“W' W V ' ~v~.-. ‘ ‘ Qt],- ‘ ‘ .: AQEfiARiA WNES GK THE LEFTAKE G? MS'ENEC 74 WEI-£3 SGDLIZQM ARSEM'EE 3V CQMFF‘QNEM’S CF AR AQUAHC ECQSYg‘fEM masts: £90 “to Dogma «if M: 5. MICHEGAN STETE WIN The mag Gordon Bah: 19 6 6 THESIS LIBRARY Michigan Static ' University ABSTRACT AQUARIA STUDIES ON THE UPTAKE OF ARSENIC 74 LABELED SODIUM ARSENITE BY COMPONENTS OF AN AQUATIC ECOSYSTEM by Thomas Gordon Bahr Aquaria experiments were designed to determine move— ment of sodium arsenite through an aquatic ecosystem using A574 as a tracer. Uptake studies with different substrate types, plants, fish, and invertebrates are presented. The uptake of arsenic into bottom sediments appears to be a function of particle size, fine substrates exhibit— ing higher uptake rates than more course sediments. The mechanism is probably that of physical adsorption. Higher aquatic plants quickly incorporate arsenic and retain it during decay following death. Uptake into plants occurred before the soil had concentrated significant amounts of the chemical. Transport of arsenic from the water to plants was probably through the leaves and/or stems rather than via the roots. Fish do not appear to concentrate the isotope directly from the water. However, they may take up the chemical by ingestion of radioactive plant material. Some Thomas Gordon Bahr crayfish and snails picked up significant amounts of the isotope. Water concentrations of the isotope were about 25 percent of initial levels after 60 days. It was suspected that an equilibrium between arsenic in the water and sedi- ments was being approached. Thus low levels of arsenic might persist in the water for extended periods of time. Qlessr AQUARIA STUDIES ON THE UPTAKE OF ARSENIC 74 LABELED SODIUM ARSENITE BY COMPONENTS OF AN AQUATIC ECOSYSTEM BY Thomas Gordon Bahr 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 1966 ACKNOWLEDGMENTS I extend my sincere thanks to Dr. Robert C. Ball whose helpful guidance and assistance during the course of my research and in preparation of this thesis was most invaluable. I wish to express my gratitude to Messrs. Leonard P. Sohacki, Jack D. Bails, Eugene H. Buck, Jerry L. Hamelink, and Walter Haney for their aid in collection of data for the manuscript, and also to the many other graduate students for their helpful comments. My wife Judy should receive special thanks for her many hours spent assisting me with the numerous tasks associated with this study. Collection and analysis of data for this study was sponsored by the Atomic Energy Commission, contract AT(ll—l) 655, administered by Drs. R. C. Ball and F. F. HOOper. Writing of this thesis was performed during the tenure of a Predoctoral Research Fellowship (5-Fl-WP-26,004-O3) spon— sored by the Division of Water Supply and Pollution Control of the United States Public Health Service. ***** ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 Selection of the Isotope . . . . . . . . . . . . 3 METHODS 0 O O O O O 0 0 O O O O O O 0 O O O O O O O 5 General . . . . . . . . . . . . . . . . . . . . 5 Counting Methods . . . . . . . . . . . . . . . . 6 Machine Conversions and Related Calculations . . 7 Design of the Aquaria System . . . . . . . . . . 15 Substrate experiments . . . . . . . . . . . 15 Plant experiments . . . . . . . . . . . . . 16 Fish eXperiments . . . . . . . . . . . . . . l6 Invertebrate experiments . . . . . . . . . . 16 Complete Ecosystem experiments . . . . . . . 17 Water experiments . . . . . . . . . . . . . l7 PROCEDURES . . . . . . . . . . . . . . . . . . . . . 19 Addition of the Isotope . . . . . . . . . . . . 19 Sampling and Processing . . . . . . . . . . . . 19 Plants . . . . . . . . . . . . . . . . . . . 20 Substrate . . . . . . . . . . . . . . . . . 21 Water . . . . . . . . . . . . . . . . . . . 22 Fish . . . . . . . . . . . . . . . . . . . . 23 Crayfish . . . . . . . . . . . . . . . . . . 24 Odonata . . . . . . . . . . . . . . . . . . 24 Snails . . . . . . . . . . . . . . . . . . . 24 Glass slides . . . . . . . . . . . . . . . . 25 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 26 Substrate Aquaria . . . . . . . . . . . . . . . 26 Water . . . . . . . . . . . . . . . . . . . 26 Substrate . . . . . . . . . . . . . . . . . 29 Plant Aquaria . . . . . . . . . . . . . . . . . 38 Water . . . . . . . . . . . . . . . . . . . 38 Substrate . . . . . . . . . . . . . . . . . 42 iii Page Complete Ecosystem Aquaria . . . . . . . . . . . 47 Water . . . . . . . . . . . . . . . . . . . 47 Substrate . . . . . . . . . . . . . . . . . 47 Uptake Into Plants . . . . . . . . . . . . . . . 55 Potamogeton praelongus . . . . . . . . . . . 55 Elodea canadensis . . . . . . . . . . . . . 61 Isoetes sp. . . . . . . . . . . . . . . . . 62 Uptake Into Fish . . . . . . . . . . . . . . . . 69 Green sunfish . . . . . . . . . . . . . . . 