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Preventing the Forward Contamination of Mars 2 Policies and Practices in Planetary Protection Planetary protection policy stems from international treaty, but it is implemented through practices within the national agencies that deploy probes to Mars. Planetary protection policies and practices are not static; they change considerably over time to reflect advances in scientific knowledge, new technologies, and the practical experiences of space agencies that launch planetary missions. This chapter discusses the history of planetary protection policies as promulgated by COSPAR and implemented in the U.S. space program. It explains the central concepts that link planetary protection policies, mission requirements, and standard practices, and it shows how COSPAR policies are translated into detailed processes of spacecraft preparation intended to prevent the forward contamination of Mars. This chapter also highlights how advances in science and technology have contributed to modifications in all three areas: the policies themselves; the specific requirements imposed by agencies to implement these policies; and the accepted practices or methods used by mission personnel to meet particular requirements. PLANETARY PROTECTION POLICY Historical Review During the early years of the space program, forward contamination controls for missions to Mars were guided by a probabilistic approach as the framework for developing quarantine standards.1 COSPAR Resolution No. 26, which COSPAR issued at its 1964 Scientific Assembly in Florence, Italy, accepted “a sterilization level such that the probability of a single viable organism aboard any spacecraft intended for planetary landing or atmospheric penetration would be less than 1 × 10–4, and a probability limit for accidental planetary impact by unsterilized flyby or orbiting spacecraft of 3 × 10–5 or less” (COSPAR, 1964, p. 26). In 1969 COSPAR agreed to “a probability of no more than 1 × 10–3 that a planet will be contaminated during the period of biological exploration” and adopted a formal probabilistic approach focused on the probability of contamination, Pc (COSPAR, 1969, p. 15). 1 For the early development of the probabilistic approach to planetary protection, see Sagan and Coleman (1965, 1966). Early thinking about planetary protection may be traced in two reports by the COSPAR Committee on Contamination by Extraterrestrial Exploration (CETEX, 1958, 1959), and in studies by Lederberg and Cowie (1958), Lederberg (1960), Brown et al. (1962), Atwood (1966), Hall (1968, 1971), and DeVincenzi et al. (1998), and references therein.
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Preventing the Forward Contamination of Mars The 1964 COSPAR policy also incorporated the notion that “all practical steps should be taken to ensure that Mars be not biologically contaminated until such time as this search [for extraterrestrial life] can have been satisfactorily carried out …” (COSPAR, 1964, p. 26). To that end, COSPAR called for limiting the probability of an accidental planetary impact by unsterilized spacecraft as well as reducing spacecraft microbial bioburdens to specified levels. NASA policy incorporated the requirement that “outbound automated spacecraft and planetary exploration programs shall not, within [established] probabilities … transport terrestrial life to planets until it is determined that life does or does not exist on the planet and the character of existing life is explored” (NASA, 1967, p. 1). A finite period of time into the future by which the search for extraterrestrial life on Mars would be completed became known as the “period of biological exploration” (COSPAR, 1969). During the 1960s and 1970s, the period of biological exploration was described in two different ways: (1) it was estimated as the time span it would take either to send a certain number of spacecraft to, or conduct a certain number of experiments on, Mars (Sagan and Coleman, 1965, 1966) and (2) it was translated into an absolute number of years, for example, the 20-year period from 1968 to 1988 (COSPAR, 1969).2 According to Hall (1968), studies at that time indicated that accidental impacts of spacecraft on the martian surface and premature entry of orbiting vehicles into the martian atmosphere represented principal forward contamination concerns. Concerns about non-nominal (accidental) impact and the requirement for orbital spacecraft to achieve on-orbit lifetimes of at least 50 years are still reflected in planetary protection policies today, although time spans are rolling time limits now that are reset for each mission.3 Probability of Contamination and Probability of Growth Historically, the approach used in establishing planetary protection requirements for spacecraft sent to Mars was to require that the probability of contamination (Pc ) with terrestrial microorganisms—that is, the probability that Earth microorganisms introduced to Mars would then reproduce in situ on Mars—be below some threshold. One approach was to require that Pc multiplied by the total number of missions expected to be sent to Mars during the period of biological exploration would remain small compared with 1, that is, that the probability of contamination summed over all missions would remain small. Thus, Pc was set to 10–3 for all spacefaring nations, with different nations then being allotted fractions of this probability (COSPAR, 1969). This approach depends on efforts to estimate Pg, the probability that Earth microorganisms will grow in the martian environment. The probability of contamination can be written as (2.1) where N0 is the total number of organisms present on the spacecraft prior to bioburden reduction steps, R is the bioburden reduction factor achieved by any pre-launch sterilization procedures, PS is the probability of surviving exposure to radiation, vacuum, temperature fluctuations, and so on during spaceflight and entry onto the planet’s surface, PI is the probability of impact on the surface (of interest for flybys or orbiters intended to avoid the surface, or for landers that risk non-nominal impacts with the surface), and PR is the probability of release of microbes into the environment.4 As written, Equation 2.1 assumes that Pc is small compared to unity.5 Therefore, 2 For example, “60 landers and 30 flyby and orbiter missions and a total of 1200 biological experiments on Mars” (Sagan and Coleman, 1965). Also, “the period of unmanned martian exploration shall be assumed not to extend beyond the year 2000, followed by manned exploration” and “the years 1966 to 2000 shall be considered the period for unmanned exploration … a total of 64 interplanetary flights toward Mars are expected” (Light et al., 1967). 3 That is, 20- and 50-year time periods of relevance to planetary protection requirements set for each mission upon mission launch; see NPR 8020.12C (NASA, 2005a), p. 63. Available at <planetaryprotection.nasa.gov/pp/index.htm>. 4 The particular form of Equation 2.1 varies from study to study, depending on which factors are collected into a single variable or broken out for individual assessment (e.g., PS, the probability of surviving spaceflight, is sometimes written as a product of PVT, the probability of surviving exposure to space vacuum and temperature, and PUV, the probability of surviving exposure to ultraviolet light, during the voyage). For alternate conventions in writing Equation 2.1 see, for example, Klein (1991) and NRC (1978, 1992). The formulation given here is perhaps closest to that used by Stabekis, as described in P.D. Stabekis, Lessons learned from Viking, presentation to the Committee on Preventing the Forward Contamination of Mars, February 27, 2004. 5 For an early exact formulation, see Sagan and Coleman (1965, 1966).
