THE UNIVERSITY OF MICHIGAN School of Natural Resources Department of Wildlife and Fisheries Technical Progress Report NUTRIENT CYCLING AND PRODUCTIVITY OF DYSTROPHIC LAKE-BOG SYSTEMS (PART A) Frank F. Hooper ORA Project 01644 under contract with: UoS, ATOMIC ENERGY COMMISSION CHICAGO OPERATIONS OFFICE CONTRACT NO. AT(ll1-)-1771 ARGONNE, ILLINOIS admi nistered through: OFFICE OF RESEARCH ADMINISTRATION ANN ARBOR December 1970

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TABLE OF CONTENTS Page SCOPE OF WORK 1 APPENDIX A. TRANSFORMATION OF HYPOLIMNETIC PHOSPHORUS BY A PERIPHYTON COMMUNITY 5 Methods 8 Uptake of Phosphorus Fractions 11 Transformation Rates 13 Discussion 15 Summary 16 References 17 APPENDIX B. MECHANISMS OF 32p UPTAKE BY INVERTEBRATES 19 Biological Transport of an Element in an Aquatic Ecosystem 21 The Mode of Radiophosphorus Uptake 22 Methods 26 Results 29 Direct Counting Experiments 29 Summary 50 References 51 APPENDIX C. DISSOLVED ORGANIC PHOSPHORUS COMPOUNDS OF A BOG LAKE ECOSYSTEM 55 Literature Review-The Organic Phosphorus of Aquatic Systems 57 Cellular Phosphorus Compounds 57 The Quantity of Phosphorus Fractions in Living Cells 60 Regeneration of Phosphorus From Organic Compounds 61 Identification of Dissolved Organic Compounds 66 Methods 67 Results 68 Discussion 70 References 71 APPENDIX Do CHEMICAL DATA, NORTH GATE LAKE, 1970 77 iii

SCOPE OF WORK December 1, 1969 - November 30, 1970 In the spring of 1970, I continued work on data from the in situ flowthrough experiments conducted during the summer of 1969, and I completed a manuscript describing this work (Appendix A). Mr. Imes continued experiments dealing with phosphorus uptake by the phantom midge Chaoborus. These studies substantiated previous evidence that bacteria were a major source of phosphorus uptake by Chaoborus and were probably important in the biological transport of this element in the lake. A manuscript summarizing these data is attached (Appendix B). 52 A second P labeling experiment designed to provide additional data on phosphorus transport and the phosphorus cycle of the upper water was conducted in early May. A mixture of 32P and rhodamine WT was injected into the 7M zone from a boat after the spring overturn. This experiment was less successful than the 1970 treatment in isolating the tracer in the bottom water. In 1969 some vertical mixing took place which moved small amounts of our tracer into the upper water. Thus there were two possible sources of 32p —(1) from Chaoborus transport into the upper water, and (2) from vertical mixing. The extent of the vertical mixing can be estimated from our rhodamine data. Hence it may be possible to quantitatively assign a fraction of activity to each of these two sources. An extensive set of data on the phosphorus were collected in spring and summer of 1970 after labeling. These included both stable and 52P activity (1) in the plankton, (2) in Chaoborus, (3) in Umbra (the only fish present), and (4) in the bog mat. Very few of these data have been compiled at the time of writing thEis report but are now being processed for computer analysis; hence most of the past summer's work has not been included. We have provided certain.: chemical data for the lake required by Professor Griffing in his analysis of the calcium cycle (Appendix D). To further evaluate the role of the bog mat in the regulation of the chemical economy of the system, we collected several sets of samples from the following locations: (1) The mat-water interface, and (2) within the mat at distances of 3, 5, 8, and 15 meters from the open water. To collect a vertical series of uncontaminated water samples at each of these locations, we devised a simple but effective device for sampling. It consisted of a section of heavy plastic tubing sealed at one end (Fig. 1). The sealed end of the tube was easily forced into the bog mat. At the site of the tube a few inches behind the sealed end, we provided a small window covered with plastic screening. During penetration of the mat, this window was sealed by 1

an inflated rubber balloon. The balloon was deflated when the sampler was in place at the proper depth. This allowed the sample tube to fill with water. We collected samples within the bog mat down to a depth of 4M with this device. Filtered and unfiltered samples were analyzed for stable phosphorus, 52p activity, and for soluble and total iron using the bathophenanthroline procedure. Representative data from this work are included (Appendix D). Work continued on the identification of bog organic phosphorus compounds O During the spring I completed a literature survey dealing with the identification, origin, and regeneration of organic phosphorus within freshwater systems, Mrs. Kay Terry perfected a new procedure for isolating and analyzing the phosphorus compounds, and in August we used this procedure in the analysis of samples from three locations within the basin. This literature survey and the results of the past summer's sampling are reported in a third report (Appendix C). 2

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APPENDIX A TRANSFORMATION OF HYPOLIMNETIC PHOSPHORUS BY A PERIPHYTON COMMUNITY Frank F. Hooper School of Natural Resources The University of Michigan 5

TRANSFORMATION OF HYPOLIMNETIC PHOSPHORUS BY A PERIPHYTON COMMUNITY Frank F. Hooper School of Natural Resources The University of Michigan The redistribution of chemicals from the hypolimnetic zone during the vernal overturn is vital to the biological productivity of lakes. Gross features of this process have been adequately described (Welch, 1952; Hutchinson, 1957); however, certain aspects have been poorly investigated. These are (1) the nature of the chemical transformations that take place when organic phosphorus compounds originating in the hypolimnion are cycled in the surface water, and (2) the rate at which orthophosphate and organic residues arising in the hypolimnion are incorporated into the plant biomass of the surface water. To determine the fate of hypolimnetic phosphorus carried to the photosynthetic zone, we conducted a series of experiments in July, 1969, in which bottom water of a bog lake, labeled with 32p, was pumped to the surface, where it was passed through a plastic tank containing a culture of periphyton and bacteria. This paper describes the transformations of the hypolimnetic phosphorus carried out by this system. In our analysis of the phosphorus cycle of North Gate Lake, a small bog lake in northern Michigan, the dissolved organic phosphorus (DOP) was of particular interest because of its sporadic occurrence in the lake both in time and in space. During the summer of 1968 there was an abundance of DOP at all depths below the thermocline. That year, thermal stratification was normal and there appeared to have been some mixing throughout the entire water column throughout the entire summer. In the spring of 1969 there was an incomplete vernal overturn and a strong thermocline developed which largely prevented excursions of surface water into the deeper layers. This year, DOP was absent or very low in both the 6M and 7M strata during most of the summer. However, it increased strongly at all depths in late July when there apparently had been vertical mixing. This pulse of DOP was short-lived, however, and soon decreased or disappeared at all depths, To test the hypothesis that within the anaerobic zone of the lake DOP is produced by the aeration arising from mixing, was one purpose served by the present experiment. The vernal overturn may take place in a few days. It is often difficult to analyze the processes taking place because the critical biological conversions may occur not only in a short span of time, but also, within restricted zones of the lake basin. By artificially pumping hypolimnetic water to the surface, we have previously demonstrated its effect upon primary production (Hooper, Ball, and Tanner, 1953). Of somewhat more fundamental interest, however, is the fate of specific phosphorus residues and their conversion into the organic phosphorus of the biota. 7

Recently there has been wide-spread interest in de-stratification of lakes and reservoirs for the purpose of (1) improving water quality, and (2) providing benefits to the fisheries (Fast, 1968; Mayhew, 1963; Koberg, 1964; Johnson, 1967). During aeration large quantities of nutrients are released and de-stratification represents a form of auto-fertilization. In view of the practical interest in redistribution of hypolimnetic water, it is of value to explore more deeply the transformations of phosphorus that take placeO Methods The tank used in these experiments contained four compartments separated by plastic partitions. During passage, the water made contact with periphyton substrates suspended in the two center compartments as well as with the plastic walls of the container. Water was pumped through the system by means of an aquarium air-lift located in the downstream compartment (Figure 1). Flow from the air-lift entered a plastic tube which returned the water to the 5M depth. As water was removed from the downstream compartment, it was replaced by a flow of 7M water entering the upstream compartment. The flow could be varied within wide limits; however, during experiments described in this paper and during the equilibration process a flow of 60 ml/min was maintained. At this rate the residence time of water in the system was 9 hr. Each compartment of the tank was supplied with a flow of compressed air. This aerated the water and circulated the flow during its passage through each unit, Three weeks before our first experiment the system was filled with 7M water and innoculated with a net plankton sample collected from the surface water, The innoculum consisted of the catch made by a 20 4 net when 50 1 of surface water were poured through at a rate of 5 1/mino The plankton of the lake at this time was sparse and the innoculum was estimated to contain less than 5 fg (dry weight) of plankton. Qualitatively it consisted of a variety of algae, including diatoms, dinoflagellates, chryosomonads, blue-green and green algae, and a few copepods, The intake of the tank system was screened with 20 { nytex to prevent loss of zooplankters, Within a week after the flow of 7M water was started, a dense growth of periphyton developed on the substrates and the walls of the tank. During the second and third week the growth increased. By the start of the experiment, the dry weight of the periphyton crop was estimated to 2789 p-gor 558 times the initial innoculum. Measurements of phosphorus transformation by the system were first made on July 3, three weeks after the unit was set in operation. A second set of measurements was made on July 10. On each of these dates two sets of measurements were made. The first was made at 6 aom., after the system had been operating in the dark for approximately 9 hro The second set was made at 3 pom. after approximately 9 hr. of sunlight. Measurements of the affluent and 8

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and effluent flows were made on aliquots from 3-1 samples siphoned from the intake and discharge. Measurement of orthophosphate, dissolved organic phosphate (DOP), and particulate unreactive phosphorus (PUP) were made on three 100-ml aliquotso Radioactivity was measured by evaporating to dryness 50 ml of the solution used for stable phosphorus measurement and counting with a low background beta system. Small but significant differences in the stable phosphorus of filtered samples from 7M before and after digestion indicated that DOP was present. To determine whether or not the DOP of the affluent and effluent were labeled, we extracted the heteropoly blue complex with 50 ml of hexanol after stable phosphorus had been measured, using the procedure given by Strickland and Parsons (1965). We then evaporated the aqueous phase to dryness and counted the residue. Since we did not detect a significant amount of activity in the water phase of the affluent samples we concluded that DOP of the 7M water was unlabeled. A small but detectable amount of activity present in the effluent indicated that labeled DOP was produced by the system, Measurements of stable and radioactive phosphorus of samples from the 7M stratum were made at frequent intervals for a period of three months before transformation experiments were undertaken. During this period orthophosphate varied from 66 to 84 ig/1. The specific activity of filtered samples (corrected for decay) increased from 180 pci/~g under the ice on March 21 to a maximum of 234 pci/jg at the time of the spring overturn. After the overturn, specific activity fell slightly and remained close to the pre-overturn level. Mean specific activity values during May and June were not significantly different from the March 21 values. The filterable phosphorus fraction of the 7M stratum therefore appeared to be in isotopic equilibrium. Direct calculation of the specific activity of the particulate phosphorus was not possible since in many cases measurements of activity and stable phosphorus were made on sets of samples collected on different dates. The 14 sets of measurements of particulate phosphorus between March 21 and June 29 can be compared with 11 measurements of particulate activity. In these measurements the percentage of the total stable phosphorus removed by filtration (24.2 ~ 9.1%) agreed well with the percentage of activity removed by filtration (25.6 ~ 8.1%). Although there was considerable sample-to-sample variation in the percentage of both particulate activity and particulate phosphorus, these variations were random and there were no detectable trends during this period. This agreement in the percentage of particulate activity and the percentage of stable particulate phosphorus indicates that the specific activity of particulates was relatively stable. At the time of the transformation experiments, the flora of the system consisted chiefly of bacteria and the green alga Oocystis. Two species of epiphytic diatoms and the chrysophycian Dionbryon were also present. Counts of periphyton made at frequent intervals indicated that forms other than 10

Oocystis and large numbers of unidentified bacteria were qualitatively unimportant. Uptake of Phosphorus Fractions A major fraction of the orthophosphate was removed by the tank system. This fraction varied from 60 to 94/o (Table I). A larger fraction of orthoTable I. Uptake (-) and export (+) of the fractions of labeled fractions of 7M water during passage through aerated tank at lake surface containing bacteria and periphyton. (Averages of 3 samples collected from affluent and effluent flow.) July 3 July 10 a.m. p.m. a.m. p.m. F Net Net Net Net F'raction tg/i Neg/1l change; g change g/l change g/ change or or or or o a affluent r affluent r affluent affluent pci/i pci/1 pci/i pci/1 cone. conc. conc. conc. Stable Phosphorus (ig/l) Orthophosphate -52 0.60 -82 0.82 -77 0.83 -94 0.86 Dissolved Organic +18 6.00 +12 4.0 +23 3.3 +26 6.5 Particulate +25 o.96 +11 1.1 +27 0.64 +24 2.61 Storage -11. 096 -59 0.52 -27 0.19 -21 0.16 Estimated labeled dissolved phosphorus in effluent* 24 17 9 6 32p Activity (pci/1) Dissolved -79 0.89 -75 0.99 -35 0.92 -36 0.92 Particulate + 7 2.9 + 7 7.2 +32 1.9 +14 1.8 *For calculation, see text. phosphate was removed from the afternoon samples than from the corresponding morning samples on both July 3 and July 10. The difference between the means of morning and afternoon samples was significant at the 950 level on both dates. 11

