The objective of the quarantine testing and biocharacterization portion of the Apollo medical program was to test appropriate representative lunar samples for the possible presence of agents that might be infectious or toxic for plants, man, and other animals. The goal of the laboratory was to provide safety clearance for lunar samples within a period of approximately 30 days. Lunar materials were analyzed in an isolated environment. These analyses were performed immediately after the lunar samples were unpacked in the Lunar Receiving Laboratory (LRL) at the Johnson Space Center. Small but representative samples of lunar material were used to assess whether they contained microorganisms, and to ensure that the lunar materials were nonhazardous to the selected test species.

The quarantine testing included a wide variety of biological species. Approximately 500 gm of lunar material were required for each investigation. Analyses of data from the Apollo 11, 12, and 14 missions indicated that no microbial life forms had been recovered from the lunar material. For subsequent missions, the containment aspects of the postflight quarantine were omitted and the biocharacterization or preliminary biomedical evaluation of lunar materials was initiated. The aims were to characterize the lunar material with respect to its ability to stimulate biological activity, and to measure possible microbial contamination of lunar samples.For the Apollo 15 mission, the number of biological tests was reduced to one-third of those performed on previous missions. Further reduction in the scope of the program occurred after the Apollo 15 mission. Except for the exposure of animal tissue culture cells to suspensions of lunar material, animal test systems were omitted from the Apollo 16 and 17 protocols.

All test protocols were extensively reviewed by the scientific community, the American Institute of Biological Sciences, and the Interagency Committee on Back-Contamination. The Interagency Committee was formed of representatives of various agencies of the Federal Government for the purpose of reviewing protocols to assure that the biosphere would not be contaminated with organisms from the moon. The aim was to use as many different kinds of organisms as possible. The organisms chosen were well known research tools, including mice, oysters, paramecia, and fishes.

The results of the first experiments in several completely new fields, namely lunar agriculture, lunar soil microbiology, and ecology of lunar soil on contact with terrestrial organisms, are presented in this chapter. It should be stated at the outset that the implication of the findings reported here are largely speculative because of limited experimentation. However, findings are consistent with the generally accepted hypothesis that the lunar surface is now, and has always been, sterile.

Botanical Investigations

The botanic quarantine studies at the Lunar Receiving Laboratory were designed to determine whether lunar material contained any agent capable of generating an epidemic disease in representative species of the plant kingdom. These tests were conducted under conditions which would ensure confinement of any infectious agents that might be found in the lunar materials or generated in the lunar-exposed plants (Walkinshaw et al., 1970). Class III biological glove boxes were used to achieve the required protective containment (Kemmerer et al., 1969).

A total of 35 plant species were exposed to lunar material returned during the Apollo 11 and 12 missions (table 1). Four test systems were employed. These included liquid or solid cultures of algal cells, germinating spores and seeds, actively growing seedlings, and tissue cultures on solid media.

Lunar samples used in Apollo 11, 12, and 14 studies were composites of representative rock fragments and surface fines; samples used in Apollo 15, 16, and 17 postflight studies were composites of surface fines. The samples were handled and analyzed as described by Johnson and co-workers (1972). Descriptions of the terrestrial controls may also be found in the work by Johnson and his associates.

Treatment of algal cultures with lunar material inhibited growth in dense cellular suspensions and stimulated growth in cultures grown on semisolid mineral media. Growth promotion was evident by marked increase in cell density in areas adjacent to lunar particles. Treatment of algal cells by exposure to lunar material suspended via gentle agitation resulted in cultures having higher respiration rates than untreated controls. Microscopic examination of treated cultures revealed no significant differences between lunar- and terrestrial-treated cells.

The fern, Onoclea sensibililis L., which was tested with each composite sample, appeared to be the most sensitive plant for demonstrating that lunar material can act as a source of nutrients for plants. Clumps of spores germinating on lunar material placed within a well cut into mineral agar showed a severalfold increase in mass. The resulting gametophytes were also greener than those treated with terrestrial basalts. Other lower plants, such as Lycopodium cernuum L. and Marchantia polymorpha L. (liverwort), exhibited similar stimulation. Measurements of chlorophyll a in the treated plants showed significantly higher concentrations of that pigment than of chlorophyll b or carotenoids.

