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CHAPTER 13 LARGE-SCALE BIOLOGICAL EXPERIMENTS USING RADIOACTIVE TRACERSl MILNER B. SCHAEFER, Inter-American Tropical Tuna Commission, Scripps Institution of Oceanography, La Jolla, California ONE OF the major difficulties in evaluating the probable results of the introduction of radio- active materials into the sea is the lack of ade- quate knowledge respecting the effects of the organisms in the sea on the distribution and transport of such materials. Some information, which has been reviewed in earlier sections of this report, has been obtained on the uptake and excretion of elements by different kinds of marine organisms. This information is, however, not sufficiently extensive. The even more important problems of the quantitative interrelationships and movements of the popu- lations of organisms at the several trophic levels are among the least understood biological phe- nomena of the oceans. These, together with physical factors, will determine the fluxes of the radioactive materials. Measurements of the fluxes of materials through physical-biological systems, or ecosys- tems in the sea are of vast and fundamental importance not only for evaluating the probable distribution of radio-active products introduced into the sea, but also as a basis of evaluating the sea as a source of food and other biological products for the use of mankind. With the approaching full utilization of the land, in- creasing attention is being directed to the sea as a source of such products, but the basic bio- logical knowledge for realistic evaluation of the potential harvest of the sea is quite inadequate. The availability of rather large quantities of radioactive materials, as by-products of the de- velopment and utilization of nuclear energy, makes possible the study, in situ, of the biologi- cal and ecological processes in the sea by the use of tracer techniques. A start has been made, in connection with the introduction of radio- isotopes into the marine and fresh waters by 1 Contribution from the Scripps Institution of Oceanography, New Series, No. 903a. weapons tests and by the disposal of low-level wastes, but the opportunities for obtaining use- ful information by these means have not been fully exploited. Also it should be possible by introducing radioisotopes in a planned, con- trolled, and purposive fashion to obtain even better information than is possible through ob- servation of introductions ancillary to opera- tions having a different primary purpose. Observation in connection with weapons tests Observations in connection with weapons tests have the advantages that (1) very large quantities of radioisotopes are introduced into the sea, sometimes over a rather large area, so that radioactivity is sufficiently high to be de- tected in the sea waters and organisms over a considerable time after the event, and (2) the difficulty of being certain that the organisms have actually remained in the water containing the isotopes is minimized. On the other hand, the determination of exact amounts of isotopes introduced, of their spatial distribution, and of their physical state presents some difficulty. Biological studies, in connection with the various weapons tests in the Western Pacific ocean, have been primarily directed toward de- termining the concentration of gross activity in different organisms, the localization of such activity in different parts of the organism, and the rates of decline of activity with time. There has also been limited determination of the isotopes concerned. The most extensive data are from the lagoons of the atolls at and near the test sites. In the open sea, outside the lagoons, usually only limited collections of or- ganisms have been made, incidental to other operations. Following the test series of 1954, however, two rather extensive surveys were made of the distribution of activity in the sea, and in organ- 133

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134 Atomic Radiation and Oceanography and Fisheries isms at different trophic levels, over a large sea area at intervals of approximately 4 months and 13 months after the test. These observations have been directed pri- marily to possible human hazards through con- tamination of edible marine products. Only minor attention has been given to ecological processes, probably because of lack of facilities for the extensive, systematic collecting required. Soon after the underwater test in the Eastern Pacific in the spring of 1955, some collections were made that indicate which organisms in the food chain are the primary concentrators of certain radioisotopes, and that give some indi- cation of the time scale in passage to the next step of the food chain. Unfortunately, it was not possible to follow the passage of isotopes farther through the system. Following a weapons test a series of obser- vations and collections taken in a carefully planned pattern in space and time could pro- vide information on the time scale involved in the passage of material through the system of prey and predators, and on the efficiency of this transfer from one stage to another, two of the little understood basic problems in marine ecol- ogy. Data from experiments with radioactive tracers, together with more limited field data, indicate that the transfer efficiencies are differ- ent for different elements. In those situations, following weapons tests, where there is a fairly extensive body of water containing radioisotopes at some particular level, say at the surface, it should be possible by means of collections at various depths over a period of time to obtain worthwhile information on the vertical migrations of organisms, and also to determine how the feeding and excretion patterns of such