The DOP concentration was from 3 to 6 times greater in the effluent than in the affluent, but the difference in production of DOP between morning and afternoon samples was not significantly different. The specific activity of the particulates entering the system was not significantly different from the specific activity of the filterable fraction of the affluent (Table II). This confirmed the earlier indications that the water and the solids of the 7M stratum had arrived at isotopic equilibrium. Table II. Specific activity of affluent and effluent fractions of flow-through system during experiment of July 3 and July 10 (pci/Ig). Affluent Effluent Date Dissolved Particulate Dissolved Particulate Mean and S.E. Mean and S.E. Mean and S.E. Mean and S.E. July 3 a.m. 0.97 + 0.14 o.94 + 0.065 0.27 ~ 0.092 0.75 ~ 0.19 p.m. 0.75 + 0.065 0.11 ~ 0.11 0.96 ~ o.014 July 10 a.m. 0.52 ~ 0.076 0.59 ~ 0.195 o.o4 + 0.02 1.59 + 1.2 p.m. 0.50 ~ 0.061 0.55 ~ 0.033 O.o6 ~ o.o6 1.98 + 1.2 In two experiments on July 3 the specific activity of particulates in the effluent was not significantly different from the activity of entering filterable or particulate fractions. However, on July 10, the mean specific activity of effluent particulates was threefold greater than that of the entering particulates. Considerable variation, however, was evident in the particulate activity of the effluent. On this date, despite this large difference in means, the mean specific activity of the afternoon effluent samples was not significantly different from the mean of the affluent particulates in either the morning or afternoon (Table II). The mean of the morning samples of effluent particulates was significantly different from the morning and afternoon affluent. Thus the specific activity of the soluble fraction decreased during passage through the tank. All effluent means were significantly lower than affluent means at the 95% level of confidence or above. The decrease in specific activity of the orthophosphate passing through the system can be either (1) the result of contamination from an undetermined source of unlabeled phosphorus, (2) production of unlabeled orthophosphate by discriminatory breakdown and release of an unlabeled fraction of particulate phosphorus entering the tank, or (3) an unlabeled polyphosphate or other complex inorganic phosphorus compound present in the 7M water that was hydrolyzed to orthophosphate during analysis. All of the orthophosphate should 12

have been labeled, however a complex inorganic phosphorus component would not be labeled. Such a fraction can be hydrolyzed by the acid molybdate reagent during analysis (Olson, 1966) and would give high values of orthophosphate in the affluent and effluent. This fraction would have been less susceptible to uptake than the orthophosphate fraction and if it appeared as unlabeled phosphorus in the effluent would give an apparent decrease in specific activityo Since a major fraction of soluble activity was removed, a small change in unlabeled phosphorus would give a large decrease in specific activity. This fraction would be less susceptible to biological uptake than orthophosphate, and would give; an increase in the percentage of unlabeled soluble phosphorus in the effluent and an apparent decrease in specific activity. Monitoring the 7M water before the experiment did not indicate any additions of unlabeled phosphorus as required by alternative (1) above. To assess alternative (2) above, I calculated the expected amount of unlabeled compound in the effluents as follows: 32P e OP OP - - x OP U e 52 a a where: OPU = the unlabeled orthophosphate fraction, OPe = the effluent orthophosphate, OPa = the affluent orthophosphate, 32pe = the soluble activity of the effluent, and 5pa = the soluble activity of the affluent. These estimates indicated that on July 3 the expected unlabeled fraction in the effluent was greater than the quantity of particulate phosphorus entering the system (Table I). Thus alternative (2) does not appear likely since at least a part of the affluent particulate phosphorus was labeled and, had a specific activity that was not significantly different from the soluble fraction (Table I)o Alternative (3) is the most attractive explanation. Labile phosphorus fractions were separated from the 7M stratum in 1969 and in 1970. In 1970 this fraction was separated by column chromatography and recovered by gradient elution with formic acid. It appeared as a UV absorption peak distinct from orthophosphate that corresponded to polymers of orthophosphate. It was hydrolized by 1 N HC1 and it comprised 36% of the inorganic phosphorus of the 7M stratum (Hooper and Terry, 1970). Transformation Rates The system removed virtually all of the orthophosphate and utilized this phosphorus in the production of DOP and PUP. Production of DOP was in all cases about equal to production of PUP; the mean increase in DOP was not significantly different from the mean increase in PUP in any of the four 15

(a) boI hD i PcT -H 1 H -l H oD 0 a)! t'-.ON 0-P b 0 0 0 rc1 h * 40 I i \ X 0 ot m t C( *Hd D H;rd CQ Er. O 4- A 0D d P-1 0 I0 l b 0 0 H d -t k O cn - no. O f- Co n% b.>0. r r40 O I D O. 0 I t rn j i I rd O\ cO -U 1HecO Oc~ vP p P- O 0 0 bO I O ~ H 1 ft ^.a). 0Q C 0 0H 0C O N *H. P H P (D (^ *HN bO a4 r) a 0 0 Od u 0 Ed Od co O — CH - CH H: 4 a)l % CHP4 -P r"d ()'O C HM l w O O ON ( H ^^^ oj 20 CU H HL, g r-1 0 0 -p-p T ]0 U) 1CH 4 U) (D~ bO^^ r^! +3 0~~~~~~~~~~~~c3?1r uS cQ ^ ^ 0 O^ S -P 0 ~ ~0 ft3 H- t- ^D ~ ~ C F3 1i

experiments. This close agreement is perhaps purely fortuitous. Rates of transformation of DOP into organic compounds were calculated from uptake measurements (Table III), Rates were expressed in terms of the biomass of periphyton in the tank system. Biomass estimates used were those made after the experiment of July 3. Further biomass measurements were not made on July 11 because substrates removed one week later (July 20) had approximately the same biomass as the July 5 substrates. The uptake rates of orthophosphate varied from 0,1 to 0.127 4ig P/hr/mg of periphyton biomass. This uptake equaled 1.38 to 1.76% of the phosphorus present in the system per hour (Table III). From 21 to 71% of the phosphorus uptake was stored in the tank and apparently went into new growth of cells. The remaining fraction (0.51 to 1.31% of phosphorus present) was incorporated into organic compounds of the export. In addition to DOP, the export contained some living cells, but consisted chiefly of dead cells and debris with its covering of epiphytic bacteria. Thus the export consists of phosphorus being turned over within the system through the autolysis and replacement of old cells as well as phosphorus released by metabolic turnover. Discussion Most of the experimental systems used for the analysis of phosphorus cycling in the past have either involved the addition of a tracer (Hutchinson and Bowen, 1950; Hayes, et al., 1952) or have enriched the system by the addition of the stable element creating a perturbation which provides the basis for a dynamic analysis. Transformations of a chemical cannot be studied effectively in a standing water system when the system is under steady state conditions, even if a tracer is used. In a flow-through type system, the recycling is minimized and the net change between import and export provides a measure of total system response. Tracer experiments using small microcosms such as aquarium systems have identified the direction of movements of tracers between pools of the stable elements within the biological system. Experiments with 52p such as those conducted by Hayes and Phillips (1958) and Phillips (1964) have indicated the sites of active exchange, but are of doubtful validity when used as a measure of uptake and flow rates because the movement of a tracer is not necessarily a quantitative measure of the movement of the stable element when there is the possibility of absorption and isotopic exchange. Rates of uptake measured under these circumstances invariably show a very rapid flow of 32p into bacteria of the water phase or into the bottom substrate. Flow rates calculated under these conditions assume uni-directional movement of the tracer and stable elements. Such a model does not allow for isotopic exchange arising from concentration gradients of the tracer. 15

Turnover rates of the order of 5 min suggested by Hayes and Phillips (1958) for aquaria containing algae and bacteria are an order of magnitude greater than the rates indicated by the present study (approximately 1-2% of the total phosphorus per hour). This turnover rate is greater than that reported for the marine copepod Calanus (10%/day; Conover, 1961), but less than excretion rate for mixed marine plankton (about 1000/day; Pomeroy, et al., 1963) and freshwater plankton (30 to 1200o of body phosphorus per day; Barlow and Bishop, 1965). Figures from the tank study must include the phosphorus released by cell replacement in addition to that arising from metabolic turnover. In this study, recycling cannot be excluded but any recycling taking place must have been principally reutilization of orthophosphate. It is doubtful if the flowthrough time (9 hr) would have permitted significant recycling of particulates and DOP. The production of DOP by the aerated tank tends to substantiate the hypothesis that DOP is produced from orthophosphate whenever bottom water is aerated. Phillips (1964) and Hayes and Phillips (1958) found that bacteria produced DOP during a decline in cell numbers but not during the growth phase. Rates of production in the present study presumably represent an equilibrium condition when both senescent and growing cells are present. Summary During recycling of labeled hypolimnetic water through a tank containing periphyton suspended at the surface, there was nearly complete uptake of the labeled orthophosphateO The smaller uptake of stable inorganic phosphorus compared to the corresponding uptake of 32P indicated that an unlabeled and notiexchangeable complek inorganic phosphate fraction was present in the hypolimnetic water. When the tank system operated under approximately steady state conditions, it removed between 89 and 99% of the hypolimnetic orthophosphate and exported approximately equal -quantities of DOP and PUP. Uptake rates ranged between o.38 and 1.76% of the system's phosphorus per hour. Phosphorus was exported from the system as DOP a:d as living and dead periphyton and bacteria at rates of from 0.51 to 1.51% of the system's phosphorus per hour. 16

References Barlow, J. P., and J. W. Bishop. 1965. Phosphate regeneration by zooplankton in Cayuga Lake. Limnol. Oceanog., 10(suppl.):R15-R24. Conover, R. J. 1961. The turnover of phosphorus by Calanus finmarchicus. Addundum to: Marshall, S.M., and A. P. Orr. 1961. On the biology of Calanus finmarchicus. J. Mar. Biol. Assoc. U. K., 41:484-488. Fast, A. W. 1968. Artificial destratification of El Capitan Reservoir by aeration. Part I. Effects on chemical and physical parameters. California Dept. of Fish and Game. Fish Bull., 141. Hayes, F. R., J. A. McCarter, M. L. Cameron, and D. A. Livingstone. 1952. On the kinetics of phosphorus exchange in lakes. J. Ecol., 40:202-216. Hayes, F. R., and J. E. Phillips. 1958. Lake water and sediments: IV. Radiophosphorus equilibrium with mud, plants, and bacteria under oxidized and xeduced conditions. Limnol. Oceanog., 5:459-475. Hooper, F. F., R. C. Ball, and H. A. Tanner. 1955. An experiment in the artificial circulation of a small Michigan lake. Amer. Fish. Soc., Trans., 82:222-241. Iooper, F. F., and K. Terry. Dissolved organic phosphorus compounds of a bog lake ecosystem. Tech. Prog. Rep., submitted to A. E. C., Nov. 1970. Hutchinson, G. E. 1957. A treatise on limnology. John Wiley & Sons, New York. 1015 PP. Hutchinson, G. E., and V. T. Bowen. 1950. Limnological studies in Connecticut. IX. A quantitative radiochemical study of the phosphorus cycle in Linsley Pond. Wash. Dept. Fish., Fish. Res. Pap., 2:5-15. Johnson, R. C. 1967. The effects of artificial circulation of a thermally stratified lake. Wash. Dept. Fish., Fish. Res. Pap., 2:5-15. Koberg, G. E. 1964. Elimination of thermal stratification in Lake Wohlford, California. U. S. Geol. Surv., Prof. Pap., (501-D):D 190-D 192. Mayhew, J. 1963. Thermal stratification and its effect on fish and fishing in Red Haw Lake, Iowa. Iowa Cons. Comm., Biol. Sec. (April):1-24. 17

Phillips, J. E. 1964. The ecological role of phosphorus in waters with special reference to microorganisms. In: Principles and applications in aquatic microbiology (Huekelekian, H., and N. C. Dendero, eds.). J. Wiley & Sons, N. Y. pp. 61-81. Pomeroy, L. R., H. M. Mathews, and H. S. Min. 1965. Excretion of phosphate and soluble organic phosphorus compounds by zooplankton. Limnol. Oceanog., 8:50-55. Strickland, J. D. H., and T. R. Parsons. 1965. A manual of sea water analysis. Fish. Res. Board of Canada., Bull., 125:37-53. Welsh, P.S. 1952. Limnology. McGraw-Hill Co., N. Y. (2nd Ed.), 538 pp. 18

APPENDIX B MECHANISMS OF 32P UPTAKE BY INVERTEBRATES David G. Imes School of Natural Resources The University of Michigan 19

MECHANISMS OF 32p UPTAKE BY INVERTEBRATES David G. Imes School of Natural Resources The University of Michigan Laboratory experiments were performed with aquatic larvae of Chaoborus americanus, a midge, in an attempt to determine the mode of radiophosphorus uptake. The results of these experiments to date will be presented in this paper. Also, I will review the literature dealing with biological transport of phosphorus in aquatic systems; discuss the problem of radiophosphorus uptake-ingestion, absorption, and adsorption by freshwater invertebrates involved; and discuss the techniques that I used in laboratory experiments in relation to the techniques used by other workers for similar problems. Biological Transport of an Element in an Aquatic Ecosystem Hasler and Likens (1963) have summarized earlier work on biological transport, and from their research on iodine state: It seems reasonable, therefore, that if aquatic organisms can take up and transport trace elements such as radioiodine, they certainly would be able to concentrate and transport appreciable quantities of the radionuclides of the more abundant protoplasmic constituents. Thus, biologically significant quantities of radionuclides could be transported from the deep and relatively inaccessible part of a lake to the surface and thence to the terrestrial environmento The biological transport of an element such as phosphorus could be important because it might make a lake more productive. Phosphorus from the bottom of the hypolimnion could be taken to the epilimnion where it could be used by phytoplankton and bacteria. Conversely, phosphorus can be transported from the epilimnion to the hypolimnion or to the terrestrial environment, thereby decreasing the productive potential of the lake. Movement of phosphorus from the hypolimnion to the epilimnion is evidently more exceptional.: Aquatic insects appear to be of the greatest importance in the transport of nutrients from the hypolimnion to the epilimnion and to the terrestrial environment. Since the mass of adult dipteran larvae emerging from a lake is almost entirely derived from the lake bottom, Vallentyne (1952) believed that a depletion of the chemical energy of the lake basin must result. He therefore attempted to determine the quantity of chemicals removed by insects and found that from July 1 to July 15 the loss of phosphorus from an Indiana lake basin was 21