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Preventing the Forward Contamination of Mars once Pc is fixed by policy, a given estimate of Pg then places a limit that the intervening product of factors must meet for that Pc to be achieved. In particular, estimates of Pg drive the requirements for bioburden reduction R. Current NASA practices for estimating bioburden (be it N0 or N0R, the bioburden present after bioburden reduction measures) on spacecraft are by proxy (as measured by colony-forming units after heat shock at 80°C followed by incubation for 72 h; see Appendix C). This procedure has served as a cornerstone for estimating microbial burden on spacecraft and will continue to do so in the near term. However, recent advances in microbial ecology reveal two significant limitations to these spore-based estimates of numbers of bacteria on the spacecraft. As detailed in Chapter 5, molecular diversity surveys demonstrate that cultivation techniques fail to recover as many as 99 percent of the microbes in a microbial population (Pace, 1997). By logical extension, the technologies on which current NASA estimates of bioburden are based will not detect the majority of heat-resistant organisms on the spacecraft. More important, spore-based estimates tell little about the cellular physiology or genomic diversity of organisms on the spacecraft surface or within enclosed components, both of which directly influence Pg and provide valuable new information on possible strategies for reducing bioburden (see Chapter 6). The heat treatment protocol means that those colony-forming units that are found are likely due to spore-forming organisms capable of surviving heat shock. However, this spore proxy can record only what grows within a few days under a given set of laboratory conditions; it does not consider what might grow under different environmental conditions or over protracted periods. That is, it does not survey many organisms that may occur in the clean-room environment but about which little is known. The use of proxies in estimating spacecraft contamination therefore brings with it inherent risks. Reasonably robust models could estimate levels of bioburden reduction during flight (PS) by considering the presence of radiation-tolerant and heat-tolerant microorganisms, if these data were available. Estimates of PI may be derived from actual crash data, and engineering models may assign values to PR. In contrast, there is little scientific basis for estimates of Pg, and estimates of Pg have in fact varied by as much as 10 orders of magnitude during the period from 1964 to 1978 (Klein, 1991) (see Appendix D). The Viking program, consisting of two orbiters and two landers that were launched in the mid-1970s, provided for the first time significant in situ scientific data on the martian environment. Following analysis of the Viking results, NASA asked the National Research Council (NRC) to evaluate Pg comprehensively, based on available knowledge of planetary conditions and the limits of life known at the time. The NRC report Recommendations on Quarantine Policy for Mars, Jupiter, Saturn, Uranus, Neptune, and Titan (1978) addressed planetary protection policy for exploratory missions to solar system locations that had been launched or planned for launch between 1974 and 1994. In that report Pc was stipulated as less than 1 × 10–3 for each planet, and Pg values were set separately for three different regions on Mars based on a “comparison between the known physical and chemical limits to terrestrial growth and the known and inferred conditions [on Mars]” (NRC, 1978, p. 4). The Pg values for Mars were set at <10–10, 10–8, and 10–7 for above- and below-surface subpolar areas and the polar caps, respectively. Although the report stipulated quantitative values for Pg, the values were arrived at subjectively and became a matter for debate.6 In fact, the 1992 NRC report noted, “It is clear that considerable uncertainty has been engendered by the probabilistic approach to planetary protection. This concern has been restated over the years by virtually every group that has analyzed the problem, and indeed by NASA” (NRC, 1992, p. 44). The debate over the probabilistic approach to planetary protection continued in the years following the 1978 report and set the stage for a subsequent significant overhaul of planetary protection policy.7 6 As noted in NRC 1992: “Although the  committee expressed a reluctance in recommending a particular value for Pg, they argued that while the Pg for Mars is exceedingly low, the probability is not zero” (p. 44). The 1978 report had stated: “And yet a numerical value for Pg is required in order to determine what procedures are needed to reduce the microbial burden on future spacecraft to Mars to levels that fulfill current COSPAR quarantine policy. Reluctantly, then, we recommend for these purposes, and these purposes alone, that NASA adopt a value of Pg less than 10–10 for the subpolar region …” (italics in original). 7 No Mars missions were ever flown whose forward contamination polices were based on the recommendations of the 1978 NRC report.