organisms transport radioiso- topes from one level to another. These and similar studies would require the assignment of a vessel, with necessary equip- ment and a team of scientists, to the exclusive pursuit of such studies. Since results will de- pend on systematic, serial observations, the ves- sel must be available to take them when and where required, which precludes the commit- ment of the vessel to other activities. Although a sizable cost is involved, it is believed that the results to be obtained are of sufficient value to more than justify it. It should also be pointed out that effective planning of such studies requires considerable knowledge of the types of organisms to be en- countered in the test area, the sizes of their populations, and some knowledge of their mi- gration patterns, as well as data on the currents and other physical parameters to be considered. A pre-survey of the test areas by standard methods of biological investigation is, therefore, an important element in the adequate planning and execution of post-test investigations by means of the radioisotopes produced by the test. Observations in connection with waste disposal The disposal of wastes from the fission in- dustry by introduction into the marine en- vironment offers another means of studying the uptake of elements by aquatic organisms, their fluxes in the ecosystem, and their effects on the organisms concerned. Advantages over weapons tests are: (1) the wastes are usually introduced in such a manner that their amount, distribution and physical state can be readily determined, (2) disposal is usually continuous, even though not of constant magnitude, thus permitting systematic study over considerable periods of time. Disposal in the United States has consisted of relatively low-level wastes introduced into fresh waters by the Hanford works on the Columbia River, the Oak Ridge National Lab- oratory, and the Plant on the Savannah River. At the first named locality, field observations, supplemented by laboratory experiments, are being made on the uptake of radioisotopes by organisms, their fluxes through the food chain, and their distribution in the river as the result of the combined effects of physical and bio- logical processes. The phosphorous cycle has been investigated in particular detail. At the Oak Ridge Laboratory, observations were made over a period of years on the uptake of fission products by various organisms, the sites of deposition of radioisotopes in the organisms and the effects on some of their populations. Continuous disposal into marine waters is not practiced at present in this country. Reports by H. Seligman, H. J. Dunster, D. R. R. Fair and A. J. McLean at the 1955 Geneva Con- ference on Peaceful Uses of Atomic Energy describe introduction of low-level wastes into the Irish Sea, and briefly review studies of the uptake of various isotopes by different kinds of organisms.

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Chapter 13 135 Large-Scale Biological Experiments with Tracers With the exception of limited work at Han- ford and Oak Ridge, it appears in all these cases that primary attention has been concentrated on monitoring aspects, that is measurement of the quantity and distribution of radioisotopes to insure against hazards to human or other animal populations. The work of Richard Foster and others on the radiophosphorus cycle in the Columbia River, and the work of Louis A. Krumholz on seasonal variations in quantities of fission products in different groups of organ- isms, indicate however, that locations where wastes are being continuously introduced into aquatic environments offer a good opportunity to study the ecological processes of the aquatic populations through the tracers provided by the introduced isotopes. It may be expected with the development of the fission industry in the next few years, that there will be disposal of some low-level wastes into marine waters, which will provide opportunities to investigate the ecology of estuaries and inshore ocean waters by these means. These introductions also constitute large-scale experiments on both the direct and genetic effects of long-term exposure of marine organ- isms to atomic radiations. It is important that these effects be carefully investigated, because it is possible that the larger organisms in the sea, which are subjected to much lower rates of natural radiation than terrestrial forms (due to the shielding effects of water on cosmic rays, as well as to the low gamma-ray activity per unit volume of sea water compared with the rock and soil of the land), may show propor- tionally a greater genetic effect from a given amount of radiation. Planned experiments Much useful information may be obtained by well conceived biological observations in con- nection with weapons tests and routine disposal of industrial atomic wastes. Much more pre- cise information could be obtained, however, by planned experiments introducing measured quantities of known isotopes into the marine environment in a controlled manner. Further- more, it is evident that the fluxes of different elements through the ecosystem vary according to their abundance in the sea and their physio- logical roles in the organisms. Some of the most important elements biologically are not fission products, nor are they present in wastes in appreciable quantity. The outstanding ex- ample is carbon. The energy which supports most of the life in the sea, as on the land, is fixed as chemical energy of complex carbon compounds synthesized by plants. To study the flux of energy through the different trophic levels of the ecosystem it is necessary, therefore, to measure directly or indirectly the flux of carbon. One of the most promising possibili- ties, discussed further below, is