69 Fathead minnow . . . . . . . . . . . . . . . 7O Bullhead . . . . . . . . . . . . . . . . . . 70 Golden shiner fry . . . . . . . . . . . . . 72 Uptake Into Crayfish . . . . . . . . . . . . . . 72 Uptake Into Snails . . . . . . . . . . . . . . . 75 SUMMARY . . . . . . . . . . . . . . . . . . . . . . 77 LITERATURE CITED . . . . . . . . . . . . . . . . . . 79 APPENDICES . . . . . . . . . . . . . . . . . . . . . 82 iv 10. ll. 12. LIST OF TABLES Composition of experimental aquarium systems . . . . . . . . . . . . . . . . . . . Rate of disappearance of water radio- activity in aquarium experiments following treatment with labeled sodium arsenite . . . Rate of uptake of radioactivity by the tOp 2 cm and bottom 2 cm of the substrata of aquarium eXperiments . . . . . . . . . . . . Activity-density (pc/g) of g, praelongus following application of the isotOpe . . . . Adsorbed activity of P, praelongus following application of the isotope . . . . . . . . . Activity-density (pc/g) of Elodea canadensis following application of the isotope . . . . .Adsorbed activity of Elodea canadensis following application of the isotOpe . . . . Activity-density (pc/g) of Isoetes sp. following application of the isotOpe . . . . Adsorbed activity of Isoetes sp. following application of the isotope . . . . . . . . . Fish that exhibited radioactivity during the study . . . . . . . . . . . . . . . . . . Uptake of radioarsenic by crayfish . . . . . Uptake of radioarsenic by Physa sp. . . . . . Page 18 54 55 56 58 62 65 66 69 71 74 75 10. LIST OF FIGURES Log plot of A574 decay curve . . . . . . . . Water radioactivity of three aquaria used in substrate eXperiments following treat- ment with labeled sodium arsenite . . . . . Water radioactivity of three aquaria used in substrate experiments after treatment with labeled sodium arsenite. Log plot . . Activity-density (pc/g) of top and bottom 2 cm fractions of mud cores from three aquaria used in substrate experiments . . . Water radioactivity measurements from three aquaria used in plant experiments following treatment with labeled sodium arsenite . . . Radioactivity in the top and bottom 2 cm fractions of sand taken from two aquaria used in plant experiments after treatment with labeled sodium arsenite . . . . . . . . Radioactivity in top 2 cm of fine gravel taken from the plant aquarium having gravel substrate 0 O O O O O O O O O O O O O O O O Radioactivity measurements of water used in complete ecosystem experiments after treat- ment with labeled sodium arsenite . . . . . Log plot of water radioactivity in complete ecosystem eXperiments . . . . . . . . . . . Radioactivity measurements in top and bottom 2 cm of sand from aquaria used in complete ecosystem experiments . . . . . . . . . . . vi Page 10 28 31 33 4O 44 46 49 51 53 Figure 11. 12. 13. Page Measurements of adsorbed radioactivity of the surface of g, praelongus following treatment with labeled sodium arsenite . . . . 60 Measurements of adsorbed radioactivity of the surface of Elodea canadensis following treatment with labeled sodium arsenite . . . . 64 Measurements of adsorbed radioactivity on the surface of Isoetes sp. following treat- ment with labeled sodium arsenite . . . . . . 68 vii INTRODUCTION Sodium arsenite has been used as an aquatic herbi— cide for almost forty years. In 1926 Domogalla first used the chemical for controlling aquatic weeds in a recreational area. Sodium arsenite was first used as a herbicidal tool in fisheries management by Surber in 1929.. Since this time use of the chemical drew wide acceptance throughout the United States. In fact, prior to World War II sodium arse— nite was one of the few chemical herbicides in use. After the war, however, many new and effective herbicides appeared on the market, but in spite of these, sodium arsenite re- tained its popularity. This popularity is perhaps linked to its low cost and efficiency in controlling aquatic weeds with a minimum of harm to fish. Despite the fact that this herbicide has been used for so many years, little is known about its effect on the aquatic ecosystem or the ultimate fate of the chemical fol- lowing treatment in a lake or pond. Also important are the long term effects following prolonged treatment such as in the case of many Wisconsin lakes which have been continu— ously treated for over ten years. Dupree (1960) studied the arsenic content of various portions of a pond ecosystem following treatments with 4 ppm of As He found that 203. considerable amounts of arsenic were retained by the muds and then slowly released to the water upon draining and refilling the ponds. He also found that certain plankton organisms concentrated the herbicide in amounts up to 7,200 ppm thirty days after treatment, and that fish contained only trace amounts of the