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Preventing the Forward Contamination of Mars Mission Categories After release of the 1978 NRC report, NASA undertook studies aimed at simplifying planetary protection procedures for upcoming missions and exploring ways to minimize the use of mathematical models and quantitative analyses. In 1982, NASA submitted a report to COSPAR suggesting that the previous quantitative policy be replaced by an entirely new approach (DeVincenzi et al., 1983). In 1984, COSPAR formally accepted the proposed approach and adopted a new policy centered on target-mission categorizations (COSPAR, 1984), an approach that has served since as the framework for planetary protection policy. This approach has also accommodated revisions to policy based on new scientific information about Mars obtained since 1984. Table 2.1 summarizes current COSPAR mission categories. Although the probabilistic quantitative approach to determining Pc was eliminated in 1984 as the central concept in COSPAR policy, current Category IV and V missions still require a contamination analysis plan that focuses on many of the same variables that were the focus of earlier planetary protection policies (e.g., probability of impact, orbital lifetimes, microbial densities and bioburden, time-temperature sterilization requirements, and so on).8 These are summarized in the section below titled “Implementation Requirements.” NASA adopted the revised COSPAR policy for application to all solar system exploration missions beginning with the Galileo mission in 1989. In 1990, in anticipation of upcoming robotic missions to Mars, NASA requested that the NRC revisit the matter of the potential forward contamination of Mars and provide recommendations that could become the basis for updating the requirements for forward contamination controls for Mars landers. The NRC report Biological Contamination of Mars: Issues and Recommendations (NRC, 1992) endorsed the use of categories rather than probability values as a “significant step forward in the process of simplifying and implementing planetary protection procedures” (NRC, 1992, p. 45), noting that “it is difficult, if not impossible” to estimate the potential for biological contamination of Mars (NRC, 1992, p. 48). The report also asserted that since “the Pg value for terrestrial organisms on Mars is so small as to be of no consequence … the need for severe reduction of spacecraft bioload solely to prevent the spread of replicating terrestrial organisms on Mars is no longer paramount” (NRC, 1992, p. 46). However, the report emphasized that the reduction of bioburden on all lander missions to Mars must continue to be addressed out of concern that life-detection measurements on future missions could be jeopardized through contamination of Mars by previous missions. The report recommended a revised approach for planetary protection controls whose stringency was based on whether or not a mission carried instruments for in situ life-detection experiments. Those recommendations for planetary protection controls made explicit reference to the levels of cleanliness used in the Viking missions of the mid-1970s. Thus, biological contamination levels for spacecraft without life-detection experiments would be “subject to at least Viking-level pre-sterilization procedures—such as clean-room assembly and cleaning of all components—for reduction of bioburden, but such spacecraft need not be sterilized” (NRC, 1992, p. 47). In contrast, missions “carrying instrumentation for in situ investigation of extant martian life [were] subject to at least Viking-level sterilization procedures.”9 (Requirements for meeting Viking pre-sterilization and post-sterilization bioburden reduction levels are summarized in Table 2.2.) This approach freed planetary protection requirements from any explicit reliance on Pg, although it should be remembered that the policy requirement that Pc lie below a certain value, coupled with a choice (e.g., Viking levels) for R makes an implicit assumption, described by Equation 2.1, about the value of Pg that is valid for Mars. COSPAR approved the recommendations proposed in the 1992 NRC report and refined the planetary protection policy for Mars missions to allow for different requirements on missions with and without life-detection 8 In current contamination analysis plans, some numerical values of important parameters and specifications are assigned by the NASA planetary protection officer at initiation of the project rather than calculated as they were long ago. For example, based on the current policy for robotic extraterrestrial missions, the period of biological exploration is interpreted as 50 years and is reflected in the probability-of-impact values and assigned orbital lifetimes currently used. Specifically, “Orbit characteristics shall be such that PI max for a mission shall be met until 20 years from launch of the mission. Between 20 and 50 years from launch, the spacecraft shall remain in orbit with an assurance of 0.95” (NPR 8020.12C; NASA, 2005a). Documents such as NPR 8020.12C are available at <planetaryprotection.nasa.gov/pp/index.htm>. 9 Viking post-sterilization bioloads begin with N = 3 × 105 spores allowed (Viking pre-sterilization level) and apply a sterilization process to reduce the total N by 4 orders of magnitude—equivalent to 30 surface spores. See Table 2.2.