the use of radio- carbon in tracer experiments on a scale larger than the present laboratory-type experiments. The need for large scale experiments under natural conditions arises because we require knowledge concerning the quantitative interre- lationships of the various populations of or- ganisms, and it is not possible to reproduce natural marine communities, especially the pe- lagic elements, in the laboratory. It is probably not possible yet to study some aspects of open- sea communities by radioactive tracers, either, but it may be possible to improve on present techniques by larger scale in situ experiments than have been attempted. Large scale experiments, employing either mixed fission products or single isotopes iso- lated from mixed fission products, appear feasi- ble (at least in selected locations in the open sea) to determine what organisms take up which elements and the quantitative aspects of how these elements are passed through the food chain. It may also be feasible to introduce sufficient quantities of radioisotopes in particu- lar situations to make possible a study of the transport of such elements by migrations of organisms. In general, however, in the open sea, it will be necessary to confine attention to those elements which are naturally present in seawater in very small concentrations, so that the organisms may be expected to take up a relatively large fraction of the isotope in ques- tion. In the case of elements such as carbon, only a small fraction of which is taken up by the organisms, experiments in unconfined vol- umes of open sea would appear to require larger quantities of the radioisotope than are feasible on a cost basis, and experiments there- fore will have to be limited, in the near future at least, to small enclosed arms of the sea or artificially bounded volumes of water in the open sea. In order to conduct experiments in the open sea it is necessary to (1) introduce the radioiso- topes into an area sufficiently large so that it can be located and followed, to insure the or-

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136 Atomic Radiation and Oceanography and Fisheries ganisms under study being in it over a known period of time, and (2) have a sufficiently high radioactivity that it may be followed from ship- board. If we use only fission products which organisms concentrate; then, since longer count- ing periods are feasible for samples of the organisms than are feasible for the equipment used to locate and follow the water mass, the radioactivity required to determine the position of the contaminated water mass is expected to be the limiting factor in the experiment. Revelle, Folsom, Goldberg and Isaacs (1955) have indicated that, in the slow-mixing levels of the sea below the thermocline, vertical mix- ing is almost negligible, so it may be expected that while the area in which the isotopes can be detected spreads over a radius of 4.1 km., vertically it will be limited to about 1 meter. In these circumstances, it has been calculated that 10 curies of gamma emitter may be detected until it has spread laterally to a radius of 4 km., or a mean concentration of about 2 x 10'7 curies per cubic meter. They do not specify the time involved, but it may be presumed to be of the order of one week to one month. For biological experiments, it would be necessary to make observations over a longer period of time, also we cannot commence significant biological ob- servations until the contaminated area is suffi- ciently large to ensure knowledge of which animals are or have been in the active water. For these reasons the time involved should perhaps be increased by a factor of 10. If the diffusion of the contaminated water, both ver- tically and horizontally follows the "random walk" law, the volume containing the activity will increase linearly with time, and, in conse- quence, about 100 curies of gamma activity will be required. Experiments in the upper mixed layer will require much larger quantities of fission prod- ucts. Mixing to the top of the thermodine is very rapid; according to the authors above cited the lower boundary of radioactive water moves down at about 10-1 cm/second. If we select an area, such as that off Central America where there is a fairly shallow sharp thermo- cline at a mean depth of about 20 meters, mix- ing down to the top of the thermocline would be complete in less than ten hours. Thereafter downward mixing should be negligible. Recent experiments suggest that the radius over which the water spreads laterally is increased as about the 0.8 power of time. In Bikini lagoon it has been found that the radius of the radioactive area increased to 4 kilometers in 3 days. If we ran an experiment for 90 days, which is probably the time necessary to follow the flux of radioelements through two or more trophic levels, we would, then, expect the radius to approximate r=4(30)'9 = 60 kilometers. The volume would then be (with a 20 meter thermocline) 7rx36x109x20 cubic meters or about 225 x 109 cubic meters To be still detectible at this dilution, using the above estimate of 2 x 10-T curies/cubic meter, an initial quantity of some 4 x 104 curies would be required. The logistics of handling large quantities of fission products will be difficult, but not perhaps impossible. Because of the smaller volume of water to be dealt with, it may be most desirable, at least initially, to conduct such experiments in a small enclosed arm of the sea. Such an environment is different in many respects from the open ocean, but much useful information about fluxes of radioelements through the several trophic levels could be obtained. It would not be diffi- cult to select a small bay, with a narrow, shal- low entrance, which could be cut off temporarily from the sea for