0.15 mg P/m2/day. This figure cannot be used to compute an annual loss. Chaoborus was one of the insects responsible for phosphorus removal. Although Vallentyne (1952) found a greater rate of phosphorus sedimentation, the removal of phosphorus by insects could be important in small lake systems containing a high density of insects. Whitten and Goodnight (1969) studied accumulation of radiophosphorus by tubificid worms from water, bacteria and sediment. Because of their abundance in some ecosystems they believed that tubificid worms may have ain important part in the transfer of radioactive phosphorus from water and detritus to other components of the ecosystem such as fish. These worms acting in conjunction with bacteria. recycle radiophosphorus from detritus and sediments. In stratified lakes with an oxygenated hypolimnion, fish might transport phosphorus from the hypolimnion to the epilimnion by feeding on tubificids and other benthic organisms in the sediments. If aquatic organisms are important in nutrient cycling, further studies should be undertaken to ascertain the magnitude and mechanisms of their nutrient transport. Without labeled elements, it is difficult to determine the source of a chemical within the basin. A labeled element in part of a lake system, such as the hypolimnion or epilimnion, might easily indicate whether certain organisms are obtaining a certain element in these areas. The Mode of Radiophosphorus Uptake The problem of determining how an organism becomes radioactive often is difficult even though only a few possible mechanisms can be involved. Radiophosphorus may be taken up by an aquatic organism via absorption through membranes, via ingestion of food or inert particles, or via adsorption to exposed surfaces. External, as well as internal, bacteria of the larger aquatic organism may become radioactive. Determination of which of the possible mechanisms involved in radiophosphorus uptake occurs will probably not be made with certainty until the mechanisms themselves are better understood. Determination of the movement of a stable element from data of a radioisotope is often difficult. Uptake of radiophosphorus by an organism only means that some radioactive atoms have been taken into the organism's phosphorus "pool"; it does not necessarily indicate that a certain quantity of stable phosphorus has also entered the organism's phosphorus "pool." Some scientists have probably incorrectly calculated the movement of stable phosphorus from one "pool" to another from their radioisotope data. The reason for the error is that a steady state distribution of an element usually exists between aquatic organisms and their environment; with an addition of a radioisotope in one part of the system, the radioisotope will tend to reach isotopic equilibrium with the other part(s) of the system. However, isotopic equilibrium can 22

be reached via isotopic exchange, in which the total content of the element in the organism is not affected, but there is replacement of the stable isotope by its radioisotope or vice versa (Polikarpov, 1966). At various times certain phosphorus pools, or individual parts of the pool may have a net uptake of phosphorus. Phosphorus may be incorporated into insoluble tissue components, but there is no way of knowing if radiophosphorus atoms have been used as raw material for synthesis, or if the atoms have merely been partners in exchange reactions. Waddington (1959) states that "atoms may be utilized for the de novo synthesis of some fixable compound, such as a protein. But secondly, there is the possibility that the atom may enter such a compound as a consequence of an'exchange reaction' in which parts of an already-formed macro-molecule become liberated from it and their place taken by a similar atomic group, containing the tracer, without any increase in the macro-molecular species " Organisms become radioactive by adsodrption when the radioactive substance physically adheres to the Quter surface of the organism (Seymour, 1959)> The outer surface also includes the surface of internal organs. During absorption the material passes through a living membrane; absorption of a substance may occur externally or internally. Absorbed materials do not have to be assimilated in tissues and may remain within an organism only temporarily. Absorption of a material can occur against an electrochemical gradient via active transport, or it can occur passively via diffusion. The material may have been brought into the digestive system selectively or by chance; the material may be in live or dead organisms, soluble organic matter, or inorganic mattero It may pass through the animal without being changed and/or used. An animal may appear to be radioactive, when in actuality the radioactivity may be in some foreign matter in its gut. The ingested material could also be adsorbed to internal organso The presence of ingested material in itself does not indicate that the organism uses the material. Whittaker (1961) gave an interesting appraisal of the problem of phosphorus uptake in organisms: "When, as in this experiment and presumably in most natural water bodies, surfaces are coated with films of microorganisms, then adsorption onto and uptake by these organisms are difficult to separate. Of surface effects in general it may be said that adsorption, absorption, and other processes are so interrelated that they cannot be sharply distinguished in practice, and perhaps should not be regarded as distinct and separate in their significance in the functioning ecosystem." Rice (1953) presents two ways in which phosphorus may enter a cell. First, by diffusion of inorganic orthophosphate through the cell membraneo In this case, the inorganic phosphate then combines with the intracellular orthophosphateo Inorganic orthophosphate is considered the source of phosphorus for the 23

various organic phosphates in the cello The second possible method is the entry of phosphorus into the cell through esterification at the cellular interface The breakdo of organic hosphte would then produce intracellular inorganic orthophosphate. Working with yeast, Kamen and Speigelman (1948) concluded that the primary mechanism of the entrance of phosphate is by esterificationo Rice (1953) thought that the mechanism of entry was important in phosphorus exchange studies because it might require that phosphorus remain on the surface of the cell for some time. Phosphorus would probably not adsorb on cells if it could diffuse into the cello Esterification, however, might require that phosphorus remain on the surface of the cello Rice (19535) states that "there has not been sufficient consideration of the part adsorption plays in phosphorus exchange in microorganisms." He could not distinguish the phosphorus exchanged from the cell surface from that exchanged from inside the cells. Ussing (1947) proposed that one-to-one exchange of ions across a membrane could occur by exchange-diffusion, in which the ions are supposed to saturate a carrier, in combination with which they pass across the membrane which they cannot traverse in the free state. Mitchell (1954) states that an enzyme may be that carrier. Roberts and Roberts (1950) found one-to-one exchange of inorganic radiophosphorus occurred across the cell membrane of Bacterium coli. They found the cell membrane freely permeable to phosphate ions, but that the internal inorganic phosphate was not free; it was adsorbed on a number of acidlabile sites within the cells. All of the mechanisms involved in moving phosphorus in and out of cells may be considered part of the transport processo The transport mechanism for phosphorus going into a cell is not necessarily the same for phosphorus coming out of the cell. Christensen (1962) defines transport as "the mode by which a solute passes from one phase to another, appearing in the same state in both phases." Conway (1954) states that "active transport across a membrane connotes movement of the solute or ion across the membrane dependent on the activity or energy change of another system. Passive transport is equivalent to free diffusion or'exchange-diffusion,' where the energy of the net movement comes from the same system. " However, active transport of some material may set up a gradient for passive transport of some other material. Active transport is generally defined as transport that takes place against an electrochemical gradient. In passive transport an electrical or chemical gradient may exist so that material moves "downhillo." The diffusion rate is determined by the nature of the membrane and by the concentration gradient. Entry into cells of ions against a concentration gradient (i.e., active transport) requires energy from metabolic processes (Stumpf, 1952) Since phosphate.molecules.may be involved at several points in the process of energyyielding reactions required to drive the active transport, studying phosphorus transport is difficult (Mitchell, 1954)o 24

If an inorganic phosphate is incorporated into an organic compound at the plasma membrane of a tissue, and then a few hours later broken down into inorganic phosphate, true transport has not taken placeO Christensen (1962) would consider this as "functional compartmentalizationo" If inorganic phosphate enters a cell by forming ATP and, successively, one or two other phosphoryl compounds before appearing seconds later as inorganic phosphate, then transport does occuro In this transport a phosphoryl translocation may prove to be the decisive stepo The time lag is what differentiates transport from compartmentalization. Conover (1_961) states that "radioactive phosphorus must be presumed to pass through several different physiological or chemical'pools, each with a different turnover time, before an equilibrium is approached" He found evidence for an additional phosphorus pool in;termedi.a-te between external phosphorus and the large, stable pool of body phcsphorus, Undoubtedly phosphorus passes through the membranes of organisms in several different wayso There is a difference in the rate of radiophosphorus exchange among different animal tissues (Greenberg, 1952) It has been. demonstrated that there is no chemical exchange between the inorganic radiopphosphrus and atoms bound in organic compoundso Incorporation of radiophosphorus into these compounds must be through metabolic reactions (Chipman, 1959; Gourley, 1952) No methods are now known that clearly differentiate absorption by metabolic T processes and adsorption by physical and ion-exchange phenomena (Cushing and Watson, 1968)> Adsorption may also occur within a cell as well as on t4he outer surface of an organismo When considering radiophosphorus uptake by invertebhtates, the role of bacteri.a must be consideredo In experiments with the beach' flea. Gamnarus, HEarris (1957) found that the absence of bacteria inhibited radiophosphorus absorptiono Hayes (1963) states, "Evidently direct absorption of inorganic phosphorus does not occur appreciably either through the body wall, intestine, or gills.o.higher invertebrates obtain their food by digestion of pa:rticulate matter rathhe r than by direct absorption of inorganic or organic solion Bond (1955) conluded that in general marine invertebrates were impermeable.-to water, salts, and organic soluteso The only way for such. organisms to obtain radioactive phosphorus is by ingestion of water or particulate material. Harris (1957) believed that Gammarus obtained radiophosphorus from bacteria. Stuart, McPherson, and Cooper (1935) found that the cladoceran Morina macrocopa was unable to subsist.n dissolved organic material, but could be cultured indefinitely on living bacteriao Fox (1952) reports that some crustaceans are known to swallow water and thus may absorb some phosphate. However, for most invertebrates phosphorus seems to be obtained from foodo Johannes (1964b) found uptake of 32P by sterile amphipods (Lembos intermedius). Nonsterile animals, however, had greater uptake than sterile ones. The medium used by the nonsterile animals was initially sterile, so there appeared to be no ingestion of labeled bacteriao Radiophosphorus could have been taken up by exchange between the amphipod and the medium, however, Johannes' results suggest that a large fraction of the radiophosphorus taken up by the 25_

nonsterile amphipods was by way of intestinal bacteria, thus making the phosphorus available to the animal. However, Rigler (1961), working with Daphnia magna, attributed the.uptake of inorganic radiophosphorus to bacteria on the exoskeleton, and concluded that this phosphorus could not be used by the cladocerans. Both Rigler and Johannes found that almost 990 of the phosphorus taken up by the animals was obtained from their food. Hassett and Jenkins (1951) found that mosquito larvae obtained most of their radiophosphorus from food, but that in the fourth instar the anal papillae functioned in the absorption of phosphates (as well as chlorides). Although the anal papillae may only be an auxiliary path for phosphorus uptake, they apparently are capable of phosphorus absorption. The reasons for the concentrations of radioactive substances by organisms has been summarized by Davis and Foster (1958) as follows: (1) The mode of uptake which includes adsorption to exposed areas, absorption into tissues, and assimilation of ingested material. (2) Retention, which is the function of the biochemistry of the particular elements and components involved, the site of deposition, the turnover rate, and the radioactive half-lifeo (3) The mode of elimination, which may involve ion exchange, diffusion, excretion, and defecation. Methods In February several thousand Chaoborus larvae, mainly Chaoborus americanus, were collected from George Pond, on the Edwin So George Reserve in Livingston County, Michigan. These larvae were kept in a jar and in two aquaria at 40~F in a cold room. The larvae were not exposed to light. Beginning on February 26, 1970, several experiments were undertaken to study radiophosphorus uptake in sterile media. Both direct counting and autoradiography techniques were used. The first experiment was carried out with 60 larvae in 1 liter of nonsterile, George Pond water containing 2.5 jCi of 52p to see if the larvae would become radioactive. After 18 hrs the larvae were removed from the medium. One aliquot was plated directly on planchets, one was washed in water before plating, and a third was washed in water and in dilute HC1 (< 1% HCl) before plating. The larvae were dried and counted with a GM counter (efficiency of 19O with 1.17 Mev standard beta source). Untreated larvae from the storage jar were used to determine background counts. 26

Although rinsing is often used to measure adsorbed phosphorus, Whittaker (1961) states that "the problem is not simply solved by rinsingo On the one hand, some adsorbed phosphate enters chemical combinations on cell surfaces and resists removal without repeated rinses; on the other hand, repeated rinses may remove substantial, but variable, fractions of the more labile phosphate in the cells " Thus, washing might remove phosphorus inside the organismo Whittaker (1961) measured the radioactivity of organisms held in radioactive media for various periods of time. He found that most of the initial uptake of radiophosphorus was adsorptive. This was followed by absorptive uptake~ The absorption curve eventually reached a plateau as isotopic equilibrium was approachedo Adsorption and absorption can occur simultaneously but the length of time the organism is exposed to the medium determines the relative importance of the -two mechanisms. In March, 1970, several experiments were initiated to study the importance of bacteria in radiophosphorus uptakeo The procedure used was adapted from Rigler (1961). Chaoborus larvae were placed in a sterilized nutrient solution containing 100 units of penicillin/ml and 100 ig of streptomycin/ml The Chaoborus were left in this antibiotic solution for 90 min, put into sterilized nutrient solution for 24 hr, returned to fresh antibiotic solution for 90 min, and cultured in radioactive sterilized nutrient medium for various time periodsO All media were maintained at 40~F or 4o5~C. Pomeroy, et al. (19635), found that a combination of penicillin and streptomycin did not interfere with phosphorus analysis, and that they were effective against a broad spectrum of microorganismso Plotz and Davis (1962) believed that penicillin and streptomycin had a synergistic action; penicillin lowered the threshold concentration of streptomycin and hastened the onset of killing by streptomycin. Penicillin interferes with synthesis of the cell wall, and streptomycin may cause membrane damageo However, streptomycin has a two-stage mechanism in which membrane damage precedes binding of the drug to intracellular sites. Streptomycin and penicillin do not always sterilizeo Pomeroy, et alo (1965), believed that sterility was not achieved because of continual seeding of the culture with viable bacteria from feces of the zooplankton, and antibiotics only maintained bacteria numbers at or below the number found in the sea watero Spencer (1952) could not obtain axenic cultures of diatoms with streptomycin and penicillin. Johannes (1964a) used a solution of penicillin, streptomycin, chloromycetin, terramycin, and polymixin B along with ultraviolet light in order to produce bacteria-free algae cultureso He found that the antibiotics did not kill, but only inhibited the growth of some species of bacteria~ Therefore, he used antibiotics during, as well as preceding, his experiments. Antibiotics are simply antimetabolites whose influence on bacterial metabolism is somewhat greater than that on the metabolism of higher organismso Pomeroy (1963) states that "metabolic rates of higher organisms also may be affected by antibiotics, and the difference observed in phosphate tulrnover may not be entirely the result of inhibition of bacterial metabolismo 27