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Preventing the Forward Contamination of Mars TABLE 2.1 COSPAR Planetary Protection Policy Categories for Solar System Bodies and Types of Missions (and Specific Requirements for Mars) Category I II III IV Mission type Any but Earth return (flyby, orbiter, lander) Any but Earth return (flyby, orbiter, lander) No direct contact (flyby, some orbiters) Direct contact (lander, probe, some orbiters) Target body Venus; Moon; undifferentiated, metamorphosed asteroids; others TBD Comets, carbonaceous chrondite asteroids, Jupiter, Saturn, Uranus, Neptune, Pluto/Charon, Kuiper Belt objects, others TBD Mars, Europa, others TBD Mars, Europa, others TBD Degree of concern None Record of planned impact probability and contamination control measures Limit on impact probability Passive bioload control Limit on probability of non-nominal impact Limit on bioload (active control) Representative range of requirements None Documentation only: planetary protection plan, pre-launch report, post-launch report, post-encounter report, end-of-mission report Documentation (Category II) plus contamination control, organics inventory (as necessary); implementing procedures such as trajectory biasing, clean room, bioload reduction (as necessary) Mars orbiters will not be required to meet orbital lifetime requirementsb if they achieve bioburden levels equivalent to the Viking lander pre-sterilization total bioburden. Documentation (Category II) plus Pc analysis plan, microbial reduction plan, microbial assay plan, organics inventory; implementing procedures such as trajectory biasing, clean room, bioload reduction, partial sterilization of contacting hardware (as necessary), bioshield monitoring of bioload via bioassay aA special region is defined as a region within which terrestrial organisms are likely to propagate, or a region that is interpreted to have a high potential for the existence of extant martian life forms. Given current understanding, this definition applies to regions where liquid water is present or may occur. Specific examples include but are not limited to (1) subsurface access in an area and to a depth where the presence of liquid water is probable, (2) penetrations into the polar caps, and (3) areas of hydrothermal activity.
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Preventing the Forward Contamination of Mars IVa IVb IVc V Earth return Mars Mars Mars Mars, Europa, others TBD If restricted Earth return: no impact on Earth or the Moon, returned hardware sterile, containment of any sample Outbound Lander systems not carrying instruments for the investigations of extant martian life are restricted to a biological burden no greater than Viking lander pre-sterilization levels. For lander systems designed to investigate extant martian life, all of the requirements of Category IVa apply, along with the following requirement: The entire landed system must be sterilized at least to Viking post-sterilization biological burden levels, or to levels of biological burden reduction driven by the nature and sensitivity of the particular life-detection experiments, whichever are more stringent, or the subsystems that are involved in the acquisition, delivery, and analysis of samples used for life detection must be sterilized to these levels, and a method of preventing recontamination of the sterilized subsystems and the contamination of the material to be analyzed is in place. For missions that investigate martian special regions,a even if they do not include life-detection experiments, all of the requirements of Category IVa apply, along with the following requirement: Case 1: If the landing site is within the special region, the entire landed system shall be sterilized at least to the Viking post-sterilization biological burden levels. Case 2: If the special region is accessed through horizontal or vertical mobility, either the entire landed system shall be sterilized to the Viking post-sterilization biological burden levels, or the subsystems that directly contact the special region shall be sterilized to these levels, and a method of preventing their recontamination before accessing the special region shall be provided. Same category as target body/outbound mission Inbound If restricted Earth return: Documentation (Category II) plus P c analysis plan, microbial reduction plan, microbial assay plan, trajectory biasing, sterile or contained returned hardware, continual monitoring of project activities, project advanced studies/research If unrestricted Earth return: none b Defined as 20 years after launch at greater than or equal to 99 percent probability, and 50 years after launch at greater than or equal to 95 percent probability. SOURCE: Reprinted from COSPAR (2003), copyright 2003, with permission from Elsevier.
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Preventing the Forward Contamination of Mars TABLE 2.2 Current Bioburden Requirements for Mars Landers (Viking Pre- and Post-Sterilization Levels for Surface and Embedded Bioburden) IVa: Viking IVb: Viking Pre-Sterilization Pre-Sterilization with Impacting Hardware Post-Sterilization Surface spore density 300 spores/m2 300 spores/m2 No explicit requirement Total surface spores 3 × 105 3 × 105 30a Total spores (including surface, mated, and embedded) __b 5 × 105 __b NOTE: Embedded or encapsulated bioburden refers to bioburden buried inside nonmetallic spacecraft material. aNo surface spore assays required; number of spores established by the application of a certified bioburden reduction method (dry heat). bNo numerical requirements on embedded bioburden except for spacecraft with impacting hardware (e.g., heat shields). Embedded bioburden is assumed to remain embedded under nominal operations. SOURCES: Perry Stabekis, The Windermere Group, and Jack Barengoltz, Jet Propulsion Laboratory, personal communication, January 2005; NPR 8020.12C (NASA, 2005a). instruments (Categories IVa and IVb) (DeVincenzi et al., 1994). In 2002, due to concerns about ensuring adequate control of forward contamination during exploration, COSPAR again modified the planetary protection policy by adding a new category, Category IVc, for missions that investigate martian “special regions,” even if those missions do not include life-detection experiments. A special region was defined as “a region within which terrestrial organisms are likely to propagate, or a region which is interpreted to have a high potential for the existence of extant Martian life forms” (COSPAR, 2003, p. 71).10 The changes made at that time in COSPAR policy stipulated additional bioburden reduction and sterilization requirements for landers or components that would come into contact with special regions or have a high probability of involving other than nominal conditions (see Table 2.1). Current planetary protection practices for U.S. missions to Mars incorporate all the revisions to COSPAR policy through 2002.11 NASA continues to work actively with COSPAR and the international community in considering whether and how COSPAR policies and associated implementing regulations should be revised to reflect rapidly changing understanding of both Mars and microbial life. IMPLEMENTATION REQUIREMENTS Historically, the various NRC recommendations on modifications to planetary protection policies (see Appendix B) have been adopted for use in revisions to COSPAR’s policies. In practical terms, they have been translated into forward contamination controls for NASA missions in the form of procedural requirements for actions under the control of mission designers, spacecraft and equipment builders, and planetary protection technicians. The current overall planetary protection policy is specified in NASA Policy Directive (NPD) 8020.7F (NASA, 1999), which applies to both robotic and human missions.12 NASA Procedural Requirements (NPR) document 8020.12C 10 The text added, “Given current understanding, this is to apply to regions where liquid water is present or may occur. Specific examples include, but are not limited to: Subsurface access in an area and to a depth where the presence of liquid water is probable; Penetrations into the polar caps; [and] Areas of hydrothermal activity.” 11 The formal incorporation of Category IVc (missions to special regions) into NASA policy received final administrative approval in NASA NPR 8020.12C in 2005. 12 NPD 8020.7F provides details on NASA’s policy on “Biological Contamination Control for Outbound and Inbound Planetary Spacecraft.” Although current implementing regulations apply only to robotic spacecraft, the NPD specifically applies to human spaceflight as well: NASA “will ensure that applicable standards and procedures established under this policy, and detailed in subordinate implementing documents, are incorporated into human missions.”
