6
Sample Containment and Biohazard Evaluation

As outlined in previous reports, there is a broad consensus in the scientific community that samples collected on Mars and returned to Earth must be contained and treated as potentially biologically hazardous until they are declared safe by applying recommended protocols, including rigorous physical and chemical characterization, life detection analyses, and biohazard testing. It is important to emphasize that the high level of containment recommended for the handling and testing of martian samples is based on a deliberate decision to take a conservative approach to planetary protection and not because of the anticipated nature of pristine martian materials or organisms. If anything, however, the discoveries over the past decade about Mars and about terrestrial extremophiles have supported an enhanced potential for liquid water habitats and, perhaps, microbial life on Mars, thus making it appropriate to continue this conservative approach.

A factor potentially complicating the policies and protocols relating to sample containment and biohazard evaluation is the de facto internationalization of a Mars sample return mission. All serious planning for Mars sample return is founded on the premise that the scope, complexity, and cost of such a mission are beyond the likely resources of any one space agency. Although no major issues have arisen to date, the international character of Mars sample return raises the possibility that differences in national policies and legal frameworks might complicate issues relating to sample quarantine policies and biohazard certification.

SAMPLE CONTAINMENT

Samples collected on Mars and returned to Earth pose a unique set of containment requirements. They must be contained in ways that will protect Earth from any potential martian hazards and will protect the samples from terrestrial contamination in order to maintain their scientific integrity.1 The NRC’s 1997 report Mars Sample Return: Issues and Recommendations divides sample containment into two distinct and separate components.2 First, there is a Mars sample return spacecraft subsystem—the sample canister—that houses the samples during their journey from Mars to Earth. Second, there is a containment laboratory—a sample-receiving facility (SRF)—to which the still-sealed sample canister is taken following recovery on Earth. Once inside an SRF, the sample canister is opened so that the samples can undergo initial characterization and biohazard testing.

Once martian materials have been placed inside the sample canister it must be sealed to preserve the scientific integrity of its contents and to ensure that its potentially hazardous contents do not contaminate the terrestrial environment. A critical issue relating to the design of the sample canister concerns the means by which those



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6 Sample Containment and Biohazard Evaluation As outlined in previous reports, there is a broad consensus in the scientific community that samples collected on Mars and returned to Earth must be contained and treated as potentially biologically hazardous until they are declared safe by applying recommended protocols, including rigorous physical and chemical characterization, life detection analyses, and biohazard testing. It is important to emphasize that the high level of containment recom- mended for the handling and testing of martian samples is based on a deliberate decision to take a conservative approach to planetary protection and not because of the anticipated nature of pristine martian materials or organ- isms. If anything, however, the discoveries over the past decade about Mars and about terrestrial extremophiles have supported an enhanced potential for liquid water habitats and, perhaps, microbial life on Mars, thus making it appropriate to continue this conservative approach. A factor potentially complicating the policies and protocols relating to sample containment and biohazard evaluation is the de facto internationalization of a Mars sample return mission. All serious planning for Mars sample return is founded on the premise that the scope, complexity, and cost of such a mission are beyond the likely resources of any one space agency. Although no major issues have arisen to date, the international charac- ter of Mars sample return raises the possibility that differences in national policies and legal frameworks might complicate issues relating to sample quarantine policies and biohazard certification. SAMPLE CONTAINMENT Samples collected on Mars and returned to Earth pose a unique set of containment requirements. They must be contained in ways that will protect Earth from any potential martian hazards and will protect the samples from terrestrial contamination in order to maintain their scientific integrity.1 The NRC’s 1997 report Mars Sample Return: Issues and Recommendations divides sample containment into two distinct and separate components. 2 First, there is a Mars sample return spacecraft subsystem—the sample canister—that houses the samples during their journey from Mars to Earth. Second, there is a containment laboratory—a sample-receiving facility (SRF)—to which the still-sealed sample canister is taken following recovery on Earth. Once inside an SRF, the sample canister is opened so that the samples can undergo initial characterization and biohazard testing. Once martian materials have been placed inside the sample canister it must be sealed to preserve the scientific integrity of its contents and to ensure that its potentially hazardous contents do not contaminate the terrestrial environment. A critical issue relating to the design of the sample canister concerns the means by which those 0