Another source of bacterial contamination is the radiophosphorus solution added to cultures. Hayes (1963) noted that 60% of the carrier-free phosphate in Normal HC1 will within a few days after it is diluted with distilled water, be converted to an organic form. In my experiments, both the time of exposure of larvae and the strength of the antibiotic solution were greater than employed by Rigler (1961). In one experiment, antibiotics were introduced directly into the radioactive medium. In a number of experiments I made autoradiographs of the larvae. After removal from the radioactive medium, larvae were frozen with dry iceo When all larvae were ready, they were taken to a dark room and allowed to thaw on a piece of absorbtive paper. They were then placed on Kodak no-screen medical X-ray film (NS54T and RPS7) in a manner which enabled them to be identified after film developmento Bruce-Chwatt and Hayward (1956) and Hassett and Jenkins (1951) used X-ray film to make autoradiographs of whole mosquito larvae that had been exposed to radiophosphorus, and Johannes (1964b) determined that 32p was deposited in the nuclei of the muscle and hypodermis cells of amphipods by microautoradiography. Other workers have used 32p for making autoradiographs but resolution is ordinarily poor in microautoradiography when X-ray film is used (Chapman-Andresen, 1959). These films have the advantage of speed, and may be useful to determine whether or not a cell has radioactive material. The resolution of an autoradiograph is affected more by the nature of the isotope than by the type of film. Although various types of film will give different resolution,. 32P will never give the quality of tritium because of differences in beta energy. Lamerton and Harriss (1954) defined resolution as the distance, d, if, the images of two uniformly active cylindrical sources of diameter d can just be resolved when the centers are separated by a distance 2d. The most important factor in making good autoradiographs is to choose the right techniqueo Gude (1968), Boyd (1955), and Rogers (1967) thoroughly discuss the theory, problems and advantages of various autoradiographic techniqueso Quimby and Feitelberg (1965), Chapman-Andresen (1959), Waddington -(1959), Messier and Leblond (1957), and Leblond, et alo (1960), go into finer detail. A variety of studies have been undertaken to isolate areas of absorption and diffusiono Koch (1938) and Wigglesworth (195533) found that the anal papillae of mosquito larvae served as osmoregulators and that many different ions could move in and out of the cells in the anal papillaeo Wigglesworth could not determine whether phosphate could diffuse into these cellso Hasset and Jenkins (1951.) found that the anal papillae of mosquito larvae functioned in the absorption of phosphorus. With ligatured larvae (human hair and/or fine silk thread) Schaller (1949) studied the osmotic regulation of Chaoborus and found that the anal papillae were the only salt and water permeable surfaces on the body. I attempted similar experiments with Chaoborus o Human hair was tied around 28

various segments of larvae. The larvae were then put in a sterile radiophosphorus nutrient medium. Later larvae were removed and autoradiographs were attempted. Results DIRECT COUNTING EXPERIMENTS In the data presented, the' activities of larvae have not been corrected for 32p decay, but have been corrected for background. Experiments are not numbered in the order performed. Experiments numbered 1-4 were performed in nonsterile media; those numbered 5-12 were in supposedly sterile media. Experiment 1 Sixty Chaoborus were put in 1 liter of George Pond water which contained 2.5 Sac of radiophosphorus. They were stored in this solution for 18 hr, removed and put on planchetso One group of Chaoborus was first washed in distilled water, and a second was washed in both distilled water and dilute HC1 (< 1% HC1). The difference between washed and unwashed samples indicates adsorption (Table )o. There was only a small residual activity after washing, however some activity may have diffused out in the washing processo Table Io Activity of Chaoborus larvae after 18 hours in George Pond water containing 2.5 ~Ci of 32P. The larvae were prepared for counting by three different methodso Number of Plating Method Chaoborus Counts/10 Min Direct 10 160 ~ 16 10 132 ~ 16 10 148 + 16 10 130 ~ 15 Water wash 10 31 + 12 10 36 ~ 12 10 35 ~ 12 10 18 ~ 11 Water and HC1 wash 10 2 ~ 11 10 7 ~ 11 10o 6 + 11 10 0 ~ 10 29

Experiment 2 Radiophosphorus was added to George Pond water to give 370 cpm/ml of medium. The same procedure as above was used. Most of the larvae were used in autoradiography, but some were saved for direct counting. Larvae were maintained in the medium for 10 dayso Results again show a large decrease in radioactivity with washing, but a significant amount of radioactivity remained (Table II). Table II. Activity of Chaoborus larvae after 10 days in George Pond water with activity 570 cpm/ml Number of Plating Method Chaoborus Counts/10 Min Direct 10 485 ~ 24 Water wash 10 287 ~ 20 Water and HC1 wash 10 188 ~ 17 Experiment 3 The medium used in the previous experiment was again used. Chaoborus were kept in the medium for 9 days. This experiment shows only a small loss of radiophosphorus with washing (Table II). Chaoborus were only rinsed once. Table III. Activity of Chaoborus larvae after 9 days in George Pond water used in Experiment 2 (Table II). Number of Plating Method Chaoborus Counts/10 Min Direct 10 211 ~ 18 10 249 ~ i9 Water Wash 10 244 ~ -19 10 181 ~ 17 Experiment 4 The medium used in the previous two experiments was again used A few Chaoborus removed and washed on March 50 were returned to the medium for eight more days (thus a total of 17 days in the medium). A significant amount of 35

radioactivity could not be washed from the larvae (Table IV), but a large difference in activities existed between the two samples. Table IV. Activity of Chaoborus larvae after 17 days in George Pond water. Larvae were removed after 9 days, washed and returned to the medium used in Experiment 3. Number of Plating Method Chaoborus Counts/10 Min Water and HC1 wash 10 357 + 22 10 134 ~ 16 Experiment 5 This experiment used normal Chaoborus and Chaoborus that had been treated with antibiotic according to Rigler's procedure (Rigler, 1961). Normal and treated Chaoborus were put in separate 1 liter flasks. The medium was the same in both flasks, except that the flask containing the larvae had twice as much radioactivity. Also, the density of normal larvae was greater than treated larvae. The larvae were in the media for 6 days. Sterilized nutrient media were used in this experiment and all the following experiments. Results were similar for normal and treated larvae, except for a single high reading for water and acid-washed larvae (Table V). Since the treated Chaoborus were fewer in number and had more radiophosphorus available, they should have had higher activities than the normal larvae. Anwar and Middleton (1963) found the number of mosquito larvae per ml of solution limited the uptake of radiophosphorus. This experiment therefore indicates a decrease in uptake when larvae are treated for bacteria. 31

Table V. Activity of treated and normal Chaoborus larvae after 6 days in initially sterile nutrient media. Treated larvae were in a medium with 464 cpm/ml while normal larvae were in a medium with 192 cpm/rml. Number of oi.t'__ lO 0 Min Plating Method Chaoborus Normal Treated Direct 10 254: 19 118 + 15 10 1453 16 176 ~ 1'7 10 84 + 14 Water wash 10 4.4 + 12 64 ~ 15 10 58 ~ 15 45: 12 10 58 ~ 11 Water and HC1 wash 10 40 ~ 12 200 ~ 17 10 60: 13 59 - 11 4 ~ 11 Experiment 6 Treated Chaoborus were put in radioactive nutrient media which had various amounts of stable phosphorus. The radioactivity of each medium was approximately the same, with a range of 930-1200 cpm/ml. Normal Chaoborus were put in one medium as a control. The experiment lasted for 7 dayso The exact number of individuals put in each medium was not noted, but more normal Chaoborus were used per ml of solution than treated Chaoboruso The amount of penicillin and the length of time in the antibiotics were increased from the previous experiment. This experiment showed a large difference between normal and treated Cha.oborus (Table VI). The normal Chaoborus had more activity for both washed and dire^ctly-plated larvae. The medium containing the normal Chaoborus not only had more larvae, it also had slightly less radioactivity before the start of the experiment. Thus a larger difference might have been expected with better control. These data'would indicate an antibiot(ic effect either direct or indirect via epiphytic and/or endophytic bacteriao There was also a correlation between activity (for both washed and direct-piate larvae) and amount of stable phosphorus presents The exact number of treated larvae in each medium was not recorded although the numbers were very similar because I attempted to add the same volume of larvae (not numbers) in each trial. 52

Table VI. Activity of normal and treated Chaoborus larvae after 7 days in sterile nutrient media containing 930-1200 cpm/ml. Phosphorus carrier added to give differences in P content. P in Medium Number of Counts/10 Min Plating Method ppbP Chaoborus Normal Treated Direct 0 10 35153 21 0 10 240 ~ 19 Water and HC1 wash 0 10 164 ~ 17 0 10 163 ~ 17 Direct 37 10 895 ~ 32 37 10 829 ~ 31 Water and HC1 wash 37 10 234 ~ 19 37 10 210 + 18 Direct 186 10 7359 ~ 86 2788 + 54 186 10 6588 ~ 82 3756 ~ 62 Water and HC1 wash 186 10 2698 ~ 53 288 + 20 186 10 2176 ~ 48 298 + 20 Experiment 7 The normal, washed Chaoborus used in Experiment 6 were returned to the medium for 8 days. The larvae had slightly more activity (Table VII) than in the previous study, but the difference would be larger if 32p decay were calculated since the activity had decreased by 153 between the counting times. Table VII. Activity of normal Chaoborus larvae after 8 days in the medium used in the previous Experiment 6. P in Medium Number of Counts/10 Min Plating Method ppbP Chaoborus (Normal) Water and HC1 wash 186 10 2948 ~ 55 Experiment 8 Treated and normal Chaoborus larvae were put in media containing various amounts of stable phosphorus. More streptomycin was used than in was used than in earlier experimentso The same amount of radioactive solution (9 microcuries) was in each medium. The larvae were incubated for 26 hr. 5533

For the normal, washed Chaoborus, it is apparent that the:re was no significant difference between the media, (Table VIII). For treated Chaoborus there was no distinct -trend in either washed or directly-plated Chaoborus although there were differences between mediao However, there was again a difference between normal and treated Chaoborus, especia.lly for the washed larvaeo Table VIII. Activity of Chaoborus larvae after 26 hours in i.nitial ly sterile media containing 9LCi. 32po Phosphorus carrier adde..i to giA- Differences in P content~ P in Medium Number of Count-s/10 Min Plating Method ppbP Cha,oborus Norma~ Trea ted Diirect 186 10 645 ~: 27 7 ~ 20 186 10 570 ~ 26 Wate-r and HC1 wash 186 10 182 1 17 65 ~ 13 186 10 147 + 16 Direct 372 10 * 224 ~ 18 372 10 177 + 17 Water and HCI wash 372 10 190 ~ 1 17 17 15 372 10 189 + 17 Direct 558 10 865 ~ 51 429: 23 558 10 480 + 24 Water and HC1 wash 558 10 155 ~ l6 53 + 12 558 10 164 ~ 17 *Sample lost. Exper iment 9 This experiment used the media and organisms from Experiment 80 After the larvae were washed, they were retfurned to their respective media, for 8 dayso Again there was a large diff-erence bet,ween treated and normal Chaobor-us in uptake (Table IX). There had been a large increase in uptake by normal Chaoborus aft er Experiment 8 and a significant increase by treated Chaoboruso The data indicate that isotopic equilibrium was not established in 26 hr (Experiment 8). The Ibw radioaetivity of the treat;ed C usd Chaobou indicate that most of the bacteria were killed instead of inhibitedo However, the antibiotics might have affected uptake by the anal papil:lae or by some other part of the bodyo 4

Table IXo Activity of normal and treated washed Chaoborus larvae from Experiment 8 put back in media for 8 additional dayso P in Medium Number of Plating Method ppbP Chaoborus Counts/10 Min Normal Chaoborus Water and HC1 wash 1.86 10 1412 + 39 186 10 1294 + 38 Water and HC1 wash 372 10. 681 ~ 28 372 10 455 ~ 24 Water and HC1 wash 558 10 675 ~ 28 Treated Chaoborus Water and HC1 wash 558 10 195 ~ 17 Experiment 10 Two 2-liter containers with the same nutrient medium and the same radioactivity (920 cpm/ml) were prepared; 170 normal Chaoborus were put in one container and 170 treated Chaoborus were put in the second containero Treated Chaoborus were prepared with weaker antibiotic solutions and exposed to the solution for a shorter period than in all earlier experiments except Experiment 5. Larvae were removed from the media after various exposures and were counted. Since some radioactive medium was always present when the direct plating technique was used, these counts probably include some radiophosphorus from the medium and not adsorbed to the larvaeo For the normal Chaoborus the direct plating counts start low, reach a maximum in 2 hr, and then decrease to a plateau level at 8 hr (Table X). The normal washed larvae show little activity until 8 hr, after which they show a steady increase,(Figo 1)o The directplated, treated larvae increase in radioactivity more slowly and reach a maximum in 8 hr, after which they decrease to a plateau level at 16 hr (Table X). The washed, treated larvae contain little activity until the 4th hour (Fig. 1); then activity steadily increases, but they did not become as radioactive as the normal Chaoborus except early in the experiment. These results indicate radiophosphorus uptake via absorptiono 3~

