THE TRANS LOCATION 05 RADIO-"PH cam-Imus THROUGH AN AQUATIC ECO-SYSTEM Thesis {or Hue Degree of M. S. MICHIGAN STATE UNIVERSITY Allen Warner Knight 1961 I. Iovv ."W 32 :1 m RAH-i LIBRARY Michigan State University 947061 THE TRANSLOCATION OF RADIOPHOSPHORUS THROUGH AN AQUATIC ECOSYSTEM By ALLEN WARNER KNIGHT AN ABSTRACT Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1961 Approved @5/Qbufd Elba/I ABSTRACT On July 8, 1959, twenty-one (21) millicuries of radioactive phosphorus were added to the West Branch of the Sturgeon River (Section 21, T. 33 N., R. 3 w.), Cheboygan County, Michigan. The main objective was to determine the fate of radioactive phosphorus released into the stream ecosystem and the manner by which the communities and pOpulations participated in the distribution of the radio- activity. The uptake of such a tracer will differ accord? ing to anatomical characteristics, physiological prOpertiea life histories and the relationships of the organisms to each other and their environment. The radiOphosphorus was first removed by suspended materials including the phytOplankton and bacteria. These forms, in turn, distributed the isotOpe to other members of the system through P32 'feedrback'. The P32 uptake by the periphyton portion of the primary producers reached a peak in approximately #.hours after isotope treatment, whereas the larger aquatic plants reached peak activity within h to 2% hours after isotope treatment. The uptake of radio- phosphorus by primary consumer organisms was the result of these herbivorous forms ingesting plant material. The primary consumers reached a maximum activity level within u weeks after isotope treatment. Uptake of P32 by the secondary consumer organisms was a result of these organisms ingesting the herbivores as food. The carnivores reached peak activity within 6 to 8 weeks from the date of isotope treatment. The uptake of radiOphosphorus by aquatic organisms may occur by absorption, as in aquatic plants; through mem- branes exposed to the surrounding water; and through in- gestion of food or inert particles, as in the case of aquatic animals, which contain the radiOphosphorus. The accumulation of radiOphosphorus, it is concluded, follows-~for the most part-~a definite and orderly pattern. The activity in the water is removed by primary producer organisms which are then fed upon by primary consumers; these in turn are fed upon by secondary consumers. From a population estimate of the standing crOp of fish and invertebrates in a 1,000-yard section of the study area, it was found that the trout and invertebrate pro— duction in the West Branch of the Sturgeon River is considered unproductive. A. W. K. THE TRANSLOCATION OF RADIOPHOSPHORUS THROUGH AN AQUATIC ECOSYSTEM BY ALLEN VARNER KNIGHT A.THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1961 Ci /5’/57 Q /"/ 73/2.- ( ACKNOWLEDGMENTS In the study of the West Branch of the Sturgeon River, the writer was variously aided by different persons to whom he wishes to eXpress his sincere gratitude: Mr. Frank Beekman and Mr. Kenneth Osborn for their assistance in the field; Mr. Hugh Clifford, a fellow graduate student, for his assistance in the field and laboratory; Dr. W. Carl Latta and his Pigeon River Staff, the writer is indebted to for the many courtesies, conveniences, facilities and direct aid; above all the writer owes his greatest indebt- edness to Dr. Robert C. Ball, under whose guidance and direction this study was carried out; Dr. Frank HOOper, who is co-investigator in the overall project, which was sponsored by the Atomic Energy Commission; to his wife, Barbara, for her assistance in the exacting task of proofa reading, typing and unending support. This study was made possible by a Graduate Research Fellowship from the Institute for Fisheries Research of the Michigan Department of Conservation. 11 TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . Description of the Study Area . . Sampling Stations . . . . . . . . Station 3 . . . . . . . . . Station 8 . . . . . . . . . Station 12 . . . . . . . . . Station 14 . . . . . . . . . METHODS AND PROCEDURES . . . . . . . . Radiological Techniques . . . . . The Introduction of IsotOpe Measurement of Activity . . Counting Equipment . . Calculation of Results Background . . . . Volume Factor . . Decay Factor . . . Flow Data . . . . . . . . . . . . Fertilization . . . . . . . . . . Water . . . . . . . . . . . . . . Field Procedures . . . . . . Laboratory Procedures . . . Periphyton . . . . . . . . . . . Field Procedures . . . . . . 111 Page -4 -a -q ox cn \» 10 10 1o 11 11 11 12 12 12 in 15 15 15 19 20 20 Laboratory Procedures . Aquatic Plants . . . . . . . Field Procedures . . . Laboratory Procedures . Biomass Estimate . . . Aquatic Invertebrates . . . Field Procedures . . . Hand Picking . . . Logs . . . . Moss and Deposits . Stones . . . Aquatic Vegetation Terrestrial Vegetation Trash . . . . Surber Square-foot Sampler . Direct Current Fish Shocker Laboratory Procedures . Biomass Estimate . . . Fish and Lamprey . . . . . . Field Procedures . . . Laboratory Procedures . Biomass Estimate . . . RESULTS AND DISCUSSION . . . . . water . ... . . . . . . . . Periphyton . . . . . . . . . Initial Uptake of RadiOphosphorus iv Page 21 23 23 23 24 25 25 26 26 27 27 27 28 29 29 3O 3O 31 33 33 35 36 38 39 51 Page Exchange and Regeneration of Radiophosphorus . . . . . . . . . . . 58 Larger Aquatic Plants . . . . . . . . . . . 63 IsotOpe Uptake and Accumulation . . . . 6} Larger Aquatic Plant Biomass Estimate . 82 Aquatic Invertebrates . . . . . . . . . . . 83 IsotOpe Uptake and Accumulation . . . . 83 Invertebrates Studied . . . . . . . . . 8” Variation in Radiophosphorus Uptake by Invertebrates . . . . . . . . . . 101 Invertebrate Biomass Estimate . . . . . 107 Fish and Lamprey . . . . . . . . . . . . . . 108 Radiophosphorus Accumulation in Various Tissues of the Brown Trout . 121 Biomass Estimate . . . . . . . . . . . 123 Translocation of RadiOphosphorus Through the Ecosystem . . . . . . . . . . 125 Water . . . . . . . . . . . . . . . . . 129 Primary Producers . . . . . . . . . . . 129 Primary Consumers . . . . . . . . . . . 129 Secondary Consumers . . . . .o. . . . . 130 Aquatic Insects . . . . . . . . . 130 Fish . . . . . . . . . . . . . . . 130 SUMMARY . . . . . . . . . . . . . . . . . . . . . 131 APPENDIX . . . . . . . . . . . . . . . . . . . . 136 LITERATURE CITED . . . . . . . . . . . . . . . . 1&2 Table 1. LIST OF TABLES Page Water activity (corrected counts per minute per milliliter) at different points in the stream channel during passage of isotOpe dose . . . . . . . . . . M7 POpulation and biomass estimates of fish in 1,000 yards of the West Branch of the Sturgeon River . . . . . . . 123 A comparison of standing crop estimates of fish, invertebrates and aquatic plants for various streams . . . . . . . . 12% Highest activity values recorded in aquatic organisms from the West Branch of the Sturgeon River after the addition of radiophosphorus . . . . . . 137 A list of organisms found in the West Branch of the Sturgeon River, 1959. The organisms presented.have been identified as far as possible or practical in the limited time avail- able. This list is based on the taxonomic keys presented by Pennak (1953), Burks (1953), Usin er (1956) and Hubbs and Laglar (1958? . . . . . . . . 138 vi LIST OF FIGURES Figure Page 1. Map of the West Branch of the Sturgeon River area, showing the sampling stations and site of isotope entry . . . 9 2. Daily staff gage readings at Station 8 on the West Branch of the Sturgeon River, during the period June 29 to August 19, 1959 . . . . . . . . . . . . l7 3. Total water activity, and the activity of water-washed and acid-washed solids filtered from stream water by milli- pore filter. Counts were corrected for background and decay . . . . . . . . . . #3 A. Total water activity at various collecting stations during passage of isotOpe. All counts were cor- rected for background and decay . . . . #6 5. Mean water activity at various collecting stations during the passage of isotope. All counts were corrected for background and decay . . . . . . . . . . . . . . . . . 50 6. Activity of periphyton taken from artificial substrates at Stations 3, 8, 12 and In, for the entire study period. Counts were corrected for background and decay . . . . . . . . . . 53 7. Activity of periphyton taken from artificial substrates at Stations 3, S, 12 and 14, for the entire study period. Counts were corrected for background and decay . . . . . . . . . . 57 8. Comparison of activity of periphyton substrates at Stations 3 and S. Counts were corrected for back- ground and decay . . . . . . . . . . . . 62 9. Activity of stream vegetation on July 8, 1959. Counts were corrected for background and decay . . . . . . . . 66 vii Figure Page 10. Activity of Potamo eton at Stations 3, 8, 12 aha In, for the entire study period. Counts were corrected for background.and decay . . . . . . . . 69 11. Activity of Fontinalis s . at Stations 3, 8, 12 EHHII¢T_TEr e entire study period. Counts were corrected for background and decay . . . . . . . . . . 71 12. Activity of Chara at Stations 3, 8, 12 'and 14, for tHe entire study period. Counts were corrected for background and decay . . . . . . . . . . . . . . . 73 13. Activity of Batrachospermum at Stations 3, 8, 12 and”I¢,_durihg July 1959 . . . 77 1%. Activity of plants collected at Station 8. The dotted lines represent the extrapolation of the logarithmic rate of decrease of activity during early stages of the experiment. Counts were corrected.for background and decay . . . 81 15. Activity recorded for Station 3 invertebrates representing four food niches. Counts were corrected for background and decay . . . . . . . . f 87 16. Activity recorded for Station 8 invertebrates representing four food niches. Counts were corrected for background and decay . . . . . . . . 89 17. Activity recorded for Station 12 invertebrates representing four food niches. Counts were corrected for background and decay . . . . . . . . 91 18. Activity recorded for Station 1h invertebrates representing four food niches. Counts were corrected for background and decay . . . . . . . . 93 19. Activity of Physa at all stations. Counts were corrected for back- ground and decay . . . . . . . . . . . . 103 viii Figure ‘ Page 20. Activity of invertebrate organisms following treatment of the stream with p32. The variation of sub— samples within routine samples is shown. Counts were-corrected for background and decay . . . . . . . . . . 106 21. Activity of American brook lamprey at all stations. Counts were corrected for background and decay . . . . . . . . 111 22. Activity of the sculpin at all stations. Counts were corrected for background and decay . . . . . . . . 113 23. Activity of brown trout at all stations. Counts were corrected for background and decay . t . . . . . . 116 an. Activity of brown trout and sculpin following treatment of the stream with radiOphosphorus. Counts were corrected for background and decay . . . 119 25. Movement of radioactive phosphorus into the different components of a lotic ecosystga, following a single addition of P to the water. Counts were corrected for background and decay . . . . . . . . . . . . . . . . . 127 ix Dedicated to M! FATHER INTRODUCTION When radioisotOpes are released into the environment, they quite often become diapersed and diluted, but may also undergo unexpected movements and concentrations. At the present time very little is known about the mechanisms of uptake, concentration, retention, and excretion of radiOphosphorus by fresh-water organisms. Even less is known about the fate of radiOphosphorus released into lotic ecosystems and the manner by which the ecological communities and.p0pu1ations of such a system control the distribution of radioactivity. The necessity of obtaining such information is becoming increasingly urgent as more and more power-producing reactors are put into Operation. In most cases the only ready means of disposal of large quantities of liquid effluent, by these reactors, is into fresh water. The nearby rivers or lakes will receive the discharge of radioactive wastes and, even though an isotope might be diluted to a relatively harmless level on release into the environment, it might become concentrated by aquatic organisms or a series of organisms to a point where it would be critical. In addition to valuable knowledge of a potential human health hazard, radiOphosphorus can also be used to trace the metabolism of phosphorus through the entire ecosystem and thus provide information available by no other neans. It is the purpose of this paper to obtain information on: (1) the modes of transfer, accumulation and transla- cation of radiophosphorus between various components of a rapid stream biOIOgical system; (2) the interrelationships between particular species; (3) the metabolism and fate of nutrient material added to such an aquatic environment. Description of the_Study Argg The West Branch of the Sturgeon River is a cold water river, flowing through Cheboygan, Otsego and Charlevoix Counties, Michigan (T. 33 N., R. 3 W.). The West Branch of the Sturgeon River has its origin in Hoffman Lake, a hard water lake with an approximate area of 128 acres. The river flows from the northeast end of Hoffman Lake and continues in a northeasterly direction, through a narrow valley with steep and rolling glacial morainic hills. The river flows for about lur miles before confluence with the Sturgeon River, at the town of Wolverine, Michigan. The vegetation in the valley is chiefly birch, aspen, cedar, tamarack and balsam fir. Along the stream margin the vegetation is primarily cedar, tamarack, aspen, tag alder and ninebark. The water temperature of the stream remains cool throughout the summer, due to water entry by way of springs and tributaries, as well as, shading by overhanging trees and.mixing of the water, due to general turbulence. The summer water temperature remains between 52°F. and 58°F. throughout the summer (Clifford 1959). The stream flow through the study area has a mean of A3.75 cubic feet per second.1 The water level of the 1Courtesy of Vannote and Carr, 1959. n, stream remained relatively stable throughout the summer. Temporary turbidity is quite frequent and is caused by rains. Unless the rains have been very severe, the turbidity lasts only a few hours. The stream water in the study area has a total alkalinity of approximately 180 p.p.m., and a total phosphorus concentration of approx- imately 7 p.p.b. (Borgeson 1959). The thorough churning and mixing of water with air insures a high dissolved oxygen content in the stream water. In addition to the churning, the low water temperature permits the absorption of greater quantities of oxygen. The area of the stream covered by this study is about 2,650 yards long (Figure 1). The stream bottom in this area varies from sand and gravel to silt and detritus. For the first 250 yards, the vegetation is abundant with beds of Chara 22,; the water moss, Fontinalis antipyretica; mareis-tail, Hippuris vulgaris, with occa- sional growths of water cress, Nasturtium officinale. The stream bottom is gravel and.sand down the middle with an occasional silt bed.along the shore. The next 300 yards are almost devoid of any vegetation. The stream in this section becomes almost entirely riffle area with only a sparse growth of pondweed, Potamogeton pectinatus; water moss, Fontinalis antipyreticg; mare's-tail, Hippuris vulgaris, and occasional beds of Chara.gp. The stream bottom is predominately sand and gravel in this area. The stream for the remainder of the study area supports a luxuriant growth of plants. The predominant plants are: ‘th£§_gp,, which grows in beds of silt and detritus; Potamoggton pectinatus, which grows in riffle areas and in swift water near the shore; Batrachospermum moniliformg, which grows attached to rocks and logs; Oedogonium s ., which grows in filamentous strands attached to twigs, logs and rocks in the