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Preventing the Forward Contamination of Mars (NASA, 2005a) and NPR 5340.1 (NASA, 1980)13 together describe the various elements in NASA’s planetary protection implementation requirements, as well as the standard methods used to implement those controls. Planetary Protection Plan Currently, missions to Mars fall into one of two COSPAR categories—Category III for orbiters and flybys, and Category IV for “direct contact” missions using, for example, landers, penetrators, and airplanes. Both Category III and IV missions require the development and approval of a planetary protection plan that provides information on all aspects of the mission, from pre-launch preparations through end-of-mission reports. NPR 8020.12C (NASA, 2005a) stipulates that for a flight project to demonstrate compliance with NASA planetary protection requirements, the mission team must develop a planetary protection plan and obtain approval of the plan from the NASA planetary protection officer. Based on the category assigned by NASA to a particular mission, different implementation guidelines and specific requirements outlined in the NPR apply to trajectory biasing, clean-room assembly, microbial reduction and assaying, organics inventory and archiving, and recontamination control. In addition, the NPR establishes requirements for documentation and schedules for reviews, and it provides assigned quantitative values for specifications on a wide variety of parameters.14 Specific information required in mission plans includes analysis of the probability of impact, estimates of microbial bioburden, a contamination analysis plan, microbiological assay plans, a microbial reduction plan if contemplated, and reporting plans and schedules. In addition, Category IV landers and probes must collect and archive an organics inventory, and document measures for avoidance of recontamination (e.g., bioshield monitoring) before launch. The requirements and plans that can have the most significant implications for the design, development, assembly, and cost of the mission are typically those related to reducing and assessing the bioburden on the spacecraft. The requirements for missions to Mars fall into the following important areas: (1) reduction of biological contamination from various sources on the spacecraft hardware; (2) consideration of non-nominal impact avoidance; (3) use of required assay methods for verifying bioburden reduction on the spacecraft and maintenance of clean-room conditions; (4) documentation to show that a spacecraft subjected to cleaning and bioburden reduction has not been recontaminated up until launch time; (5) development and maintenance of inventories of bioburden and organic constituents of the spacecraft and its components; and (6) planning and scheduling of required documentation extending from pre- and post-launch plans through end of mission reports. Four of those six areas—microbial reduction and bioburden control, use of standard assay methods, development and maintenance of archived inventories, and impact avoidance—have particularly important implications for the current implementation of forward contamination controls for missions to Mars. Table 2.3 provides descriptions of all six areas. Bioburden Reduction Bioburden or microbial reduction15 is of great importance because it significantly affects the densities of microbial contaminants on a spacecraft and/or its component parts before launch, thereby reducing the potential for forward contamination of a planet. Bioburden reduction may be accomplished in a variety of ways—performed on either the entire spacecraft or its component parts and pieces. Depending on the COSPAR category assigned to 13 NASA’s NPR 8020.12C (NASA, 2005a) provides the detailed planetary protection provisions for robotic extraterrestrial missions. NPR 5340.1 (NASA, 1980) is currently being revised and provides standard procedures for the microbial examination of space hardware and associated clean-room assembly and pre-launch environments. NPR 5340.1C (NASA, 2005b) is an informal reissue of NHB 5340.1B. A revised NPR 5340.1D is pending formal approval at the time of this writing. 14 Various pertinent parameters and specifications are used to address how contamination controls will be implemented on a particular mission. Appendix B of NPR 8020.12C (NASA, 2005a) provides quantitative values and acceptable ranges for specifications related to clean-room requirements, probability of accidental impacts, material-related microbial densities, microbial burden assays, sterilization time-temperature specifications, and planet-specific requirements (e.g., orbital lifetimes). 15 The term “microbial reduction,” which has the same meaning as bioburden reduction, is used in this chapter to be consistent with the terminology used in the NASA requirements documents discussed throughout the chapter. Other sections of this report use the term “bioburden reduction.”