Seeds germinated in the presence of lunar materials grew vigorously and absorbed significant quantities of aluminum, chromium, iron, titanium (Walkinshaw & Johnson, 1971), and a variety of elements including rare-earth elements. In addition, cabbage and brussels sprouts absorbed large amounts of manganese. Lettuce seedlings generally thrived in the presence of lunar material. Germ-free bean, citrus, corn, sorghum, soybean, tobacco, and tomato plants showed no deleterious effects when their leaves or roots were treated with 0.2 gm/specimen of lunar material (figure 1). Citrus, corn, and soybean plants appeared to grow consistently better if treated in the sand-water culture system originally described by Walkinshaw and co-workers (1970). Histological specimens taken from lunar-treated plants revealed no deleterious effects.

The twelve plant tissue culture system used in the biocharacterization program appeared to be the most useful for studying cell/lunar particle interactions (Walkinshaw et al., 1973). Lunar-treated tobacco cells accumulated approximately 30 percent more total chlorophyll a than did untreated ones (Weete & Walkinshaw, 1972). Relative and absolute concentrations of fatty acids and sterols were changed by lunar treatment (Weete, Walkinshaw & Laseter, 1972). Pine cells, on the other hand, exhibited a remarkable increase in accumulation of tannin but not of fatty acids or sterols. Both stationary and suspension cultures of tobacco tissue cultures treated with lunar material exhibited an increased maturation of chloroplasts and apparent secretary activity (Baur et al., 1973).

In summary, a number of beneficial effects were observed to be associated with the use of lunar soil cultivation, and none of these effects was found to be associated with an infectious process. The absence of microorganisms or any harmful substance suggests that lunar material could be used as a support medium for the growth of many plants. The tests conducted at the Johnson Space Center indicate that ferns, liverworts, and tobacco cultures utilize lunar material as a source of nutrients (Walkinshaw et al., 1970).

Virological Investigations

Virological studies of the lunar material obtained during the Apollo missions consisted primarily of analyses for replicating agents, principally those able to reproduce. The materials tested and the systems challenged are presented in table 2. The fluid obtained from centrifuging 50 percent weight per volume (W/V) suspensions of lunar material in sterile media was used to inoculate the test systems. Mammalian and avian cultures were re-inoculated ten and twenty days later. Fish cell cultures were re-inoculated in 15 days. Cell cultures in the final passage were tested for infection. All systems were tested to make sure they would react with known viruses. African green monkey kidney (GMK) cultures were challenged with enteric cytopathogenic human orphan virus type 11; mammalian and avian cultures were challenged with pancreatic necrosis virus. Embryonated eggs were inoculated by way of the yolk sac, the chorioallantoic membrane, and the amniotic and allantoic sacs. Extracts of lunar material were inoculated into the brain and the body cavity of mice (figure 2). Materials from tissue cultures, embryonated eggs, and suckling mice were tested for hemaglutinins using chicken, guinea pig, and human type O red blood cells. Viral passage materials were processed for light- and electron-microscope examinations. Standard mycoplasma isolation procedures were used. No evidence of replicating agents was found in any of the systems used.

Additional studies were performed on the Apollo 15 lunar material to measure changes in the ability to infect the host cells. The green monkey kidney cell cultures were exposed to extracts (20 percent W/V) of lunar material and were challenged with parainfluenza and rubella viruses. The ability of the cell cultures to support virus replication was not affected. To determine the effect on growth, metabolism, and colony morphology of Mycoplasma pneumoniae, the organism was grown in suspensions of lunar material (ten percent W/V), in mycoplasma broth medium, and in agar containing 0.75 percent lunar material. No significant differences were observed between terrestrial basalt used to simulate lunar material and lunar material suspensions. Colonies grown on agar containing lunar material were similar to those grown on agar medium alone or on agar containing simulated lunar material.

Another study was performed to determine the effect of lunar materials on the stability of poliomyelitis virus. Fifty-percent suspensions of lunar material from the Apollo 11, 12, 14, and 15 missions were inoculated with poliomyelitis virus and incubated at 277° K (~ 4° C). Virus-inoculated balanced salt solution and suspensions of simulated lunar material served as controls. Aliquots were removed for viral assay periodically. The number of virus particles in the suspensions of the lunar material was significantly lower than the number in the balanced salt solution. However, no significant differences were detected between simulated and lunar material suspensions.

Zoological Investigations

Following the Apollo 11, 12, 14, and 15 missions, 15 species of animals representing five phyla were exposed to untreated lunar material (table 3). These tests were complementary to the other protocols and were designed to detect any viable or replicating agents capable of infecting and multiplying in animals. The lunar material used for these tests came from the pooled biosamples (Long et al., 1972).