stream; mare's-tail, Hippuris vulgarig, which grows in silt and detritus beds near the stream bank; and tape-grass, Vallisneria 53., which grows occasionally in silt beds along the bank, as does occasional solid tangled masses of water cress, Nasturtium officinale. The stream bottom in this area varies from sand and gravel to silt and detritus. The fish present are typical cold water forms: eastern slimy sculpin, Cottus cognatus; northern mottled sculpin, Cottus bairdii; brown trout, Salmg_trutta; rainbow trout, Salmo gairdnerii; and brook trout, Salvelinus fontinalis. The aquatic insects present are represented by the orders: Odonata, Ephemeroptera, PleCOptera, Trichoptera, Diptera, Megaloptera, Coleoptera, and Hemiptera--in general, only such insect types as require a high degree of oxygen- ation. Because of the currents, the biota is further limited to species that are either strong swimmers or strong clingers, the latter directly by means of structural adaptations (claws, suckers) or indirectly by means of clinging devices constructed by the various species (webs, Jellies, attached cases). The insects will be discussed more fully elsewhere. Other invertebrates present are the annelids, gastrOpods and pelecypods. Sampling Stations Sampling stations were established within the prin- cipal study area and were so located as to most nearly. represent the various situations that were found to exist within the eXperimental area. Such factors as shade, stream velocity, bottom type and vegetation composition were the variables taken into account when each station was established. The sampling stations that were desig- nated as permanent stations were numbers 3, 8, l2 and 1% (Figure 1). These stations were the permanent sites for collections of aquatic plants, periphyton, fish, lamprey and aquatic invertebrates throughout the study period. Station 3. A well shaded area 300 yards below the isotope entry was selected for this station. The water depth was quite shallow with an average depth of 12.8 inches (Zettelmaier, unpublished). The stream bottom, at this station, was mainly of sand with isolated areas of gravel. The water flow at this site was somewhat retarded, due to a log obstruction immediately below this sampling range. Vegetation in this area was sparse and limited mainly to moss and Chara J3. Station 8. This site was situated in a straightaway portion of the stream, 900 yards below the point of isotOpe entry, where there was a great deal of streamside vegeta— tion which did not shade the stream to any extent. The mean depth of the water at this station was found to be 17.2 inches (Zettelmaier, ibid,) and flowing rather rapidly over a predominately gravel midstream channel with luxuri- ant beds of Chara bordering this main channel. Station 12. The site chosen for this sampling range was a river bend, 2,200 yards below the point of isotope entry, with a gravel bottom, except for silt beds located near the shore. This site was devoid of any shade and was obstructed in part by a stream deflector. The mean water depth at this range was 13.3 inches (Zettelmaier, 121d.) with a rather rapid flow, due to the stream deflector. The vegetation consisted of huge beds of 99222.22! and a few patches of Potamggeton pectinatus. Station 14. The site chosen for this station was a very rapid.riffle area, some 2,650 yards below the point of isotOpe entry. There was little shading of the stream by streamside vegetation and only sparse aquatic plant growth. The bottom of this area ranged from gravel to huge boulders. The mean water depth was 12.2 inches (Zettelmaier, ibid.). .RApco smouomd no spam was mcoaumpm madaaamm can mmdxonm .mmmm cmbdm commaspm can go nommnm pmmz map no as: .H shaman mi: WM/ - I .. I mz.mw>i_os> 00 cam 60 3\ ' ..JD t .9 \\ \ .\ , A emeeq _ _maoiom_ 8:05 3358 1e 59:22 .5560 89885 .>> mix ..2 mm H __mn_maxm / J Figure l METHODS AND PROCEDURES Radiological Techniques The Introduction of Isotope The choice of a tracer to be used for a particular experiment is dependent on the specific nature of the experiment. A tracer with a very short half-life pre- cludes the possibilities of shipment for appreciable distances, of synthesis of compounds, and of lengthy ex- periments. The tracer that was chosen for the present study was radiOphosphorus (P32) which is a beta ray emitter with a maximum energy of radiation of 1.712 mev. Radio- phosphorus is further desirable because of ease of counting and possession of a satisfactory half-life (1h.3 days). The radioisotope for this study was supplied by the Oak Ridge National Laboratory, Oak Ridge, Tennessee, as phosphate (POh) dissolved in weak hydrochloric acid. The 1.1 ml. solution of radiophosphorus was assayed at 8:00 AM, July 6, 1959, and was found to have an activity of 23.29 i 31 millicuries. The addition of isotOpe was made in Section 21, T. 33 N., R. 3 W. (Figure 1), on July 8, 1959. The activity of the radioisotOpe at the time of intro- duction into the river was calculated to be 21.17 i 3% millicuries. The method whereby the isotope was released into the West Branch of the Sturgeon River has been described in 10 11 detail by Borgeson (ibi§,) and Clifford (ibi§.), but it is felt that a brief description at this time would be de- sirable. The 1.1 ml. solution of P32 was thoroughly mixed with 50 gallons of stream water in a 55-gallon drum. The diluted isotOpe was siphoned from the drum by way of a polyethylene tube. The rate of siphoning was controlled in such a way that the isotope entered the river at a constant rate over a 33-minute period, commencing at 9:33 AM and terminating at 10:06 AM. The flow of the stream at the point of isotope introduction was approximately 38 cubic feet per second. Using this stream flow and.the flow rate of the diluted isotOpe given above, the theoret- ical concentration of isotOpe in the water was calculated to be approximately 1.22 x 10’5 microcuries per milliliter. Measurement of Activity Counting_§guipment. Radioactivity was measured with a Nuclear measurements, internal flow prOportional counter converter, PCC—lOA, and coupled to a decade sealer (model PC-lA). Each day a 15-minute count was made using an empty counting chamber. This value was plotted on a back- ground control chart. The background varied very little from day to day, generally from 50 to 5% cpm with a mean value of 52 cpm. Each sample was counted for a minimum of three minutes. Calculation of Results. To convert the relative value of counts to an absolute value of corrected cpm, 12 ,uc/gm., or uc/ml., it is necessary to apply various cor- rection factors to the raw counts (the correction factors are from Robeck, gt_gl,, 195A). The factors are: Background: the natural or instrument background count is caused by cosmic radiation and radioactive sub- stances in or near the counter. The determination of back- ground was carried out each day by Operating the counter without a sample for 15 minutes. The mean background count was 52 cpm for the entire experimental period. This background count was subtracted from all observed counts to correct for any radioactivity arising from sources other than the one directly under consideration. Volume factor: due to the various organisms and materials samples, tremendous variation in sample size resulted. To correct for this difference the observed cpm minus background was divided by the weight of the sample in grams, or, as in the case of water, it was divided by the volume in milliliters. Decay factor: due to the decrease, with time, of the number of radioactive atoms in a sample as a result of their spontaneous transformation, counts must be corrected for this decay. The influence of this decay is very im- portant when using a short-lived isotOpe, such as radio- phosphorus, with a half-life of 1#.3 days when sample trans- fer and preparation time is large. Kinsman (1957) presents a radiological decay table giving the fraction of radio- activity remaining at a given time. The table for 13 radiOphosphorus can be used conveniently for the recorded time unit and calculations were made for times which fell beyond the time span given in the table. The value ob- tained from the table was divided into the observed.counts corrected for background and volume factors. The recip- rocal of the decay value obtained from the table can be used and, instead of dividing, one multiplies the corrected observed counts by this reciprocal value. Various other correction factors are given by Robeck, gt_§1,, ($2193): but if all observed counts were corrected for background, volume and decay, the resulting values would be meaningful without multiplication by correction factors remaining constant throughout the study period. In this study the correction factors used were: Background.a variable from day to day, generally (8G) 50 to 5“ cpm with a mean value of 52 0pm. Volume factor = variable depending upon the size (VF) of the sample. Decay factor = variable with time between time (BF) isotope was introduced into the river and counting ime. Activity density : (cpm - SO) x (VF) x (DP) 3 corrected cpm. In order to facilitate comparisons with other investi- gators and determination of maximum permissible concen- trations set down by the National Committee on Radiation 1% Protection, sponsored by the National Bureau of Standards (1953), it becomes necessary to convert corrected cpm into microcuries. It is known that (Robeck, 31 31,, ibid.): 1 curie (c) 3.7 x 1010 disintegrations per second (dps) 3.7 x 10” ape 2.22 x 105 dpm l microcurie (uc) 1 dpm 1/2.22 x 106 = 4.5 x 10-7 uc Therefore Conversion Factor (CF) 3 u.5 x 10-7 If results are desired in terms of microcuries, then: microcuries = (cpm - BC) x (VF) x (DF) x (h.5 x 10‘?) Flow Data Fluctuation of the water level in the West Branch of the Sturgeon River for the entire study period is shown in Figure 2. The measurements were made from a river staff gage located ten yards above Station 8 (Figure l). The greatest fluctuation was recorded on August 17 and was due to a severe storm. The river, except for one or two dates, maintained a stable water level. Flow data obtained on July 7, 1959, indicated a uni- form progressive increase in water velocity as one proceeds l progressively downstream through the experimental area. The flow recorded at various locations in the eXperimental 1Courtesy of Vannote and Carr, 1959. 15 area in cubic feet per second is as follows: Station 1, 38.17; Station 5, 38.73; Station a, A3.n6; Station 11, Hu.85; 100 yards below Station 12, #7.53; and bridge below station 15, A9.72 (Figure 1). Fertilization In the preliminary investigation of the stream in 1958 (Clifford, ibid.), a low level of periphyton production was revealed. It was assumed that a similar situation was operative in June of 1959. It was thus deemed necessary to ”prime” the experimental area with inorganic fertilizer in order to insure a rapid uptake of isotope and provide a sufficient amount of periphyton for radiological examina- tion on the isotOpe entry date. The inorganic fertilizer used was a commercial 12-12-12 fertilizer. At the point of isotOpe release, 200 pounds of the fertilizer were ap- plied continuously during the period June 29 to July 6, 1959. Station 8 (Figure 1) received 100 pounds of the fertilizer continuously during the period July 3 through July 6, 1959. Water Field Procedures In order to obtain water samples that would be re- presentative of the detectable activity present during the entire period of isotOpe passage through the study area, samples were taken at intervals varying from 5 to 30 minutes. 16 Figure 2. Daily staff gage readings at Station 8 on the West Branch of the Sturgeon River, during the period June 29 to August 19, 1959. 17 2i 1 A A n A A A2223 7 11 15 19 l A A 1 L A A bi A L L A i is 11: 18 22 26 3o 3 Figure 2 July 14 LI 5 h». 3 2 1. 0 9 C O O O O O 0 Ac ,o /o ,0 ,o ,0 a; knock a no masoniccov unmaon hobaa ho coapcsposah 8. 18 ' The 5-minute interval samples were obtaineds-at some of the stations near the isotOpe release-~when it was hypothesized that the isotope was flowing past that particular station. Stations maintaining a 10—minute sampling schedule were those stations located in the lower portion of the study area. At the expiration of the initial hour of sampling, the frequency of sampling was maintained at lS-minute intervals. At some of the more remote downstream stations, the sampling interval eventually lapsed into 30-minute periods. The water sampling stations maintained in this study were Stations 3, 5, 8, ll, 12, 14 and 16. The seven sam- pling stations were located between the point of isotOpe entry and the State Highway Park adjacent to US-27 (Station 16, Section 1A of T. 33 N., R. 3 W.), a distance of approx- imately 3.3 miles (Figure 1). Experiments were conducted (Borgeson, 131$.) in 1958 with fluorescein dye to determine flow time between stations and the feasibility of using such a dye as a visual indi- cator of the actual movement of isotOpe mass through.the experimental area. This method proved satisfactory as such an indicator and the assumption was made that the isotOpe would perform physically in the same manner as the dye. The fluorescein dye was released into the water at the isotope entry point 10 minutes previous to the actual isotOpe entry and again at the termination of isotOpe re- lease. The arrival of the dye at each station instigated 19 the water sampling. It was necessary to add additional dye at Stations 8 and 12, in order to supplement the fading color, due to dilution as it was transported.through the study period. The water samples were procured using 1H0 ml. poly- ethylene bottles, except for sixteen 500 ml. water samples taken while the isotope was moving downstream, which were rinsed for 30 seconds prior to obtaining each water sample. Samples were taken in the main current at each sampling station. The samples thus obtained were capped and trans- ported to the laboratory for radiological analysis. Laboratory Procedures The preparation of water samples for radiological examination was carried out as suggested by Robeck, g£_gl, (1212,), except for minor modifications. Three milliliters of concentrated nitric acid were I introduced into the sample bottle containing 140 ml. of water for analysis, whereupon the bottle was thoroughly shaken. A 50 m1. subsample was placed in a.l50 ml. beaker and allowed to evaporate to a small volume. The contents of the 150 m1. beaker were transferred to a stainless steel counting planchet. The 150 m1. beaker was then washed with 2N nitric acid and the washings thus obtained were placed in the planchet. This material was evaporated to dryness and placed in a muffle furnace for about one minute at 600°C. The planchet was removed from the furnace and 2O allowed to cool in an aluminum transfer pan, whereupon the samples were placed in the counting equipment for activity determination. The 500 ml. water samples were filtered through a type HA, 47 mm. “millipore” filter. Solids collected on the filter were washed with one-tenth normal hydrochloric acid in some instances and washed with only water in other cases, then placed in planchets. These were dried in a drying oven, set at 900-10006. for 10 minutes. The plan- chets were cooled and placed in the counting equipment for activity determination. Periphyton Field Procedureg Plexiglass plates with a total exposed area of 1.4 decimeters (2' x 5”) and a thickness of 7 millimeters were used to collect the periphyton growth. The plates were attached to a horizontal crossbar with 3/4” metal screws. The crossbar was attached to a steel post, by means of bolts and wing nuts, which was driven into the stream bed (Grzenda and Brehmer, 1960). The crossbar was lowered to approximately 8 inches below the stream surface when in the exposure position. A total of 35 plexiglass plates (2' x 5') were distributed on stands at each sampling station. In addition to the above plates, one large (4' x 10') plexiglass plate was also placed at each station. These large plates were attached to logs, roots and branches 21 below the water surface. The sampling stations maintained.in the periphyton study were Stations 3, 8, 12 and 14 (Figure l). The schedule for the plexiglass plate removal was carried out as follows: ten minutes after the second dye marker passed each station (signifying the completion of isotope addition), 4 hours, 24 hours and 96 hours after the dye marker passed each station, samples were collected. There- after, samples were obtained each week on Wednesday for the remainder of the study period. The total number of plexiglass plates removed each sample date was 5 at each station. Four of the small (2” x 5”) plates and one large (4' x 10') comprised a periphyton sample at each station. In addition to the above sampling procedure, a series of substrates that had been maintained at Stations 3 and 8 were removed from the stream temporarily during the period when the isotOpe pulse was passing through the eXperimental area and.replaced immediately after the isotOpe dosage had passed the station. The total period during which periphyton analysis was conducted was 35 days after the isotope was introduced into the stream. The plexiglass plates were transported to the laboratory as soon as possible for immediate radiological analysis.' All animal forms, such as blackfly larvae and other invertebrates, were picked from the plexiglass plates immediately upon removal from the stream. 