Table Xo Activity of normal and treated Chaoborus larvae after various t:imes in initially sterile media with 920 cpm/ml. Time in Medium Number of Counts/lO Min and Plating Method Chaoborus Normal Treated 30 minutes Direct 10 89 ~ 14 114 ~ 15 Hp0 and HCi wash 10 0 ~ 10 0 ~ 10 1 hour Direct 10 287 ~ 20 105 ~ 15 H2O and HC1 wash 10 19 ~ 11 4 ~ 11 2 hours Direct 10 1503 ~ 38 472 ~ 24 H.O and HC1 wash 10 15 ii 20 t 11 4 hours Direct 10 653 3 28 588 ~ 22 H20 and HC1 wash 10 9 ~ 11 55 ~ 15 8 hours Direct 10 408 8 ~ 2 918 ~ 32 f1IQ and HC1 wash i0 114 15 64 ~ 13 16 hours Direct 10 486 + 24 552 ~ 26 HI2 and HCi wash 10 1.30 ~ 15 80 ~ 14 48 hours Direct 10 416 ~ 23 475 ~ 24 HnO and HC1 wash 10 17 17 134 ~ 16 Experiment 11 Seventy Chaoborus were put in media with identonical nutrients and with 850 cpm/ml One medium contained normal Chaoborus, the other medium contained treated Chaoboruso The treated larvae were prepared with large doses of antibiotics, and in this experiment the solution with the treated Cha(bcrus contained antibioticso The larvae were incubated in their respective media for 7 dayso No larvae died in any of the media. This experimions were ident s corole in that he ond s wee denical in the beginning, except that one medium had normal Chaoborus and the other had treated Chaoborus and a.ntibioticsB The differences between activities of normral and treated Chaoborus (Table XI) strcrngly suggest that bacteria account for most of the activity in Chaoborus larvaeo If there were no bacteria in the treated larvae, the uptake of 32p by the washed larvae would have occurred 56

00 \ \-' \ 4-4 \r-4.t-'\~~~~ \~o ~~0 Xi \ Q' \ *' (vl o) ^ \ \'N ^rd \D a) co o co0 I3?C..~~~~~~~~~~~~~~~3 1-r4 000000000O000 OQ 000 0 CON- (0 N 0 M CN- 0 re') N - S3inNIV 01/ SILNnOo 57

chiefly via adsorption. The adsorbed phosphorus of treated larvae is small compared to the adsorbed phosphorus of normal larvae. These data suggest that both endophytic and epiphytic bacteria are responsible for the principal activity-of Chaoborus larvae. Table XI. Activity of Chaoborus larvae after 7 days in initially sterile nutrient media with 850 cpm/ml. Number of Counts/10 Min Plating Method Chaoborus Normal Treated Direct 10 1337 + 38 187 + 17 10 1560 ~ 41 225 ~ 18 10 1190 ~ 36 170 ~ 17' Water and HC1 wash 10 647 + 28 37 + 12 10 603 + 27 82 + 14 10 487 + 24 84 + 14 Experiment 12 Three identical media with 750 cpm/ml of radiophosphorus were prepared for normal Chaoborus. One medium was kept at 45~C for 24 hr, one was kept at 21~F for 24 hr, and one medium kept at 45~C for 16 hr and at 210C for 8 hr. Larvae were removed and plated at 8 hr intervals. This experiment showed no increase, but perhaps a slight decrease, in radioactivity of washed larvae, (Table XII). The washed larvae in the warm medium for 24 hr had much more radioactivity than those in the cold medium (Fig. 2). This suggests a higher metabolic uptake at higher temperatures. No other patterns seem significant, except that the larvae plated directly decreased in radioactivity in the cold medium. The larvae that were changed from cold room to warm room showed a slight increase in radioactivity in the warm room. Table XII. Activity of Chaoborus larvae in three initially sterile nutrient media with 750 cpm/ml at 8-hour intervals at 4.5~C and 21~C. Counts/10 Min Time Plating Method 24 hr, 4.5~C 16 hr, 4.5~C; 8 hr, 21~C 24 hr, 21~C 8 hours Direct 343 + 21 156 + 16 315 ~ 21 H20 and HC1 wash 65 + 13 26 ~ 12 180 + 17 16 hours Direct 202 ~ 18 153 ~ 16 288 ~ 20 H20 and HC1 wash 26 + 12 26 + 12 188 ~ 17 24 hours Direct 95 ~ 14 268 + 19 446 + 24 H20 and HC1 wash 25 ~ 12 33 ~ 12 172 ~ 17 38

I cV I 11 N H o ~ r4 0) /..r < i ~ ^Cd0 U/ 0 | / -<M I ~X L0_ a) -/ 0 / I ~o / o I. - H | N N-~~~~~~~~~~r In 0 I 0 I 0 C. 0 \n 0 C(J C In N a \ n N NI - - \ S3iLnNIN O / SNnO3 39

Autoradiography. Chacborus larvae from Experiments 2,, 4, 6, 7, 8, 9, and a ligature experiment were autoradiographed. Several films did not produce good autcradiographs, but all the experiments except the ligature experiment are represented in Figso 3-70 Since the autoradiograph of the ligatured larvae was extremely poor, a heavier film ioe., one with a thicker emulsion, was used for all succeeding autoradiographs (Figso 5-7). These results were not as good as the autoradiographs taken with lighter film (FigSo 3 and 4). The autoradiographs in Figs. 3 and 4 were made with a one-week exposure to the radioactive larvae while the autoradiographs in Figso 5-7 were made with a six-month exposure to the radioacti.ve larvaeo Also, in Figs. 3 and 4 a picture of the larvaewas taken by light as well as by radiopophosphoruso Since the larvae did not change form, a change in position of the larvae, as in Figo 4, is advantageous beca us; the area of radioactivity is better knowno In. Fig. 3, two washed larvae from Experiment 2 have a concentrati.on of:radiophosphorus in th.e head region. Other larvae have distinct black spots, but these were caused by the t.ansparent body segments act:ing as a lens and bringing light4 rays to a focal pointo Regardless of the experiment, the head region usually had the highest concentration of radiophosph r..uso Although the pictures (Figso 5-7) are not as clear as t.Jhe X-ray film itself for determining areas of radiophosphorus concentrations, one can. see from Fig. 4 that the head region is frequently the only noticeable radioactive part of the organismo The entire body of normal, washed Chaoborus larvae from Experiment 6 is radioactive (row C, Figo 4). These larvae were very radioactive (Table VI) compared with the other larvae used in this autoradiograph. Since th e washed larvae should not have much adsorbed phosphorus, this autoradiograph shows that the absorbed phosphorus has been distributed. throughout the body of one groupo When there is not much radioactivity, the head region has the highest concentration of radiophosphorus. Sin.e the autoradiographs in Figs. 5-7 were exposed to radiation for 6 months, some radioactivity appears in all the sampleso However, normal Chaoborus larvae from the storage jar in the cold room were put on these three films, and they are not at all visible. In Fi.g 7 the nonradioactive larvae are above row Ao The activities of Larvae within rows appear to be similar. The larvae from Experiment 4, however, show differences within a row (Fig. 6, row B) The large observed differences in counting (Table IV) are verified by variation in the activity of individual la-rvae. Other experiments produced equal or greater variation; however the similarity of activities within a row and the difference between rows is clear. The normal Chaoborus with the greatest activit;ies pr oduced the Largest autoreadiographs, i.e., largest black spots (Figso 4-7). The treated Chaoborus 40

Fig. 5. Autoradiograph of washed Chaoborus larvae maintained in George Pond water for 10 days ( Experiment 2). 41~~~~~~~~~~~~~~~~~~~~~~~~~~

Fig. 40 Autoradiograph of washed Chaoborus larvae from Experiments 3, 6, and 8. Ao Expt. 6- Treated, washed, 37 ppb P. Bo Expt 6 - Normal, washed, 8 ppb P. C. Expt 6 - Normal, washed, 186 ppb P. Do Expto 6 - Treated, washed, 186 ppb Po E. Expto 3 - Normal washed. F. Expto 8 - Treated, washed, 558 ppb Po Go Expt. 8 - Normal, washed, 558 ppb P. Ho Expt, 8 - Normal, washed, 186 ppb Po I. Expt 8 - Treated, washed, 372 ppb P. J. Expt. 8 - Treated, washed, 186 ppb P. Ko Expt. 8 - Normal, washed, 372 ppb Po ltr

D F " H K Fig. 4.

Fig. 5. Activity of normal and treated, directly plated and washed, Chaoborus larvae from Experiment 9. Ao Expt. 9- Treated, washed, 372 ppb P. B, Expt. 9 - Normal, washed, 372 ppb P. C. Expt. 9 - Normal, plated, 372 ppb P. D. Expto 9- Normal, plated, 186 ppb P. Eo Expt. 9 - Normal, washed, 186 ppb PO Fo Expt. 9 - Treated, plated, 558 ppb P. G. Expt. 9- Treated, washed, 558 ppb Po 44

I~~~~~~~~~~~~~~~~~~~~ A A B Fig. 5. E F G: —:::::::_i:- ~ —:i-::::a4: ~-:~-~:i:~~,::;::-:::::-::::~~::'V aa~~~~~~-.K;,y:Fig_ 5:i::::s:

Figo 6. Autoradiograph of normal and treated, directly plated and washed Chaoborus larvae from Experiments 4, 7, and 9 A. Expto 9 - Normal, plated, 558 ppb P0 B. Expt. 4 - Normal, washed. Co Expt. 9 - Treated, washed, 186 ppb P. D. Expt. 9 - Treated, plated, 186 ppb P0 E. Expto 7 - Normal, washed, 186 ppb P, F. Expt. 4 - Normal, platedo Go Expto 7 - Normal, plated, 186 ppb P0 46

A ~ ~~~K''Lia~'::_:: A B C D,i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~w~~ —-:i:ii 4~~~~4 s j Xmi~l~al~$~&Fig. 6. C li~~~~~~~ii~~91-i14

Fig. 7. Activity of normal, washed, and directly plated Chaoborus larvae from Experiments 4, 7, and 9. A. Expt, 9 - Washed, normal, 558 ppb P. Bo Expt. 4 - Washed, normal. C. Expt. 9 - Plated, normal, 558 ppb Po Do Expt. 4 - Plated, normal. Eo Expt, 7 - Washed, normal, 186 ppb P. F. Expto 7 - Plated, normal, 186 ppb P. 48

B'' 7~~~~~ ~ F g 7.' ~~~r~4 I$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~77" ~i t~~~~~~7 k~7"7'77 7" 7' "'7~~:A'7 ""'77 "77,,77""7'7'77'777'777 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~777'" 777,7777',, 777~~~~~~~~~~~~2::i~-iiir-'ii~~Bi 7' "''7'7',~~~~~~~~~~~~~~~-i B """"""'~~~~~~~~~il",77777~ -~~ili~~;~Ii~~77':: - -:_~~-~-~~~~:-,:-, ~:-r-i-::iii Fig. 7. )49

produced autoradiographs similar to the autoradiographs of normal Chaoborus with equivalent activities; thus the autoradiographs can be used to determine relative activities Summary Direct counting and autoradiography experiments indicated that bacteria play a major role in radiophosphorus uptake by the midge larvae Chaoborus americanuso The initial radioactivity uptake of normal Chaoborus put in radioactive nutrient media was predominantly 32p adsorption. Uptake by absorption became more important with time. Epiphytic bacteria did not play a major role in phosphorus absorption; however, endophytic bacteria are very important even when food, i.e., particulate matter, is not presento In several experiments, the amount of adsorbed phosphorus reached a maximum, then decreased or remained constanto However there was no decrease in activity in any experiment with washed larvae (and thus absorbed 32p)o Chaoborus treated with antibiotics picked up less radioactivity than untreated Chaoborus, except at the beginning of an experiment. The antibiotics apparently killed bacteria. and/or inhibited bacterial metabolism, but they did not produce bacteria-free conditions because treated larvae slowly increased in activity. The rate of 32p uptake was temperature-dependent, and thus probably depends upon metabolic rate, Autoradiography indicated that the head region concentrated radiophosphorus more than any other part of the bodyo Many normal (untreated) larvae had 32P distributed throughout their bodies, but the head appeared to be the most active regiono These results suggest that Chaoborus have large numbers of bacteria in the mouth and foregut where the food is broken down, These bacteria apparently survive during extended periods when food is not supplied.

Ref eren es Anwar, Mahmood and Lawrence Jo Middletono 1963. The effect of radioactive phosphorus on the development of Aedes aegypti larvaeo Proco Hawaii Ento Soco 19(3):361-366. Bond, Ro M, 19533 A contribution to the study of the natural food cycle in aquatic environments, Bullo Bingham Oceanography Collection, 4,1-89. Boyd, George, Ao 1955. Autoradiography in biology and medicine. Academic Press Inc., New York. 399 pp. Bruce-Chwatt, Lo JO, and Jo Haywardo 1956. Uptake of radioactive phosphorus by larvae and adults of mosquitoeso Natureo 177:661-662o Chapman-Andresen, Cicilyo 1959. Autoradiographic techniques as applied to unicellular organismsO Pubblo Staz. Zoolo Napoli 31 (supplo):100-114. Chipman, Wo Ao J1959 The use of radioisotopes in studies of the foods and feeding activities of marine animalso Pubblo Stazo Zoolo Napoli 31 (suppl ):154-175o Christensen, Halver No 19620 Biological transport. W. Ao Benjamin, Inco, New Yorko 133 pPConover, Ro Jo 1961. The turnover of phosphorus by Calanus finmarchicus. Jo Marine Biol. Assoc. U.Ko, 41: 484-488. Conway, Edward J. 19540 Some aspects of ion transport through membranes. In Active transport and Secretion edited by Ro Brown and Jo Fo Danielli. Academic Press Inc., New York:297-324. Cushing, C. Eo, and Do Go Watson. 1968. Accumulation of 32p and 65Zn by living and killed plankton. Oikos 19();:143-145. Davis, J. Jo. and R, Fo Fostero 1958. Bioaccumulation of radioisotopes through aquatic food chains. Ecology 39:5530-555 Fox, H. Mo 1952. Anal and oral water intake by crustacea. Nature (London) i69: 1051-1052. Gourley, Do Ro Ho 1952. Failure of 32P to exchange with organic phosphorus compoundso Nature 169:192o 51

Greenberg, David M. 1952. The acid-soluble phosphates in animal metabolism. In: Phosphorus metabolism Vol. II. edited by W. D. McElroy and B. Gloss. Johns Hopkins Press, Baltimore. Gude, William, D. 1968. Autoradiographic techniques. Prentice-Hall, Inc., Englewood Cliffs, N.J. 113 pp. Harris, E. 1957. Radiophosphorus metabolism in zooplankton and microorganisms. Can. J. Zool., 35:769-782. Hasler, Arthur D., and Gene E. Likens. 1963. Biological and physical transport of radionuclides in stratified lakes. In: Radioecology edited by Vincent Schultz and Alfred W. Klement, Jr. Reinhold Publishing Corp., U.S.A.: 463-470. Hasset, C. C., and D. W. Jenkins. 1951. The uptake and effect of radiophosphorus in mosquitoes. Physiol. Zool., 24:257-266. Hayes, E. R. 1963. The role of bacteria in the mineralization of phosphorus in lakes. Symposium on Marine Microbiology, Chap. 61:654-663. Johannes, R. E. 1964a. Uptake and release of dissolved organic phosphorus by representatives of a coastal marine ecosystem. Limnol. Oceanogr. 9(2): 224-234.. 1964b. Uptake and release of phosphorus by a benthic marine amphipod. Limnol. and Oceanogr. 9(2):235-242. Kamen, Martin D., and S. Spiegelman. 1948. Studies on the phosphate metabolism of some unicellular organisms. Cold. Spr. Harb. Symp. Quant. Biol. 13:151163. Koch, H. J. 1938. The absorption of chloride ions by the anal papillae of diptera larvae. J. Exptl. Biol. 15:152-160. Lamerton, L. F., and E. B. Harriss. 1954. Resolution and sensitivity considerations in autoradiography. J. Photograph. Science Vol. 2. 135-144. Leblond, C. P., B. Kopriwa, and B. Messier. 1960. Radioautography as a histochemical tool. 1st International Cong. Histochem. Cytochem. (Paris). Messier, B., and C. P. Leblond. 1957. Preparation of coated radioautographs by dipping sections in fluid emulsion. Proc. Soc. Exp. Biol. Med. 96:7-10. Mitchell, P. D. 1954. Transport of phosphate through an internal barrier. In: Active transport and secretion. Symp. Soc. Exp. Biol. 8:254-261. Plotz, Paul H., and Bernard D. Davis. 1962. Synergism between streptomycin and penicillin: a proposed mechanism. Science, 155:1067-1068. 52

Pclikarpov, Go G. 1966. Radioecology of aquatic organisms. Edited by Vincent Schultz and Alfred W. Klement, Jr. Reinhold Book Division, New Yorko 314 pp. Pomeroy, L. Ro 1960. Residence time of dissolved phosphate in natural waters. Science 131:1731-1732o o 19635 Experimental studies of the turnover of phosphate in marine environmentso In: Radioecology edited by Vincent Schultz and Alfred W. Klement, Jro Reinhold Publishing Corp. UoS.A:163-166. Ho Mo Mathews and Hong Shik Min. 19635 Excretion of phosphate and soluble organic phosphorus compounds by zooplankton. Limnol. Oceanog. 8:50-~55 Quimby, Edith H., and Sergei Feitelberg. 1963. Radioactive isotopes in medicine and biology: basic physics and instrumentation. Lea and Febiger, Philadelphia, 343 pp. Rice, To Ro 1953 Phosphorus exchange in marine phytoplanktono Fish. Bull. Fish and Wildlife Serv. 54(80):77-89. Rigler, Ro Ho 1961. The uptake and release of inorganic phosphorus by Daphnia magna Straus. Limnolo and Oceanogr. 6(2):165-174. Roberts, Rc.hard Bo, and Irena Roberts. 1950o Potassium metabolism in Excherichia colio III Interrelationship of potassium and phosphorus metabolism. J. Cello Compo Physiol. 36:15-39. Rogers, Andrew W. 1967. Techniques of autoradiography. Elsevier Publishing Company, New YorkO 5355 Schaller, Friedrich. 1949. Osmoregulation und Wasserhaushalt der Larvae von Corethra plumicornis mit besonderer Beraksichtigung der Vorgance am Darmkanal. Zeit fu*r vergleichende Physiol 31:684-695. Seymour, Allyn, H. 1959. The distribution of radioisotopes among marine organisms in the West Central Pacific. Pubblo Staz. Zool. Napoli, 31 (supplE): 24-33 Spencer, Co Po 1952o On the use of antibiotics for isolating bacteria free cultures of marine phytoplankton organismso Jo Mar. Biolo Assoco U.K. 31: 97-106o Stuart, Co Ao, McPherson, M., and Cooper, H. Jo 1931- Studies on the bacteriiologically sterile Moina macrocopa and their food requirementso Physiolo Zoolo 4:87-100o Stumpf, Po K.o 952. Phosphate assimilation in higher plants. In: Phosphorus Metaboc.ism Volo IIo W. Do McElroy and Bo Glass (Eds)o Johns Hopkins Press, Balt imoreo

Ussing, E. H. 1947. Interpretation of the exchange of radio-sodium in isolated muscleo Nature, Lond. L60:262-263. Vallentyne, J. Ro 1952. Insect removal of nitrogen and phosphorus compounds from lakes. Ecology. 33:573-577, Waddington, Co H. 1959. Autoradiography and some of its uses. Pubblo Staz. Zoolo Napoli, 31 (suppl.):~115-122. Whittaker, R. Ho 1961. Experiments with radiophosphorus tracer in aquarium microcosms. Ecological Monogr. 31'(2):157-188. Whitten, Bert K., and Clarence J. Goodnighto 1969. The role of tubificid worms in the transfer of radioactive phosphorus in an aquatic ecosystem. In: Symposium in radioecblogy edited by Daniel J. Nelson and Francis Co Evanso (TID-45000):270-277o Wigglesworth, Vo B. 1933. The effect of salts on the anal gills of the mosquito larva. Jo Expto Biol. 10:1-15. 5)L

APPEND IX C DISSOLVED ORGANIC PHOSPHORUS COMPOUNDS OF A BOG LAKE ECOSYSTEM Frank F. Hooper School of Natural Resources The University of Michigan and Kay Terry Department of Genetics and Cell Biology Unriversity of Min.esota 55

DISSOLVED ORGANIC PHOSPHORUS COMPOUNDS OF A BOG LAKE ECOSYSTEM Frank F. Hooper School of Natural Resources The University of Michigan and Kay Terry Department of Genetics and Cell Biology University of Minnesota In an earlier report we presented preliminary analyses of dissolved organic phosphorus compounds (DOP) of North Gate Lake, a small Michigan bog lake (Hooper, 1969). These studies have indicated that the phosphorus of nucleic acids and polyphosphates were important fractions of the DOP in this ecosystem. In the present report, we wish (1) to review the literature dealing with the quantity and chemical nature of dissolved organic phosphorus compounds, their possible biochemical origin and function, and their relationship to the phosphorus cycle of aquatic systems, and (2) to report additional data on the dissolved organic compounds of North Gate Lake. Literature Review-The Organic Phosphorus of Aquatic Systems CELLULAR PHOSPHORUS COMPOUNDS The particulate phosphorus found in natural waters consists chiefly of cellular phosphorus within a variety of microorganisms including planktonic algae and bacteria, and also nonliving detritus particles undergoing decomposition. During the degradation process within natural waters, many of these organic compounds may be released at least temporarily, into the water in a free state. In their free state, the cellular products constitute a part of the "dissolved" fraction. To adequately review the role of organic phosphorus in natural waters, it is of value to first discuss the intracellular compounds, consider their function in the cell, and review the present state of our knowledge regarding their metabolism. Phosphorus is involved in the entire range of metabolic processes. It is connected in an essential way with virtually all energy transformation systems in the living cells. Phosphorus enters the cell as inorganic phosphate by means of an active transport process, and once inside the cell it becomes incorporated (1) into a variety of organic phosphorus compounds, and (2) into 57

corLdensed inorganic phosphate (polyphosphate). In algae, the disposition of phosphorus into these compounds arises from reactions iInIvolved in two major energy-yielding chains present in all living plant cells. These are (1) the procless of photophosphorylation within chloroplasts whereby light energy is used for esterificatio: of inorganic phosphate into ATP (adenosine triphosphate), a.d (2) phosphorylation coupled with respiration which is associated with mitochondria (oxidative phosphorylation). In all organisms, most of the energy-consuming reactions are coupled with the hydrolysis of the energy-rich phosphate bond of ATP. In these reactions phosphate groups are actively transferred by the action of enzymes. The ATP arising from photophosphorylatioCn is used by the plant cells to support gr owth anid all energy-requiring metabolism such as CO2 assimilation, uptake of various organtic and inorganic compounds, synthesis of amino acids, synthesis of proteins and nucleic acids, and synthesis of inorganic polyphosphateso This close coupling of phosphorus with cell energetics and photosynthesis makes photosynthetic production dependent upon available phosphates. It is clear that a phosphate deficiency in algae sooe reduces photosynthesis anrd other metabolic reactions (Pirson, 1955), and may lead to unusual accumulat~io. s of fat, starch, and other cell wall substanceso This, in turn, suggests a secondary inrterference with the nitrogen metabolism of the plant (Bergmann, 1955) Of the numerous intracellular phosphorus compounds of bacteria and algae, polyphosphates have received a great deal of attention because for many years they were believed to be the compounds used for energy storage within the cell (Hoffman-Ostenhof, 1962)o Polyphosphates are one of three types of condensed inor.ganic phosphate compounds containing pentavalent phosphorus, i.nt which various numbers of tetrahedral P04 groups are linked together by oxygen bridges. The chain length of polyphosphates ranges from 2 units (pyrophosphates) to the inrsoluble Maddrell's salts of chain length. up to 104o Only the molecules of lower chain lengthl. have been identified as indiv.idual compounds. Nearly all the polyphosphates from.Living cells are regarded as mi.xtures of various proporti:on: s of polyphosphates of different molecular size. Polyphosphates cf vartying chain lengths have been extracted from yeast cells using a variety of solvents and pH ranges. Fractio-..s have tvaried i. c.haina length. from 4 to 260, and molecular weights range from 550 to 30,700 (Langen, Liss, and Lohmann, 1.962). Columi. and paper chromatography have been used to separate polyphosphates anid metaphosphates of shorter chain lengthrs from' living cellular materialso PclyphospLihates appear to be characteristic of microcrganisms and have been isolat ced from a wide variety of bacteria, fungi, protozoa, and all major groups of algae (Harold, 1966; K2ul, 1962), In addition, they have been identified from some higher plants and amina.ls (Miyachi, 1961; Lynn anld Brown, 19653) Some controversy ex:ists regarding the chemical state of polyphosphates withi.rn cells. Since some polyphosphates cannot be extrao. ted with acid, for 58

many years they were believed to be bound with protein or ribonucleoprotein complexes (Katchman and von Wazer, 1954). Harold (1966) now favors the view that complexes do not exist in vivo but are formed during the extraction process and that polyphosphates exist free in living cells. In bacterial cells much of the polyphosphate may be localized in granules that stain metachromatically with basic dyes. These granules can be identified in cells with either the light or electron microscope and have been called volutin granules. In addition to polyphosphates, volutin granules may contain RNA, lipid, protein and Mg+2 (Widra, 1959). In yeast and in Chlamydomonas phosphates may be localized in vacuoles. The biosynthesis of polyphosphate is accomplished in the living cell by the addition of the terminal phosphoryl group of ATP to the chain according to the following reaction: ATP + (Polyphosphate)' ADP + (Polyphosphate)+ n n+l The discovery by Kornberg (1957) of the polyphosphate kinase catalyzing the above reaction led to the hypothesis that polyphosphate was in some way related to energy process within the cell. The above reaction appears to be reversible at low ATP-ADP ratios. Other enzymes have been isolated from cellular material which bring about the transfer of phosphate from polyphosphate to adenosine monophosphate (Winder and Denneny, 1955) and which bring about the phosphorylation of glucose and fructose (Szymona, et al., 1962; 1966). A number of enzymes which have been isolated from living tissues will hydrolyze pyrophosphates, tripolyphosphates, tetrapolyphosphates, metapolyphosphates, and polyphosphates of high molecular weight to orthophosphate (Grossman and Lang, 1962; Muhammed, 1961). At this time the evidence is not clear whether hydrolysis is direct to orthophosphate or through intermediates such as ATP. The polyphosphate content of living cells has been shown to fluctuate within a wide range. Polyphosphates usually cannot be detected in phosphorusstarved cells. They are low or undetectable during the exponential growth phase of cells. On the other hand, they accumulate to very high levels in cells with nutritional deficiencies (Wilkinson and Duguid, 1960), up to 20%o of the dry weight of yeast cells (Liss and Langen, 1962). When sulfur, carbon, or nitrogen is limiting, Nityosomonas converts nucleic acid into polyphosphate. When the limiting element is restored, polyphosphate is converted back into nucleic acid (Terry and Hooper, 1970). The cycle of polyphosphate in Aerobacter aerogenes and its genetic control has been outlined by Harold (1964, 1965), and Harold and Harold (1965). Briefly, it appears that in growing cells, the synthesis of nucleic acid inhibits polyphosphate synthesis and stimulates the degradation of polyphosphate. Consequently little or no polyphosphate is deposited. If growth and nucleic acid synthesis stop because, of the exhaustion of a nutrient, net polyphosphate synthesis is promoted. This is presumed to occur because competition for ATP is relieved 59

