National Aeronautics and Space Administration Planetary Protection Office

A Draft Test Protocol for Detecting Possible Biohazards in Martian Samples Returned to Earth (pdf)

J.D. Rummel, M.S.Race, D.L. DeVincenzi, P. J. Schad, P.D. Stabekis, M.
Viso & S. E. Acevedo (eds.), NASA/CP-2002-211842, Washington DC (2002)

Workshop Summary & Documents

Summary of the Draft Test Protocol
In anticipation of missions to Mars that will involve the return of martian materials, the NASA Planetary Protection officer convened a series of workshops to produce a Draft Protocol by which returned martian samples could be assessed for biological hazards and examined for evidence of life (extant or extinct), while safeguarding the samples from possible terrestrial contamination. At the end of the workshop series, a working draft of the protocol was subsequently reviewed and revised by an external oversight committee to yield the Draft Protocol.

The Draft Protocol is intended to be just that; it is neither practical nor useful for the Draft Protocol to be developed into a final form at this time. The Draft protocol provides a proof-of-concept model of the final protocol, demonstrating one approach (and more importantly, a sufficient approach) to testing returned Mars samples for possible biohazards or biological activity of martian origin. It provides “a sequential series of tests that can be applied to martian samples to provide data that can be used to make decisions about the release of unsterilized samples from containment-either wholly or partially-while allowing for an earlier release of samples subjected to a decontamination process (‘sterilization’) to ensure they are safe for analyses outside of containment.”

The Draft Protocol is expected to evolve both in content and implementation in coming years as a result of new or improved analytical methodologies or expanded states of knowledge in physical, chemical, and biological research. It is anticipated that the final protocol will receive its final review at or about the time the first samples leave the martian surface.

Overview of the Draft Test Protocol

The Draft Protocol has one basic purpose – to ensure that a representative set of sub-samples undergoes sufficient testing to evaluate them against the release criteria. The Draft Protocol has three main segments: Physical/Chemical (P/C) processing, Life Detection (LD) testing, and Biohazard (BH) testing.

The overall process is as follows: the samples will be removed from the Sample Return Canister (SRC) under maximum biocontainment in gloveboxes containing an inert gas atmosphere and housed within a combination cleanroom/biosafety lab. After initial documentation, samples will undergo preliminary characterization, splitting, and detailed examination using a variety of different methodologies. Ultimately, data from LD and BH testing will be used to determine whether to release materials from biocontainment. All sample materials not selected for further testing will be archived in sealed containers in an inert atmosphere glovebox within the lab for future scientific purposes (the ‘bank’).

Physical/Chemical Testing

The overall objective for P/C processing is to specify information about the samples required to enable effective LD and BH testing, and curation, as well as to provide information needed for sample preservation purposes. All returned materials will be examined at least minimally to help avoid a worst case scenario where an obviously biogenic sample could be stored unexamined an only discovered after nominal LD/BH tests were complete. P/C processing can be divided into three phases in roughly sequential order:

  1. Pre-processing, before preliminary examination of the samples
  2. Preliminary examination and screening of gas, fines, and solids, to permit informed choices about samples for later detailed testing, banking, or curation
  3. Sub-division of samples selected for Life Detection and Biohazard tests.

Preliminary examination and screening will be different for the three types of samples: gases, homogeneous particulate samples, and inherently inhomogeneous samples like rocks, rock cores, and regolith cores. Each of these sample types follows a different track through preliminary examination and screening. All characterization procedures in the P/C processing are nominally non-contact and non-destructive.

Because it is likely that the returned samples will not be exactly as we imagine them now, treatment of potentially complex samples cannot be defined in advance and will require a mechanisms such as an SRF oversight committee to adjust the final protocol to fit the actual samples.

Life Detection Testing

The proposed Life Detection (LD) analyses will use a broad definition of and criteria for life and an approach for detecting life not intended to be limited by the specific features of life as we know it on Earth. This approach will begin with, and rely on, “signatures” of various types that encompass all known terrestrial life, and that might encompass non-terrestrial life ( e.g., structures, structural and biosynthetic chemistry, isotopic patterns, and geochemical features)

Analytical methods are divided into those that facilitate a wide survey of a representative portion of different sample types, and those that facilitate a more focused, but high-resolution, examination of areas or interest. Survey methods, which are less destructive of samples, include microscopy, broad-band fluorescence, surface scanning and chemistry, tomography, and isotope release experiments. These methods seek structural and basic chemical signatures, and local inhomogeneities. Higher resolution methods, which are generally more destructive, include mass spectroscopic methods, combustion, isotope analysis, and electron microprobe procedures for elemental mapping. Standard microbiological examinations and culturing will also be used, despite their known limitations for detecting many terrestrial organisms.

There are three possible outcomes of the Life Detection procedures:

  1. Failure to detect any of the biosignatures described above, and absence of any carbon or complex carbon in representative samples. This result would lead to proposals for downgrading of the containment level for controlled distribution.
  2. Clear and overwhelming evidence of living organisms that appears to be of non-terrestrial origin. This finding could result in the continued containment of all unsterilized samples for an indefinite period of time, until the living organisms are better understood. Biological experimentation and biohazard assessment would be given highest priority.
  3. The third and most likely scenario lies between these extremes, where clear evidence of life or its absence is not forthcoming. An example would be a situation in which complex carbon-containing compounds were detected in the sample, but without other evidence of life or biosignatures.

If viable cells are found in the samples, and especially in cultures taken from samples, it will be important to address the possibility of terrestrial microbial contamination using phenotypic and genotypic analyses on detected cells to identify contaminants quickly.

As methods mature and new approaches become available, these sample testing requirements may change. It is anticipated that survey methods can be completed within weeks to months, while other parts of the protocol such as enrichment culture experiments may extend for many months.

Biohazard Testing

The biohazard testing process is intended to determine if samples from Mars pose any threat to terrestrial organisms or ecosystems, regardless of whether the samples are found to contain life forms or non-replicative hazards. Since potential hazards could take one or more of a multitude of forms, the spectrum of tests selected is deliberately diverse. In practical terms, Biohazard testing should allow a determination of whether the samples contain any biohazard and whether to distribute sub-samples while providing a reasonable assurance that the samples will not put humans or other terrestrial organisms at risk.

The pathway of experiments for biohazard testing includes a combination of in vitro- , in vivo- and molecular biology tests as well as model ecosystem tests. A general approach for Biohazard testing, rather than a specific list of tests, was considered the most useful and responsible approach for deliberations at this time. Broad spectrum biohazard tests will likely include a combination of direct culture, exposure of cellular and ‘small models, molecular and biological tests to detect proteins and metabolites, assays to detect genetic damage (mutagenesis, DNA damage, altered gene expression), whole organisms exposure, and model ecosystem tests.

The data from biohazard testing will be used in combination with those from Life Detection and Physical/Chemical testing to determine what level of containment, if any, will be required for the further study of the samples. If sufficient data are gathered to rule out concerns about human virulence and infection, a decision could be made later to allow subsequent work at a lower containment level. The biohazard testing process is designed to allow for gradual decontainment or adjustment to less stringent containment levels if justified. Biohazard testing will be conducted within containment at the primary receiving facility or at other secure containment facilities. Since neither all the necessary scientific expertise, nor all of the required high-end scientific instrumentation are located at a single facility, there may be a need to allow samples to be distributed for study and curation at facilities other than the initial receiving laboratory.

Other Issues

The Draft Protocol also discusses several key issues that must be addressed to prepare for the handling and testing process. These include containment, sterilization and criteria for release.

Containment in the Sample Receiving Facility (SRF): In addition to satisfying both biosafety and cleanliness needs, the SRF will need to provide different types of laboratory environments for carrying out the various aspects of protocol testing. It is envisaged that all samples initially returned from Mars will be placed in a single SRF and held there through the preliminary examination phase. The use of multiple containment facilities to accomplish different aspects of the protocol was not ruled out. A new term “Planetary Protection Level” (PPL) was developed for the purpose of categorizing and describing the different combinations of containment and cleanliness conditions required. The four PPLs are described as follows:

PPL-α – for incoming samples and archived samples; maximum biocontainment (BSL-4) and cleanliness; maintains sample in an inert gas environment and Mars-like conditions;

PPL-β – maintains maximum biocontainment and protection for workers and the environment; maximum cleanliness, but allows exposure to ambient terrestrial conditions;?

PPL-γ – maintains maximum biocontainment with moderate cleanliness and ambient terrestrial conditions (i.e., for animal testing scenarios); and

PPL-δ -maintains BSL-3-Agriculture standards for containment conditions, with less emphasis on cleanliness, and under ambient terrestrial conditions.

Sterilization of Samples: Only two methods of sterilization are considered viable options for martian samples-dry heat and gamma radiation, either alone or in combination. Within reason, every effort should be made to develop and implement a method(s) of sterilization that protects the scientific integrity of the samples under appropriate conditions, while ensuring an adequate margin of safety for destroying putative martian organisms.

Criteria for Release: The following criteria govern the release of samples evaluated using this Draft Protocol:

  1. No solid sample shall be released from containment until it or its parent sample undergoes preliminary examination, baseline description, cataloguing, and any necessary repackaging. Samples to be used for Life Detection procedures or to be released from containment will be screened for radioactivity and potential chemical hazards. Additionally, samples to be used for Biohazard testing will be screened for known toxicity to bacterial and eukaryotic cells.
  2. Samples containing any active martian form of life, be it hazardous or not, will be kept under the appropriate level of containment, or be thoroughly sterilized before release.
  3. Samples providing indications of life-related molecules, including proteins, nucleic acids, or molecular chirality, will require more extensive testing, including additional Biohazard testing, prior to their release. Samples may be released if they are first subjected to a sterilizing process involving heat, radiation, or a combination of these agents, to ensure they are safe for analyses outside of containment. A sample that is “safe” is stipulated to be free of any viable self-replicating entities or entities able to be amplified.
  4. Samples may be released if Biohazard testing does not yield evidence of live, extraterrestrial, self-replicating entities, or of harmful effects on terrestrial life forms or environments under Earth-like conditions. Biohazard testing will involve assays for replication and effect or growth on various cultures, whole organisms, and ecosystems. Basic biohazard testing will be required even in the absence of evidence of organic carbon in a martian sample.

Sample Receiving Facility

Based on experience following receipt of lunar samples, the primary SRF should be designed to be expandable and allow great flexibility in functions as needed. The primary SRF should be designed to allow continuous and long-term operation in addition to accomplishing its primary goal of receiving the Mars samples and implementing the final protocol. The overall planning and development timetable for the SRF is based on the following assumptions:

  1. The protocol must be fully and successfully tested before the actual handling of martian samples.
  2. It is estimated that a complete Experiment Verification Test (EVT) will last approximately 6 months and at least one complete EVT must be demonstrated successfully before actual handling of the returned samples.
  3. These EVTs are consistent with the recommendation of the SSB (1997) that the SRF be operational two years before the arrival of the actual Mars samples.
  4. Based on experiences at other BSL-4 laboratories, no less than one year is required to staff and properly train the technical and scientific personnel
  5. Commissioning the SRF will require at least 18 months.
  6. Construction of the facility must be finished 3 years before actual operations in order to accommodate the staffing, training and commissioning requirements.
  7. Development of design specifications and plans will take three years and, based on experience, construction of the facility will also require 3 years

To accommodate all the activities necessary to design, build and operate an SRF, the entire process must begin fully ten years in advance of sample return.