22 Laboratory Procedures The plexiglass substrates, upon arrival at the labo- ratory, were scraped into a large beaker, using a polished glass slide, thus removing the periphyton community which had accrued thereon. The substrates were then rinsed to remove any periphyton that might be present after the scraping procedure. The resulting mixture of periphyton and water was filtered through a type HA, 47 mm. Millipore filter. While the filter pad (previously weighed) remained in place, .3 cc. of 0.01 N hydrochloric acid was introduced into the filter apparatus. The acid rinse was followed with a rinse of approximately 5 cc. of distilled water. The filter apparatus was allowed to remain in Operation ten seconds after the filter pad was observed to be free of all visible moisture. The filter pad was then removed from the filter apparatus and.p1aced in a previously weighed.plan- chet. The wet weight of the periphyton was Obtained by subtracting the weight of the planchet plus the filter pad from the total weight of the combined planchet, filter pad and periphyton. The material in the planchet was digested, using 5 ml. of concentrated nitric acid and.then placing this under a heat lamp until completely digested. The digestate was placed in a muffle furnace set at 600°C. and remained therein until the planchet was heated to red heat. The samples were then cooled and removed to the counting room for activity determination. The digestion procedure given above was adapted from the method outlined by Robeck, et al., for the preparation of filamentous algae samples. Aquatic Plants Field Procedureg The collection Of aquatic plants was done manually and routinely at Stations 3, 8, 12 and 14. The collection schedule was as follows: 4, 24 and 96 hours after the isotOpe arrived at each station; thereafter a weekly col- lection on Wednesday was maintained for a total of 7 weeks after the introduction of the radiOphosphorus. The aquatic plants collected at each station were: gh§£g_s ., Potamogeton pectinatus and Fontinalis antipyretica. The plant samples consisted of the entire plant, less roots, where such were present. The samples were rinsed in the stream water to remove adhering material or organisms and placed in 500 cc. polyethylene bottles for transport to the laboratory. Laboratory Procedures A l-to-2 gram subsample of each plant type to be pro- cessed was washed.with distilled water and dried with blotting paper. This blotted sample was placed in a pre- viously weighed evaporating dish. The total weight was then determined, using a beam balance. The weight of the evaporating dish was subtracted from the total weight and the resulting weight recorded as the wet weight of the plant sample. Concentrated nitric acid-~enough to cover the plant sample—'was added and the sample was placed on a hot plate 23 24 until completely digested. The digestate was placed in a muffle furnace, set at 600°C. The sample was removed from the muffle furnace and allowed to cool. After the sample had been allowed to cool, approximately 20 drOps of 2N nitric acid were introduced into the evaporating dish con- taining the ashed sample. The introduction of the acid solution and the scraping facilitated the removal of residue adhering to the evaporating dish. This scraping and wash- ing with acid solution was continued until complete re- moval of the residue was completed. The material obtained in the rinsing and scraping procedure was transferred into a stainless steel planchet. The planchet and contents were placed on a hot plate and the sample evaporated to dryness. The planchet and dry sample were placed in a muffle furnace, set at 600°C.; the planchet was heated to red heat, removed from the fur- nace and allowed to cool. The planchet containing the sample was transferred to the counting room for activity determination. Biomass Estimate On August 15, 1959, a quantitative estimate of the aquatic vegetation biomass was conducted on 1,000 yards of the study area. The area was delimited by Station 1 and Station 8 (Figure 1). Numbers were taken from a Table of Random Numbers and numbers thus selected were designated as the cross-stream transects. The procedure for the aquatic 25 plants was exactly as for the aquatic insects, except for the collecting equipment, discussed elsewhere under aquatic insect biomass estimate, to which the reader is referred for a detailed discussion Of the random sampling technique. In order to isolate a square foot of aquatic vegeta- tion, a metal frame measuring exactly one foot square was attached to a wooden handle. The sample was placed firmly against the stream bottom at the random collection site. Thereupon, a complete removal of all vegetation enclosed by the sampler was completed at each site. Each square foot sample of vegetation was blotted to remove excess moisture and weighed. NO attempt was made to ascertain the individual weights of the several different plant types found in the samples. The results of the vegetation were recorded as pounds per square foot. Aquatic Invertebrates Field Procedures The collection of aquatic insects for radiological analysis was made at Stations 3, 8, 12 and 14 (Figure l). The sampling schedule for aquatic invertebrates was as follows: 4, 24 and 96 hours after the introduction of the radiophosphorus with weekly sampling every Wednesday there“ after, for a total of 7 weeks. In a tracer study such as the present one, it is de- sirable to select those aquatic invertebrates for radio- logical purposes which are abundant, consistently and easily 26 collected. The animals were also selected.as representa- tives of various trOphic levels, in order to Obtain data that were representative of both primary and secondary consumer invertebrates. With this in mind, the following invertebrates were Obtained and processed throughout the study period: the stonefly nymph, Pteronargys s ., black- fly larvae, Simulium 22,; mayfly nymph, Hexggenia 52.; snipe fly larvae, Atherix variegata Walker; caddisfly larvae, Brachycentrus 22.; fishfly larvae, Chauliodes 32,; and the pouch snail, Phygg_gp. Inasmuch as specific organisms were used in the radiological study, it was neces- sary that collection procedures obtain predetermined organ- isms consecutively at each collection site. In order to accomplish this, many specific methods were used. The fol- lowing is a discussion of aquatic invertebrate collecting procedures used in the present study. Hand Picking Logs: Logs in the water are very rich sources of insect larvae. The crevices and cracks of logs usually harbor many insect larvae. The exterior surface of the log serves as a highway on which many organisms scurry back and forth in the acquisition of food. The pupal form of some insects can also be found in logs. Logs were either lifted from the water and examined, or were placed upon the shore for examination. As soon as the log was exposed to the atmosphere, many immatures crawled and ran from their 27 hiding places. The loose strips and rotten slivers were stripped from the log; thus, many insect larvae of the crawling type were located. If the log was in water that Just covered it, one could, by using forceps, pick many larvae from it while it remained under water. Moss and deposits: The water moss, Fontinalis antipyretioa, growing on logs is an excellent habitat for many insect larvae. As one removes the moss from the log or stone, care should be exercised in obtaining those forms hiding under the tuft of moss. Once the moss was separated from its substrate and exposed to the atmosphere, insects crawled from it, making procurement easy. The moss tuft was then carefully dismantled, removing larvae in the pro- cess. Marly deposits on logs and rocks are also the hiding place for many species. Stones: Stones were found to have many organ- isms adhering tO, crawling on and clinging to them, as well as, many forms hiding under them. Some stones are of such a nature that hook and claw bearing larvae can cling to them in very fast water. Pupal cases and net and case bearing larvae also make use of rocks as a substrate for clinging and attachment. Some organisms use rocks as an attachment from which food is either filtered from the water, or Obtained from the surface of the rock. Aquatic vegetation: The aquatic vegetation Offers many insects a substrate on which to cling in rapid water. For others, the vegetation is a protected place to 28 hide and prey on other insects and small organisms. Vege- tation is also a place for other forms to feed on algae and diatoms. Some forms even feed on the aquatic plant itself. The aquatic plant of greatest abundance was the stonewort, 92352.52, EEEEE grows in submerged gardens or in large mats upon silt and debris. In order to obtain insect larvae from such a growth, it was necessary to isolate a portion of it from the bottom and from the rest Of the QEEEE mat. The method found to produce the best results was to remove a large portion of 92352, roots, silt, debris, and all, and throw it out on the stream bank or on a log. As the water drained from the pile of ghgrg, insects began to crawl from it. In order to separate some of the forms from the silt and debris, the splashing of stream water over the 92253 that had been placed on the shore was necessary. The above method was slightly modified later in using a shovel instead of hands to remove the £2355 from the stream. The 92252 produced.great quantities of im- mature insect forms, along with some burrowing forms from the silt and debris, disengaged with the ghggg. The pondweed, Potamogeton pectinatug, grows rooted to the bottom, submerged in swift water. This pondweed af- forded the collector with many clinging forms, especially those with cases. Terrestrial vegetation: The stream edge, for the most part, was pOpulated with overhanging trees and shrubs. The shrubs, tag alder and ninebark, Occasionally dipped 29 their branches into the water. Clinging forms adhered to the branches and leaves of these shrubs dipping into the swift water. Trash: In a swift stream such as the one in the present study, one finds trash in the form of leaves, algae, twigs and numerous other materials caught in bundles on projections or deflectors in the stream. These collections of trash Offer an ideal habitat for many forms of aquatic insects. The forms range from the clinging forms to those that crawl and climb. This niche Offered one of the rich- est habitats (with the exception of ghggg) found in the stream as far as different types of organians were con- cerned. The trash was pulled from its snag point and spread upon the shore or on a log. The organisms readily crawled from the trash. By picking over each piece of trash, many additional forms were isolated. The cover of the trash Offers protection and a continually collecting food supply for carnivorous, herbivorous and omnivorous forms. Surber Square-foot Sampler The Surber sampler was used as a nonquantitative sampler. The midrchannel river bottom was, for the most part, of a gravel and rock character. The Surber sampler was used as a water not by holding it close to the bottom of the stream while gravel, stones and trash were disturbed as the collector moved upstream, scuffing the bottom with his boots. Numerous clinging and case forming insects were collected in this manner. 30 Direct Current Fish Shocker The collection of certain immature insects with a fish shocker was found effective. The positive electrodes were thrust into ghgag mats or silt and debris beds. This method was used primarily to obtain the burrowing mayfly, Hexagenia _p, Thrusting the positive electrode into a silt or debris bank produced the burrowing mayfly in great quantities, floating up from the substrate. The mayflies were easily scOOped up in a mesh net. The invertebrate organisms thus Obtained were taken immediately to the laboratory for radiological processing. Laboratory Procedures The procedure for the preparation of aquatic inverte- brates was modified from the method presented by Robeck, g£_gl, (ibidJ. The organisms that were to comprise a sample were rinsed with 0.01 N hydrochloric acid and placed in wire centrifuge baskets. The basket, plus sample, was placed in a centrifuge and Spun at 1,840 rpm for 15 seconds. At the termination of the l5—second.period, the propelling power was shut Off and the samples were allowed to run to a complete stOp. It was felt that this procedure would produce the most consistent moisture re— moval from all types of organisms. The organisms thus pre- pared were weighed on an electric Mettler balance. Upon weighing the sample, it was transferred into a stainless steel planchet for the digestion procedure. Concentrated 31 nitric acid (0.1 to 1 ml.) was placed over the organisms and the planchet and sample were placed under a heat lamp until complete digestion had taken place. The digestate thus Obtained was transferred.to a muffle furnace set at 600°C. for 5 to 10 minutes. The planchet and residue were removed from the furnace and.sllowed to cool. The planchet was then removed to the counting room for activity determination. Biomass Estimate On August 31 and September 1, 1959, the author made a quantitative survey of stream bottom organisms in the West Branch of the Sturgeon River. The survey was made with a Surber square-foot sampler. Inasmuch as the survey area is predominantly riffle area, it was felt that the Surber sampler could be used in a random sample of the area. The area of the stream used for this survey was de~ limited to 1,000 yards (Station 1 to Station 8). In order to eliminate bias or prejudice and to insure that the com- ponents of sample were completely independent from one another, random sampling techniques were brought into play. To facilitate the drawing of random samples, the Table Of Random Numbers was used. Since the survey area of the stream was 1,000 yards long, a maximum of four digits, groups of four numbers, were used.