and polyphosphate accumulates at a rate determined. by the level of polyphosphate ki'ase. It has also beenL shown that cells subject to prior phosphate starvation contain elevated levels of polyphosphate kinase and are th.us capable of very rapid polyphosphate synthesis when orthophosphate is providedo Thus in Aerobacter, under favorable conditions for growth, net polyphosphate synthesis occurs at a very low rate and n-rcleic aciLd synthesis proceeds, whereas under unfavorable envilronmental circumstances, polyphosphate synthesis takes place and rapid accumulat;i on occurs, In algae, Kuhl (1960) found that there is rapid incorporation of orthophosphate into polyphosphate in the presence of light. In the dark, incorporation is depressed but not abolishedo Thus in algae it appears that the formation of polyphosphate is stimulated by light but is not totally dependent on it. In algae as in other microorganisms the polyphlosphate content is a function o)f growt.h ph.ase and is lowest.i.n the exponential plhase and hi.g:hest in old cultur es. There also appears to be a reciprocal relationsLip between RNA and thie polyphosphate content (Smillie and Krotov, 1960)0 Harold (1966) reviews theories on the function of polyphosphate in living cells. His review raises doubts as to the validity of the earlier hypothesi.s that polypphosphates are stres e-bound energy from which phosphoryl groups may be transfe.rred to ADP from ATP, He provides evidence that polyphosphates serve as a phosplhate reserve and storage center for phosphorus with'in cells from which rapid b.iosynthesis of nucleic acid and phospholipids can take place. This leads to the general concept of polyphosphates acting as regulatory agents of cellular phosphorus. On the one hand, there is the cyclic regereration of ATP and its participation in rumerous reactionis i. which metabolic energy is produced and utilized. On the other hand, in growing cells ATP is conti;.nually drawn into nucleic acids an.d phospholipids. There appear to be valid biochemical reasons for believing cells must control the steady state concentr ation of orthophosphates, ADP, and ATPo Polyphosphate appears'to be the compound regulating these levels by serving, in effect, as a metabolLic buffer. THE QUANTITY OF PHOSPHORUS FRACTIONS IN LIVING CELLS The percentages of the various phosphorus fractions within bacterial and algal cells fluctuate considerably depe.nding on ligh.t conditions, concentration of the phosphorus in the environment, pH, and temperature. Overbeck (1963) prov ides data on four of the above fractions in Scenedesmus quadricauda. The TCA soluble fraction made up 27o of the phosphorus of normal Scen.edesmus cells. About half of this faction was orthobhosphate and half was 7-mi. hydrolyzable phosphate (acid-soluvble polyphosph.ate)o The TCA insoluble phosphorus made up the remaining 735 of the cell phosphorus. This on:sisted of a fraction hydrolyzable by the 7Tm.i hydrolysis (50eGo of cell phosphorus) and stable organic phosphorus (22o4%)o 60

Correll (1965) fractionated particulate material of two size categories collected fromAntarctic waters into five chemical fractions. The large size particles consisted chiefly of zooplankters while the smaller category was chiefly diatoms. The larger size particles contained higher quantities of RNA and polyphosphate (27-550 of total phosphorus) than the smaller size particles. On the other hand, the smaller category of particle was richer in phospholipids (13-29% of total phosphorus). Both classes of particle contained more orthophosphate, oligopolynucleotides, and oligophosphates than healthy laboratory plankton, which indicated that enzymatic degradation of cells had occurredo By incubating aliquots of these two samples with 32p, rates of incorporation of the label into various fractions were observed. As expected, the smaller particles took up radioactivity about six times faster than the larger size fraction. The RNA-polyphosphate fraction was labeled more rapidly than any of the other four organic fractions separated. However, phospholipi'ds were also labeled rapidly and reached a high specific activity. REGENERATION OF PHOSPHORUS FROM ORGANIC COMPOUNDS Demonstration that the stores of soluble phosphate measured in the open water of the photosynthetic zones of lakes and the ocean were inadequate to provide the necessary phosphorus for production of phytoplankton for any extended period (Hutchinson, 1941; Juday, et al, 1928; Redfield, Smith, and Ketchum, 1937) made it necessary to hypothesize that (1) phosphorus was being brought into open water from greater depths and/or bottom sediments, or (2) that major stores of phosphorus were regenerated from the phosphorus of the phytoplankton, zooplankton, or the dissolved organic phosphoruso It is now clear that recycling the phosphorus may be rapid and that regeneration can account for a major share of the needs of the system. In the sea and in lakes it is difficult or impossible to determine the origin of sol-uble phosphorus compounds that appear in the open water since these compounds may arise by (1) in situ decomposition of the phytoplankton and zooplankton organisms themselves, (2) excretion by the plankton organisms, (3) regenerationr. from bettom sediments and transport to the photosynthetic areas, or (4) in situ release of dissolved organic compounds by algae and bacteria, and subsequent breakdown of the organic compounds into soluble phosphate. All of these processes may be operating simultaneously or one process may predominate to the exclusion of others. Perhaps the most successful approach to the study of cycling of phosphorus in shallow systems where there are several regeneration processes operating simultaneously has been made by the use of radiophosphoruso Kinetic analysis of the movement of the label from the water into the various "pools" or "compartments" within the system has shown a logarithmic decline followed by a decrease at a much lower rate as the tracer is returned to the open water from pools. Pomeroy (1960) reviewed data on the residence time and turnover 61

rate from 32p experiments in water systems and found the residence time range to be from 0.05 to 200 hr. He suggests that a short residence time indicates either depleted phosphorus resources, or high metabolic activity, or a combination of these two conditions. He found that turnover rates fell between 0.1 and'1.0 g P/m5/hr regardless of the phosphate concentration of the water except in biologically active systems where values between 1.0 and 20 ig P/m3/hr are encountered. Turnover time was considered more important than phosphate concentration in maintaining high productivity. In many unpolluted freshwater systems, rapid regeneration within the water is believed to take place because the concentration of orthophosphates is frequently 1 mg/m3 or lower and variations in quantity of orthophosphate may be small despite large changes in plankton biomass (Juday, et al., 1928). The transient state of orthophosphate can be demonstrated in a striking way by simple aquarium experiments in which radiophosphorus is added as a tracer (Hayes and Phillips, 1958; Phillips, 1964). Aquarium experiments as well as in situ lake measurements by Rigler (1956) suggest that the rate of turnover in bacterial cells is a matter of a few minutes and that turnover of the dissolved organic phosphorus pool-in the water may be a matter of a few hours. There can be little doubt that the major share of the phosphorus of the hypolimnia of stratified lakes comes from regeneration of the phosphorus from plankton settling below the thermocline. Radiophosphorus added to the epilimnion promptly appears as a soluble phosphorus in bottom strata (Hutchinson and Bowen, 1950; McCarter, Hayes, Jodney, and Cameron, 1952). However even in the deepest layers of lakes regeneration is a complex problem since in this zone phosphorus may also come from sediments. The classical study by Mortimer (1941, 1942) in Esthwaite Water demonstrates that regeneration of phosphate from bottom mud proceeds when the surface layer of the mud becomes chemically reduced and an iron colloid layer is broken down. Mortimer's studies indicate that the dissolved phosphate which moves from the mud into water may be transported vertically in the hypolimnion by currents arising from internal waves. In the open ocean a major share of the phytoplankton cells produced in the euphotic zone is consumed by herbivorous zooplankters living in this zone, and is not lost by sedimentation (Ketchum, 1962). After digestion and assimilation by zooplankton it is subsequently excreted into the water. Ketchum and Corwin (1965) recorded changes in the phosphorus fractions within the euphotic zone during a plankton bloom in the Gulf of Maine. The magnitude of the regeneration process can be deduced from their field measurements. During the tenday period in which the bloom was studied, 14.2 ig- at P/m2 was removed from the 0-50 M stratum. This phosphorus gave an observed increase of 7.58 Mg-at /m2 of new particulate phosphorus (plankton) and 1.05 4g-at/m2 increase in dissolved organic phosphorus. All of the organic phosphorus was liberated in the upper 25 M. Below the euphotic zone (137 M) 2.7 4g-at/m2 of inorganic phosphorus appeared in the water, presumably from regeneration of particulate matter sinking to this zone. The overall rate of regeneration of phosphorus 62

in this study was 4.0 x 105-3 g-at/m3/day. This rate under bloom conditions can be compared to rates in the Gulf of Maine under non-bloom situations of 2.0 x 10-3 pg/at/m3/day, and the average, 0.01 x 10-3, and the maximum 0.30 x 10-53 pg-at/m3/day, for the North Atlantic ocean given by Riley (1951). In field studies such as the above, it is seldom possible to identify the source of the organic material being decomposed. Ketchum and Corwin (1965) point out that the dissolved and organic phosphorus appearing in their study may have come from excretion of phytoplankton as well as zooplankton. Cultures of phytoplankton cells may liberate up to 25% of the organic carbon fixed during the 24-hr-period (Hellebust, 1965). Carbon excretion presumably would be accompanied by sizable loss in organic phosphorus. However autolysis and bacterial degradation of phytoplankton and zooplankton together with excretion of living zooplankton organisms are believed to account for a major share of regeneration in the sea. The relative importance of autolysis as compared to bacterial decomposition of phytoplankton and zooplankton cells has not been adequately evaluated. There are several laboratory studies in which the breakdown of plankton has been followed. Hoffman (1956) found that 20-25%o of the inorganic phosphorus and 30-40% of the organic phosphorus was liberated shortly after death, and between 80 and 90o was liberated within 24 hr. Marshall and Orr (1961) found a complete breakdown of the phosphorus of dead marine copepods of the genus Calanus within 2 days. The rapidity of decomposition led the above authors to conclude that autolysis, rather than bacterial action or the action of free enzymes, was the most important regenerative process. Grill and Richards (1964) followed the regeneration of phosphorus from a culture of phytoplankton (chiefly a centric diatom) which had been incubated in sea water and then stored in the dark. During the first day they noted that a sizable fraction of the inorganic phosphorus passed into particulate form, presumably uptake by bacteria. The increase in particulate phosphorus continued for approximately 8 days, at which time there was a sudden increase in the dissolved organic phosphorus and a decrease in particulate phosphorus. This change, they believe, was the result of autolytic release similar to that described by Hoffman. The dissolved organic phosphorus liberated was quickly acted upon by the bacteria population which was rapidly increasing so that after 17 days it was almost completely reassimilated into particulate matter. After 4 weeks there was again decomposition of particulate phosphorus and an increase in inorganic phosphorus. This continued until the end of the experiment. By plotting the decay of various components, Grill and Richards developed a simple kinetic model of the mineralization process. The process was approximated by the resultant of the first order decay rates of two labile organic fractions plus a refractory fraction which did not change during the experiment. They believed the initial decay rate represented organic phosphorus released from diatoms. Decay of this fraction gave a second fraction which appeared to be the phosphorus contained within the bacterial cells. This fraction, in turn, gave rise to (1) inorganic phosphorus and (2) refractory organic products of bacterial matabolism and the undecomposed particulates of bacterial cells. Cooper (1935) observed the release of phosphorus from the decomposition of a mixed culture of marine zooplankters and from a mixed cluture of marine diatoms. He observed a large release of inorganic phosphorus from the zoo63

plank'ton -within the first 12 hr. An amount of phosphorus equivalent to that in the plan'kton "was set free wit;hin a period of 6 days. Regeneration of phosph:orus from the diatom culture was somewhat delayed. Very little phosphate appeared within tahbe first 3 days but thereafter rapid liberation took place, reaching a maximum amount in approximately one month, After this maximum there was a slow decrease in phosphate for a 4-mo.nith period. Although. there are few or no data on excretion rates of the zooplankters un.der field conditions, there are data from laboratory studies and from in situ fi.eld measurements in closed systems. The excretion rates of herbivores depend upon feeding rates, density of herbivores, density and quality of the food, temperature, and other variables. A wide variation in regeneration rates is encounteredo Although most attention has been. paid both in marine and fresh.water studies to the larger herbivorous zooplarkters, the mi crozoopla.nkton may be of great significance. Johannres (1964) demonstrated with a series of marine animals of varying sizes that. t4he rate of excretion of dissolved phosphorus per unit of body weight increased as the body weight decreased. This suggests that the smaller shlort-lived species may play a very important role in mineralization. The large -. umbers of protozoa, rotifers, and other small metazoa inhabiting the mud-water.interface as well as the open water are capable of liberating large quantitlies of phosphoruso Excretion of phosphorus is not confined to forms grazing upon living cells. Many of the microzooplankters as well as benthic filter feeders may ingest both living and dead cells (Edmanson, 1957). Dead cells, together with their covering of epiphytic bactera may con.stitute a large fraction of the organic material in many freshwater ecosystemso However, the microplankton may release inorganic phosphorus in the absence of bacteria. Hooper and Elliot (19553) demonstrated the release of inorganic phosphorus from bacteria-free cultures of the ciliate Tetrahymena pyriformes that contained autoclaved extracts of the organic matter from lake mud, Margalef (1951) observed release of inorganic plhosphorus from cultures con....taining cladocera and +th.e algae Srenedesmus. Release of phosphorus ir..creased as thr..e number of cells,of Scenedesmus increasedo Margalef believed tI.at the algae stimulated phosphotaase activit+y in'the digesti.ve tract of the clado.era. Rigler (1961) verified Margalef s findin.g that liv.ing crustacea sec-:ete phosphateses into the medium and Overbeck (1961) found that a bacterial suspe.rsion, releases phosphate enzymatlc.ally from sodium glycerophosphate and makes thi.s phosphorus available to the alga Scenedesmus quadricauda. The early experience of Gardiner (1957) using mixed cultures of the larger marin-e zooplankters demonstrated that large stores of inorganic phosphate are excreted by these animals wi.th..ir a 3-hr test period. These and later studies'by Marshall avid Orr (1955) provide estimates of excretion rates of cultured ani-mals under differenit conditions of feeding. More refined measures of excretio:n rates come from studies in which diatoms labeled with 32P were fed to the marine copepod Calanus and the rate of loss of metabolized label from the medium was measured (Marshall and Orr, 1961). In these studies the biological 64