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 SAMPLE CONTAINMENT AND BIOHAZARD EVALUATION charged with implementing a Mars sample return mission can demonstrate the integrity of the canister’s seal. A discussion of the technical means by which this containment is achieved is beyond the scope of this report. What is of concern to the committee is that an overly prescriptive requirement may be counterproductive (see the section “Conclusions and Recommendations” below) With respect to an SRF, there is a long, well-documented history of successful biocontainment of pathogenic and infectious organisms in biosafety laboratories and under biosafety conditions. However, such facilities typi- cally use negative-pressure gradients to prevent harmful materials from getting out.3 That is, they are designed to leak in and as a result are usually “dirty” both chemically and biologically. Similarly, there is a record of success- ful containment for maintaining the integrity of extraterrestrial and planetary materials. However, these facilities typically use positive-pressure gradients to prevent contaminants from getting in.4 That is, they are designed to leak out and as a result are useless for containing hazardous materials. Currently, no single facility exists that combines containment for both biological and planetary materials as required for an SRF for martian materials. Nevertheless, the integration of functions for handling and testing returned martian materials in a single facility seems both appropriate and feasible, as outlined in the existing draft protocol 5 (see next section). Issues relating to an SRF are discussed in Chapter 7. BIOHAZARDS TESTING Following publication of the 1997 NRC report Mars Sample Return: Issues and Recommendations,6 two additional reports, the NRC’s The Quarantine and Certification of Martian Samples7 and NASA’s A Draft Test Protocol for Detecting Possible Biohazards in Martian Samples Returned to Earth, 8 were published in 2002. The latter report provided a set of protocol release criteria indicating when and under what conditions martian samples could be released from containment in an SRF. Conditions for release include: 1. Self-replicating extraterrestrial life forms or indications of life-related molecules are not present, and 2. No harmful effects to terrestrial organisms and environments are evident in biohazard tests. Sample materials that have not met these criteria must remain in containment, or first be subjected to a steril- ization process involving heat, radiation, or a combination of these agents, to ensure that they are safe for further analyses outside containment. Samples that fail to meet these requirements must remain in containment, and all pristine samples released from containment (regardless of the outcome of biohazards testing) must be properly sterilized. Although detailed protocol planning is not within the scope of its charge, the committee agrees with these general recommendations. The committee was, however, verbally encouraged by NASA’s planetary protection officer to raise topics that should be considered in greater depth by subsequent planning groups. The committee’s digressions in this area focus on general approaches to sample characterization in the context of future protocol testing and decisions about the intentional release of pristine martian samples from containment. Although the reports mentioned above identified general requirements for sample handling, containment, testing, and certification for release of martian samples, important details are still being discussed, and conflicting recommendations remain that are in need of resolution. To further refine and expand the Mars sample return draft test protocol, NASA and its international partners plan to undertake a follow-up series of workshops, building on all earlier studies and the initial steps taken at the Mars Sample Return Sample Receiving Facility Workshop, held at the European Space Technology Center in the Netherlands in February 2009. While a detailed discussion of Mars sample return protocols is beyond the scope of the present report, in the course of its discussions the committee identified several topics for more detailed consideration by future protocol planning groups. The Challenge of Biohazards Testing The testing of potentially biohazardous (especially disease-causing) biological materials has become a some- what routine procedure in biocontainment laboratories worldwide. Once sufficient information is available for characterizing and understanding the biological materials in question, informed decisions can be made to down-