‘ The random numbers were selected by starting at the beginning of the table and proceeding downward until 32 30 numbers had been selected. The 30 numbers thus selected were designated as the cross-stream transects, starting at the upper end of the survey area and proceeding downstream. Each of these.cross-stream transects was randomly sub- divided. Each cross-stream transect was designated as 100 percent of stream width. Random numbers were selected in two digits. The number selected was converted into a per- cent. The percent selected at random was converted into a number in feet, corresponding to the percentage of the total width of each transect. This number (in feet), measured from the stream shore on the transect, was the sampling point. In selecting the sample point in this manner, the author believes the possibility of bias and prejudice has been held as low as possible, within the limitations of human error and equipment thus used. In using this method the sample points were selected on a random basis: i.e., any number of possible transects may have been selected for sampling and any number of points may have been selected on each transect. As each cross-stream transect was located, the width of the stream at this point was measured and recorded. The random predetermined percentage of this width was computed and, when the resulting measurement was located on the cross-stream transect, it became the collection site. The frame Of the Surber square-foot sampler was placed in po~ sition on the bottom, and.the enclosed pebbles, gravel, rubble, sand and silt or other material were carefully gone 33 over with the fingers to dislodge the specimen. The ma. terial thus obtained was emptied into a collecting jar and labeled for sorting later in the day. In the laboratory the samples were sorted, using a floatation method adapted from the one described by Anderson (1959). The organisms removed by the floatation process were transferred to preservation jars in which 70 percent alcohol was added and the sample was prOperly labeled. In the laboratory, also, the organisms were sorted into groups and identified to order and-~in some cases--to family. Each group was individually counted, then placed in a wire basket and spun for a consistent period of 30 seconds to remove surface moisture. Each group was then weighed on a Mettler multipurpose balance. The organisms were re- ported as number per square foot and the weights given in grams per square foot. Fish and Lamprey Field Procedures The collection of fish and lampreys for radiological examination was made at Stations 3, 8, 12 and 14 (Figure l). The fish and lampreys were collected, using a direct cur- rent fish shocker. The direct current power was furnished by a 230-volt, 2,500-watt, gasoline driven generator. The total weight of the generator is about 135 pounds. A sheet of OOpper, approximately 14 inches by 8 feet, mounted on 34 the bottom Of the flat bottom boat used to float the shocker, served as the negative electrode. The two posi- tive, hand held electrodes consisted of wooden handles, at the extreme end of which was affixed a piece of copper cable. The electrodes were thrust under the overhanging stream banks, under logs and other Obstructions which fur- nish cover for fish. Lampreys were Obtained in great numbers by thrusting the electrode into a gh§£§_or silt bed. The collection schedule for fish and lampreys was as follows: the first sample was obtained 48 hours after isotope treatment; thereafter, sampling was at weekly in- tervals with collections on each Wednesday. The fish and lampreys collected at each station were transported to the laboratory for radiological examination. The brown trout, S5123 trutta {£512 Linnaeus, and the Eastern slimy sculpin, Cottus cognatus gracilis Heckel, were the fish collected.and examined throughout the study period. The American brook lamprey, Entogphenus lamottenii lamottenii (LeSueur), was the lamprey collected and examined throughout the study period. The brown trout and the sculpin were the fish of choice, primarily because of their availability and because neither of these fish are stocked regularly in the West Branch of the Sturgeon River. The weight Of the brown trout collected varied from approximately 0.37 to 122 grams, while the sculpins col- lected.ranged.from 1.13 to 5.06 grams. The weight of the lamprey samples ranged from 0.69 to 4.42 grams. In addition 35 to the above collections, several brown trout were Obtained for the purpose of determining the radioactivity levels in various organs and other parts of fish. Laboratory Procedures The lamprey samples were prepared and processed in the exact manner as were the fish, so the procedure is given as one method, as modified from Robeck, st 31. (121g). The sample to be examined was washed with distilled water, blotted with blotting paper and weighed, using a beam balance. If the sample weight was in excess of 2 to 3 grams (exclusive of lamprey), the sample was placed in a Waring blender and ground up, thus facilitating the removal Of an aliquot of approximately 2 to 3 grams. The aliquot or entire organism, as the case may be, was placed in an evaporating dish, flooded with concen- trated nitric acid and placed under an infra-red.heater until dry. When the sample became dry, it was transferred to a muffle furnace, heated to 600°C. and allowed to ash for 5 to 10 minutes. This step was repeated until the sample was completely ashed (white ash) and then removed and allowed to cool. When sufficiently cool, about 20 drops of 2N nitric acid was introduced into the evaporating dish the the residue was scraped into a planchet, using a glass stirring rod. The addition of acid.and the scraping pro- cedure were continued until complete removal of all re- sidue was realized. 36 The planchet and material contained therein were trans- ferred to a hot plate and complete evaporation to dryness was completed. The planchet bearing the dry sample was placed in a muffle furnace heated to 600°C. and allowed to remain until the planchet was heated to red heat. The sample so treated was allowed to cool, whereupon it was transferred to the counting room for activity determination. Biomass Estimate POpulation estimates for fish in the West Branch of the Sturgeon River were made using an electric fish shocker. The shocker was Operated by a field party throughout 1,000 yards (point of isotOpe release to Station 8) of stream section on August 25 and 28, 1959. On the first ”run“ through the stream section, fish.were captured, marked by fin-clipping and released, giving a known number of marked fish (m) present. 0n the second ”run” through the same stream section, the number of recaptures (r) of previously marked fish and the number of unmarked fish (u) were re- corded. The numerical computation of the pOpulation was estimated, using the Petersen formula for pOpulation estimate, and is as follows: P=m(ur‘r)/r where: P = total fish pOpulation estimate, m = number of fish captured and marked and re- leased on the first run, u I total number of unmarked fish captured, r = number of marked fish recaptured on the second run. 37 In order to establish a weight-length relationship and obtain a mean weight value with which to calculate the biomass, a third ”run” through the stream section was necessary on September 1, 1959. Each captured trout in the section was weighed and measured. Inasmuch as the sculpin pOpulation was extremely large, only the sculpins obtained from 100 yards of stream were used in the cal- culation of mean weight and length. The section of stream for which the biomass was estimated was 1,000 yards long and the average stream width in this section as determined on the basis of 30 random measurements was found to be 25.5 feet. The area of stream used in the fish biomass estimate was computed to be 1.76 acres. The fish biomass was cal- culated by multiplying the mean weight of the fish by the total estimated fish pOpulation and then computing the result in terms of pounds of fish per acre. RESULTS AND DISCUSSION When radioisotOpes are released into an aquatic en- vironment, they quite often become dispersed and diluted, but they may also undergo unexpected movements and concen- trations. The accumulation of radioactive materials in an aquatic system follws a definite_pattern. It is this pattern that will be discussed in the balance of this report. It should be emphasized at this time that a tracer study such as the one under consideration is designed in such a way that the amount of radiOphosphorus introduced is extremely small in comparison to the amount of non-radio- active phosphorus already present in the system. Thus there is no increase in the phosphorus present in the system, one is simply following the behavior of a few marked atoms. Therefore, neither the radioactivity nor the extra ions of phosphorus disturb the system; what happens to the tracer simply reflects what is normally happening to naturally occuring phosphorus as a continuous exchange is going on between the water, plants, animals and bottom complex of the stream. The mode of uptake by which radiometerials may become associated with fresh-water organisms occurs in one of three ways: through adsorption to surface area; through absorption from the surrounding medium; or through ingestion as food (Krumholz and Foster, 1957). 38 39 The fate of radiOphosphorus released into the stream system and the manner by which the ecological communities and pOpulations control the distribution of radioactivity will be discussed in detail in the following sections. ease. The addition of a single dose of radiOphosphorus into the stream ecosystem resulted in immediate uptake of the isotOpe (Figure 4). Inasmuch as the isotOpe introduced in- to the stream was in the inorganic form, adsorption and absorption of P32 by plants occurs almost instantaneously. All of the nutrient materials and thus the biologically important phosphorus, as well as, radiOphosphorus, that are metabolized by plants are absorbed directly from the en- vironment. Adsorption of radiOphosphorus directly from water, however, cannot be neglected. The absorption and adsorption are the primary mechanisms by which inorganic materials are acquired by aquatic plants, which are the food sources of the animals. It is hypothesized that immediately upon addition, the radiOphosphorus was adsorbed or absorbed by particulate solids made up of--possibly--nanOplankton, bacteria and diatoms. It is seen that upon addition of P32 to water the immediate reaction within minutes is a transfer of the isotOpe through the bodies of unicellular floating forms of life. In view of this reaction, these forms must occupy a very important position in the distribution Of phosphorus 40 (P32) to other pools Of the system. Bacteria and algae, according to Rigler (1956), are the two groups of planktonic organisms known to take up phOSphate (P32) from solution in sufficient amounts. The bacteria (Krumholz and Foster, 1213,) may have the greatest powers for concentrating radiomaterials Of any freshpwater organism; their concen- tration factor for certain isotOpes may exceed 1,000,000. Hayes and Phillips (1958) indicate that bacteria are con- tinuously putting inorganic phosphate rapidly through their bodies, changing most of it enroute to the organic form. There is at the same time a regeneration of the inorganic phosphate by breakdown of the organic fraction. Hayes and Phillips (1212:) further indicate that in their study, water bacteria rapidly incorporated 50% of the P32, which was introduced as inorganic POh, as part of their body protOplasm. As a result of the analysis of the filtered solids rinsed.with.dilute acid in the present study, it was found that only a small fraction Of the activity was removed (Figure 3), lending supporting evidence that most of the filtered solids incorporated the tracer as part Of their body protOplasm. The results Obtained at Station 3 indicated that approximately two-thirds of the water activity was in the form Of solids, and at Station 8--sampled after the peak activity had.passede-the data indicated that the solids made up 50% of the total activity. The data Obtained at Station 12 appeared impossible, since the activity of the 41 solids was greater than the total water activity. This difference was probably due to sampling error. The water sample used to determine the total water activity was not from the sample used for solids determination. It is hy- pothesized that although the samples were taken from the same station at the same time, a different collection site was selected.for the actual sampling procedure and a slight time difference in collection Of these two samples was involved. In conjunction with the radiOphosphorus adsorption and absorption by the particulate solids, great quantities Of radiOphosphorus were also taken up by the periphyton and aquatic plants which possessed a mechanism of initial phosphorus uptake that might be due to physical processes unconnected with active cell metabolism (Coffin, e£_§1., 1949). This uptake by periphyton and aquatic plants is discussed more fully elsewhere in this paper. Water activity curves (Figure 4) show the water acti- vity for the various stations throughout the period.when the fisotOpe was moving through the experimental area. The maximum activity value reached was approximately 12.4 cpm/ml. and was recorded at Station 5. Station 3 reached nearly the same value and may have surpassed this value between sample periods. A similar trend was common to the water curves at all stations. As the isotOpe arrived at the sampling station, there was an increase in activity until peak activity was recorded, in all cases, somewhat past the 42 .hsome can ecsoammomo cow OsgooaaOo one: mumsoo .AOpHHu mcomaaaaa so cones anthem song concuaam menace Oommsauuaos one connmslcmpca co hpa>apoc on» use .hpa>auoc mops: deuce .m omswam 43 O'Q'O'O'O'O'O'O'O'O' >0000000,0,0.0 0 0. 20202020202 .1 Wo’rer solids Wo’rer solids ( 500mlsomple ocud washed ) ( SOOmlsomple, water wash ed ) (50ml. samples Figure 3 E Total woferootivi’gy RN >'O'O'O'O'O‘O'v'vvvvvvv v v v v v v . ...... 0099099009099 90909.9.9... >000000000000000000.0.,.., . entrainment“: o 2.2.: 2 l I l | I I I I I mmNQOlDVMN— Jed ainugui Jed slunooMuAuov l2— Il— EIO— (JGIIIIIII 44 midpoint of the time span. This was followed by a reduction in activity to almost background level at the expiration of 70 minutes after the isotOpe arrived at each station. Station 3 reached a peak water activity of 12.3 cpm/m1. approximately 40 minutes after the isotOpe arrived at this station. At Station 5, as previously mentioned, the maxi- mum water activity value of 12.4 cpm/ml. was recorded approximately 35 minutes after the arrival of the isotOpe. Station 8 showed a somewhat reduced water activity when compared with the previous stations. The peak water acti- vity of 7.1 Opm/ml. was recorded approximately 40 minutes after the radiOphosphorus was detected. Station 11 initi- ates a series of four stations in the lower portion of the study area. Station 11 reached a peak activity Of nearly 3 cpm/ml. 30 minutes after the arrival of P32 at this station. Station 12 reached a peak of 2.7 cpm/ml. 30 min- utes after arrival of the isotOpe. A second peak of nearly equal magnitude as the first was recorded nearly 20 minutes after the first. Station 14 reached a peak of 1.6 cpm/ml. approximately 40 minutes after the arrival of the isotope. The theory advanced in an attempt to explain the nearly symmetrical curve of activity resulting during the passage of isotOpe past each station prOposes a purely physical process. The initial entry of radiOphosphorus in- to the stream resulted in low concentration, due to tremen- dous dilution as it proceeded downstream. Water receiving a dosage of radiOphosphorus at progressively later times l*5 .msOmc use canosmxomn sou ucpommnoo ohms «assoc HH< .OQOpoma no Ommmmmo madame mcoapmpm manpoca taco nsoams> pm hpa>apom capes Hence .: tarmac "OIlDIS L._._§_. 