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Preventing the Forward Contamination of Mars TABLE 2.3 Elements of Planetary Protection Forward Contamination Controls Planetary Protection Elements Requirements, Parameters/ Methodsa Standard Methodsb Comments Bioburden Control 1. Facility: clean-room requirements Xc X Contamination control, microbial culture assays, and monitoring must be demonstratebly effective; facility certification; current assay methods based on culture growth for 72 h under varied conditions 2. Hardware decontamination methods 2a. Dry-heat/sterilization procedure (preferred method) Xc Time-temperature conditions and D-valuesd specified for bioburden of exposed, mated, and encapsulated materials 2b. Alternative methods for hardware decontamination Xe Must demonstrate effectiveness in reducing bioburden; approval by the planetary protection officer; no standard certification process exists for new methods Non-Nominal Impact Avoidance X Total probability of any accidental impact by hardware other than probe or lander modules must not exceed 10–4 Assay Methods 1. Standard assay methods to determine hardware microbial burden X Methods based on culturing and colony growth for 72 h under varied specified conditions 2. Alternative procedures for assaying Xe May be proposed, but no standard certification process exists for new methods Protection from Recontamination Assaying and monitoring Use of microbial barriers Macro-organism control Contingency planning X Specifications for shrouds, filters, seals, and so on, are provided in the requirements document. Assay methods same as standard assay methods (1) above Bulk Organics Inventory a. Parts and materials lists b. Samples of organic compounds c. Location of landing and impact points d. Condition of landed spacecraft (to track spread of organics) X Archiving planetary-protection-related information: Flight program office must provide for collection and storage of information for at least 20 years from the launch of the spacecraft. No requirement currently exists for the collection and storage of microbial assay information Documentation Required Planetary protection plan/requirements compliance Office Schedules Pre-launch planetary protection report Post-launch planetary protection report End-of-mission report X Administrative requirements of Planetary Protection aStipulated in NPR 8020.12C (NASA, 2005a). bDescribed in NPR 5430.1C (NASA, 2005b). c Appendix A of NPR 8020.12C. dA D-value in the context of spacecraft sterilization is the time required at a specific temperature to cause a 1 log decrease in the spore population. eAllowed, but not specified.
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Preventing the Forward Contamination of Mars a mission, the mission team is tasked with ensuring that spacecraft bioburden does not exceed the levels specified for that category during the entire process encompassing assembly, test, and launch operations (ATLO). As outlined in NASA requirements (NPR 8020.12C; NASA, 2005a), microbial reduction for an entire planetary spacecraft, including entry probes and landing capsules, may be accomplished by any qualified process approved by the planetary protection officer. At present, the dry-heat cycle is considered the preferred method for conditioning spacecraft to a sterile or near-sterile condition, and it is the only NASA-certified method for bioburden reduction.16 A dry-heat cycle involves heating the entire spacecraft or particular components to specified elevated temperatures and atmospheric conditions for defined lengths of time. Mission managers may use alternative methods of microbial reduction provided that no undue reduction of hardware reliability occurs and the method is supported by rigorous data demonstrating biological effectiveness and reproducibility. Because many components of modern spacecraft are particularly sensitive to heat (e.g., electronics, some nonmetallic portions), it is often desirable to reduce the severity of the subsequent heat sterilization by precleaning individual parts and components. In those cases, certain elements of hardware are subjected to separate microbial reduction processes prior to their assembly into the entire spacecraft. The microbial reduction methods used on such hardware components and pieces may differ from those used for the entire spacecraft. In all cases, however, the methods themselves must be preapproved to ensure biological qualification, quality assurance of the method, and demonstration of nondegradation of parts to ensure that they are able to withstand any subsequent microbial reduction performed on the entire spacecraft. Although alternative methods may be approved for use on hardware components, there is no standard process for certifying new methods as qualified bioburden reduction procedures. Chapter 6 details several alternatives to heat sterilization, including chemical and radiative techniques. According to NASA requirements (NPR 8020.12C; NASA, 2005a), calculations of the microbial reduction process must be supported by detailed information on the methods used throughout the cleaning processes (so-called parameter values and specifications), as well as data from reproducible laboratory tests or technical references supplied by the project team. Important parameters in the calculations include factors such as clean-room conditions, encapsulated and surface microbial density, sterilization cycle times, temperature constraints, process atmospheric conditions, D-values17 for various bioburden categories (exposed, mated, and encapsulated), minimum number of spores per assay, and survival of hardy organisms after nominal cycles. Appendix E outlines the approaches taken to bioburden reduction on several past lander missions to Mars. Standard Microbiological Assays for Assessing Microbial Contamination Levels NASA’s NPR 5340.1 (NASA, 1980) defines the accepted standard procedures for assessing the amount of microbial burden on space hardware and in associated ATLO facility environments. Specifically, it describes “uniform microbiological assay procedures that shall be used to: (a) Assess the degree of microbiological contamination of intramural environments where spacecraft hardware is assembled, tested and launched [and] (b) Assess the level of microbiological contamination of spacecraft hardware in relation to the known or anticipated environments of the target planets” (NASA, 1980, p. 3). In addition, NPR 5340.1 (NASA, 1980) provides protocols for preparation and sterilization of both equipment and culture media used in association with the assay methods. Appendix C of this report summarizes methods for 16 Other acceptable and approved methods (e.g., wiping with alcohol) are also used to reduce microbial bioburden on surfaces; however, these other processes are not certified and must be followed by standard assay methods to document the cleanliness levels achieved. A “certified process” is one for which a “credit for microbial reduction” is granted by virtue of using the certified process alone, with no further assaying required. Dry-heat microbial reduction is the only certified microbial reduction process; its efficacy has been fully documented. Because the dry-heat process is associated with strict specifications and assigned parameter values, microbial reduction is assumed and no further assaying is required. 17 The D-value for bioburden on exposed surfaces is defined as the “time required to destroy 90% of the microbial spore population on surfaces subjected to sterilizing dry heat at a temperature of 125° C at an absolute humidity corresponding to a relative humidity of less than 25% referenced to the standard conditions of 0°C and 760 torr pressure” (NPR 8020.12C; NASA, 2005a).