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 ASSESSMENT OF PLANETARY PROTECTION REQUIREMENTS FOR MARS SAMPLE RETURN MISSIONS grade or even eliminate containment requirements, if deemed appropriate. However, it is worth pointing out that high-level biocontainment laboratories (e.g., those classified as biosafety level (BSL) 3 and 4) do not routinely test “unknowns.” They test materials that have some indication of being involved in causing disease or symptoms. This is a significant distinction. BSL-3 and BSL-4 facilities do not routinely test soils, rocks, water, and other materials for pathogens unless they are implicated in some form or fashion in a disease. In addition, the testing of pathogens becomes increasingly limited to viral agents of a somewhat known molecular basis once it has reached the highest containment level. The committee was charged with reviewing earlier criteria for sample release and providing suggestions that can guide protocol-planning activities in the years ahead. It is assumed that subsequent protocol planning will define a rigorous battery of tests that will combine physical and chemical characterization, life detection, and biohazards testing. It is also assumed that the criteria for the release of samples from containment, or for bypass- ing certain tests, will eventually be specified in detail as well. However, at this juncture a key question concerns whether specific, safe, and scientifically justified approaches can be identified that would allow selected martian materials to be released prior to the completion of rigorous biohazards testing. The NRC’s 1997 Mars report indicated that pristine samples of martian materials can be released from con- tainment, prior to completion of the entire battery of tests, provided they are first sterilized. However, a detailed comparison of criteria for release in all subsequent reports (see Table 7.1) reveals conflicting statements with regard to both the approach to be used and the specific criteria for sample release. For example, NASA’s draft sample- handling protocol allows the release of filtered, contained gases from an SRF, with no further requirement for processing or sterilization. However, the NRC’s 2002 report The Quarantine and Certification of Martian Samples says nothing about gases, instead focusing entirely on solid samples. 9 It indicates that if solid samples contain no detectable carbon compounds and no evidence of past or present biological activity, smaller subsamples (aliquots) of untreated samples may be released from SRF containment. In contrast, NASA’s draft protocol takes a more conservative approach in stating that, regardless of the outcome of physical and chemical tests or life detection studies, all solid samples must undergo complete biohazard testing before release—unless first sterilized. Such discrepancies will need careful resolution in future protocol-planning efforts. The present committee further supports the NRC’s 1997 recommendation that, once samples have been deliv- ered to an SRF, NASA maintain a conservative approach in implementing the protocol and in making decisions about the intentional release of pristine martian samples from containment. Presumably, in the time leading up to sample return, there will be continuing refinements in methods for sample handling and in the development of new analytical instrumentation for characterizing and testing samples. There will thus be an ongoing need for periodic review of these advances and their potential impacts on NASA’s draft protocol and associated criteria for releasing samples from containment. Future protocol planning groups, scientific advisory committees, the Centers for Disease Control and Prevention and other biosafety and biosecurity agencies, and international partners associated with Mars sample return and an SRF will play important roles in these reviews. The Problem of Sample Heterogeneity NASA’s draft protocol indicates that martian samples that are shown to contain organic molecules (e.g., amino acids, proteins, and so on) must undergo extensive testing before being released from containment. It is presumed that biohazard testing will require the selection of aliquots taken from larger samples. This raises concerns about how best to obtain representative samples that will yield reliable results during testing. This problem is exacer- bated by the fact that the biosignatures of rocks, soils, and ices typically show highly heterogeneous distributions within samples at microscopic scales of observation. This heterogeneity arises from spatial variations in mineral (and elemental) compositions; the sizes, shapes, and sorting of grains; and variations in the sizes, shapes, and distributions of void spaces (e.g., intergranular porosity, dissolution voids, vesicles, and interconnected networks of microfractures; see Figures 2.3, 4.2, 6.1, and 6.2, for example). Because the living microorganisms contained in rocks and mineral samples typically reside within voids, or in association with particular mineral phases, sampling