half-life of phosphorus was estimated to be 14 days. A kinetic analysis of Marshall and Orr's data by Conover (1961) indicated that the phosphorus of Calanus existed in two pools, one of labile phosphorus with a short biological half-life (approximately 0.375 days), and a second pool of more stable phosphorus with a half-life of approximately 13 days. It was estimated that between 94 and 99% of the total phosphorus in Calanus was in, the stable form and that the mean turnover rate from both pools was about 10%o of the body phosphorus per day. Ketchum (1962) summarizes unpublished data of Conover on excretion rates of a variety of marine organisms. The rates vary from 0.03 to 6.9/0 of the total phosphorus content of the organisms per day. Pomeroy, Mathews, and Min (1963) found much higher excretion rates for estuarine plankton than reported above. Net plankton excreted an amount of phosphorus nearly equal to its total phosphorus content in a day. Slightly more than half of the excreted phosphorus was phosphate; the remainder was insoluble organic compounds. Rigler (1961) found that Daphnia magna excreted inorganic phosphorus into the medium at the rate of 8.4 x 105-3 g/animal/hour. This loss was independent of epizostic bacteria, and was independent of the production of feces. He was not able to detect loss of organic phosphorus. His excretion rates for Daphnia appear to be much higher than those reported by Gardiner (1957) and are slightly higher than those reported by Marshall and Orr for Calanus. Barlow and Bishop (1965) followed the regeneration of phosphorus from zooplankton by collecting net plankton from the epilimnion (5 M) and from the hypolimnion (45 M) of Cayuga Lake and re-suspending the plankton at 100 times its original concentration in bottles of water taken from the depth from which the plankton was collected. Excretion rates ranged from 30 to 120% of body phosphorus per day and were considerably higher than those reported by Ketchum (1962) but within the range reported by Pomeroy, et al. The epilimnetic zooplankton population of Cayuga Lake consisted chiefly of cladocera while the hypolimnetic plankton consisted chiefly of copepods. The latter were, on the average, of the larger size. Excretion rates were consistently lower for the epilimnetic zooplankton as compared to the hypolimnetic forms of the same body size. The fecal pellets produced by herbivorous zooplankton have been suggested as a source of debris that may be converted into phosphorus compounds. Marshall and Orr (1961) found that Calanus produced pellets at the rate of one pellet every 5 to 6 min. However these authors believe the phosphorus content of pellets is low and their studies on digestion indicate that the phosphorus assimilation efficiency of Calanus is high. Thus the role of fecal pellets in the regeneration cycle of phosphorus is not clear. The role of bacteria in the regeneration process appears to be complex. Both the tracer studies on aquatic microcosms (Hayes and Phillips, 1958; and Phillips, 1964) and the direct measurements of decomposition in sea water (Grill and Richards, 1964) indicate that living bacteria not only incorporate phosphorus rapidly, but following uptake much of the phosphorus is released as dissolved organic phosphorus. Although most of the direct evidence for 65

bacterial production of dissolved organic phosphorus comes from experiments with laboratory microcosms, much of the dissolved organic phosphate in nature may be of bacterial origino In following the uptake of 32P by bacteria, Phillips (1964) noted that normally there is not a release of dissolved organic phosphorus during the growth phase of cells and that release is usually associated with a decline in. cell numberso He concluded that release was associated with death of microorganisms. Bacteria also appeared to be the agents responsible for the conversion of dissolved organic phosphorus to inorganic phosphateO Phillips (1964) added labeled dissolved organic phosphorus to sea water under sterile and nonsterile conditions. With bacteria present, labeled dissolved organic phosphorus declined rapidly and the label entered the particc ulate phase (bacteria) O Later, inorganic phosphorus appeared. On the other han-d, the label remained in organic for m in sterile cultureso From these experiments, Phillips concluded that there was:no con.ivexrsion, of dissolved organ.ic phosphorus to inorganic phosphate without bacteriao Johan-ines (1964b) found that marine bacteria utilized 8P0o of the dissolved organic phosphorus released by an amphipod. He also found that a large fraction of this organic phosphorus was hydrolyzed in a sterile medium, He noted a release of organic phosphorus by bacteria-free diatoms after thLeiJr growth phase had ceased, but was unable to report regeneration of dissolved inorganic phosphorus from dissolved organic phosphorus in the presence of bacteria; contrary to the findings in freshwater systems, he believes that marine bacteria (living or dead) released little dissolved organic phosphorusO Under anoxic conditions, cultures of bacteria and other organisms have been shown to release large quantities of orthophosphate very rapidly (Shapiro, 1967) When oxygen is restored to the medium, much of this phosphorus is reabsorbed. The phosphorus liberated appears to have been cellular, since release was inhibited by mercuric chloride and by 2, 4-di.nitrophenolo The fracti ons of the phosphorus compounds in. the systems at various times during the:release anld uptake process indicated t.hat the phosphorus liberated during th,.e early stage of release was phosphorus that could be extracted by acid. Later in the release process RNA and DNA fractions were produced. There did not appear to be a release from phospholipids or phospi..-.oproteins. IDENI\TIFICATION OF DISSOLVED ORGANIC COMPOUNDS Despite the wealth of data on the quantity of dissolved organic phosphorus in marine and freshwater systems, only fragmentary and isolated efforts have been made toward the identification of these compounds, More important, little or no effor-t has gone toward linking these orgar.{nic fract ions with the metabolic actirities of bacterial and plant cells from wh-ich- many of these materials must arise. Other than suggestions that the dissolved organic phosphorus represents the more refractory compounds that decompose slowly (Ketchum, 1962; Grill and 66

Richards, 1964) and that they are frequently liberated by bacterial cells, there are few guides as to their role in the biochemical cycles of either marine or freshwater systems. Phillips (1964) separated the dissolved organic phosphorus of sea water that had been equilibrated with 32p into 6 fractionso Three were identified as nucleotides or polynucleotides on the basis of adsorption of Norit A charcoal. Three other chromatographic peaks were not absorbed and were believed to represent phosphorylated hydrocarbons. The fractionation of dissolved organic phosphorus of bog water samples (Hooper, 1969) showed that in the aphotic zone, between 0.3 and 4.2% of the total phosphorus was absorbed by charcoal and presumably was nucleic acid. This fraction, however, was not present in the euphotic zone. From 1.7 to 12.35 of the total phosphorus was labile inorganic phosphorus, presumably polyphosphate or pyrophosphate. This fraction was present at all depths. The identified fractions however represent a small portion of the dissolved organic phosphorus since 55 to 470 of the organic phosphorus remained unidentified. Since algal and bacterial cells are known to accumulate large quantities of polyphosphates, the question arises whether or not these fractions exist free in the euphotic zone since they would be expected to be liberated by the lysis of cellular material. Solorzano and Strickland (1968) and Armstrong and Tibbitts (1968) found only small quantities of polyphosphates in unpolluted coastal waters. Solorzano and Strickland suggest that polyphosphate does not accumulate to any extent in unpolluted situations since it appears to be taken up rapidly by algal cells even in the presence of bacteria. However since it accumulates when there are deficiencies of other nutrients (Harold, 1966) and under adverse environmental conditions, it may occur in tropholytic waters and its presence in the euphotic zone may be an indication of a limiting micronutrient. Because little of the organic phosphorus of sea water is easily hydrolyzed by enzyme phosphate esters Sol6zano and Strickland suggest that the organic phosphorus isolated from sea water is mainly nucleic acid, Particulate DNA has been isolated from sea water by Holm-Hansen, Sutcliffe, and Sharp, (1968). However, Armstrong, Williams, and Strickland (1966) found that nucleic acids were easily degradable by ultraviolet light, and thus might not be expected in the surface water but might appear in the aphotic layers, as reported for bog lakes (Hooper, 1969). Methods Analyses made during 1969 were on samples concentrated by flash evaporationo This concentration procedure produced substances that interfered with the reduction of the molybdate complex in orthophosphate measurement. Removal of the interfering materials proved difficult and time-consuming (Hooper, 1969). 67

In 1970, we perfected a procedure for concentrating the organic residues on an ion-exchange resin. This proved more efficient and more reliable. For these analyses we collected 600-ml water samples in clean polyethylene bottles from three locations in the lake: (1) the center of the lake at a depth of 1 M, (2) the center of the lake at a depth of 7 M, (3) at a depth of 0.2 M in the bog mat at the southeast corner of the lake, 5.5 M from the edge of the open water. The 600-mi samples were applied to a 2 x 15 cm column of Dowex 1 x 8 anion exchange resin, 100-200 mesh, formate form. The effluent and the subsequent water washes of the resin were colorless, contained no phosphate, and did not absorb light at 260 nm, as compared to a water blank. A gradient elution was set up between 0.01 and 0.5 formic acid and allowed to flow at a rate of 0.5 ml/min. Measured fractions were collected on a Buchler fraction collector. Fractions were checked for absorbance at 260 nm against appropriate formic blank. Phosphate was analyzed using the procedure given by Strickland and Parsons (1965). Selected fractions were assayed for ribose content, using the orcinol method (Ashwell, 1957) and UV absorption spectra were determined on a Beckmann DB-G scanning spectrophotometer. Some fractions were assayed for labile phosphorus by adjusting the pH to 1.0 with HC1, boiling for 10 min, and assaying for orthophosphate. Fractions were digested with perchloric acid to determine total phosphorus content. Results Phosphorus compounds such as phosphoric acid, polyphosphoric acid, nucleotides, and nucleic acid can be absorbed on an anion exchange resin. Elution with increasing normality of acid or salt can successively elute and separate various compounds (Cohn, 1957). When lakewater from a depth of 7 M was analyzed in this manner, it was shown to contain several different ribose and phosphate-containing compounds which absorbed in the 250-260 nm, wavelength region (Table I). Such properties are characteristic of ribose nucleic acids. It was not determined whether these compounds were ribose nucleotides or polymers. Free ribose nucleosides, which also absorb in the 250-260 nm range, do not absorb to the resin and would have been detected in the pre-elution wash. Nucleosides were not present in these samples. Mat water and lake water from a depth of 1 M contained smaller amounts of RNA than the 7 M water. All three samples had a clearly defined orthophosphate peak and a second inorganic peak that corresponded in order of elution to polymers of orthophosphate (Beukencamp, et al., 1954). Since earlier work has shown that this lake water contains a labile inorganic phosphate, similar in properties to polyphosphate, this peak is described in Table II as polyphosphate. 68

O X o OH u0 L' J 0J0J a ra P-I r - - r-r- 00 H 0 0.r-4 a) 0 P. I U'\ -J "lo\0 0 ~t H H ~ro 0 *. r- I o H H *H ~ SCH o o * * N Jl CH -H o 0l 0 r 0 r-.r13 t N. r-I Or,O rdrrj H < 2 X 0 0 H P 0 d d 9 C 0 CT D o MU\ n t o I P \10 I - r <P-l D, ^n O\ o ^ L\L r. — OD -t \o 69

Table II RNA* Orthosphate, Polyphosphate, Sample amplemol/1 fg P/1 kg/1i g/l Lake water, 7 M 1.56 48.3 22.7 13o0 Lake water, 1 M 0.52 16.1 6.6 4.3 Mat water 0.40 12.4 32.8 5.0 *Based on UV absorption, using an average extinction coefficient of 14,000 cmr-lM-. P content assumes molar equivalence between phosphate content and base content, Discussion There were sharp differences between samples from the three locations in the amount and character of the 260 nm absorbing materials. This may indicate differences of origin of the RNA fractions or differences in state of degradationo Peaks in the 250-260 nm range were broad and lacked prominent shoulders, which indicated that the material was not single nucleotides but some size of polymeric form. The lack of agreement in stoichiometry between UV absorption, ribose, and phosphorus leaves some uncertainties. These differences arose because we were below the desirable range of accuracy in certain of the procedures. The larger quantity of RNA at 7 M than at 1 M may in part arise from the greater degradability of nucleic acids in light (Armstrong, Williams, and Strickland, 1966). However, larger quantities of RNA might be expected at 7 M from autolysis of cells settling into this layer. The lower RNA content of the mat water compared to 7 M and 1 M suggests a limnetic origin of this RNA since water exchange takes place between the mat and the lake. However, differences in character of the RNA fraction were also apparent; therefore, origin in the mat cannot be excludedo 70

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APPENDIX D CHEMICAL DATA, NORTH GATE LAKE, 1970. 77

n) V ) CL /O rf)0 0 N L0 Oco) -rH 1 x/ 0 -d N (,- (0' C 0D ed 0 ~ ~ (Sti3131N) HldJa 79

CD: O- ID \ ~ N'N d 6 ~~~~~~~r~~~~~~~~~~~~~~~~) ~ ~ ~ ~ ~ ~ ~ z z (S\3ln \ HJ~d3G 8o 80

(D ^i 0 I 10 --- T1 ~/~~~~~~~~O OD N N /<{ 113 0 OD N O~~~~~~~~~~~~~~~~~~O!l ~~~~~~~~~~~~~~~~~~~OD od~~~~C Nl) 0 w 0 z~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ D D ct3 (x) LA~~~~~~~~~~~~~~~~~~c tc)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~a cw )/ \, / /^\ ~> p~~~~~~~~~~~~~~~~- ~ /// / \~ -i < 0~~ (S\3i3 ) Hid3G 81

111\ I< — U) \\= ~l OD u -'k0 -N s (00 CDCC ~ ~ ~ ~O 1C~,,-', _ - <~ NN R D LOz r 00 (Sa313JN) HId3a 82

'0 4 ~.~ ~ ~ ~ ~ ~rQ C 0 0 0 4~ -p C D~~~~~~D~~~~~ a,~~~~(O 1 - / \ ~ _ CM- C o 0) 0 l —------ l —-- I 0 H- oj (sjaau) 8~daa 83