chemical. Lawrence (1958) found that sodium arsenite markedly reduced fish production in treated ponds and killed most of the micro-crustaceans and rotifers. He claims that this lack of fish food partly explains reduced numbers and poor growth in the fish popula— tion. Rigg (1955) investigated the effect of several herbi— cides (including sodium arsenite) on a series of aquatic plants and described some possible modes of action which sodium arsenite had on these plants. Attempts to the present time at accurately tracing sodium arsenite through an aquatic ecosystem have been hin— dered by the lack of a good analytical technique for arsenic analysis. Conventional methods are very inefficient and time consuming. It was the purpose of this study to utilize a radioactive isotope of arsenic as a tag to determine the movement of arsenic after its introduction into an aquatic system. It was hoped that by using radioactive counting methods, a degree of analytical precision could be obtained far and above that of conventional methods. The overall isotope translocation study was divided into two phases. The first was a study of the movement of the tagged herbicide through the ecosystem of a small pond. The second was a parallel laboratory study carried out by means of a series of aquaria experiments in which conditions were carefully controlled. The research was carried out under AEC contract AT(1l-l) 655. Collection of data for this thesis was done during the summer of 1963 at the Mich- igan State University Experimental Station at Lake City, Michigan. Selection of the Isotope Since radioactive tracer techniques were to be employed in the proposed study, selection of a suitable iso— tope was vital to the study. For an isotope to be of value in this type of biological translocation study, the isotope needed the following characteristics: 1. The half-life of the isotope must be long enough to not completely decay before termination of the study. 2. The radioactive emissions should be of high enough energy to be easily detected with conven- tional counting equipment. 3. No biological discrimination between the stable and radioactive forms of the element should occur . There are ten different radioactive isotopes of arsenic. Of these, only two have half—lifes long enough to be suitable for this study. Shorter lived isotopes could have been used only if the initial activity was extremely high, however, this could lead to undesirable radiation health haz ards. The two isotopes with sufficiently long half-lifes are arsenic 74 and arsenic 73. Arsenic 73 has a 76 day half-life, but emissions are of very low energy. Arsenic 74 on the other hand has a 17.5 day half—life and emits two levels of gamma radiation and two levels of beta radiation, 0.635 and 0.596 Mev, and 0.69 and 1.36 Mev respec— tively. The fact that two types of emissions are given off from this isotope meant that gamma and/or beta counting equipment could be employed. Considering the above criteria, arsenic 74 appeared the isotope of choice for this study. METHODS General The incorporation of arsenic into the various compo- nents of the aquaria system was measured by the level of radioactivity emitted from any given sample. Using the technique of isotopic dilution the uptake of stable arsenic is directly proportional to the amount of the tagged arsenic in the sample. Back calculation from activity levels in a sample gives the amount of stable arsenic present. The iso- topic dilution was done by adding enough labled arsenic to the commercial herbicide to give sufficient activity for counting accuracy while maintaining the level of total arse- nic at a concentration high enough to kill the higher aquatic plants. Fifty millicuries of A374 were obtained in the form of sodium arsenite. This isotope which was dissolved in an alkaline solution was supplied by the Oak Ridge National Laboratory, Oak Ridge, Tennessee. Initial dilutions of the tracer and the herbicide were made in a fifty gallon drum lined with polyethylene. Pre-measured quantities of double distilled water were used for the dilutions. After diluting and mixing the isotOpe-herbicide solution, a period of 12 hours was allowed for equilibration of the stable and radio— active forms of arsenic. When 10 ml of the final solution were dissolved in one gallon of aquarium water a specific activity of 6 disintegrations per second per milliliter and a concentration of 8 ppm of A3203 were obtained. Counting Methods Two counters were used in the study. The first was a Tracerlab Omni/Guard Low Background Counting System which was used primarily for counting beta radiation. The counter was used in conjunction with a SC—88 Auto/Computer. This system employed a printed tape readout which gave sample activities in counts per minute corresponding