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 SAMPLE CONTAINMENT AND BIOHAZARD EVALUATION 5.1 from word.eps FIGURE 6.1 Thin-section photomicrograph showing mineral heterogeneity. Hematite (orange) and opaline silica (white) are found within host halite crystal from Lake Polaris,bitmap Australia. Such minerals may provide nutrients for organisms, Western image thereby controlling their microscale distribution in samples. SOURCE: Photograph courtesy of Kathleen Benison, Central Michigan University. for biohazard testing must carefully consider heterogeneities in the spatial distribution of such features and the impact on the distribution of microorganisms and their by-products within samples. Especially important as a source of spatial heterogeneity in the microscale distribution of habitable environ- ments that could support living organisms are fluid inclusionssmall quantities (~microliters) of aqueous and/or gaseous fluids that are captured during mineral formation. Such inclusions are especially common in aqueous sediments formed by primary chemical precipitation (e.g., evaporites, hot spring sinters, sedimentary cements, fracture fills, ices, and so on—lithologies that have been given a high priority for Mars sample return) or during later aqueous alteration of a mineral. Fluid inclusions found in such deposits frequently contain viable microor- ganisms or their by-products (see Figures 4.1, 6.1, and 6.2). Thus, it is very important that samples subjected to biohazard testing be acquired from the spatial locations in samples that have the highest potential for containing life or its biosignatures. The following steps in sample characterization are intended to define a general approach to creating a proper context for subsample selection for biohazards testing: 1. Under primary (SRF) containment, sample exteriors are tested for organic compounds and any released (e.g., possibly biogenic) gases;

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 ASSESSMENT OF PLANETARY PROTECTION REQUIREMENTS FOR MARS SAMPLE RETURN MISSIONS FIGURE 6.2 Examples of heterogeneity in martian and Mars-analog samples from extremely acid saline lakes in Western Aus- 5.2 from word.eps tralia, as well as living microorganisms and microfossils trapped within such rocks: (a) martian sedimentary rock composed of hematite concretions in a matrix of sand-sized volcanic grains and reworked sulfate grains (see also Figure 2.2); (b) sedimentary bitmap image rock near acid saline lake in Western Australia composed of red hematite concretions in a matrix of reworked grains of gypsum and quartz coated with hematite; (c) gypsum crystal with included bands of hematite mud, with c1 showing Fe-oxide/Fe-sili- cate-coated pollen and wood extracted from gypsum and c2 showing microbes within a fluid inclusion in gypsum; (d) interior of hematite concretion, including reworked gypsum (large white grain near top) and halite (white cubic grain), with d1 show- ing a dark mass consisting of fossil bacteria/archaea and sulfate crystals incorporated in a crystal of halite that formed within the interior of a hematite concretion, and d2 shows a microorganism within a fluid inclusion in a halite crystal. SOURCE: (a) Photograph courtesy of NASA/Jet Propulsion Laboratory and Cornell University; (b, c, c2, d, d1, d2) photographs courtesy of Kathleen Benison, Central Michigan University; (c1) photograph courtesy of Stacy Story, Purdue University. 2. Samples are transported in secondary containers to outside laboratory facilities where nondestructive methods of analysis (e.g., scanning x-ray imaging,10 tomographic imaging of samples by micro-CT scanning,11 laser confocal imaging,12 and synchrotron x-ray photoelectron emission microscopy (X-PEEM)13) could be used to map the microscale spatial distributions of minerals and biological elements in samples; and 3. Contained samples are returned to the SRF and microsamples are acquired for biohazards testing from microscopic areas within samples that have been targeted based on compositional and microtextural mapping, obtained in step 2, above. Given the small sample volumes that are likely to be returned from Mars, it will be important to identify early on the most appropriate approaches for the nondestructive characterization of samples and to support the develop- ment of appropriate laboratory facilities. However, implementation of many of the advanced characterization and

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 SAMPLE CONTAINMENT AND BIOHAZARD EVALUATION analytical techniques may not be feasible within the constraints of an SRF. Therefore, a crucial step in defining approaches for sample characterization outside an SRF will be the design of secondary containers for transporting samples to outside laboratory facilities where they can be analyzed (under containment) by a potentially wider range of instruments. Early consideration of the best analytical capabilities will be needed to ensure that flexible, safe designs for secondary containers will be available to ensure proper interfaces with a limited variety of non- SRF laboratory instruments. Optimal designs are likely to require end-to-end testing of a variety of Mars analog materials to refine instrument designs, define necessary instrument sensitivities, and determine minimum sample volumes needed for obtaining reliable results with different types of materials. CONCLuSIONS AND RECOMMENDATIONS Planetary protection considerations require that martian materials be securely contained within a sample canister for their journey from Mars, through collection and retrieval on Earth, and subsequent transport to, and confinement in, a sample-receiving facility. With respect to the journey from Mars to an SRF, the NRC’s 1997 report Mars Sample Return: Issues and Recommendations concluded that the integrity of the seal of the sample canister should be verified and monitored during all phases of a Mars sample return mission. The committee found this requirement to be overly prescriptive. Establishing the technical means to verify containment en route has proven to be a stumbling block in past mission studies. Elaborate steps must be taken to guarantee containment at every stage of the mission. Resources might be better spent in simply improving containment (e.g., by using multiple seals) rather than designing elaborate means of monitoring. The first priority should be to ensure that the samples remain reliably contained until opened in an SRF. The means by which this result is achieved will best be determined by those designing the implementation of a Mars sample return mission. Recommendation: The canister(s) containing material returned from Mars should remain sealed during all mis- sion phases (launch, cruise, re-entry, and landing) through transport to a sample-receiving facility where it (they) can be opened under strict containment. No facility currently exists that combines all of the characteristics required for an SRF. However, the com- mittee found that there is a long, well-documented history of both the successful biocontainment of pathogenic and infectious organisms and the maintenance of the scientific integrity of extraterrestrial and planetary materi- als. Thus, the committee concluded that the requirement for handling and testing returned martian materials in a single facility combining biocontainment and integrity-maintaining functions is both appropriate and technically feasible, albeit challenging. In addition, the use of specialty instruments at other facilities may be considered as long as appropriate containment is designed for interfacility transport of pristine materials. Changes to the requirements for sample containment or criteria for sample release were an issue of concern in the NRC’s 1997 Mars report, which recommended that: “The planetary protection measures adopted for the first Mars sample-return mission should not be relaxed for subsequent missions without thorough scientific review and concurrence by an appropriate independent body” (p. 4). The present committee concurs with the spirit of that recommendation with three provisos: first, that the protocols for sample containment, handling, testing, and release be articulated in advance of Mars sample return; second, that the protocols be reviewed regularly to update them to reflect the newest standards; and third, that international partners be involved in the articulation and review of the protocols. Recommendation: Detailed protocols for sample containment, handling, and testing, including criteria for release from a sample-receiving facility (SRF), should be clearly articulated in advance of Mars sample return. The pro- tocols should be reviewed periodically as part of the ongoing SRF oversight process that will incorporate new laboratory findings and advances in analytical methods and containment technologies. International partners involved with the implementation of a Mars sample return mission should be a party to all necessary consulta- tions, deliberations, and reviews.