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Preventing the Forward Contamination of Mars each type of assay for the various categories of material and locations assayed (e.g., clean-room air and surfaces, spacecraft hardware by size categories, and so on). In all cases, suitable controls are also specified for the assay processes and procedures. Assays play two important roles before the launch of a spacecraft: (1) they allow monitoring by flight program or project personnel for microbial contamination throughout the ATLO process (if surface contamination is detected, particular parts are removed and recleaned and then replaced on the spacecraft); and (2) they certify the required cleanliness levels of environments, facilities, and flight hardware for the planetary protection officer at particular phases of the ATLO process. Assays are often performed by a technical organization designated by the planetry protection officer. In accounting for the bioburden of pre-launch spacecraft, attention is focused mainly on two types of microbial burden—surface and encapsulated (embedded)18—based on the location on or in the spacecraft. Surface bioburdens are actually measured through standard microbial assays, whereas values for encapsulated microbial densities inside nonmetallic materials or portions of the spacecraft are assigned as parameter values (NPR 8020.12C; NASA, 2005a). Because embedded or encapsulated bioburden cannot be accessed or measured, there is currently no quantitative indication of its phylogenetic diversity or the densities of the various types of microbes. In addition, the assigned parameter values are based on pre-Viking data,19 and so they may not reflect values appropriate for current (4 decades later) spacecraft materials or manufacturing processes. Archived Information and Organics Inventories The primary assay data for each mission are compiled and submitted to the planetary protection officer as part of the planetary protection plan for each mission, providing verification of a mission’s having met planetary protection requirements. For all current and past missions, such data are obtained from standard swab and culture assays, rather than from more sensitive modern molecular methods. This means that although there are archival data on levels of contaminants from previous outbound missions, they provide almost no information on the phylogenetic diversity or actual density of the individual types of microbial bioburden. Using contemporary molecular techniques, microbiologists have successfully cultivated between 0.1 and 1 percent of the different kinds of organisms from complex microbial communities. Historically, microbial cultures have not been required to be retained routinely. Today, despite the fact that planetary protection controls are being implemented on all missions whose categorizations require it, the controls do not require a comprehensive understanding of the actual bioburden levels or of the phylogenies of the microbes on the spacecraft. Thus, a comprehensive understanding of potential forward contamination of Mars remains elusive. Finally, in attending to the forward contamination control requirements for Category IV landers, the flight program office must provide for collection and storage of the bulk (>1 kg) organic constituents of all launched hardware that is intended to directly contact Mars or might accidentally do so. Parts and materials lists, actual samples, and information on landing and impact points must be maintained for at least 20 years after spacecraft launch. Given recent scientific findings about Mars and the fact that the period of biological exploration continues to be extended, this requirement may be insufficient for archiving scientifically important information. Impact Avoidance Although the meaning of the “period of biological exploration” has changed over time, the term remains in current policies and implementation requirements. In particular, Category III Mars spacecraft (flybys and orbiters) have to be able to guarantee orbital lifetimes of 20 and 50 years with probabilities of impacting Mars over those time periods of less than 1 and 5 percent, respectively. If the orbiter cannot meet those requirements, it has to meet 18 Mated bioburden is also mentioned in planetary protection requirements, but quantitative standards are stipulated only for surface and encapsulated and embedded bioburden. 19 See NPR 8020.12C (NASA, 2005a), pp. 39-41. Standards for average encapsulated microbial density are referenced to the Planetary Quarantine Advisory Panel Review, September 28, 1971, Denver, Colo.