Because of the differences in maintenance techniques for the aquatic and terrestrial species, the methods of providing exposure to the lunar samples differed. The aquatic and protozoan species were exposed by adding lunar material to the medium in which the animals were living. For the Apollo 14 tests, oysters were exposed by introduction of lunar material into the shell cavity through a 0.32 cm (1/8 in.) hole drilled in the shell. Exposure of the insect species was accomplished by mixing the lunar samples with their food. The mice were exposed by inoculation into the body cavity (intraperitoneally) or the skin (subcutaneously). The guinea pigs used for evaluating pulmonary response to lunar material were exposed by inoculating this suspension into the respiratory tract (trachea). The quail were exposed by intraperitoneal inoculation.

Results of exposure of the various animal species were uniformly negative (Simmonds et al., 1972; and Benschoter et al., 1970). No viable or replicating agents, other than identifiable terrestrial microorganisms, were ever recovered or observed in the test animals. Exposure of the animals to the lunar material resulted in some minor and temporary inhibition or toxicity.

Following relaxation of the quarantine requirements after the Apollo 14 mission, lifespan studies were initiated with germ-free mice inoculated with lunar material. The response of these mice to both intraperitoneal and subcutaneous injections of aqueous suspensions of lunar material was evaluated on a long-term basis (Holland & Simmonds, 1973). Classical inflammatory reactions were noted in both intraperitoneal and subcutaneous inoculations, and the lunar material was observed to persist for the life of the animal (20 months). A low-grade inflammatory reaction and the absence of significant fibroplasia (fibrous tissue development) characterized the lesion. These observations suggest that the lunar material was relatively insoluble in tissue and that, although acting as a low-grade irritant, it has little tendency to evoke reactive fibrosis. The significance of such a chronic low-level stimulus and the various factors governing the retention, the elimination, and the turnover of lunar material in mammalian tissue have yet to be determined.

Bacteriological and Mycological Investigations

A variety of samples from all six lunar exploration missions was examined for the presence of biological forms or viable organisms (Taylor & Wooley, 1973). To evaluate lunar material for the presence of viable organisms, aliquots of each sample were inoculated into an array of culture media and incubated at several temperatures [277°, 297°, 308°, 328° K (~ 4° , 24°, 35° and 55° C)] in three gaseous environments (sterile nitrogen, 10 percent carbon dioxide in air, and air) (Taylor & Ferguson, 1970). No evidence of viable organisms was obtained from any of the analyses.

Following incubation of the lunar material in the culture media complexes, microbial growth dynamics studies were performed with known test species to evaluate the possible presence of toxic factors. Only extracts of culture media that had been in contact with a mixture of lunar material from both Apollo 11 core tubes proved to be toxic to all species tested (Taylor et al., 1971; and Taylor, Ellis et al., 1970). Attempts to reproduce this toxic effect with individual Apollo 11 core samples obtained at other parts of the core tube and analyzed under somewhat different conditions were unsuccessful. The mechanism causing this microbial death has not been determined. In all, 48 different lunar samples, collected to a depth of 297 cm (117 in.) from six different landing sites, were examined.

Summary

The likelihood that life existed on the moon was considered quite remote by most members of the scientific community and by NASA officials, but the extensive testing described above was conducted to ensure the safety of all life on Earth. The plants and animals which were exposed to lunar material were carefully observed for prolonged periods to determine if any mutation or changes in growing characteristics and behavior occurred. The quarantine testing was terminated after the Apollo 14 flight when it became apparent that previously returned lunar material contained no potentially harmful agents. Further biological experimentation with the lunar material was conducted to determine its chemical, physical, and nutritional qualities.

References

Anderson, D.H.: Numbering System for Moon Samples. Science, vol. 167, no. 3918, Jan. 1970, p. 781.

Baur, P.S.; Walkinshaw, C.H.; Halliwell, R.S.; and Scholes, V.E.: Morphology of Nicotiana tabacum Cells Grown in Contact with Lunar Material. Can. J. Botany, vol. 51, Jan. 1973, pp. 151-156.

Benschoter, C.A.; Allison, T.C.; Boyd, J.F.; Brooks, M.A.; Campbell, J.W.; Groves, R.O.; Heimpel, A.M.; Mills, H.E.; Ray, S.M.; Warren, J.W.; Wolf, K.E.; Wood, E.M.; Wrenn, R.T.; and Zein-Eldin, A.: Apollo 11: Exposure of Lower Animals to Lunar material. Science, vol. 169, no. 3943, July 1970, pp. 470-472.

Carrier, W.D., III; Johnson, S.W.; Werner, R.A.; and Schmidt, R.: Disturbance in Samples Recovered with the Apollo Core Tubes. Proceedings of the Second Lunar Science Conference, vol. 3, 1971, pp. 1959-1972.

Carrier, W.D., III; Johnson, S.W.; Werner, R.A.; and Schmidt, R.: Core Sample Depth Relationships: Apollo 14 and 15. Proceedings of the Third Lunar Science Conference, vol. 3, 1972, pp. 3213-3221.

Holland, J.,M.; and Simmonds, R.C.: The Mammalian Response to Lunar Particulates. Space Life Sciences, vol. 4, no. 1, Jan. 1973, pp. 97-109.

Johnson, P.H.; Walkinshaw, C.H.; Martin, J.R.; Nance, W.B.; and Bennett, A.D.: Elemental Analysis of Apollo 15 Surface Fines Used in Biological Studies at the Lunar Receiving Laboratory. Bioscience, vol. 22, no. 2, Feb. 1972, pp. 96-99.

Kemmerer, W.W., Jr.; Mason, J.A., and Wooley, B.C.: Physical, Chemical, and Biological Activities at the Lunar Receiving Laboratory. Bioscience, vol. 19, no. 8, Aug. 1969, pp. 712-715.

Long, R.; Ellis, W.; and Schneider, H.: Biopreparation of Apollo 14 Lunar Material. Tex. J. Science, vol. 24, 1972, pp. 262-263.

Simmonds, R.C.; Holland, J.M.; Young, E.L.; and Boyd, J.F.: Animal Maintenance for Biomedical Evaluation of Lunar Material. J. Amer. Vet. Med. Assn., vol. 161, no. 6, Sept. 1972, pp. 720-727.

Taylor, G.R.; Ellis, W.L.; Arredondo, M.; and Mayhew, B.: Growth Response of Pseudomonas aeroginosa (ATCC 15442) to the Presence of Lunar Material. Bacteriol. Proceedings, 1970, p. 42.

Taylor, G.R.; Ellis, W.; Johnson, P.H.; Kropp, K.; and Groves, T.: Microbial Assay of Lunar Samples. Proceedings of the Second Lunar Conference, vol. 2, 1971, pp. 1939-1949.

Taylor, G.R.; Ferguson, J.K.; and Truby, C.P.: Methods Used to Monitor the Microbial Load of Returned Lunar Material. Appl. Microbiol., vol. 20, no. 2, Aug. 1970, pp. 271-272.

Taylor, G.R.; and Wooley, B.C.: Evaluations of Lunar Samples for the Presence of Viable Organisms. Proceedings of the Fourth Lunar Science Conference, Geochim Cosmochim Acta, vol. 2, 1973, pp. 2267-2274.

Walkinshaw, C.H.; Sweet, H.C.; Venketeswaran, S.; and Home, W.H.: Results of Apollo 11 and 12 Quarantine Studies on Plants. Bioscience, vol. 20, no. 24, Dec. 1970, pp. 1297-1302.

Walkinshaw, C.H.; and Johnson, P.H.: Analysis of Vegetable Seedlings Grown in Contact with Apollo 14 Lunar Surface Fines. J. Hort. Science, vol. 6, 1971, pp. 532-535.

Walkinshaw, C.H.; Venketeswaran, S.; Baur, P.S.; Hall, R.H.; Croley, T.E.; Weete, J.D.; Scholes, V.E.; and Halliwell, R.H.: Effect of Lunar Materials on Plant Tissue Culture. Space Life Sciences, vol. 4, no. 1, Jan. 1973, pp. 78-89.

Walkinshaw, C.H.; Wooley, B.C.; and Bozarth, G.A.: Technology Advancements in the Growth of Germ-free Plants at the Manned Spacecraft Center. Germ-free Research: Biological Effect of Gnotobiotic Environments. Academic Press, 1973.

Weete, J.D.; and Walkinshaw, C.H.: Apollo 12 Lunar Material: Effects on Plant Pigments. Can. J. Botany, vol. 50, Jan. 1972, pp. 101-104.

Weete, J.D.; Walkinshaw, C.H.; and Laseter, J.L.: Apollo 12 Lunar Material: Effects on Lipid Levels of Tobacco Tissue Cultures. Science, vol. 175, no. 4021, Feb. 1972, pp. 623-624.

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