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 ASSESSMENT OF PLANETARY PROTECTION REQUIREMENTS FOR MARS SAMPLE RETURN MISSIONS The NRC’s 1997 Mars report recommended that: “Controlled distribution of unsterilized materials returned from Mars should occur only if rigorous analyses determine that the materials do not contain a biological hazard. If any portion of the sample is removed from containment prior to completion of these analyses, it should first be sterilized” (p. 4). Subsequent NRC and NASA reports have made related, but in some cases conflicting, statements. Irrespective of these conflicts, there are critical issues concerning the selection of the aliquots for biohazard testing and the nature of the tests to be employed. The discussion of advances in geobiology and biosignature detection in Chapter 4 raises the possibility that viable organisms might be preserved over a prolonged span of time within certain geological deposits. The distri- bution of extant and fossil organisms and biomolecules in rocks, soils, and ices is heterogeneous at microscopic scales of observation, and this heterogeneity requires careful consideration because it complicates the selection of representative aliquots for biohazards testing. Recommendation: Future protocol guidelines should carefully consider the problems of sample heterogeneity in developing strategies for life detection analyses and biohazards testing in order to avoid sampling errors and false negatives. The limited amount of material likely to be returned from Mars demands that nondestructive means of analysis be employed to the maximum extent possible in sample characterization and biohazards testing. Recommendation: The best nondestructive methods must be identified for mapping the microscale spatial distribu- tions of minerals, microstructures, and biologically important elements within returned martian samples. It is highly likely that many of the appropriate nondestructive methods will require the use of techniques whose implementation is not feasible within the confines of an SRF. Thus, a critical issue concerns the design of secondary containers for transporting samples to outside laboratory facilities where they can be analyzed (under containment) using advanced analytic techniques. Recommendation: Sample characterization in laboratories outside the primary sample-receiving facility will require the design of secondary containers for safely transporting samples and interfacing with a potentially wider variety of instruments. NOTES 1 . National Research Council, The Quarantine and Certification of Martian Samples, National Academy Press, Wash- ington, D.C., 2002, pp. 52-54. 2 . National Research Council, Mars Sample Return: Issues and Recommendations, National Academy Press, Washing- ton, D.C., 1997. 3 . The atmospheric pressure inside the contained volume is slightly lower than ambient. 4 . The atmospheric pressure inside the contained volume is slightly higher than ambient. 5 . J.D. Rummel, M.S. Race, D.L. DeVincenzi, P.J. Schad, P.D. Stabekis, M. Viso, and S.E. Acevedo, eds., A Draft Test Protocol for Detecting Possible Biohazards in Martian Samples Returned to Earth, NASA/CP-20-02-211842, NASA Ames Research Center, Moffett Field, Calif., 2002. 6 . National Research Council, Mars Sample Return: Issues and Recommendations, National Academy Press, Washing- ton, D.C., 1997. 7 . National Research Council, The Quarantine and Certification of Martian Samples, National Academy Press, Wash- ington, D.C., 2002. 8 . J.D. Rummel, M.S. Race, D.L. DeVincenzi, P.J. Schad, P.D. Stabekis, M. Viso, and S.E. Acevedo, eds., A Draft Test Protocol for Detecting Possible Biohazards in Martian Samples Returned to Earth, NASA/CP-20-02-211842, NASA Ames Research Center, Moffett Field, Calif., 2002. 9 . National Research Council, The Quarantine and Certification of Martian Samples, National Academy Press, Wash- ington, D.C. 2002.

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 SAMPLE CONTAINMENT AND BIOHAZARD EVALUATION 10 . L. Lemelle, M. Salome, M. Fialin, A. Simionovici, and P. Gillet, “In Situ Identification and X-ray Imaging of Micro- organisms Distribution on the Tatahouine Meteorite,” Spectrochimica Acta Part B-Atomic Spectroscopy 59:1703-1710, 2004. 11 . A.A. Kilfeather and J.J.M. van der Meer, “Pore Size, Shape and Connectivity in Tills and Their Relationship to Deformation Processes,” Quaternary Science Reviews 27:250-266, 2008. 12 . K. Kamburoglu, S.F. Barenboim, T. Ariturk, and I. Kaffe, “Quantitative Measurements Obtained by Micro-computed Tomography and Confocal Laser Scanning Microscopy,” Dentomaxillofacial Radiology 37:385-391, 2008. 13 . G. De Stasio, B. Gilbert, B.H. Frazer, K.H. Nealson, P.G. Conrad, V. Livi, M. Labrenz, and J.F. Banfield, “The Mul- tidisciplinarity of Spectromicroscopy: From Geomicrobiology to Archaeology,” Journal of Electron Spectroscopy and Related Phenomena 114-116:997-1003, 2001.