this purpose. A body of, say, one square kilometer with an average depth of ten meters might be used, giving a volume of 107 cubic meters. Since the problem of locating the water mass is eliminated, and fairly large volumes of water can be filtered for organisms, rather small quantities of fission products, which would not be hazardous, could be employed. One curie would be ample, and the contamina- tion of the water itself would be within safe levels for human hazards. It was noted earlier that one of the important fundamental ecological problems is to measure the flux of carbon through different trophic levels. Since the fraction of the carbon taken up by plants is a very small part of the total in the sea water, experiments with radio-carbon in the open sea are not feasible. Experiments using samples in bottles have been conducted in situ in recent years, but these have two de- ficiencies: (1) the surface and other effects of the container modify the environment so that the resulting computations for photosynthesis probably are not those that would have occurred

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Chapter 13 137 Large-Scale Biological Experiments with Tracers naturally in the sea and (2) only the uptake of carbon at the phytoplankton level is meas- ured. It seems feasible to improve on the ex- periments in bottles by conducting experiments in small lagoons, or by employing larger partly- enclosed volumes in the open sea. From experience with such experiments in bottles, it can be shown that there is sufficient uptake of carbon by the phytoplankton, if grown in a concentration of 0.3 micro-curie per liter for one day, to measure it if a one liter sample is filtered and the radioactivity of the filtered plants determined in a counter of 20 per cent efficiency. By increasing either the counting time or the volume of water filtered, the initial concentration of C14 can be decreased correspondingly. For an experiment in a lagoon, we might use a body of water of, say, 500 meters long by 200 meters wide with an average depth of 10 meters, giving a volume of Ix100 cubic meters or 1 x 109 liters. By filtering 100 liters of water for phytoplankton, C14 at a concen- tration in the water of 3x10~* curies per liter would suffice, or 3 curies for the experiment. Since there is probably between a 50 per cent and 90 per cent loss at each step up the food chain, correspondingly larger volumes would have to be strained for the higher forms, but this is a simple problem by the use of standard nets, etc. To get improved measurements of the uptake of carbon by phytoplankton in the open sea, and the passage of carbon to the smaller grazing organisms, it is suggested that a moderately large rubber tank open at the surface be em- ployed to isolate a piece of the top of the sea, yet have a sufficiently small surface-to-volume ratio that the processes will more nearly ap- proach normal conditions than is obtained in bottle experiments. We might employ such an apparatus of 20 meters diameter by 10 meters deep, having a volume of -n 102 cubic meters, or Trx10' liters. By filtering 10 liter samples for phytoplankton, with 20 per cent efficient counting equipment, we would need to provide about 3 x10-5 curies per liter, or a total of about 1/10 curie of C". Some cost and logistic considerations For the two experiments with C14, discussed immediately above, the problems of handling the amounts of activity involved present no particular difficulty. Since C14 is a pure beta emitter, the shielding problem for even the experiment requiring 3 curies is a simple matter. The cost of the isotope, however is fairly high; at present about $30,000 per curie. This might be reduced somewhat if the present demand were to increase. The cost, notwithstanding, however, the information to be gained is well worth the outlay. In the case of an experiment using gamma emitters in the slow-mixing layer below the thermocline, where about 100 curies would be required, it is suggested that mixed fission products from wastes from processing of reactor fuel elements be used. A large quantity of such wastes will be available, probably at no charge. If one used HNO2 salted waste product from a natural uranium-plutonium reactor, after 100 days "cooling," the reactor waste will contain about 200 curies/gallon. Approximately half a gallon will be needed, requiring about 10" of lead shielding for transportation and han- dling. A cubical container will require 10.05 cubic feet of lead, weighing 7,175 pounds. This is feasible to handle by freight and on shipboard. For the kilocurie quantities required for an experiment in the upper mixed layer of the sea, the handling problem reaches a different order of magnitude. It becomes quite infeasible to handle waste liquids in the volume required. It may be possible, because of the much higher activity per unit volume to employ slugs of U225 from a reactor, which, after 30 per cent burning and 100 days "cooling" have about 2x105 curies per kilo of fairly long term gamma activity. Even then some 2/10 kilos of "used" U225 would be required. The prob- lems of transporting and handling this are somewhat difficult as are methods of dissolving and liberating the material at sea, but probably feasible. Further detailed consideration needs to be given to this problem. It may, of course, be that the use of an explosive reaction — a small nuclear detonation for oceanographic and biological experimental purposes — is the only logistically feasible method. REFERENCES REVELLE, R., T. R. FOLSOM, E. D. GOLDBERG, and J. D. ISAACS. 1955. Nuclear Science and Oceanography. United Nations Inter- national Conf. on Peaceful Uses of Atomic Energy, Geneva, Paper no. 277:22 pp.

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