47 was also diluted by the stream water but was subjected to additional activity entering the main course of the stream by way of backwater, eddies and areas near stream bottom and surface. This additional activity built up higher and higher concentrations of activity passing a given point until the conclusion of the isotOpe entry, at which time a steady decline in activity was noted. The decrease in activity was due to the fact that activity was no longer being introduced into the stream. The tail of activity appeared as the isotOpe continued to re—enter the stream from backwater and embayment. In order to demonstrate horizontal variation in water activity, a series of water samples were Obtained on a transect across the stream. These samples were obtained at Station 8, daring the passage of the isotope, just after the peak activity had passed this station. The data Ob- tained indicated that the greatest activity occurred along the stream margin and that lower activity levels were re- corded in the main current near midstream (Table 1). Table 1. Water activity (corrected counts per minute per milliliter) at different points in the stream channel during passage of isotOpe dose. :— B Left Bank Left Center Right Center Right Bank ..- 3.0 2.1 2.3 3.3 48 Inasmuch as the peak activity had passed this station prior to sampling, the higher activity along the stream margin would be reentering the main channel of the stream, forming the tail of the activity curve. Current velocity is not uniform in all parts of the transverse section of a stream (Welch 1952), but is reduced at and near the surface because of surface tension, and diminished as the bottom and sides of the channel are approached, owing to frictional effects. Welch (1213‘) further states --- ”the distribution of velocities in natural streams is determined by several different factors Operating simultaneously, such as, shape of channel, rough- ness Of channel, size of channel and lepe of channel”. It can readily be seen from the above that it would be possible for many situations to develOpe which might sidetrack and detain portions of water containing a considerable amount of isotOpe, allowing it to reenter the main channel of the stream sometime later. Figure 5 shows the mean activity value for each station during the period of isotOpe passage. From this curve, with some error expected, when using the mean value, it is pos- sible to Obtain the mean uptake of activity at each station during the passage of isotOpe. This activity curve indi- cates a nearly logarithmic decrease between Stations 5 and 11; thereafter, the downstream stations decreased at a re- duced rate. The uptake pattern was rather uniform as the isotOpe proceeded downstream. The removal Of isotOpe from 49 Figure 5. Mean water activity at various col- lecting stations during the passage of isotOpe. All counts were corrected for background and decay. 5o l6 A.Ha non commas moo upcsoo Omuoosmoov soasaooa 1h... 1 12 l 11 1 [do 1.5 l3 6 5 ..u. 3 2 0 Stations Figure 5 51 the water was nearly complete in passing through the ex- perimental area. This depletion Of activity from the water is not an actual loss of the isotOpe, but rather the tracer has become distributed into the various phosphorus pools of the ecosystem. The movement of the isotOpe from one com- partment (water) into other compartments (vegetation, bot- tom silt, fish, etc.) illustrates the dynamic state Of phosphorus in the ecosystem (Foster 1959). The only true loss of isotOpe will be through the tracer being swept out of the study area, through the harvest of fish or other craps which have concentrated the isotOpe, through the emergence of aquatic insects and through radiological decay. Periphyton Igitial Uptake of Radiophosphorus The periphyton biocenosis accruing on the artificial substrates in the West Branch of the Sturgeon River is made up almost entirely Of diatoms (Clifford, 1213.). According to Clifford, Synedra ulgg accounts for the great- est portion Of the periphyton complex. In lesser numbers, Cymbellg 522., Navicula 522., Cocconeig 522 and Gomphonemg 32p. were the other principal diatoms comprising the peri- phyton complex. Figure 6 indicates that the uptake of radiOphosphorus, ten minutes after the isotOpe passage, was greatest at Station 3 and progressively less at the downstream stations. This is undoubtedly due to the fact that Station 3 was 52 Figure 6. Activity of periphyton taken from artificial substrates at Stations 3, 8, l2 and 14. Counts were corrected for background and decay. Activity (Corrected counts per minute per gram) 53 3000 10 minutes after isotope —-—- 4 hours after isotOpe 2800 ---- 24 hours after isotOpe 2600 ° 2400 2200 ~2000 — 1800 — 1600 — 1400 — 1200 — 1000 — 800 l— 600 — 400-— 200-— ) . _ Stations Figure 6 exposed to a greater water activity and, as the isotOpe proceeded downstream, the activity remaining in the water became progressively less due to uptake by the stream vege» tation and dilution, The uptake of radiOphosphorus by the peiiphyton showed a somewhat different trend when sampled four hours after the passage of the isotOpe dose. The maximum uptake at this time appeared at Station 8 and upa take was greatly reduced at Station 3, as well as, the stations downstream from Station 8. The uptake curve obe tained 24 hours after the passage of the isotOpe dosage indicates that the maximum activity was recorded at Station 8 with decreased activity values recorded for stations up- stream and downstream from this point. The initial radiOphosphorus uptake in periphyton was extremely rapid, reaching a peak at Station 8 some 4 hours after the initial dose. The isotope during this initial period apparently was entering what plant physiologists call the 1"outer space“ of the plant, that is adsorbing onto the surface of the cell and is not incorporated into protOm plasm (Odum, 23.92:, 1958). Apparently this initial uptake might be due to physical processes unconnected with active cell metabolism. Some of the P32 taken up at Station 3 was apparently returned to the water and taken up by the vege= tation progressively downstream. Inasmuch as Station 8 was the first station sampled below Station 3, it showed the greatest activity level of all other stations. Station 8 apparently utilized the radiOphosphorus released from the 55 upstream stations, accumulating the isotOpe until a peak h hours after the isotOpe arrived at this station. Station 3 is located in a well shaded area, while Station 8 is fully exposed to direct sunlight. This difference in light intensity reaching the plants may have a pronounced effect on the periphyton community metabolism, and thus may be a factor in the greater P32 uptake at Station 8, when comp pared with Station 3. This phenomenon is shown in Figure 7. As was observed in Figure 7, Station 3 reached its peak activity 10 minutes after the isotope dosage, while Station 8 reached its peak fl hours after isotOpe passage. Station 12 (Figure 7) did not reach peak activity until an hours after the isotOpe arrived at this station. The activity curve at Station 1“ indicates a double activity peak: the first, 4 hours, and the second, 96 hours, after the isotope passage. This delayed peak activity develOping progressively downstream indicates that release of the isotOpe at an up- stream station was followed by a downstream uptake. This proceeded progressively downstream until it ultimately passed beyond the final periphyton collecting station. The magnitude of peak activity recorded at each station de- creased progressively downstream in all cases except Station 3. The explanation for this might lie in the fact that the isotOpe entry point is only 300 yards upstream from Station 3 and the initial isotOpe uptake was greatest at this station, in comparison to the other stations, 10 minutes after the passage of the isotOpe dose because of exposure 56 Figure 7. Activity of periphyton taken from arti- ficial substrates at Stations 3, 8, 12 and in, for the entire study period. Counts were corrected for background and decay. 2000 1000 U 0 O 00 2000 1000 Activity (Corrected counts per minute per gram) 5? Station 3 Station 8 0L“ ' l 7““ 1 L J 2000 Station 12 91 1000 - 0 2000L Station In I 1000 - o k r 8 12 15 29 5 12 19 26 ' A232§t 58 to the greatest concentration of activity. The reason for Station 8 exhibiting a peak of greater activity than Station 3 was probably due to the fact that Station 8 re- ceived additional activity released from upstream vegeta- tion, while Station 3 apparently received little activity from the area preceding it. This may also be due to in- complete mixing of the isotOpe. Complete mixing of the isotOpe probably was not completed until very close to Station 3. The transport of the isotOpe prior to complete mixing wouldp-for the most part-~follow the main midchannel of the stream where very little vegetation grows and thus little Opportunity was afforded the area above Station 3 for the uptake of the radiOphosphorus. Conversely, little P32 wand be available for exchange. Exchange and Regeneration of RadiOphosphorus Figure 7 shows that periphyton activity at Station 3 decreased only slightly during the first week, and at a greatly reduced rate thereafter. The periphyton activity trend at Station 8 shows a sharp decrease at a logarithmic rate during the first week; thereafter, at a reduced.rate. Station 12 periphyton activity shows a rapid decline in activity, after the initial peak, until the end of the second week; thereafter, at a reduced rate. Station 1% shows a trend similar to Station 12, inasmuch as the activity decrease was rather noticeable until the end of 59 the second week when a greatly reduced rate of activity de- crease was observed. This much reduced rate of periphyton activity after the initial period of rapid.uptake and re- lease of radiOphosphorus occurred at each of the sampling stations and indicates that periphyton apparently reached a plateau of activity. This plateau phenomenon, according to Hayes, 33 El: (1952), can be explained as a function of radiophosphorus exchange between water and plants. They further state that when a number of marked atoms are placed in the water, they will tend to leave the water exponentially and enter the plants. At the same time there is a return of material from the plants, which also proceeds exponen- tially. During the initial uptake period after the addi- tion of radiOphosphorus, little isotope can return from the plants because little has yet entered them. As soon as a significant fraction of the isotOpe has entered the solids, there will be a ”feedrback” into the water (Foster, EEES')° This equilibration or plateau phenomenon may be the result of isotOpe uptake when radiOphosphorus reentered the water as a result of phosphorus exchange, coupled with the con- tinual release of radiOphosphorus, due to regeneration of the isotope as a result of decomposing organisms. Supporting evidence of this equilibration level of regenerated or exchanged radiOphosphorus comes from measure- ments made of the activity of substrates removed from the stream during the isotOpe passage, but replaced immediately after the isotOpe dosage had passed the station (Figure 83. 60 Substrates thus exposed tend to show isotOpe uptake, but in a greatly reduced rate in comparison to those substrates remaining in the stream during isotope treatment. The radiOphosphorus picked up by the substrates placed in the water after treatment was the result of periphyton uptake of regenerated or exchanged isotope reentering the water with subsequent uptake by the periphyton introduced after isotOpe treatment. This plateau or equilibration level ex- hibited by the periphyton may be the so-called minimum phosphate content of the periphyton components and may be designated as ”bound phosphate” (Goldberg, g£_gl., 1951), or the phosphate which is not readily exchangeable with that of the surroundings. Such a condition as this must exist whenever the phosphate (radiOphosphorus) is incor- porated irreversibly into the cell protOplasm. The immense ‘initial isotOpe uptake shown by the stations remaining in the water during treatment (Figure 8) probably represents- in part--the labile or readily exchangeable phosphate in the organisms and only the activity or the substrates remaining out or the water during treatment are representa- tive of the ”bound phosphate", or that which is stored or taken up and incorporated into protOplasm. Once the peri- phyton cells have equilibrated (lost the labile P32), further decline in activity can be attributed to three causes. First, a loss of P32 would.occur as a result of isotOpe “feedrback” between the periphyton and the water. Secondly, as might be expected, the initial mass of cells 61 .hwomu use ccsouwxomn you copooa taco out: mpcsoo .w and m ecoapmpm pa mmpmAmeSm coumnmaumd go hua>apom do comaammaoo .w madmam 62 w onsmah 52.28 89.02 Etc misc: mm. om. mm Na. me E 13x33 0’. I. I. I. I. ’0 ’0’,- I'O'ol .'.'.'.l.'-'-' .....l O EmEBm: 05:6 $63 5% 8765 IIIII E9509: octet 8.62, do SOLm cozfim ..I..l +58sz octau 3.63 c Tm c0765 ..... EmEBmt 05.3 .20; .6 .SO..@ c0765 ’ a z r oom v o H... .A.. com ,M \o/ . O 000. m In? S 003 w .. w. 08.. m we. .0 oo~.~ m 5 m 003 w ( 63 containing P32 would be increased by biological dilution as a result of the addition of new cells, growth of cells and loss of old cells. This would have the effect of diluting the sample taken for analysis. Thirdly, the concentration of isotope would diminish as a result of radioactive decay. Larger Aquatic Plants_ IsotOpe Uptake and Accumulation The variations in activity for the larger aquatic plants, by sampling stations, are shown in Figures 10, 11 and 12. The larger plants readily concentrate radiOphos- phorus, but, in most cases, to a lesser degree than peri- phyton. Maximum values found were approximately 1,700 counts per minute per gram, as against approximately 2,600 counts per minute per gram for periphyton. The curves showing the decrease in activity with.time for ghgga, Potamogeton and Fontinalis, are strikingly similar to the activity curves for periphyton. All of these aquatic plants show a rapid initial uptake of isotOpe (Figure 9). A very interesting situation developed when the ability of a gram of the different plant species to ”absorb" the tracer was compared. The initial uptake of isotOpe for Station 3 in Potamogeton was twice that recorded for Fontinalis, and over four times greater than that recorded for EEEEE Figure 9). The downstream stations show a similar uptake pattern, except for Stations 12 and 1%. The uptake re- corded for these stations show activity values that do not 61L vary to any degree from each other. The uptake values at the downstream stations may reflect physical factors, such as exposure of certain areas of vegetation to greater or lesser amounts of water activity levels. Plants growing in the main water course (ghagg, for example) might have been exposed to a greater water activity in some areas than other plant species, and, therefore, exhibited an uptake rate similar to the other plants even though isotOpe up- take was slower than for other plants. The upstream sta- tions (3 and 8) may have shown the plant isotope uptake when there was a greater concentration of tracer in the stream water. This difference in the ability of different species to ”absorb” the tracer may be due to surface-per- volume ratios discussed by Odum, 33.21. ($232,). They suggest that tracer amounts of P32 and, therefore, phos- phorus, in general, are absorbed at a rate determined by the inherent structural surface area features of the plants. High surface-per—volume ratio increases the uptake rate. 0n the basis of this surface-per—volume ratio, the values recorded for the present study indicate that the plants would rank as follow: periphyton, Potamogeton, Fontinalis and 05352, in descending order. In addition to the surface- per-volume ratio, the differential plant species uptake may be limited by the fact that each organism in each en- vironment has specific requirements for the different chemical elements. In order to understand and interpret the role played by phosphorus in the metabolic processes, 65 Figure 9. Activity of stream vegetation on July 8, 1959. Counts were corrected for background and decay. 66 e——Chara getog __._. -——-—-Fontinalis \ 8 Stations _ _ _ . _ _ _ _ _ O O nu o O 0 O o 0 0 O O O O 0 0 O O 0 l .l. l 1.. Aamhm hon saunas you muczoo bopomhhoov andsapod Figure 9 67 the specific phosphorus requirements and chemical composi- tion of each plant species must be obtained. This informa- tion is not available for the organisms utilized in the present study. The activity curves for Potamogeton are shown in Figure 10. The maximum value found for Potmmggeton was approximately 1,700 counts per minute per gram at Station 3,on July 8. The peak activity for the stations below Station 3 decreased progressively downstream, apparently because there was less P32 in the water to be picked up by the plants, to a peak value of slightly over 100 counts per minute per gram at Station 1%. Stations 3 and 8 showed an initial logarithmic decrease in activity until July 12, followed thereafter by an equilibration phase in which there was little or no rise in activity until a slight increase on July 5. Stations 12 and 1% showed a brief logarithmic de- crease in activity until July 12, followed thereafter by a sharp rise in activity on July 15 at both stations. This increase in activity was apparently due to “back diffusion“, or exchange of P32 between the organisms (periphyton and aquatic plants) and the water with immediate uptake at the downstream stations. The increased activity at Stations 12 and 1% took place at precisely the base of the logarithmic phase and the onset of the equilibration phase of Potamogeton at the upstream stations. The slightly in- creased activity levels at Stations 8, l2 and 1% on August 5 and at weekly intervals progressively downstream also 68 Figure 10. Activity of Potamogeton at Stations 3, 8, 12 and 14, for the entire sfumv period. Counts were corrected for background and decay. 69 2000 Station 3 1000 — 0L H I I 4 T? I W» Station 8 Activity (Corrected.counts per minute per gram) station 1” 100 _. o H I I I I I I 8 12 15 22 29 5 12 19 2 July August Figure 10 70 Figure 11. Activity of Fontinalis s . at Stations 3, 6, l2 and 14, for the en ire study period. Counts were corrected for background and decay. 71 1600 i _ 1#00 _' Station 3 1200 - 1000 -‘ 800 _ 60m- 11 l l l J l l l l __ 8 c> § °§o T .p:f I Station 8 600 — JL AA 500 — § I 300 .- 200 100 - - )— h p P _ _ u— — 300 Station 12 200 Activity (Corrected counts per minute per gram) 100 l 1 0 II I l l l l 300 j Station 1% l 200 100 12 19 2 Au st Figure 11 72 Figure 12. Activity of Chara at Stations 3, 8, 12 and 14, for the entire study period. Counts were corrected for background and decay. 73 600 r- Station 3 I 500 - #00 h- 300 - 200 - 100- _‘,.a*‘“ (>l_riu L,J. I“"—7. fi—T I __J 36 A 00 Station 8 two- a. Ignoo — E300- s 3200~ 100 r Station 12 ] Activity (Corrected counts p r4 to x» f? c: C) <3 <3 c> c> c> <3<3 Station 14 100 Figure 12 74 indicate ”feedrback” with downstream uptake. The activity curves for Fontinalig are shown in Figure 11. Maximum activity value recorded was approximately 1,600 counts per'minute per'gram at Station 3 on July 9. The peak activity value decreased progressively downstream (probably due to the fact that there was less 933 in the water to be picked up by the plants) to a peak value of approximately 300 counts per minute per gram, recorded at Stations 12 and 1#. The peak activity at Station 3 did not, however, occur until 24 hours after the introduction of the tracer; This may indicate, as did.Figure 9, that Fontinalis did not take up radiOphosphorus as rapidly as ggtamogeton. The activity loss observed at all of the stations did not follow that of Potamogeton. The activity curves at Stations 8 and 1% showed an initial loss of activity followed by a slight rise on July 12, followed by an equilibration phase. The reason for the belated acti- vity peak exhibited by the Fontinalis at Station 3 is not known. The answer may lie in the fact that the sample col- lected on July 9 was obtained in an area that received a greater concentration of P32 during the isotOpe treatment than did the plant sample taken on July 8. Inasmuch as Station 3 is located rather close to the isotOpe entrance point, the major portion of tracer may have passed this station in the main channel of the stream, leaving areas exposed to lesser amounts of tracer. Station 12 showed a rather scanty initial P32 uptake with an increased uptake, 75 reaching a peak on July 12. This peak was apparently due to 'feedpback” of radiOphosphorus from upstream plants. The activity curves for Chara are shown in Figure 12. The maximum activity value for 9233; was approximately 560 cpm/gm., recorded on July 8 and 9 at Stations 8 and 3, re- spectively. The activity trends for Stations 3 and 8 are very similar except for the activity recorded for Station 3 on July 8. The activity value decreased progressively downstream (probably due to the fact that there was less P32 in the water to be picked up by the plants) to a peak of about 100 cpm/gm., recorded for Station in. Stations 12 and l“ showed.an initial isotope activity value rela- tively close to the equilibration level. The activity curve of the red alga, Batrachospermum, is shown in Figure 13. This red alga was not sampled as extensively as were the other larger aquatic plants. This plant was discovered only while obtaining the routine plant samples reported in this paper. This alga was first pro- cessed for radiological analysis on July 12, four days after the isotope treatment date. It is suspected that the activity values for Batrachospermum greatly exceeded any value reported for the routine larger aquatic plants and periphyton. If this red.alga followed a similar pattern of isotOpe uptake as the other algae and larger plants-«as it apparently did-~the theoretical activity value may have been as high as #,000 cpm/gm. or more during the initial uptake period, Just after isotOpe treatment. The activity 76 Figure 13. Activity of Batrachos ermum at Stations 3, 6, 12 and 1%, during JuIy I959. Counts were corrected for background and decay. Activity (Corrected counts per minute per 77 R; *' " I; 2% C) i? 2? c: <3 0 OO 0 O H O O O 800 600 ii <3 200 July 12, 1959 ----- July 15. 1959 '——" “‘ July 22, 1959 Stations Figure 13 78 curve indicates that Batrachospermum released the isotOpe rather rapidly as an equilibration phase apparently was present on July 22 (Figure 13). In the study of phosphorus exchange in Bluff Lake. Hayes, EE.E£3 (ibid.) indicates that the alga, Batrachospermum, displayed a rapid P32 up- take from the first hours. The uptake and accumulation of radiOphosphorus by plant cells are characterized by two features. First, the living cells can accumulate ionic material, g.g., radio- phosphorus. That is, they can continue to take up the isotope even if the concentration level of this tracer in- side the cells is far above the concentration of the same ionic species (P32) in the external medium. From the point of view of diffusion, the accumulation of phosphorus (and presumably P32) by a cell is, not only unorthodox, but impossible. According to Bonner and Galston (1952), when substances penetrate into cells in response to a diffusion gradient, they may-~at most-~attain an internal concentra- tion equal to the external concentration. It may be con- cluded, therefore, that the entrance into, and the in- creased internal concentration of, ionic materials (and presumably P32) by cells is not a simple diffusion process. On the contrary, phosphorus accumulation is a process which requires the expenditure of energy by the plant--energy to do the osmotic work involved in moving the phosphorus (and presumably P32) against a concentration gradient (Bonner and Galston, ibid.). Rice (1953) discusses the uptake and 79 accumulation of P32 in algae. He discusses the entry of radiophosphorus into the cell, indicating that P32 may enter algae by diffusion through the cell membrane or by entry of P32 into the cell through esterification at the cellular interface, or a combination of these two methods. The process of phosphorus (and presumably P32) accumulation, which makes possible the uptake and retention by the plant of large quantities of phosphorus (and presumably P32), is-- at least in part-~an energy—requiring process driven by, and wholly dependent on, the energy liberated in respira- tory metabolism (Bonner and Galston, ibig.). The second feature of radiophosphorus accumulation is that of equilibration between water and the aquatic plants which occurs within approximately one week. As soon as a significant fraction of isotope has entered the aquatic plants, there will be a “feed-back” of 932 into the water (Foster, £212.). According to Foster, as more and more isotOpe enters the aquatic plants, the ”feedeback' will increase until an equilibrium is established such that the amount of isotOpe leaving the water and entering the plants is balanced by the amount returned to the water from the solids. The data for Station 8 is shown in Figure 1h, de- monstrating exchange and equilibrium. In Figure 14 the logarithmic rate of decrease during the first week has been extrapolated to the X axis and a smooth curve has been fitted to the activity values by inspection. Had there been no I'feed.--back" of radiophosphorus between the solids 80 Figure 1M. Activity of plants collected at Station 8. The dotted lines represent the extrapolation of the logarithmic rate of decrease of activity during early stages of the experiment. Counts were cor- rected for background and decay.- 81 B A A mmA t e 8 .1 O a a: nagA t o u m n O F a m 3F 22 29 s 12 15 C- oc- 10c- sc— 6c— he. 20- .. mama 1000 800 600 #000— o “scam and speeds use upcsoo couooanoov auaeduod Auggst Figure 13'. 82 and the water, one would expect the decline in the concen- tration of isotope in the plants.to follow the broken line. The increase with time in the amount of regenerated P32 re- turned from the water to the solids is evident from the space between the two curves (Foster, £212.). 0n the basis of these data, the amount of regenerated P32 taken up by Fontinalis during the equilibration phase of the activity curve is greater than twice that taken up by either Potamogeton or Chara (Figure 1%). Once the aquatic plants have equilibrated, further decrease in activity can be attributed to: (l) a decrease of P32 as a result of iso- tope loss from the plants back into the water; (2) in- crease in initial cell mass due to cell growth, new cell addition and loss of old cells, causing biological dilution; and (3) the concentration of isotope would diminish as a result of radioactive decay. Larger Aquatic Plant Biomass Estimate An estimate was made of the standing crOp of larger aquatic plants for the first 1,000 yards of the stream below the point of isotope entry. The area encompassed by this area was from the point of isotOpe release to Station 8 (Figure l), and.represented an area of approximately 1.76 acres. Riffle area occupied a major portion of this section of the experimental area with a vast area of scanty vege- tational growth observed in the middle portion of the sampling area. The mean plant biomass computed for this 33 portion of the experimental area was 21,780.0 pounds per acre. The dominant aquatic plant observed in the plant samples was Chara. Aquatic Invertebrates Isotope Uptake and Accumulation Radioactive materials are taken into the body of an organism through physiological processes and incorporated directly into the tissues of the organism. The principal mode of accumulation of radiomaterials by invertebrate aquatic organisms is by ingestion of food. In their studies of the Columbia River, Robeck, at 21. (ibid.), indicate that activity levels in most aquatic invertebrates are de- pendent upon their metabolic rates and the radioactivity values of the materials upon which they feed. In his work with Gammarus, Harris (1957) indicates that Gammarus does not take up appreciable quantities of phosphorus by direct absorption through the body wall, intestine or gills. It is assumed in the present study that no appreciable radio- phosphorus entered the aquatic invertebrates by direct absorption. 0n the basis that the isotOpe enters the in- vertebrates through ingestion, it appears quite logical that the invertebrate organisms, in the present study, could be discussed most effectively if they were subdivided on the basis of their food habits. The seven organisms collected and analyzed throughout the study period were subdivided into five food habit 8% categories, as follow: filter feeders, periphyton scrapers, detritus feeders, predators and an example of an organism with an omnivorous feeding habit. Even though the followb ing section will subdivide the organisms into food habits, it is well recognized that precisely such a partitioning is impossible, due to various factors in nature. Environmental conditions in fast streams are strenuous; the strong current in particular makes life somewhat precarious and selective feeding difficult (Muttkowski 1929). It is recognized also that aquatic insects in rapid streams may become Oppor- tunists in regards to food and, out of necessity, may eat whatever is available. Invertebrates Studied The blackfly, Simulium 32,, was selected to represent the filter feeder group. The activity curve for the black- fly larvae is shown in Figures 15, l6, l7 and 18. The maximum blackfly radiophosphorus concentration of approx- imately 6,000 cpm/gm. was recorded at Station 3 on July 12, four days after the isotope treatment. The peak acti- vity at Station 3 was the lowest peak reported at any of the sampling.stations. The maximum activity value of 6,000 cpm/gm. recorded at Station 8 was double the peak activity recorded for periphyton, indicating that the blackfly was either feeding on a material with (a much higher activity concentration than anything analyzed in the present study or the blackfly is capable of concentrating great amounts m \J I of the radiomaterial. The blackfly larvae possess two prominent fanlike structures located at the extreme ante~ rior end of the organism. These anterior fans strain plankton and organic debris from the water for food (Pennak 1953). The material strained from the water by the blackfly larvae possibly might include bacteria and diatoms, containing a great concentration of radiOphos- phorus. According to Robeck, at 31. (ibid.), radioactivity density of organisms is dependent upon the rate of meta- bolism and the activity of the organisms upon which they feed. If blackfly larvae follow the pattern of other organisms in that metabolism per gram decreases with in- creasing size of the individual, the small blackfly larvae would utilize relatively large quantities of food material which might be high in radiophosphorus and possibly con— centrate P32 at a rate many times greater than the larger invertebrate organisms. The activity curves for the blackfly larvae at all of the sample stations show strikingly similar trends. An initial period of increasing P32 concentration built up to a peak on July 12 at Stations 3 and 8, and on July 15 at Stations 12 and 14. Following the maximum activity level, the blackfly activity at all stations decreased at a loga- rithmio rate, indicating there was little uptake from re- generated materials and its source of activity had nearly disappeared. Studies on radiophosphorus metabolism by Harris (ibid.) indicate that bacteria in that particular 86 Figure 15. Activity recorded for Station 3 invertebrates representing four food niches. Counts were corrected for background and decay. 6000 5000 r: 900 3000 2000 p O O O O\ O O 00 WNW gm 0 0 O O O O O O O O O O O O O 000 H (UN-F3 O O O O O O O O 0 Activity (Corrected counts per minute per gram) 0 OS 0 O O 4000' 3000 2000 1000 5? T— I I I l I Filter Feeder Simulium ‘ I I I I ~re ‘1:-—~=g_ . Periphyton Scraper _:_Brachycentrus 1 I l 1 *Jl ‘r er Detritus Feeders Onarcya L— - Hexagenia . ----_ -—--——-—4——--+----+----¢ Predators Atherix A _ l/ \\ // \\ _ A / \\Chauliodes ’\ ,..—AP-~.._A___._.A/ 39-.....5 LI—II ~A’" ] I «8 12 15 22 29 5 12 19 2 July u at Figure 15 88 Figure 16. Activity recorded for Station 8 in- vertebrates representing four food niches. Counts were corrected for background and decay. Activity (Corrected counts per minute per gram) 6000 5000 #000 3000 2000 89 g Filter Feeder j Simulium ll 1 1 Periphyton Scraper 2! Brachycentrus Detritus Feeders Pteronarcys Hexagenia sék ’ \‘ -—-+--—+--—+" . Predators Atherix Chauliodes -———A—""“‘. A.----L~~~'A""-A.“~~A I I l 22 29 5 12 19 2 Age“ Figure 16 90 Figure 17. Activity recorded for Station 12 invertebrates representing four food niches. Counts were corrected for background and decay. Activity (Corrected counts per minute per gram) 0 NW 0 o 5 O O O O O O 0 91 Filter feeder 42‘ F- —. Simulium J i 41 1 i 1 1 l I Periphyton Scraper Brachycentrus I Pteronarcys ”us—"4% j Predators F - Atherix hu— - Chauliodes xA‘~\ _ //’ 'N‘f;____ . a” '” ___c A... I - ’ "fl”. l -1 J 8 12 15 22 a 29 5 12 19 2 July August 92 Figure 18. Activity recorded for Station 14 invertebrates representing four food niches. Counts were corrected for background and decay. 5000 1: Filter Feeder 4000 - 3000 - 2000 - Simulium 1000 O. .p O O O I T l R H 1 Periphyton Scraper S a m a 3000 3. Brachycentrus m 2000 p 3 «(1000 a 53 o :‘1000 +3 Detritus 5 800 " Pteronarcys Feeders 8 «a 600 ‘- 3 . 0 400 - g Hexa enia //’A""--A ‘4 - ~~ / 8 200 - ”new" “a w ‘5"; r o a, s 1 .p g 3000 S Predators 2- 2000 Atherix 1000 ,’A\‘\\ Chauliodes /’ ‘A _____ _A_-‘~- £31! Apgug: Figure 18 94 study required nearly 40 hours before they began to equili= brats with the inorganic radiophosphorus in the solution. Harris found that about the same length of time was re- quired before gggggggg began to take up the radiophosphorus more quickly. Harris (ibid.) attributed this to the fact that there are more bacteria containing radiophosphorus after about 40 hours, so that animals feeding on bacteria at a constant rate will take up more radiOphosphorus in the bodies of the bacteria after 40 hours. Such a situation may have been operating in the present study, causing the initial relatively slow uptake of radiOphosphorus by the blackflies. This initial slow uptake may be a result of the time interval required to accumulate P32 by way of _“food organisms. The stations downstream from Station 3 show somewhat higher peak activity values. This situation probably develOped as a result of downstream "Weed-back"a of P32. The blackfly at the downstream stations would be in a position to filter out activity released from the upstream solids. Further evidence of this is indicated in the fact that peak activity values for the downstream Stations 12 and 14 were recorded three days after peak levels were re- corded at the upstream Station 3 and 8. The caddisfly, Brachycentrus gp., was selected as a representative of a periphyton scraper. The Brachycentrus 22° attaches its case to rocks or higher aquatic plants in exposed places and faces upecurrent so that the water 95 sweeps directly into the case. According to Morgan (1930), the larvae first live upon diatoms and than upon other algae; but when about six weeks old, it adds to this a diet of mayflies, water mites, midge larvae and small crustaceans. Muttkowski (ibid.) found that the diet of Brachycentrus consisted of 18.3% animal food, 72.7% plant food and 9% detritus. The activity curves for the Brachycentrus are shown in Figures l5, l6, l7 and 18. The maximum activity of the caddisfly larvae was approximately 6,000 cpm/gm. and was recorded on July 29, three weeks after isotope treatment. The activity for B_rachycentrus at the four sanpling stations all show similar activity trends. There is an initial period of gradual isotope concentration increase until a peak activity is reached on July 29, three weeks after isotope treatment. Apparently, by feeding on the periphyton on rocks and higher plants, the Brachycentrus was able to maintain a rather high activity plateau or equilibration shortly after peak activity was recorded. RadioisotOpes will be deposited.and.retained in the or- ganism according to the physiological behavior of the particular element involved and, in this case, phosphorus may be so tightly fixed.that little loss occurs, except by radioactive decay and.b1010gical dilution (Krumholz and Foster, 33113.). The stonefly nymph, Pteronarcys, and the mayfly nymph, Hexagenia, were selected to represent detritus feeders. 96 The Pteronarcys nymph is herbivorous feeding on algae and vegetable debris (Pennak, ibid.). The activity curves for Pteronarcys is shown in Figures l5, l6, l7 and 18. The maximum activity recorded for the stonefly was nearly 4,000 cpm/gm. collected at Station 3 on August 5, four weeks after isotOpe treatment. The activity curves of the stone- fly show a trend in complete reverse of the aquatic in- vertebrates heretofore discussed. The peak activity re- corded for the stonefly was collected at Station 3 and the activity peaks decreased progressively downstream. This downstream decrease in activity peaks was demonstrated.pre- viously when.discussing the larger aquatic plants. The stonefly isot0pe activity curves show a lag period in which uptake was slow at precisely the period when larger aquatic plants exhibited.their greatest uptake (Figures 10, 11 and 12). At the time when the larger aquatic plants showed the greatest decrease in activity, the stoneflies exhibited a sharp activity uptake trend. Inasmuch as the stonefly inhabits and.reeds on collections of plant debris, this trend is not too surprising. In his study of the ecology of trout streams in Yellowstone National Park, Muttkowski (ibid.) observed some small Pteronarcys nymphs feeding on blackfly larvae. Whether or not the stoneflies in this study fed on blackfly larvae is not known. It is interesting to note that once the peak activity was reached, the stonefly activity never decreased below 1,000 cpm/gm. in any of the collections (except for Station 14) for the 97 remainder of the study period. This indicates that the stonefly nymphs continued to pick up activity for a con- siderable period. The mayfly, Hexagenia £23: feeds on vegetable detritus and microsc0pic aquatic organisms, principally diatoms (Burks 1953), which it obtains from the rich ooze through which they plow their way (Morgan, ibid.). The activity curves of Hexageni§_are shown in Figures l5, 16, 17 and 18. The maximum activity uptake by Hexagenia was approximately 700 opm/gm. recorded on August 19, six weeks after the. isotOpe treatment, in the sample obtained at Station at The activity trend recorded for all of the stations followed a continuous uptake pattern throughout the study period. This perhaps was due to a greater amount of radioactivity reaching the detritus and sediments as the study period progressed. The Hexagenia obtained radioactive phosphorus from diatoms and other plant materials as a result of fall out to the sediment surface in back water areas. The activity actually available to the mayfly was very much reduced due to various causes. In the first place, much of the plant material reaching the bottom sediments may be dead material sloughed off upstream and deposited in back waters downstream. For the most part, any plant material reaching the ooze would have lost the majority of its activity (corrected counts) due to radiological decay. RadiOphosphorus would be introduced into the sur- face layers of the detritus beds by the fall out of 98 plankton which have died and fecal pellets of animals which have eaten materials containing radiOphosphorus. A possible explanation for the reduced radiOphosphOrus uptake by the mayfly_may lie in the fact that very little activity actually entered the ooze and silt beds. According to Hayes and Phillips (ibid.), a fall out of organisms to the sediment surface is followed by a bacterial breakdown to inorganic P32, so that the P32 is restored to the water. The snipe fly larvae, Atherix variegat§_walker, and the fishfly larvae, Chauliodes s ., were selected as re— presentatives of the predator feeding habit. The snipe fly larvae activity curves are shown in Figures 15, 16, 17 and 18. The maximum activity recorded for the snipe fly was approximately 6,400 cpm/gm. for the sample collected at Station 12 on August 19, six weeks after isotOpe treatment. The dates on which the other stations were observed to reach peak activity varied from July 22 for Station 14 to August 19 for Stations 8 and 12. The activity curves show a great deal of variability in trends as might be expected for predacious organisms. The food eaten would depend upon what became available. Thus the activity curves would be expected to indicate an erratic pattern from station to station, as well as, at the same station, indicating the organism was feeding on food mar terial of various activity levels. It appears possible that the snipe fly larvae at Station 12 may have found a certain type of food so much to their liking that they may 99 have preyed upon only certain individuals. It may be that a certain organism or several organisms high in activity became available at Station 12 shortly after July 29, thus bringing about a peak in the activity curve of the snipe fly. Similar situations may have been Operative at Stations 3, 8 and 12, which show trends in their activity curves that may indicate variations in feeding habits. The activity curves for the fishfly larvae, Chauliodes 22., are shown in Figures 15, l6, l7 and 18. The maximum activity recorded for fishfly larvae was approximately 2,500 cpm/gm., recorded for the sample collected at Station 12 on August 12, five weeks after isotOpe treatment. Even though the fishfly larvae are predacious, as are the snipe fly larvae, the isotOpe uptake did not show similar acti- vity patterns. The peak activity of the fishfly larvae was less than one-half the peak value recorded for the snipe fly larvae, indicating that the two larvae either did not feed upon the same organisms or did not concentrate P32 in a like manner, due to variation in physiological make-up or phosphorus metabolism rate. The P32 uptake by the fish- fly larvae showed a slow initial uptake period. Within 2 to 4 weeks-~depending upon the station--there was an in- crease in P32 uptake, followed by an equilibration period with little activity variation from one sample date to the next. . The pouch snail, 22122.223' was selected as a re- presentative of an organism with an omnivorous feeding 100 habit. {hygg is a scavenger and is essentially omnivorous, eating materials that range from living and dead plant material to dead.animal material (Pennak, ibid.). The activity curves for 22133 are shown in Figure 19. The maximum activity of approximately 8,500 cpm/gm. was recorded for the sample taken at Station 8 on July 15, one week after isotope treatment. The omnivorous feeding habit of this organism would lead one to suspect that the activity recorded on the various sample dates would actually re- present the various activity levels of the food previously eaten by the snail. The activity curves show a bimodal type trend at each sample station. The explanation for this may lie in terms of trOphic level shifting of P32 activity with time from an initial plant uptake to a later animal uptake of radiOphosphorus. It appears probable that-the first mode was a result of the snail feeding on periphyton and other living and dead plant material. As the plant material decreased in activity, the snail activity decreased in response to the decreased activity of its food. The dead animal material that the snail fed upon during the first peak was low in activity but, with the passage of time, the animal material available to the snail contained more and more activity, thus accounting for the second peak activity in mid to late August. The snail, because of its omnivorous food habit, concentrates radiOphosphorus from two trOphic levels. The initial uptake of F32, presumably, was a result ofd 101 feeding upon primary producers and the second uptake as a result of feeding upon dead consumer organisms (dead animal material). Variation in_had10phosphorus uptake by Invertebrates In order to determine to what extent the concentration of P32 varied among organisms of the same species collected at the same sampling station at precisely the same time, a duplicate sample was obtained. The duplicate sample was processed for radiological determination exactly as was the routine sample. The data thus obtained indicated that in some organisms a rather large variation occurred between duplicate samples. The activity curves shown in Figure 20 illustrate this variation of duplicate samples. No attempt is made to show such variability for all samples or all organisms for the entire study period. A thorough treat- ment of such a subject would encompass a_paper in itself. In lieu of such extensive treatment, it is believed that Figure 20 will suffice as an indication of variability in isotope concentration, Operating in the present study. Several factors may be responsible for the variation of activity in some of the invertebrate forms. Foremost of these factors is the fact that some aquatic inverte- brates in rapid streams may become opportunists in regard to food and eat whatever becomes available under certain circumstances. Environmental conditions in streams with strong current makes life somewhat precarious and selective 102 Figure 19. Activity of Physa at all Stations Counts were corrected for Background and decay. 103 Station 3 ] 2000 FStation 12 1000 - ~ Activity (Corrected counts per minut c> —_ 0L4 2000f Station 14 ‘ I 1000 Figure 19 104 feeding at times may be difficult. The activity of Fbod eaten just prior to collection, as well as the condition of the digestive tract at the time of sampling, would have a great bearing on the activity of the organism. It is apparent that an organism with a digestive tract gorged with material of great isotope concentration would tend to indicate a greater tracer concentration over a similar or- ganism that had not eaten for several hours prior to col- lection. The factor of age may play an important part in activity uptake. It is generally agreed that younger, more rapidly growing individuals accumulate relatively greater amounts of radioactivity than the older, more slowly growb ing ones. This phenomenon is apparently a reflection of _the more rapid anabolism that accompanies the growth of younger individuals. If a sample contained young individuals, a rather great variance in isotope accumulation might result over samples containing older individuals. Still another factor Operating to cause variation may arise as a result of drift materials. Dendy (1944) presents results of his investigation on drift in three Northern Michigan streams. He found that 71 different kinds of macroscOpic animals, representing 7 phyla, occurred in the drift during the summer months, as well as the fact that the presence of macroscOpic animals, although highly variable in kind and quantity, was a constant feature. Dendy further indicates that in one of the streams, all species represented in bottom fauna samples were sooner or later found in the drift 105 Figure 20. Activity of invertebrate organisms at Station 14, following treatment of the stream with P33. The variation of subsamples within routine samples is shown. Counts were corrected for background and decay. Mm 7000 Brachycentrus 6000 5000 4000 3000 2000 - ) am )4 O O (D Maximum C) 2000 Pteronarcys lCHDC) L H l l 0 SCH) Hexagenia 5 o ‘3CN3 2CND 113C) ’ w’dfier "\ \ age \\ Maximum Activity (Corrected counts per minute per gr 0 100!) Figure 20 107 of that stream. 0n the basis of this information, it ap- pears safe to assume that organisms collected at a station may have originated further upstream. In some cases a certain organism swept downstream and relocating would contain a greater or lesser accumulation of activity than a similar organism native to the area. In such a situation a great variation in isotOpe accumulation could occur and thus distort the actual isotOpe accumulation value repre- sentative of that area. It is not known just how much this stream drift phenomenon distorted recorded activity values. Invertebrate Biomass Estimate A random sample estimate of invertebrate standing crop was obtained for 1,000 yards of the experimental area (Station 1 to Station 8) on August 31 and September 1, 1959. The mean live weight of invertebrates was calculated to be 0.377 grams per square foot, or 36 pounds per acre. The mean number of organisms was found to be 109 per square foot. The organisms dominating the samples on a weight basis were mayfly nymphs (40%), caddisfly larvae (21%), stonefly nymphs (12%), fishfly larvae (10%) and Oligochaetes (8%). The dominant organisms on a numerical basis were the caddisfly (39%), Oligochaetes (28%) and mayfly (11%). Fish and Lampreyg Assimilation of ingested materials is the chief means by which many radioactive materials accumulate in animals, 108 since the bulk of their essential elements is obtained from their food (Davis and Foster, 1958). The principal mode of accumulation of radiomaterials by fish is through in- gestion (Krumholz and Foster, ibigt). Direct absorption of P32 by fish apparently is inconsequental. According to Krumholz and Foster (ibid.), in fish that live downstream from the Hanford reactors, sorption of radioactive materials directly from the effluents account for only about 1.5% of the total radioactivity. The activity curves for the American brook lamprey, Entosphenu§_lamottenii lamottenii (LeSueur), are shown in Figure 21. The maximum activity for the lamprey was ape proximately 305 cpm/gm. recorded in the sample collected at Station 12 on August 19, 1959. The peak activity for Stations 3 and 8 were quite similar (about 150 cpm/gm.) but occurred on August 5 and 26, respectively. The peak acti- vity recorded for Station 14 was about 215 cpm/gm., about two-thirds the peak activity recorded at Station 12. The activity of the brook lamprey was somewhat higher for the samples obtained at the downstream stations (12 and 14). This may be due to the fact that the detritus at the down- stream stations possessed a greater activity due to I”fall out“ material from upstream areas. This increased activity at the lower stations, the low level of activity at its maximum and somewhat slow activity increase are suggestive of detritus feeders. The material in the digestive tract of numerous brook lampreys was found to contain large tat: 109 quantities of sand, decaying plant material and other de- bris. The general activity trends-although somewhat vari- able--Of the brook lamprey was a more-or-less steady in- crease after isotope treatment. The activity curves of the Eastern slimy sculpin, Cottus cognatus gracilis Heckel, are shown in Figure 22. A maximum activity of about 7,400 cpm/gm. was recorded for the sculpin sample collected at Station 3 on August 23, 1959. The activity curves for the sculpin indicate a great deal of variation and thus mask, for the most part, any uptake pattern that might have been present. The activity pattern, even though somewhat variable, does show a general pattern of uptake throughout the study period. The varia- bility of activity from one sample date to the next appar- ently was due to the activity of food consumed. As has been shown earlier in this paper, the various fish food organisms present in the stream showed considerable dif- ference in radiOphosphorus accumulation. On this basis the variation in sculpin activity probably reflects nothing more than difference in the past feeding experience of the individual fish. This becomes even more certain when one examines the food ingested by sculpins. The sculpin is an Opportunist as far as food habits are concerned. According to Roster (1937), the bulk of the sculpin diet is made up of insect larval stages. The larval forms of Diptera, TricOptera, EphemerOptera and Plecoptera are the chief insect forms. In addition to the insects, the sculpin may 110 I Figure 21. Activity of American brook lamprey at all stations. Counts were corrected for background and decay. 111 200 Station 3 100 r' o 200 vs Station 8 E {:0 h 100 — O Q. 0 ‘s’ c 0 .... a 330° _ station 12 On Q is 5 200 e 0 Id 0) .p O 2'; 100 _- a O 3 >5 :3 0L? 1 5 300 p ‘2 Station 14 200 — 100 — O P I I l I l I I 10 15 22 29 5 12 19 25 July August Figure 21 112 Figure 22. Activity of the sculpin at all stations. Counts were corrected for background and decay. 113 i800 Station 3 600 Station 8 \N O O 8000 Station 12 Station 14 Activity (Corrected counts per minute per gram) July Augugt Figure 22 26 114 ems... eat a considerable amount of ghgga and Oligochaetes. The stomach contents of approximately 200 sculpins taken from the West Branch of the Sturgeon River were examined in an attempt to determine the food habit of the sculpin in the experimental area. The food items found in the digestive tracts of the sculpin were represented chiefly by aquatic insect larvae. The immature insect forms occurring most frequently were: caddisfly larvae, mayfly nymphs, biting midge larvae, blackfly Larvae, stonefly nymphs and dragon- fly nymphs. Organisms occurring less frequently were Oligochaetes, pouch snails and fingernail clams. In addi- tion to the items listed above the sculpin intestinal tracts were observed to contain great quantities of detritus and other debris. The writer realizes that studies based on contents of digestive tracts merely show what an animal will eat and has eaten shortly before capture. However, such facts do illustrate the fact that the sculpins, in the West Branch of the Sturgeon River, are apparently oppor- tunists in regard to food and eat whatever becomes available. The accumulation of P32, in the case of sculpins,:§rou1d be as variable as the type of food ingested. The sculpins may, however, prefer to feed in a particular region and stay there until satiated. Thus, when feeding from a .92232 bed, for example, they may eat whatever they can find there; and once they begin to feed from a ghggg_bed, they continue feeding there until their hunger is satisfied. Thus it was common to find stomachs filled with dozens of 115 . Figure 23. Activity of brown trout at all stations. Counts were corrected for background and decay. Activity (Corrected counts per minute per 116 Station 3 6000 - 4000 h 2000 — 0 8000 F'- Station 8 Station 12 3000 — 2000 e 1000 r- oi—~e1 I 6000 I Station 14 5000 — 4000 - 3000 2000 1000 0 l 1 l 12 19 26 August par L__Jee 117 individuals of one type of food. This behavioristic type of feeding would inject a great deal of variability into the activity pattern of the sculpin. The variability be- tween sculpins collected at the same station at the same time showed great variability between each individual sculpin (Figure 24). For example, a sculpin that had fed on blackfly larvae or stonefly nymphs just previous to collection might show an activity of nearly 7,000 cpm/gm.; whereas a sculpin that had been feeding upon Hexagenia nymphs might exhibit an activity of less than 100 cpm/gm. Still another factor in the variability of activity between individuals may be attributable to age. Among the fishes, it has been established by Olsen and Foster (1952) that the younger, more rapidly growing individuals accumu- late relatively greater amounts of radioactivity than the older, more slowly growing ones. This phenomenon isgpro- bably a reflection of the more rapid anabolism that accompanies the growth of younger fish. The activity curves for the brown trout, Sglgg_trutta .fggig Linnaeus, are shown in Figure 23. The maximum accumu- lation of P32 was 7,100 cpm/gm. reported for the collection at Station 8 on August 5, 1959, four weeks after isotOpe treatment. The initial uptake of P32 in the brown trout is somewhat slow at all stations until July 29, followed by a rapid uptake (except for Station 3) leading to a peak on August 5. The lag in initial P32 uptake may possibly be due to the fact that the brown trout is almost entirely 118 Figure 24. Activity of brown trout and sculpins following treatment of the stream with radiOphos- phorus. Counts were corrected for background and decay. Activity (Corrected counts per minute per gram) 119 lagooF Brown Trout - Station 8 Specimen with higher activity 11000 " Specimen with lower activity ----- 10000 - 9000 - 8000 - 7000 -' 6000 - 5000 - 4000 - 3000 - ' /’ 2000 "' \ /~_____\\ I, \\ / \\\ / 1000 — \\ ,’ ~-———-_/ \ / I 0 l i \i- -—_ i’ J l l I l _l 9000 ...: Sculpins - Station 8 8000 - Specimen with higher activity —e Specimen with lower 7000 _- activity ----- 6000 — 5000 F 4000 — 3000 — -- | 2000 — I 1000 L- , ____./ 0 r~-—4-——*r”' l I I if I 15 22 29 5 12 19 26 1 Jul Figure 24 120 carnivorous (Pentelow 1932). HadiOphosphorus did not ac- cumulate in the animals comprising the trout diet until the F32 was taken up by the primary producers and then accumu- lated in the trout only as a result of ingestion of plant material or other organisms. The appearance of a peak in activity, at all stations on August 5, further indicates the brown trout was feeding primarily on animal organisms which were probably reaching peak activity during this same period. The activity curves for the brown trout indicate a pattern. The higher activity values occurred at Stations 3 and 8 and decreased progressively downstream, a pattern similar to that found in many of the stream invertebrates. The P32 activity values recorded for individual brown brout showed considerable variation. The variation between individual brown trout is shown in Figure 24. This variation is undoubtedly due to feeding habit and to the age of the individual. The influence age has on radio- phosphorus uptake and accumulation in fish was discussed at length in the section on sculpin uptake and accumulation of P32, and need not be repeated here. According to Muttkowski (1212'): fish will take food that is easily captured and which is accessible. In general, trout are Opportunists as far as their food is concerned. They eat what animal food is available, regardless of the origin. Neil (1938) found that the major items in the brown trout diet were the blackfly larvae, mayfly nymphs, caddisfly 121 nymphs, stonefly nymphs, leeches and other fish. Pentelow (1223,) indicates somewhat similar food items for brown trout in his work on the Tees and Itchen Rivers. He found immature insects to be the item most frequently found in brown trout digestive tracts. The immature forms found by Pentelow (ibid.) were chiefly representatives of the stone- fly, caddisfly and mayfly groups. He found water plants to be in complete absence in the brown trout diet. In view of the tremendous variability in food items, it is little wonder that the activity of the individual brown trout varied.considerably. It is further felt that variability between individuals may be due to feeding prior to capture. Because the samples were prepared from whole specimens of trout, the amount and kind of food material contained in the stomach would have some effect on the re- sults in individual cases. For example, if a brown trout that had been feeding on the mayfly, Hexagenia, was cap~ tured and processed, the activity level would probably be greatly reduced when compared to an individual processed with its stomach gorged.with stoneflies. Radio hos horus Accumulation in Various Tissues of the rown .rout Highest activity values in the brown trout were found in the bone, head and gills, and viscera, with much lower results in the muscle. The maximum value of 6,375 cpm/gm. (2.87 x 10’3‘uc/gm.) was found in the bone of the brown trout. The value found in the head and gills was 2,111 cpm/gm. 122 (9.5 x 10'” uc/gm.), while the value recorded for the brown trout viscera was 1,986 cpm/gm. (8.9 x 10"4 uc/gm.). The maximum value of 826 cpm/gm. (3.7 x 10‘” uc/gm.) was found in the muscle of the brown trout. As trout may concentrate radioactive phosphorus many times above the level found in water, the use of trout for human consumption presents a potential public health.prob- 1cm (Robeck, 33.91,, gpgg.). Permissible levels of P32 of fish used for human consumption have been discussed by Donaldson and Foster (1957). They calculated the amount of radiophosphorus that would be expected in edible parts of fish, assuming an intake of 3 pa per week. This figure was based upon uptake from water containing the maximum permissible concentration for drinking water (International Committee on Radiation Protection). Donaldson and Foster‘s suggested maximum level is 7 x 10’“ us P32 per gram for fish flesh, a figure which apparently includes a safety factor of 10. This value is substantially greater than the maximum recorded in trout muscle (3.7 x lO‘u,pc/gm.) in the present study. Bone, viscera and head and gills of some of the fish in the present study exceeded this activity level slightly. It is pointed out, however, that the portion of the fish with the greatest activity value is not considered the edible part of the trout. Further, the fish are not eaten immediately after being caught, some time elapsing for cleaning, cooking and other preparation. Thus the actual permissible intake would be greater than that computed (Robeck, 33 21,, ibid.). 123 Biomass Estimate An estimate was made of the standing crOp of fish in 1,000 yards of the West Branch of the Sturgeon River (isotOpe entry to Station 8) on August 25 and 28, 1959. The area encompassed by this section was calculated to be 1.76 acres. The pOpulation estimate yielded the following data: Table 2. POpulation and biomass estimates of fish in 1,000 yards of the West Branch of the Sturgeon River. Fish Type Pounds Per Acre POpulation (Number of Fish) Brown Trout 9.79 5,103 Brook Trout 1.12 91 Rainbow Trout 9.13 1,010 Sculpin 106.6 17,484 Standing crop estimates of fish, invertebrates and vegetation in various stream systems are given in Table 3. 0n the basis of the data presented in Table 3, it becomes apparent that the West Branch of the Sturgeon River supported a very low standing crop of trout in 1959. The reason for this is not known but, according to professional sources acquainted with trout in this section of the river, the pOpulation of trout in 1959 was extremely low. The answer for this low standing crop may lie in factors Table 3. 124 A comparison of standing crOp estimates of fish, invertebrates and aquatic plants for various streams. Pounds of Stream Invertebrates —. Pounds of Trout Per Acre :Pounds of Aquatic Plants Per Acre Pounds of All Fish Per Acre West Branch, Sturgeon River (1958) (Bryant 1960) 53 West Branch, Sturgeon River (1959) Hunt Creek, Michigan (Shetter and Leonard, 1942) 72 Houghton Creek Mich. (Ellis and Gowing, 1957) Big Spring Creek, Va. (Surber 1937) 483.9 36.58 138-411 Prickly Pear Creek, Mont. (Sept. 1949) (Stefanich 1951) Prickly Pear Creek, Mont. (Sept. 1950) (Stefanich 1951) 86 20 94 120-127 T 101.8 126.6 21,780 104 92.3 58.9 associated with the severe winter present in 1959. What- ever the cause, it affected the trout to a greater degree than invertebrates. The invertebrate estimate in 1959 is 125 about 68% of that reported for 1958, while the trout stand- ing crop estimate is about 25% of that reported for 1958 (Bryant, 1233. ). The estimate for all fish in 1959 shows a greater standing crop than in 1958 or in any of the other streams in Table 3. This elevated value is due to a large estimated sculpin standing crap in 1959. Due to a scanty return of marked sculpin, a great deal of weight cannot be given the sculpin estimate in 1959. Translocation of radio hos horus Thrdugfi the Ecosystem The accumulation of radiOphosphorus by aquatic organisms follows a definite pattern. The mean activity level of aquatic organisms and water, computed for all of the sampling stations, is shown in Figure 25. This figure provides a good picture of the fate of P32 as it passes along the food.chain. In order to demonstrate the trans- location of P32 along the food chain, the organisms are arranged‘according to broad trOphic levels, represented by producers (plants), primary consumers (plant eaters) and secondary consumers (carnivores which eat the herbivores). The P32 in the water reached a peak activity in 40 minutes after its arrival at the sampling stations. 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