to the plan— chet number. We originally intended to use this system for gamma detection by using a 2" scintillation crystal (flat- type). The machine efficiencies encountered with this gamma counter were quite low, therefore we replaced the scintilla- tion detecter with a Geiger—Muller tube and used the system for beta counting. The Geiger-Muller detecting system had an anti-coincidence circuit which improved the count to back- ground ratio. The counting efficiency of this beta counter was much higher than the gamma system. The second counter used was a scintillation well- type counter for use as a gamma detecter. The system was composed of a 3" NaI well crystal embedded in a shielded well. The crystal and photomultiplier were connected to a variable single channel spectrum analyzer. This allowed only those emissions of desired energy to be registered on the decade scaler, and excluded emissions from sources other than arsenic 74. During the study the spectrometer was adjusted to include emissions plus or minus 0.05 Mev from the 0.635 Mev gamma peak. Three times a day throughout the study the level of background radioactivity was counted on each machine. Blank planchets and counting vials were used in these determina— tions. The mean background activities for each day were subtracted from sample counts obtained on that same day. Background activity measured in the well counter (gamma counts) averaged about 20 counts per minute. Background activity measured in the Omni/Guard (beta counts) was less than 2 counts per minute. Machine Conversions and Related Calculations Two A574 standards with the same specific activity were made using a portion of the original isotope. Each standard contained 0.9800 uc. The standards were counted in their respective machines from time to time throughout the duration of the study. Data obtained from these counts were used in decay corrections and machine efficiency calcu- lations. To obtain the actual activity of a sample given a certain observed activity, one needs to know the machine efficiency. Using a standard as previously described, the machine efficiency is readily obtained by dividing the ob- served activity of the standard by the actual activity. However, before this calculation can be made, the physical decay of the isotope must be taken into consideration. This is done by equating all observed activities to some reference time, namely, when the isotope was made. The calculations are given below. The loglO of the observed activity of the standard(s) is plotted against time in days. This plot is shown in Figure 1. Using the least squares method of regression analysis, equations for the decay of radioarsenic as observed in the two counters are as follows: Omni/Guard: Log Y -0.013 X + 4.701 -0.0l6 X + 4.447 Well counter: Log Y Extrapolation of these two curves to time zero gives the observed activity at this time. Observed activity for the Omni/Guard at time zero is 50,200 Cpm and for the well counter, 27,900 cpm. Conversion of counts per minute into microcuries is accomplished by dividing the counts per min- ute by 2.22 x 106. (2.22 x 106 cpm = luc.) The units now being the same the machine efficiency can be calculated by dividing the observed activity by the actual activity. Figure 1. Log plot of A574 decay curve. Radioactivity measurements in counts per minute of two identical standards are shown when counted in the Omni/Guard counter and well counter. Lines are calculated by methods of least squares. 10 m>muw H mN anmmmuu Hm>muw N OH mHHmcm mcoz H mmumunmunw>cH OOH muwcHnm Hm>muw N mN mpmmnHHsm Hm>muo N mN SmHmcsm Hm>mnw m cmHm NH coummOEmuom NH mmumomH Hm>mu0 H NH mmUOHm ocmm N m mucmHm 652 m mumuumnsm EsHum5q¢ mom mHmEHc¢ mUQMHm mumuumnsm mHHmsq< mucmEHHmmxm mEchmmHO mo HmQEDZ mamumxm EdHHmsqm HmucmEHnmmxm mo :oHuHmomEOO .H mHnma PROCEDURES Addition of the Isotope After the radioarsenic had been mixed with the com- mercial herbicide and allowed to equilibrate, aliquots were measured into a 100 ml graduated cylinder and added directly to each of the aquaria. Because the activity of the isotope was rather high in its concentrated form, remote handling tongs were used to transport the isotope to the aquaria. Shelves and the floors of the laboratory were protected from accidental spills with special absorbent paper. Other safety precautions concerning the use of radioisotopes were observed at all times. Sampling and Processing Prior to the actual sampling of experimental orga- nisms, representatives of each plant and animal used in the study were counted to determine the natural radioactivity level of these organisms. None of these pre-experimental organisms taken from the same area as the experimental orga- nisms had activity significantly greater than background. 19 20 Plants Plants from both the Complete Ecosystem and Plant aquaria were collected and processed identically. Each of these aquaria was sampled at intervals of two hours, twenty- four hours, and one week after addition of the isotope. Whole plants, two of each species, were taken from each aquarium. They were processed for counting in the following manner: 1. After removal from the water each plant was blotted with absorbent paper to remove excess moisture. 2. Plants were then weighed (live wt.) to the near- est 10 mg. 3. Each plant was placed in a beaker and washed with 10 ml of 0.01 N HCl. 4. The acid rinse was poured into a stainless steel planchet and evaporated at 600 C in a drying oven. 5. Each whole plant was placed in a plastic counting vial. 6. The acid rinse was counted in the Omni/Guard and plants were counted in the well counter. In addition to the scheduled sampling periods mentioned above, occasional sampling of dead or dyring plant material was done later in the study during the process of mineraliza- tion. Because of the fragility of the decaying plant mate— rial no acid rinse was attempted for fear of dissolving the 21 entire plant. These additional samples were limited to Potamogeton and Isoetes. Elodea rapidly disintegrated and could not be sampled. An alternative sampling method whereby the leaves, stems, and roots would be counted separately was given con- sideration, however, it was not known whether enough plant material could have been obtained in this manner to obtain significant counts. There was also a very large backlog of uncounted samples by this time due to the limitations of counter availability. Had this alternative method been used, the backlog would have been greater. Since the half-life of the isotope is short,some of the samples might have lost activity by the time they were counted. Substrate Sand from the Complete Ecosystem and Plant aquaria and mud from the Substrate aquaria were sampled at the same time and processed identically. Samples were taken on the first day, second day, and then once a week after addition of the isotope. One cylindrical core, chosen randomly from the bottom of each aquarium, comprised a sample. At no time was the same spot in the aquarium floor sampled twice. This was quite easy to avoid because a spot which had been sam- pled once left a slight depression in the substrata. The core sampler was made by using an open-ended plastic vial 2 cm in diameter. To the closed end of the 22 vial a short length of hollow glass tubing was attached by forcing the tubing through a small hole. The purpose of the tubing was to furnish a handle for the sampler and also to provide an escape route for the water as the vial was pushed into the mud. By pushing the open end of the vial into the mud while holding the glass tubing, water is allowed to escape from above the core and out through the tubing. The core was removed from the substrate by pulling upward on the glass tubing while having a finger over the hole at the end of the tube. This created a partial vacuum above the core itself, preventing it from dropping out of the vial upon removal from the substrate. The plastic vial containing the core was quickly frozen, then sliced into two 2cm sections. Each section was placed into a plastic vial, dried, weighed, and then counted in the well counter for gamma activity. Water Water samples were taken every day for the first week, then three times a week for the duration of the study. This sampling interval applied to the Plant, Complete Eco- system,Substrate, and Water Only aquaria. Other aquaria were sampled only when some other component of that aquaria was sampled. Water was taken from the approximate center of each aquarium using a 10 m1 volumetric pipette. The water was drained from the pipette into stainless steel planchets and 23 allowed to dry at 600 C in a drying oven. The planchets were then counted in the Omni/Guard. Occasionally, water was sampled from different depths in the Water Only aquaria, but no differences in activity were noticed. No attempt was made to fractionate the water samples into bacterial and planktonic fractions. This method would have been preferred but it required a larger volume of water per sample than could have been supplied by the aquaria over the sixty day period. Fish Fish from the Complete Ecosystem aquaria were sam- pled twice during the study, two days and six weeks after application of the isotope. All other fish in other aquaria were sampled once a week for the first month, then once every two weeks until termination of the study. Three fish were taken from each aquarium during a sampling period. Each was blotted, weighed, and then rinsed with 0.01 N HCl. The fish were placed in plastic counting vials, and the acid rinse placed in stainless steel planchets to evaporate in the drying ovens. Fish were counted in the well counter and the acid rinse was counted in the Omni/Guard. Some of the fish were counted alive but most of them quickly died.) If the well counter had been large enough to accommodate larger counting vials perhaps this procedure could have been done successfully. 24 Crayfish Duplicate samples of crayfish were taken from the two<2rayfish aquaria three times per week, starting three days after addition of the isotOpe. They were processed and counted identically to the fish. The crayfish from the Com- plete Ecosystem aquaria were sampled at two week intervals and processed in a similar manner. Live counting of these organisms was also attempted. The mortality was much lower than in the fish but still high to warrant_disbontinuance of the procedure. Odonata Dragonfly naiads in the Complete Ecosystem aquaria could never be recovered. They were probably eaten or may have all died. Naiads from the aquaria containing only Odonata were to be sampled weekly but most of them died before many samples could be taken. Snails Physa was sampled once every two weeks throughout the study. They were counted alive and replaced in the same aquaria. No acid rinse was attempted for fear of killing them. .Again, significant mortality occurred during the live counting process. 25 Glass slides The glass slides from aquaria which contained only water were sampled three times during the study. They were rinsed with 0.01 N HCl. The rinse liquid and glass slides were then counted separately. No activity was found in either fraction. RESULTS AND DISCUSSION Water from the Plant, Complete Ecosystem, and Sub- strate aquaria showed significant decreases in radioarsenic activity during the 60 days of the study. Radioarsenic levels in all of the other aquaria did not decrease in this period to any measurable extent. One should note that none of these latter aquaria contained sand or'pond mud substrata but rather contained gravel of relatively large size. Small amounts of activity occasionally occurred in some of the fish and invertebrates but this uptake did not noticeably decrease the water activity in the respective aquaria. Results of changes in the water activity in the Plant, Complete Ecosystem, and Substrate aquaria are given below. Substrate Aquaria Water Changes in water activity of the three Substrate aquaria are shown in Figure 2. The activity has been cor- rected for background, decay, and machine efficiency and is expressed in units of pc/ml. Each point represents the activity-density in one 10 m1 sample. Results from the three aquaria are combined into one graph and the curve is an occular estimate of the regression. 26 Figure 2. 27 Water radioactivity of three aquaria used in substrate experiments following treatment with labeled sodium arsenite. Each point is the activity of one 10 ml sample. Data corrected for background, decay, and machine efficiency. Bottom substrate was pond mud. 28 200 50)- 30 4o 50 DA YS 20 IO 29 The initial activity of about 200 pc/ml decreased to half that amount in approximately two weeks. The time required for the second "half-life" is much longer, in this case taking over sixty days. That is, it takes more than sixty days for the initial concentration to decrease by 75 percent. The flatness of the curve at the termination of the study suggests that an equilibrium between the arsenic in the water and the arsenic in the muds is being approached. Whether or not the activity in the water would become com- pletely lost is uncertain, but it is evident from the curve that terminal levels of radioarsenic should persist for some time. Figure 3 is a logarithmic plot of Figure 2. In this case the log of the corrected activity is plotted against time. Each point represents the mean of three sample activ- ities. The curve is fitted by eye. This curve is still concave upward which means that a greater percentage of available activity is being lost early in the study and a lower percentage later. Had this percentage been constant, the curve would have been linear. Substrate Figure 4 is a plot of the uptake of radioarsenic into the muds of the three Substrate aquaria. Each point represents the mean corrected activity of three cores, one from each aquarium. The top curve represents the activity— 30 Figure 3. Water radioactivity of three aquaria used in sub- strate experiments after treatment with labeled sodium arsenite. Each point is the log of the mean of three water samples. Data corrected for background, decay, and machine efficiency. Bottom substrate was pond mud. 31 m>._._>:.o< 1.3.0... 0000. 0000 009 _ _ L 03.00.". 000 - 9 All/\llOV oasaosovv. 0‘90-] banana: 61 necessary that a sufficient amount of radioarsenic would have to be concentrated in the soil surrounding the roots. As was shown in an earlier section, radioarsenic levels in the soil had not yet reached high concentrations. Elodea canadensis Elodea was the most susceptible plant to the toxic action of the herbicide. Death occurred in about three days and in less than one week most of the plants from the three Plant aquaria were so fragile that further sampling was impossible. Plants from the Complete Ecosystem aquaria were also extremely fragile, but with care, samples could be taken at the one week sampling period. As in the case of Potamogeton, uptake in Elodea occurred almost immediately. In two hours, levels of activ- ity in Elodea had exceeded 1,000 pc/g and in Complete Eco- system aquarium No. 1 levels had exceeded 2,000 pc/g. .After twenty-four hours of exposure to the isotope, further in- creases were apparent. One week later, even greater in— creases were observed. In one case, plant activity—density had reached 10,000 pc/g. Results of uptake into Elodea- are shown in Table 6. 62 Table 6. Activity—density (pc/g) of Elodea canadensis following application of the isotope Complete Aquaria Plant Aquaria Time No. 1 No. 2 No. 3 No. 1 No. 2 No. 3 2 hr. 2492 1407 628 542 1554 0 2365 1903 1621 1775 844 385 24 hr. 4038 1742 3282 1432 5307 462 3554 1444 1599 887 1598 433 168 hr. 6734 766 10213 .... .... ... 4701 2130 7657 .... .... ... Adsorbed activity from Elodea also appeared to be a function of the total radioactivity in the plant. These results are shown in Table 7. .A log plot of the percent adsorbed activity versus total activity of the isotope is shown in Figure 12. .Again a straight line relationship is indicated. Isoetes sp. Radioarsenic was also immediately incorporated into these plants. .After two hours, plants in five out of the six aquaria sampled contained activity levels greater than 400 pc/g. The initial uptake into this species was slightly lower than in the case of Potamogeton and Elodea. After twenty-four hours of exposure to the isotOpe, plants in most of the aquaria showed noticeable gains in activity. V'w“. 7W1 Figure 12. 63 Measurements of adsorbed radioactivity on surface of Elodea canadensis following treatment with labeled sodium arsenite. The log of the percent of total plant activity which was ad- sorbed is plotted as a function of total plant activity. Plants were from complete ecosystem and plant eXperiments. 64 On. I >._._>_._.O< |_<._.O._. OOOM OOON OOO_ H _ T _ _ 0030959935004“: N— All/\llOV oasaosav °/.°'9m 65 Table 7. Adsorbed activity of Elodea canadensis following application of the isotope. Each value is a percent of the total activity in the plant Complete Aquaria Plant Aquaria Time No. 1 No. 2 No. 3 No. 1 No. 2 No. 3 2 hr. 22.4 25.6 52.9 75.9 41.5 .... 19.1 26.8 56.9 43.6 38.1 44.2 24 hr. 34.3 37.2 24.3 49.0 19.2 60.3 36.9 40.3 44.4 59.0 34.4 73.6 168 hr. 15.9 61.4 13.0 .... .... .... 26.7 29.4 20.0 .... .... .... After one week, uptake was again apparent, activity levels greatly exceeding 1000 pc/g. The maximum observed activity density occurred in the Complete Ecosystem aquarium No. 1 where one plant had a concentration of over 9000 pc/g. For the most part, however, activity levels were about 50 per— cent less than this figure after one week. Uptake of radio- arsenic into Isoetes is summarized in Table 8. Isoetes was not asfragile as Elodea during the decay- ing process and was sampled again after twenty-five days. One whole plant was taken from each aquarium and counted. No outstanding increases in activity density were observed from this sampling. These data are also shown in Table 8. 66 Table 8. Activity-density (pc/g) of Isoetes sp. following application of the isotope Complete Aquaria Plant Aquaria Time No. 1 No. 2 No. 3 No. 1 No. 2 No. 3 2 hr. 2447 408 278 1002 1302 423 1566 676 127 496 976 918 24 hr. 2080 1159 148 1544 1264 156 735 1634 903 1421 1337 3347 168 hr. 9540 1992 6541 3196 6580 2804 8962 2770 2350 5319 8594 999 25 days 7020 6698 6916 3339 7081 2137 Table 9 shows the percent of the total activity in Isoetes which had adsorbed to the surface of the plant. The relation between total activity and the log of the percent adsorbed activity is shown in Figure 13. high degree of appeared to be uncertain, activity which total activity but curvilinear. increases. This plot had a variability and instead of being linear The significance of this is again it is shown that the percent of the is adsorbed in fact does decrease when the Figure 13. 67 Measurements of adsorbed radioactivity on the surface of Isoetes sp. following treatment with labeled sodium arsenite. The log of the percent of total plant activity which was adsorbed is plotted against total plant activity. Plants were from both the complete ecosystem experiments and plant experiments. «...-.4 68 l I l l l 00. co.