Environmental and Health Monitoring and Safety

Procedures for monitoring the health and safety of personnel of the SRF and the environment in and around the SRF (as well as secondary sites if used) must be developed and implemented as part of the final protocol. These will require a consideration of monitoring over time and an assessment of how long to continue monitoring, beginning prior to the arrival of Mars samples and continuing during work on the samples at the SRF and at secondary sites, and for some time thereafter. The monitoring plan should allow for cross-correlation of the data from the Life Detection and biohazard testing with the data from monitoring.

Five categories of hazards were considered: physical hazards, potential chemical hazards from non-biological toxins, biological hazards, psychological hazards (for both workers and the public risk perception), and loss of containment itself.

Physical hazards from radiation and equipment and chemical hazards from non-biological toxins will be assessed early in the P/C testing using standard methods, procedures, training and maintenance.

Standard methods for monitoring and correcting containment problems can be adapted for use in the SRF in cases of breaches inside the SRF or to the external environment. Procedures for handling different types of breaches should be considered in response plans.

Monitoring the environment around the SRF should be undertaken before Mars sample arrival, during sample handling at the SRF, and after completion of LD and BH testing. Environmental monitoring may also include surveillance of humans in the nearby population if warranted by the SRF location.

Monitoring of the SRF personnel. Baseline medical evaluations and training of personnel should be done prior to the arrival of samples from Mars. Regular evaluations of personnel should also be done during sample handling as well as after the completion of life detection/biohazard testing.

Monitoring at Secondary Sites: The level of monitoring to be used at secondary sites receiving and working on portions of the martian samples should be based on the results of LD and BH testing. Monitoring of environment and personnel at secondary sites should use the same protocols as at the SRF.

Database Issues: A central database with data analysis capabilities and procedures should be used for environmental data, personnel data, secondary site data and sample tracking data. Access to, and confidentiality of data should be defined and assured; and consideration should be given to requirements for ethical review and approval for any research protocols.

Personnel Management Considerations: The future operations and staffing of the SRF can be accomplished in a number of ways (ex. permanent staff, competitive process, or a combination of the two approaches). It’s likely that a variety of personnel selection processes will be used. A conceptual approach and plan was developed for considering staffing and organizational needs. While the scenario for the operation and staffing of the SRF is yet to be determined, it is apparent that planning should begin at least 10 years in advance in order to meet future needs.

Contingency Planning for Different Outcomes

Contingency plans for different outcomes of the final protocol must be developed in advance. Recommend responses to various likely scenarios should be considered (presence of organic carbon; evidence of extant life or biomarkers; or non-Earth life confirmed) . Although it is premature to develop specific recommendations at this time, it is possible to identify three categories of issues that will need discussion in advance-science and testing issues, facility and technological concerns, and policy and administrative concerns. Special consideration should also be given in advance to how contradictory or inconsistent results will be handled, how release criteria will be applied, and how breaches of containment will be handled.

Maintaining and Updating the Protocol

A protocol implementation and update process will be required that includes the involvement of key oversight and review committees to re-evaluate proposed plans at key points in time before sample return. Open communication should be maintained with scientists, international partners and the public regarding risks, benefits and plans. Evaluations of the protocol or revisions should be reviewed both domestically and internationally by broad scientific and public audiences, particularly since changes in scientific methodology and instrumentation are inevitable due to the long development time anticipated.

Advisory committees and expert panels should be appointed to provide appropriate expertise in reviewing technical processes, scientific procedures, and safety/biosafety issues, as well as other areas of concern. Among the other items that must be considered in advance are: development of a proactive communication plan to inform the public and scientific community about results and findings; development of flow charts and timelines to coordinate the complex series of interrelated procedures ahead; scheduling of workshops to review plans and designs at appropriate times in advance of sample arrival; and preparations and processes for decision making about release of samples.