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Preventing the Forward Contamination of Mars the Category III bioburden requirement; that is, it must have its bioburden reduced to below 5 × 105 total aerobic spores (COSPAR, 2003; also see Table 2.1). But if the orbiter can meet these requirements—that is, if the probability is high that its impact will occur subsequent to the implicit period of biological exploration—then no special bioburden reductions are required beyond standard assembly in a class-100,000 clean room, and so forth (see Table 2.2). MAINTAINING CLEANLINESS DURING LAUNCH Preventing contamination of a Mars-bound spacecraft does not end with spacecraft assembly and bioburden reduction. Payloads, once cleaned, are kept in clean-room facilities that can maintain the level of cleanliness required for the spacecraft. If higher sterilization levels are achieved than can be ensured in the clean-room facilities, the payloads are put in a biobarrier to prevent recontamination. The biobarriers are not removed until the flight system is launched into space. For U.S. Mars-bound spacecraft, the payload fairing20 (otherwise referred to as the launch vehicle shroud) is installed in final-assembly clean rooms at Kennedy Space Center. Once the payload is within the payload fairing, it is under continuous conditioned air or gaseous nitrogen purge, which means that a positive pressure gradient within the fairing is maintained with the nitrogen exiting the fairing through high-efficiency particulate air (HEPA) filters. This state is maintained during the mating of the spacecraft to the launch vehicle and the movement of the mated payload out to the pad. If engineers require access through the payload fairing to reach the spacecraft while on the pad, the positive pressure is maintained, with the nitrogen flowing continuously out the access door. The nitrogen source is disconnected at the moment of launch, but outflow continues as the launch vehicle ascends, owing to the decrease in atmospheric pressure with altitude. The payload fairing is jettisonned at approximately 400,000 ft after launch, where little or no atmospheric contamination remains. The payload fairing separation mechanisms are designed and tested so that virtually no particle debris from the jettison operation will contact the spacecraft. In summary, little or no recontamination of the spacecraft should occur during launch. As an additional precaution, starting with the launch of the Mars Science Laboratory (MSL), scheduled for 2009, payload fairing manufacturers will be required to adhere to planetary protection cleanliness requirements for all inside surfaces of the shroud. These measures are being required to minimize any spore migration within the shroud. CURRENT LIMITATIONS OF STANDARD METHODS AND IMPLEMENTING REQUIREMENTS Although COSPAR’s planetary protection policies and NASA’s implementation requirements have been modified over time, the standard NASA methods and practices for cleaning, sterilizing, and assaying spacecraft in preparation for launch have remained largely unchanged for nearly 3 decades. Even the 1992 NRC report on Mars forward contamination accepted the continuing use of established practices while encouraging the development and adoption of more modern molecular methods for assaying spacecraft and spacecraft assembly clean rooms (see Appendix B). For the most part, the standard methods and practices were developed through an extensive research program during the early years of the space program, particularly before the launch of the Viking landers (Bionetics, 1990). In addition, there is no standard certification process for approval of new methods, whether for microbial reduction or assaying, both of which currently require extensive documentation of process effectiveness, reproducibility, and equipment reliability before approval as alternate methods. There are no requirements for 20 The payload fairing (also referred to as the launch vehicle shroud) is the uppermost section of the launch vehicle that encloses the payload, protecting it from atmospheric dynamic pressure and heating during the initial ascent through the atmosphere. The payload fairing is typically designed in two sections resembling a clamshell. At burnout of the first stage of the launch that occurs around 400,000 ft, the payload fairing is jettisoned with explosive bolts that separate the fairing at the clamshell seam and at its base, where it interfaces with the launch vehicle adapter. The two halves fall away from the launch vehicle before the second stage is ignited and the launch flight continued.
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Preventing the Forward Contamination of Mars developing and maintaining information on microbial phylogenetic diversity and densities in clean-room environments or on prelaunch spacecraft. Moreover, the period required for retaining organic inventory archival information is relatively brief in relation to the lengthening period of biological exploration. The committee has identified a number of limitations to the NASA standard methods and planetary protection requirements. In particular, improvements in implementation practices and/or additional research are needed in areas related to (1) bioburden reduction and sterilization methods; (2) assigned parameter values and specifications used in contamination control planning; (3) microbial assay methods based on quicker, more sensitive molecular techniques that yield greater resolution of actual microbial bioburdens; (4) assessment of embedded bioburdens in contemporary spacecraft hardware; (5) characterization and inventory of the phylogenetic diversity and true microbial densities on spacecraft and in clean rooms before launch; and (6) time requirements for maintaining organics inventory information and materials. These improvements are especially needed in light of ambitious plans for future Mars exploration, new knowledge about the martian environment relative to life, and the limits of life on Earth that are discussed in the following chapters. REFERENCES Atwood, K.C. 1966. 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Preventing the Forward Contamination of Mars National Research Council (NRC). 1978. Recommendations on Quarantine Policy for Mars, Jupiter, Saturn, Uranus, Neptune and Titan. National Academy Press, Washington, D.C. NRC. 1992. Biological Contamination of Mars: Issues and Recommendations. National Academy Press, Washington, D.C. Pace, N.R. 1997. A molecular view of microbial diversity and the biosphere. Science 276: 734-740. Sagan, C., and S. Coleman. 1965. Spacecraft sterilization standards and contamination of Mars. J. Astronaut. Aeronaut. 3: 22-27. Sagan, C., and S. Coleman. 1966. Decontamination standards for martian exploration programs. Pp. 470-481 in Biology and the Exploration of Mars, C.S. Pittendrigh, W. Vishniac, and J.P.T. Pearman, eds. NRC Publication 1296. National Academy of Sciences, Washington, D.C.
Representative terms from entire chapter: