoire au . tude Medic lcs 1969 1969-71 (C.P.E.M.), Univcr itc de ;rcnobl , Fr- nc Biology, California State University, Los alifornia T ching A istant, I partm nt of Biological lif rnia, Los 1 n Univer ity of outh rn Angel 1969-7 Graduate . tudent, Univcr ity of outhern alifornia 1971 Biome

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Creator Morel, Nicole Marie Louise (author)

Core Title Action of DDT on two clones of a marine green alga Pyramimonas

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Biological Sciences

Degree Conferral Date 1975-10

Publication Date 01/12/1976

Defense Date 01/12/1976

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Algae,DDT (Insecticide),marine ecology,OAI-PMH Harvest

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC25323

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